Intracellular labile iron is a key regulator of hepcidin expression and iron metabolism.

BACKGROUND AND AIMS: Liver iron loading can induce hepatic expression of hepcidin and regulate iron metabolism. However, the mechanism by which hepatocyte senses iron loading and further regulates iron metabolism remains unclear. Intracellular labile iron is nonferritin-bound and redox active; it is transitory, and it serves as a crossroads of cellular iron metabolism, the effect of intracellular labile iron in iron metabolism regulation is particularly poorly understood. METHODS: An intracellular labile iron overload cell model was established using ferric ammonium citrate (FAC) and the lipophilic iron chelator 8-hydroxyquinoline (8HQ/FAC). RNA-Seq was performed to screen the genes that were highly expressed exclusively in 8HQ/FAC-treated HepG2 cells. High-iron-diet mice model and Hfe knockout hemochromatosis mice were used to investigate the importance of tumor necrosis factor α (TNFα) in iron metabolism. RESULTS: Intracellular labile iron in hepatocytes had a dual function in iron metabolism: It induced hepatocytes to express hepcidin via endoplasmic reticulum stress-induced transcription factors, and it stimulated expression of bone morphogenic protein 6 (BMP6, regulator of iron metabolism) in liver sinusoidal endothelial cells (LSECs) via promoting the secretion of TNFα by the hepatocytes. Blockade of TNFα dysregulated iron metabolism during iron overload. Furthermore, administration of TNFα could reduce iron burden in Hfe knockout hemochromatosis mice. CONCLUSIONS: Our findings reveal the importance of intracellular labile iron in iron metabolism, and propose that TNFα might be a novel therapeutic target for HFE-associated hemochromatosis.

In vivo pharmacokinetic enhancement of monomeric Fc and monovalent bispecific designs through structural guidance.

In a biologic therapeutic landscape that requires versatility in targeting specificity, valency and half-life modulation, the monomeric Fc fusion platform holds exciting potential for the creation of a class of monovalent protein therapeutics that includes fusion proteins and bispecific targeting molecules. Here we report a structure-guided approach to engineer monomeric Fc molecules to adapt multiple versions of half-life extension modifications. Co-crystal structures of these monomeric Fc variants with Fc neonatal receptor (FcRn) shed light into the binding interactions that could serve as a guide for engineering the half-life of antibody Fc fragments. These engineered monomeric Fc molecules also enabled the generation of a novel monovalent bispecific molecular design, which translated the FcRn binding enhancement to improvement of in vivo serum half-life.

MHC Phosphopeptides: Promising Targets for Immunotherapy of Cancer and Other Chronic Diseases.

Major histocompatibility complex-associated peptides have been considered as potential immunotherapeutic targets for many years. MHC class I phosphopeptides result from dysregulated cell signaling pathways that are common across cancers and both viral and bacterial infections. These antigens are recognized by central memory T cells from healthy donors, indicating that they are considered antigenic by the immune system and that they are presented across different individuals and diseases. Based on these responses and the similar dysregulation, phosphorylated antigens are promising candidates for prevention or treatment of different cancers as well as a number of other chronic diseases.

An engineered human Fc domain that behaves like a pH-toggle switch for ultra-long circulation persistence.

The pharmacokinetic properties of antibodies are largely dictated by the pH-dependent binding of the IgG fragment crystallizable (Fc) domain to the human neonatal Fc receptor (hFcRn). Engineered Fc domains that confer a longer circulation half-life by virtue of more favorable pH-dependent binding to hFcRn are of great therapeutic interest. Here we developed a pH Toggle switch Fc variant containing the L309D/Q311H/N434S (DHS) substitutions, which exhibits markedly improved pharmacokinetics relative to both native IgG1 and widely used half-life extension variants, both in conventional hFcRn transgenic mice and in new knock-in mouse strains. engineered specifically to recapitulate all the key processes relevant to human antibody persistence in circulation, namely: (i) physiological expression of hFcRn, (ii) the impact of hFcγRs on antibody clearance and (iii) the role of competing endogenous IgG. DHS-IgG retains intact effector functions, which are important for the clearance of target pathogenic cells and also has favorable developability.

