RÉSUMÉ
Cytokine engineering has shown promise as a means to create novel immunomodulatory agents or to improve upon the therapeutic potential of natural cytokines. NL-201, a de novo, hyperstable, IL2 receptor alpha (IL2Rα)-independent agonist of the receptors for IL2 and IL15, elicits robust preclinical activity in syngeneic murine cancer models, including those resistant to immune checkpoint inhibitors (ICI). Here, we report that NL-201 monotherapy converts 'cold' tumor microenvironments (TME) to immunologically 'hot' states by driving pro-inflammatory gene expression, enhancing IFNγ-dependent MHC-I expression, and expanding both T-cell number and clonal diversity. In addition, the combination of NL-201 and anti-PD-1 resulted in complementary antitumor activity in the immunologically 'cold' and ICI resistant B16F10, EMT6, and Renca syngeneic models. In the B16F10 model, treatment with NL-201 plus anti-PD-1 increased the abundance of CD4+ and CD8+ effector T cells in the TME. These findings reveal an important mechanistic basis for the antitumor activity of NL-201 both as a monotherapy and in combination with PD-1 antagonists, and provide further context for the role of IL2Rα-based signaling in ICI-resistant tumors.
Sujet(s)
Tumeurs , Souris , Humains , Animaux , Lymphocytes T/métabolisme , Cytokines/usage thérapeutique , Récepteurs aux antigènes des cellules T/usage thérapeutique , Microenvironnement tumoral , Lymphocytes T CD8+ , Lignée cellulaire tumoraleRÉSUMÉ
Microproteins (MPs) are a potentially rich source of uncharacterized metabolic regulators. Here, we use ribosome profiling (Ribo-seq) to curate 3,877 unannotated MP-encoding small ORFs (smORFs) in primary brown, white, and beige mouse adipocytes. Of these, we validated 85 MPs by proteomics, including 33 circulating MPs in mouse plasma. Analyses of MP-encoding mRNAs under different physiological conditions (high-fat diet) revealed that numerous MPs are regulated in adipose tissue in vivo and are co-expressed with established metabolic genes. Furthermore, Ribo-seq provided evidence for the translation of Gm8773, which encodes a secreted MP that is homologous to human and chicken FAM237B. Gm8773 is highly expressed in the arcuate nucleus of the hypothalamus, and intracerebroventricular administration of recombinant mFAM237B showed orexigenic activity in obese mice. Together, these data highlight the value of this adipocyte MP database in identifying MPs with roles in fundamental metabolic and physiological processes such as feeding.
Sujet(s)
Adipocytes blancs , Tissu adipeux brun , Humains , Animaux , Souris , Adipocytes blancs/métabolisme , Tissu adipeux brun/métabolisme , Cadres ouverts de lecture/génétique , Tissu adipeux blanc/métabolisme , Adipocytes bruns/métabolisme ,RÉSUMÉ
Antibody-drug conjugates (ADC) rely on the target-binding specificity of an antibody to selectively deliver potent drugs to cancer cells. IgG antibody half-life is regulated by neonatal Fc receptor (FcRn) binding. Histidine 435 of human IgG was mutated to alanine (H435A) to explore the effect of FcRn binding on the pharmacokinetics, efficacy, and tolerability of two separate maytansine-based ADC pairs with noncleavable linkers, (c-DM1 and c-H435A-DM1) and (7v-Cys-may and 7v-H435A-Cys-may). The in vitro cell-killing potency of each pair of ADCs was similar, demonstrating that H435A showed no measurable impact on ADC bioactivity. The H435A mutant antibodies showed no detectable binding to human or mouse FcRn in vitro, whereas their counterpart wild-type IgG ADCs were found to bind to FcRn at pH = 6.0. In xenograft bearing SCID mice expressing mouse FcRn, the AUC of 7v-Cys-may was 1.6-fold higher than that of 7v-H435A-may, yet the observed efficacy was similar. More severe thrombocytopenia was observed with 7v-H435A-Cys-may as compared to 7v-Cys-may at multiple dose levels. The AUC of c-DM1 was approximately 3-fold higher than that of c-H435A-DM1 in 786-0 xenograft bearing SCID mice, which led to a 3-fold difference in efficacy by dose. Murine FcRn knockout, human FcRn transgenic line 32 SCID animals bearing 786-0 xenografts showed an amplified exposure difference between c-DM1 and c-H435A-DM1 as compared to murine FcRn expressing SCID mice, leading to a 10-fold higher dose required for efficacy despite a 6-fold higher AUC of the c-H435A-DM1. The accelerated clearance observed for the noncleavable maytansine ADCs with the H435A FcRn mutation led to reduced efficacy at equivalent doses and exacerbation of clinical pathology parameters (decreased tolerability) at equivalent doses. The results show that reduced ADC clearance mediated by FcRn modulation can improve therapeutic index.
