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Macrophage-stimulating protein (MSP) is a plasminogen-related growth factor and ligand for the receptor tyrosine kinase RON. The MSP/RON system promotes wound healing and invasive tumor growth and suppresses proinflammatory immune response. MSP binding to RON requires proteolytic conversion of the inactive single-chain form (pro-MSP) into the disulfide-linked α/β heterodimer. The pro-MSP cleavage sequence (Ser-Lys-Leu-Arg483↓Val484) closely matches the substrate recognition sequences of hepsin, a type II transmembrane serine protease, that is overexpressed in several cancers. Here, we show that recombinant hepsin cleaves pro-MSP at the consensus site Arg483-Val484 with superior efficiency compared with the known activators MT-SP1 and hepatocyte growth factor activator (HGFA). At least 50% of pro-MSP was processed within 1 hour at a hepsin concentration of 2.4 nmol/L and at a molar enzyme to substrate ratio of 1:500. An uncleavable single-chain variant of MSP weakly bound to a RON–Fc fusion protein, whereas hepsin-cleaved MSP bound with a KD of 10.3 nmol/L, suggesting that the high-affinity binding site in MSP β-chain was properly formed. LNCaP prostate cancer cells overexpressing hepsin on the cell surface efficiently activated pro-MSP, which was blocked by a specific anti-hepsin antibody. Incubation of pro-MSP with hepsin led to robust RON-mediated phosphorylation of mitogen-activated protein kinase, ribosomal S6 protein, and Akt in human A2780 ovarian carcinoma cells stably expressing RON protein. In macrophages, pro-MSP with hepsin induced chemotaxis and attenuated lipopolysaccharide-dependent production of nitric oxide. These findings suggest that the MSP/RON signaling pathway may be regulated by hepsin in tissue homeostasis and in disease pathologies, such as in cancer and immune disorders. Mol Cancer Res; 9(9); 1175–86. ©2011 AACR.

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BackgroundNK cells expanded on membrane-bound (mb) IL-15 and 41BBL expressing K562 stimulatory cells (NKSTIM) for clinical use can be genetically modified to express activating chimeric receptors.1 2 3 NK cells activated in the presence of IL-12, IL-15 and IL-18 develop cytokine induced memory-like (CIML) phenotype and function; these cells have shown clinical promise.4 Additionally, HSCT AML transplants using NK KIR Haplotype Group B donors with better and best Group B profiles (≥2 activating genes) show better survival.5 6 Here we investigate whether KIR profiles impact healthy allogeneic donor NK cell function and phenotype when these cells are expanded on NKSTIM in the presence of IL-12 and IL-18 (12–18).MethodsHealthy donor PBMC NK were genotyped for HLA and KIR and expanded on K562-mbIL15-41BBL stimulatory cells with IL-2 alone or with IL-2 plus IL-12 and IL-18 (12–18). Expanded NK were transduced with CAR constructs including CD19, and then evaluated for NK cell expansion, cytokine secretion, RNA profiles, cytotoxicity against tumor lines, and cell surface phenotypes. Expanded CD19 NK donors with varying numbers of activating KIR vs inhibitory KIR were tested for effector function, and these donors were then tested for in vivo efficacy and pharmacokinetics. A KIR ranking score was developed by considering both the number of activating and inhibitory KIR genes expressed by each donor. This score was correlated with functional properties of CAR NK cells.ResultsAddition of 12–18 to the K562-mbIL15-41BBL stimulatory cells improves CD19-CAR NK potency 2-fold relative to the stimulatory cell line alone (P=.02) while NK cell expansion is unchanged. 12–18 also drove an increase in effector cytokine accumulation on exposure of CAR-NK to CD19 tumor. CIML CAR NK cells from donors with higher KIR scoring also had higher cytotoxicity (Pearson’s R=0.74, P=0.006); this correlation was not observed following expansion in the absence of 12–18. 12–18 also drove more potent in vivo activity against tumor with an increased presence of circulating NK cells over 4 weeks in the mice.ConclusionsCIML CAR NK cells derived from donors with favorable KIR scoring have greater cytotoxic activity, effector cytokine production, and in vivo pharmacokinetics and efficacy. These findings may provide an important criterion for donor selection in the development of more robust and potent engineered NK cells for clinical use.ReferencesLapteva N, Durett AG, Sun J, Rollins LA, Huye LL, Fang J, Dandekar V, Mei Z, Jackson K, Vera J, Ando J, Ngo MC, Coustan-Smith E, Campana D, Szmania S, Garg T, Moreno-Bost A, Vanrhee F, Gee AP, Rooney CM. Large-scale ex vivo expansion and characterization of natural killer cells for clinical applications. Cytotherapy 2012;14(9):1131–1143.Chihaya I, Iwamoto S, Campana D. Genetic modification of primary natural killer cells overcomes inhibitory signals and induces specific killing of leukemic cells. Blood 2005;106:376–383.Yang Y, Connolly J, Shimasaki N, Mimura K, Kono K, Campana D. A Chimeric Receptor with NKG2D Specificity Enhances Natural Killer Cell Activation and Killing of Tumor Cells. Cancer Res 2013;73(6):1777–1786.Romee R, Rosario M, Berrien-Elliott MM, Wagner JA, Jewell BA, Schappe T, Leong JW, Abdel-Latif S, Schneider SE, Willey S, Neal CC, Yu L, Oh ST, Lee YS, Mulder A, Claas F, Cooper MA, Fehniger TA. Cytokine-induced memory-like natural killer cells exhibit enhanced responses against myeloid leukemia. Sci Trans Med 2016;8(357): 357ra123.Cooley S, Weisdorf DJ, Guethlein LA, Klein JP, Wang T, Le CT, Marsh SGE, Geraghty D, Spellman S, Haagenson MD, Ladner M, Trachtenberg E, Parham P, and Miller JS. Donor selection for natural killer cell receptor genes leads to superior survival after unrelated transplantation for acute myelogenous leukemia. Blood 2010;116(14):2414–2419.Cooley S, Weisdorf DJ, Guethlein LA, Klein JP, Wang T, Marsh SGE, Spellman S, Haagenson MD, Saeturn K, Ladner M, Trachtenberg E, Parham P, and Miller JS. Donor Killer Cell Ig-like Receptor B Haplotypes, Recipient HLA-C1, and HLA-C Mismatch Enhance the Clinical Benefit of Unrelated Transplantation for Acute Myelogenous Leukemia. JI, 2014;192(10):4592–600.Ethics ApprovalAnimal studies were conducted with IACUC approval.

