Better Peptides via Chemical Glycosylation: Somatostatin Analogues Having a Human Complex-Type N-Glycan with Improved Drug Properties.

Somatostatin (somatotropin release-inhibiting factor, SRIF) is a growth hormone inhibitory factor in the form of a 14- or 28-amino acid peptide. SRIF affects several physiological functions through its action on five distinct SRIF receptor subtypes (sst1-5). Native SRIF has only limited clinical applications due to its rapid degradation in plasma. To overcome this obstacle, we have developed glycosylated SRIF analogues that possess not only metabolic stability but also high affinity to all five receptor subtypes by attaching human complex-type oligosaccharides. Such glycosylated SRIF analogues with improved pharmacokinetic profiles could be potent and novel therapeutic drugs for SRIF-related diseases in which several SRIF receptor subtypes are closely involved, and also shed light on new indications. Our results show that chemical glycosylation can be a powerful tool for the development of peptide and protein analogues superior to the original molecules with enhanced drug properties.

Hair Follicle Regeneration by Transplantation of a Bioengineered Hair Follicle Germ.

Hair follicle morphogenesis is first induced by epithelial-mesenchymal interactions in the developing embryo. In the hair follicle, various stem-cell populations are maintained in specialized niches to promote repetitive hair follicle-morphogenesis, which is observed in the variable lower region of the hair follicle as a postnatal hair cycle. In contrast, the genesis of most organs is induced only once during embryogenesis. We developed a novel bioengineering technique, the Organ Germ Method, that employs three-dimensional stem cell culture for regenerating various organs and reproducing embryonic organogenesis. In this chapter, we describe a protocol for hair follicle germ reconstitution using adult follicle-derived epithelial stem cells and dermal papilla cells with intracutaneous transplantation of the bioengineered hair-follicle organ germ. This protocol can be useful not only for the clinical study of hair regeneration but also for studies of stem cell biology and organogenesis.

Chemical synthesis of erythropoietin glycoforms for insights into the relationship between glycosylation pattern and bioactivity.

The role of sialyloligosaccharides on the surface of secreted glycoproteins is still unclear because of the difficulty in the preparation of sialylglycoproteins in a homogeneous form. We selected erythropoietin (EPO) as a target molecule and designed an efficient synthetic strategy for the chemical synthesis of a homogeneous form of five EPO glycoforms varying in glycosylation position and the number of human-type biantennary sialyloligosaccharides. A segment coupling strategy performed by native chemical ligation using six peptide segments including glycopeptides yielded homogeneous EPO glycopeptides, and folding experiments of these glycopeptides afforded the correctly folded EPO glycoforms. In an in vivo erythropoiesis assay in mice, all of the EPO glycoforms displayed biological activity, in particular the EPO bearing three sialyloligosaccharides, which exhibited the highest activity. Furthermore, we observed that the hydrophilicity and biological activity of the EPO glycoforms varied depending on the glycosylation pattern. This knowledge will pave the way for the development of homogeneous biologics by chemical synthesis.

Hypothermic temperature effects on organ survival and restoration.

A three-dimensional multicellular organism maintains the biological functions of life support by using the blood circulation to transport oxygen and nutrients and to regulate body temperature for intracellular enzymatic reactions. Donor organ transplantation using low-temperature storage is used as the fundamental treatment for dysfunctional organs. However, this approach has a serious problem in that donor organs maintain healthy conditions only during short-term storage. In this study, we developed a novel liver perfusion culture system based on biological metabolism that can maintain physiological functions, including albumin synthesis, bile secretion and urea production. This system also allows for the resurrection of a severely ischaemic liver. This study represents a significant advance for the development of an ex vivo organ perfusion system based on biological metabolism. It can be used not only to address donor organ shortages but also as the basis of future regenerative organ replacement therapy.

Functional tooth restoration by next-generation bio-hybrid implant as a bio-hybrid artificial organ replacement therapy.

