NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 12 DECEMBER 2007 1411

Albinterferon α-2b: a genetic fusion protein for the treatment of chronic hepatitis CG Mani Subramanian1, Michele Fiscella1, Araba Lamousé-Smith1, Stefan Zeuzem2 & John G McHutchison3

Treatment regimens based on the use of interferon-α (IFN-α) remain the cornerstone of therapy for chronic hepatitis C virus infection, which affects nearly 170 million people worldwide. Treatment options include unmodified IFN-α given three times weekly or pegylated IFNs given once weekly. The albumin-fusion platform takes advantage of the long half-life of human albumin to provide a new treatment approach that allows the dosing frequency of IFN-α to be reduced in individuals with chronic hepatitis C. Albinterferon α-2b (alb-IFN), a recombinant polypeptide composed of IFN-α2b genetically fused to human albumin, has an extended half-life and early evidence indicates that it is efficacious and well tolerated. Pharmacodynamic modeling supports treatment with alb-IFN at 2- or 4-week intervals. Phase 3 registration trials are in progress. The albumin-fusion platform is currently being applied to other important bioactive peptides with short half-lives. These fusion proteins, which are at present in different phases of clinical development, might lead to improved therapies across a broad range of diseases.

The therapeutic potential of several bioactive peptides and proteins is limited by their rapid clearance from the body, resulting in the need for frequent treatment of individuals to maintain drug levels1. Drug devel-opment technologies that delay clearance of drugs, to allow increased drug exposure and thereby to reduce dosing frequency, might improve patient compliance with and tolerability of therapies. Although the pharmacokinetic profile of a drug, and the area under the concen-tration curve to optimize pharmacology, are the primary drivers for improving clinical outcomes, increased compliance with dosing regi-mens also increases the likelihood of a favorable clinical outcome2,3. A genetic technology has been developed to enhance the pharmacokinetic properties of bioactive peptides and proteins by exploiting the long half-life of human albumin, a naturally occurring, widely distributed and enzymatically inert serum protein. Genetic fusion of the comple-mentary DNA that encodes human albumin to a cDNA that encodes a therapeutically active peptide or protein results in the creation of a polypeptide with a markedly enhanced pharmacokinetic profile. These

recombinant, long-acting protein drugs might offer important thera-peutic alternatives for the treatment of many diseases.

This article describes the scientific and clinical rationale that underlie the development of the human albumin-fusion platform and provides a brief description of other strategies that are designed to improve the pharmacokinetics of therapeutically important proteins and peptides. The effectiveness of the human albumin-fusion technology platform is discussed in the context of alb-IFN, the genetic fusion product of human albumin and IFN-α2b. Data on the biological characteriza-tion of alb-IFN and an overview of its clinical development for the treatment of individuals with chronic hepatitis C (CHC) are provided. Given the potent antiviral, immunomodulatory and antiproliferative effects of IFN-α, this article also addresses the potential therapeutic benefits of alb-IFN for the treatment of other disorders, including cer-tain cancers.

Approaches for enhancing polypeptide pharmacokineticsSeveral technologies have been developed to enhance the pharma-cokinetics of polypeptides (Table 1), including chemical/covalent modification, micro-encapsulation and the use of protease-resistant variants4–22.

Efforts to reduce the renal clearance of small therapeutic molecules have focused on increasing their molecular size through covalent link-age to larger molecules, such as polyethylene glycol10,23,24 and albu-min25. As well as increasing elimination half-life, covalent linkage to these larger molecules might reduce immunogenicity, improve solubil-ity and bioavailability, and enhance resistance to proteolysis. However, the covalent modification of bioactive molecules for therapeutic goals is limited by a potential loss of biological activity, such as reduced recep-tor binding due to steric hindrance and product heterogeneity due to integration of the inert molecule at one or more sites of the bioactive peptide. Structural heterogeneity of the final product is often associated with functional heterogeneity and a consequent reduction in biological or enzymatic activity.

Micro-encapsulation is an alternative method that can be used to enhance the pharmacokinetic profile of therapeutic agents26,27. The active moiety is surrounded by a continuous, polymeric layer that degrades over time, resulting in slow and sustained release of the bio-active compound from the microspheres. Although this established technique has been widely used to improve drug stability and provide targeted drug release, technical issues associated with the drug encap-sulation process for proteins and peptides, and the efficiency of release from the resulting microspheres, have presented the biopharmaceutical industry with several pharmacological challenges27.

1Human Genome Sciences, Inc., 14200 Shady Grove Road, Rockville, Maryland 21042, USA. 2J.W. Goethe University Hospital, Theodor-Stern-Kai 7, D-60590 Frankfurt, Germany. 3Duke Clinical Research Institute, PO Box 17969, Durham, North Carolina 27715, USA. Correspondence should be addressed to G.M.S. (Mani_Subramanian@hgsi.com).

Published online 7 December 2007; doi:10.1038/nbt1364

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1412 VOLUME 25 NUMBER 12 DECEMBER 2007 NATURE BIOTECHNOLOGY

Proteolysis of biotherapeutic molecules is an important obstacle to the advancement of drug-delivery technologies. Shortly after injection, bioactive polypeptides are readily degraded by endogenous serum or tissue proteases, reducing their therapeutic potential. In addition, pro-teolysis facilitates the clearance of the degraded protein, further limiting drug exposure. The introduction of one or more proteolysis-resistant mutations to the bioactive molecule is currently a focus of drug design and development14. In vivo stabilization can also be achieved through the introduction of artificial amino-acid analogs (β-amino acids)28. These modifications produce subtle conformational changes that can result in enhanced resistance to proteolytic degradation11,22.

