IL-12p40 Homodimer Ameliorates Experimental Autoimmune Arthritis.

IL-23 is the key cytokine that induces the expansion of Th17 cells. It is composed of p19 and p40 subunits of IL-12. The p40 subunit binds competitively to the receptor of IL-23 and blocks its activity. Our aim was to assess the preventive and therapeutic effect of the IL-12p40 homodimer (p40)2 subunit in autoimmune arthritis animal models. In the current study, using IL-1R antagonist-knockout mice and a collagen-induced arthritis model, we investigated the suppressive effect of (p40)2 on inflammatory arthritis. We demonstrated that the recombinant adenovirus-expressing mouse (p40)2 model prevented the development of arthritis when given before the onset of arthritis. It also decreased the arthritis index and joint erosions in the mouse model if transferred after arthritis was established. (p40)2 inhibited the production of inflammatory cytokines and Ag-specific T cell proliferation. It also induced CD4(+)CD25(+)Foxp3 regulatory T (Treg) cells in vitro and in vivo, whereas the generation of retinoic acid receptor-related organ receptor γt and Th17 cells was suppressed. The induction of Treg cells and the suppression of Th17 cells were mediated via activated STAT5 and suppressed STAT3. Our data suggest that (p40)2 suppressed inflammatory arthritis successfully. This could be a useful therapeutic approach in autoimmune arthritis to regulate the Th17/Treg balance and IL-23 signaling.

Crucial roles of interleukin-7 in the development of T follicular helper cells and in the induction of humoral immunity.

UNLABELLED: T follicular helper (Tfh) cells are specialized providers of cognate B cell help, which is important in promoting the induction of high-affinity antibody production in germinal centers (GCs). Interleukin-6 (IL-6) and IL-21 have been known to play important roles in Tfh cell differentiation. Here, we demonstrate that IL-7 plays a pivotal role in Tfh generation and GC formation in vivo, as treatment with anti-IL-7 neutralizing antibody markedly impaired the development of Tfh cells and IgG responses. Moreover, codelivery of mouse Fc-fused IL-7 (IL-7-mFc) with a vaccine enhanced the generation of GC B cells as well as Tfh cells but not other lineages of T helper cells, including Th1, Th2, and Th17 cells. Interestingly, a 6-fold-lower dose of an influenza virus vaccine codelivered with Fc-fused IL-7 induced higher antigen-specific and cross-reactive IgG titers than the vaccine alone in both mice and monkeys and led to markedly enhanced protection against heterologous influenza virus challenge in mice. Enhanced generation of Tfh cells by IL-7-mFc treatment was not significantly affected by the neutralization of IL-6 and IL-21, indicating an independent role of IL-7 on Tfh differentiation. Thus, IL-7 holds promise as a critical cytokine for selectively inducing Tfh cell generation and enhancing protective IgG responses. IMPORTANCE: Here, we demonstrate for the first time that codelivery of Fc-fused IL-7 significantly increased influenza virus vaccine-induced antibody responses, accompanied by robust expansion of Tfh cells and GC B cells as well as enhanced GC formation. Furthermore, IL-7-mFc induced earlier and cross-reactive IgG responses, leading to striking protection against heterologous influenza virus challenge. These results suggest that Fc-fused IL-7 could be used for inducing strong and cross-protective humoral immunity against highly mutable viruses, such as HIV and hepatitis C virus, as well as influenza viruses.

Safety and immunogenicity of therapeutic DNA vaccine with antiviral drug in chronic HBV patients and its immunogenicity in mice.

