Michael J. Grace

Structural and biological characterization of pegylated recombinant interferon alpha-2b and its therapeutic implications

The type I interferon alpha family consists of small proteins that have clinically important anti-infective and anti-tumor activity. Interferon alpha-2b (Intron A) combination therapy with ribavirin is the current standard of care for the treatment of chronic hepatitis C virus infection. A drawback to the therapy however, is the short serum half-life and rapid clearance of the interferon alpha protein. Schering-Plough has developed a semi-synthetic form of Intron A by attaching a 12-kDa mono-methoxy polyethylene glycol to the protein (PEG Intron) which fulfills the requirements of a long-acting interferon alpha protein while providing significant clinical benefits. A detailed physicochemical and biological characterization of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Though pegylation appeared to decrease the specific activity of the interferon alpha-2b protein, the potency of PEG Intron, independent of protein concentration, was comparable to the Intron A standard at both the molecular and cellular level. Importantly, PEG Intron has demonstrated an enhanced pharmacokinetic profile in both animal and human studies. Recently, PEG Intron in combination with ribavirin has been shown to be very effective in reducing hepatitis C viral load and maintaining effective sustained viral suppression in patients. Because of the improved clinical benefits, it is anticipated that the PEG Intron plus ribavirin combination therapy will become the new standard of care for the treatment of chronic hepatitis C.

Interferon-α as an immunotherapeutic protein

Interferon‐α (IFN‐α) has proven to be a clinically effective antiviral and antineoplastic therapeutic drug for more than 16 years. During this time, evidence from in vitro laboratory studies and the clinical arena has supported the concept that IFN‐α is an immunotherapeutic drug. By regulating a diverse set of cytokines and their receptors, IFN‐α is uniquely positioned to prime the host immune response and provide an effective antineoplastic‐ and antiviral‐immune response. IFN‐α stimulates the innate cell‐mediated response and then participates in the transition of the initial host innate response into an effective adaptive‐immune response. IFN‐α also drives the adaptive cell‐mediated CD8+ T‐cell response and helps to maintain a CD4+ Th1‐cell population balance for an effective antineoplastic and antiviral host defense. This review will describe the current state of knowledge of IFN‐α as an immunoregulatory protein and address specific issues of IFN‐α as an immunotherapeutic for antineoplastic and antiviral diseases.

Structural and Biologic Characterization of Pegylated Recombinant IFN-α2b

The type I interferon-α (IFN-α) family is a family of natural small proteins that have clinically important anti-infective and antitumor activity. We have developed a semisynthetic protein-polymer conjugate of IFN-α2b (Intron® A) by attaching a 12,000-Da monomethoxypolyethylene glycol (PEG-12000) polymer to the protein. PEG conjugation is thought to increase the serum half-life and thereby prolong patient exposure to IFN-α2b without altering the biologic potency to the protein. Matrix-assisted laser desorption ionization/mass spectrometry (MALDI-MS), high-performance size exclusion chromatography (HPSEC), circular dichroism (CD) analysis and tryptic digestion peptide analysis of PEG Intron demonstrated that the IFN-α2b protein was approximately 95% monopegylated and that the primary, the secondary, and the tertiary structures were unaltered. Pegylation did not affect the epitope recognition of antibodies used for Intron A quantitation. An extensive analysis of the pegylated positional isomers revealed tha...

Pegylated Interferons for the Treatment of Chronic Hepatitis C Infection

BACKGROUND Interferon (IFN) alfa is a clinically effective therapy used in a wide range of viral infections and cell-proliferative disorders. Combination therapy with IFN alfa-2b and ribavirin is the current standard of care for the treatment of chronic hepatitis C (CHC) infection. However, standard IFN alfa has the drawbacks of a short serum half-life and rapid clearance. To overcome this problem, 2 pegylated forms of IFN have been developed and tested clinically. OBJECTIVE This article reviews the development and properties of pegylated IFN alfa-2b and pegylated IFN alfa-2a, and presents safety and efficacy data from recent clinical trials. METHODS Relevant clinical studies were identified through a MEDLINE search from 1966 through the present using the key words hepatitis C and interferon. Studies of the pegylated IFNs in humans were then selected. RESULTS Pegylated IFN alfa-2b is formed by covalent conjugation of a 12-kd mono-methoxy polyethylene glycol (PEG) molecule to IFN alfa-2b, and pegylated IFN alfa-2a by covalent conjugation of a 40-kd branched mono-methoxy PEG molecule to IFN alfa-2a. The 2 pegylated IFNs differ in the mixture of pegylation isomers resulting from their conjugation chemistry. Pegylated IFN alfa-2b has a prolonged serum half-life (40 hours) relative to standard IFN alfa-2b (7-9 hours). The greater polymer size of pegylated IFN alfa-2a acts to reduce glomerular filtration, markedly prolonging its serum half-life (72-96 hours) compared with standard IFN alfa-2a (6-9 hours). In clinical studies, once-weekly dosing of the pegylated IFNs was associated with a sustained virologic response in patients infected with hepatitis C virus (HCV). Once-weekly dosing with either of the pegylated IFNs was more effective than the respective thrice-weekly regimen of IFN alfa, with a comparable safety profile. The combination of once-weekly pegylated IFN and ribavirin effectively reduced HCV viral load and sustained viral suppression. CONCLUSIONS Once-weekly dosing with either pegylated IFN alfa-2b or pegylated IFN alfa-2a has been shown to produce significantly higher rates of viral eradication than standard thrice-weekly IFN alfa therapy without compromising safety. With respect to the treatment of CHC, the greatest anti-HCV efficacy has been achieved with the combination of once-weekly pegylated IFN and ribavirin.

