Search

Crigler-Najjar syndrome is an ultra-rare monogenic recessive liver disease caused by UGT1A1 gene mutations. Complete UGT1A1 deficiency results in severe unconjugated hyperbilirubinemia in newborns that, if not treated, may lead to brain damage and death. Treatment is based on intensive phototherapy, but its efficacy decreases with age, rendering liver transplantation the only curative option. Adeno-associated virus (AAV)-mediated gene therapy has shown long-term correction in adult patients, but loss of viral DNA and therapeutic efficacy are expected in younger patients associated with liver growth. Effective vector re-administration is hindered by anti-AAV neutralizing antibodies generated during the first administration. Here, we investigated AAV vector re-administration by modulating the immune response with rapamycin-loaded nanoparticles (ImmTOR) in Gunn rats ( Ugt1a -/- ) and Ugt1a -/- mice. We administered a liver-specific AAV8 vector expressing a codon-optimized h UGT1A 1 cDNA (1.0E11 vg/kg) in P25-P28 mutant animals and, upon loss of efficacy after 3 to 5 weeks, a higher second dose (1.0E12 or 5.0E12 vg/kg) was given. ImmTOR co-administration reduced anti-AAV neutralizing antibodies and immunoglobulin Gs generation in male animals of both models allowing effective re-dosing, underscored by a significant and long-term decrease in plasma bilirubin, although efficacy was affected by low-titer residual anti-AAV antibodies suggesting that re-administration in patients may require combination with other methods., Competing Interests: T.K.K. is an employee and shareholder of Selecta Bioscience; F.M., G.R., F.C., G.B., and A.F.M. are inventors in patents describing the AAV technology and gene therapy-based treatments for Crigler-Najjar syndrome., (© 2024 The Authors.)

Tolerogenic ImmTOR nanoparticles encapsulating rapamycin have been demonstrated to mitigate immunogenicity of adeno-associated virus (AAV) gene therapy vectors, enhance levels of transgene expression, and enable redosing of AAV at moderate vector doses of 2 to 5E12 vg/kg. However, recent clinical trials have often pushed AAV vector doses 10-fold to 50-fold higher, with serious adverse events observed at the upper range. Here, we assessed combination therapy of ImmTOR with B cell-targeting drugs for the ability to increase the efficiency of redosing at high vector doses. The combination of ImmTOR with a monoclonal antibody against B cell activation factor (aBAFF) exhibited strong synergy leading to more than a 5-fold to 10-fold reduction of splenic mature B cells and plasmablasts while increasing the fraction of pre-/pro-B cells. In addition, this combination dramatically reduced anti-AAV IgM and IgG antibodies, thus enabling four successive AAV administrations at doses up to 5E12 vg/kg and at least two AAV doses at 5E13 vg/kg, with the transgene expression level in the latter case being equal to that observed in control animals receiving a single vector dose of 1E14 vg/kg. Similar synergistic effects were seen with a combination of ImmTOR and a Bruton's tyrosine kinase inhibitor, ibrutinib. These results suggest that ImmTOR could be combined with B cell-targeting agents to enable repeated vector administrations as a potential strategy to avoid toxicities associated with vector doses above 1E14 vg/kg., (© The Author(s) 2023. Published by Oxford University Press on behalf of National Academy of Sciences.)

Interleukin-2 (IL-2) therapies targeting the high affinity IL-2 receptor expressed on regulatory T cells (Tregs) have shown promising therapeutic benefit in autoimmune diseases through nonselective expansion of pre-existing Treg populations, but are potentially limited by the inability to induce antigen-specific Tregs, as well as by dose-limiting activation of effector immune cells in settings of inflammation. We recently developed biodegradable nanoparticles encapsulating rapamycin, called ImmTOR, which induce selective immune tolerance to co-administered antigens but do not increase total Treg numbers. Here we demonstrate that the combination of ImmTOR and an engineered Treg-selective IL-2 variant (termed IL-2 mutein) increases the number and durability of total Tregs, as well as inducing a profound synergistic increase in antigen-specific Tregs when combined with a target antigen. We demonstrate that the combination of ImmTOR and an IL-2 mutein leads to durable inhibition of antibody responses to co-administered AAV gene therapy capsid, even at sub-optimal doses of ImmTOR, and provides protection in autoimmune models of type 1 diabetes and primary biliary cholangitis. Importantly, ImmTOR also increases the therapeutic window of engineered IL-2 molecules by mitigating effector immune cell expansion and preventing exacerbation of disease in a model of graft-versus-host-disease. At the same time, IL-2 mutein shows potential for dose-sparing of ImmTOR. Overall, these results establish that the combination of ImmTOR and an IL-2 mutein show synergistic benefit on both safety and efficacy to provide durable antigen-specific immune tolerance to mitigate drug immunogenicity and to treat autoimmune diseases., Competing Interests: Declaration of competing interest T.K.K., M.F., A.M., G.R., C.R., T.C., N.N., N.L., L.D., F.F. P.G.T., S.S.L., and P.O.I. are employees and shareholders of Selecta Biosciences, a company developing ImmTOR and ImmTOR-IL. Selecta Biosciences have filed a patent application (WO2022217095A1) on the combination of ImmTOR with engineered Treg-selective IL-2 and have licensed F5111 IC from Johns Hopkins University and the University of California San Francisco. D.VD. and J.B.S. are inventors of a patent application (WO2020264318A1) on F5111 IC., (Copyright © 2023 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Introduction: SEL-212 is a developmental treatment for uncontrolled gout characterized by serum uric acid (sUA) levels ≥ 6 mg/dl despite treatment. It comprises a novel PEGylated uricase (SEL-037; also called pegadricase) co-administered with tolerogenic nanoparticles containing sirolimus (rapamycin) (SEL-110; also called ImmTOR ® ), which mitigates the formation of anti-drug antibodies (ADAs) against uricase and SEL-037 (PEGylated uricase), thereby enabling sustained sUA control (sUA < 6 mg/dl). The aim of this study was to identify appropriate dosing for SEL-037 and SEL-110 for use in phase 3 clinical trials., Methods: This open-label phase 2 study was conducted in adults with symptomatic gout and sUA ≥ 6 mg/dl. Participants received five monthly infusions of SEL-037 (0.2 or 0.4 mg/kg) alone or in combination with three or five monthly infusions of SEL-110 (0.05-0.15 mg/kg). Safety, tolerability, sUA, ADAs, and tophi were monitored for 6 months., Results: A total of 152 adults completed the study. SEL-037 alone resulted in rapid sUA reductions that were not sustained beyond 30 days in most participants due to ADA formation and loss of uricase activity. Levels of ADAs decreased with increasing doses of SEL-110 up to 0.1 mg/kg, with anti-uricase titers < 1080 correlating with sustained sUA control and reductions in tophi. Overall, 66% of evaluable participants achieved sUA control at week 20 following five monthly doses of SEL-037 0.2 mg/kg + SEL-110 0.1-0.15 mg/kg, whereas only 26% achieved sUA control at week 20 when SEL-110 was withdrawn after week 12. Compared to other dose combinations, SEL-037 0.2 mg/kg + SEL-110 0.15 mg/kg achieved the greatest sUA control at week 12 and was well-tolerated with no safety concerns., Conclusion: Results provide continued support for the use of multiple monthly administrations of SEL-037 0.2 mg/kg + SEL-110 0.1-0.15 mg/kg in clinical trials for SEL-212., Trial Registration: ClinicalTrials.gov identifier, NCT02959918., (© 2023. The Author(s).)

