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1.
Appl Immunohistochem Mol Morphol ; 26(1): 6-12, 2018 Jan.
Article in English | MEDLINE | ID: mdl-29189265

ABSTRACT

Nivolumab is a monoclonal antibody that blocks the interaction between programmed cell death 1 (PD1) and programmed cell death 1-ligand 1 (PD-L1), resulting in enhanced antitumor activity by the immune system. Nivolumab is currently approved by the US Food and Drug Administration (FDA) for melanoma, non-small cell lung cancer (NSCLC), renal cell carcinoma, classical Hodgkin lymphoma, squamous cell carcinoma of the head and neck, and urothelial carcinoma. PD-L1 IHC 28-8 pharmDx is FDA-approved as a complementary diagnostic for immunohistochemical (IHC) detection of PD-L1 in non-squamous NSCLC and melanoma. We report validation of PD-L1 IHC 28-8 pharmDx for PD-L1 detection on formalin-fixed, paraffin-embedded human melanoma specimens using Autostainer Link 48. A prevalence assessment of 104 melanoma specimens indicated that PD-L1 was detected across the full expression level range (0% to 100% of tumor cells). Assay robustness and precision studies were conducted at Agilent Technologies, with additional reproducibility studies performed at 3 external laboratories. Precision studies evaluated at ≥1% and ≥5% expression levels revealed a range of average negative agreement from 89.5%, 95% CI (83.2, 93.6) to 100%, 95% CI (97.3, 100), and average positive agreement from 85.5%, 95% CI (77.6, 90.9) to 100%, 95% CI (97.9, 100). For external reproducibility, precise results were obtained. These results demonstrate PD-L1 IHC 28-8 pharmDx is a precise, robust, and reproducible assay for determining PD-L1 expression in melanoma. This is the first PD-L1 IHC test to receive FDA approval as a complementary diagnostic in melanoma patients whereby positive PD-L1 expression is correlated with the magnitude of nivolumab treatment effect.


Subject(s)
B7-H1 Antigen/metabolism , Immunohistochemistry/methods , Melanoma/diagnosis , Melanoma/drug therapy , Nivolumab/therapeutic use , Humans , Reproducibility of Results , Treatment Outcome
3.
Bioorg Med Chem ; 24(10): 2215-34, 2016 05 15.
Article in English | MEDLINE | ID: mdl-27085672

ABSTRACT

One of the challenges for targeting B-Raf(V600E) with small molecule inhibitors had been achieving adequate selectivity over the wild-type protein B-Raf(WT), as inhibition of the latter has been associated with hyperplasia in normal tissues. Recent studies suggest that B-Raf inhibitors inducing the 'DFG-in/αC-helix-out' conformation (Type IIB) likely will exhibit improved selectivity for B-Raf(V600E). To explore this hypothesis, we transformed Type IIA inhibitor (1) into a series of Type IIB inhibitors (sulfonamides and sulfamides 4-6) and examined the SAR. Three selectivity indices were introduced to facilitate the analyses: the B-Raf(V600E)/B-Raf(WT) biochemical ((b)S), cellular ((c)S) selectivity, and the phospho-ERK activation ((p)A). Our data indicates that α-branched sulfonamides and sulfamides show higher selectivities than the linear derivatives. We rationalized this finding based on analysis of structural information from the literature and provided evidence for a monomeric B-Raf-inhibitor complex previously hypothesized to be responsible for the desired B-Raf(V600E) selectivity.


Subject(s)
Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Purines/chemistry , Purines/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Amination , Crystallography, X-Ray , Drug Design , Humans , Models, Molecular , Point Mutation , Protein Conformation, alpha-Helical/drug effects , Proto-Oncogene Proteins B-raf/chemistry , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Structure-Activity Relationship
4.
Cancer Res ; 71(17): 5818-26, 2011 Sep 01.
Article in English | MEDLINE | ID: mdl-21742770

ABSTRACT

Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors. As expected, KRAS downregulation decreased the survival of KRAS-dependent cells. In contrast, STK33 downregulation or dominant mutant overexpression had no effect on KRAS signaling or survival of these cells. Similarly, a synthetic lethal siRNA screen conducted in a broad panel of KRAS wild-type or mutant cells identified KRAS but not STK33 as essential for survival. We also obtained similar negative results using small molecule inhibitors of the STK33 kinase identified by high-throughput screening. Taken together, our findings refute earlier proposals that STK33 inhibition may be a useful therapeutic approach to target human KRAS mutant tumors.


