ABSTRACT
Administration of a novel and selective small molecule integrin αvß6 inhibitor, MORF-627, to young cynomolgus monkeys for 28 days resulted in the rapid induction of epithelial proliferative changes in the urinary bladder of 2 animals, in the absence of test agent genotoxicity. Microscopic findings included suburothelial infiltration by irregular nests and/or trabeculae of epithelial cells, variable cytologic atypia, and high mitotic rate, without invasion into the tunica muscularis. Morphologic features and patterns of tumor growth were consistent with a diagnosis of early-stage invasive urothelial carcinoma. Ki67 immunohistochemistry demonstrated diffusely increased epithelial proliferation in the urinary bladder of several monkeys, including those with tumors, and αvß6 was expressed in some epithelial tissues, including urinary bladder, in monkeys and humans. Spontaneous urothelial carcinomas are extremely unusual in young healthy monkeys, suggesting a direct link of the finding to the test agent. Inhibition of integrin αvß6 is intended to locally and selectively block transforming growth factor beta (TGF-ß) signaling, which is implicated in epithelial proliferative disorders. Subsequent in vitro studies using a panel of integrin αvß6 inhibitors in human bladder epithelial cells replicated the increased urothelial proliferation observed in monkeys and was reversed through exogenous application of TGF-ß. Moreover, analysis of in vivo models of liver and lung fibrosis revealed evidence of epithelial hyperplasia and cell cycle dysregulation in mice treated with integrin αvß6 or TGF-ß receptor I inhibitors. The cumulative evidence suggests a direct link between integrin αvß6 inhibition and decreased TGF-ß signaling in the local bladder environment, with implications for epithelial proliferation and carcinogenesis.
Subject(s)
Carcinoma, Transitional Cell , Integrins , Urinary Bladder Neoplasms , Animals , Humans , Mice , Carcinoma, Transitional Cell/chemically induced , Integrins/antagonists & inhibitors , Integrins/metabolism , Macaca fascicularis , Transforming Growth Factor beta/metabolism , Urinary Bladder Neoplasms/chemically inducedABSTRACT
CD137 (4-1BB) is a co-stimulatory receptor on immune cells and Nectin-4 is a cell adhesion molecule that is overexpressed in multiple tumor types. Using a series of poly(ethylene glycol) (PEG)-based linkers, synthetic bicyclic peptides targeting CD137 were conjugated to Bicycles targeting Nectin-4. The resulting bispecific molecules were potent CD137 agonists that require the presence of both Nectin-4-expressing tumor cells and CD137-expressing immune cells for activity. A multipronged approach was taken to optimize these Bicycle tumor-targeted immune cell agonists by exploring the impact of chemical configuration, binding affinity, and pharmacokinetics on CD137 agonism and antitumor activity. This effort resulted in the discovery of BT7480, which elicited robust CD137 agonism and maximum antitumor activity in syngeneic mouse models. A tumor-targeted approach to CD137 agonism using low-molecular-weight, short-acting molecules with high tumor penetration is a yet unexplored path in the clinic, where emerging data suggest that persistent target engagement, characteristic of biologics, may lead to suboptimal immune response.
Subject(s)
Neoplasms , Animals , Cell Adhesion Molecules , Mice , Nectins , Neoplasms/drug therapy , Tumor Necrosis Factor Receptor Superfamily, Member 9/metabolismABSTRACT
Ephrin receptor A2 (EphA2) is a member of the Ephrin/Eph receptor cell-to-cell signaling family of molecules, and it plays a key role in cell proliferation, differentiation, and migration. EphA2 is overexpressed in a broad range of cancers, and its expression is in many cases associated with poor prognosis. We recently developed a novel EphA2-targeting antibody-directed nanotherapeutic encapsulating a labile prodrug of docetaxel (EphA2-ILs-DTXp) for the treatment of EphA2-expressing malignancies. Here, we characterized the expression of EphA2 in bladder cancer using immunohistochemistry in 177 human bladder cancer samples and determined the preclinical efficacy of EphA2-ILs-DTXp in four EphA2-positive patient-derived xenograft (PDX) models of the disease, either as a monotherapy, or in combination with gemcitabine. EphA2 expression was detected in 80-100% of bladder cancer samples and correlated with shorter patient survival. EphA2 was found to be expressed in tumor cells and/or tumor-associated blood vessels in both primary and metastatic lesions with a concordance rate of approximately 90%. The EphA2-targeted antibody-directed nanotherapeutic EphA2-ILs-DTXp controlled tumor growth, mediated greater regression, and was more active than free docetaxel at equitoxic dosing in all four EphA2-positive bladder cancer PDX models. Combination of EphA2-ILs-DTXp and gemcitabine in one PDX model led to improved tumor growth control compared to monotherapies or the combination of free docetaxel and gemcitabine. These data demonstrating the prevalence of EphA2 in bladder cancers and efficacy of EphA2-ILs-DTXp in PDX models support the clinical exploration of EphA2 targeting in bladder cancer.