Bio-hybrid artificial organs are an attractive concept to restore organ function through precise biological cooperation with surrounding tissues in vivo. However, in bio-hybrid artificial organs, an artificial organ with fibrous connective tissues, including muscles, tendons and ligaments, has not been developed. Here, we have enveloped with embryonic dental follicle tissue around a HA-coated dental implant, and transplanted into the lower first molar region of a murine tooth-loss model. We successfully developed a novel fibrous connected tooth implant using a HA-coated dental implant and dental follicle stem cells as a bio-hybrid organ. This bio-hybrid implant restored physiological functions, including bone remodelling, regeneration of severe bone-defect and responsiveness to noxious stimuli, through regeneration with periodontal tissues, such as periodontal ligament and cementum. Thus, this study represents the potential for a next-generation bio-hybrid implant for tooth loss as a future bio-hybrid artificial organ replacement therapy.

Chemical synthesis of homogeneous human glycosyl-interferon-ß that exhibits potent antitumor activity in vivo.

Chemical synthesis of homogeneous human glycoproteins exhibiting bioactivity in vivo has been a challenging task. In an effort to overcome this long-standing problem, we selected interferon-ß and examined its synthesis. The 166 residue polypeptide chain of interferon-ß was prepared by covalent condensation of two synthetic peptide segments and a glycosylated synthetic peptide bearing a complex-type glycan of biological origin. The peptides were covalently condensed by native chemical ligation. Selective desulfurization followed by deprotection of the two Cys(Acm) residues gave the target full-length polypeptide chain of interferon-ß bearing either a complex-type sialyl biantennary oligosaccharide or its asialo form. Subsequent folding with concomitant formation of the native disulfide bond afforded correctly folded homogeneous glycosyl-interferon-ß. The chemically synthesized sialyl interferon-ß exhibited potent antitumor activity in vivo.

Definitive evidence that a single N-glycan among three glycans on inducible costimulator is required for proper protein trafficking and ligand binding.

Glycosylation is a widespread post-translational modification found in glycoproteins. Glycans play key roles in protein folding, quality control in the endoplasmic reticulum (ER) and protein trafficking within cells. However, it remains unclear whether all positions of protein glycosylation are involved in glycan functions, or if specific positions have individual roles. Here we demonstrate the integral involvement of a specific N-glycan from amongst the three glycans present on inducible costimulator (ICOS), a T-cell costimulatory molecule, in proper protein folding and intracellular trafficking to the cell surface membrane. We found that glycosylation-defective mutant proteins lacking N-glycan at amino-acid position 89 (N89), but not proteins lacking either N23 or N110, were retained within the cell and were not detected on the cell surface membrane. Additional evidence suggested that N89 glycosylation was indirectly involved in ICOS ligand binding. These data suggest that amongst the three putative ICOS glycosylation sites, N89 is required for proper ICOS protein folding in the ER, intracellular trafficking and ligand binding activity. This study represents a substantial contribution to the current mechanistic understanding of the necessity and potential functions of a specific N-glycan among the multiple glycans of glycoproteins.

JTA-009, a fully human antibody against human AILIM/ICOS, ameliorates graft-vs-host reaction in SCID mice grafted with human PBMCs.

OBJECTIVE: Activation-inducible lymphocyte immunomediatory molecule (AILIM; also referred to as inducible costimulator [ICOS]) is the third molecule identified in the CD28 family participating in T-cell activation. AILIM/ICOS has been implicated in both effector and pathogenic T-cell functions, as evidenced by the beneficial effects of AILIM/ICOS blockade in several murine disease models. In the present study, the role of human AILIM/ICOS in T-cell function was investigated using a fully human monoclonal antibody specific to human AILIM/ICOS (JTA-009). MATERIALS AND METHODS: The effect of JTA-009 on allogenic T-cell proliferation was examined using human mixed lymphocyte reaction (MLR). To investigate the efficacy of AILIM/ICOS blockade in vivo, a graft-vs-host disease (GVHD) model, in which severe combined immunodeficient (SCID) mice were grafted with human peripheral blood mononuclear cells (PBMCs), was used. RESULTS: In MLR, suppressive effect of JTA-009 on allogenic T-cell proliferation was detected with comparable potency to CD28 blockade by cytotoxic T-lymphocyte antigen 4 (CTLA4)-Ig at an intermediate culture phase. JTA-009 acts as a blocking antibody in vivo and inhibited binding of human AILIM/ICOS to mouse AILIM/ICOS ligand (B7h). Treatment with JTA-009 significantly prolonged survival of mice, with reductions of human interferon-gamma levels in blood and number of human cells in spleens. CONCLUSION: These results demonstrate that human AILIM/ICOS plays a role in the GVHD pathogenesis mediated by human T cells, and its blockade is attractive for abrogating undesired T-cell responses as is well-documented in mice.