Subject(s)
Neoplasms/enzymology , Neoplasms/pathology , Protein Serine-Threonine Kinases/metabolism , Proto-Oncogene Proteins/genetics , ras Proteins/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Gene Knockdown Techniques , Humans , Neoplasms/genetics , Protein Kinase Inhibitors/pharmacology , Protein Serine-Threonine Kinases/antagonists & inhibitors , Protein Serine-Threonine Kinases/genetics , Proto-Oncogene Proteins p21(ras) , RNA Interference
5.
Toxicol Pathol ; 39(5): 809-22, 2011 Aug.
Article in English | MEDLINE | ID: mdl-21677315

ABSTRACT

Seven novel and potent Raf small molecule kinase inhibitors (C1-7) were evaluated in seven-day oral repeat dose rat toxicity studies. All compounds tested induced hyperplasia in multiple tissues. Consistently affected was stratified squamous epithelium at a number of sites and transitional epithelium of urinary bladder and kidney. A seven-day time course study in rats showed morphologic evidence of epithelial proliferation in the nonglandular stomach within four to five hours after a single dose of C-1. Similar indications of cellular proliferation were observed in the urinary bladder by day 2 and in the heart, kidney, and liver by day 3. Transcriptional evidence of proliferation in the urinary bladder was detected within four to five hours after a single dose consistent with activation of the PI3K/AKT and ERK/MAPK pathways. In a twenty-eight-day rat toxicity study of C-1, hyperplasia was observed in the esophagus, nonglandular stomach, skin, urinary bladder, kidney, and heart. Hyperplasia of transitional epithelium of the urinary bladder was particularly severe and in one female rat was accompanied by the presence of a transitional cell carcinoma. These results suggest that these Raf inhibitors induce early transcriptional changes driving unchecked cell proliferation, resulting in marked tissue hyperplasia that can progress to carcinoma within a short time frame.


Subject(s)
Protein Kinase Inhibitors/toxicity , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Urinary Bladder Neoplasms/chemically induced , Urinary Bladder Neoplasms/enzymology , Animals , Cell Proliferation/drug effects , Cluster Analysis , Female , Histocytochemistry , Hyperplasia , MAP Kinase Signaling System/drug effects , Male , Mutation , Myocardium/pathology , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Rats , Rats, Sprague-Dawley , Sciatic Nerve/drug effects , Sciatic Nerve/pathology , Stomach/drug effects , Stomach/pathology , Tissue Distribution , Toxicity Tests, Chronic , Urinary Bladder/drug effects , Urinary Bladder/pathology , Urinary Bladder Neoplasms/metabolism , Urinary Bladder Neoplasms/pathology
6.
Mol Cancer Ther ; 9(8): 2399-410, 2010 Aug.
Article in English | MEDLINE | ID: mdl-20663930

ABSTRACT

Raf inhibitors are under clinical investigation, specifically in patients with tumor types harboring frequent activating mutations in B-Raf. Here, we show that cell lines and tumors harboring mutant B-Raf were sensitive to a novel series of Raf inhibitors (e.g., (V600E)B-Raf A375, IC(50) on cells = 2 nmol/L; ED(50) on tumor xenografts = 1.3 mg/kg). However, in cells and tumors with wild-type B-Raf, exposure to Raf inhibitors resulted in a dose-dependent and sustained activation of mitogen-activated protein kinase signaling. In some of these cell lines, Raf inhibition led to entry into the cell cycle, enhanced proliferation, and significantly stimulated tumor growth in vivo. Inhibition with structurally distinct Raf inhibitors or isoform-specific small interfering RNA knockdown of Raf showed that these effects were mediated directly through Raf. Either A-Raf or C-Raf mediated the Raf inhibitor-induced mitogen-activated protein kinase pathway activation in an inhibitor-specific manner. These paradoxical effects of Raf inhibition were seen in malignant and normal cells in vitro and in vivo. Hyperplasia of normal epithelial cells in the esophagus and the stomach was evident in mice with all efficacious Raf inhibitors (n = 8) tested. An implication of these results is that Raf inhibitors may induce unexpected normal cell and tumor tissue proliferation in patients.


Subject(s)
Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Animals , Cell Cycle/drug effects , Cell Line, Tumor , Cell Proliferation/drug effects , Cell Survival/drug effects , Enzyme Activation/drug effects , Epithelium/drug effects , Epithelium/pathology , Gene Expression Regulation, Neoplastic/drug effects , Humans , Hyperplasia , Intercellular Signaling Peptides and Proteins/pharmacology , Isoenzymes/metabolism , MAP Kinase Signaling System/drug effects , MAP Kinase Signaling System/genetics , Mice , Mutant Proteins/metabolism , Neoplasms/enzymology , Protein Kinase Inhibitors/administration & dosage , Proto-Oncogene Proteins B-raf/metabolism , Xenograft Model Antitumor Assays
7.
J Med Chem ; 52(20): 6189-92, 2009 Oct 22.
Article in English | MEDLINE | ID: mdl-19764794

ABSTRACT

The discovery and optimization of a novel series of aminoisoquinolines as potent, selective, and efficacious inhibitors of the mutant B-Raf pathway is presented. The N-linked pyridylpyrimidine benzamide 2 was identified as a potent, modestly selective inhibitor of the B-Raf enzyme. Replacement of the benzamide with an aminoisoquinoline core significantly improved kinase selectivity and imparted favorable pharmacokinetic properties, leading to the identification of 1 as a potent antitumor agent in xenograft models.


