Differential Leukocyte MicroRNA Responses Following Pan T Cell, Allorecognition and Allosecretome-Based Therapeutic Activation.

Effective immunomodulation of T-cell responses is critical in treating both autoimmune diseases and cancer. Our previous studies have demonstrated that secretomes derived from control or methoxypolyethylene glycol mixed lymphocyte alloactivation assays exerted potent immunomodulatory activity that was mediated by microRNAs (miRNA). The immunomodulatory effects of biomanufactured miRNA-based allo-secretome therapeutics (SYN, TA1, IA1 and IA2) were compared to Pan T-cell activators (PHA and anti-CD3/CD28) and lymphocyte alloactivation. The differential effects of these activation strategies on resting peripheral blood mononuclear cells (PBMC) were assessed via T-cell proliferation, subset analysis and miRNA expression profiles. Mitogen-induced PBMC proliferation (> 85%) significantly exceeded that arising from either allostimulation (~ 30%) or the pro-inflammatory IA1 secretome product (~ 12%). Consequent to stimulation, the ratio of CD4 to CD8 cells of the resting PBMC (CD4:CD8; 1.7 ± 0.1) decreased in the Pan T cell, allrecognition and IA1 activated cells (averages of 1.1 ± 0.2; 1.2 ± 0.1 and 1.0 ± 0.1). These changes arose consequent to the expansion of both CD4+CD8+ and CD4-CD8- populations as well as the shrinkage of the CD4 subset and the expansion of the CD8 T cells. Importantly, these activation strategies induced vastly different miRNA expression profiles which were associated with significant differences in cellular differentiation and biological function. These findings support the concept that the "differential patterns of miRNA expression" regulate the biologic immune response in a "lock and key" manner. The biomanufacturing of miRNA-enriched secretome biotherapeutics may be a successful therapeutic approach for the systemic treatment of autoimmune diseases (TA1) and cancer (IA1).

Enhancing the pro-inflammatory anti-cancer T cell response via biomanufactured, secretome-based, immunotherapeutics.

T lymphocytes play a critical role in the pro-inflammatory anti-cancer response; hence, significant pharmacologic efforts have been made to enhance the endogenous T cell response. Unfortunately, significant toxicity arises consequent to pan T cell activation. In contrast, the less robust T cell alloresponse has also demonstrated an anti-cancer effect, but poses an inherent risk of GvHD. To overcome the GvHD risk, an acellular pro-inflammatory agent (IA1) has been biomanufactured from the secretome of the allorecognition response. To assess IA1's immunomodulatory activity, T cell proliferation and differentiation were determined in vitro. The pro-inflammatory properties of the IA1 therapeutic were mediated by the miRNA-enriched fractions. Moreover, cross-species efficacy was observed consequent to the evolutionary conservation of miRNA. IA1 exerted no toxicity to resting PBMC but induced significant proliferation of resting CD3+ (CD4+ and CD8+) T cells and skewed the response towards a pro-inflammatory state (i.e., increased Teff:Treg ratio). Crucially, IA1-activated PBMC demonstrated a potent inhibition of cancer cell (HeLa and SH-4 melanoma) proliferation relative to the resting PBMC. The anti-proliferation effect of IA1-activated PBMC was noted within ˜12 h versus 4-5 days for resting cells. A second biomanufactured therapeutic (IA2; produced using HeLa cells) surprisingly demonstrated direct toxicity to cancer cells but was less effective than IA1 in inducing a cell-mediated response. This study demonstrates that miRNA-enriched therapeutics can be biomanufactured from the secretome and can induce a potent pro-inflammatory, anti-cancer, effect on resting lymphocytes.

Effects of methoxypoly (Ethylene glycol) mediated immunocamouflage on leukocyte surface marker detection, cell conjugation, activation and alloproliferation.

Tissue rejection occurs subsequent to the recognition of foreign antigens via receptor-ligand contacts between APC (antigen presenting cells) and T cells, resulting in initialization of signaling cascades and T cell proliferation. Bioengineering of donor cells by the covalent attachment of methoxypolyethylene glycol (mPEG) to membrane proteins (PEGylation) provides a novel means to attenuate these interactions consequent to mPEG-induced charge and steric camouflage. While previous studies demonstrated that polymer-mediated immunocamouflage decreased immune recognition both in vitro and in vivo, these studies monitored late events in immune recognition and activation such as T cell proliferation. Consequently little information has been provided concerning the early cellular events governing this response. Therefore, the effect of PEGylation was assessed by examining initial cell-cell interactions, changes to activation pathways, and apoptosis to understand the role that each may play in the decreased proliferative response observed in modified cells during the course of a mixed lymphocyte reaction (MLR). The mPEG-modified T cells resulted in significant immunocamouflage of lymphocyte surface proteins and decreased interactions with APC. Furthermore, mPEG-MLR exhibited decreased NFκB pathway activation, while exhibiting no significant differences in degree of cell death compared to the control MLR. These results suggest that PEGylation may prevent the direct recognition of foreign alloantigens by decreasing the stability and duration of initial cell-cell interactions.