The albumin-fusion technology platformAlbumin-fusion technology represents a simple and flexible alterna-tive platform for the production of proteins with extended circulatory

half-lives (Table 1). Human albumin is the most prevalent serum pro-tein (35–40 g l–1) in the circulatory system29. Several features that are unique to albumin make it an ideal candidate for integration into a drug-design platform, including its unusually long half-life (~19 d), wide distribution, negligible potential for confounding enzymatic or immunological function and its physiological role as a carrier of blood substances30,31.

The albumin-fusion technology involves the translation of a recom-binant, long-acting therapeutic protein from a cDNA produced from a plasmid representing the human albumin cDNA fused to either the carboxy or the amino terminus of a cDNA encoding a therapeutically active protein (Fig. 1a). Accordingly, this technology has the potential to produce long-acting proteins that are effective and beneficial in the treatment of a wide range of human diseases. The applicability of the albumin-fusion platform to proteins with varied molecular weights and diverse biological functions is shown in Table 2 (refs.4,15,16,21,32,33). Larger proteins (~20 kDa) that are widely used in clinical practice, including cytokines (for example, IFNs and interleukins) and growth factors (for example, granulocyte colony-stimulating factor, erythro-poietin and growth hormone) are candidates for this technology. Other candidates include small bioactive peptide hormones, such as gluca-gon-like peptide-1 and β-natriuretic peptide, which are being evalu-ated for the treatment of diabetes mellitus and congestive heart failure,

Table 1 Currently available approaches to enhance polypeptide pharmacokineticsApproach Advantages Challenges Examples

Chemical/covalent modification • Increased in vivo half-life • Protection from degradation • Reduced renal clearance • Increased solubility/stability

• Decreased tissue uptake • Immunogenicity • Functional heterogeneity • Maintenance of functional activity;

depends on chemistry used

PEGylation (PEG-hGH7, PEG-IFN-β18 and PEG-IFN-α-2b9), albumin and fatty acid acylated insulin13

Microsphere/nanoparticle delivery • Sustained/targeted drug release • Protection from degradation

• Encapsulation efficiency • Efficiency of drug release from microspheres • Functional heterogeneity • Maintenance of functional activity

Poly L-Glu nanoparticle, IFN-α-XL6, poly(lactic-co-glycolic acid) (PLGA) microsphere, IFN-α19, human growth hormone5 and PEG-insulin12

Protease-resistant variants • Improved stability • Protection from degradation

• Maintenance of functional activity • Immunogenicity

Ala20Pro-RNaseA14, MART-1 with β amino acid substitution11, T-cell mimotopes22 and G15A growth hormone releasing hormone20

Albumin fusion • Increased in vivo half-life • No modification required • Design flexibility • Reduced renal clearance • Increased solubility/stability

• Maintenance of functional activity • Immunogenicity

alb-IFN-α4, alb-GLP-1, alb-insulin8, alb-GH16, alb-IL-215 and alb-BNP21

0 2 4 7 9 11 16 18 21 23 2514

The

rape

utic

inde

x

Efficacy region

28 30

Time (d)

Unmodified IFN dosing (TIW)

alb-IFN dosing (Q2w)

Design flexibility

Reduced dosing frequency and sustained exposure

N-terminal fusion

HA GENE X

C-terminal fusion

HAGENE X

Large cytokines

Transcription/translation

HA

66.5 kDa

19.2 kDa

Small peptides

HA

66.5 kDa

GLP-1IFNa

3.8 kDa

Upper limit

Lower limit

a

b

Figure 1 Key attributes of the albumin-fusion technology platform. (a) Design flexibility. Albumin fusion technology permits the creation of recombinant, long-acting protein drugs by fusing the cDNA (gene X) encoding human albumin (HA) to cDNAs encoding therapeutically active proteins. Fusion can occur at either the N- or C-terminal of HA (arrows indicate transcription initiation). Molecular models depicting HA fusion to a large cytokine (IFN-α-2b) or a small peptide (glucagon-like peptide 1) illustrate the adaptability of the albumin fusion platform to biologically active compounds of varying sizes. (b) Reduced dosing frequency and sustained exposure. The observed therapeutic index of alb-IFN versus unmodified IFN is used as an example to illustrate the sustained exposure and reduced dosing frequency associated with the albumin-fusion technology platform. Unmodified IFN reaches peak levels shortly after administration, followed by a rapid decline to undetectable levels at the end of each dosing interval (TIW (three times weekly) dosing, on Monday, Wednesday and Friday). By contrast, alb-IFN administered Q2w (every two weeks) provides sustained drug exposure that lies within the range of therapeutic efficacy throughout each dosing interval. Sustained drug exposure accompanied by infrequent dosing might improve tolerability.

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NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 12 DECEMBER 2007 1413

respectively. At the molecular level, there is significant design simplicity and flexibility wherein C- or N-terminal fusion construct variants can be generated without interven-ing linker sequences (Fig. 1a). Thus, this platform can potentially be used to produce a broad spectrum of bioactive molecules, and its applicability is not restricted by their size or biological activity.