BackgroundPeripheral blood natural killer (NK) cells are mature cytotoxic innate lymphocytes possessing an inherent capacity for tumor cell killing, thus making them attractive candidates for adoptive cell therapy. These NK cells are also amenable to CRISPR and chimeric antigen receptor (CAR) genomic engineering for enhanced functions. Moreover, NK cells possess an inherent capacity for off-the-shelf therapy since they are not known to cause graft-versus-host disease, unlike T cells. Presently, approved CAR cell therapy is custom-made from each patient‘s own T cells, a process that can limit patient pool, narrow therapeutic window, and contribute to product variability. In this study, we investigate whether peripheral blood NK cells from a selected donor can be edited, engineered, and expanded sufficiently for off-the-shelf use in a wide patient population.MethodsUsing the CRISPR/Cas9 system, we knocked out CISH expression in isolated peripheral blood NK cells from 3 healthy donors. Subsequently, we expanded edited NK cells by using IL-2 and sequential stimulations using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added twice during expansion to drive memory-like NK cell differentiation. We transduced the expanded NK cells to express engineered CD19-targeted CAR and mbIL-15 during an interval between the first and second NKSTIM pulses. We assessed NK cell cytotoxicity against Nalm6 target cells by IncuCyte.ResultsIsolated peripheral blood NK cells from 3 healthy donors were successfully edited using CRISPR/Cas9, engineered to express high levels of CAR, extensively expanded using a series of NKSTIM pulses in the presence of IL-2, and differentiated into memory-like NK cells using IL-12 and IL-18. Interestingly, NK cells from the 3 donors exhibited distinct outcomes. NK cells from one donor reached a peak expansion limit of approximately 7-million-fold before undergoing contraction whereas NK cells from two donors continued to expand over the length of the study surpassing 100-million-fold expansion, without appearing to have reached a terminal expansion limit. At the end of the study, NK cells from one donor exceeded 1-billion-fold expansion and maintained 88% cytolytic activity compared to Nkarta’s standard process control in a 72-hour IncuCyte assay.ConclusionsIn this study, we demonstrate that healthy donor-derived peripheral blood NK cells are capable of expanding over billion-fold while maintaining potency. These results provide a rationale for the development of off-the-shelf CAR NK cell therapies using NK cells from donors selected to provide optimal product characteristics.Ethics ApprovalHuman samples were collected with written informed consent by an approved vendor.

BackgroundPeripheral blood natural killer (NK) cells are attractive candidates for adoptive cell therapy. NK cells possess innate ability for tumor cell killing and are also amenable to genomic engineering for enhanced functions. Moreover, NK cells possess an inherent capacity for allogeneic, off-the-shelf therapy since, unlike T cells, they are neither HLA-restricted nor known to cause graft-versus-host disease. Cytokine inducible SH2-containing protein (CISH) is a negative regulator of interleukin 15 (IL-15) signaling in natural killer (NK) cells. Here we show the potential application of CISH gene-knockout CAR NK cells targeting CD70 and expressing a membrane-bound form of IL-15. CD70 is an antigen that is aberrantly expressed in a variety of malignant settings, including renal cell carcinoma (RCC), while its expression in normal tissues is restricted to a subset of lymphoid cell types.MethodsTo target CD70 on RCC cells, we generated CD70-CAR NK cells with CISH deletion. Using the CRISPR/Cas9 system, we knocked out CISH expression in isolated peripheral blood NK cells from healthy donors. Since CD70 expression is present on activated NK cells, we also targeted CD70 for CRISPR knockout to avoid fratricide. We then expanded these edited NK cells by using IL-2 and stimulation using NKSTIM, a modified K562 stimulatory cell line expressing membrane-bound form of IL-15 (mbIL-15) and 4-1BBL. IL-12 and IL-18 were added during expansion to drive memory-like NK cell differentiation. We transduced the expanded NK cells to express engineered CD70-targeted CAR and mbIL-15. We assessed CAR expression, NK cell persistence, and NK cell activity against RCC target cells using end-point cytotoxicity assays and IncuCyte.ResultsCISH gene-knockout CD70-CAR NK cells could be produced efficiently and exhibited extended persistence in culture. After engineering and expansion, CD70-CAR transduction efficiency was 60–80%. CD70-CAR NK cells displayed potent cytotoxicity against CD70-expressing renal cancer derived cell lines. Interestingly, cytotoxicity assays demonstrated that CISH gene-knockout CD70-CAR NK cells were partially resistant to TGFß and adenosine inhibition of cytotoxicity. Furthermore, CISH gene-knockout CD70-CAR NK cells maintained their activity during prolonged culture.ConclusionsIn summary, we show CISH gene-knockout CD70-CAR NK cells demonstrate potent anti-tumor activity against relevant solid tumor cell lines and partially provide resistance to tumor microenvironment inhibition. These data support the further exploration of CISH gene-knockout CD70 CAR NK cells for clinical application.

Introduction: Chimeric antigen receptors (CARs) have been used successfully to retarget T cells in patients with hematologic malignancies. Natural killer (NK) cells offer an alternative to T cells for cellular immunotherapy and are suitable for allogeneic use as they are not HLA-restricted and patients who receive NK cell treatment do not develop graft-versus-host disease (GVHD). Therefore, NK cells can provide an attractive alternative for “off-the-shelf” cellular therapy. Here, we investigated multiple approaches to modify and enhance CD19 CAR expression on NK cells. It has been previously reported that costimulatory domains play an important role in proliferation, efficacy, and persistence of CAR-T cells both in vitro and in vivo. To understand how CAR structure effects the functional behavior of NK cells, we assessed the capability of various costimulatory signaling domains, including CD28, OX40, CD28-41BB, etc., all coupled to CD3ζ, to enhance CD19 CAR signaling and cytotoxicity in NK cells. We demonstrated that NK activity and persistence can be elevated by simultaneous expression of chimeric constructs directing the expression of a CD19 CAR and a membrane-bound form of IL-15 (mbIL-15). Methods: NK cells were generated by coculture of peripheral blood mononuclear cells (PBMC) with genetically modified irradiated K562 feeder cells. NK cells were transduced at a MOI of 1-2 with a gamma-retrovirus encoding a CD19 CAR and mbIL-15. NK expansion and CAR expression were evaluated by flow cytometry. In vitro cytotoxicity of transduced NK cells was measured using both flow cytometry and the IncuCyte S3 live cell analysis system. The in vivo activity of engineered NK cells was further assessed in a xenograft tumor model in NSG mice, using a Nalm6 leukemia cell line. Results: CD19 CAR constructs containing various costimulatory domains were all expressed in expanded and transduced NK cells. CAR expression in multiple donor NK cells was typically between 60-90%. In cytotoxicity assays, CD19 CAR constructs containing the costimulatory domains of OX40, CD28, or CD27 generally exhibited the greatest cytotoxic activity against Nalm6 and Raji tumor cell lines in vitro. High expression of these three CD19 CAR constructs was also maintained over the course of 4 weeks in addition to sustained NK proliferation and cell numbers, indicating an increase in NK cell persistence. In comparing these constructs in an in vivo Nalm6 tumor model, all three constructs demonstrated greater antitumor efficacy relative to unmodified NK cells. The CD19 CAR construct containing the OX40 costimulatory domain showed moderately enhanced efficacy than the CD28 or CD27 variants in vivo. Conclusion: Transduction of NK cells with CD19-OX40-CD3ζ CAR and mbIL15 increases their cytotoxic activity and persistence. Based on these data, further development of NK CD19 CAR for clinical use will be pursued. Citation Format: Luxuan G. Buren, Chao Guo, Yanying Fan, Alexander Aronov, Xiumin Wu, Daofeng Liu, Ming-Hong Xie, Sasha Lazetic, Ivan H. Chan, James B. Trager. Coexpression of a CD19-OX40-CD3ζ CAR with membrane-bound IL-15 enhances natural killer cell function [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B64.

