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1.
J Immunol Methods ; 451: 20-27, 2017 12.
Article in English | MEDLINE | ID: mdl-28803843

ABSTRACT

With current available assay formats using either immobilized protein (ELISA, enzyme-linked immunosorbent assay) or immunostaining of fixed cells for primary monoclonal antibody (mAb) screening, researchers often fail to identify and characterize antibodies that recognize the native conformation of cell-surface antigens. Therefore, screening using live cells has become an integral and important step contributing to the successful identification of therapeutic antibody candidates. Thus the need for developing high-throughput screening (HTS) technologies using live cells has become a major priority for therapeutic mAb discovery and development. We have developed a novel technique called Multiplexed Fluorescent Cell Barcoding (MFCB), a flow cytometry-based method based upon the Fluorescent Cell Barcoding (FCB) technique and the Luminex fluorescent bead array system, but is applicable to high-through mAb screens on live cells. Using this technique in our system, we can simultaneously identify or characterize the antibody-antigen binding of up to nine unique fluorescent labeled cell populations in the time that it would normally take to process a single population. This has significantly reduced the amount of time needed for the identification of potential lead candidates. This new technology enables investigators to conduct large-scale primary hybridoma screens using flow cytometry. This in turn has allowed us to screen antibodies more efficiently than before and streamline identification and characterization of lead molecules.


Subject(s)
Antibodies, Monoclonal/metabolism , Antigens, Surface/immunology , Cell Separation/methods , Flow Cytometry/methods , Fluorescent Dyes/chemistry , High-Throughput Screening Assays/methods , Hybridomas/metabolism , Animals , Antibodies, Monoclonal/biosynthesis , Antibodies, Monoclonal/immunology , Antibody Formation , Antibody Specificity , Antigens, Surface/genetics , Binding Sites, Antibody , CHO Cells , Cricetulus , Female , HEK293 Cells , Humans , Hybridomas/immunology , Mice, Inbred C57BL , Predictive Value of Tests , Protein Binding , Reproducibility of Results , Transfection
2.
Biochem Mol Biol Educ ; 44(4): 326-35, 2016 Jul 08.
Article in English | MEDLINE | ID: mdl-26923186

ABSTRACT

Understanding how basic structural units influence function is identified as a foundational/core concept for undergraduate biological and biochemical literacy. It is essential for students to understand this concept at all size scales, but it is often more difficult for students to understand structure-function relationships at the molecular level, which they cannot as effectively visualize. Students need to develop accurate, 3-dimensional mental models of biomolecules to understand how biomolecular structure affects cellular functions at the molecular level, yet most traditional curricular tools such as textbooks include only 2-dimensional representations. We used a controlled, backward design approach to investigate how hand-held physical molecular model use affected students' ability to logically predict structure-function relationships. Brief (one class period) physical model use increased quiz score for females, whereas there was no significant increase in score for males using physical models. Females also self-reported higher learning gains in their understanding of context-specific protein function. Gender differences in spatial visualization may explain the gender-specific benefits of physical model use observed. © 2016 The Authors Biochemistry and Molecular Biology Education published by Wiley Periodicals, Inc. on behalf of International Union of Biochemistry and Molecular Biology, 44(4):326-335, 2016.


Subject(s)
Biochemistry/education , Comprehension , Learning , Microtubule-Associated Proteins/chemistry , Minor Histocompatibility Antigens/chemistry , Models, Anatomic , Recognition, Psychology , Students/psychology , Computer Graphics , Female , Humans , Imaging, Three-Dimensional , Male , Protein Structure, Secondary , Structure-Activity Relationship
3.
Cancer Res ; 75(24): 5329-40, 2015 Dec 15.
Article in English | MEDLINE | ID: mdl-26631267

ABSTRACT

Antibody-drug conjugates (ADC) target cytotoxic drugs to antigen-positive cells for treating cancer. After internalization, ADCs with noncleavable linkers are catabolized to amino acid-linker-warheads within the lysosome, which then enter the cytoplasm by an unknown mechanism. We hypothesized that a lysosomal transporter was responsible for delivering noncleavable ADC catabolites into the cytoplasm. To identify candidate transporters, we performed a phenotypic shRNA screen with an anti-CD70 maytansine-based ADC. This screen revealed the lysosomal membrane protein SLC46A3, the genetic attenuation of which inhibited the potency of multiple noncleavable antibody-maytansine ADCs, including ado-trastuzumab emtansine. In contrast, the potencies of noncleavable ADCs carrying the structurally distinct monomethyl auristatin F were unaffected by SLC46A3 attenuation. Structure-activity experiments suggested that maytansine is a substrate for SLC46A3. Notably, SLC46A3 silencing led to relative increases in catabolite concentrations in the lysosome. Taken together, our results establish SLC46A3 as a direct transporter of maytansine-based catabolites from the lysosome to the cytoplasm, prompting further investigation of SLC46A3 as a predictive response marker in breast cancer specimens.