The platform-production approaches that are widely used in the biopharmaceutical industry can be exploited for the production of distinct albumin-fusion proteins34–36. By circumventing the secondary processing steps that are required for the covalent modification or encapsulation of therapeutic target proteins, the human albumin-fusion platform approach offers a scalable and efficient strategy for consistent production of high-quality products. A common yeast host strain has typically been used for albumin-fusion expression. During the fermentation process, the gene is transcribed and translated as a single polypeptide, which is then secreted into the fermentation broth. The fermentation broth is harvested and clari-fied using centrifugation and/or filtration techniques, and standard chromatography techniques are used to purify the drug. As discussed below, alb-IFN, the first clinically validated product of this technology, is a promising option for the treatment of CHC.

Hepatitis C infection and rationale for alb-IFNCHC is one of the most common causes of chronic liver disease, with ~3% of the world’s population (or as many as 170 million people) affected37. Approximately 30% of individuals with human immuno-deficiency virus type 1 (HIV-1) infection are also infected with the hepatitis C virus (HCV), which is one of the leading causes of morbidity in this co-infected population38. Between 60% and 80% of individuals

who are acutely infected with HCV develop CHC2,39, and 20% eventu-ally develop cirrhosis. CHC is also associated with the development of hepatocellular carcinoma and is the primary cause of liver transplanta-tion worldwide40.

The therapeutic goal of treatment for CHC is sustained virologic response (SVR), which is defined as viral clearance 24 weeks after the end of treatment (treatment duration is 24 weeks for genotypes 2 or 3, and 48 weeks for genotype 1, the latter being the most prevalent HCV genotype worldwide and also the most difficult to treat)41. Other important virologic measurements include rapid virologic response (HCV RNA below the limit of quantification (LOQ) at week 4), early virologic response (≥2-log reduction in HCV RNA at week 12) and undetectable HCV RNA (below LOQ at week 12). There is substantial evidence that early viral clearance (HCV RNA negativity at weeks 4 and 12) has a high positive predictive value for achieving SVR41–44. Thus, therapeutic agents that maximize early antiviral response and maintain viral suppression for the duration of therapy have the highest probability of achieving SVR or lasting viral eradication.

The past two decades have witnessed an evolution in the use of IFN (first discovered 50 years ago45) for the treatment of CHC. The lower

Table 2 Clinical studies with albumin-fusion products

Albumin-fusion productsFusion partner

MW (kDa) Development-stage Indication

Alb-growth hormone (GH) 22 Phase 1/2 Growth hormone deficiency

Alb-granulocyte colony-stimulating factor (GCSF)

19.6 Phase 1/2 Neutropenia

Alb-interferon (IFN-α) 19.2 Phase 3 Chronic hepatitis C

Alb-interleukin 2 (IL2) 15.5 Phase 1/2 Malignancy

Alb-glucagon-like peptide 1 (GLP-1) 3.8 Phase 1/2 Diabetes mellitus

Alb-β-natriuretic peptide (BNP) 3.5 Phase 1/2 Congestive heart failure

Unt

reat

ed 0

.1 n

g m

l–1

1 n

g m

l–1

10

ng m

l–1

1 n

g m

l–1

10

ng m

l–1

100

ng

ml–1

IFNa-2b alb-IFN

Unt

reat

ed 0

.1 n

g m

l–1

1 n

g m

l–1

10

ng m

l–1

1 n

g m

l–1

10

ng m

l–1

100

ng

ml–1

IFNa-2b alb-IFN

10–15 10–1410–13 10–1210–11 10–10 10–9 10–8 10–7 10–6 10–5

Drug concentration g ml–1

Rep

orte

r ou

tput

(S

EA

P u

nits

)

1,000

800

600

400

0

200

PEG-IFNa-2aIFNa-2b

190 215 240 265 290 315 340

Wavelength (nm)

Mol

ar e

llipt

icity

(de

g cm

2 dm

ol–1

) Molar ellipticity (deg cm

2 dmol –1)

–5,000

–10,000

–15,000

–25,000

–20,000

–40

–80

–120

–200

–240

–160

Spectral sumalb-IFN

alb-IFN

Structural properties Signaling through ISRE Gene expressiona b c

Figure 2 Biochemical characterization of alb-IFN. (a) Near- and far-ultraviolet circular dichroism (UV-CD) spectra of alb-IFN. This panel shows the spectra of alb-IFN compared with the spectral sums of human albumin and IFN-α. The near- and far- UV-CD spectra indicate that the tertiary and secondary structures of IFN-α are retained in the fusion protein52. (b) Signaling through IFN-specific response element (ISRE). Albinterferon α-2b activates the ISRE pathway, demonstrating IFN-stimulated gene (ISG) induction characteristics similar to PEG-IFN-α-2a4. RLU, relative luminescence unit. (c) Induction of IFN-specific genes measured using cDNA microarray analysis. Albinterferon α-2b induces a global transcription profile similar to that observed with native IFN-α. The expression profiles of this set of genes are shown in pseudocolor (green indicates upregulation), ranked by the average treated/control sample ratios54.

