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.

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-4023's 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.

Patents on enhancing the potency and longevity of highly valuable protein hormones.

One major issue regarding the clinical use of many peptides is their short half-life span in the body, due to the rapid clearance from the circulation. Thus, at the clinical level, there is a need for a regime of frequent injections of the peptides into the patients to overcome this low stability factor. The major strategies for overcoming this problem by pharmaceutical companies are based on chemical techniques and using specific peptidase inhibitors or cocktails. For this purpose, the cassette gene contains the sequence of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin ß subunit which was ligated to the coding sequence of follitropin (FSH), erythropoietin (EPO), growth hormone (GH) and thus to increase the longevity and bioactivity of these proteins in vivo. Interestingly, FSH-CTP and GH-CTP were found to be not immunogenic in humans. FSH-CTP was approved by The European Commission. In addition, GH-CTP is not toxic and it passed successfully clinical trials Phase II in adults. Thus, using this technology seems to be promising in designing long acting peptides. Development of long acting peptides will diminish the cost of these drugs and perhaps reduce the number of injections in the clinical protocols. The article also summarizes some relevant patents.

Designing a Long Acting Erythropoietin by Fusing Three Carboxyl-Terminal Peptides of Human Chorionic Gonadotropin ß Subunit to the N-Terminal and C-Terminal Coding Sequence.

A new analog of EPO was designed by fusing one and two CTPs to the N-terminal and C-terminal ends of EPO (EPO-(CTP)(3)), respectively. This analog was expressed and secreted efficiently in CHO cells. The in vitro test shows that the activity of EPO-(CTP)(3) in TFI-1 cell proliferation assay is similar to that of EPO-WT and commercial rHEPO. However, in vivo studies indicated that treatment once a week with EPO-(CTP)(3) (15 µg/kg) dramatically increased (~8 folds) haematocrit as it was compared to rHuEPO. Moreover, it was found that EPO-(CTP)(3) is more effective than rHuEPO and Aranesp in increasing reticulocyte number in mice blood. The detected circulatory half-lives of rHuEPO, Aranesp, and EPO-(CTP)(3) following IV injection of 20 IU were 4.4, 10.8, and 13.1 h, respectively. These data established the rational for using this chimera as a long-acting EPO analog in clinics. The therapeutic efficacy of EPO-CTP analog needs to be established in higher animals and in human clinical trials.

Designing a long-acting human growth hormone (hGH) by fusing the carboxyl-terminal peptide of human chorionic gonadotropin beta-subunit to the coding sequence of hGH.

Chimeric genes were constructed by fusing of human GH (hGH) cDNA to one, two, or three cassettes of the carboxyl-terminal peptide (CTP) of human chorionic gonadotropin (hCG)-beta-subunit. hGH variant genes were inserted into the pCI-DHFR plasmid, transfected into DG44 cells, and stable clones were selected. Bioactivity and pharmacokinetic studies were performed in hypophysectomized Sprague Dawley derived male rats. The results indicated that sc injections of GH-wild-type (WT), Biotropin (commercial), GH-CTP, or CTP-GH (0.6 mg/kg) once every 5 d for 11 d (total dose of 1.2 mg/kg) resulted in an increased weight gain by 4, 4.9, 5.1, and 7 g, respectively. Treatment with CTP-GH-CTP-CTP (GH-LA) or CTP-GH-CTP (0.6 mg/kg) once every 5 d for 11 d or with Biotropin (0.12 mg/kg) daily for 11 d (total dose 1.2 mg/kg) resulted in a dramatic increase in weight gain of 16.5, 16.8, and 17 g, respectively. Repeated injections with different doses of GH-LA, 0.6, 1.8 mg/kg every 4 d or daily injection of 0.12 mg/kg of Biotropin increased the weight gain by 16, 28, and 18 gr, respectively. In addition, the cumulative serum levels of IGF-I after injection of GH-LA was significantly higher than that detected after injection of Biotropin. Pharmacokinetic studies indicated that the half-life, mean residence time, area under the curve, time of maximal plasma concentration, and maximal plasma concentration of GH-LA are dramatically increased compared with Biotropin. This may suggest that the mechanism of GH metabolic clearance is affected by the presence of CTP. These data establish a rationale for using this chimera as a long-acting GH analog.