ABSTRACT
Combinations of chemotherapy with immunotherapy have seen recent clinical success, including two approvals of anti-PD-1/L1 agents in combination with taxane-based chemotherapy in non-small cell lung cancer and triple-negative breast cancer. Here, we present a study on the combination activity and mechanistic rationale of a novel EphA2-targeted liposomal taxane (EphA2-ILs-DTXp) and anti-PD-1. This combination was highly active in mouse syngeneic tumor models, with complete responses observed in 3 of 5 models. In the EMT-6 tumor model, combination of EphA2-ILs-DTXp with anti-PD-1 resulted in a 60% complete response rate, with durable responses that were resistant to rechallenge. These responses were not observed in the absence of CD8+ T cells. Characterization of the immune infiltrates in EMT-6 tumors reveals increased CD8+ T cells, increased CD8+ IFNγ+ CTLs, and an increased CD8/regulatory T-cell (Treg) ratio. These immunomodulatory effects were not observed in mice treated with a combination of docetaxel and anti-PD-1. Pharmacokinetic analysis revealed that the AUC of docetaxel was increased 15 times, from 52.1 to 785 ng/mL/hour, when delivered by EphA2-ILs-DTXp. A dose reduction study of EphA2-ILs-DTXp showed a dose-response relationship for both tumor growth inhibition and the CD8/Treg ratio. Our data indicate that synergism between docetaxel and anti-PD-1 is achievable with nanoliposomal delivery.
Subject(s)
Bridged-Ring Compounds/therapeutic use , Neoplasms/drug therapy , Programmed Cell Death 1 Receptor/antagonists & inhibitors , Receptor, EphA2/metabolism , Taxoids/therapeutic use , Animals , Bridged-Ring Compounds/pharmacology , Disease Models, Animal , Female , Humans , Mice , Neoplasms/pathology , Taxoids/pharmacologyABSTRACT
Tumor necrosis factor receptor 2 (TNFR2) is the alternate receptor for TNF and can mediate both pro- and anti-inflammatory activities of T cells. Although TNFR2 has been linked to enhanced suppressive activity of regulatory T cells (Tregs) in autoimmune diseases, the viability of TNFR2 as a target for cancer immunotherapy has been underappreciated. Here, we show that new murine monoclonal anti-TNFR2 antibodies yield robust antitumor activity and durable protective memory in multiple mouse cancer cell line models. The antibodies mediate potent Fc-dependent T cell costimulation and do not result in significant depletion of Tregs Corresponding human agonistic monoclonal anti-TNFR2 antibodies were identified and also had antitumor effects in humanized mouse models. Anti-TNFR2 antibodies could be developed as a novel treatment option for patients with cancer.
Subject(s)
Antibodies, Monoclonal/immunology , Antibodies, Monoclonal/therapeutic use , CD8-Positive T-Lymphocytes/drug effects , CD8-Positive T-Lymphocytes/metabolism , Receptors, Tumor Necrosis Factor, Type II/antagonists & inhibitors , Receptors, Tumor Necrosis Factor, Type II/immunology , Animals , Colonic Neoplasms/immunology , Colonic Neoplasms/metabolism , Colonic Neoplasms/therapy , Disease Models, Animal , Female , Humans , Lymphocyte Activation/drug effects , Mice , Mice, Inbred BALB C , Mice, Inbred C57BLABSTRACT
Antibody-mediated tumour targeting and nanoparticle-mediated encapsulation can reduce the toxicity of antitumour drugs and improve their efficacy. Here, we describe the performance of a nanotherapeutic encapsulating a hydrolytically sensitive docetaxel prodrug and conjugated to an antibody specific for EphA2-a receptor overexpressed in many tumours. Administration of the nanotherapeutic in mice led to slow and sustained release of the prodrug, reduced exposure of active docetaxel in the circulation (compared with administration of the free drug) and maintenance of optimal exposure of the drug in tumour tissue. We also show that administration of the nanotherapeutic in rats and dogs resulted in minimal haematological toxicity, as well as the absence of neutropenia and improved overall tolerability in multiple rodent models. Targeting of the nanotherapeutic to EphA2 improved tumour penetration and resulted in markedly enhanced antitumour activity (compared with administration of free docetaxel and non-targeted nanotherapeutic controls) in multiple tumour-xenografted mice. This nanomedicine could become a potent and safe therapeutic alternative for cancer patients undergoing chemotherapy.