Comparative efficacy of blood cell immunocamouflage by membrane grafting of methoxypoly(ethylene glycol) and polyethyloxazoline.

The grafting of low-immunogenic polymers to cells dramatically reduces antigenic recognition and immunogenicity of allogeneic donor cells consequent to steric and charge camouflage (i.e., immunocamouflage). While methoxypoly(ethylene glycol) [mPEG] has historically been utilized for the immunocamouflage of cells, other low-immunogenic polymers such as polyethyloxazoline propionic acid (PEOZ) may also be capable of conferring immunoprotection. Moreover, PEOZ may have attributes that could have enhanced pharmacological and biological utility relative to mPEG. To evaluate the immunocamouflage efficacy of PEOZ relative to mPEG, human red blood cells (RBC) and leukocytes were modified with mPEG or PEOZ. The differential effects of mPEG and PEOZ was assessed via grafting efficacy, cell morphology and viability, immunocamouflage of surface antigens, and the prevention of in vitro immune recognition (RhD and HLA). Although membrane grafting of mPEG and PEOZ were similar, mPEG demonstrated superior immunocamouflage efficacy as measured by antibody binding and phagocytosis of opsonized RBC while PEOZ showed improved RBC morphology. While mPEG appears to be superior to PEOZ in the immunocamouflage of cells, PEOZ may still be a valuable addition to our repertoire of immunomodulatory polymers. Moreover, our results demonstrate the importance of indirect immunocamouflage of antigens found in membrane protein complexes.

Influence of polymer architecture on antigens camouflage, CD47 protection and complement mediated lysis of surface grafted red blood cells.

Hyperbranched polyglycerol (HPG) and polyethylene glycol (PEG) polymers with similar hydrodynamic sizes in solution were grafted to red blood cells (RBCs) to investigate the impact of polymer architecture on the cell structure and function. The hydrodynamic sizes of polymers were calculated from the diffusion coefficients measured by pulsed field gradient NMR. The hydration of the HPG and PEG was determined by differential scanning calorimetry analyses. RBCs grafted with linear PEG had different properties compared to the compact HPG grafted RBCs. HPG grafted RBCs showed much higher electrophoretic mobility values than PEG grafted RBCs at similar grafting concentrations and hydrodynamic sizes indicating differences in the structure of the polymer exclusion layer on the cell surface. PEG grafting impacted the deformation properties of the membrane to a greater degree than HPG. The complement mediated lysis of the grafted RBCs was dependent on the type of polymer, grafting concentration and molecular size of grafted chains. At higher molecular weights and graft concentrations both HPG and PEG triggered complement activation. The magnitude of activation was higher with HPG possibly due to the presence of many hydroxyl groups per molecule. HPG grafted RBCs showed significantly higher levels of CD47 self-protein accessibility than PEG grafted RBCs at all grafting concentrations and molecular sizes. PEG grafted polymers provided, in general, a better shielding and protection to ABO and minor antigens from antibody recognition than HPG polymers, however, the compact HPGs provided greater protection of certain antigens on the RBC surface. Our data showed that HPG 20 kDa and HPG 60 kDa grafted RBCs exhibited properties that are more comparable to the native RBC than PEG 5 kDa and PEG 10 kDa grafted RBCs of comparable hydrodynamic sizes. The study shows that small compact polymers such as HPG 20 kDa have a greater potential in the generation of functional RBC for therapeutic delivery applications. The intermediate sized polymers (PEG or HPG) which showed greater antigen camouflage at lower grafting concentrations have significant potential in transfusion as universal red blood donor cells.

Immunocamouflage of latex surfaces by grafted methoxypoly(ethylene glycol) (mPEG): proteomic analysis of plasma protein adsorption.

