HK1178549A - Control of protein glycosylation and compositions and methods relating thereto - Google Patents

Description

Control of protein glycosylation and compositions and methods related thereto

Cross Reference to Related Applications

This application claims priority from U.S. provisional patent application No. 61/170,897, filed on 20/4/2009, which is incorporated herein by reference in its entirety.

Background

The present invention relates to methods for controlling protein glycosylation and compositions and methods related thereto. The invention further relates to methods of reducing glycosylation and/or glycan site occupancy (occupancy) of glycoproteins to thereby affect their biological characteristics and/or function. The invention also relates to compositions prepared using the methods of the invention to control glycosylation and uses thereof.

Proteins and polypeptides have become increasingly important therapeutic agents. In most cases, these proteins and polypeptides are produced in cell culture from cells engineered and/or selected to produce abnormally high levels of a particular protein or polypeptide of interest. Control and optimization of cell culture conditions is of paramount importance for successful commercial production of proteins and polypeptides.

Many proteins and polypeptides produced in cell culture are glycoproteins that contain covalently linked sugar structures, including oligosaccharide chains. These oligosaccharide chains are linked to proteins in the endoplasmic reticulum and golgi apparatus by either N-or O-bonds. The oligosaccharide chains may comprise a significant portion of the mass of the glycoprotein. Oligosaccharide chains are believed to play key roles in the function of glycoproteins, including facilitating proper folding of glycoproteins, mediating protein-protein interactions, conferring stability, conferring favorable pharmacodynamic and/or pharmacokinetic properties, inhibiting proteolytic digestion, targeting glycoproteins to the appropriate secretory pathway, and targeting glycoproteins to a specific organ or organs.

Typically, N-linked oligosaccharide chains are added to nascent translocator proteins In the lumen of the endoplasmic reticulum (see Alberts et al, 1994, In: Molecular Biology of the Cell, incorporated herein by reference). The oligosaccharide is added to an amino group on the side chain of an asparagine residue contained within the target consensus sequence Asn-X-Ser/Thr, where X can be any amino acid other than proline. Specific glycosidases normally passed in the endoplasmic reticulum trim the original oligosaccharide chains, resulting in short branched core oligosaccharides consisting of 2N-acetylglucosamine and 3 mannose residues.

Following initial processing in the endoplasmic reticulum, glycoproteins are shuttled through vesicles to the golgi apparatus where the oligosaccharide chains are further processed and then secreted to the cell surface. The trimmed N-linked oligosaccharide chain can be modified by the addition of several mannose residues, resulting in a high mannose oligosaccharide. Alternatively, one or more monosaccharide units of N-acetylglucosamine may be added to the core mannose subunits to form complex oligosaccharides (complex oligosaccharides). Galactose may be added to the N-acetylglucosamine subunits, and sialic acid subunits may be added to the galactose subunits, resulting in chains terminated with sialic acid, galactose, or N-acetylglucosamine residues. In addition, fucose residues may be added to the N-acetylglucosamine residues of the core oligosaccharide. Each of these additions is catalyzed by a specific glycosyltransferase.

In addition to modifying glycoproteins through the N-linked glycosylation pathway, glycoproteins may also be modified by the addition of O-linked oligosaccharide chains to specific serine or threonine residues when they are processed in the Golgi apparatus. One O-linked oligosaccharide residue at a time is added and the addition of each residue is catalyzed by a specific enzyme. The consensus amino acid sequence for O-linked glycosylation is less well defined than for N-linked glycosylation.

Glycosylation of proteins, particularly immunoglobulins, has been shown to have a significant impact on their biological function. For example, for immunoglobulins, glycosylation has been shown to affect effector function, structural stability and secretion rate of antibody-producing cells (Leatherbarrow et al, 1985, mol. Immunol.22: 407). The glycosyl groups responsible for these properties are typically attached to the constant (C) region of the antibody. For example, the full capacity of IgG to activate the classical pathway of complement-dependent cytolysis is required at C_HGlycosylation of IgG at asparagine 297 in domain 2 (Tao and Morrison, 1989, J.Immunol.143: 2595). At C_HGlycosylation of IgM at asparagine 402 in domain 3 is essential for proper assembly and cytolytic activity of the antibody (Muraoka and Shulman, 1989, J.Immunol.142: 695).Removal of C such as IgA antibody_H1 and C_HThe glycosylation sites at positions 162 and 419 in the 3 domain lead to intracellular degradation and inhibition of secretion by at least 90% (Taylor and Wall, 1988, mol.cell.biol.8: 4197).

Glycosylation of the immunoglobulin in the variable (V) region containing the antigen binding site was also observed. Sox and Hood (1970, Proc. Natl. Acad. Sci. USA 66: 975) report that about 20% of human antibodies are glycosylated in the V region. Glycosylation of the V domain is believed to be caused by the occasional occurrence of the N-linked glycosylation signal Asn-Xaa-Ser/Thr in the V region sequence, and is not believed in the art to play an important role in immunoglobulin function.

Recently, glycosylation in the antigen binding site of mouse antibodies specific for alpha- (1-6) glucan at CDR2 of the heavy chain has been shown to increase their affinity for glucan (Wallick et al, 1988, J.exp.Med.168: 1099; and Wright et al, 1991, EMBO J.10: 2717). Mutations that remove glycosylation sites within the antigen binding site of anti-CD 33 antibodies have also been demonstrated, more specifically, removal of N-linked glycosylation sites within the framework region of the V domain of antibody M195 enhances antibody binding to antigen (Co et al, U.S. patent No. 6,350,861). However, the N-linked glycosylation sites present in the Fc portion of the antibody are not removed and are preferably glycosylated to maintain effector functions of the molecule.

In addition, glycolytic inhibitory substances have been added to cell culture media to reduce the accumulation of the metabolic waste, lactic acid, thereby increasing cell viability and protein production of antibodies. See, for example, international patent application No. PCT/US2007/083473, which is now published as WO2008/055260 on 8/5/2008 (addition of glycolytic inhibitory compounds to cell cultures reduces lactate concentration and increases production of growth differentiation factor 8(GDF-8) -specific antibodies). Although glycolytic inhibition can also affect the level of protein glycosylation, the effect of inhibitors on protein glycosylation is not evaluated or even discussed. Furthermore, anti-GDF-8 antibodies produced by cell culture do not contain any potential glycosylation sites in the antigen binding site (see WO2008/055260, page 49, paragraph 170, cited in Veldman et al, U.S. patent publication No. 2004/0142382, describing anti-GDF-8 antibodies Myo22, Myo28, and Myo 29). Thus, even assuming that glycosylation of the anti-GDF-8 antibody at the heavy chain constant domain is affected under the disclosed culture conditions, the effect of altered glycosylation of the antigen binding site cannot be assessed, as it appears that the resulting antibody lacks potential glycosylation sites in the antigen binding region (e.g., V domain).

A major problem with protein therapeutics is reduced or low affinity for ligands or antigens. The loss or reduced affinity is highly undesirable and requires more therapeutic protein to be injected into the patient, at higher cost and with a greater risk of side effects. Even more seriously, proteins with reduced affinity may have reduced biological functions such as complement lysis, antibody-dependent cellular cytotoxicity, and viral neutralization. Furthermore, the reduced competition between the antigen or binding partner and the therapeutic protein as compared to the endogenous ligand or binding partner that is intended to inhibit its interaction makes it possible for the protein to have reduced antagonist function. For example, loss of affinity in the partially humanized antibody HUVHCAMP can result in its loss of all its ability to mediate complement lysis (see, Riechmann et al, 1988, Nature, 332, 323-Asca 327, Table 1).

Furthermore, given the unpredictability of protein conformation, even mutation of a single amino acid, particularly at the antigen-binding site or ligand-binding site of a protein, can have a dramatic impact on the biological activity of a potential therapeutic protein.

Thus, there is a need in the art for therapeutic proteins with altered affinity for ligands, or in the case of antibodies, altered affinity for antigens, particularly increased affinity and/or increased specificity for antigens or ligands, as well as the desired potential for lower immunogenicity and improved effector function conferred by glycosylation of naturally occurring constant regions. Alternatively, there is a need for therapeutic proteins, particularly antibodies or Fc fusion proteins comprising immunoglobulin constant regions, or portions thereof, that have increased binding affinity and/or specificity for an antigen or ligand, but have reduced or no effector function mediated by glycosylation of glycosylation sites present in the heavy chain constant region of the antibody. It is also desirable to inhibit or remove glycosylation of an antigen or ligand binding site of a therapeutic protein without the need to introduce any mutations into the amino acid sequence of the antigen or ligand binding site. Thus, there is a need in the art for methods of increasing potency and reducing the required dose of immunoglobulins and other therapeutically important proteins, and for therapeutic proteins produced by such methods, which are met by the present invention without requiring the introduction of any mutations into the antigen-binding site of the antibody or the ligand-binding site of the therapeutic protein.

Despite the importance of therapeutic glycoproteins and advances in cell culture methods, there remains an unmet need for new methods for producing such proteins under conditions in which glycosylation of the ligand binding site can be controlled, without requiring the introduction of amino acid mutations into such important protein portions. The present invention satisfies this need.

Summary of The Invention

The present invention includes methods for reducing the level of glycosylation of a protein. The method comprises expressing a protein comprising at least one glycosylation site in a host cell grown in culture in the presence of a glycosylation inhibiting amount of a glycosylation inhibitor, wherein the protein comprises a lower level of glycosylation as compared to an otherwise identical protein produced in an otherwise identical host cell grown under otherwise identical conditions in the absence of the inhibitor, thereby controlling the level of glycosylation of the protein.

In one aspect, the glycosylation site is in the ligand binding site or antigen binding site.

In another aspect, the glycosylation level is selected from the glycan site occupancy at the glycosylation site and the degree of glycosylation at the glycosylation site.

In another aspect, the glycosylation sites are selected from the group consisting of O-linked glycosylation sites and N-linked glycosylation sites.

In another aspect, the glycosylation site is an N-linked glycosylation site, and further wherein the glycosylation site comprises the amino acid sequence asparagine-X-serine or asparagine-X-threonine, wherein X is any amino acid except proline.

In another aspect, the glycosylation inhibitor is at least one selected from the following inhibitors: tunicamycin, tunicamycin (tunicamycin) homolog, streptovirins, brazidomycin, amfomycin, ziromycin, antibiotic 24010, antibiotic MM 19290, bacitracin, corynetoxin, oxytetracycline, chlortetracycline (duimycin), 1-deoxymannose nojirimycin (1-deoxymannanojirimycin), deoxynojirimycin, N-methyl-1-deoxymannose nojirimycin, brefeldin A, glucose analogs, mannose analogs, 2-deoxy-D-glucose, 2-deoxyglucose, D- (+) -mannose, D- (+) galactose, 2-deoxy-2-fluoro-D-glucose, 1, 4-dideoxy-1, 4-imino-D-mannitol (DIM), fluoroglucose, doxycycline, Fluoromannose, UDP-2-deoxyglucose, GDP-2-deoxyglucose, mevalonate monoacyl-CoA reductase inhibitor, 25-hydroxycholesterol, swainsonine, actinone, puromycin, actinomycin D, monensin, carbonyl cyanide metachlorophenylhydrazone (m-chlorocarbanyl-cyanophenylhydrazone, CCCP), compactin, polyterpene long-alcohol-phosphoryl-2-deoxyglucose (dolichyl-2-deoxyglucide), N-acetyl-D-glucosamine, hypoxanthine (xanthine), thymidine, cholesterol, glucosamine, mannosamine, castanospermine, glutamine, bromocyclohexenetetraol (bromocyclododecanol), cyclohexenetetraol epoxide (conduritol epoxide), cyclohexenetetraol derivative, glycosylmethyl-p-nitrophenyltriazene (glyco-p-triazene), Beta-hydroxynorvaline, threo beta-fluoroaspartamide, D- (+) -glucono delta-lactone, di (2-ethylhexyl) phosphate, tributyl phosphate, dodecyl phosphate, 2-dimethylaminoethyl (benzhydryl) -phosphate, [2- (diphenylphosphinoxy) ethyl ] trimethylammonium iodide, iodoacetate and fluoroacetate.

In another aspect, the glycosylation inhibitor is 2-deoxy-D-glucose.

In another aspect, the glycosylation inhibiting amount ranges from about 0.5g/L to about 3 g/L.

In another aspect, the method further comprises maintaining the glucose concentration in the culture in an amount ranging from about 0.05g/L to 10 g/L.

In another aspect, the host cell is selected from the group consisting of a yeast cell, an insect cell, and a mammalian cell.

In another aspect, the mammalian host cell is selected from the group consisting of CHO cells, NS0 cells, NS0/1, Sp2/0, human cells, HEK 293, BHK, COS, Hep G2, PER. C6, COS-7, TM4, CV1, VERO-76, MDCK, BRL 3A, W138, MMT 060562, TR1, MRC5, and FS 4.

In another aspect, the host cell is a CHO cell.

In another aspect, the glycosylation site is at least one glycosylation site comprising an amino acid sequence selected from the group consisting of asparagine-isoleucine-threonine (NIT), asparagine-glycine-serine (NGS), asparagine-serine-threonine (NST), and asparagine-threonine-serine (NTS).

The present invention encompasses a protein produced according to a method for reducing the glycosylation level of a protein, wherein the method comprises expressing in a host cell grown in culture a protein comprising at least one glycosylation site in the presence of a glycosylation inhibiting amount of a glycosylation inhibitor, wherein the protein comprises a lower level of glycosylation as compared to an otherwise identical protein produced in an otherwise identical host cell grown under otherwise identical conditions in the absence of the inhibitor, thereby controlling the glycosylation level of the protein, wherein the glycosylation site is a ligand binding site or an antigen binding site, wherein the glycosylation level is selected from the group consisting of glycan occupancy at the glycosylation site and degree of glycosylation at the glycosylation site, wherein the glycosylation site is selected from the group consisting of an O-linked glycosylation site and an N-linked glycosylation site, wherein the glycosylation site is at least one glycosylation site comprising an amino acid sequence selected from the group consisting of asparagine-isoleucine-threonine (NIT), wherein the glycosylation inhibitor is 2-deoxy-D-glucose, the glycosylation inhibiting amount ranges from about 0.5g/L to 3g/L, the method comprising maintaining a glucose concentration in the culture in an amount ranging from about 0.05g/L to 10g/L, wherein the host cell is selected from the group consisting of yeast cells, insect cells, and mammalian cells.

In one aspect, the mammalian cell is a CHO cell and asparagine-glycine-serine (NGS), asparagine-serine-threonine (NST), and asparagine-threonine-serine (NTS).

In another aspect, the protein is an antibody or antigen-binding portion thereof, and further wherein the antibody is an anti-human IgE antibody.

In another aspect, the antibody is a human antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and the light chain variable region has the amino acid sequence of SEQ ID NO: 10.

In another aspect, the antibody comprises at least one N-linked glycosylation site that is unoccupied and/or comprises at least one sugar moiety less than an otherwise identical antibody produced in the absence of the glycosylation inhibitor, wherein the site is selected from the group consisting of heavy chain relative to SEQ ID NO: 8 at asparagine 73(N73) and asparagine 301 (N301).

In another aspect, the antibody comprises an unoccupied N-linked glycosylation site at asparagine 73.

In another aspect, the protein comprises a Ligand Binding Site (LBS) for receptor for advanced glycation end products (RAGE), the ligand binding site comprising a sequence selected from the group consisting of SEQ ID NO: 13. SEQ ID NO: 14 and SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof.

In another aspect, the protein is a RAGE fusion protein comprising the amino acid sequence of SEQ ID NO: 1, said signal sequence comprising amino acid residue number 1 to amino acid residue number 23, wherein said fusion protein lacks a terminal lysine residue (Lys 438).

In another aspect, the RAGE fusion protein comprises at least one N-linked glycosylation site that is unoccupied and/or comprises at least one sugar moiety less than an otherwise identical RAGE fusion protein produced in the absence of the glycosylation inhibitor, wherein the site is at least one site selected from the group consisting of asparagine at amino acid residue No. 2 (N2), asparagine at amino acid residue No. 58 (N58), and asparagine at amino acid residue No. 288 (N288), all relative to the sequence of SEQ ID NO: 1.

In one aspect, the protein comprises at least two N-linked glycosylation sites described below that are unoccupied and/or comprise at least one sugar moiety less.

In another aspect, the protein comprises three N-linked glycosylation sites, at least one of which is unoccupied and/or comprises at least one carbohydrate moiety less.

In another aspect, the protein exhibits increased binding to an antigen as compared to an otherwise identical protein produced in the absence of the inhibitor.

In another aspect, the protein exhibits increased binding to a RAGE ligand as compared to an otherwise identical protein produced in the absence of the inhibitor.

In one aspect, the method further comprises a temperature transition from a temperature in the range of about 34 ℃ to 39 ℃ to a temperature in the range of about 28 ℃ to 33 ℃.

In another aspect, the method includes a temperature transition from a temperature in the range of about 34 ℃ to 37 ℃ to a temperature in the range of about 30.5 ℃ to 31.5 ℃.

The present invention relates to a pharmaceutical composition comprising a protein produced according to a method for reducing the glycosylation level of a protein, wherein the method comprises expressing in a host cell grown in culture a protein comprising at least one glycosylation site in the presence of a glycosylation inhibiting amount of a glycosylation inhibitor, wherein the protein comprises a lower level of glycosylation compared to an otherwise identical protein produced in an otherwise identical host cell grown under otherwise identical conditions in the absence of the inhibitor, wherein the glycosylation site is in a ligand binding site or an antigen binding site, thereby controlling the glycosylation level of the protein, wherein the glycosylation level is selected from the group consisting of glycan site occupancy at the glycosylation site and degree of glycosylation at the glycosylation site, wherein the glycosylation site is an N-linked glycosylation site comprising the amino acid sequence asparagine-X-serine or asparagine-X-threonine, wherein X is any amino acid other than proline, wherein the glycosylation inhibitor is 2-deoxy-D-glucose, wherein the glycosylation inhibiting amount ranges from about 0.5g/L to 3g/L, and the method further comprises maintaining the glucose concentration in the culture in an amount ranging from about 0.05g/L to 10g/L, and wherein the host cell is a CHO cell.

In another aspect, the glycosylation site is at least one glycosylation site comprising an amino acid sequence selected from the group consisting of asparagine-isoleucine-threonine (NIT), asparagine-glycine-serine (NGS), asparagine-serine-threonine (NST), and asparagine-threonine-serine (NTS).

The present invention relates to compositions comprising an amount of a fusion protein, wherein the fusion protein comprises a RAGE polypeptide linked to an immunoglobulin polypeptide, wherein the RAGE polypeptide comprises a fragment of human RAGE (SEQ ID NO: 3), wherein the fragment of human RAGE comprises a ligand binding site and at least one amino acid residue that may be glycosylated, wherein the immunoglobulin polypeptide comprises C of an immunoglobulin_H2 domain or part C_H2 domain and immunoglobulin C_H3 domain, and wherein the N-terminal residue of the immunoglobulin polypeptide is linked to the C-terminal residue of the RAGE polypeptide; and wherein at least 0.5% of the amount of the fusion protein is aglycosylated (aglycosylated).

In one aspect, at least 30% of the total amount of the fusion protein is non-glycosylated.

In another aspect, the percentage of the amount of the fusion protein in the fully glycosylated form is less than the percentage of the amount of the fusion protein in all the non-fully glycosylated forms.

In another aspect, the fusion protein comprises at least three amino acid residues that may be glycosylated, wherein a first potential site of glycosylation is an amino acid residue of the RAGE ligand binding site, a second potential site of glycosylation is an amino acid residue of the RAGE polypeptide, and a third potential site of glycosylation is an amino acid residue of the immunoglobulin polypeptide.

The present invention relates to a composition comprising a protein comprising at least one potential glycosylation site, wherein the amount of fully glycosylated protein is less than the amount of less than fully glycosylated protein, and further comprising a pharmaceutically acceptable carrier.

In one aspect, the protein comprises two potential glycosylation sites, and wherein the amount of fully glycosylated protein is less than the sum of the amount of protein comprising one site that is not glycosylated and/or is less glycosylated and the amount of protein comprising two sites that are not glycosylated and/or are less glycosylated.

Alternative embodiments of the invention are described below, such as embodiments employing various culture conditions, glycoproteins, and involving different glycosylation inhibitors.

Drawings

The foregoing summary, as well as the following detailed description of the invention, will be better understood when read in conjunction with the appended drawings. For the purpose of illustrating the invention, there is shown in the drawings embodiments which are presently preferred. It should be understood, however, that the invention is not limited to the precise arrangements and instrumentalities shown.

In the figure:

FIG. 1 is a diagram showing the construction of sRAGE-Ig fusion protein. The receptor for advanced glycation end products (RAGE) is shown with its three extracellular domains (V, C1 and C2), transmembrane domain, and intracellular domain presumed to be responsible for RAGE signaling. The soluble or secreted forms of RAGE encompassed by the terms "soluble RAGE" and "sRAGE" are shown to lack transmembrane and intracellular domains. sRAGE can act as a decoy receptor, and is shown in the figure as a RAGE extracellular domain only. Full-length human heavy chain IgG1 molecules are shown, showing a variable binding domain (V)_H) And the first constant domain C_H1 together form an antibody binding fragment (Fab) which is connected by a flexible hinge region to the second and third constant domains (C) of the IgG1 molecule_H2 and C_H3). The figure also shows the linkage of the V and C1 domains of human RAGE to the C of an IgG1 molecule_H2 and C_H3 domains to construct sRAGE-Ig fusion proteins with or without (shown) an immunoglobulin hinge region.

FIG. 2, comprising FIGS. 2A and 2B, shows the amino acid and nucleic acid sequences of sRAGE-Ig fusion proteins of the present invention. FIG. 2A shows the amino acid sequence of an sRAGE-Ig fusion protein of the present invention (SEQ ID NO: 1). The sRAGE-Ig protein sequence comprises 461 amino acids, which includes a 23 amino acid signal peptide/leader sequence, which in some cases may be 22 amino acids, cleaved during expression to yield a mature protein of 438 amino acids (or 437 amino acids when the N-terminal lysine is cut off). For the purposes of this example, the numbering of amino acid residues does not include a 23 amino acid leader sequence, so that the sRAGE-Ig fusion protein sequence has a glutamine at amino acid 1(Q1), which can be cyclized to form pyroglutamic acid (pE) at amino acid 1 (pE 1). The human RAGE sequence is derived from GenBank accession No. NM _ 001136. The three underlined asparagines (N) at positions N2, N58 and N288 represent potential sites for N-linked glycosylation. The V domain of RAGE spans from amino acid residue 1 to amino acid residue E109. The C1 domain of RAGE overlaps the C-terminal sequence of the V domain by about 10 amino acids and spans about amino acids G99 to E220. The underlined amino acids RALR from amino acid residue 195 to amino acid residue 198 represent the consensus sequence for furin cleavage. The approximately 8 amino acid residues at the N-terminus of the C2 domain of sRAGE (PEGGAVAP) overlap with the C-terminal sequence of the C1 domain, spanning approximately amino acids L212 to P228 at the C1/C2 junction. C of IgG1 Fc _H2 and C_HThe 3 domain is shown to encompass about P229 to K438, where the terminal lysine (K) is not clipped. In the illustrated embodiment, the fusion protein does not comprise the entire human IgG1 hinge domain. In another embodiment, the fusion protein may comprise a hinge or any portion thereof, including wherein the hinge, if present, may be modified, for example, to alter any effector function associated with the unmodified hinge region. Figure 2B shows a sequence encoding a polypeptide comprising SEQ ID NO: 1 (SEQ ID NO: 2). The coding sequence 1-753 highlighted in bold encodes the RAGE N-terminal protein sequence, while the sequence 754-1386 encodes the hinge-free human IgG Fc (. gamma.1) protein sequence.

FIG. 3 is a diagram of SDS-PAGE analysis of sRAGE-Ig fusion protein, demonstrating the effect of digestion with PNGaseF and furin. sRAGE-Ig fusion proteins expressed in cells grown in shake flask culture were purified by mabselect and digested with PNGaseF or furin, and then analyzed on reduced 4-12% gradient Bis-Tris gels. Each lane contained 2.7. mu.g of protein. Molecular weights expressed as Kd are shown on the left. Lanes 1 and 6 contain untreated fusion protein. Lane 2 contains the fusion protein incubated with PNGaseF digestion buffer alone. Lane 3 contains the fusion protein digested with PNGaseF. Lane 4 contains the fusion protein incubated with furin digestion buffer alone. Lane 5 contains the fusion protein digested with furin. FLM ═ full length monomer.

