AU2019200306B2 - Immunoglobulin chimeric monomer-dimer hybrids - Google Patents

Abstract

The invention relates to a chimeric monomer-dimer hybrid protein wherein said protein comprises a first and a second polypeptide chain, said first polypeptide chain comprising at least a portion of an immunoglobulin constant region and a biologically active molecule, and said second polypeptide chain comprising at least a portion of an immunoglobulin constant region without the biologically active molecule of the first chain. The invention also relates to methods of using and methods of making the chimeric monomer-dimer hybrid protein of the invention.

Description

IMMUNOGLOBULIN CHIMERIC MONOMER-DIMER HYBRIDS

[001] This application is a divisional of Australian patent application no.

2016244273, filed on 13 October 2016 which is a divisional of 2013245463, filed on

October 2013, which is a divisional of Australian patent application no.

2013204787, filed on 12 April 2013, which is a divisional of Australian patent

application no. 2012200470, filed on 27 January 2012, which is a divisional of

Australian patent application no. 2004252422, filed on 6 May 2004, and is related to

International patent application no. PCT/US2004/014064, filed on 6 May 2004 and

claiming priority from United States patent application no. 60/469,600, filed on 6 May

2003, United States patent application no. 60/487,964, filed on 17 July 2003 and

United States patent application no. 60/539,207, filed on 26 January 2004; each of

which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[002] The invention relates generally to therapeutic chimeric proteins,

comprised of two polypeptide chains, wherein the first chain is comprised of a

therapeutic biologically active molecule and the second chain is not comprised of the

therapeutic biologically active molecule of the first chain. More specifically, the

invention relates to chimeric proteins, comprised of two polypeptide chains, wherein

both chains are comprised of at least a portion of an immunoglobulin constant region

wherein the first chain is modified to further comprise a biologically active molecule,

and the second chain is not so modified. The invention, thus relates to a chimeric

protein that is a monomer-dimer hybrid, i.e., a chimeric protein having a dimeric aspect

and a monomeric aspect, wherein the dimeric aspect relates to the fact that it is

comprised of two polypeptide chains each comprised of a portion of an

immunoglobulin constant region, and wherein the monomeric aspect relates to the fact that only one of the two chains is comprised of a therapeutic biologically active molecule. Figure 1 illustrates one example of a monomer-dimer hybrid wherein the

[Text continues on page 2.]

la biologically active molecule is erythropoietin (EPO) and the portion of an immunoglobulin constant region is an igG Fc region.

BACKGROUND OF THE INVENTION

[003] Immunoglobulins are comprised of four polypeptide chains, two to form heavy chains and two light chains, which associate via disulfide bonds

tetramers. Each chain is further comprised of one variable region and one constant while the region. The variable regions mediate antigen recognition and binding,

constant regions, particularly the heavy chain constant regions, mediate a variety of

effector functions, e.g., complement binding and Fc receptor binding (see, e.g., U.S.

Patent Nos.: 6,086,875; 5,624,821; 5,116,964).

[004] The constant region is further comprised of domains denoted CH

(constant heavy) domains (CH1, CH2, etc.). Depending on the isotype, (i.e. IgG, or four CH IgM, IgA IgD, gE) the constant region can be comprised of three

domains. Some isotypes (e.g. IgG) constant regions also contain a hinge region

Janeway et al. 2001,Immunobiology, Garland Publishing, N.Y., N.Y.

[005] The creation of chimeric proteins comprised of immunoglobulin

constant regions linked to a protein of interest, or fragment thereof, has been et al. described (see, e.g., U.S. Patent Nos. 5,480,981 and 5,808,029; Gascoigne

1987, Proc. Natl. Acad. Sci. USA 84:2936; Capon et al. 1989, Nature 337:525;

Traunecker et al. 1989, Nature 339:68; Zettmeissl et al. 1990, DNA Cell Biol. USA

9:347; Byrn et al. 1990, Nature 344:667; Watson et al. 1990, J. Cell. Biol. 110:2221;

Watson et al. 1991, Nature 349:164; Aruffo et al. 1990, Cell 61:1303; Linsley et al. Stamenkovic 1991, J. Exp. Med. 173:721; Linsley et al. 1991, J. Exp. Med. 174:561;

et al., 1991, Cell 66:1133; Ashkenazi et al. 1991, Proc. Nati. Acad. Sci. USA et al. 1991, J. 88:10535; Lesslauer et al. 1991, Eur. J. Immunol. 27:2883; Peppel Chem. 266:23060; Kurschner et al. Exp. Med. 174:1483; Bennett et al. 1991, J. Biol. Proc. Nati. Acad. Sci. USA 1992, J. Biol. Chem. 267:9354; Chalupny et al. 1992, 1448; Zheng 89:10360; Ridgway and Gorman, 1991, J. Cell. Biol. 115, Abstract No. possess both the et al. 1995, J.Immun. 154:5590). These molecules usually interest as well as the biological activity associated with the linked molecule of associated with the effector function, or some other desired characteristic immunoglobulin constant region (e.g. biological stability, cellular secretion). depending on

[006] The Fc portion of an immunoglobulin constant region, and CH4 domains, as well as the immunoglobulin isotype can include the CH2, CH3, Fc portion of an immunoglobulin the hinge region. Chimeric proteins comprising an including increased bestow several desirable properties on a chimeric protein 1989, Nature 337:525) as well stability, increased serum half life (see Capon et al. (FcRn) (U.S. Patent as binding to Fc receptors such as the neonatal Fc receptor

Nos. 6,086,875, 6,485,726, 6,030,613; WO 03/077834; US2003-0235536A1). and expressed in the lumen of

[007] FcRn is active in adult epithelial tissue surfaces, colon and rectal the intestines, pulmonary airways, nasal surfaces, vaginal of FcRn binding surfaces (U.S. Patent No. 6,485,726). Chimeric proteins comprised shuttled across epithelial barriers partners (e.g. IgG, Fc fragments) can be effectively to systemically administer a desired by FcRn, thus providing a non-invasive means FcRn binding therapeutic molecule. Additionally, chimeric proteins comprising an the FcRn. But instead of being marked partner are endocytosed by cells expressing again, thus for degradation, these chimeric proteins are recycled out into circulation

increasing the in vivo half life of these proteins.

[008] Portions of immunoglobulin constant regions, e.g., FcRn binding interactions, partners typically associate, via disulfide bonds and other non-specific invention is with one another to form dimers and higher order multimers. The instant proteins based in part upon the surprising discovery that transcytosis of chimeric by the molecular weight of comprised of FcRn binding partners appears to be limited less the chimeric protein, with higher molecular weight species being transported

efficiently.

[009] Chimeric proteins comprised of biologically active molecules, once

administered, typically will interact with a target molecule or cell. The instant

invention is further based in part upon the surprising discovery that monomer-dimer portions of an immunoglobulin hybrids, with one biologically active molecule, but two be transported constant region, e.g., two FcRn binding partners, function and can or more effectively than homodimers, also referred to herein simply as "dimers" biologically active molecule. higher order multimers with two or more copies of the This is due in part to the fact that chimeric proteins, comprised of two or more

biologically active molecules, which exist as dimers and higher order multimers, can due to the be sterically hindered from interacting with their target molecule or cell, in close proximity to one presence of the two or more biologically active molecules for itself. another and that the biologically active molecule can have a high affinity proteins

[010] Accordingly one aspect of the invention provides chimeric across the epithelium comprised of a biologically active molecule that is transported comprised barrier. An additional aspect of the invention provides chimeric proteins

of at least one biologically active molecule that is able to interact with its target

molecule or cell with little or no steric hindrance or self aggregation.

comprising

[011] The aspects of the invention provide for chimeric proteins at least a portion of a first and second polypeptide chain, the first chain comprising immunoglobulin constant immunoglobulin constant region, wherein the portion of an active molecule and the second region has been modified to include a biologically region, wherein the chain comprising at least a portion of immunoglobulin constant has not been so modified to include portion of an immunoglobulin constant region the biologically active molecule of the first chain. SUMMARYOFTHE INVENTION one biologically

[012] The invention relates to a chimeric protein comprising of an immunoglobulin active molecule and two molecules of at least a portion

constant region. The chimeric protein is capable of interacting with a target chimeric protein comprised molecule or cell with less steric hindrance compared to a a portion of two of at least two biologically active molecules and at least to a chimeric protein immunoglobulin constant regions. The invention also relates and two molecules of at least a comprising at least one biologically active molecule that is transported across an portion of an immunoglobulin constant region homodimer, i.e., wherein epithelium barrier more efficiently than a corresponding invention, thus both chains are linked to the same biologically active molecule. The polypeptide chain linked relates to a chimeric protein comprising a first and a second biologically active molecule and at together, wherein said first chain comprises a said second chain least a portion of an immunoglobulin constant region, and constant region, but no comprises at least a portion of an immunoglobulin active molecule immunoglobulin variable region and without any biologically

attached.

and a

[013] The invention relates to a chimeric protein comprising a first a second polypeptide chain linked together, wherein said first chain comprises

biologically active molecule and at least a portion of an immunoglobulin constant of an immunoglobulin region, and said second chain comprises at least a portion active constant region without an immunoglobulin variable region or any biologically any molecule molecule and wherein said second chain is not covalently bonded to kD. In one having a molecular weight greater than I kD, 2 kD, 5 kD, 10 kD, or 20

embodiment, the second chain is not covalently bonded to any molecule having a chain is not molecular weight greater than 0-2 kD. In one embodiment, the second than 5-10 kD. covalently bonded to any molecule having a molecular weight greater molecule in one embodiment, the second chain is not covalently bonded to any

having a molecular weight greater than 15-20 kD. first and a

[014] The invention relates to a chimeric protein comprising a a second polypeptide chain linked together, wherein said first chain comprises constant biologically active molecule and at least a portion of an immunoglobulin portion of an immunoglobulin region, and said second chain comprises at least a portion of an constant region not covalently linked to any other molecule except the

immunoglobulin of said first polypeptide chain. first and a

[015] The invention relates to a chimeric protein comprising a comprises a second polypeptide chain linked together, wherein said first chain constant biologically active molecule and at least a portion of an immunoglobulin of an immunoglobulin region, and said second chain consists of at least a portion

constant region and optionally an affinity tag.

a first and a

[016] The invention relates to a chimeric protein comprising chain comprises a second polypeptide chain linked together, wherein said first constant biologically active molecule and at least a portion of an immunoglobulin of at least a portion of an region, and said second chain consists essentially

immunoglobulin constant region and optionally an affinity tag. a first and a

[017] The invention relates to a chimeric protein comprising chain comprises a second polypeptide chain linked together, wherein said first immunoglobulin constant biologically active molecule and at least a portion of an a portion of an immunoglobulin region, and said second chain comprises at least or any biologically active constant region without an immunoglobulin variable region 10 kD, 5 kD, 2 molecule and optionally a molecule with a molecular weight less than a molecule less than kD or I kD. In one embodiment, the second chain comprises a molecule less than 5 15-20 kD. In one embodiment, the second chain comprises a molecule less than 1-2 10 kD. In one embodiment, the second chain comprises

kD. comprising a first and

[018] The invention relates to a chimeric protein a biologically active second polypeptide chain, wherein said first chain comprises region, and at least a first molecule, at least a portion of an immunoglobulin constant partner, and wherein domain, said first domain having at least one specific binding constant said second chain comprises at least a portion of an immunoglobulin said second domain is a specific region, and at least a second domain, wherein variable region or a binding partner of said first domain, without any immunoglobulin

biologically active molecule.

protein

[019] The invention relates to a method of making a chimeric the first polypeptide chain comprising a first and second polypeptide chain, wherein

and the second polypeptide chain are not the same, said method comprising

transfecting a cell with a first DNA construct comprising a DNA molecule encoding a and at least a first polypeptide chain comprising a biologically active molecule a linker, and a second portion of an immunoglobulin constant region and optionally chain DNA construct comprising a DNA molecule encoding a second polypeptide without any comprising at least a portion of an immunoglobulin constant region

biologically active molecule or an immunoglobulin variable region, and optionally a chain encoded linker, culturing the cells under conditions such that the polypeptide encoded by the by the first DNA construct is expressed and the polypeptide chain comprised second DNA construct is expressed and isolating monomer-dimer hybrids

of the polypeptide chain encoded by the first DNA construct and the polypeptide

chain encoded by the second DNA construct. protein

[020] The invention relates to a method of making a chimeric the first polypeptide chain comprising a first and second polypeptide chain, wherein first and the second polypeptide chain are not the same, and wherein said at least a portion of an polypeptide chain comprises a biologically active molecule,

immunoglobulin constant region, and at least a first domain, said first domain, having chain at least one specific binding partner, and wherein said second polypeptide region and a second comprises at least a portion of an immunoglobulin constant of said first domain, wherein said second domain, is a specific binding partner variable domain, without any biologically active molecule or an immunoglobulin a first DNA construct region, said method comprising transfecting a cell with comprising a DNA molecule encoding said first polypeptide chain and a second DNA construct comprising a DNA molecule encoding, said second polypeptide chain, culturing the cells under conditions such that the polypeptide chain encoded by the first DNA construct is expressed and the polypeptide chain encoded by the second

DNA construct is expressed and isolating monomer-dimer hybrids comprised of the

polypeptide chain encoded by the first DNA construct and polypeptide chain

encoded by the second DNA construct.

[021] The invention relates to a method of making a chimeric protein of the

invention said method comprising transfecting a cell with a first DNA construct

comprising a DNA molecule encoding a first polypeptide chain comprising a

biologically active molecule and at least a portion of an immunoglobulin constant

region and optionally a linker, culturing the cell under conditions such that the

polypeptide chain encoded by the first DNA construct is expressed, isolating the

polypeptide chain encoded by the first DNA construct and transfecting a cell with a

second DNA construct comprising a DNA molecule encoding a second polypeptide

chain comprising at least a portion of an immunoglobulin constant region without any

biologically active molecule or immunoglobulin variable region, culturing the cell

under conditions such that the polypeptide chain encoded by the second DNA

construct is expressed, isolating the polypeptide chain, encoded by the second DNA

construct, combining the polypeptide chain, encoded by the first DNA construct and

the polypeptide chain encoded by the second DNA construct under conditions such

that monomer-dimer hybrids comprising the polypeptide chain encoded by the first

DNA construct and the polypeptide chain encoded by the second DNA construct

form, and isolating said monomer-dimer hybrids.

[022] The invention relates to a method of making a chimeric protein

comprising a first and second polypeptide chain, wherein the first polypeptide chain

and the second polypeptide chain are not the same, said method comprising

transfecting a cell with a DNA construct comprising a DNA molecule encoding a

polypeptide chain comprising at least a portion of an immunoglobulin constant

region, culturing the cells under conditions such that the polypeptide chain encoded

by the DNA construct is expressed with an N terminal cysteine such that dimers of

the polypeptide chain form and isolating dimers comprised of two copies of the

polypeptide chain encoded by the DNA construct and chemically reacting the

isolated dimers with a biologically active molecule, wherein said biologically active

molecule has a C terminus thioester, under conditions such that the biologically

active molecule reacts predominantly with only one polypeptide chain of the dimer

thereby forming a monomer-dimer hybrid.

[023] The invention relates to a method of making a chimeric protein

comprising a first and second polypeptide chain, wherein the first polypeptide chain

and the second polypeptide chain are not the same, said method comprising

transfecting a cell with a DNA construct comprising a DNA molecule encoding a

polypeptide chain comprising at least a portion of an immunoglobulin constant

region, culturing the cells under conditions such that the polypeptide chain encoded

by the DNA construct is expressed with an N terminal cysteine such that dimers of

the polypeptide chains form, and isolating dimers comprised of two copies of the

polypeptide chain encoded by the DNA construct, and chemically reacting the

isolated dimers with a biologically active molecule, wherein said biologically active

molecule has a C terminus thioester, such that the biologically active molecule is linked to each chain of the dimer, denaturing the dimer comprised of the portion of the immunoglobulin linked to the biologically active molecule such that monomeric chains form, combining the monomeric chains with a polypeptide chain comprising at least a portion of an immunoglobulin constant region without a biologically active molecule linked to it, such that monomer-dimer hybrids form, and isolating the monomer-dimer hybrids.

[024] The invention relates to a method of making a chimeric protein

comprising a first and second polypeptide chain, wherein the first polypeptide chain

and the second polypeptide chain are not the same, said method comprising

transfecting a cell with a DNA construct comprising a DNA molecule encoding a

polypeptide chain comprising at least a portion of an immunoglobulin constant

region, culturing the cells under conditions such that the polypeptide chain encoded

by the DNA construct is expressed as a mixture of two polypeptide chains, wherein

the mixture comprises a polypeptide with an N terminal cysteine, and a polypeptide

with a cysteine in close proximity to the N terminus, isolating dimers comprised of

the mixture of polypeptide chains encoded by the DNA construct and chemically

reacting the isolated dimers with a biologically active molecule, wherein said

biologically active molecule has an active thioester, such that at least some

monomer-dimer hybrid forms and isolating the monomer-dimer hybrid from said

mixture.

[025] The invention relates to a method of treating a disease or condition

comprising administering a chimeric protein of the invention thereby treating the

disease or condition.

[026] Additional objects and advantages of the invention will be set forth in

part in the description which follows, and in part will be obvious from the description,

or may be learned by practice of the invention. The objects and advantages of the

invention will be realized and attained by means of the elements and combinations

particularly pointed out in the appended claims.

[027] It is to be understood that both the foregoing general description and

the following detailed description are exemplary and explanatory only and are not

restrictive of the invention, as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

[028] Figure 1 is a schematic diagram comparing the structure of an EPO

Fc homodimer, or dimer, and the structure of an Epo-FC monomer-dimer hybrid.

[029] Figure 2a is the amino acid sequence of the chimeric protein Factor

VIl-Fc. Included in the sequence is the signal peptide (underlined), which is cleaved

by the cell and the propeptide (bold), which is recognized by the vitamin K

dependent y carboxylase which modifies the Factor VII to achieve full activity. The

sequence is subsequently cleaved by PACE to yield Factor VII-Fc.

[030] Figure 2b is the amino acid sequence of the chimeric protein Factor

IX-Fc. Included in the sequence is the signal peptide (underlined) which is cleaved

by the cell and the propeptide (bold) which is recognized by the vitamin K-dependent

y carboxylase which modifies the Factor IX to achieve full activity. The sequence is

subsequently cleaved by PACE to yield Factor IX-Fc.

[031] Figure 2c is the amino acid sequence of the chimeric protein FNa-Fc.

Included in the sequence is the signal peptide (underlined), which is cleaved by the

cell resulting in the mature IFNa-Fc.

[032] Figure 2d is the amino acid sequence of the chimeric protein IFNa-Fc

A linker. Included in the sequence is the signal peptide (underlined) which is

cleaved by the cell resulting in the mature IFNa- Fc A linker.

[033] Figure 2e is the amino acid sequence of the chimeric protein Flag-Fc.

Included in the sequence is the signal peptide (underlined), which is cleaved by the

cell resulting in the mature Flag-Fc.

[034] Figure 2f is the amino acid sequence of the chimeric protein Epo

CCA-Fc. Included in the sequence is the signal peptide (underlined), which is cleaved by the cell resulting in the mature Epo-CCA-Fc. Also shown in bold is the acidic coiled coil domain.

[035] Figure 2g is the amino acid sequence of the chimeric protein CCB-Fc. Included in the sequence is the signal peptide (underlined), which is cleaved by the

cell resulting in the mature CCB-Fc. Also shown in bold is the basic coiled coil

domain.

[036] Figure 2h is the amino acid sequence of the chimeric protein Cys-Fc.

Included in the sequence is the signal peptide (underlined), which is cleaved by the

cell resulting in the mature Cys-Fc. When this sequence is produced in CHO cells a

percentage of the molecules are incorrectly cleaved by the signal peptidase such

that two extra amino acids are left on the N terminus, thus preventing the linkage of

a biologically active molecule with a C terminal thioester (e.g., via native ligation).

When these improperly cleaved species dimerize with the properly cleaved Cys-Fc

and are subsequently reacted with biologically active molecules with C terminal

thioesters, monomer-dimer hybrids form.

[037] Figure 2i is the amino acid sequence of the chimeric protein IFNa

GS15-Fc. Included in the sequence is the signal peptide (underlined) which is

cleaved by the cell resulting in the mature IFNa- GS15-Fc.

[038] Figure 2j is the amino acid sequence of the chimeric protein Epo-Fc.

Included in the sequence is the signal peptide (underlined), which is cleaved by the

cell resulting in the mature Epo-Fc. Also shown in bold is the 8 amino acid linker.

[039] Figure 3a is the nucleic acid sequence of the chimeric protein Factor

VII-Fc. Included in the sequence is the signal peptide (underlined) and the

propeptide (bold) which is recognized by the vitamin K-dependent y carboxylase which modifies the Factor VII to achieve full activity. The translated sequence is subsequently cleaved by PACE to yield mature Factor ViI-Fc.

[040] Figure 3b is the nucleic acid sequence of the chimeric protein Factor

IX-Fc. Included in the sequence is the signal peptide (underlined) and the

propeptide (bold) which is recognized by the vitamin K-dependent y carboxylase

which modifies the Factor IX to achieve full activity. The translated sequence is

subsequently cleaved by PACE to yield mature Factor IX-Fc.

[041] Figure 3c is the nucleic acid sequence of the chimeric protein IFNa

Fc. Included in the sequence is the signal peptide (underlined), which is cleaved by

the cell after translation resulting in the mature IFNa-Fc.

[042] Figure 3d is the nucleic acid sequence of the chimeric protein IFNa

Fc A linker. Included in the sequence is the signal peptide (underlined) which is

cleaved by the cell after translation resulting in the mature IFNa- Fc A linker.

[043] Figure 3e is the amino acid sequence of the chimeric protein Flag-Fc.

Included in the sequence is the signal peptide (underlined), which is cleaved by the

cell after translation resulting in the mature Flag-Fc.

[044] Figure 3f is the nucleic acid sequence of the chimeric protein Epo

CCA-Fc. Included in the sequence is the signal peptide (underlined), which is

cleaved by the cell after translation resulting in the mature Epo-CCA-Fc. Also shown

in bold is the acidic coiled coil domain.

[045] Figure 3g is the nucleic acid sequence of the chimeric protein CCB

Fc. Included in the sequence is the signal peptide (underlined), which is cleaved by

the cell after translation resulting in the mature CCB-Fc. Also shown in bold is the

basic coiled coil domain.

[046] Figure 3h is the nucleic acid sequence of the chimeric protein Cys-Fc.

Included in the sequence is the signal peptide (underlined), which is cleaved by the

cell after translation resulting in the mature Cys-Fc.

[047] Figure 3i is the nucleic acid sequence of the chimeric protein IFNa

GS15-Fc. Included in the sequence is the signal peptide (underlined) which is

cleaved by the cell after translation resulting in the mature IFNa-GS15-Fc.

[048] Figure 3j is the nucleic acid sequence of the chimeric protein Epo-Fc.

Included in the sequence is the signal peptide (underlined), which is cleaved by the

cell after translation resulting in the mature Epo-Fc. Also shown in bold is a nucleic

acid sequence encoding the 8 amino acid linker.

[049] Figure 4 demonstrates ways to form monomer-dimer hybrids through

native ligation.

[050] Figure 5a shows the amino acid sequence of Fc MESNA (SEQ ID

NO:4).

[051] Figure 5b shows the DNA sequence of Fc MESNA (SEQ ID NO:5).

[052] Figure 6 compares antiviral activity of IFNa homo-dimer (i.e.

comprised of 2 IFNa molecules) with an IFNa monomer-dimer hybrid (i.e. comprised

of 1 IFNa molecule).

[053] Figure 7 is a comparison of clotting activity of a chimeric monomer

dimer hybrid Factor Vila-Fc (one Factor VII molecule) and a chimeric homodimer

Factor Vlla-Fc (two Factor VIImolecules).

[054] Figure 8 compares oral dosing in neonatal rats of a chimeric

monomer-dimer hybrid Factor VIla-Fc (one Factor VI1 molecule) and a chimeric

homodimer Factor Vlla-Fc (two Factor V Imolecules).

[055] Figure 9 compares oral dosing in neonatal rats of a chimeric

monomer-dimer hybrid Factor IX-Fc (cie Factor IX molecule) with a chimeric

homodimer.

[056] Figure 10 is a time course study comparing a chimeric monomer

dimer hybrid Factor IX-Fc (one Factor IX molecule) administered orally to neonatal

rats with an orally administered chimeric homodimer.

[057] Figure 11 demonstrates pharmokinetics of Epo-Fc dimer compared to

Epo-Fc monomer-dimer hybrid in cynomolgus monkeys after a single pulmonary

dose.

[058] Figure 12 compares serum concentration in monkeys of

subcutaneously administered Epo-Fc monomer-dimer hybrid with subcutaneously

administered Aranesp* (darbepoetin alfa).

[059] Figure 13 compares serum concentration in monkeys of intravenously

administered Epo-Fc monomer-dimer hybrid with intravenously administered

Aranesp (darbepoetin alfa) and Epogen* (epoetin alfa).

[060] Figure 14 shows a trace from a Mimetic Red 2TM column (ProMetic

LifeSciences, Inc., Wayne, NJ) and an SDS-PAGE of fractions from the column

containing EpoFc monomer-dimer hybrid, EpoFc dimer, and Fc. EpoFc monomer

dimer hybrid is found in fractions 11, 12, 13, and 14. EpoFc dimer is found in

fraction 18. Fc is found in fractions 1/2.

[061] Figure 15 shows the pharmacokinetics of IFNpFc with an 8 amino

acid linker in cynomolgus monkeys after a single pulmonary dose.

[062] Figure 16 shows neopterin stimulation in response to the IFNp-Fc

homodimer and the IFNp-Fc N297A monomer-dimer hybrid in cynomolgus monkeys.

[063] Figure 17a shows the nucleotide sequence of interferon p-Fc; Figure

17b shows the amino acid sequence of interferon p-Fc.

[064] Figure 18 shows the amino acid sequence of T20(a); T21(b) and

T1249(c).

DESCRIPTION OF THE EMBODIMENTS

A. Definitions

[065] Affinity tag, as used herein, means a molecule attached to a second

molecule of interest, capable of interacting with a specific binding partner for the

purpose of isolating or identifying said second molecule of interest.

[066] Analogs of chimeric proteins of the invention, or proteins or peptides

substantially identical to the chimeric proteins of the invention, as used herein,

means that a relevant amino acid sequence of a protein or a peptide is at least 70%,

%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% identical to a given

sequence. By way of example, such sequences may be variants derived from

various species, or they may be derived from the given sequence by truncation,

deletion, amino acid substitution or addition. Percent identity between two amino

acid sequences is determined by standard alignment algorithms such as, for

example, Basic Local Alignment Tool (BLAST) described in Altschul et al. 1990, J.

Mol. Biol., 215:403-410, the algorithm of Needleman et al. 1970, J. Mol. Biol.,

48:444-453; the algorithm of Meyers et al. 1988, Comput. AppL. Biosci., 4:11-17; or

Tatusova et al. 1999, FEMS Microbiol. Lett., 174:247-250, etc. Such algorithms are

incorporated into the BLASTN, BLASTP and "BLAST 2 Sequences" programs (see

www.ncbi.nlm.nih.gov/BLAST). When utilizing such programs, the default

parameters can be used. For example, for nucleotide sequences the following settings can be used for "BLAST 2 Sequences": program BLASTN, reward for match

2, penalty for mismatch -2, open gap and extension gap penalties 5 and 2 For amino acid respectively, gap xdropoff 50, expect 10, word size 11, filter ON.

sequences the following settings can be used for "BLAST 2 Sequences": program

BLASTP, matrix BLOSUM62, open gap and extension gap penalties 11 and 1

respectively, gap x_dropoff 50, expect 10, word size 3, filter ON. a

[067] Bloavailability, as used herein, means the extent and rate at which

substance is absorbed into a living system or is made available at the site of

physiological activity. a non

[0681 Biologically active molecule, as used herein, means

immunoglobuin molecule or fragment thereof, capable of treating a disease or

condition or localizing or targeting a molecule to a site of a disease or condition in

the body by performing a function or an action, or stimulating or responding to a

function, an action or a reaction, in a biological context (e.g. in an organism, a cell,

or an in vitro model thereof). Biologically active molecules may comprise at least

one of polypeptides, nucleic acids, small molecules such as small organic or

inorganic molecules. of

[0691 A chimeric protein, as used herein, refers to any protein comprised

a first amino acid sequence derived from a first source, bonded, covalently or non

covalently, to a second amino acid sequence derived from a second source, wherein

the first and second source are not the same. A first source and a second source

that are not the same can include two different biological entities, or two different

proteins from the same biological entity, or a biological entity and a non-biological

entity. A chimeric protein can include for example, a protein derived from at least 2 different biological sources. A biological source can include any non-synthetically produced nucleic acid or amino acid sequence (e.g. a genomic or cDNA sequence, a plasmid or viral vector, a native virion or a mutant or analog, as further described herein, of any of the above). A synthetic source can include a protein or nucleic acid sequence produced chemically and not by a biological system (e.g. solid phase synthesis of amino acid sequences). A chimeric protein can also include a protein derived from at least 2 different synthetic sources or a protein derived from at least one biological source and at least one synthetic source. A chimeric protein may also comprise a first amino acid sequence derived from a first source, covalently or non covalently linked to a nucleic acid, derived from any source or a small organic or inorganic molecule derived from any source. The chimeric protein may comprise a linker molecule between the first and second amino acid sequence or between the first amino acid sequence and the nucleic acid, or between the first amino acid sequence and the small organic or inorganic molecule.

[070] Clotting factor, as used herein, means any molecule, or analog

thereof, naturally occurring or recombinantly produced which prevents or decreases

the duration of a bleeding episode in a subject with a hemostatic disorder. In other

words, it means any molecule having clotting activity.

[071] Clotting activity, as used herein, means the ability to participate in a

cascade of biochemical reactions that culminates in the formation of a fibrin clot

and/or reduces the severity, duration or frequency of hemorrhage or bleeding

episode.

[072] Dimer as used herein refers to a chimeric protein comprising a first

and second polypeptide chain, wherein the first and second chains both comprise a biologically active molecule, and at least a portion of an immunoglobulin constant region. A homodimer refers to a dimer where both biologically active molecules are the same.

[073] Dimerically linked monomer-dimer hybrid refers to a chimeric

protein comprised of at least a portion of an immunloglobulin constant region, e.g. an

Fc fragment of an immunoglobulin, a biologically active molecule and a linker which

links the two together such that one biologically active molecule is bound to 2

polypeptide chains, each comprising a portion of an immunoglobulin constant region.

Figure 4 shows an example of a dimerically linked monomer-dimer hybrid.

[074] DNA construct, as used herein, means a DNA molecule, or a clone of

such a molecule, either single- or double-stranded that has been modified through

human intervention to contain segments of DNA combined in a manner that as a

whole would not otherwise exist in nature. DNA constructs contain the information

necessary to direct the expression of polypeptides of interest. DNA constructs can

include promoters, enhancers and transcription terminators. DNA constructs

containing the information necessary to direct the secretion of a polypeptide will also

contain at least one secretory signal sequence.

[075] Domain, as used herein, means a region of a polypeptide (including

proteins as that term is defined) having some distinctive physical feature or role

including for example an independently folded structure composed of one section of

a polypeptide chain. A domain may contain the sequence of the distinctive physical

feature of the polypeptide or it may contain a fragment of the physical feature which

retains its binding characteristics (i.e., it can bind to a second domain). A domain may be associated with another domain. In other words, a first domain may naturally bind to a second domain.

[076] A fragment, as used herein, refers to a peptide or polypeptide

comprising an amino acid sequence of at least 2 contiguous amino acid residues, of

at least 5 contiguous amino acid residues, of at least 10 contiguous amino acid

residues, of at least 15 contiguous amino acid residues, of at least 20 contiguous

amino acid residues, of at least 25 contiguous amino acid residues, of at least 40

contiguous amino acid residues, of at least 50 contiguous amino acid residues, of at

least 100 contiguous amino acid residues, or of at least 200 contiguous amino acid

residues or any deletion or truncation of a protein, peptide, or polypeptide.

[077] Hemostasis, as used herein, means the stoppage of bleeding or

hemorrhage; or the stoppage of blood flow through a blood vessel or body part.

[078] Hemostatic disorder, as used herein, means a genetically inherited or

acquired condition characterized by a tendency to hemorrhage, either spontaneously

or as a result of trauma, due to an impaired ability or inability to form a fibrin clot.

[079] Linked, as used herein, refers to a first nucleic acid sequence

covalently joined to a second nucleic acid sequence. The first nucleic acid

sequence can be directly joined or juxtaposed to the second nucleic acid sequence

or alternatively an intervening sequence can covalently join the first sequence to the

second sequence. Linked as used herein can also refer to a first amino acid

sequence covalently, or non-covalently, joined to a second amino acid sequence.

The first amino acid sequence can be directly joined or juxtaposed to the second

amino acid sequence or alternatively an intervening sequence can covalently join

the first amino acid sequence to the second amino acid sequence.

[080] Operatively linked, as used herein, means a first nucleic acid

sequence linked to a second nucleic acid sequence such that both sequences are

capable of being expressed as a biologically active protein or peptide.

[081] Polypeptide, as used herein, refers to a polymer of amino acids and

does not refer to a specific length of the product; thus, peptides, oligopeptides, and

proteins are included within the definition of polypeptide. This term does not exclude

post-expression modifications of the polypeptide, for example, glycosylation,

acetylation, phosphorylation, pegylation, addition of a lipid moiety, or the addition of

any organic or inorganic molecule. Included within the definition, are for example,

polypeptides containing one or more analogs of an amino acid (including, for

example, unnatural amino acids) and polypeptides with substituted linkages, as well

as other modifications known in the art, both naturally occurring and non-naturally

occurring.

