WO2012146630A1

WO2012146630A1 - N-terminal acylated polypeptides, methods for their production and uses thereof

Info

Publication number: WO2012146630A1
Application number: PCT/EP2012/057592
Authority: WO
Inventors: Stefan Jenewein; Stefan Klostermann; Erhard Kopetzki; Stefan Lorenz; Joerg Thomas Regula; Georg Tiefenthaler
Original assignee: F. Hoffmann-La Roche Ag
Priority date: 2011-04-29
Filing date: 2012-04-26
Publication date: 2012-11-01

Abstract

Herein is reported a fusion protein comprising in N- to C-terminal direction a hepatitis-B-virus preSl -domain derived amino acid sequence, a first optional linker polypeptide, an antibody heavy chain Fc-region, a second optional linker polypeptide, and optionally a biologically active polypeptide as well as its production and use.

Description

N-terminal acylated polypeptides, methods for their production and uses thereof Herein are reported N-terminal acylated polypeptides as well as a method for their production, especially by in vitro acylation, and uses of the N-terminally acylated polypeptides.

Background of the Invention

An important hepatotropic component of the Hepatitis-B-virus (HBV) is encoded within the hydrophilic and myristoylated preSl -domain of the large viral envelope/surface protein (L-protein) (Neurath, A.R., et al, Cell 46 (1986) 429- 436).

During synthesis, the L-protein is myristoylated at the glycine residue penultimate to the N-terminal methionine of the preSl -domain and translocated through the endoplasmic reticulum (Persing, D.H., et al, J. Virol. 61 (1987) 1672-1677). This posttranslational modification and the integrity of the preSl -domain are essential for HBV infectivity but not virion assembly (Gripon, P., et al, Virology 213 (1995) 292-299). Mutation of this penultimate glycine at amino acid position 2, as well as the next glycine, at amino acid position 13 did not interfere with secretion (WO 2008/103380). Numbering of the preSl-domain amino acids refer to genotype

A of the HBV.

Specifically, previous studies have reported a cryptic N-glycosylation site motif (NLS) at amino acid position 15 (Hepatitis-B-virus genotype A subtype adw2) (Bruss, V. and Vieluf, K., J. Virol. 69 (1995) 6652-6657). Mutation of this asparagine to glutamine did not have an observable effect on secretion

(WO 2008/103380).

Furthermore, myristoylation was occurring during secretion on immunoadhesins containing the wild-type (wt) preS 1(1-119)-domain fused N-terminally to the Fc-domain of a rabbit immunoglobulin heavy chain but blocked on mutated preSl-forms (G2A, G13A) (Chai, N., et al, J. Virol. 81 (2007) 4912-4918). Both mutated immunoadhesins were tested for their ability to be labeled with ³H myristoic acid. Only the wild-type form was labeled. Also, it was observed that a C-terminally truncated (deletion of aa position 60-119) preSl(l-59)-domain was both myristoylated and secreted. It has been shown that peptides derived from the preSl -domain of the HBV large viral surface protein are able to interfere with infection of primary hepatocytes and of HepaRG cells, a cell line made from a differentiated liver tumor (Gripon, P., et al, J. Virol. 79 (2005) 1613-1622, Gripon, P., et al, Proc. Natl. Acad. Sci. USA 99 (2002) 15655-15660). Furthermore, it is known that N-terminal acylation of the preSl -domain potently enhanced virus entry inhibition ability of such peptides (Gripon, P., et al, J. Virol. 79 (2005) 1613-1622). Another known interfering N-terminally acylated synthetic peptide is derived from the preSl -domain of HBV genotype D and subtype ayw which corresponds to amino acid position 2-48 and which is myristoylated at the N-terminal glycine as reported by Engelke, M., et al., in Hepatology 43 (2006) 750-760.

In Chinese patent application CN 101045156 a specially targeting medicine and its application is reported. WO 2009/092611 and WO 2009/092396 report hydrophobic modified preS-derived peptides of hepatitis-B-virus and their use as HBV and HDV entry inhibitors. Hepatitis-B-virus compositions and methods of use are reported in WO 2008/103380. In WO 02/094866 a pre-S protein of hepatitis-B-virus as an adjuvant and a composition of HBV vaccine are reported. Chai, N., et al. report that immunoadhesins containing pre-S domains of hepatitis B virus large envelope protein are secreted and inhibit virus infection (J. Virol. 81 (2007) 4912-4918.

Summary of the Invention

It has been found that it is advantageous for enzymatic in vitro N-terminal acylation of polypeptides to remove (potential) N-glycosylation sites present within fifty amino acid residues from the site of acylation. Thus, herein is reported an N-terminal peptide derived from hepatitis-B-virus preSl -domain that can be acylated enzymatically in vitro, especially at the N-terminus. Herein are also reported fusion proteins comprising this peptide.

One aspect as reported herein is a method for the production of an (N-terminally) acylated polypeptide comprising the step of: - acylating the polypeptide (N-terminally) in vitro and thereby producing an

N-terminal acylated polypeptide, whereby the polypeptide has no actual or potential N-glycosylation site within the first fifty N-terminal amino acids. In one embodiment the actual or potential N-glycosylation site is removed by determining the position of actual or potential N-glycosylation sites in the amino acid sequence of the polypeptide within the first fifty N-terminal amino acid residues of the polypeptide's amino acid sequence. In another embodiment the removing of the actual or potential N-glycosylation site is by modifying the

N-glycosylation site motif (Asn-X-Ser/Thr, sequon) by changing the amino acid residue asparagine to a non-asparagine amino acid residue, or by changing the amino acid residue serine or threonine to a non-serine or non-threonine residue. In one embodiment the modifying is by changing the codon encoding the asparagine residue in the N-glycosylation site motif to a codon encoding a different, non- asparagine amino acid residue, or by changing the codon encoding the serine residue in the N-glycosylation site motif to a codon encoding a different, non- serine amino acid residue, or by changing the codon encoding the threonine residue in the N-glycosylation site motif to a codon encoding a different, non-threonine amino acid residue .

In one embodiment the polypeptide has no actual or potential N-glycosylation site within the first twenty N-terminal amino acid residues.

In one embodiment the amino acid residue asparagine is changed to a neutral hydrophilic amino acid residue. In one embodiment the neutral hydrophilic amino acid residue is glutamine.

In one embodiment the codon encoding the asparagine residue of the actual or potential N-glycosylation site is modified to encode a neutral hydrophilic amino acid residue. In also an embodiment the neutral hydrophilic amino acid residue is glutamine. In one embodiment the polypeptide is a fusion protein comprising at the

N-terminus a hepatitis-B-virus preSl -domain derived polypeptide.

In another embodiment the polypeptide is a fusion protein comprising an antibody heavy chain Fc-region.

In one embodiment the polypeptide is a fusion protein comprising in N- to C- terminal direction a hepatitis-B-virus preSl -domain derived amino acid sequence, optionally a first linker polypeptide, an antibody heavy chain Fc-region, optionally a second linker polypeptide, and a biologically active polypeptide.

In one embodiment a) the preSl -domain derived polypeptide has an amino acid sequence selected from SEQ ID NO: 01, or SEQ ID NO: 02, or SEQ ID NO: 30, and/or b) the first and second polypeptide linker have an amino acid sequence independently selected from SEQ ID NO: 03 to SEQ ID NO: 08, and/or c) the antibody heavy chain Fc-region has an amino acid sequence selected from SEQ ID NO: 16 to SEQ ID NO: 20, and/or d) the biologically active polypeptide has the amino acid sequence of SEQ ID NO: 21.

In one embodiment the acylating of the polypeptide is by incubating the polypeptide in vitro with an acyl transferase and an activated carbonic acid. In one embodiment the acyl transferase is yeast N-myristoyl transferase (NMT) or a variant thereof. In one embodiment the activated carbonic acid is myristoyl-CoA.

Also an aspect as reported herein is a preSl -domain derived polypeptide that has the amino acid sequence of SEQ ID NO: 30.

Also an aspect as reported herein is an N-terminal acylated polypeptide comprising as N-terminal amino acid residues/part a preSl -domain derived polypeptide that has the amino acid sequence of SEQ ID NO: 01, or SEQ ID NO: 02, or SEQ ID NO: 30.

Another aspect as reported herein is an N-terminal acylated fusion protein comprising an N-terminal amino acid sequence of SEQ ID NO: 01, or SEQ ID NO: 02, or SEQ ID NO: 23, or SEQ ID NO: 24, or SEQ ID NO: 25, or SEQ ID NO: 26, or SEQ ID NO: 27, or SEQ ID NO: 28, or SEQ ID NO: 29.

In one embodiment the N-terminal acylated fusion protein further comprises an antibody heavy chain Fc-region with an amino acid sequence selected from SEQ ID NO: 16 to 20.

In one embodiment the N-terminal acylated fusion protein further comprises the amino acid sequence of SEQ ID NO: 21.

Also an aspect as reported herein is an N-myristoyl transferase variant that has the amino acid sequence of SEQ ID NO: 22. Another aspect as reported herein is a complex comprising an N-terminal acylated protein, which is a fusion protein comprising an N-terminal amino acid sequence of SEQ ID NO: 23, and a polypeptide, which comprises an amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18. Another aspect as reported herein is a complex comprising an N-terminal acylated protein, which is a fusion protein comprising an N-terminal amino acid sequence of SEQ ID NO: 24, and a polypeptide, which comprises an amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18.

Another aspect as reported herein is a complex comprising an N-terminal acylated protein, which is a fusion protein comprising an N-terminal amino acid sequence of SEQ ID NO: 26, and a polypeptide, which comprises an amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18.

Another aspect as reported herein is a complex comprising an N-terminal acylated protein, which is a fusion protein comprising an N-terminal amino acid sequence of SEQ ID NO: 27, and a polypeptide, which comprises an amino acid sequence of

SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18.

Another aspect as reported herein is a complex comprising an N-terminal acylated fusion protein that has the amino acid sequence of SEQ ID NO: 27 and an N- terminal acylated fusion protein that has the amino acid sequence of SEQ ID NO: 28.

Another aspect as reported herein is a complex comprising an N-terminal acylated fusion protein that has the amino acid sequence of SEQ ID NO: 27 and a polypeptide, which comprises an amino acid sequence of SEQ ID NO: 36.

Another aspect as reported herein is a complex comprising an N-terminal acylated fusion protein that has the amino acid sequence of SEQ ID NO: 28 and a polypeptide, which comprises an amino acid sequence of SEQ ID NO: 37.

A further aspect as reported herein is the use of an N-terminal acylated fusion protein as reported herein for the treatment of hepatitis virus infection.

In one embodiment the hepatitis virus infection is a hepatitis-B-virus infection. In one embodiment the hepatitis virus infection is a hepatitis-C-virus infection. In one embodiment the hepatitis virus infection is a hepatitis-D-virus infection. In one embodiment the hepatitis virus infection is an acute or a chronic hepatitis-B- virus infection, or hepatitis-C-virus infection, or hepatitis-D-virus infection.

The invention further provides an isolated nucleic acid encoding the fusion protein as reported herein. Also provided are isolated nucleic acids encoding an antibody heavy chain as reported herein. Further provided is an isolated nucleic acid encoding the antibody light chain as reported herein.

The invention also provides a host cell comprising one or more of the nucleic acids as reported herein.

Also provided is a method of producing a fusion protein as reported herein comprising culturing a host cell as reported herein so that the fusion is produced. In one embodiment the method comprises the following steps: (a) providing a cell as reported herein, (b) cultivating the provided cell, (c) recovering the fusion protein from the cell or the cultivation medium and thereby producing the fusion protein.