Subject(s)
Isoquinolines/pharmacology , Isoquinolines/pharmacokinetics , MAP Kinase Signaling System/drug effects , Mutant Proteins/antagonists & inhibitors , Mutation , Proto-Oncogene Proteins B-raf/antagonists & inhibitors , Administration, Oral , Animals , Biological Availability , Cell Line, Tumor , Drug Discovery , Humans , Isoquinolines/administration & dosage , Isoquinolines/chemical synthesis , Male , Mice , Models, Molecular , Molecular Conformation , Mutant Proteins/chemistry , Mutant Proteins/genetics , Mutant Proteins/metabolism , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacokinetics , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/chemistry , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins B-raf/metabolism , Rats , Substrate Specificity
8.
Mol Immunol ; 44(6): 1331-41, 2007 Feb.
Article in English | MEDLINE | ID: mdl-16814387

ABSTRACT

Epratuzumab is a humanized anti-CD22 monoclonal antibody currently in clinical trials for treatment of non-Hodgkin lymphoma (NHL) and certain autoimmune diseases. Here we report the results of investigations of epratuzumab's mode of action in comparison to and in combination with the anti-CD20 mAb, rituximab. In vitro cell growth inhibition, induction of apoptosis, and the ability of the mAbs to mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) were evaluated. We also investigated the potential activity of epratuzumab in the regulation of B-cell antigen receptor (BCR) activation. Epratuzumab and rituximab displayed very distinct modes of action; epratuzumab acts as an immunomodulatory agent, while rituximab is an acutely cytotoxic therapeutic antibody. Epratuzumab has distinct effects on cell growth from rituximab. For example, rituximab+anti-human IgG Fcgamma yielded marked inhibition of proliferation in human NHL cell lines, while epratuzumab had little or no effect in this assay. However, when cells were immobilized and stimulated with anti-IgM, epratuzumab, but not rituximab, caused a significant antiproliferative effect. Unlike rituximab, no CDC could be detected, and ADCC was modest but significant with epratuzumab. Importantly, combining rituximab and epratuzumab did not decrease rituximab's ability to induce apoptosis, CDC, and ADCC. In fact, the combination is more effective than rituximab alone in inhibiting proliferation of Daudi Burkitt lymphoma cells in the presence of second antibody, and at least equally effective to rituximab in the absence of crosslinking. These observations suggest that it may be possible to enhance clinical efficacy by combination therapy comprised of anti-CD20 and anti-CD22 mAbs.


Subject(s)
Antibodies, Monoclonal/pharmacology , Recombinant Proteins/pharmacology , Sialic Acid Binding Ig-like Lectin 2/immunology , Sialic Acid Binding Ig-like Lectin 2/metabolism , Antibodies, Monoclonal, Humanized , Antibodies, Monoclonal, Murine-Derived , Antigens, Neoplasm/biosynthesis , Antigens, Neoplasm/immunology , Antineoplastic Agents/pharmacology , Apoptosis/immunology , Burkitt Lymphoma/immunology , Burkitt Lymphoma/pathology , Burkitt Lymphoma/therapy , Cell Line, Tumor , Humans , Rituximab
9.
Toxicol Pathol ; 32(3): 275-94, 2004.
Article in English | MEDLINE | ID: mdl-15204970

ABSTRACT

Artemin (ART) signals through the GFR alpha-3/RET receptor complex to support sympathetic neuron development. Here we show that ART also influences autonomic elements in adrenal medulla and enteric and pelvic ganglia. Transgenic mice over-expressing Art throughout development exhibited systemic autonomic neural lesions including fusion of adrenal medullae with adjacent paraganglia, adrenal medullary dysplasia, and marked enlargement of sympathetic (superior cervical and sympathetic chain ganglia) and parasympathetic (enteric, pelvic) ganglia. Changes began by gestational day 12.5 and formed progressively larger masses during adulthood. Art supplementation in wild type adult mice by administering recombinant protein or an Art-bearing retroviral vector resulted in hyperplasia or neuronal metaplasia at the adrenal corticomedullary junction. Expression data revealed that Gfr alpha-3 is expressed during development in the adrenal medulla, sensory and autonomic ganglia and their projections, while Art is found in contiguous mesenchymal domains (especially skeleton) and in certain nerves. Intrathecal Art therapy did not reduce hypalgesia in rats following nerve ligation. These data (1) confirm that ART acts as a differentiation factor for autonomic (chiefly sympathoadrenal but also parasympathetic) neurons, (2) suggest a role for ART overexpression in the genesis of pheochromocytomas and paragangliomas, and (3) indicate that ART is not a suitable therapy for peripheral neuropathy.