Natural Killer Cell Inhibition by HLA-E Molecules on Induced Pluripotent Stem Cell-Derived Retinal Pigment Epithelial Cells.

Purpose: To determine whether human induced pluripotent stem (iPS) cell-derived retinal pigment epithelial (RPE) cells (iPS-RPE) can suppress natural killer (NK) cell activation. Methods: iPS-RPE cells were cocultured with peripheral blood mononuclear cells (PBMCs) or purified NK cells from healthy donors after stimulation with cytokines. To confirm expression of NK cell-specific markers, flow cytometry and quantitative RT-PCR (qRT-PCR) were performed. NK cells (or PBMCs) cocultured with iPS-RPE cells were assessed for proliferation by Ki-67 expression with flow cytometry, and NK suppression by RPE cells was assessed for granzyme B production with ELISA. Human leukocyte antigen (HLA) expression including HLA-E on iPS-RPE cells was evaluated with flow cytometry and qRT-PCR. The effect of HLA-E downregulation was also investigated using small interfering RNA (siRNA) systems. Following iPS-RPE cell transplantation in vivo, we evaluated NK cell invasion in the retina with immunohistochemistry. Results: Activated NK cells expressed NK-related markers such as CD16, CD56, and CD11b, and NK cells produced cytotoxic agents such as granzyme B, perforin, and TNF-α. Human iPS-RPE cells inhibited cell proliferation and production of these cytotoxic agents by activated NK cells in vitro. iPS-RPE cells constitutively expressed HLA-E and suppressed NK cell activation through an interaction between HLA-E and CD94/NKG2A. Moreover, immunohistochemical evaluation of monkey RPE transplantation into in vivo immune rejection models showed no NK cell invasion in the retina in allografts or xenografts except for one xenografted eye. Conclusions: Cultured iPS cell-derived RPE cells greatly suppress NK cell activation. Thus, NK cells might be inactivated when exposed to this type of retinal cell.

In vivo depletion of serum IgG by an affibody molecule binding the neonatal Fc receptor.

Lowering the total level of Immunoglobulin G (IgG) in circulation is a promising general treatment option for many autoimmune diseases driven by pathogenic autoantibodies. The half-life of IgG in circulation is unusually long as a consequence of its interaction with the neonatal Fc receptor (FcRn), which protects it from lysosomal degradation by cells in contact with blood. Blocking the IgG/FcRn interaction prevents FcRn-mediated rescue, which may lead to increased catabolism and a lowering of the total IgG level. Here, we find that an engineered alternative scaffold protein, an affibody molecule, interacting specifically with FcRn, is able to block the IgG/FcRn interaction in vitro. The affibody molecule (ZFcRn) was expressed alone or as a fusion to an albumin binding domain (ABD), to extend its half-life in circulation, in both cases with retained affinity and blocking potential. Repeated i.v. injections in mice of ZFcRn and ZFcRn-ABD were found to result in an up to 40% reduction of the IgG serum-level after 5 days. Potential applications of ZFcRn as a general treatment modality for autoimmune diseases are discussed.

Engineered clearing agents for the selective depletion of antigen-specific antibodies.

Here we have designed a novel class of engineered antibody-based reagents ('Seldegs') that induce the selective degradation of antigen-specific antibodies. We demonstrate the rapid and specific clearance of antibodies recognizing the autoantigen, myelin oligodendrocyte glycoprotein and tumour target, HER2. Seldegs have considerable potential in multiple areas, including the treatment of antibody-mediated autoimmunity and diagnostic imaging.

Single chain antibody fragments with pH dependent binding to FcRn enabled prolonged circulation of therapeutic peptide in vivo.