FIG. 4 shows the results of analysis of sRAGE-Ig fusion protein by Size Exclusion Chromatography (SEC). The purified sRAGE-Ig fusion protein was diluted to 3mg/mL in mobile phase (350mM citrate, pH 6.0) and analyzed by Size Exclusion Chromatography (SEC) using Superdex^TM20010/300GL column, ambient temperature, 0.75 mL/min flow rate, 20 μ L injection volume and 40 min run time, monitored at 280 nm. SEC enables relative quantification of homodimers, heterodimers, Fc dimers and high molecular weight species (species) with peak results shown in the upper left-hand insert.

FIG. 5 shows the results of reverse phase-HPLC, which shows the glycan site occupancy spectrum of the sRAGE-Ig fusion protein. The superimposed chromatograms were sRAGE-Ig fusion protein (S1) and FLM from fractionated samples (S1F1, S1F2, and S1F 3). The concentration of fraction S1F3 was very low and therefore it was not included in the subsequent ELISA binding assay. The upper right hand insert shows the approximate content of each glycan, as a percentage of each sample/fraction.

Fig. 6 is a graph showing peak property confirmation by mass spectrometry of the peaks S1F1 and S1F2 shown in fig. 5. Mass spectral data identified peaks S1F1 as being of the 3-glycan type and S1F2 as being of the 2-glycan type.

FIG. 7 is an identification chart showing normal phase HPLC analysis of N-linked glycans released from sRAGE-Ig fusions along with peaks shown on the chromatogram. The relative abundances of the various types are shown in table 1.

FIG. 8 is a graph showing the reverse phase separation results of tryptic digestion of sRAGE-Ig. The inset shows the region where the N-linked tryptic peptide fragments elute.

FIG. 9 shows the identification and theoretical monoisotopic molecular weights of trypsin-digested peptide fragments containing a common N-linked glycosylation site. The figure shows the amino acid sequences of three predicted tryptic digest glycopeptides from a tryptic digested sRAGE-Ig fusion protein, and shows the theoretical monoisotopic molecular weight of each tryptic digest (digest). The tryptic glycopeptide was as follows: t1pE (pENITAR) having a mass of about 638.36 Da; t10(VLPNGSLFLPAVGIQDEGIFR) having a molecular weight of about 2241.22 Da; and T31(EEQYNSTYR) having a molecular weight of about 1188.50 Da.

FIG. 10 is a graph showing reverse phase-HPLC, which shows the glycan site occupancy profile of sRAGE-Ig fusion protein expressed in the presence of tunicamycin. The superimposed chromatograms were sRAGE-Ig fusion protein (S2) from tunicamycin-treated cells and three fraction samples (S2F1, S2F1, and S2F 3). The inset in the upper right corner shows the relative percentage of each glycan in each sample or fraction thereof.

Fig. 11 is a confirmation diagram showing peak identification by mass spectrometry of the peaks S2F1, S2F2, and S2F3 shown in fig. 10. Mass spectral data identified peaks S2F1 as being of the 3-glycan type, S2F2 as being of the 2-glycan type and S2F3 as being of the 0-glycan type.

Fig. 12 is a diagram showing exemplary biantennary glycoforms typically present in immunoglobulin heavy chain constant regions. According to the invention, "G0" refers to a biantennary structure in which no terminal sialic acid (NeuAc) or Gal is present, "G1" refers to a biantennary structure with one Gal and no NeuAc, and "G2" refers to a biantennary structure with two terminal gals and no NeuAc.

Fig. 13, comprising fig. 13A through 13D, shows the sequences of the heavy and light chains of anti-IgE antibody 5.948.1. FIG. 13A shows the amino acid sequence of the heavy chain of mAb 5.948.1 (SEQ ID NO: 8). Variable domains are shown in lower case letters, while constant domains are shown in upper case letters. Complementarity Determining Regions (CDRs) are underlined, and potential N-linked glycosylation sites at asparagine 73 of framework 3 in the heavy chain variable region are shown in capital letters ("NTS"). The N-linked glycosylation site ("NST") Asn301 in the heavy chain constant domain is underlined. FIG. 13B shows the nucleic acid sequence encoding the heavy chain of mAb 5.948.1 (SEQ ID NO: 9). FIG. 13C shows the amino acid sequence of the light chain of mAb 5.948.1 (SEQ ID NO: 10). Variable domains are shown in lower case letters, while constant domains are shown in upper case letters. Complementarity Determining Regions (CDRs) are underlined. FIG. 13D shows the nucleic acid sequence encoding the light chain of mAb 5.948.1 (SEQ ID NO: 11).

Fig. 14 is a diagram showing 4 possible glycan site occupying variants of the heavy chain of antibody 5.948.1. From left to right, the figure shows 0-glycan occupying variants that do not contain glycans at Asn73 or Asn 301; 1-glycan occupying variants comprising only one glycan at Asn 301; 1-glycan occupying variants comprising only one glycan at Asn 73; and fully occupied 2-glycan occupied site variants comprising glycans at both Asn73 and Asn 301.

Fig. 15 is a graph showing a glycan profile of antibody 5.948. The heavy chain was resolved into 3 peaks, showing 2-glycan and 1-glycan variants. No 0-glycan occupying variants were detected. The 1-glycan occupancy peak can be further resolved to identify variants that are glycosylated in the Fc region (more generally) and in the Fab region of the antibody heavy chain. The data show that fully glycosylated 2-glycans occupy about 98% of the total heavy chain produced under the control culture conditions, while 1-glycans occupy about 2% of the total heavy chain produced under the control culture conditions.

Fig. 16 is a graph showing glycan profiles of monoclonal antibody 5.948.1(mAb 5.948.1) along with a sample of 5.948.1 antibody deglycosylated with ethylene glycol-N-glycanase and a sample denatured with SDS and BME. As seen previously (fig. 15, supra), the heavy chain of the native 5.948.1 antibody resolved into 3 major peaks, showing 2-glycan and 1-glycan variants. No 0-glycan occupying variants were detected. The 1-glycan occupancy peak can be further resolved to identify variants glycosylated in the Fc region and variants glycosylated in the Fab region of the antibody heavy chain. The heavy chain of the naturally deglycosylated 5.948.1 antibody shows a profile containing predominantly 1-glycan types with some 0-glycan types. The heavy chain of the denatured deglycosylated 5.948.1 antibody showed a profile containing predominantly 0-glycan types.

FIG. 17 is a graph showing glycan profiles of antibody 5.948.1 produced under control conditions (i.e., in the absence of a glycosylation inhibitor), wherein a sample was taken from the culture on day 14 (sample 7; S7). The heavy chain was resolved into 3 peaks, showing 2-glycan and 1-glycan variants. No 0-glycan occupying variants were detected. The 1-glycan occupancy peak can be further resolved to identify variants that are glycosylated in the Fc region (more generally) and in the Fab region of the antibody heavy chain. The data show that fully glycosylated 2-glycans occupy about 98% of the total heavy chain produced under the control culture conditions, while 1-glycans occupy about 2% of the total heavy chain produced under the control culture conditions.

FIG. 18 is a graph showing a glycan profile of antibody 5.948.1 produced in the presence of the glycosylation inhibitor 2-deoxy-D-glucose, with a sample taken from the culture on day 14 (sample 11; S11). The heavy chain was resolved into 3 peaks, showing 2-glycans and 1-glycans. 0-glycan occupying variants are readily detected. The 1-glycan occupancy peak can be further resolved to identify variants that are glycosylated in the Fc region (more generally) and in the Fab region of the antibody heavy chain.

Fig. 19 is a graph of a spectrum overlay comparing the glycan profile of mAb 5.948.1 produced under control conditions (in the absence of glycosylation inhibitor) and mAb 5.948.1 produced under glycosylation control conditions (i.e., in the presence of glycosylation inhibitor 2-deoxy-D-glucose), wherein samples (S7 and S11, respectively) were taken from each culture on day 14. The heavy chain was resolved into 4 peaks, showing 2-glycan, 1-glycan and 0-glycan variants. 0-glycan occupying variants were readily detected only in S11. The 1-glycan occupancy peak can be further resolved to identify variants that are glycosylated in the Fc region (more generally) and in the Fab region of the antibody heavy chain. The data show that the 1-glycan Fab variant heavy chain is greatly increased relative to the 1-glycan Fc variant in the 2-deoxy-D-glucose sample (S11) compared to the control sample (S7) in which the 1-glycan variant consists essentially of the Fc variant.

Fig. 20, comprising fig. 20A and 20B, is a detailed profile comparison graph comparing glycan profiles of antibody 5.948.1 produced in the presence and absence of a glycosylation inhibitor. Fig. 20A is a graph showing the glycan profile of mAb 5.948.1 produced under control conditions (in the absence of glycosylation inhibitors), with samples taken from the cultures on day 14 (S7). Fig. 20B is a graph showing the glycan profile of mAb 5.948.1 produced in the presence of 2-deoxy-D-glucose, with samples taken from the cultures on day 14 (S11). Comparison of fig. 20A and 20B shows that 0-glycan occupying variants are readily detectable in S11 alone. The 1-glycan occupancy peak can be further resolved to identify variants that are glycosylated in the Fc region (more generally) and in the Fab region of the antibody heavy chain. The data show a large increase in 1-glycan Fab variant heavy chains relative to the 1-glycan Fc variant in the 2-deoxy-D-glucose sample (S11; FIG. 20B) compared to the control sample (S7; FIG. 20A) in which the 1-glycan variant consists essentially of the Fc variant.

Fig. 21 is a graph showing glycan profiles of mAb 5.948.1 produced under control conditions (i.e., in the absence of the glycosylation inhibitor 2-deoxy-D-glucose) over the time course of the method. The figure shows 3 curves showing glycan profiles of samples taken on days 7 (S5), 12 (S6) and 14 (S7) of culture. The data show that the relative amount of 1-glycan heavy chain glycosylated at the Fc region (Asn301) of the protein increases over time compared to the amount of 1-glycan heavy chain glycosylated at the Fab site (Asn 73). The data also confirmed that no 0-glycan heavy chain was detected in any sample taken at any time.

Fig. 22 is a graph showing glycan profiles of mAb 5.948.1 produced in the presence of the glycosylation inhibitor 2-deoxy-D-glucose over the time course of the method. The figure shows 3 curves showing glycan profiles of samples taken on days 7 (S9), 12 (S10) and 14 (S11) of culture. The data show that the relative amount of 1-glycan heavy chain protein increases over time compared to the amount of 2-glycan heavy chain protein. In addition, the data show that the amount of 1-glycan heavy chain protein glycosylated at the Fab region (Asn73) of the protein increases over time as compared to the amount of 1-glycan heavy chain glycosylated at the Fc site (Asn 301). This is in contrast to what was observed in the control sample, where Fc 1-glycans were increased relative to Fab 1-glycans during incubation. The data also demonstrate that the production of 0-glycan heavy chain increases over time under glycosylation-inhibiting conditions.

Fig. 23 is a graph showing glycan profiles of native antibody 5.948.1(mAb 5.948.1) produced under control conditions (i.e., in the absence of glycosylation inhibitors) together with equivalent amounts of 5.948.1 antibody samples deglycosylated with ethylene glycol-N-glycanase and samples denatured with SDS and BME, where the samples were taken on day 14 of culture (S7). As seen previously, the heavy chain of the native 5.948.1 antibody resolved into 3 major peaks, showing 2-glycan and 1-glycan variants. In the absence of the glycosylation inhibitor 2-deoxy-D-glucose, no 0-glycan occupying variants of this sample were detected.

FIG. 24 is a graph showing the glycan profile of the native antibody 5.948.1(mAb 5.948.1) produced in the presence of the glycosylation inhibitor 2-deoxy-D-glucose (solid line) compared to an equivalent sample of 5.948.1 antibody deglycosylated with ethylene glycol-N-glycanase (- - ● -) and a sample denatured with SDS and BME (- -), wherein the sample was taken on day 14 of culture (S7). The heavy chain of the native 5.948.1 antibody was resolved into 4 peaks, showing 2-glycan, 1-glycan, and 0-glycan variants. The 0-glycan occupying variants of this sample were readily detected and further confirmed the production of the 0-glycan heavy chain in the presence of the glycosylation inhibitor 2-deoxy-D-glucose.

Detailed Description

Unless defined otherwise herein, scientific and technical terms used in the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular. In general, the nomenclature used and the techniques used in cell and tissue culture, molecular biology, immunology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly employed in the art.

Unless otherwise indicated, the methods and techniques of the present invention are generally performed according to methods well known in the art and as described in various general and more specific references that are cited and discussed in the present specification. Such references include, for example, Sambrook and Russell, 2001, In: molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY; ausubel et al, 2002, In: current Protocols in Molecular Biology, John Wiley & Sons, NY; and Harlow and Lane, 1990, In: antibodies: a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, which is incorporated herein by reference. Enzymatic reactions and purification techniques were performed according to the manufacturer's instructions, as is commonly done in the art or as described herein. The nomenclature used in analytical chemistry, organic synthetic chemistry, cellular and molecular biology, immunology and medicinal chemistry, and the laboratory procedures and techniques thereof, described herein, are those well known and commonly employed in the art. Standard techniques are used for chemical synthesis, chemical analysis, pharmaceutical preparation, formulation and delivery, and treatment of patients.

Definition of

As used herein, each of the following terms in this section has its associated meaning.

The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. For example, "an element" means one element or more than one element.

As used herein, 20 conventional amino acids and their abbreviations follow conventional usage. See Immunology- -A Synthesis (2nd Edition, E.S. Golub and D.R.Gren, eds., Sinauer Associates, Sunderland, Mass. (1991)), incorporated herein by reference.

As used herein, amino acids are represented by their full name, their corresponding three letter code, or their corresponding one letter code, as shown in the following table:

a "conservative amino acid substitution" is one in which an amino acid residue is substituted with another amino acid residue having a side chain R group with similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions do not substantially alter the functional properties of the protein. In the case where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upward to correct for the conservation of the substitution. Methods for making such adjustments are well known to those skilled in the art. See, e.g., Pearson, 1994, Methods mol. biol. 243: 307-31).

Examples of groups of amino acids having side chains with similar chemical properties include: 1) aliphatic side chain: glycine, alanine, valine, leucine, and isoleucine; 2) aliphatic-hydroxy side chain: serine and threonine; 3) amide-containing side chains: asparagine and glutamine; 4) aromatic side chain: phenylalanine, tyrosine and tryptophan; 5) basic side chain: lysine, arginine and histidine; 6) acidic side chain: aspartic acid and glutamic acid; and 7) a sulfur-containing side chain: cysteine and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamic acid-aspartic acid and asparagine-glutamine.

Alternatively, conservative substitutions are described in Gonnet et al, 1992, Science 256: 1443-1445 (which is incorporated herein by reference) discloses PAM250 log-likelihood matrices having any change in positive value. A "moderately conservative" substitution is any change that has a non-negative value in the PAM250 log-likelihood matrix.

Preferred amino acid substitutions are those which: (1) reduced susceptibility to proteolysis, (2) reduced susceptibility to oxidation, (3) altered binding affinity for formation of protein complexes, and (4) other physicochemical and functional properties that confer or modify such analogs. Analogs containing substitutions, deletions, and/or insertions can include various muteins having sequences other than the specific peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in a particular sequence (preferably in a portion of the polypeptide outside the domain forming the intermolecular contacts, e.g., outside the CDRs). Conservative amino acid substitutions should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to open a helix that exists in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence). Examples of secondary and tertiary Structures of polypeptides known in the art are, for example, Proteins, Structures and Molecular Principles (Creighton, Ed., W.H.Freeman and Company, New York (1984)); introduction to Protein Structure (C.Branden and J.Tooze, eds., Garland Publishing, New York, N.Y. (1991)); and Thornton et al, 1991, Nature 354: 105) each of which is incorporated herein by reference.

Sequence similarity of polypeptides is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity due to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For example, the Genetics Computer Group (GCG available from Genetics Computer Group, inc.), also known as Wisconsin Package, is an integrated software Package of over 130 programs for accessing, analyzing, and manipulating nucleotide and protein sequences. GCG contains programs such as "Gap" and "Bestfit" which can be used to determine by default parameters sequence similarity, homology and/or sequence identity between closely related polypeptides, such as homologous proteins from different species of organisms, or between a wild-type protein and its mutant protein. See, for example, GCG version 6.1, version 7.0, version 9.1, and version 10.0.

Polypeptide sequences can also be compared using the program FASTA in GCG, using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percentage sequence identity of regions of optimal overlap between query and search sequences (Pearson, 1990, Methods enzymol.183: 63-98; Pearson, 2000, Methods mol.biol.132: 185-219). Another preferred algorithm when comparing the sequences of the invention to a database containing a large number of sequences from different organisms is the computer program BLAST, in particular blastp or tblastn, using default parameters. See, e.g., Altschul et al, 1990, j.mol.biol.215: 403-; altschul et al, 1997, nucleic acids res.25: 3389-; which is incorporated herein by reference.

The polypeptide sequences are described herein using conventional notation: the left end of the polypeptide sequence is an amino terminal; the right end of the polypeptide sequence is the carboxyl end.

An intact "antibody" comprises at least two heavy (H) chains and two light (L) chains that are linked to each other by disulfide bonds. See, generally, Fundamental Immunology, ch.7(Paul, w., 1989, ed., 2nd ed. raven Press, n.y.) (which is incorporated herein by reference in its entirety for all purposes). Each heavy chain is composed of a heavy chain variable region (HCVR or V)_H) And heavy chain constant region (C)_H) And (4) forming. The heavy chain constant region consists of 3 domains, CH1, CH2, and CH 3. Each light chain is composed of a light chain variable region (LCVR or V)_L) And a light chain constant region. The light chain constant region consists of a domain C_LAnd (4) forming. Can be combined with V_HAnd V_LThe regions are further subdivided into hypervariable regions, known as Complementarity Determining Regions (CDRs), interspersed with regions that are more conserved, known as Framework Regions (FRs). Each V_HAnd V_LConsists of 3 CDRs and 4 FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. According to Kabat, 1987and 1991, In: sequences of Proteins of Immunological Interest (national institutes of Health, Bethesda, Md.) or Chothia &Lesk，1987，J.Mol.Biol.196：901-917; chothia et al, 1989, Nature 342: 878-883 assigns amino acids to each domain. The constant region of an antibody can mediate the binding of an immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component of the classical complement system (Clq). Within the light and heavy chains, the variable and constant regions are connected by a "J" region of about 12 or more amino acids, while the heavy chain also includes a "D" region of about 10 or more amino acids. See generally, Fundamental Immunology ch.7(Paul, w., ed., 2nd ed. raven Press, n.y. (1989)).

The term "ligand binding site" as used herein refers to a portion of a polypeptide or protein, or fragment thereof, that specifically binds to another molecule, also referred to as a binding partner or cognate ligand (cognate ligand). Preferably, the ligand binding site ("LBS") may, but need not, comprise at least one glycan site (also referred to herein as a "potential glycosylation site") that may, but need not, be occupied by a sugar moiety. Examples of ligand binding sites are the ligand binding site of RAGE, which comprises a glycan site at amino acid residue No. 2 asparagine (Asn2 or N2) and further comprises a second glycan site at N58, wherein LBS specifically binds to RAGE ligands including, but not limited to, amyloid beta (a β), serum amyloid a (saa), S100, Carboxymethyllysine (CML), amphoterin, and CD11b/CD 18.

The terms "antigen binding site" or "antigen binding portion" are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., IgE). It was confirmed that the antigen binding function of the antibody can be performed by a fragment of the full-length antibody. Examples of binding fragments encompassed within the term "antigen binding site" of an antibody include (i) Fab fragments, consisting of V_L、V_H、C_LAnd C_H1 domain; (ii) f (ab')₂A fragment comprising a bivalent fragment of two Fab fragments linked by a disulfide bond at the hinge region; (iii) from V_HAnd C_H1 domainA composed Fd fragment; (iv) v with one arm consisting of antibody_LAnd V_H(iii) an Fv fragment consisting of a domain; (v) dAb fragments (Wardet et al, 1989, Nature 341: 544-546) consisting of V_HDomain composition; and (vi) an isolated Complementarity Determining Region (CDR). Furthermore, although the two domains V of the Fv fragment_LAnd V_HEncoded by different genes, but they can be joined by recombinant methods through synthetic linkers that enable them to be used as a single protein chain, where V_LAnd V_HPairing to form monovalent molecules (known as single chain fv (scfv)); see, e.g., Bird et al, 1988, Science 242: 423-426; and Huston et al, 1988, proc.natl.acad.sci.usa 85: 5879-5883. Such single chain antibodies are also encompassed within the term "antigen-binding portion" of an antibody. Other forms of single chain antibodies, such as diabodies (diabodies), are also contemplated. Diabodies are bivalent, bispecific antibodies in which V is expressed on a single polypeptide chain _HAnd V_LDomains, but use a linker that is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on the other chain and creating two antigen binding sites (see, e.g., Holliger et al, 1993, Proc. Natl. Acad. Sci. USA 90: 6444-.

In addition, an antibody or antigen-binding portion thereof can be part of a larger immunoadhesion molecule formed by covalent or non-covalent linking of the antibody or antibody portion to one or more other proteins or peptides. Examples of such immunoadhesion molecules include the generation of tetrameric scFv molecules with the streptavidin core region (Kipriyanov et al, 1995, Human Antibodies and hybrids 6: 93-101) and the generation of bivalent and biotinylated scFv molecules with cysteine residues, marker peptides and C-terminal polyhistidine tags (Kipriyanov et al, 1994, mol. immunol.31: 1047-1058). Other examples include where one or more CDRs from an antibody are incorporated covalently or non-covalently into a molecule to make it specifically bind to a molecule of interest such as IgE Immunoadhesins of antigens. In such embodiments, the CDRs may be incorporated as part of a larger polypeptide chain, may be covalently linked to another polypeptide chain, or may be non-covalently incorporated. Such as Fab and F (ab')₂The antibody portion of the fragment is, for example, the entire antibody is digested with papain or pepsin, respectively. Furthermore, antibodies, antibody portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques, as described herein.

When reference is made herein to an "antibody" in relation to the present invention, it is to be understood that an antigen binding portion thereof may also be used. The antigen binding portion competes with the intact antibody for specific binding. See generally, paul.w., ed., 1989, In: fundamental Immunology, ch.7(2nd ed., Raven Press, N.Y.) (which is incorporated herein by reference in its entirety for all purposes). The antigen binding portion may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of an intact antibody. In some embodiments, an "antigen binding portion" includes Fab, Fab ', F (ab')₂Fd, Fv, dAb, and Complementarity Determining Region (CDR) fragments, single chain antibodies (scFv), chimeric antibodies, diabodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen binding to the polypeptide. In embodiments having one or more binding sites, the binding sites may be the same as each other or may be different.

The term "monoclonal antibody" or "monoclonal antibody composition" as used herein refers to a preparation of antibody molecules of a single molecular composition. Monoclonal antibody compositions exhibit a single binding specificity and affinity for a particular epitope.

As used herein, the term "human antibody" or "fully human antibody" includes antibodies having variable regions in which both framework and CDR regions are derived from human germline (germline) immunoglobulin sequences. Furthermore, if the antibody contains a constant region, the constant region is also derived from a human germline immunoglobulin sequence. The human antibodies, or antigen-binding portions thereof, of the present disclosure can include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, as used herein, the term "human antibody" does not include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, are grafted to human framework sequences.

The term "human monoclonal antibody" or "fully human monoclonal antibody" refers to an antibody exhibiting a single binding specificity, which has variable regions in which both the framework and CDR regions are derived from human germline immunoglobulin sequences. In one embodiment, the human monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, wherein the B cell is fused to an immortalized cell.

As used herein, the term "recombinant human antibody" includes all human antibodies made, expressed, produced or isolated by recombinant methods, such as (a) antibodies isolated from an animal (e.g., a mouse) transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom (described further below); (b) antibodies isolated from host cells transformed to express human antibodies, e.g., isolated from transfectomas; (c) antibodies isolated from a library of recombinant, combinatorial human antibodies; and (d) antibodies prepared, expressed, produced or isolated by any other method including splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable regions in which both framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when animals transgenic for human Ig sequences are used, in vivo somatic mutagenesis), and thus the V's of the recombinant antibodies_HAnd V_LThe amino acid sequence of the region is derived from human germline V_HAnd V_LSequences are related to, but may not naturally occur within the human antibody germline repertoire (reportire) in vivo.

