Eyal Fima

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.

Differential effects of IL-1 alpha and IL-1 beta on tumorigenicity patterns and invasiveness

In this study, we show that distinct compartmentalization patterns of the IL-1 molecules (IL-1α and IL-1β), in the milieu of tumor cells that produce them, differentially affect the malignant process. Active forms of IL-1, namely precursor IL-1α (pIL-1α), mature IL-1β (mIL-1β), and mIL-1β fused to a signal sequence (ssIL-1β), were transfected into an established fibrosarcoma cell line, and tumorigenicity and antitumor immunity were assessed. Cell lines transfected with pIL-1α, which expresses IL-1α on the membrane, fail to develop local tumors and activate antitumor effector mechanisms, such as CTLs, NK cells, and high levels of IFN-γ production. Cells transfected with secretable IL-1β (mIL-1β and ssIL-1β) were more aggressive than wild-type and mock-transfected tumor cells; ssIL-1β transfectants even exhibited metastatic tumors in the lungs of mice after i.v. inoculation (experimental metastasis). In IL-1β tumors, increased vascularity patterns were observed. No detectable antitumor effector mechanisms were observed in spleens of mice injected with IL-1β transfectants, mock-transfected or wild-type fibrosarcoma cells. Moreover, in spleens of mice injected with IL-1β transfectants, suppression of polyclonal mitogenic responses (proliferation, IFN-γ and IL-2 production) to Con A was observed, suggesting the development of general anergy. Histologically, infiltrating mononuclear cells penetrating the tumor were seen at pIL-1α tumor sites, whereas in mIL-1β and ssIL-1β tumor sites such infiltrating cells do not penetrate inside the tumor. This is, to our knowledge, the first report on differential, nonredundant, in vivo effects of IL-1α and IL-1β in malignant processes; IL-1α reduces tumorigenicity by inducing antitumor immunity, whereas IL-1β promotes invasiveness, including tumor angiogenesis, and also induces immune suppression in the host.

PKCη enhances cell cycle progression, the expression of G1 cyclins and p21 in MCF-7 cells

Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. However, not much is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that the expression of PKCη in MCF-7 cells, under the control of a tetracycline-responsive inducible promoter, enhanced cell growth and affected the cell cycle at several points. The induced expression of another PKC isoform, PKCδ, in MCF-7 cells had opposite effects and inhibited their growth. PKCη expression activated cellular pathways in these cells that resulted in the increased expression of the G1 phase cyclins, cyclin D and cyclin E. Expression of the cyclin-dependent kinase inhibitor p21WAF1 was also specifically elevated in PKCη expressing cells, but its overall effects were not inhibitory. Although, the protein levels of the cyclin-dependent kinase inhibitor p27KIP1 were not altered by the induced expression of PKCη, the cyclin E associated Cdk2 kinase activity was in correlation with the p27KIP1 bound to the cyclin E complex and not by p21WAF1 binding. PKCη expression enhanced the removal of p27KIP1 from this complex, and its re-association with the cyclin D/Cdk4 complex. Reduced binding of p27KIP1 to the cyclin D/Cdk4 complex at early time points of the cell cycle also enhanced the activity of this complex, while at later time points the decrease in bound p21WAF1 correlated with its increased activity in PKCη-expressing cells. Thus, PKCη induces altered expression of several cell cycle functions, which may contribute to its ability to affect cell growth.

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.

PKCη associates with cyclin E/Cdk2 complex in serum-starved MCF-7 and NIH-3T3 cells

Abstract Protein kinase C (PKC) encodes a family of enzymes implicated in cellular differentiation, growth control, and tumor promotion. However, very little is known with respect to the molecular mechanisms that link protein kinase C to cell cycle control. Here we report that PKCη associates with the cyclin E/Cdk2 complex. This is shown for the ectopically overexpressed PKCη in NIH-3T3 cells, the inducibly expressed PKCη in MCF-7 cells (under control of the tetracycline-responsive promoter), and the endogenously expressed PKCη in mouse mammary epithelial HC11 cells. Subcellular cell fractionation experiments revealed that the complex with cyclin E is formed mostly in the nuclear fractions, although in these cells PKCη is predominantly expressed in the cytosolic fractions. The complex of PKCη and cyclin E was studied at various phases of the cell cycle, in serum-starved quiescent cells and in cells stimulated with serum to reenter the cell cycle. Interestingly, the interaction between PKCη and cyclin E was most prominent in serum-starved cells and was disintegrated when cells entered the cells cycle. Immunofluorescence staining demonstrated that in serum-starved cells PKCη is concentrated at the perinuclear zone, which is also the site of its colocalization with cyclin E. Colocalization of PKCη and cyclin E in the perinuclear region was observed in serum-starved cells, and less in proliferating cells. These experiments suggest that the interaction between PKCη and cyclin E is carefully regulated, and is correlated with the inactivated form of the cyclin E/Cdk2 complex. Thus, our studies support an important link between PKC and cell cycle control.

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.

MOD-4023, a long-acting carboxy-terminal peptide-modified human growth hormone: results of a Phase 2 study in growth hormone-deficient adults

Objective Growth hormone (GH) replacement therapy currently requires daily injections, which may cause distress and low compliance. C-terminal peptide (CTP)-modified growth hormone (MOD-4023) is being developed as a once-weekly dosing regimen in patients with GH deficiency (GHD). This study’s objective is to evaluate the safety, pharmacokinetics (PK), pharmacodynamics (PD) and efficacy of MOD-4023 administered once-weekly in GHD adults. Design 54 adults with GHD currently treated with daily GH were normalized and randomized into 4 weekly dosing cohorts of MOD-4023 at 18.5%, 37%, 55.5% or 123.4% of individual cumulative weekly molar hGH dose. The study included 2 stages: Stage A assessed the effectiveness and PK/PD profiles of the 4 dosing regimens of MOD-4023. Stage B was an extension period of once-weekly MOD-4023 administration (61.7% molar hGH content) to collect further safety data and confirm the results from Stage A. Results Dose-dependent response was observed for both PK and PD data of weekly MOD-4023 treatment. Insulin-like growth factor I (IGF-I) SDS levels were maintained within normal range. The 18.5% cohort was discontinued due to low efficacy. MOD-4023 was well tolerated and exhibited favorable safety profile in all dose cohorts. The reported adverse events were consistent with known GH-related side effects. Conclusions Once-weekly MOD-4023 administration in GHD adults was found to be clinically effective while maintaining a favorable safety profile and may obviate the need for daily injections. Weekly GH injections may improve compliance and overall outcome. The promising results achieved in this Phase 2 study led to a pivotal Phase 3 trial, which is currently ongoing.

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.).