[082] High stringency, as used herein, includes conditions readily

determined by the skilled artisan based on, for example, the length of the DNA.

Generally, such conditions are defined in Sambrook et al. Molecular Cloning: A

Laboratory Manual, 2 ed. Vol. 1, pp. 1.101-104, Cold Spring Harbor Laboratory

Press (1989), and include use of a prewashing solution for the nitrocellulose filters

X SSC, 0.5% SDS, 1.0 mM EDTA (PH 8.0), hybridization conditions of 50%

formamide, 6X SSC at 420C (or other similar hybridization solution, such as Stark's

solution, in 50% formamide at 420C, and with washing at approximately 68°C, 0.2X

SSC, 0.1% SDS. The skilled artisan will recognize that the temperature and wash

solution salt concentration can be adjusted as necessary according to factors such

as the length of the probe.

[083] Moderate stringency, as used herein, include conditions that can be

readily determined by those having ordinary skill in the art based on, for example,

the length of the DNA. The basic conditions are set forth by Sambrook et al.

Molecular Cloning: A Laboratory Manual, 2d ed. Vol. 1, pp. 1.101-104, Cold Spring

Harbor Laboratory Press (1989), and include use of a prewashing solution for the

nitrocellulose filters 5X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization

conditions of 50% formamide, 6X SSC at 420C (or other similar hybridization

solution, such as Stark's solution, in 50% formamide at 42°C), and washing

conditions of 60°C, 0.5X SSC, 0.1% SDS.

[084] A small inorganic molecule, as used herein means a molecule

containing no carbon atoms and being no larger than 50 kD.

[085] A small organic molecule, as used herein means a molecule

containing at least one carbon atom and being no larger than 50 kD.

[086] Treat, treatment, treating, as used herein means, any of the

following: the reduction in severity of a disease or condition; the reduction in the

duration of a disease course; the amelioration of one or more symptoms associated

with a disease or condition; the provision of beneficial effects to a subject with a

disease or condition, without necessarily curing the disease or condition, the

prophylaxis of one or more symptoms associated with a disease or condition.

B. Improvements Offered by Certain Embodiments of the Invention

[087] The invention provides for chimeric proteins (monomer-dimer hybrids)

comprising a first and a second polypeptide chain, wherein said first chain comprises

a biologically active molecule and at least a portion of an immunoglobulin constant

region, and said second chain comprises at least a portion of an immunoglobulin constant region without any biologically active molecule or variable region of an immunoglobulin. Figure 1 contrasts traditional fusion protein dimers with one example of the monomer-dimer hybrid of the invention. In this example, the biologically active molecule is EPO and the portion of an immunoglobulin is IgG Fc region.

[088] Like other chimeric proteins comprised of at least a portion of an

immunoglobuin constant region, the invention provides for chimeric proteins which

afford enhanced stability and increased bioavailability of the chimeric protein

compared to the biologically active molecule alone. Additionally, however, because

only one of the two chains comprises the biologically active molecule, the chimeric

protein has a lower molecular weight than a chimeric protein wherein all chains

comprise a biologically active molecule and while not wishing to be bound by any

theory, this may result in the chimeric protein being more readily transcytosed

across the epithelium barrier, e.g., by binding to the FcRn receptor thereby

increasing the half-life of the chimeric protein. In one embodiment, the invention

thus provides for an improved non-invasive method (e.g. via any mucosal surface,

such as, orally, buccally, sublingually, nasally, rectally, vaginally, or via pulmonary or

occular route) of administering a therapeutic chimeric protein of the invention. The

invention thus provides methods of attaining therapeutic levels of the chimeric

proteins of the invention using less frequent and lower doses compared to previously

described chimeric proteins (e.g. chimeric proteins comprised of at least a portion of

an immunoglobulin constant region and a biologically active molecule, wherein all

chains of the chimeric protein comprise a biologically active molecule).

[089] In another embodiment, the invention provides an invasive method,

e.g., subcutaneously, intravenously, of administering a therapeutic chimeric protein

of the invention. Invasive administration of the therapeutic chimeric protein of the

invention provides for an increased half life of the therapeutic chimeric protein which

results in using less frequent and lower doses compared to previously described

chimeric proteins (e.g. chimeric proteins comprised of at least a portion of an

immunoglobulin constant region and a biologically active molecule, wherein all

chains of the chimeric protein comprise a biologically active molecule).

[090] Yet another advantage of a chimeric protein wherein only one of the

chains comprises a biologically active molecule is the enhanced accessibility of the

biologically active molecule for its target cell or molecule resulting from decreased

steric hindrance, decreased hydrophobic interactions, decreased ionic interactions,

or decreased molecular weight compared to a chimeric protein wherein all chains

are comprised of a biologically active molecule.

C. Chimeric Proteins

[091] The invention relates to chimeric proteins comprising one biologically

active molecule, at least a portion of an immunoglobulin constant region, and

optionally at least one linker. The portion of an immunoglobulin will have both an N,

or an amino terminus, and a C, or carboxy terminus. The chimeric protein may have

the biologically active molecule linked to the N terminus of the portion of an

immunoglobulin. Alternatively, the biologically active molecule may be linked to the

C terminus of the portion of an immunoglobuin. In one embodiment, the linkage is a

covalent bond. In another embodiment, the linkage is a non-covalent bond.

[092] The chimeric protein can optionally comprise at least one linker; thus,

the biologically active molecule does not have to be directly linked to the portion of

an immunoglobulin constant region. The linker can intervene in between the

biologically active molecule and the portion of an immunoglobulin constant region.

The linker can be linked to the N terminus of the portion of an immunoglobulin

constant region, or the C terminus of the portion of an immunoglobulin constant

region. If the biologically active molecule is comprised of at least one amino acid the

biologically active molecule will have an N terminus and a C terminus and the linker

can be linked to the N terminus of the biologically active molecule, or the C terminus

the biologically active molecule.

[093] The invention relates to a chimeric protein of the formula X-La-F:F or

F:F-La-X, wherein X is a biologically active molecule, L is an optional linker, F is at

least a portion of an immunoglobulin constant region and, a is any integer or zero.

The invention also relates to a chimeric protein of the formula Ta-X-La-F:F or Ta-F:F

La-X, wherein X is a biologically active molecule, L is an optional linker, F is at least

a portion of an immunoglobulin constant region, a is any integer or zero, T is a

second linker or alternatively a tag that can be used to facilitate purification of the

chimeric protein, e.g., a FLAG tag, a histidine tag, a GST tag, a maltose binding

protein tag and (:) represents a chemical association, e.g. at least one non-peptide

bond. In certain embodiments, the chemical association, i.e., (:) is a covalent bond.

In other embodiments, the chemical association, i.e., (:) is a non-covalent

interaction, e.g., an ionic interaction, a hydrophobic interaction, a hydrophilic

interaction, a Van der Waals interaction, a hydrogen bond. It will be understood by the skilled artisan that when a equals zero X will be directly linked to F. Thus, for example, a may be 0, 1, 2, 3, 4, 5, or more than 5.

[094] In one embodiment, the chimeric protein of the invention comprises

the amino acid sequence of figure 2a (SEQ ID NO:6). In one embodiment, the

chimeric protein of the invention comprises the amino acid sequence of figure 2b

(SEQ ID NO:8). In one embodiment, the chimeric protein of the invention comprises

the amino acid sequence of figure 2c (SEQ ID NO:10). In one embodiment, the

chimeric protein of the invention comprises the amino acid sequence of figure 2d

(SEQ ID NO:12). In one embodiment, the chimeric protein of the invention

comprises the amino acid sequence of figure 2e (SEQ ID NO:14). In one

embodiment, the chimeric protein of the invention comprises the amino acid

sequence of figure 2f (SEQ ID NO:16). In one embodiment, the chimeric protein of

the invention comprises the amino acid sequence of figure 2g (SEQ ID NO:18). In

one embodiment, the chimeric protein of the invention comprises the amino acid

sequence of figure 2h (SEQ ID NO:20). In one embodiment, the chimeric protein of

the invention comprises the amino acid sequence of figure 2i (SEQ ID NO:22). In

one embodiment, the chimeric protein of the invention comprises the amino acid

sequence of figure 2j (SEQ ID NO:24). In one embodiment, the chimeric protein of

the invention comprises the amino acid sequence of figure 17b (SEQ ID NO:27).

1. Chimeric Protein Variants

[095] Derivatives of the chimeric proteins of the invention, antibodies against

the chimeric proteins of the invention and antibodies against binding partners of the

chimeric proteins of the invention are all contemplated, and can be made by altering

their amino acids sequences by substitutions, additions, and/or deletions/truncations or by introducing chemical modification that result in functionally equivalent molecules. It will be understood by one of ordinary skill in the art that certain amino acids in a sequence of any protein may be substituted for other amino acids without adversely affecting the activity of the protein.

[096] Various changes may be made in the amino acid sequences of the

chimeric proteins of the invention or DNA sequences encoding therefore without

appreciable loss of their biological activity, function, or utility. Derivatives, analogs,

or mutants resulting from such changes and the use of such derivatives is within the

scope of the present invention. In a specific embodiment, the derivative is

functionally active, i.e., capable of exhibiting one or more activities associated with

the chimeric proteins of the invention, e.g., FcRn binding, viral inhibition, hemostasis,

production of red blood cells. Many assays capable of testing the activity of a

chimeric protein comprising a biologically active molecule are known in the art.

Where the biologically active molecule is an HIV inhibitor, activity can be tested by

measuring reverse transcriptase activity using known methods (see, e.g., Barre

Sinoussi et al. 1983, Science 220:868; Gallo et al. 1984, Science 224:500).

Alternatively, activity can be measured by measuring fusogenic activity (see, e.g.,

Nussbaum et al. 1994, J. Virol. 68(9):5411). Where the biological activity is

hemostasis, a StaCLot FVlla-rTF assay can be performed to assess activity of

Factor VIla derivatives (Johannessen et al. 2000, Blood Coagulation and Fibrinolysis

I1:S159).

[097] Substitutes for an amino acid within the sequence may be selected

from other members of the class to which the amino acid belongs (see Table 1).

Furthermore, various amino acids are commonly substituted with neutral amino acids, e.g., alanine, leucine, isoleucine, valine, praline, phenylalanine, tryptophan, and methionine (see, e.g., MacLennan et al. 1998, Acta Physiol. Scand. Suppl.

643:55-67; Sasaki et al. 1998, Adv. Biophys. 35:1-24).

TABLE 1

Original Exemplary Typical Residues Substitutions Substitutions Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gin, Asn Lys Asn (N) Gin Gin Asp (D) Glu GIu Cys (C) Ser, Ala Ser Gin (Q) Asn Asn Gly (G) Pro, Ala Ala His (H) Asn, Gin, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Leu Phe, Norleucine Leu (L) Norleucine, lie, Val, lie Met, Ala, Phe Lys (K) Arg, Arg 1,4-Diamino-butyric Acid, Gin, Asn Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, lie, Ala, Tyr Leu Pro (P) Ala Gly Ser (S) Thr, Ala, Cys Thr Thr (T) Ser Ser Trp (W) Tyr, Phe Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Met, Leu, Phe, Ala, Leu Norleucine

2. Biologically Active Molecules

[098] The invention contemplates the use of any biologically active molecule

as the therapeutic molecule of the invention. The biologically active molecule can be

a polypeptide. The biologically active molecule can be a single amino acid. The

biologically active molecule can include a modified polypeptide.

[099] The biologically active molecule can include a lipid molecule (e.g. a

steroid or cholesterol, a fatty acid, a triacylglycerol, glycerophospholipid, or

sphingolipid). The biologically active molecule can include a sugar molecule (e.g.

glucose, sucrose, mannose). The biologically active molecule can include a nucleic

acid molecule (e.g. DNA, RNA). The biologically active molecule can include a small

organic molecule or a small inorganic molecule.

a. Cytokines and Growth Factors

[0100] In one embodiment, the biologically active molecule is a growth

factor, hormone or cytokine or analog or fragment thereof. The biologically active

molecule can be any agent capable of inducing cell growth and proliferation. In a

specific embodiment, the biologically active molecule is any agent which can induce

erythrocytes to proliferate. Thus, one example of a biologically active molecule

contemplated by the invention is EPO. The biologically active molecule can also

include, but is not limited to, RANTES, MIP1a, MIP1p, IL-2, IL-3, GM-CSF, growth

hormone, tumor necrosis factor (e.g. TNFa orp).

[0101] The biologically active molecule can include interferon a, whether

synthetically or recombinantly produced, including but not limited to, any one of the

about twenty-five structurally related subtypes, as for example interferon-a2a, now

commercially available for clinical use (ROFERON@, Roche) and interferon-a2b also approved for clinical use (INTRON@, Schering) as well as genetically engineered versions of various subtypes, including, but not limited to, commercially available consensus interferon a (INFERGEN@, Intermune, developed by Amgen) and consensus human leukocyte interferon see, e.g., U.S. Patent Nos.: 4,695,623;

4,897,471, interferon P, epidermal growth factor, gonadotropin releasing hormone

(GnRH), leuprolide, follicle stimulating hormone, progesterone, estrogen, or

testosterone.

[0102] A list of cytokines and growth factors which maybe used in the

chimeric protein of the invention has been previously described (see, e.g., U.S.

Patent Nos. 6,086,875, 6,485,726, 6,030,613; WO 03/077834; US2003-0235536A1).

b. Antiviral Agents

[0103]In one embodiment, the biologically active molecule is an antiviral

agent, including fragments and analogs thereof. An antiviral agent can include any

molecule that inhibits or prevents viral replication, or inhibits or prevents viral entry

into a cell, or inhibits or prevents viral egress from a cell. In one embodiment, the

antiviral agent is a fusion inhibitor. In one embodiment, the antiviral agent is a

cytokine which inhibits viral replication. In another embodiment, the antiviral agent is

interferon a.

[0104] The viral fusion inhibitor for use in the chimeric protein can be any

molecule which decreases or prevents viral penetration of a cellular membrane of a

target cell. The viral fusion inhibitor can be any molecule that decreases or prevents

the formation of syncytia between at least two susceptible cells. The viral fusion

inhibitor can be any molecule that decreases or prevents the joining of a lipid bilayer

membrane of a eukaryotic cell and a lipid bilayer of an enveloped virus. Examples of enveloped virus include, but are not limited to HIV-1, HIV-2, SIV, influenza, parainfluenza, Epstein-Barr virus, CMV, herpes simplex 1, herpes simplex 2 and respiratory syncytia virus.

[0105] The viral fusion inhibitor can be any molecule that decreases or

prevents viral fusion including, but not limited to, a polypeptide, a small organic

molecule or a small inorganic molecule. In one embodiment, the fusion inhibitor is a

polypeptide. In one embodiment, the viral fusion inhibitor is a polypeptide of 3-36

amino acids. In another embodiment, the viral fusion inhibitor is a polypeptide of 3

amino acids, 10-65 amino acids, 10-75 amino acids. The polypeptide can be

comprised of a naturally occurring amino acid sequence (e.g. a fragment of gp4l)

including analogs and mutants thereof or the polypeptide can be comprised of an

amino acid sequence not found in nature, so long as the polypeptide exhibits viral

fusion inhibitory activity.

[0106] In one embodiment, the viral fusion inhibitor is a polypeptide, identified

as being a viral fusion inhibitor using at least one computer algorithm, e.g.,

ALLMOTI5,107x178x4 and PLZIP (see, e.g., U.S. Patent Nos.: 6,013,263;

6,015,881; 6,017,536; 6,020,459; 6,060,065; 6,068,973; 6,093,799; and 6,228,983).

[0107] In one embodiment, the viral fusion inhibitor is an HIV fusion inhibitor.

In one embodiment, HIV is HIV-1. In another embodiment, HIV is HIV-2. In one

embodiment, the HIV fusion inhibitor is a polypeptide comprised of a fragment of the

gp4l envelope protein of HIV-1. The HIV fusion inhibitor can comprise, e.g., T20

(SEQ ID NO:1) or an analog thereof, T21 (SEQ ID NO:2) or an analog thereof,

T1249 (SEQ ID NO:3) or an analog thereof, NccGgp41 (Louis et al. 2001, J. Biol.

Chem. 276:(31)29485) or an analog thereof, or 5 helix (Root et al. 2001, Science

291:884) or an analog thereof.

[0108] Assays known in the art can be used to test for viral fusion inhibiting

activity of a polypeptide, a small organic molecule, or a small inorganic molecule.

These assays include a reverse transcriptase assay, a p24 assay, or syncytia

formation assay (see, e.g., U.S. Patent No. 5,464,933).

[0109] A list of antiviral agents which may be used in the chimeric protein of

the invention has been previously described (see, e.g., U.S. Patent Nos. 6,086,875,

6,485,726, 6,030,613; WO 03/077834; US2003-0235536A1).

c. Hemostatic Agents

[0110] In one embodiment, the biologically active molecule is a clotting factor

or other agent that promotes hemostasis, including fragments and analogs thereof.

The clotting factor can include any molecule that has clotting activity or activates a

molecule with clotting activity. The clotting factor can be comprised of a polypeptide.

The clotting factor can be, as an example, but not limited to Factor VIII, Factor IX,

Factor XI, Factor XII, fibrinogen, prothrombin, Factor V, Factor VII, Factor X, Factor

XIII or von Willebrand Factor. In one embodiment, the clotting factor is Factor VII or

Factor Vla. The clotting factor can be a factor that participates in the extrinsic

pathway. The clotting factor can be a factor that participates in the intrinsic pathway.

Alternatively, the clotting factor can be a factor that participates in both the extrinsic

and intrinsic pathway.

[0111] The clotting factor can be a human clotting factor or a non-human

clotting factor, e.g., derived from a non-human primate, a pig or any mammal. The

clotting factor can be chimeric clotting factor, e.g., the clotting factor can comprise a portion of a human clotting factor and a portion of a porcine clotting factor or a portion of a first non-human clotting factor and a portion of a second non-human clotting factor.

[0112] The clotting factor can be an activated clotting factor. Alternatively, the

clotting factor can be an inactive form of a clotting factor, e.g., a zymogen. The

inactive clotting factor can undergo activation subsequent to being linked to at least

a portion of an immunoglobulin constant region. The inactive clotting factor can be

activated subsequent to administration to a subject. Alternatively, the inactive

clotting factor can be activated prior to administration.

[0113] In certain embodiments an endopeptidase, e.g., paired basic amino

acid cleaving enzyme (PACE), or any PACE family member, such as PCSK1-9,

including truncated versions thereof, or its yeast equivalent Kex2 from S. cerevisiae

and truncated versions of Kex2 (Kex2 1-675) (see, e.g., U.S. Patent Nos. 5,077,204;

,162,220; 5,234,830; 5,885,821; 6,329,176) may be used to cleave a propetide to

form the mature chimeric protein of the invention (e.g. factor VII, factor IX).

d. Other Proteinaceous Biologically Active Molecules

[0114] In one embodiment, the biologically active molecule is a receptor or a

fragment or analog thereof. The receptor can be expressed on a cell surface, or

alternatively the receptor can be expressed on the interior of the cell. The receptor

can be a viral receptor, e.g., CD4, CCR5, CXCR4, CD21, CD46. The biologically

active molecule can be a bacterial receptor. The biologically active molecule can be

an extra-cellular matrix protein or fragment or analog thereof, important in bacterial

colonization and infection (see, e.g., U.S. Patent Nos.: 5,648,240; 5,189,015;

,175,096) or a bacterial surface protein important in adhesion and infection (see, e.g., U.S. Patent No. 5,648,240). The biologically active molecule can be a growth factor, hormone or cytokine receptor, or a fragment or analog thereof, e.g., TNFa receptor, the erythropoietin receptor, CD25, CD122, or CD132.

[0115] A list of other proteinaceous molecules which may be used in the

chimeric protein of the invention has been previously described (see, e.g., U.S.

Patent Nos. 6,086,875; 6,485,726; 6,030,613; WO 03/077834; US2003-0235536A1).

e. Nucleic Acids

[0116] In one embodiment, the biologically active molecule is a nucleic acid,

e.g., DNA, RNA. In one specific embodiment, the biologically active molecule is a

nucleic acid that can be used in RNA interference (RNAi). The nucleic acid

molecule can be as an example, but not as a limitation, an anti-sense molecule or a

ribozyme or an aptamer.

[0117] Antisense RNA and DNA molecules act to directly block the translation

of mRNA by hybridizing to targeted mRNA and preventing protein translation.

Antisense approaches involve the design of oligonucleotides that are

complementary to a target gene mRNA. The antisense oligonucleotides will bind to

the complementary target gene mRNA transcripts and prevent translation. Absolute

complementarily, is not required.

[01181A sequence "complementary" to a portion of an RNA, as referred to

herein, means a sequence having sufficient complementarity to be able to hybridize

with the RNA, forming a stable duplex; in the case of double-stranded antisense

nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex

formation may be assayed. The ability to hybridize will depend on both the degree

of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be). One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

[0119] Antisense nucleic acids should be at least six nucleotides in length,

and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length.

In specific aspects, the oligonucleotide is at least 10 nucleotides, at least 17

nucleotides, at least 25 nucleotides or at least 50 nucleotides.

[0120] The oligonucleotides can be DNA or RNA or chimeric mixtures or

derivatives or modified versions thereof, single-stranded or double-stranded. The

oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate

backbone, for example, to improve stability of the molecule, hybridization, etc. The

oligonucleotide may include other appended groups such as polypeptides (e.g. for

targeting host cell receptors in vivo), or agents facilitating transport across the cell

membrane (see, e.g., Letsinger et al. 1989, Proc. Nati. Acad. Sci. USA 86:6553;

Lemaitre et al. 1987, Proc. Nati. Acad. Sci. USA 84:648; WO 88/09810,) or the

blood-brain barrier (see, e.g., WO 89/10134), hybridization-triggered cleavage

agents (see, e.g., Krol et al. 1988, BioTechniques 6:958) or intercalating agents

(see, e.g., Zon 1988, Pharm. Res. 5:539). To this end, the oligonucleotide may be

conjugated to another molecule, e.g., a polypeptide, hybridization triggered cross

linking agent, transport agent, or hybridization-triggered cleavage agent.

[0121] Ribozyme molecules designed to catalytically cleave target gene

mRNA transcripts can also be used to prevent translation of target gene mRNA and, therefore, expression of target gene product. (See, e.g., WO 90/11364; Sarver et al.

1990, Science 247,1222-1225).

[0122] Ribozymes are enzymatic RNA molecules capable of catalyzing the

specific cleavage of RNA. (See Rossi 1994, Current Biology 4:469). The

mechanism of ribozyme action involves sequence specific hybridization of the

ribozyme molecule to complementary target RNA, followed by an endonucleolytic

cleavage event. The composition of ribozyme molecules must include one or more

sequences complementary to the target gene mRNA, and must include the well

known catalytic sequence responsible for mRNA cleavage. For this sequence, see,

e.g., U.S. Pat. No. 5,093,246.

[0123] In one embodiment, ribozymes that cleave mRNA at site specific

recognition sequences can be used to destroy target gene mRNAs. In another

embodiment, the use of hammerhead ribozymes is contemplated. Hammerhead

ribozymes cleave mRNAs at locations dictated by flanking regions that form

complementary base pairs with the target mRNA. The sole requirement is that the

target mRNA have the following sequence of two bases: 5'-UG-3'. The construction

and production of hammerhead ribozymes is well known in the art and is described

more fully in Myers 1995, Molecular Biology and Biotechnology: A Comprehensive

Desk Reference, VCH Publishers, New York, and in Haseloff and Gerlach 1988,

Nature, 334:585.

f. Small Molecules

[0124] The invention also contemplates the use of any therapeutic small

molecule or drug as the biologically active molecule in the chimeric protein of the

invention. A list of small molecules and drugs which may be used in the chimeric protein of the invention has been previously described (see, e.g., U.S. Patent Nos.

6,086,875; 6,485,726; 6,030,613; WO 03/077834; US2003-0235536A1).

2. Immunoglobulins

[0125] The chimeric proteins of the invention comprise at least a portion of an

immunoglobulin constant region. Immunoglobulins are comprised of four protein

chains that associate covalently-two heavy chains and two light chains. Each

chain is further comprised of one variable region and one constant region.

Depending upon the immunoglobulin isotype, the heavy chain constant region is

comprised of 3 or 4 constant region domains (e.g. CH1, CH2, CH3, CH4). Some

isotypes are further comprised of a hinge region.

[0126] The portion of an immunoglobulin constant region can be obtained

from any mammal. The portion of an immunoglobulin constant region can include a

portion of a human immunoglobulin constant region, a non-human primate

immunoglobulin constant region, a bovine immunoglobulin constant region, a

porcine immunoglobulin constant region, a murine immunoglobulin constant region,

an ovine immunoglobulin constant region or a rat immunoglobulin constant region.

[0127] The portion of an immunoglobulin constant region can be produced

recombinantly or synthetically. The immunoglobulin can be isolated from a cDNA

library. The portion of an immunoglobulin constant region can be isolated from a

phage library (See, e.g., McCafferty et al. 1990, Nature 348:552, Kang et al. 1991,

Proc. Natl. Acad. Sci. USA 88:4363; EP 0 589 877 B1). The portion of an

immunoglobulin constant region can be obtained by gene shuffling of known

sequences (Mark et al. 1992, Bio/Technol. 10:779). The portion of an

immunoglobulin constant region can be isolated by in vivo recombination

(Waterhouse et al. 1993, Nucl. Acid Res. 21:2265). The immunoglobulin can be a

humanized immunoglobulin (U.S. Patent No. 5,585,089, Jones et al. 1986, Nature

332:323).

[0128] The portion of an immunoglobulin constant region can include a portion

of an IgG, an IgA, an IgM, an IgD, or an IgE. In one embodiment, the

immunoglobulin is an IgG. In another embodiment, the immunoglobulin is IgG1. In

another embodiment, the immunoglobulin is IgG2.

[0129] The portion of an immunoglobulin constant region can include the

entire heavy chain constant region, or a fragment or analog thereof. In one

embodiment, a heavy chain constant region can comprise a CH1 domain, a CH2

domain, a CH3 domain, and/or a hinge region. In another embodiment, a heavy

chain constant region can comprise a CH1 domain, a CH2 domain, a CH3 domain,

and/or a CH4 domain.

[0130] The portion of an immunoglobulin constant region can include an Fc

fragment. An Fc fragment can be comprised of the CH2 and CH3 domains of an

immunoglobulin and the hinge region of the immunoglobulin. The Fc fragment can

be the Fc fragment of an IgG1, an IgG2, an IgG3 or an IgG4. In one specific

embodiment, the portion of an immunoglobulin constant region is an Fc fragment of

an IgG1. In another embodiment, the portion of an immunoglobulin constant region

is an Fc fragment of an IgG2.

[0131] In another embodiment, the portion of an immunoglobulin constant

region is an Fc neonatal receptor (FcRn) binding partner. An FcRn binding partner

is any molecule that can be specifically bound by the FcRn receptor with consequent

active transport by the FcRn receptor of the FcRn binding partner. Specifically bound refers to two molecules forming a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity. Typically, binding is considered specific when the affinity constant KA is higher than 106 M 1 , or more preferably higher than 108 M 1. If necessary, non-specific binding can be reduced without substantially affecting specific binding by varying the binding conditions. The appropriate binding conditions such as concentration of the molecules, ionic strength of the solution, temperature, time allowed for binding, concentration of a blocking agent (e.g. serum albumin, milk casein), etc., may be optimized by a skilled artisan using routine techniques.

[0132] The FcRn receptor has been isolated from several mammalian species

including humans. The sequences of the human FcRn, monkey FcRn rat FcRn, and

mouse FcRn are known (Story et al. 1994, J. Exp. Med. 180:2377). The FcRn

receptor binds igG (but not other immunoglobulin classes such asIgA, IgM, IgD, and

IgE) at relatively low pH, actively transports the IgG transcellularly in a luminal to

serosal direction, and then releases the IgG at relatively higher pH found in the

interstitial fluids. It is expressed in adult epithelial tissue (U.S. Patent Nos.

6,485,726, 6,030,613, 6,086,875; WO 03/077834; US2003-0235536A1) including

lung and intestinal epithelium (Israel et al. 1997,Immunology 92:69) renal proximal

tubular epithelium (Kobayashi et al. 2002, Am. J. Physiol. Renal Physiol. 282:F358)

as well as nasal epithelium, vaginal surfaces, and biliary tree surfaces.

[0133] FcRn binding partners of the present invention encompass any

molecule that can be specifically bound by the FcRn receptor including whole IgG, the Fc fragment of IgG, and other fragments that include the complete binding region of the FcRn receptor. The region of the Fc portion of IgG that binds to the FcRn receptor has been described based on X-ray crystallography (Burmeister et al. 1994,

Nature 372:379). The major contact area of the Fc with the FcRn is near the

junction of the CH2 and CH3 domains. Fc-FcRn contacts are all within a single Ig

heavy chain. The FcRn binding partners include whole IgG, the Fc fragment of IgG,

and other fragments of IgG that include the complete binding region of FcRn. The

major contact sites include amino acid residues 248, 250-257, 272, 285, 288, 290

291, 308-311, and 314 of the CH2 domain and amino acid residues 385-387, 428,

and 433-436 of the CH3 domain. References made to amino acid numbering of

immunoglobulins or immunoglobulin fragments, or regions, are all based on Kabat et

al. 1991, Sequences of Proteins ofImmunological Interest, U.S. Department of

Public Health, Bethesda, MD.

[0134] The Fc region of IgG can be modified according to well recognized

procedures such as site directed mutagenesis and the like to yield modified IgG or

Fc fragments or portions thereof that will be bound by FcRn. Such modifications

include modifications remote from the FcRn contact sites as well as modifications

within the contact sites that preserve or even enhance binding to the FcRn. For

example, the following single amino acid residues in human IgG1 Fc (Fcyl) can be

substituted without significant loss of Fc binding affinity for FcRn: P238A, S239A,

K246A, K248A, D249A, M252A, T256A, E258A, T260A, D265A, S267A, H268A,

E269A, D270A, E272A, L274A, N276A, Y278A, D280A, V282A, E283A, H285A,

N286A, T289A, K290A, R292A, E293A, E294A, Q295A, Y296F, N297A, S298A,

Y300F, R301A, V303A, V305A, T307A, L309A, Q311A, D312A, N315A, K317A,

E318A, K320A, K322A, S324A, K326A, A327Q, P329A, A330Q, P331A, E333A,

K334A, T335A, S337A, K338A, K340A, Q342A, R344A, E345A, Q347A, R355A,

E356A, M358A, T359A, K360A, N361A, Q362A, Y373A, S375A, D376A, A378Q,

E380A, E382A, S383A,N384A, Q386A, E388A, N389A, N390A, Y391F, K392A,

L398A, S400A, D401A, D413A, K414A, R416A, Q418A, Q419A, N421A, V422A,

S424A, E430A, N434A, T437A, Q438A, K439A, S440A, S444A, and K447A, where

for example P238A represents wildtype proline substituted by alanine at position

number 238. As an example, one specifc embodiment, incorporates the N297A

mutation, removing a highly conserved N-glycosylation site. In addition to alanine

other amino acids may be substituted for the wildtype amino acids at the positions

specified above. Mutations may be introduced singly into Fc giving rise to more than

one hundred FcRn binding partners distinct from native Fc. Additionally,

combinations of two, three, or more of these individual mutations may be introduced

together, giving rise to hundreds more FcRn binding partners. Moreover, one of the

FcRn binding partners of the monomer-dimer hybrid may be mutated and the other

FcRn binding partner not mutated at all, or they both may be mutated but with

different mutations. Any of the mutations described herein, including N297A, may

be used to modify Fc, regardless of the biologically active molecule (e.g., EPO, IFN,

Factor IX, T20).

[0135] Certain of the above mutations may confer new functionality upon the

FcRn binding partner. For example, one embodiment incorporates N297A,

removing a highly conserved N-glycosylation site. The effect of this mutation is to

reduce immunogenicity, thereby enhancing circulating half life of the FcRn binding

partner, and to render the FcRn binding partner incapable of binding to FcyRI,

FcyRIlA, FcyRIlIB, and FcyRIIA, without compromising affinity for FcRn (Routledge

et al. 1995, Transplantation 60:847; Friend et al. 1999, Transplantation 68:1632;

Shields et al. 1995, J. Biol. Chem. 276:6591). As a further example of new

functionality arising from mutations described above affinity for FcRn may be

increased beyond that of wild type in some instances. This increased affinity may

reflect an increased "on" rate, a decreased "off" rate or both an increased "on" rate

and a decreased "off" rate. Mutations believed to impart an increased affinity for

FcRn include T256A, T307A, E380A, and N434A (Shields et al. 2001, J. Biol. Chem.

276:6591).

[0136] Additionally, at least three human Fc gamma receptors appear to

recognize a binding site on IgG within the lower hinge region, generally amino acids

234-237. Therefore, another example of new functionality and potential decreased

immunogenicity may arise from mutations of this region, as for example by replacing

amino acids 233-236 of human IgG1 "ELLG" to the corresponding sequence from

IgG2 "PVA" (with one amino acid deletion). It has been shown that FcyRI, FcyRIl,

and FcyRilIl, which mediate various effector functions will not bind to IgG1 when

such mutations have been introduced. Ward and Ghetie 1995, Therapeutic

Immunology 2:77 and Armour et al.1999, Eur. J. Immunol. 29:2613.