Background & Aims: Gene therapy using recombinant adeno-associated virus (rAAV) vector carrying multidrug resistance protein 3 (MDR3) coding sequence (AAV8-MDR3) represents a potential curative treatment for progressive familial intrahepatic cholestasis type 3 (PFIC3), which presents in early childhood. However, patients with the severest form of PFIC3 should receive treatment early after detection to prevent irreversible hepatic fibrosis leading ultimately to liver transplantation or death. This represents a challenge for rAAV-based gene therapy because therapeutic efficacy is expected to wane as rAAV genomes are lost owing to hepatocyte division, and the formation of AAV-specific neutralising antibodies precludes re-administration. Here, we tested a strategy of vector re-administration in infant PFIC3 mice with careful evaluation of its oncogenicity - a particular concern surrounding rAAV treatment., Methods: AAV8-MDR3 was re-administered to infant Abcb4 -/- mice 2 weeks after a first dose co-administered with tolerogenic nanoparticles carrying rapamycin (ImmTOR) given at 2 weeks of age. Eight months later, long-term therapeutic efficacy and safety were assessed with special attention paid to the potential oncogenicity of rAAV treatment., Results: Co-administration with ImmTOR mitigated the formation of rAAV-specific neutralising antibodies and enabled an efficacious second administration of AAV8-MDR3, resulting in stable correction of the disease phenotype, including a restoration of bile phospholipid content and healthy liver function, as well as the prevention of liver fibrosis, hepatosplenomegaly, and gallstones. Furthermore, efficacious repeat rAAV administration prevented the appearance of liver malignancies in an animal model highly prone to developing hepatocellular carcinoma., Conclusions: These outcomes provide strong evidence for rAAV redosing through co-administration with ImmTOR, as it resulted in a long-term therapeutic effect in a paediatric liver metabolic disorder, including the prevention of oncogenesis., Impact and Implications: Redosing of gene therapy for inborn hepatobiliary disorders may be essential as effect wanes during hepatocyte division and renewal, particularly in paediatric patients, but the approach may carry long-term risks of liver cancer. Viral vectors carrying a therapeutic gene exerted a durable cure of progressive familial intrahepatic cholestasis type 3 in infant mice and reduced the risk of liver cancer only following a second administration., Competing Interests: NDW, IRG, JPC, and GGA are employees and stockholders of Vivet Therapeutics. TKK is an employee of Selecta Biosciences. All other authors declare no conflict of interest. Please refer to the accompanying ICMJE disclosure forms for further details., (© 2023 The Author(s).)

Biologic drugs have transformed the standard of care for many diseases. However, many biologics induce the formation of anti-drug antibodies (ADAs), which can compromise their safety and efficacy. Preclinical studies demonstrate that biodegradable nanoparticles-encapsulating rapamycin (ImmTOR), but not free rapamycin, mitigate the immunogenicity of co-administered biologic drugs. Here we report the outcomes from two clinical trials for ImmTOR. In the first ascending dose, open-label study (NCT02464605), pegadricase, an immunogenic, pegylated uricase enzyme derived from Candida utilis, is assessed for safety and tolerability (primary endpoint) as well as activity and immunogenicity (secondary endpoint); in the second single ascending dose Phase 1b trial (NCT02648269) composed of both a double-blind and open-label parts, we evaluate the safety of ImmTOR (primary endpoint) and its ability to prevent the formation of anti-drug antibodies against pegadricase and enhance its pharmacodynamic activity (secondary endpoint) in patients with hyperuricemia. The combination of ImmTOR and pegadricase is well tolerated. ImmTOR inhibits the development of uricase-specific ADAs in a dose-dependent manner, thus enabling sustained enzyme activity and reduction in serum uric acid levels. ImmTOR may thus represent a feasible approach for preventing the formation of ADAs to a broad range of immunogenic biologic therapies., (© 2022. The Author(s).)

In recent months as vaccines against the SARS-CoV-2 virus continue to rollout across the globe, there has been a renewed interest in ways to activate or ignite the immune system. For a vaccine to be effective, it must be immunogenic and specific to provoke the body's defenses to mount an effective response that protects the host from disease. However, there are other situations wherein the immune system mounts an unwanted immune response that can be detrimental to health, either directly, by causing an autoimmune disease, or indirectly, by compromising the safety and/or efficacy of biologic drugs. In these scenarios, it would be desirable to have a 'tolerogenic vaccine' that could selectively and effectively mitigate these unwanted immune responses. ImmTORTM, a nanoparticle technology, is being developed to address the issue of immunogenicity for gene therapy vectors and other biologic drugs. By targeting antigen-presenting cells, ImmTORTM has the potential to amplify the efficacy of biologic therapies and unlock the full potential of such treatments to improve the lives of those who suffer from serious and debilitating diseases., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society and the Royal Society of Biology.)

Current treatments for unwanted antibody responses largely rely on immunosuppressive drugs compromising overall immunity. New approaches to achieve antigen-specific tolerance are desirable to avoid unwanted side effects. Several nanoparticle-based approaches that utilize different mechanisms to tolerize the B or T cell arms of the humoral immune response have shown promise for induction of antigen-specific tolerance, raising the possibility that they could work synergistically if combined. Earlier we showed that Siglec-engaging tolerance-inducing antigenic liposomes (STALs) that display both an antigen (Ag) and glycan ligands of the inhibitory co-receptor CD22 (CD22L) lead to robust antigen-specific B cell tolerance to protein antigens in naive mice. In another approach, administration of free Ag with poly(lactic- co -glycolic acid)-rapamycin nanoparticles (PLGA-R) induced robust antigen-specific tolerance through production of regulatory T cells. Here we illustrate that coadministration of STALs together with PLGA-R to naive mice induced more robust tolerance to multiple antigen challenges than either nanoparticle alone. Moreover, in K/BxN mice that develop spontaneous autoimmune arthritis to the self-antigen glucose-6-phosphate-isomerase (GPI), co-delivery of GPI-LP-CD22L and PLGA-R delayed onset of disease and in some mice prevented the disease indefinitely. The results show synergy between B cell-tolerizing STALs and T cell-tolerizing PLGA-R and the potential to induce tolerance in early stage autoimmune disease.

A major barrier to adeno-associated virus (AAV) gene therapy is the inability to re-dose patients due to formation of vector-induced neutralizing antibodies (Nabs). Tolerogenic nanoparticles encapsulating rapamycin (ImmTOR) provide long-term and specific suppression of adaptive immune responses, allowing for vector re-dosing. Moreover, co-administration of hepatotropic AAV vectors and ImmTOR leads to an increase of transgene expression even after the first dose. ImmTOR and AAV Anc80 encoding the methylmalonyl-coenzyme A (CoA) mutase (MMUT) combination was tested in a mouse model of methylmalonic acidemia, a disease caused by mutations in the MMUT gene. Repeated co-administration of Anc80 and ImmTOR was well tolerated and led to nearly complete inhibition of immunoglobulin (Ig)G antibodies to the Anc80 capsid. A more profound decrease of plasma levels of the key toxic metabolite, plasma methylmalonic acid (pMMA), and disease biomarker, fibroblast growth factor 21 (FGF21), was observed after treatment with the ImmTOR and Anc80-MMUT combination. In addition, there were higher numbers of viral genomes per cell (vg/cell) and increased transgene expression when ImmTOR was co-administered with Anc80-MMUT. These effects were dose-dependent, with the higher doses of ImmTOR providing higher vg/cell and mRNA levels, and an improved biomarker response. Combining of ImmTOR and AAV can not only block the IgG response against capsid, but it also appears to potentiate transduction and enhance therapeutic transgene expression in the mouse model., Competing Interests: P.O.I., A.M.M., G.L.R., C.J.R., S.S.L., S.L.E., T.C., A.C., L.P.M.J., and T.K.K. are employees and shareholders of Selecta Biosciences. E.A.-M is an employee and holds stocks of Akouos, Inc. L.H.V. received consulting fees and research funding from Selecta Biosciences and holds equity in and serves on the Scientific Advisory Board of Akouos. He is an inventor of Anc80L65, licensed to biopharmaceutical companies, including Selecta Biosciences, from which he receives royalties. C.P.V. received research funding from Selecta Biosciences. R.J.C., L.L., I.M., and C.P.V. are co-inventors on patents and patent applications filed by the NIH on their behalf.