Sujet(s)
Anticorps/métabolisme , Antigènes d'histocompatibilité de classe I/métabolisme , Immunoconjugués/pharmacologie , Immunoglobuline G/métabolisme , Récepteur Fc/métabolisme , Animaux , Anticorps/génétique , Antigènes CD70/métabolisme , Lignée cellulaire , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Femelle , Antigènes d'histocompatibilité de classe I/génétique , Humains , Immunoconjugués/composition chimique , Maitansine/métabolisme , Souris , Souris SCID , Récepteur Fc/génétiqueRÉSUMÉ
FLT3(ITD) subtype acute myeloid leukemia (AML) has a poor prognosis with currently available therapies. A number of small molecule inhibitors of FLT3 and/or CDK4/6 are currently under development. A more complete and quantitative understanding of the mechanisms of action of FLT3 and CDK4/6 inhibitors may better inform the development of current and future compounds that act on one or both of the molecular targets, and thus may lead to improved treatments for AML. In this study, we investigated in both subcutaneous and orthotopic AML mouse models, the mechanisms of action of three FLT3 and/or CDK4/6 inhibitors: AMG925 (Amgen), sorafenib (Bayer and Onyx), and quizartinib (Ambit Biosciences). A composite model was developed to integrate the plasma pharmacokinetics of these three compounds on their respective molecular targets, the coupling between the target pathways, as well as the resulting effects on tumor burden reduction in the subcutaneous xenograft model. A sequential modeling approach was used, wherein model structures and estimated parameters from upstream processes (e.g. PK, cellular signaling) were fixed for modeling subsequent downstream processes (cellular signaling, tumor burden). Pooled data analysis was employed for the plasma PK and cellular signaling modeling, while population modeling was applied to the tumor burden modeling. The resulting model allows the decomposition of the relative contributions of FLT3(ITD) and CDK4/6 inhibition on downstream signaling and tumor burden. In addition, the action of AMG925 on cellular signaling and tumor burden was further studied in an orthotopic tumor mouse model more closely representing the physiologically relevant environment for AML.
Sujet(s)
Kinase-4 cycline-dépendante/antagonistes et inhibiteurs , Kinase-6 cycline-dépendante/antagonistes et inhibiteurs , Leucémie aigüe myéloïde/traitement médicamenteux , Inhibiteurs de protéines kinases/pharmacologie , Transduction du signal/effets des médicaments et des substances chimiques , Charge tumorale/effets des médicaments et des substances chimiques , Tyrosine kinase-3 de type fms/antagonistes et inhibiteurs , Animaux , Benzothiazoles/pharmacologie , Lignée cellulaire tumorale , Kinase-4 cycline-dépendante/métabolisme , Kinase-6 cycline-dépendante/métabolisme , Modèles animaux de maladie humaine , Leucémie aigüe myéloïde/métabolisme , Souris , Nicotinamide/analogues et dérivés , Nicotinamide/pharmacologie , Phénylurées/pharmacologie , Sorafénib , Tyrosine kinase-3 de type fms/métabolismeRÉSUMÉ
We describe the structural optimization of a lead compound 1 that exhibits dual inhibitory activities against FLT3 and CDK4. A series of pyrido[4',3':4,5]pyrrolo[2,3-d]pyrimidine derivatives was synthesized, and SAR analysis, using cell-based assays, led to the discovery of 28 (AMG 925), a potent and orally bioavailable dual inhibitor of CDK4 and FLT3, including many FLT3 mutants reported to date. Compound 28 inhibits the proliferation of a panel of human tumor cell lines including Colo205 (Rb(+)) and U937 (FLT3(WT)) and induced cell death in MOLM13 (FLT3(ITD)) and even in MOLM13 (FLT3(ITD, D835Y)), which exhibits resistance to a number of FLT3 inhibitors currently under clinical development. At well-tolerated doses, compound 28 leads to significant growth inhibition of MOLM13 xenografts in nude mice, and the activity correlates with inhibition of STAT5 and Rb phosphorylation.