Herein is also provided a pharmaceutical formulation comprising the fusion protein as reported herein and a pharmaceutically acceptable carrier.

The invention further provides the fusion protein as reported herein for use as a medicament.

The invention also provides the fusion protein as reported herein for use in treating hepatitis-B-virus infection. The invention also provides the fusion protein as reported herein for use in delivering an anti-viral cytokine to hepatitis-B-virus infected hepatocytes.

The invention also provides the use of the fusion protein as reported herein in the manufacture of a medicament. In one embodiment the medicament is for the treatment of hepatitis-B-virus infection. In a further embodiment the hepatitis-B- virus infection is a chronic infection. In also an embodiment the medicament is for delivering an anti-viral cytokine to hepatitis-B-virus infected hepatocytes.

The invention provides a method of treating an individual having a hepatitis-B- virus infection comprising administering to the individual an effective amount of the fusion protein as reported herein. The invention also provides a method of delivering an anti-viral cytokine to hepatitis-B-virus infected hepatocytes in an individual comprising administering to the individual an effective amount of the fusion protein as reported herein to deliver an anti-viral cytokine to hepatitis-B-virus infected hepatocytes.

One aspect as reported herein is a dimeric antibody Fc-region fusion polypeptide comprising an antibody Fc-region, a hepatitis-B-virus preSl -domain derived polypeptide, and a biologically active polypeptide. In one embodiment the dimeric

Fc-region fusion polypeptide comprises exactly one, i.e. a single, hepatitis-B-virus preSl -domain derived polypeptide, and exactly one, i.e. a single, biologically active polypeptide.

In one embodiment the antibody Fc-region comprises two antibody heavy chain fragments starting with the amino acid sequence DKTHT in the antibody heavy chain hinge region and ending with the C-terminal amino acid of an antibody CH3 domain.

In one embodiment the biologically active polypeptide is human interferon alpha 2a. In one embodiment a hepatitis-B-virus preSl -domain derived polypeptide is fused independently of each other either directly or via a linker polypeptide to the N- terminus of each of the chains of the dimeric Fc-region fusion polypeptide.

In one embodiment the preSl -domain derived polypeptide is fused to the N- terminus of only one chain of the Fc-region polypeptides. In one embodiment the biologically active polypeptide is fused either directly or via a linker polypeptide to the N-terminus or the C-terminus of one chain of the Fc- region polypeptides.

In one embodiment the biologically active polypeptide is fused to the C-terminus of one chain of the dimeric Fc-region fusion polypeptide. In one embodiment the preSl -domain derived polypeptide is fused to the N- terminus and the biologically active polypeptide is fused to the respective C- terminus of the same chain of the dimeric Fc-region fusion polypeptide.

In one embodiment the preSl -domain derived polypeptide is fused to the N- terminus and the biologically active polypeptide is fused to the respective C- terminus of different chains of the dimeric Fc-region fusion polypeptide. In one embodiment the dimeric Fc-region fusion polypeptide comprises knob mutation in one antibody heavy chain fragment and a hole mutation in the respective other antibody heavy chain fragment.

Detailed Description of the Invention I. DEFINITIONS

The term "nucleic acid" denotes a polymeric molecule consisting of the individual nucleotides (also called bases) a, c, g, and t (or u in RNA), for example, DNA or R A or modifications thereof. This polynucleotide molecule can be a naturally occurring polynucleotide molecule or a synthetic polynucleotide molecule or a combination of one or more naturally occurring polynucleotide molecules with one or more synthetic polynucleotide molecules. Also encompassed by this definition are naturally occurring polynucleotide molecules in which one or more nucleotides are changed (e.g. by mutagenesis), deleted, or added. A nucleic acid can either be isolated, or integrated in another nucleic acid, e.g. in an expression cassette, a plasmid, or the chromosome of a host cell. A nucleic acid is likewise characterized by its nucleic acid sequence consisting of individual nucleotides.

To a person skilled in the art procedures and methods are well known to convert an amino acid sequence, e.g. of a polypeptide, into a corresponding nucleic acid sequence encoding this amino acid sequence. Therefore, a nucleic acid is characterized by its nucleic acid sequence consisting of individual nucleotides and likewise by the amino acid sequence of a polypeptide encoded thereby.

The term "glycosylated" or grammatical equivalents thereof denote that the respective polypeptide comprises at least one saccharide residue covalently linked to an amino acid of the amino acid backbone of the polypeptide. The term "N-glycosylation site" (sequon) denotes an N-glycosylation site amino acid motifs comprising the amino acid sequence asn-X-thr, asn-X-ser, or asn-X-cys, wherein X can be any amino acid residues but not proline (pro, P).

The term "amino acid" denotes the group of carboxy a-amino acids, which directly or in form of a precursor can be encoded by a nucleic acid. The individual amino acids are encoded by nucleic acids consisting of three nucleotides, so called codons or base-triplets. Each amino acid is encoded by at least one codon. This is known as "degeneration of the genetic code". The term "amino acid" as used within this application denotes the naturally occurring carboxy a-amino acids comprising alanine (three letter code: ala, one letter code: A), arginine (arg, R), asparagine (asn, N), aspartic acid (asp, D), cysteine (cys, C), glutamine (gin, Q), glutamic acid (glu, E), glycine (gly, G), histidine (his, H), isoleucine (ile, I), leucine (leu, L), lysine (lys, K), methionine (met, M), phenylalanine (phe, F), proline (pro, P), serine (ser, S), threonine (thr, T), tryptophan (trp, W), tyrosine (tyr, Y), and valine

(val, V).

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Met, Ala, Val, Leu, Ile;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu;

(4) basic: His, Lys, Arg;

(5) aromatic: Trp, Tyr, Phe.

An "expression cassette" refers to a nucleic acid construct that contains the necessary regulatory elements, such as promoter and polyadenylation site, for expression of at least the contained encoding nucleic acid in a cell. An "expression plasmid" is a nucleic acid providing all required elements for the expression of the contained expression cassette(s) in a (host) cell. Typically, an expression plasmid comprises a prokaryotic plasmid propagation unit, e.g. for E. coli, comprising an origin of replication, and a selectable marker, an eukaryotic selection marker, and one or more expression cassettes for the expression of the structural gene(s) of interest each comprising a promoter, a structural gene, and a transcription terminator including a polyadenylation signal. Gene expression is usually placed under the control of a promoter, and such a structural gene is said to be "operably linked to" the promoter. Similarly, a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.

"Operably linked" refers to a juxtaposition of two or more components, wherein the components so described are in a relationship permitting them to function in their intended manner. For example, a promoter and/or enhancer are operably linked to a coding sequence, if it acts in cis to control or modulate the transcription of the linked sequence. Generally, but not necessarily, the DNA sequences that are "operably linked" are contiguous and, where necessary to join two protein encoding regions such as a secretory leader and a polypeptide, contiguous and in (reading) frame. A "complete antibody heavy chain" comprises a variable domain (variable region) (generally the amino terminal portion), which comprises binding regions that are able to interact with an antigen, and a constant region (generally the carboxyl terminal portion). The constant region of the heavy chain mediates the binding of the antibody i) to cells bearing a Fc gamma receptor (FcyR), such as phagocytic cells, or ii) to cells bearing the neonatal Fc receptor (FcRn) also known as Brambell receptor. It also mediates the binding to some factors including factors of the classical complement system such as component (Clq). Depending on the amino acid sequence of the constant region of the heavy chains, antibodies (immunoglobulins) are divided in the classes: IgA, IgD, IgE, IgG, and IgM. Some of these classes are further divided into subclasses (isotypes), i.e. IgG in IgGl, IgG2, IgG3, and IgG4, or IgA in IgAl and IgA2. According to the immunoglobulin class to which an antibody belongs are the heavy chain constant regions of immunoglobulins are called oc(IgA), δ (IgD), ε (IgE), γ (IgG), and μ (IgM), respectively. An "Fc-region" is a term well known to the skilled artisan and defined on basis of the papain cleavage of antibodies. In one embodiment the Fc-region is of a human antibody of the subclass IgG4 or a human antibody of the subclass IgGl, IgG2, or IgG3. In one embodiment the Fc-region is of a human antibody of the subclass IgGl with mutations L234A and L235A (numbering according to Kabat (see e.g. Kabat, E.A., et al., Sequences of Proteins of Immunological

Interest, 5^th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242, Vols. 1-3). In another embodiment the Fc-region is of a human antibody of the subclass IgGl with mutations L234A, L235A, and P329G (numbering according to Kabat). In one embodiment the Fc-region is of a human antibody of the subclass IgG4 with mutations S228P and L235E. While

IgG4 shows reduced Fey receptor (FcyRIIIa) binding, antibodies of other IgG subclasses show strong binding. However Pro238, Asp265, Asp270, Asn297 (loss of Fc carbohydrate), Pro329, Leu234, Leu235, Gly236, Gly237, Ile253, Ser254, Lys288, Thr307, Gln311, Asn434, or/and His435 are residues which, if altered, provide also reduced Fey receptor binding (see e.g. Shields, R.L., et al, J. Biol. Chem. 276 (2001) 6591-6604; Lund, J., et al, FASEB J. 9 (1995) 115-119; Morgan, A., et al, Immunology 86 (1995) 319-324; EP 0 307 434).

Interferon, in particular interferon a2, is a pharmaceutically active protein, which has anti-viral and anti-proliferative activity. For example, interferon is used to treat hairy cell leukemia and Kaposi's sarcoma, and is active against hepatitis. In order to improve stability and solubility, and reduce immunogenicity, pharmaceutically active proteins such as interferon may be conjugated to the polymer polyethylene glycol (PEG) (see EP-B 0 809 996). The amino acid sequence of human interferon a2a is shown in SEQ ID NO: 21. In one embodiment human interferon alpha 2a has the amino acid sequence of SEQ ID NO: 21.

The term "anti-viral cytokine" denotes a cytokines that mediates the establishment of an anti-viral response after infection and recruits inflammatory cells to the site of infection. Anti-viral cytokines comprise type I (interferon (IFN)-a and IFN-β), type II (IFN-γ) and type III (IFN-λ or interleukin (IL)-28/29) interferon. Interferon α, β, γ and λ are important interferons produced in the innate immune response to viral infections. The terms "antibody heavy chain Fc-region" or "IgG Fc-region" denote a part of a full length antibody heavy chain, which comprises the hinge region, the CH2 domain and the CH3 domain. An IgG Fc-region is a dimeric polypeptide comprising two disulfide-linked polypeptide chains, whereby the chains have the same or a different amino acid sequence and each of the polypeptide chains is a C- terminal fragment of a full length antibody heavy chain. Thus, an Fc-region is a dimeric polypeptide comprising two disulfide-linked C-terminal antibody heavy chain polypeptides whereby each C-terminal antibody heavy chain polypeptide comprises at least a part of an antibody hinge-region containing at least one cysteine residue, an antibody CH2 domain, and an antibody CH3 domain. Alternatively the antibody heavy chain Fc-region can be defined to be the fragment obtained by Papain cleavage of a full length antibody. In particular, the term "Fc- region" denotes the C-terminal region of an immunoglobulin heavy chain that contains at least a portion of the constant region. The term includes native sequence Fc-regions and Fc-regions variants. In one embodiment, a human IgG heavy chain Fc-region extends from about amino acid residue 226 (Cys), or from about amino acid residue 230 (Pro), to the carboxy-terminus of the heavy chain. However, the C-terminal lysine residue (Lys447) of the Fc-region may or may not be present. Unless otherwise specified herein, numbering of amino acid residues of antibody light and heavy chains is according to the EU numbering system, also called the EU index, as described in Kabat, E.A., et al, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication No. 91-3242, Vols. 1-3. The Fc- region is a dimeric molecule comprising two antibody heavy chain C-terminal regions linked via one or more disulfide bonds. The terms "host cell", "host cell line", and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants", "transformed cells" and "transfected cells", which include the primary transformed cell and progeny derived therefrom without regard to the number of passages.