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1414 VOLUME 25 NUMBER 12 DECEMBER 2007 NATURE BIOTECHNOLOGY

SVR rates associated with unmodified IFN monotherapy improved considerably with the addition of the oral antiviral agent ribavirin to the CHC treatment regimen46. The introduction of pegylated IFN-α−2a (PEG-IFN-α−2a) or IFN-α-2b (PEG-IFN-α-2b) to combination therapy with ribavirin has resulted in improved SVR and patient-reported health-related quality of life (HRQOL) over standard IFN-α; these agents represent the current standard of care for the treatment of CHC42,43,47–49. Indeed, >50% of individuals with CHC can now expect to achieve SVR after combination treatment with PEG-IFN-α and ribavirin50. Moreover, compliance with therapy has been shown to be important in maximizing SVR rates3,43. Given the dependence of treatment success on patience adherence, the development of lon-ger-acting IFN formulations with improved pharmacokinetic profiles (sustained drug exposure) that permit reduced dosing frequencies is an important focus of HCV therapy. As new direct antiviral drugs (for example, inhibitors of the HCV replication machinery) are developed and concerns over the emergence of resistant viral variants assume greater prominence51, long-acting IFN formulations that maintain viral suppression over longer dosing intervals (theoretically minimizing the development of viral resistance) are likely to remain an integral com-ponent of therapy for CHC in the foreseeable future.

The pharmacological rationale that underlies the development of alb-IFN is to optimize drug exposure so as to provide maximal sustained viral suppression, with the enhanced convenience of an improved dos-ing schedule. Unmodified IFN-α reaches peak serum concentrations shortly after administration, followed by a rapid decline to undetectable levels toward the end of its dosing interval, which allows significant viral rebound (Fig. 1b). Moreover, wide and frequent variations in drug levels (high peak-to-trough ratio) associated with administration three times a week might exacerbate the IFN-related symptoms that often manifest after drug administration, thereby diminishing HRQOL.

A longer-acting IFN, such as alb-IFN, which provides sustained expo-sure (low peak-to-trough ratio), might reduce viral rebounds and, furthermore, improve patient-reported HRQOL owing to the longer dosing interval.

Preclinical characterization of alb-IFNSeveral converging lines of in vitro and preclinical in vivo evidence sup-port the clinical development of alb-IFN. In vitro studies have shown that the physiochemical properties, binding characteristics and appar-ent biological activity of IFN-α are preserved in the alb-IFN fusion protein (Figs. 2,3). Using a representation of near- and far-ultraviolet circular dichroism spectra as an indication of tertiary and secondary structure, respectively, the spectral signature of the fusion protein was found to be highly similar to the theoretical sum of the individual spec-tra of each native (unmodified) component52 (Fig. 2a).

The IFN-α molecule mediates its immunomodulatory and antiviral actions by binding to its cell-surface receptor (comprising the chains of IFN-α/β-receptors 1 and 2 (IFNAR1 and IFNAR2)), thereby activating a complex, intracellular signaling cascade that culminates in the tran-scription of genes that encode IFN-inducible proteins. Surface plasmon resonance studies (a standard method for assessing protein-protein interactions)53 have revealed that the binding properties of alb-IFN to IFNAR2 are retained, as alb-IFN binds to the recombinant extracellular domain of IFN-α/β-receptor chain 2 with an affinity (KD, the equilib-rium constant determined by the ratio of the kinetic rate constants kd/ka) of 24 ± 8 nM, which is at least comparable to that of PEG-IFN-α-2a (116 ± 16 nM) (Yun Hee Cho, Andy Garcia and Stephen Ullrich, unpublished data; Human Genome Sciences, Rockville, MD, USA).

The ability of alb-IFN to activate the IFN-specific signal-transduc-tion pathway has been tested using an IFN-α-responsive mammalian cell line stably transfected with a chemiluminescent reporter under the control of an IFN-specific response element (Fig. 2b)4. The albumin-fusion and pegylated IFN-α-2a produced similar activation. In a com-plementary approach, cDNA microarray analysis using cultured human lymphoma cells has revealed that alb-IFN induces a global transcription profile similar to that induced by unmodified recombinant IFN-α (Fig. 2c)54. The enhanced pharmacokinetic profile has been demonstrated in cynomolgus monkeys, where alb-IFN was detected for >7 d after a single dose, with a concomitant induction of the IFN-responsive gene OAS1 (2′-5′ oligoadenylate synthetase 1)17.

Albinterferon α-2b shows antiviral activity in various cell-cul-ture assays (Fig. 3). Figure 3a illustrates the comparative antiviral activity of several IFN formulations (modified and unmodified) in Madin-Darby bovine kidney cells infected with vesicular stomatitis virus, a system that is widely used to assess the potency of IFN-α and

Antiviral activity against VSV in MDBK cells

Suppression of HCV replication in Huh-7 replicon cells

10 100 1,000 10,000 100,0002,500

5,000

7,500

10,000

12,500

15,000

[IFN] (fmol)

RF

Us

alb-IFN

IFNα-2b

PEG-IFNα-2b

IFNα-2a

PEG-IFNα-2a

alb-IFN(ng mL–1)

IFNa-2b(U mL–1)

PEG-IFNa-2b(ng mL–1)

PEG-IFNa-2a(ng mL–1)