Subject(s)
Autonomic Nervous System/embryology , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Neuroprotective Agents/metabolism , Peripheral Nerves/embryology , Adult , Animals , Autonomic Nervous System/drug effects , Autonomic Nervous System/physiology , Autonomic Nervous System Diseases/pathology , Blotting, Southern , Cells, Cultured , Female , Glial Cell Line-Derived Neurotrophic Factor Receptors , Humans , Male , Mice , Mice, Transgenic , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/pharmacology , Neurons/drug effects , Neuroprotective Agents/pharmacology , Peripheral Nerves/drug effects , Peripheral Nerves/physiology , Peripheral Nervous System Diseases/pathology , Polymerase Chain Reaction , Proto-Oncogene Proteins , Proto-Oncogene Proteins c-ret , Rats , Receptor Protein-Tyrosine Kinases , Signal Transduction/physiology
10.
Clin Cancer Res ; 9(10 Pt 2): 3982S-90S, 2003 Sep 01.
Article in English | MEDLINE | ID: mdl-14506197

ABSTRACT

PURPOSE: Epratuzumab is a novel humanized antihuman CD22 IgG1 antibody that has recently shown promising clinical activity, both as a single agent and in combination with rituximab, in patients with non-Hodgkin's lymphomas (NHL). In an attempt to better understand the mode of action of epratuzumab, the antibody was tested in vitro in a variety of cell-based assays similar to those used to evaluate the biological activity of other therapeutic monoclonal antibodies, including rituximab. In this report, we present epratuzumab activities as they relate to binding, signaling, and internalization of the receptor CD22. METHODS: Chinese hamster ovary-expressed CD22 extracellular domain was used to measure epratuzumab affinity on Biacore. CD22 receptor density and internalization rate were measured indirectly using a monovalently labeled, noncompeting (with epratuzumab) anti-CD22 antibody on Burkitt lymphoma cell lines, primary B cells derived from fresh tonsils, and B cells separated from peripheral blood samples obtained from patients with chronic lymphocytic leukemia or healthy volunteers. Epratuzumab-induced CD22 phosphorylation was measured by immunoprecipitation/Western blot and compared with that induced by anti-IgM stimulation. RESULTS: Epratuzumab binds to CD22-extracellular domain, with an affinity of K(D) = 0.7 nM. Binding of epratuzumab to B cell lines, or primary B cells from healthy individuals and patients with NHL, results in rapid internalization of the CD22/antibody complex. Internalization appears to be faster at early time points in cell lines than in primary B cells and NHL patient-derived B cells, but the maximum internalization reached is comparable for all B cell populations after several hours of treatment and appears to reach saturation at antibody concentrations of 1-5 micro g/ml. Finally, epratuzumab binding results in modest but significant CD22 phosphorylation. CONCLUSIONS: Epratuzumab represents an excellent anti-CD22 ligating agent, highly efficacious in inducing CD22 internalization, and can induce phosphorylation. Although we cannot unequivocally demonstrate here that epratuzumab-induced internalization and signaling of CD22 directly contribute to its therapeutic efficacy, these properties are the fundamental characteristics of the target CD22 and its interaction with epratuzumab. Similar results were observed when epratuzumab was tested in vitro on Burkitt B cell lines as well as on primary normal B cells and neoplastic B cells separated from fresh peripheral blood samples from patients with chronic lymphocytic leukemia.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antigens, CD/biosynthesis , Antigens, Differentiation, B-Lymphocyte/biosynthesis , Cell Adhesion Molecules , Lectins/biosynthesis , Animals , Antibodies, Monoclonal, Humanized , Antibodies, Monoclonal, Murine-Derived , Antigens, CD/genetics , Antigens, CD19/biosynthesis , Antigens, CD20/biosynthesis , Antigens, Differentiation, B-Lymphocyte/genetics , Blotting, Western , CHO Cells , Cell Line , Cell Line, Tumor , Cloning, Molecular , Cricetinae , Humans , Immunoglobulin M/chemistry , In Vitro Techniques , Kinetics , Lectins/genetics , Microscopy, Confocal , Phosphorylation , Precipitin Tests , Protein Binding , Protein Structure, Tertiary , Rituximab , Sialic Acid Binding Ig-like Lectin 2 , Time Factors
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