BACKGROUND & AIMS: Here, we evaluated the safety and immunogenicity of hepatitis B virus (HBV) DNA vaccine, HB-110, in mice and Korean patients with chronic hepatitis B (CHB) undergoing adefovir dipivoxil (ADV) treatment. METHODS: For animal study, mice (BALB/c or HBV transgenic) were immunized with mHB-110, and T-cell and antibody responses were evaluated. For clinical study, 27 patients randomly received either ADV alone or ADV in combination with HB-110. Liver function tests, serum HBV DNA levels and the presence of HBeAg/anti-HBe were analysed. T-cell responses were estimated by ELISPOT and FACS analysis. RESULTS: mHB-110 induced higher T-cell and antibody responses than mHB-100 in mice. No adverse effects were observed by HB-110 cotreated with ADV. HBV-specific T-cell responses were induced in a portion of patients in medium to high dose of HB-110. Interestingly, HB-110 exhibited positive effects on ALT normalization and maintenance of HBeAg seroconversion. One patient, who received high dose of HB-110 exhibited HBeAg seroconversion during vaccination, which correlated with vaccine-induced T-cell responses without ALT elevation. CONCLUSIONS: HB-110 was safe and tolerable in CHB patients. In contrast to results in animal models, HB-110 in Korean patients exhibited weaker capability of inducing HBV-specific T-cell responses and HBeAg seroconversion than HB-100 in Caucasian patients. As Asian patients, who are generally infected via vertical transmission, appeared to have higher level of immune tolerance than Caucasian, novel approaches for breaking immune tolerance rather than enhancing immunogenicity may be more urgently demanded to develop effective therapeutic HBV DNA vaccines.

Arterial Distension Monitoring Scheme Using FPGA-Based Inference Machine in Ultrasound Scanner Circuit System.

This paper presents an arterial distension monitoring scheme using a field-programmable gate array (FPGA)-based inference machine in an ultrasound scanner circuit system. An arterial distension monitoring requires a precise positioning of an ultrasound probe on an artery as a prerequisite. The proposed arterial distension monitoring scheme is based on a finite state machine that incorporates sequential support vector machines (SVMs) to assist in both coarse and fine adjustments of probe position. The SVMs sequentially perform recognitions of ultrasonic A-mode echo pattern for a human carotid artery. By employing sequential SVMs in combination with convolution and average pooling, the number of features for the inference machine is significantly reduced, resulting in less utilization of hardware resources in FPGA. The proposed arterial distension monitoring scheme was implemented in an FPGA (Artix7) with a resource utilization percentage less than 9.3%. To demonstrate the proposed scheme, we implemented a customized ultrasound scanner consisting of a single-element transducer, an FPGA, and analog interface circuits with discrete chips. In measurements, we set virtual coordinates on a human neck for 9 human subjects. The achieved accuracy of probe positioning inference is 88%, and the Pearson coefficient (r) of arterial distension estimation is 0.838.

Biochemical characterization of a new recombinant TNF receptor-hyFc fusion protein expressed in CHO cells.

The currently used Tumor Nectosis Factor (TNF)-α blockers such as infliximab, adalimumab and etanercept have Fc regions of the human IgG1 subtype have advantages in terms of in vivo half-life, however these could raise potential concerns for unwanted effector-mediated effects, such as antibody dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). To address this issue, we constructed a novel hybrid protein with decreased ADCC and CDC potentials by fusing the TNF receptor to a hybrid Fc (hyFc) containing CH2 and CH3 regions of IgG4 and highly flexible hinge regions of IgD which neither has ADCC and CDC activities. The resulting fusion protein, TNFR-hyFc, was over-expressed in CHO cells. For use as a pre-clinical material in pharmacology, PK and toxicological evaluations were carried out for biochemical characterization which was then compared with etanercept that has similarity in structure. Amino acid composition analysis and peptide mapping showed that the expressed TNFR-hyFc matched the theoretical composition derived from the DNA sequence. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) showed that TNFR-hyFc is 2.9 kDa larger than etanercept. MALDI-TOF after removal of N-glycans by PNGase treatment showed that TNFR-hyFc is 3.9 kDa larger than etanercept. Isoelectric focusing and monosaccharide analysis showed that TNFR-hyFc is slightly more acidic than etanercept. N-terminal amino acid sequencing showed that N-terminal heterogeneity is present in both TNFR-hyFc and etanercept, although the ratios are somewhat different. Glycan analysis showed that the main glycan form is bi-antennary, similar to etanercept.

The pharmacology study of a new recombinant TNF receptor-hyFc fusion protein.