As used herein, "isotype" or "class" refers to the class of antibodies (e.g., IgM or IgG) encoded by the heavy chain constant region gene. Although the constant domain of an antibody is not involved in binding to an antigen, it exhibits various effector functions. Depending on the amino acid sequence of the heavy chain constant region, a given human antibody or immunoglobulin can be classified into one of five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM. The structure and three-dimensional configuration of different classes of immunoglobulins are well known. Among the various human immunoglobulin classes, only human IgG1, IgG2, IgG3, IgG4 and IgM are known to activate complement. Human IgG1 and IgG3 are known to mediate ADCC in humans.

As used herein, "subclass" refers to further divisions within the isotype of the heavy chain constant region genes, for example, the IgG1, IgG2, IgG3, or IgG4 subclasses within the IgG isotype.

The phrases "antibody that recognizes an antigen" and "antigen-specific antibody" are used interchangeably herein with the term "antibody that specifically binds to an antigen".

The term "antibody-dependent cellular cytotoxicity" or "ADCC" refers to a cell-mediated reaction in which non-specific cytotoxic cells (e.g., NK cells, neutrophils, macrophages, etc.) recognize antibodies bound to target cells, which subsequently causes lysis of the target cells. Such cytotoxic cells that mediate ADCC typically express Fc receptors (fcrs). Primary cells (NK cells) that mediate ADCC express Fc γ RIII, while monocytes express Fc γ RI, Fc γ RII, Fc γ RIII and/or Fc γ RIV. FcR expressed on hematopoietic cells such as ravech and Kinet, annu. 9: 457-92 (1991). To assess ADCC activity of a molecule, an in vitro ADCC assay may be performed as described in U.S. patent No. 5,500,362 or 5,821,337. Effector cells useful for such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Alternatively or additionally, ADCC activity of a molecule of interest may be assessed in vivo, for example in animal models, such as Clynes et al, proc.natl.acad.sci. (USA)95: 652-.

The term "Fc receptor" or "FcR" is used to describe a receptor that binds to the Fc region of an antibody, wherein the Fc region comprises the hinge region of a heavy chain and C_H2 and C_H3 domain. For example, the FcR may be a native sequence human FcR. An FcR may be a receptor (gamma receptor) that binds an IgG antibody and includes receptors of the Fc γ RI, Fc γ RII, Fc γ RIII, and Fc γ RIV subclasses, including allelic variants and alternatively spliced forms of these receptors. Fc γ RII receptors include Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor Fc γ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, Daeron, 1997, Annu. Rev. Immunol.15: 203-234). In ravechand Kinet, 1991, annu, rev, immunol.9: 457-92; capel et al, 1994, immunology 4: 25-34; and de Haas et al, 1995, j.lab.clin.med.126: the fcrs are reviewed in 330-341. The term "FcR" herein encompasses other fcrs, including those identified in the future. The term also includes the neonatal receptor FcRn, which is responsible for the transfer of maternal IgG to the fetus (Guyer et al, 1976, immunol.117: 587) and Kim et al, 1994, j.immunol.24: 249). The major FcR binding site on the Fc fragment of immunoglobulins is located at C _H1 and C_H2 in the hinge region between. This hinge region interacts with FcR1-3 on various leukocytes and triggers these cells to attack the target. (Wines et al, 2000, J.Immunol.164: 5313-5318). Hinge regions include, but are not limited to, the sequences described in U.S. patent No. 6,165,476.

The term "capable of inducing antibody-dependent cellular cytotoxicity" refers to the ability of a substance (such as an antibody) to cause ADCC as measured by assays known to those skilled in the art. This activity is generally characterized by binding of the Fc region to various fcrs. Without being limited to any particular mechanism, one skilled in the art will recognize that the ability of an antibody to cause ADCC may, for example, by virtue of its subclass (e.g., IgG1 or IgG3), by introducing mutations in the Fc region, or by virtue of modifications to the glycoform in the Fc region of the antibody. Such modifications are described in U.S. patent publication No. 2007/0092521.

The term "human antibody derivative" refers to any modified form of a human antibody, e.g., a conjugate of an antibody with another substance or antibody.

The term "humanized antibody" refers to an antibody in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted to human framework sequences. Additional framework region modifications can be made within the human framework sequences.

As used herein, the phrase "specifically binds" refers to a compound, e.g., a protein, nucleic acid, antibody, etc., that recognizes and binds a particular molecule, but does not substantially recognize or bind other molecules in a sample. For example, an antibody or peptide inhibitor that recognizes and binds a cognate ligand (e.g., an anti-IgE antibody that binds IgE to its cognate antigen) in a sample, but does not substantially recognize or bind other molecules in the sample. Thus, under the specified assay conditions, a particular binding moiety (e.g., an antibody or antigen-binding portion thereof) preferably binds to a particular target molecule, such as IgE, and does not bind in significant amounts to other components present in the test sample. Various assay formats can be used to select antibodies that specifically bind to the molecule of interest. For example, among the many assays that can be used to identify antibodies specifically reactive with IgE are solid phase ELISA immunoassays, immunoprecipitation, BIAcore, FACS and western blot analysis. Typically, the specific or selective reaction will be at least 2 times background signal or noise, and more typically more than 10 times background, even more particularly when the equilibrium dissociation constant (K) is high _D) An antibody is said to "specifically bind" to an antigen at ≦ 1 μ M, preferably ≦ 100nM, and most preferably ≦ 10 nM.

As used herein, the term "k_on"refers to the binding rate (on-rate) or association rate (association rate) of a particular antibody-antigen or receptor-ligand interaction, and as used herein, the term" k "refers to_off"refers to specific antibody-antigen/receptor-ligand interactionsOff-rate or dissociation rate. As used herein, the term "K_D"refers to the dissociation constant, obtained from k_offAnd k is_onRatio of (i.e., k)_off/k_on) And is expressed as molar concentration (M). K of an antibody or other binding partner can be determined using methods established in the art_DThe value is obtained. For determining K_DBy using surface plasmon resonance, usually with biosensor systems, such as BiacoreProvided is a system.

The term "chimeric antibody" as used herein means an antibody comprising regions from two or more different antibodies. In certain embodiments, a "chimeric antibody" comprises a variable region sequence derived from one species and a constant region sequence derived from another species, such as an antibody, wherein the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody. In one embodiment, one or more of the CDRs are derived from a mouse anti-human IgE antibody. In another embodiment, all CDRs are derived from a mouse anti-human IgE antibody. In another embodiment, the CDRs from more than one mouse anti-human IgE antibody are combined in a chimeric human antibody. For example, a chimeric antibody can comprise a CDR1 from the light chain of a first mouse anti-IgE antibody, a CDR2 from the light chain of a second mouse anti-human IgE antibody, and a CDR3, and a CDR3 from the light chain of a third mouse anti-human IgE antibody, and the CDRs from the heavy chain can be derived from one or more other anti-IgE antibodies. Furthermore, the framework regions may be derived from one of the same mouse anti-human IgE antibody or from one or more different mice.

Furthermore, as previously discussed herein, chimeric antibodies include antibodies that comprise portions of germline sequences derived from more than one species.

The sugar moieties of the invention will be described with reference to the usual nomenclature used to describe oligosaccharides. An overview of sugar chemistry using this nomenclature is found in Hubbard and Ivatt (1981) ann.rev.biochem.50: 555-. Such nomenclature includes, for example, Man, which stands for mannose; GicNAc, which represents 2-N-acetylglucosamine; gal, which represents galactose; fuc, which is fucose; and Glc, which represents glucose. Sialic acids are described by shorthand notation as follows: NeuNAc is 5-N-acetylneuraminic acid, and NeuNGc is 5-glycolylneuraminic acid (IUB-IUPAC Joint Commission on Biochemical Nomenclature, 1982, J.biol.chem.257: 3347-3351, (1982) J.biol.chem.257: 3352).

The sugar structures of the invention are present on proteins expressed as N-linked oligosaccharides. "N-linked glycosylation" refers to the attachment of a sugar moiety to an asparagine residue in a polypeptide chain by GlcNAc. The N-linked sugars all contain a common Man 1-6(Man1-3) Man β 1-4GlcNAc β 1-4GlcNAc β -R core structure. Thus, in the core structure, R represents the asparagine residue of the resulting glycoprotein. The sequence of the protein produced will contain asparagine-X-serine, asparagine-X-threonine, and asparagine-X-cysteine, where X is any amino acid other than proline (Asn-Xaa-Ser/Thr). In contrast, "O-linked" sugars are characterized by a common core structure, which is GalNAc linked to the hydroxyl group of threonine or serine, but no consensus sequence is required. The most important of the N-linked sugars are "complex" N-linked sugars, such as the "biantennary" structures described herein.

The skilled artisan will recognize that the glycoprotein immunoglobulin G (IgG) is associated with three types of complex biantennary structures containing 0, 1 or 2 galactose residues (Wormland et al, 1997, Biochemistry 36: 1370-1380), commonly referred to as G0, G1 and G2, respectively. With respect to human antibody molecules of the IgG class, each having an N-linked oligosaccharide with an amide side chain attached to Asn 297, Asn 297 is located at the β -4 turn of the inner surface of the CH2 domain of the Fc region (Beale and Feinstein, 1976, Q.Rev.Biophys.9: 253-259; Jefferis et al, 1995, immunol.letters.44: 111-117). The oligosaccharide moiety Asn 297 attached to the IgG CH2 domain is of the complex biantennary type with an identified core structure of a polyhexose and variable external sugar residues (see Jefferis et al, 1997, supra; Wys and Wagner, 1996, Current Opinions in Biotech.7: 409-. The core structure (GIcNAc2Man3GIcNAc) is a typical biantennary oligosaccharide and can be represented as the following scheme:

since each core structure can have bisecting N-acetylglucosamines, core fucose and galactose or sialic acid external sugars, a total of 36 structurally distinct oligosaccharides that can occupy the site Asn 297 (Jefferis and Lund, supra). It will also be appreciated that within a particular CH2 domain, glycosylation at Asn 297 may be asymmetric due to the different oligosaccharide chains attached at any Asn 297 residue within the Fc domains of the two chains. For example, while a heavy chain synthesized within a single antibody-secreting cell may be homogeneous in its amino acid sequence, it is generally differentially glycosylated, resulting in a large number of structurally distinct Ig glycoforms.

The main types of complex oligosaccharide structures found in the CH2 domain of IgG are shown on page 7 of international patent publication No. WO 99/22764.

According to the invention, G0 refers to a biantennary structure in which no terminal sialic acid (NeuAc) or Gal is present, G1 refers to a biantennary structure with one Gal and no NeuAc, and G2 refers to a biantennary structure with two terminal gals and no NeuAc. See, for example, FIG. 12 which shows exemplary structures of G0, G1, G-1, and G2.

"glycoform" refers to a linked complex oligosaccharide structure comprising various saccharide units. Such structures are described in essences of Glycobiology Varki et al, eds., Cold Spring Harbor laboratory Press, Cold Spring Harbor, NY (1999), which also provides a review of standard Glycobiology nomenclature. Such glycoforms include, but are not limited to, G2, G1, G0, G-1, and G-2 (see, for example, International patent publication Nos. WO98/58964 and WO 99/22764).

The terms "glycan site occupancy" or "glycan occupancy" include that potential N-linked or O-linked glycosylation sites in a protein may or may not comprise covalently linked sugar moieties (i.e., glycan sites are occupied). When there are at least two potential glycosylation sites on the polypeptide, 0 (occupied by 0-glycan sites), 1 (occupied by 1-glycan sites) or 2 (occupied by 2-glycan sites) can be occupied by a sugar moiety.

"glycosylation pattern" is defined as a pattern of saccharide units covalently linked to a protein (e.g., glycoform) and to a site through which the glycoform is covalently linked to the peptide backbone of the protein, more specifically to an immunoglobulin.

Proteins, including antibodies, expressed by different cell lines or in transgenic animals may have different glycan site occupancy, glycoform and/or glycosylation patterns compared to each other. However, all glycoproteins encoded by the nucleic acid molecules provided herein, or all glycoproteins comprising the amino acid sequences provided herein, including antibodies, are part of the invention, regardless of the glycosylation, glycan occupancy, or glycoform pattern of the glycoprotein.

The term "glycosylation inhibitor" or "glycosylation inhibiting compound" as used herein refers to a substance or compound wherein a polypeptide or protein produced in the presence of the substance or compound comprises at least one unglycosylated (unglycosylated) site or contains at least one sugar moiety at the same site less than an otherwise identical polypeptide or protein produced by an otherwise identical cell under otherwise identical conditions but in the absence of the substance or compound. Glycosylation inhibitors include, but are not limited to, tunicamycin homologs, streptovirins, cladosporin, amfomycin, ziromycin, antibiotic 24010, antibiotic MM 19290, bacitracin, corynetoxin, pyrophyllomycin, chlortetracycline, 1-deoxymannose nojirimycin, deoxynojirimycin, N-methyl-1-deoxymannose nojirimycin, brerafenidin A, glucose and mannose analogs, 2-deoxy-D-glucose, 2-deoxyglucose, D- (+) -mannose, D- (+) galactose, 2-deoxy-2-fluoro-D-glucose, 1, 4-dideoxy-1, 4-imino-D-mannitol (DIM), fluoroglucose, fluoromannose, UDP-2-deoxyglucose, GDP-2-deoxyglucose, mevalonate monoacyl-CoA reductase inhibitors, 25-hydroxycholesterol, swainsonine, actinone, puromycin, actinomycin D, monensin, carbonyl cyanide metachlorophenyl hydrazone (CCCP), compactin, polyterpene long alcohol-phosphoryl-2-deoxyglucose, N-acetyl-D-glucosamine, hypoxanthine, thymidine, cholesterol, glucosamine, mannosamine, spermine, glutamine, bromocyclohexatetraol, cyclohexatetraol epoxide and cyclohexatetraol derivatives, glycosylmethyl paranitrotrine, beta-hydroxynorvaline, threo beta-fluoroaspartamide, D- (+) -glucono delta-lactone, alpha-glucono delta-lactone, Di (2-ethylhexyl) phosphate, tributyl phosphate, dodecyl phosphate, 2-dimethylaminoethyl (benzhydryl) -phosphate, [2- (diphenylphosphinoxy) ethyl ] trimethylammonium iodide, iodoacetate, and/or fluoroacetate. One of ordinary skill in the art will readily recognize or be able to ascertain, without undue experimentation, glycosylation inhibiting materials that can be used in accordance with the methods and compositions of the present invention.

The term "glycosylation inhibiting amount" as used herein refers to an amount of a substance or compound wherein the polypeptide or protein produced in the presence of the substance or compound comprises a detectable reduction in glycosylation as compared to an otherwise identical polypeptide or protein produced in the absence of the substance or compound. That is, the polypeptide or protein comprises at least one more unglycosylated site (unoccupied glycan site) or less at least one sugar moiety at the same potential glycosylation site as compared to an otherwise identical polypeptide or protein produced by the cell under otherwise identical conditions but in the absence of the substance or compound.

"reduced glycosylation," "less glycosylation," or "inhibited glycosylation" used interchangeably herein includes polypeptides or proteins that contain at least one more unglycosylated (i.e., non-glycosylated) site, i.e., a completely unoccupied glycan site without an attached sugar moiety, or at least one less sugar moiety at the same potential glycosylation site, as compared to an otherwise identical polypeptide or protein produced by a cell under otherwise identical conditions but in the absence of a glycosylation inhibiting substance or compound.

As used herein, the term "effective amount" means an amount that, when administered to a cell, results in a detectable reduction in the mediation of glycosylation of a polypeptide or protein of interest produced by the cell as compared to the glycosylation of an otherwise identical polypeptide or protein produced by an otherwise identical cell under otherwise identical conditions in the absence of the substance or compound. Glycosylation of a polypeptide or protein can be assessed by a number of methods well known in the art, including, for example, such methods as disclosed herein.

The skilled artisan will appreciate that the effective amount of a compound or composition administered herein will vary and can be readily determined based on a number of factors, such as the cell type, the number of potential glycosylation sites in the polypeptide or protein, the cell culture conditions used, and the like.

As used herein, with respect to an antibody or ligand: a receptor binding pair, the term "competes" means that a first antibody, or antigen-binding portion thereof, or a receptor protein competes with a second antibody, or antigen-binding portion thereof, or a cognate ligand of said receptor protein, wherein binding of the first antibody to its cognate epitope in the presence of the second antibody is detectably reduced as compared to binding of the first antibody in the absence of the second antibody. Alternatives may be present but are not required where binding of the second antibody to its epitope in the presence of the first antibody is also detectably reduced. That is, the first antibody may inhibit binding of the second antibody to its epitope while the second antibody does not inhibit binding of the first antibody to its corresponding epitope. However, when each antibody detectably inhibits the binding of other antibodies to their cognate epitope or ligand, the antibodies are said to "cross-compete" with each other for binding to their respective epitope, whether to the same, greater or lesser extent. For example, a cross-competing antibody may bind to an epitope or portion of an epitope bound by an antibody of the invention. The invention includes competitive and cross-competitive antibodies. Whether such competition or cross-competition occurs by any mechanism (e.g., steric hindrance, conformational change, or binding to a common epitope or portion thereof, etc.), the skilled artisan, based on the teachings provided herein, will appreciate that such competing and/or cross-competing antibodies are encompassed herein and may facilitate the methods disclosed herein.

The term "instructional material" as used herein includes a publication, a record, a figure, or any other expression medium that can be used to communicate the use of the methods, compounds, combinations, and/or compositions of the invention in a kit for affecting, ameliorating or treating various diseases or conditions recited herein or for using a novel method disclosed herein. Optionally or alternatively, the instructional material may describe one or more methods of producing a therapeutic protein and/or alleviating a disease or condition in a cell, tissue or mammal, comprising using the protein of the invention as disclosed elsewhere herein.

For example, the instructional materials of the kit can be attached to or shipped with a container containing the compound and/or composition of the invention. Alternatively, the instructional material may be shipped separately from the container of the invention and the recipient used the instructional material and the compound in conjunction.

Unless otherwise indicated, the terms "patient" or "subject" are used interchangeably and refer to mammals, such as human patients and non-human primates; and veterinary subjects such as rabbits, rats, and mice; and other animals. Preferably, the patient refers to a human.

The polypeptide sequences are described herein using conventional notation: the left end of the polypeptide sequence is an amino terminal; the right end of the polypeptide sequence is the carboxyl end.

As used herein, the phrase "specifically binds" refers to a compound, e.g., a protein, nucleic acid, antibody, etc., that recognizes and binds a particular molecule, but does not substantially recognize or bind other molecules in a sample. For example, an antibody or peptide receptor that recognizes and binds a cognate ligand or binding partner in a sample (e.g., an anti-IgE antibody that binds its cognate antigen IgE, or one of the advanced glycation end product Receptor (RAGE) and its ligand, such as amyloid beta peptide or S100), but does not substantially recognize or bind other molecules in the sample. Thus, under the specified assay conditions, a specific binding moiety (e.g., an antibody or antigen-binding portion thereof, or a receptor or ligand-binding portion thereof) preferably binds to a particular target molecule and does not bind in significant amounts to other components present in the test sample. Various assay formats can be used to select antibodies or peptides that specifically bind to the molecule of interest. For example, among the many assays that can be used to identify antibodies specifically reactive with an antigen, or receptors or ligand-binding portions thereof that specifically bind to a cognate ligand or binding partner, are solid-phase ELISA immunoassays, immunoprecipitations, BIAcore ^TM(GE Healthcare，Piscataway，NJ)、FACS、Octet^TM(Forte Bio, Inc., Menlo Park, CA) and Western blot analysis. Typically, the specific or selective reaction will be at least 2 times background signal or noise, and more typically more than 10 times background, even more particularly when the equilibrium dissociation constant (K) is high_D) An antibody is said to "specifically bind" to an antigen at ≦ 1 μ M, preferably ≦ 100nM, and most preferably ≦ 10 nM.

As used herein, "substantially pure" means that the substance of interest (species) is the predominant species present (i.e., it is more abundant in the composition on a molar basis than any other single species), and preferably, the substantially purified fraction is a composition in which the substance of interest (e.g., a glycoprotein, including an antibody or receptor) comprises at least about 50% of all macromolecular species present (on a molar basis). Generally, a substantially pure composition will comprise more than about 80%, more preferably more than about 85%, 90%, 95%, and 99% of all macromolecular species present in the composition. Most preferably, the target species is purified to substantial homogeneity (contaminants are not detectable in the composition by conventional detection methods), wherein the composition consists essentially of a single macromolecular species.

As used herein, "treating" means reducing the frequency with which a patient experiences symptoms of a disease (i.e., tumor growth and/or metastasis, or other effects mediated by the number and/or activity of immune cells, etc.). The term includes administering a compound or substance of the invention to prevent or delay the onset of the symptoms, complications, or biochemical indicators of a disease, to alleviate the symptoms, or to retard or inhibit the further development of the disease, disease condition, or disorder. Treatment may be prophylactic (preventing or delaying the onset of disease, or arresting the manifestation of clinical or subclinical symptoms) or therapeutic inhibition or alleviation of symptoms following disease manifestation.

"combination therapy" includes the administration of a first therapeutic agent and another therapeutic agent as part of a particular treatment regimen, optionally including a maintenance phase, intended to provide a beneficial effect from the combined action of these therapeutic agents. The beneficial effects of the combination include, but are not limited to, pharmacokinetic or pharmacodynamic co-action resulting from the combination of the therapeutic agents. The combined administration of these therapeutic agents is typically carried out over a specified time (typically minutes, hours, days or weeks, depending on the combination selected). "combination therapy" generally does not include the administration of two or more of these therapeutic agents as part of separate monotherapy regimens that incidentally or arbitrarily result in a combination of the invention.

"combination therapy" includes administration of these therapeutic agents in a sequential manner, i.e., wherein each therapeutic agent is administered at a different time; and administering the therapeutic agents or at least two therapeutic agents in a substantially simultaneous manner. Any suitable route may affect sequential or substantially simultaneous administration of each therapeutic agent, including, but not limited to, oral routes, intravenous routes, intramuscular, subcutaneous routes, and direct absorption through mucosal tissues. The therapeutic agents may be administered by the same route or by different routes. For example, a first therapeutic agent (e.g., a chemotherapeutic agent) may be administered orally, and a second substance (e.g., an antibody or other glycoprotein) may be administered intravenously. Furthermore, the first therapeutic agent of the selected combination may be administered by intravenous injection, whereas the other therapeutic agents of the combination may be administered orally. Alternatively, for example, the two therapeutic agents may be administered by intravenous or subcutaneous injection.

In this specification, unless otherwise indicated, the term "sequential" means characterized by a regular sequence or order, for example, if an administration regimen includes administration of a therapeutic protein (e.g., an antibody, glycoprotein, etc.) and a chemotherapeutic agent, a sequential administration regimen may include administration of the therapeutic protein prior to, simultaneously, substantially simultaneously, or after administration of the chemotherapeutic agent, but the two substances are administered in a regular sequence or order. Unless otherwise indicated, the term "separate" means separating one from another. The term "simultaneously", unless otherwise indicated, means occurring or completed at the same time, i.e., the compounds of the invention are administered at the same time. The term "substantially simultaneously" means that the compounds are administered within minutes of each other (e.g., within 10 minutes of each other) and includes combined administration as well as continuous administration, but if the administration is continuous, it is only separated in time by a short period (e.g., the time it takes for a physician to administer the two compounds separately). As used herein, concurrent administration and substantially simultaneous administration are used interchangeably. Sequential administration refers to the temporally separate administration of a therapeutic protein and a chemotherapeutic agent.

"combination therapy" may also include the administration of a therapeutic agent as described above in further combination with other bioactive ingredients (such as, but not limited to, a second and different therapeutic protein, vaccine, etc.) and non-drug therapy (such as, but not limited to, surgery or radiation therapy). When the combination therapy further includes radiation therapy, the radiation therapy can be administered at any suitable time so long as the beneficial effect from the combined action of the therapeutic agent and the radiation therapy is obtained. For example, where appropriate, beneficial effects are still obtained when radiation therapy is removed from administration of the therapeutic agent over time, possibly for days or even weeks.

The term "batch culture" as used herein refers to a method of culturing cells in which all components that will ultimately be used in culturing the cells, including the culture medium as well as the cells themselves, are provided at the beginning of the culturing process. Batch cultures are usually terminated at some point, and the cells and/or components in the culture medium are harvested and optionally purified.

