RESUMEN
Cytokine release syndrome (CRS) is a serious and potentially life-threatening complication typically associated with biological drug products. Pre-clinical testing in vitro and in vivo studies using non-human primates had failed to reliably predict CRS. To determine if bone marrow-thymus-liver (BLT) humanized mice with a fully engrafted human immune system or a CD34-humanized mouse model could predict CRS, we tested an anti-CD28 monoclonal antibody (mAb) similar to TGN1412. This TGN1412 analogue (TGN1412A) was initially tested in vitro and found to produce significant dose-dependent increases in cytokine production. For in vivo studies, adalimumab, an anti-tumor necrosis factor-alpha antibody known not to cause CRS, served as a negative control. We evaluated immune cell activation and cytokine expression in three independent experiments. In BLT humanized mice, significant increases in levels of human cytokines were identified in animals treated with anti-CD28 mAb. As expected, CD28+ cell detection was strongly reduced in the anti-CD28 treated group. Increased T cell activation was also observed. The control group did not show reductions in CD28+ T-cells and did not experience increased cytokine levels. Responses by CD34-humanized mice showed no significant differences between adalimumab and anti-CD28 treatment at doses used to test BLT-humanized mice. These results suggest that the TGN1412A produces similar results in vitro to the original TGN1412 monoclonal antibody. The BLT immune humanized mice but not the CD34 humanized mice produce both robust and specific cytokine responses and may represent a pre-clinical model to identify CRS.
Asunto(s)
Anticuerpos Monoclonales Humanizados/toxicidad , Antígenos CD28/antagonistas & inhibidores , Síndrome de Liberación de Citoquinas/etiología , Citocinas/sangre , Linfocitos T/efectos de los fármacos , Animales , Antígenos CD34/inmunología , Antígenos CD28/sangre , Antígenos CD28/inmunología , Células Cultivadas , Síndrome de Liberación de Citoquinas/sangre , Síndrome de Liberación de Citoquinas/inmunología , Citocinas/inmunología , Femenino , Trasplante de Células Madre Hematopoyéticas , Humanos , Trasplante de Hígado , Ratones Endogámicos NOD , Ratones SCID , Medición de Riesgo , Linfocitos T/inmunología , Linfocitos T/metabolismo , Timo/embriología , Timo/trasplanteRESUMEN
Toll-like receptors 7 and 8 are involved in modulating the adaptive and innate immune responses, and their activation has shown promise as a therapeutic strategy in the field of immuno-oncology. While systemic exposure to TLR7/8 agonists can result in poor tolerance, combination therapies and targeted delivery through antibody-drug conjugates (ADCs) can help mitigate adverse effects. Described herein is the identification of a novel and potent series of pyrazolopyrimidine-based TLR7/8 agonists with tunable receptor selectivity. Representative agonists from this series were successfully able to induce the production of various proinflammatory cytokines and chemokines from human peripheral blood mononuclear cells. Anti-HER2-25 and anti-HER2-26 ADCs made from this class of payloads demonstrated mechanism-based activation of TLR7/8 in a THP1/N87 coculture system.
RESUMEN
LAG3 and PD1 are both immunomodulatory receptor that act by inhibiting activation of T cells, producing a more immunosuppressive environment. Even with the recent clinical success of PD1 and LAG3 co-blockade, signal transduction downstream of LAG3 remains largely unknown. We have leveraged an engineered Jurkat (T cell) and Raji (APC) co-culture system to assess simultaneous blockade of PD1 and LAG3 pathways using antibodies. RNA-Seq analysis of cell pellets individually treated with LAG3 or PD1 antibodies revealed modest immune activation however, 5-fold more genes were upregulated upon combination treatment. There were increases in costimulatory genes like CD28, CD5, CD6 as well as intracellular signaling molecules like LCP2 and ITK. Given the role of ERK in immune activation of T cells, pERK levels of Jurkat cells in the assay were evaluated. A very modest activation of pERK was observed with anti-LAG3 compared to anti-PD1 but a combination treatment resulted in prolonged ERK phosphorylation. Treatment of Jurkat cells with a commercial phosphatase inhibitor NSC87877 which can impact many phosphatases resulted in immune activation, measured by increased IL2 levels, only in the presence of LAG3. When NSC87877 was combined with the PD1 antibody, it could phenocopy combination benefit of PD1 and LAG3 blocking antibodies. CD28 has a recognized role in PD1 signaling but the impact on LAG3 signaling remains unknown. CD28 knockout in Jurkat cells affected overall IL-2 response of both LAG3 and PD1 antibody treatment but still retained combination benefit. Taken together this reductionist system highlights differences in downstream effects of LAG3 and PD1 blockade and we believe that the assay may have further utility to dissect convergence of both signaling pathways and augment studies in primary cells.