In order to achieve three-dimensional automatic splicing from the multi-angle, the paper presents a new splicing method. The method utilizes the invariance of the spatial characteristics of the characteristic landmarks and then marks the characteristic landmarks by introducing the angle collection between the characteristic landmarks, and match in space for the characteristic landmarks within two perspective regional based on this characteristic collection. This method eliminates the tedious encoding process of the landmarks, so improves the spatial matching rate and ensures the matching accuracy. In addition, in the process of striking the three-dimensional coordinates of the landmarks, this paper strikes image coordinate of the characteristic signs circle center by introducing the regional connectivity feature flag. This method eliminates interference from unwanted areas, and distinguish between the various signs round independence and improves the strike rate of the circle center. At the end rotation matrix R and translation vector T of the coordinate system normalization are solved by the quadruple method. Examples show that the algorithm has strong robustness, can realize quickly, automatic stitching for the visual data, and has the very good practical value.

Macrophage-stimulating protein (MSP) is a plasminogen-related growth factor and ligand for the receptor tyrosine kinase RON. The MSP/RON system promotes wound healing and invasive tumor growth and suppresses proinflammatory immune response. MSP binding to RON requires proteolytic conversion of the inactive single-chain form (pro-MSP) into the disulfide-linked α/β heterodimer. The pro-MSP cleavage sequence (Ser-Lys-Leu-Arg483↓Val484) closely matches the substrate recognition sequences of hepsin, a type II transmembrane serine protease, that is overexpressed in several cancers. Here, we show that recombinant hepsin cleaves pro-MSP at the consensus site Arg483-Val484 with superior efficiency compared with the known activators MT-SP1 and hepatocyte growth factor activator (HGFA). At least 50% of pro-MSP was processed within 1 hour at a hepsin concentration of 2.4 nmol/L and at a molar enzyme to substrate ratio of 1:500. An uncleavable single-chain variant of MSP weakly bound to a RON–Fc fusion protein, whereas hepsin-cleaved MSP bound with a KD of 10.3 nmol/L, suggesting that the high-affinity binding site in MSP β-chain was properly formed. LNCaP prostate cancer cells overexpressing hepsin on the cell surface efficiently activated pro-MSP, which was blocked by a specific anti-hepsin antibody. Incubation of pro-MSP with hepsin led to robust RON-mediated phosphorylation of mitogen-activated protein kinase, ribosomal S6 protein, and Akt in human A2780 ovarian carcinoma cells stably expressing RON protein. In macrophages, pro-MSP with hepsin induced chemotaxis and attenuated lipopolysaccharide-dependent production of nitric oxide. These findings suggest that the MSP/RON signaling pathway may be regulated by hepsin in tissue homeostasis and in disease pathologies, such as in cancer and immune disorders. Mol Cancer Res; 9(9); 1175–86. ©2011 AACR.

In order to get the exact water cutting of the complexity shape, the 6-DOF robot kinematics model is put forward. The gesture of every point can be calculated in real-time through NC interpolation algorithm of the expected discrete points on the track. On the basis of the matrix of position and gesture, a precise control method of 6-DOF water cutting robot trajectory and attitude of the end is proposed which comes from the improved NC-based code and could get any angle cutting surface, which is easy to program off-line. Finally, the simulation verity of NC interpolation control is made by VC and MATLAB tools.

Activation of the co-stimulatory receptor GITR (Glucocorticoid-Induced Tumor Necrosis Factor Receptor) by GITR-Ligand (GITRL) promotes proliferation and activation of effector T cells (T eff) and inhibits suppressive activity of regulatory T cells (Treg). Here, we have further characterized the mechanism of action of a single-gene GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc, 336B3) by examining pharmacodynamic (PD) biomarkers in time course studies. Mice bearing CT26.WT colon tumors were treated with weekly GITRL-Fc and sacrificed 24 hours, 7 and 14 days after the first dose. Immuno-phenotyping of tumor-associated immune cells revealed a reduction in Treg frequency in tumor by 24 hours post-dose that was maintained at 7 and 14 days. Furthermore, GITRL-Fc treatment increased activation markers on tumor-associated CD4+ and CD8+ T cells, suggesting an increased cytotoxic environment within the tumor. This was supported by significant and sustained increase in CD8+ T cell:Treg ratio in the tumor after GITRL-Fc treatment. To determine whether intratumoral (IT) injection of GITRL-Fc is an effective route of administration, we compared efficacy, pharmacodynamic (PD) markers and pharmacokinetics in IT- and intraperitoneal (IP)-injected mice bearing bilateral CT26.WT tumors. Both routes of administration showed similar tumor growth inhibition (TGI) and PD markers in both the treated and the abscopal tumors, but IT injection resulted in a significantly lower serum GITRL-Fc concentration, suggesting that IT administration may be an alternative route of administration to IP with similar efficacy. The GITRL-Fc molecule 336B3 is effector function competent and able to induce cell-mediated cytotoxicity upon binding. To determine whether this effector function is required for GITRL-Fc-induced TGI, we treated CT26.WT tumor-bearing mice with 336B3 and 336B22, a GITRL-Fc molecule deficient in effector function. The effector function-competent 336B3 induced significant TGI and a more robust activation of Teff cells and reduction in Treg frequency, when compared to 336B22, suggesting that effector function is important for efficacy. To identify biomarkers for GITRL-Fc, we performed microarray analyses on multiple syngeneic mouse models treated with GITRL-Fc and developed GITRL gene signatures from blood and from tumors. We also developed multiplexed immunohistochemistry panels designed to quantify frequency of GITR and GITRL expression (GITR+CD8, GITR+FOXP3) in tumors. In conclusion, we have examined the effects of GITRL-Fc on preclinical mouse models. Biomarker analysis showed that loss of Tregs, activation of T cells and Fc-mediated effector function are key elements in the mechanism of action of the molecule. We have identified potential biomarkers to be used for PD and potential predictive analysis in clinical trial patient samples. Citation Format: Gretchen M. Argast, Belinda Cancilla, Fiore Cattaruzza, Pete Yeung, Reyhaneh Lahmy, Erwan Le Scolan, Rose Harris, Alayne Brunner, Min Wang, Fumiko Axelrod, Jorge Monteon, Jennifer Elechko, Andrew Lam, MingHong Xie, Earth Light Lowe, Gilbert O'Young, Austin Gurney, Ann M. Kapoun. GITRL-Fc biomarker and mechanism study: GITRL-Fc reduces Treg frequency in tumors and requires effector function for inhibition of tumor growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3826.