Subject(s)
Antineoplastic Agents, Phytogenic/metabolism , Immunoconjugates/metabolism , Maytansine/metabolism , Membrane Transport Proteins/metabolism , Antineoplastic Agents, Phytogenic/administration & dosage , Cell Line, Tumor , Cytoplasm/metabolism , Drug Delivery Systems , Humans , Immunoconjugates/administration & dosage , Lysosomes/metabolism , Maytansine/administration & dosage
4.
Mol Cancer Ther ; 14(7): 1614-24, 2015 Jul.
Article in English | MEDLINE | ID: mdl-25931519

ABSTRACT

Epidermal growth factor receptor variant III (EGFRvIII) is a cancer-specific deletion mutant observed in approximately 25% to 50% of glioblastoma multiforme (GBM) patients. An antibody drug conjugate, AMG 595, composed of the maytansinoid DM1 attached to a highly selective anti-EGFRvIII antibody via a noncleavable linker, was developed to treat EGFRvIII-positive GBM patients. AMG 595 binds to the cell surface and internalizes into the endo-lysosomal pathway of EGFRvIII-expressing cells. Incubation of AMG 595 with U251 cells expressing EGFRvIII led to potent growth inhibition. AMG 595 treatment induced significant tumor mitotic arrest, as measured by phospho-histone H3, in GBM subcutaneous xenografts expressing EGFRvIII. A single intravenous injection of AMG 595 at 17 mg/kg (250 µg DM1/kg) generated complete tumor regression in the U251vIII subcutaneous xenograft model. AMG 595 mediated tumor regression in the D317 subcutaneous xenograft model that endogenously expresses EGFRvIII. Finally, AMG 595 treatment inhibited the growth of D317 xenografts orthotopically implanted into the brain as determined by magnetic resonance imaging. These results demonstrate that AMG 595 is a promising candidate to evaluate in EGFRvIII-expressing GBM patients.


Subject(s)
Antibodies, Monoclonal/pharmacology , Brain Neoplasms/drug therapy , ErbB Receptors/immunology , Glioblastoma/drug therapy , Immunoconjugates/pharmacology , Xenograft Model Antitumor Assays , Animals , Antibodies, Monoclonal/administration & dosage , Antibodies, Monoclonal/immunology , Brain Neoplasms/immunology , Brain Neoplasms/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Dose-Response Relationship, Drug , ErbB Receptors/genetics , ErbB Receptors/metabolism , Female , Glioblastoma/immunology , Glioblastoma/metabolism , Humans , Immunoconjugates/administration & dosage , Immunoconjugates/immunology , Immunohistochemistry , Injections, Intravenous , Maytansine/analogs & derivatives , Maytansine/immunology , Maytansine/pharmacology , Mice, Nude , Mice, SCID , Mutation , Reverse Transcriptase Polymerase Chain Reaction , Treatment Outcome , Tumor Burden/drug effects , Tumor Burden/immunology
5.
Br J Haematol ; 140(1): 46-58, 2008 Jan.
Article in English | MEDLINE | ID: mdl-17991300

ABSTRACT

CD19 and CD21 (CR2) are co-receptors found on B-cells and various B-cell lymphomas, including non-Hodgkin lymphoma. To evaluate their suitability as targets for therapy of such lymphomas using internalization-dependent antibody-drug conjugates [such as antibody-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, (N2'-deacetyl-N2'-(3-mercapto-1-oxopropyl)-maytansine) (MCC-DM1) conjugates, which require lysosomal degradation of the antibody moiety for efficacy], we examined uptake of antibodies to CD19 and CD21 in a panel of B-cell lines. Anti-CD21 antibodies were not sufficiently internalized even in the highest CD21-expressing Raji cells, resulting in lack of efficacy with anti-CD21-MCC-DM1 conjugates. Anti-CD19 antibody uptake was variable, and was unexpectedly negatively correlated with CD21 expression. Thus, high CD21-expressing Raji, ARH77 and primary B-cells only very slowly internalized anti-CD19 antibodies, while CD21-negative or low expressing cells, including Ramos and Daudi, rapidly internalized these antibodies in clathrin-coated vesicles followed by lysosomal delivery. Anti-CD19-MCC-DM1 caused greater cytotoxicity in the faster anti-CD19-internalizing cell lines, implying that the rate of lysosomal delivery and subsequent drug release is important. Furthermore, transfection of Ramos cells with CD21 impeded anti-CD19 uptake and decreased anti-CD19-MCC-DM1 efficacy, suggesting that CD21-negative tumours should respond better to such anti-CD19 conjugates. This may have possible clinical implications, as anti-CD21 immunohistochemistry revealed only approximately 30% of 54 diffuse large B-cell lymphoma patients lack CD21 expression.