"bioreactor" includes any vessel that facilitates the growth of a cell culture. The bioreactor may be of any size as long as it facilitates the cultivation of the cells. Generally, the bioreactor is at least 1 liter and may be 10, 100, 250, 500, 1,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume in between. Internal conditions of the bioreactor are optionally controlled during the culturing, including but not limited to pH and temperature. The bioreactor may be comprised of any material suitable for containing a cell culture suspended in a culture medium under the culture conditions of the present invention, including glass, plastic, or metal. The term "production bioreactor" as used herein refers to the final bioreactor used in the production of a polypeptide or protein of interest. The volume of the production bioreactor is typically at least 500 liters and may be 1,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume in between. One of ordinary skill in the art will know and be able to select a suitable bioreactor for use in the practice of the present invention.

The term "cell density" as used herein refers to the number of cells present in a given volume of culture medium.

As used herein, "cell viability" refers to the ability of a cell in culture to survive under given culture conditions or experimental variables. The term as used herein also refers to the fraction of viable cells in a culture at a particular time relative to the total amount of viable and dead cells in the culture at that time.

The terms "medium", "cell culture medium" and "culture medium" as used herein refer to a solution containing nutrients that nourish growing cells. In certain embodiments, the culture medium facilitates growth of mammalian cells. In general, the culture medium provides essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements required for minimal growth and/or survival of the cells. The culture medium may also contain supplementary components that increase growth and/or survival above a minimum level, including but not limited to hormones and/or other growth factors, specific ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds typically present at very low final concentrations), amino acids, lipids, and/or glucose or other energy source. In certain embodiments, the culture medium is advantageously formulated to a pH and salt concentration that is optimal for cell survival and proliferation. In certain embodiments, the medium is a feed medium added after the start of cell culture. In certain embodiments, the cell culture medium is a mixture of the starting nutrient solution and any feed medium added after the start of cell culture.

The term "complex medium" as used herein refers to a medium containing at least one component of unknown or uncontrolled nature or quantity.

The terms "culture" and "cell culture" as used herein refer to a population of cells suspended in a culture medium (defined elsewhere herein) under conditions suitable for the survival and/or growth of the cell population. It will be clear to one of ordinary skill in the art that these terms as used herein also refer to a combination comprising a population of cells and a culture medium in which the population is suspended. In certain embodiments, the cell culture is a mammalian cell culture.

The term "defined medium" as used herein refers to a medium wherein the composition of the medium is known and controlled.

The term "fed-batch culture" as used herein includes methods of culturing cells wherein additional components are provided to the culture at one or more times after the start of the culturing process. The provided components typically comprise a nutritional supplement for cells depleted during the culture process. Additionally or alternatively, the additional components may include supplemental components (defined elsewhere herein). In certain embodiments, additional components may be provided in the feed medium. The fed-batch culture is usually terminated at some point, and the cells and/or components in the culture medium are harvested and optionally purified.

The term "modified fed-batch" or "mixed mode culture process" as used herein includes processes for culturing cells using a combination of perfusion and/or batch and/or fed-batch processes during the inoculum phase and/or the final bioreactor production phase. Exemplary improved fed-batch processes are discussed, but are not limited to, the processes in U.S. patent application publication No. US 2009/0042253 (published 2/12 2009), which is incorporated herein by reference in its entirety.

As used herein, "feed medium" refers to a solution containing nutrients that nourish growing mammalian cells that is added after the start of cell culture. The feed medium may contain the same components as provided by the starting cell culture medium. Alternatively, the feed medium may contain one or more additional components in addition to those provided by the initial cell culture medium. Additionally or alternatively, the feed medium may lack one or more components provided by the initial cell culture medium. In certain embodiments, one or more components of the feed medium are provided at the same or similar concentrations or levels as those provided for the initial cell culture medium. In certain embodiments, one or more components of the feed medium are provided at a concentration or level that is different from the concentration or level of these components provided in the initial cell culture medium. In certain embodiments, the feed medium contains supplemental components.

The term "supplemental components" as used herein encompasses components that enhance growth and/or survival above a minimum level, including, but not limited to, hormones and/or other growth factors, specific ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds typically present at very low final concentrations), amino acids, lipids, and/or glucose or other energy source. In certain embodiments, supplemental components are added to the initial cell culture. In certain embodiments, the supplemental components are added after the cell culture has begun.

The term "fragment" as used herein refers to a polypeptide and is defined as any discrete portion of a given polypeptide that is unique to or characteristic of that polypeptide. The term as used herein also refers to any discrete portion of a given polypeptide that retains at least the activity of the full-length polypeptide. In certain embodiments, the portion that retains activity is at least 10% of the activity of the full-length polypeptide. In certain embodiments, the portion of retained activity is at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the activity of the full-length polypeptide. In certain embodiments, the portion of retained activity is at least 95%, 96%, 97%, 98%, or 99% of the activity of the full-length polypeptide. In certain embodiments, the portion that retains activity is 100% or more of the activity of the full-length polypeptide. Alternatively or additionally, the term as used herein also refers to any portion of a given polypeptide that includes at least one defined sequence element found in the full-length polypeptide. In certain embodiments, the sequence element spans at least about 4-5, 10, 15, 20, 25, 30, 35, 40, 45, 50 or more amino acids of the full-length polypeptide.

The term "gene" as used herein refers to any nucleotide sequence, DNA or RNA, at least some portion of which encodes a discrete end product, typically but not limited to a polypeptide. Optionally, the term refers not only to the coding sequence encoding the polypeptide or other discrete end product, but may also include regions preceding and/or following the coding sequence that regulate the basal level of expression (see definition of "genetic control elements" below) as well as intervening sequences ("introns") between individual coding segments ("exons").

The term "genetic control element" as used herein refers to any sequence element that regulates the expression of the product of a gene to which it is operably linked. Genetic control elements may function by increasing or decreasing the expression level of a gene product, and may be located before, within, or after a coding sequence. The genetic control elements may function at any stage of gene expression, for example, to regulate initiation, extension, or termination of transcription, mRNA splicing, mRNA editing, mRNA stability, intracellular mRNA localization, initiation, extension, or termination of translation, or any other stage of gene expression. The genetic control elements may function alone or in combination with one another.

The term "host cell" as used herein refers to a cell that is grown in culture to produce a protein or polypeptide of interest according to the present invention. In certain embodiments, the host cell is a mammalian cell.

The term "hybridoma" as used herein includes cells or progeny of cells obtained by fusing an immortalized cell with an antibody-producing cell. The resulting hybridomas are antibody-producing immortalized cells. The individual cells used to produce the hybridomas can be from any mammalian source, including but not limited to rat, pig, rabbit, sheep, goat, and human. The term also includes trioma cell lines which are obtained when progeny of a heterohybrid myeloma fusion (the product of a fusion between a human cell and a mouse myeloma cell line) are subsequently fused with a plasma cell. In addition, the term includes any antibody-producing immortalized hybrid cell line, e.g., a tetragenic hybridoma (quadroma) (see, e.g., Milstein et al, 1983, Nature 537: 3053).

The term "polypeptide" as used herein refers to a sequential chain of amino acids linked together by peptide bonds. The term is used to refer to a chain of amino acids of any length, but those of ordinary skill in the art will appreciate that the term is not limited to long chains, and may refer to the smallest chain comprising two amino acids linked together by peptide bonds. The polypeptide may be processed and/or modified as known to those skilled in the art. For example, the polypeptide may be glycosylated. The polypeptide expressed according to the invention may be a therapeutic polypeptide. A therapeutic polypeptide is a polypeptide that has a biological effect on a part of the body on which it acts or acts remotely through an intermediate. Examples of therapeutic polypeptides are discussed in more detail below.

The term "protein" as used herein refers to one or more polypeptides that function as discrete units. The terms "polypeptide" and "protein" are used interchangeably if a single polypeptide is a discrete functional unit and does not require permanent or temporary physical association with other polypeptides to form a discrete functional unit. The term "protein" as used herein refers to a plurality of polypeptides that are physically coupled and function together as a discrete unit if the discrete functional unit is comprised of a plurality of polypeptides that are physically associated with each other. The protein expressed according to the present invention may be a protein therapeutic. Protein therapeutics are proteins that have a biological effect on a part of the body that acts on the part of the body or acts remotely through an intermediary. Examples of protein therapeutics are discussed in more detail below.

As used herein, "recombinantly expressed polypeptide" and "recombinant polypeptide" refer to a polypeptide expressed from a host cell that is manipulated to express the polypeptide. In certain embodiments, the host cell is a mammalian cell. In certain embodiments, such manipulations may comprise one or more genetic modifications. For example, a host cell may be genetically modified by the introduction of one or more heterologous genes encoding the polypeptide to be expressed. The heterologous recombinantly expressed polypeptide may be the same or similar to the polypeptide normally expressed in the host cell. Heterologous recombinant expression of a polypeptide may also be unrelated to the host cell, e.g., heterologous to the polypeptide normally expressed in the host cell. In certain embodiments, the heterologous, recombinantly expressed polypeptide is chimeric. For example, portions of the polypeptide may contain amino acid sequences that are the same as or similar to those of a polypeptide normally expressed in a host cell, while other portions contain amino acid sequences unrelated to the host cell. Additionally or alternatively, the polypeptide may contain amino acid sequences from two or more different polypeptides, both of which are normally expressed in the host cell. In addition, the polypeptide may contain amino acid sequences from two or more polypeptides that are not associated with the host cell. In certain embodiments, the host cell is genetically modified by activation or upregulation of one or more endogenous genes.

The term "titer" as used herein refers to the total amount of recombinantly expressed polypeptide or protein produced by a cell culture in a given volume of medium. Titers are usually expressed in units of milligrams or micrograms of polypeptide or protein per milliliter of culture medium.

Method for controlling glycosylation

The present invention provides improved methods and media formulations for the production of glycosylated proteins and/or glycosylated polypeptides in cell culture, wherein the protein or polypeptide comprises reduced glycosylation. In certain embodiments, the invention provides methods of reducing glycosylation of a protein in cell culture.

Previous work has demonstrated that the metabolic waste, lactic acid, is detrimental to cell growth, viability and/or protein production or quality. Previous work has also demonstrated that low lactate levels in cell cultures can be maintained by maintaining low glucose levels during culture to reduce glycolysis (Cruz et al, 1999, Biotechnology and Bioengineering 66: 104-113). However, since continuous monitoring and adjustment of glucose levels is not practical for large-scale production of proteins or polypeptides, previous work has demonstrated that the addition of glycolytic inhibitory substances to cell culture media can reduce glycolysis to improve protein production (international patent application No. PCT/US2007/083473, published as WO 2008/055260 on 8/5 of 2008). More specifically, it was demonstrated that the addition of the glycolytic inhibitor 2-deoxy-D-glucose reduces the lactate level in the cell culture medium and, at certain concentrations, it also increases the production of the protein of interest by the cell.

The present invention provides improved methods and media formulations for producing glycoproteins and/or polypeptides comprising reduced glycosylation by cell culture that do not require continuous monitoring and adjustment of glucose levels in the culture. The present invention provides improved methods and media formulations for producing glycoproteins or polypeptides comprising reduced glycosylation, wherein the glycoprotein or polypeptide comprises a potential glycosylation site in the ligand binding site or antigen binding site. In certain embodiments, glycoproteins or polypeptides comprising reduced glycosylation demonstrate improved biological characteristics, including, but not limited to, improved binding (e.g., increased specificity and/or avidity) to a cognate ligand or binding partner. In certain embodiments, the cell culture is a batch or fed-batch culture.

Certain compositions of the invention include a cell culture medium comprising a glycosylation inhibiting material. In certain embodiments, such glycosylation inhibiting substances include tunicamycin, tunicamycin homologs, streptovirins, brazidomycin, amfomycin, ziromycin, antibiotic 24010, antibiotic MM 19290, bacitracin, corynetoxin, oxytetracycline, chlortetracycline, 1-deoxymannose nojirimycin, deoxynojirimycin, N-methyl-1-deoxymannose nojirimycin, brefeldin A, glucose and mannose analogs, 2-deoxy-D-glucose, 2-deoxyglucose, D- (+) -mannose, D- (+) galactose, 2-deoxy-2-fluoro-D-glucose, 1, 4-dideoxy-1, 4-imino-D-mannitol (DIM), Fluoro-glucose, fluoro-mannose, UDP-2-deoxyglucose, GDP-2-deoxyglucose, mevalonate monoacyl-CoA reductase inhibitor, 25-hydroxycholesterol, swainsonine, actinone, puromycin, actinomycin D, monensin, carbonyl cyanide metachlorophenylhydrazone (CCCP), compactin, polyterpene long alcohol-phosphoryl-2-deoxyglucose, N-acetyl-D-glucosamine, hypoxanthine, thymidine, cholesterol, glucosamine, mannosamine, castanospermine, glutamine, bromocyclohexatetraol, cyclohexatetraol epoxide and cyclohexatetraol derivatives, glycosylmethyl p-nitrophenyltriazene, beta-hydroxynorvaline, threo beta-fluoro-asparagine, D- (+) -glucono delta-lactone, Di (2-ethylhexyl) phosphate, tributyl phosphate, dodecyl phosphate, 2-dimethylaminoethyl (benzhydryl) -phosphate, [2- (diphenylphosphinoxy) ethyl ] trimethylammonium iodide, iodoacetate and/or fluoroacetate. The skilled artisan, having the benefit of the teachings provided herein, will appreciate that many useful potential glycosylation inhibitors are known in the art that inhibit N-linkage, O-linkage, or both glycosylation and that their use in the novel methods of the invention can be determined. See, e.g., Elbein, 1985, Methods in enzymol.98: 135-; elbein, 1987, ann.rev.biochem.56: 497-534.

According to certain embodiments, the level of glycosylation of a recombinant protein produced by cell culture (e.g., the number of glycan sites occupied on the protein or peptide, the size and/or complexity of the glycoform at such sites, etc.) is lower than the level of glycosylation of a protein produced in an otherwise identical medium lacking such glycolysis inhibiting substances under otherwise identical conditions. According to certain embodiments, the glycosylation level of the culture is lower than the glycosylation level produced under otherwise identical conditions in an otherwise identical medium lacking such glycosylation inhibiting substances.

Other embodiments of the invention are discussed in detail below. However, one of ordinary skill in the art appreciates that various modifications of the embodiments are within the scope of the appended claims. The claims and their equivalents define the scope of the invention, which is not limited or should not be limited by this description of certain embodiments.

Cells

Any host cell that can allow cell culture and expression of a protein or polypeptide can be utilized in accordance with the present invention. In certain embodiments, the host cell is a mammal. Mammalian cell lines useful as hosts for expression are well known in the art and include many immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese Hamster Ovary (CHO) cells, HEK, human cervical cancer cells (HeLa, ATCC CCL 2), Baby Hamster Kidney (BHK) cells, monkey kidney Cells (COS), and human hepatocellular carcinoma cells (e.g., Hep G2). Other non-limiting examples of mammalian cells that can be used in accordance with The present invention include human retinoblasts (per. c6(CruCell, Leiden, The Netherlands)); SV 40-transformed monkey kidney CV1 line (COS-7, ATCC CRL 1651); human embryonic kidney line 293 or 293 cells subcloned for growth in suspension culture (Grahamat al, 1977, J.Gen Virol.36: 59); baby hamster kidney cells (BHK, ATCC CCL 10); mouse support cells (TM4, Mather, 1980, biol. reprod.23: 243-251); monkey kidney cells (CV1ATCC CCL 70); vero cells (VERO-76, ATCC CRL-1587); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TR1 cells (Mather et al, 1982, Annals N.Y.Acad.Sci.383: 44-68); MRC 5 cells; FS4 cells; human liver cancer line (Hep G2); and a number of myeloma cell lines including, but not limited to, BALB/c mouse myeloma line (NS0/1, ECACC No: 85110503), NS0 cells and Sp2/0 cells.

In addition, any number of commercially available and non-commercially available hybridoma cell lines expressing a polypeptide or protein may be utilized in accordance with the present invention. One skilled in the art will appreciate that hybridoma cell lines can have different nutritional requirements and/or can require different culture conditions for optimal growth and polypeptide or protein expression, and that conditions can be modified as desired.

The present invention includes any eukaryotic expression system known in the art or disclosed herein for producing a protein of interest, such as expression in an insect cell system, a yeast expression system, or a mammalian cell system, for example, but not limited to, CHO cells. That is, where the protein produced is otherwise glycosylated and where reduced glycosylation (N-linked and/or O-linked) is desired, any cell culture system may be used in the novel methods of the invention.

In certain embodiments, a yeast expression system is used and glycosylation control using glycosylation inhibitors reduces or eliminates N-linkage, O-linkage, or both types of glycosylation. This is particularly useful because the yeast system can mediate unwanted N-linked glycosylation, and more particularly unwanted O-linked glycosylation. Thus, the novel methods disclosed herein provide useful methods for producing proteins with reduced levels of N-linked and/or O-linked glycosylation in yeast. The resulting less glycosylated or non-glycosylated proteins may thus exhibit useful characteristics such as, but not limited to, less heterogeneity, more standardized characteristics of improved batch/lot results, improved binding characteristics, and the like.

Other cell lines that may be used are insect cell lines such as Sf9 cells. Plant host cells include, for example, tobacco, Arabidopsis, duckweed, maize, wheat, potato, and the like. Bacterial host cells include escherichia coli (e.coli) and Streptomyces species (Streptomyces species). Yeast host cells include Schizosaccharomyces pombe (Schizosaccharomyces pombe), Saccharomyces cerevisiae (Saccharomyces cerevisiae), and Pichia pastoris (Pichia pastoris).

Nucleic acid molecules encoding proteins of interest, expression vectors containing these nucleic acid molecules may be used for transfection of suitable mammalian, plant, bacterial or yeast host cells. Transformation can be performed by any known method for introducing a polynucleotide into a host cell. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene (polybrene) -mediated transfection, protoplast fusion, electroporation, encapsulation of the polynucleotide into liposomes, and direct microinjection of DNA into the nucleus. In addition, the nucleic acid molecule can be introduced into a mammalian cell by a viral vector. Methods for transforming plant cells are well known in the art and include, for example, Agrobacterium-mediated transformation, biolistic (biolistic) transformation, direct injection, electroporation, and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.

The expression vector can also be delivered to an expression system using a DNA gene gun, wherein the plasmid is precipitated onto microscopic particles, preferably gold, and the particles are pushed into the target cell or expression system. DNA Gene Gun technology is well known in the art, and devices such as "Gene guns" are commercially available for delivering microparticles into cells (e.g., Helios Gene Gun, Bio-Rad labs., Hercules, CA) and skin (PMED Device, PowderMed ltd., Oxford, UK).

Protein expression from a production cell line can be increased using a number of known techniques. For example, the glutamine synthetase gene expression system (GS system) and the protoplasmic-encoded neomycin resistance system (plasma-encoded) are common methods for increasing expression under certain conditions.

As noted above, in many cases cells will be selected or engineered to produce high levels of a protein or polypeptide of interest. Typically, cells are manipulated to produce high levels of a protein, for example, by introducing a gene encoding a protein or polypeptide of interest and/or by introducing control elements that regulate the expression of the gene encoding the polypeptide or protein of interest (whether endogenous or introduced).

Certain polypeptides may have a deleterious effect on cell growth, cell viability, or some other characteristic of the cell, in some way ultimately limiting production of the polypeptide or protein of interest. Even in a particular type of cell population engineered to express a particular polypeptide, there may be variability within the cell population such that certain individual cells will grow better and/or produce more of the polypeptide of interest. In certain embodiments, the practitioner empirically selects a cell line that is robust to growth under the particular conditions selected for culturing the cells. In certain embodiments, individual cells engineered to express a particular polypeptide are selected for large-scale production based on cell growth, final cell density, percent cell viability, titer of the expressed polypeptide, or any combination of these or any other condition deemed important by the practitioner.

Culturing cells

The present invention may be used with any cell culture method or system suitable for expressing a polypeptide. For example, the cells may be grown in batch or fed-batch culture, wherein the culture is terminated after sufficient expression of the polypeptide, and the expressed polypeptide is then harvested and optionally purified. Alternatively, the cells may be grown in perfusion culture, wherein the culture is not terminated and new nutrients and other components are added to the culture periodically or continuously during which the expressed polypeptide is harvested. In other embodiments, the cells are grown in a modified fed-batch process, such as the method described in U.S. patent application publication No. US 2009/0042253.

Cells can be grown in any convenient volume chosen by the practitioner. For example, cells may be grown in small scale reaction vessels ranging in volume from a few milliliters to a few liters. Alternatively, cells may be grown in large-scale commercial bioreactors having volumes ranging from about a minimum of 1 liter to 10, 100, 250, 500, 1,000, 2,500, 5,000, 8,000, 10,000, 12,000 liters or more, or any volume in between.

The temperature of the cell culture is selected primarily based on the temperature range at which the cell culture remains viable, produces high levels of polypeptide, minimizes the production or accumulation of metabolic waste products, and/or any combination of these or other factors deemed important by the practitioner. As a non-limiting example, CHO cells grow well at about 37 ℃ and produce high levels of protein or polypeptide. Generally, most mammalian cells grow well and/or can produce high levels of protein or polypeptide in the range of about 25 ℃ to 42 ℃, although the methods taught by the present disclosure are not limited to these temperatures. Certain mammalian cells grow well in the range of about 35 ℃ to 40 ℃ and/or can produce high levels of protein or polypeptide. In certain embodiments, the cell culture is grown at a temperature of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 ℃ for one or more times during the cell culture process. One of ordinary skill in the art will be able to select the appropriate temperature or temperatures for growing the cells based on the needs of the cells and the production requirements of the practitioner.

In addition, the culture may be subjected to one or more temperature shifts during the culturing process. The temperature shift may be relatively gradual when the temperature of the culture is shifted. For example, it may take several hours or days to complete the temperature change. Alternatively, the temperature change may be relatively abrupt. The temperature can be steadily increased or decreased during the culture. Alternatively, the temperature may be raised or lowered in discrete amounts at different times during the incubation process. The subsequent temperature or temperature range may be lower or higher than the initial or previous temperature or temperature range. One of ordinary skill in the art will appreciate that these embodiments encompass a plurality of discrete temperature transitions. For example, the temperature may be switched once (to a higher or lower temperature or temperature range), the cells maintained at this temperature or temperature range for a certain time, and then the temperature may be switched again to a new temperature or temperature range, which may be higher or lower than the previous temperature or temperature range. The temperature of the culture may be constant after each discrete transformation, or may be maintained within a certain temperature range.

As with the initial temperature or temperature range, the temperature or temperature range of the cell culture after the temperature shift is selected based primarily on the temperature at which the cell culture remains viable, the range at which high levels of polypeptides or proteins are produced, the range at which production or accumulation of metabolic waste products is minimized, and/or any combination of these or other factors deemed important by the practitioner. In general, most mammalian cells remain viable and produce high levels of protein or polypeptide in the range of about 25 ℃ to 42 ℃, although the methods taught by the present disclosure are not limited to these temperatures. In certain embodiments, mammalian cells remain viable and produce high levels of protein or polypeptide in the range of about 25 ℃ to 35 ℃. In certain embodiments, the cell culture is grown at a temperature of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 ℃ for one or more times after the temperature shift. One of ordinary skill in the art will be able to select the appropriate temperature or temperature range for growing cells after a temperature shift according to the particular needs of the cell and the particular production requirements of the practitioner. The cells may be grown for any amount of time, depending on the needs of the practitioner and the needs of the cell itself. The person skilled in the art will be able to select the exact point in time at which to switch the culture to a lower temperature based on the characteristics of the cell line used, the characteristics of the protein or polypeptide to be produced, the presence or absence of other components in the culture medium or any other factors that may be desirable for his or her experiments and/or other needs.

In certain embodiments, the cell culture is grown at a temperature ranging from about 32 ℃ to about 42 ℃ and the temperature is switched to about 25 ℃ to about 31 ℃; more preferably, the cell culture is grown at a temperature ranging from about 33 ℃ to about 37 ℃ and the temperature is switched to about 28 ℃ to about 31 ℃; more preferably, the cell culture is grown at a temperature in the range of about 34 ℃ to 37 ℃ and the temperature is switched to about 29 ℃ to about 31 ℃; more preferably, the cell culture is grown at a temperature of about 34 ℃ and the temperature is switched to about 31 ℃.