TNF-α-blocking agents such as infliximab, adalimumab and etanercept are widely used for the treatment of severe inflammatory diseases including rheumatoid arthritis and psoriasis. The currently used TNF-α blockers have Fc regions of the human IgG1 subtype, which is advantageous in terms of in vivo half-life but also raise the potential for unwanted effector-mediated effects, such as antibody dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). To address this issue, we constructed a novel hybrid protein by fusing the TNF receptor (TNFR) with a hybrid Fc (hyFc) consisting of the CH2 and CH3 regions of IgG4 and the highly flexible hinge regions of IgD which would not have ADCC and CDC activity. The resulting fusion protein, TNFR-hyFc, was over-expressed in CHO and pharmacological characteristics were evaluated in comparison with the structurally similar etanercept. TNFR-hyFc effectively neutralized TNF-α in L929 bioassay and showed a 1.5-fold higher neutralizing activity compared to etanercept. In a pharmacokinetic study in cynomolgus monkeys, TNFR-hyFc showed plasma half-life and AUC comparable to etanercept. In a mouse collagen induced arthritis model, TNFR-hyFc showed significant amelioration of arthritis compared to etanercept or vehicle control. In an LPS-induced septic shock model, TNFR-hyFc showed a similar level of protection against mortality as etanercept. These results confirm the feasibility of the TNFR-hyFc as an effective TNF-α blocker for the treatment of inflammatory diseases.

A single administration of hIL-7-hyFc induces long-lasting T-cell expansion with maintained effector functions.

Interleukin-7 (IL-7) is an essential cytokine for T-cell homeostatic proliferation and maintenance. Clinical studies have shown the potential benefits of IL-7 therapy in various diseases associated with lymphopenia. However, the kinetics of the T-cell response to a single administration of IL-7 in humans have not been fully elucidated. Here, we investigated the effects of Fc-fused long-acting recombinant human IL-7 (hIL-7-hyFc, efineptakin alfa) on lymphocytes in healthy adults after a single subcutaneous or intramuscular administration. Administration of hIL-7-hyFc increased the CD8+ and CD4+ T-cell numbers up to 2.5-fold, with corresponding upregulation of Ki-67 and Bcl-2 expression, peaking at day 3 or 7. Regulatory T cells (Tregs) did not expand. Among CD8+ and CD4+ T cells, all T-cell subsets (TN, TEM, TCM, TEMRA, and TSCM) increased for 56 days. The T-cell receptor repertoire diversity of naive CD8+ and CD4+ T cells was increased by hIL-7-hyFc, whereas the memory T-cell subsets did not differ between day 56 and day 0. Transcriptomic analysis revealed that hIL-7-hyFc induced robust T-cell expansion without changes in gene expression profiles associated with T-cell functions or genes related to T-cell exhaustion, senescence, and anergy. The effector functions of antigen-specific CD8+ T cells were preserved after hIL-7-hyFc administration. Our results suggest that hIL-7-hyFc administration induced a sustained increase in the numbers of CD8+ and CD4+ T cells, but not Tregs, without qualitative changes. These results support the potential of hIL-7-hyFc as a treatment for patients with compromised T-cell immunity or as a vaccine adjuvant.

Long-Acting Recombinant Human Interleukin-7, NT-I7, Increases Cytotoxic CD8 T Cells and Enhances Survival in Mouse Glioma Models.

PURPOSE: Patients with glioblastoma (GBM) are treated with radiotherapy (RT) and temozolomide (TMZ). These treatments may cause prolonged systemic lymphopenia, which itself is associated with poor outcomes. NT-I7 is a long-acting IL7 that expands CD4 and CD8 T-cell numbers in humans and mice. We tested whether NT-I7 prevents systemic lymphopenia and improves survival in mouse models of GBM. EXPERIMENTAL DESIGN: C57BL/6 mice bearing intracranial tumors (GL261 or CT2A) were treated with RT (1.8 Gy/day × 5 days), TMZ (33 mg/kg/day × 5 days), and/or NT-I7 (10 mg/kg on the final day of RT). We followed the mice for survival while serially analyzing levels of circulating T lymphocytes. We assessed regulatory T cells (Treg) and cytotoxic T lymphocytes in the tumor microenvironment, cervical lymph nodes, spleen, and thymus, and hematopoietic stem and progenitor cells in the bone marrow. RESULTS: GBM tumor-bearing mice treated with RT+NT-I7 increased T lymphocytes in the lymph nodes, thymus, and spleen, enhanced IFNγ production, and decreased Tregs in the tumor which was associated with a significant increase in survival. NT-I7 also enhanced central memory and effector memory CD8 T cells in lymphoid organs and tumor. Depleting CD8 T cells abrogated the effects of NT-I7. Furthermore, NT-I7 treatment decreased progenitor cells in the bone marrow. CONCLUSIONS: In orthotopic glioma-bearing mice, NT-I7 mitigates RT-related lymphopenia, increases cytotoxic CD8 T lymphocytes systemically and in the tumor, and improves survival. A phase I/II trial to evaluate NT-I7 in patients with high-grade gliomas is ongoing (NCT03687957).

