RESUMEN
TG02 is a novel cyclin-dependent kinase (CDK) inhibitor and thought to act mainly via CDK-9 inhibition-dependent depletion of short-lived oncoproteins such as MCL-1 or c-MYC. We studied the activity of TG02 in 9 human long-term glioma cell lines (LTC) and 5 glioma-initiating cell lines (GIC) using various cell death assays in vitro and in the LN-229 LTC and ZH-161 GIC models in vivo. TG02 exhibits strong anti-tumor cell activity with EC50 concentrations in the nanomolar range. Median survival in the LN-229 and ZH-161 models was moderately prolonged by TG02. Neither constitutive CDK levels nor those of MCL-1 or c-MYC correlated with sensitivity to TG02. Cdk-9 or cdk-5 gene silencing alone did not fully reproduce the effects of TG02. C-myc gene silencing inhibited cell growth, but did not modulate TG02 activity. Electron microscopy revealed cell death to be essentially apoptotic. High concentrations of TG02 induced annexin V binding and minor caspase 3 cleavage, but the pan-caspase inhibitor, zVAD-fmk, or BCL-2 or MCL-1 gene transfer only moderately attenuated TG02-induced cell death, and caspase inhibition did not prevent loss of MCL-1 or c-MYC. TG02 activity was independent of O6 -methylguanine DNA methyltransferase expression. Repetitive exposure to TG02 did not generate an acquired TG02 resistance phenotype, but accumulation of MCL-1, loss of c-MYC, or senescence. TG02 is a highly potent apoptosis-inducing agent in glioma cells in vitro. Caspase inhibition does not rescue TG02-treated cells and repetitive exposure fails to confer acquired resistance, supporting the clinical evaluation of TG02 in glioblastoma.
Asunto(s)
Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Animales , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Metilasas de Modificación del ADN/genética , Metilasas de Modificación del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Resistencia a Antineoplásicos , Femenino , Expresión Génica/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Compuestos Heterocíclicos de 4 o más Anillos/farmacocinética , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Distribución Tisular , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Despite clinical evidence of antitumor activity, the development of cytokine therapies has been hampered by a narrow therapeutic window and limited response rates. Two cytokines of high interest for clinical development are interleukin 2 (IL2) and interleukin 12 (IL12), which potently synergize to promote the activation and proliferation of T cells and NK cells. However, the only approved human IL2 therapy, Proleukin, is rarely used in the clinic due to systemic toxicities, and no IL12 product has been approved to date due to severe dose-limiting toxicities. Here, we describe CLN-617, a first-in-class therapeutic for intratumoral (IT) injection that co-delivers IL2 and IL12 on a single molecule in a safe and effective manner. CLN-617 is a single-chain fusion protein comprised of IL2, leukocyte-associated immunoglobulin-like receptor 2 (LAIR2), human serum albumin (HSA), and IL12. LAIR2 and HSA function to retain CLN-617 in the treated tumor by binding collagen and increasing molecular weight, respectively. We found that IT administration of a murine surrogate of CLN-617, mCLN-617, eradicated established treated and untreated tumors in syngeneic models, significantly improved response to anti-PD1 checkpoint therapy, and generated a robust abscopal response dependent on cellular immunity and antigen cross-presentation. CLN-617 is being evaluated in a clinical trial in patients with advanced solid tumors (NCT06035744).