The immune checkpoint co-inhibitory receptor TIGIT (T cell immunoreceptor with Ig ITIM domain) is expressed on regulatory T cells (Tregs) and on activated CD4+ T, CD8+ T, and NK cells. We have reported that by blocking TIGIT activity with an IgG2a anti-TIGIT antibody (313R12), CD8+ and CD4+ Tcells and NK cells were activated, resulting in dose-dependent tumor growth inhibition (TGI) in multiple syngeneic mouse models. To explore the pharmacodynamics (PD) and mechanism of action of tumor growth inhibition (TGI) by anti-TIGIT antibodies, we examined the kinetics of immune cell frequency and activation in tumor by flow cytometry, qPCR and immunohistochemistry (IHC). We performed in vivo time course studies in the CT26.WT colon carcinoma model using weekly dosing at 0.1, 0.5 and 12.5 mg/kg anti-TGIT. Mice were sacrificed at 24 hours, 7 days and 14 days after the first dose for biomarker analysis. After 24 hours of treatment, Tregs in the tumor decreased and this reduction of Tregs was sustained at 7 and 14 days. Markers of immune cell activation and exhaustion such as CD69, PD1 and intracellular cytokines were modulated during the course of the study, suggesting a more cytotoxic intratumoral environment after 313R12 treatment. In addition, CD226, a binding partner of TIGIT, was significantly upregulated in T cells, Tregs and NK cells throughout the study, reflecting a feedback loop activated by inhibiting TIGIT activity. The anti-TIGIT antibody used in these studies, 313R12, is effector function competent and is able to induce cell-mediated cytotoxic effector functions upon binding. In order to determine whether effector function is necessary for anti-TIGIT antibody activity, we compared 313R12 with an effector function-deficient molecule, 313R13, in CT26.WT tumors. After 7 days, only 313R12 showed significant TGI compared to control-treated animals, suggesting that effector function is required for efficacy. While the effector function-deficient molecule 313R13 was able to similarly induce some changes in PD biomarkers, including immune cell activation, it required a higher dose than 313R12 to do so. To develop biomarkers for anti-TIGIT, we used microarray analyses to identify anti-TIGIT gene signatures in tumors and blood from multiple syngeneic models. In addition, we developed multiplexed IHC panels (e.g., TIGIT+CD8, TIGIT+FOXP3) to quantify expression of TIGIT and TIGIT ligand-positive immune cells in the tumor and surrounding stroma, and we profiled a panel of 80 human tumors with these panels. In summary, we examined the effects of anti-TIGIT antibodies on preclinical mouse models. Biomarker analysis demonstrated loss of Tregs and activation of T cells and NK cells, as well as effector function, as part of the mechanism of action of the molecule. We have also identified biomarkers that can be used for PD and potential predictive analysis in clinical trial samples. Citation Format: Gretchen M. Argast, Belinda Cancilla, Fiore Cattaruzza, Pete Yeung, Erwan le Scolan, Rose Harris, Reyhaneh Lahmy, Alayne Brunner, Min Wang, Gilbert O'Young, Earth Light Lowe, Fumiko Axelrod, Jorge Monteon, Jennifer Elechko, Andrew Lam, MingHong Xie, Austin Gurney, John Lewicki, Ann Kapoun. Anti-TIGIT biomarker study: Inhibition of TIGIT induces loss of Tregs from tumors and requires effector function for tumor growth inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5627.

Glucocorticoid-induced TNFR-related protein (GITR) is a member of the TNF receptor superfamily. GITR, a co-stimulatory surface receptor, is activated upon binding to GITR Ligand (GITRL) and mediates co-stimulation of T-cell and NK responses and inhibits the suppressive activity of regulatory T-cells (Tregs). As such, GITR is an attractive immuno-oncology target for activation via GITR agonists. OMP-336B11 is a single-gene recombinant fusion protein consisting of two trimeric human GITRLs and a human immunoglobulin (IgG1) Fc domain. Murine preclinical studies using a surrogate GITRL-Fc fusion protein demonstrated robust antitumor efficacy. Functional characterization of OMP-336B11 in various human immune cell assays is presented here. In human peripheral blood mononuclear cells (PBMC), OMP-336B11 stimulated IL-2 cytokine release in a dose-dependent manner. In activated human T-cells, OMP-336B11 enhanced cell proliferation in a dose-responsive fashion. OMP-336B11 also augmented IL-2 induced IFNγ from human NK cells. To elicit NK mediated cytotoxicity of high GITR expressing cells (i.e., Tregs), OMP-336B11 is designed with an IgG1 Fc domain. Co-incubation of primary human NK cells (effector) and GITR expressing cells (target) resulted in an OMP-336B11 dependent dose-titratable increase in target cytotoxicity. Furthermore, OMP-336B11 agonistic activity was compared to anti-GITR agonist antibodies. By measuring cell proliferation and IFNγ from activated T-cells, OMP-336B11 demonstrated superior activity compared to the other agonist anti-GITR antibodies. In conclusion, OMP-336B11 is designed to induce effective GITR activation and also to mediate depletion of GITR-high cells. OMP-336B11 is currently undergoing phase I clinical study. Citation Format: Ivan H. Chan, Ming-Hong Xie, Andrew Lam, Fumiko T. Axelrod, Jennifer Elechko, Angie I. Park, Austin Gurney. In vitro functional activity of OMP-336B11, a GITRL-Fc fusion protein, on primary human immune cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2726.

Meeting abstracts Blocking DLL4, a Notch ligand, effectively inhibits tumor growth by increasing non-functional angiogenesis and decreasing the cancer stem cell (CSC) population. Preclinical studies have demonstrated inhibition of tumor growth by anti-DLL4 treatment and have led us to enter

The angiopoietin family of secreted factors is functionally defined by the C-terminal fibrinogen (FBN)-like domain, which mediates binding to the Tie2 receptor and thereby facilitates a cascade of events ultimately regulating blood vessel formation. By screening expressed sequence tag data bases for homologies to a consensus FBN-like motive, we have identified ANGPTL3, a liver-specific, secreted factor consisting of an N-terminal coiled-coil domain and the C-terminal FBN-like domain. Co-immunoprecipitation experiments, however, failed to detect binding of ANGPTL3 to the Tie2 receptor. A molecular model of the FBN-like domain of ANGPTL3 was generated and predicted potential binding to integrins. This hypothesis was experimentally confirmed by the finding that recombinant ANGPTL3 bound to alpha(v)beta(3) and induced integrin alpha(v)beta(3)-dependent haptotactic endothelial cell adhesion and migration and stimulated signal transduction pathways characteristic for integrin activation, including phosphorylation of Akt, mitogen-activated protein kinase, and focal adhesion kinase. When tested in the rat corneal assay, ANGPTL3 strongly induced angiogenesis with comparable magnitude as observed for vascular endothelial growth factor-A. Moreover, the C-terminal FBN-like domain alone was sufficient to induce endothelial cell adhesion and in vivo angiogenesis. Taken together, our data demonstrate that ANGPTL3 is the first member of the angiopoietin-like family of secreted factors binding to integrin alpha(v)beta(3) and suggest a possible role in the regulation of angiogenesis.