*

**

*

7 U ml–1EC50

0 1.0 10 20 40 100 0 0.08 1.11.4 5.0 200 0 16 22 30 50 80 0 19 25 30 50 100

1.1 ng ml–1 18 ng ml–1 15 ng ml–1

1.2

1.0

0.8

0.6

0.4

0.2

0

Rel

ativ

e H

CV

RN

A le

vel

a

b

Figure 3 In vitro antiviral activity. (a) Antiviral activity against vesicular stomatitis virus (VSV) in Madin-Darby bovine kidney (MDBK) cells. The relative antiviral potency on a molar basis (given the differences in molecular weight) of several IFN formulations were compared in MDBK epithelial cells infected with VSV. Whereas alb-IFN was less potent on a molar basis than IFN-α (50% maximal effective concentration (EC50) 3,813 fmol versus 476 fmol), it was roughly equivalent to PEG-IFN-α-2a (EC50 3,813 fmol versus 4,401 fmol). RFU, relative fluorescence unit. (b) Suppression of HCV replication in liver (Huh7) replicon cells. Compared with other modified IFNs, alb-IFN exhibits greater anti-HCV activity at clinically relevant serum concentrations in Huh7 replicon cells. The graph represents the normalized data, with glyceraldehyde 3-phosphate dehydrogenase (GADPH) as the internal control. Asterisks denote mean maximum serum concentrations for the corresponding IFNs based on published data55. The EC50 values for IFN-α-2b, PEG-IFN-α-2b, PEG-IFN-α-2a and alb-IFN were 7 U ml–1, 1.1 ng ml–1, 18 ng ml–1 and 15 ng ml–1, respectively.

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NATURE BIOTECHNOLOGY VOLUME 25 NUMBER 12 DECEMBER 2007 1415

related products. Whereas alb-IFN was less potent on a molar basis than IFN-α (50% maximal effective concentration (EC50): 3,813 fmol ver-sus 476 fmol), it was approximately equivalent to PEG-IFN-α−2a (EC50: 3,813 fmol versus 4,401 fmol; Jonathan Zmuda, unpublished data, Human Genome Sciences). A comparison of the anti-HCV activity of unmodified IFN-α with that of three modified IFN-α products in human hepatocellular carcinoma cells (Huh7) revealed that alb-IFN inhibits HCV replication in replicon cells (Fig. 3b), with an EC50 comparable to that of PEG-IFN-α-2a55. Moreover, at clinically relevant serum concentrations, alb-IFN exhibits greater antiviral activity than the PEG-IFNs55.

Clinical development of alb-IFNThe clinical development program for alb-IFN includes the following: first, dose-ranging, limited-exposure studies (phase 1/2) of alb-IFN to assess its safety, pharmacokinetics and pharmacodynamic param-eters56,57; second, dose-ranging studies (phase 2) of alb-IFN in combi-nation with ribavirin to assess its safety and antiviral efficacy; and third, pivotal phase 3 studies to establish efficacy and safety for regulatory approval of alb-IFN (Fig. 6).

From phase 1 to phase 2a. A phase 1/2a evaluation of 22 escalating doses of alb-IFN (10–1,200 µg), administered subcutaneously as a single dose or as two injections administered 14 days apart, was per-formed in 119 individuals with CHC who had failed clear HCV RNA with previous IFN-α-based therapy57. This limited-exposure study was

performed to assess the pharmacokinetics and safety of alb-IFN and to obtain an estimate of its antiviral activity. The results showed that alb-IFN had a favorable safety profile at doses up to 1,200 µg; there were no discontinuations associated with adverse events. Reduced clearance resulted in a mean elimination half-life of 159 h, which supports dosing at 2- to 4-week intervals. Induction of the IFN-specific gene OAS1 was maintained for ≥28 d after a single injection of alb-IFN, and significant antiviral activity was observed in individuals who received alb-IFN doses ≥120 µg57,58.

A phase 2, open-label, dose-ranging study was conducted to assess antiviral response in IFN-α-naive individuals infected with CHC geno-type 1 (ref. 56). Consistent with the presence of high drug levels fol-lowing the administration of two injections 14 days apart (Fig. 4a), most individuals showed continued viral decline over a 14- to 28-day interval after the second dose. As shown in Figure 4b, antiviral activ-ity was greatest in the 900- and 1,200-µg dose groups, with a mean HCV RNA reduction of 3.2 log10 infectious units (IU) per ml at week 4. Mathematical modeling of the HCV RNA decline according to the viral dynamics model of Neumann et al.59 revealed a biphasic response in all treatment groups with an inflection between days 1 and 4. The first-phase HCV RNA reduction indicates the direct antiviral activity of blocking virion production and shows dose dependence with 1.7 log10 IU ml–1 for the high-dose groups (Fig. 4c)56. Similarly, the second-phase HCV RNA decline slope, which indicates killing of virus-infected cells, is also dose dependent (P < 0.02; Fig. 4d)56. Importantly, 24 of 26 individuals (92%) in the higher-dose groups had a second-phase

a b

c d

Study day Study day0

1

–4.0

–3.5

–3.0

–2.5

–2.0

–1.5

–1.0

–0.5

0.0

0.5 0.5

0.0

–0.5

–1.0

–1.5

–2.0

–4

–3

–2

–1

0

10

100

7 14 21 28 35 42 49 0 4 7 14 18 21 28 35 42

Con

cent

ratio

n (n

g m

l–1)

Mea

n H

CV

RN

A r

educ

tion

(log 10

IU/m

l)

P < .003 P < .02

1st phase response 2nd phase slope

1st p

hase

dec

line

(log)

2nd

phas

e de

clin

e sl

ope

(log

per

wee

k)