Asunto(s)
Anticuerpos , Antígenos CD28 , Humanos , Antígenos CD28/genética , Células Jurkat , Anticuerpos Bloqueadores , FenotipoRESUMEN
Targeted immune agonist (TIA) comprising a TLR7 agonist conjugated to tumor-targeting antibodies have been shown to induce potent anti-tumor responses in various preclinical models. However, the clinical proof-of-concept of a TIA has been hampered by systemic dose-limiting immune-related toxicities, including rapid induction of anti-drug antibodies in patients. We have developed ELISPOT-based assay to measure activation of antibody-secreting cells (ASCs), intended to simulate the interaction between TIA and peripheral B cells as a tool to pre-clinically de-risk tumor target-independent peripheral B-cell activation by TIA. This method has proven to be robust and has fast turn-around time to evaluate the induction of spontaneous B-cell activation by TIA in a tumor target- and FcγR-independent manner. This novel ASC assay platform may serve as a preclinical tool to de-risk TIAs that can potentially induce immune-related adverse effects in the clinic.
Asunto(s)
Neoplasias , Receptor Toll-Like 7 , Humanos , Adyuvantes Inmunológicos , Anticuerpos , Activación de LinfocitosRESUMEN
LAG3 is an important regulator of T cell homeostasis and studies in mouse tumor models have demonstrated that simultaneously antagonizing LAG3 and PD1 can augment tumor-specific T cell responses and induce tumor rejection. The combined use of LAG3 antagonist antibodies with established anti-PD1 therapies is currently being evaluated in human clinical trials. A functional assay for human LAG3 was developed by co-culture of a Jurkat T-cell lymphoma line overexpressing LAG3 with a Raji B-cell lymphoma line in the presence of staphylococcal enterotoxins. Reversal of LAG3 repression was measured as an increase in IL-2 production or NFAT activation in response to treatment with MK-4280, an anti-human LAG3 antagonist antibody. Changes in cytokines, chemokines, and other mRNA transcripts were in agreement with published in vitro and in vivo models for LAG3 biology which highlights the physiological relevance of the Jurkat functional assay. Additional engineering of PD1 and PDL1 components into the LAG3 assay resulted in a bi-functional assay that is capable of inducing a 10-fold response to individual antibodies blocking either PD1 or LAG3. Importantly, when MK-4280 and pembrolizumab were combined to block both pathways, a synergistic 50-fold increase in response was observed.
Asunto(s)
Anticuerpos Bloqueadores/análisis , Antígenos CD/inmunología , Ingeniería Celular , Receptor de Muerte Celular Programada 1/inmunología , Transducción de Señal/inmunología , Anticuerpos Bloqueadores/inmunología , Antígenos CD/genética , Citometría de Flujo , Perfilación de la Expresión Génica , Humanos , Células Jurkat , Receptor de Muerte Celular Programada 1/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Transducción de Señal/genética , Linfocitos T/inmunología , Células Tumorales Cultivadas , Proteína del Gen 3 de Activación de LinfocitosRESUMEN
GITR is a T-cell costimulatory receptor that enhances cellular and humoral immunity. The agonist anti-mouse GITR antibody DTA-1 has demonstrated efficacy in murine models of cancer primarily by attenuation of Treg-mediated immune suppression, but the translatability to human GITR biology has not been fully explored. Here, we report the potential utility of MK-4166, a humanized GITR mAb selected to bind to an epitope analogous to the DTA-1 epitope, which enhances the proliferation of both naïve and tumor-infiltrating T lymphocytes (TIL). We also investigated the role of GITR agonism in human antitumor immune responses and report here the preclinical characterization and toxicity assessment of MK-4166, which is currently being evaluated in a phase I clinical study. Expression of human GITR was comparable with that of mouse GITR in tumor-infiltrating Tregs despite being drastically lower in other human TILs and in many human peripheral blood populations. MK-4166 decreased induction and suppressive effects of Tregsin vitro In human TIL cultures, MK-4166 induced phosphorylation of NFκB and increased expression of dual specificity phosphatase 6 (DUSP6), indicating that MK-4166 activated downstream NFκB and Erk signaling pathways. Furthermore, MK-4166 downregulated FOXP3 mRNA in human tumor infiltrating Tregs, suggesting that, in addition to enhancing the activation of TILs, MK-4166 may attenuate the Treg-mediated suppressive tumor microenvironment. Cancer Res; 77(16); 4378-88. ©2017 AACR.