ImmTOR biodegradable nanoparticles encapsulating rapamycin have been shown to induce a durable tolerogenic immune response to co-administered biologics and gene therapy vectors. Prior mechanism of action studies have demonstrated selective biodistribution of ImmTOR to the spleen and liver following intravenous (IV) administration. In the spleen, ImmTOR has been shown to induce tolerogenic dendritic cells and antigen-specific regulatory T cells and inhibit antigen-specific B cell activation. Splenectomy of mice resulted in partial but incomplete abrogation of the tolerogenic immune response induced by ImmTOR. Here we investigated the ability of ImmTOR to enhance the tolerogenic environment in the liver. All the major resident populations of liver cells, including liver sinusoidal endothelial cells (LSECs), Kupffer cells (KC), stellate cells (SC), and hepatocytes, actively took up fluorescent-labeled ImmTOR particles, which resulted in downregulation of MHC class II and co-stimulatory molecules and upregulation of the PD-L1 checkpoint molecule. The LSEC, known to play an important role in hepatic tolerance induction, emerged as a key target cell for ImmTOR. LSEC isolated from ImmTOR treated mice inhibited antigen-specific activation of ovalbumin-specific OT-II T cells. The tolerogenic environment led to a multi-pronged modulation of hepatic T cell populations, resulting in an increase in T cells with a regulatory phenotype, upregulation of PD-1 on CD4 + and CD8 + T cells, and the emergence of a large population of CD4 - CD8 - (double negative) T cells. ImmTOR treatment protected mice in a concanavalin A-induced model of acute hepatitis, as evidenced by reduced production of inflammatory cytokines, infiltrate of activated leukocytes, and tissue necrosis. Modulation of T cell phenotype was seen to a lesser extent after administration by empty nanoparticles, but not free rapamycin. The upregulation of PD-1, but not the appearance of double negative T cells, was inhibited by antibodies against PD-L1 or CTLA-4. These results suggest that the liver may contribute to the tolerogenic properties of ImmTOR treatment., Competing Interests: All authors are employees and shareholders in Selecta Biosciences, Inc, (Copyright © 2021 Ilyinskii, Roy, LePrevost, Rizzo and Kishimoto.)

Systemic AAV (adeno-associated virus) gene therapy is a promising approach for the treatment of inborn errors of metabolism, but questions remain regarding its potency and durability. Tolerogenic ImmTOR nanoparticles encapsulating rapamycin have been shown to block the formation of neutralizing anti-capsid antibodies, thereby enabling vector re-administration. Here, we further demonstrate that ImmTOR admixed with AAV vectors also enhances hepatic transgene expression at the initial dose of AAV vector, independent of its effects on adaptive immunity. ImmTOR enhances AAV trafficking to the liver, resulting in increased hepatic vector copy numbers and transgene mRNA expression. Enhanced transgene expression occurs through a mechanism independent of the AAV receptor and cannot be replicated in vivo with free rapamycin or empty nanoparticles. The multipronged mechanism of ImmTOR action makes it an attractive candidate to enable more efficient transgene expression at first dose while simultaneously inhibiting adaptive responses against AAV to enable repeat dosing., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Ornithine transcarbamylase deficiency (OTCD) is an X-linked liver disorder caused by partial or total loss of OTC enzyme activity. It is characterized by elevated plasma ammonia, leading to neurological impairments, coma, and death in the most severe cases. OTCD is managed by combining dietary restrictions, essential amino acids, and ammonia scavengers. However, to date, liver transplantation provides the best therapeutic outcome. AAV-mediated gene-replacement therapy represents a promising curative strategy. Here, we generated an AAV2/8 vector expressing a codon-optimized human OTC cDNA by the α1-AAT liver-specific promoter. Unlike standard codon-optimization approaches, we performed multiple codon-optimization rounds via common algorithms and ortholog sequence analysis that significantly improved mRNA translatability and therapeutic efficacy. AAV8-hOTC-CO (codon optimized) vector injection into adult OTC Spf-Ash mice (5.0E11 vg/kg) mediated long-term complete correction of the phenotype. Adeno-Associated viral (AAV) vector treatment restored the physiological ammonia detoxification liver function, as indicated by urinary orotic acid normalization and by conferring full protection against an ammonia challenge. Removal of liver-specific transcription factor binding sites from the AAV backbone did not affect gene expression levels, with a potential improvement in safety. These results demonstrate that AAV8-hOTC-CO gene transfer is safe and results in sustained correction of OTCD in mice, supporting the translation of this approach to the clinic., Competing Interests: F.M. is currently an employee of Spark Therapeutics, a Roche company. P.I. and T.K.K. are employees of Selecta Biosciences., (© 2020 The Authors.)

The development of anti-drug antibodies (ADAs) is a common cause for treatment failure and hypersensitivity reactions for many biologics. The focus of this review is the development of ImmTOR, a platform technology designed to prevent the formation of ADAs that can be applied broadly across a wide variety of biologics by inducing immunological tolerance with ImmTOR nanoparticles encapsulating rapamycin. The induction of tolerance is antigen-specific and dependent on the incorporation of rapamycin in nanoparticles and the presence of the antigen at the time of administration of ImmTOR. Evidence for the induction of specific immune tolerance vs. general immune suppression is supported by the findings that: (1) ImmTOR induces regulatory T cells specific to the co-administered antigen; (2) tolerance can be transferred by adoptive transfer of splenocytes from treated animals to naïve recipients; (3) the tolerance is durable to subsequent challenge with antigen alone; and (4) animals tolerized to a specific antigen are capable of responding to an unrelated antigen. ImmTOR nanoparticles can be added to new or existing biologics without the need to modify or reformulate the biologic drug. The ability of ImmTOR to mitigate the formation of ADAs has been demonstrated for coagulation factor VIII in a mouse model of hemophilia A, an anti-TNFα monoclonal antibody in a mouse model of inflammatory arthritis, pegylated uricase in hyperuricemic mice and in non-human primates, acid alpha-glucosidase in a mouse model of Pompe disease, recombinant immunotoxin in a mouse model of mesothelioma, and adeno-associated vectors in a model of repeat dosing of gene therapy vectors in mice and in non-human primates. Human proof-of concept for the mitigation of ADAs has been demonstrated with SEL-212, a combination product consisting of ImmTOR + pegadricase, a highly immunogenic enzyme therapy for the treatment of gout. ImmTOR represents a promising approach to preventing the formation of ADAs to a broad range of biologic drugs., (Copyright © 2020 Kishimoto.)

Gene therapy mediated by recombinant adeno-associated virus (AAV) vectors is a promising treatment for systemic monogenic diseases. However, vector immunogenicity represents a major limitation to gene transfer with AAV vectors, particularly for vector re-administration. Here, we demonstrate that synthetic vaccine particles encapsulating rapamycin (SVP[Rapa]), co-administered with AAV vectors, prevents the induction of anti-capsid humoral and cell-mediated responses. This allows successful vector re-administration in mice and nonhuman primates. SVP[Rapa] dosed with AAV vectors reduces B and T cell activation in an antigen-selective manner, inhibits CD8 + T cell infiltration in the liver, and efficiently blocks memory T cell responses. SVP[Rapa] immunomodulatory effects can be transferred from treated to naive mice by adoptive transfer of splenocytes, and is inhibited by depletion of CD25 + T cells, suggesting a role for regulatory T cells. Co-administration of SVP[Rapa] with AAV vector represents a powerful strategy to modulate vector immunogenicity and enable effective vector re-administration.

