Ahuva Bar-Ilan

Membrane-Associated Heparan Sulfate Proteoglycans Are Involved in the Recognition of Cellular Targets by NKp30 and NKp46

Lysis of virus-infected and tumor cells by NK cells is mediated via natural cytotoxicity receptors (NCRs). We have recently shown that the NKp44 and NKp46 NCRs, but not the NKp30, recognize viral hemagglutinins. In this study we explored the nature of the cellular ligands recognized by the NKp30 and NKp46 NCRs. We demonstrate that target cell surface heparan sulfate proteoglycans (HSPGs) are recognized by NKp30 and NKp46 and that 6-O-sulfation and N-acetylation state of the glucose building unit affect this recognition and lysis by NK cells. Tumor cells expressing cell surface heparanase, CHO cells lacking membranal heparan sulfate and glypican-1-suppressed pancreatic cancer cells manifest reduced recognition by NKp30 and NKp46 and are lysed to a lesser extent by NK cells. Our results are the first clue for the identity of the ligands for NKp30 and NKp46. Whether the ligands are particular HSPGs, unusual heparan sulfate epitopes, or a complex of HSPGs and either other protein or lipid moieties remains to be further explored.

H5-Type Influenza Virus Hemagglutinin Is Functionally Recognized by the Natural Killer-Activating Receptor NKp44

ABSTRACT Antiviral immune defenses involve natural killer (NK) cells. We previously showed that the NK-activating receptor NKp44 is involved in the functional recognition of H1-type influenza virus strains by NK cells. In the present study, we investigated the interaction of NKp44 and the hemagglutinin of a primary influenza virus H5N1 isolate. Here we show that recombinant NKp44 recognizes H5-expressing cells and specifically interacts with soluble H5 hemagglutinin. H5-pseudotyped lentiviral particles bind to NK cells expressing NKp44. Following interaction with target cells expressing H5, pseudotyped lentiviral particles, or membrane-associated H5, NK cells show NKp44-mediated induced activity. These findings indicate that NKp44-H5 interactions induce functional NK activation.

Dengue Virus Replicon Expressing the Nonstructural Proteins Suffices To Enhance Membrane Expression of HLA Class I and Inhibit Lysis by Human NK Cells

ABSTRACT Many viruses escape the cellular immune response by downregulating cell surface expression of major histocompatibility complex (MHC) class I molecules. However, infection of cells with flaviviruses can upregulate the expression of these molecules. In this study we analyzed the expression of MHC class I in K562 and THP-1 human cell lines that were stably transfected with self-replicating subgenomic dengue virus RNA (replicons) and express all the dengue virus nonstructural proteins together. We show that MHC class I expression is upregulated in the dengue virus replicon-expressing cells and that the binding of natural killer (NK) inhibitory receptors to these cells is augmented. This upregulation results in reduced susceptibility of the dengue virus replicon-expressing cells to NK lysis, indicating a possible mechanism for evasion of the dengue virus from NK cell recognition. Visualizing MHC class I expression in replicon-containing K562 and THP-1 cells by confocal microscopy demonstrated aggregation of MHC class I molecules on the cell surface. Finally, replicon-expressing K562 cells manifested increased TAP (transporter associated with antigen processing) and LMP (low-molecular-mass protein) gene transcription, while replicon-expressing THP-1 cells manifested increased NF-κB activity and MHC class I transcription. We suggest that expression of dengue virus nonstructural proteins is sufficient to induce MHC class I upregulation through both TAP-dependent and -independent mechanisms. Additionally, aggregation of MHC class I molecules on the cell membrane also contributes to significantly higher binding of low-affinity NK inhibitory receptors, resulting in lower sensitivity to lysis by NK cells.