Subject(s)
Antibodies, Neoplasm/immunology , Antigens, CD19/immunology , Immunoconjugates/therapeutic use , Lymphoma, B-Cell/therapy , Receptors, Complement 3d/metabolism , Apoptosis/immunology , Cell Line, Tumor , Clathrin/pharmacology , Cytotoxicity, Immunologic/immunology , Flow Cytometry , Humans , Immunohistochemistry , Lymphoma, B-Cell/immunology
6.
Proc Natl Acad Sci U S A ; 103(21): 8215-20, 2006 May 23.
Article in English | MEDLINE | ID: mdl-16698938

ABSTRACT

Deposition of plaques containing amyloid beta (Abeta) peptides is a neuropathological hallmark of Alzheimer's disease (AD). Here we demonstrate that neuronal overexpression of the epsilon isozyme of PKC decreases Abeta levels, plaque burden, and plaque-associated neuritic dystrophy and reactive astrocytosis in transgenic mice expressing familial AD-mutant forms of the human amyloid precursor protein (APP). Compared with APP singly transgenic mice, APP/PKCepsilon doubly transgenic mice had decreased Abeta levels but showed no evidence for altered cleavage of APP. Instead, PKCepsilon overexpression selectively increased the activity of endothelin-converting enzyme, which degrades Abeta. The activities of other Abeta-degrading enzymes, insulin degrading enzyme and neprilysin, were unchanged. These results indicate that increased neuronal PKCepsilon activity can promote Abeta clearance and reduce AD neuropathology through increased endothelin-converting enzyme activity.


Subject(s)
Amyloid/chemistry , Aspartic Acid Endopeptidases/metabolism , Metalloendopeptidases/metabolism , Protein Kinase C-epsilon/physiology , Animals , Brain/metabolism , Endothelin-Converting Enzymes , Endothelium, Vascular/cytology , Gliosis/pathology , Hippocampus/metabolism , Humans , Mice , Mice, Transgenic , Neurodegenerative Diseases/pathology , Neurons/metabolism , Phosphorylation
7.
J Neurosci ; 22(22): 9905-11, 2002 Nov 15.
Article in English | MEDLINE | ID: mdl-12427847

ABSTRACT

Conventional gene targeting is a powerful tool to study the influence of specific genes on behavior. However, conclusions relevant for adult animals are limited by consequences of gene loss during development. Mice lacking protein kinase C epsilon (PKCepsilon) consume less alcohol and show greater acute sensitivity to alcohol than do wild-type mice. There are no selective inhibitors of PKCepsilon that can be administered systemically and cross the blood-brain barrier to test whether these phenotypes result from loss of PKCepsilon during development or in adulthood. Here we used conditional expression of PKCepsilon in the basal forebrain, amygdala, and cerebellum to rescue wild-type responses to alcohol in adult PKCepsilon(-/-) mice. Subsequent suppression of transgenic PKCepsilon restored PKCepsilon(-/-) behaviors. These findings establish that PKCepsilon signaling in the adult brain regulates alcohol consumption and sensitivity. If this extends to humans, then PKCepsilon inhibitors might prove useful as novel therapeutics for alcoholism.


Subject(s)
Drug Resistance/genetics , Ethanol/pharmacology , Isoenzymes/genetics , Isoenzymes/metabolism , Protein Kinase C/genetics , Protein Kinase C/metabolism , Sleep/drug effects , Age Factors , Alcohol Drinking/drug therapy , Alcohol Drinking/genetics , Amygdala/enzymology , Animals , Behavior, Animal/drug effects , Cerebellum/enzymology , Choice Behavior/drug effects , Doxycycline/pharmacology , Gene Expression/drug effects , Isoenzymes/deficiency , Mice , Mice, Inbred Strains , Mice, Knockout , Mice, Transgenic , Organ Specificity/genetics , Phenotype , Promoter Regions, Genetic , Prosencephalon/enzymology , Protein Kinase C/deficiency , Protein Kinase C-epsilon , Receptors, GABA-A/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Transgenes/drug effects
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