Progeny may not be completely identical in nucleic acid content to a parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein. The term "pharmaceutical formulation" refers to a preparation which is in such form as to permit the biological activity of an active ingredient contained therein to be effective, and which contains no additional components which are unacceptably toxic to a subject to which the formulation would be administered.

A "pharmaceutically acceptable carrier" refers to an ingredient in a pharmaceutical formulation, other than an active ingredient, which is nontoxic to a subject. A pharmaceutically acceptable carrier includes, but is not limited to, a buffer, excipient, stabilizer, or preservative.

As used herein, "treatment" (and grammatical variations thereof such as "treat" or "treating") refers to clinical intervention in an attempt to alter the natural course of the individual being treated, and can be performed either for prophylaxis or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, preventing occurrence or recurrence of disease, alleviation of symptoms, diminishment of any direct or indirect pathological consequences of the disease, preventing metastasis, decreasing the rate of disease progression, amelioration or palliation of the disease state, and remission or improved prognosis. In some embodiments, antibodies of the invention are used to delay development of a disease or to slow the progression of a disease.

The term "type I interferon" denotes interferons that bind to the cell surface receptor complex which consists of IFNAR1 and IFNAR2 protein chains (the IFN-a receptor, IFNAR). The type I interferons present in humans comprise interferon a, interferon β and interferon ω.

The term "type II interferon" denotes interferons that bind to the interferon-gamma receptor (IFNGR). The type II interferons present in humans comprise interferon γ. The term "type III interferon" denotes interferons that signal through a receptor complex consisting of class II cytokine receptor (CIICR) IL10R2 and IFNLR1. The type III interferon group consists of 3 IFN-λ molecules called IFN-λΙ, ΙΚΝ-λ2 and ΙΚΝ-λ3 (also called interleukin-29, interleukin-28A and interleukin-28B, respectively).

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents a cellular function and/or causes cell death or destruction. Cytotoxic agents include, but are not limited to, radioactive isotopes (e.g. At²¹¹, 1¹³¹, 1¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu); chemotherapeutic agents or drugs (e.g. methotrexate, adriamicin, vinca alkaloids

(vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents); growth inhibitory agents; enzymes and fragments thereof such as nucleolytic enzymes; antibiotics; toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, including fragments and/or variants thereof; and the various antitumor or anticancer agents disclosed below.

"Effector functions" refer to those biological activities attributable to the Fc region of an antibody, which vary with the antibody isotype. Examples of antibody effector functions include: Clq binding and complement dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity

(ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor); and B cell activation.

An "effective amount" of an agent, e.g. a pharmaceutical formulation, refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic or prophylactic result.

An "immunoconjugate" is a fusion protein conjugated to one or more heterologous molecule(s), including but not limited to a cytotoxic agent.

An "individual" or "subject" is a mammal. Mammals include, but are not limited to, domesticated animals (e.g. cows, sheep, cats, dogs, and horses), primates (e.g. humans and non-human primates such as monkeys), rabbits, and rodents (e.g. mice and rats). In certain embodiments, the individual or subject is a human.

The term "package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, combination therapy, contraindications and/or warnings concerning the use of such therapeutic products.

"Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, % amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was authored by Genentech, Inc., and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, California, or may be compiled from the source code. The ALIGN-2 program should be compiled for use on a UNIX operating system, including digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is employed for amino acid sequence comparisons, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (which can alternatively be phrased as a given amino acid sequence A that has or comprises a certain % amino acid sequence identity to, with, or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and where Y is the total number of amino acid residues in B. It will be appreciated that where the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless specifically stated otherwise, all % amino acid sequence identity values used herein are obtained as described in the immediately preceding paragraph using the ALIGN -2 computer program. The term "vector," as used herein, refers to a nucleic acid molecule capable of propagating another nucleic acid to which it is linked. The term includes the vector as a self-replicating nucleic acid structure as well as the vector incorporated into the genome of a host cell into which it has been introduced. Certain vectors are capable of directing the expression of nucleic acids to which they are operatively linked. Such vectors are referred to herein as "expression vectors".

II. COMPOSITIONS AND METHODS

Herein is reported a method taking advantage of the fact that for in vitro N-terminal acylation of polypeptides one or more (actual or potential) N-glycosylation sites present within fifty amino acid residues from the site of acylation, i.e. the N-terminus, should be removed in order to allow in vitro acylation to proceed properly.

One aspect as reported herein is a method for the production of an (N-terminally) acylated polypeptide that comprises the step of: acylating the polypeptide (N-terminally) in vitro and thereby producing an N-terminal acylated polypeptide, whereby in the amino acid sequence of the polypeptide any actual or potential N-glycosylation site within the first fifty N-terminal amino acid residues of the amino acid sequence of the polypeptide has been removed.

Thus, in one embodiment the method as reported herein comprises one or more of the following further steps: a) providing a nucleic acid sequence encoding the polypeptide, and/or b) determining whether one or more actual or potential N-glycosylation sites are present in the encoded amino acid sequence, and if present determining the position of the N-glycosylation sites and removing the N-glycosylation site motif (Asn-X-Ser/Thr, sequon), e.g. by modifying the codon encoding the asparagine residue of the glycosylation site motif to a codon that encodes a different non-asparagine amino acid residue, and/or c) transforming a eukaryotic cell with the modified nucleic acid, optionally after the modified nucleic acid encoding the polypeptide has been introduced in an expression cassette or an expression plasmid, d) cultivating a mammalian cell comprising the modified nucleic acid, optionally within an expression cassette or an expression vector, and recovering the polypeptide from the cell or the cultivation medium, and/or e) acylating the polypeptide N-terminally in vitro by incubating the polypeptide with an acylase and an activated carbonic acid, and thereby producing an N-terminal acylated polypeptide. The acylated polypeptide as reported herein is useful, e.g. for the treatment of subjects infected with hepatitis-B-virus, or hepatitis-C-virus, or hepatitis-D-virus either as chronic infection, or acute infection.

A further aspect as reported herein is an N-terminal acylated polypeptide comprising an amino acid sequence of SEQ ID NO: 01, or SEQ ID NO: 02 as the N-terminal amino acid sequence.

Also an aspect as reported herein is an N-myristoyl transferase variant comprising the amino acid sequence of SEQ ID NO: 22.

A. Exemplary fusion protein comprising a preSl-derived polypeptide, an Fc-region, and optionally an anti-viral cytokine In one aspect herein is reported a fusion protein comprising in N-terminal to

C-terminal direction a preSl -domain derived polypeptide, in which potential N-glycosylation sites within the first fifty N-terminal amino acid residues have been removed, an antibody heavy chain Fc-region, and optionally a biologically active polypeptide. In one embodiment the preSl -domain derived polypeptide comprises an amino acid sequence of SEQ ID NO: 01, or SEQ ID NO: 02, or SEQ ID NO: 30, or is a variant thereof comprising at most five amino acid changes.

In one embodiment the preSl -domain derived polypeptide comprises an amino acid sequence having at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100 % sequence identity to the amino acid sequence of SEQ ID

NO: 01, or SEQ ID NO: 02, or SEQ ID NO: 30. In one embodiment the preSl- domain derived polypeptide comprises an amino acid sequence having at least 95 %, at least 98 %, or more than 99 % sequence identity to the amino acid sequence of SEQ ID NO: 01, or SEQ ID NO: 02, or SEQ ID NO: 30.1n certain embodiments an amino acid sequence having at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % identity contains substitutions (e.g. conservative substitutions), insertions, or deletions relative to the reference sequence, but retains the ability of the parent amino acid sequence.

In one embodiment the Fc-region is of human origin. In another embodiment the Fc-region comprises the amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18, or SEQ ID NO: 19, or SEQ ID NO: 20.

In one embodiment the Fc-region comprises an amino acid sequence having at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, 99 %, or 100 % sequence identity to the amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18, or SEQ ID NO: 19, or SEQ ID NO: 20. In one embodiment the Fc- region comprises an amino acid sequence having at least 95 %, at least 98 %, or more than 99 % sequence identity to the amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18, or SEQ ID NO: 19, or SEQ ID NO: 20. In certain embodiments an amino acid sequence having at least 90 %, 91 %, 92 %, 93 %, 94 %, 95 %, 96 %, 97 %, 98 %, or 99 % identity contains substitutions (e.g. conservative substitutions), insertions, or deletions relative to the reference sequence, but retains the ability of the parent amino acid sequence.

In one embodiment the fusion protein comprises in N-terminal to C-terminal direction a preSl -domain derived polypeptide, in which potential N-glycosylation sites within the first fifty N-terminal amino acid residues have been removed, and an Fc-region has the amino acid sequence of SEQ ID NO: 24, or of SEQ ID NO:

25, or of SEQ ID NO: 27.

In one embodiment the fusion protein comprising in N-terminal to C-terminal direction a preSl -domain derived polypeptide, in which potential N-glycosylation sites within the first fifty N-terminal amino acid residues have been removed, an Fc-region, and a biologically active polypeptide has the amino acid sequence of

SEQ ID NO: 26, or SEQ ID NO: 28.

One aspect as reported herein is a complex comprising i) at least one fusion protein comprising in N-terminal to C-terminal direction a preSl -domain derived polypeptide, in which potential N-glycosylation sites within the first fifty N-terminal amino acid residues have been removed, an Fc-region, and a biologically active polypeptide that has an amino acid sequence selected from SEQ ID NO: 26 or SEQ ID NO: 28, and ii) and at least one polypeptide that has an amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18, or SEQ ID NO: 27. In one embodiment the complex comprises a fusion protein that has the amino acid sequence of SEQ ID NO: 28 and a polypeptide that has the amino acid sequence of SEQ ID NO: 27. In one embodiment the complex comprises a fusion protein that has the amino acid sequence of SEQ ID NO: 27 and a polypeptide that has the amino acid sequence of SEQ ID NO: 36. In one embodiment the complex comprises a fusion protein that has the amino acid sequence of SEQ ID NO: 28 and a polypeptide that has the amino acid sequence of SEQ ID NO: 37.

In one embodiment the complex is a covalent complex. In one embodiment the complex comprises one, two, three, four or five disulfide bonds between the fusion protein and the polypeptide.

Mono-myristoylated complex as reported herein can be produced more easily as the complexity of the obtained product mixture after recombinant production is reduced. Additionally, mono-myristoylated complexes, without being bound by theory, are expected to bind more specifically to liver cells compared to di- myristoylated complexes.