200 µg 450 µg 670 µg 900 µg 1,200 µg

200 µg 450 µg 670 µg 900 µg 1,200 µg 200 µg 450 µg 670 µg 900 µg 1,200 µg

alb-IFN dose alb-IFN dose

Median values

Figure 4 Pharmacokinetics and viral kinetics of alb-IFN. (a) Mean concentration-time profiles were evaluated after two injections (on days 0 and 14; dashed lines) of alb-IFN. The administration of 200–1,200 µg of alb-IFN resulted in detectable serum concentrations of drug across the 42-day sampling period in all participants. Serum concentrations were dose-related and dose-proportional. Maximum serum concentrations increased in a linear fashion over the evaluated dosage range56. (b) Mean HCV RNA (log10 IU ml–1) change from baseline investigated in genotype 1 IFN-naive individuals after two alb-IFN injections administered at days 0 and 14 (dashed lines). At day 42, the mean HCV RNA reduction in the higher dose (900 µg and 1,200 µg) cohorts was significantly greater (P < 0.05) than in the 200-µg cohort56. (c) First-phase HCV RNA reduction (log10IU ml–1) in the low-dose (200–670 µg) or high-dose (900–1,200 µg) treatment groups. The mean (± s.d.) HCV RNA reductions (horizontal bar) for the low- and high-dose groups are –1.0 and –1.7 log10 IU ml–1, respectively. The dashed line represents the clinically significant first-phase response (HCV RNA reduction >0.5 log10IU ml–1). This dose-dependent, first phase decline is related to direct inhibition of virus production56,60. (d) Second-phase HCV RNA reduction slopes (log10IU ml–1) in low-dose (200 µg to 670 µg) and high-dose (900 µg to 1,200 µg) treatment groups. The mean (± s.d.) HCV RNA reductions (horizontal bar) for the low- and high-dose groups are –0.45 and –0.75 log per week, respectively. The dashed line represents the clinically significant second-phase slope of 0.3 log per week. This second slope of viral decline represents killing of infected cells that is essential for HCV viral clearance and SVR56,60.

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1416 VOLUME 25 NUMBER 12 DECEMBER 2007 NATURE BIOTECHNOLOGY

decline of >0.3 log10 IU per week, which is a strong predictor of SVR60. In summary, the pharmacodynamic profiles of the 900- and 1,200-µg doses provide the greatest level of viral suppression, with minimal HCV RNA rebound at the end of the dosing interval, by maintaining drug levels over the EC90 in most individuals. This observation provided the rationale for further evaluation of alb-IFN administered at 2- or 4-week intervals in combination with ribavirin.

Dose-ranging phase 2 studies. Three phase 2 trials were conducted to evaluate the efficacy and safety of alb-IFN in combination with ribavi-rin therapy for the treatment of CHC. These included studies in IFN-experienced (non-responders to IFN-α-based therapy) and IFN-naive (genotypes 1, 2 or 3) individuals.

The long-term safety and efficacy of alb-IFN combined with oral rib-avirin was evaluated in 115 individuals with CHC who were defined as

non-responders on the basis of their failure to clear HCV RNA following therapy with previ-ous IFN-α-based regimens61. Individuals were initially randomized into three alb-IFN treat-ment cohorts (900 or 1,200 µg every 2 weeks or 1,200 µg every 4 weeks) in combination with oral weight-based ribavirin (1,000–1,200 mg d–1). Following the evaluation of safety data, which showed acceptable tolerability, two higher-dose treatment groups of alb-IFN (1,500 and 1,800 µg every two weeks) were enrolled. The higher dose cohorts contained a higher proportion of individuals with baseline characteristics associated with poor response (for example, pretreatment HCV RNA, pre-vious PEG-IFN/ribavirin non-response, African-American race). The overall safety profile for doses up to 1,800 µg every two weeks was acceptable. Of note, hematological reductions reached a plateau by weeks 4 to 8 of therapy, with complete recovery following treatment cessation. The antiviral response was greatest in the 1,500- and 1,800-µg biweekly groups in the subgroup of genotype 1-infected non-responders to prior PEG-IFN/ribavirin therapy compared with lower dose groups (Fig. 5a) (Nelson, D.R., et al., personal communication, The Liver Meeting, Boston, Massachusetts, 2–6 November, 2007). Drug accumulation was observed in the biweekly cohorts between weeks 2 and 12, with mini-mal change in concentrations after week 12 (Fig. 5b). No drug accumulation was observed in individuals undergoing treatment with 1,200 µg every four weeks.

The efficacy and safety of alb-IFN was com-pared with those of PEG-IFN-α-2a in 458 IFN-naive individuals with genotype 1 CHC62. The four treatment arms included PEG-IFN-α-2a (180 µg every week) and three alb-IFN groups (900 and 1,200 µg every two weeks, and 1,200 µg every four weeks). The primary effi-cacy endpoint was SVR; secondary endpoints included antiviral response profiles at weeks 4 and 12, which have previously been shown to be highly predictive of SVR41. The individu-als receiving 1,200 µg every two weeks showed the highest levels of antiviral activity, with the greatest proportion of individuals achieving HCV RNA below LOQ at weeks 4 and 12 (Fig. 5c). As early as week 2 after a single dose of alb-IFN, the proportion of individuals who achieved a reduction of ≥2 log in HCV RNA was significantly higher in the group receiving