Sujet(s)
Kinase-4 cycline-dépendante/antagonistes et inhibiteurs , Composés hétérocycliques 3 noyaux/synthèse chimique , Naphtyridines/synthèse chimique , Inhibiteurs de protéines kinases/synthèse chimique , Tyrosine kinase-3 de type fms/antagonistes et inhibiteurs , Animaux , Kinase-6 cycline-dépendante/antagonistes et inhibiteurs , Cytochrome P-450 CYP3A , Inhibiteurs du cytochrome P-450 CYP3A , Chiens , Découverte de médicament , Composés hétérocycliques 3 noyaux/pharmacologie , Humains , Macaca fascicularis , Naphtyridines/pharmacologie , Inhibiteurs de protéines kinases/pharmacocinétique , Inhibiteurs de protéines kinases/pharmacologie , Rats , Relation structure-activité , Cellules U937 , Tyrosine kinase-3 de type fms/génétiqueRÉSUMÉ
Acute myeloid leukemia (AML) remains a serious unmet medical need. Despite high remission rates with chemotherapy standard-of-care treatment, the disease eventually relapses in a major proportion of patients. Activating Fms-like tyrosine kinase 3 (FLT3) mutations are found in approximately 30% of patients with AML. Targeting FLT3 receptor tyrosine kinase has shown encouraging results in treating FLT3-mutated AML. Responses, however, are not sustained and acquired resistance has been a clinical challenge. Treatment options to overcome resistance are currently the focus of research. We report here the preclinical evaluation of AMG 925, a potent, selective, and bioavailable FLT3/cyclin-dependent kinase 4 (CDK4) dual kinase inhibitor. AMG 925 inhibited AML xenograft tumor growth by 96% to 99% without significant body weight loss. The antitumor activity of AMG 925 correlated with the inhibition of STAT5 and RB phosphorylation, the pharmacodynamic markers for inhibition of FLT3 and CDK4, respectively. In addition, AMG 925 was also found to inhibit FLT3 mutants (e.g., D835Y) that are resistant to the current FLT3 inhibitors (e.g., AC220 and sorafenib). CDK4 is a cyclin D-dependent kinase that plays an essential central role in regulating cell proliferation in response to external growth signals. A critical role of the CDK4-RB pathway in cancer development has been well established. CDK4-specific inhibitors are being developed for treating RB-positive cancer. AMG 925, which combines inhibition of two kinases essential for proliferation and survival of FLT3-mutated AML cells, may improve and prolong clinical responses.
Sujet(s)
Kinase-4 cycline-dépendante/antagonistes et inhibiteurs , Composés hétérocycliques 3 noyaux/administration et posologie , Leucémie aigüe myéloïde/traitement médicamenteux , Naphtyridines/administration et posologie , Inhibiteurs de protéines kinases/administration et posologie , Tyrosine kinase-3 de type fms/antagonistes et inhibiteurs , Animaux , Apoptose/effets des médicaments et des substances chimiques , Cycle cellulaire/effets des médicaments et des substances chimiques , Lignée cellulaire tumorale , Prolifération cellulaire/effets des médicaments et des substances chimiques , Relation dose-effet des médicaments , Composés hétérocycliques 3 noyaux/pharmacocinétique , Composés hétérocycliques 3 noyaux/usage thérapeutique , Humains , Leucémie aigüe myéloïde/génétique , Leucémie aigüe myéloïde/anatomopathologie , Souris , Souris nude , Naphtyridines/pharmacocinétique , Naphtyridines/usage thérapeutique , Tumeurs expérimentales , Nicotinamide/analogues et dérivés , Nicotinamide/pharmacologie , Phénylurées/pharmacologie , Pipérazines/pharmacologie , Inhibiteurs de protéines kinases/pharmacocinétique , Pyridines/pharmacologie , Transduction du signal/effets des médicaments et des substances chimiques , Sorafénib , Cellules U937 , Tests d'activité antitumorale sur modèle de xénogreffeRÉSUMÉ
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to death receptors 4 and 5 (DR4, DR5) to transduce apoptotic signals. Conatumumab (AMG 655) is an investigational, fully human monoclonal agonist antibody (IgG(1)) to human DR5, which induces apoptosis via caspase activation. In this study, we demonstrate that conatumumab binds to DR5, activating intracellular caspases in vitro in the presence of a cross-linker. We also show that conatumumab has activity in vivo and inhibits tumor growth in colon (Colo205 and HCT-15), lung (H2122) and pancreatic (MiaPaCa2/T2) xenograft models. Conatumumab also enhances the antitumor activity of chemotherapeutics in vivo. Caspase activation in Colo205 tumors is dose-dependent and correlated with serum concentrations of conatumumab. We demonstrate for the first time that increases in serum caspase-3/7 activity and levels of M30 (neoepitope of caspase-cleaved cytokeratin-18) are linked to activation of the extrinsic apoptotic pathway using conatumumab in a preclinical model. These data suggest that conatumumab has potential as a therapeutic agent for treating patients with multiple tumor types, and that serum caspase-3/7 and M30 levels may serve as biomarkers of conatumumab activity.