[0137]In one embodiment, the FcRn binding partner is a polypeptide

including the sequence PKNSSMISNTP (SEQ ID NO:26) and optionally further

including a sequence selected from HQSLGTQ (SEQ ID NO:27), HQNLSDGK (SEQ

ID NO:28), HQNISDGK (SEQ ID NO:29), or VISSHLGQ (SEQ ID NO:30) (U.S.

Patent No. 5,739,277).

0138] Two FcRn receptors can bind a single Fc molecule. Crystallographic

data suggest that each FcRn molecule binds a single polypeptide of the Fc

homodimer. In one embodiment, linking the FcRn binding partner, e.g., an Fc

fragment of an IgG, to a biologically active molecule provides a means of delivering

the biologically active molecule orally, buccally, sublingually, rectally, vaginally, as

an aerosol administered nasally or via a pulmonary route, or via an ocular route. In

another embodiment, the chimeric protein can be administered invasively, e.g.,

subcutaneously, intravenously.

[0139]The skilled artisan will understand that portions of an immunoglobulin

constant region for use in the chimeric protein of the invention can include mutants

or analogs thereof, or can include chemically modified immunoglobulin constant

regions (e.g. pegylated), or fragments thereof (see, e.g., Aslam and Dent 1998,

Bioconjugation: Protein Coupling Techniques For the Biomedical Sciences Macmilan

Reference, London). In one instance, a mutant can provide for enhanced binding of

an FcRn binding partner for the FcRn. Also contemplated for use in the chimeric

protein of the invention are peptide mimetics of at least a portion of an

immunoglobulin constant region, e.g., a peptide mimetic of an Fc fragment or a

peptide mimetic of an FcRn binding partner. In one embodiment, the peptide

mimetic is identified using phage display or via chemical library screening (see, e.g.,

McCafferty et a. 1990, Nature 348:552, Kang et al. 1991, Proc. Nati. Acad. Sci. USA

88:4363; EP 0 589 877 B1).

3. Optional Linkers

[0140] The chimeric protein of the invention can optionally comprise at least

one linker molecule. The linker can be comprised of any organic molecule. In one embodiment, the linker is polyethylene glycol (PEG). In another embodiment, the linker is comprised of amino acids. The linker can comprise 1-5 amino acids, 1-10 amino acids, 1-20 amino acids, 10-50 amino acids, 50-100 amino acids, 100-200 amino acids. In one embodiment, the linker is the eight amino acid linker

EFAGAAAV (SEQ ID NO:31). Any of the linkers described herein may be used in

the chimeric protein of the invention, e.g., a monomer-dimer hybrid, including

EFAGAAAV, regardless of the biologically active molecule (e.g. EPO, IFN, Factor

IX).

[0141] The linker can comprise the sequence Gn. The linker can comprise the

sequence (GA), (SEQ ID NO:32). The linker can comprise the sequence (GGS)n

(SEQ ID NO:33). The linker can comprise the sequence (GGS)n(GGGGS)n (SEQ ID

NO:34). In these instances, n may be an integer from 1-10, i.e., 1, 2, 3, 4, 5, 6, 7, 8,

9, 10. Examples of linkers include, but are not limited to, GGG (SEQ ID NO:35),

SGGSGGS (SEQ ID NO:36), GGSGGSGGSGGSGGG (SEQ ID NO:37),

GGSGGSGGGGSGGGGS (SEQ ID NO:38), GGSGGSGGSGGSGGSGGS (SEQ ID

NO:39). The linker does not eliminate or diminish the biological activity of the

chimeric protein. Optionally, the linker enhances the biological activity of the

chimeric protein, e.g., by further diminishing the effects of steric hindrance and

making the biologically active molecule more accessible to its target binding site.

[0142] In one specific embodiment, the linker for interferon a is 15-25 amino

acids long. In another specific embodiment, the linker for interferon a is 15-20 amino

acidslong. In another specific embodiment, the linker for interferon a is 10-25 amino

acids long. In another specific embodiment, the linker for interferon a is 15 amino

acidslong. In one embodiment, the linker for interferon a is (GGGGS)n (SEQ ID

NO:40) where G represents glycine, S represents serine and n is an integer from 1

10. In a specific embodiment, n is 3.

[0143] The linker may also incorporate a moiety capable of being cleaved

either chemically (e.g. hydrolysis of an ester bond), enzymatically (i.e. incorporation

of a protease cleavage sequence) or photolytically (e.g.,a chromophore such as 3

amino-3-(2-nitrophenyl) proprionic acid (ANP)) in order to release the biologically

active molecule from the Fc protein.

4. Chimeric Protein Dimerization Using Specific Binding Partners

[0144] In one embodiment, the chimeric protein of the invention comprises a

first polypeptide chain comprising at least a first domain, said first domain having at

least one specific binding partner, and a second polypeptide chain comprising at

least a second domain, wherein said second domain, is a specific binding partner of

said first domain. The chimeric protein thus comprises a polypeptide capable of

dimerizing with another polypeptide due to the interaction of the first domain and the

second domain. Methods of dimerizing antibodies using heterologous domains are

known in the art (U.S. Patent Nos.: 5,807,706 and 5,910,573; Kostelny et al. 1992, J.

Immunol. 148(5):1547).

[0145] Dimerization can occur by formation of a covalent bond, or

alternatively a non-covalent bond, e.g., hydrophobic interaction, Van der Waal's

forces, interdigitation of amphiphilic peptides such as, but not limited to, alpha

helices, charge-charge interactions of amino acids bearing opposite charges, such

as, but not limited to, lysine and aspartic acid, arginine and glutamic acid. In one

embodiment, the domain is a helix bundle comprising a helix, a turn and another

helix. In another embodiment, the domain is a leucine zipper comprising a peptide having several repeating amino acids in which every seventh amino acid is a leucine residue. In one embodiment, the specific binding partners are fos/jun. (see Branden et al. 1991,Introduction To Protein Structure, Garland Publishing, New York).

[01461 In another embodiment, binding is mediated by a chemical linkage

(see, e.g., Brennan et al. 1985, Science 229:81). In this embodiment, intact

immunoglobulins, or chimeric proteins comprised of at least a portion of an

immunoglobulin constant region are cleaved to generate heavy chain fragments.

These fragments are reduced in the presence of the dithiol complexing agent

sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide

formation. The fragments generated are then converted to thionitrobenzoate (TNB)

derivatives. One of the TNB derivatives is then reconverted to the heavy chain

fragment thiol by reduction with mercaptoethylamine and is then mixed with an

equimolar amount of the other TNB derivative to form a chimeric dimer.

D. Nucleic Acids

[0147] The invention relates to a first nucleic acid construct and a second

nucleic acid construct each comprising a nucleic acid sequence encoding at least a

portion of the chimeric protein of the invention. In one embodiment, the first nucleic

acid construct comprises a nucleic acid sequence encoding a portion of an

immunoglobulin constant region operatively linked to a second DNA sequence

encoding a biologically active molecule, and said second DNA construct comprises a

DNA sequence encoding an immunoglobulin constant region without the second

DNA sequence encoding a biologically active molecule.

[0148] The biologically active molecule can include, for example, but not as a

limitation, a viral fusion inhibitor, a clotting factor, a growth factor or hormone, or a receptor, or analog, or fragment of any of the preceding. The nucleic acid sequences can also include additional sequences or elements known in the art (e.g., promoters, enhancers, poly A sequences, affinity tags). In one embodiment, the nucleic acid sequence of the second construct can optionally include a nucleic acid sequence encoding a linker placed between the nucleic acid sequence encoding the biologically active molecule and the portion of the immunoglobulin constant region.

The nucleic acid sequence of the second DNA construct can optionally include a

linker sequence placed before or after the nucleic acid sequence encoding the

biologically active molecule and/or the portion of the immunoglobulin constant

region.

[0149] In one embodiment, the nucleic acid construct is comprised of DNA. In

another embodiment, the nucleic acid construct is comprised of RNA. The nucleic

acid construct can be a vector, e.g., a viral vector or a plasmid. Examples of viral

vectors include, but are not limited to adeno virus vector, an adeno associated virus

vector or a murine leukemia virus vector. Examples of plasmids include but are not

limited to pUC, pGEM and pGEX.

[0150]In one embodiment, the nucleic acid construct comprises the nucleic

acid sequence of figure 3a (SEQ ID NO:7). In one embodiment, the nucleic acid

construct comprises the nucleic acid sequence of figure 3b (SEQ ID NO:9 ). In one

embodiment, the nucleic acid construct comprises the nucleic acid sequence of

figure 3c (SEQ ID NO:11). In one embodiment, the nucleic acid construct comprises

the nucleic acid sequence of figure 3d (SEQ ID NO:13). In one embodiment, the

nucleic acid construct comprises the nucleic acid sequence of figure 3e (SEQ ID

NO:15). In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 3f (SEQ ID NO:17). In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 3g (SEQ ID NO:19). In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 3h (SEQ ID NO:21). In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 3i (SEQ ID NO:23). In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 3 (SEQ ID NO:25). In one embodiment, the nucleic acid construct comprises the nucleic acid sequence of figure 17a (SEQ ID NO:27).

[0151] Due to the known degeneracy of the genetic code, wherein more than one codon can encode the same amino acid, a DNA sequence can vary from that shown in SEQ ID NOS:7, 9, 11, 13, 15, 17,19, 21, 23, 25 or 27 and still encode a polypeptide having the corresponding amino acid sequence of SEQ ID NOS:6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26 respectively. Such variant DNA sequences can result from silent mutations (e.g. occurring during PCR amplification), or can be the product of deliberate mutagenesis of a native sequence. The invention thus provides isolated DNA sequences encoding polypeptides of the invention, chosen from: (a) DNA comprising the nucleotide sequence of SEQ ID NOS:7, 9, 11, 13, 15, 17, 19, 21, 23, 25 or 27; (b) DNA encoding the polypeptides of SEQ ID NOS:6, 8, 10, 12, 14, 16, 18, 20, 22, 24 or 26; (c) DNA capable of hybridization to a DNA of (a) or (b) under conditions of moderate stringency and which encodes polypeptides of the invention; (d) DNA capable of hybridization to a DNA of (a) or (b) under conditions of high stringency and which encodes polypeptides of the invention, and (e) DNA which is degenerate as a result of the genetic code to a DNA defined in (a),

(b), (c), or (d) and which encode polypeptides of the invention. Of course,

polypeptides encoded by such DNA sequences are encompassed by the invention.

[0152] In another embodiment, the nucleic acid molecules comprising a

sequence encoding the chimeric protein of the invention can also comprise

nucleotide sequences that are at least 80% identical to a native sequence. Also

contemplated are embodiments in which a nucleic acid molecules comprising a

sequence encoding the chimeric protein of the invention comprises a sequence that

is at least 90% identical, at least 95% identical, at least 98% identical, at least 99%

identical, or at least 99.9% identical to a native sequence. A native sequence can

include any DNA sequence not altered by the human hand. The percent identity

may be determined by visual inspection and mathematical calculation. Alternatively,

the percent identity of two nucleic acid sequences can be determined by comparing

sequence information using the GAP computer program, version 6.0 described by

Devereux et al. 1984, Nucl. Acids Res. 12:387, and available from the University of

Wisconsin Genetics Computer Group (UWGCG). The preferred default parameters

for the GAP program include: (1) a unary comparison matrix (containing a value of I

for identities and 0 for non identities) for nucleotides, and the weighted comparison

matrix of Gribskov and Burgess 1986, Nuc. Acids Res. 14:6745, as described by

Schwartz and Dayhoff, eds. 1979, Atlas of Protein Sequence and Structure, National

Biomedical Research Foundation, pp. 353-358; (2) a penalty of 3.0 for each gap and

an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end

gaps. Other programs used by one skilled in the art of sequence comparison may

also be used.

E. Synthesis of Chimeric Proteins

[0153] Chimeric proteins comprising at least a portion of an immunoglobulin

constant region and a biologically active molecule can be synthesized using

techniques well known in the art. For example, the chimeric proteins of the invention

can be synthesized recombinantly in cells (see, e.g., Sambrook et al. 1989,

Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y. and

Ausubel et al. 1989, Current Protocols in Molecular Biology, Greene Publishing

Associates and Wiley Interscience, N.Y.). Alternatively, the chimeric proteins of the

invention can be synthesized using known synthetic methods such as solid phase

synthesis. Synthetic techniques are well known in the art (see, e.g., Merrifield, 1973,

Chemical Polypeptides, (Katsoyannis and Panayotis eds.) pp. 335-61; Merrifield

1963, J. Am. Chem. Soc. 85:2149; Davis et al. 1985, Biochem. Intl. 10:394; Finn et

al. 1976, The Proteins (3d ed.) 2:105; Erikson et al. 1976, The Proteins (3d ed.)

2:257; U.S. Patent No. 3,941,763. Alternatively, the chimeric proteins of the

invention can be synthesized using a combination of recombinant and synthetic

methods. In certain applications, it may be beneficial to use either a recombinant

method or a combination of recombinant and synthetic methods.

[0154] Nucleic acids encoding a biologically active molecule can be readily

synthesized using recombinant techniques well known in the art. Alternatively, the

peptides themselves can be chemically synthesized. Nucleic acids of the invention

may be synthesized by standard methods known in the art, e.g., by use of an

automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.). As examples, phosphorothioate oligonucleotides may be

synthesized by the method of Stein et al. 1988, Nuc/. Acids Res. 16:3209, methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports as described in Sarin et al. 1988, Proc. Nat/. Acad. Sci. USA

:7448. Additional methods of nucleic acid synthesis are known in the art. (see,

e.g., U.S. Patent Nos. 6,015,881; 6,281,331; 6,469,136).

[0155] DNA sequences encoding immunoglobulin constant regions, or

fragments thereof, may be cloned from a variety of genomic or cDNA libraries known

in the art. The techniques for isolating such DNA sequences using probe-based

methods are conventional techniques and are well known to those skilled in the art.

Probes for isolating such DNA sequences may be based on published DNA

sequences (see, for example, Hieter et al. 1980, Cell22:197-207). The polymerase

chain reaction (PCR) method disclosed by Mullis et al. (U.S. Patent No. 4,683,195)

and Mullis (U.S. Patent No. 4,683,202) may be used. The choice of library and

selection of probes for the isolation of such DNA sequences is within the level of

ordinary skill in the art. Alternatively, DNA sequences encoding immunoglobulins or

fragments thereof can be obtained from vectors known in the art to contain

immunoglobulins or fragments thereof.

[0156] For recombinant production, a first polynucleotide sequence encoding

a portion of the chimeric protein of the invention (e.g. a portion of an immunoglobulin

constant region) and a second polynucleotide sequence encoding a portion of the

chimeric protein of the invention (e.g. a portion of an immunoglobulin constant region

and a biologically active molecule) are inserted into appropriate expression vehicles,

i.e. vectors which contains the necessary elements for the transcription and

translation of the inserted coding sequence, or in the case of an RNA viral vector, the necessary elements for replication and translation. The nucleic acids encoding the chimeric protein are inserted into the vector in proper reading frame.

[0157] The expression vehicles are then transfected or co-transfected into a

suitable target cell, which will express the polypeptides. Transfection techniques

known in the art include, but are not limited to, calcium phosphate precipitation

(Wigler et al. 1978, Cell14:725) and electroporation (Neumann et al. 1982, EMBO,

J. 1:841), and liposome based reagents. A variety of host-expression vector

systems may be utilized to express the chimeric proteins described herein including

both prokaryotic or eukaryotic cells. These include, but are not limited to,

microorganisms such as bacteria (e.g. E. coli) transformed with recombinant

bacteriophage DNA or plasmid DNA expression vectors containing an appropriate

coding sequence; yeast or filamentous fungi transformed with recombinant yeast or

fungi expression vectors containing an appropriate coding sequence; insect cell

systems infected with recombinant virus expression vectors (e.g. baculovirus)

containing an appropriate coding sequence; plant cell systems infected with

recombinant virus expression vectors (e.g. cauliflower mosaic virus or tobacco

mosaic virus) or transformed with recombinant plasmid expression vectors (e.g. Ti

plasmid) containing an appropriate coding sequence; or animal cell systems,

including mammalian cells (e.g. CHO, Cos, HeLa cells).

[0158] When the chimeric protein of the invention is recombinantly

synthesized in a prokaryotic cell it may be desirable to refold the chimeric protein.

The chimeric protein produced by this method can be refolded to a biologically active

conformation using conditions known in the art, e.g., denaturing under reducing

conditions and then dialyzed slowly into PBS.

[0159] Depending on the expression system used, the expressed chimeric

protein is then isolated by procedures well-established in the art (e.g. affinity

chromatography, size exclusion chromatography, ion exchange chromatography).

[0160] The expression vectors can encode for tags that permit for easy

purification of the recombinantly produced chimeric protein. Examples include, but

are not limited to vector pUR278 (Ruther et al. 1983, EMBO J. 2:1791) in which the

chimeric protein described herein coding sequences may be ligated into the vector in

frame with the lac z coding region so that a hybrid protein is produced; pGEX

vectors may be used to express chimeric proteins of the invention with a glutathione

S-transferase (GST) tag. These proteins are usually soluble and can easily be

purified from cells by adsorption to glutathione-agarose beads followed by elution in.

the presence of free glutathione. The vectors include cleavage sites (thrombin or

Factor Xa protease or PreScission ProteaseTM (Pharmacia, Peapack, N.J.)) for easy

removal of the tag after purification.

[0161] To increase efficiency of production, the polynucleotides can be

designed to encode multiple units of the chimeric protein of the invention separated

by enzymatic cleavage sites. The resulting polypeptide can be cleaved (e.g. by

treatment with the appropriate enzyme) in order to recover the polypeptide units.

This can increase the yield of polypeptides driven by a single promoter. When used

in appropriate viral expression systems, the translation of each polypeptide encoded

by the mRNA is directed internally in the transcript; e.g., by an internal ribosome

entry site, IRES. Thus, the polycistronic construct directs the transcription of a

single, large polycistronic mRNA which, in turn, directs the translation of multiple,

individual polypeptides. This approach eliminates the production and enzymatic processing of polyproteins and may significantly increase yield of polypeptide driven by a single promoter.

[0162] Vectors used in transformation will usually contain a selectable marker

used to identify transformants. In bacterial systems, this can include an antibiotic

resistance gene such as ampicillin or kanamycin. Selectable markers for use in

cultured mammalian cells include genes that confer resistance to drugs, such as

neomycin, hygromycin, and methotrexate. The selectable marker may be an

amplifiable selectable marker. One amplifiable selectable marker is the DHFR gene.

Another amplifiable marker is the DHFR cDNA (Simonsen and Levinson 1983, Proc.

Nat. Acad. Sci. USA 80:2495). Selectable markers are reviewed by Thilly

(Mammalian Cell Technology, Butterworth Publishers, Stoneham, MA) and the

choice of selectable markers is well within the level of ordinary skill in the art.

[0163] Selectable markers may be introduced into the cell on a separate

plasmid at the same time as the gene of interest, or they may be introduced on the

same plasmid. If on the same plasmid, the selectable marker and the gene of

interest may be under the control of different promoters or the same promoter, the

latter arrangement producing a dicistronic message. Constructs of this type are

known in the art (for example, U.S. Pat. No. 4,713,339).

[0164] The expression elements of the expression systems vary in their

strength and specificities. Depending on the host/vector system utilized, any of a

number of suitable transcription and translation elements, including constitutive and

inducible promoters, may be used in the expression vector. For example, when

cloning in bacterial systems, inducible promoters such as pL of bacteriophage A,

plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used; when cloning in insect cell systems, promoters such as the baculovirus polyhedron promoter may be used; when cloning in plant cell systems, promoters derived from the genome of plant cells (e.g. heat shock promoters; the promoter for'the small subunit of

RUBISCO; the promoter for the chlorophyll a/b binding protein) or from plant viruses

(e.g. the 35S RNA promoter of CaMV; the coat protein promoter of TMV) may be

used; when cloning in mammalian cell systems, promoters derived from the genome

of mammalian cells (e.g. metallothionein promoter) or from mammalian viruses (e.g.

the adenovirus late promoter; the vaccinia virus 7.5 K promoter) may be used; when

generating cell lines that contain multiple copies of expression product, SV40-, BPV

and EBV-based vectors may be used with an appropriate selectable marker.

[0165] In cases where plant expression vectors are used, the expression of

sequences encoding linear or non-cyclized forms of the chimeric proteins of the

invention may be driven by any of a number of promoters. For example, viral

promoters such as the 35S RNA and 19S RNA promoters of CaMV (Brisson et al.

1984, Nature 310:511-514), or the coat protein promoter of TMV (Takamatsu et al.

1987, EMBO J. 6:307-311) may be used; alternatively, plant promoters such as the

small subunit of RUBISCO (Coruzzi et al. 1984, EMBO J. 3:1671-1680; Broglie et al.

1984, Science 224:838-843) or heat shock promoters, e.g., soybean hsp17.5-E or

hsp17.3-B (Gurley et al. 1986, Mol. Cell. Biol. 6:559-565) may be used. These

constructs can be introduced into plant cells using Ti plasmids, Ri plasmids, plant

virus vectors, direct DNA transformation, microinjection, electroporation, etc. For

reviews of such techniques see, e.g., Weissbach &Weissbach 1988, Methods for

Plant Molecular Biology, Academic Press, NY, Section VIII, pp. 421-463; and

Grierson & Corey 1988, Plant Molecular Biology, 2d Ed., Blackie, London, Ch. 7-9.

{0166] In one insect expression system that may be used to produce the.

chimeric proteins of the invention, Autographa californica nuclear polyhidrosis virus

(AcNPV) is used as a vector to express the foreign genes. The virus grows in

Spodoptera frugiperda cells. A coding sequence may be cloned into non-essential

regions (for example, the polyhedron gene) of the virus and placed under control of

an AcNPV promoter (for example, the polyhedron promoter). Successful insertion of

a coding sequence will result in inactivation of the polyhedron gene and production

of non-occluded recombinant virus (i.e. virus lacking the proteinaceous coat coded

for by the polyhedron gene). These recombinant viruses are then used to infect

Spodoptera frugiperda cells in which the inserted gene is expressed. (see, e.g.,

Smith et al. 1983, J. Virol. 46:584; U.S. Patent No. 4,215,051). Further examples of

this expression system may be found in Ausubel et al., eds. 1989, Current Protocols

in Molecular Biology, Vol. 2, Greene Publish. Assoc. &Wiley Interscience.

[0167] Another system which can be used to express the chimeric proteins of

the invention is the glutamine synthetase gene expression system, also referred to

as the "GS expression system" (Lonza Biologics PLC, Berkshire UK). This

expression system is described in detail in U.S. Patent No. 5,981,216.

[0168] In mammalian host cells, a number of viral based expression systems

may be utilized. In cases where an adenovirus is used as an expression vector, a

coding sequence may be ligated to an adenovirus transcription/translation control

complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene

may then be inserted in the adenovirus genome by in vitro or in vivo recombination.

Insertion in a non-essential region of the viral genome (e.g. region El or E3) will

result in a recombinant virus that is viable and capable of expressing peptide in infected hosts (see, e.g., Logan & Shenk 1984, Proc. Natl. Acad. Sci. USA 81:3655).

Alternatively, the vaccinia 7.5 K promoter may be used (see, e.g., Mackett et al.

1982, Proc. Natl. Acad. Sci. USA 79:7415; Mackett et al. 1984, J. Virol. 49:857;

Panicali et al. 1982, Proc. Nat. Acad. Sci. USA 79:4927).

[0169] In cases where an adenovirus is used as an expression vector, a

coding sequence may be ligated to an adenovirus transcription/translation control

complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene

may then be inserted in the adenovirus genome by in vitro or in vivo recombination.

Insertion in a non-essential region of the viral genome (e.g. region El or E3) will

result in a recombinant virus that is viable and capable of expressing peptide in

infected hosts (see, e.g., Logan &Shenk 1984, Proc. Nat. Acad. Sci. USA 81:3655).

Alternatively, the vaccinia 7.5 K promoter may be used (see, e.g., Mackett et al.

1982, Proc. Nat!. Acad. Sci. USA 79:7415; Mackett et al. 1984, J. Virol. 49:857;

Panicali et al. 1982, Proc. Nat/. Acad. Sci. USA 79:4927).

[0170] Host cells containing DNA constructs of the chimeric protein are grown

in an appropriate growth medium. As used herein, the term "appropriate growth

medium" means a medium containing nutrients required for the growth of cells.

Nutrients required for cell growth may include a carbon source, a nitrogen source,

essential amino acids, vitamins, minerals and growth factors. Optionally the media

can contain bovine calf serum or fetal calf serum. In one embodiment, the media

contains substantially no IgG. The growth medium will generally select for cells

containing the DNA construct by, for example, drug selection or deficiency in an

essential nutrient which is complemented by the selectable marker on the DNA

construct or co-transfected with the DNA construct. Cultured mammalian cells are generally grown in commercially available serum-containing or serum-free media

(e.g. MEM, DMEM). Selection of a medium appropriate for the particular cell line

used is within the level of ordinary skill in the art.

[0171] The recombinantly produced chimeric protein of the invention can be

isolated from the culture media. The culture medium from appropriately grown

transformed or transfected host cells is separated from the cell material, and the

presence of chimeric proteins is demonstrated. One method of detecting the

chimeric proteins, for example, is by the binding of the chimeric proteins or portions

of the chimeric proteins to a specific antibody recognizing the chimeric protein of the

invention. An anti-chimeric protein antibody may be a monoclonal or polyclonal

antibody raised against the chimeric protein in question. For example, the chimeric

protein contains at least a portion of an immunoglobulin constant region. Antibodies

recognizing the constant region of many immunoglobulins are known in the art and

are commercially available. An antibody can be used to perform an ELISA or a

western blot to detect the presence of the chimeric protein of the invention.

[0172] The chimeric protein of the invention can be synthesized in a

transgenic animal, such as a rodent, cow, pig, sheep, or goat. The term "transgenic

animals" refers to non-human animals that have incorporated a foreign gene into

their genome. Because this gene is present in germline tissues, it is passed from

parent to offspring. Exogenous genes are introduced into single-celled embryos

(Brinster et al. 1985, Proc. Natl. Acad. Sc. USA 82:4438). Methods of producing

transgenic animals are known in the art, including transgenics that produce

immunoglobulin molecules (Wagner et al. 1981, Proc. Nat. Acad. Sci. USA 78:6376;

McKnight et al. 1983, Cell 34:335; Brinster et al. 1983, Nature 306:332; Ritchie et al.

1984, Nature 312:517; Baldassarre et al. 2003, Theriogenology 59:831; RobI et al.

2003, Theriogenology 59:107; Malassagne et al. 2003, Xenotransplantation

(3):267).

[0173] The chimeric protein of the invention can also be produced by a

combination of synthetic chemistry and recombinant techniques. For example, the

portion of an immunoglobulin constant region can be expressed recombinantly as

described above. The biologically active molecule, can be produced using known

chemical synthesis techniques (e.g. solid phase synthesis).

[0174] The portion of an immunoglobulin constant region can be ligated to the

biologically active molecule using appropriate ligation chemistry and then combined

with a portion of an immunoglobulin constant region that has not been ligated to a

biologically active molecule to form the chimeric protein of the invention. In one

embodiment, the portion of an immunoglobulin constant region is an Fc fragment.

The Fc fragment can be recombinantly produced to form Cys-Fc and reacted with a

biologically active molecule expressing a thioester to make a monomer-dimer hybrid.

In another embodiment, an Fc-thioester is made and reacted with a biologically

active molecule expressing an N terminus Cysteine (Figure 4).

[0175] In one embodiment, the portion of an immunoglobulin constant region

ligated to the biologically active molecule will form homodimers. The homodimers

can be disrupted by exposing the homodimers to denaturing and reducing conditions

(e.g. beta-mercaptoethanol and 8M urea) and then subsequently combined with a

portion of an immunoglobulin constant region not linked to a biologically active

molecule to form monomer-dimer hybrids. The monomer-dimer hybrids are then renatured and refolded by dialyzing into PBS and isolated, e.g., by size exclusion or affinity chromatography.

[0176] In another embodiment, the portion of an immunoglobulin constant

region will form homodimers before being linked to a biologically active molecule. In

this embodiment, reaction conditions for linking the biologically active molecule to

the homodimer can be adjusted such that linkage of the biologically active molecule

to only one chain of the homodimer is favored (e.g. by adjusting the molar

equivalents of each reactant).

[0177] The biologically active molecule can be chemically synthesized with an

N terminal cysteine. The sequence encoding a portion of an immunoglobulin

constant region can be sub-cloned into a vector encoding intein linked to a chitin

binding domain (New England Biolabs, Beverly, MA). The intein can be linked to the

C terminus of the portion of an immunoglobulin constant region. In one

embodiment, the portion of the immunoglobulin with the intein linked to its C

terminus can be expressed in a prokaryotic cell. In another embodiment, the portion

of the immunoglobulin with the intein linked to its C terminus can be expressed in a

eukaryotic cell. The portion of immunoglobulin constant region linked to intein can

be reacted with MESNA. In one embodiment, the portion of an immunoglobulin

constant region linked to intein is bound to a column, e.g., a chitin column and then

eluted with MESNA. The biologically active molecule and portion of an

immunoglobulin can be reacted together such that nucleophilic rearrangement

occurs and the biologically active molecule is covalently linked to the portion of an

immunoglobulin via an amide bond. (Dawsen et al. 2000, Annu. Rev. Biochem.

69:923). The chimeric protein synthesized this way can optionally include a linker peptide between the portion of an immunoglobulin and the biologically active molecule. The linker can for example be synthesized on the N terminus of the biologically active molecule. Linkers can include peptides and/or organic molecules

(e.g. polyethylene glycol and/or short amino acid sequences). This combined

recombinant and chemical synthesis allows for the rapid screening of biologically

active molecules and linkers to optimize desired properties of the chimeric protein of

the invention, e.g., viral inhibition, hemostasis, production of red blood cells,

biological half-life, stability, binding to serum proteins or some other property of the

chimeric protein. The method also allows for the incorporation of non-natural amino

acids into the chimeric protein of the invention which may be useful for optimizing a

desired property of the chimeric protein of the invention. If desired, the chimeric

protein produced by this method can be refolded to a biologically active

conformation using conditions known in the art, e.g., reducing conditions and then

dialyzed slowly into PBS.

[0178] Alternatively, the N-terminal cysteine can be on the portion of an

immunoglobulin constant region, e.g., an Fc fragment. An Fc fragment can be

generated with an N- terminal cysteine by taking advantage of the fact that a native

Fc has a cysteine at position 226 (see Kabat et al. 1991, Sequences of Proteins of

Immunological Interest, U.S. Department of Public Health, Bethesda, MD).

[0179] To expose a terminal cysteine, an Fc fragment can be recombinantly

expressed. In one embodiment, the Fc fragment is expressed in a prokaryotic cell,

e.g., E.coli. The sequence encoding the Fc portion beginning with Cys 226 (EU

numbering) can be placed immediately following a sequence endcoding a signal

peptide, e.g., OmpA, PhoA, STI. The prokaryotic cell can be osmotically shocked to release the recombinant Fc fragment. In another embodiment, the Fc fragment is produced in a eukaryotic cell, e.g., a CHO cell, a BHK cell. The sequence encoding the Fc portion fragment can be placed directly following a sequence encoding a signal peptide, e.g., mouse IgK light chain or MHC class I Kb signal sequence, such that when the recombinant chimeric protein is synthesized by a eukaryotic cell, the signal sequence will be cleaved, leaving an N terminal cysteine which can than be isolated and chemically reacted with a molecule bearing a thioester (e.g. a C terminal thioester if the molecule is comprised of amino acids).

[0180] The N terminal cysteine on an Fc fragment can also be generated

using an enzyme that cleaves its substrate at its N terminus, e.g., Factor X',

enterokinase, and the product isolated and reacted with a molecule with a thioester.

[0181] The recombinantly expressed Fc fragment can be used to make

homodimers or monomer-dimer hybrids.

[0182] In a specific embodiment, an Fc fragment is expressed with the human

a interferon signal peptide adjacent to the Cys at position 226. When a construct

encoding this polypeptide is expressed in CHO cells, the CHO cells cleave the signal

peptide at two distinct positions (at Cys 226 and at Val within the signal peptide 2

amino acids upstream in the N terminus direction). This generates a mixture of two

species of Fc fragments (one with an N-terminal Val and one with an N-terminal

Cys). This in turn results in a mixture of dimeric species (homodimers with terminal

Val, homodimers with terminal Cys and heterodimers where one chain has a

terminal Cys and the other chain has a terminal Val). The Fc fragments can be

reacted with a biologically active molecule having a C terminal thioester and the

resulting monomer-dimer hybrid can be isolated from the mixture (e.g. by size exclusion chromatography). It is contemplated that when other signal peptide sequences are used for expression of Fc fragments in CHO cells a mixture of species of Fc fragments with at least two different N termini will be generated.

[0183] In another embodiment, a recombinantly produced Cys-Fc can form a

homodimer. The homodimer can be reacted with peptide that has a branched linker

on the C terminus, wherein the branched linker has two C terminal thioesters that

can be reacted with the Cys-Fc. In another embodiment, the biologically active

molecule has a single non-terminal thioester that can be reacted with Cys-Fc.

Alternatively, the branched linker can have two C terminal cysteines that can be

reacted with an Fc thioester. In another embodiment, the branched linker has two

functional groups that can be reacted with the Fc thioester, e.g., 2-mercaptoamine.

The biologically active molecule may be comprised of amino acids. The biologically

active molecule may include a small organic molecule or a small inorganic molecule.

