RESUMO
Small interfering RNA (siRNA) is gaining momentum as a therapeutic modality with six approved products. Since siRNA has the potential to elicit undesired immune responses in patients, immunogenicity assessment is required during clinical development by regulatory authorities. In this study, anti-siRNA polyclonal antibodies were generated through animal immunization. These cross-reactive polyclonal antibodies recognized mostly the N-acetylgalactosamine (GalNAc) moiety with a small fraction against sequence-independent epitopes. We demonstrate that the polyclonal antibodies can be utilized as immunogenicity assay positive controls for the same class of GalNAc-conjugated siRNAs. In addition, anti-GalNAc mAbs showed desired sensitivity and drug tolerance, supporting their use as alternative surrogate positive controls. These findings can guide positive control selection and immunogenicity assay development for GalNAc-conjugated siRNAs and other oligonucleotide therapeutics.
Assuntos
Acetilgalactosamina , Oligonucleotídeos , Animais , Humanos , RNA Interferente Pequeno/genética , Anticorpos MonoclonaisRESUMO
Biotherapeutics have the potential to trigger undesired immune responses in the patients. For therapeutic proteins, immunogenicity is manifested as anti-drug antibodies (ADA). Because ADA could compromise pharmacokinetics, efficacy, and safety, regulatory agencies require immunogenicity assessment during clinical development. A tiered bioanalytical approach is recommended to monitor clinical immunogenicity, and neutralizing antibodies (NAb) are studied in Tier 4 if the molecule is immunogenic. Although cell-based assays, which reflect the pharmacological mechanism of action, are in some cases the preferred assay format for detecting NAbs, they are associated with operational complexity and sometimes suboptimal assay performance. Alternatively, non-cell-based assays have also been developed and implemented. In our current study, a competitive ligand binding assay (CLBA) was developed to detect NAbs for insulin efsitora alfa (efsitora, basal insulin Fc, LY3209590), a novel fusion protein being studied for the treatment of Type 1 diabetes and Type 2 diabetes. The CLBA demonstrated acceptable sensitivity, drug tolerance, precision, and robustness, and thus provides a suitable approach for detecting NAbs against efsitora.
Assuntos
Anticorpos Neutralizantes , Diabetes Mellitus Tipo 2 , Humanos , Insulina , Ligantes , Tolerância a Medicamentos , Ligação CompetitivaRESUMO
Merestinib is an oral multi-kinase inhibitor targeting a limited number of oncokinases including MET, AXL, RON and MKNK1/2. Here, we report that merestinib inhibits neurotrophic receptor tyrosine kinases NTRK1/2/3 which are oncogenic drivers in tumors bearing NTRK fusion resulting from chromosomal rearrangements. Merestinib is shown to be a type II NTRK1 kinase inhibitor as determined by x-ray crystallography. In KM-12 cells harboring TPM3-NTRK1 fusion, merestinib exhibits potent p-NTRK1 inhibition in vitro by western blot and elicits an anti-proliferative response in two- and three-dimensional growth. Merestinib treatment demonstrated profound tumor growth inhibition in in vivo cancer models harboring either a TPM3-NTRK1 or an ETV6-NTRK3 gene fusion. To recapitulate resistance observed from type I NTRK kinase inhibitors entrectinib and larotrectinib, we generated NIH-3T3 cells exogenously expressing TPM3-NTRK1 wild-type, or acquired mutations G595R and G667C in vitro and in vivo. Merestinib blocks tumor growth of both wild-type and mutant G667C TPM3-NTRK1 expressing NIH-3T3 cell-derived tumors. These preclinical data support the clinical evaluation of merestinib, a type II NTRK kinase inhibitor (NCT02920996), both in treatment naïve patients and in patients progressed on type I NTRK kinase inhibitors with acquired secondary G667C mutation in NTRK fusion bearing tumors.