Grafting of methoxypoly(ethylene glycol) (mPEG) to cells and biomaterials is a promising non-pharmacological immunomodulation technology. However, due to the labile nature of cells, surface-plasma interactions are poorly understood; hence, a latex bead model was studied. PEGylation of beads resulted in a density and molecular weight dependent decrease in total adsorbed protein with a net reduction from (159.9±6.4) ng cm(-2) on bare latex to (18.4±0.8) and (52.3±5.3) ng cm(-2) on PEGylated beads (1 mmol L(-1) of 2 or 20 kD SCmPEG, respectively). SDS-PAGE and iTRAQ-MS analysis revealed differential compositions of the adsorbed protein layer on the PEGylated latex with a significant reduction in the compositional abundance of proteins involved in immune system activation. Thus, the biological efficacy of immunocamouflaged cells and materials is mediated by both biophysical obfuscation of antigens and reduced surface-macromolecule interactions.

The potential utility of methoxypoly(ethylene glycol)-mediated prevention of rhesus blood group antigen RhD recognition in transfusion medicine.

Red blood cell (RBC) transfusions are an important clinical intervention. However, RBC express hundreds of non-ABO antigens making alloimmunization a significant risk. RhD expression is the most immunologically important non-ABO antigen. Availability of RhD(-) blood, often problematic in North America and Europe, is a significant issue in Asia and Africa where RhD(-) blood is uncommon (<0.5% of supply). The immunocamouflage of RhD is readily accomplished by the covalent grafting of methoxypoly(ethylene glycol) [mPEG] to the RBC membrane. To determine if RhD immunocamouflage would inhibit its immunologic recognition, an in vitro RhD-sensitized antigen presentation assay using PBMC and dendritic cells (DC) from RhD-sensitized women was used. The immunological effects of polymer grafting to an immunodominant RhD peptide, purified RhD protein and intact RhD(+) RBC were examined via T cell proliferation and cytokine release assays. At Day 11, PEGylation significantly attenuated T cell proliferation arising from RhD peptide (~80 → 5%), protein (36 → 0.2%) and intact RBC (33 → 1.4%). Cytokine secretion was similarly blunted following PEGylation of the purified protein or intact RBC. These data support the immunomodulatory effects of PEGylation and the potential utility of this technology in transfusion medicine - especially in situations where RhD(-) blood is rare or in short supply.

The effect of grafted methoxypoly(ethylene glycol) chain length on the inhibition of respiratory syncytial virus (RSV) infection and proliferation.

Respiratory syncytial virus (RSV) is a significant cause of morbidity in humans. To date, no effective treatments exist and current prophylactic therapy access is limited and is only approximately 50% effective. To attenuate the risk of RSV infection, we hypothesized that bioengineering of either the virus particle or host cell via the covalent grafting of methoxypoly(ethylene glycol) [mPEG] would prevent infection. To this end, the anti-viral effects of grafting concentration, linker chemistry and polymer length on RSV infection was assessed. For viral modification, short chain polymers (2 kDa) were significantly more effective than long chain (20 kDa) polymers. In contrast, modification of host cells with small polymers provided no (approximately 0%) protection while long chain polymers effectively prevented infection. For example, at 48 hours post-infection at a multiplicity of infection of 0.5 and grafting concentrations of 5, 7.5, and 15 mm, 20 kDa mPEG decreased infection by 45, 83, and 91%, respectively. Importantly, both viral and host cell PEGylation strategies were able to provide near complete protection against RSV infection of both non-polarized and polarized cells. In conclusion, mPEG-modification of either RSV or the host cell is a highly effective prophylactic strategy for preventing viral infection.

Immunocamouflage: the biophysical basis of immunoprotection by grafted methoxypoly(ethylene glycol) (mPEG).

Development of novel approaches for the immunomodulation of donor cells would have significant utility in transfusion and transplantation medicine. Immunocamouflage of cell surfaces by covalently grafted methoxypoly(ethylene glycol) (mPEG) (PEGylation) has emerged as a promising approach. While previous studies demonstrated the in vitro and in vivo efficacy of immunocamouflaged allogeneic blood cells, the biophysical mechanisms of immunoprotection have not been well-defined due to the fragility of intact cells. To overcome this limitation, polystyrene beads (1.2 and 8.0 microm) were used to elucidate the biophysical effects of polymer size, density and linker chemistry on charge camouflage and protein adsorption. These findings were correlated with biological studies using red blood cells and lymphocytes. Charge camouflage of both beads and cells was best achieved with long polymers. However, protein adsorption studies demonstrated an unexpected effect of target size. For 1.2 microm beads, decreased protein adsorption was best achieved with short (2 kDa) polymers whereas long chain (20 kDa) polymers were optimal for 8.0 microm particles. The biophysical findings correlated well with biological immunocamouflage as measured by particle electrophoresis and the inhibition of antibody-antigen (CD3, CD4 and CD28) recognition. Moreover, it was observed that antigen topography (CD28 vs. CD4) was of significance in selecting the appropriate polymer size. The biophysical interactions of PEGylated surfaces and macromolecules involve complex mechanisms dependent on the molecular weight, grafting concentration, target size and surface complexity. Cellular PEGylation strategies must be customized to account for target cell size, membrane complexity and antigen density and height.