Characterization of Replication-Competent Adenovirus Isolates from Large-Scale Production of a Recombinant Adenoviral Vector

Replication-deficient adenoviral vectors have been developed for the delivery of DNA sequences encoding a variety of proteins intended for the management of disease through gene therapy. One concern is the occurrence of replication-competent adenovirus (RCA) in the population of replication-deficient adenoviral vectors as a result of recombination or contamination. To address this concern, it is necessary to determine the frequency of occurrence and to fully characterize the molecular structure and biological infectivity of RCA. rAd/p53 is a pIX-deleted p53 gene therapy vector that is designed to lower the RCA occurrence and to deliver the tumor suppressor gene p53 for treatment of various cancers. Multiple preparations of the replication-deficient adenoviral vector rAd/p53 were tested for the presence of RCA, employing a sensitive biological assay. Single plaques from RCA-positive preparations of rAd/p53 were isolated for molecular characterization. All of the RCA isolates displayed a single unique structure that contains the complete E1 sequence of adenovirus type 5 but lacks the p53 sequence. The detailed sequence analysis of the RCA suggests that it is most likely generated as a result of recombination events between the rAd/p53 DNA and the 293 host adenoviral sequence. Results from viral infectivity analysis by flow cytometry demonstrate no substantial difference in infectivity of RCA, rAd/p53, and wild-type adenovirus type 5 in 293 cells.

Structure, biology, and therapeutic implications of pegylated interferon alpha-2b.

Derivatization of protein-based therapeutics with polyethylene glycol (pegylation) can often improve pharmacokinetic and pharmacodynamic properties of the proteins and thereby, improve efficacy and minimize dosing frequency. This review will provide an overview of pegylation technology and pegylated protein-based drugs being used or investigated clinically. The novel therapeutic, PEG Intron(R), formed by attaching a 12-kDa mono-methoxy polyethylene glycol (PEG) to the interferon alpha-2b protein, will be discussed in detail in terms of its structure, biological activities, pharmacokinetic properties, and clinical efficacy for the treatment of chronic hepatitis C. Detailed physicochemical and biological characterization studies of PEG Intron revealed its composition of pegylated positional isomers and the specific anti-viral activity associated with each of them. Pegylation of Intron A at pH 6.5 results in a mixture of > or = 95% mono-pegylated isoforms with the predominant species (approximately 50%) derivatized to the His(34) residue with the remaining positional isomers pegylated at various lysines, the N-terminal cysteine, as well as serine, tyrosine, and another histidine residue. The anti-viral activity for each pegylated isomer showed that the highest specific activity (37%) was associated with the His(34)-pegylated isomer. Though pegylation decreases the specific activity of the interferon alpha-2b protein in vitro, the potency of PEG Intron was comparable to the Intron A standard at both the molecular and cellular level. The substituted IFN had an enhanced pharmacokinetic profile in both animal and human studies, and, when combined with ribavirin, was very effective in reducing hepatitis C viral load and maintaining sustained viral suppression in patients.

Pharmacodynamics of PEG‐IFN α differentiate HIV/HCV coinfected sustained virological responders from nonresponders

Pegylated interferon (PEG‐IFN) has become standard therapy for hepatitis C virus (HCV) infection. We evaluated whether PEG‐IFN pharmacodynamics and pharmacokinetics account for differences in treatment outcome and whether these parameters might be predictors of therapeutic outcome. Twenty‐four IFN‐naïve, HCV/human immunodeficiency virus–coinfected patients received PEG‐IFN α‐2b (1.5 μg/kg) once weekly plus daily ribavirin (1,000 or 1,200 mg) for up to 48 weeks. HCV RNA and PEG‐IFN α concentrations were obtained from samples collected frequently after the first 3 PEG‐IFN doses. We modeled HCV kinetics incorporating pharmacokinetic and pharmacodynamic parameters. Although PEG‐IFN concentrations and pharmacokinetic parameters were similar in sustained virological responders (SVRs) and nonresponders (NRs), the PEG‐IFN α‐2b concentration that decreases HCV production by 50% (EC50) was lower in SVRs compared with NRs (0.04 vs. 0.45 μg/L [P = .014]). Additionally, the median therapeutic quotient (i.e., the ratio between average PEG‐IFN concentration and EC50[C̄/EC50]), and the PEG‐IFN concentration at day 7 divided by EC50 (C(7)/EC50) were significantly increased in SVRs compared with NRs after the first (10.1 vs. 1.0 [P = .012], 2.8 vs. 0.3 [P = .007], respectively) and second (14.0 vs. 1.1 [P = .016], 5.4 vs. 0.4 [P = .02], respectively) PEG‐IFN doses. All 3 parameters may be used to identify NRs. In conclusion, PEG‐IFN concentrations and pharmacokinetic parameters do not differ between SVRs and NRs. In contrast, pharmacodynamic measurements—namely EC50, the therapeutic quotient, and C(7)/EC50—are different in coinfected SVRs and NRs. These parameters might be useful predictors of treatment outcome during the first month of therapy. (HEPATOLOGY 2006;43:943–953.)