F. Methods of Using Chimeric Proteins

[0184] The chimeric proteins of the invention have many uses as will be

recognized by one skilled in the art, including, but not limited to methods of treating a

subject with a disease or condition. The disease or condition can include, but is not

limited to, a viral infection, a hemostatic disorder, anemia, cancer, leukemia, an

inflammatory condition or an autoimmune disease (e.g. arthritis, psoriasis, lupus

erythematosus, multiple sclerosis), or a bacterial infection (see, e.g., U.S. Patent

Nos. 6,086,875, 6,030,613, 6,485,726; WO 03/077834; US2003-0235536A1).

1. Methods of Treating a Subject with a Red Blood Cell Deficiency

[0185] The invention relates to a method of treating a subject having a

deficiency of red blood cells, e.g., anemia, comprising administering a therapeutically effective amount of at least one chimeric protein, wherein the chimeric protein comprises a first and a second polypeptide chain, wherein the first chain comprises at least a portion of an immunoglobulin constant region and at least one agent capable of inducing proliferation of red blood cells, e.g., EPO, and the second polypeptide chain comprises at least a portion of an immunoglobulin without the agent capable of inducing red blood cell proliferation of the first chain.

2. Methods of Treating a Subject with a Viral Infection

[01861The invention relates to a method of treating a subject having a viral

infection or exposed to a virus comprising administering a therapeutically effective

amount of at least one chimeric protein, wherein the chimeric protein comprises a

first and a second polypeptide chain, wherein the first chain comprises at least a

portion of an immunoglobulin constant region and at least one antiviral agent, e.g., a

fusion inhibitor or interferon a and the second polypeptide chain comprises at least a

portion of an immunoglobulin without the antiviral agent of the first chain. In one

embodiment, the subject is infected with a virus which can be treated with IFNa, e.g.,

hepatitis C virus. In one embodiment, the subject is infected with HIV, such as HIV

1 or HIV-2.

[0187]In one embodiment, the chimeric protein of the invention inhibits viral

replication. In one embodiment, the chimeric protein of the invention prevents or

inhibits viral entry into target cells, thereby stopping, preventing, or limiting the

spread of a viral infection in a subject and decreasing the viral burden in an infected

subject. By linking a portion of an immunoglobulin to a viral fusion inhibitor the

invention provides a chimeric protein with viral fusion inhibitory activity with greater

stability and greater bioavailability compared to viral fusion inhibitors alone, e.g.,

T20, T21, T1249. Thus, in one embodiment, the viral fusion inhibitor decreases or

prevents HIV infection of a target cell, e.g., HIV-1.

a. Conditions That May Be Treated

[0188] The chimeric protein of the invention can be used to inhibit or prevent

the infection of a target cell by a hepatitis virus, e.g., hepatitis virus C. The chimeric

protein may comprise an anti-viral agent which inhibits viral replication.

[0189] In one embodiment, the chimeric protein of the invention comprises a

fusion inhibitor. The chimeric protein of the invention can be used to inhihit or

prevent the infection of any target cell by any virus (see, e.g., U.S. Patent Nos.

6,086,875, 6,030,613, 6,485,726; WO 03/077834; US2003-0235536A). In one

embodiment, the virus is an enveloped virus such as, but not limited to HIV, SIV,

measles, influenza, Epstein-Barr virus, respiratory syncytia virus, or parainfluenza

virus. In another embodiment, the virus is a non-enveloped virus such as rhino virus

or polio virus

[0190] The chimeric protein of the invention can be used to treat a subject

already infected with a virus. The subject can be acutely infected with a virus.

Alternatively, the subject can be chronically infected with a virus. The chimeric

protein of the invention can also be used to prophylactically treat a subject at risk for

contracting a viral infection, e.g., a subject known or believed to in close contact with

a virus or subject believed to be infected or carrying a virus. The chimeric protein of

the invention can be used to treat a subject who may have been exposed to a virus,

but who has not yet been positively diagnosed.

[0191] In one embodiment, the invention relates to a method of treating a

subject infected with HCV comprising administering to the subject a therapeutically effective amount of a chimeric protein, wherein the chimeric protein comprises an Fc fragment of an IgG and a cytokine, e.g., IFNa.

[0192] In one embodiment, the invention relates to a method of treating a

subject infected with HIV comprising administering to the subject a therapeutically

effective amount of a chimeric protein wherein the chimeric protein comprises an Fc

fragment of an IgG and the viral fusion inhibitor comprises T20.

3. Methods of Treating a Subject Having a Hemostatic Disorder

[01931The invention relates to a method of treating a subject having a

hemostatic disorder comprising administering a therapeutically effective amount of at

least one chimeric protein, wherein the chimeric protein comprises a first and a

second chain, wherein the first chain comprises at least one clotting factor and at

least a portion of an immunoglobulin constant region, and the second chain

comprises at least a portion of an immunoglobulin constant region.

[0194] The chimeric protein of the invention treats or prevents a hemostatic

disorder by promoting the formation of a fibrin clot. The chimeric protein of the

invention can activate any member of a coagulation cascade. The clotting factor can

be a participant in the extrinsic pathway, the intrinsic pathway or both. In one

embodiment, the clotting factor is Factor VII or Factor VIla. Factor VIla can activate

Factor X which interacts with Factor Va to cleave prothrombin to thrombin, which in

turn cleaves fibrinogen to fibrin. In another embodiment, the clotting factor is Factor

IX or Factor IXa. In yet another embodiment, the clotting factor is Factor Vill or

Factor Villa. In yet another embodiment, the clotting factor is von Willebrand Factor,

Factor XI, Factor XII, Factor V, Factor X or Factor XIII.

a. Conditions That May Be Treated

[0195] The chimeric protein of the invention can be used to treat any

hemostatic disorder. The hemostatic disorders that may be treated by

administration of the chimeric protein of the invention include, but are not limited to,

hemophilia A, hemophilia B, von Willebrand's disease, FactorX deficiency (PTA

deficiency), Factor XII deficiency, as well as deficiencies or structural abnormalities

in fibrinogen, prothrombin, Factor V, Factor VII, Factor X, or Factor XIII.

[0196] In one embodiment, the hemostatic disorder is an inherited disorder.

In one embodiment, the subject has hemophilia A, and the chimeric protein

comprises Factor VI IIor Factor VIlla. In another embodiment, the subject has

hemophilia A and the chimeric protein comprises Factor VII or Factor Vila. In

another embodiment, the subject has hemophilia B and the chimeric protein

comprises Factor IX or Factor IXa. In another embodiment, the subject has

hemophilia Band the chimeric protein comprises Factor VII or Factor VIla. In

another embodiment, the subject has inhibitory antibodies to FactorVIII or Factor

VIlIa and the chimeric protein comprises Factor VII or Factor Vila. Inyetanother

embodiment, the subject has inhibitory antibodies against Factor IX or Factor IXa

and the chimeric protein comprises Factor VI Ior Factor VIla.

[0197] The chimeric protein of the invention can be used to prophylactically

treat a subject with a hemostatic disorder. The chimeric protein of the invention can

be used to treat an acute bleeding episode in a subject with a hemostatic disorder

[0198] In one embodiment, the hemostatic disorder is the result of a

deficiency in a clotting factor, e.g., Factor IX, Factor Vill. In another embodiment, the hemostatic disorder can be the result of a defective clotting factor, e.g., von

Willebrand's Factor.

[0199] In another embodiment, the hemostatic disorder can be an acquired

disorder. The acquired disorder can result from an underlying secondary disease or

condition. The unrelated condition can be, as an example, but not as a limitation,

cancer, an autoimmune disease, or pregnancy. The acquired disorder can result

from old age or from medication to treat an underlying secondary disorder (e.g.

cancer chemotherapy).

4. Methods of Treating a Subject In Need of a General Hemostatic Agent

[0200] The invention also relates to methods of treating a subject that does

not have a hemostatic disorder or a secondary disease or condition resulting in

acquisition of a hemostatic disorder. The invention thus relates to a method of

treating a subject in need of a general hemostatic agent comprising administering a

therapeutically effective amount of at least one chimeric protein, wherein the

chimeric protein comprises a first and a second polypeptide chain wherein the first

polypeptide chain comprises at least a portion of an immunoglobulin constant region

and at least one clotting factor and the second chain comprises at least a portion of

an immunoglobulin constant region without the clotting factor of the first polypeptide

chain.

a. Conditions That May Be Treated

[0201] In one embodiment, the subject in need of a general hemostatic agent

is undergoing, or is about to undergo, surgery. The chimeric protein of the invention

can be administered prior to or after surgery as a prophylactic. The chimeric protein of the invention can be administered during or after surgery to control an acute bleeding episode. The surgery can include, but is not limited to, liver transplantation, liver resection, or stem cell transplantation.

[0202] The chimeric protein of the invention can be used to treat a subject

having an acute bleeding episode who does not have a hemostatic disorder. The

acute bleeding episode can result from severe trauma, e.g., surgery, an automobile

accident, wound, laceration gun shot, or any other traumatic event resulting in

uncontrolled bleeding.

5. Treatment Modalities

[0203] The chimeric protein of the invention can be administered

intravenously, subcutaneously, intra-muscularly, or via any mucosal surface, e.g.,

orally, sublingually, buccally, sublingually, nasally, rectally, vaginally or via

pulmonary route. The chimeric protein can be implanted within or linked to a

biopolymer solid support that allows for the slow release of the chimeric protein to

the desired site.

[0204] The dose of the chimeric protein of the invention will vary depending

on the subject and upon the particular route of administration used. Dosages can

range from 0.1 to 100,000 pg/kg body weight. In one embodiment, the dosing range

is 0.1-1,000 pg/kg. The protein can be administered continuously or at specific

timed intervals. In vitro assays may be employed to determine optimal dose ranges

and/or schedules for administration. Many in vitro assays that measure viral

infectivity are known in the art. For example, a reverse transcriptase assay, or an rt

PCR assay or branched DNA assay can be used to measure HIV concentrations. A

StaClot assay can be used to measure clotting activity. Additionally, effective doses

may be extrapolated from dose-response curves obtained from animal models.

[0205] The invention also relates to a pharmaceutical composition comprising

a viral fusion inhibitor, at least a portion of an immunoglobulin and a

pharmaceutically acceptable carrier or excipient. Examples of suitable

pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by

E.W..Martin. Examples of excipients can include starch, glucose, lactose, sucrose,

gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,

talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and

the like. The composition can also contain pH buffering reagents, and wetting or

emulsifying agents.

[0206] For oral administration, the pharmaceutical composition can take the

form of tablets or capsules prepared by conventional means. The composition can

also be prepared as a liquid for example a syrup or a suspension. The liquid can

include suspending agents (e.g. sorbitol syrup, cellulose derivatives or hydrogenated

edible fats), emulsifying agents (lecithin or acacia), non-aqueous vehicles (e.g.

almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and

preservatives (e.g. methyl or propyl -p-hydroxybenzoates or sorbic acid). The

preparations can also include flavoring, coloring and sweetening agents.

Alternatively, the composition can be presented as a dry product for constitution with

water or another suitable vehicle.

[0207] For buccal and sublingual administration the composition may take the

form of tablets, lozenges or fast dissolving films according to conventional protocols.

[0208] For administration by inhalation, the compounds for use according to

the present invention are conveniently delivered in the form of an aerosol spray from

a pressurized pack or nebulizer (e.g. in PBS), with a suitable propellant, e.g.,

dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoromethane, carbon

dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit

can be determined by providing a valve to deliver a metered amount. Capsules and

cartridges of, e.g., gelatin for use in an inhaler or insufflator can be formulated

containing a powder mix of the compound and a suitable powder base such as

lactose or starch.

[0209] The pharmaceutical composition can be formulated for parenteral

administration (i.e. intravenous or intramuscular) by bolus injection. Formulations for

injection can be presented in unit dosage form, e.g., in ampoules or in multidose

containers with an added preservative. The compositions can take such forms as

suspensions, solutions, or emulsions in oily or aqueous vehicles, and contain

formulatory agents such as suspending, stabilizing and/or dispersing agents.

Alternatively, the active ingredient can be in powder form for constitution with a

suitable vehicle, e.g., pyrogen free water.

[0210] The pharmaceutical composition can also be formulated for rectal

administration as a suppository or retention enema, e.g., containing conventional

suppository bases such as cocoa butter or other glycerides.

6. Combination Therapy

[0211] The chimeric protein of the invention can be used to treat a subject

with a disease or condition in combination with at least one other known agent to

treat said disease or condition.

[0212] In one embodiment, the invention relates to a method of treating a

subject infected with HIV comprising administering a therapeutically effective amount

of at least one chimeric protein comprising a first and a second chain, wherein the

first chain comprises an HIV fusion inhibitor and at least a portion of an

immunoglobulin constant region and the second chain comprises at least a portion

of an immunoglobulin without an HIV fusion inhibitor of the first chain, in combination

with at least one other anti-HIV agent. Said other anti-HIV agent can be any

therapeutic with demonstrated anti-HIV activity. Said other anti-HIV agent can

include, as an example, but not as a limitation, a protease inhibitor (e.g.

Amprenavir*, Crixivan*, Ritonivir*), a reverse transcriptase nucleoside analog (e.g.

AZT, DDI, D4T, 3TC, Ziagen), a nonnucleoside analog reverse transcriptase

inhibitor (e.g. Sustiva*), another HIV fusion inhibitor, a neutralizing antibody specific

to HIV, an antibody specific to CD4, a CD4 mimic, e.g., CD4-IgG2 fusion protein

(U.S. Patent Application 09/912,824) or an antibody specific to CCR5, or CXCR4, or

a specific binding partner of CCR5, or CXCR4.

[0213] In another embodiment, the invention relates to a method of treating a

subject with a hemostatic disorder comprising administering a therapeutically

effective amount of at least one chimeric protein comprising a first and a second

chain, wherein the first chain comprises at least one clotting factor and at least a

portion of an immunoglobulin constant region and the second chain comprises at least a portion of an immunoglobulin constant region without the clotting factor of the first chain, in combination with at least one other clotting factor or agent that promotes hemostasis. Said other clotting factor or agent that promotes hemostasis can be any therapeutic with demonstrated clotting activity. As an example, but not as a limitation, the clotting factor or hemostatic agent can include Factor V, Factor

VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, Factor X111, prothrombin, or

fibrinogen or activated forms of any of the preceding. The clotting factor of

hemostatic agent can also include anti-fibrinolytic drugs, e.g., epsilon-amino-caproic

acid, tranexamic acid.

7. Methods of Inhibiting Viral Fusion With a Target Cell

[0214} The invention also relates to an in vitro method of inhibiting HIV fusion

with a mammalian cell comprising combining the mammalian cell with at least one

chimeric protein, wherein the chimeric protein comprises a first and a second chain,

wherein the first chain comprises at least a portion of an immunoglobulin constant

region and an HIV inhibitor and the second chain comprises at least a portion of an

immunoglobulin constant region without the HIV inhibitor of the first chain. The

mammalian cell can include any cell or cell line susceptible to infection by HIV

including but not limited to primary human CD4* T cells or macrophages, MOLT-4

cells, CEM cells, AA5 cells or HeLa cells which express CD4 on the cell surface.

G. Methods of Isolating Chimeric Proteins

[0215] Typically, when chimeric proteins of the invention are produced they

are contained in a mixture of other molecules such as other proteins or protein

fragments. The invention thus provides for methods of isolating any of the chimeric

proteins described supra from a mixture containing the chimeric proteins. It has been determined that the chimeric proteins of the invention bind to dye ligands under suitable conditions and that altering those conditions subsequent to binding can disrupt the bond between the dye ligand and the chimeric protein, thereby providing a method of isolating the chimeric protein. In some embodiments the mixture may comprise a monomer-dimer hybrid, a dimer and at least a portion of an immunoglobulin constant region, e.g., an Fc. Thus, in one embodiment, the invention provides a method of isolating a monomer-dimer hybrid. In another embodiment, the invention provides a method of isolating a dimer.

[0216] Accordingly, in one embodiment, the invention provides a method of

isolating a monomer-dimer hybrid from a mixture, where the mixture comprises

a) the monomer-dimer hybrid comprising a first and second polypeptide

chain, wherein the first chain comprises a biologically active molecule, and at least a

portion of an immunoglobulin constant region and wherein the second chain

comprises at least a portion of an immunoglobulin constant region without a

biologically active molecule or immunoglobulin variable region;

b) a dimer comprising a first and second polypeptide chain, wherein the first

and second chains both comprise a biologically active molecule, and at least a

portion of an immunoglobulin constant region; and

c) a portion of an immunoglobulin constant region; said method comprising

1) contacting the mixture with a dye ligand linked to a solid support

under suitable conditions such that both the monomer-dimer hybrid and the dimer

bind to the dye ligand;

2) removing the unbound portion of an immunoglobulin constant

region;

3) altering the suitable conditions of 1) such that the binding

between the monomer-dimer hybrid and the dye ligand linked to the solid support is

disrupted;

4) isolating the monomer-dimer hybrid.

In some embodiments, prior to contacting the mixture with a dye ligand, the mixture

may be contacted with a chromatographic substance such as protein A sepharose or

the like. The mixture is eluted from the chromatographic substance using an

appropriate elution buffer (e.g. a low pH buffer) and the eluate containing the mixture

is then contacted with the dye ligand.

[0217] Suitable conditions for contacting the mixture with the dye ligand may

include a buffer to maintain the mixture at an appropriate pH. An appropriate pH

may include a pH of from, 3-10, 4-9, 5-8. In one embodiment, the appropriate pH is

8.0. Any buffering agent known in the art may be used so long as it maintains the

pH in the appropriate range, e.g., tris, HEPES, PIPES, MOPS. Suitable conditions

may also include a wash buffer to elute unbound species from the dye ligand. The

wash buffer may be any buffer which does not disrupt binding of a bound species.

For example, the wash buffer can be the same buffer used in the contacting step.

[0218] Once the chimeric protein is bound to the dye ligand, the chimeric

protein is isolated by altering the suitable conditions. Altering the suitable conditions

may include the addition of a salt to the buffer. Any salt may be used, e.g., NaCl,

KCl. The salt should be added at a concentration that is high enough to disrupt the

binding between the dye ligand and the desired species, e.g., a monomer-dimer

hybrid.

[0219] In some embodiments where the mixture is comprised of an Fc, a

monomer-dimer hybrid, and a dimer, it has been found that the Fc does not bind to

the dye ligand and thus elutes with the flow through. The dimer binds more tightly to

the dye ligand than the monomer-dimer hybrid. Thus a higher concentration of salt

is required to disrupt the bond (e.g. elute) between the dimer and the dye ligand

compared to the salt concentration required to disrupt the bond between the dye

ligand and the monomer-dimer hybrid.

[0220] In some embodiments NaCl may be used to isolate the monomer

dimer hybrid from the mixture. In some embodiments the appropriate concentration

of salt which disrupts the bond between the dye ligand and the monomer-dimer

hybrid is from 200-700 mM, 300-600 mM, 400-500 mM. In one embodiment, the

concentration of NaCl required to disrupt the binding between the dye ligand the

monomer-dimer hybrid is 400 mM.

[0221] NaCl may also be used to isolate the dimer from the mixture.

Typically, the monomer-dimer hybrid is isolated from the mixture before the dimer.

The dimer is isolated by adding an appropriate concentration of salt to the buffer,

thereby disrupting the binding between the dye ligand and the dimer. In some

embodiments the appropriate concentration of salt which disrupts the bond between

the dye ligand and the dimer is from 800 mM to 2 M, 900 mM tol.5 M, 950 mM to

1.2 M. In one specific embodiment, I M NaCl is used to disrupt the binding between

the dye ligand and the dimer.

[0222] The dye ligand may be a bio-mimetic. A bio-mimetic is a human

made substance, device, or system that imitates nature. Thus in some

embodiments the dye ligand imitates a molecule's naturally occurring ligand. The dye ligand may be chosen from Mimetic RedI TM,Mimetic Red 2TM, Mimetic Orange

1TMMimetic Orange 2TM, Mimetic Orange 3TMMimetic Yellow 1TM, Mimetic Yellow

2 TMMimetic Green IT11, Mimetic BlueI TM, and Mimetic Blue 2TM (Prometic

Biosciences (USA) Inc., Wayne, NJ). In one specific embodiment, the dye ligand is

Mimetic Red 2TM (Prometic Biosciences (USA) Inc., Wayne, NJ). In certain

embodiments the dye ligand is linked to a solid support, e.g., from Mimetic Red

IA6XLTM,Mimetic Red 2 A6XL TM, Mimetic Orange I A6XL TM, Mimetic Orange 2

A6XL TM,Mimetic Orange 3 A6XL ,Mimetic Yellow 1 A6XL , Mimetic Yellow 2 TM TM

TM A6XL TM,Mimetic Green I A6XL TM, Mimetic Blue I A6XL , and Mimetic Blue 2

A6XL T M(Prometic Biosciences (USA) Inc., Wayne, NJ).

[0223] The dye ligand may be linked to a solid support. The solid support

may be any solid support known in the art (see, e.g., www.seperationsNOW.com).

Examples of solid supports may include a bead, a gel, a membrane, a nanoparticle,

or a microsphere. The solid support may comprise any material which can be linked

to a dye ligand (e.g. agarose, polystyrene, sepharose, sephadex). Solid supports

may comprise any synthetic organic polymer such as polyacrylic, vinyl polymers, comprise a acrylate, polymethacrylate, and polyacrylamide. Solid supports may also

carbohydrate polymer, e.g., agarose, cellulose, or dextran. Solid supports may magnesia comprise inorganic oxides, such as silica, zirconia, titania, ceria, alumina,

(i.e., magnesium oxide), or calcium oxide. Solid supports may also comprise

combinations of some of the above-mentioned supports including, but not limited to,

dextran-acrylamide.

Examples

Example 1: Molecular Weight Affects FcRn Mediated Trancytosis

[0224] Chimeric proteins comprised of various proteins of interest and IgG

Fc were recombinantly produced (Sambrook et al. Molecular Cloning: A Laboratory

Manual, 2 ed., Cold Spring Harbor Laboratory Press, (1989)) or in the case of

contactin-Fc, MAB-@-gal, (a complex of a monoclonal antibody bound to P-gal)

(Biodesign International, Saco, ME) and MAB-GH (a complex of monoclonal

antibody and growth hormone)(Research Diagnostics, Inc. Flanders, NJ) were

purchased commercially. Briefly, the genes encoding the protein of interest were

cloned by PCR, and then sub-cloned into an Fc fusion expression plasmid. The

plasmids were transfected into DG44 CHO cells and stable transfectants were

selected and amplified with methotrexate. The chimeric protein homodimers were

purified over a protein A column. The proteins tested included interferon a, growth

hormone, erythropoietin, follicle stimulating hormone, Factor IX, beta-galactosidase,

contactin, and Factor VIII. Linking the proteins to immunoglobulin portions, including

the FcRn receptor binding partner, or using commercially available whole antibody

(including the FcRn binding region)-antigen complexes permitted the investigation of

transcytosis as a function of molecular weight (see U.S. Patent No. 6,030,613). The

chimeric proteins were administered to rats orally and serum levels were measured

2-4 hours post administration using an ELISA for recombinantly produced chimeric

proteins and both a western blot and ELISA for commercially obtained antibody

complexes and chimeric proteins. Additionally, all of the commercially obtained

proteins or complexes as well as Factor VIII-Fc, Factor IX-Fc and Epo-Fc controls

were iodinated using IODO beads (Pierce, Pittsburgh, PA). The results indicated serum levels of Fc and monoclonal antibody chimeric proteins orally administered to rats are directly related to the size of the protein. The apparent cutoff point for orally administered Fc chimeric proteins is between 200-285 kD. (Table 2).

TABLE 2

Protein Size(kD) Transcytosis IFNa-Fc 92 ++++ GH-Fc 96 +++ Epo-Fc 120 +++ FSH-Fc 170 +++ MAB:GH 172-194 +++ FIX-Fc 200

+ MAB:pGal 285-420 Contactin-Fc 300 | FVIIIA-Fc 380 | _

Example 2: Cloning of pcDNA 3.1-Flag-Fe

[0225] The sequence for the FLAG peptide (Asp-Tyr-Lys-Asp-Asp-Asp-Asp

Lys), a common affinity tag used to identify or purify proteins, was cloned into the

pcDNA 3.1-Fc plasmid, which contains the mouse IgK signal sequence followed by

the Fc fragment of human IgGI (amino acids 221-447, EU numbering). The

construct was created by overlapping PCR using the following primers:

FlagFc-FI: 5'- GCTGGCTAGCCACCATGGA -3'(SEQ ID NO:41)

FlagFc-R1: 5'- CTTGTCATCGTCGTCCTTGTAGTCGTCA CCAGTGGAACCTGGAAC -3'(SEQ ID NO:42)

FlagFc-F2: 5'- GACTACAAGG ACGACGATGA CAAGGACAAA ACTCACACAT GCCCACCGTG CCCAGCTCCG GAACTCC -3'(SEQ ID NO:43)

FlagFc-R2: 5'- TAGTGGATCCTCATTTACCCG -3'(SEQ ID NO:44)

[0226] The pcDNA 3.1-Fc template was then added to two separate PCR

reactions containing 50 pmol each of the primer pairs FlagFc-F1/RI or FlagFc-F2/R2

in a 50 pl reaction using Pfu Ultra DNA polymerase (Stratagene, CA) according to manufacturer's standard protocol in a MJ Thermocycler using the following cycles:

°C 2 minutes; 30 cycles of (95°C 30 seconds, 52°C 30 seconds, 720C 45

seconds), followed by 72°C for 10 minutes. The products of these two reactions

were then mixed in another PCR reaction (2 pl each) with 50 pmol of FlagFc-FI and

FlagFc-R2 primers in a 50 pl reaction using Pfu Ultra DNA polymerase (Stratagene,

CA) according to manufacturer's standard protocol in a MJ Thermocycler using the

following cycles: 950C 2 minutes; 30 cycles of (950C 30 seconds, 52°C 30 seconds,

720C 45 seconds), followed by 720C for 10 minutes. The resulting fragment was gel

purified, digested and inserted into the pcDNA 3.1-Fc plasmid Nhel-Bam HI. The

resulting plasmid contains contains the mouse IgK signal sequence producing the

FlagFc protein.

Example 3: Cloning of -Factor VII-Fc construct

[0227] The coding sequence for Factor VII, was obtained by RT-PCR from

human fetal liver RNA (Clontech, Palo Alto, CA). The cloned region is comprised of

the cDNA sequence from bp 36 to bp 1430 terminating just before the stop codon. A

Sbfl site was introduced on the N-terminus. A BspEl site was introduced on the C

terminus. The construct was cloned by PCR using the primers:

Downstream: 5'GCTACCTGCAGGCCACCATGGTCTCCCAGGCCCTCAGG 3'(SEQ ID NO:45)

Upstream: 5'CAGTTCCGGAGCTGGGCACGGCGGGCACGTGTGAGTTT TGTCGGGAAAT GG 3'(SEQ ID NO:46)

and the following conditions: 95°C for 5 minutes followed by 30 cycles of 950C for 30

seconds, 550C for 30 seconds, 720C for 1 minute and 45 seconds, and a final

extension cycle of 720C for 10 minutes.

[0228] The fragment was digested Sbfl - BspE I and inserted into pED.dC-Fc

a plasmid encoding for the Fc fragment of an IgG1.

Example 4: Cloning of Factor IX-Fc construct

[0229] The human Factor IX coding sequence, including the prepropeptide

sequence, was obtained by RT-PCR amplification from adult human liver RNA using

the following primers:

natFIX-F: 5'-TTACTGCAGAAGGTTATGCAGCGCGTGAACATG- 3'(SEQ ID NO:47)

F9-R: 5'-TTTTTCGAATTCAGTGAGCTTTGTTTTTTCCTTAATCC- 3'(SEQ ID NO:48)

[0230] 20 ng of adult human liver RNA (Clontech, Palo Alto, CA) and 25

pmol each primer were added to a RT-PCR reaction using the SuperScript.TM One

Step RT-PCR with PLATINUM@ Taq system (Invitrogen, Carlsbad, CA) according to

manufacturers protocol. Reaction was carried out in a MJ Thermocycler using the

following cycles: 50 0C 30 minutes; 94 0C 2 minutes; 35 cycles of (94 0C 30 seconds,

58 0C 30 seconds, 720C 1 minute), and a final 720 C 10 minutes. The fragment was

gel purified using Qiagen Gel Extraction Kit (Qiagen, Valencia, CA), and digested

with Pstl-EcoR, gel purified, and cloned into the corresponding digest of the

pED.dC.XFc plasmid.

Example 5: Cloning of PACE construct

[0231] The coding sequence for human PACE (paired basic amino acid

cleaving enzyme), an endoprotease, was obtained by RT-PCR. The following

primers were used:

PACE-Fl: 5'- GGTAAGCTTGCCATGGAGCTGAGGCCCTGGTTGC -3'(SEQ ID NO:49)

PACE-RI: 5'- GTTTTCAATCTCTAGGACCCACTCGCC -3'(SEQ ID NO:50)

PACE-F2: 5'- GCCAGGCCACATGACTACTCCGC -3'(SEQ ID NO:51)

PACE-R2: 5'- GGTGAATTCTCACTCAGGCAGGTGTGAGGGCAGC -3'(SEQ ID NO:52)

[0232] The PACE-F1 primer adds a HindIll site to the 5'end of the PACE

sequence beginning with 3 nucleotides before the start codon, while the PACE-R2

primer adds a stop codon after amino acid 715, which occurs at the end of the

extracellular domain of PACE, as well as adding an EcoRi site to the 3' end of the

stop codon. The PACE-R1 and -F2 primers anneal on the 3'and 5'sides of an

internal BamHI site, respectively. Two RT-PCR reactions were then set up using 25

pmol each of the primer pairs of PACE-F1/R1 or PACE-F2/R2 with 20 ng of adult

human liver RNA (Clontech; Palo Alto, CA) in a 50 pl RT-PCR reaction using the

SuperScript. TM One-Step RT-PCR with PLATINUM@ Taq system (Invitrogen,

Carlsbad, CA) according to manufacturers protocol. The reaction was carried out in

a MJ Thermocycler using the following cycles: 501C 30 minutes; 94°C 2 minutes; 30

cycles of (94°C 30 seconds, 58°C 30 seconds, 72°C 2 minutes), followed by 72°C 10

minutes. These fragments were each ligated into the vector pGEM T-Easy

(Promega, Madison, WI) and sequenced fully. The F2-R2 fragment was then

subcloned into pcDNA6 V5/His (Invitrogen, Carlsbad, CA) using the BamHI/EcoRI

sites, and then the F-RI fragment was cloned into this construct using the

HindIll/BamHI sites. The final plasmid, pcDNA6-PACE, produces a soluble form of

PACE (amino acids 1-715), as the transmembrane region has been deleted. The

sequence of PACE in pcDNA6-PACE is essentially as described in Harrison et al.

1998, Seminars in Hematology 35:4.

Example 6: Cloning of IFNa-Fc eight amino acid linker construct

[0233] The human interferon a 2b (hlFNa) coding sequence, including the

signal sequence, was obtained by PCR from human genomic DNA using the

following primers:

IFNa-Sig-F: 5'-GCTACTGCAGCCACCATGGCCTTGACCTTTGCTTTAC 3'(SEQ ID NO:53)

IFNa-EcoR-R: 5'-CGTTGAATTCTTCCTTACTTCTTAAACTTTCTTGC 3'(SEQ ID NO:54)

[0234] Genomic DNA was prepared from 373MG human astrocytoma cell

line, according to standard methods (Sambrook et al. 1989, Molecular Cloning: A

LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press). Briefly,

approximately 2 x 105 cells were pelleted by centrifugation, resuspended in 100 pl

phosphate buffered saline pH 7.4, then mixed with an equal volume of lysis buffer

(100 mM Tris pH 8.0/ 200 mM NaCl / 2% SDS / 5 mM EDTA). Proteinase K was

added to a final concentration of 100 pg/ml, and the sample was digested at 37°C for

4 hours with occasional gentle mixing. The sample was then extracted twice with

phenol:chloroform, the DNA precipitated by adding sodium acetate pH 7.0 to

100 mM and an equal volume of isopropanol, and pelleted by centrifugation for 10

min at room temperature. The supernatant was removed and the pellet was washed

once with cold 70% ethanol and allowed to air dry before resuspending in TE

(10 mM Tris pH 8.0 / 1 mM EDTA).

[0235] 100 ng of this genomic DNAwas then used in a 25 pi PCR reaction

with 25 pmol of each primer using Expand High Fidelity System (Boehringer

Mannheim, Indianapolis, IN) according to manufacturer's standard protocol in a MJ

Thermocycler using the following cycles: 940C 2 minutes; 30 cycles of (940C 30 seconds, 500C 30 seconds, 720C 45 seconds), and finally 720C 10 minutes. The expected sized band (-550 bp) was gel purified with a Gel Extraction kit (Qiagen,

Valencia, CA), digested with Pstl/EcoRI, gel purified again, and cloned into the

Pstl/EcoRl site of pED.dC.XFc, which contains an 8 amino acid linker (EFAGAAAV)

followed by the Fc region of human IgG1.