In vitro chelating, cytotoxicity, and blood compatibility of degradable poly(ethylene glycol)-based macromolecular iron chelators.

Desferrioxamine (DFO) is used to treat an excess accumulation of iron in the body and is currently the most commonly used iron chelator for the treatment of 'iron overload' disorder. However, the disadvantages of DFO surround its high toxicity and very short plasma half-life. Here, the detailed in vitro evaluation of a novel class of high molecular weight iron chelators based on DFO and polyethylene glycol methacrylate is reported. Reversible addition fragment chain transfer (RAFT) copolymerization afforded polymer conjugates (P-DFO) with well-controlled molecular weight (27-127 kDa) and substitution of DFO (5-26 units per chain) along the copolymer. Human umbilical vein endothelial cell (HUVEC) based cell viability assays showed that the cytotoxicity of P-DFO decreased more than 100-fold at identical concentrations of DFO. The hemocompatibilities of various P-DFO samples were determined by measuring prothrombin time (PT), activated partial thromboplastin time (APTT), thrombelastograph parameters (TEG), complement activation, platelet activation, and red blood cell aggregation. Furthermore, the iron binding properties and chelating efficiency of P-DFO were compared to DFO by measuring the spectral properties upon binding to iron(III), while the prevention of iron(III) mediated oxidation of hemoglobin was also determined. Degradation of the P-DFO conjugates via cleavable ester linkages between the polymer backbone and the PEG side chains was evaluated using gel permeation chromatography (GPC) and NMR. Since the chelating ability of DFO remains intact after conjugation to the copolymer backbone, these macromolecular, blood compatible and degradable conjugates are promising candidates as long circulating, non-toxic iron chelators.

Immune complex binding by immunocamouflaged [poly(ethylene glycol)-grafted] erythrocytes.

Immune complexes (IC) are constantly formed at low levels in normal individuals. In humans, the red blood cell (RBC) complement receptor 1 (CR1) plays the dominant role in the IC binding and clearance. Over the last several years, we have investigated the potential utility of immunocamouflaged (methoxypoly(ethylene glycol) [mPEG] grafted) RBC to attenuate the risk of alloimmunization. Because the grafted polymer nonspecifically camouflages membrane proteins, its effects on CR1 detection and IC binding were assessed. The dose dependent (0-2.5 mM) effects of activated mPEG (CmPEG, 5 kDa; and BTCmPEG, 5 and 20 kDa) on CR1 detection and the binding of artificially generated IC [C3b coated alkaline phosphatase and antialkaline phosphatase complexes] to control and pegylated RBC was investigated by flow cytometry. In contrast to selected non-ABO blood group antigens, grafted mPEG did not effectively camouflage CR1. Surprisingly, however, even very low grafting concentrations of mPEG (>or=0.3 mM) resulted in a >or=95% loss in IC binding. Further reductions in grafting concentration (0.15 and 0.03 mM mPEG) still yielded decreased IC binding of approximately 60 and 40%, respectively. Importantly, unactivated mPEG had minimal effects on IC binding. These data demonstrate that even small amounts of grafted mPEG interfere with the multivalent CR1-IC interaction necessary for high affinity IC binding, hence large volume transfusions of mPEG-RBC may be contraindicated in patients with pre-existing IC disease. Whether this concern is of clinical significance in healthy humans is less clear due to dilutional effects and the presence of secondary clearance pathways.

H2O2 injury in beta thalassemic erythrocytes: protective role of catalase and the prooxidant effects of GSH.