The neonatal Fc receptor for IgG (FcRn) is considered critical for the regulation of endogenous IgG and serum albumin (SA) and their circulation half-life in vivo. Both IgG and SA can bind to FcRn tightly at acidic pH but not so much at neutral pH. Here we reported a few novel single chain antibody fragments (scFv) obtained based on screening of a phage library. FnAb-8 and FnAb-12 can bind to human FcRn with higher affinities than IgG at acidic pH but similar or lower affinities than IgG at pH7.4. Fusion proteins consisted of the therapeutic peptide, GLP-1 (Glucagon-like peptide-1) connected to the N-terminus of FnAb-8 and FnAb-12, named as G8 and G12, were shown to retain the pH-dependent binding capabilities to FcRn while also bound to the GLP-1 receptor. In vivo efficacy studies in diet induced diabetes mice confirmed the GLP-1 receptor (GLP-1R) agonist activities and sustained blood sugar lowering effect. In vivo pharmacokinetics (PK) studies were performed in nonhuman primates and FnAb-8 was found to have circulation half-life several folds longer than what have been reported for scFvs. G8 may be developed into long acting GLP-1R agonists with great potentials in clinical applications.

VEGFR2 targeted antibody fused with MICA stimulates NKG2D mediated immunosurveillance and exhibits potent anti-tumor activity against breast cancer.

Binding of MHC class I-related chain molecules A and B (MICA/B) to the natural killer (NK) cell receptor NK group 2, member D (NKG2D) is thought critical for activating NK-mediated immunosurveillance. Angiogenesis is important for tumor growth and interfering with angiogenesis using the fully human IgG1 anti-VEGFR2 (vascular endothelial growth factor receptor 2) antibody (mAb04) can be effective in treating malignancy. In an effort to make mAb04 more effective we have generated a novel antibody fusion protein (mAb04-MICA) consisting of mAb04 and MICA. We found that mAb04-MICA maintained the anti-angiogenic and antineoplastic activities of mAb04, and also enhanced immunosurveillance activated by the NKG2D pathway. Moreover, in human breast tumor-bearing nude mice, mAb04-MICA demonstrated superior anti-tumor efficacy compared to combination therapy of mAb04 + Docetaxel or Avastin + Docetaxel, highlighting the immunostimulatory effect of MICA. In conclusion, mAb04-MICA provided new inspiration for anti-tumor treatment and had prospects for clinical application.

Antitumor immunity. A shed NKG2D ligand that promotes natural killer cell activation and tumor rejection.

Immune cells, including natural killer (NK) cells, recognize transformed cells and eliminate them in a process termed immunosurveillance. It is thought that tumor cells evade immunosurveillance by shedding membrane ligands that bind to the NKG2D-activating receptor on NK cells and/or T cells, and desensitize these cells. In contrast, we show that in mice, a shed form of MULT1, a high-affinity NKG2D ligand, causes NK cell activation and tumor rejection. Recombinant soluble MULT1 stimulated tumor rejection in mice. Soluble MULT1 functions, at least in part, by competitively reversing a global desensitization of NK cells imposed by engagement of membrane NKG2D ligands on tumor-associated cells, such as myeloid cells. The results overturn conventional wisdom that soluble ligands are always inhibitory and suggest a new approach for cancer immunotherapy.

Enhanced prevalence of plasmatic soluble MHC class I chain-related molecule in vascular pregnancy diseases.

The major histocompatibility complex class I related chain (MIC) is a stress-inducible protein modulating the function of immune natural killer (NK) cells, a major leukocyte subset involved in proper trophoblast invasion and spiral artery remodeling. Aim of the study was to evaluate whether upregulation of soluble MIC (sMIC) may reflect immune disorders associated to vascular pregnancy diseases (VPD). sMIC was more frequently detected in the plasma of women with a diagnostic of VPD (32%) than in normal term-matched pregnancies (1.6%, P < 0.0001), with highest prevalence in intrauterine fetal death (IUDF, 44%) and vascular intrauterine growth restriction (IUGR, 39%). sMIC levels were higher in preeclampsia (PE) than in IUFD (P < 0.01) and vascular IUGR (P < 0.05). sMIC detection was associated with bilateral early diastolic uterine notches (P = 0.037), thrombocytopenia (P = 0.03), and high proteinuria (P = 0.03) in PE and with the vascular etiology of IUGR (P = 0.0038). Incubation of sMIC-positive PE plasma resulted in downregulation of NKG2D expression and NK cell-mediated IFN-γ production in vitro. Our work thus suggests that detection of sMIC molecule in maternal plasma may constitute a hallmark of altered maternal immune functions that contributes to vascular disorders that complicate pregnancy, notably by impairing NK-cell mediated production of IFN-γ, an essential cytokine favoring vascular modeling.