Marked enhancement of antigen-specific T-cell responses by IL-7-fused nonlytic, but not lytic, Fc as a genetic adjuvant.

IL-7 plays a crucial role in the homeostatic proliferation, differentiation and survival of T cells, as well as in the survival and proliferation of precursor B cells. Here, we demonstrated that utilizing nonlytic Fc-fused IL-7 (IL-7-Fc(m)) as a genetic adjuvant significantly enhanced not only CD4(+) but also CD8(+) T-cell responses by E7 DNA immunization, in addition to improving protection against TC-1-induced tumors in comparison to IL-7 alone. Similar results were obtained in OT-1 adoptive transfer experiments with OVA DNA injection, suggesting independence from antigenic nature and experimental conditions. In particular, the increased frequency of CD8(+) T cells was mainly due to enhanced T-cell proliferation in T-cell priming, and not to decreased cellular apoptosis. Interestingly, the enhanced adjuvant effect was not seen in the co-delivery of lytic Fc-fused IL-7 (IL-7-Fc) which increases T-cell apoptosis as well as T-cell proliferation, suggesting that the T-cell proliferative effect may be neutralized by T-cell apoptosis. Thus, our findings suggest that nonlytic Fc, in contrast to lytic Fc, fusion to cytokines may provide an insight in designing a potent genetic adjuvant for inducing CD4(+) and CD8(+) T-cell responses.

hIL-7-hyFc, A Long-Acting IL-7, Increased Absolute Lymphocyte Count in Healthy Subjects.

A low lymphocyte count puts immune-compromised patients at risk of mortality. hIL-7-hyFc is a homodimeric interleukin-7 (IL-7), a potent T-cell amplifier, fused to the hybridizing IgD/IgG4 immunoglobulin domain. We performed a randomized, double-blind, placebo-controlled, dose-escalation, phase I study to assess the pharmacokinetic, pharmacodynamic, safety, tolerability, and immunogenicity profiles of hIL-7-hyFc administered s.c. and i.m. to healthy volunteers. Thirty subjects randomly received hIL-7-hyFc or its matching placebo in an 8:2 ratio at 20, 60 µg/kg s.c., or 60 µg/kg i.m. The hIL-7-hyFc was slowly absorbed and its terminal half-life was 63.26 hours after i.m. administration. The hIL-7-hyFc increased absolute lymphocyte count, mostly in T-cells, which peaked 3 weeks after administration and then lasted for several additional weeks. The hIL-7-hyFc was well-tolerated after a single s.c. and i.m. administration. Injection site reaction was the most common treatment-emergent adverse event, which resolved spontaneously without treatment. The hIL-7-hyFc can be developed into a beneficial treatment option for patients with compromised T-cell immunity. This trial was registered at www.clinicaltrials.gov as #NCT02860715.

Increased in vivo immunological potency of HB-110, a novel therapeutic HBV DNA vaccine, by electroporation.

Pulse-induced permeabilization of cellular membranes, generally referred to as electroporation (EP), has been used for years as a tool to increase macromolecule uptake in tissues, including nucleic acids, for gene therapeutic applications, and this technique has been shown to result in improved immunogenicity. In this study, we assessed the utility of EP as a tool to improve the efficacy of HB-110, a novel therapeutic DNA vaccine against chronic hepatitis B, now in phase 1 of clinical study in South Korea. The potency of HB-110 in mice was shown to be improved by EP. The rapid onset of antigen expression and higher magnitude of humoral and cellular responses in electric pulse-treated mice revealed that EP may enable a substantial reduction in the dosage of DNA vaccine required to elicit a response similar in magnitude to that achievable via conventional administration. This study also showed that EP-based vaccination at 4-week-intervals elicited a cellular immune response which was about two-fold higher than the response elicited by conventional vaccination at 2-week intervals. These results may provide a rationale to reduce the clinical dose and increase the interval between the doses in the multidose vaccination schedule. Electric pulsing also elicited a more balanced immune response against four antigens expressed by HB-110: S, preS, Core, and Pol.