Redox-mediated injury is an important pathway in the destruction of beta thalassemic red blood cells (RBC). Because of the autoxidation of the unstable hemoglobin chains and subsequent release of globin free heme and iron, significant amounts of superoxide (O2-) and, more importantly, hydrogen peroxide (H2O2) are generated intracellularly. Hence, catabolism of H2O2 is crucial in preventing cellular injury. Removal of H2O2 is mediated via two primary pathways: GSH-dependent glutathione peroxidase or catalase. Importantly, both pathways are ultimately dependent on NADPH. In the absence of any exogenous oxidants, model thalassemic RBC demonstrated significantly decreased GSH levels (P < 0.001 at 20 h). Perhaps of greater pathophysiologic importance, however, was the finding that the model thalassemic RBC exhibited significantly (P < 0.001) decreased catalase activity. Following 20 h incubation at 37 degrees C only 61.5 +/- 2.9% of the initial catalase activity remained in the alpha-hemoglobin chain-loaded cells versus 104.6 +/- 4.5 and 108.2 +/- 3.2% in the control and control-resealed cells, respectively. The mechanism underlying the loss of both catalase activity and GSH appears to be the same in that both catabolic pathways require adequate NADPH levels. As shown in this study, model beta thalassemic cells are unable to maintain a normal ( approximately 1.0) NADPH/NADP(total) ratio and, after 20 h, the model beta thalassemic cells have a significantly (P < 0.001) lower ratio ( approximately 0.5) which is quite similar to a G6PD-deficient RBC. In support of these findings, direct inactivation of catalase gives rise to significantly increased oxidant damage. In contrast, GSH depletion is not closely associated with oxidant sensitivity. Indeed, the consumption of GSH noted in the thalassemic RBC may be via a prooxidant pathway as augmentation of cellular GSH levels actually enhances alpha-hemoglobin chain-mediated injury.

Inactivation of prion proteins via covalent grafting with methoxypoly(ethylene glycol).

Transmissible spongiform encephalopathies (TSE) such as bovine spongiform encephalitis (BSE), Creutzfeld-Jakob disease (CJD) as well as other proteinaceous infectious particles (prions) mediated diseases have emerged as a significant concern in transfusion medicine. This concern is derived from both the disease causing potential of prion contaminated blood products but also due to tremendous impact of the active deferral of current and potential blood donors due to their extended stays in BSE prevalent countries (e.g., the United Kingdom). To date, there are no effective means by which infectious prion proteins can be inactivated in cellular and acellular blood products. Based on current work on the covalent grafting of methoxypoly(ethylene glycol) [mPEG] to proteins, viruses, and anuclear, and nucleated cells, it is hypothesized that the conversion of the normal PrP protein to its mutant conformation can be prevented by the covalent grafting of mPEG to the mutant protein. Inactivation of infective protein particles (prions) in both cellular blood products as well as cell free solutions (e.g., clotting factors) could be of medical/commercial value. It is hypothesized that consequent to the covalent modification of donor-derived prions with mPEG the requisite nucleation of the normal and mutant PrP proteins is inhibited due to the increased solubility of the modified mutant PrP and that the conformational conversion arising from the mutant PrP is prevented due to obscuration of protein charge by the heavily hydrated and neutral mPEG polymers, as well as by direct steric hindrance of the interaction due to the highly mobile polymer graft.

Separation and purification of methoxypoly(ethylene glycol) grafted red blood cells via two-phase partitioning.

Alloimmunization to donor blood group antigens remains a significant problem in transfusion medicine. To attenuate the risk of alloimmunization, we have pioneered the membrane grafting of methoxypoly(ethylene glycol) (mPEG) to produce immunocamouflaged red blood cells (RBC). Grafting of the mPEG was accomplished using cyanuric chloride activated mPEG (CmPEG; M(r) = 5000), benzotriazole carbonate methoxyPEG (BTCmPEG; M(r) = 2000, 5000 or 20000); or N-hydroxysuccinimidyl ester of mPEG propionic acid (SPAmPEG; M(r) = 5000, or 20000). Because of the heterogeneity of grafting, a crucial tool in developing the stealth RBC is an ability to purify the modified RBC from unmodified (immunogenic) donor cells. As demonstrated, a (5, 4) dextran:PEG aqueous two-phase polymer partitioning system cleanly separated the immunologically silent mPEG-grafted human RBC from control or lightly modified cells. Cell mixing experiments employing varying ratios of mPEG-modified and control RBC confirmed the purification efficacy of the phase partitioning system. Proportional changes in PEG-rich phase partitioning were achieved by increasing either the quantity of surface mPEG or the mPEG molecular weight. The biological viability of purified mPEG-RBC (BTCmPEG; [M(r) = 20000) was demonstrated by their normal in vivo survival at immunoprotective grafting concentrations (