Subject(s)
Antineoplastic Agents/therapeutic use , Nanoparticles/therapeutic use , Receptor, EphA2/metabolism , Animals , Antineoplastic Agents/pharmacology , Bridged-Ring Compounds/pharmacology , Bridged-Ring Compounds/therapeutic use , Cell Line, Tumor , Cell Proliferation/drug effects , Disease Models, Animal , Docetaxel/blood , Docetaxel/chemistry , Docetaxel/pharmacokinetics , Docetaxel/therapeutic use , Humans , Liposomes , Mice, Inbred NOD , Mice, SCID , Taxoids/pharmacology , Taxoids/therapeutic use , Tissue Distribution/drug effects , Tumor Burden/drug effects , Xenograft Model Antitumor AssaysABSTRACT
MM-302 is an anti-HER2 antibody-targeted pegylated liposomal doxorubicin designed to deliver doxorubicin specifically to HER2-expressing solid tumors. The delivery and activity of MM-302 were evaluated in orthotopic, transgenic, and intravenous breast cancer models expressing varying levels of HER2 that metastasize to some of the most common sites of dissemination for breast cancer, namely, lung, liver, and brain. Metastatic burden was quantified by gross evaluation, immunohistochemistry (IHC), and bioluminescent imaging. Liposome delivery was quantified by IHC and ex vivo fluorescent imaging. Unlike its non-targeted counterpart, pegylated liposomal doxorubicin (PLD), MM-302 showed activity at controlling both primary and metastatic tumor burden in all models tested. The effect of HER2-targeting was greatest in the lung where lymphatic vessel density and MM-302 delivery were highest. Our data indicate that the therapeutic advantage of actively targeting a nanoliposome with an antibody is influenced by both target expression and the tumor microenvironment.
Subject(s)
Antibiotics, Antineoplastic/administration & dosage , Breast Neoplasms/drug therapy , Doxorubicin/analogs & derivatives , Immunoconjugates/chemistry , Liposomes/chemistry , Single-Chain Antibodies/chemistry , Animals , Antibiotics, Antineoplastic/therapeutic use , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Disease Progression , Doxorubicin/administration & dosage , Doxorubicin/chemistry , Doxorubicin/therapeutic use , Drug Delivery Systems , Female , Mice , Polyethylene Glycols/administration & dosage , Polyethylene Glycols/therapeutic use , Receptor, ErbB-2/metabolism , Tumor Microenvironment/drug effectsABSTRACT
Human epidermal growth factor receptor 2 (HER2) is an important biomarker for breast and gastric cancer prognosis and patient treatment decisions. HER2 positivity, as defined by IHC or fluorescent in situ hybridization testing, remains an imprecise predictor of patient response to HER2-targeted therapies. Challenges to correct HER2 assessment and patient stratification include intratumoral heterogeneity, lack of quantitative and/or objective assays, and differences between measuring HER2 amplification at the protein versus gene level. We developed a novel immunofluorescence method for quantitation of HER2 protein expression at the single-cell level on FFPE patient samples. Our assay uses automated image analysis to identify and classify tumor versus non-tumor cells, as well as quantitate the HER2 staining for each tumor cell. The HER2 staining level is converted to HER2 protein expression using a standard cell pellet array stained in parallel with the tissue sample. This approach allows assessment of HER2 expression and heterogeneity within a tissue section at the single-cell level. By using this assay, we identified distinct subgroups of HER2 heterogeneity within traditional definitions of HER2 positivity in both breast and gastric cancers. Quantitative assessment of intratumoral HER2 heterogeneity may offer an opportunity to improve the identification of patients likely to respond to HER2-targeted therapies. The broad applicability of the assay was demonstrated by measuring HER2 expression profiles on multiple tumor types, and on normal and diseased heart tissues.