Polypeptide variants

In certain embodiments, amino acid sequence variants of the polypeptides comprised in the fusion protein provided herein are contemplated. For example, it may be desirable to improve the biological properties of one of the polypeptides. Amino acid sequence variants may be prepared by introducing appropriate modifications into the nucleotide sequence encoding the polypeptide, or by peptide synthesis. Such modifications include, for example, deletions from, and/or insertions into and/or substitutions of residues within the amino acid sequences of the polypeptide. Any combination of deletion, insertion, and substitution can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics, e.g. antigen-binding. a) Substitution, Insertion, and Deletion Variants

In certain embodiments, fusion proteins comprising a polypeptide variant having one or more amino acid substitutions with respect to the parent polypeptide are provided. Conservative substitutions are shown in Table 1 under the heading of "preferred substitutions". More substantial changes are provided in Table 1 under the heading of "exemplary substitutions", and as further described below in reference to amino acid side chain classes. Amino acid substitutions may be introduced into the polypeptide and the products screened for a desired activity, e.g. retained/improved antigen binding, decreased immunogenicity, or improved ADCC or CDC.

TABLE 1

Amino acids may be grouped according to common side-chain properties:

(1) hydrophobic: Norleucine, Met, Ala, Val, Leu, He;

(2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gin;

(3) acidic: Asp, Glu; (4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Gly, Pro;

(6) aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

One type of substitutional variant involves substituting one or more amino acid residues of a parent polypeptide. Generally, the resulting variant(s) selected for further study will have modifications (e.g. improvements) in certain biological properties (e.g. increased affinity, reduced immunogenicity) relative to the parent polypeptide and/or will have substantially retained certain biological properties of the parent polypeptide.

In certain embodiments, substitutions, insertions, or deletions may occur everywhere within the polypeptide so long as such alterations do not substantially reduce the biological activity of the polypeptide. For example, conservative alterations (e.g. conservative substitutions as provided herein) that do not substantially reduce the biological activity may be made.

Amino acid sequence insertions include amino- and/or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing a hundred or more residues, as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include an N-terminal methionyl residue. b) Glycosylation variants

In certain embodiments, the fusion proteins provided herein comprise a polypeptide and/or an Fc-region that is altered to increase or decrease the extent to which the polypeptide and/or Fc-region is glycosylated. Addition or deletion of one or more glycosylation sites may be conveniently accomplished by altering the amino acid sequence such that one or more glycosylation sites are created or removed. The generation of new glycosylation sites is excluded for/new glycosylation sites are not formed within the first fifty N-terminal amino acid residues of the fusion proteins, i.e. within the preSl -derived polypeptide. Native Fc-regions produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc-region (see, e.g. Wright, A. et al., TIBTECH 15 (1997) 26-32). The oligosaccharide may include various carbohydrates, e.g. mannose, N-acetyl glucosamine (GlcNAc), galactose, and sialic acid (NANA, Neu5Ac), as well as a fucose attached to a GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, modifications of the oligosaccharide in an Fc-region may be made in order to create variants with certain improved properties.

In one embodiment, the Fc-region has a carbohydrate structure that lacks attached fucose (directly or indirectly). For example, the amount of fucose in such an Fc-region may be from 1 % to 80 %, or from 1 % to 65 %, or from 5 % to 65 %, or from 5 % to 20 %, or from 20 % to 40 %. The amount of fucose is determined by calculating the average amount of fucose within the sugar chain at Asn297

(numbering according to Kabat), relative to the sum of all glycostructures attached to Asn 297 (e.g. complex, hybrid and high mannose structures) as measured by MALDI-TOF mass spectrometry, as reported in WO 2008/077546, for example. Asn297 refers to the asparagine residue located at about position 297 in the Fc-region (EU numbering of Fc-region residues). However, Asn297 may also be located about ± 3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to minor sequence variations in antibodies. Such fucosylation variants may have improved ADCC function (see, e.g. US 2003/0157108 and US 2004/0093621). Examples of publications related to "defucosylated" or "fucose-deficient" antibody variants include:

US 2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US 2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US 2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO 2005/035586; WO 2005/035778; WO 2005/053742; WO 2002/031140; Okazaki, A., et al, J. Mol. Biol. 336 (2004) 1239-1249; Yamane-Ohnuki, N., et al,

Biotech. Bioeng. 87 (2004) 614-622. Examples of cell lines capable of producing defucosylated antibodies include Led 3 CHO cells deficient in protein fucosylation (Ripka, J., et al, Arch. Biochem. Biophys. 249 (1986) 533-545; US 2003/0157108; WO 2004/056312, especially at Example 11), and knockout cell lines, such as alpha- 1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see, e.g. Yamane-

Ohnuki, N., et al, Biotech. Bioeng. 87 (2004) 614-622; Kanda, Y., et al, Biotechnol. Bioeng. 94 (2006) 680-688; WO 2003/085107).

Further fusion proteins are provided comprising an Fc-region with bisected oligosaccharides, e.g. in which a biantennary oligosaccharide attached to the Fc-region is bisected by GlcNAc. Such variants may have reduced fucosylation and/or improved ADCC function. Examples of such variants are described, e.g. in WO 2003/011878; US 6,602,684; and US 2005/0123546. Fusion proteins comprising an Fc-region with at least one galactose residue in the oligosaccharide attached to the Fc-region are also provided. Such variants may have improved CDC function. Such variants are described, e.g. in WO 1997/30087; WO 1998/58964; and WO 1999/22764. c) Fc-region variants

In certain embodiments, one or more amino acid modifications may be introduced into the Fc-region of the fusion protein as reported herein, thereby generating an Fc-region variant. The Fc-region variant may comprise an Fc-region sequence of human origin (e.g. a human IgGl, IgG2, IgG3 or IgG4 Fc-region) comprising an amino acid modification (e.g. a substitution) at one or more amino acid positions.

In certain embodiments a fusion protein comprising an Fc-region variant that possesses some but not all effector functions, which make it a desirable candidate for applications in which the half-life of the polypeptide in vivo is important yet certain effector functions (such as complement and ADCC) are unnecessary or deleterious are contemplated. In vitro and/or in vivo cytotoxicity assays can be conducted to confirm the reduction/depletion of CDC and/or ADCC activities. For example, Fc receptor (FcR) binding assays can be conducted to ensure that the antibody lacks FcyR binding (hence likely lacking ADCC activity), but retains FcRn binding ability. The primary cells for mediating ADCC, NK cells, express

FcyRIII only, whereas monocytes express FcyRI, FcyRII and FcyRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch, J.V. and Kinet, J.P. (Annu. Rev. Immunol. 9 (1991) 457-492). Non-limiting examples of in vitro assays to assess ADCC activity of a molecule of interest is reported in US 5,500,362 (see, e.g. Hellstrom, I., et al, Proc. Natl. Acad.

Sci. USA 83 (1986) 7059-7063) and Hellstrom, I., et al, Proc. Natl. Acad. Sci. USA 82 (1985) 1499-1502; US 5,821,337; Brueggemann, M., et al, J. Exp. Med. 166 (1987) 1351-1361). Alternatively, non-radioactive assays methods may be employed (see, for example, ACTI™ non-radioactive cytotoxicity assay for flow cytometry (CellTechnology, Inc. Mountain View, CA), and CytoTox 96^® non-radioactive cytotoxicity assay (Promega, Madison, WI). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g. in an animal model such as that disclosed in Clynes, R., et al, Proc. Natl. Acad. Sci. USA 95 (1998) 652-656. Clq binding assays may also be carried out to confirm that the fusion protein is unable to bind Clq and hence lacks CDC activity (see, e.g. Clq and C3c binding ELISA in WO 2006/029879 and WO 2005/100402). To assess complement activation, a CDC assay may be performed (see, e.g. Gazzano-Santoro, H., et al, J. Immunol. Methods 202 (1997) 163-171; Cragg, M.S., et al, Blood 101 (2003) 1045-1052; and Cragg, M.S. and M.J. Glennie, Blood 103 (2004) 2738-2743). FcRn binding and in vivo clearance/half-life determinations can also be performed using methods known in the art (see, e.g. Petkova, S.B., et al, Int. Immunol. 18 (2006) 1759- 1769). Fusion proteins with reduced effector function include those with substitution of one or more of Fc-region residues 238, 265, 269, 270, 297, 327 and 329 (see, e.g. US 6,737,056, numbering according to Kabat). Such Fc-region mutants include Fc mutants with substitutions at two or more of amino acid positions 265, 269, 270, 297 and 327, including the so-called "DANA" Fc mutant with substitution of residues 265 and 297 to alanine (see US 7,332,581).

Certain Fc-region variants with improved or diminished binding to FcRs are reported (see, e.g. US 6,737,056; WO 2004/056312; Shields, R.L., et al, J. Biol. Chem. 276 (2001) 6591-6604).

In certain embodiments, the Fc-region comprises one or more amino acid substitutions which improve ADCC, e.g. substitutions at positions 298, 333, and/or

334 of the Fc-region (EU numbering of residues).

In some embodiments, alterations are made in the Fc-region that result in altered (i.e., either improved or diminished) Clq binding and/or Complement Dependent Cytotoxicity (CDC), e.g. as reported in US 6,194,551, WO 99/51642, and Idusogie, E.E., et al, J. Immunol. 164 (2000) 4178-4184.

Fusion proteins with increased half-lives and improved binding to the neonatal Fc receptor (FcRn), which is responsible for the transfer of maternal IgGs to the fetus (Guyer, R.L., et al, J. Immunol. 117 (1976) 587-593; Kim, J.K., et al, Eur. J. Immunol. 24 (1994) 2429-2434), are reported in US 2005/0014934. Those fusion proteins comprise an Fc-region with one or more substitutions therein which improve binding of the Fc-region to FcRn. Such Fc variants include those with substitutions at one or more of Fc-region residues: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or 434, e.g. substitution of Fc-region residue 434 (US 7,371,826). See also Duncan, A.R. and Winter, G., Nature 332 (1988) 738-740; US 5,648,260; US 5,624,821; and WO 94/29351 concerning other examples of Fc-region variants.

B. Recombinant Methods and Compositions

The fusion proteins as reported herein may be produced using recombinant methods and compositions, e.g. as reported in US 4,816,567 for antibodies. In one embodiment, one or more isolated nucleic acids encoding a fusion protein as reported herein are provided. Such nucleic acid may encode an amino acid sequence comprising the preSl -domain derived polypeptide and an amino acid sequence comprising the Fc-region of the fusion protein. In a further embodiment, one or more vectors (e.g. expression vectors) comprising such nucleic acid are provided. In a further embodiment, a host cell comprising such nucleic acid is provided. In one such embodiment, a host cell comprises (e.g. has been transformed or transfected with): (1) a vector comprising a nucleic acid that encodes an amino acid sequence comprising the preSl -domain derived polypeptide and an amino acid sequence comprising the Fc-region, or (2) a first vector comprising a nucleic acid that encodes a fusion protein as reported herein comprising a preSl -domain derived polypeptide and an Fc-region and a second vector comprising a nucleic acid that encodes a second polypeptide comprising an Fc-region. In one embodiment, the host cell is eukaryotic, e.g. a Chinese Hamster Ovary (CHO, CHO-K1 , CHO-DG44) cell or a Baby Hamster Kidney (BHK) cell or a Human Embryonic Kidney (HEK) cell, or lymphoid cell (e.g. Y0, NS0, Sp2/0 cell). In one embodiment, a method of making a fusion protein as reported herein is provided, wherein the method comprises culturing a host cell comprising a nucleic acid encoding the fusion protein, as provided above, under conditions suitable for expression of the fusion protein, and optionally recovering the fusion protein from the host cell (or host cell culture medium).

For recombinant production of a fusion protein as reported herein, nucleic acid encoding the fusion protein, e.g. as described above, is isolated and inserted into one or more vectors for further cloning and/or expression in a host cell. Such nucleic acid may be readily isolated and sequenced using conventional procedures.