In certain embodiments, once the expressed polypeptide reaches a sufficiently high titer, the batch and fed-batch reactions are terminated, as determined by the practitioner's needs. By way of non-limiting example, cell culture can be terminated when the polypeptide titer is 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 2000mg/L or higher. One of ordinary skill in the art will be able to select one or more suitable titers at which batch and/or fed-batch cultures can be harvested. Additionally or alternatively, the batch and fed-batch reactions are terminated once the cells reach a sufficiently high density, as determined by the practitioner's needs. For example, once the cells reach 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 99% of maximum viable cell density, the culture can be terminated.

In certain embodiments, batch and/or fed-batch cell culture is terminated to prevent the production or accumulation of undesirable metabolic wastes, such as lactate and ammonium. In certain embodiments, the cell culture is terminated before lactic acid accumulates to an undesirable level in the culture. As non-limiting examples, cell culture may be terminated before lactate reaches 8, 7, 6, 5, 4, 3, 2, or 1 g/L.

In certain embodiments, once the cell density reaches a sufficiently high level, the batch and fed-batch reactions are terminated, as determined by the practitioner's needs. For example, once the cell density reaches 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 × 10⁶The cell culture may be terminated per mL or more. In certain embodiments, a cell density of 1X 10 is achieved⁶Batch and fed-batch reactions were terminated before individual cells/mL.

In certain instances, it may be beneficial or necessary to supplement the cell culture with nutrients or other media components that have been depleted or metabolized by the cells in a subsequent production phase. As non-limiting examples, it may be beneficial and necessary to supplement the cell culture with hormones and/or other growth factors, specific ions (such as sodium, chloride, calcium, magnesium and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds typically present at very low final concentrations), amino acids, lipids, or glucose or other energy sources. These supplementary components may be added all at once to the cell culture, or they may be provided to the cell culture by a series of additions.

One of ordinary skill in the art will be able to formulate specific cell culture conditions to optimize certain characteristics of the cell culture, including but not limited to the degree of glycosylation of the protein of interest, the cell growth rate, the cell viability, the final cell density of the cell culture, the final concentration of harmful metabolic byproducts such as lactate and ammonium, the final titer of the expressed polypeptide, or any combination of these, or other conditions deemed important by the practitioner.

Culture medium composition

Any of a variety of growth media may be used in accordance with the present invention. In certain embodiments, the cells are grown in any of a variety of chemically-defined media, wherein the composition of the media is known and controlled. In certain embodiments, cells are grown in any of a variety of complex media, wherein the composition of not all of the media is known and/or controlled.

During the last decades, chemically defined growth media for cell culture, including chemically defined growth media for mammalian cell culture, have been widely developed and disclosed. Defined media all components are well characterized and therefore do not contain complex additives such as serum or hydrolysates. Early media formulations were developed to allow cells to grow and maintain viability with little or no concern for protein production. Recently, media formulations have been developed for the purpose of supporting expression of high producing cell cultures producing recombinant proteins and/or polypeptides.

Defined media typically consists of approximately 50 chemical entities in water at known concentrations. Most defined media also contain one or more well-characterized proteins, such as insulin, IGF-1, transferrin, or BSA, but others do not require a protein component, and are therefore referred to as protein-free defined media. The chemical composition of defined media generally falls into five major categories: amino acids, vitamins, inorganic salts, trace elements and other classes that are difficult to classify in order.

All media, defined or complex media, include the energy source for growing the cells. Typically, the energy source is glucose, having the formula C₆H₁₂O₆Simple monosaccharides. Traditional media formulations containing relatively high levels of glucose include commercially available media such as Ham's F10(Sigma), minimal essential media ([ MEM)]Sigma), RPMI-1640(Sigma) and Darber's modified eagle's medium ([ DMEM)]Sigma). It has traditionally been thought that abundant glucose is required because it is the primary metabolic energy source for the cell. However,the rapid consumption of glucose leads to increased glycosylation. Because reduced glycosylation can provide improved biological functions and characteristics to the glycoprotein as demonstrated herein, it can be desirable to reduce and/or control the level of glucose in cell culture. Accordingly, the present invention includes reducing and/or controlling the amount of glucose in the cell culture medium. In one embodiment, the glucose concentration is maintained in the range of about 100 μ g/L to about 10g/L, more preferably, about 500 μ g/L to about 5g/L, even more preferably, about 500 μ g/L to about 3g/L, even more preferably, about 1g/L to about 3g/L, more preferably, about 1g/L to about 2g/L, and more preferably, 0.05g/L to 0.5 g/L. Most preferably, the glucose concentration is maintained at about 1.5 g/L.

The present invention includes the discovery that certain cell culture methods and media formulations minimize or even completely inhibit glycosylation of glycoproteins. Tunicamycin is an antibiotic which is a glycosylation inhibitor that inhibits the glycosyltransferase that transfers phosphate-N-acetylglucosamine (P-GlcNAc) from Uridine Diphosphate (UDP) -GlcNAc to form dolichol phosphate (Dol-P) -GlcNAc. 2-deoxy-D-glucose is a structural analog of glucose in which the hydroxyl group at the 2' position of the sugar is replaced with a hydrogen moiety. Thus, without wishing to be bound by any particular theory, it appears that the mechanism of glycosylation inhibition is unimportant in glycoproteins with lower levels of glycosylation resulting from reduced glycosylation of the protein by either inhibitor. More surprisingly, reducing glycosylation of a protein comprising a glycosylation site located within a ligand binding site mediates improved binding of the protein to a cognate ligand, despite glycosylation inhibitors used to inhibit glycosylation in cell culture.

The present disclosure demonstrates that media formulations containing glycosylation inhibiting substances, including but not limited to 2-deoxy-D-glucose and tunicamycin, result in reduced glycosylation of glycoproteins when used to grow cells in cell culture. Furthermore, the disclosure provided herein surprisingly demonstrates that when a glycosylation site is located within a ligand binding site of a glycoprotein or an antigen binding site of an antibody, reduced glycosylation at that site mediates improved biological functions of the glycoprotein (e.g., improved ligand or antigen binding) as compared to the same glycoprotein comprising higher levels of glycosylation and produced in the absence of glycosylation inhibiting substances under otherwise identical cell culture conditions. Without wishing to be bound by any particular theory, it is possible to reduce glycosylation by providing such glycosylation inhibiting substances in the cell culture medium, thereby affecting the ability of the binding site to bind its cognate ligand, thus increasing the binding affinity or specificity of the protein.

Furthermore, the disclosure provided herein surprisingly demonstrates that when the glycosylation site is located within the antigen binding site of an antibody, the novel methods disclosed herein can reduce the level of glycosylation of the antibody at both the glycosylation site located within the antigen binding site and the glycosylation site located in the constant region of the heavy chain of the antibody. Moreover, reducing glycosylation at the mediated site can improve the biological function of the antibody (e.g., improve antigen binding) as compared to the same antibody comprising higher levels of glycosylation and produced in the absence of glycosylation inhibiting substances under otherwise identical cell culture conditions.

Accordingly, the present invention provides novel methods for affecting the glycosylation level and/or binding characteristics of glycoproteins, including antibodies, without altering the amino acid sequence of the glycoprotein.

In certain embodiments, the glycosylation inhibiting material to be used according to the invention comprises 2-deoxy-D-glucose. In certain embodiments, the 2-deoxy-D-glucose is provided at a concentration of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50 or more grams per liter. In certain embodiments, the ratio of 2-deoxy-D-glucose to glucose in the cell culture is 1/50, 1/45, 1/40, 1/39, 1/38, 1/37, 1/36, 1/35, 1/34, 1/33, 1/32, 1/31, 1/30, 1/29, 1/28, 1/27, 1/26, 1/25, 1/24, 1/23, 1/22, 1/21, 1/20, 1/19, 1/18, 1/17, 1/16, 1/15, 1/14, 1/13, 1/12, 1/11, 1/10, 1/9, 1/8, 1/7, 1/6, glucose, 1/5, 1/4, 1/3, 1/2, 1/1 or any ratio higher or lower than these. One of ordinary skill in the art will appreciate that the concentration and ratio of 2-deoxy-D-glucose in the cell culture medium described above can be achieved using batch, fed-batch, perfusion, and/or modified fed-batch cultures.

The invention also includes media formulations in which the concentration of glucose is limited. In certain embodiments, the concentration of glucose is maintained in the range of about 100 μ g/L to about 10g/L, more preferably, about 100 μ g/L to about 5g/L, even more preferably, about 100 μ g/L to about 3g/L, more preferably, about 100ug/L to about 1.5g/L, more preferably, about 100ug/L to 1g/L, and most preferably, about 50ug/L to 0.5 g/L.

The present disclosure teaches that several factors may be important for reducing glycosylation of a protein of interest to provide the protein with improved biological characteristics including improved binding to cognate ligands. Such factors include, but are not limited to, the addition of glycosylation inhibitors to the cell culture, the level of glucose in the culture, the temperature of the culture including, but not limited to, subjecting the cell culture to a temperature shift, and the medium used to culture the cells.

Particular cell lines may produce different levels of glycosylation and/or glycoforms present at the glycosylation site. Regardless, utilization of the inventive methods and media compositions described herein in any given cell culture results in lower overall glycosylation of the glycoprotein as compared to the level of glycosylation of the protein produced in the absence of the glycosylation inhibitor under otherwise identical conditions.

When necessary or desired, the inventive media formulations disclosed herein may be optionally supplemented with hormones and/or other growth factors, specific ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds typically present at very low final concentrations), amino acids, lipids, protein hydrolysates, or glucose or other energy source. In certain embodiments of the invention, it may be beneficial to supplement the culture medium with a chemical inducer (indutant), such as hexamethylene-bis (acetamide) ("HMBA") and/or sodium butyrate ("NaB"). These optional supplements may be added at the beginning of the culture, or may be added later to replenish depleted nutrients or for other reasons. One of ordinary skill in the art will know of any desirable or necessary supplements that can be included in the media formulations of the present invention, and will be able to select which particular supplements to add based on his or her experimentation and/or other needs.

Polypeptides

Any polypeptide comprising a potential glycosylation site that is expressible in a host cell can be produced according to the methods and compositions disclosed herein. Even more preferably, the polypeptide comprises a potential glycosylation site in the ligand binding site of the polypeptide, such as, but not limited to, the antigen binding site of an antibody, the ligand binding site of a receptor, and the like.

The polypeptide may be expressed from an endogenous gene of the host cell, or from a heterologous gene introduced into the host cell. The polypeptide may be a naturally occurring polypeptide or, alternatively, may have a sequence that has been engineered or selected. The polypeptide to be produced may be assembled from polypeptide fragments that exist alone in nature. Additionally or alternatively, the engineered polypeptide may comprise one or more fragments that are not naturally occurring.

Exemplary glycoproteins that can be produced according to the novel methods provided herein are well known in the art and include, but are not limited to, the extensive list provided by international patent publication No. PCT/US2004/011494 filed 4/9 of 2004, and published 11/18 of 2004 as WO 2004/099231 (see, e.g., fig. 28A through 28 CC).

Practitioners of the invention will select the polypeptide of interest and will know the exact amino acid sequence. Any given protein to be expressed according to the invention will have its own specific characteristics, can influence the level of glycosylation by the cultured cells, and can be expressed at a lower level than another polypeptide or protein grown under the same culture conditions. One of ordinary skill in the art will be able to appropriately modify the inventive media and methods described herein to control the level of glycosylation of any given expressed polypeptide or protein.

In certain embodiments, the polypeptide comprises a potential glycosylation site. That is, the skilled artisan will appreciate that the polypeptide comprises features that indicate that the protein can be glycosylated by cells, including certain amino acid sequences. In certain embodiments, a potential glycosylation site is an O-linked glycosylation site comprising a serine or threonine. See Peter-Katalinic, 2005, Methods in enzymol.405: 139-171. In other embodiments, the polypeptide comprises at least one N-linked glycosylation site. See Medzihradszky, 2005, Methods in enzymol.405: 116-138. One skilled in the art will understand that cells recognize certain amino acid sequences as glycosylation signals, thereby linking glycans to certain asparagine residues. In certain embodiments, the N-linked glycosylation site comprises the amino acid sequence asparagine-X-serine (N-X-S) or asparagine-X-threonine, wherein X can be any amino acid other than proline. In certain embodiments, the N-linked glycosylation site comprises an amino acid sequence including, but not limited to, asparagine-isoleucine-threonine (NIT), asparagine-glycine-serine (NGS), asparagine-serine-threonine (NST), and asparagine-threonine-serine (NTS). Methods for identifying potential and actual Protein glycosylation sites are known in the art and include, but are not limited to, post-translational modification prediction software available at no charge at the Protein Analysis Expert System (Expert Protein Analysis System) proteomics server of Swiss Institute of bioinformatics, URLhttp:// www.expasy.ch/tools/. These various programs include, but are not limited to, DictyOGlyc (prediction of GlcNAc O-glycosylation sites in DictyoStolium; available for free at http:// www.cbs.dtu.dk/services/DictyOGlyc /); netcmlyc (prediction of C-mannosylation sites in mammalian proteins; available at http:// www.cbs.dtu.dk/services/Netcmlyc/free); NetOGlyc (prediction of O-GalNAc, mucin-type glycosylation sites in mammalian proteins; available at http:// www.cbs.dtu.dk/services/NetOGlyc/free); NetGlycate (prediction of glycation of the epsilon amino group of lysine in human proteins; available for free at http:// www.cbs.dtu.dk/services/NetNGlyc /); OGPET (prediction of O-GalNAc, mucin-type glycosylation sites in eukaryotic (non-protozoan) proteins; freely available at http:// OGPET. ute. edu/OGPET.); and YinOYang (prediction of the O-beta-GlcNAc junction site in eukaryotic protein sequences, available at http:// www.cbs.dtu.dk/services/YinOYang/free). In certain embodiments, proteins may comprise O-linked and N-linked glycosylation sites, and such proteins are well known in the art or can be readily identified by one of skill.

The polypeptides that may be desired to be expressed according to the invention are generally selected on the basis of interesting or useful biological or chemical activities. In certain embodiments, protein therapeutics or polypeptide therapeutics are expressed using the methods and/or compositions of the invention. For example, the present invention may be employed to express any pharmaceutically or commercially relevant receptor, antibody, enzyme, hormone, regulatory factor, antigen, binding agent, and the like. The following list of polypeptides and proteins that can be produced according to the invention is merely exemplary in nature and is not intended to be a limiting description. One of ordinary skill in the art will appreciate that any polypeptide or protein can be expressed in accordance with the present invention and that the particular polypeptide to be produced can be selected based on his or her particular needs.

Receptors

One class of polypeptides that have proven effective as pharmaceutical and/or commercial agents and that can be expected to be produced according to the teachings of the present invention includes receptors. The production of these molecules according to the methods and compositions of the present invention is of particular interest in view of the biological importance of the receptors and their importance as potential therapeutic agents. Receptors are typically transmembrane glycoproteins that function by recognizing extracellular signaling ligands. In addition to the ligand recognition domain, receptors typically have a protein kinase domain. This protein kinase domain initiates signaling pathways by phosphorylating target intracellular molecules when bound to ligands, resulting in developmental or metabolic changes within the cell.

In certain embodiments, the systems and methods according to the present invention express receptor for advanced glycation end products (RAGE). Among the exemplary RAGE proteins that may be expressed are provided in: international patent application No. PCT/US2005/027694, filed on 3.8.2005, now published as WO 2006/017643 on 16.2.2006; international patent application No. PCT/US2005/027705, filed on 3.8.2005, now published as WO 2006/017647 on 16.2.2006; international patent application No. PCT/US2007/001686, filed on 23.1.2007, now published as WO 2007/094926 on 23.8.2007; international patent application No. PCT/US2007/010125, filed on 25/4/2007, now published as WO2007/130302 on 15/11/2007; and international patent application No. PCT/US2008/001786 filed on 11/2/2008, now published as WO 2008/100670 on 21/8/2008 (each incorporated herein by reference in its entirety). In one embodiment, the RAGE ligand binding site comprises about amino acid 24(Gln 24; Q24) to amino acid residue number 52 (Lys 52; K52) of the amino acid sequence of full-length human RAGE (SEQ ID NO: 3), including the signal sequence. In other embodiments, the ligand binding site comprises the RAGE V-domain from about amino acid 24(Gln 24; Q24) to amino acid residue number 116 (Arg 116; R116) of the amino acid sequence of full-length human RAGE (SEQ ID NO: 3), including the signal sequence. The full-length amino acid sequence of human RAGE is shown below, the signal sequence is in lower case letters, and the two potential N-linked glycosylation sites are underlined:

Full-length human RAGE (SEO ID NO: 3)

The nucleic acid sequence encoding amino acids 1-251 of human RAGE (SEQ ID NO: 4) is as follows:

typically, the first 22 or 23 amino acids (in lower case letters) comprise a signal sequence and are not present in the mature RAGE expressed by mammalian cells. Furthermore, when the first 23 amino acids comprise a signal sequence and the mature RAGE comprises a glutamine amino acid residue (Q) at position 1, the glutamine (Q) is typically cyclized to pyroglutamic acid (shown as "pE"), such that the ligand binding site of human RAGE comprises the following sequence:

in certain embodiments, the RAGE polypeptide comprises a V-like domain of RAGE comprising the amino acid sequence:

from the disclosure provided herein, those skilled in the art will appreciate that the ligand binding site of human RAGE may comprise amino acids of any length, ranging from SEQ ID NO: 14 to SEQ id no: 16. In each example, the ligand binding site of RAGE comprises at least one N-linked glycosylation site located within the amino acid sequence of SEQ ID No: 13 and 16 (wherein the signal sequence is 22 amino acids in length), or Asn3 and 59 in the sequence of SEQ ID NO: 14. 15, 17 and 18, Asn2 and/or Asn58 (with a signal sequence of 23 amino acids in length).

According to certain embodiments, the advanced glycation end product receptor comprises soluble rage (srage). That is, sRAGE does not contain the transmembrane and cytoplasmic domains of RAGE. See Neeper et, 1992, j.biol.chem.267: 14998-15004; park et al, 1998, Nature med.4: 1025-1031. In certain embodiments, the RAGE comprises a soluble RAGE-Ig fusion protein, e.g., RAGE-Fc, comprising a portion of RAGE comprising a ligand binding site covalently linked to a portion of an immunoglobulin ("Ig"). In certain embodiments, the sRAGE fusion protein comprises an Fc of an immunoglobulin (where the Fc typically comprises, for example, the hinge region, C, of an IgG_H2 domain and C_H3 domain). In certain embodiments, sRAGE-Ig comprises the hinge portion of human Ig, wherein the hinge domain spans from the first heavy chain constant domain (C)_H1) To the second heavy chain constant domain (C)_H2) The open-ended amino acid sequence of (a). See, for example, Strom&Zhen (amino acid sequences of hinge domains of human and mouse immunoglobulins are shown in the sequence joining the bottom of column 4 to the top of column 5). In certain embodiments, a human sRAGE-Ig fusion protein comprising a hinge region encompasses a human sRAGE-Fc as shown in FIG. 3A as described in International patent application No. PCT/US2003/025996 of WO 2004/016229, filed 8/18/2003, now published 2/16/2004. In other embodiments, the sRAGE-Ig fusion protein comprises a 6 amino acid linker (i.e., IEGRMD) with a human IgG constant domain (human IgG) ₁Amino acids Pro100 to Lys330) of human RAGE extracellular domain (SEQ ID NO: 3 amino acid residues No. 1 to 344). See product number 1145-RG, R&D Systems, Minneapolis, MN. Furthermore, sRAGE-Fc fusions comprising a hinge region encompass the mouse sRAGE-Fc as described in WO 2004/016229 FIG. 1B, and can be derived from R&D Systems (Minneapolis, MN) commercially available mouse and rat sRAGE-Fc fusion proteins as described in product Nos. 1179-RG and 1616-RG, respectively. In other embodiments, the RAGE fusion protein comprises a soluble domain of mouse RAGE fused to a human Ig domain. In other embodiments, the RAGE fusion protein comprises human RAGE fused to a constant heavy chain domain from a human IgG4 immunoglobulinsRAGE part. See, for example, International patent publication No. PCT/US2008/066956 (the amino acid sequence of a human sRAGE-Fc fusion protein comprising the Fc region of human IgG4 is shown as Table 2 on page 12, paragraph 055; the amino acid sequence of a sRAGE-Fc IgG4 fusion protein comprising the 7 amino acid linker sequence GSGSGSGSGSGSGSG, which connects the sRAGE moiety to the human IgG4Fc, is shown as Table 4 on page 13, paragraph 0059; the amino acid sequence of a sRAGE-Fc fusion protein comprising the amino acid sequence of human sRAGE linked to the Fc region of human IgG4, which further comprises two point mutations at or near the putative furin cleavage site of human RAGE, is shown as Table 6 on page 15, paragraph 0063; and the amino acid sequence of a sRAGE-Fc 4 fusion protein of sRAGE-Fc fusion protein comprising the 7 amino acid linker sequence GSGSG, is shown as Table 8 on page 17, paragraph 0067, the linker connects the sRAGE to a human IgG4Fc portion, and the sRAGE-Fc IgG4 fusion protein further comprises two point mutations at or near the putative furin cleavage site of human RAGE).

In certain embodiments, sRAGE-Ig comprises an immunoglobulin moiety lacking a hinge moiety, wherein C_H2, and wherein the hinge region is replaced with a linker sequence within the RAGE domain or a sequence at least 70, 75, 80, 85, 90, 95, 97, 98 or 99% identical thereto.

In certain embodiments, the RAGE-Ig fusion protein comprises the extracellular portion of RAGE fused to a portion of Ig lacking a hinge region. In one embodiment, the extracellular portion of human RAGE comprising the V-domain of RAGE is fused to a partially human IgG1, said partially human IgG1 comprising the portion C of human IgG1_H2 domain and entire C_H3 domain and lacks a hinge region. See, e.g., WO2006/017643 and WO 2006/017647 (incorporated herein by reference in their entirety and as shown in FIG. 4, which provides sRAGE-Ig fusion proteins comprising 3 domains). In another embodiment, the RAGE-Ig fusion protein comprises 4 domains, a RAGE V-domain, a RAGE C1 domain, a partially human IgG1C_H2 Domain and entire C of human IgG1_H3 domain, and a hinge region. See, e.g., WO2006/017643 and WO 2006/017647 schemes5; and WO 2008/100470 (figures 4B and 4D provide 3-domain sRAGE-Fc fusion proteins, and as shown in figures 4A and 4C, 4-domain sRAGE-Ig fusion proteins) (each incorporated herein in its entirety) all lack a hinge region. Furthermore, in certain embodiments, sRAGE fusion proteins comprise fusion proteins comprising the extracellular domain, i.e., the soluble domain, of canine RAGE fused at the carboxy terminus to a 6-histidine tag (product number 4750-RG, R) &D Systems，Minneapolis，MN)。

Certain RAGE fusion proteins are exemplified herein. However, the present invention is not limited to these or any other RAGE polypeptides, but encompasses any RAGE protein or fragment thereof comprising a ligand binding site, preferably comprising a potential glycosylation site, or a sequence at least 70, 75, 80, 85, 90, 95, 97, 98 or 99% identical thereto.

In certain embodiments, the RAGE-fusion protein comprises a ligand binding site for RAGE starting at alanine 23 (the 22 amino acid signal sequence is not in the mature peptide) or glutamine (Q) or pyroglutamic acid (pE) at residue 24 (the 23 amino acid signal sequence is not in the mature peptide and glutamine cyclization to pE), respectively, and is set forth in SEQ ID NO: 3, Lys30 or Lys 29. In other embodiments, the RAGE portion of the fusion protein comprises the V-domain of RAGE from about amino acid alanine 23 (the 22 amino acid signal sequence is not in the mature peptide) or glutamine (Q) or pyroglutamic acid at residue 24 (the 23 amino acid signal sequence is not in the mature peptide and glutamine is cyclized to pE) and is present in SEQ ID NO: 3 (including the leader peptide). See WO2007/094926, e.g., FIG. 1D.

In other embodiments, where the RAGE-Ig fusion protein comprises a C-terminal lysine (K) amino acid residue, one of skill in the art will appreciate that the lysine residue may be truncated, resulting in a fusion protein lacking the C-terminal lysine residue.