Harnessing soluble NK cell killer receptors for the generation of novel cancer immune therapy

The natural cytotoxic receptors (NCRs) are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The NCRs, which include three members; NKp46, NKp44 and NKp30, are critically involved in NK cytotoxicity against different targets, including a wide range of tumor cells derived from various origins. Even though the tumor ligands of the NCRs have not been identified yet, the selective manner by which these receptors target tumor cells may provide an excellent basis for the development of novel anti-tumor therapies. To test the potential use of the NCRs as anti-tumor agents, we generated soluble NCR-Ig fusion proteins in which the constant region of human IgG1 was fused to the extracellular portion of the receptor. We demonstrate, using two different human prostate cancer cell lines, that treatment with NKp30-Ig, dramatically inhibits tumor growth in vivo. Activated macrophages were shown to mediate an ADCC response against the NKp30-Ig coated prostate cell lines. Finally, the Ig fusion proteins were also demonstrated to discriminate between benign prostate hyperplasia and prostate cancer. This may provide a novel diagnostic modality in the difficult task of differentiating between these highly common pathological conditions.The natural cytotoxic receptors (NCRs) are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. The NCRs, which include three members; NKp46, NKp44 and NKp30, are critically involved in NK cytotoxicity against different targets, including a wide range of tumor cells derived from various origins. Even though the tumor ligands of the NCRs have not been identified yet, the selective manner by which these receptors target tumor cells may provide an excellent basis for the development of novel anti-tumor therapies. To test the potential use of the NCRs as anti-tumor agents, we generated soluble NCR-Ig fusion proteins in which the constant region of human IgG1 was fused to the extracellular portion of the receptor. We demonstrate, using two different human prostate cancer cell lines, that treatment with NKp30-Ig, dramatically inhibits tumor growth in vivo. Activated macrophages were shown to mediate an ADCC response against the NKp30-Ig coated prostate cell lines. Finally, the Ig fusion proteins were also demonstrated to discriminate between benign prostate hyperplasia and prostate cancer. This may provide a novel diagnostic modality in the difficult task of differentiating between these highly common pathological conditions.

In Vitro and in Vivo Characterization of MOD-4023, a Long-Acting Carboxy-Terminal Peptide (CTP)-Modified Human Growth Hormone.

MOD-4023 is a novel long-acting version of human growth hormone (hGH), containing the carboxy-terminal peptide (CTP) of human chorionic gonadotropin (hCG). MOD-4023 is being developed as a treatment for adults and children with growth hormone deficiency (GHD), which would require fewer injections than currently available GH formulations and thus reduce patient discomfort and increase compliance. This study characterizes MOD-4023s binding affinities for the growth hormone receptor, as well as the pharmacokinetic and pharmacodynamics, toxicology, and safety profiles of repeated dosing of MOD-4023 in Sprague-Dawley rats and Rhesus monkeys. Although MOD-4023 exhibited reduced in vitro potency and lower affinity to the GH receptor than recombinant hGH (rhGH), administration of MOD-4023 every 5 days in rats and monkeys resulted in exposure comparable to daily rhGH, and the serum half-life of MOD-4023 was significantly longer. Repeated administration of MOD-4023 led to elevated levels of insulin-like growth factor 1 (IGF-1), and twice-weekly injections of MOD-4023 resulted in larger increase in weight gain with fewer injections and a lower accumulative hGH dose. Thus, the increased half-life of MOD-4023 in comparison to hGH may increase the frequency of protein-receptor interactions and compensate for its decreased in vitro potency. MOD-4023 was found to be well-tolerated in rats and monkeys, with minimal adverse events, suggesting an acceptable safety profile. These results provide a basis for the continued clinical development of MOD-4023 as a novel treatment of GHD in children and adults.

Correction: Harnessing Soluble NK Cell Killer Receptors for the Generation of Novel Cancer Immune Therapy

The funding information for this paper is missing. It should read: This work was supported by the Israel Science Foundation (O.M.), the Binational Science Foundation (O.M.), the AICR (O.M), the ICRF (O.M.), the European Commission (LSHC-CT-2002-518178 and MRTN-CT-2005 to O.M.), the Israel Ministry of Health (A.P.), the Cooperation Program in Cancer Research of the Deutsches Krebsforschungszentrum (DKFZ), and Israelis Ministry of Science and Technology (MOST) (A.P.).