Suitable host cells for cloning or expression of fusion protein-encoding vectors include prokaryotic or eukaryotic cells as reported herein. For example, the fusion protein may be produced in bacteria, in particular when glycosylation and Fc effector function are not needed. For expression of fragments and polypeptides in bacteria, see, e.g. US 5,648,237, US 5,789,199, and US 5,840,523, also see Charlton, K.A., In: Methods in Molecular Biology, Vol. 248, Lo, B.K.C. (ed.), Humana Press, Totowa, NJ, (2003), pp. 245-254, reporting expression of antibody fragments in E. coli. After expression, the fusion protein may be isolated from the bacterial cell paste in a soluble fraction and can be further purified. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for fusion protein-encoding vectors, including fungi and yeast strains whose glycosylation pathways have been "humanized", resulting in the production of a fusion protein with a partially or fully human glycosylation pattern (see Gerngross, T.U., Nat. Biotech. 22 (2004) 1409- 1414; Li, H., et al, Nat. Biotech. 24 (2006) 210-215).

Suitable host cells for the expression of glycosylated fusion proteins are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. Numerous baculoviral strains have been identified which may be used in conjunction with insect cells, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures can also be utilized as hosts (see, e.g. US 5,959,177, US 6,040,498, US 6,420,548, US 7,125,978, and US 6,417,429).

Vertebrate cells may also be used as hosts. For example, mammalian cell lines that are adapted to grow in suspension may be useful. Other examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7), human embryonic kidney line (293 or 293 cells as reported, e.g. in Graham, F.L., et al, J. Gen Virol. 36 (1977) 59-74), baby hamster kidney cells (BHK), mouse Sertoli cells (TM4 cells as reported, e.g. in Mather, J.P., Biol. Reprod. 23 (1980) 243-252), monkey kidney cells (CV1), African green monkey kidney cells (VERO-76), human cervical carcinoma cells (HELA), canine kidney cells (MDCK), buffalo rat liver cells (BRL 3A), human lung cells (W138), human liver cells (Hep G2), mouse mammary tumor cells (MMT 060562), TRI cells, as reported, e.g. in Mather, J.P., et al, Annals N.Y. Acad. Sci. 383 (1982) 44-68, MRC 5 cells, and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR^" CHO cells (Urlaub, G., et al, Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines such as Y0, NS0 and Sp2/0. For a review of certain mammalian host cell lines suitable for fusion protein production, see, e.g. Yazaki, P.J. and Wu, A.M., Methods in Molecular Biology, Vol. 248 (B.K.C. Lo, ed., Humana Press, Totowa, NJ), pp. 255-268 (2003). C. Pharmaceutical Formulations

Pharmaceutical formulations of a fusion protein as reported herein are prepared by mixing a fusion protein having the desired degree of purity with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences, 16th ed., Osol, A. (ed.) (1980)), in the form of lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the dosages and concentrations employed, and include, but are not limited to: buffers such as phosphate, citrate, and other organic acids, antioxidants including ascorbic acid and methionine, preservatives (such as octadecyl dimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenol, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol), low molecular weight (less than about 10 residues) polypeptides, proteins, such as serum albumin, gelatin, or immunoglobulins, hydrophilic polymers such as poly vinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine, monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins, chelating agents such as EDTA, sugars such as sucrose, mannitol, trehalose or sorbitol, salt-forming counter-ions such as sodium, metal complexes (e.g. Zn-protein complexes), and/or non-ionic surfactants such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers herein further include interstitial drug dispersion agents such as soluble neutral-active hyaluronidase glycoproteins (sHASEGP), for example, human soluble PH-20 hyaluronidase glycoproteins, such as rhuPH20 (HYLENEX^®, Baxter International, Inc.). Certain exemplary sHASEGPs and methods of use, including rhuPH20, are reported in

US 2005/0260186 and US 2006/0104968. In one aspect, a sHASEGP is combined with one or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized formulations are reported in US 6,267,958. Aqueous formulations include those reported in US 6,171,586 and WO 2006/044908, the latter formulations including a histidine-acetate buffer.

The formulation herein may also contain more than one active ingredients as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. Such active ingredients are suitably present in combination in amounts that are effective for the purpose intended. Active ingredients may be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethyl cellulose or gelatin-microcapsules and polymethylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example liposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 16^th ed., Osol, A. (ed.) (1980).

Sustained-release preparations may be prepared. Suitable examples of sustained- release preparations include semi permeable matrices of solid hydrophobic polymers containing the fusion protein, which matrices are in the form of shaped articles, e.g. films, or microcapsules.

The formulations to be used for in vivo administration are generally sterile. Sterility may be readily accomplished, e.g. by filtration through sterile filtration membranes. D. Therapeutic Methods and Compositions

Any of the fusion proteins provided herein may be used in therapeutic methods.

In one aspect, the invention provides for the use of a fusion protein in the manufacture or preparation of a medicament.

In one aspect, the invention provides a method for treating hepatitis-B-virus infection, or hepatitis-C-virus infection, or hepatitis-D-virus infection.

In one aspect, the invention provides pharmaceutical formulations comprising any of the fusion proteins provided herein, e.g. for use in any of the above therapeutic methods. In one embodiment, a pharmaceutical formulation comprises any of the fusion proteins provided herein and a pharmaceutically acceptable carrier. In another embodiment, a pharmaceutical formulation comprises any of the fusion proteins provided herein and at least one additional therapeutic agent, e.g. as described below.

Fusion proteins of the invention can be used either alone or in combination with other agents in a therapy. For instance, a fusion protein of the invention may be co-administered with at least one additional therapeutic agent. Such combination therapies noted above encompass combined administration (where two or more therapeutic agents are included in the same or separate formulations), and separate administration, in which case, administration of the fusion protein of the invention can occur prior to, simultaneously, and/or following, administration of the additional therapeutic agent and/or adjuvant.

A fusion protein of the invention (and any additional therapeutic agent) can be administered by any suitable means, including parenteral, intrapulmonary, and intranasal, and, if desired for local treatment, intralesional administration. Parenteral infusions include intramuscular, intravenous, intraarterial, intraperitoneal, or subcutaneous administration. Dosing can be by any suitable route, e.g. by injections, such as intravenous or subcutaneous injections, depending in part on whether the administration is brief or chronic. Various dosing schedules including but not limited to single or multiple administrations over various time -points, bolus administration, and pulse infusion are contemplated herein. Fusion proteins of the invention would be formulated, dosed, and administered in a fashion consistent with good medical practice. Factors for consideration in this context include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, the site of delivery of the agent, the method of administration, the scheduling of administration, and other factors known to medical practitioners. The fusion protein need not be, but is optionally formulated with one or more agents currently used to prevent or treat the disorder in question. The effective amount of such other agents depends on the amount of fusion protein present in the formulation, the type of disorder or treatment, and other factors discussed above. These are generally used in the same dosages and with administration routes as described herein, or about from 1 % to 99 % of the dosages described herein, or in any dosage and by any route that is empirically/clinically determined to be appropriate.

For the prevention or treatment of disease, the appropriate dosage of a fusion protein of the invention (when used alone or in combination with one or more other additional therapeutic agents) will depend on the type of disease to be treated, the severity and course of the disease, whether the fusion protein is administered for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the fusion protein, and the discretion of the attending physician. The fusion protein is suitably administered to the patient at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μ§/1¾ to 15 mg/kg (e.g. 0.1 mg/kg-10 mg/kg) of fusion protein can be an initial candidate dosage for administration to the patient, whether, for example, by one or more separate administrations, or by continuous infusion. One typical daily dosage might range from about 1 μg/kg to 100 mg/kg or more, depending on the factors mentioned above. For repeated administrations over several days or longer, depending on the condition, the treatment would generally be sustained until a desired suppression of disease symptoms occurs. One exemplary dosage of the fusion protein would be in the range from about 0.05 mg/kg to about 10 mg/kg. Thus, one or more doses of about 0.5 mg/kg, 2.0 mg/kg, 4.0 mg/kg or 10 mg/kg (or any combination thereof) may be administered to the patient. Such doses may be administered intermittently, e.g. every week or every three weeks (e.g. such that the patient receives from about two to about twenty, or e.g. about six doses of the antibody). An initial higher loading dose, followed by one or more lower doses may be administered. E. Articles of Manufacture

In another aspect of the invention, an article of manufacture containing materials useful for the treatment, prevention and/or diagnosis of the disorders described above is provided. The article of manufacture comprises a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, IV solution bags, etc. The containers may be formed from a variety of materials such as glass or plastic. The container holds a composition which is by itself or combined with another composition effective for treating, preventing and/or diagnosing the condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a fusion protein as reported herein. The label or package insert indicates that the composition is used for treating the condition of choice. Moreover, the article of manufacture may comprise (a) a first container with a composition contained therein, wherein the composition comprises a fusion protein as reported herein, and (b) a second container with a composition contained therein, wherein the composition comprises a further therapeutic agent. The article of manufacture in this embodiment of the invention may further comprise a package insert indicating that the compositions can be used to treat a particular condition. Alternatively, or additionally, the article of manufacture may further comprise a second (or third) container comprising a pharmaceutically-acceptable buffer, such as water for injection (WFI), bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

The following examples, sequence listing and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

III. DESCRIPTION OF THE SEQUENCES

SEQ ID NO: 01 amino acid sequence of the N15Q-mutant preSl(l-62; N15Q) polypeptide (genotype A)

SEQ ID NO: 02 amino acid sequence of the consensus-N17Q-mutant preSl(l-61 ;

N17Q; consensus) polypeptide

SEQ ID NO: 03 linker 1 amino acid sequence

SEQ ID NO: 04 linker 2 amino acid sequence

SEQ ID NO: 05 linker 3 amino acid sequence

SEQ ID NO: 06 linker 4 amino acid sequence

SEQ ID NO: 07 linker 5 amino acid sequence

SEQ ID NO: 08 linker 6 amino acid sequence

SEQ ID NO: 09 human kappa light chain

SEQ ID NO: 10 human lambda light chain

SEQ ID NO: 11 IgGl (Caucasian allotype) constant region amino acid sequence

SEQ ID NO: 12 IgGl (afro-american allotype) constant region amino acid sequence

SEQ ID NO: 13 IgGl LALA variant (Caucasian allotype) constant region amino acid sequence

SEQ ID NO: 14 IgG4 constant region amino acid sequence

SEQ ID NO: 15 IgG4 SPLE variant constant region amino acid sequence

SEQ ID NO: 16 Fc-region IgGl (Caucasian allotype) amino acid sequence

SEQ ID NO: 17 Fc-region IgGl (afro-american allotype) amino acid sequence SEQ ID NO: 18 Fc-region IgGl LALA variant (Caucasian allotype) amino acid sequence

SEQ ID NO: 19 Fc-region IgG4 amino acid sequence

SEQ ID NO: 20 Fc-region IgG4 SPLE variant amino acid sequence

SEQ ID NO: 21 amino acid sequence of mature human interferon-a2a SEQ ID NO: 22 amino acid sequence of yeast NMT (N-myristoyl transferase) variant

SEQ ID NO: 23 amino acid sequence of the mature preS 1(2-62; wt)-Fc fusion SEQ ID NO: 24 amino acid sequence of the mature preS 1(2-62; N15Q)-Fc fusion SEQ ID NO: 25 amino acid sequence of the mature preS 1(2-62; consensus,