Critical role of activation-inducible lymphocyte immunomediatory molecule/inducible costimulator in the effector function of human T cells: a comparative in vitro study of effects of its blockade and CD28 blockade in human beings and monkeys.

Activation-inducible lymphocyte immunomediatory molecule (AILIM; also referred to as inducible costimulator, ICOS) is the third homolog of the "professional" costimulatory molecule, CD28. To date, the characteristics and role of AILIM/ICOS, especially in effector function of T cells, have been determined through numerous studies in vitro and in vivo using mice. Considering potential differences among species, whether the AILIM/ICOS blockade acts as an efficacious immunomodulator for human diseases remains to be elucidated. In the present study, ability of AILIM/ICOS blockade to modulate immune responses of human and monkey cells was investigated using a fully human antibody (JTA-009), comparing the effect of CD28 blockade. JTA-009 blocked the response of human and monkey T cells co-stimulated with anti-CD3 and AILIM/ICOS ligand, B7h. AILIM/ICOS and CD28 blockade both inhibited human mixed lymphocyte reaction in different fashions, as well as cytokine production in T helper (Th) 1-/Th2-type recall responses. In monkeys however, CD28 blockade by CTLA4-Ig effectively prevented mixed lymphocyte reaction to a greater extent than AILIM/ICOS blockade. These results suggest that AILIM/ICOS blockade is valuable for suppressing both primary allogenic response and recall responses of T cell in human beings, and that there are differences between human and monkey use preferences for costimulatory molecules.

AILIM/ICOS-mediated elongation of activated T cells is regulated by both the PI3-kinase/Akt and Rho family cascade.

T-cell migration and movement is a critical component of a fully functional immune system. Activation-inducible lymphocyte immunomediatory molecule/inducible co-stimulator (AILIM/ICOS), which is a member of CD28 co-stimulatory receptor family, induces both activated T-cell migration underneath tumor necrosis factor alpha-treated human umbilical vein endothelial cell layers and also the morphological polarization of activated T cells. In our current study, we have investigated the signaling mechanisms underlying the morphological polarization of activated T cells, initiated by AILIM/ICOS signaling. AILIM/ICOS signaling induces the activation of phosphoinositide-3 (PI3)-kinase, the product of which, phosphatidylinositol 3,4,5-trisphosphate (PIP3), was found to be localized in the lamellipodia at the front part of the cells. Phosphorylated Akt is also co-localized with PIP3 and filamentous actin in lamellipodia and the PI3-kinase/Akt signaling cascade has critical roles in T-cell polarization and lamellipodia formation via the re-organization of the actin cytoskeleton. Rho family members and their downstream effectors, Rho-associated kinase and p21-activated kinase (PAK), are also involved in AILIM/ICOS-mediated elongation. The PAK family members are serine/threonine kinase downstream effectors of both Rac and Cdc42. PAK3 is induced by the activation of T cells, whereas PAK1 is constitutively expressed in both naive and activated T cells. During the elongation, not only PAK1 but also PAK3 play an essential role through the phosphorylation of their conservative autophosphorylation sites and catalytic domain. Ser-244 phosphorylation, which is a putative Akt phosphorylation site, on PAK3 but not on PAK1 also regulates the morphological polarization of activated T cells by AILIM/ICOS signaling. Both the PI3-kinase/Akt and Rho family cascades operate coordinately to induce the forward migration of activated T cells by AILIM/ICOS signaling.

Anergic lymphocytes generated by blocking CD28 and ICOS pathways in vitro prolong rat cardiac graft survival.

