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1.
Int J Cancer ; 137(3): 710-20, 2015 Aug 01.
Article in English | MEDLINE | ID: mdl-25556716

ABSTRACT

CD98 is expressed on several tissue types and specifically upregulated on fast-cycling cells undergoing clonal expansion. Various solid (e.g., nonsmall cell lung carcinoma) as well as hematological malignancies (e.g., acute myeloid leukemia) overexpress CD98. We have identified a CD98-specific mouse monoclonal antibody that exhibits potent preclinical antitumor activity against established lymphoma tumor xenografts. Additionally, the humanized antibody designated IGN523 demonstrated robust tumor growth inhibition in leukemic cell-line derived xenograft models and was as efficacious as standard of care carboplatin in patient-derived nonsmall lung cancer xenografts. In vitro studies revealed that IGN523 elicited strong ADCC activity, induced lysosomal membrane permeabilization and inhibited essential amino acid transport function, ultimately resulting in caspase-3 and -7-mediated apoptosis of tumor cells. IGN523 is currently being evaluated in a Phase I clinical trial for acute myeloid leukemia (NCT02040506). Furthermore, preclinical data support the therapeutic potential of IGN523 in solid tumors.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Fusion Regulatory Protein-1/antagonists & inhibitors , Amino Acids/metabolism , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Antibodies, Monoclonal, Humanized/administration & dosage , Antibodies, Monoclonal, Humanized/immunology , Antibodies, Monoclonal, Humanized/pharmacology , Antibody-Dependent Cell Cytotoxicity/immunology , Antineoplastic Agents/administration & dosage , Biological Transport , Caspases/metabolism , Cell Line, Tumor , Cell Membrane Permeability , Complement System Proteins/immunology , Cytotoxicity, Immunologic , Disease Models, Animal , Drug Evaluation, Preclinical , Female , Humans , Lysosomes/metabolism , Mice , Models, Biological , Protein Binding , Tumor Burden/drug effects , Xenograft Model Antitumor Assays
2.
J Immunol Methods ; 405: 1-14, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24380699

ABSTRACT

The diversity of a panel of antibodies that target a specific antigen can be established in various assay formats. In conventional epitope binning assays purified antibodies are tested in a pairwise manner: two antibodies that compete with each other for binding to an antigen are grouped into the same cluster or bin, while they are assigned to two different clusters when they do not compete. Here we present a high through put assay that enables grouping of crude hybridoma supernatants without a need for antibody purification. In addition, the assay does not require recombinant protein, because it is conducted on cells that express the antigen of interest. Hence, one can use the antibody-clustering assay for cell surface proteins that are not amenable to purification. Heavy chain variable region (VH) sequencing shows that VH composition within clusters is conserved. Finally, the assay is in good agreement with a conventional epitope binning assay with purified antigen.


Subject(s)
Antibodies, Monoclonal/immunology , Binding Sites, Antibody/immunology , Cluster Analysis , Epitopes/immunology , Animals , Antibodies, Monoclonal/classification , Antigens/immunology , Cell Line, Tumor , Culture Media, Conditioned/metabolism , Enzyme-Linked Immunosorbent Assay , Epitopes/classification , Humans , Hybridomas/immunology , Hybridomas/metabolism , Immunoglobulin G/immunology , Immunoglobulin G/metabolism , Immunoglobulin Variable Region/classification , Immunoglobulin Variable Region/immunology , Mice , Mice, 129 Strain
3.
Cancer Lett ; 328(2): 261-70, 2013 Jan 28.
Article in English | MEDLINE | ID: mdl-23041621

ABSTRACT

Recent evidence suggests that Notch signaling may play a role in regulation of cancer stem cell (CSC) self-renewal and differentiation hence presenting a promising target for development of novel therapies for aggressive cancers such as triple negative breast cancer (TNBC). We generated Notch1 monoclonal antibodies (mAbs) that specifically bind to the negative regulatory region of human Notch1. Notch1 inhibition in TNBC Sum149 and patient derived xenograft (PDX) 144580 models led to significant TGI particularly in combination with docetaxel. More interestingly, Notch1 mAbs caused a reduction in mammosphere formation and CD44+/CD24-/lo cell population. It also resulted in decreased tumor incidence upon re-implantation and delay in tumor recurrence. Our data demonstrated a potent antitumor efficacy of Notch1 mAbs, with a remarkable activity against CSCs. These findings suggest that anti-Notch1 mAbs may provide novel therapies to improve the efficacy of conventional therapies by directly targeting the CSC niche. They may also delay tumor recurrence and hence have a major impact on cancer patient survival.


