Nano-pulse stimulation™ therapy (NPS™) is superior to cryoablation in clearing murine melanoma tumors.

Background: Nano-Pulse Stimulation™ Therapy (NPS™) is a new, bioelectric modality that applies ultrashort pulses of electric energy to trigger regulated cell death in treated tissues. Instead of initiating necrosis by heating or freezing, NPS therapy permeabilizes intracellular organelles to activate the cell's own self-destruct pathway of programmed or regulated cell death. Unlike cryotherapies that can both damage structural tissues and diffuse into the periphery beyond the margins of the lesion, NPS only affects cells within the treated zone leaving surrounding tissue and acellular components unaffected.

Antibody targeting of phosphatidylserine for the detection and immunotherapy of cancer.

Phosphatidylserine (PS) is a negatively charged phospholipid in all eukaryotic cells that is actively sequestered to the inner leaflet of the cell membrane. Exposure of PS on apoptotic cells is a normal physiological process that triggers their rapid removal by phagocytic engulfment under noninflammatory conditions via receptors primarily expressed on immune cells. PS is aberrantly exposed in the tumor microenvironment and contributes to the overall immunosuppressive signals that antagonize the development of local and systemic antitumor immune responses.

First-in-Man Evaluation of I-PGN650: A PET Tracer for Detecting Phosphatidylserine as a Biomarker of the Solid Tumor Microenvironment.

Purpose: PGN650 is a F(ab') antibody fragment that targets phosphatidylserine (PS), a marker normally absent that becomes exposed on tumor cells and tumor vasculature in response to oxidative stress and increases in response to therapy. PGN650 was labeled with I to create a positron emission tomography (PET) agent as an in vivo biomarker for tumor microenvironment and response to therapy. In this phase 0 study, we evaluated the pharmacokinetics, safety, radiation dosimetry, and tumor targeting of this tracer in a cohort of patients with cancer.

Phosphatidylserine Sensing by TAM Receptors Regulates AKT-Dependent Chemoresistance and PD-L1 Expression.

Tyro3, Axl, and Mertk (collectively TAM receptors) are three homologous receptor tyrosine kinases that bind vitamin K-dependent endogenous ligands, Protein S (ProS), and growth arrest-specific factor 6 (Gas6), and act as bridging molecules to promote phosphatidylserine (PS)-mediated clearance of apoptotic cells (efferocytosis). TAM receptors are overexpressed in a vast array of tumor types, whereby the level of expression correlates with the tumor grade and the emergence of chemo- and radioresistance to targeted therapeutics, but also have been implicated as inhibitory receptors on infiltrating myeloid-derived cells in the tumor microenvironment that can suppress host antitumor immunity. In the present study, we utilized TAM-IFNγR1 reporter lines and expressed TAM receptors in a variety of epithelial cell model systems to show that each TAM receptor has a unique pattern of activation by Gas6 or ProS, as well as unique dependency for PS on apoptotic cells and PS liposomes for activity.

Phosphatidylserine is a global immunosuppressive signal in efferocytosis, infectious disease, and cancer.

Apoptosis is an evolutionarily conserved and tightly regulated cell death modality. It serves important roles in physiology by sculpting complex tissues during embryogenesis and by removing effete cells that have reached advanced age or whose genomes have been irreparably damaged. Apoptosis culminates in the rapid and decisive removal of cell corpses by efferocytosis, a term used to distinguish the engulfment of apoptotic cells from other phagocytic processes.

Formation, clearance, deposition, pathogenicity, and identification of biopharmaceutical-related immune complexes: review and case studies.

Vascular inflammation, infusion reactions, glomerulopathies, and other potentially adverse effects may be observed in laboratory animals, including monkeys, on toxicity studies of therapeutic monoclonal antibodies and recombinant human protein drugs. Histopathologic and immunohistochemical (IHC) evaluation suggests these effects may be mediated by deposition of immune complexes (ICs) containing the drug, endogenous immunoglobulin, and/or complement components in the affected tissues. ICs may be observed in glomerulus, blood vessels, synovium, lung, liver, skin, eye, choroid plexus, or other tissues or bound to neutrophils, monocytes/macrophages, or platelets.

Measuring response to therapy by near-infrared imaging of tumors using a phosphatidylserine-targeting antibody fragment.

Imaging tumors and their response to treatment could be a valuable biomarker toward early assessment of therapy in patients with cancer. Phosphatidylserine (PS) is confined to the inner leaflet of the plasma membrane in normal cells but is externalized on tumor vascular endothelial cells (ECs) and tumor cells, and PS exposure is further enhanced in response to radiation and chemotherapy. In the present study, we evaluated the potential of a PS-targeting human F(ab')2 antibody fragment, PGN650, to detect exposure of PS in tumor-bearing mice.