In another aspect, the present invention provides a composition comprising an amount of a protein according to any one of the embodiments herein, wherein at least 0.5% of the protein is non-glycosylated. The percentage of protein mass relates to the number of protein molecules in the sample and, where appropriate, takes into account the effect of glycosylation on protein molecular weight. In another embodiment, the percentage of the protein in fully glycosylated form is less than 67%, or less than 65%, or less than 60%, or less than 55%, or less than 50%, or less than 45% of the total amount of protein. In another embodiment, the percentage of the mass of protein in fully glycosylated form is less than the sum of the mass of protein in all non-fully glycosylated forms. For example, where a protein has 2 potential sites for glycosylation, the percentage of the amount of sRAGE-Ig fusion protein in a fully glycosylated form (i.e., double glycosylation) is less than the sum of the amounts of protein present in the mono-glycosylated and non-glycosylated forms. In another embodiment, the invention provides a composition comprising an amount of a sRAGE-Ig fusion protein as described in any one of the embodiments herein, wherein at least 0.5% of the sRAGE-Ig fusion protein is aglycosylated. In further embodiments of any of the embodiments in this paragraph, the site may be considered non-glycosylated, wherein the number of saccharide residues attached to the site is 3 or less, or 2 or less, or 1 or less.

When used within the context of a composition comprising an amount of sRAGE-Ig fusion protein, the "amount of sRAGE-Ig fusion protein" is an amount detectable by typical analytical detection methods for proteins. In one embodiment, the amount of sRAGE-Ig fusion protein is greater than 0.01 mg. In another embodiment, the amount of sRAGE-Ig fusion protein is greater than 0.1 mg. In another embodiment, the amount of sRAGE-Ig fusion protein is greater than 1.0 mg.

In another embodiment, the percentage of the amount of sRAGE-Ig fusion protein in an unglycosylated form is at least 1%, or at least 2%, or at least 3%, or at least 4%, or at least 5%, or at least 6%, or at least 7%, at least 8%, or at least 9%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%, or at least 20%, or at least 22%, or at least 24%, or at least 26%, or at least 28%, or at least 30% of the total amount of sRAGE-Ig fusion protein. In further embodiments of any of the embodiments in this paragraph, the site may be considered non-glycosylated, wherein the number of saccharide residues attached to the site is 3 or less, or 2 or less, or 1 or less.

In another embodiment, the percentage of the fully glycosylated form of the sRAGE-Ig fusion protein is less than 67%, or less than 65%, or less than 60%, or less than 55%, or less than 50%, or less than 45% of the total amount of sRAGE-Ig fusion protein. In another embodiment, the percentage of the amount of sRAGE-Ig fusion protein in a fully glycosylated form is less than the sum of the amounts of sRAGE-Ig fusion protein in all non-fully glycosylated forms. For example, where the sRAGE-Ig fusion protein has 3 potential sites for glycosylation, the percentage of the amount of sRAGE-Ig fusion protein in fully glycosylated form (i.e., triglycosylated) is less than the sum of the amounts of sRAGE-Ig fusion protein in di-glycosylated, mono-glycosylated, and non-glycosylated forms. Thus, when the number of potential glycosylation sites is n and n is an integer greater than or equal to 1, the amount of sRAGE-Ig fusion protein containing n glycosylation sites is less than the total amount of sRAGE-Ig fusion protein present in all forms having less than or equal to n-1 glycosylation sites. In further embodiments of any of the embodiments in this paragraph, the site may be considered non-glycosylated, wherein the number of saccharide residues attached to the site is 3 or less, or 2 or less, or 1 or less.

In another aspect, the invention provides a composition comprising an amount of a fusion protein, wherein the fusion protein comprises a RAGE polypeptide linked to an immunoglobulin polypeptide,

a) wherein the RAGE polypeptide comprises human RAGE (SEQ ID NO: 3) wherein the fragment of human RAGE comprises a ligand binding site and at least one amino acid residue that may be glycosylated,

b) wherein the immunoglobulin polypeptide comprises C of an immunoglobulin_H2 domain or part C_H2 domain and immunoglobulin C_H3 domain, and

c) wherein the N-terminal residue of the immunoglobulin polypeptide is linked to the C-terminal residue of the RAGE polypeptide; and

wherein at least 0.5% of the amount of the fusion protein is non-glycosylated. In one embodiment, the fusion protein does not include a signal sequence for a RAGE polypeptide. The signal sequence may be SEQ id no: 4, 1-18, 1-22 or 1-23. In another embodiment, the RAGE polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 13. SEQ ID NO: 14 and SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof. In another embodiment, the RAGE polypeptide comprises a sequence selected from the group consisting of SEQ ID NOs: 16. SEQ ID NO: 17 and SEQ ID NO: 18, or a pharmaceutically acceptable salt thereof. In another embodiment, the N-terminal sequence of the fusion protein is selected from SEQ ID NO: 13. SEQ ID NO: 14 and SEQ ID NO: 15. in another embodiment, the RAGE polypeptide comprises the amino acid sequence of SEQ ID NO: 3, 23-116, 23-118, 23-136, 23-230, 23-256, 23-305, 23-321, 23-330, or 23-344.

When used within the context of a composition comprising an amount of a fusion protein comprising a RAGE polypeptide linked to an immunoglobulin polypeptide, the "amount of fusion protein" is an amount detectable by typical analytical detection methods for proteins. In one embodiment, the amount of the fusion protein is greater than 0.01 mg. In another embodiment, the amount of the fusion protein is greater than 0.1 mg. In another embodiment, the amount of the fusion protein is greater than 1.0 mg.

In another embodiment, the percentage of the amount of fusion protein in the unglycosylated form is at least 1%, or at least 2%, or at least 3%, or at least 4%, or at least 5%, or at least 6%, or at least 7%, at least 8%, or at least 9%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%, or at least 20%, or at least 22%, or at least 24%, or at least 26%, or at least 28%, or at least 30% of the total amount of sRAGE-Ig fusion protein. In further embodiments of any of the embodiments in this paragraph, the site may be considered non-glycosylated, wherein the number of saccharide residues attached to the site is 3 or less, or 2 or less, or 1 or less.

In another embodiment, the percentage of the amount of fusion protein in a fully glycosylated form is less than 67%, or less than 65%, or less than 60%, or less than 55%, or less than 50%, or less than 45% of the total amount of sRAGE-Ig fusion protein. In another embodiment, the percentage of the amount of the fusion protein in the fully glycosylated form is less than the percentage of the amount of the fusion protein in all non-glycosylated forms. For example, where the fusion protein has 3 potential sites for glycosylation, the percentage of the amount of the fusion protein in the fully glycosylated form (i.e., triglycosylation) is less than the percentage of the sum of the amounts of the fusion protein in the doubly glycosylated, singly glycosylated and non-glycosylated forms. In further embodiments of any of the embodiments in this paragraph, the site may be considered non-glycosylated, wherein the number of saccharide residues attached to the site is 3 or less, or 2 or less, or 1 or less.

In another embodiment, the fusion protein comprises at least 2 amino acid residues that can be glycosylated. In another embodiment, the fusion protein comprises at least 3 amino acid residues that can be glycosylated. In another embodiment, the number of potential sites for glycosylation in the fusion protein is 2. In another embodiment, the number of potential sites for glycosylation in the fusion protein is 3. In another embodiment, the RAGE ligand binding site comprises amino acid residues that may be glycosylated. In another embodiment, wherein the fusion protein comprises at least 2 amino acid residues that can be glycosylated, the first potential site of glycosylation is an amino acid residue of a RAGE ligand binding site, and the second potential site of glycosylation is an amino acid residue of an immunoglobulin polypeptide. In another embodiment, wherein the fusion protein comprises at least 3 amino acid residues that can be glycosylated, the first potential site of glycosylation is an amino acid residue of a RAGE ligand binding site, the second potential site of glycosylation is an amino acid residue of a RAGE polypeptide, and the third potential site of glycosylation is an amino acid residue of an immunoglobulin polypeptide. In further embodiments of any of the embodiments in this paragraph, the site may be considered non-glycosylated, wherein the number of saccharide residues attached to the site is 3 or less, or 2 or less, or 1 or less.

When used in the context of comprising an amount of a fusion protein comprising a RAGE polypeptide linked to an immunoglobulin polypeptide, an "immunoglobulin polypeptide" is included in the C of the immunoglobulin_H2 and C_H3 domain, and a protected FcRn scavenger receptor binding site at the junction of the domains. The immunoglobulin polypeptide may be a fragment or an entire heavy chain from any of the known heavy chain isotypes: IgG (. gamma.), IgM (. mu.), IgD (. delta.), IgE (. epsilon.) or IgA (. alpha.). Furthermore, the heavy chain (or portion thereof) may be derived from any one of the known heavy chain subtypes: IgG1(γ 1), IgG2(γ 2), IgG3(γ 3), IgG4(γ 4), IgA1(α 1), IgA2(α 2) or mutated or biologically active subtypes of these isotypes. In one embodiment, the immunoglobulin polypeptide may comprise portions or domains of different immunoglobulin isotypes and/or heavy chain subtypes. In one embodiment, the immunoglobulin polypeptide is of the IgG1 subtype. In another embodiment, the immunoglobulin polypeptide is of the IgG2 subtype. In another embodiment, the immunoglobulin polypeptide is of the IgG3 subtype. In another embodiment, the immunoglobulin polypeptide is of the IgG4 subtype. In another embodiment, the immunoglobulin polypeptide comprises a fragment of a heavy chain fragment of an Fc fragment of an immunoglobulin. In another embodiment, the immunoglobulin polypeptide comprises a heavy chain of an immunoglobulin. In another embodiment, the immunoglobulin polypeptides and fusion proteins do not include the hinge region of an immunoglobulin.

In certain embodiments, tumor necrosis factor inhibitors in the form of tumor necrosis factor alpha and beta receptors (TNFR-1; EP 417,563, published 3/20 1991; TNFR-2, EP 417,014, published 3/20 1991, each incorporated herein by reference in its entirety) are expressed in accordance with the systems and methods of the present invention (for review see Naismith and Sprang, 1995-96, J. Inflamm.47: 1-7, incorporated herein by reference in its entirety). According to certain embodiments, the tumor necrosis factor inhibitor comprises a soluble TNF receptor. In certain embodiments, the tumor necrosis factor inhibitor comprises soluble TNFR-Ig. In certain embodiments, the TNF inhibitors of the present invention are TNFRI and TNFRII soluble forms. In other embodiments, the TNF inhibitor comprises a mouse soluble TNFRII-Fc fusion protein. See, for example, WO 2004/016229 fig. 2B). In certain embodiments, the TNF inhibitor of the present invention is a soluble TNF binding protein. In certain embodiments, the TNF inhibitors of the invention are TNFR-Ig fusion proteins, e.g., TNFR-Fc or etanercept. As used herein, "etanercept" refers to TNFR-Fc, which is a dimer of two molecules of the extracellular portion of the p75 TNF-alpha receptor, each molecule consisting of the 235 amino acid Fc portion of human IgG 1.

In certain embodiments, the receptor to be produced according to the present invention is a Receptor Tyrosine Kinase (RTK). The RTK family includes receptors critical to a variety of functions and Cell types (see, e.g., Yarden and Ullrich, 1988, Ann. Rev. biochem. 57: 433-254; Ullrich and Schlessinger, 1990, Cell 61: 243-254, incorporated herein by reference). Non-limiting examples of RTKs include tumor necrosis factor alpha and beta receptors (TNFR-1; EP 417,563, published 3/20 1991; and TNFR-2; EP 417,014, published 3/20 1991; for review see Naismuth and Sprang, incorporated herein by reference), members of the Fibroblast Growth Factor (FGF) receptor family, members of the epidermal growth factor receptor (EGF) family, platelet-derived growth factor (PDGF) receptors, tyrosine kinase-I (TIE-1) and TIE-2 receptors having immunoglobulin and EGF homology domains (Sato et al, 1995, Nature 376: 70-74, incorporated herein by reference), and c-Met receptors, some of which are suggested to directly or indirectly promote angiogenesis (Muston and Alitalo, 1995, J.cell biol.129: 895-898). Other non-limiting examples of RTKs include fetal liver kinase 1(FLK-1) (sometimes referred to as kinase insert domain-containing receptor (KDR) (Terman et al, 1991, Oncogene 6: 1677-83) or vascular endothelial growth factor receptor 2(VEGFR-2)), fms-like tyrosine kinase-1 (Flt-1) (DeVries et al, 1992, Science 255; 989. sup. 991; Shibuya et al, 1990, Oncogene 5: 519. sup. 524), sometimes referred to as vascular endothelial growth factor receptor 1(VEGFR-1), neuropilin-1, endothelial factor (endoglin), endosialin, and Ax 1. One of ordinary skill in the art will know of other receptors that may be expressed according to certain methods and compositions of the present invention.

In certain embodiments, the receptor to be produced according to the invention is a G protein-coupled receptor (GPCR). GPCRs are the primary target of drug action and development, leading to more than half of the drugs currently known (Drews, 1996, Nature Biotechnology, 14: 1516), and represent the most important target for therapeutic intervention, with 30% of clinical prescriptions antagonizing or antagonizing GPCRs (Milligan, G.and Rees, S., TIPS, 20: 118-. Production of GPCRs in accordance with the present invention is also of particular interest because of the established, proven history of these receptors as therapeutic targets.

Glutamate receptors form a group of GPCRs important in neurotransmission. Glutamate is The major neurotransmitter In The CNS and is believed to play an important role In neuronal plasticity, cognition, memory, learning, and In some neurological disorders such as epilepsy, stroke, and neurodegeneration (Watson, S, and S. arknstall, In: The G-Protein Linked receptors books Book, Academic Press, San Diego calif, pp.130-132, 1994). The Vasoactive Intestinal Peptide (VIP) family is a group of related polypeptides, the action of which is also mediated by GPCRs. The major members of this family are VIP itself, secretin and growth hormone releasing factor (GRF). VIP has a wide range of physiological effects including relaxation of smooth muscle, stimulation or inhibition of secretion in various tissues, modulation of various immune cell activities, and various excitatory and inhibitory activities in the CNS. Secretin stimulates the secretion of kinases and ions in the pancreas and intestine, and is also present in small amounts in the brain.

Antibodies

Antibodies are proteins that have the ability to specifically bind to a particular antigen. Given the large number of antibodies currently used or studied as pharmaceutical or other commercial agents, the production of antibodies according to the methods and compositions of the present invention is of particular interest.

Any antibody that can be expressed in a host cell can be used in accordance with the present invention. More preferably, the antibody comprises a potential glycosylation site in or near the antigen binding site of the antibody. In one embodiment, the antibody comprises a glycosylation site in the variable domain. In another embodiment, the glycosylation site can be located in a CDR or FR, and even more preferably, the site occupancy of the glycosylation site affects binding of the antibody to its antigen.

In certain embodiments, the antibody to be expressed is a monoclonal antibody. In certain embodiments, the monoclonal antibody is a chimeric antibody. As is known in the art, chimeric antibodies contain amino acid fragments derived from more than one organism. Chimeric antibody molecules can include, for example, the antigen binding domain of an antibody from a mouse, rat, or other species, as well as human constant regions. Various methods for making chimeric antibodies have been described. See, e.g., Morrison et al, 1985, proc.natl.acad.sci.u.s.a.81: 6851; takeda et al, 1985, Nature 314: 452; cabilly et al, U.S. patent No. 4,816,567; boss et al, U.S. patent No. 4,816,397; tanaguchi et al, european patent publication EP 171496; european patent publication No. 0173494, british patent No. GB 2177096B, each incorporated herein by reference in its entirety.

In certain embodiments, the monoclonal antibody is a humanized antibody. Humanized antibodies are chimeric antibodies in which a majority of the amino acid residues are derived from human antibodies, thereby minimizing any possible immune response when delivered to a human subject. In humanized antibodies, amino acid residues in the hypervariable region are replaced by residues from non-human species which confer the desired antigen specificity or affinity. In certain embodiments, the humanized antibody has an amino acid sequence that is at least 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% identical or higher to a human antibody. In certain embodiments, the humanized antibody is optimized by introducing conservative substitutions, consensus substitutions, germline substitutions and/or back mutations. Such altered immunoglobulin molecules can be prepared by any of several techniques known in the art, (e.g., Teng et al., 1983, Proc. Natl. Acad. Sci. U.S.A.80: 7308-7312; Kozboret al, 1983, Immunology Today 4: 7279; Olsson et al, 1982, meth. enzymol.92: 3-16), and can be prepared according to the teachings of PCT publication WO92/06193 or EP 0239400, each of which is incorporated herein by reference in its entirety).

In certain instances, the antibodies of the present disclosure are human monoclonal antibodies. Such human monoclonal antibodies can be produced using transgenic or transchromosomal mice carrying portions of the human immune system rather than the mouse system. These transgenic and transchromosomal mice include the HuMAbMouse, referred to herein separately as HuMAbMouseAnd KM MouseAnd are collectively referred to herein as "human Ig mice".

HuMAb Mouse(MedarexInc.) contains a human immunoglobulin gene minilocus (minioci) encoding unrearranged human heavy (mu and gamma) and kappa light chain immunoglobulin sequences, as well as targeted mutations that inactivate endogenous mu and kappa chain loci (see, e.g., Lonberg, et al., 1994, Natu, et al.)re 368: 856-859). Thus, the mouse exhibits reduced mouse IgM or kappa expression and, in response to the immune response, class switching and somatic mutation of the introduced human heavy and light chain transgenes to generate high affinity human IgG kappa monoclonals (Lonberg et al, 1994; reviewed in Lonberg, 1994, Handbook of Experimental Pharmacology 113: 49-101; Lonberg and Huszar, 1995, Intern.Rev.Immunol.13: 65-93, Harding and Lonberg, 1995, Ann.N.Y.Acad.Sci.764: 536-. See further, Taylor et al, 1992, Nucleic Acids Research 20: 6287-6295; chen et al, 1993, International Immunol.5: 647-656; tuaillon et al, 1993, proc.natl.acad.sci.usa 90: 3720-3724; choi et al, 1993, NatureGenetics 4: 117-; chen et al, 1993, EMBO j.12: 821-830; tuaillon et al, 1994, j.immunol.152: 2912-2920; taylor et al, 1994, international immunology 6: 579-; and fisherworld et al, 1996, Nature Biotechnology 14: 845-; U.S. Pat. nos. 5,545,806 to Lonberg and Kay, all; 5,569,825; 5,625,126, respectively; 5,633,425, respectively; 5,789,650, respectively; 5,877,397, respectively; 5,661,016, respectively; 5,814, 318; 5,874,299, respectively; and 5,770,429; U.S. patent No. 5,545,807 to Surani et al; PCT publications WO 92/03918, WO 93/12227, WO 94/25585, WO 97/13852, WO 98/24884 and WO 99/45962, all Lonberg and Kay; and PCT publication No. WO01/14424 to Korman et al.

In another case, human antibodies of the disclosure can be produced using mice carrying human immunoglobulin sequences on transgenes and transchromosomes (transchromosomes), such as mice carrying human heavy chain transgenes and human light chain transchromosomes. The term "KM mica" as used herein is described in detail in PCT publication WO 02/43478 to Ishida et al^TM"of such a mouse.

In addition, alternative transgenic animal systems expressing human immunoglobulin genes are available in the art and can be used to produce the antibodies of the present disclosure. For example, an alternative transgenic system known as Xenomouse (Abgenix, Inc.); for example, U.S. Pat. nos. 5,939,598 to Kucherlapati et al; 6,075,181; 6,114,598, respectively; 6,150,584 and 6,162,963 describe such mice.

In addition, alternative transchromosomal animal systems for expressing human immunoglobulin genes are available in the art and can be used to produce the antibodies of the present disclosure. For example, a mouse carrying a human heavy chain transchromosome and a human light chain transchromosome, referred to as a "TC mouse," can be used; such mice are as described in Tomizuka et al, 2000, proc.natl.acad.sci.usa 97: 722-727. Furthermore, cows carrying human heavy and light chain transchromosomes have been described in the art (Kuroiwa et al, 2002, Nature Biotechnology 20: 889-.

Human monoclonal antibodies of the disclosure can also be prepared using SCID mice into which human immune cells have been reconstituted so that a human antibody response can be generated when immunized. Such mice are described, for example, in Wilson et al, U.S. Pat. Nos. 5,476,996 and 5,698,767.

The human monoclonal antibodies of the present disclosure can also be prepared using phage display methods for screening human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are established in the art. See, for example: U.S. Pat. Nos. 5,223,409 to Ladner et al; 5,403,484; and 5,571,698; dower et al, U.S. patent nos. 5,427,908 and 5,580,717; McCafferty et al, U.S. Pat. Nos. 5,969,108 and 6,172,197; and U.S. patent No. 5,885,793 to Griffiths et al; 6,521,404; 6,544,731, respectively; 6,555,313, respectively; 6,582,915, and 6,593,081.

In certain embodiments, an antibody of the invention comprises at least one potential glycosylation site in the variable domain of the antibody. In certain embodiments, the glycosylation site is located in the CDR, while in other embodiments, the glycosylation site is located in the FR or in both the CDR and the FR. In other embodiments, the antibody comprises at least one glycosylation site in the constant domain. Preferably, the antibody comprises a glycosylation site in the heavy chain constant domain.

In another aspect, the present invention provides a composition comprising an amount of a protein according to any one of the embodiments herein, wherein at least 0.5% of the protein is non-glycosylated. The percentage of protein mass relates to the number of protein molecules in the sample and, where appropriate, takes into account the effect of glycosylation on protein molecular weight. In another embodiment, the percentage of the fully glycosylated form of the protein is less than 98%, or less than 97%, or less than 96%, or less than 95%, or less than 94%, or less than 93%, or less than 92%, or less than 91%, or less than 90%, or less than 87%, or less than 85%, or less than 80%, or less than 75%, or less than 70%, or less than 67%, or less than 65%, or less than 60%, or less than 55%, or less than 50%, or less than 45% of the total amount of the protein. In another embodiment, the percentage of the mass of protein in fully glycosylated form is less than the sum of the mass of protein in all non-glycosylated forms. For example, where a protein has 2 potential sites for glycosylation, the percentage of the mass of the protein in fully glycosylated form (i.e., both sites are fully glycosylated, with a full glycoform present) is less than the added percentage of the mass of the protein present in both singly non-glycosylated (one site is non-glycosylated) and fully non-glycosylated (both sites are non-glycosylated), where "non-glycosylated" includes where the glycoform present at a site contains at least 1, at least 2, at least 3 sugar moieties less than the full glycoform present at that site when the protein is fully glycosylated. In another embodiment, the present invention provides a composition comprising an amount of a protein according to any one of the embodiments herein, wherein at least 0.5% of the protein is non-glycosylated. In further embodiments of any of the embodiments in this paragraph, the site may be considered non-glycosylated, wherein the number of saccharide residues attached to the site is 3 or less, or 2 or less, or 1 or less.

When used within the context of a composition comprising an amount of protein, the "protein mass" is the amount detectable by typical analytical detection methods for proteins. In one embodiment, the protein mass is greater than 0.01 mg. In another embodiment, the protein mass is greater than 0.1 mg. In another embodiment, the protein mass is greater than 1.0 mg.

In another embodiment, the percentage of the mass of protein in the unglycosylated form is at least 1%, or at least 2%, or at least 3%, or at least 4%, or at least 5%, or at least 6%, or at least 7%, at least 8%, or at least 9%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%, or at least 20%, or at least 22%, or at least 24%, or at least 26%, or at least 28%, or at least 30% of the total amount of protein. In further embodiments of any of the embodiments in this paragraph, the site may be considered non-glycosylated, wherein the number of saccharide residues attached to the site is 3 or less, or 2 or less, or 1 or less.

In another embodiment, the percentage of the protein in fully glycosylated form is less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, and less than 67%, or less than 65%, or less than 60%, or less than 55%, or less than 50%, or less than 45% of the total amount of protein. In another embodiment, the percentage of the mass of protein in fully glycosylated form is less than the sum of the mass of protein in all non-glycosylated forms. For example, where a protein has 3 potential sites for glycosylation, the percentage of the mass of the protein in fully glycosylated form (i.e., 3 sites fully glycosylated) is less than the sum of the percentages of the masses of the protein in the doubly glycosylated (2 sites fully glycosylated), singly glycosylated (1 site fully glycosylated) and unglycosylated (all 3 sites unglycosylated) forms. Thus, when the number of potential glycosylation sites is n and n is an integer greater than or equal to 1, the mass of protein comprising n glycosylation sites is less than the total amount of protein present in all forms having less than or equal to n-1 glycosylation sites. In further embodiments of any of the embodiments in this paragraph, the site may be considered non-glycosylated, wherein the number of saccharide residues attached to the site is 3 or less, or 2 or less, or 1 or less.