Epitope-specific regulation of memory programming by differential duration of antigen presentation to influenza-specific CD8(+) T cells.

Memory CD8(+) T cells are programmed during the primary response for robust secondary responsiveness. Here we show that CD8(+) T cells responding to different epitopes of influenza virus received qualitatively different signals during the primary response that altered their secondary responsiveness. Nucleoprotein (NP)-specific CD8(+) T cells encountered antigen on CD40-licensed, CD70-expressing, CD103(-)CD11b(hi) dendritic cells (DCs) at later times in the primary response. As a consequence, they maintained CD25 expression and responded to interleukin-2 (IL-2) and CD27, which together programmed their robust secondary proliferative capacity and interferon-γ (IFN-γ)-producing ability. In contrast, polymerase (PA)-specific CD8(+) T cells did not encounter antigen-bearing, CD40-activated DCs at later times in the primary response, did not receive CD27 and CD25 signals, and were not programmed to become memory CD8(+) T cells with strong proliferative and cytokine-producing ability. As a result, CD8(+) T cells responding to abundant antigens, like NP, dominated the secondary response.

Extending serum half-life of albumin by engineering neonatal Fc receptor (FcRn) binding.

A major challenge for the therapeutic use of many peptides and proteins is their short circulatory half-life. Albumin has an extended serum half-life of 3 weeks because of its size and FcRn-mediated recycling that prevents intracellular degradation, properties shared with IgG antibodies. Engineering the strictly pH-dependent IgG-FcRn interaction is known to extend IgG half-life. However, this principle has not been extensively explored for albumin. We have engineered human albumin by introducing single point mutations in the C-terminal end that generated a panel of variants with greatly improved affinities for FcRn. One variant (K573P) with 12-fold improved affinity showed extended serum half-life in normal mice, mice transgenic for human FcRn, and cynomolgus monkeys. Importantly, favorable binding to FcRn was maintained when a single-chain fragment variable antibody was genetically fused to either the N- or the C-terminal end. The engineered albumin variants may be attractive for improving the serum half-life of biopharmaceuticals.

MULT1E/mIL-12: a novel bifunctional protein for natural killer cell activation.

Natural killer (NK) cells have the potential to be effective killers of tumor cells. They are governed by inhibitory and activating receptors like NKG2D, whose ligands are normally upregulated in cells that are stressed, like cancer cells. Advanced cancer cells, however, have ways to reduce these ligands' expression, leaving them less detectable by NK cells. Along with these receptors, NK cells also require activating cytokines, like interleukin 12 (IL-12). The goal of this study is to develop a novel bi-functional fusion protein for enhanced NK cell activation. The proposed protein combines the extracellular domain of the NKG2D ligand Mouse UL-16-binding protein-like transcript 1 (MULT1E) and mouse IL-12 (mIL-12). It is hypothesized that when expressed by tumor cells, the protein will activate NK and other killer cells using the NKG2D receptor, and deliver mIL-12 to the NK cells where it can interact with the IL-12R and enhance cytotoxicity. The fusion protein, when expressed by engineered tumor cells, indeed activated NK cells in vitro as assayed by increased production of interferon-γ and cytotoxicity and significantly reduced tumor growth in vivo. Although the study is preliminary, the data suggest that the MULT1E/mIL-12 bi-functional fusion protein is an effective activator of NK cells for cancer treatment.

Deletion of naïve T cells recognizing the minor histocompatibility antigen HY with toxin-coupled peptide-MHC class I tetramers inhibits cognate CTL responses and alters immunodominance.