We previously reported that synthetic vaccine particles (SVP) encapsulating antigens and TLR agonists resulted in augmentation of immune responses with minimal production of systemic inflammatory cytokines. Here we evaluated two different polymer formulations of SVP-encapsulated antigens and tested their ability to induce cytolytic T lymphocytes (CTL) in combination with SVP-encapsulated adjuvants. One formulation led to efficient antigen processing and cross-presentation, rapid and sustained CTL activity, and expansion of CD8+ T cell effector memory cells locally and centrally, which persisted for at least 1-2 years after a single immunization. SVP therapeutic dosing resulted in suppression of tumor growth and a substantial delay in mortality in several syngeneic mouse cancer models. Treatment with checkpoint inhibitors and/or cytotoxic drugs, while suboptimal on their own, showed considerable synergy with SVP immunization. SVP encapsulation of endosomal TLR agonists provided superior CTL induction, therapeutic benefit and/or improved safety profile compared to free adjuvants. SVP vaccines encapsulating mutated HPV-16 E7 and E6/E7 recombinant proteins led to induction of broad CTL activity and strong inhibition of TC-1 tumor growth, even when administered therapeutically 13-14 days after tumor inoculation in animals bearing palpable tumors. A pilot study in non-human primates showed that SVP-encapsulated E7/E6 adjuvanted with SVP-encapsulated poly(I:C) led to robust induction of antigen-specific T and B cell responses., Competing Interests: All authors are employees and shareholders of Selecta Biosciences and SelectaRUS. This does not alter our adherence to PLOS ONE policies on sharing data and materials.

T cells reacting to self-components can promote tissue damage when escaping tolerogenic control mechanisms which may result in autoimmune disease. The current treatments for these disorders are not antigen (Ag) specific and can compromise host immunity through chronic suppression. We have previously demonstrated that co-administration of encapsulated or free Ag with tolerogenic nanoparticles (tNPs) comprised of biodegradable polymers that encapsulate rapamycin are capable of inhibiting Ag-specific transgenic T cell proliferation and inducing Ag-specific regulatory T cells (Tregs). Here, we further show that tNPs can trigger the expansion of endogenous Tregs specific to a target Ag. The proportion of Ag-specific Treg to total Ag-specific T cells remains constant even after subsequent Ag challenge in combination with a potent TLR7/8 agonist or complete Freund's adjuvant. tNP-treated mice do not develop experimental autoimmune encephalomyelitis (EAE) after adoptive transfer of encephalitogenic T cells; furthermore, tNP treatment provided therapeutic protection in relapsing EAE that was transferred to naïve animals. These findings describe a potent therapy to expand Ag-specific Tregs in vivo and suppress T cell-mediated autoimmunity.

Antigen-specific immune tolerance has been a long-standing goal for immunotherapy for the treatment of autoimmune diseases and allergies and for the prevention of allograft rejection and anti-drug antibodies directed against biologic therapies. Nanoparticles have emerged as powerful tools to initiate and modulate immune responses due to their inherent capacity to target antigen-presenting cells (APCs) and deliver coordinated signals that can elicit an antigen-specific immune response. A wide range of strategies have been described to create tolerogenic nanoparticles (tNPs) that fall into three broad categories. One strategy includes tNPs that provide antigen alone to harness natural tolerogenic processes and environments, such as presentation of antigen in the absence of costimulatory signals, oral tolerance, the tolerogenic environment of the liver, and apoptotic cell death. A second strategy includes tNPs that carry antigen and simultaneously target tolerogenic receptors, such as pro-tolerogenic cytokine receptors, aryl hydrocarbon receptor, FAS receptor, and the CD22 inhibitory receptor. A third strategy includes tNPs that carry a payload of tolerogenic pharmacological agents that can "lock" APCs into a developmental or metabolic state that favors tolerogenic presentation of antigens. These diverse strategies have led to the development of tNPs that are capable of inducing antigen-specific immunological tolerance, not just immunosuppression, in animal models. These novel tNP technologies herald a promising approach to specifically prevent and treat unwanted immune reactions in humans. The first tNP, SEL-212, a biodegradable synthetic vaccine particle encapsulating rapamycin, has reached the clinic and is currently in Phase 2 clinical trials.

Protein-based drugs are very active in treating cancer, but their efficacy can be limited by the formation of neutralizing antidrug antibodies (ADAs). Recombinant immunotoxins are proteins that are very effective in patients with leukemia, where immunity is suppressed, but induce ADAs, which compromise their activity, in patients with intact immunity. Here we induced a specific, durable, and transferable immune tolerance to recombinant immunotoxins by combining them with nanoparticles containing rapamycin (SVP-R). SVP-R mitigated the formation of inhibitory ADAs in naïve and sensitized mice, resulting in restoration of antitumor activity. The immune tolerance is mediated by colocalization of the SVP-R and immunotoxin to dendritic cells and macrophages in the spleen and is abrogated by depletion of regulatory T cells. Tolerance induced by SVPs was not blocked by checkpoint inhibitors or costimulatory agonist monoclonal antibodies that by themselves enhance ADA formation., Competing Interests: Conflict of interest statement: T.K.K. is an employee and shareholder of Selecta Biosciences. All other authors declare no competing interests.

A major obstacle to enzyme replacement therapy (ERT) with recombinant human acid-α-glucosidase (rhGAA) for Pompe disease is the development of high titers of anti-rhGAA antibodies in a subset of patients, which often leads to a loss of treatment efficacy. In an effort to induce sustained immune tolerance to rhGAA, we supplemented the rhGAA therapy with a weekly intravenous injection of synthetic vaccine particles carrying rapamycin (SVP-Rapa) during the first 3 weeks of a 12-week course of ERT in GAA-KO mice, and compared this with three intraperitoneal injections of methotrexate (MTX) per week for the first 3 weeks. Empty nanoparticles (NP) were used as negative control for SVP-Rapa. Co-administration of SVP-Rapa with rhGAA resulted in more durable inhibition of anti-rhGAA antibody responses, higher efficacy in glycogen clearance in skeletal muscles, and greater improvement of motor function than mice treated with empty NP or MTX. Body weight loss was observed during the MTX-treatment but not SVP-Rapa-treatment. Our data suggest that co-administration of SVP-Rapa may be an innovative and safe strategy to induce durable immune tolerance to rhGAA during the ERT in patients with Pompe disease, leading to improved clinical outcomes.

The development of antidrug antibodies (ADAs) is a common cause for the failure of biotherapeutic treatments and adverse hypersensitivity reactions. Here we demonstrate that poly(lactic-co-glycolic acid) (PLGA) nanoparticles carrying rapamycin, but not free rapamycin, are capable of inducing durable immunological tolerance to co-administered proteins that is characterized by the induction of tolerogenic dendritic cells, an increase in regulatory T cells, a reduction in B cell activation and germinal centre formation, and the inhibition of antigen-specific hypersensitivity reactions. Intravenous co-administration of tolerogenic nanoparticles with pegylated uricase inhibited the formation of ADAs in mice and non-human primates and normalized serum uric acid levels in uricase-deficient mice. Similarly, the subcutaneous co-administration of nanoparticles with adalimumab resulted in the durable inhibition of ADAs, leading to normalized pharmacokinetics of the anti-TNFα antibody and protection against arthritis in TNFα transgenic mice. Adjunct therapy with tolerogenic nanoparticles represents a novel and broadly applicable approach to prevent the formation of ADAs against biologic therapies.

Current treatments to control pathological or unwanted immune responses often use broadly immunosuppressive drugs. New approaches to induce antigen-specific immunological tolerance that control both cellular and humoral immune responses are desirable. Here we describe the use of synthetic, biodegradable nanoparticles carrying either protein or peptide antigens and a tolerogenic immunomodulator, rapamycin, to induce durable and antigen-specific immune tolerance, even in the presence of potent Toll-like receptor agonists. Treatment with tolerogenic nanoparticles results in the inhibition of CD4+ and CD8+ T-cell activation, an increase in regulatory cells, durable B-cell tolerance resistant to multiple immunogenic challenges, and the inhibition of antigen-specific hypersensitivity reactions, relapsing experimental autoimmune encephalomyelitis, and antibody responses against coagulation factor VIII in hemophilia A mice, even in animals previously sensitized to antigen. Only encapsulated rapamycin, not the free form, could induce immunological tolerance. Tolerogenic nanoparticle therapy represents a potential novel approach for the treatment of allergies, autoimmune diseases, and prevention of antidrug antibodies against biologic therapies.