In another aspect, the invention encompasses a composition comprising a protein, wherein the protein is an antibody comprising a polypeptide chain comprising two potential glycosylation sites, wherein the amount of antibody in fully glycosylated form (i.e., both sites are occupied and there is a complete glycoform at each site) is less than the sum of the amounts of antibody in all non-fully glycosylated forms. As understood by the skilled person, a typical antibody typically comprises two heavy chains and two light chains. A fully glycosylated antibody may comprise 4 glycosylation sites when the heavy chain comprises two glycosylation sites (an antibody comprising one glycosylation site in the variable domain and one glycosylation site in the constant domain as schematically shown in fig. 14), or when the heavy and light chains each comprise one glycosylation site, or when each light chain comprises two glycosylation sites. Thus, in the case of a typical antibody, the antibody may contain 4 potential sites for glycosylation, two on each heavy chain, or one on each heavy and light chain, or two on the light chain, and the invention includes a composition in which the percentage of the amount of antibody in fully glycosylated form (i.e., 4 sites are fully glycosylated) is less than the sum of the percentages of the amounts of antibody present in single non-glycosylated form (one site in one chain and two on the other chain are fully glycosylated), double non-glycosylated form (two sites in one chain or one site on each chain are fully glycosylated), triple non-glycosylated form (only one site in one chain is fully glycosylated), and fully non-glycosylated form (all 4 sites are non-glycosylated). In further embodiments of any of the embodiments in this paragraph, the site may be considered non-glycosylated or incompletely glycosylated, wherein the number of saccharide residues attached to the site is 3 or less, or 2 or less, or 1 or less.

In another aspect, the present invention provides a composition comprising an amount of protein, said protein comprising an antibody, wherein at least 0.5% of the amount of said protein is non-glycosylated.

When used in the context of an amount of protein, including antibodies, the "protein mass" is the amount detectable by typical analytical detection methods for proteins. In one embodiment, the protein mass is greater than 0.01 mg. In another embodiment, the protein mass is greater than 0.1 mg. In another embodiment, the protein mass is greater than 1.0 mg.

In another embodiment, the percentage of the mass of protein in the unglycosylated form is at least 1%, or at least 2%, or at least 3%, or at least 4%, or at least 5%, or at least 6%, or at least 7%, at least 8%, or at least 9%, or at least 10%, or at least 12%, or at least 14%, or at least 16%, or at least 18%, or at least 20%, or at least 22%, or at least 24%, or at least 26%, or at least 28%, or at least 30% of the total amount of protein. In further embodiments of any of the embodiments in this paragraph, the site may be considered non-glycosylated, wherein the number of saccharide residues attached to the site is 3 or less, or 2 or less, or 1 or less.

In another embodiment, the percentage of the mass of protein in fully glycosylated form is less than 97%, less than 96%, less than 95%, less than 94%, less than 93%, less than 92%, less than 91%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 67%, or less than 65%, or less than 60%, or less than 55%, or less than 50%, or less than 45% of the total amount of protein in the composition. In another embodiment, the percentage of the mass of protein in the fully glycosylated form in the sample is less than the percentage of the mass of protein in all the non-fully glycosylated forms. For example, where a protein has 3 potential sites for glycosylation, the percentage of the mass of protein in the fully glycosylated form (i.e., triglycosylation) in the sample is less than the sum of the masses of the proteins in the di-, mono-, and non-glycosylated forms. In further embodiments of any of the embodiments in this paragraph, the site may be considered non-glycosylated, wherein the number of saccharide residues attached to the site is 3 or less, or 2 or less, or 1 or less.

In another embodiment, the protein comprises at least two amino acid residues that can be glycosylated. In another embodiment, the protein comprises at least three amino acid residues that can be glycosylated. In another embodiment, the number of potential sites for glycosylation in the protein is 2. In another embodiment, the number of potential sites for glycosylation in the protein is 3. In another embodiment, the protein comprises a ligand binding site comprising amino acid residues that can be glycosylated. In another embodiment, wherein the protein comprises at least two amino acid residues that can be glycosylated, the first potential site of glycosylation is an amino acid residue of a ligand binding site and the second potential site of glycosylation is an amino acid residue in a non-ligand binding site of the protein. In another embodiment, wherein the protein comprises at least three amino acid residues that can be glycosylated, the first potential site of glycosylation is an amino acid residue in the ligand binding site of the protein, the second potential site of glycosylation is also an amino acid residue in the ligand binding site of the protein, and the third potential site of glycosylation is an amino acid residue that is not in the ligand binding site of the protein. In further embodiments of any of the embodiments in this paragraph, the site may be considered non-glycosylated, wherein the number of saccharide residues attached to the site is 3 or less, or 2 or less, or 1 or less. In a further embodiment of any of the embodiments in this paragraph, the protein encompasses a polypeptide chain of an antibody and the ligand binding site comprises an antigen binding site of the antibody.

As one non-limiting example, an antibody that can be produced according to the present teachings is an anti-human IgE antibody. anti-IgE antibodies are a particularly promising potential therapeutic approach in the treatment of asthma and other IgE-mediated diseases such as, but not limited to, allergic rhinitis and food allergy.

anti-IgE antibodies are potentially promising therapeutic approaches for asthma because once IgE antibodies bind to an antigen, they can prevent IgE binding to fcsri, thereby preventing cross-linking of fcsri receptors. Preventing cross-linking of IgE antibodies bound to fcsri receptors inhibits the triggering of histamine release in potential allergic reactions. Thus, anti-IgE antibodies useful in the treatment of, for example, asthma, ideally bind IgE and inhibit its interaction with the fcsri receptor, but do not bind IgE that has bound to the receptor, thereby ensuring that the antibody cannot cross-link the receptor and trigger an allergic response (referred to as "non-allergic antibodies").

Examples of antibodies that can be employed in the present invention and methods for producing them are described in international application No. PCT/US2008/004286, published as WO 2008/123999 at 1/4/2008, 2008 and 16/2008, which is incorporated herein by reference. Whilst information is provided herein about the amino acid sequence of this antibody, further information can be found in WO 2008/123999; the sequences shown in these applications are incorporated herein by reference.

Certain uses of these antibodies for the treatment of various IgE-mediated diseases, including but not limited to asthma, allergic rhinitis and food allergy, are discussed in WO 2008/123999, which is incorporated herein by reference in its entirety.

In certain embodiments, the anti-IgE antibody, or antigen-binding portion thereof, comprises a potential glycosylation site. The glycosylation site can be in the CDRs, in the FRs, or in the CDRs and FRs of the antibody or antigen binding portion thereof.

In certain embodiments, an anti-IgE antibody or portion thereof directed against human IgE has an IC50 of 0.5 μ g/mL or less as measured by its ability to reduce IgE binding in a cell binding assay that utilizes a RBL-2H3 cell line transfected with human fcer 1.

In another embodimentIn an embodiment, the anti-IgE antibody or portion thereof has an IC50 of 0.5 μ g/mL or less as measured by its ability to inhibit IgE-mediated degranulation of a human fcer 1-transfected RBL-2H3 cell line, wherein RBL-2H3 (fcer 1) cells are incubated with the anti-IgE antibody and human IgE for 48 hours, washed to remove anti-IgE: IgE complexes, leaving IgE bound to fcer 1, and then stimulated with polyclonal anti-IgE antibody that cross-links the bound IgE, resulting in IgE-mediated degranulation. In another embodiment, the IC ₅₀Less than 0.2. mu.g/mL, less than 0.1. mu.g/mL, less than 0.08. mu.g/mL, or less than 0.02. mu.g/mL.

In another embodiment, the antibody or antigen-binding portion thereof directed against human IgE does not cross-link receptor-bound IgE and does not stimulate IgE-dependent degranulation of RBL-2H3(Fc ε R1) cells that are cultured with human IgE for 48 hours and then washed to remove unbound IgE. The antibodies or antigen binding portions thereof directed against human IgE of the present invention have no agonist activity against isolated RBL-2H3(Fc ε R1)

In certain embodiments, the antibody or antigen-binding portion thereof directed against human IgE does not cross-link receptor-bound IgE and does not stimulate IgE-dependent degranulation of human blood basophils that are cultured overnight with human IgE. The antibodies or antigen-binding portions thereof directed against human IgE of the present invention have no agonist activity against isolated human basophils.

In other embodiments, IgE is highly selected for antibodies or antigen-binding portions thereof to human IgE relative to human IgA, IgGI, and lgG 3.

In certain embodiments, the antibody, or antigen-binding portion thereof, binds to full-length human IgE with an affinity constant K of 15nM or less, as measured by surface plasmon resonance (BIAcore) ₀。

In certain embodiments, the non-allergic anti-IgE antibody is mAb 5.948.1 as disclosed in WO 2008/123999. 5.948.1 the antibody contains a potential N-linked glycosylation site at amino acid residue 73 of the heavy chain variable domain. More specifically, the glycosylation site is located in the third framework region (FR3) of the variable domain.

In certain embodiments, the anti-IgE antibody or portion thereof comprises a potential glycosylation site in the antigen binding domain. In certain embodiments, the antibodies comprise potential glycosylation sites in the CDRs or FRs of the variable domains.

In another embodiment, an anti-IgE antibody or portion thereof, such as antibody 5.948.1, binds to the same epitope of human IgE.

In certain embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable region that hybridizes to SEQ ID NO: 8, and a heavy chain variable domain amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 97%, even more preferably at least 98%, more preferably at least 99% sequence identity to SEQ id no: 10, a light chain variable domain amino acid sequence having at least 90%, preferably at least 95%, more preferably at least 97%, even more preferably at least 98%, more preferably at least 99% sequence identity. In another embodiment, the antibody, or antigen-binding portion thereof, comprises an amino acid sequence that is identical to SEQ ID NO: 8 and a heavy chain variable domain amino acid sequence identical to SEQ ID NO: 10, and a light chain variable domain amino acid sequence.

In certain embodiments, the antibody, or antigen-binding portion thereof, comprises a heavy chain variable domain comprising the amino acid sequence of SEQ ID NO: 8, and further comprising a light chain variable domain comprising the CDR of SEQ ID NO: 10.

In certain embodiments, the antibody, or antigen-binding portion thereof, comprises an anti-CD 33 antibody M195 as disclosed in Co et al, U.S. patent nos. 5,714,350 and 6,350,861, each incorporated herein by reference in its entirety. M195 comprises a glycosylation site within the antigen-binding portion of the antibody; more specifically, M195 comprises an N-linked glycosylation site within the third framework region of the heavy chain variable domain. That is, in the humanization process of the mouse parent antibody, a glycosylation site was introduced into FR3 of the heavy chain variable domain. Removal of glycosylation sites by mutagenesis increases binding of the antibody to CD 33. Although about 20% of antibodies are known to contain glycosylation sites within the variable domain, it is believed that glycosylation in the antigen-binding fragment of an antibody has no effect on antigen binding. See Sox & Hood, 1970, proc.natl.acad.sci.usa 66: 975. indeed, it has been reported that glycosylation at CDR2 actually increases the binding of an antibody to its antigen α - (1, 6) -glucan (Wallick et al, 1988, J.Exp.Med.168: 1099; Wright et al, 1991, EMBO J.10: 2717). Thus, once the disclosure provided herein is employed, the skilled artisan will understand that the antibodies of interest should be evaluated for the presence of potential glycosylation sites. Such assessment can be performed using a variety of methods well known in the art, including but not limited to the methods provided herein, such as amino acid sequence analysis and/or chromatographic techniques that can identify the presence of glycosylation in a protein.

Once it has been determined that an antibody comprises potential glycosylation sites in the variable domain or antigen binding portion, one skilled in the art will appreciate that the present invention encompasses reducing the level of glycosylation of the antibody according to the novel methods provided herein, which do not require alteration of the amino acid sequence of the antibody. One of skill in the art, having the benefit of the teachings provided herein, will further appreciate that the novel methods provided herein allow for the reduction of the glycosylation level of an antibody without altering the amino acid sequence of the variable region. Furthermore, those skilled in the art will appreciate that altering even a single amino acid in an antibody variable domain can have dramatic detrimental effects on antigen binding. Thus, the novel methods provided herein enable the reduction of glycosylation of an antibody, which may improve the binding characteristics of the antibody, but without altering the amino acid sequence of the variable domain.

In certain embodiments, reducing or eliminating glycosylation of a protein increases binding of the protein to at least one ligand or antigen of a receptor or antibody, respectively, as compared to binding of an otherwise identical but fully glycosylated protein to the same ligand or antigen.

In other embodiments, the skilled artisan will appreciate that the production of non-glycosylated proteins may provide important manufacturing advantages, even though the biological activity of the less glycosylated protein may not be affected as compared to an otherwise identical but more or fully glycosylated protein. That is, particularly but not exclusively, the production of proteins comprising at least two glycans and/or when glycan occupancy levels and/or glycoforms present at each site may be difficult to control, the novel methods provide useful methods for producing controlled glycans to meet specifications (e.g., aglycosylation) and/or reduce batch and/or batch failures. Thus, once subjected to the teachings provided herein, the skilled artisan will appreciate that the present invention provides novel methods to increase the robustness of the cell culture manufacturing process of glycoproteins, even if no detectable difference is obtained in reducing the biological activity of the glycosylated/non-glycosylated protein.

In another embodiment, proteins of the invention that contain lower levels of glycosylation can be useful for x-ray crystallography and trypsin digestion studies (see, e.g., Chaplin et al, 1991, GBH Monographs 15 (Protein): 279-282).

Alternatively or additionally, when the protein of the invention comprises an immunoglobulin domain, e.g. the protein is an antibody or a fusion protein comprising a portion Ig (e.g. an Fc portion), it may be advantageous to combine the amino acid modification with one or more further amino acid modifications that alter the biological characteristics of the protein.

In addition to modifications made within the framework or CDR regions, or alternatively, the glycoproteins of the present disclosure may be engineered to include modifications within the Fc region, typically in order to alter one or more functional properties of the antibody, such as serum half-life, complement fixation, Fc receptor binding, and/or antigen-dependent cellular cytotoxicity. In addition, the antibodies of the present disclosure may be chemically modified (e.g., one or more chemical moieties may be attached to the antibody) or modified to alter the glycosylation thereof, again in order to alter one or more functional properties of the antibody. Each of these aspects is described in further detail below. The numbering of residues in the Fc region is that of the EU index of Kabat.

In one instance, the hinge region of CH1 is modified such that the number of cysteines in the hinge region is altered, e.g., increased or decreased. Such a process is further described in U.S. Pat. No. 5,677,425 to Bodmer et al. For example, the number of cysteine residues in the hinge region of CH1 is altered to facilitate assembly of the light and heavy chains or to increase or decrease the stability of the antibody.

In another instance, the Fc hinge region of an antibody is mutated to reduce the biological half-life of the antibody. More specifically, one or more amino acid mutations are introduced into the C of the Fc-hinge fragment_H2-C_H3 domain interface region, such that the antibody has impaired staphylococcal (staphylococcus) protein a (SpA) binding relative to native Fc-hinge domain SpA binding. This method is described in further detail in U.S. Pat. No. 6,165,745 to Ward et al.

In another instance, the antibody is modified to increase its biological half-life. Various methods are possible. For example, as described in U.S. Pat. No. 6,277,375 to Ward, one or more of the following mutations may be introduced: T252L, T254S, T256F. Alternatively, to increase biological half-life, as described in U.S. Pat. Nos. 5,869,046 and 6,121,022 to Presta et al, can be at C _H1 or CL regions to contain salvage receptor binding epitopes from C of the Fc region of IgG_H2 domain.

In another instance, the Fc region is altered by replacing at least one amino acid residue with a different amino acid residue to alter the effector function of the antibody. For example, one or more amino acids selected from amino acid residues 234, 235, 236, 237, 297, 318, 320 and 322 may be replaced with a different amino acid residue, such that the antibody has altered affinity for an effector ligand, but retains the antigen binding ability of the parent antibody. The effector ligand for which affinity is altered may be, for example, an Fc receptor or the C1 component of complement. Such methods are described in further detail in U.S. Pat. Nos. 5,624,821 and 5,648,260 to Winter et al.

In another instance, one or more amino acids selected from amino acid residues 329, 331 and 322 can be replaced with a different amino acid residue, such that the antibody has altered C1q binding and/or reduced or disrupted Complement Dependent Cytotoxicity (CDC). This method is described in further detail in U.S. Pat. No. 6,194,551 to Idusogene et al.

In another example, one or more amino acid residues within amino acid positions 231 and 239 are altered in order to thereby alter the ability of the antibody to fix complement. This process is further described in PCT publication WO 94/29351 to Bodmer et al.

In another example, the Fc region is modified to increase the ability of the antibody to mediate antibody-dependent cellular cytotoxicity (ADCC) and/or to increase the affinity of the antibody for fey receptors by modifying one or more amino acids at the following positions: 238. 239, 248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276, 278, 280, 283, 285, 286, 289, 290, 292, 293, 294, 295, 296, 298, 301, 303, 305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333, 334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414, 416, 419, 430, 434, 435, 437, 438 or 439. This process is further described in PCT publication WO 00/42072 to Presta. Furthermore, binding sites for Fc γ R1, Fc γ RII, Fc γ RIII and FcRn have been mapped on human IgG1 and variants with improved binding have been described (see Shield et al, 2001 J.biol.chem.276: 6591-6604). Specific mutations at positions 256, 290, 298, 333, 334 and 339 were shown to enhance binding to Fc γ RIII. Furthermore, the following combination mutants were demonstrated to improve Fc γ RIII binding: T256A/S298A, S298A/E333A, S298A/K224A and S298A/E333A/K334A. For example, the 22B5/DLE antibody described herein uses the IgG1 subclass of the IgG isotype, but has incorporated the following mutations (as compared to the wild-type IgG1 subclass) by site-directed mutagenesis: S247D; a 338L; and I340E. Similarly, such S247D, a338L, and I340E mutations have been introduced into the 24C7, 1D9, and 2D2 monoclonal antibodies, as described in more detail below. As further described in the examples, such mutations may increase the affinity of the antibody for the Fc γ receptor, and thus increase its effector function. Accordingly, the invention encompasses an antibody comprising at least one mutation in the Fc region and having a detectably greater ADCC response than an otherwise identical antibody not comprising said mutation.

In certain embodiments, a monoclonal, chimeric, single chain, or humanized antibody described above can contain amino acid residues that do not naturally occur in any antibody in any species in nature. For example, these exogenous residues may be utilized to confer new or modified specificity, affinity, or effector function to a monoclonal, chimeric, single-chain, or humanized antibody.

Blood coagulation Factor (Clotting Factor)

Coagulation factors have been shown to be effective as pharmaceutical and/or commercial agents. In view of the importance of recombinant coagulation factors in the treatment of diseases such as hemophilia, it is of particular interest that the methods and compositions according to the invention optimize the expression of recombinantly produced coagulation factors. One non-limiting example of a coagulation factor that can be produced according to the present invention is coagulation factor IX (factor IX, or "FIX"). FIX is a single chain glycoprotein, the deficiency of which results in hemophilia B, a condition in which the patient's blood cannot coagulate. Thus, any small wound that causes bleeding is a potentially life-threatening event.

FIX has multiple glycosylation sites, including N-linked and O-linked sugars. FIX produced by chinese hamster ovary ("CHO") cells in cell culture showed some variability in the oligosaccharide chain of serine 61. These different glycoforms and other potential glycoforms may have different abilities to induce coagulation and/or may have different stability in the blood when administered to a human or animal, resulting in less effective coagulation.

Hemophilia a, which is clinically indistinguishable from hemophilia B, is caused by a deficiency in another glycoprotein, human factor VIII, which is synthesized as a single-chain zymogen and then processed into a double-chain active form. The present invention may also be employed to control or alter the glycosylation pattern of factor VIII to modulate its clotting activity. Other glycoprotein coagulation factors whose glycosylation pattern can be produced and controlled or altered in accordance with the present invention include, for example, but are not limited to, tissue factor and von willebrand factor.

Enzyme

Another class of polypeptides that have proven effective as pharmaceutical and/or commercial agents and that can be expected to be produced according to the teachings of the present invention include enzymes. In view of the importance of recombinant enzymes in disease therapy and other commercial and pharmaceutical applications, the production of enzymes according to the invention is of particular interest.

As a non-limiting example, a deficiency in Glucocerebrosidase (GCR) results in a disease condition known as gaucher's disease, which is caused by the accumulation of glucocerebrosidase in the lysosomes of certain cells. Subjects with gaucher disease exhibit a range of symptoms including splenomegaly, hepatomegaly, skeletal disorders, thrombocytopenia, and anemia. Friedman and Hayes demonstrated that recombinant GCR (rGCR) containing a single substitution in the primary amino acid sequence exhibited altered glycosylation patterns, particularly increased fucose and N-acetylglucosamine residues as compared to naturally occurring GCR (see U.S. Pat. No. 5,549,892, incorporated herein by reference in its entirety). Therefore, it is contemplated to produce GCR according to the method of the present invention. One of ordinary skill in the art will know of other desirable enzymes that can be produced according to the methods of the present invention.

Growth factors and other signaling molecules

Another class of polypeptides that have proven effective as pharmaceutical and/or commercial agents and that can be expected to be produced according to the teachings of the present invention include growth factors and other signal molecules. In view of the biological importance of growth factors and other signal molecules and their importance as potential therapeutic agents, the production of these molecules according to the methods and compositions of the present invention is of particular interest. Growth factors are generally glycoproteins secreted by cells and bind to and activate receptors on other cells, triggering metabolic or developmental changes in the recipient cell.

Non-limiting examples of mammalian growth factors and other signaling molecules include cytokines; epidermal Growth Factor (EGF); platelet Derived Growth Factor (PDGF); fibroblast Growth Factor (FGF), such as aFGF and bFGF; transforming Growth Factors (TGF), such as TGF-alpha and TGF-beta, including TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, or TGF-beta 5; insulin-like growth factors-I and-II (IGF-I and IGF-II); des (1-3) -IGF-I (brain IGF-I), insulin-like growth factor binding protein; CD proteins, such as CD-3, CD-4, CD-8 and CD-19; erythropoietin; an osteoinductive factor; an immunotoxin; bone Morphogenetic Protein (BMP); interferons, such as interferon- α, - β, and- γ; colony Stimulating Factors (CSF), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (TL), e.g., IL-1 through IL-10; tumor Necrosis Factor (TNF) alpha and beta; an insulin a chain; insulin B chain; proinsulin; follicle stimulating hormone; a calcitonin; luteinizing hormone; glucagon; coagulation factors, such as factor VIIIC, factor IX, tissue factor and von willebrand factor; anti-coagulation factors, such as protein C; atrial natriuretic peptides; a pulmonary surfactant; plasminogen activators, such as urokinase or human urine or tissue type plasminogen activator (t-PA); bombesin; thrombin, hematopoietic growth factors; enkephalinase; RANTES (modulation activates normal T cell expression and secretion factors); human macrophage inflammatory protein (MIP-1-alpha); a Muller inhibitor; a relaxin a chain; a relaxin B chain; (ii) prorelaxin; mouse gonadotropin-related peptides; neurotrophic factors, such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5 or-6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factors, such as NGF-beta. One of ordinary skill in the art will know of other growth factors or signal molecules that may be expressed according to the present invention.

Introduction of nucleic acids for expression of polypeptides into host cells

In certain embodiments, the nucleic acid molecule introduced into the cell encodes a polypeptide that is desired to be expressed according to the present invention. In certain embodiments, the nucleic acid molecule can encode a gene product that induces the cell to express the desired polypeptide. For example, the introduced genetic material may encode a transcription factor that activates transcription of an endogenous or heterologous polypeptide. Alternatively or additionally, the introduced nucleic acid molecule may increase translation or stability of the polypeptide expressed by the cell.