Alloreactive T-cell responses directed against minor histocompatibility (H) antigens, which arise from diverse genetic disparities between donor and recipient outside the MHC, are an important cause of rejection of MHC-matched grafts. Because clinically significant responses appear to be directed at only a few antigens, the selective deletion of naïve T cells recognizing donor-specific, immunodominant minor H antigens in recipients before transplantation may be a useful tolerogenic strategy. We have previously demonstrated that peptide-MHC class I tetramers coupled to a toxin can efficiently eliminate specific TCR-transgenic T cells in vivo. Here, using the minor histocompatibility antigen HY as a model, we investigated whether toxic tetramers could inhibit the subsequent priming of the two H2-D(b)-restricted, immunodominant T-cell responses by deleting precursor CTL. Immunization of female mice with male bone marrow elicited robust CTL activity against the Uty and Smcy epitopes, with Uty constituting the major response. As hypothesized, toxic tetramer administration prior to immunization increased survival of cognate peptide-pulsed cells in an in vivo CTL assay, and reduced the frequency of corresponding T cells. However, tetramer-mediated decreases in either T-cell population magnified CTL responses against the non-targeted epitope, suggesting that D(b)-Uty(+) and D(b)-Smcy(+) T cells compete for a limited common resource during priming. Toxic tetramers conceivably could be used in combination to dissect manipulate CD8(+) T-cell immunodominance hierarchies, and to prevent the induction of donor-specific, minor H antigen CTL responses in allotransplantation.

Foxp3+ regulatory T cells are activated in spite of B7-CD28 and CD40-CD40L blockade.

Costimulatory signals are required for priming and activation of naive T cells, while it is less clear how they contribute to induction of regulatory T (Treg)-cell activity. We previously reported that the blockade of the B7-CD28 and CD40L-CD40 interaction efficiently suppresses allogeneic T-cell activation in vivo. This was characterized by an initial rise in Foxp3(+) cells, followed by depletion of host-reactive T cells. To further investigate effects of costimulatory blockade on Treg cells, we used an in vitro model of allogeneic CD4(+) cell activation. When CTLA-4Ig and anti-CD40L mAb (MR1) were added to the cultures, T-cell proliferation and IL-2 production were strongly reduced. However, Foxp3(+) cells proliferated and acquired suppressive activity. They suppressed activation of syngeneic CD4(+) cells much more efficiently than did freshly isolated Treg cells. CD4(+) cells activated by allogeneic cells in the presence of MR1 and CTLA-4Ig were hyporesponsive on restimulation, but their response was restored to that of naive CD4(+) cells when Foxp3(+) Treg cells were removed. We conclude that natural Treg cells are less dependent on B7-CD28 or CD40-CD40L costimulation compared with Foxp3(-) T cells. Reduced costimulation therefore alters the balance between Teff and Treg-cell activation in favor of Treg-cell activity.

Blocking the natural killer cell inhibitory receptor NKG2A increases activity of human natural killer cells and clears hepatitis B virus infection in mice.

BACKGROUND & AIMS: We studied the functions of natural killer (NK) cells and the role of the NK cell inhibitory receptor (NKG2A) during hepatitis B virus (HBV) infection in patients and mice. METHODS: We analyzed levels of NKG2A on peripheral blood NK cells from 42 patients with active chronic hepatitis B (CHB), 31 patients with inactive CHB, and 35 healthy volunteers (controls). Five patients with CHB treated with antiviral therapy were also included to evaluate changes in NK cells after HBV titers decreased. We examined the effects of blocking antibodies against NKG2A or its ligand Qa-1 (equivalent to HLA-E in humans) in immunocompetent mice that express HBV from a plasmid and are positive for serum hepatitis B surface antigen (a mouse model of HBV infection). RESULTS: A higher percentage of NK cells from patients with active CHB were positive for NKG2A (38.47%) than from patients with inactive CHB (19.33%; P < .01) or controls (27.96%; P < .05). The percentage of NKG2A(+) cells correlated with serum viral load (r = 0.5457; P < .001). The percentage of NKG2A(+) cells decreased along with HBV load in patients that received antiviral therapy (P < .05). Blocking NKG2A interaction with HLA-E in peripheral NK cells from patients with active CHB increased their cytotoxicity in vitro. NK cells of HBV carrier mice also had higher percentages of NK cells that expressed NKG2A compared with control mice; NKG2A was likely to be up-regulated by production of interleukin-10 by hepatic regulatory CD4(+)CD25(+) T cells. Blocking Qa-1 in these mice promoted viral clearance in an NK cell-dependent manner. CONCLUSIONS: Infection with HBV increases levels of the inhibitory receptor NKG2A on NK cells in mice and humans, and reduces their ability to clear HBV. Reagents designed to block the interaction between NKG2A and HLA-E might be developed to treat CHB infection.