N17Q)-Fc fusion

SEQ ID NO: 26 amino acid sequence of the mature preSl(2-62; N15Q)-Fc-IFN- a2a fusion protein

SEQ ID NO: 27 amino acid sequence of the knob-into-hole mature preS 1(2-62;

N15Q; hole)-Fc fusion protein

SEQ ID NO: 28 amino acid sequence of the knob-into-hole mature preS 1(2-62;

N15Q; knob)-Fc-IFN-a2a fusion protein

SEQ ID NO: 29 amino acid sequence of the wild-type preSl(l-62; wt) peptide

(genotype A)

SEQ ID NO: 30 consensus amino acid sequence of the preSl (1-62, wt) peptide

SEQ ID NO: 31 pro-sequence for protein initiation of translation in E.coli

SEQ ID NO: 32 poly-histidine-tag

SEQ ID NO: 33 Avi-tag amino acid sequence

SEQ ID NO: 34 IgA protease cleavage site

SEQ ID NO: 35 murine IgG signal amino acid sequence

SEQ ID NO: 36 amino acid sequence of the mature -Fc-IFN-a2a fusion protein with a knob mutation

SEQ ID NO: 37 amino acid sequence of the Fc-region with a hole mutation

IV. DESCRIPTION OF THE FIGURES Figure 1 Schematic diagram of the knob-into-hole preSl-Fc-IFN fusion molecule: The knob-into-hole preSl(2-62; N15Q)-Fc-IFN-a2a fusion protein is composed of the HBV preSl -domain of genotype A from amino acid position 2 to 62 including a N15Q mutation, the human Fc-gamma-1 -heavy chain constant region (Hinge-CH2-CH3) including the mutations L234A and L235A and mature human interferon alpha-2a. The IFN is fused to the C-terminal ends of the Fc constant domains via a glycine-serine linker. The peptide sequence for an exemplary linker consisting of two Gly₄Ser repeats is shown. The C-terminal lysine of the Fc constant domain is removed. Fc-region hole mutations: T366S,

L368A, Y407V, and optionally Y349C; Fc-region knob mutations: T366W, and optionally S354C.

Figure 2 Myristoylation kinetics of the different protein constructs according to Example la (open circles) and Example lb (open triangles). Absorption at 412 nm is shown as a function of reaction time (min). Increase in absorption correlates with degree of myristoylation due to coupling of myristoylation with an indicator Ellman's reaction (see Example 3).

Figure 3 Myristoylation kinetics of the different protein constructs according to Example lb (open triangles), Example Id (crosses), and Example le (stars). Absorption at 412 nm is shown as a function of reaction time (min). Increase in absorption correlates with degree of myristoylation due to coupling of myristoylation with an indicator Ellman's reaction (see Example 3).

Figure 4 Myristoylation kinetics of the different protein constructs according to Example Id (crosses), Example le (stars), Example If (open squares), and Example lg (open diamonds). Absorption at 412 nm is shown as a function of reaction time (min). Increase in absorption correlates with degree of myristoylation due to coupling of myristoylation with an indicator Ellman's reaction (see Example 3).

V. EXAMPLES

Materials and Methods

Recombinant DNA techniques

Standard methods were used to manipulate DNA as described in Sambrook, J., et al., Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, (1989). The molecular biological reagents were used according to the manufacturer's instructions.

Gene synthesis

Desired gene segments were prepared by chemical synthesis at Geneart GmbH (Regensburg, Germany). The synthesized gene fragments were cloned into an E. coli plasmid for propagation/amplification. The DNA sequence of the subcloned gene fragments were verified by DNA sequencing. Protein determination

The protein concentration of purified polypeptides was determined by determining the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence of the polypeptide. Example 1

Generation of the expression plasmids for the preSl-Fc fusion proteins a) Generation of the expression plasmid for a wild-type preS 1(2-62; wf)-Fc fusion protein

The SS-preS 1(2-62; wt)-Fc fusion gene was assembled by fusing a chemically synthesized DNA fragment coding for a murine immunoglobulin heavy chain signal sequence (SS; MGWSCIILFLVATATGVHS (SEQ ID NO: 35) and a HBV preSl peptide from amino acid residues 2-62 (i.e. excluding the starting methionine) of genotype A to a human Fc-gamma-1 -heavy chain constant region (Hinge-CH2-CH3; for exemplary sequences see SEQ ID NO: 16 to 20 and also US 2005/0008642).

The expression plasmid for the transient expression of a wild-type preS 1(2-62; wt)- Fc fusion protein in HEK293 cells comprised besides the SS-preS 1(2-62; wt)-Fc expression cassette an origin of replication from the vector pUC18 which allows replication of this plasmid in E. coli, and a beta-lactamase gene which confers ampicillin resistance in E. coli. The transcription unit of the SS-preS 1(2-62; wt)-Fc fusion gene comprises the following functional elements:

- the immediate early enhancer and promoter from the human cytomegalovirus (P-CMV) including intron A,

- a human heavy chain immunoglobulin 5 '-untranslated region (5 'UTR), - a murine immunoglobulin heavy chain signal sequence (SS),

- a HBV preSl -domain of genotype A from amino acid position 2-62 (preS 1(2-62)),

- a human Fc-gamma-1 -heavy chain constant region (Hinge-CH2-CH3) and the bovine growth hormone polyadenylation sequence (BGH poly A signal sequence). The amino acid sequence of the mature preS 1(2-62; wt)-Fc fusion protein is shown in SEQ ID NO: 23. b) Generation of the expression plasmid for the N15Q-mutant preSl(2-62; N15Q)- Fc fusion protein

The expression plasmid for the transient expression of the N15Q-mutant preSl(2- 62; N15Q)-Fc fusion protein in HEK293 cells was derived from the expression vector described before. It differentiated in the DNA sequence coding for the N15Q-mutation of the preSl(2-62; N15Q)-Fc fusion protein. The amino acid sequence of the mature preSl(2-62; N15Q)-Fc fusion protein is shown in SEQ ID NO: 24. c) Generation of the expression plasmid for the consensus-N17Q-mutant preSl 2- 62; N17Q; consensusVFc fusion protein

The expression plasmid for the transient expression of the consensus-N17Q-mutant preSl(2-62; N17Q; consensus)-Fc fusion protein in HEK293 cells can be derived from the expression vector described above under item a). It differentiates only in the DNA segment encoding the consensus-N17Q-mutant preSl(2-62; N17Q; consensus) peptide. The amino acid sequence of the mature preS 1(2-62; N17Q; consensus)-Fc fusion protein is shown in SEQ ID NO: 25. d) Generation of the expression plasmid for the dimeric preS 1(2-62; N15Q)-Fc- IFN-a2a fusion protein

The SS-preS 1(2-62; N15Q)-Fc-IFN-a2a fusion gene was assembled by fusing a chemically synthesized DNA fragment coding for mature human IFN-a2a and a glycine-serine linker consisting of two Gly₄Ser repeats (C-terminus of heavy chain- LSPG~GGGSGGGGS-IFNoc2a) to the 3^* end of the SS-preS 1(2-62; N15Q)-Fc gene wherein the human gamma- 1 heavy chain constant region was truncated (removal of the last natural lysine amino acid residue).

The expression plasmid for the transient expression of the preSl(2-62; N15Q)-Fc- IFN-a2a fusion protein in HEK293 cells was derived from the expression vector reported above under item b) by insertion of a DNA fragment coding for mature human IFN-a2a, a glycine-serine linker and the C-terminal Lys-truncated human gamma- 1 heavy chain. The amino acid sequence of the mature preS 1(2-62; N15Q)- Fc-IFN-a2a fusion protein is shown in SEQ ID NO: 26. e) Generation of the "knob-into-hole" expression plasmids for the dimeric preS 1(2- 62; N15Q; holeVFc / preSl(2-62; N15Q; knob -Fc-IFN-a2a fusion protein

The expression plasmid for the transient expression of the preS 1(2-62; N15Q; hole)-Fc fusion protein in HEK293 cells was derived from the expression vector described above under item b). It differentiated therefrom in the DNA sequence coding for the Fc-region with hole mutations T366S, L368A, Y407V, Y349C and the Fc effector functions reducing mutations L234A and L235A within the human gamma- 1 heavy chain constant region. The amino acid sequence of the mature preSl(2-62; N15Q; hole)-Fc fusion protein is shown in SEQ ID NO: 27. The expression plasmid for the transient expression of the preS 1(2-62; N15Q; knob)-Fc-IFN-a2a fusion protein in HEK293 cells was derived from the expression vector reported above under item d). It differentiated in the DNA sequence coding for the Fc-region knob mutations T366W and S354C and the Fc effector functions reducing mutations L234A and L235A within the human gamma-1 heavy chain constant region. The amino acid sequence of the mature preSl(2-62; N15Q; knob)-

Fc-IFN-a2a fusion protein is shown in SEQ ID NO: 28. f) Generation of the "knob-into-hole" expression plasmids for the monomeric myristoylated preSl(2-62; N15Q; hole; mono-myristoylated)-Fc / (knob)-Fc-IFN- a2a fusion protein The expression plasmid for the transient expression of the preS 1(2-62; N15Q; hole)-Fc fusion protein in HEK293 cells was identical to the first expression plasmid described above under item e) which was derived from the expression vector described above under item b). It differentiated therefrom in the DNA sequence coding for the Fc-region hole mutations T366S, L368A, Y407V, Y349C and the Fc effector functions reducing mutations L234A and L235A within the human gamma-1 heavy chain constant region. The amino acid sequence of the mature preSl(2-62; N15Q; hole)-Fc fusion protein is shown in SEQ ID NO: 27.

The expression plasmid for the transient expression of the (knob)-Fc-IFN-a2a fusion protein in HEK293 cells was derived from the second expression vector reported above under item e). It differs in the DNA sequence in that it lacks the region coding for preS 1(2-62; N15Q). The amino acid sequence of the mature (knob)-Fc-IFN-a2a fusion protein is shown in SEQ ID NO: 36 g) Generation of the "knob-into-hole" expression plasmids for the (hole)-Fc / monomeric myristoylated preSl(2-62; N15Q; knob; mono-myristoylated)-Fc-IFN- g2a fusion protein

The expression plasmid for the transient expression of the (hole)-Fc protein in HEK293 cells was derived from the first expression vector described above under item e). It differs in the DNA sequence in that it lacks the region coding for preSl(2-62; N15Q). The amino acid sequence of the mature (hole)-Fc protein is shown in SEQ ID NO 37.

The expression plasmid for the transient expression of the preS 1(2-62; N15Q; knob)-Fc-IFN-a2a fusion protein in HEK293 cells was identical to the second expression plasmid described above under item e) which was derived from the expression vector reported above under item d). It differentiated in the DNA sequence coding for the Fc-region knob mutations T366W and S354C and the Fc effector functions reducing mutations L234A and L235A within the human gamma- 1 heavy chain constant region. The amino acid sequence of the mature preSl(2-62; N15Q; knob)-Fc-IFN-a2a fusion protein is shown in SEQ ID NO: 28.

Example 2

Transient expression, purification and analytical characterization of the preSl-Fc and preSl-Fc-IFN-a2a fusion proteins The Fc-fusion proteins were generated by transient transfection of HEK293 cells

(human embryonic kidney cell line 293 -derived) cultivated in F17 Medium (Invitrogen Corp.). For transfection "293 -Free" Transfection Reagent (Novagen) was used. The knob-into-hole Fc-fusion proteins were expressed from two different plasmids using an equimolar plasmid ratio upon transfection. Transfections were performed as specified in the manufacturer's instructions. Fc-fusion protein- containing cell culture supematants were harvested seven days after transfection. Supematants were stored at reduced temperature until purification.