Subject(s)
Antibodies, Monoclonal/pharmacology , Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Neoplastic Stem Cells/drug effects , Neoplastic Stem Cells/metabolism , Receptor, Notch1/metabolism , Signal Transduction/drug effects , Animals , Antibodies, Monoclonal/administration & dosage , Antineoplastic Agents/administration & dosage , Breast Neoplasms/pathology , Cell Line, Tumor , Cell Proliferation/drug effects , Docetaxel , Drug Synergism , Female , Humans , Mice , Mice, Nude , Recurrence , Spheroids, Cellular/drug effects , Taxoids/pharmacology , Tumor Burden , Tumor Cells, Cultured , Xenograft Model Antitumor Assays
4.
Eur J Pharmacol ; 627(1-3): 16-25, 2010 Feb 10.
Article in English | MEDLINE | ID: mdl-19850035

ABSTRACT

Protein kinase C (PKC) family members such as PKCbetaII may become activated in the hyperglycemic state associated with diabetes. Preclinical and clinical data implicate aberrant PKC activity in the development of diabetic microvasculature abnormalities. Based on this potential etiological role for PKC in diabetic complications, several therapeutic PKC inhibitors have been investigated in clinical trials for the treatment of diabetic patients. In this report, we present the discovery and preclinical evaluation of a novel class of 3-amino-pyrrolo[3,4-c]pyrazole derivatives as inhibitors of PKC that are structurally distinct from the prototypical indolocarbazole and bisindolylmaleimide PKC inhibitors. From this pyrrolo-pyrazole series, several compounds were identified from biochemical assays as potent, ATP-competitive inhibitors of PKC activity with high specificity for PKC over other protein kinases. These compounds were also found to block PKC signaling activity in multiple cellular functional assays. PF-04577806, a representative from this series, inhibited PKC activity in retinal lysates from diabetic rats stimulated with phorbol myristate acetate. When orally administered, PF-04577806 showed good exposure in the retina of diabetic Long-Evans rats and ameliorated retinal vascular leakage in a streptozotocin-induced diabetic rat model. These novel PKC inhibitors represent a promising new class of targeted protein kinase inhibitors with potential as therapeutic agents for the treatment of patients with diabetic microvascular complications.


Subject(s)
Diabetes Complications/metabolism , Drug Discovery , Protein Kinase C/antagonists & inhibitors , Protein Kinase Inhibitors/pharmacology , Retinal Diseases/metabolism , Retinal Vessels/drug effects , Signal Transduction/drug effects , Administration, Oral , Animals , Cattle , Cell Line , Diabetes Complications/drug therapy , Diabetes Complications/enzymology , Disease Models, Animal , Humans , Male , Protein Kinase C/metabolism , Protein Kinase Inhibitors/administration & dosage , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacokinetics , Pyrazoles/administration & dosage , Pyrazoles/chemistry , Pyrazoles/pharmacokinetics , Pyrazoles/pharmacology , Rats , Retinal Diseases/drug therapy , Retinal Diseases/enzymology , Retinal Vessels/metabolism , Retinal Vessels/pathology , Substrate Specificity
5.
Oncogene ; 21(46): 7067-76, 2002 Oct 10.
Article in English | MEDLINE | ID: mdl-12370829

ABSTRACT

The receptor-like protein tyrosine phosphatase DEP1, also known as CD148, is expressed predominantly in epithelial cells, in a variety of tumor cell lines, and in lymphocytes. Expression of DEP1 is enhanced at high cell density, and this observation suggests that DEP1 may function in the regulation of cell adhesion and possibly contact inhibition of cell growth. In order to investigate the function of DEP1, substrate-trapping mutants of the phosphatase were used to identify potential substrates. GST-fusion proteins containing the DEP1 catalytic domain with a substrate-trapping D/A mutation were found to interact with p120(ctn), a component of adherens junctions. DEP1 also interacted with other members of the catenin gene family including beta-catenin and gamma-catenin. The interaction with p120(ctn) is likely to be direct, as the interaction occurs in K562 cells lacking functional adherens junctions and E-cadherin expression. Catalytic domains of the tyrosine phosphatases PTP-PEST, CD45, and PTPbeta did not interact with proteins of the catenin family to detectable levels, suggesting that the interaction of DEP1 with these proteins is specific. DEP1 expression was concentrated at sites of cell-cell contact in A549 cells. p120(ctn) was found to colocalize with these structures. Together these data suggest an important role for DEP-1 in the function of cell-cell contacts and adherens junctions.


Subject(s)
Cell Adhesion Molecules/metabolism , Phosphoproteins/metabolism , Protein Tyrosine Phosphatases/physiology , Catenins , Cell Communication , Cells, Cultured , Cytoskeletal Proteins/metabolism , Humans , Phosphorylation , Protein Tyrosine Phosphatases/analysis , Receptor-Like Protein Tyrosine Phosphatases, Class 3 , Tyrosine/metabolism , alpha Catenin , Delta Catenin
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