In vivo kinetics and biodistribution of HB-110, a novel HBV DNA vaccine, after administration in mice.

This study investigated the pharmacokinetic profile and biodistribution of HB-110, a novel HBV therapeutic vaccine candidate, in mice. HB-110 was rapidly degraded in the blood after i.v. injection with a half-life of 1.9+/-0.083 min, and was no longer detected at 60 min except in one individual near the detection limit. In the i.m. injection, plasmid DNA was detectable at the injection site until 11 days after administration, but the amounts were just above the detection limit. The blood concentration of HB-110 showed a maximum of 604 pg/mL at 15 min after i.m. injection, which was followed by degradation to undetectable levels at 90 min. The plasmid DNA in tissues peaked at 90 min after administration. The highest concentration of plasmid DNA was detected in the liver (24.172 pg/mg tissue), and considerable amounts were also observed in the lung (9.467 pg/mg tissue) and spleen (7.688 pg/mg tissue). The amount of plasmid DNA in tissues was 2 to 3 orders of magnitude lower than in the injection site at the same time points. The HB-110 concentration in tissues, including gonads, decreased rapidly and was undetectable 24 h after administration.

(p40)2-Fc reduces immune-inflammatory response through the activation of T cells in collagen induced arthritis mice.

IL-12p40 homodimer, a natural antagonist of IL-12 and IL-23, performs an important role in the expression of proinflammatory cytokines that is essential for Th1 and Th17 immune responses. Here, we reveal the therapeutic and immunosuppressive effect of the IL-12p40 subunit ((p40)2-Fc) in an experimental autoimmune arthritis model. We hypothesized that (p40)2-Fc may reduce the inflammatory response and the activation of T cells. In this study, we intraperitoneally injected (p40)2-Fc into collagen induced arthritis (CIA) mice to identify whether (p40)2-Fc attenuates CIA severity. (p40)2-Fc reduced the development of CIA, joint inflammation and cartilage destruction. (p40)2-Fc also significantly decreased the concentration of serum immunoglobulin as well as the number of T cells and C II specific T cells. In addition, osteoclastogenesis in (p40)2-Fc treated mice was down-regulated compared to the mice treated with (p40)2-Fc control. We observed that (p40)2-Fc treatment alleviates arthritis in mice with CIA, reducing inflammation and osteoclast differentiation. These findings suggest that (p40)2-Fc can be a potential therapeutic approach for autoimmune arthritis.

HCV core protein promotes liver fibrogenesis via up-regulation of CTGF with TGF-beta1.

Liver cirrhosis is one of the major complications of hepatitis C virus (HCV) infection, but the mechanisms underlying HCV-related fibrogenesis are still not clear. Although the roles of HCV core protein remain poorly understood, it is supposed to play an important role in the regulation of cellular growth and hepatocarcinogenesis. The aim of this study was to examine the role of HCV core protein on the hepatic fibrogenesis. We established an in vitro co-culture system with primary hepatic stellate cell (HSC) isolated from rats, and a stable HepG2-HCV core cell line which had been transfected with HCV core gene. The expressions of fibrosis-related molecules transforming growth factor beta1 (TGF-beta1), transforming growth factor beta receptor II (TGFbetaRII), alpha-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF) were analyzed via histological or molecular methods. In addition, the expression levels of matrix metaloprotinase-2 (MMP-2) and collagen type I (Col I) from the co-cultured media were measured by zymogram and ELISA, respectively. The expressions of alpha-SMA, TGF-beta1, Col I, TGFbetaRII and MMP-2 were significantly increased in the co-culture of stable HepG2-HCV core with HSC. Moreover, the significant increases of CTGF and TGF-beta1 in the HCV core-expressing cells were observed by either Northern or Western blot analysis. These results suggest that HCV core protein may contribute to the hepatic fibrogenesis via up-regulation of CTGF and TGF-beta1.

Hepatitis C virus core inhibits the Fas-mediated p38 mitogen activated kinase signaling pathway in hepatocytes.