Subject(s)
Genetic Heterogeneity , Neoplasms/classification , Neoplasms/metabolism , Receptor, ErbB-2/metabolism , Single-Cell Analysis/methods , Animals , Breast Neoplasms/classification , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Line, Tumor , Cluster Analysis , Female , Fluorescent Antibody Technique , Humans , Mice , Mice, Nude , Neoplasms/pathology , Reference Standards , Reproducibility of Results , Stomach Neoplasms/classification , Stomach Neoplasms/metabolism , Stomach Neoplasms/pathology , Tissue Array Analysis , Urinary Bladder Neoplasms/classification , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathologyABSTRACT
The prevalence of ErbB2 amplification in breast cancer has resulted in the heavy pursuit of ErbB2 as a therapeutic target. Although both the ErbB2 monoclonal antibody trastuzumab and ErbB1/ErbB2 dual kinase inhibitor lapatinib have met with success in the clinic, many patients fail to benefit. In addition, the majority of patients who initially respond will unfortunately ultimately progress on these therapies. Activation of ErbB3, the preferred dimerization partner of ErbB2, plays a key role in driving ErbB2-amplified tumor growth, but we have found that current ErbB2-directed therapies are poor inhibitors of ligand-induced activation. By simulating ErbB3 inhibition in a computational model of ErbB2/ErbB3 receptor signaling, we predicted that a bispecific antibody that docks onto ErbB2 and subsequently binds to ErbB3 and blocks ligand-induced receptor activation would be highly effective in ErbB2-amplified tumors, with superior activity to a monospecific ErbB3 inhibitor. We have developed a bispecific antibody suitable for both large scale production and systemic therapy by generating a single polypeptide fusion protein of two human scFv antibodies linked to modified human serum albumin. The resulting molecule, MM-111, forms a trimeric complex with ErbB2 and ErbB3, effectively inhibiting ErbB3 signaling and showing antitumor activity in preclinical models that is dependent on ErbB2 overexpression. MM-111 can be rationally combined with trastuzumab or lapatinib for increased antitumor activity and may in the future complement existing ErbB2-directed therapies to treat resistant tumors or deter relapse.
Subject(s)
Antibodies, Bispecific/pharmacology , Neoplasms/drug therapy , Neuregulin-1/pharmacology , Receptor, ErbB-2/antagonists & inhibitors , Receptor, ErbB-3/antagonists & inhibitors , Animals , Antibodies, Bispecific/metabolism , Antibodies, Bispecific/pharmacokinetics , Antineoplastic Agents/metabolism , Antineoplastic Agents/pharmacokinetics , Antineoplastic Agents/pharmacology , Blotting, Western , Cell Line, Tumor , Cell Proliferation/drug effects , Cyclin D1/metabolism , Drug Design , Female , Humans , Inhibitory Concentration 50 , Mice , Mice, Inbred NOD , Mice, Nude , Mice, SCID , Multiprotein Complexes/metabolism , Neoplasms/metabolism , Neoplasms/pathology , Phosphatidylinositol 3-Kinases/metabolism , Protein Binding , Receptor, ErbB-2/metabolism , Receptor, ErbB-3/metabolism , Signal Transduction/drug effects , Xenograft Model Antitumor AssaysABSTRACT
The monomeric form of IgM, also known as low molecular weight IgM, is found in increased concentrations in patients chronically infected with a variety of viral and bacterial pathogens or suffering from various autoimmune diseases. Whether monomeric IgM contributes to the disease process, however, is not known. To address this question, transgenic mice were created that secreted elevated levels of IgM monomers. In normal mice (C57BL/6), the presence of IgM monomers did not alter the composition of the immune system significantly: lymphocyte subsets and serum antibody levels were normal, with the exception of increased levels of IgM due to the presence of the monomers. Immune responses also appeared to be normal. Transgenic mice did develop antinuclear antibodies (ANA) earlier than non-transgenic littermates, but did not develop further indications of autoimmune disease. When the transgene was expressed in the autoimmune-prone strain of mice, B6.MRL-Tnfrsf6(lpr) (B6/lpr), these mice developed autoimmune manifestations more rapidly than non-transgenic littermates, including hypergammaglobulinemia, splenomegaly, and ANA production. Transgenic mice also displayed earlier evidence of immune complex deposition in the kidneys. From these results, we conclude that monomeric IgM does not induce autoimmune disease, but its presence can accelerate the onset of autoimmune manifestations in otherwise autoimmune prone animals.
