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1.
Nat Biomed Eng ; 4(2): 195-206, 2020 02.
Article in English | MEDLINE | ID: mdl-31819155

ABSTRACT

Micropatterned nickel titanium (commonly known as nitinol) thin films with complex designs, high structural resolution and excellent biocompatibility can be cheaply fabricated using magnetron sputtering. Here, we show that these benefits can be leveraged to fabricate micromesh implants that are loaded with tumour-specific human chimeric antigen receptor (CAR)-T cells for the treatment of solid tumours. In a mouse model of non-resectable ovarian cancer, the cell-loaded nitinol thin films spatially conformed to the implantation site, fostered the rapid expansion of T cells, delivered a high density of T cells directly to the tumour and significantly improved animal survival. We also show that self-expandable stents that were coated with T-cell-loaded films and implanted into subcutaneous tumours in mice improved the duration of stent patency by delaying tumour ingrowth. By providing direct access to tumours, CAR-T-cell-loaded micropatterned nitinol thin films can improve the effects of cell-based therapies.


Subject(s)
Alloys , Immunotherapy, Adoptive/instrumentation , Immunotherapy, Adoptive/methods , Infusion Pumps, Implantable , Ovarian Neoplasms/therapy , Receptors, Chimeric Antigen/therapeutic use , Animals , Cell Line, Tumor , Cell Movement , Female , Humans , Mice , Ovarian Neoplasms/immunology , T-Lymphocytes/physiology
2.
J Clin Invest ; 130(10): 5127-5141, 2020 10 01.
Article in English | MEDLINE | ID: mdl-32831296

ABSTRACT

Proteins created from recurrent fusion genes like CBFB-MYH11 are prevalent in acute myeloid leukemia (AML), often necessary for leukemogenesis, persistent throughout the disease course, and highly leukemia specific, making them attractive neoantigen targets for immunotherapy. A nonameric peptide derived from a prevalent CBFB-MYH11 fusion protein was found to be immunogenic in HLA-B*40:01+ donors. High-avidity CD8+ T cell clones isolated from healthy donors killed CBFB-MYH11+ HLA-B*40:01+ AML cell lines and primary human AML samples in vitro. CBFB-MYH11-specific T cells also controlled CBFB-MYH11+ HLA-B*40:01+ AML in vivo in a patient-derived murine xenograft model. High-avidity CBFB-MYH11 epitope-specific T cell receptors (TCRs) transduced into CD8+ T cells conferred antileukemic activity in vitro. Our data indicate that the CBFB-MYH11 fusion neoantigen is naturally presented on AML blasts and enables T cell recognition and killing of AML. We provide proof of principle for immunologically targeting AML-initiating fusions and demonstrate that targeting neoantigens has clinical relevance even in low-mutational frequency cancers like fusion-driven AML. This work also represents a first critical step toward the development of TCR T cell immunotherapy targeting fusion gene-driven AML.


Subject(s)
Leukemia, Myeloid, Acute , Animals , Carcinogenesis , Core Binding Factor beta Subunit/genetics , Humans , Leukocytes , Mice , Mutation , Myosin Heavy Chains/genetics , Oncogene Proteins, Fusion/genetics
3.
Mol Cancer Ther ; 2(10): 1067-78, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14578472

ABSTRACT

Lysophosphatidic acid acyltransferase beta (LPAAT-beta) is an intrinsic membrane protein that catalyzes the synthesis of phosphatidic acid (PA) from lysoPA. Given that PA is a cofactor in a number of signaling cascades that are constitutively active in tumors, we evaluated the role of PA produced by LPAAT-beta in Xenopus oocyte meiotic maturation assays and an isoform-specific inhibitor of LPAAT-beta in mammalian cell assays. We found that ectopic overexpression of LPAAT-beta cooperates in activation of the Ras/Raf/Erk pathway in Xenopus oocytes and that inhibition of LPAAT-beta inhibits signaling in both the Ras/Raf/Erk and PI3K/Akt pathways. When LPAAT-beta activity is suppressed by CT32228 (N-(4-bromo-phenyl)-6-(5-chloro-2-methyl-phenyl)-[1,3,5]triazine-2,4-diamine), an isoform-specific noncompetitive inhibitor, tumor cells undergo mitotic catastrophe while most normal cells simply arrest or become quiescent. The data presented here suggest that PA produced by LPAAT-beta plays an important role in signaling pathways critical to tumor cell survival.