Example 7: Cloning of IFNaFc Alinker construct

[0236] 1 pg of purified pED.dC.native human IFNaFc DNA, from Example 6,

was then used as a template in a 25 pl PCR reaction with 25 pmol of each primer

IFNa-Sig-F and the following primer:

hlFNaNoLinkFc-R: 5'CAGTTCCGGAGCTGGGCACGGCGGG

CACGTGTGAGTTTTGTCTTCCTTACTTCTTAAACTTTTTGCAAGTTTG- 3'(SEQ ID NO:55)

[0237] The PCR reaction was carried out using Expand High Fidelity System

(Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's standard

protocol in a RapidCycler thermocycler (Idaho Technology, Salt Lake City, UT),

denaturing at 940C for 2 minutes followed by 18 cycles of 95°C for 15 seconds, 55C

for 0 seconds, and 72°C for 1 minute with a slope of 6, followed by 72°C extension

for 10 minutes. A PCR product of the correct size (-525 bp) was gel purified using a

Gel Extraction kit (Qiagen; Valencia, CA), digested with the Pstl and BspEl

restriction enzymes, gel purified, and subcloned into the corresponding sites of a

modified pED.dC.XFc, where amino acids 231-233 of the Fc region were altered

using the degeneracy of the genetic code to incorporate a BspEl site while

maintaining the wild type amino acid sequence.

Example 8: Cloning of IFNaFc GS15 linker construct

[0238] A new backbone vector was created using the Fc found in the Alinker

construct (containing BspEl and RsrIl sites in the 5' end using the degeneracy of the

genetic code to maintain the amino acid sequence), using this DNA as a template for

a PCR reaction with the following primers:

5' B2xGGGGS: 5'gtcaggatccggcggtggagggagcgacaaaactcacacgtgccc 3'(SEQ ID NO:56) 3'GGGGS: 5'tgacgcggccgctcatttacccggagacaggg 3'(SEQ ID NO:57)

[0239] A PCR reaction was carried out with 25 pmol of each primer using

Pfu Turbo enzyme (Stratagene, La Jolla, CA) according to manufacturer's standard

protocol in a MJ Thermocycler using the following method: 95°C 2 minutes; 30

cycles of (95°C 30 seconds, 540C 30 seconds, 72°C 2 minutes), 720C 10 minutes.

The expected sized band (-730 bp) was gel purified with a Gel Extraction kit

(Qiagen, Valencia CA), digested BamHl/Notl; gel purified again, and cloned into the

BamHl/Notl digested vector of pcDNA6 ID, a version of pcDNA6 with the IRES

sequence and dhfr gene inserted into Notl/Xbal site.

[0240] 500 ng of purified pED.dC.native human IFNaFc DNA was then used

as a template in a 25 pl PCR reaction with the following primers:

5' IFNa for GGGGS: 5'ccgctagcctgcaggccaccatggccttgacc 3'(SEQ ID NO:58)

3' IFNa for GGGGS: 5'ccggatccgccgccaccttccttactacgtaaac 3'(SEQ ID NO:59)

[0241] A PCR reaction was carried out with 25 pmol of each primer using

Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN) according to

manufacturer's standard protocol in a MJ Thermocycler using the following cycles:

950C 2 minutes; 14 cycles of (940C 30 seconds, 480C 30 seconds, 720C 1 minute),

720C 10 minutes. The expected sized band (-600 bp) was gel purified with a Gel

Extraction kit (Qiagen, Valencia CA), digested Nhel/BamHl, gel purified again, and

cloned into the NheI/BamHI site of the pcDNA6 ID/Fc vector, above, to create an

IFNa Fc fusion with a 10 amino acid Gy/Ser linker (2xGGGGS), pcDNA6 ID/IFNa

GS10-Fc.

[0242] A PCR reaction was then performed using 500 ng of this pcDNA6

ID/IFNa-GS1-Fc with the following primers

5'B3XGGGGS:5'(SEQ ID NO:60)

gtcaggatccggtggaggcgggtccggcggtggagggagcgacaaaactcacacgtgccc 3'(SEQ ID NO:61)

fccIv-R: 5'atagaagcctttgaccaggc 3'(SEQ ID NO:62)

[0243] A PCR reaction was carried out with 25 pmol of each primer using

Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN) according to

manufacturer's standard protocol in a MJ Thermocycler using the following cycles:

950C 2 minutes; 14 cycles of (94°C 30 seconds, 480C 30 seconds, 72°C 1 minute),

720C 10 minutes. The expected sized band (504 bp) was gel purified with a Gel

Extraction kit (Qiagen, Valencia CA), digested BamHI/BspE, the 68 bp band was gel

purified, and cloned into the BamHI/BspE site of the pcDNA6 ID/IFNa-GS10-Fc

vector, above, to create an IFNa Fc fusion with a 15 amino acid Gly/Ser linker

(3xGGGGS), pcDNA6 ID/IFNa-GS15-Fc.

Example 9: Cloning of a Basic Peptide Construct

[0244] The hinge region of the human IgG1 Fc fragment from amino acid

221-229 (EU numbering) was replaced with a basic peptide (CCB).

Four overlapping oligos were used (IDT, Coralville, IA):

1. CCB-Fc Sense 1:

'GCC GGC GAA TTC GGT GGT GAG TAC CAG GCC CTG AAG AAG AAG GTG GCC CAG CTG AAG GCC GAAGC CAG GCC CTG AAG AAG AAG 3'(SEQ ID NO:63)

2. CCB-Fc Sense 2:

'GTG GCC CAG CTG AAG CAC AAG GGC GGC GGC CCC GCC CCA GAG CTC CTG GGC GGA CCG A 3'(SEQ ID NO:64)

3. CCB-Fc Anti-Sense 1:

'CGG TCC GCC CAG GAG CTC TGG GGC GGG GGGCC GCC CTT GTG CTT CAG CTG GGC CAC CTT CTT CTT CAG GGC CTG GTT CTT G 3'(SEQ ID NO:65)

4. CCB-Fc Anti-Sense 2:

'GCC TTC AGC TGG GCC ACC TTC TTC TTC AGG GCC TGG TAC TCA CCA CCG AAT TCG CCG GCA 3'(SEQ ID NO:66)

[0245] The oligos were reconstituted to a concentration of 50 pM with dH 0. 2

pl of each oligo were annealed to each other by combining in a thin walled PCR

tube with 2.2 pl of restriction buffer #2 (i.e. final concentration of 10 mM Tris HCI pH

7.9, 10 mM MgCl 2 , 50 mM Na CI, 1 mM dithiothreitol) (New England Biolabs,

Beverly, MA) and heated to 950C for 30 seconds and then allowed to anneal by

cooling slowly for 2 hours to 25°C. 5 pmol of the now annealed oligos were ligated

into a pGEM T-Easy vector as directed in the kit manual. (Promega, Madison WI).

The ligation mixture was added to 50 pl of DH5a competent E. coli cells (Invitrogen,

Carlsbad, CA) on ice for 2 minutes, incubated at 370 C for 5 minutes, incubated on

ice for 2 minutes, and then plated on LB+100 pg/L ampicillin agar plates and placed

at 370C for 14 hours. Individual bacterial colonies were picked and placed in 5 ml of

LB+100 pg/L ampicillin and allowed to grow for 14 hours. The tubes were spun down at 2000xg, 40C for 15 minutes and the vector DNA was isolated using Qiagen miniprep kit (Qiagen, Valencia, CA) as indicated in the kit manual. 2 pg of DNA was digested with NgoM IV-Rsr-l. The fragment was gel purified by the Qiaquick method as instructed in the kit manual (Qiagen, Valencia, CA) and ligated to pED.dcEpoFc with NgoM IV/Rsr II. The ligation was transformed into DH5a competent E. coli cells and the DNA prepared as described for the pGEM T-Easy vector.

Example 10: Cloning of the erythropoietin-acidic peptide Fc construct

[0246] The hinge region of the human IgG1 Fc fragment in EPO-Fc from

amino acid 221-229 (EU numbering) was replaced with an acidic peptide (CCA).

Four overlapping oligos were used (IDT, Coralville, IA):

1. Epo-CCA-Fc Sense 1:

'CCG GTG ACA GGG AAT TCG GTG GTG AGT ACC AGG CCC TGG AGA AGG AGG TGG CCC AGC TGG AG 3'(SEQ ID NO:67)

2. Epo-CCA-Fc Sense 2:

'GCC GAG AAC CAG GCC CTG GAG AAG GAG GTG GCC CAG CTG GAG CAC GAG GGT GGT GGT CCC GCT CCA GAG CTG CTG GGC GGA CA 3'(SEQ ID NO:68)

3. Epo-CCA-Fc Anti-Sense 1:

'GTC CGC CCA GCA GCT CTG GAG CGG GAC CAC CAC CCT CGT GCT CCA GCT GGG CCA C 3'(SEQ ID NO:69)

4. Epo-CCA-Fc Anti-Sense 2:

'CTC CTT CTC CAG GGC CTG GTT CTC GGC CTC CAG CTG GGC CAC CTC CTT CTC CAG GGC CTG GTA CTC ACC ACC GAA TTC CCT GTC ACC GGA 3'(SEQ ID NO:70)

[0247] The oligos were reconstituted to a concentration of 50 pM with dH 20.

pl of each oligo were annealed to each other by combining in a thin walled PCR

tube with 2.2 pl of restriction buffer No. 2 (New England Biolabs, Beverly, MA) and

heated to 95°C for 30 seconds and then allowed to cool slowly for 2 hours to 25°C.

pmol of the now annealed oligos were ligated into a pGEM T-Easy vector as

directed in the kit manual. (Promega, Madison, WI). The ligation mixture was added

to 50 pl of DH5a competent E. coli cells (Invitrogen, Carlsbad, CA) on ice for 2

minutes, incubated at 37 0C 5 minutes, incubated on ice for 2 minutes, and then

plated on LB+100 pg/L ampicillin agar plates and placed at 370 C for 14 hours.

Individual bacterial colonies were picked and placed in 5 ml of LB+100 pg/L

ampicillin and allowed to grow for 14 hours. The tubes were spun down at 2000xg,

4°C for 15 minutes and the vector DNA was prepared using Qiagen miniprep kit

(Qiagen, Valencia, CA) as indicated in the kit manual. 2 pg of DNA was digested

with Age I-Rsr-II. The fragment was gel purified by the Qiaquick method as

instructed in the kit manual (Qiagen, Valencia, CA) and ligated into pED.Epo Fc.1

Age l-Rsr 11. The ligation was transformed into DH5a competent E. coli cells and

DNA prepped as described above.

Example 11: Cloning of Cys-Fc construct

[0248] Using PCR and standard molecular biology techniques (Sambrook et

al. 1989, Molecular Cloning: A Laboratoiy Manual, 2 nd ed., Cold Spring Harbor

Laboratory Press), a mammalian expression construct was generated such that the

coding sequence for the human IFNa signal peptide was directly abutted against the

coding sequence of Fc beginning at the first cysteine residue (Cys 226, EU

Numbering). Upon signal peptidase cleavage and secretion from mammalian cells, an Fc protein with an N-terminal cysteine residue was thus generated. Briefly, the primers

IFNa-Sig-F (IFNa-Sig-F: 5'-GCTACTGCAGCCACCATGGCCTTGACCTT

TGCTTTAC-3')(SEQ ID NO:71) and Cys-Fc-R

(5'-CAGTTCCGGAGCTGGGCACGGCGGA

GAGCCCACAGAGCAGCTTG-3') (SEQ ID NO:72) were used in a PCR reaction to

create a fragment linking the IFNa signal sequence with the N terminus of Fc,

beginning with Cys 226. 500 ng of pED.dC.native hFNa Alinker was added to 25

pmol of each primer in a PCR reaction with Expand High Fidelity System

(Boehringer Mannheim, Indianapolis, IN) according to manufacturer's standard

protocol. The reaction was carried out in a MJ Thermocycler using the following

cycles: 94°C 2 minutes; 30 cycles of (94°C 30 seconds, 50°C 30 seconds, 72°C 45

seconds), and finally 72°C 10 minutes. The expected sized band (-112 bp) was gel

purified with a Gel Extraction kit (Qiagen, Valencia CA), digested with the Pstl and

BspEl restriction enzymes, gel purified, and subcloned into the corresponding sites

pED.dC.native hIFNa Alinker to generate pED.dC.Cys-Fc (Figure 5).

Example 12: Protein Expression and Preparation of Fc-MESNA

[0249] The coding sequence for Fc (the constant region of human IgG1) was

obtained by PCR amplification from an Fc-containing plasmid using standard

conditions and reagents, following the manufacturer's recommended procedure to

subclone the Fc coding sequence Ndel/Sapl. Briefly, the primers 5'- GTGGTCATA

TGGGCATTGAAGGCAGAGGCGCCGCTGCGGTCG - 3'(SEQ ID NO:73) and 5'

GGTGGTTGC TCTTCCGCAAAAACCCGGAGACAGGGAGAGACTCTTCTGCG - 3'

(SEQ ID NO:74)were used to amplify the Fc sequence from 500 ng of the plasmid

pED.dC.Epo-Fc using Expand High Fidelity System (Boehringer Mannheim, Basel

Switzerland) in a RapidCylcler thermocycler (Idaho Technology Salt Lake City,

Utah), denaturing at 95 0C for 2 minutes followed by 18 cycles of 95°C for 0 sec,

550C for 0 sec, and 72°C for 1 minute with a slope of 4, followed by 720C extension

for 10 minutes. The PCR product was subconed into an intermediate cloning vector

and sequenced fully, and then subcloned using the Ndel and Sapl sites in the

pTWINI vector following standard procedures. Sambrook, J., Fritsch, E.F. and

Maniatis, T. 1989, Molecular Cloning: A Laboratoty Manual, 2 nd ed.; Cold Spring

Harbor, New York: Cold Spring Harbor Laboratory Press. This plasmid was then

transformed into BL21(DE3) pLysS cells using standard methods. Id. A 1 liter

culture of cells was grown to an absorbance reading of 0.8 AU at 37°C, induced with

1 mM isopropyl beta-D-1-thiogalactopyranoside, and grown overnight at 25C. Cells

were pelleted by centrifugation, lysed in 20 mM Tris 8.8/1% NP40/0.1 mM

phenylmethanesulfonyl fluoride/ ! pg/ml Benzonase (Novagen Madison, WI), and

bound to chitin beads (New England Biolabs; Beverly, MA) overnight at 41C. Beads

were then washed with several column volumes of 20 mM Tris 8.5/ 500 mM NaCl/ 1

mM EDTA, and then stored at -80°C. Purified Fc-MESNA was generated by eluting

the protein from the beads in 20 mM Tris 8.5/ 500 mM NaCl / 1 mM EDTA / 500 mM

2-mercapto ethane sulfonic acid (MESNA), and the eluate was used directly in the

coupling reaction, below.

Example 13: Factor VII-Fc monomer-dimer hybrid expression and purification

[0250] CHO DG-44 cells expressing Factor VII-Fc were established. CHO

DG-44 cells were grown at 370C, 5% C02, in MEM Alpha plus nucleoside and ribonucleosides and supplemented with 5% heat-inactivated fetal bovine serum until transfection.

[0251] DG44 cells were plated in 100 mm tissue culture petri dishes and

grown to a confluency of 50%- 60%. A total of 10 pg of DNA was used to transfect

one 100 mm dish: 7.5 pg of pED.dC.FVII-Fc + 1.5 pg pcDNA3/Flag-Fc + 1 pg of

pcDNA6-PACE. The cells were transfected as described in the Superfect

transfection reagent manual (Qiagen, Valencia, CA). The media was removed from

transfection after 48 hours and replaced with MEM Alpha without nucleosides plus

% dialyzed fetal bovine serum and 10 pg/ml of Blasticidin (Invitrogen, Carlsbad,

CA) and 0.2 mg/ml geneticin (Invitrogen, Carlsbad, CA). After 10 days, the cells

were released from the plate with 0.25% trypsin and transferred into T25 tissue

culture flasks, and the selection was continued for 10-14 days until the cells began

to grow well as stable cell lines were established. Protein expression was

subsequently amplified by the addition 25 nM methotrexate.

[0252] Approximately 2 x107 cells were used to inoculate 300 ml of growth

medium in a 1700 cm 2 roller bottle (Corning, Corning, NY) supplemented with 5

pg/ml of vitamin K 3 (menadione sodium bisulfite) (Sigma, St Louis, MO). The roller

bottles were incubated in a 5% CO 2 at 370 C for 72 hours. Then the growth medium

was exchanged with 300 ml serum-free production medium (DMEM/F12 with 5 pg/ml

bovine insulin and 10 pg/ml Gentamicin) supplemented with 5 pg/L of vitamin K 3 .

The production medium (conditioned medium) was collected every day for 10 days

and stored at 40 C. Fresh production medium was added to the roller bottles after

each collection and the bottles were returned to the incubator. Pooled media was

first clarified using a Sartoclean glass fiber filter (3.0 pm + 0.2 pm) (Sartorious Corp.

Gottingen, Germany) followed by an Acropack 500 filter (0.8 pm + 0.2 pm) (Pall

Corp., East Hills, NY). The clarified media was then concentrated approximately 20

fold using Pellicon Biomax tangential flow filtration cassettes (10 kDa MWCO)

(Millipore Corp., Billerica, MA).

[0253] Fc chimeras were then captured from the concentrated media by

passage over a Protein A Sepharose 4 Fast Flow Column (AP Biotech, Piscataway,

NJ). A 5 x 5 cm (100 ml) column was loaded with 5 mg Fc protein per mi column

volume at a linear flow rate of 100 cm/hour to achieve a residence time of 3

minutes. The column was then washed with >5 column volumes of IX DPBS to

remove non-specifically bound proteins. The bound proteins were eluted with

100 mM Glycine pH 3.0. Elution fractions containing the protein peak were then

neutralized by adding I part 1 M Tris-HCL, pH 8 to 10 parts elute fraction.

[0254] To remove FLAG-Fc homodimers (that is, chimeric Fc dimers with

FLAG peptide expressed as fusions with both Fc molecules) from the preparation,

the Protein A Sepharose 4 Fast Flow pool was passed over a Unosphere S cation

exchange column (BioRad Corp., Richmond, CA). Under the operating conditions

for the column, the FLAG-Fc monomer-dimer hybrid is uncharged (FLAG-Fc

theoretical pl=6.19) and flows through the column while the hFVII-Fc constructs are

positively charged, and thus bind to the column and elute at higher ionic strength.

The Protein A Sepharose 4 Fast Flow pool was first dialyzed into 20 mM MES,

mM NaCl, pH 6.1. The dialyzed material was then loaded onto a 1.1 x 11 cm

(9.9 ml) column at 150 cm/hour. During the wash and elution, the flow rate was

increased to 500 cm/hour. The column was washed sequentially with 8 column

volumes of 20 mM MES, 20 mM NaCl, pH 6.1 and 8 column volumes of 20 mM

MES, 40 mM NaCl, pH 6.1. The bound protein was eluted with 20 mM MES,

750 mM NaCl, pH 6.1. Elution fractions containing the protein peak were pooled

and sterile filtered through a 0.2 pm filter disc prior to storage at -80°C.

[0255] An anti-FLAG MAB affinity column was used to separate chimeric Fc

dimers with hFVIl fused to both Fc molecules from those with one FLAG peptide and

one hFVil fusion. The Unosphere S Eluate pool was diluted 1:1 with 20 mM Tris,

mM NaCl, 5 mM CaCl 2 , pH 8 and loaded onto a 1.6 x 5 cm M2 anti-FLAG

sepharose column (Sigma Corp., St. Louis, MO) at a linear flow rate of 60 cm/hour.

Loading was targeted to < 2.5 mg monomer-dimer hybrid /ml column volume. After

loading the column was washed with 5 column volumes 20 mM Tris, 50 mM NaCl,

mM CaCl 2 , pH 8.0, monomer-dimer hybrids were then eluted with 100 mM Glycine,

pH 3.0. Elution fractions containing the protein peak were then neutralized by

adding I part I M Tris-HCl, pH 8 to 10 parts eluate fraction. Pools were stored at

-80°C.

Example 14: Factor IX-Fc homodimer and monomer-dimer hybrid expression and purification

[0256] CHO DG-44 cells expressing Factor IX-Fc were established. DG44

cells were plated in 100 mm tissue culture petri dishes and grown to a confluency of

%- 60%. A total of 10 pg of DNA was used to transfect one 100 mm dish: for the

homodimer transfection, 8 pg of pED.dC.Factor IX-Fc + 2 pg of pcDNA6-PACE was

used; for the monomer-dimer hybrid transfection, 8 pg of pED.dC.Factor IX-Fc + 1

pg of pcDNA3-FlagFc +1 pg pcDNA6-PACE was used. The cells were transfected

as described in the Superfect transfection reagent manual (Qiagen, Valencia, CA).

The media was removed from transfection after 48 hours and replaced with MEM

Alpha without nucleosides plus 5% dialyzed fetal bovine serum and 10 pg/ml of

Blasticidin (Invitrogen, Carlsbad, CA) for both transfections, while the monomer

dimer hybrid transfection was also supplemented with 0.2 mg/ml geneticin

(Invitrogen, Carlsbad, CA). After 3 days, the cells were released from the plate with

0.25% trypsin and transferred into T25 tissue culture flasks, and the selection was

continued for 10-14 days until the cells began to grow well as stable cell lines were

established. Protein expression was subsequently amplified by the addition 10 nM

or 100 nM methotrexate for the homodimer or monomer-dimer hybrid, respectively.

[0257] For both cell lines, approximately 2 x107 cells were used to inoculate

300 ml of growth medium in a 1700cm 2 roller bottle (Corning, Corning, NY),

supplemented with 5 pg/L of vitamin K3 (menadione sodium bisulfite) (Sigma, St.

Louis, MO). The roller bottles were incubated in a 5% CO 2 at 37°C for

approximately 72 hours. The growth medium was exchanged with 300 ml serum

free production medium (DMEM/F12 with 5 pg/ml bovine insulin and 10 pg/ml

Gentamicin), supplemented with 5 pg/L of vitamin K 3. The production medium

(conditioned medium) was collected everyday for 10 days and stored at 4°C. Fresh

production medium was added to the roller bottles after each collection and the

bottles were returned to the incubator. Prior to chromatography, the medium was

clarified using a SuporCap-100 (0.8/0.2 pm) filter (Pall Gelman Sciences, Ann Arbor,

MI). All of the following steps were performed at 40 C. The clarified medium was

applied to Protein A Sepharose, washed with 5 column volumes of IX PBS (10 mM

phosphate, pH 7.4,2.7 mM KCI, and 137 mM NaCl), eluted with 0.1 M glycine, pH

2.7 , and then neutralized with 1/10 volume of 1 M Tris-HCI, pH 9.0. The protein

was then dialyzed into PBS.

[0258] The monomer-dimer hybrid transfection protein sample was subject

to further purification, as it contained a mixture of FIX-Fc:FIX-Fc homodimer, FIX

Fc:Flag-Fc monomer-dimer hybrid, and Flag-Fc:Flag-Fc homodimer. Material was

concentrated and applied to a 2.6 cm x 60 cm (318 ml) Superdex 200 Prep Grade

column at a flow rate of 4 ml/minute (36 cm/hour) and then eluted with 3 column

volumes of 1X PBS. Fractions corresponding to two peaks on the UV detector were

collected and analyzed by SDS-PAGE. Fractions from the first peak contained

either FIX-Fc:FIX-Fc homodimer or FIX-Fc:FlagFc monomer-dimer hybrid, while the

second peak contained FlagFc:FlagFc homodimer. All fractions containing the

monomer-dimer hybrid but no FlagFc homodimer were pooled and applied directly to

a 1.6 x 5 cm M2 anti-FLAG sepharose column (Sigma Corp., St. Louis, MO) at a

linear flow rate of 60 cm/hour. After loading, the column was washed with 5 column

volumes PBS. Monomer-dimer hybrids were then eluted with 100 mM Glycine,

pH 3.0. Elution fractions containing the protein peak were then neutralized by

adding 1/10 volume of 1 M Tris-HCI, and analyzed by reducing and nonreducing

SDS-PAGE. Fractions were dialyzed into PBS, concentrated to 1-5 mg/ml, and

stored at -800 C.

Example 15: IFNa homodimer and monomer-dimer hybrid expression and purification

[0259] CHO DG-44 cells expressing hlFNa were established. DG44 cells

were plated in 100 mm tissue culture petri dishes and grown to a confluency of 50%

60%. A total of 10 pg of DNA was used to transfect one 100 mm dish: for the

homodimer transfection, 10 pg of the hlFNaFc constructs; for the monomer-dimer

hybrid transfection, 8 pg of the hlFNaFc constructs + 2 pg of pcDNA3-FlagFc. The cells were transfected as described in the Superfect transfection reagent manual

(Qiagen, Valencia, CA). The media was removed from transfection after 48 hours

and replaced with MEM Alpha without nucleosides plus 5% dialyzed fetal bovine

serum, while the monomer-dimer hybrid transfection was also supplemented with

0.2 mg/ml geneticin (Invitrogen, Carlsbad, CA). After 3 days, the cells were released

from the plate with 0.25% trypsin and transferred into T25 tissue culture flasks, and

the selection was continued for 10-14 days until the cells began to grow well and

stable cell lines were established. Protein expression was subsequently amplified

by the addition methotrexate: ranging from 10 to 50 nM. 7

[0260] For all cell lines, approximately 2 x 10 cells were used to inoculate

300 ml of growth medium in a 1700cm 2 roller bottle (Corning, Corning, NY). The

roller bottles were incubated in a 5% CO 2 at 37°C for approximately 72 hours. Then

the growth medium was exchanged with 300 ml serum-free production medium

(DMEM/F12 with 5 pg/mI bovine insulin and 10 pg/ml Gentamicin). The production

medium (conditioned medium) was collected every day for 10 days and stored at

C. Fresh production medium was added to the roller bottles after each collection

and the bottles were returned to the incubator. Prior to chromatography, the

medium was clarified using a SuporCap-I00 (0.8/0.2 pm) filter from Pall Gelman

Sciences (Ann Arbor, MI). All of the following steps were performed at 40C. The

clarified medium was applied to Protein A Sepharose, washed with 5 column

volumes of IX PBS (10 mM phosphate, pH 7.4, 2.7 mM KC, and 137 mM NaCl),

eluted with 0.1 M glycine, pH 2.7, and then neutralized with 1/10 volume of I M Tris

HCI, pH 9.0. The protein was then dialyzed into PBS.

[0261] The monomer-dimer hybrid transfection protein samples were then

subject to further purification, as it contained a mixture of IFNaFc:FNaFc

homodimer, IFNaFc:FlagFc monomer-dimer hybrid, and FlagFc:FlagFc homodimer

(orAlinkerorGS15 linker). Material was concentrated and applied to a2.6 cmx60

cm (318 ml) Superdex 200 Prep Grade column at a flow rate of 4 ml/min (36 cm/hr)

and then eluted with 3 column volumes of 1X PBS. Fractions corresponding to two

peaks on the UV detector were collected and analyzed by SDS-PAGE. Fractions

from the first peak contained either IFNaFc:IFNaFc homodimer or IFNaFc:FlagFc

monomer-dimer hybrid, while the second peak contained FlagFc:FlagFc homodimer.

All fractions containing the monomer-dimer hybrid, but no FlagFc homodimer, were

pooled and applied directly to a 1.6 x 5 cm M2 anti-FLAG sepharose column (Sigma

Corp., St. Louis, MO) at a linear flow rate of 60 cm/hour. After loading the column

was washed with 5 column volumes PBS monomer-dimer hybrids were then eluted

with 100 mM Glycine, pH 3.0. Elution fractions containing the protein peak were

then neutralized by adding 1/10 volume of 1 M Tris-HCI, and analyzed by reducing

and nonreducing SDS-PAGE. Fractions were dialyzed into PBS, concentrated to 1

mg/ml, and stored at -80°C.

Example 16: Coiled coil protein expression and purification

[0262] The plasmids, pED.dC Epo-CCA-Fc and pED.dC CCB-Fc will be

transfected either alone or together at a 1:1 ratio into CHO DG44 cells. The cells will

be transfected as described in the Superfect transfection reagent manual (Qiagen,

Valencia, CA). The media will be removed after 48 hours and replaced with MEM

Alpha w/o nucleosides plus 5% dialyzed fetal bovine serum. Purification will be

done by affinity chromatography over a protein A column according to methods known in the art. Alternatively, purification can be achieved using size exclusion chromatography.

Example 17: Cys-Fc expression and purification

[0263] CHO DG-44 cells expressing Cys-Fc were established. The

pED.dC.Cys-Fc expression plasmid, which contains the mouse dihydrofolate

reductase (dhfr) gene, was transfected into CHO DG44 (dhfr deficient) cells using

Superfect reagent (Qiagen; Valencia, CA) according to manufacturer's protocol,

followed by selection for stable transfectants in aMEM (without nucleosids) tissue

culture media supplemented with 5% dialyzed FBS and penicillin/stroptomycin

antibiotics (Invitrogen; Carlsbad, CA) for 10 days. The resulting pool of stably

transfected cells were then amplified with 50 nM methotrexate to increase

expression. Approximately 2 x 107 cells were used to inoculate 300 ml of growth

medium in a 1700 cm 2 roller bottle (Corning, Corning, NY). The roller bottles were

incubated in a 5% CO 2 at 370 C for approximately 72 hours. The growth medium

was exchanged with 300 ml serum-free production medium (DMEM/F12 with 5 pg/ml

bovine insulin and 10 pg/ml Gentamicin). The production medium (conditioned

medium) was collected every day for 10 days and stored at 40 C. Fresh production

medium was added to the roller bottles after each collection and the bottles were

returned to the incubator. Prior to chromatography, the medium was clarified using

a SuporCap-100 (0.8/0.2 pm) filter from Pall Gelman Sciences (Ann Arbor, MI). All

of the following steps were performed at 4°C. The clarified medium was applied to

Protein A Sepharose, washed with 5 column volumes of 1X PBS (10 mM phosphate,

pH 7.4, 2.7 mM KCI, and 137 mM NaCI), eluted with 0.1 M glycine, pH 2.7, and then neutralized with 1/10 volume of I M Tris-HC, pH 9.0. Protein was dialyzed into PBS and used directly in conjugation reactions.

Example 18: Coupling of T20-thioesters to Cys-Fc

{0264] Cys-Fc (4 mg, 3.2 mg/ml final concentration) and either T20-thioester

or T20-PEG-thioester (2 mg, approximately 5 molar equivalents) were incubated for

16 hours at room temperature in 0.1 M Tris 8/ 10 mM MESNA. Analysis by SDS

PAGE (Tris-Gly gel) using reducing sample buffer indicated the presence of a new

band approximately 5 kDa larger than the Fc control (>40-50% conversion to the

conjugate). Previous N-terminal sequencing of Cys-Fc and unreacted Cys-Fc

indicated that the signal peptide is incorrectly processed in a fraction of the

molecules, leaving a mixture of (Cys)-Fc, which will react through native ligation with

peptide-thioesters, and (Val)-(Gly)-(Cys)-Fc, which will not. As the reaction

conditions are insufficient to disrupt the dimerization of the Cys-Fc molecules, this

reaction generated a mixture of T20-Cys-Fc:T20-Cys-Fc homodimers, T20-Cys-Fc:

Fc monomer-dimer hybrids, and Cys-Fc:Cys-Fc Fc-dimers. This protein was purified

using size exclusion chromatography as indicated above to separate the three

species. The result was confirmed by SDS-PAGE analysis under nonreducing

conditions.

Example 19: Antiviral assay for IFNa activity

[0265] Antiviral activity (IU/m) of IFNa fusion proteins was determined using

a CPE (cytopathic effect) assay. A549 cells were plated in a 96 well tissue culture

plate in growth media (RPMI 1640 supplemented with 10% fetal bovine serum (FBS)

and 2 mM L-glutamine) for 2 hours at 370 C, 5% CO .2 IFNa standards and IFNa

fusion proteins were diluted in growth media and added to cells in triplicate for 20 hours at 37°C, 5% C02. Following incubation, all media was removed from wells, encephalomyocarditis virus (EMC) virus was diluted in growth media and added

(3000 pfu/well) to each well with the exception of control wells. Plates were

incubated at 37°C, 5% C02for 28 hours. Living cells were fixed with 10% cold

trichloroacetic acid (TCA) and then stained with Sulforhodamine B (SRB) according

to published protocols (Rubinstein et al. 1990, J. Nat. Cancer Inst. 82, 1113). The

SRB dye was solubilized with 10 mM Tris pH 10.5 and read on a spectrophotometer

at 490 nm. Samples were analyzed by comparing activities to a known standard

curve World Health Organization IFNa 2b international Standard ranging from 5 to

0.011 IU/ml. The results are presented below in Table 3 and Figure 6 and

demonstrate increased antiviral activity of monomer-dimer hybrids.

TABLE 3: INTERFERON ANTIVIRAL ASSAY HOMODIMER V. MONOMER-DIMER HYBRID

Protein Antiviral Activity Std dev (IU/nmol) IFNaFc 8aa linker homodimer 0.45 x 10 0.29 x 105 IFNaFc 8aa linker:FlagFc 4.5 x 10I 1.2 x 10i monomer-dimer hybrid IFNaFc A linker homodimer 0.22 x 10 0.07 x 10' IFNaFc A delta linker: FlagFc 2.4 x 10 0.0005 x 10 monomer-dimer hybrid _________0 _______

IFNaFc GS15 linker 2.3x105 1.0x105 homodimer IFNaFc GS15 linker 5.3x105 0.15x100 monomer-dimer hybrid

Example 20: FVlla Clotting Activity Analysis

[0266] The StaClot FVila-rTF assay kit was purchased from Diagnostica

Stago (Parsippany, NJ) and modified as described in Johannessen et al. 2000,

Blood Coagulation and Fibrinoysis 11:S159. A standard curve was preformed with

the FVIla World Health Organization standard 89/688. The assay was used to

compare clotting activity of monomer-dimer hybrids compared to homodimers. The

results showed the monomer-dimer hybrid had four times the clotting activity

compared to the homodimer (Figure 7).