Regulatory cells may play a pivotal role in inducing and maintaining transplantation tolerance. We investigated the mechanism of anergic lymphocytes with regulatory cell potential generated in vitro by ICOS and CD 28 co-stimulatory blockades as a source of cellular therapy for treating allograft rejection. Anergic lymphocytes were generated by a mixed lymphocyte reaction consisting of DA splenocytes as the stimulator and Lewis splenocytes as the responder in the presence of anti-ICOS mAb and rCTLA-4I g. Immunoregulatory effects of these lymphocytes were evaluated by secondary MLR and using other various stimulations. DA heart was transplanted into 7.5 Gy-irradiated Lewis rat after intravenous administration of these cells and/or Lewis spleen lymphocytes. We observed that these lymphocytes were not only anergic to alloantigen and polyclonal stimulations but also exhibited regulative activity to inhibit the alloreactive T-cell response. Our adoptive transfer studies revealed that irradiated recipients that received both anergic lymphocytes and naIve Lewis lymphocytes had significantly prolonged DA cardiac graft survival (mean 17.5 days) compared with a group that received Lewis lymphocytes alone (mean 10.8 days). Furthermore, some of the recipients accepted the graft indefinitely after receiving anergic lymphocytes alone (>100 days). These results demonstrated that anergic lymphocytes with regulatory activities can be generated through blocking co-stimulatory signals, CD 28 and ICOS, simultaneously in vitro, and may advance a new immunomodulatory strategy for preventing allorejection in organ transplantation.

Expression and function of inducible co-stimulator in patients with systemic lupus erythematosus: possible involvement in excessive interferon-gamma and anti-double-stranded DNA antibody production.

Inducible co-stimulator (ICOS) is the third member of the CD28/cytotoxic T-lymphocyte associated antigen-4 family and is involved in the proliferation and activation of T cells. A detailed functional analysis of ICOS on peripheral blood T cells from patients with systemic lupus erythematosus (SLE) has not yet been reported. In the present study we developed a fully human anti-human ICOS mAb (JTA009) with high avidity and investigated the immunopathological roles of ICOS in SLE. JTA009 exhibited higher avidity for ICOS than a previously reported mAb, namely SA12. Using JTA009, ICOS was detected in a substantial proportion of unstimulated peripheral blood T cells from both normal control individuals and patients with SLE. In CD4+CD45RO+ T cells from peripheral blood, the percentage of ICOS+ cells and mean fluorescence intensity with JTA009 were significantly higher in active SLE than in inactive SLE or in normal control individuals. JTA009 co-stimulated peripheral blood T cells in the presence of suboptimal concentrations of anti-CD3 mAb. Median values of [3H]thymidine incorporation were higher in SLE T cells with ICOS co-stimulation than in normal T cells, and the difference between inactive SLE patients and normal control individuals achieved statistical significance. ICOS co-stimulation significantly increased the production of IFN-gamma, IL-4 and IL-10 in both SLE and normal T cells. IFN-gamma in the culture supernatants of both active and inactive SLE T cells with ICOS co-stimulation was significantly higher than in normal control T cells. Finally, SLE T cells with ICOS co-stimulation selectively and significantly enhanced the production of IgG anti-double-stranded DNA antibodies by autologous B cells. These findings suggest that ICOS is involved in abnormal T cell activation in SLE, and that blockade of the interaction between ICOS and its receptor may have therapeutic value in the treatment of this intractable disease.

Alteration of phosphatidylinositol 3-kinase cascade in the multilobulated nuclear formation of adult T cell leukemia/lymphoma (ATLL).

Adult T cell leukemia/lymphoma (ATLL) has been characterized as one of the most aggressive human neoplasias and its incidence is thought to be caused by both genetic and epigenetic alterations to the host cellular genes of T cells infected with human T cell leukemia virus type I (HTLV-I). A multilobulated nuclear appearance is an important diagnostic marker of ATLL, and we have now identified that the molecular mechanisms underlying these formations occur through microtubule rearrangement via phosphatidylinositol 3-kinase (PI3-kinase) activation by AILIM/ICOS signaling. We also show that PTEN and/or SHIP-1, which are PIP3 inositol phosphatases that inhibit the activation of downstream effectors of the PI3-kinase cascade, are disrupted in both ATLL neoplasias and in multilobulated nuclei-forming Jurkat cells. This down-regulation of PTEN was found to be essential for the formation of ATLL-type nuclear lobules. Furthermore, PI3-kinase and PTEN activities were observed to be closely associated with cellular proliferation. Thus, our results suggest that alteration of PI3-kinase signaling cascades, as a result of the down-regulation of inositol phosphatases, induces ATLL-type multilobulated nuclear formation and is also associated with the cellular proliferation of malignant T cell leukemias/lymphomas.