Phase 2 clinical pharmacology: IFN nonresponders and naive CHC patients

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Figure 5 Clinical pharmacology in IFN non-responders and treatment-naive individuals with CHC. (a,b) Responses to alb-IFN of non-responders to PEG-IFN plus ribavirin (RBV) therapy. (a) HCV RNA reduction over 24 weeks in HCV genotype (Gt) 1-infected PEG-IFN+RBV non-responders (5 treatment groups). Antiviral response was greater in the higher dose alb-IFN groups (1,500 µg and 1,800 µg every two weeks) than in the lower dose groups, despite including a greater proportion of hard-to-treat individuals61. (b) Albinterferon-α-2b accumulation through week 24 of treatment after administration of 1,500 µg every two weeks. Dashed lines denote mean drug concentrations for individuals at each time point. There was a minimal increase in alb-IFN concentration after week 12 (ref. 61). (c,d) Responses of IFN-naive individuals (Gt 1 HCV) to alb-IFN. (c) Summary of virologic response at weeks 2, 4 and 12 in IFN-naive, HCV Gt 1 participants (4 treatment groups). Response rate is represented as either percentage less than lower limit of quantification (LOQ = 43 IU ml–1) or as percentage ≥2 log decrease. The group receiving 1,200 µg alb-IFN every two weeks showed the greatest response at weeks 2 and 4 (rapid virological response; RVR4) and 12 (early virological response; EVR12). The group receiving 900 µg alb-IFN every two weeks had an EVR profile comparable to that of individuals receiving PEG-IFN. The group receiving 1,200 µg alb-IFN every four weeks showed lower RVR4 and EVR1262. (d) Hematological reductions (shown by reduction in absolute neutrophil count (ANC)) reached a plateau by week 8 and recovered rapidly after the end of treatment. These reductions were lowest in the group receiving 1,200 µg alb-IFN every four weeks and were comparable across the remaining cohorts (alb-IFN every two weeks and PEG-IFN)62. F12 and F24 (12 and 24 weeks after the end of treatment) include all data, including early discontinuations.

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1,200 µg alb-IFN every two weeks than in those receiving PEG-IFNα-2a. Individuals receiving 900-µg alb-IFN every two weeks had an overall antiviral response profile that was similar to that of individuals receiving PEG-IFN-α-2a. The SVR rates were comparable between individuals receiving alb-IFN every two weeks and those receiving PEG-IFN-α-2a, but slightly lower in individuals receiving 1,200 µg alb-IFN every four weeks62. Hematological reductions, assessed by reductions in absolute neutrophil counts, reached a plateau by week 8 and recovered rapidly after the end of treatment (Fig. 5d). Of note, absolute neutrophil count reductions were lowest in the group receiving 1,200 µg alb-IFN every four weeks, and were comparable between individuals receiving alb-IFN every two weeks and PEG-IFN-α-2a. The incidence and severity of adverse events were similar for individuals receiving 900 µg alb-IFN every two weeks and those receiving PEG-IFN-α-2a, but slightly higher in those receiving 1,200 µg alb-IFN every two weeks. This study showed significantly lower rates of immunogenicity with alb-IFN than with PEG-IFN-α-2a (P < 0.001)63. Although the clinical relevance of this finding remains unclear, the data indicate that genetic fusion to albumin with a resultant novel polypeptide molecule might be associ-

ated with reduced immunogenicity. Last, on the basis of assessments at weeks 12 and 24 (the main decision points for continuing treatment) using the validated medical outcomes survey-Short Form 36, version 2 (SF-36 v2) and missed workdays, alb-IFN (especially the 900 µg every two weeks regimen) also showed potential to provide better HRQOL than PEG-IFN-α-2a (Pianko, S., et al., personal communication, The Liver Meeting, Boston, Massachusetts, 2–6 November 2007).

To investigate the safety and efficacy of a higher dose of alb-IFN (1,500 µg every four weeks), a study was conducted in IFN-naive indi-viduals with genotype 2 or 3 HCV64. The observed SVR rate of 77% in individuals receiving 1,500 µg every four weeks and acceptable safety profile further justified continued evaluation of this dosing regimen. Although phase 3 studies are now underway to enable the approval of alb-IFN administered every two weeks, a dose-finding study to evaluate the feasibility of higher alb-IFN doses administered every four weeks is also planned.

Phase 3 program. Treatment with 900 µg or 1,200 µg alb-IFN every two weeks is currently being evaluated in two global, randomized,

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Phase: 2Design: RandomizedPopulation: IFN naive, Gt 1N: 56alb-IFN dose: 200-1,200 µgStudy duration: 42 d (2 injections: d 0 and 14); 5 dose cohortsSafety, PK/PD Publication: Bain et al. 2006 (ref. 56) Phase: 2

Design: RandomizedPopulation: IFN naive, Gt 2/3N: 43alb-IFN dose: 1,500 µg Q4w and Q2w†

Treatment duration: 24 wk; follow-up 24 wkSafety and efficacy (SVR)

Phase: 2Design: Randomized with dose escalationPopulation: IFN nonrespondersN: 115alb-IFN dose: 900 and 1,200 µg Q2w, 1,200 µg Q4w (randomized);

dose escalation, 1,500 and 1,800 µg Q2w†

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Phase: 2bDesign: Active controlled, randomizedPopulation: IFN naive, Gt 1N: 458alb-IFN dose: 900 and 1,200 µg Q2w, 1,200 µg Q4w† Treatment duration: 48 wk; follow-up 24 wkActive control: PEG-IFNα-2a 180 µg Qw†

Safety and efficacy (SVR)

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Phase: 1/2Design: Sequential dose escalationPopulation: IFN experiencedN: 119alb-IFN dose: 10-1,200 µgStudy duration: 56-70 d (2 injections: d 0 and 14); 22 dose cohortsSafety, PK/PD Publication: Balan et al., 2006 (ref. 57)