General information regarding the recombinant expression of human immunoglobulins in e.g. HEK293 cells is given in: Meissner, P., et al, Biotechnol. Bioeng. 75 (2001) 197-203.

The Fc-fusion protein-containing culture supematants were filtered and purified by two chromatographic steps. The Fc-fusion proteins were captured by affinity chromatography using HiTrap MabSelectSuRe (GE Healthcare) equilibrated with PBS (1 mM KH₂P0₄, 10 niM Na₂HP0₄, 137 niM NaCl, 2.7 mM KC1), pH 7.4. Unbound proteins were removed by washing with equilibration buffer, and the fusion protein was recovered with 0.1 M citrate buffer, pH 2.8, immediately after elution neutralized to pH 6.0 with 1 M Tris-base, pH 9.0. Size exclusion chromatography on Superdex 200™ (GE Healthcare) was used as second purification step. The size exclusion chromatography was performed in 20 mM histidine buffer, 0.14 M NaCl, pH 6.0. The eluted Fc-fusion proteins were concentrated with an Ultrafree -CL centrifugal filter unit equipped with a Biomax- SK membrane (Millipore, Billerica, MA) and stored at -80 °C. The protein concentrations of the Fc-fusion proteins were determined by measuring the optical density (OD) at 280 nm, using the molar extinction coefficient calculated on the basis of the amino acid sequence. Purity and proper dimer formation of Fc-fusion proteins were analyzed by SDS-PAGE in the presence and absence of a reducing agent (5 mM 1. 4-dithiotreitol) and staining with Coomassie brilliant blue. Aggregate content of the Fc-fusion protein preparations was determined by high-performance SEC using a Superdex 200TM analytical size- exclusion column (GE Healthcare). The integrity of the amino acid backbone of reduced Fc fusion proteins were verified by Nano Electrospray QTOF mass spectrometry after removal of N-glycans by enzymatic treatment with a combination of neuraminidase, O-glycanase and peptide-N-glycosidase F (Roche

Applied Science).

Example 3

Acylation of the preSl-Fc and preSl-Fc-IFN-a2a fusion proteins

The Fc fusion proteins were enzymatically myristoylated using a yeast N-myristoyl transferase (NMT) variant (SEQ ID NO: 22) in neutral to weakly basic environment (pH 7.5-8.0) as described in Farazi, T.A., et al., Biochemistry 40 (2001) 9177-9186).

In order to monitor the kinetics of myristoylation, a coupling to a colorimetric detectable (absorption at 412 nm) reaction was used: the disulfide bond of DTNB (Ellman^'s reagent; Dithiobis-2-nitrobenzoic acid) is cleaved via the free thiol group of released coenzyme A after the transfer of the myristoyl group from myristoyl- coenzyme A (MyrCoA) to proteins.

The myristoylation proceeded with protein concentrations of 22 μΜ (constructs according to Example la to le) or 44 μΜ (constructs according to Example If and lg) in the presence of 100 μΜ MyrCoA (myristoic acid coupled to coenzyme A), 0.5 mM DNTB in 50 mM HEPES buffer (pH 7.6), 0.1 % (w/v) Zwittergent 3-14 at room temperature. The reaction was initiated by adding NMT to a final concentration of 100 nM. The reaction mixture without NMT was used as a blank for absorption measurements at 412 nm. The absorption was measured in distinct intervals, the reaction samples were incubated in the dark.

Reference reactions without addition of NMT showed no absorption at 412 nm above blank absorption.

The absorption was plotted as a function of reaction time (see Figures 2 to 4). The reaction time at which 75% of the absorption maximum was reached was used as a parameter for the accessibility of the various constructs for myristoylation (see Table 2).

TABLE 2

RT₇₅: reaction time after which 75 % of the maximum absorption value was reached

n.r.: maximum absorption value not reached Example 4

Purification and analytical characterization of myristoylated preSl-Fc and preSl-Fc-IFN-a2a fusion proteins

After completion of the acylation, size exclusion chromatography on Superdex 200™ (GE Healthcare) was used as first purification step combined with buffer exchange. The size exclusion chromatography was performed in 3 mM KH2P04, pH 7.5, 0.1 Zwittergent 3-14. Fraction of myristoylated protein could be enriched via ion exchange chromatography on CHT™ Ceramic Hydroxyapatite (Bio-Rad Laboratories). The sample was loaded onto the in buffer A (3 mM KH2P04, pH 7.5, 0.1 Zwittergent 3-14) pre-equilibrated column (2.5 ml) and washed with buffer A until the absorbance signal at 280 nm reached back close to baseline. The protein was eluted with a linear gradient from 0 to 50 % buffer B (500 mM KH₂PO₄, pH 7.5, 0.1 Zwittergent 3-14) in 40 column volumes, at which myristoylated proteins eluted at a higher elution volume. A size exclusion chromatography on Superdex 200™ (GE Healthcare) was used as polishing step combined with buffer exchange into storage buffer. The size exclusion chromatography was performed in 50 mM HEPES buffer, pH 7.6, 0.1 % Zwittergent 3-14. The eluted Fc-fusion proteins were concentrated with an Ultrafree -CL centrifugal filter unit equipped with a Biomax-SK membrane (Millipore, Billerica, MA) and stored at -80 °C.

The myristoylated fusion proteins were analyzed by reversed phase UPLC (RP-UPLC). 0.5 - 2 mg/ml protein was incubated in the presence of 10 mM DTT for 2 h at room temperature. 2 - 10 μg of the sample was injected onto an Acquity UPLC BEH300 C4 (2.1 x 100 mm; Waters) RP-UPLC column pre-equilibrated with 0.1 % TFA in water. Protein was eluted by a linear gradient from 100 % buffer A (water, 0.1 % TFA) to 100 % buffer B (acetonitrile, 0.085 % TFA) at a flow rate of 0.6 ml/min. Myristoylation of the PreSl sequence translated into a retardation in the elution volume. An additional peak could be detected between both peaks representing a mono-myristoylated dimeric fusion protein. Example 5

Generation of the E. coli N-myristoyl transferase (NMT) expression plasmid

The catalytic domain of the yeast Saccharomyces cerevisiae NMT(32-455) was expressed in E. coli as a fusion protein including the following N-terminal modifications (in N- to C-terminal direction): the pre-sequence MRGS (SEQ ID NO: 31) for optimal protein initiation of translation in E.coli,

the poly-histidine tag HHHHHH (SEQ ID NO: 32),

a GS linker,

- an Avi-tag with the amino acid sequence LNDIFEAQKIEWHE (SEQ

ID NO: 33) for BirA mediated biotinylation,

a second GS linker,

the IgA protease cleavage site PRPPTP (SEQ ID NO: 34) for optional proteolytic release of the catalytic NMT domain, and

- the S. cerevisiae catalytic domain of NMT(32-455) from amino acid position 32-455 (Farazi et al, see supra).

The amino acid sequence of the yeast NMT variant protein is shown in SEQ ID NO: 22.

The expression plasmid for the expression of the NMT variant protein in E. coli was derived from the E. coli expression vector 4980 (pBRori-URA3-LACI-SAC as reported in EP 10187663.9) by replacing the 435 bp long core-streptavidin encoding EcoRI/Celll-fragment with a chemically prepared 1495 bp long NMT variant encoding EcoRI/Celll-fragment.

The E.coli expression plasmid comprised the following elements: - the origin of replication from the vector pBR322 which allows replication of the plasmid in E. coli,

- the URA3 gene of Saccharomyces cerevisiae coding for orotidine 5'- phosphate decarboxylase (Rose, M., et al. Gene 29 (1984) 113-124) which allows plasmid selection by complementation of E.coli pyrF mutant strains (uracil auxotrophy),

- the lacl repressor gene from E. coli (Farabaugh, P. J., Nature 274 (1978) 765-769), and

- the yeast NMT variant expression cassette comprising the T5 hybrid promoter (T5-PN25/03/04 hybrid promoter according to Bujard, H., et al, Methods Enzymol. 155 (1987) 416-433, Stueber, D., et al., Immunol. Methods IV (1990) 121-152) including a synthetic ribosomal binding site according to Stueber, D., et al. (see before),

- the DNA segment encoding the yeast NMT variant protein as described above, and

- two bacteriophage-derived transcription terminators, the λ-ΤΟ terminator (Schwarz, E., et al, Nature 272 (1978) 410-414) and the fd-terminator (Beck, E. and Zink, B., Gene 1-3 (1981) 35-58).

Example 6

Expression and purification of the yeast NMT variant gene/protein

For the expression of the Saccharomyces cerevisiae NMT variant an E.coli host-vector system was employed enabling an antibiotic-free plasmid selection by complementation of an E.coli auxotrophy (PyrF) (see US 6,291,245).

E. coli host strain

The yeast NMT variant gene was expressed in the E.coli strain CSPZ-2 (leuB, proC, trpE, thi-1, ApyrF).

Transformation and cell culturing by complementation of a pyrF auxotrophy in selective medium

The E.coli K12 strain CSPZ-2 (leuB, proC, trpE, thi-1, ApyrF) was transformed with the NMT expression plasmid as reported above. The transformed E. coli cells were first grown at 37 °C on agar plates and subsequently in a shaking culture in M9 minimal medium containing 0.5 % casamino acids (Difco) up to an optical density at 550 nm (OD₅₅₀) of 0.4 and subsequently cooled down to 25 °C. Expression was induced by the addition of IPTG (1-5 mmol/1 final concentration) at an OD550 of 1.0-1.2. After an induction phase of 4 to 16 hours at 25 °C, the cells were harvested by centrifugation, washed with PBS buffer (10 mM Na₂HP0₄, 1 mM KH₂P0₄, 137 mM NaCl, 2.7 mM KC1, pH 7.4) and stored at -20 °C until further processing.

Expression analysis

For expression analysis cell pellets from 1 OD₅₅onm unit (1 OD₅₅o_nm = 1 ml cell suspension with an OD at 550 nm of 1) of centrifuged culture medium were resuspended in 0.25 ml 1 x SDS sample buffer (50 mmol/1 Tris-HCl, pH 6.8, 1 % (w/v) SDS, 6 mol/1 urea, 50 mmol/1 DTT, 10 % (w/v) glycerol, 0.001 % (w/v) bromophenol blue).

To reveal if the S. cerevisiae NMT variant is expressed in inclusion bodies 1 OD of centrifuged culture medium was resuspended in 0.2 ml PBS buffer and the cells were lysed by ultrasonic treatment (two pulses of 30 s at 50 % intensity). The lysate was centrifuged at 14,000 g for 5 min. The pellet (resembling the inclusion body fraction) was resuspended in 0.2 ml 1 x SDS sample buffer, whereas to the supernatant (soluble fraction) 50 μΐ of 5 x SDS sample buffer was added.

The samples were incubated for 5 min. at 95 °C. Subsequently, the proteins were separated by SDS polyacrylamide gel electrophoresis (PAGE) (Laemmli, U.K.,

Nature 227 (1970) 680-685) and stained with Coomassie Brilliant Blue R dye.