Example 21: FVlla-Fc Oral dosing in day 10 rats

[0267] 25 gram day 9 newborn Sprague Dawley rats were purchased from

Charles River (Wilmington, MA) and allowed to acclimate for 24 hours. The rats

were dosed orally with FVIlaFc homodimer, monomer-dimer hybrid or a 50:50 mix of

the two. A volume of 200 pl of a FVIlaFc solution for a dose of 1 mg/kg was

administered. The solution was composed of a Tris-HCI buffer pH 7.4 with 5 mg/m

soybean trypsin inhibitor. The rats were euthanized with C02 at several time points,

and 200 pl of blood was drawn by cardiac puncture. Plasma was obtained by the

addition of a 3.8% sodium citrate solution and centrifugation at room temperature at

a speed of 1268xg. The plasma samples were either assayed fresh or frozen at

200C. Orally dosed monomer-dimer hybrid resulted in significantly higher maximum

(Cmax) serum concentrations compared to homodimeric Factor VII (Figure 8).

Example 22: Factor IX-Fc Oral dosing of neonatal rats

[0268] Ten-day old neonatal Sprague-Dawley rats were dosed p.o. with

200 pl of FIX-Fc homodimer or FIX-Fc: FlagFc monomer-dimer hybrid at

approximately equimolar doses of 10 nmol/kg in 0.1 M sodium phosphate buffer, pH

6.5 containing 5 mg/mI soybean trypsin inhibitor and 0.9% NaCl. At 1, 2, 4, 8, 24,

48, and 72 hours post injection, animals were euthanized with C02, blood was

drawn via cardiac puncture and plasma was obtained by the addition of a 3.8% sodium citrate solution and centrifugation at room temperature at a speed of 1268xg.

Samples were then sedimented by centrifugation, serum collected and frozen at

-20°C until analysis of the fusion proteins by ELISA.

Example 23: Factor IX-Fc ELISA

[0269] A 96-well Immulon 4HBX ELISA plate (Thermo LabSystems, Vantaa,

Finland) was coated with 100 pl/well of goat anti-Factor IX IgG (Affinity Biologicals,

Ancaster, Canada) diluted 1:100 in 50 mM carbonate buffer, pH 9.6. The plates

were incubated at ambient temperature for 2 hours or overnight at 40 C sealed with

plastic film. The wells were washed 4 times with PBST, 300 pl/well using the

TECAN plate washer. The wells were blocked with PBST + 6% BSA, 200 pl/well,

and incubated 90 minutes at ambient temperature. The wells were washed 4 times

with PBST, 300 pl/well using the TECAN plate washer. Standards and blood

samples from rats described in Example 18 were added to the wells, (100 pl/well),

and incubated 90 minutes at ambient temperature. Samples and standards were

diluted in HBET buffer (HBET: 5.95 g HEPES, 1.46 g NaCI, 0.93 g Na2EDTA, 2.5 g

Bovine Serum Albumin, 0.25 ml Tween-20, bring up to 250 ml with dH 20, adjust pH

to 7.2). Standard curve range was from 200 ng/m to 0.78 ng/ml with 2 fold dilutions

in between. Wells were washed 4 times with PBST, 300 pl/well using the TECAN

plate washer. 100 pl/well of conjugated goat anti-human IgG-Fc-HARP antibody

(Pierce, Rockford, IL) diluted in HBET 1:25,000 was added to each well. The plates

were incubated 90 minutes at ambient temperature. The wells were washed 4 times

with PBST, 300 pl/well using the TECAN plate washer. The plates were developed

with 100 pl/well of tetramethylbenzidine peroxidase substrate (TMB) (Pierce,

Rockford, IL)was added according to the manufacturer's instructions. The plates were incubated 5 minutes at ambient temperature in the dark or until color developed. The reaction was stopped with 100 pl/well of 2 M sulfuric acid.

Absorbance was read at 450 nm on SpectraMax plusplate reader (Molecular

Devices, Sunnyvale, CA). Analysis of blood drawn at 4 hours indicated more than a

fold difference in serum concentration between Factor IX-Fc monomer-dimer

hybrids compared to Factor IX Fc homodimers (Figure 9). The results indicated

Factor IX-Fc monomer-dimer hybrid levels were consistently higher than Factor IX

Fc homodimers (Figure 10).

Example 24: Cloning of Epo-Fc

[0270] The mature Epo coding region was obtained by PCR amplification

from a plasmid encoding the mature erythropoietin coding sequence, originally

obtained by RT-PCR from Hep G2 mRNA, and primers hepoxba-F and hepoeco-R,

indicated below. Primer hepoxba-F contains an Xbal site, while primer hepoeco-R

contains an EcoRI site. PCR was carried out in theIdaho Technology RapidCycler

using Vent polymerase, denaturing at 950C for 15 seconds, followed by 28 cycles

with a slope of 6.0 of 95°C for 0 seconds, 55°C for 0 seconds, and 72°C for 1 minute

seconds, followed by 3 minute extension at 72C. An approximately 514 bp

product was gel purified, digested with Xbal and EcoRl, gel purified again and

directionally subcloned into an Xbal/EcoRl-digested, gel purified pED.dC.XFc vector,

mentioned above. This construct was named pED.dC.EpoFc.

[0271] The Epo sequence, containing both the endogenous signal peptide

and the mature sequence, was obtained by PCR amplification using an adult kidney

QUICK-clone cDNA preparation as the template and primers Epo+Pep-Sbf-F and

Epo+Pep-Sbf-R, described below. The primer Epo+Pep-Sbf-F contains an Sbfl site upstream of the start codon, while the primer Epo+Pep-Sbf-R anneals downstream of the endogenous SbfIsite in the Epo sequence. The PCR reaction was carried out in the PTC-200 MJ Thermocycler using Expand polymerase, denaturing at 94°C for

2 minutes, followed by 32 cycles of 940C for 30 seconds, 570C for 30 seconds, and

720C for 45 seconds, followed by a 10 minute extension at 72C. An approximately

603 bp product was gel isolated and subcloned into the pGEM-T Easy vector. The

correct coding sequence was excised by Sbl digestion, gel purified, and cloned into

the Pstl-digested, shrimp alkaline phosphatase (SAP)-treated, gel purified

pED.dC.EpoFc plasmid. The plasmid with the insert in the correct orientation was

initially determined by Kpnl digestion. A Xmnl and Pvull digestion of this construct

was compared with pED.dC.EpoFc and confirmed to be in the correct orientation.

The sequence was determined and the construct was named pED.dC.natEpoFc.

PCR Primers:

hepoxba-F (EPO-F): 5'-AATCTAGAGCCCCACCACGCCTCATCTGTGAC-3'(SEQ ID NO:75)

hepoeco-R (EPO-R) 5'-TTGAATTCTCTGTCCCCTGTCCTGCAGGCC-3'(SEQ ID NO:76)

Epo+Pep-Sbf-F: 5'-GTACCTGCAGGCGGAGATGGGGGTGCA-3'(SEQ ID NO:77)

Epo+Pep-Sbf-R: 5'-CCTGGTCATCTGTCCCCTGTCC-3'(SEQ ID NO:78)

Example 25: Cloning of Epo-Fc

[0272] An alternative method of cloning EPO-Fc is described herein.

Primers were first designed to amplify the full length Epo coding sequence, including

the native signal sequence, as follows:

Epo-F: 5'-GTCCAACCTG CAGGAAGCTTG CCGCCACCAT GGGAGTGCAC GAATGTCCTG CCTGG- 3'(SEQ ID NO:79)

Epo-R: 5'-GCCGAATTCA GTTTTGTCGA CCGCAGCGG CGCCGGCGAA CTCTCTGTCC CCTGTTCTGC AGGCCTCC- 3'(SEQ ID NO:80)

[0273] The forward primer incorporates an Sbfl and HindIll site upstream of

a Kozak sequence, while the reverse primer removes the internal Sbfl site, and adds

an 8 amino acid linker to the 3'end of the coding sequence (EFAGAAAV) (SEQ ID

NO:81) as well as Sall and EcoRI restriction sites. The Epo coding sequence was

then amplified from a kidney cDNA library (BD Biosciences Clontech, Palo Alto, CA)

using 25 pmol of these primers in a 25 pl PCR reaction using Expand High Fidelity

System (Boehringer Mannheim, Indianapolis, IN) according to manufacturer's

standard protocol in a MJ Thermocycler using the following cycles: 940 C 2 minutes;

cycles of (94°C 30 seconds, 58°C 30 seconds, 720C 45 seconds), followed by

72°C for 10 minutes. The expected sized band (641 bp) was gel purified with a Gel

Extraction kit (Qiagen, Valencia, CA) and ligated into the intermediate cloning vector

pGEM T-Easy (Promega, Madison, WI). DNA was transformed into DH5a cells

(Invitrogen, Carlsbad, CA) and miniprep cultures grown and purified with a Plasmid

Miniprep Kit (Qiagen, Valencia, CA) both according to manufacturer's standard

protocols. Once the sequence was confirmed, this insert was digested out with

Sbfl/EcoRI restriction enzymes, gel purified, and cloned into the Pst/EcoRI sites of

the mammalian expression vector pED.dC in a similar manner.

[0274] Primers were designed to amplify the coding sequence for the

constant region of human IgG1 (the Fc region, EU numbering 221-447) as follows:

Fc-F: 5'-GCTGCGGTCG ACAAAACTCA CACATGCCCA CCGTGCCCAG CTCCGGAACT CCTGGGCGGA CCGTCAGTC- 3'(SEQ ID NO:82)

Fc-R 5'-ATTGGAATTC TCATTTACCC GGAGACAGGG AGAGGC- 3'(SEQ ID NO:83)

The forward primer incorporates a Sall site at the linker-Fc junction, as well as

introducing BspEl and RsrIl sites into the Fc region without affecting the coding

sequence, while the reverse primer adds an EcoRi site after the stop codon. The Fc

coding sequence was then amplified from a leukocyte cDNA library (BD Biosciences

Clontech, Palo Alto, CA) using 25 pmol of these primers in a 25 pl PCR reaction

using Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN)

according to manufacturer's standard protocol in a MJ Thermocycler using the

following cycles: 940C 2 minutes; 30 cycles of (94 0C 30 seconds, 58°C 30 seconds,

72°C 45 seconds), followed by 72 0C for 10 minutes. The expected sized band (696

bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia, CA) and ligated into

the intermediate cloning vector pGEM T-Easy (Promega, Madison, WI). DNA was

transformed into DH5a cells (Invitrogen, Carlsbad, CA) and miniprep cultures grown

and purified with a Plasmid Miniprep Kit (Qiagen, Valencia, CA), both according to

manufacturer's standard protocols. Once the sequence was confirmed, this insert

was digested out with Sal/EcoRI restriction enzymes, gel purified, and cloned into

the Sall/EcoRI sites of the plasmid pED.dC.Epo (above) in a similar manner, to

generate the mammalian expression plasmid pED.dC.EpoFc. In another experiment

this plasmid was also digested with Rsrl/Xmal, and the corresponding fragment

from pSYN-Fc-002, which contains the Asn 297 Ala mutation (EU numbering) was

cloned in to create pED.dC.EPO-Fc N297A (pSYN-EPO-004). Expression in

mammalian cells was as described in Example 26. The amino acid sequence of

EpoFc with an eight amino acid linker is provided in figure 2j. During the process of

this alternative cloning method, although the exact EpoFc amino acid sequence was

preserved (figure 2J), a number of non-coding changes were made at the nucleotide level (figure 3J). These are G6A (G at nucleotide 6 changed to A) (eliminate possible secondary structure in primer), G567A (removes endogenous SbfI site from

Epo), A582G (removes EcoRi site from linker), A636T and T639G (adds unique

BspEl site to Fc), and G651C (adds unique Rsrli site to Fc). The nucleotide

sequence in figure 3J is from the construct made in Example 25, which incorporates

these differences from the sequence of the construct from Example 24.

Example 26: EPO-Fc Homodimer And Monomer-dimer Hybrid Expression And Purification

[0275] DG44 cells were plated in 100 mm tissue culture petri dishes and

grown to a confluency of 50%-60%. A total of 10 pg of DNA was used to transfect

one 100 mm dish: for the homodimer transfection,10 pg of pED.dC.EPO-Fc; for the

monomer-dimer hybrid transfection, 8 pg of pED.dC.EPO-Fc + 2 pg of pcDNA3

FlagFc. The constructs used were cloned as described in Example 24. The cloning

method described in Example 25 could also be used to obtain constructs for use in

this example. The cells were transfected as described in the Superfect transfection

reagent manual (Qiagen, Valencia, CA). Alternatively, pED.dC.EPO-Fc was

cotransfected with pSYN-Fc-016 to make an untagged monomer. The media was

removed from transfection after 48 hours and replaced With MEM Alpha without

nucleosides plus 5% dialyzed fetal bovine serum for both transfections, while the

monomer-dimer hybrid transfection was also supplemented with 0.2 mg/ml geneticin

(Invitrogen, Carlsbad, CA). After 3 days, the cells were released from the plate with

0.25% trypsin and transferred into T25 tissue culture flasks, and the selection was

continued for 10-14 days until the cells began to grow well as stable cell lines were established. Protein expression was subsequently amplified by the addition methotrexate.

[0276] For both cell lines, approximately 2 x 107 cells were used to inoculate

300 ml of growth medium in a 1700 cm 2 roller bottle (Corning, Corning, NY). The

roller bottles were incubated in a 5% C02 at 37°C for approximately 72 hours. The

growth medium was exchanged with 300 ml serum-free production medium

(DMEM/F12 with 5 pg/ml bovine insulin and 10 pg/mI Gentamicin). The production

medium (conditioned medium) was collected every day for 10 days and stored at

C. Fresh production medium was added to the roller bottles after each collection

and the bottles were returned to the incubator. Prior to chromatography, the

medium was clarified using a SuporCap-100 (0.8/0.2 pm) filter from Pall Gelman

Sciences (Ann Arbor, MI). All of the following steps were performed at 4C. The

clarified medium was applied to Protein A Sepharose, washed with 5 column

volumes of IX PBS (10 mM phosphate, pH 7.4, 2.7 mM KCl, and 137 mM NaC),

eluted with 0.1 M glycine, pH 2.7, and then neutralized with 1/10 volume of 1 M Tris

HCI, pH 9.0. Protein was then dialyzed into PBS.

[0277] The monomer-dimer hybrid transfection protein sample was subject

to further purification, as it contained a mixture of EPO-Fc:EPO-Fc homodimer,

EPO-Fc:Flag-Fc monomer-dimer hybrid, and Flag-Fc:Flag-Fc homodimer. Material

was concentrated and applied to a 2.6 cm x 60 cm (318 ml) Superdex 200 Prep

Grade column at a flow rate of 4 m/min (36 cm/hour) and then eluted with 3 column

volumes of 1X PBS. Fractions corresponding to two peaks on the UV detector were

collected and analyzed by SDS-PAGE. Fractions from the first peak contained

either EPO-Fc:EPO-Fc homodimer or EPO-Fc:FlagFc monomer-dimer hybrid, while the second peak contained FlagFc:FlagFc homodimer. All fractions containing the monomer-dimer hybrid but no FlagFc homodimer were pooled and applied directly to a 1.6 x 5 cm M2 anti-FLAG sepharose column (Sigma Corp.) at a linear flow rate of cm/hour. After loading the column was washed with 5 column volumes PBS.

Monomer-dimer hybrids were then eluted with 100 mM Glycine, pH 3.0. Elution

fractions containing the protein peak were then neutralized by adding 1/10 volume of

1 M Tris-HCI, and analyzed by reducing and nonreducing SDS-PAGE. Fractions

were dialyzed into PBS, concentrated to 1-5 mg/ml, and stored at -80°C.

[0278] Alternatively, fractions from first peak of the Superdex 200 were

analyzed by SDS-PAGE, and only fractions containing a majority of EpoFc

monomer-dimer hybrid, with a minority of EpoFc homodimer, were pooled. This

pool, enriched for the monomer-dimer hybrid, was then reapplied to a Superdex 200

column, and fractions containing only EpoFc monomer-dimer hybrid were then

pooled, dialyzed and stored as purified protein. Note that this alternate purification

method could be used to purify non-tagged monomer-dimer hybrids as well.

Example 27: Administration of EpoFc Dimer and Monomer-Dimer Hybrid With an Eight Amino Acid Linker to Cynomolqus Monkeys

[0279] For pulmonary administration, aerosols of either EpoFc dimer or

EpoFc monomer-dimer hybrid proteins (both with the 8 amino acid linker) in PBS, pH

7.4 were created with the Aeroneb Pro TM (AeroGen, Mountain View, CA) nebulizer,

in-line with a Bird Mark 7A respirator, and administered to anesthetized naive

cynomolgus monkeys through endotracheal tubes (approximating normal tidal

breathing). Both proteins were also administered to naive cynomolgus monkeys by

intravenous injection. Samples were taken at various time points, and the amount of

Epo-containing protein in the resulting plasma was quantitated using the Quantikine

IVD Human Epo Immunoassay (R&D Systems, Minneapolis, MN). Pharmacokinetic

parameters were calculated using the software WinNonLin. Table 4 presents the

bioavailability results of cynomolgus monkeys treated with EpoFc monomer-dimer

hybrid or EpoFc dimer.

TABLE 4: ADMINISTRATION OF EPOFC MONOMER-DIMER HYBRID AND EPOFC DIMER TO MONKEYS

Approx. Route Deposited Cmax Cmax t112 t1 /2 avg Protein Monkey P e RDose' (ng/mi) (fmollml) (hr) (hr) (pglkg) C06181 pulrn 20 72.3 1014 23.6 C06214 pulm 20 50.1 703 23.5 25.2 EpoFo C07300 pulm 20 120 1684 36.2 monomer- C07332 pulm 20 100 1403 17.5 IV 25 749 10508 21.3 mr hybrid C07285 C07288 IV 25 566 7941 23 22.6 C07343 IV 25 551 1014 23.5 DD026 pulm 15 10.7 120 11.5 DD062 pulm 15 21.8 244 27.3 DD046 pulm 15 6.4 72 21.8 22.1 DD015 pulm 15 12.8 143 20.9 DD038 pulm 35 27 302 29 EpoFc F4921 IV 150 3701 41454 15.1 dimer 96Z002 IV 150 3680 41219 15.3 1261CQ IV 150 2726 30533 23.6 14.6 127-107 IV 150 4230 47379 15.0 118-22 IV 150 4500 50403 8.7 126-60 IV 150 3531 39550 9.8 Based on 15% deposition fraction of nebulized dose as determined by gammascintigraphy

[0280] The percent bioavailability (F) was calculated for the pulmonary

doses using the following equation:

F= (AUC pulmonary / Dose pulmonary) / (AUC IV / Dose IV) * 100

TABLE 5: CALCULATION OF PERCENT BIOAVAILABILITY FOR EPOFC MONOMER-DIMER HYBRID V. DIMER AFTER PULMONARY ADMINISTRATION TO NAIVE CYNOMOLGUS MONKEYS

Approx. AUC Bioavailability2 Average Protein Monkey # Dose' ngAhr/mL (F) Bioavailabiity (deposited) EpoFc C06181 20 pg/kg 3810 25.2% monomer- C06214 20 pg/kg 3072 20.3% 34.9% dimer C07300 20 pg/kg 9525 63.0% hybrid C07332 20 pg/kg 4708 31.1% DD026 15 pg/kg 361 5.1% EpoFc DD062 15 pg/kg 1392 19.6% dimer DD046 15 pg/kg 267 3.8% 10.0% DD015 15 pg/kg 647 9.1% 1 DD038 35 pg/kg 2062 12.4% SBased on 15% deposition fraction of nebulized dose as determined by gamma scintigraphy 2Mean AUC for IV EpoFc monomer-dimer hybrid = 18,913 ng-hr/mL (n=3 monkeys), dosed at pg/kg. Mean AUC for IV EpoFc dimer = 70, 967 ng-hr/mL (n=6 monkeys), dosed at 150 pgkg

[0281] The pharmacokinetics of EpoFc with an 8 amino acid linker

administered to cynomolgus monkeys is presented in figure 11. The figure

compares the EpoFc dimer with the EpoFc monomer-dimer hybrid in monkeys after

administration of a single pulmonary dose. Based on a molar comparison

significantly higher serum levels were obtained in monkeys treated with the

monomer-dimer hybrid compared to the dimer.

Example 28: Subcutaneous Administration of EPOFc Monomer-dimer Hybrid

[0282] To compare serum concentrations of known erythropoietin agents

with EPOFc monomer-dimer hybrids, both EPOFc monomer-dimer hybrid and

Aranesp* (darbepoetin alfa), which is not a chimeric fusion protein, were

administered subcutaneously to different monkeys and the serum concentration of

both was measured overtime.

[0283] Cynomolgus monkeys (n = 3 per group) were injected

subcutaneously with 0.025 mg/kg EpoFc monomer-dimer hybrid. Blood samples were collected predose and at times up to 144 hours post dose. Serum was prepared from the blood and stored frozen until analysis by ELISA (Human Epo

Quantikine Immunoassay) (R & D Systems, Minneapolis, MN). Pharmacokinetic

parameters were determined using WinNonLina software (Pharsight,

Mountainview, CA).

[0284] The results indicated the serum concentrations of both EPOFc

monomer-dimer hybrid and Aranesp* (darbepoetin alfa) were equivalent overtime,

even'though the administered molar dose of Aranesp* (darbepoetin alfa) was

slightly larger (Table 6) (figure 12).

TABLE 6

Route Dose Dose Cmax AUC T1

% ([pg/kg) (nmol/kg) (ng/mL) (ngohremL1 ) (hr) Bioavaildbility (F) EpoFc Subcutaneous 25 0.3 133 10,745 26 57 ± 17 Monomer- 34 3,144 t5 dimer hybrid 53± 8 Aranesp* Subcutaneous 20 0.54 83 ± 11 5390 ±747 22 ±2

Example 29: Intravenous Administration of EPOFc Monomer-dimer Hybrid

[0285] To compare serum concentrations of known erythropoietin agents

with EPOFc monomer-dimer hybrids, EPOFc monomer-dimer hybrid, Aranesp*

(darbepoetin alfa), and Epogen* (epoetin alfa), neither of which is a chimeric fusion

protein, were administered intravenously to different monkeys and the serum

concentration of both was measured overtime.

[0286] Cynomolgus monkeys (n = 3 per group) were injected intravenously

with 0.025 mg/kg EpoFc monomer-dimer hybrid. Blood samples were collected

predose and at times up to 144 hours post dose. Serum was prepared from the blood and stored frozen until analysis by ELISA (Human Epo Quantikine

Immunoassay) (R &D Systems, Minneapolis, MN). Pharmacokinetic parameters

were determined using WinNonLinA software (Pharsight, Mountainview, CA).

[0287] The results indicated the serum concentration versus time (AUC) of

EPOFc monomer-dimer hybrid was greater than the concentrations of either

Epogen* (epoetin alfa) or Aranesp* (darbepoetin alfa), even though the monkeys

received larger molar doses of both Epogen* (epoetin alfa) and Aranesp*

(darbepoetin alfa) (Table 7) (Figure 13).

TABLE 7

Route Dose Dose Cmax AUC T2 ( tg/kg) (nmol/kg) (ng/mL) (ngehromL-) (hr) EpoFc Intravenous 25 0.3 622± 18,913± 23 ± 1 Monomer- 110 3,022 dimer hybrid Aranesp* Intravenous 20 0.54 521 ±8 10,219+ 20± 1 298 Epogen Intravenous 20 0.66 514± 3936 ±636 6.3 ±0.6 172

Example 30: Alternative Purification of EpoFc Monomer-dimer Hybrid

[0288] Yet another alternative for purifying EPO-Fc is described herein. A

mixture containing Fc, EpoFc monomer-dimer hybrid, and EpoFc dimer was applied

to a Protein A Sepharose column (Amersham, Uppsala, Sweden). The mixture was

eluted according to the manufacturer's instructions. The Protein A Sepharose

eluate, containing the mixture was buffer exchanged into 50 mM Tris-Cl (pH 8.0).

The protein mixture was loaded onto an 8 mL Mimetic Red 2 XL column (ProMetic

Life Sciences, Inc., Wayne, NJ) that had been equilibrated in 50 mM Tris-Cl (pH

8.0). The column was then washed with 50 mM Tris-CI (pH 8.0); 50 mM NaCl. This step removed the majority of the Fc. EpoFc monomer-dimer hybrid was specifically eluted from the column with 50 mM Tris-CI (pH 8.0); 400 mM NaCi. EpoFc dimer canbe eluted and the column regenerated with column volumes ofi 1 MNaOH.

Eluted fractions from the column were analyzed by SDS-PAGE (Figure 14).

Example 31: Cloning of lqK signal sequence - Fc construct for making untaqqed Fc alone.

[0289] The coding sequence for the constant region of IgG1 (EU # 221-447;

the Fc region) was obtained by PCR amplification from a leukocyte cDNA library

(Clontech, CA) using the following primers:

rcFc-F 5'- GCTGCGGTCGACAAAACTCACACATGCCCACCGTGCCCAGCTCC

GGAACTCCTGGGCGGACCGTCAGTC -3'(SEQ ID NO: 84)

rcFc-R 5'- ATTGGAATTCTCATTTACCCGGAGACAGGGAGAGGC -3'(SEQ ID

NO: 85)

[0290] The forward primer adds three amino acids (AAV) and a Sall cloning

site before the beginning of the Fc region, and also incorporates a BspEl restriction

site at amino acids 231-233 and an Rsrl restriction site at amino acids 236-238

using the degeneracy of the genetic code to preserve the correct amino acid

sequence (EU numbering). The reverse primer adds an EcoRI cloning site after the

stop codon of the Fc. A 25 pl PCR reaction was carried out with 25 pmol of each

primer using Expand High Fidelity System (Boehringer Mannheim, Indianapolis, IN)

according to the manufacturer's standard protocol in a MJ Thermocycler using the

following cycles: 94 0C 2 minutes; 30 cycles of (941C 30 seconds, 58°C 30 seconds,

72 0C 45 seconds), 72°C 10 minutes. The expected sized band (-696 bp) was gel purified with a Gel Extraction kit (Qiagen, Valencia CA), and cloned into pGEM T

Easy (Promega, Madison, WI) to produce an intermediate plasmid pSYN-Fc-001

(pGEM T-Easy/Fc).

[0291] The mouse IgK signal sequence was added to the Fc CDS using the

following primers:

rc-IgK Sig seq-F: 5'-TTTAAGCTTGCCGCCACCATGGAGACAGACACACTCC

TGCTATGGGTACTGCTGCTCTGGGTTCCAGGTTCCACTGGTGACAAAACT

CACACATGCCCACCG -3'(SEQ ID NO: 86)

Fc-noXma-GS-R: 5'- GGTCAGCTCATCGCGGGATGGG -3'(SEQ ID NO: 87)

Fc-noXma-GS-F: 5'- CCCATCCCGCGATGAGCTGACC -3'(SEQ ID NO: 88)

[0292] The rc-IgK signal sequence-F primer adds a Hindill restriction site to

the 5'end of the molecule, followed by a Kozak sequence (GCCGCCACC) (SEQ ID

NO: 89) followed by the signal sequence from the mouse IgK light chain, directly

abutted to the beginning of the Fc sequence (EU# 221). The Fc-noXma-GS-F and

R primers remove the internal Xmal site from the Fc coding sequence, using the

degeneracy of the genetic code to preserve the correct amino acid sequence. Two

pl PCR reactions were carried out with 25 pmol of either rc-IgK signal sequence-F

and Fc-noXma-GS-R or Fc-noXma-GS-F and rcFc-R using Expand High Fidelity

System (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's

standard protocol in a MJ Thermocycler. The first reaction was carried out with 500

ng of leukocyte cDNA library (BD Biosciences Clontech, Palo Alto, CA) as a 0 template using the following cycles: 940C 2 minutes; 30 cycles of (94 C 30 seconds,

550C 30 seconds, 720C 45 seconds), 720C 10 minutes. The second reaction was carried out with 500 ng of pSYN-Fc-001 as a template (above) using the following cycles: 940C 2 minutes; 16 cycles of (940C 30 seconds, 580C 30 seconds, 72°C 45 seconds), 720C 10 minutes. The expected sized bands (-495 and 299 bp, respectively) were gel purified with a Gel Extraction kit (Qiagen, Valencia CA), then combined in a PCR reaction with 25 pmol of rc-IgK signal sequence-F and rcFc-R primers and run as before, annealing at 580 C and continuing for 16 cycles. The expected sized band (-772 bp) was gel purified with a Gel Extraction kit (Qiagen,

Valencia CA) and cloned into pGEM T-Easy (Promega, Madison, WI) to produce an

intermediate plasmid pSYN-Fc-007 (pGEM T-Easy/lgK sig seq-Fc). The entire IgK

signal sequence-Fc cassette was then subcloned using the HindIll and EcoRI sites

into either the pEE6.4 (Lonza, Slough, UK) or pcDNA3.1 (Invitrogen, Carlsbad, CA)

mammalian expression vector, depending on the system to be used, to generate

pSYN-Fc-009 (pEE6.4/IgK Sig seq-Fc) and pSYN-Fc-015 (pcDNA3/lgK sig seq-Fc).

Example 32: Cloning of lqK signal sequence - Fc N297A construct for making untagged Fc N297A alone.

[0293] In orderto mutate Asn 297 (EU numbering) of the Fcto an Ala

residue, the following primers were used:

N297A-F 5'- GAGCAGTACGCTAGCACGTACCG -3'(SEQ ID NO: 90)

N297A-R 5'- GGTACGTGCTAGCGTACTGCTCC -3'(SEQ ID NO: 91)

[0294] Two PCR reactions were carried out with 25 pmol of either rc-IgK

signal sequence-F and N297A-R or N297A-F and rcFc-R using Expand High Fidelity

System (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's

standard protocol in a MJ Thermocycler. Both reactions were carried out using 500 ng of pSYN-Fc-007 as a template using the following cycles: 94 C 2 minutes; 16 cycles of (94 0C 30 seconds, 480C 30 seconds, 720C 45 seconds), 72°C 10 minutes.

The expected sized bands (-319 and 475 bp, respectively) were gel purified with a

Gel Extraction kit (Qiagen, Valencia CA), then combined in a PCR reaction with 25

pmol of rc-IgK signal sequence-F and rcFc-R primers and run as before, annealing at

58°C and continuing for 16 cycles. The expected sized band (-772 bp) was gel

purified with a Gel Extraction kit (Qiagen, Valencia CA) and cloned into pGEM T

Easy (Promega, Madison, WI) to produce an intermediate plasmid pSYN-Fc-008

(pGEM T-Easy/lgK sig seq-Fc N297A). The entire IgK signal sequence-Fc alone

cassette was then subcloned using the Hindlli and EcoRI sites into either the

pEE6.4 (Lonza, Slough, UK) or pcDNA3.1 (Invitrogen, Carlsbad, CA) mammalian

expression vector, depending on the system to be used, to generate pSYN-Fc-010

(pEE6.4/lgK sig seq-Fc N297A) and pSYN-Fc-016 (pcDNA3/lgK sig seq-Fc N297A).

[0295] These same N297A primers were also used with rcFc-F and rcFc-R

primers and pSYN-Fc-001 as a template in a PCR reaction followed by subcloning

as indicated above to generate pSYN-Fc-002 (pGEM T Easy/Fc N297A).

Example 33:Cloning of EpoFc and Fc into single plasmid for double gene vectors for making EpoFc wildtype or N297A monomer-dimer hybrids, and expression.

[0296] An alternative to transfecting the EpoFc and Fc constructs on

separate plasmids is to clone them into a single plasmid, also called a double gene

vector, such as used in the Lonza Biologics (Slough, UK) system. The Rsrl/EcoR

fragment from pSYN-Fc-002 was subcloned into the corresponding sites in pEE12.4

(Lonza Biologics, Slough, UK) according to standard procedures to generate pSYN

Fc-006 (pEE12.4/Fc N297A fragment). The pSYN-EPO-004 plasmid was used as a template for a PCR reaction using Epo-F primer from Example 25 and the following primer:

EpoRsr-R: 5'- CTGACGGTCCGCCCAGGAGTTCCG

GAGCTGGGCACGGTGGGCATG TGTGAGTTTTGTCGACCGCAGCGG -3'(SEQ

ID NO: 91)

[0297] A PCR reaction was carried out using Expand High Fidelity System

(Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's standard

protocol in a MJ Thermocycler as indicated above, for 16 cycles with 55°C annealing

temperature. The expected sized band (-689 bp) was gel purified with a Gel

Extraction kit (Qiagen, Valencia CA) and cloned into pSYN-Fc-006 using the

HindIll/Rsril restriction sites, to generate pSYN-EPO-005 (pEE12.4/EpoFc N297A).

The double gene vector for the EpoFc N297A monomer-dimer hybrid was then

constructed by cloning the Not/BamHl fragment from pSYN-Fc-010 into the

corresponding sites in pSYN-EPO-005 to generate pSYN-EPO-008 (pEE12.4

6.4/EpoFc N297A/Fc N297A).

[0298] The wild type construct was also made by subconing the wild type Fc

sequence from pSYN-Fc-001 into pSYN-EPO-005 using the RsrlI and EcoRI sites,

to generate pSYN-EPO-006 (pEE12.4/EpoFc). The double gene vector for the

EpoFc monomer-dimer hybrid was then constructed by cloning the Not/BamHl

fragment from pSYN-Fc-009 into the corresponding sites in pSYN-EPO-006 to

generate pSYN-EPO-007 (pEE1,2.4-6.4/EpoFc /Fc).