T cell immunoreceptor with Ig and ITIM domains (TIGIT) is a co-inhibitory molecule containing an immunoreceptor tyrosine-based inhibition motif (ITIM) within its cytoplasmic tail, and is highly expressed on regulatory T cells and activated CD4+ T, CD8+ T, and NK cells. TIGIT competes with CD226, which contains an immunoreceptor tyrosine-based activation motif (ITAM) within its cytoplasmic tail for ligands poliovirus receptor (PVR) and poliovirus receptor-related 2 (PVRL2), with higher affinity to PVR. The ligands are expressed on the surface of antigen presenting cells and at high levels on most tumors. Therefore, when TIGIT is present, the ligands preferentially engage TIGIT rather than CD226, leading to cell suppression. We have generated antibodies against TIGIT that blocks ligand binding and inhibits TIGIT signaling. The clinical candidate, OMP-313M32 binds human TIGIT but not rodent and non-human primate TIGIT. Therefore, a surrogate antibody was generated for pre-clinical assessments in mice. Antibody 313R12 is an anti-mouse TIGIT antibody that can block mouse PVR ligand binding and inhibit TIGIT signaling in a manner similar to the clinical candidate OMP-313M32. 313R12 inhibited the growth of syngeneic colon and kidney tumors in immune competent mice. In some cases, anti-TIGIT antibody 313R12 caused complete tumor regression and a potent anti-tumor immune memory response as demonstrated by the lack of tumor growth upon re-challenge of mice that remained tumor-free after prior anti-TIGIT treatment. Mechanistically, anti-TIGIT antibody 313R12 was shown to induce a Th1 response and increase cytotoxic T lymphocyte (CTL) activity. By in vivo depletion of T cell populations, we have shown that CD8 T cell depletion completely abrogated the anti-TIGIT therapeutic effect, whereas CD4 T cell depletion led to partial reversal of efficacy of anti-TIGIT. Therefore, both CD4+ and CD8+ T cells are critical for anti-TIGIT-mediated immune responses. Using mice reconstituted with human hematopoietic stem cells, we also demonstrated that the clinical candidate OMP-313M32 inhibits patient-derived melanoma tumor growth. Taken together, these data demonstrate that anti-TIGIT therapy suppresses tumor growth and generates long-term immunological memory against multiple tumors. Citation Format: Angie Inkyung Park, Minu Srivastava, Erin Mayes, Hyun-Bae Jie, Rui Yun, Christopher Murriel, Ming-hong Xie, Andrew Lam, May Ji, Fumiko Axelrod, Jorge Monteon, John Lewicki, Tim Hoey, Austin Gurney. Antibody against TIGIT (T cell immunoreceptor with Ig and ITIM domains) induces anti-tumor immune response and generates long-term immune memory [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2003. doi:10.1158/1538-7445.AM2017-2003

TIGIT (T cell immunoreceptor with Ig and ITIM domains) has been recently described as an inhibitory receptor which blocks CD8 T cell-mediated anti-tumor immune responses. We have generated an anti-mouse TIGIT antibody (313R12) to evaluate drug efficacy and mechanism of action in pre-clinical tumor models. Anti-TIGIT as a single agent promoted an anti-tumor immune response in multiple syngeneic mouse tumor models. Anti-TIGIT enhanced tumor specific T cell responses, particularly of the Th1 type and reduced Th2 type responses and also increased the function of cytotoxic T cells. Furthermore, anti-TIGIT displayed combination activity with immune checkpoint inhibitors anti-PD1 and anti-PDL1 in inhibiting tumor growth, promoting complete tumor rejection and significantly increasing mouse survival in the murine CT26 colon carcinoma model as compared to controls and single agents alone. Mice “cured” with anti-TIGIT/anti-PDL1 or anti-TIGIT/anti-PD1 combination treatments did not form tumors upon subsequent re-challenges with increasing number of CT26 tumor cells, suggesting the existence of immunologic memory. IL2 and tumor-specific IFN-γ production by splenic T cells were increased in mice who responded to combination treatment compared to controls. Additionally, both effector and memory CD8+ T cell frequencies were increased within the total CD8+ T cell population in responding mice. We also demonstrated a systemic increase in tumor-specific CD8 T cells after anti-TIGIT/anti-PDL1 combination treatment compared to controls. Therefore, these results suggest that co-targeting of TIGIT and PD1 or PDL1 may be an effective and durable cancer therapy by increasing T cell-mediated anti-tumor immune responses and promoting long-term immunological memory. Citation Format: Minu K. Srivastava, Rui Yun, Erin Mayes, Janice Yu, Hyun-Bae Jie, Fumiko Axelrod, Ming-Hong Xie, Jorge Monteon, Andrew Lam, May Ji, Yuwang Liu, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. Anti-Tigit induces T cell mediated anti-tumor immune response and combines with immune checkpoint inhibitors to enhance strong and long term anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2612. doi:10.1158/1538-7445.AM2017-2612

We report the identification of a novel human cytokine, distantly related to interleukin (IL)-10, which we term IL-22. IL-22 is produced by activated T cells. IL-22 is a ligand for CRF2-4, a member of the class II cytokine receptor family. No high affinity ligand has yet been reported for this receptor, although it has been reported to serve as a second component in IL-10 signaling. A new member of the interferon receptor family, which we term IL-22R, functions as a second component together with CRF2-4 to enable IL-22 signaling. IL-22 does not bind the IL-10R. Cell lines were identified that respond to IL-22 by activation of STATs 1, 3, and 5, but were unresponsive to IL-10. In contrast to IL-10, IL-22 does not inhibit the production of proinflammatory cytokines by monocytes in response to LPS nor does it impact IL-10 function on monocytes, but it has modest inhibitory effects on IL-4 production from Th2 T cells.

MuSK is a tyrosine kinase localized to the postsynaptic surface of the neuromuscular junction. We have searched for modulators of MuSK function using a library of human single chain variable region antibodies (scFv) that can be displayed on M13 phage or expressed as soluble protein. A panel of 21 independent MuSK-specific scFv, identified in a screen for binding to MuSK-Fc immunoadhesin, were examined for ability to induce proliferation in a factor dependent cell line (Ba/F3) through a chimeric receptor, MuSK-Mpl. Four of the scFv induced a proliferative response, suggesting an ability to induce dimerization of MuSK. These scFv were also able to induce tyrosine phosphorylation of full-length MuSK and retained this ability when re-engineered to be expressed as authentic (and dimeric) human IgG molecules. Addition of agonist scFv to a cultured myotube cell line induced AChR clustering and tyrosine phosphorylation. These results provide direct evidence that MuSK activation is capable of triggering a key event in neuromuscular junction formation and further demonstrate that large libraries of phage-displayed scFv provide a robust method for generating highly specific agonist agents.

Secretion of anionic endo- and xenobiotics is essential for the survival of animal and plant cells; however, the underlying molecular mechanisms remain uncertain. To better understand one such model system--i.e., secretion of bile acids by the liver--we utilized a strategy analogous to that employed to identify the multidrug resistance (mdr) genes. We synthesized the methyl ester of glycocholic acid (GCE), which readily enters cells, where it is hydrolyzed to yield glycocholic acid, a naturally occurring bile acid. The rat hepatoma-derived HTC cell line gradually acquired resistance to GCE concentrations 20-fold higher than those which inhibited growth of naive cells, yet intracellular accumulation of radiolabel in resistant cells exposed to [14C]GCE averaged approximately 25% of that in nonresistant cells. As compared with nonresistant cells, resistant cells also exhibited (i) cross-resistance to colchicine, a known mdr substrate, but not to other noxious substances transported by hepatocytes; (ii) increased abundance on Northern blot of mRNA species up to 7-10 kb recognized by a probe for highly conserved nucleotide-binding domain (NBD) sequences of ATP-binding cassette (ABC) proteins; (iii) increased abundance, as measured by RNase protection assay, of mRNA fragments homologous to a NBD cRNA probe; and (iv) dramatic overexpression, as measured by Western blotting and immunofluorescence, of a group of 150- to 200-kDa plasma membrane proteins recognized by a monoclonal antibody against a region flanking the highly conserved NBD of mdr/P-glycoproteins. Finally, Xenopus laevis oocytes injected with mRNA from resistant cells and incubated with [14C]GCE secreted radiolabel more rapidly than did control oocytes. Enhanced secretion of glycocholic acid in this cell line is associated with overexpression of ABC/mdr-related proteins, some of which are apparently novel and are likely to include a bile acid transport protein.