RESUMO
Purpose Approximately 3% of lung cancer bears mutations leading to MET exon 14 skipping, an oncogenic driver which is further evidenced by case reports of patient response to MET kinase inhibitor treatment. Approximately 15% of tumors harboring MET exon14 skipping have concurrent MET amplification. Experimental Design Merestinib is a type II MET kinase inhibitor. Emibetuzumab, a bivalent anti-MET antibody, internalizes MET receptor. Each single agent and the combination were evaluated in the Hs746t gastric cancer line bearing MET exon14 skipping and MET amplification. Results Merestinib inhibited Hs746t cell proliferation (IC50=34 nM) and totally eliminated pMET at 100nM. Emibetuzumab showed little anti-proliferative activity against Hs746t cells (IC50>100nM), did not reduce pMET, and slightly reduced cell surface MET. In the Hs746t xenograft model, dose dependent differences in durability of response were seen with merestinib including durable tumor regression (91.8%) at 12 mg/kg qd. Emibetuzumab treatment (10mg/kg qw) provided transient tumor regression (37.7%), but tumors re-grew while on treatment. Concurrent combination of merestinib (6 mg/kg qd) and emibetuzumab resulted in 85% tumor regression, while a sequential combination (initiating merestinib first) resulted in longer duration of treatment response. Conclusions Data in this study support a clinical evaluation of merestinib in patients with MET exon 14 skipping (NCT02920996). As a type II MET kinase inhibitor, merestinib may provide a therapeutic option to treatment naïve patients or to patients who progress on type I MET inhibitor treatment. Data also support clinical evaluation of the sequential combination of merestinib with emibetuzumab when patients progress on single agent merestinib.
Assuntos
Anticorpos Biespecíficos/farmacologia , Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos/farmacologia , Éxons/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Camundongos , Camundongos Nus , Neoplasias Gástricas/tratamento farmacológico , Neoplasias Gástricas/metabolismoRESUMO
Background Circulating tumor cells (CTCs) and chemokine (C-X-C motif) receptor 4 (CXCR4) expression in CTCs and tumor tissue were evaluated as prognostic or predictive markers of CXCR4 peptide antagonist LY2510924 plus carboplatin-etoposide (CE) versus CE in extensive-stage disease small cell lung cancer (ED-SCLC). Methods This exploratory analysis of a phase II study evaluated CXCR4 expression in baseline tumor tissue and peripheral blood CTCs and in post-treatment CTCs. Optimum cutoff values were determined for CTC counts and CXCR4 expression in tumors and CTCs as predictors of survival outcome. Kaplan-Meier estimates and hazard ratios were used to determine biomarker prognostic and predictive values. Results There was weak positive correlation at baseline between CXCR4 expression in tumor tissue and CTCs. Optimum cutoff values were H-score ≥ 210 for CXCR4+ tumor, ≥7% CTCs with CXCR4 expression (CXCR4+ CTCs), and ≥6 CTCs/7.5 mL blood. Baseline H-score for CXCR4+ tumor was not prognostic of progression-free survival (PFS) or overall survival (OS). Baseline CXCR4+ CTCs ≥7% was prognostic of shorter PFS. CTCs ≥6 at baseline and cycle 2, day 1 were prognostic of shorter PFS and OS. None of the biomarkers at their respective optimum cutoffs was predictive of treatment response of LY2510924 plus CE versus CE. Conclusions In patients with ED-SCLC, baseline CXCR4 expression in tumor tissue was not prognostic of survival or predictive of LY2510924 treatment response. Baseline CXCR4+ CTCs ≥7% was prognostic of shorter PFS. CTC count ≥6 at baseline and after 1 cycle of treatment were prognostic of shorter PFS and OS.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Biomarcadores Tumorais/sangue , Carboplatina/farmacologia , Etoposídeo/farmacologia , Células Neoplásicas Circulantes , Peptídeos Cíclicos/farmacologia , Receptores CXCR4/antagonistas & inibidores , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carboplatina/uso terapêutico , Contagem de Células , Intervalo Livre de Doença , Etoposídeo/uso terapêutico , Humanos , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/sangue , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Peptídeos Cíclicos/uso terapêutico , Prognóstico , Receptores CXCR4/metabolismo , Carcinoma de Pequenas Células do Pulmão/sangue , Carcinoma de Pequenas Células do Pulmão/tratamento farmacológico , Carcinoma de Pequenas Células do Pulmão/metabolismoRESUMO