CD4T cells play a key role in humoral immunity by providing help to B cells, enabling effective antibody class switching and affinity maturation. Some vaccines may generate a poor response due to a lack of effective MHC class II epitopes, resulting in ineffective helper T cell activation and recall and consequently poor humoral immunity. It may be beneficial to provide a CD4T cell helper peptide with a vaccine particularly in the case of a poorly immunogenic antigen. Such a T cell helper peptide must be promiscuous in its ability to bind a broad range of MHC class II alleles due to broad allelic variation in the human population. We designed a chimeric MHC class II peptide (TpD) with epitopes from tetanus toxoid and diphtheria toxoid, separated by an internal cathepsin cleavage site. TpD was capable of inducing a memory recall response in peripheral blood mononuclear cells from 20/20 human donors. T cells responding to TpD showed a central memory phenotype. Immunization of mice with a synthetic nicotine nanoparticle vaccine containing TpD showed that the peptide was required for robust antibody production and resulted in a long term CD4 memory T cell recall response. As a pre-clinical model two non-human primate species, rhesus macaques and cynomolgus monkeys, were immunized with a nicotine nanoparticle vaccine and evaluated for an anti-nicotine antibody response and TpD specific memory T cells. We found that 4/4 rhesus monkeys had both sustained antibody production and TpD memory T cells for the duration of the experiment (119 days). In addition 30/30 cynomolgus monkeys dosed with nicotine vaccine nanoparticles showed dose-dependent antibody generation and T cell recall response compared to saline injected controls. In summary we have developed a potent universal memory T cell helper peptide (TpD) that is active in vitro in human PBMCs and in vivo in mice and non-human primates., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Augmentation of immunogenicity can be achieved by particulate delivery of an antigen and by its co-administration with an adjuvant. However, many adjuvants initiate strong systemic inflammatory reactions in vivo, leading to potential adverse events and safety concerns. We have developed a synthetic vaccine particle (SVP) technology that enables co-encapsulation of antigen with potent adjuvants. We demonstrate that co-delivery of an antigen with a TLR7/8 or TLR9 agonist in synthetic polymer nanoparticles results in a strong augmentation of humoral and cellular immune responses with minimal systemic production of inflammatory cytokines. In contrast, antigen encapsulated into nanoparticles and admixed with free TLR7/8 agonist leads to lower immunogenicity and rapid induction of high levels of inflammatory cytokines in the serum (e.g., TNF-a and IL-6 levels are 50- to 200-fold higher upon injection of free resiquimod (R848) than of nanoparticle-encapsulated R848). Conversely, local immune stimulation as evidenced by cellular infiltration of draining lymph nodes and by intranodal cytokine production was more pronounced and persisted longer when SVP-encapsulated TLR agonists were used. The strong local immune activation achieved using a modular self-assembling nanoparticle platform markedly enhanced immunogenicity and was equally effective whether antigen and adjuvant were co-encapsulated in a single nanoparticle formulation or co-delivered in two separate nanoparticles. Moreover, particle encapsulation enabled the utilization of CpG oligonucleotides with the natural phosphodiester backbone, which are otherwise rapidly hydrolyzed by nucleases in vivo. The use of SVP may enable clinical use of potent TLR agonists as vaccine adjuvants for indications where cellular immunity or robust humoral responses are required., (Copyright © 2014. Published by Elsevier Ltd.)

Heparins have been reported to cause elevations in serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) but have not been associated with clinically significant liver injury. The mechanisms underlying these benign laboratory abnormalities are unknown. Forty-eight healthy men were randomized to receive subcutaneous injections of unfractionated heparin (UFH; 150 U/kg), enoxaparin sodium (1 mg/kg), dalteparin sodium (120 IU/kg), or adomiparin sodium (125 IU/kg; a novel heparin) every 12 h for 4.5 days. Asymptomatic elevations in serum ALT or AST were observed in >90% of the subjects. Elevations were also observed in the levels of serum sorbitol dehydrogenase (SDH), glutamate dehydrogenase (GLDH), miR-122, high-mobility group box-1 protein (including the acetylated form), full-length keratin 18, and DNA. Keratin 18 fragments, which are apoptosis biomarkers, were not detected. Biomarker profiles did not differ significantly across heparin treatments. We conclude that heparins as a class cause self-limited and mild hepatocyte necrosis with secondary activation of an innate immune response.

A series of size-defined low-molecular-weight heparins were generated by regioselective chemical modifications and profiled for their in vitro and in vivo activities. The compounds displayed reduced anti-coagulant activity, demonstrated varying affinities toward angiogenic growth factors (fibroblast growth factor-2, vascular endothelial growth factor and stromal cell-derived factor-1α), inhibited the P-selectin/P-selectin glycoprotein ligand-1 interaction and, notably, exhibited anti-tumor efficacy in a murine melanoma experimental metastasis model. Our results demonstrate that modulating specific sequences, especially the N-domains (-NS or -NH(2) or -NHCOCH(3)) in these polysaccharide sequences, has a major impact on the participation in a diverse range of biological activities. These results also suggest that the 6-O-sulfates, but not the 2-O-sulfates, critically affect the binding of a desulfated derivative to certain angiogenic proteins as well as its ability to inhibit P-selectin-mediated B16F10 melanoma metastases. Furthermore, N-desulfation followed by N-acetylation regenerates the affinity/inhibition properties to different extents in all the compounds tested in the in vitro assays. This systematic study lays a conceptual foundation for detailed structure function elucidation and will facilitate the rational design of targeted heparan sulfate proteoglycan-based anti-metastatic therapeutic candidates.

Heparan sulfate proteoglycans (HSPGs) play a key role in shaping the tumor microenvironment by presenting growth factors, cytokines, and other soluble factors that are critical for host cell recruitment and activation, as well as promoting tumor progression, metastasis, and survival. M402 is a rationally engineered, non-cytotoxic heparan sulfate (HS) mimetic, designed to inhibit multiple factors implicated in tumor-host cell interactions, including VEGF, FGF2, SDF-1α, P-selectin, and heparanase. A single s.c. dose of M402 effectively inhibited seeding of B16F10 murine melanoma cells to the lung in an experimental metastasis model. Fluorescent-labeled M402 demonstrated selective accumulation in the primary tumor. Immunohistological analyses of the primary tumor revealed a decrease in microvessel density in M402 treated animals, suggesting anti-angiogenesis to be one of the mechanisms involved in-vivo. M402 treatment also normalized circulating levels of myeloid derived suppressor cells in tumor bearing mice. Chronic administration of M402, alone or in combination with cisplatin or docetaxel, inhibited spontaneous metastasis and prolonged survival in an orthotopic 4T1 murine mammary carcinoma model. These data demonstrate that modulating HSPG biology represents a novel approach to target multiple factors involved in tumor progression and metastasis.

Early detection of tumors can significantly improve the outcome of tumor treatment. One of the most frequently asked questions in cancer imaging is how many cells can be detected non-invasively in a live animal. Although many factors limit such detection, increasing the light emission from cells is one of the most effective ways of overcoming these limitations. Here, we describe development and utilization of a lentiviral vector containing enhanced firefly luciferase (luc2) gene. The resulting single cell clones of the mouse mammary gland tumor (4T1-luc2) showed stable light emission in the range of 10,000 photons/sec/cell. In some cases individual 4T1-luc2 cells inserted under the skin of a nu/nu mouse could be detected non-invasively using a cooled CCD camera in some cases. In addition, we showed that only few cells are needed to develop tumors in these mice and tumor progression can be monitored right after the cells are implanted. Significantly higher luciferase activity in these cells allowed us to detect micrometastases in both, syngeneic Balb/c and nu/nu mice.

