ABSTRACT
Preclinical evaluation of Retrocyclins (RC-100, RC-101) and Protegrin-1 (PG-1) antimicrobial peptides (AMPs) is important because of their therapeutic potential against bacterial, fungal and viral infections. Human mast cells (HMCs) play important roles in host defense and wound healing but the abilities of retrocyclins and protegrin-1 to harness these functions have not been investigated. Here, we report that chemically synthesized RC-100 and PG-1 caused calcium mobilization and degranulation in HMCs but these responses were not blocked by an inhibitor of formyl peptide receptor-like 1 (FPRL1), a known receptor for AMPs. However, RC-100 and PG-1 induced degranulation in rat basophilic leukemia (RBL-2H3) cells stably expressing Mas related G protein coupled receptor X2 (MrgX2). Chemical synthesis of these AMPs is prohibitively expensive and post-synthesis modifications (cyclization, disulfide bonds, folding) are inadequate for optimal antimicrobial activity. Indeed, we found that synthetic RC-100, which caused mast cell degranulation via MrgX2, did not display any antimicrobial activity. Green-fluorescent protein (GFP)-tagged RC-101 (analog of RC-100) and GFP-tagged PG-1 purified from transgenic plant chloroplasts killed bacteria and induced mast cell degranulation. Furthermore, GFP-PG1 bound specifically to RBL-2H3 cells expressing MrgX2. These findings suggest that retrocyclins and protegrins activate HMCs independently of FPRL1 but via MrgX2. Harnessing this novel feature of AMPs to activate mast cell's host defense/wound healing properties in addition to their antimicrobial activities expands their clinical potential. Low cost production of AMPs in plants should facilitate their advancement to the clinic overcoming major hurdles in current production systems.
Subject(s)
Anti-Bacterial Agents/pharmacology , Antimicrobial Cationic Peptides/pharmacology , Cell Degranulation/drug effects , Defensins/pharmacology , Immunologic Factors/pharmacology , Mast Cells/drug effects , Animals , Antimicrobial Cationic Peptides/biosynthesis , Antimicrobial Cationic Peptides/genetics , Bacteria/drug effects , Bacteria/growth & development , Calcium Signaling/drug effects , Cell Line, Tumor , Defensins/biosynthesis , Defensins/genetics , Dose-Response Relationship, Drug , Humans , Immunologic Factors/biosynthesis , Immunologic Factors/genetics , Mast Cells/immunology , Mast Cells/metabolism , Microbial Viability/drug effects , Nerve Tissue Proteins/drug effects , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Plants, Genetically Modified/genetics , Plants, Genetically Modified/metabolism , Rats , Receptors, G-Protein-Coupled/drug effects , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Receptors, Neuropeptide/drug effects , Receptors, Neuropeptide/genetics , Receptors, Neuropeptide/metabolism , Recombinant Fusion Proteins/pharmacology , Signal Transduction/drug effects , Time Factors , TransfectionABSTRACT
NY-ESO-1 and LAGE-1 are cancer testis antigens with an ideal profile for tumor immunotherapy, combining up-regulation in many cancer types with highly restricted expression in normal tissues and sharing a common HLA-A*0201 epitope, 157-165. Here, we present data to describe the specificity and anti-tumor activity of a bifunctional ImmTAC, comprising a soluble, high-affinity T-cell receptor (TCR) specific for NY-ESO-1157-165 fused to an anti-CD3 scFv. This reagent, ImmTAC-NYE, is shown to kill HLA-A2, antigen-positive tumor cell lines, and freshly isolated HLA-A2- and LAGE-1-positive NSCLC cells. Employing time-domain optical imaging, we demonstrate in vivo targeting of fluorescently labelled high-affinity NYESO-specific TCRs to HLA-A2-, NY-ESO-1157-165-positive tumors in xenografted mice. In vivo ImmTAC-NYE efficacy was tested in a tumor model in which human lymphocytes were stably co-engrafted into NSG mice harboring tumor xenografts; efficacy was observed in both tumor prevention and established tumor models using a GFP fluorescence readout. Quantitative RT-PCR was used to analyze the expression of both NY-ESO-1 and LAGE-1 antigens in 15 normal tissues, 5 cancer cell lines, 10 NSCLC, and 10 ovarian cancer samples. Overall, LAGE-1 RNA was expressed at a greater frequency and at higher levels than NY-ESO-1 in the tumor samples. These data support the clinical utility of ImmTAC-NYE as an immunotherapeutic agent for a variety of cancers.
Subject(s)
Antigens, Neoplasm/immunology , Antigens, Surface/immunology , Membrane Proteins/immunology , Receptors, Antigen, T-Cell/immunology , Recombinant Fusion Proteins/pharmacology , T-Lymphocytes/immunology , Animals , Antibodies, Bispecific/immunology , Antibodies, Bispecific/pharmacology , Antigens, Neoplasm/biosynthesis , Antigens, Surface/biosynthesis , CD3 Complex/immunology , Cell Line, Tumor , Epitopes/immunology , Female , HLA-A2 Antigen/immunology , Humans , Immunoglobulin Fragments/immunology , Lung Neoplasms/immunology , Lung Neoplasms/metabolism , Melanoma/immunology , Melanoma/metabolism , Membrane Proteins/biosynthesis , Mice , Mice, Inbred NOD , Mice, SCID , Ovarian Neoplasms/immunology , Ovarian Neoplasms/metabolism , Random Allocation , Recombinant Fusion Proteins/immunology , Xenograft Model Antitumor AssaysABSTRACT
Along with useful microorganisms, there are some that cause potential damage to the animals and plants. Detection and identification of these harmful organisms in a cost and time effective way is a challenge for the researchers. The future of detection methods for microorganisms shall be guided by biosensor, which has already contributed enormously in sensing and detection technology. Here, we aim to review the use of various biosensors, developed by integrating the biological and physicochemical/mechanical properties (of tranducers), which can have enormous implication in healthcare, food, agriculture and biodefence. We have also highlighted the ways to improve the functioning of the biosensor.