The p38 mitogen activated kinase (MAPK) signaling pathway plays an essential role in regulating many cellular processes, including inflammation, cell differentiation, and cell death. Here, we report that the hepatitis C virus (HCV) core inhibits the Fas-mediated p38 signaling pathway. The Fas-mediated p38 activation is suppressed in core-expressing HepG2 cell lines, as well as in the hepatocytes of transgenic mice. In addition, core protein blocked the Fas-mediated activation of apoptosis signal-regulating kinase 1 (ASK1), a major upstream MAPKKK of p38. Treatment of a specific p38 inhibitor (SB203580) or overexpression of a kinase-defective mutant, ASK1 (K709R), promoted Fas-mediated cell death in HepG2 cells. This suggests that the p38 and ASK1 activation is required for cell survival against Fas-mediated cell death. In addition, we observed that the HCV core protein enhances Fas-mediated liver injury and lethality in transgenic mice. Collectively, our findings suggest that the HCV core inhibits the Fas-mediated p38 signaling pathway, which results in accelerated Fas-mediated cell death.

[The role of hepatitis C virus core protein on liver fibrogenesis: a study using an in vitro co-culture system].

BACKGROUND/AIMS: The study of liver fibrogenesis by hepatitis C virus (HCV) has been limited due to the lack of an efficiency in vitro culture systems. In the present study, we investigated whether or not HCV core protein is directly related to liver fibrogenesis through stimulation of hepatic stellate cells (HSC). METHODS: Human and rat HSC were isolated and we established an in vitro co-culture system of a stable HepG2-HCV core cell line which was transfected with HCV core gene and primary HSC. We performed immunocytochemical staining and Western and Northern blot analysis in the stimulated HSC by HCV core protein to identify the expression of transforming growth factor beta1 (TGF-beta1), transforming growth factor beta receptor II (TGFbeta R II), alpha-smooth muscle actin (alpha-SMA) and connective tissue growth factor (CTGF). The expression of matrix metalloproteinase-2 (MMP-2) and collagen type I (Col I) in the culture media were measured by zymogram and ELISA, respectively. RESULTS: The expression of TGF-beta1 and CTGF was significantly higher in the stable HepG2-HCV core cell line than in HepG2 cells. Furthermore, the makers related to fibrosis such as alpha-SMA, TGF-beta1, Col I, TGFRII and MMP-2 were highly expressed in the co-culture of stable HepG2-HCV core with HSC. CONCLUSIONS: HCV core protein may play a direct role in the fibrogenesis of chronic liver disease with HCV infection.

A long-acting erythropoietin fused with noncytolytic human Fc for the treatment of anemia.

The Fc fusion technology has been introduced to generate long-acting antagonistic drugs such as Enbrel, Orencia and Amevive. Here, Genexine created a novel noncytolytic hybrid Fc (hyFc) as a carrier of agonistic protein drugs using naturally existing IgD and IgG4 Fcs without any mutation in the hyFc region. The erythropoietin (EPO) fused with hyFc exhibited little binding activity to FcγR and C1q molecules that are main mediators for death of target cells. The EPO-hyFc showed higher in vitro and in vivo bioactivities than EPOIgG1 Fc and highly glycosylated EPO (Aranesp). Phase I clinical trial with EPO-hyFc is currently undergoing in Korea.

Dendritic cell internalization of α-galactosylceramide from CD8 T cells induces potent antitumor CD8 T-cell responses.

Dendritic cells (DC) present α-galactosylceramide (αGalCer) to invariant T-cell receptor-expressing natural killer T cells (iNKT) activating these cells to secrete a variety of cytokines, which in turn results in DC maturation and activation of other cell types, including NK cells, B cells, and conventional T cells. In this study, we showed that αGalCer-pulsing of antigen-activated CD8 T cells before adoptive transfer to tumor-bearing mice caused a marked increase in donor T-cell proliferation, precursor frequency, and cytotoxic lymphocyte activity. This effect was interleukin (IL)-2 dependent and involved both natural killer T cells (NKT) and DCs, as mice lacking IL-2, NKTs, and DCs lacked any enhanced response to adoptively transferred αGalCer-loaded CD8 T cells. iNKT activation was mediated by transfer of αGalCer from the cell membrane of the donor CD8 T cells onto the αGalCer receptor CD1d which is present on host DCs. αGalCer transfer was increased by prior activation of the donor CD8 T cells and required AP-2-mediated endocytosis by host DCs. In addition, host iNKT cell activation led to strong IL-2 synthesis, thereby increasing expansion and differentiation of donor CD8 T cells. Transfer of these cells led to improved therapeutic efficacy against established solid tumors in mice. Thus, our findings illustrate how αGalCer loading of CD8 T cells after antigen activation in vitro may leverage the therapeutic potential of adoptive T-cell therapies.