Subject(s)
Acyltransferases/antagonists & inhibitors , Acyltransferases/chemistry , Apoptosis , Animals , Cell Division , Cell Line, Tumor , Cell Membrane/metabolism , Cell Survival , Cells, Cultured , Dose-Response Relationship, Drug , Flow Cytometry , Humans , Immunoblotting , Inhibitory Concentration 50 , MAP Kinase Signaling System , Mitosis , Models, Chemical , Oocytes/metabolism , Phosphorylation , Protein Isoforms , Protein Transport , Signal Transduction , Subcellular Fractions , Xenopus
4.
MAbs ; 7(3): 562-70, 2015.
Article in English | MEDLINE | ID: mdl-25898160

ABSTRACT

Accurate measurement and functional characterization of antibody Fc domain N-linked glycans is critical to successful biosimilar development. Here, we describe the application of methods to accurately quantify and characterize the N-linked glycans of 2 IgG1 biosimilars with effector function activity, and show the potential pitfalls of using assays with insufficient resolution. Accurate glycan assessment was combined with glycan enrichment using lectin chromatography or production with glycosylation inhibitors to produce enriched pools of key glycan species for subsequent assessment in cell-based antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity effector function assays. This work highlights the challenges of developing high-quality biosimilar candidates and the need for modern biotechnology capabilities. These results show that high-quality analytics, combined with sensitive cell-based assays to study in vivo mechanisms of action, is an essential part of biosimilar development.


Subject(s)
Antibodies, Monoclonal/chemistry , Antibody-Dependent Cell Cytotoxicity , Biological Assay , Biosimilar Pharmaceuticals/chemistry , Immunoglobulin G/chemistry , Polysaccharides/chemistry , Cell Line , Humans , Immunoglobulin Fc Fragments/chemistry
5.
Nat Genet ; 41(4): 415-23, 2009 Apr.
Article in English | MEDLINE | ID: mdl-19270708

ABSTRACT

A principal task in dissecting the genetics of complex traits is to identify causal genes for disease phenotypes. We previously developed a method to infer causal relationships among genes through the integration of DNA variation, gene transcription and phenotypic information. Here we have validated our method through the characterization of transgenic and knockout mouse models of genes predicted to be causal for abdominal obesity. Perturbation of eight out of the nine genes, with Gas7, Me1 and Gpx3 being newly confirmed, resulted in significant changes in obesity-related traits. Liver expression signatures revealed alterations in common metabolic pathways and networks contributing to abdominal obesity and overlapped with a macrophage-enriched metabolic network module that is highly associated with metabolic traits in mice and humans. Integration of gene expression in the design and analysis of traditional F(2) intercross studies allows high-confidence prediction of causal genes and identification of pathways and networks involved.


Subject(s)
Carrier Proteins/genetics , Glutathione Peroxidase/genetics , Glycoproteins/genetics , Nerve Tissue Proteins/genetics , Obesity/genetics , Abdomen/anatomy & histology , Adipose Tissue/anatomy & histology , Animals , Disease Models, Animal , Female , Gene Expression Profiling , Genetic Variation , Humans , Liver/physiology , Male , Mice , Mice, Knockout , Mice, Transgenic , Muscle, Skeletal/anatomy & histology , Phenotype , Reproducibility of Results , Transcription, Genetic , Vesicular Transport Proteins
6.
Expert Opin Ther Targets ; 7(5): 643-61, 2003 Oct.
Article in English | MEDLINE | ID: mdl-14498826

ABSTRACT

Phosphatidic acid (PA) is a component of cellular membranes that is also a mediator of certain cell signalling functions associated with oncogenesis. These include ras/raf/Erk and Akt/mTor [1-3]. The authors have investigated whether it would be possible to interrupt these known oncogenic pathways through the inhibition of lysophosphatidic acid acyltransferase (LPAAT), an enzyme that catalyses the biosynthesis of PA. The expression and activity of the LPAAT-beta isoform are elevated in human tumours, and the respective gene displays transforming capacity when overexpressed in vitro. Inhibition by either genetic means or by isoform-specific small molecules results in a block to cell signalling pathways and apoptosis. Furthermore, the small-molecule inhibitors of LPAAT-beta are not cytotoxic to a number of normal cell types, including primary bone marrow progenitors, indicating a differential dependence of tumour cells on LPAAT-beta function. These discoveries indicate that LPAAT-beta represents a potential novel cancer therapy target.


Subject(s)
Acyltransferases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Drug Design , Neoplasms/drug therapy , Acylation/drug effects , Acyltransferases/genetics , Acyltransferases/physiology , Animals , Antineoplastic Agents/therapeutic use , Carcinoma, Lewis Lung/drug therapy , Cell Division/drug effects , Cell Division/physiology , Cell Line, Tumor/drug effects , Cell Transformation, Neoplastic , Chromosomes, Human, Pair 9/genetics , Drug Evaluation, Preclinical , Genes, ras , Humans , Hydrocarbons, Halogenated/pharmacology , Hydrocarbons, Halogenated/therapeutic use , Lung Neoplasms/drug therapy , Mice , Neoplasm Proteins/antagonists & inhibitors , Neoplasm Proteins/physiology , Neoplasms/pathology , Phosphatidic Acids/physiology , Protein Conformation , Protein Processing, Post-Translational/drug effects , Signal Transduction/drug effects , Signal Transduction/physiology , Triazines/pharmacology , Triazines/therapeutic use
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