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.

Characterization of the heparin/heparan sulfate binding site of the natural cytotoxicity receptor NKp46.

NKp46 is a member of a group of receptors collectively termed natural cytotoxicity receptors (NCRs) that are expressed by natural killer (NK) cells. NCRs are capable of mediating direct killing of tumor and virus-infected cells by NK cells. We have recently shown that NKp46 recognizes the heparan sulfate moieties of membranal heparan sulfate proteoglycans (HSPGs), thus enabling lysis of tumor cells by NK cells. In the current study, we further examined the residues in NKp46 that may be involved in heparan sulfate binding on tumor cells. On the basis of both the electrostatic potential map and comparison to the heparin binding site on human fibronectin, we predicted a continuous region containing the basic amino acids K133, R136, H139, R142, and K146 to be involved in NKp46 binding to heparan sulfate. Mutating these amino acids on NKp46D2 to noncharged amino acids retained its virus binding capacity but reduced its binding to tumor cells with a 10-100 fold lower K(D) when tested for direct binding to heparin. The minimal length of the heparin/heparan sulfate epitope recognized by NKp46 was eight saccharides as predicted from the structure and proven by testing heparin oligomers. Testing selectively monodesulfated heparin oligomers emphasized the specific contributions of O-sulfation, N-sulfation, and N-acetylation to epitope recognition by NKp46. The characterization of heparan sulfate binding region in NKp46 offers further insight into the identity of the ligands for NKp46 and the interaction of NK and cancers.

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-1alpha and IL-1beta), in the milieu of tumor cells that produce them, differentially affect the malignant process. Active forms of IL-1, namely precursor IL-1alpha (pIL-1alpha), mature IL-1beta (mIL-1beta), and mIL-1beta fused to a signal sequence (ssIL-1beta), were transfected into an established fibrosarcoma cell line, and tumorigenicity and antitumor immunity were assessed. Cell lines transfected with pIL-1alpha, which expresses IL-1alpha on the membrane, fail to develop local tumors and activate antitumor effector mechanisms, such as CTLs, NK cells, and high levels of IFN-gamma production. Cells transfected with secretable IL-1beta (mIL-1beta and ssIL-1beta) were more aggressive than wild-type and mock-transfected tumor cells; ssIL-1beta transfectants even exhibited metastatic tumors in the lungs of mice after i.v. inoculation (experimental metastasis). In IL-1beta tumors, increased vascularity patterns were observed. No detectable antitumor effector mechanisms were observed in spleens of mice injected with IL-1beta transfectants, mock-transfected or wild-type fibrosarcoma cells. Moreover, in spleens of mice injected with IL-1beta transfectants, suppression of polyclonal mitogenic responses (proliferation, IFN-gamma 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-1alpha tumor sites, whereas in mIL-1beta and ssIL-1beta 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-1alpha and IL-1beta in malignant processes; IL-1alpha reduces tumorigenicity by inducing antitumor immunity, whereas IL-1beta promotes invasiveness, including tumor angiogenesis, and also induces immune suppression in the host.

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

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 PKCeta associates with the cyclin E/Cdk2 complex. This is shown for the ectopically overexpressed PKCeta in NIH-3T3 cells, the inducibly expressed PKCeta in MCF-7 cells (under control of the tetracycline-responsive promoter), and the endogenously expressed PKCeta 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 PKCeta is predominantly expressed in the cytosolic fractions. The complex of PKCeta 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 PKCeta 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 PKCeta is concentrated at the perinuclear zone, which is also the site of its colocalization with cyclin E. Colocalization of PKCeta 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 PKCeta 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.