Natural form of noncytolytic flexible human Fc as a long-acting carrier of agonistic ligand, erythropoietin.

Human IgG1 Fc has been widely used as a bioconjugate, but exhibits shortcomings, such as antibody- and complement-mediated cytotoxicity as well as decreased bioactivity, when applied to agonistic proteins. Here, we constructed a nonimmunogenic, noncytolytic and flexible hybrid Fc (hyFc) consisting of IgD and IgG4, and tested its function using erythropoietin (EPO) conjugate, EPO-hyFc. Despite low amino acid homology (20.5%) between IgD Fc and IgG4 Fc, EPO-hyFc retained "Y-shaped" structure and repeated intravenous administrations of EPO-hyFc into monkeys did not generate EPO-hyFc-specific antibody responses. Furthermore, EPO-hyFc could not bind to FcγR I and C1q in contrast to EPO-IgG1 Fc. In addition, EPO-hyFc exhibited better in vitro bioactivity and in vivo bioactivity in rats than EPO-IgG1 Fc, presumably due to the high flexibility of IgD. Moreover, the mean serum half-life of EPO-hyFc(H), a high sialic acid content form of EPO-hyFc, was approximately 2-fold longer than that of the heavily glycosylated EPO, darbepoetin alfa, in rats. More importantly, subcutaneous injection of EPO-hyFc(H) not only induced a significantly greater elevation of serum hemoglobin levels than darbepoetin alfa in both normal rats and cisplatin-induced anemic rats, but also displayed a delayed time to maximal serum level and twice final area-under-the-curve (AUC(last)). Taken together, hyFc might be a more attractive Fc conjugate for agonistic proteins/peptides than IgG1 Fc due to its capability to elongate their half-lives without inducing host effector functions and hindering bioactivity of fused molecules. Additionally, a head-to-head comparison demonstrated that hyFc-fusion strategy more effectively improved the in vivo bioactivity of EPO than the hyperglycosylation approach.

Increase of Plasma IL-12/p40 Ratio Induced by the Combined Therapy of DNA Vaccine and Lamivudine Correlates with Sustained Viremia Control in CHB Carriers.

BACKGROUND: We previously reported that IFN-gamma producing T cell responses induced by the combined therapy of DNA vaccine and lamivudine for one year are important for the induction of sustained virological response (SVR). However, IFN-gamma production is not sufficient to predict sustained viremia control in chronic hepatitis B (CHB) carriers treated. METHODS: Twelve CHB carriers were intramuscularly immunized 12 times at a 4-week interval with 8 mg of HBV DNA vaccine during the standard lamivudine treatment (100 mg/daily/1 year). The level of cytokines during and after the combined therapy in plasma of all 12 CHB carriers treated was determined by each ELISA kit. Six out of 12 CHB carriers revisited the clinic, and their HBV DNA levels were examined. RESULTS: The combined therapy increased plasma IL-12 and IL-12/p40 ratio during the treatment (baseline vs. peak level: 41.8+/-8.3 vs. 163.1+/-29.2 pg/ml; p<0.01 and 0.96+/-0.25 vs. 3.58+/-0.86; p<0.01, espectively), and the peak level of plasma IL-12 and IL-12/p40 ratio was evoked at 6 to 10 months during the combined therapy. In particular, CHB carriers with SVR had two and three-fold higher level of the peak plasma IL-12 and plasma IL-12/p40 ratio than non-virological responders (NVRs), respectively (218.0+/-41.4 vs. 108.1+/-28.6 pg/ml; p=0.09 and 5.35+/-1.38 vs. 1.80+/-0.29; p<0.05, respectively), while p40 level was consistent during the combined therapy. In addition, there was no significant temporal correlation between the peak IL-12/p40 ratio and the elevation of serum alanine aminotransferase (ALT) in this study, contrast to IFN-alpha therapy which induced peak IL-12 level following ALT flares. CONCLUSION: Our results indicate that the combined therapy induces the increase of plasma IL-12 and IL-12/p40 ratio, which are associated with long-term SVR in CHB carriers.