The initial choice of anticoagulant therapy administered in emergency departments for acute coronary syndromes (ACS) has important consequences for subsequent patient care, as neither unfractionated heparin (UFH) nor low-molecular-weight heparin (LMWH) are ideally suited for all potential clinical treatment pathways. UFH remains widely used for surgical interventions because of the ability to rapidly reverse its anticoagulant activity. However, the unpredictable pharmacokinetic profile of UFH presents safety issues, and the low subcutaneous bioavailability limits the utility of UFH for patients who are medically managed. LMWH has superior pharmacokinetic properties, but its anticoagulant activity cannot be effectively monitored or reversed during surgery. There is an unmet medical need for a baseline anticoagulant therapy that addresses these shortcomings while retaining the beneficial properties of both UFH and LMWH. We describe here M118, a novel LMWH designed specifically for use in the treatment of ACS. M118 shows broad anticoagulant activity, including potent activity against both factor Xa (~240 IU/mg) and thrombin (factor IIa; ~170 IU/mg), low polydispersity, high (78%) subcutaneous bioavailability in rabbits, and predictable subcutaneous and intravenous pharmacokinetics. Additionally, the anticoagulant activity of M118 is monitorable by standard coagulation assays and is reversible with protamine. M118 demonstrates superior activity to conventional LMWH in a rabbit model of abdominal arterial thrombosis without increasing bleeding risk, and is currently being evaluated in a phase II clinical trial evaluating efficacy and safety in patients undergoing percutaneous coronary intervention.

Background: In January 2008, the Centers for Disease Control and Prevention began a nationwide investigation of severe adverse reactions that were first detected in a single hemodialysis facility. Preliminary findings suggested that heparin was a possible cause of the reactions., Methods: Information on clinical manifestations and on exposure was collected for patients who had signs and symptoms that were consistent with an allergic-type reaction after November 1, 2007. Twenty-one dialysis facilities that reported reactions and 23 facilities that reported no reactions were included in a case-control study to identify facility-level risk factors. Unopened heparin vials from facilities that reported reactions were tested for contaminants., Results: A total of 152 adverse reactions associated with heparin were identified in 113 patients from 13 states from November 19, 2007, through January 31, 2008. The use of heparin manufactured by Baxter Healthcare was the factor most strongly associated with reactions (present in 100.0% of case facilities vs. 4.3% of control facilities, P<0.001). Vials of heparin manufactured by Baxter from facilities that reported reactions contained a contaminant identified as oversulfated chondroitin sulfate (OSCS). Adverse reactions to the OSCS-contaminated heparin were often characterized by hypotension, nausea, and shortness of breath occurring within 30 minutes after administration. Of 130 reactions for which information on the heparin lot was available, 128 (98.5%) occurred in a facility that had OSCS-contaminated heparin on the premises. Of 54 reactions for which the lot number of administered heparin was known, 52 (96.3%) occurred after the administration of OSCS-contaminated heparin., Conclusions: Heparin contaminated with OSCS was epidemiologically linked to adverse reactions in this nationwide outbreak. The reported clinical features of many of the cases further support the conclusion that contamination of heparin with OSCS was the cause of the outbreak., (2008 Massachusetts Medical Society)

Background: There is an urgent need to determine whether oversulfated chondroitin sulfate (OSCS), a compound contaminating heparin supplies worldwide, is the cause of the severe anaphylactoid reactions that have occurred after intravenous heparin administration in the United States and Germany., Methods: Heparin procured from the Food and Drug Administration, consisting of suspect lots of heparin associated with the clinical events as well as control lots of heparin, were screened in a blinded fashion both for the presence of OSCS and for any biologic activity that could potentially link the contaminant to the observed clinical adverse events. In vitro assays for the activation of the contact system and the complement cascade were performed. In addition, the ability of OSCS to recapitulate key clinical manifestations in vivo was tested in swine., Results: The OSCS found in contaminated lots of unfractionated heparin, as well as a synthetically generated OSCS reference standard, directly activated the kinin-kallikrein pathway in human plasma, which can lead to the generation of bradykinin, a potent vasoactive mediator. In addition, OSCS induced generation of C3a and C5a, potent anaphylatoxins derived from complement proteins. Activation of these two pathways was unexpectedly linked and dependent on fluid-phase activation of factor XII. Screening of plasma samples from various species indicated that swine and humans are sensitive to the effects of OSCS in a similar manner. OSCS-containing heparin and synthetically derived OSCS induced hypotension associated with kallikrein activation when administered by intravenous infusion in swine., Conclusions: Our results provide a scientific rationale for a potential biologic link between the presence of OSCS in suspect lots of heparin and the observed clinical adverse events. An assay to assess the amidolytic activity of kallikrein can supplement analytic tests to protect the heparin supply chain by screening for OSCS and other highly sulfated polysaccharide contaminants of heparin that can activate the contact system., (Copyright 2008 Massachusetts Medical Society.)

The beta(2) integrin LFA-1 (CD11a/CD18) is a leukocyte-specific adhesion molecule that mediates leukocyte extravasation, antigen presentation, and T-cell-mediated cytolysis through its interaction with its counter-receptors, ICAM-1, ICAM-2, and ICAM-3. We have recently described a small molecule antagonist of LFA-1 (BIRT 377) that inhibits LFA-1/ICAM-1 molecular interactions, LFA-1-dependent adhesion assays, antigen-induced proliferation of T-cells, and superantigen-induced production of IL-2 in vivo in mice. We have also recently described a unique monoclonal antibody, R3.1, which competes with BIRT 377 and its analogs for binding to both purified full-length LFA-1 and the purified recombinant I domain module. In this manuscript, we extend these studies to cell-based systems and utilize this unique reagent for the development of a receptor occupancy assay. Exploiting these observations, we have designed and validated an assay that allows us to measure receptor occupancy in vitro on monkey and human peripheral blood leukocytes and ex vivo in whole blood from monkeys dosed with small molecule LFA-1 antagonists. Further refinement of these reagents has led to the development of a Fab-based assay that allows rapid and reproducible analysis of whole blood samples. These optimized reagents allow for quantification of the number of receptors expressed on the cell surface and a more accurate quantitation of receptor occupancy.

Objective: HIV-1 infection in humans has been reported to lead to a shift in the cytokine balance, with a relative decrease in T helper 1 type cytokines, especially IL-2. On the basis of the expression of CD45RA, in combination with homing markers CD62L or alpha4beta7, T helper cells can be sub-divided into naive, activated naive, central memory and effector memory cells as well as gut-homing subpopulations. In addition, each subset may have the potential to express distinct cytokines. At present it is unclear whether the changes in cytokine expression observed in HIV-1-infected individuals are secondary to changes within the composition of CD4 T cell subsets or are caused by changes in cytokine expression within each subset., Materials and Methods: A new technique was developed to detect cytokine expression in phorbol 12-myristate 13-acetate/ionomycin-activated CD62L and alpha4beta7-expressing CD4 T cell subsets, using the protease inhibitor KD-IX-73-4., Results: In SIV-infected macaques that develop AIDS a marked decrease in IL-2 expression was found within central, effector, or gut-homing memory cell subsets, whereas the expression of IL-2 in naive T cell subsets remained unaffected. This reduced IL-2 expression by memory cells and not a loss of the frequency of CD4 memory cells accounted for the reduced expression of IL-2 by CD4 T cells during SIV infection., Conclusion: As defined by the cell surface markers utilized, it appears that progression to AIDS is associated with functional impairment of memory cells, but not changes in lymphocyte circulation patterns.