[0299] Each plasmid was transfected into CHOKISV cells and positive

clones identified and adpated to serum-free suspension, as indicated in the Lonza

Biologics Manual for Standard Operating procedures (Lonza Biologics, Slough, UK),

and purified as indicated for the other monomer-dimer constructs.

Example 34: Cloning of human IFNDFc, IFND-Fc N297A with eight amino acid linkers and lqK-Fc-6His constructs

[03001 10 ng of a human genomic DNA library from Clontech (BD

Biosciences Clontech, Palo Alto, CA) was used as a template to isolate human IFNp

with its native signal sequence using the following primers:

IFNp-F H3/SbfI: 5'- CTAGCCTGCAGGAAGCTTGCCGCCACCATGACCA ACAAGTGTCTCCTC -3'(SEQ ID NO: 92)

IFNp-R (EFAG) Sal: 5'TTTGTCGACCGCAGCGGCGCCGGCGAACTCGTTTCGG AGGTAACCTGTAAG -3'(SEQ ID NO: 93)

[0301] The reverse primer was also used to create an eight amino acid linker

sequence (EFAGAAAV) (SEQ ID NO: 94) on the 3'end of the human IFNs

sequence. The PCR reaction was carried out using the Expand High Fidelity

System (Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's

standard protocol in a Rapid Cycler thermocycler (Idaho Technology, Salt Lake City,

UT). A PCR product of the correct size (-607 bp) was gel purified using a Gel

Extraction kit (Qiagen; Valencia, CA), cloned into TA cloning vector (Promega,

Madison, WI) and sequenced. This construct was named pSYN-IFNp-002. pSYN

IFNp-002 was digested with Sbfl and SaIl and cloned into pSP72 (Promega) at Pst

and Sall sites to give pSYN-IFNp-005.

[0302] Purified pSYN-Fc-001 (0.6 pg) was digested with Sall and EcoR

and cloned into the corresponding sites of pSYN-FNp-005 to create the plasmid

pSYN-IFNp-006 which contains human IFNs linked to human Fc through an eight amino acid linker sequence. pSYN-IFNp-006 was then digested with Sbfl and EcoR and the full-length IFNp-Fc sequence cloned into the Pstl and EcoRi sites of pEDdC.sig to create plasmid pSYN-IFNP-008.

[0303] pSYN-Fc-002 containing the human Fc DNA with a single amino acid

change from asparagine to alanine at position 297 (N297A; EU numbering) was

digested with BspEl and Xmal to isolate a DNA fragment of -365 bp containing the

N297A mutation. This DNA fragment was cloned into the corresponding sites in

pSYN-lFNp-008 to create plasmid pSYN-IFNp-009 that contains the IFNp-Fc

sequence with an eight amino acid linker and an N297A mutation in Fc in the

expression vector, pED.dC.

[0304] Cloning of IgK signal sequence-Fc N297A - 6His. The following

primers were used to add a 6xHis tag to the C terminus of the Fc N297A coding

sequence:

Fc GS-F: 5'- GGCAAGCTTGCCGCCACCATGGAGACAGACACACTCC -3'(SEQ ID

NO: 95)

Fc.6His-R: 5'- TCAGTGGTGATGGTGATGATGTTTACCCGGAGACAGGGAG -3'

(SEQ ID NO: 96)

Fc.6His-F: 5'- GGTAAACATCATCACCATCACCACTGAGAATTCC

AATATCACTAGTGAATTCG -3'(SEQ ID NO: 97)

Sp6+T-R: 5'- GCTATTTAGGTGACACTATAGAATACTCAAGC -3'(SEQ ID NO: 98)

[0305] Two PCR reactions were carried out with 50 pmol of either Fc GS-F

and Fc.6His-R or Fc.6His-F and Sp6+T-R using the Expand High Fidelity System

(Boehringer Mannheim, Indianapolis, IN) according to the manufacturer's standard

protocol in a MJ Thermocycler. Both reactions were carried out using 500 ng of pSYN-Fc-008 as a template in a 50 pl reaction, using standard cycling conditions.

The expected sized bands (-780 and 138 bp, respectively) were gel purified with a

Gel Extraction kit (Qiagen, Valencia CA), then combined in a 50 pl PCR reaction

with 50 pmol of Fc GS-F and Sp6+T-R primers and run as before, using standard

cycling conditions. The expected sized band (-891 bp) was gel purified with a Gel

Extraction kit (Qiagen, Valencia CA) and cloned into pcDNA6 V5-His B using the

Hindill and EcoRI sites to generate pSYN-Fc-014 (pcDNA6/gK Sig seq-Fc N297A-6

His).

Example 35: Expression and purification of IFNpFc, IFN-Fc N297A homodimer and IFNp-Fc N297A monomer-dimer hybrid

[0306] CHO DG44 cells were plated in 100 mm tissue culture dishes and

grown to a confluency of 50%-60%. A total of 10 pg of DNA was used to transfect a

single 100mm dish. For the homodimer transfection, 10 pg of the pSYN-FNp-008 or

pSYN-IFNp-009 construct was used; for the monomer-dimer hybrid transfection, 8

pg of the pSYN-lFNp-009 + 2 pg of pSYN-Fc-014 construct was used. The cells

were transfected using Superfect transfection reagents (Qiagen, Valencia, CA)

according to the manufacturer's instructions. 48 to 72 hours post-transfection,

growth medium was removed and cells were released from the plates with 0.25%

trypsin and transferred to T75 tissue culture flasks in selection medium (MEM Alpha

without nucleosides plus 5% dialyzed fetal bovine serum). The selection medium for

the monomer-dimer hybrid transfection was supplemented with 5 pg/ml Blasticidin

(Invitrogen, Carlsbad, CA). Selection was continued for 10-14 days until the cells

began to grow well and stable cell lines were established. Protein expression was

subsequently amplified by the addition methotrexate: ranging from 10 to 50 nM.

[0307] For all cell lines, approximately 2 x 107 cells were used to inoculate

300 ml of growth medium in a 1700 cm 2 roller bottle (Corning, Corning, NY). The

roller bottles were incubated in a 5% CO 2 incubator at 37°C for approximately 72

hours. The growth medium was then exchanged with 300 ml serum-free production

medium (DMEM/F12 with 5 pg/ml human insulin). The production medium

(conditioned medium) was collected every day for 10 days and stored at 4°C. Fresh

production medium was added to the roller bottles after each collection and the

bottles were returned to the incubator. Prior to chromatography, the medium was

clarified using a SuporCap-100 (0.8/0.2 pm) filter from Pall Gelman Sciences (Ann

Arbor, MI). All of the following steps were performed at 4C. The clarified medium

was applied to Protein A Sepharose, washed with 5 column volumes of 1X PBS (10

mM phosphate, pH 7.4, 2.7 mM KCl, and 137 mM NaC), eluted with 0.1 M glycine,

pH 2.7, and then neutralized with 1/10 volume of I M Tris-HCI pH 8.0, 5 M NaCL.

The homodimer proteins were further purified over a Superdex 200 Prep Grade

sizing column run and eluted in 50 mM sodium phosphate pH 7.5, 500 mM NaCI,

% glycerol.

[0308] The monomer-dimer hybrid protein was subject to further purification

since it contained a mixture of IFNpFc N297A:IFNpFc N297A homodimer, IFNpFc

N297A: Fc N297A His monomer-dimer hybrid, and Fc N297A His: Fc N297A His

homodimer. Material was applied to a Nickel chelating column in 50 mM sodium

phosphate pH 7.5, 500 mM NaCl. After loading, the column was washed with 50

mM imidazole in 50 mM sodium phosphate pH 7.5, 500 mM NaCI and protein was

eluted with a gradient of 50 - 500 mM imidazole in 50 mM sodium phosphate pH

7.5, 500 mM NaCL Fractions corresponding to elution peaks on a UV detector were collected and analyzed by SDS-PAGE. Fractions from the first peak contained

IFNpFc N297A: Fc N297A His monomer-dimer hybrid, while the second peak

contained Fc N297A His: Fc N297A His homodimer. All fractions containing the

monomer-dimer hybrid, but no Fc homodimer, were pooled and applied directly to a

Superdex 200 Prep Grade sizing column, run and eluted in 50 mM sodium

phosphate pH 7.5, 500 mM NaCl, 10% glycerol. Fractions containing IFNp-Fc

N297A:Fc N297A His monomer-dimer hybrids were pooled and stored at -801C.

Example 36: Antiviral assay for IFNs activity

[0309] Antiviral activity (IU/mI) of IFNs fusion proteins was determined using

a CPE (cytopathic effect) assay. A549 cells were plated in a 96 well tissue culture

plate in growth media (RPMI 1640 supplemented with 10% fetal bovine serum (FBS)

and 2 mM L-glutamine) for 2 hours at 370C, 5% C0 2 . IFNs standards and IFNs

fusion proteins were diluted in growth media and added to cells in triplicate for 20

hours at 370C, 5% C02. Following incubation, all media was removed from wells,

encephalomyocarditis virus (EMCV) was diluted in growth media and added (3000

pfu/well) to each well with the exception of control wells. Plates were incubated at

37 0C, 5% C02for 28 hours. Living cells were fixed with 10% cold trichloroacetic acid

(TCA) and then stained with Sulforhodamine B (SRB) according to published

protocols (Rubinstein et al. 1990, J. Natl. Cancer Inst. 82, 1113). The SRB dye was

solubilized with 10 mM Tris pH 10.5 and read on a spectrophotometer at 490 nm.

Samples were analyzed by comparing activities to a known standard curve ranging

from 10 to 0.199 IU/ml. The results are presented below in Table 8 and demonstrate

increased antiviral activity of monomer-dimer hybrids.

TABLE 8: INTERFERON BETA ANTIVIRAL ASSAY HOMODIMER V. MONOMER-DIMER HYBRID

Protein Antiviral Std dev Activity (IU/nmol) IFNp-Fc 8aa linker homodimer 4.5 x 100 0.72 x 105 IFNpFc N297A 8aa linker homodimer 3.21 x 10 0.48 x 10 IFNpFc N297A 8aa linker: Fc His 12.2 x 105 2 x 10 monomer-dimer hybrid

Example 37: Administration of IFNpFc Homodimer and Monomer-Dimer Hybrid With an Eight Amino Acid Linker to Cynomolqus Monkeys

[0310] For pulmonary administration, aerosols of either IFNPFc homodimer

or IFNpFc N297A monomer-dimer hybrid proteins (both with the 8 amino acid linker)

in PBS, pH 7.4, 0.25% HSA were created with the Aeroneb ProT (AeroGen,

Mountain View, CA) nebulizer, in-line with a Bird Mark 7A respirator, and

administered to anesthetized naive cynomolgus monkeys through endotracheal

tubes (approximating normal tidal breathing). Blood samples were taken at various

time points, and the amount of IFNp-containing protein in the resulting serum was

quantitated using a human IFNs Immunoassay (Biosource International, Camarillo,

CA). Pharmacokinetic parameters were calculated using the software WinNonLin.

Table 9 presents the results of cynomogus monkeys treated with IFNpFc N297A

monomer-dimer hybrid or IFNpFc homodimer.

TABLE 9: ADMINISTRATION OF IFNpFC N297A MONOMER-DIMER HYBRID AND IFNpFC HOMODIMER TO MONKEYS

Approx. AUC Cmax (hr*ng/ml) t 1/ 2 t112avg Protein Monkey Route Deposited Dose' (hr) (hr) P (ng/ml) (pg/kg) IFNpFc C07308 pulm 20 23.3 987.9 27.6 N297A C07336 pulm 20 22.4 970.6 25.6 27.1 monomer- pulm 20 1002.7 dimer hybrid C07312 1 21.2 28.0 IFNpFc C07326 pulm 20 2.6 94.6 11.1 4 homodimer C07338 pulm 20 5.0 150.6 11.7 1 Based on 15% deposition fraction of nebulized dose as determined by gamma scintigraphy

[0311] The pharmacokinetics of IFNpFc with an 8 amino acid linker

administered to cynomolgus monkeys is presented in figure 15. The figure

compares the IFNpFc homodimer with the IFNpFc N297A monomer-dimer hybrid in

monkeys after administration of a single pulmonary dose. Significantly higher serum

levels were obtained in monkeys treated with the monomer-dimer hybrid compared

to the homodimer.

[03121 Serum samples were also analyzed for neopterin levels (a biomarker

of IFNs activity) using a neopterin immunoassay (MP Biomedicals, Orangeburg,

NY). The results for this analysis are shown in figure 16. The figure compares

neopterin stimulation in response to the IFNp-Fc homodimer and the IFNP-Fc N297A

monomer-dimer hybrid. It can be seen that significantly higher neopterin levels were

detected in monkeys treated with IFNP-Fc N297A monomer-dimer hybrid as

compared to the IFNP-Fc homodimer.

[0313] All numbers expressing quantities of ingredients, reaction conditions,

and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." Accordingly, unless indicated to the contrary, the numerical parameters set forth in the specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0314] All references cited herein are incorporated herein by reference in

their entirety and for all purposes to the same extent as if each individual publication or

patent or patent application was specifically and individually indicated to be

incorporated by reference in its entirety for all purposes. To the extent publications and

patents or patent applications incorporated by reference contradict the disclosure

contained in the specification, the specification is intended to supercede and/or take

precedence over any such contradictory material.

[0315] Many modifications and variations of this invention can be made

without departing from its spirit and scope, as will be apparent to those skilled in the

art. The specific embodiments described herein are offered by way of example only

and are not meant to be limiting in any way. It is intended that the specification and

examples be considered as exemplary only, with a true scope and spirit of the

invention being indicated by the following claims.

[0316] The term 'comprise' and variants of the term such as 'comprises' or

comprising' are used herein to denote the inclusion of a stated integer or stated

integers but not to exclude any other integer or any other integers, unless in the

context or usage an exclusive interpretation of the term is required.

[0317] Any reference to publications cited in this specification is not an admission

that the disclosures constitute common general knowledge in Australia.

[0318] In a first embodiment there is provided a chimeric protein comprising a first

polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain

comprises (i) a clotting factor, which comprises Factor IX, which is fused to (ii) at least a

portion of an immunoglobulin constant region which is a neonatal Fc receptor (FcRn) binding

partner, and

the second polypeptide chain comprises at least a portion of an immunoglobulin

constant region, which is an FcRn binding partner, without the clotting factor of the first

polypeptide chain and without an immunoglobulin variable domain, and

wherein the first polypeptide chain and the second polypeptide chain are associated.

[0319] In a second embodiment there is provided a chimeric protein having the

formula:

X-L-F:F or F:F-L-X

wherein F is a portion of an immunoglobulin constant region comprising an FcRn

binding partner, L is a linker or a direct bond, X is Factor IX, and (:) is a covalent bond or a

non-covalent bond.

[0320] In a third embodiment there is provided a pharmaceutical composition

comprising the chimeric protein of the first or second embodiments and a pharmaceutically

acceptable carrier.

[0321] In a fourth embodiment there is provided the use of the chimeric protein of

the first or second embodiments or the pharmaceutical composition of the third embodiment for

the manufacture of a medicament for treating hemophilia B.

[0322] In a fifth embodiment there is provided a method of treating hemophilia B in

a subject in need thereof comprising administering to the subject a therapeutically effective

amount of the chimeric protein of the first or second embodiments or the pharmaceutical

composition of the third embodiment.

Claims (1)

1. A chimeric protein comprising a first polypeptide chain and a second polypeptide chain, wherein the first polypeptide chain comprises (i) a clotting factor, which comprises Factor IX, which is fused to (ii) at least a portion of an immunoglobulin constant region which is a neonatal Fc receptor (FcRn) binding partner, and the second polypeptide chain comprises at least a portion of an immunoglobulin constant region, which is an FcRn binding partner, without the clotting factor of the first polypeptide chain and without an immunoglobulin variable domain, and wherein the first polypeptide chain and the second polypeptide chain are associated.

2. The chimeric protein of claim 1, wherein the clotting factor is fused to the portion of the immunoglobulin constant region by a linker.

3. The chimeric protein of claim 2, wherein the linker comprises about 1 to about 20 amino acids.

4. The chimeric protein of any one of claims 1-3, wherein the portion of the immunoglobulin constant region of the first polypeptide chain is an Fc fragment.

5. The chimeric protein of any one of claims 1-4, wherein the portion of the immunoglobulin constant region of the second polypeptide chain is an Fc fragment.

6. The chimeric protein of any one of claims 1-5, wherein the portion of the immunoglobulin constant region of the first polypeptide chain and the portion of the immunoglobulin constant region of the second polypeptide chain are identical.

7. The chimeric protein of any one of claims 1-6, wherein the first polypeptide chain and the second polypeptide chain are associated covalently or non-covalently.

8. The chimeric protein of any one of claims 1-6, wherein the first polypeptide chain and the second polypeptide chain are associated via a disulfide bond.

9. The chimeric protein of any one of claims 1-8, wherein the Factor IX is fused to the N terminus of the FcRn binding partner of the first polypeptide chain.

10. The chimeric protein of any one of claims 1-9, wherein the second polypeptide chain consists of the portion of the immunoglobulin constant region, which is an FcRn binding partner.

11. The chimeric protein of claim 10, wherein the FcRn binding partner of the first polypeptide chain and the FcRn binding partner of the second polypeptide chain are identical and are associated via a disulfide bond.

12. A chimeric protein having the formula: X-L-F:F or F:F-L-X wherein F is a portion of an immunoglobulin constant region comprising an FcRn binding partner, L is a linker or a direct bond, X is Factor IX, and (:) is a covalent bond or a non-covalent bond.

13. The chimeric protein of claim 12, wherein the F is an Fc fragment.

14. The chimeric protein of claim 12 or 13, wherein the F in X-L-F and the F in F are identical.

15. The chimeric protein of any one of claims 12-14, wherein the (:) is a disulfide bond.

16. A pharmaceutical composition comprising the chimeric protein of any one of claims 1 to and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16, which comprises an effective amount of the chimeric protein for treatment of a disease or disorder.

18. The pharmaceutical composition of claim 16 or 17, wherein the pharmaceutical composition comprising the chimeric protein is suitable for administration intravenously, subcutaneously, orally, buccally, sublingually, nasally, parenterally, rectally, vaginally or via a pulmonary route.

19. The pharmaceutical composition of claim 17 or 18, wherein said disease or disorder is a hemostatic disorder.

20. The pharmaceutical composition of claim 19, wherein said disease or disorder is hemophilia B.

21. The pharmaceutical composition of any one of claims 17 to 20, wherein the effective amount is 0.1-1,000 pg/kg.

22. Use of the chimeric protein of any one of claims 1 to 15 or the pharmaceutical composition of any one of claims 16 to 21 for the manufacture of a medicament for treating hemophilia B.

23. A method of treating a hemophilia B in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the chimeric protein of any one of claims 1 to 15 or the pharmaceutical composition of any one of claims 16 to 21.

24. The method of claim 23, wherein the chimeric protein or the pharmaceutical composition treats an acute bleeding episode.

25. The method of claim 23 or 24, wherein the chimeric protein or the pharmaceutical composition is administered intravenously, subcutaneously, intramuscularly, orally, sublingually, buccally, nasally, rectally, vaginally or via a pulmonary route.

26. The method of any one of claims 23 to 25, wherein the treatment is prophylactic.

27. The method of any one of claims 23 to 25, wherein the chimeric protein or composition is administered prior to, during, or after surgery.

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1/27

Fig. 2A

Factor VII -Fc amino acid sequence (signal peptide underlined, propeptide in bold) 1 MVSQALRLLC LLLGLQGCLA AVFVTQEEAH GVLHRRRRAN AFLEELRPGS 51 LERECKEEQC SFEEAREIFK DAERTKLFWI SYSDGDQCAS SPCQNGGSCK 101 DQLQSYICFC LPAFEGRNCE THKDDQLICV NENGGCEQYC SDHTGTKRSC 151 RCHEGYSLLA DGVSCTPTVE YPCGKIPILE KRNASKPQGR IVGGKVCPKG 201 ECPWQVLLLV NGAQLCGGTL INTIWVVSAA HCFDKIKNWR NLIAVLGEHD

251 LSEHDGDEQS RRVAQVIIPS TyVPGTTNHD IALLRLHQPV VLTDHVVPLC 2019200306

301 LPERTFSERT LAFVRFSLVS GWGQLLDRGA TALELMVLNV PRLMTQDCLQ 351 QSRKVGDSPN ITEYMFCAGY SDGSKDSCKG DSGGPHATHY RGTWYLTGIV 401 SWGQGCATVG HFGVYTRVSQ YIEWLQKLMR SEPRPGVLLR APFPDKTHTC 451 PPCPAPELLG GPSVFLFPPK PKDTLMISRT PEVTCVVVDV SHEDPEVKFN 501 WYVDGVEVHN AKTKPREEQY NS':!'YRVVSVL 'l'\)LHQDW1JNG KExKCKItSNK

551 ALPAPIEKTI SKAKGQPREP QVYTLPPSRD ELTKNQVSLT CLVKGFYPSD 601 IAVEWESNGQ ,PENNYKTTPP VLDSDGSFFL YSKLTVDKSR WQQGNVFSCS 651 VMHEALHNHY TQKSLSLSPG K

Fig. 2B

Factor ix -Fc amino acid sequence (signal peptide underl ined, propeptide in bold) 1 MQRVNMlMAE SPGLITICLL GYLLSAECTV FLDHENANKI LNRPKRYNSG 51 KLEEFVQGNL ERECMEEKCS FEEAREVFEN TERTTEFWKQ YVDGDQCESN 101 PCLNGGSCKD DINSYECWCP FGFEGKNCEL DVTCNIKNGR CEQFCKNSAD

151 NKVVCSCTEG YRLAENQKSC EPAVPFPCGR VSVSQTSKLT RAETVFPDVD

201 yVNSTEAETI LDNITQSTQS FNDFTRVVGG EDAKPGQFPW QVVLNGKVDA 251 FCGGSIVNEK WIVTAAHCVE TGVKITVVAG EHNIEETEHT EQKRNVIRII 301 PHHNYNAAIN KYNHDIALLE LDEPLVLNSY VTPICIADKE YTNIFLKFGS 351 GYVSGWGRVF HKGRSALVLQ YLRVPLVDRA TCLRSTKFTI YNNMFCAGFH 401 EGGRDSCQGD SGGPHVTEVE GTSFLTGIIS WGEECAMKGK YGIYTKVSRY 451 VNWIKEKTKL TEFAGAAAVD KTHTCPPCPA PELLGGPSVF LFPPKPKDTL 501 MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR 551 VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL 601 PPSRDELTKN QVSLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD 651 GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL SLSPGK

2/27

Fig. 2C

IFNa-Fc amino acid sequence (8 amino acid linker) (signal sequence underlined) 1 MALTFALLVA LLVLSCKSSC SVGCDLPQTH SLGSRRTLML LAQMRRlSLF 51 SCLKDRHDFG FPQEEFGNQF QKAETIPVLH EMIQQIFNLF STKDSSAAWD 101 ETLLDKFYTE LYQQLNDLEA CVIQGVGVTE TPLMKEDSIL AVRKYFQRIT 151 LYLKEKKYSP CAWEVVRAEI MRSFSLSTNL QESLRSKEEF AGAAAVDKTH 201 TCPPCPAPEL LGGPSVFLFP PKPKDTLMIS RTPEVTCVVV DVSHEDPEVK 251 FNWYVDGVEV HNAKTKPREE QYNSTYRVVS VLTVLHQDWL NGKEYKCKVS 2019200306

301 NKALPAPIEK TISKAKGQPR EPQVYTLPPS RDELTKNQVS LTCLVKGFYP 351 SDIAVEWESN GQPENNYKTT PPVLDSDGSF FLYSKLTVDK SRWQQGNVFS 401 CSVMHEALHN HYTQKSLSLS PGK

Pig. 2D

IFNa-Fc ~ linker amino acid sequence (signal sequence underlined) 1 MALTFALLVA LLVLSCKSSC SVGCDLPQTH SLGSRRTLML LAQMRRI SLF 52 SCLKDRHDFG FPQEEFGNQF QKAETIPVLH EMIQQIFNLF STKDSSAAWD ~Ol ETLLDKFYTE LYQQLNDL~. CVIQGVGVTE TPLMKEDSIL AVRKYFQRIT 151 LYLKEKKYSP CAWEVVRAEI MRSFSLSTNL QESLRSKEDK THTCPPCPAP 201 ELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPE VKFNWYVDGV 251 EVHNAKTKPR EEQYNSTYRV VSVLTVLHQD WLNGKEYKCK VSNKALPAPI 301 EKTISKAKGQ PREPQVYTLP PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE 351 SNGQPENNYK TTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL 401 HNHYTQKSLS LSPGK

Fig. 2E

FlagFc amino acid sequence (signal sequence underlined)

LLLWVPGSTG KTHTCPPCPA DDYKDDDDKD PELLGGPSVF 1 METDTLLLWV

MISRTPEVTC VVVDVSHEDP EVKFNWYVDG VEVHNAKTKP 51 LFPPKPKDTL VVSVLTVLHQ DWLNGKEYKC KVSNKALPAP IEKTISKAKG 101 REEQYNSTYR QVSLTCLVKG FYPSDIAVEW ESNGQPENNY 151 QPREPQVYTL PPSRDELTKN

GSFFLYSKLT VDKSRWQQGN VFSCSVMHEA LHNHYTQKSL 201 KTTPPVLDSD 251 SLSPGK

3/27

Fig. 2F

Epo-CCA-Fc amino acid sequence (Kb signal sequence underlined, acidic coiled coil in bold)

1 MVPCTLLLLL AAALAPTQTR AGSRAPPRLI CDSRVLQRYL LEAKEAENIT 51 TGCAEHCSLN ENITVPDTKV NFYAWKRMEV GQQAVEVWQG LALLSEAVLR 2019200306

101 GQALLVNSSQ PWEPLQLHVD KAVSGLRSLT TLLRALGAQK EAISPPDAAS 151 AAPLRTITAD TFRKLFRVYS NFLRGKLKLY TGEACRTGDR EFGGEYQALE 201 KEVAQLEAEN QALEKEVAQL EHE~GGPAPE LLGGPSVFLF PPKPKDTLMI 251 SRTPEVTCVV VDVSHEDPEV KFNWYVDGVE VHNAKTKPRE EQYNSTYRVV

301 SVLTVLHQDW LNGKEYKCKV SNKALPAPIE KTISKAKGQP REPQVYTLPP 351 SRDELTKNQV SLTCLVKGFY PSDIAVEWES NGQPENNYKT TPPVLDSDGS 401 FFLYSKLTVD KSRWQQGNVF SCSVMHEALH NHYTQKSLSL SPGK

Fig. 2G

CCB-Fc amino acid sequence (Kb signal sequence underlined; basic coiled coil in bold)

1 MVPCTLLLLL AAALAPTQTR AGEFGGEYQA LKKKVAQLKA KNQALKKKVA 51 QLKHKGGGPA PELLGGPSVF LFPPKPKDTL MISRTPEVTC VVVDVSHEDP 101 EVKFNWYVDG VEVHNAKTKP REEQYNSTYR VVSVLTVLHQ DWLNGKEYKC 151 KVSNKALPAP IEKTIS~.KG QPREPQVYTL PPSRDELTKN QVSLTCLVKG 201 FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLYSKLT VDKSRWQQGN 251 VFSCSVMHEA LHNHYTQKSL SLSPGK

Fig. 2H

CysFC amino acid sequence (hIFNU signal sequence underlined) ELLGGPSVFL FPPKPKDTLM 1 MALTFALLVA LLVLSCKSSC SVGCPPCPAP EVHNAKTKPR EEQYNSTYRV 51 I SRTPEVTCV VVDVSHEDPE VKFNWYVDGV PREPQVYTLP 101 VSVLTVLHQD WLNGKEYKCK VSNKALPAPI EKTISKAKGQ TTPPVLDSDG SNGQPENNYK 151 PSRDELTKNQ VSLTCLVKGF YPSDIAVEWE HNHYTQKSLS LSPGK 201 SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL

4/27

Fig. 21

1FNa GS15 Fe protein sequence (signal sequence underlined) :

1 MALTFALLVA LLVLSCKSSC SVGCDLPQTH SLGSRRTLML LAQMRRISLF 51 SCLKDRHDFG FPQEEFGNQF QKAET1PVLH EM1QQIFNLF STKDSSAAWD

101 ETLLDKFYTE LYQQLNDLEA CVIQGVGVTE TPLMKEDSIL AVRKYFQRIT 151 LYLKEKKYSP CAWEVVRAEI MRSFSLSTNL QESLRSKEGG GGSGGGGSGG 201 GGSDKTHTCP PCPAPELLGG PSVFLFPPKP KDTLMISRTP EVTCVVVDVS 2019200306

251 HEDPEVKFNW YVDGVEVHNA KTKPREEQYN STYRVVSVLT VLHQDWLNGK 301 EYKCKVSNKA LPAPIEKTIS KAKGQPREPQ VYTLPPSRDE LTKNQVSLTC 351 LVKGFYPSDI AVEWESNGQP ENNYKTTPPV LDSDGSFFLY SKLTVDKSRW 401 QQGNVFSCSV MHEALHNHYT QKSLSLSPGK

Fig. 2J'

EpoFc amino ~çid sequence (signal sequence underlined, linker in bold) 1 MGVHECPAWL WLLLSLLSLP LGLPVLGAPP RLl CDSRVLE RYLLEAKEAE 51 NITTGCAEHC SLNENITVPD TKVNFYAWKR MEVGQQAVEV WQGLALLSEA 101 VLRGQALLVN SSQPWEPLQL HVDKAVSGLR SLTTLLRALG AQKEA1SPPD 151 AASAAPLRTI TADTFRKLFR VYSNFLRGKL KLYTGEACRT GDREFAGAAA

201 VDKTHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV TCVVVDVSHE 251 DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL HQDWLNGKEY 301 KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT KNQVSLTCLV 351 KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK LTVDKSRWQQ 401 GNVFSCSVMH EALHNHYTQK SLSLSPGK

5/27

Fig. 3A

Factor VII-Fc nucleotide sequence (signal peptide underlined, propeptide in bold)

at qgt c t cccaggc c ct caggct cctct gcct t c tgct tgggct t cagggctgcctggct 9 c ag tc t tegta acccagg agga agcccacggcgtcc tgcaccggcgccggcgcgeeaaegegt tect gg agga 9 ct geg9 e cggget ccct ggag aggga gt gcaaggagga gcagt get eet t eg a ggag geecggg ag a t et t c aagga cgcggagagga cgaagetgt tct gga t t t et t acagt ga tgggg aecagt gtgect eaagtecatgceagaatggggget eetgcaaggaeeagctccagt ccta tat 2019200306

ctgct t ct gcetccctgeet t cgagggceggaactgtgagacgcacaagga tgaceagctga tc t gt gt 9 a a eg ag a a cggcgget gt gageagt a et geagtga cc a ea egggca ccaagcgct c ct gt cggt 9 ceaegaggggt actetctgct ggeagaeggggtgt cetgcacacecacagt tg aa t a t cea t gt ggaaaaa t accta t t ct agaaaaa agaaa tgeeagcaaaceceaaggccgaa t tgtg ggggg ca a ggtgt gecccaaaggggagtgt ee a tggcaggt cetgt tgt tggtgaa tggaget c agt tgtgtggggggaeeetgat eaacacca tctgggtggtct ecgcggcecaetgt t tcgacaa aa t caaga actggaggaacct gat cgcggt get gggcgagca egaect cagegageacgacggg gat gag c a gagccgg cgggt gg eg ca ggt ca t ca t eccc a gc a cgt acgt eccgggcacea c ea acca cga ca t egeget get ccgeetgeaecagcccgtggt cct ca ct gacca tgtggtgeecet etgectgccegaaeggacgt tctctgagaggaegctggcet t cgtgegct tctcat tggtcage ggct ggggceagctgct ggaccgtggcgeeacggccetggaget cat ggt eeteaaegtgecce ggct 9 a tgacccaggactgcct gcagcagt eacggaaggt gggaga et ceccaaata t caegga gt a ea t gt t ctgtgccggct act cgga t ggeag caaggaet cetg eaagggggacagt 99a99c ce aLa t gc ca ceca et a ce9gggca eg t ggt ace t ga eggg e at egt e age t gggg eeaggget gcgeaa ecgtgggec act t tggggt gtaeaecagggt ct cceagt aca t egagtggctgcaaaa get ca tgcgct cagagceacgcccaggagtcetcctgcgageccca t t tcccgaeaaaactcac a cg tgcccgecgtgcecagct ecggaact gctgggcgga e cgt cagt ct t cct ct t ccccccaa aa c cca aggaca ccct ca tga t ct ccc9gaccect gaggt ca ca t gcgt 99t9gtggacgtgag eca cg aagaecct gaggt caagt t eaaet ggt acgt ggacggcgt ggaggtgcataa tgce aag a c a a age cg cggg agg ag ca gt a c aa cagc a cgt a ecgt gt gg t c a 9 cgt cet ea c egt e et 9 c a cc aggact ggct gaa tggeaaggagtaeaagtgcaaggt ct ceaacaaagcectcccagcccc ea t ega g5aaa cea t ct eeaa agceaaagggcag ecccg5ga a ceacaggtgtacaecct 9 ecc eca t c ecggga t gagct gacca agaaccaggt ea gcct gacct gcct ggt caaagget t et ate ccagcgaea t cgc cgt ggagt gggagagcaa t gggcagccggagaa c a act acaagacc acgcc t eccgtgt tggaetecgacggct cct tet teet et aCâgeaaget caccgtggacaagagcagg tggcagcaggggaacgt ct t ctcatgctccgtgatgcatgaggctctgcacaaccaet acaegc agaagageet ct ecct gt et ccgggt aaa t ga