The expressed NMT variant protein was found to be mainly expressed as soluble protein. The NMT variant protein was the most dominant protein present in the soluble fraction. Purification of the NMT variant protein

NMT variant protein was purified by metal chelate affinity chromatography (IMAC) and cation ion exchange chromatography using published standard protocols. Briefly, an E.coli cell pellet obtained from one liter shaking culture of E. coli K12 CSPZ-2 transformed with the NMT expression plasmid was resuspended in 30 ml ice-cold NiA buffer (50 mmol/1 TRIS, 300 mM NaCl, 5 mM imidazole containing 1 complete EDTA-free protease inhibitor cocktail tablet, pH 8; Roche Applied Science). Subsequently, the cells were lysed by ultrasonic treatment (3 pulses of 60 s at 100 % intensity) with a Sonifier Cell Disruptor B15 from the Branson Company (Heusenstamm, Germany). The insoluble cell components were sedimented by centrifugation (Sorvall centrifuge, SS34 rotor, 19,000 rpm, 15 min) and the cleared lysate supernatant was loaded onto a 13 ml Ni Sepharose™ 6 Fast Flow column pre-equilibrated with a NiA-buffer at a flow of 1 ml/min using an AKTA explorer 100 system (GE Health Care, Uppsala, Sweden). The column was washed with NiA buffer until the UV reading reaches back close to baseline. The NMT variant protein was eluted with a 5 mM to 300 mM linear imidazole gradient in 50 mmol/1 TRIS and 300 mM NaCl, pH 8 in 10 column volumes.

In order to remove still remaining traces of E. coli derived thioesterases, which potentially could cleave acyl-CoA substrates, the NMT variant protein was further purified by cation exchange chromatography on SP-Sepharose. The pooled peak fractions of the Ni Sepharose column were dialyzed against SpA buffer (20 mM K₂HPO₄, 1 mM EDTA, 1 mM DTT, pH 7.4) and loaded onto a 30 ml SP-Sepharose™ Fast Flow column (GE Healthcare) pre-equilibrated with SpA buffer, pH 7.4 at a flow of 2 ml/min. After washing the column with about

5 volumes of SpA buffer the NMT variant protein was eluted with a 0-500 mM linear NaCl gradient in SpA buffer, pH 7.4 in 10 column volumes at a flow of 3 ml/min. The NMT variant protein eluted at around 200 mM NaCl.

The homogeneity of the purified NMT variant protein was analyzed by reducing SDS polyacrylamide gel electrophoresis (PAGE) and staining with Coomassie

Brilliant Blue R dye. The NMT variant protein migrated as a single band with an apparent molecular weight of about 55 kDa.

The aggregate content of the NMT variant protein preparation was analyzed by high-performance SEC using a Superdex 200TM analytical size-exclusion column (GE Healthcare). The purified NMT variant protein was monomeric and free of aggregates. The integrity of the amino acid backbone of the reduced NMT variant protein was verified by Nano Electrospray QTOF mass spectrometry.

Before storage at -80°C, the protein was dialyzed against 50 mM HEPES, 1 mM EDTA and 1 mM DTT, pH 7.4.

Claims

Patent Claims

A fusion protein comprising a human IgG Fc-region, characterized in that to one or both N-termini of the human IgG Fc-region one hepatitis-B-virus preSl -domain derived amino acid sequence is fused, and in that to one or both C-termini of the human IgG Fc-region a single biologically active polypeptide is fused, whereby each of the fusions is independently of each other either directly or with an intermediate linker polypeptide.

The fusion protein according to claim 1, characterized in that to one N- terminus of the human IgG Fc-region a single hepatitis-B-virus preSl- domain derived amino acid sequence is fused, and in that to one C-terminus of the human IgG Fc-region a single biologically active polypeptide is fused, whereby the fusion is independently of each other either directly or with an intermediate linker polypeptide.

The fusion protein according to any one of the preceding claims, characterized in that

a) the preSl -domain derived polypeptide has an amino acid sequence selected from SEQ ID NO: 01, or SEQ ID NO: 02, or SEQ ID NO: 30, and/or

b) the first and second polypeptide linker has an amino acid sequence independently selected from SEQ ID NO: 03 to SEQ ID NO: 08, and/or c) the antibody heavy chain Fc-region has an amino acid sequence selected from SEQ ID NO: 16 to SEQ ID NO: 20, and/or

d) the biologically active polypeptide has the amino acid sequence of SEQ ID NO: 21.

An N-terminal acylated fusion protein comprising an N-terminal amino acid sequence of SEQ ID NO: 01, or SEQ ID NO: 02, or SEQ ID NO: 23, or SEQ ID NO: 24, or SEQ ID NO: 26, or SEQ ID NO: 27, or SEQ ID NO: 28, or SEQ ID NO: 29.

A polypeptide that has the amino acid sequence of SEQ ID NO: 30 or is a variant thereof.

6. An N-myristoyl transferase variant that has the amino acid sequence of SEQ ID NO: 22.

7. A complex comprising an N-terminal acylated fusion protein comprising an amino acid sequence of SEQ ID NO: 23 and a polypeptide comprising an amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18. 8. A complex comprising an N-terminal acylated fusion protein comprising an amino acid sequence of SEQ ID NO: 24 and a polypeptide comprising an amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18.

9. A complex comprising an N-terminal acylated fusion protein comprising an amino acid sequence of SEQ ID NO: 26 and a polypeptide comprising an amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO: 18.

10. A complex comprising an N-terminal acylated fusion protein comprising an amino acid sequence of SEQ ID NO: 27 and a polypeptide comprising an amino acid sequence of SEQ ID NO: 16, or SEQ ID NO: 17, or SEQ ID NO:

18.

11. A complex comprising an N-terminal acylated fusion protein comprising an amino acid sequence of SEQ ID NO: 27 and a polypeptide comprising an amino acid sequence of SEQ ID NO: 28. 12. A method for the production of an (N-terminally) acylated polypeptide comprising the step of:

acylating the polypeptide (N-terminally) in vitro and thereby producing an N-terminal acylated polypeptide, whereby in the amino acid sequence of the polypeptide any potential N-glycosylation site within the first fifty N-terminal amino acid residues of the amino acid sequence of the polypeptide has been removed.

13. The method according to claim 12, characterized in that the potential N- glycosylation site is removed by determining the position of potential N- glycosylation sites in the amino acid sequence of the polypeptide within the first fifty N-terminal amino acid residues of the polypeptide's amino acid sequence and modifying the N-glycosylation site motif (Asn-X-Ser/Thr, sequon) by changing the amino acid residue asparagine to a non-asparagine amino acid residue.

14. The method according to any one of claims 12 to 13, characterized in that the modifying is by changing the codon encoding the asparagine residue in the N-glycosylation site motif to a codon encoding a different, non-asparagine amino acid residue.

15. The method according to any one of claims 12 to 14, characterized in that any potential N-glycosylation site within the first twenty N-terminal amino acid residues of the polypeptide has been removed.

16. The method according to any one of claims 12 to 15, characterized in that the amino acid residue asparagine is changes to a neutral hydrophilic amino acid residue.

17. The method according to claim 16, characterized in that the neutral hydrophilic amino acid residue is glutamine.

18. The method according to any one of claims 12 to 16, characterized in that the codon encoding the asparagine residue of the potential N-glycosylation site is modified to encode a neutral hydrophilic amino acid residue.

19. The method according to claim 18, characterized in that the neutral hydrophilic amino acid residue is glutamine.

20. The method according to any one of claims 12 to 19, characterized in that the polypeptide is a fusion protein comprising at the N-terminus a hepatitis-B- virus preSl -domain derived polypeptide.

21. The method according to any one of claims 12 to 20, characterized in that the polypeptide is a fusion protein comprising an antibody heavy chain Fc-region.

22. The method according to any one of claims 12 to 21, characterized in that the polypeptide is a fusion protein comprising in N- to C-terminal direction a hepatitis-B-virus preSl -domain derived amino acid sequence, a first optional linker polypeptide, an antibody heavy chain Fc-region, a second optional linker polypeptide, and a biologically active polypeptide.

23. The method according to claim 22, characterized in that a) the preSl -domain derived polypeptide has an amino acid sequence selected from SEQ ID NO: 01, or SEQ ID NO: 02, or SEQ ID NO: 30, and/or

24. The method according to any one of claims 12 to 23, characterized in that the acylating of the polypeptide is by incubating the polypeptide in vitro with an acyl transferase and an activated carbonic acid.

25. The method according to claim 24, characterized in that the acyl transferase is yeast N-myristoyl transferase (NMT) or a variant thereof.

26. The method according to any one of claims 24 to 25, characterized in that the activated carbonic acid is myristoyl-CoA.

27. Use of an N-terminal acylated fusion protein according to any one of claim 1 to 5 or of a complex according to any one of claims 7 to 11 for the treatment of hepatitis virus infection.

28. The use according to claim 27, characterized in that the hepatitis virus infection is a hepatitis-B-virus infection.

29. The use according to claim 27, characterized in that the hepatitis virus infection is a hepatitis-C-virus infection. 30. The use according to claim 27, characterized in that the hepatitis virus infection is a hepatitis-D-virus infection.

31. The use according to any one of claims 28 to 30, characterized in that the hepatitis virus infection is an acute or a chronic hepatitis-B-virus, or hepatitis-C-virus, or hepatitis-D-virus infection. 32. An isolated nucleic acid encoding the fusion protein according to any one of claim 1 to 5.

33. A method of producing a fusion protein as reported herein comprising culturing a host cell as reported herein so that the fusion protein is produced.

34. A pharmaceutical formulation comprising a fusion protein according to any one of claim 1 to 5 or of a complex according to any one of claims 7 to 11.

35. A fusion protein according to any one of claim 1 to 5 or of a complex according to any one of claims 7 to 11 for use as a medicament.

36. The use according to claim 35, characterized in that the medicament is for delivering an anti-viral cytokine to hepatitis-B-virus, or hepatitis-C-virus, or hepatitis-D-virus infected hepatocytes.

37. A fusion protein according to any one of claim 1 to 5 or of a complex according to any one of claims 7 to 11 for use in treating hepatitis-B-virus infection, or hepatitis-C-virus infection, or hepatitis-D-virus infection.

38. A fusion protein according to any one of claim 1 to 5 or of a complex according to any one of claims 7 to 11 for use in delivering an anti-viral cytokine to hepatitis-B-virus infected hepatocytes, or hepatitis-C-virus infected hepatocytes, or hepatitis-D-virus infected hepatocytes.

39. Use of a fusion protein according to any one of claim 1 to 5 or of a complex according to any one of claims 7 to 11 in the manufacture of a medicament.

40. The use according to claim 39, characterized in that the medicament is for the treatment of hepatitis-B-virus infection, or hepatitis-C-virus infection, or hepatitis-D-virus infection.

41. The use according to claim 40, characterized in that the infection is a chronic infection.

42. A method of treating an individual having a hepatitis-B-virus infection, or a hepatitis-C-virus infection, or a hepatitis-D-virus infection comprising administering to the individual an effective amount of the fusion protein according to any one of claim 1 to 5 or of a complex according to any one of claims 7 to 11.

43. A method of delivering an anti-viral cytokine to hepatitis-B-virus infected hepatocytes, or hepatitis-C-virus infected hepatocytes, or hepatitis-D-virus infected hepatocytes in an individual comprising administering to the individual an effective amount of the fusion protein according to any one of claim 1 to 5 or of a complex according to any one of claims 7 to 11 to deliver an anti-viral cytokine to hepatitis-B-virus infected hepatocytes, or hepatitis- C-virus infected hepatocytes, or hepatitis-D-virus infected hepatocytes.