Asunto(s)
Interleucina-12 , Interleucina-2 , Animales , Femenino , Humanos , Ratones , Línea Celular Tumoral , Interleucina-12/metabolismo , Interleucina-2/uso terapéutico , Interleucina-2/farmacología , Ratones Endogámicos C57BL , Neoplasias/inmunología , Neoplasias/terapia , Neoplasias/tratamiento farmacológico , Proteínas Recombinantes de Fusión/farmacología , Proteínas Recombinantes de Fusión/uso terapéutico , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
BACKGROUND: Despite significant progress in the development of T cell-engaging therapies for various B-cell malignancies, a high medical need remains for the refractory disease setting, often characterized by suboptimal target levels. METHODS: To address this issue, we have developed a 65-kDa multispecific antibody construct, CLN-978, with affinities tuned to optimize the killing of low-CD19 expressing tumor cells. CLN-978 bound to CD19 on B cells with picomolar affinity, and to CD3ε on T cells with nanomolar affinity. A serum albumin binding domain was incorporated to extend serum half-life. In this setting, we biophysically characterize and report the activities of CLN-978 in cell co-culture assays, multiple mouse models and non-human primates. RESULTS: Human T cells redirected by CLN-978 could eliminate target cells expressing less than 300 copies of CD19 on their surface. The half-life extension and high affinity for CD19 led to significant antitumor activity in murine lymphoma models at very low doses of CLN-978. In primates, we observed a long serum half-life, deep and sustained depletion of normal B cells, and remarkable tolerability, in particular, reduced cytokine release when CLN-978 was administered subcutaneously. CONCLUSIONS: CLN-978 warrants further exploration. An ongoing clinical phase 1 trial is investigating safety, pharmacokinetics, pharmacodynamics, and the initial therapeutic potential of subcutaneously administered CLN-978 in patients with non-Hodgkin's lymphoma.
Asunto(s)
Linfoma no Hodgkin , Neoplasias , Humanos , Animales , Ratones , Semivida , Proteínas Adaptadoras Transductoras de Señales , Anticuerpos , Antígenos CD19RESUMEN
BACKGROUND: In lymphoid malignancies, the introduction of chimeric antigen receptor T (CAR-T) cells and bispecific antibodies (bsAbs) has achieved remarkable clinical success. However, such immunotherapeutic strategies are not yet established for acute myeloid leukemia (AML), the most common form of acute leukemia in adults. Common targets in AML such as CD33, CD123, and CLEC12A are highly expressed on both AML blasts and on normal myeloid cells and hematopoietic stem cells (HSCs), thereby raising toxicity concerns. In B-cell acute lymphoblastic leukemia (B-ALL), bsAbs and CAR-T therapy targeting CD19 and CD22 have demonstrated clinical success, but resistance via antigen loss is common, motivating the development of agents focused on alternative targets. An attractive emerging target is FLT3, a proto-oncogene expressed in both AML and B-ALL, with low and limited expression on myeloid dendritic cells and HSCs. METHODS: We developed and characterized CLN-049, a T cell-activating bsAb targeting CD3 and FLT3, constructed as an IgG heavy chain/scFv fusion. CLN-049 binds the membrane proximal extracellular domain of the FLT3 protein tyrosine kinase, which facilitates the targeting of leukemic blasts regardless of FLT3 mutational status. CLN-049 was evaluated for preclinical safety and efficacy in vitro and in vivo. RESULTS: CLN-049 induced target-restricted activation of CD4+ and CD8+ T cells. AML cell lines expressing a broad range of surface levels of FLT3 were efficiently lysed on treatment with subnanomolar concentrations of CLN-049, whereas FLT3-expressing hematopoietic progenitor cells and dendritic cells were not sensitive to CLN-049 killing. Treatment with CLN-049 also induced lysis of AML and B-ALL patient blasts by autologous T cells at the low effector-to-target ratios typically observed in patients with overt disease. Lysis of leukemic cells was not affected by supraphysiological levels of soluble FLT3 or FLT3 ligand. In mouse xenograft models, CLN-049 was highly active against human leukemic cell lines and patient-derived AML and B-ALL blasts. CONCLUSIONS: CLN-049 has a favorable efficacy and safety profile in preclinical models, warranting evaluation of its antileukemic activity in the clinic.