PURPOSE: MET, the receptor for hepatocyte growth factor (HGF), has been implicated in driving tumor proliferation and metastasis. High MET expression is correlated with poor prognosis in multiple cancers. Activation of MET can be induced either by HGF-independent mechanisms such as gene amplification, specific genetic mutations, and transcriptional upregulation or by HGF-dependent autocrine or paracrine mechanisms. EXPERIMENTAL DESIGN/RESULTS: Here, we report on LY2875358, a novel humanized bivalent anti-MET antibody that has high neutralization and internalization activities, resulting in inhibition of both HGF-dependent and HGF-independent MET pathway activation and tumor growth. In contrast to other bivalent MET antibodies, LY2875358 exhibits no functional agonist activity and does not stimulate biologic activities such as cell proliferation, scattering, invasion, tubulogenesis, or apoptosis protection in various HGF-responsive cells and no evidence of inducing proliferation in vivo in a monkey toxicity study. LY2875358 blocks HGF binding to MET and HGF-induced MET phosphorylation and cell proliferation. In contrast to the humanized one-armed 5D5 anti-MET antibody, LY2875358 induces internalization and degradation of MET that inhibits cell proliferation and tumor growth in models where MET is constitutively activated. Moreover, LY2875358 has potent antitumor activity in both HGF-dependent and HGF-independent (MET-amplified) xenograft tumor models. Together, these findings indicate that the mechanism of action of LY2875358 is different from that of the one-armed MET antibody. CONCLUSIONS: LY2875358 may provide a promising therapeutic strategy for patients whose tumors are driven by both HGF-dependent and HGF-independent MET activation. LY2875358 is currently being investigated in multiple clinical studies.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Anticorpos Neutralizantes/farmacologia , Fator de Crescimento de Hepatócito/metabolismo , Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Neutralizantes/administração & dosagem , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Regulação para Baixo , Ativação Enzimática/efeitos dos fármacos , Feminino , Humanos , Macaca fascicularis , Masculino , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Fosforilação , Transporte Proteico , Proteólise , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Lung cancer is the leading cause of cancer-related death worldwide. Sustained activation, overexpression, or mutation of the MET pathway is associated with a poor prognosis in a variety of tumors, including non-small cell lung cancer (NSCLC), implicating the MET pathway as a potential therapeutic target for lung cancer. Previously, we reported on the development of LY2801653: a novel, orally bioavailable oncokinase inhibitor with MET as one of its targets. Here, we discuss the evaluation of LY2801653 in both preclinical in vitro and in vivo NSCLC models. Experimental Design/ RESULTS: Treatment with LY2801653 showed tumor growth inhibition in tumor cell lines and patient-derived tumor xenograft models as a single agent (37.4%-90.0% inhibition) or when used in combination with cisplatin, gemcitabine, or erlotinib (66.5%-86.3% inhibition). Mechanistic studies showed that treatment with LY2801653 inhibited the constitutive activation of MET pathway signaling and resulted in inhibition of NCI-H441 cell proliferation, anchorage-independent growth, migration, and invasion. These in vitro findings were confirmed in the H441 orthotopic model where LY2801653 treatment significantly inhibited both primary tumor growth (87.9% inhibition) and metastasis (64.5% inhibition of lymph node and 67.7% inhibition of chest wall). Tumor-bearing animals treated with LY2801653 had a significantly greater survival time (87% increase compared with the vehicle-treated mice). In the MET-independent NCI-H1299 orthotopic model, treatment with LY2801653 showed a significant inhibition of primary tumor growth but not metastasis. CONCLUSIONS: Collectively, these results support clinical evaluation of LY2801653 in NSCLCs and suggest that differences in the MET activation of tumors may be predictive of response.