Asunto(s)
Anticuerpos/farmacología , Proteína Relacionada con TNFR Inducida por Glucocorticoide/inmunología , Linfocitos T Reguladores/inmunología , Animales , Anticuerpos/inmunología , Línea Celular Tumoral , Femenino , Proteína Relacionada con TNFR Inducida por Glucocorticoide/agonistas , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C3H , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Microambiente TumoralRESUMEN
The aryl hydrocarbon receptor (AhR) is a ligand-dependent transcription factor that requires heterodimerization with its partner, the Ah receptor nuclear translocator (Arnt), for activation of transcription. The heterodimer specifically recognizes the dioxin response element (DRE), which contains a core sequence (5'-TNGCGTG-3'). This AhR/Arnt/DRE complex has been well characterized and can be observed readily by the gel shift assay. Human AhR and Arnt with a C-terminal histidine tag have been expressed functionally using a baculovirus expression system. However, after purification of these proteins using the metal resin, they are not able to bind the response element in a ligand-dependent manner unless crude extracts, such as the rabbit reticulocyte lysate (RRL), are reconstituted with these proteins. Proteins in the RRL are responsible for this restoration of the gel shift complex because the activity is sensitive to both heat and proteolytic treatments. We have examined whether hsp90 and p23 are among the protein factors in the RRL that are responsible for this activity. By performing fractionation studies using filtration devices and immunodepletion studies, we have selectively fractionated these proteins. Among all the fractions, the centricon-10 retentate, which contains 100% of p23 but no hsp90, possessed the most enriched activity. Purified bacterial-expressed p23 restored the gel shift complex; the mechanism was mediated at the heterodimerization step and was hsp90-dependent.
Asunto(s)
Proteínas de Unión al ADN , ADN/metabolismo , Chaperonas Moleculares/metabolismo , Fosfoproteínas/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Translocador Nuclear del Receptor de Aril Hidrocarburo , Ensayo de Cambio de Movilidad Electroforética , Proteínas HSP90 de Choque Térmico/metabolismo , Humanos , Prostaglandina-E Sintasas , Factores de Transcripción/metabolismoRESUMEN
Checkpoint kinase 1 (CHK1) is an essential serine/threonine kinase that responds to DNA damage and stalled DNA replication. CHK1 is essential for maintenance of replication fork viability during exposure to DNA antimetabolites. In human tumor cell lines, ablation of CHK1 function during antimetabolite exposure led to accumulation of double-strand DNA breaks and cell death. Here, we extend these observations and confirm ablation of CHK2 does not contribute to these phenotypes and may diminish them. Furthermore, concomitant suppression of cyclin-dependent kinase (CDK) activity is sufficient to completely antagonize the desired CHK1 ablation phenotypes. These mechanism-based observations prompted the development of a high-content, cell-based screen for γ-H2AX induction, a surrogate marker for double-strand DNA breaks. This mechanism-based functional approach was used to optimize small molecule inhibitors of CHK1. Specifically, the assay was used to mechanistically define the optimal in-cell profile with compounds exhibiting varying degrees of CHK1, CHK2, and CDK selectivity. Using this approach, SCH 900776 was identified as a highly potent and functionally optimal CHK1 inhibitor with minimal intrinsic antagonistic properties. SCH 900776 exposure phenocopies short interfering RNA-mediated CHK1 ablation and interacts synergistically with DNA antimetabolite agents in vitro and in vivo to selectively induce dsDNA breaks and cell death in tumor cell backgrounds.