Targeting of humanized antibody D93 to sites of angiogenesis and tumor growth by binding to multiple epitopes on denatured collagens.

A humanized, affinity-matured IgG1 antibody, called D93, and its parental murine IgM HUI77 have been shown to specifically bind denatured collagens and thereby inhibit angiogenesis and tumor growth in various animal models. In this study, we have identified epitopes for both HUI77 and D93 on human collagen type IV. Several tryptic D93-binding peptides were identified by Western blot analysis and protein sequencing.

Novel anti-denatured collagen humanized antibody D93 inhibits angiogenesis and tumor growth: An extracellular matrix-based therapeutic approach.

Matrix metalloproteases (MMPs) secreted by both tumor and endothelial cells proteolytically degrade collagen during tumor growth and neo-vascularization. This exposes cryptic binding sites on collagen with functional relevance for angiogenesis. In this report, we characterized a novel humanized monoclonal IgG1 antibody, D93.

Rapid vascular regrowth in tumors after reversal of VEGF inhibition.

Inhibitors of VEGF signaling can block angiogenesis and reduce tumor vascularity, but little is known about the reversibility of these changes after treatment ends. In the present study, regrowth of blood vessels in spontaneous RIP-Tag2 tumors and implanted Lewis lung carcinomas in mice was assessed after inhibition of VEGF receptor signaling by AG-013736 or AG-028262 for 7 days. Both agents caused loss of 50%-60% of tumor vasculature.

Intratracheal administration of interleukin 12 plasmid-cationic lipid complexes inhibits murine lung metastases.

Administration of plasmid/lipid complexes to the lung airways for the treatment of metastatic pulmonary diseases represents a new strategy of gene therapy. In this study we present evidence that intratracheal administration of a plasmid encoding murine IL-12 complexed with N-[1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride:cholesterol inhibits the growth of lung metastases, using a renal cell carcinoma model. Instillation of pIL-12/lipid complexes resulted in expression of biologically active IL-12 (170-240 pg/ml) and IFN-gamma (100-190 pg/ml) in the bronchoalveolar lavage fluid.

Ultrasonic nebulization of cationic lipid-based gene delivery systems for airway administration.

Purpose: This study relates to the development of gene therapies for the treatment of lung diseases. It describes for the first time the use of ultrasonic nebulization for administration of plasmid/lipid complexes to the lungs to transfect lung epithelial cells.

Methods: Plasmid complexed to cationic liposomes at a specific stoichiometric ratio was nebulized using an ultrasonic nebulizer.

Target specific optimization of cationic lipid-based systems for pulmonary gene therapy.

Purpose: Cationic lipids are capable of transferring foreign genes to the pulmonary epithelium in vivo. It is becoming increasingly clear that factors other than lipid molecular structure also influence efficiency of delivery using cationic lipid systems. This study is aimed at evaluating the effect of formulation variables such as cationic lipid structure, cationic lipid/DNA ratio, particle size, co-lipid content and plasmid topology on transgene expression in the lung.

Cationic lipids enhance cytokine and cell influx levels in the lung following administration of plasmid: cationic lipid complexes.

Administration of plasmid/lipid complexes to the lung airways may be associated, in addition to expression of transgene, with a range of other responses. We report here the induction of cytokines and cellular influx in the lung airway following intratracheal administration of an N-[1-(2-3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride/cholesterol/plasmid positively charged complex in mice. We show that 1) the appearance of the Th1-associated cytokines IFN-gamma and IL-12 in bronchoalveolar lavage fluid is caused by unmethylated CpG dinucleotide sequences present within the plasmid, and is enhanced by the lipid formulation; 2) cationic lipids by themselves do not induce IL-12 or IL-12p40; 3) TNF-alpha is rapidly induced by cationic lipids and plasmid/lipid complex, but not by plasmid alone; 4) an acute cellular influx is induced by cationic lipid alone and by a plasmid/lipid complex, but to a much lesser extent by plasmid alone; and 5) plasmid methylation does not influence the degree of inflammatory cell influx.

Isothiazolones interfere with normal matrix metalloproteinase activation and inhibit cartilage proteoglycan degradation.

A series of isothiazolones that inhibit pro-(matrix metallo-proteinase) (proMMP) activation but do not inhibit the active enzyme are effective as cartilage protectants in bovine nasal cartilage organ culture, preventing interleukin-1 (IL-1)-induced proteoglycan (aggrecan) degradation without affecting its synthesis. These compounds were found to bind to prostromelysin (proMMP-3) in a non-dialysable and stoichiometric manner. Preincubation with cartilage-protectant isothiazolones prevented the binding of [14C]iodoacetamide to Cys75 of the MMP-3 propeptide, suggesting that the activity of these compounds involves their binding to the Cys75 of the MMP zymogen.

