RESUMO
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
The immunotoxic potential of drug candidates is assessed through the examination of results from a variety of studies and endpoints. While the functional assessment of CD8+ cytotoxic T-lymphocytes (CTL) is well-characterized in the clinic, the lack of a robust macaque CTL functional assay has been an important hurdle in evaluating and accurately quantifying cell-mediated CD8+ T-cell effector responses in the nonclinical setting. This paper describes the development of an assay to measure CTL activity in peripheral blood mononuclear cells (PBMC) isolated from Cynomolgus macaques. A human EGFR/CD3 Bispecific T-cell Engager (BiTE®) was used to mount a robust CD8+ T-cell response in the presence of target-expressing cells. Upon target engagement, degranulation of CD107a and production of interferon (IFN)-γ both reliably indicated a robust functional response in CD8+ T-cells. The BiTE®-mediated stimulation method proved to be favorable when compared to other methods of stimulation in the absence of target cells. These studies demonstrated acceptable longitudinal variability of the functional assay and sensitivity to dexamethasone-mediated immunosuppression. Taken together, the results indicated an assay leveraging CD3-bispecific antibodies and target-expressing cells can provide a robust approach to the in vitro or ex vivo assessment of CTL function in Cynomolgus macaques. Because the impairment of CTL activity by immunomodulators is recognized to be an important contributor to decreased antiviral defense and increased carcinogenicity risk, we believe that this novel assay to be a valuable addition to the immunotoxicology assessment of therapeutic drug candidates.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Imunoensaio/métodos , Macaca fascicularis/imunologia , Animais , Anticorpos Biespecíficos/metabolismo , Complexo CD3/imunologia , Células Cultivadas , Avaliação Pré-Clínica de Medicamentos , Receptores ErbB/imunologia , Humanos , Interferon gama/metabolismo , Leucócitos Mononucleares/citologia , Ativação Linfocitária , Proteína 1 de Membrana Associada ao Lisossomo/metabolismo , Variações Dependentes do ObservadorRESUMO
OBJECTIVE: To determine whether a combination of B cell depletion and BAFF blockade is more effective than monotherapy in treating models of spontaneous or accelerated systemic lupus erythematosus (SLE) in (NZB × NZW)F1 mice. METHODS: Clinical parameters such as disease progression-free survival, proteinuria, and renal injury were assessed in models of spontaneous, interferon-α (IFNα)-accelerated, or pristane-accelerated lupus in (NZB × NZW)F1 mice. Treatment arms included anti-CD20 (B cell depletion), B lymphocyte stimulator receptor 3 fusion protein (BR-3-Fc) (BAFF blockade), the combination of anti-CD20 and BR-3-Fc, isotype control, or cyclophosphamide. In models of spontaneous, IFNα-accelerated, or pristane-accelerated lupus, mice were treated for 24 weeks, 8 weeks, or 12 weeks, respectively. Peripheral and resident B cell subsets and various autoantibodies were examined. RESULTS: Compared to B cell depletion or BAFF blockade alone, combined therapy significantly improved disease manifestations in all 3 lupus models. In addition, marginal zone B cells, plasmablasts, and circulating and tissue plasma cells were decreased more effectively. Dual B cell immunotherapy also reduced multiple classes of pathogenic autoantibodies, consistent with its observed effectiveness in reducing immune complex-mediated renal injury. CONCLUSION: Dual immunotherapy via B cell depletion and BAFF blockade is more efficacious than single agent immunotherapy in murine SLE models, and this combination treatment is predicted to be an effective strategy for immunotherapy in human SLE.