GITRL (Glucocorticoid-Induced Tumor Necrosis Factor Receptor Ligand, TNFSF18) is a member of the TNF superfamily and naturally exists as a membrane-anchored type II protein that self assembles as a trimer. GITRL activates the co-stimulatory receptor GITR. GITR is found primarily on activated T effector (Teff) cells and regulatory T (Treg) cells. Co-stimulation of GITR by agonist agents is hypothesized to promote anti-tumor immunity by enhancing Teff cell activity and inhibiting Treg suppression. We generated a novel single-gene GITRL trimer fused to an immunoglobulin Fc domain (GITRL-Fc). GITRL-Fc activated GITR signaling more effectively than prototype GITR agonist antibody DTA-1. GITRL-Fc promoted a robust anti-tumor immune response in multiple syngeneic mouse tumor models. GITRL-Fc enhanced tumor specific T-cell responses, particularly of the Th1 type, and also led to reduction in Treg-mediated immunesuppressive activity. GITRL-Fc displayed single agent activity in inhibiting tumor growth and promoting complete tumor rejection in the murine CT26 colon carcinoma model and combination activity with anti-PDL1 as compared to anti-PDL1 and control IgG2a alone. Mice “cured” with GITRL or GITRL/anti-PDL1 combination treatments were protected from re-challenge with tumor cells, suggesting the existence of immunologic memory. More mice were protected from tumor re-challenge with the combination of GITRL-Fc and anti-PDL1, as compared to GITRL-Fc alone. Our results demonstrate that agonist GITRL-Fc induces potent T cell responses, overcomes Treg inhibition, and promotes anti-tumor activity in preclinical models as a single agent or in combination with anti PDL1. The mechanism of tumor eradication and induction of long-term immune memory response by the combination is under investigation and will be discussed at the presentation. Citation Format: Minu K. Srivastava, Rui Yun, Erin Mayes, Hyun_Bae Jie, Fumiko Axelrod, Jorge Monteon, Ming-Hong Xie, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. GITR ligand fusion protein (GITRL-Fc) induces T cell mediated anti-tumor immune response and can combine with anti-PDL1 to enhance anti-tumor immunity and long-term immune memory. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2214.

Thrombopoietin (TPO), the ligand for c-mpl, is a novel cytokine comprising an amino terminal domain with homology to erythropoietin and a glycosylated carboxyl terminal domain that does not bear overall homology to other known proteins. We report the cloning of cDNAs encoding the porcine and murine TPO and the characterization of the human TPO gene. The cDNA for an additional splice form (TPO-2) with a four-amino-acid deletion within the erythropoietin-like domain has been isolated and is conserved between humans, pigs, and mice. Species comparison of TPO shows that the amino terminal erythropoietin-like domain is highly conserved, while the carboxyl terminal domain is less conserved. Recombinant murine TPO and human TPO are each able to activate both the murine and human c-mpl receptors, indicating an absence of strict species specificity. Human TPO is encoded by a single gene consisting of six exons and located on chromosome 3q271–28.

A variety of hormonal agonists activate transmembrane Na+ and Ca2+ flux in hepatocytes, but the responsible mechanisms are poorly understood. We employed microfluorimetric and patch clamp recording techniques in hepatocytes to determine the effect of the hormone vasopressin on cytosolic Na+ concentration ([Na+]i) and to identify the transmembrane Na+ transport pathways activated by this agonist. Under basal conditions, [Na+]i, measured using the Na(+)-sensitive fluorophore sodium-binding benzofuran isophthalate, averaged 12.1 +/- 1.6 mM. Exposure to vasopressin rapidly increased [Na+]i by 8.3 +/- 0.9 mM. This increase was attributable to activation of Na+ influx. It occurred in the absence of solutes co-transported with Na+ and was not associated with activation of Na+/H+ antiport. In cell-attached membrane patches, vasopressin activated ion channels that carried inward positive current at the resting membrane potential. Further characterization in excised membrane patches revealed two classes of ion channels, with conductances of 16.0 +/- 2.8 and 30.9 +/- 3.1 picosiemens, respectively. Single channel currents reversed near 0 mV, and ion substitution studies demonstrated that each channel type was permeable to Na+, Ca2+, and K+ but not Cl-. These observations in hepatocytes indicate that vasopressin increases [Na+]i and activates cation-selective channels, which likely accounts for vasopressin-activated Na+ and Ca2+ influx.

We have examined the hypothesis that a regulatory interplay between pH-regulated plasma membrane K+ conductance (gK+) and electrogenic Na+/HCO3- cotransport contributes importantly to regulation of intracellular pH (pHi) in hepatocytes. In individual cells, membrane depolarization produced by transient exposure to 50 mM K+ caused a reversible increase in pHi in the presence, but not absence, of HCO3-, consistent with voltage-dependent HCO3- influx. In the absence of HCO3-, intracellular alkalinization and acidification produced by NH4Cl exposure and withdrawal produced membrane hyperpolarization and depolarization, respectively, as expected for pHi-induced changes in gK+. By contrast, in the presence of HCO3-, NH4Cl exposure and withdrawal produced a decrease in apparent buffering capacity and changes in membrane potential difference consistent with compensatory regulation of electrogenic Na+/HCO3- cotransport. Moreover, the rate of pHi and potential difference recovery was several-fold greater in the presence as compared with the absence of HCO3-. Finally, continuous exposure to 10% CO2 in the presence of HCO3- produced intracellular acidification, and the rate of pHi recovery from intracellular acidosis was inhibited by Ba2+, which blocks pHi-induced changes in gK+, and by 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid, which inhibits Na+/HCO3- cotransport. These findings suggest that in hepatocytes, changes in transmembrane electrical potential difference, mediated by pH-sensitive gK+, play a central role in regulation of pHi through effects on electrogenic Na+/HCO3- cotransport.

Recent studies in hepatocytes indicate that Na(+)-coupled HCO3- transport contributes importantly to regulation of intracellular pH and membrane HCO3- transport. However, the direction of net coupled Na+ and HCO3- movement and the effect of HCO3- on Na+ turnover and Na+/K+ pump activity are not known. In these studies, the effect of HCO3- on Na+ influx and turnover were measured in primary rat hepatocyte cultures with 22Na+, and [Na+]i was measured in single hepatocytes using the Na(+)-sensitive fluorochrome SBFI. Na+/K+ pump activity was measured in intact perfused rat liver and hepatocyte monolayers as Na(+)-dependent or ouabain-suppressible 86Rb uptake, and was measured in single hepatocytes as the effect of transient pump inhibition by removal of extracellular K+ on membrane potential difference (PD) and [Na+]i. In hepatocyte monolayers, HCO3- increased 22Na+ entry and turnover rates by 50-65%, without measurably altering 22Na+ pool size or cell volume, and HCO3- also increased Na+/K+ pump activity by 70%. In single cells, exposure to HCO3- produced an abrupt and sustained rise in [Na+]i from approximately 8 to 12 mM. Na+/K+ pump activity assessed in single cells by PD excursions during transient K+ removal increased congruent to 2.5-fold in the presence of HCO3-, and the rise in [Na+]i produced by inhibition of the Na+/K+ pump was similarly increased congruent to 2.5-fold in the presence of HCO3-. In intact perfused rat liver, HCO3- increased both Na+/K+ pump activity and O2 consumption. These findings indicate that, in hepatocytes, net coupled Na+ and HCO3- movement is inward and represents a major determinant of Na+ influx and Na+/K+ pump activity. About half of hepatic Na+/K+ pump activity appears dedicated to recycling Na+ entering in conjunction with HCO3- to maintain [Na+]i within the physiologic range.