2,5-Diarylisothiazolone: novel inhibitors of cytokine-induced cartilage destruction.

A series of 2,5-diarylisothiazolones is reported that inhibit the IL-1 beta-induced breakdown of bovine nasal septum cartilage in an organ culture assay. The synthesis and preliminary SAR of these compounds are described. These compounds represent a novel, nonpeptide lead series approach to the mediation of the chronic cartilage breakdown associated with arthritic disease.

Metabolism resistant isothiazolone inhibitors of cartilage breakdown.

A series of 2-(arylmethyl)pyridoisothiazolones is reported that inhibit the IL-1 beta induced breakdown of bovine nasal septum cartilage in an organ culture assay. The synthesis and preliminary SAR of these compounds are described. These compounds represent a novel, non-peptide lead series approach to the mediation of the chronic cartilage breakdown associated with arthritic disease.

Multiple sites of the propeptide region of human stromelysin-1 are required for maintaining a latent form of the enzyme.

Latency of matrix metalloproteinase 3 (MMP-3) is regulated by the interaction of a free cysteine residue (Cys-75) in the conserved amino acid sequence Pro-Arg-Cys-Gly-Val-Pro-Asp located in the COOH-terminal portion of the propeptide with a chelated zinc atom in the active site of the catalytic domain. Proteolytic activation of full-length human pro-MMP-3 involves the removal of approximately 35 amino acids from the NH2-terminal portion of the propeptide, forming a 53-kDa unstable intermediate that undergoes intermolecular autocatalysis to form the 45-kDa mature active enzyme. In this study, we have evaluated the contribution of the NH2-terminal 35 amino acids to the maintenance of latency.

Heteroaryl-fused 2-phenylisothiazolone inhibitors of cartilage breakdown.

The synthesis, biological evaluation, and structure-activity relationships of a series of N-phenyl heteroaryl-fused isothiazolones are described. These isothiazolones have been shown to exhibit potent, dose-dependent inhibition of IL-1 beta-induced breakdown of proteoglycan in a cartilage organ culture assay. This effect is likely due to inhibition of MMP activation and a consequent reduction in MMP activity following IL-1 beta stimulation.

Production of human matrix metalloproteinase 3 (stromelysin) in Escherichia coli.

Full-length human matrix metalloproteinase 3 (prostomelysin or proMMP-3) was produced in Escherichia coli as an intracellular insoluble aggregate that could be solubilized and refolded to yield an activatable proenzyme. The refolded protein was purified to > 95% homogeneity. The recombinant proMMP-3 (re-proMMP-3) could be activated by agents known to stimulate self-catalyzed cleavage of native fibroblast proMMP-3.

IL-1 and its role in rat carrageenan pleurisy.

The carrageenan pleurisy model, which is characterized by cellular influx and oedema, has been used to examine the effects of anti-inflammatory compounds such as naproxen. Interleukin-1alpha and beta (IL-1) are known to be pro-inflammatory mediators, and their roles in this model are unknown. Intrapleural injection of 1% viscarin carrageenan or saline was administered to male Lewis rats.

Quantitative characterization of the intrinsic ligand-binding affinity of the interleukin 2 receptor beta chain and its modulation by the alpha chain and a second affinity-modulating element.

The interleukin 2 receptor is a multisubunit receptor known to consist of at least two IL-2 binding subunits, alpha and beta. We report here kinetic evidence defining the contribution of an affinity-modulating element(s) intimately involved in modulation of the ligand-binding affinity of the beta chain and alpha/beta complex. The principal effect of this modulating element on the beta chain is to slow the dissociation of IL-2 more than 150-fold and thus raise its low intrinsic IL-2 binding affinity (Kd = 70 nM) as defined in transfected fibroblast cells to the level observed in lymphoid cells (Kd = 1.

Functional expression of native and chimeric human and murine IL-2 receptor p70 chains in an IL-3-dependent murine cell line.

Full length cDNAs encoding the human and murine p70 genes were isolated using polymerase chain reaction (PCR) and conventional cDNA library screening techniques, respectively. To validate their functional potential, expression vectors containing human, murine and chimeric human/murine p70 cDNAs were transfected into the murine IL-3-dependent cell line FDC-P1. Transfected cells expressed a combination of high and low-affinity IL-2 binding sites while parental cells displayed only the low-affinity sites associated with expression of endogenous p55 IL-2 receptor chains.