Lymphocyte function-associated antigen (LFA)-1/intercellular adhesion molecule (ICAM)-1 interactions mediate several important steps in the evolution of an immune response. LFA-1 is normally expressed in a quiescent state on the surface of leukocytes and interacts weakly with its ligands ICAM-1, -2, and -3. LFA-1 activity may be regulated by receptor clustering and by increasing the affinity of LFA-1 for its ligands. Affinity modulation of LFA-1 has been shown to occur via a conformational change in the LFA-1 heterodimer that can be detected by using monoclonal antibody 24 (mAb24). We have recently described a small-molecule antagonist of LFA-1, BIRT 377, that demonstrates selective in vitro and in vivo inhibition of LFA-1/ICAM-1-mediated binding events. We now demonstrate that BIRT 377 blocks the induction of the mAb24 reporter epitope on LFA-1 on the surface of SKW-3 cells treated with various agonists known to induce high-affinity LFA-1. These data imply that BIRT 377 exerts its inhibitory effects by preventing up-regulation of LFA-1 to its high-affinity conformation.

Neutrophil recruitment at sites of inflammation is regulated by a series of adhesion and activation events. L-selectin (CD62L) is a leukocyte expressed adhesion protein that is important for neutrophil accumulation and rolling along the vascular endothelium. L-selectin is unique from other adhesion molecules involved in leukocyte transmigration in that its adhesiveness appears to be regulated partly by rapid endoproteolysis. Cleavage of L-selectin occurs within a membrane-proximal region that results in ectodomain shedding and retention of a 6-kDa transmembrane fragment. The cleavage domain of L-selectin has been well characterized through mutational analysis. Whether the cytoplasmic domain of L-selectin also plays a role in regulating shedding is controversial. We have previously shown that the Ca(2+)-sensing protein calmodulin (CaM) constitutively associates with the cytoplasmic domain of L-selectin in transfected cell lines. However, in the absence of mapping and mutational analysis of the CaM-binding region of L-selectin, there remains no direct evidence that this interaction affects shedding. Using synthesized peptides and expressed L-selectin constructs, we demonstrate that CaM binding activity occurs in the membrane-proximal region of the cytoplasmic domain. Mutations engineered in this region that prevent CaM binding increase the proteolytic turnover of L-selectin. Moreover, we demonstrate that CaM binding to the 6-kDa transmembrane fragment is greatly reduced compared with intact L-selectin in neutrophils, suggesting that CaM binding is regulated. These data imply that the cytoplasmic domain of L-selectin can regulate shedding by a mechanism in which bound CaM may operate as a negative effector.

The release of tumor necrosis factor-alpha (TNF-alpha) from cellular membranes has been shown by different laboratories to be controlled by a disintegrin and metalloprotease, ADAM10 or ADAM17. In contrast, only ADAM17 has shown to be involved in L-selectin shedding. To determine the specific roles of ADAM10 and ADAM17 in the processing of TNF-alpha and L-selectin shedding, antisense oligonucleotides (ASO) targeting both ADAM10 and ADAM17 were identified. We show that ISIS 16337 reduces ADAM17 mRNA and ISIS 100750 reduces ADAM10 mRNA in a sequence-specific and dose-dependent manner in both Jurkat and THP-1 cells. The ADAM17 ASO (ISIS 16337) inhibited both TNF-alpha secretion in THP-1 cells and L-selectin shedding in Jurkat cells, whereas the ADAM10 ASO (ISIS 100750) did not significantly inhibit release of either protein. These results suggest that ADAM17 is one of the major metalloproteases involved in L-selectin shedding as well as TNF-alpha processing. The biologic substrates for ADAM10 in Jurkat and THP-1 cells remain to be elucidated.

Neutrophil influx into the lung is an important event in the pathogenesis of acute lung injury in gram-negative sepsis. We hypothesized that administration of a monoclonal antibody to intercellular adhesion molecule 1 (ICAM-1, CD54), a molecule mediating neutrophil adhesion to endothelial cells, would decrease neutrophil sequestration and transmigration in the lung and attenuate lung injury in Escherichia coli sepsis. Sepsis was induced in 12 baboons primed with heat-killed E. coli (1 x 10(9) CFU/kg) 12 h before infusion of live bacteria (1 x 10(10) CFU/kg). Six animals received monoclonal antibody to CD54 (1 mg/kg) intravenously at the time of live E. coli infusion. After 48 h or when blood pressure could not be maintained, tissues were harvested and bronchoalveolar lavage (BAL) samples were obtained. Median survival time was decreased in anti-CD54-treated animals. This group also had decreased mean arterial pressure, increased metabolic acidosis, and decreased urine output. Measures of lung injury including gas exchange, lung lavage protein and lactate dehydrogenase (LDH), lung thiobarbituric acid-reactive species, and lung histology, including alveolar neutrophil volumes, were unaffected by treatment. The effect of anti-CD54 on neutrophil influx into tissues as measured by myeloperoxidase was organ specific. These data show that monoclonal antibody to CD54 does not ameliorate acute lung injury in E. coli sepsis, and septic primates given anti-CD54 have worsened metabolic parameters and decreased survival.

The signaling factors that direct the rapid shedding of L-selectin from neutrophils upon chemoattractant stimulation are poorly understood. Protein kinase C (PKC) has been implicated, yet previous studies have relied on the use of phorbol esters and nonselective kinase inhibitors. We treated neutrophils with various selective kinase inhibitors to evaluate their effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP)-induced L-selectin shedding. We found that three selective inhibitors of PKC, structurally related to staurosporine, largely blocked both fMLP- and phorbol 12-myristate 13-acetate (PMA)-induced L-selectin shedding; however, these inhibitors did not affect fMLP-induced up-regulation of Mac-1 (CD11b/CD18) expression, which has been shown not to involve PKC. Other selective serine, threonine, and tyrosine kinase inhibitors were found not to block fMLP-induced L-selectin shedding. These findings provide more definitive evidence for the role of PKC in chemoattractant-induced L-selectin proteolysis. It is interesting that certain highly selective PKC inhibitors, not structurally related to staurosporine, were found to directly induce L-selectin shedding from neutrophils.

Neutrophil-endothelial cell interactions are regulated by cell adhesion molecules and their cognate ligands. It has been proposed that L-selectin and Mac-1 (CD11b/CD18), two neutrophil adhesion receptors, have sequential roles in neutrophil extravasation during inflammation. In this model, L-selectin mediates rolling and initial adherence of neutrophils to endothelial cells, while Mac-1 strengthens this initial adherence and also facilitates migration of neutrophils through endothelial cells. L-selectin and Mac-1 expression are known to be inversely regulated. Here an in vitro culture system has been developed to investigate in situ expression of L-selectin during cell-to-cell interactions between neutrophils and endothelial cell monolayers by confocal immunofluorescence analysis. Neutrophils underwent profound cell shape change from round to polarized cell morphology with pseudopod formation after 5 to 15 min coculture with IL-1-stimulated human endothelial cells. L-selectin was redistributed to the pseudopod of the polarized neutrophils in correlation with such cellular changes. During initial cell attachment, neutrophils bound to IL-1-stimulated endothelial cells expressed a high level of L-selectin in a polarized pattern. L-selectin expression decreased over time during neutrophil-endothelial cell interactions., (Copyright 1998 Academic Press.)

Expression of the L-selectin adhesion molecule is rapidly down-regulated upon cell activation through proteolysis at a membrane-proximal site. Here we demonstrate that calmodulin, an intracellular calcium regulatory protein, specifically coprecipitates with L-selectin through a direct association with the cytoplasmic domain of L-selectin. Furthermore, calmodulin inhibitors disrupt L-selectin-dependent adhesion by inducing proteolytic release of L-selectin from the cell surface. The effects of the calmodulin inhibitors on L-selectin expression and function can be prevented by cotreatment with a hydroxamic acid-based metalloprotease inhibitor. Our results suggest a novel role for calmodulin in regulating the expression and function of an integral membrane protein through a protease-dependent mechanism. These findings may have broader implications for other cell surface proteins that also undergo regulated proteolysis.