Inhibition of chronic rejection and development of tolerogenic T cells after ICOS-ICOSL and CD40-CD40L co-stimulation blockade.

BACKGROUND: Blockade of the CD40-CD40L pathway results in long-term allograft survival but does not prevent chronic rejection. ICOS-ICOSL are members of the CD28-B7 family that play an important role in T-cell activation. METHODS: The authors analyzed the effect of single or combined treatment with an anti-ICOS monoclonal antibody and the fusion molecule CD40 immunoglobulin (Ig) on acute and chronic rejection of heart allografts in rats. RESULTS: Treatment with anti-ICOS resulted in a modest but significant prolongation of allograft survival. Treatment with CD40Ig resulted in long-term graft survival but the cardiac grafts developed chronic rejection lesions. Combined CD40Ig+anti-ICOS treatment led to indefinite graft survival in all recipients and a significant decrease of chronic rejection lesions compared with CD40Ig alone. Importantly, four of the seven CD40Ig+anti-ICOS-treated recipients showed a complete absence of chronic rejection lesions, whereas all of the CD40Ig-treated recipients showed chronic rejection. The CD40Ig+anti-ICOS group also showed significant decreased graft infiltration, decreased antidonor cytotoxic T-lymphocyte activity, and decreased alloantibodies compared with the CD40Ig-treated group. Adoptive transfer of splenocytes indefinitely prolonged allograft survival, whereas those depleted of T cells did not, suggesting the development of T-regulatory mechanisms. CONCLUSIONS. These data indicate that the chronic rejection mechanisms that are CD40-CD40L independent are ICOS-ICOSL dependent. These results were obtained with conservation of cognate immune responses and development of tolerogenic T cells.

The role of ICOS in the CXCR5+ follicular B helper T cell maintenance in vivo.

ICOS is a new member of the CD28 family of costimulatory molecules that is expressed on activated T cells. Its ligand B7RP-1 is constitutively expressed on B cells. Although the blockade of ICOS/B7RP-1 interaction inhibits T cell-dependent Ab production and germinal center formation, the mechanism remains unclear. We examined the contribution of ICOS/B7RP-1 to the generation of CXCR5+ follicular B helper T (T(FH)) cells in vivo, which preferentially migrate to the B cell zone where they provide cognate help to B cells. In the spleen, anti-B7RP-1 mAb-treated or ICOS-deficient mice showed substantially impaired development of CXCR5+ T(FH) cells and peanut agglutinin+ germinal center B cells in response to primary or secondary immunization with SRBC. Expression of CXCR5 on CD4+ T cells was associated with ICOS expression. Adoptive transfer experiments showed that the development of CXCR5+ T(FH) cells was enhanced by interaction with B cells, which was abrogated by anti-B7RP-1 mAb treatment. The development of CXCR5+ T(FH) cells in the lymph nodes was also inhibited by the anti-B7RP-1 mAb treatment. These results indicated that the ICOS/B7RP-1 interaction plays an essential role in the development of CXCR5+ T(FH) cells in vivo.

Inhibition of chronic rejection and development of tolerogenic T cells after ICOS-ICOSL and CD40-CD40L co-stimulation blockade.

BACKGROUND: : Blockade of the CD40-CD40L pathway results in long-term allograft survival but does not prevent chronic rejection. ICOS-ICOSL are members of the CD28-B7 family that play an important role in T-cell activation. METHODS: : The authors analyzed the effect of single or combined treatment with an anti-ICOS monoclonal antibody and CD40 immunoglobulin (Ig) on acute and chronic rejection of heart allografts in rats. RESULTS: : Treatment with anti-ICOS resulted in a modest but significant prolongation of allograft survival. Treatment with CD40Ig resulted in long-term graft survival but the cardiac grafts developed chronic rejection lesions. Combined CD40Ig+anti-ICOS treatment led to indefinite graft survival in all recipients and a significant decrease of chronic rejection lesions compared with CD40Ig alone. Importantly, four of the seven CD40Ig+anti-ICOS-treated recipients showed a complete absence of chronic rejection lesions, whereas all of the CD40Ig-treated recipients showed chronic rejection. The CD40Ig+anti-ICOS group also showed significant decreased graft infiltration, decreased antidonor cytotoxic T-lymphocyte activity, and decreased alloantibodies compared with the CD40Ig-treated group. Adoptive transfer of splenocytes indefinitely prolonged allograft survival, whereas those depleted of T cells did not, suggesting the development of T-regulatory mechanisms. CONCLUSIONS: : These data indicate that the chronic rejection mechanisms that are CD40-CD40L independent are ICOS-ICOSL dependent. These results were obtained with conservation of cognate immune responses and development of tolerogenic T cells.