Phase: 3 Design: Multicenter, randomized Population: IFN naive, Gt 1 N: 1,278; 426/armalb-IFN dose: 900, 1,200 µg Q2w†

Treatment duration: 48 wk Active control: PEG-IFNα-2a 180 µg Qw†

Primary efficacy endpoint: SVR

Phase: 3 Design: Multicenter, randomized Population: IFN naive, Gt 2/3

alb-IFN dose: 900, 1,200 µg Q2w†

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Primary efficacy endpoint: SVR

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Figure 6 Clinical development program overview. The program for alb-IFN includes two completed phase 1/2 studies, three recently completed phase 2 studies and two ongoing pivotal phase 3 studies (ACHIEVE-1 in HCV genotype (Gt) 1 individuals and ACHIEVE-2/3 in HCV Gt 2 or 3 individuals). The ACHIEVE trials are global, randomized, active-controlled trials designed to evaluate the efficacy, safety and impact on HRQOL of alb-IFN (900 and 1,200 µg every two weeks) versus PEG-IFN-α-2a (180 µg every week) (all in combination with ribavirin). Targets of at least 426 individuals per treatment group will be randomized for the ACHIEVE-1 study and 306 individuals per treatment group for the ACHIEVE-2/3 study. This sample size will provide 90% power to establish non-inferiority of each alb-IFN dose level relative to PEG-IFN-α-2a, with an overall 1-sided α = 0.025 significance level. PK/PD, pharmacokinetics/pharmacodynamics. In phase 3 trials, all treatment arms include RBV (1,000–1,200 mg d–1 (Gt 1) or 800 mg d–1 (Gt 2/3)).

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active-controlled, phase 3 trials (Fig. 6). The objectives of these trials are to compare the efficacy, safety and impact on HRQOL of alb-IFN (900 µg and 1,200 µg every two weeks) with those of PEG-IFN-α-2a (all in combination with ribavirin). These studies will provide 90% power to establish non-inferiority of each alb-IFN dose relative to PEG-IFN-α-2a65. If non-inferiority is established for both alb-IFN groups, the primary analysis will continue sequentially to assess supe-riority, with 80% power to detect an SVR difference of 10% between alb-IFN and PEG-IFN-α-2a. In addition, these studies are adequately powered for the main secondary HRQOL endpoint analyses.

The rationale for dose selection is summarized as follows: the 900 µg every two weeks dose showed a similar antiviral response and safety profile to those of PEG-IFN-α2a, and favorable HRQOL, whereas the 1200 µg every two weeks dose provided the greatest early antiviral response compared with PEG-IFN-α2a. The efficacy of the 900 µg every two weeks regimen provides support for dose reductions in the higher dose 1,200 µg every two weeks arm to enable optimal dose titration based on the individual’s tolerability in the phase 3 program.

Conclusions and future directionsAlbinterferon α-2b represents the initial clinical validation of the albumin-fusion technology platform. Clinical results so far confirm that the efficacy and safety of alb-IFN plus ribavirin compare favor-ably with those of PEG-IFN-α-2a plus ribavirin. These promising findings are now undergoing evaluation in pivotal phase 3 trials; if they are confirmed, alb-IFN has the potential to become an impor-tant therapy for CHC66. The pharmacodynamic attributes of alb-IFN, which include the maintenance of viral suppression across a longer dosing interval, might improve compliance and prevent the devel-opment of viral resistance when combined with novel, direct anti-viral therapies67. In addition, multiple-drug combination regimens that include alb-IFN in conjunction with novel antiviral agents (for example, viral protease inhibitors) might benefit the growing num-ber of individuals who are refractory to PEG-IFN therapy and the high proportion of individuals with HIV-1 who are co-infected with HCV38,68. For these co-infected individuals, new treatment options are particularly urgent; combination regimens that include alb-IFN could help to reduce the high morbidity and mortality seen in this population.

Albinterferon α-2b also has potential clinical utility in other diseases. Combining the immunomodulatory and antiviral activity of a well-tolerated IFN with direct and potent suppression of viral replication might improve clinical outcomes in the treatment of chronic hepatitis B69; with a prevalence of more than 300 million people, this infection represents an important global issue. Advances in cancer biology are beginning to elucidate the molecular mechanisms that underlie IFN activity, including the upregulation of cell death receptors40 and modu-lation of the central apoptosis regulator nuclear factor κB (NF-κB)70,71. In preliminary studies, alb-IFN showed potent antiproliferative activity in Daudi cells (human Burkitt’s B-cell lymphoma line), indicating that this agent might be useful in the treatment of certain malignancies17. New therapeutic advances that target apoptosis directly or indirectly (for example, raf kinase inhibitors) provide a rationale for combination therapy with IFN-α72. The pharmacology of alb-IFN, with its sustained high exposure and favorable tolerability, might prove useful in com-bination therapy for various malignancies. Given the growing human proteome database and improved understanding of the role of other proteins in disease pathophysiology, albumin-fusion technology could rapidly accelerate the development of new treatment approaches across a broad spectrum of human diseases.

ACKNOWLEDGMENTSWe thank BioScience Communications, New York, for editorial support.

COMPETING INTERESTS STATEMENTThe authors declare competing financial interests: details accompany the full-text HTML version of the paper at http://www.nature.com/naturebiotechnology/.

Published online at http://www.nature.com/naturebiotechnology/Reprints and permissions information is available online at http://npg.nature.com/reprintsandpermissions

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