The effect of GM-CSF and G-CSF on human neutrophil function

A direct comparison of GM-CSF and G-CSF in a panel of in vitro neutrophil-function assays was performed to investigate any differences in activity profiles. In our modified chemotactic assay, GM-CSF rapidly increased the migratory capacity of polymorphonuclear cells (PMNs) to move toward fMLP and LTB4. In contrast, G-CSF only stimulated PMN migration towards fMLP. GM-CSF, but not G-CSF, increased PMN cytotoxic killing of C. albicans blastospores. The expression of PMN surface antigens associated with Fc- and complement-mediated cell-binding (Fc gamma R1, CR-1 and CR-3), and adhesion signalling (ICAM-1), was increased after the exposure of GM-CSF, but not to G-CSF. In contrast these CSFs demonstrated relative equipotency in their ability to induce PMN anti-bacterial phagocytosis, and to restore the Staphylococcus aureus killing capacity of dexamethasone-suppressed neutrophils. The phagocytic activity of PMNs for opsonized yeast, as well as hexose-monophosphate shunt activity, was equivalent following GM-CSF or G-CSF treatment. We discuss the significance of the difference in activity profiles in this article.

Comparison of flow cytometry and laser scanning cytometry for the intracellular evaluation of adenoviral infectivity and p53 protein expression in gene therapy.

The determination of recombinant adenoviral (rAd) infectivity and p53 protein expression is important for the evaluation of rAd vectors containing the p53 gene (rAd-CMV-p53) for gene therapy. We have previously reported that rAd5-CMV-p53 vectors can be assessed for infectivity and concomitant p53 protein expression in single- and two-color assays using intracellular staining methodology and flow cytometric analysis. We have compared the flow cytometry-based assays for rAd infectivity (hexon protein) and p53 protein expression with the new slide-based laser scanning cytometry (LSC). We report that LSC analysis of rAd-CMV-p53-infected human 293 cells correlated very well with flow cytometric analysis across a wide range of viral infectivity for both infectivity assessment (r2 = 0.97) and p53 protein expression (r2 = 0.96). Absolute values for infective titer and p53 protein expression titer from an rAd5-CMV-p53 production batch were similar and within experimental error with the two different analytical methods. Finally, bivariate format analysis of rAd-CMV-p53-infected cells revealed comparable results between LSC and flow cytometric analysis. LSC is a reliable and useful tool for the intracellular staining for adenoviral hexon protein expression for determining infectivity and for p53 protein expression from an expressed p53 transgene.

IL-4 induces neutrophilic maturation of HL-60 cells and activation of human peripheral blood neutrophils

IL‐4 is a T‐helper cell derived cytokine that has effects on myelomonocytic cell maturation and activation. We have studied the effect of IL‐4 on neutrophilic maturation using the cell line HL‐60 and found that it has a profound effect on the maturation and activation of the cell line. The treatment of HL‐60 cells with recombinant hu IL‐4 (0.15 to 15.0 ng/ml) induced a shift in the percentage of HL‐60 cells staining positive for chloroacetate esterase enzyme activity (indicating commitment to the neutrophilic lineage). IL‐4 increased surface expression of the neutrophil‐lineage antigen WEM G11, the complement receptors CR3 (CD11b) and CR1 (CD35), but not for the monocyte differentiation antigen CD14. IL‐4 treated HL‐60 cells demonstrated enhanced Fc‐ and complement‐mediated phagocytic capacity and increased hexose‐monophosphate shunt activity. In addition, IL‐4 was capable of sustaining the neutrophil maturation of HL‐60 cells that had been pre‐treated for 24h with DMSO. To investigate the effect of IL‐4 on the mature neutrophil, we studied freshly isolated and rested human peripheral blood neutrophils. In the absence of other stimuli, neutrophils were induced by IL‐4 to have significantly elevated phagocytic responses. The response was specific since treatment with anti‐human IL‐4 abolished phagocytic stimulation. Finally, IL‐4 treatment also stimulated resting neutrophils to migrate toward zymosan‐activated serum (ZAS) and human IL‐5. The results demonstrate that IL‐4 is a potent maturation factor for myelocytes to become neutrophils and that IL‐4 can stimulate resting mature neutrophils.