AILIM/ICOS signaling induces T-cell migration/polarization of memory/effector T-cells.

AILIM/ICOS has critical roles in the regulation of T-cell differentiation and effector T-cell function in various immune responses. The counter-ligand for AILIM/ICOS, B7h, is widely expressed in not only lymphoid tissue and antigen-presenting cells, but also in fibroblast and endothelial cells in various organs. Here, we demonstrate that activated human T-cells migrate beneath TNF-alpha-treated HUVEC and display morphological polarization via AILIM/ICOS signaling. AILIM/ICOS stimulation, in the absence of antigen stimulation, also induced T-cell polarization. Importantly, AILIM/ICOS-mediated polarization was evident in CD4+CD45RO+ memory T-cells and generated Th1 cells, but not in CD4+CD45RA+ naive T-cells and generated Th2 cells. Furthermore, AILIM/ICOS signaling is involved in transendothelial migration of Th1 cells, but not Th2 cells. Our data suggest that AILIM/ICOS-B7h interactions play an important role in the endothelium in controlling the entry of memory/effector T-cells into inflamed tissues in the periphery.

Significant enhancement by anti-ICOS antibody of suboptimal tacrolimus immunosuppression in rat liver transplantation.

A member of the costimulatory molecule family, inducible costimulator (ICOS), is expressed on activated T cells and plays a critical role in their primary activation and cytokine production. ICOS is involved in different immune phenomena, such as Th1-mediated autoimmune disease and graft rejection. Although blockade of ICOS costimulation theoretically may protect grafts from rejection, a single dose of anti-ICOS antibody did not result in the prolongation of rat liver allograft survival. However, in this article, we report that anti-rat ICOS antibody markedly enhanced the immunosuppressive activity of a suboptimal dose of tacrolimus (FK506). After fully allogenic DA to LEW liver transplantation, recipients received a single injection of tacrolimus (1 mg/kg, intramuscularly) with or without anti-ICOS antibody (1 mg/kg, intravenously). Recipient survival was significantly prolonged in rats treated with both the antibody and suboptimal tacrolimus (median survival time 44 days vs. 28 days with tacrolimus alone, P <.01). The extent of cell infiltration into the graft was closely associated with prolongation of recipient survival. Our findings thus demonstrate that anti-ICOS antibody immunotherapy combined with suboptimal tacrolimus has a synergistic effect in preventing hepatic allograft rejection and that it may induce long-term graft acceptance intimately associated with a marked reduction of intragraft T lymphocyte infiltration.

AdCTLA-4Ig combined with donor splenocytes, bone marrow cells and anti-ICOS antibody treatment induce tolerance in a rat heart transplantation model.

It is difficult to induce rat cardiac allograft tolerance by co-stimulator blockade of the B7-CD28 pathway with CTLA-4Ig monotherapy alone. However, combined therapies of AdCTLA-4Ig plus donor-specific spleen-cell infusion, bone marrow cell infusion, and anti-ICOS antibody have been demonstrated to effectively induce indefinite acceptance of rat cardiac allografts. In this report, we compared the tolerance of cardiac allograft tolerant recipients induced by the above three strategies. The results show that treating Lewis recipients of a DA cardiac allograft with a combination of AdCTLA4-Ig and anti-ICOS antibody, donor splenocytes or bone marrow cells produced indefinite graft survival. Second transplantation of donor type skin or heart grafts could not affect the survival of primary heart graft in anti-ICOS treated groups, but results in rejection of primary heart grafts in other two groups, and that co-stimulator blockade, CD28 and ICOS simultaneously with CTLA-4Ig and anti-ICOS antibody, facilitates the development of CD25+ CD4+ regulatory T cells and induces stable transplantation tolerance in the rat cardiac allograft model. This also provides an effective therapy in clinical transplantation for promoting permanent graft survival by generating regulatory T cells.