Asunto(s)
Leucemia Mieloide Aguda , Leucemia-Linfoma Linfoblástico de Células Precursoras , Animales , Humanos , Inmunoglobulina G/uso terapéutico , Inmunoterapia Adoptiva , Subunidad alfa del Receptor de Interleucina-3 , Lectinas Tipo C , Leucemia Mieloide Aguda/tratamiento farmacológico , Ratones , Receptores MitogénicosRESUMEN
Purpose: Glioblastoma (GBM) is highly resistant to treatment, largely due to disease heterogeneity and resistance mechanisms. We sought to investigate a promising drug that can inhibit multiple aspects of cancer cell survival mechanisms and become an effective therapeutic for GBM patients.Experimental Design: To investigate TG02, an agent with known penetration of the blood-brain barrier, we examined the effects as single agent and in combination with temozolomide, a commonly used chemotherapy in GBM. We used human GBM cells and a syngeneic mouse orthotopic GBM model, evaluating survival and the pharmacodynamics of TG02. Mechanistic studies included TG02-induced transcriptional regulation, apoptosis, and RNA sequencing in treated GBM cells as well as the investigation of mitochondrial and glycolytic function assays.Results: We demonstrated that TG02 inhibited cell proliferation, induced cell death, and synergized with temozolomide in GBM cells with different genetic background but not in astrocytes. TG02-induced cytotoxicity was blocked by the overexpression of phosphorylated CDK9, suggesting a CDK9-dependent cell killing. TG02 suppressed transcriptional progression of antiapoptotic proteins and induced apoptosis in GBM cells. We further demonstrated that TG02 caused mitochondrial dysfunction and glycolytic suppression and ultimately ATP depletion in GBM. A prolonged survival was observed in GBM mice receiving combined treatment of TG02 and temozolomide. The TG02-induced decrease of CDK9 phosphorylation was confirmed in the brain tumor tissue.Conclusions: TG02 inhibits multiple survival mechanisms and synergistically decreases energy production with temozolomide, representing a promising therapeutic strategy in GBM, currently under investigation in an ongoing clinical trial. Clin Cancer Res; 24(5); 1124-37. ©2017 AACR.
Asunto(s)
Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Neoplasias Encefálicas/tratamiento farmacológico , Glioblastoma/tratamiento farmacológico , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Apoptosis/efectos de los fármacos , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Encéfalo/patología , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Línea Celular Tumoral/trasplante , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos , Sinergismo Farmacológico , Metabolismo Energético/efectos de los fármacos , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Compuestos Heterocíclicos de 4 o más Anillos/uso terapéutico , Humanos , Ratones , Ratones Endogámicos C57BL , Temozolomida/farmacología , Temozolomida/uso terapéuticoRESUMEN
PURPOSE: ERBB3 is overexpressed in a broad spectrum of human cancers, and its aberrant activation is associated with tumor pathogenesis and therapeutic resistance to various anticancer agents. Neuregulin 1 (NRG1) is the predominant ligand for ERBB3 and can promote the heterodimerization of ERBB3 with other ERBB family members, resulting in activation of multiple intracellular signaling pathways. AV-203 is a humanized IgG1/κ ERBB3 inhibitory antibody that completed a first-in-human phase I clinical trial in patients with advanced solid tumors. The purpose of this preclinical study was to identify potential biomarker(s) that may predict response to AV-203 treatment in the clinic. EXPERIMENTAL DESIGN: We conducted in vivo efficacy studies using a broad panel of xenograft models representing a wide variety of human cancers. To identify biomarkers that can predict response to AV-203, the relationship between tumor growth inhibition (TGI) by AV-203 and the expression levels of ERBB3 and NRG1 were evaluated in these tumor models. RESULTS: A significant correlation was observed between the levels of NRG1 expression and TGI by AV-203. In contrast, TGI was not correlated with ERBB3 expression. The correlation between the levels of NRG1 expression in tumors and their response to ERBB3 inhibition by AV-203 was further validated using patient-derived tumor explant models. CONCLUSIONS: NRG1 is a promising biomarker that can predict response to ERBB3 inhibition by AV-203 in preclinical human cancer models. NRG1 warrants further clinical evaluation and validation as a potential predictive biomarker of response to AV-203.