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Indazóis/farmacologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Niacinamida/análogos & derivados , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-met/metabolismo , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Modelos Animais de Doenças , Expressão Gênica , Fator de Crescimento de Hepatócito/antagonistas & inibidores , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Indazóis/administração & dosagem , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/genética , Camundongos , Metástase Neoplásica , Niacinamida/administração & dosagem , Niacinamida/farmacologia , Proteínas Oncogênicas/antagonistas & inibidores , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Quinases/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-met/genética , Transdução de Sinais/efeitos dos fármacos , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The HGF/MET signaling pathway regulates a wide variety of normal cellular functions that can be subverted to support neoplasia, including cell proliferation, survival, apoptosis, scattering and motility, invasion, and angiogenesis. MET over-expression (with or without gene amplification), aberrant autocrine or paracrine ligand production, and missense MET mutations are mechanisms that lead to activation of the MET pathway in tumors and are associated with poor prognostic outcome. We report here preclinical development of a potent, orally bioavailable, small-molecule inhibitor LY2801653 targeting MET kinase. LY2801653 is a type-II ATP competitive, slow-off inhibitor of MET tyrosine kinase with a dissociation constant (Ki) of 2 nM, a pharmacodynamic residence time (Koff) of 0.00132 min(-1) and t1/2 of 525 min. LY2801653 demonstrated in vitro effects on MET pathway-dependent cell scattering and cell proliferation; in vivo anti-tumor effects in MET amplified (MKN45), MET autocrine (U-87MG, and KP4) and MET over-expressed (H441) xenograft models; and in vivo vessel normalization effects. LY2801653 also maintained potency against 13 MET variants, each bearing a single-point mutation. In subsequent nonclinical characterization, LY2801653 was found to have potent activity against several other receptor tyrosine oncokinases including MST1R, FLT3, AXL, MERTK, TEK, ROS1, DDR1/2 and against the serine/threonine kinases MKNK1/2. The potential value of MET and other inhibited targets within a number of malignancies (such as colon, bile ducts, and lung) is discussed. LY2801653 is currently in phase 1 clinical testing in patients with advanced cancer (trial I3O-MC-JSBA, NCT01285037).
Assuntos
Indazóis/farmacologia , Niacinamida/análogos & derivados , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Tetrazóis/farmacologia , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/química , Antineoplásicos/farmacologia , Disponibilidade Biológica , Vasos Sanguíneos/efeitos dos fármacos , Vasos Sanguíneos/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Indazóis/administração & dosagem , Indazóis/química , Camundongos , Mutação/genética , Niacinamida/administração & dosagem , Niacinamida/química , Niacinamida/farmacologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-met/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Tetrazóis/administração & dosagem , Tetrazóis/química , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The splicing factor SPF45 (RBM17) is frequently overexpressed in many solid tumors, and stable expression in HeLa cells confers resistance to doxorubicin and vincristine. In this study, we characterized stable transfectants of A2780 ovarian carcinoma cells. In a 3-day cytotoxicity assay, human SPF45 overexpression conferred 3- to 21-fold resistance to carboplatin, vinorelbine, doxorubicin, etoposide, mitoxantrone, and vincristine. In addition, resistance to gemcitabine and pemetrexed was observed at the highest drug concentrations tested. Knockdown of SPF45 in parental A2780 cells using a hammerhead ribozyme sensitized A2780 cells to etoposide by approximately 5-fold relative to a catalytically inactive ribozyme control and untransfected cells, suggesting a role for SPF45 in intrinsic resistance to some drugs. A2780-SPF45 cells accumulated similar levels of doxorubicin as vector-transfected and parental A2780 cells, indicating that drug resistance is not due to differences in drug accumulation. Efforts to identify small molecules that could block SPF45-mediated drug resistance revealed that the selective estrogen receptor (ER) modulators tamoxifen and LY117018 (a raloxifene analogue) partially reversed SPF45-mediated drug resistance to mitoxantrone in A2780-SPF45 cells from 21-fold to 8- and 5-fold, respectively, but did not significantly affect the mitoxantrone sensitivity of vector control cells. Quantitative PCR showed that ERbeta but not ERalpha was expressed in A2780 transfectants. Coimmunoprecipitation experiments suggest that SPF45 and ERbeta physically interact in vivo. Thus, SPF45-mediated drug resistance in A2780 cells may result in part from effects of SPF45 on the transcription or alternate splicing of ERbeta-regulated genes.