~7

Fig. 3B

Factor IX-Fc nucleotide sequence (signal peptide underlined, propeptide in bold) at ea egegt aaeat ateatg ca aatcacca eetcatcaceatct cctttta at atctact cagtgctgaatgtacagt t t ttct tgatca tgaaaacgccaacaaaa ttctgaatcg gccaaagaggta taat t caggtaaa t tggaag agt t tgt tcaagggaacet tgagagagaatgt atggaagaaaagtgtagt t t tgaagaagcacgagaagtt t t tgaaaacactgaaagaacaactg aat t t t ggaagcagtatgt tga t ggagat eagtgtgagtecaatccatgt tt aaatggcggcag 2019200306

t tgcaagga tgaeattaa t tecta tgaatgt tggtgtcect t tggat t tgaaggaaagaactgt gaa t taga tgtaa ea t gt aaca t t aagaa tgg eagatgegagcagt t t tgtaaaaatagtgctg a taacaaggtggt t tgct ect gt aetgaggga tat cgact tgeagaaaaccagaagtcctgtga aceageagtgceat t t eea tgtggaagagt t tetgt t teacaaact tet aagctcaeecgtgct gagactgt t t t t cetga t gt gga cta tgt aaa t t ctaet gaagct gaaacea t t t tgga t aaca teaeteaaagcacceaateat t taatgaet teactegggt tgt tggtggagaagatgecaaacc aggt caatt ccct tggcaggt t gtt t tga atgst aaagttgat gcatt ctgtggaggctctat c gt taa tgaaaaa tgga t t gt aact get geceaetgtgt tgaaaet ggt gt taaaa t tacagt tg tegcaggtgaaca taa tat tgaggaga cagaacat aeagagcaa aagcgaaa tgtga t t egaa t tat teet ca ecaeaaet a caa t gcagcta t t aa taagtacaaeca t gaea t tgecet tetggaa ctggacgø5ccct tagtgetaaaeagct a cot t nr.acctat t tqca t tgetgaeaaggaataca egaaca tct teet caaa t t tgga t ctggcta t gtaagtggetggggaagagtcttccacaaagg gagatcagct t tagt tet tcagtaeet tagagt teeaet tgt tgaccgagccacatgtct tcga t etacaaagt t caeca t ctat aacaaca tgt tetgtgetggct t ccatgaaggaggtagagat t c a tgt caagga gatagtggggg acceca tgt tact gaagtggaagggaceagt t tct taactgg aat tat taget ggggtgaagagtgt gcaa tgaaaggeaaatatggaatatataceaaggta tcc eggtatgt caactgga t t aaggaaaaaaeaaagct eaetgaat t cgecggcgeegetgcggtcg aeaaaact cacacatgecca cegtgeceagca ectgaact cetggggggaccgtcagtct t cct et teecceeaaaacceaaggaeaccet ca tga t et cecggaeccetgaggtcacatgcgtggtg gt gga cgtgagccaega agaccct gaggt c a agt t eaact ggt a cgtggaeggcgtggaggtgc at aat 9 cca ag acaaag c egeg ggagg ag c a 9 tac a acagca cg t a e cgt gt ggt c agegt c et cacegt cetgeaccagg act gg ct gaa tgg caaggagt acaagt gcaaggtct eeaaeaaagcc ct eceagcec e cat cga ga aa a eca t et cc aaagc caa agggca gcccegagaaceacaggtgt a caccct gecceca t cccgggB t gagetgacca a9 aa ccaggt cagcctgaectgcctggt caa aggct t ct atcccagcgaeatcgcegt ggagt gggagagcaat gggcagecggagaaeaactae aagaeeaegcct cccgtgt tggactccgacggct cet t ct tect ctacagcaagetcacegtgg acaag agcaggt ggcagcaggggaa cgt et t et cat get ccgt ga t gea t gaggctctgcaeaa ccaetacaegcagaagagcet et ccetgt et ecgggtaaatga

W7

Fig. 3C

IFNU-Fc nucleotide sequence (8 amino acid linker)

at ggcct tgacct t tgct t tactggtggcect cctggtgctcagctgcaagt eaagetgetetg ~t gt gat et 9 eet ea aaeeeaeagc et 9 ggt agcagg aggaeet tga t geteet ggeaea ga tgaggagaa t ct etct t t t etcctget tgaaggacagaca tgact t tgga t t tceeeagg ag gagt t tggcaaccagt t eeaaaaggetgaaacca tecetgte eteea tgaga tga teeageaga tet t eaat ct ct t cagcacaaaggactcatetgetget tgggatgagaceet eetagaeaaa t t 2019200306

ct ac a et gaa ct et acc ag c a get gaa tga eet ggaagcet gt gt gat a eagggggt gggggtg acagagaet cccet ga tgaaggaggact cea t t ctggctgtgaggaaa tact teeaaagaa tea ct ct et at ct gaaagagaagaaa t acagccct t gtgcetgggaggt tgteagageagaaa teat gagatct t t t tett tgteaacaaact tgcaagaaagt ttaagaagtaaggaagaattegeeggc gecgct geggt cgacaaaact eaca cat gceca ecgtgeccagcacct gaaeteetggggggac egt cagtet t cctct t ccecceaaaaeccaaggacaecetcatgateteecggaecectgaggt caca tgcgtggt ggtggacgtgagecacgaagaccct gaggt caagt t eaaetggtacgtggac ggcgt ggaggtgca taa t gccaagacaaagccgcgggaggagcagt a caacagcacgtaccgtg tggt cagegt cct eaccgt cctgcaceaggactggct gaa t ggcaaggagtaeaagtgcaaggt ct ccaa ea a age cet eee a gceceea t ega ga a a ace a t et ee a aag c caaagggcagceecga 9 aa cca eaggtgt a c aeect 9 eeecca t cecg99 a t gag ct gaccaag aaceaggt cag cet ga cetgcctggt caaagget t eta t cecagcgaea t cgccgt 99a9t ggg agagcaatgggcagce ggagaaeaact acaagaccacgcctcccgtgt t ggact cegacggct cet tet tcet ct aeagc aagct cacegt gga eaagag caggtggcagcaggggaaegt et t ct ca tget ccgtga tgea tg agget ctgcacaaceaetaeaegcagaagageet ct ceetgt et ccgggt aaa tga

m7

Fig, 3D

IFNU-Fc ~ linker nucleotide sequence

atggcct tgacct t tgct t tactggtggccct cctggtgctcagctgcaagtcaagetgctetg ~t gt gat ct gcct caaac c caca gect gggt agcaggagg aect tg a t get cctggea ca gatgaggagaatetetct t t tctcctgct tgaaggacagaeatgaet ttggatttceceaggag 2019200306

gagt t t ggeaaecagt tecaaa aggctgaaaccatceetgt eet eea tgagatga tecageaga t et tcaa t ctct t cageacaaa ggactca tet getget tggga tgagaccet eet agaeaaa t t ct a ea ct gaaet ct aecag c ag ct 9 aa t gaeet gg aageet gt gt gat aeagggggt gggggt 9 aeagagact ccect ga t gaagg aggaet eea t t ctggctgt 9 aggaaa tact tceaaagaa t ca etet ctatctgaaagagaagaaa t acagcect tgtgeetgggaggt t gteagageagaaat cat gagatct t t ttet t tgt caacaaact tgcaagaaagt t taagaagtaaggaagacaaaactcac acgtgcccgecgtgcccagct ccggaactgctgggcggaccgt cagt ct teetct t ecccceaa aa eccaa ggacaccet cat gat c t c ccgga eec ct 9 a ggt ca ca t gcgt ggtggt gga cgtgag ccacgaaga cr.r.t gaggt eaagt t eaaet ggtacgtggacggcgt ggaggtgea t aatgeeaag a ca a agccgegggagg ageagt a c a aeagcacgt accgt gt ggt e a 9 cgt eet eaccgt cctgc a e c a ggact ggct gaa t gg caa gga gt a c aagt gc aaggt ct eca a eaaa gccct cccagcc cc ca t cgogoaaaeca tet ceaa 80rr ñ ñ ñgggcagccccgagaaccaeaggtgtaeaceetgcee e ca t eceggg a t gaget 9 a eeaaga a ee a ggt ea gcctgaect gect ggt caaaggct t ct at c c ca 9 eg a ea t cg ccgt ggagt 999 ag ageaa t gggca geegga 9 a a c a a eta ea ag acca cgec t cecgtgt tggactcegaeggeteet t et tect etacageaagct eacegtggacaagagcagg t ggeageaggggaaegt et t et ea tgct cegt ga t gea tgaggct etgca caa ceactaca egc agaagagcctctccetgtct ccgggt aaa tga

Fig. 3E

FlagFc nucleotide sequence

a tqgaqacagacacactcctgct atgggt actgct gctctgggt tccaggt tccactggtgacg a et a caaggacgaega t gacaaggac aaaa et cacaca t gc ceaccgt gcceagct cegga act cetggggggacegt eagt ct t cct et t ccecccaaaacceaaggacaccctca tgatctcecgg a ecc ct gaggt caca t gcgtggt ggt gga cg t gagceaega agaeeet gaggt c aagt t caact ggt a egt gga eggcgtggaggt gca t aa t gc caag a caaagcegegggaggagcagtacaaeag caegt accgtgtggt cagcgt cct ea c egt c ct gc ae caggact ggct gaat ggcaaggagt ac a agtgcaaggt ct ccaacaaag ecct c ceag ecceca t cgagaaaacca t ct eeaaagccaaag ggcageceegagaaeeacaggt gt a ea ecct 9 ececca t cccggga t gagctgaceaagaacca ggt eagectgacet geet ggt caaaggct t ct at cecagcg acatcgccgtggagtgggagagc a at gggcagccggagaacaact aca ag a eea cgect cccgtgt tggact ccgacggct cet t et t cet ctaeageaagct caeegtggacaagagcaggtggcagcaggggaacgtct tetca tgctc egt ga tgca t 9 agget ctgca caa ce a et acacgcagaagagcct ct ccetgt ct ccgggtaaa tga

wn

Fig. 3F

Epo-CCA-Fc nucleotide sequence (Kb signal sequence' underlined, acidic coiled coil in bold) ~

at ggta c cgt gcacgctgct cet gct gt t ggcgg cegcce t gg ct c cg act ca ga eccgcgccg gct ctagageeecaceacgectcat etgt gacagecgagtcct geagaggt acctct tggaggc eaagg aggcegagaa ta t ca ega cggg et gt 9 et 9 aa eaet ge aget t 9 aa tg agaa ta t cact gtcceagacaceaaagt t aa t t t et a t geetggaagagga t ggaggtcgggcagcaggccgtag 2019200306

aagt ct gg cagggectggccctg ct gt cggaagct gt cet 9 eggggccaggccctgt tggt caa et ct t eccagecgtgggagccectgeagct gea t gt gga t aaagecgt eagtggect tcgcagc ct c a ce actetgct t eggget et ggg a geeea 9 a a gg aag e eat et eee et eeag at gcggcet eagctgct ceaeteegaaeaateact getgacact t tcegeiaa et ett ecgagtct act ccaa t t teet eeggggaaagetgaagetgt aeaeaggggaggeet ge aggaeeggtgacagggaat te gg tgg tg ag t a c c agg c e e tgg ag a a gg agg t ggee cage t gg a gg e eg ag aae e agge c c tgg a9 aaggagg tggc ce age t gg ag c acg a ggg tgg tggt cecg e a c c egagct 9 et gggegg a ec gt cagt ct tcct et tccccccaaaa cceaagga caccctca tga t ct cccggaeeeetgaggte aca tgegtggtggtggacgtgagceaegaaga eeet gaggt ea agt t eaaetggtacgt ggacg gcgt gg aggt 9 c at aa t 9 eea a ga c a a ag ccg eggg ag gag ea 9 t a e aa eagca cgt accgtg t ggt eagegteet eaccgt eet gcaeeaggaet ggct gaa tggca aggagtacaagtgcaaggt c t e eaa c aaag ee ct ceeagcc ccca t eg agaaaae cat ct cc a a ag c eaa agggeag ccccg ag aa eeacaggtgt aeaeect gecceea t ccegggat gaget gaccaagaaceaggt cagcctgac ct gcct ggtea a agget t eta tcceagcgaca t cgccgtggagt gggagagcaa tgggcagccg gagaaeaaetaeaagaecacgcet ecegtgt tggaeteegacggct eet tet tcctetacagca aget eacegt ggaeaagagcaggt gge agc agggg aacgt ct t et c a tgctccgt ga tgea t ga ggctctgeaeaa ceact aeacg cag a agagcct et ecctgt ct ecgggt aaa tga

Fig. 3G

CCB-Fe nucleotide sequence (signal sequence underlined, basic coiled coil in bold)

at gtacc tocac et cteet ct ttg eg ec ecetggctcc aetea acee e eeg gegaa t t eggtgg tg agtaecagg c cc t 9 aaga ag a aggtggee cage tg aaggeeaagaacc a gg eee tg aaga aga aggtggcce age t 9 aagcae aa gggeggeggccccg ceceagaget c etg ggcggaccgtcagtct t cct ct tcceeecaaaaeceaaggaeaeect ca tgateteccggacee et gaggt ea ca t gegt ggtgg t gga cg t gageeacgaaga ecet 9 aggt c aagt t caaetggta cgt ggaeggcgt ggaggt gea t aa t geeaagaeaaagccgcgggagg age agt a caaeagea cg t accgtgtggt eagcgt cet eaccgt ectgcaceaggaetggctgaa tggeaaggagtacaagt geaaggt et eeaacaaagecet ee e ag eceeca t cg agaa aacea t et ceaaagccaaagggea gccecgagaae caeaggt gt acace ctgccccca t cccggg a t gagct gaccaagaaccaggt c agcctgaeetgcctggt caaagget t ct at eeeagcgacat egccgtggagtgggagagcaa tg ggeagccggagaacaactacaagaecaegcet eeegtgt tggaet cegacggctcet tct t cet ct acageaaget cacegt ggacaag agea gg t ggcageaggggaaegt ct tet ea t get eegt 9 a tgca tg agget ct gca caa cea et a c a egcagaagagc ct et eect gt et eegggt aaa t ga

iwn

Fig. 3H

CysFc nucleotide sequence (h1FNa signal sequence underlined) at ccttqaccttt ctttact t gccctcct t ctca ctgcaa tcaa ct ctct t 999 ct 9 c ccgccgt 9 cccagct ceggaaetgetgggcggacegt eagt et teet ct t eeeec c aa a accea agg a caecet ca tg a t ct cecgga eeect gaggt ea eat gegtggtggtgga cgt 9 ag ecaeg a aga ecet 9 aggt eaagt t eaact ggt a cgtggaeggcgtggaggtgc a taa tgcc a agacaaag Ccgcgggaggagcagta caacagca cgt aeegtgtggt cagcgt cet caecgt c ct gca ccagg a et gget 9 aa t ggeaaggagta ea agt geaaggt et ecaa caaagecct eeeagce 2019200306

ceca t cga 9 a aa a cca t ct cca aagccaaa gggeageeeegagaaecaeaggt gt aeaeeet ge cecca t eccggga tgagctgaecaagaaccaggtcagcctgacctgcetggt eaaaggct tcta t cccag cg a cat cgccgtgga gt gggaga 9 c aa t gggcagcc ggag a a caact acaagaeca cg ceteccgtgt tggact ccgaeggct eet t ct tcctctacagcaagctcaccgtggacaagagca ggtggcag caggggaa cgt et t ct ca t gct cegtga tgca tg agg etctgca caaceactaeae gcagaagagcct ct cectgtct ccgggt aaa tga

Fig. 31

IFNa Gsi5 Fc nucleotide sequence (signal sequence underlined) : at cctt accttt ctttaeta~t ccetect tQetca ct caa tcaa et ctct t gggct gt ga tctgcct eaaaeccacageet gggtagcaggaggacct tgatgetcctggcaea ga t gaggagaat ct etct t t t ctcctgct tgaaggacagacatgact t tggat t tccceaggag gagt t t ggcaaccagt t ccaaaaggctgaaaccatccetgtcctccatgagatgatccageaga t et t caat ct ct t cagcacaaaggaetca t ctgctgct tggga tgagaccctcctagaeaaat t ct a ca ct ga a ct ctaecagcagct gaa t ga c ct ggaggcct gt gt gat acagggggtgggggtg acagagaet cccctgatgaaggaggact eea t tctggetgtgaggaaa tact teeaaagaatea ct et ct at ctgaaagagaaga aatacageect tgtgcetgggaggt tgtcagageagaaateat gaga t ct t t t t ct t tgtcaaeaaact tgeaagaaagt t tacgtagtaaggaaggtggeggcgga t ecggt ggagg cgggt ccggcggt 99aggga gcga caa aact caca cgtgcccgccgtgceeag ct ccggaactg ctgggcggaccgtcagt et t cct ct tcececcaaaaeccaaggacaceetcat ga t ct eccgga cccetgaggt eaca t gcgtggt ggtggacgt gagccacgaagaccct gaggt c a agt t caactggta cgtgga cggcgt ggaggt 9 cat a a tgcc aaga ca âagccgcgggaggagc agt a ca acagcacgt accgtgt ggt cagcgt cct caccgtcct gcaceaggact ggctgaa tgg caaggagt a caagtgeaaggt ct ccaa caaagccct c ccagccceca t cgagaaaa cca t ct ce a a age ca a a ggg c agccccgag a a cca ca ggt gt a c a cc ct 9 cce cc a t cccggg a t 9 a get ga ccaagaaccaggt cagectgacct geet ggt ca aagget t eta t cccagcga ca tcgecgtgga gt ggg a ga 9 eaa t gggcagccgg â 9 aa e a a ct a ea a 9 a c c a cg eet c c egt gt tggact ecg a c 99 ct cct t ct t cct ct acageaagct c a ccgt ggacaaga gcaggt ggcagcaggggaacgt et t ct catgct cegtgatgcatgaggct ct gcacaaccactacacgcagaagagcctctccctgtc tcegggtaâatga

11/27

Fig. 3J

EpoFc nucleotide sequence (signal sequence underlined, linker in bold) at gggagt gcaegaatgt eet gcetggetgtgget tetectgt ecetgetgt eget eeet etgg gc et cee agt eetgggcgeeceaeeaeg eetea t etgtgaeageegagt eetgg agaggtaeet et t ggaggcc aaggaggcegagaa ta teaegaeggget gtgetgaaeaetgeaget tgaa tgag aa t a teaetgt eceagaeaeeaaagt taa t t t etatgeetggaagagga tggaggtegggeage 2019200306

aggcegt ag a agt et ggeaggg eet ggecct getgt eggaaget gt c ct gcggggeeagg ee et gt tggt eaaetct teceageegtgggageeeetgeagetgea tgtggataaageegt cagtgge et t cgeageetcaeeaetetget t egggetetgggagceeagaaggaageea tetececteeag at geggeet eaget get eeaet ecgaaeaa t c aetgct ga caet t t ecgcaa act et tecgagt ct a et ceaa t t teet ceggggaaaget gaagct gtaeaeaggggagg eetge agaacaggggac agagagt tcgccggcge egc tgcggtcgaeaaaactea caea t gcceaeegt geccagct cegg aactcctgggcggaeegteagt et teet et tceeeecaaaaeecaaggaeacectcatga tete eeggaceeetgaggtcaca tgegt ggt ggtggaegtgagccaegaagacect gaggtcaagt t c aa e t ggt acgt gga egg egt gga ggt 9 cat aa t geeaagaca aagcegegggagg agcagt aca a eageaegtacegt gtggt cagegt eet eacegtectgcaecaggaetggctgaa tggcaagga gt aeaagtgcaaggt ct ceaacaaageeet eecageeeeca t cgagaaa acea t ctceaaagee a aagggc a geeecg a 9 aaec a eaggt gt a caee et geeecea t c c cggga t 9 agct 9 a c caaga aeeaggt eageetgaectgcct ggt caaagget t ct at cccagcga cat cgeegt ggagtggga 9 ageaa t gggcagccggagaaea a ct a eaaga ccaegcetcecgt gt t ggaet ccga cggct cc t t et tcetct aeageaaget eaccgtggaeaagageaggtggeageaggggaacgtet tet cat get eegt ga t geatgaggct et gea eaa eeaet aca cgcagaagageet ct eeetgt ct ceggg taaatga

12n7

Fig. 4 Various Ways to form monomer/dimer hybrids Through Native Ligation note that peptide could be substitued with any other small molecule. DNA, etc.

60% .10% i ... T . I~E1:';:fÌTGJçi;i::c:., ;,:..;.Gí51 OR iÍ(~.;Mv.!s(1 ç~i::C;..; ,.:. :.~~

¡~~~;:;~R¥~i çl?~G:.. ;...;, ;.GijJ Ig" sIgnal seq Fe t (others may be used) ~ protease cleavage site Fc hlFNn sIgnal seq Fc e.g. FX., enterokinase signal peptidase cleavage secretion from CHO signal peptidase cleavage secrelion from CHO (possibly E.eoli) (possibly E.coll) treatment with enzyme secretion from CHO thioester 1 add ~ihioester

.... (,H f:' f: -- N -i ~: L. ø. ø add ~At-th¡oeSier ~add IJWíjøAJ- thloester I..;;i. modified rxn conditions

1 ,dd p"""",.i-- "'".~

Figure 5a

Amino acid sequence of Fc-MESNA (produced in pTWINl vector from NEB; when Fc-Intein-CBD is eluted from chitin beads with MESNA, produces the following protein with a C-terminal thioester on the final Phe residue)

1 MGIEGRGAAA VDTSHTCPPC PAPELLGGPS VFLFPPKPKD TLMISRTPEV 2019200306

51 TCVVVDVSHE DPEVKFNWYV DGVEVHNAKT KPREEQYNST YRVVSVLTVL 101 HQDWLNGKEY KCKVSNKALP APIEKTISKA KGQPREPQVY TLPPSRDELT 151 KNQVSLTCLV KGFYPSDIAV EWESNGQPEN NYKTTPPVLD SDGSFFLYSK 201 LTVDKSRWQQ GNVFSCSVMH EALHNHYTQK SLSLSPGF

Figure 5 b Nucleotide sequence of Fc CDS in pTWINl (the final F residue, ttt, directly abuts the Mxe GyrA intein CDS in pTWIN1)

a tgggca t t gaaggcagaggcgccgctg cggt cgat actagt ca c aca tgcccaeegtgcecag eacct gaa et cctggggggaccgt cagt ct teet et t ceeeecaa aacccaaggacaccctea t ga t et cecggacccctgaggt eaca t gegtggt ggtggacgtgagcca cgaagaccetgaggtc aagt t eaa ct ggt a cgtggacggcgtggaggt gea taa tgccaagaea aagccgcgggagg age agt a c a a c ag e acgt a c cgt gt ggt cag cgt c c t ca ccgt e et gc a cc agg act gg ct 9 aa t gg eaaggagt a caagt gcaaggt ct ccaa eaaa gccet eceagcc ceca t cgagaaa a eca t etce a a ag c caa aggg cag cc ccg ag a ace a e a gg t gt a ca c ect 9 c c c ce a t cc eggg a t 9 ag ctga ccaag aaccaggt e agcctgae ctgeet ggt ea aagget t cta t cccagcgaca t egcegt gga gt gggagagcaa tgggcagccggagaacaaet aeasgaccacgcet eccgtgt tggaetecgae ggctcct t ct tcct ctacageaagctcaccgtggacaagagcaggtggcagcaggggaacgt ct t ct ea t get eegtga tgea t gagget et gea ca accaeta eacgc agaagagt ct ct ccctgte tccgggtttt

14/27

','

2019200306 16 Jan 2019

Fig. 6

Antiviral Activity of IFNa-Fc Dimer v. Hybrid Monomer-Dimer Fusions (IU/nmol) 600)(103

500x103

Õ E C 400x103 :J

.è .... ~ '5 300x103 N :g -.:i rn o ¡¡:: 13 200x103 OJ 0- l/)

10Ox103

o 0e e,t Ó"~ú flJ ¡:Q' o~ ,,~ ~~v f.'.o~ ~\~~ ~ .ò ~(jv øt .~0 lOt~ ~~oò'~ ...v l0o~'Q\\ò ~(j. G~&l0'ò ~(j ~~\\ò ~Òf Ò\~ ~ú~ ~ t\~ t\O~~ ò"'t &~ \t( o~ øt'('. '('. ...(;) t\~ d'~.~0t ''l; ~0t % t\ol0o ..~sY \Ò\l0t"& ~(j. ò" ,(( l0o~o

IFNa. fusion protein tested

I'-

0) u: 2019200306

~ cu (/) ....cP (/) ~ c:( 'Ó 0) c:

~o Ü LL ~ t: ~ -0 i :~¡;Z~~t;,,~;;:d"?i:,.,;; ,; , ..... . '0. ." YcP 9-r- CU ~~ ~ 0(,

%-

o i-

Ü --l

i c:( 0 in "'. 0 '" ~ 0 '" 0N '" 0 '" c: 00 i- i.n c -.::t c C'J "! 0 0 c (/) 0 0 0 0 0 0

OUlJ ill 1 IDI

16/27

Fig. 8 FVII.:Ag Elisa Oral uptake in Neonatal Rats

20.0

17.5

.... 15.0 ~ N -:i

12.5

~= 10.0

7.5

5.0

2.5

0.0 Monomer-Di1l1er Hybrid Dimer

Fig. 9

Cmax after single oral dose of Factor IX-Fc homodimer v. Factor IX-Fc:Flag-Fc monomer-dimer hybrid into 10 day old Sprague Dawley Rats

Ll

*1 100 ..... S' Ç) -- N -:i 0::i Ç) C1l 3- õ)

6' ::i

5' en I 10 C1l 2 3

i Factor IX-Fc:Flag Fc Factor IX.Fe: Factor l~-.¡::~.."_.__._.... ........ ..! !. ---"' - --_. --- 'rnononïër.ëiíiiiër Iiybrid . ... dfrñ-er hybrid..'. ." - .. -

Fig. 10

100

___ F1XFc:FlagFc Monomer.Dim~r Hybrid .... ___ FIXFc:FIXFc homodimer ~ Ñ -:i

10

o o ::i

~ ::i -. - ~ 65 70 75 õ' 50 55 60 ::i o 5 10 15 20 25 30 35 40 45 "0 3 Time, hr f1

3" ----------------

Fig. i) Pharmacokinetics of EpoFc Dimer v. Monomer-Dimer Hybrid in Cynomolgus Monkeys After a Single Pulmonary Dose Molar Comparison 2019200306

1800

1600

1400

1200

Plasma 1000 Concentration (1mol/ml) 800

600

400

200

o

o 20 40 60 80 100 120 140 160

Time (hr)

.-.- monkey #1: monomer.dimer, 281 pmoVkg ...... monkey #2: monomer-dimer, 281 pmoVkg -J,...... monkey #3: monomer.dimer, 281 pmollkg . ...,.... monkey #4: monomer.dimer, 281 pmollkg -0- monkey #5: dimer, 168 pmol/kg ..0.. monkey 416: dimer, 168 pmol/kg -ò- monkey #7: dimer, 392 pmoVkg -v- monkey 418: dimer, 168 pmol/kg ..0.. monkey #9: dimer, 168 pmol/kg

Fig. 12

10000 ro--o' __.________._______0_.___.___._ ..0.__0______. . Aranesp (0.54 nmollkg dose) rm EpoFc Monomer.Dimer Hybrid (IJ.3 nriliol/kg dose)

N .... 1000 Ñ -.i Serum Concentration (fmol/mL) 100 He

10 o 20 40 60 80 100 120 140 160 180 Time, hr . .' ....__., ..__.w.___w____..~ ......__~___.~_._...W_R.___ .... .".

Fig. 13

1000

N 100 ~ t1/2 = 23 hr -i

Serum Concentration 10 (ng/mL) t1l2=6~ 1 . Epogen (EJ EpoFc Monomer-Dimer Hybrid A Aranesp 0.1 140 20 40 60 80 100 120 o Time, hr .- _....~..__.- -----_........_. . L...

C') ""- o:i

~ ...- ..... :J 0..

.E

- i:i

III

._..---_...;..~ ...k_-;:-_.___---:: :'''''' ......tr; (1)

E .......-.,..._........~"' .... _......_-~-" is j i- / (1)

E o ,/ ..t: .J o. :1E 'ii

_._-......_.._.............

1"~

1 i

" / !

._._~._--------./ : () u.. .... .....~. . -_._._'), o ". " ~ Q r-.~.- ,., ~ ;: ¿ ,; ¿ -:t ,: .,.... ,"j Q 0=' ;:: t

,Ql u..

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Fig. 15

Pharmacokinetics of IFNß-008 (IFNß-Fc wt dimer) and IFNß-009/Fc-014 (IFNß-Fc N297A Monomer) In Cynomolgus Monkey Serum After a Single Pulmonary Dose of 2011g/kg

100 Å. C07308J .. C07336 monomer-dimer . C07312 hybrid (l C07326 J . @ C07338 dimer E -- 10 0)

N c .i;. -- N ¿0 -i :g .J::; c Q) () c0 0

0.1 120 140 20 40 60 80 100 o Time, hr

Fig. 16

Neopterin Levels in Cynomolgus Monkey Serum After A Single Pulmonary Dose (20lig/kg) of IFNß-008 (IFNß-Fc wt Dimer) Or IFNß-009/Fc-014 (IFNß-Fc N297A Monomer)

@ C07326l . 5 @) C07338 J dimer

... C07308J 4 ... C07336 monomer-dimer N ... C07312 hybrid Ui -- Q) N 0) -i C ro .i:: u 3 "0

.8

c .:: Q) 2 ...... 0- 0Q) z ~ a' /r ~ ~ z

o 150 200 250 o 50 100

Time, hr

Fig 17a.

IFNß-Fe nucleotide sequence (signal peptide underlined) a tgaceaacaagtgtctcctecaaat tgctctcctgt tgtgct tetccactacagctct t teca tgagctacaact tgct tgga t tcctacaaagaagcagcaa t t t teagtgt eagaagctcctgtg gcaa t tgaa tgggaggct tgaata t tgectcaaggacagga tgaact t tgaea tccctgaggag at taageagctgcagcagt tecagaaggaggaegccgca t tgacca teta tgagatgctccaga acatct ttgcta t t t tcagacaagat tcatctagcaetggctggaatgagactat tgt tgagaa 2019200306

ectcctggct aa tgtct a t ca teaga taaacca t ctgaagacagtcctggaagaaaaactggag aaagaagat t t eace aggggaaaact catgagcagt ctgcac c tgaaaagat at tatgggagg a t t ctgea t tacctgaaggccaaggagt acagt eactgtgcctggacca tagtcagagtggaaa t cctaaggaact t t tact tcat taaeagact tacaggt tacctccgaaacgagt tcgccggcgcc gctgcggt cg a eaaaact c aeac a tgcc c ac cgtgccc agct c cgg aaet c etgggcgga c cgt cagtet tcetct tecccceaaaacec aaggacaccctca tga tetcceggaccectgaggtcae a tgcgtggtggtgga cgt gagcc acg aagac cet gaggt caagt tcaae tggt acgtggacgge gtggaggtgc a taa tgccaag aeaaagccgcgggagg ageagt acaaeage a egtacegtgtgg t c agegt cct eac cgt cetg eaccaggact ggetgaa tgg caagg agt ae aagtgcaaggt etc caaeaaagce e t ce eagcceeca t cg agaaaae ea t ct c e aaagccaaaggg cagce ccgaga a ccaeaggtgt ae acectgce cec at e eeggg a tg agetga ceaagaa ee aggt eagcetg ae c t gcetggt eaaagget t et at eee ag eg aea t egcegtgg agtggg ag age aa tgggeage egg a gaaeaaetaeaagaecacgeetcccgtgt tggactccgacggetcet t ct t ectet acageaag ct caecgtgg a caag age aggtggcagc agggg aacgt et t ct ea tget e cgt ga tg ca t gagg ct ctgcacaa c ca ct aea ege ag aag agce te t c cc t gt et ccgggt a aa tga

Fig 17b. IFNß-Fc amino acid sequence (signal sequence underlined, linker sequence in bold, N297 in bold underlined) . 1 MTNKCLLQIA LLLCFSTTAL SMSYNLLGFL QRSSNFQCQK LLWQLNGRLE 51 YCLKDRMNFD IPEEIKQLQQ FQKEDAALTI YEMLQNIFAI FRQDSSSTGW 101 NETIVENLLA NVYHQINHLK TVLEEKLEKE DFTRGKLMSS LHLKRYYGRI 151 LHYLKAKEYS HCAWTIVRVE ILRNFYFINR LTGYLRNEFA GAAAVDKTHT 201 CPPCPAPELL GGPSVFLFPP KPKDTLMISR TPEVTCVVVD VSHEDPEVKF 251 NWYVDGVEVH NAKTKPREEQ YNSTYRVVSV LTVLHQDWLN GKEYKCKVSN 301 KALPAPIEKT ISKAKGQPRE PQVYTLPPSR DELTKNQVSL TCLVKGFYPS 351 DIAVEWESNG QPENNYKTTP PVLDSDGSFF LYSKLTVDKS RWQQGNVFSC 401 SVMHEALHNH YTQKSLSLSP GK

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Fig.18A YTSLlHSLlEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO: 99).

Fig. 188

NNLRAIEAQQHLLQLTVWGIKQLQARILAVERYLKOQ (SEQ 10 NO: 100) 2019200306

Fig.18C WQEWEQKITALLEQAQIQQEKNEYELQKLDKWASLWEWF (SEQ 10 NO: 101)

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