Blocking DLL4, a Notch ligand, effectively inhibits tumor growth by increasing non-functional angiogenesis and decreasing the cancer stem cells (CSC) population. We are currently testing an anti-DLL4 antibody, demcizumab, in Phase1B trials in NSCLC, pancreatic, and ovarian cancer. DLL4 is also known to modulate immune responses. In the current study we examine the impact of anti-DLL4 on anti-tumor immune responses as a single agent and in combination with the key immune checkpoint inhibitor Programmed Cell Death Protein 1 (PD1). While the recent clinical success of PD1 inhibitors represents a new and promising cancer immunotherapeutic approach, high initial response rates are often associated by a lack of long-term, durable effects in a significant number of patients. Therefore, we hypothesized that dual blockade of DLL4 and PD1 might further impact tumor growth by further enhancing anti-tumor immune immunity. Our data demonstrates that dual blockade of DLL4 and PD1 using antibodies not only reduces tumor growth, but also led to tumor rejection in ∼50% in CT26WT tumor-bearing mice, similar to those treated with anti-PD1 alone (no tumor rejection was observed with anti-DLL4 alone). Anti-PD1 increased specific CD8+ T cell-mediated IFN-γ production while decreasing IL6. Anti-DLL4 treatment reduced IL17 production. Interestingly, only the dual blockage led to increased production of IL2 by splenocytes. Since IL2 is required for secondary population expansion of CD8+ memory T cells, increased IL2 in the combination group suggests potential for increased T cell activation, maintenance and memory T cell function, as compared to single agent anti-DLL4 and anti-PD1. While anti-PD1 reduced inhibition of CD4+ T cell proliferation by Tregs, the dual blockade significantly reduced Treg-mediated CD8+ T cell suppression. Furthermore, both effector and memory CD8+ T cell frequencies were increased within the total CD8+ T cell population. Interestingly, anti-PD1 decreased granulocytic MDSCs, while anti-DLL4 reduced monocytic MDSCs. Mice cured with single-agent anti-PD1 and anti-DLL4/anti-PD1 combination treatments were protected from series of re-challenge with tumor cells, suggesting the existence of immunologic memory. Interestingly, more mice were protected from tumor re-challenge when both DLL4 and PD1 were blocked, as compared to PD1 alone. Surprisingly, mice previously treated with the anti-DLL4/anti-PD1 combination produced more IL2, clearly indicating the role of DLL4 blockade in enhancing anti-tumor immunity. Therefore, these results show that dual targeting of DLL4 and PD1 may be an effective and durable cancer therapy by increasing anti-tumor immune response and promoting long-term immunological memory. Citation Format: Minu Srivastava, Christopher L. Murriel, Julie Roda, Hyun-Bae Jie, Fumiko Axelrod, Ming-Hong Xie, Rui Yun, Erin Mayes, Trevor Bentley, Belinda Cancilla, Raymond Tam, Tracy Tang, Ann Kapoun, John Lewicki, Tim Hoey, Austin Gurney, Angie Inkyung Park. Dual targeting of Delta-like ligand 4 (DLL4) and programmed death 1(PD1) inhibits tumor growth and generates enhanced long-term immunological memory. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 255. doi:10.1158/1538-7445.AM2015-255

A large-scale effort, termed the Secreted Protein Discovery Initiative (SPDI), was undertaken to identify novel secreted and transmembrane proteins. In the first of several approaches, a biological signal sequence trap in yeast cells was utilized to identify cDNA clones encoding putative secreted proteins. A second strategy utilized various algorithms that recognize features such as the hydrophobic properties of signal sequences to identify putative proteins encoded by expressed sequence tags (ESTs) from human cDNA libraries. A third approach surveyed ESTs for protein sequence similarity to a set of known receptors and their ligands with the BLAST algorithm. Finally, both signal-sequence prediction algorithms and BLAST were used to identify single exons of potential genes from within human genomic sequence. The isolation of full-length cDNA clones for each of these candidate genes resulted in the identification of >1000 novel proteins. A total of 256 of these cDNAs are still novel, including variants and novel genes, per the most recent GenBank release version. The success of this large-scale effort was assessed by a bioinformatics analysis of the proteins through predictions of protein domains, subcellular localizations, and possible functional roles. The SPDI collection should facilitate efforts to better understand intercellular communication, may lead to new understandings of human diseases, and provides potential opportunities for the development of therapeutics.

Interleukin-22 (IL-22) (also reported as IL-10-related T cell-derived inducible factor, IL-TIF) is a recently identified cytokine found to signal through a receptor comprising the class II cytokine receptor family members IL-10Rbeta/CRF2-4 and IL-22R. Previous work has established that IL-10Rbeta, also a component of the IL10R complex, exhibits a broad distribution of mRNA expression. Here, we observe that IL-22R exhibits a restricted expression pattern, with highest levels of mRNA expression in pancreas and detectable expression in multiple other tissues, particularly liver, small intestine, colon, and kidney. We find that isolated primary pancreatic acinar cells and the acinar cell line 266-6 respond to IL-22 with activation of Stat3 and changes in gene transcription. IL-22 mediates robust induction of mRNA for pancreatitis-associated protein (PAP1)/Reg2 and osteopontin (OPN). PAP1 is a secreted protein related to the Reg family of trophic factors and was initially characterized as a protein elevated in pancreatitis. In vivo injection of IL-22 resulted in rapid induction of PAP1 in pancreas, a response not observed in mice deficient in IL-10Rbeta. These results support the conclusion that IL-10Rbeta is a required common component of both the IL-10 and IL-22 receptors and suggest that IL-22 may play a role in the immune response in pancreas.

On antigen challenge, T-helper cells differentiate into two functionally distinct subsets, Th1 and Th2, characterized by the different effector cytokines that they secrete. Th1 cells produce interleukin (IL)-2, interferon-gamma (IFN-gamma) and lymphotoxin-beta, which mediate pro-inflammatory functions critical for the development of cell-mediated immune responses, whereas Th2 cells secrete cytokines such as IL-4, IL-5 and IL-10 that enhance humoral immunity. This process of T-helper cell differentiation is tightly regulated by cytokines. Here we report a new member of the type I cytokine receptor family, designated T-cell cytokine receptor (TCCR). When challenged in vivo with protein antigen, TCCR-deficient mice had impaired Th1 response as measured by IFN-gamma production. TCCR-deficient mice also had increased susceptibility to infection with an intracellular pathogen, Listeria monocytogenes. In addition, levels of antigen-specific immunoglobulin-gamma2a, which are dependent on Th1 cells, were markedly reduced in these mice. Our results demonstrate the existence of a new cytokine receptor involved in regulating the adaptive immune response and critical to the generation of a Th1 response.

We have identified a novel fibroblast growth factor, FGF-19, the most distant member of the FGF family described to date. FGF-19 is a high affinity, heparin dependent ligand for FGFR4 and is the first member of the FGF family to show exclusive binding to FGFR4. Human FGF-19 maps to chromosome 11 q13.1, a region associated with an osteoporosis-pseudoglioma syndrome of skeletal and retinal defects. FGF-19 message is expressed in several tissues including fetal cartilage, skin, and retina, as well as adult gall bladder and is overexpressed in a colon adenocarcinoma cell line.