Subject(s)
Antibodies, Antinuclear/blood , Autoimmune Diseases/immunology , Autoimmunity/physiology , Immunoglobulin M/physiology , Amino Acid Sequence , Animals , Antibody Formation/physiology , Antigen-Antibody Complex/analysis , Antigen-Antibody Complex/metabolism , Autoimmune Diseases/genetics , Autoimmunity/genetics , Hypergammaglobulinemia/genetics , Hypergammaglobulinemia/metabolism , Immunoglobulin G/blood , Immunoglobulin J-Chains/genetics , Immunoglobulin M/genetics , Kidney/immunology , Mice , Mice, Transgenic , Molecular Sequence Data , Mutation/genetics , Splenomegaly/genetics , Splenomegaly/immunology , Transgenes/genetics , fas Receptor/geneticsABSTRACT
Structure-activity relationship studies of substituted arylsulfoanilides as antiproliferatives, which are mediated by the partial depletion of intracellular Ca(2+) stores, resulted in the identification of compounds with micromolar activity against lung cancer cells in a growth inhibition assay. Incorporating the substitution pattern of the best arylsulfoanilides onto the 3-phenyloxindole scaffold resulted in a potent arylsulfoanilide-oxindole hybrid, 27. Compound 27 inhibits cancer cell growth by partial depletion of intracellular Ca(2+) stores and phosphorylation of eIF2alpha.
Subject(s)
Aniline Compounds/chemical synthesis , Antineoplastic Agents/chemical synthesis , Indoles/chemical synthesis , Sulfonamides/chemical synthesis , Aniline Compounds/chemistry , Aniline Compounds/pharmacology , Animals , Antineoplastic Agents/chemistry , Antineoplastic Agents/pharmacology , Calcium/metabolism , Cell Line, Tumor , Drug Screening Assays, Antitumor , Humans , Indoles/chemistry , Indoles/pharmacology , Mice , Structure-Activity Relationship , Sulfonamides/chemistry , Sulfonamides/pharmacologyABSTRACT
In an effort to generate novel translation initiation inhibitors for cancer therapy, a series of 2'-benzyloxy-5'-substituted-5-benzylidene-thiazolidine-2,4-thione and dione derivatives was synthesized and evaluated for activity in translation initiation specific assays. Several candidates of the 5-benzylidene-thiazolidine-2,4-diones (3c, 3d, and 3f) and -thiones (2b, 2e, and 2j), inhibit cell growth with low microM GI(50) mediated by inhibition of translation initiation, which involves partial depletion of intracellular Ca(2+) stores and strong phosphorylation of eIF2alpha.
Subject(s)
Antineoplastic Agents/chemical synthesis , Thiazoles/chemical synthesis , Thiones/chemical synthesis , Antineoplastic Agents/pharmacology , Calcium/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Eukaryotic Initiation Factor-2/metabolism , Humans , Phosphorylation , Protein Biosynthesis/drug effects , Structure-Activity Relationship , Thiazoles/pharmacology , Thiazolidinediones , Thiones/pharmacology , Time Factors , Tumor Cells, CulturedABSTRACT
Agents that extract or sequester membrane cholesterol stimulate IkappaB degradation and lead to NF-kappaB activation in a subset of B cells. Although the extraction of cholesterol by methyl-beta-cyclodextrin is the most potent stimulus of NF-kappaB, other agents that sequester cholesterol have similar effects. B cells and B cell lines with an immature phenotype are significantly more sensitive to the effects of cholesterol perturbation than their mature B cell counterparts. NF-kappaB activation does not involve signaling from the B cell receptor complex. Instead, the disruption of membrane cholesterol activates NF-kappaB through a MyD88-dependent pathway involving the pattern recognition receptor, Toll-like receptor 4. We suggest that lipid raft microdomains may serve not only to orchestrate receptor signaling, but to sequester signaling components one from one another, which serves to prevent receptor-mediated signaling from occurring. A role for this process during B cell development is suggested.