The major high affinity ligand for P-selectin on human leukocytes is P-selectin glycoprotein ligand-1 (PSGL-1). To bind P-selectin, PSGL-1 must be modified with tyrosine sulfate and sialylated, fucosylated, core-2 O-glycan(s). The required sites for these modifications on full-length PSGL-1 have not been defined. The N-terminal region of mature PSGL-1, which begins at residue 42, includes tyrosines at residues 46, 48, and 51, plus potential sites for Thr-linked O-glycans at residues 44 and 57. We expressed full-length PSGL-1 constructs with substitutions of these residues in transfected Chinese hamster ovary cells. The cells were co-transfected with cDNAs for the glycosyltransferases required to construct sialylated and fucosylated, core-2 O-glycans on PSGL-1. The transfected cells were assayed for their abilities to bind fluid-phase P-selectin and to support rolling adhesion of pre-B cells expressing P-selectin under hydrodynamic flow. In both assays, substitution of Thr-57 with alanine eliminated binding of PSGL-1 to P-selectin without affecting sulfation of PSGL-1, whereas substitution with serine, to which an O-glycan might also be attached, did not affect binding. Binding was not altered by substituting alanines for the two amino acids on either side of Thr-57, or by substituting alanine for Thr-44. Substitution of all three tyrosines with phenylalanines markedly reduced sulfation and prevented binding to P-selectin. However, all constructs in which one or two tyrosines were replaced with phenylalanines bound P-selectin. These results suggest that full-length PSGL-1 requires an O-glycan attached to Thr-57 plus sulfation of any one of its three clustered tyrosines to bind P-selectin.

Recruitment of polymorphonuclear leukocytes (PMN) through upregulation of cellular adhesion molecules is a proposed mechanism of injury in sepsis and acute respiratory distress syndrome (ARDS). We hypothesized that pretreatment of baboons with a monoclonal antibody to human E- and L-selectin (EL-246) during sepsis would decrease PMN influx into tissues and result in less organ injury during gram-negative sepsis. We studied 14 anesthetized, ventilated adult baboons; six animals received 1 mg/kg of EL-246 before infusion of an LD100 of live Escherichia coli and six received the E. coli infusion without antibody therapy. Two other animals received 1 mg/kg of EL-246 intravenously without an infusion of bacteria. Intermittent measurements were made of circulatory pressures, cardiac output, urine output, arterial blood gases, ventilation:perfusion ratio (VA/Q), and hematologic status. The experiments were ended at 48 h or at the time of death. Tissues were harvested for pathology and biochemical measurements. The E. coli infusions were associated with a hyperdynamic state, pulmonary hypertension, systemic hypotension, decreased urine output (UOP), and metabolic acidosis. The antibody partly blocked PMN migration, but there were few significant physiologic or biochemical differences between the EL-246-treated and untreated animals. In the antibody-treated animals, UOP was decreased, metabolic acidosis was worsened, and median survival time was decreased significantly. We conclude that treatment with an antibody to E- and L-selectin in gram-negative sepsis does not improve gas exchange or protect against lung injury, and is associated with decreased survival time in primates.

Leukocytes attach to and roll on inflamed endothelium and on leukocyte monolayers that form on the endothelial cells. Leukocyte-leukocyte interactions occurring under hydrodynamic shear stress are mediated by binding of L-selectin to unknown sialomucin-like glycoproteins. We show that purified neutrophil PSGL-1, a sialomucin glycoprotein that serves as a ligand for both P- and E-selectin, can also support the attachment and rolling of free flowing neutrophils in vitro. Neutrophil rolling on PSGL-1 was abolished by the anti-L-selectin mAb DREG200 and by the anti-PSGL-1 mAb PL1, indicating that L-selectin can interact directly with PSGL-1. Neutrophil rolling on neutrophil monolayers was also blocked by PL1 (60 +/- 9% SEM inhibition); however, DREG200 blocked more efficiently (93 +/- 7% SEM inhibition), suggesting that other L-selectin ligands may exist on the neutrophil surface. These studies demonstrate that PSGL-1 on the neutrophil surface is a major functional ligand for L-selectin. The avidity of this L-selectin-dependent adhesion event was sufficient to allow individual neutrophils rolling on P-selectin to capture free flowing neutrophils, which progressed to form linear strings and discrete foci of rolling neutrophils. Neutrophil accumulation on P-selectin accelerated with time as a result of neutrophil-assisted capture of free flowing neutrophils. When neutrophil-neutrophil interactions were blocked by DREG200, neutrophils accumulated on P-selectin in a random pattern and at a uniform rate. Thus, leukocyte-assisted capture of flowing leukocytes may play an important role in amplifying the rate of initial leukocyte recruitment at sites of inflammation.

Atherosclerosis is increasingly thought to be a chronic inflammatory disease. Inflammation requires transmigration of leukocytes from the circulation to the tissues. Adhesion of leukocytes to endothelial calls is the initial event in an inflammatory response and is mediated by expression of several adhesion molecules. In this study we characterize the contribution of intercellular adhesion molecules (ICAM-1) and L-selectin in patients with different coronary artery disease syndromes. Serum concentrations of cICAM-1 and sL-selectin were measured by enzyme-linked immunosorbent assay in 31 patients with stable angina, 30 patients with unstable angina, 18 patients with acute myocardial infarction and 20 healthy subjects in a control group. All patients underwent coronary angiography. Mean (+/-SE) cICAM-1 levels were higher (p < 0.05) in patients with stable angina (249 +/- 6 ng/ml), unstable angina (260 +/- 16 ng/ml), or acute myocardial infarction (261 +/- 24 ng/ml) compared with those in subjects in the control group (171 +/- 11 ng/ml). In contrast, levels of sL-selectin were lower (p < 0.01) in patients with stable angina (1.2 +/- 0.1 microg/ml), unstable angina (1.1 +/- 0.6 microg/ml), or acute myocardial infarction (1.1 +/- 0.1 microg/ml) compared with those in subjects in the control group (1.8 +/- 0.1 microg/ml). No difference was found in cICAM-1 or sL-selectin levels among patients with stable angina, unstable angina, or acute myocardial infarction. No correlation was seen between cICAM-1 or sL-selectin levels and extent (or severity) of coronary artery disease or leukocyte count. L-selectin expression was observed to be depressed in patients with severe angina compared with that in members of the control group. To examine the mechanism of reduction in sL-selectin levels and L-selectin expression on leukocytes, leukocytes from the control group were stimulated in vitro. Stimulation of leukocytes resulted in a rapid downregulation of surface L-selectin expression, measured by flowcytometry, similar to the suppressed expression of L-selectin found on leukocytes from patients with coronary artery disease. In conclusion, altered cICAM-1 and sL-selectin levels in patients with coronary artery disease reflect the presence of a chronic inflammatory process. This inflammatory process results in downregulation of leukocyte expression of L-selectin and thus lower circulating sL-selectin levels.

The extracellular domains of a diverse group of membrane proteins are shed in response to protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA). The lack of sequence similarity in the cleavage sites suggests the involvement of many proteases of diverse specificity in this process. However, a mutant Chinese hamster ovary cell line recently isolated for being defective in PMA-activated shedding of the membrane-anchored growth factor transforming growth factor alpha precursor (proTGF-alpha) is concomitantly defective in the shedding of many other unrelated membrane proteins. Here we show that independent mutagenesis and selection experiments yield shedding mutants having the same recessive phenotype and belonging to the same genetic complementation group. Furthermore, two structurally distinct agents, TAPI-2 and 1,10-phenanthroline, which are known to inhibit metalloproteases, block PMA-activated shedding of proTGF-alpha, cell adhesion receptor L-selectin, interleukin 6 receptor alpha subunit, beta-amyloid precursor protein, and an entire set of anonymous Chinese hamster ovary cell surface proteins. Certain serine protease inhibitors prevent release of these proteins by interfering with their maturation and transport to the cell surface but do not inhibit ectodomain shedding from the cell surface. The results suggest the existence of a common system for membrane protein ectodomain shedding involving one or several proteolytic activities sensitive to metalloprotease inhibitors, whose ability to act can be disrupted by recessive mutations in a single gene.