We used the nonpeptide angiotensin II receptor antagonist DuP 753, which lacks the agonist and kinin/prostaglandin-inducing properties of saralasin and captopril, respectively, to examine the role of endogenous angiotensin II in regulating transport in the proximal convoluted tubule (PCT) and whole kidney. During in vivo microperfusion in the Munich-Wistar rat, a maximally effective dose of DuP 753 (10 mg/kg, intravenously) powerfully inhibited absorption of bicarbonate (370 +/- 3 to 200 +/- 9 pEq/mm.min, P less than .001), chloride (214 +/- 3 to 105 +/- 9 pEq/mm.min, P less than .001), and water 5.2 +/- 0.1 to 2.8 +/- 0.2 nL/mm.min, P less than .001) in the S1 subsegment of the PCT. DuP 753 was significantly more effective than captopril (3 mg/kg, intravenously) in inhibiting sodium chloride transport and is the most potent diuretic ever described in this segment. Consistent with the axial decline of angiotensin II receptor density in the PCT, DuP 753 was a less effective transport inhibitor in the S2 subsegment of the PCT, similar to captopril. Using free-flow micropuncture and clearance techniques, though inhibition in the earliest segment of the nephron is partially compensated by downstream reabsorption, DuP 753 induces a substantial diuresis, natriuresis, and chloruresis. In conclusion, DuP 753 markedly decreases S1 PCT fluid and electrolyte absorption, indicating that endogenous angiotensin II exerts significant tonic support of proximal transport in vivo.

Vertical polymer hot-carrier transistor using the low bandgap material poly(3-hexylthiophene) as both the emitter and the collector are studied. The common emitter current gain is shown to depend on the LiF thickness and the emitter thickness, with maximal value at 31. Current density as high as 31mA∕cm2 is achieved when collector voltage is −10V. For the device using blend of poly(3-hexylthiophene) and high bandgap polymer poly(9-vinylcarbazole) as the emitter, the current density rises sharply to 428mA∕cm2. The brightness of 3000cd∕m2 is obtained as a polymer light-emitting diode is driven by the transistor with the same area. The transistor can be operated at 100kHz.

Plasma membrane Na(+)-Ca2+ exchange contributes importantly to the regulation of cytosolic Ca2+ concentration ([Ca2+]i) in excitable cells. Despite extensive study in excitable tissues, the role of this transporter in the regulation of [Ca2+]i in hepatocytes is unknown, and conflicting information has been reported regarding the presence of Na(+)-Ca2+ exchange in hepatocyte plasma membrane vesicles. We have therefore assessed the role of Na(+)-dependent Ca2+ transport in the regulation of [Ca2+]i in rat hepatocytes in primary culture under basal conditions and after exposure to vasopressin, a hormone that elevates [Ca2+]i. Ca2+ efflux, measured using 45Ca, did not differ in the presence or absence of extracellular Na+, either under basal conditions or in response to vasopressin. [Ca2+]i, measured using the Ca2(+)-sensitive dye fura-2, was not altered by transient or prolonged exposure to Na(+)-free media or by exposure to ouabain in concentrations sufficient to produce a five-fold elevation in intracellular Na+ concentration. The [Ca2+]i response to vasopressin was also unaffected by Na+ removal or ouabain. By contrast, in cultured rat cardiac myocytes, cells that possess Na(+)-Ca2+ exchange, transient or prolonged Na+ removal as well as ouabain exposure produced greater than fivefold increases in [Ca2+]i compared with controls. We conclude that Na(+)-Ca2+ exchange does not contribute to the regulation of [Ca2+]i in hepatocytes.

Proximal nephron and renal effects of DuP 753, a nonpeptide angiotensin II receptor antagonist. The purpose of these studies was to quantitatively assess the role of endogenous angiotensin II activity in controlling transport in the proximal convoluted tubule (PCT) and whole nephron. We used the nonpeptide angiotensin II receptor antagonist DuP 753, which lacks the agonist and kinin/prostaglandin-inducing properties of saralasin and captopril, respectively. During in vivo microperfusion in the Munich-Wistar rat, we found that DuP 753 had a powerful inhibitory effect on bicarbonate (370 ± 3 to 200 ± 9 pEq/mm o min, P < 0.001), chloride (214 ± 3 to 105 ± 9 pEq/mm · min, P < 0.001), and water (5.2 ± 0.1 to 2.8 ± 0.2 nl/mm · min, P < 0.001) absorption in the S1 subsegment of the PCT. At maximally effective doses, DuP 753 (10 mg/kg i.v.) was significantly more effective than was captopril (3 mg/kg i.v.) in inhibiting sodium chloride transport in the S1 PCT. DuP 753 is the most potent diuretic ever described in this segment. Consistent with the axial decline of angiotensin II receptor density in the PCT, DuP 753 was a less effective transport inhibitor in the S2 subsegment of the PCT, similar to captopril. Though downstream reabsorptive elements partially compensate for the action in the earliest segment of the nephron, we also showed using free-flow micropuncture and clearance techniques that DuP 753 induces a substantial diuresis, natriuresis and chloruresis. In conclusion, the marked decrease in S1 PCT fluid and electrolyte absorption induced by DuP 753 indicates that endogenous angiotensin II exerts significant tonic support of proximal transport in vivo.

Current evidence suggests guanosine 3',5'-cyclic monophosphate (cGMP) serves as the second messenger for atrial natriuretic factor (ANF) in the kidney in vivo. We examined whether extracellular cGMP accumulation quantitatively reflected the concentration of cGMP within renal cells and whether urinary excretion of cGMP correlated with the physiological action of ANF. cGMP egression was examined in renal epithelial LLC-PK1 cells. ANF augmented intracellular cGMP concentration and extracellular cGMP appearance. Extracellular cGMP was an excellent function of the time-integrated intracellular cGMP concentration. In clearance studies in awake rats, urinary cGMP was primarily of renal cellular origin and correlated with the natriuresis induced by ANF in a time-dependent and concentration-dependent fashion. Urinary cGMP excretion may be useful as a biological marker for the renal activity of ANF in vivo.

Vertebrates and invertebrates initiate a series of defence mechanisms following infection by Gram-negative bacteria by sensing the presence of lipopolysaccharide (LPS), a major component of the cell wall of the invading pathogen1. In humans, monocytes and macrophages respond to LPS by inducing the expression of cytokines, cell-adhesion proteins, and enzymes involved in the production of small proinflammatory mediators. Under pathophysiological conditions, LPS exposure can lead to an often fatal syndrome known as septic shock2. Sensitive responses of myeloid cells to LPS require a plasma protein called LPS-binding protein and the glycosylphosphatidylinositol-anchored membrane protein CD14. However, the mechanism by which the LPS signal is transduced across the plasma membrane remains unknown3. Here we show that Toll-like receptor 2 (TLR2) is a signalling receptor that is activated by LPS in a response that depends on LPS-binding protein and is enhanced by CD14. A region in the intracellular domain of TLR2 with homology to a portion of the interleukin (IL)-1 receptor that is implicated in the activation of the IL-1–receptor-associated kinase is required for this response. Our results indicate that TLR2 is a direct mediator of signalling by LPS.