Asunto(s)
Anticuerpos Monoclonales Humanizados/farmacología , Expresión Génica , Neoplasias/genética , Neurregulina-1/genética , Receptor ErbB-3/antagonistas & inhibidores , Animales , Anticuerpos Monoclonales Humanizados/administración & dosificación , Área Bajo la Curva , Biomarcadores , Línea Celular Tumoral , Modelos Animales de Enfermedad , Resistencia a Antineoplásicos/genética , Femenino , Humanos , Ligandos , Ratones , Mutación , Neoplasias/tratamiento farmacológico , Neoplasias/metabolismo , Neoplasias/patología , Neurregulina-1/metabolismo , Pronóstico , Unión Proteica , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Reproducibilidad de los Resultados , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Dysregulated fibroblast growth factor (FGF) signaling has been implicated in the pathogenesis of human cancers. Aberrant activation of FGF receptor 2 (FGFR2) signaling, through overexpression of FGFR2 and/or its ligands, mutations, and receptor amplification, has been found in a variety of human tumors. We generated monoclonal antibodies against the extracellular ligand-binding domain of FGFR2 to address the role of FGFR2 in tumorigenesis and to explore the potential of FGFR2 as a novel therapeutic target. We surveyed a broad panel of human cancer cell lines for the dysregulation of FGFR2 signaling and discovered that breast and gastric cancer cell lines harboring FGFR2 amplification predominantly express the IIIb isoform of the receptor. Therefore, we used an FGFR2-IIIb-specific antibody, GP369, to investigate the importance of FGFR2 signaling in vitro and in vivo. GP369 specifically and potently suppressed ligand-induced phosphorylation of FGFR2-IIIb and downstream signaling, as well as FGFR2-driven proliferation in vitro. The administration of GP369 in mice significantly inhibited the growth of human cancer xenografts harboring activated FGFR2 signaling. Our findings support the hypothesis that dysregulated FGFR2 signaling is one of the critical oncogenic pathways involved in the initiation and/or maintenance of tumors. Cancer patients with aberrantly activated/amplified FGFR2 signaling could potentially benefit from therapeutic intervention with FGFR2-targeting antibodies.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Neoplasias/terapia , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/inmunología , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Secuencia de Aminoácidos , Animales , Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos , Procesos de Crecimiento Celular/efectos de los fármacos , Línea Celular Tumoral , Femenino , Amplificación de Genes , Humanos , Ratones , Ratones SCID , Datos de Secuencia Molecular , Neoplasias/enzimología , Neoplasias/genética , Neoplasias/inmunología , Fosforilación/efectos de los fármacos , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/antagonistas & inhibidores , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Transducción de Señal , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Increased Aurora A expression occurs in a variety of human cancers and induces chromosomal abnormalities during mitosis associated with tumor initiation and progression. MLN8054 is a selective small-molecule Aurora A kinase inhibitor that has entered Phase I clinical trials for advanced solid tumors. MLN8054 inhibits recombinant Aurora A kinase activity in vitro and is selective for Aurora A over the family member Aurora B in cultured cells. MLN8054 treatment results in G(2)/M accumulation and spindle defects and inhibits proliferation in multiple cultured human tumor cells lines. Growth of human tumor xenografts in nude mice was dramatically inhibited after oral administration of MLN8054 at well tolerated doses. Moreover, the tumor growth inhibition was sustained after discontinuing MLN8054 treatment. In human tumor xenografts, MLN8054 induced mitotic accumulation and apoptosis, phenotypes consistent with inhibition of Aurora A. MLN8054 is a selective inhibitor of Aurora A kinase that robustly inhibits growth of human tumor xenografts and represents an attractive modality for therapeutic intervention of human cancers.