Major histocompatibility complex class I molecules modulate embryonic neuritogenesis and neuronal polarization.

We studied cultured hippocampal neurons from embryonic wildtype, major histocompatibility complex class I (MHCI) heavy chain-deficient (K(b)D(b)-/-) and NSE-D(b) (which have elevated neuronal MHCI expression) C57BL/6 mice. K(b)D(b)-/- neurons displayed slower neuritogenesis and establishment of polarity, while NSE-D(b) neurons had faster neurite outgrowth, more primary neurites, and tended to have accelerated polarization. Additional studies with ß2M-/- neurons, exogenous ß2M, and a self-MHCI monomer suggest that free heavy chain cis interactions with other surface molecules can promote neuritogenesis while tripartite MHCI interactions with classical MHCI receptors can inhibit axon outgrowth. Together with the results of others, MHCI appears to differentially modulate neuritogenesis and synaptogenesis.

Prolonged activity of a recombinant factor VIII-Fc fusion protein in hemophilia A mice and dogs.

Despite proven benefits, prophylactic treatment for hemophilia A is hampered by the short half-life of factor VIII. A recombinant factor VIII-Fc fusion protein (rFVIIIFc) was constructed to determine the potential for reduced frequency of dosing. rFVIIIFc has an ∼ 2-fold longer half-life than rFVIII in hemophilia A (HemA) mice and dogs. The extension of rFVIIIFc half-life requires interaction of Fc with the neonatal Fc receptor (FcRn). In FcRn knockout mice, the extension of rFVIIIFc half-life is abrogated, and is restored in human FcRn transgenic mice. The Fc fusion has no impact on FVIII-specific activity. rFVIIIFc has comparable acute efficacy as rFVIII in treating tail clip injury in HemA mice, and fully corrects whole blood clotting time (WBCT) in HemA dogs immediately after dosing. Furthermore, consistent with prolonged half-life, rFVIIIFc shows 2-fold longer prophylactic efficacy in protecting HemA mice from tail vein transection bleeding induced 24-48 hours after dosing. In HemA dogs, rFVIIIFc also sustains partial correction of WBCT 1.5- to 2-fold longer than rFVIII. rFVIIIFc was well tolerated in both species. Thus, the rescue of FVIII by Fc fusion to provide prolonged protection presents a novel pathway for FVIII catabolism, and warrants further investigation.

[Effects of natural killer cells stimulated with immobilized MHC class I chain-related antigen A on activities of dendritic cells].

AIM: To observe the effects of NK cells stimulated with immobilized MHC class I chain-related antigen A (iMICA) on activities of dendritic cells (DCs). METHODS: Firstly fresh allogeneic NK cells, or iMICA-stimulated allogeneic NK cells, and autologous NK cells stimulated with IL-2 or IL-2 and iMICA were co-cultured with immature DCs (iDCs) at the ratio of 5:1 for 24 hours. Frequencies of HLA-DR positive or CD86 positive DCs were detected by flow cytometry. Next autologous NK cells stimulated with iMICA were co-cultured with iDCs at the ratio of 1:5 for 24 hours. Variation of HLA-DR or CD86 expression on dendritic cells was measured. Lastly IFN-γ neutralizing antibody was added into the NK-DCs co-culture system to observe HLA-DR or CD86 expression on DCs. RESULTS: When NK:iDCs ratio was 5:1, both fresh allogeneic and activated autologous NK cells killed iDCs efficiently. iMICA did not synergize this cytotoxicity. However, when NK:iDCs ratio was 1:5, NK cells activated by iMICA promoted HLA-DR and CD86 expression on DCs. IFN-γ antibody down-regulated HLA-DR and CD86 expression on DCs which were co-cultured with iMICA-activated NK cells. CONCLUSION: iMICA is redundant as fresh allogeneic or activated autologous NK cells killed iDCs. However, if numbers of NK cell are less than those of DCs, iMICA can stimulate NK cells to produce IFN-γ for DCs maturation.