The nucleic acid may be any nucleic acid encoding a protein comprising at least one potential glycosylation site, N-linkage or O-linkage. In other embodiments, the nucleic acid encodes a protein comprising at least one N-linked and one O-linked potential glycosylation site. The presence of potential glycosylation sites may be known for a particular protein or may be predicted based on factors well known in the art, including but not limited to the presence of typical glycosylation sites in the amino acid sequence of the protein, many freely available computer software programs for predicting glycosylation sites, and the like. In other embodiments, the proteins are known to be glycosylated, such proteins include, but are not limited to, antibodies and a number of glycoproteins, such as the glycoprotein shown in WO 2004/099231. Preferably, the glycosylation site is known to be within a portion of the protein that is associated with or involved in the biological function of the protein, such as, but not limited to, a site that mediates the interaction of the protein with another protein. Even more preferably, glycosylation of the protein is known to affect the biological function of the protein. However, any protein comprising potential glycosylation sites can be produced using the novel methods provided herein, and any effect on the function of interest, e.g., interaction with another protein, can be evaluated. That is, the method according to the present invention can be compared to a protein produced in the presence of a glycosylation inhibitor for a function of interest and an otherwise identical protein produced under otherwise identical conditions but in the absence of the glycosylation inhibitor to assess whether glycosylation affects the function of interest. When glycosylation affects the function of interest of a protein, the protein is a candidate to be produced according to the method of the invention. Thus, any therapeutic protein of interest, wherein glycosylation affects, mediates or is associated with a therapeutic function of the protein, can be produced by the novel methods of the invention.

Nucleic acid sequences encoding proteins of interest can be obtained by methods well known in the art, and many nucleic acid sequences are readily available from many databases. In certain embodiments, the sequences are known in the art. See, for example, human RAGE sequences are available from GenBank (accession No. NM — 001136), and the sequences of various RAGE-Ig fusion proteins are available in the art, as are the sequences of many other proteins of interest, including antibodies. In other embodiments, where the nucleic acid sequence encoding the protein of interest is not known, the sequence can be obtained using standard methods well known in the art. In certain embodiments, the nucleic acid sequence may, but need not, be codon optimized to increase expression of the protein in a host cell used in the methods of the invention. Techniques for codon optimization to increase host cell expression of a nucleic acid of interest are well known in the art and include, but are not limited to, the techniques described by Angov et al (2008, PloS ONE 3: e2189[ available at www.plosone.org ]) and Hatfield & Roth (2007, Biotechnol. Ann. Rev. 13: 27-42), and protein expression optimization services are also commercially available from, for example, CODA Genomics, Inc. (Laguna Hills, Calif.) and GENEART AG (Regensburg, Germany).

Once obtained, the nucleic acid encoding the therapeutic protein of the invention is then introduced into a host cell. Methods are known in the art that are suitable for introducing nucleic acids sufficient to effect expression of a polypeptide of interest into a host cell. See, e.g., Gething et al, 1981, Nature 293: 620 and 625; mantei et al, 1979, Nature, 281: 40-46; levinson et al ep 117,060; and EP 117,058, each incorporated herein by reference. For mammalian cells, common methods for introducing genetic material into cells include Graham and devan der Erb, 1978, Virology 52: 456-: 73 Lipofectamine^TM(Gibco BRL) method. Axel has described a general overview of mammalian cell host system transformation in U.S. patent No. 4,399,216. For various techniques for introducing genetic material into mammalian cells, see Keown et al, 1990, Methods in enzymology 185: 527- & gt 537; mansour et al, 1988, Nature, 336: 348 and 352; sambrook and Russell, 2001, In: molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY; and Ausubel et al, 2002, In: current Protocols in Molecular Biology, John Wiley &Sons，NY。

In certain embodiments, the nucleic acid to be introduced is in the form of a naked nucleic acid molecule. In certain aspects of these embodiments, the nucleic acid molecule introduced into the cell consists only of nucleic acid encoding the polypeptide and the necessary genetic control elements. In certain aspects of these embodiments, the nucleic acid encoding the polypeptide (including the necessary regulatory elements) is contained within a plasmid vector. Non-limiting representative examples of suitable vectors for expressing polypeptides in mammalian cells include pcDNA 1; pCD, see Okayama, et al, 1985, mol.cell biol.5: 1136-; pMClneo Poly-A; see Thomas, et al, 1987, Cell 51: 503-512; baculovirus vectors such as pAC 373 or pAC 610; CDM8(Seed, 1987, Nature 329: 840) and pMT2PC (Kaufman et al, 1987, EMBO J.6: 187-195). In certain embodiments, the nucleic acid molecule to be introduced into the cell is contained within a viral vector. For example, a nucleic acid encoding a polypeptide can be inserted into a viral genome (or a portion of a viral genome). Regulatory elements that direct the expression of the polypeptide may be included within the nucleic acid inserted into the viral genome (i.e., linked to the gene inserted into the viral genome), or may be provided by the viral genome itself.

Naked DNA can be introduced into cells by forming a precipitate containing DNA and calcium phosphate. Additionally or alternatively, naked DNA can also be introduced into cells by forming a mixture of DNA and DEAE-dextran and incubating the mixture with the cells, or by incubating the cells and DNA together in a suitable buffer and subjecting the cells to high voltage electrical pulses (i.e., by electroporation). In certain embodiments, naked DNA is introduced into a cell by mixing the DNA with a liposome suspension containing cationic lipids. The DNA/liposome complex is then incubated with the cells. Naked DNA can also be injected directly into cells by, for example, microinjection.

Additionally or alternatively, naked DNA may be introduced into cells by complexing the DNA to a cation, such as polylysine, that is coupled to a ligand for a cell surface receptor (see, e.g., Wuand Wu, 1988, J.biol. chem.263: 14621; Wilson et al., 1992, J.biol. chem.267: 963-. The DNA-ligand complex binds to the receptor, facilitating the uptake of DNA by receptor-mediated endocytosis.

The use of viral vectors, which contain a particular nucleic acid sequence, such as a cDNA encoding a polypeptide, is a common method for introducing nucleic acid sequences into cells. Infecting cells with a viral vector has the advantage that most cells receive the nucleic acid, which may avoid the need to select cells that have received the nucleic acid. In addition, molecules encoded by cDNA contained within viral vectors, such as viral vectors, are typically expressed at high levels in cells that have received the viral vector nucleic acid.

The use of defective retroviruses for gene transfer for gene therapy purposes is well characterized (for a review see Miller, A.D., Blood 76: 271, 1990). Recombinant retroviruses can be constructed having a nucleic acid encoding a polypeptide of interest inserted into the retroviral genome. In addition, portions of the retroviral genome may be removed to render the retrovirus replication defective. The replication-defective retroviruses are then packaged into viral particles that can be used to infect target cells with the helper virus by standard techniques.

The genome of an adenovirus can be manipulated so that it encodes and expresses a protein of interest, but its ability to replicate in the normal lytic viral life cycle is inactivated. See, e.g., Berkner et al, BioTechniques 6: 616, 1988; rosenfeld et al, Science 252: 431-434, 1991; and Rosenfeld et al, Cell 68: 143-155, 1992. Suitable adenoviral vectors derived from the adenoviral strain Ad 5-type dl324 or other strains of adenovirus (e.g., Ad2, Ad3, Ad7, etc.) are known in the art. The advantages of recombinant adenoviruses are that they do not require dividing cells as efficient gene delivery vectors (vehicle) and can be used to infect a variety of cell types, including airway epithelial cells (Rosenfeld et al, 1992, cited supra), endothelial cells (Lemarchand et al, Proc. Natl. Acad. Sci. USA 89: 6482-. Furthermore, the introduced adenoviral DNA (and the foreign DNA contained therein) is not integrated into the genome of the host cell, but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in cases where the introduced DNA is integrated into the host genome (e.g., retroviral DNA). Moreover, the adenoviral genome is very capable of carrying exogenous DNA (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al, cited supra; Haj-Ahmand Graham, J.Virol 57: 267, 1986). Most replication-defective adenovirus vectors currently in use lack all or part of the viral E1 and E3 genes, but retain up to 80% of the adenoviral genetic material.

Adeno-associated virus (AAV) is a naturally occurring defective virus that requires another virus, such as adenovirus or herpes virus, as a helper virus for efficient replication and production life cycle. (for a review see Muzyczka et al, curr. topics in micro. and immunol.158: 97-129, 1992). Adeno-associated virus is also one of the few viruses that can integrate their DNA into non-dividing cells and exhibit a high frequency of stable integration (see, e.g., Flotte et al, am.J.Respir.cell.mol.biol.7: 349-356, 1992; Samulski et al, J.Virol.63: 3822-3828, 1989; and McLaughlin et al, J.Virol.62: 1963-1973, 1989). Vectors containing AAV as low as 300 base pairs can be packaged and can integrate. The space of the foreign DNA is limited to about 4.5 kb. AAV vectors as described by Tratschin et al (mol.cell.biol.5: 3251-3260, 1985) can be used for introducing DNA into cells. Various nucleic acids have been introduced into different cell types using AAV vectors (see, e.g., Hermonat et al, Proc. Natl. Acad. Sci. USA 81: 6466-6470, 1984; Tratschin et al, mol. cell. biol. 4: 2072-2081, 1985; Wondsford et al, mol. Endocrinol. 2: 32-39, 1988; Tratschin et al, J. Virol. 51: 611-619, 1984; and Flotte et al, J. biol. chem. 268: 3781-3790, 1993).

In certain embodiments, the invention includes the use of mammalian artificial chromosomes to introduce a DNA or gene of interest into a host cell using, for example, Artificial Chromosome Expression (ACE) techniques to generate cell lines expressing recombinant proteins. These mammalian artificial chromosomes offer advantages over conventional techniques for cellular protein production due to their high load-bearing capacity and independence from the ability to self-replicate integrated into the host genome. Previously, it was demonstrated that large satellite DNA-based artificial chromosomes, known as ACE (artificial chromosome expression), could be readily regenerated in a variety of host cell contexts (Cska et al, 2000, J.cell Sci.113: 3207-3216; Hadlaczky, 2001, curr.Opin.mol.Ther.3: 125-132; Lindenbaum et al, 2004, Nucleic Acids Research 32: 21; Perez et al, 2004. Bioprocessing J.July/August 2004: 61-68; Stewart et al, 2002, Gene Ther.9: 719 723; Vanderbyl et al, 2005, exp. Hematol.33: 1470-14765).

When the method used to introduce the nucleic acid molecule into a population of cells results in alteration of a majority of the cells and efficient expression of the polypeptide by the cells, the altered population of cells can be used without further isolation or subcloning of individual cells within the population. That is, sufficient polypeptide production is possible by the cell population so that no further cell isolation is required, and the population can be used immediately to inoculate a cell culture for polypeptide production. In certain embodiments, it may be desirable to isolate and expand a homogenous cell population from a single cell that efficiently produces a polypeptide.

In addition to introducing a nucleic acid molecule encoding a polypeptide of interest into a cell, the introduced nucleic acid may encode another polypeptide, protein, or regulatory element that induces or increases the expression level of the protein or polypeptide endogenously produced by the cell. For example, a cell is capable of expressing a particular polypeptide, but may fail without additional treatment of the cell. Similarly, the cell may express an insufficient amount of the polypeptide for the desired purpose. Thus, agents that stimulate the expression of a polypeptide of interest can be used to induce or increase the expression of the polypeptide by a cell. For example, the introduced nucleic acid molecule may encode a transcription factor that activates or upregulates transcription of the polypeptide of interest. Expression of such transcription factors in turn leads to expression of the polypeptide of interest or more robust expression. Similarly, the introduced nucleic acid molecule may contain one or more regulatory elements that probe (titrate) one or more transcriptional repressors from the regulatory region of the polypeptide of interest.

In certain embodiments, a nucleic acid that directs the expression of a polypeptide is stably introduced into a host cell. In certain embodiments, the nucleic acid that directs the expression of the polypeptide is transiently introduced into the host cell. One of ordinary skill in the art can choose to stably or transiently introduce nucleic acids into cells based on his or her experimental needs.

The gene encoding the polypeptide of interest may optionally be linked to one or more regulatory genetic control elements. In certain embodiments, the genetic control element directs constitutive expression of the polypeptide. In certain embodiments, genetic control elements that provide inducible expression of a gene encoding a polypeptide of interest may be used. The use of inducible genetic control elements (e.g., inducible promoters) allows for the modulation of the production of a polypeptide in a cell. Non-limiting examples of potentially useful inducible genetic control elements for use in eukaryotic cells include hormone regulatory elements (see, e.g., Mader, S, and White, J.H., Proc. Natl. Acad. Sci. USA 90: 5603-. Additional cell-specific or other regulatory systems known in the art can be used in accordance with the methods and compositions described herein.

In certain embodiments, the nucleic acid sequence encoding the polypeptide is codon optimized to increase expression levels in any particular cell. Methods for codon optimization are well known in the art and include, for example, the methods described by Angov et al (2008, PloS ONE 3: e2189[ available at www.plosone.org ]) and Hatfield & Roth (2007, Biotechnol. Ann. Rev.13: 27-42). In addition, codon optimization services are commercially available from, for example, CODA Genomics, Inc. (Laguna Hills, Calif.) and GENEART AG (Regensburg, Germany).

In other embodiments, when the polypeptide comprises a signal sequence, an endogenous signal sequence naturally associated with the polypeptide can be replaced with a signal sequence not normally associated with the polypeptide to increase the level of expression of the polypeptide in cultured cells. That is, the polypeptides of the invention may be expressed as fusion protein polypeptides fused to a heterologous peptide, preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide (e.g., at Ala22 or Glu23 of human RAGE). The heterologous sequence of choice is preferably one that is recognized and processed (i.e., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native signal sequence, the signal sequence may be replaced by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leader sequence. For yeast secretion, the native signal sequence may be replaced by, for example, a yeast invertase leader, an alpha-factor leader including yeast (Saccharomyces) and Kluyveromyces (Kluyveromyces) alpha-factor leaders, or an acid phosphatase leader, a Candida albicans (C.albicans) glucoamylase leader, or a signal such as that described in WO 1990/13646. Mammalian signal sequences as well as viral secretion leader sequences are available in mammalian cell expression, e.g., the human interleukin-3 signal sequence (amino acid sequence MSRLPVLLLLQLLVRPAMA [ SEQ ID NO: 19 ]; encoded by nucleic acid sequence ATGAGCCGCCTGCCCGTCCTGCTCCTGCTCCAACTCCTGGTCCGCCCCGCCATGGCT [ SEQ ID NO: 20 ]), the herpes simplex gD signal. The nucleic acid of such precursor region is linked in-frame to a nucleic acid encoding a protein of interest.

In certain embodiments, when the protein comprises an endogenous signal sequence, the nucleic acid sequence encoding the signal sequence can be codon optimized to increase the level of expression of the protein in the host cell.

One of ordinary skill in the art would be able to select and optionally appropriately modify methods for introducing genes that result in the expression of a polypeptide of interest by a cell in accordance with the teachings of the present invention.

Isolation of expressed Polypeptides

In certain embodiments, it is desirable to isolate and/or purify a protein or polypeptide expressed according to the present invention. In certain embodiments, the expressed polypeptide or protein is secreted into the culture medium, so cells and other solids can be removed, for example, as a first step in the purification process, such as by centrifugation or filtration.

When using recombinant techniques, the protein may be produced intracellularly, in the periplasmic space, or directly secreted into the culture medium. If the antibody variant is produced intracellularly, as a first step, for example, by centrifugation or ultrafiltration, particulate debris, host cells or lysed fragments can be removed. Carter et al, 1992, Bio/Technology 10: 163-167 describes a method for isolating antibodies secreted into the periplasmic space of E.coli. Briefly, the cell mass (paste) was thawed in the presence of sodium acetate (pH 3.5), EBTA and phenylmethylsulfonyl fluoride (PMSF) for about 30 minutes. Cell debris can be removed by centrifugation.

When the protein is secreted into the culture medium, the supernatant from such expression systems is generally first concentrated using commercially available protein concentration filters, e.g., Amicon or Millipore Pellicon ultrafiltration units. Protease inhibitors such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

Polypeptides or proteins may be isolated and purified by standard Methods including, but not limited to, chromatography (e.g., ion exchange, affinity, size exclusion, and hydroxyapatite chromatography), gel filtration, centrifugation, or differential solubility, ethanol precipitation (see, e.g., Scopes, In: Protein Purification Principles and Practice, 2 d edition, Springer-Verlag, New York, 1987; Higgins & Hames, In: Protein expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher. (eds.), In: Guide to Protein Purification: Methods In Enzymology, software, 1997, each incorporated herein In its entirety), or by any other available technique for Protein Purification.

In particular for immunoaffinity chromatography, proteins can be isolated by binding the protein to an affinity column comprising antibodies directed against the protein and attached to a stationary support. Alternatively, affinity tags such as influenza virus coat sequences, polyhistidine or glutathione-S-transferase can be attached to the protein by standard recombinant techniques to allow easy purification by a suitable affinity column. One of ordinary skill in the art will know of other known affinity tags that facilitate isolation of the expressed polypeptide. Protease inhibitors such as phenylmethylsulfonyl fluoride (PMSF), leupeptin, pepstatin or aprotinin may be added at any or all stages to reduce or eliminate degradation of the polypeptide or protein during purification. The use of protease inhibitors is often advantageous when the cells must be lysed to isolate and purify the expressed polypeptide or protein.

For antibodies or proteins comprising immunoglobulin Fc domains, protein compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being a preferred purification technique. The suitability of protein A as an affinity ligand depends on the presence of the antibody or The type and isotype of any immunoglobulin Fc domain in the Fc fusion protein. Protein A can be used to purify antibodies based on human IgG1, IgG2, or IgG4 heavy chains (Lindmark et al, 1983, J.Immunol meth.62: 1-13). The G protein is recommended for all mouse isotypes and for human IgG3(Guss et al, 1986, EMBO J.5: 1567-1575). The matrix to which the affinity ligand is attached is most often agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly (styrene divinyl) benzene allow faster flow rates and shorter processing times than can be achieved with agarose. When the antibody variant comprises a CH3 domain, Bakerbond ABXTM resin (j.t.baker, phillips burg, n.j.) facilitates purification. Depending on the antibody variant to be recovered, other techniques for protein purification are also available, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, heparin Sepharose^TMChromatography on an anion or cation exchange resin (e.g., polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation.

After any preliminary purification step, the mixture containing the protein or antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography, using an elution buffer at a pH between about 2.5-4.5, preferably at a low salt concentration (e.g., about 0-0.25M salt).

One of ordinary skill in the art will appreciate that the exact purification technique may vary depending on the characteristics of the polypeptide or protein to be purified, the characteristics of the cell expressing the polypeptide or protein, and the composition of the medium in which the cell is grown.

Pharmaceutical composition

The invention encompasses the preparation and use of pharmaceutical compositions comprising as an active ingredient a polypeptide or protein of the invention. The invention further encompasses the preparation and use of pharmaceutical compositions comprising as an active ingredient a polypeptide or protein of the invention in combination with a second therapeutic agent, e.g., a chemotherapeutic agent, another antibody or protein, an immunostimulant, etc. Such pharmaceutical compositions may consist of each active ingredient alone, as a combination of at least one active ingredient (e.g., an effective dose of a therapeutic protein, an effective dose of a therapeutic agent) in a form suitable for administration to an individual, or the pharmaceutical composition may comprise the active ingredients in combination with one or more pharmaceutically acceptable carriers, one or more additional (active or inactive) ingredients, or some combination of these.

In one embodiment, the second therapeutic agent (e.g., chemotherapeutic agent, second therapeutic protein, second antibody, etc.) is administered parenterally (e.g., intravenously) in an aqueous solution concurrently with the oral administration of the second therapeutic agent in pill/capsule form. However, based on the disclosure provided herein, the skilled artisan will appreciate that the invention is not limited to these, or any other formulations, dosages, routes of administration, and the like. Rather, the invention encompasses any formulation or method of administering a protein in combination with a second therapeutic agent, including, but not limited to, administering each substance separately in different formulations by different routes of administration, as well as administering the protein and therapeutic agent in a single composition (e.g., when the second therapeutic agent is a protein, such as another antibody, a second therapeutic protein, etc.), wherein administration is intravenous in an aqueous composition), and the like. Thus, the following discussion describes various formulations for practicing the methods of the invention, including the administration of any therapeutic protein, in combination with any other therapeutic protein or compound, but the invention is not limited to these formulations, but rather includes any formulation that can be readily determined for use in the methods of the invention by one of skill in the art, such as once guided by the teachings provided herein.

The therapeutic proteins used in the methods of the invention may be incorporated into pharmaceutical compositions suitable for administration to an individual. Typically, the pharmaceutical composition comprises the protein and a pharmaceutically acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any and all physiologically compatible solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. Examples of pharmaceutically acceptable carriers include one or more of water, saline, hydrochloric acid buffered saline, glucose, glycerol, ethanol, and combinations thereof. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol (sorbitol), sorbitol or sodium chloride in the composition. Pharmaceutically acceptable substances, such as wetting or minor amounts of auxiliary substances, such as wetting or emulsifying agents, preservatives or buffers, which may increase the shelf life and effectiveness of the protein or portion thereof.

The therapeutic protein used in the present invention may be in various forms. These forms include, for example, liquid, semi-solid, and solid dosage forms, such as liquid solutions (e.g., injectable and infusible solutions), dispersions or suspensions, tablets, pills, powders, liposomes, and suppositories. The preferred form depends on the therapeutic agent, the intended mode of administration and the therapeutic application. Typically preferred compositions are in the form of injectable or infusible solutions, such as compositions similar to those used for passive immunization of humans with other antibodies. Preferred modes of administration are parenteral (e.g., intravenous, subcutaneous, intraperitoneal, intramuscular). In a preferred embodiment, the protein is administered by intravenous infusion or injection. In another preferred embodiment, the protein is administered by intramuscular or subcutaneous injection.

Therapeutic compositions must generally be sterile and stable under the conditions of manufacture and storage. The compositions may be formulated as solutions, microemulsions, dispersions, liposomes or other ordered structures suitable for high drug concentrations. Sterile injectable solutions can be prepared by incorporating the protein in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a base dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

Pharmaceutical compositions suitable for injectable use typically include sterile aqueous solutions (in which water is soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL ^TM(BASF, Parsippany, n.j.) or hydrochloric acid buffered saline (PBS). In all cases, the composition should be sterile and should flow to the extent that easy syringeability (syringability) exists. Advantageously, certain pharmaceutical formulations are stable under the conditions of manufacture and storage and must be protected against the contaminating action of microorganisms such as bacteria and fungi. In general, the relevant carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof. For example, proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In some cases, it may be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by the inclusion in the composition of a substance that delays absorption, for example, aluminum monostearate and gelatin.

Therapeutic proteins can be administered by a variety of methods known in the art including, but not limited to, oral, parenteral, mucosal, inhalation, topical, buccal, nasal, and rectal administration. For many therapeutic applications, the preferred route/mode of administration is subcutaneous, intramuscular, intravenous or infusion. Needleless injections may be used if desired. As the skilled artisan will appreciate, the route and/or mode of administration will vary depending on the desired result.

In certain embodiments, the protein may be prepared with carriers that protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. Many methods for preparing such formulations have been patented or are generally known to those skilled in the art. See, e.g., Robinson, ed., In: sustained and Controlled Release Drug Delivery Systems, Marcel Dekker, Inc., New York (1978).

The dosage regimen may be adjusted to provide the best desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the individual mammals to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention depends on and directly depends on (a) the unique characteristics of the protein and the particular therapeutic or prophylactic effect to be achieved, and (b) limitations inherent in the art of synthesizing such active compounds for sensitive therapy in an individual.

It should be noted that dosage values may vary with the type and severity of the condition being alleviated, and may include single and multiple doses. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or application of the claimed compositions.

In one embodiment, the protein is administered in an intravenous formulation containing about 1mg/ml, preferably about 5mg/ml, or more preferably about 10mg/ml, or more preferably about 15mg/ml, or even more preferably about 20mg/ml of the protein and sodium acetate, polysorbate 80 and sodium chloride as a sterile aqueous solution at a pH of about 5-6. Preferably, the intravenous formulation is a sterile aqueous solution containing 5 or 10mg/ml of the protein with 20mM sodium acetate, 0.2mg/ml polysorbate 80 and 140mM sodium chloride, pH 5.5. Furthermore, the protein-containing solution may comprise histidine, mannitol, sucrose, trehalose, glycine, polyethylene glycol, EDTA, methionine, and any combination thereof, among many other compounds known in the relevant art.

In one embodiment, a partial dose is administered by intravenous bolus injection and the remainder is infused by the protein formulation. For example, an intravenous injection of 0.01mg/kg of the protein may be administered as a bolus, and the remaining predetermined protein mass may be administered by intravenous injection. For example, a predetermined amount of the protein may be administered over a period of one-half hour to two hours to five hours.

With respect to the second therapeutic agent, when the therapeutic agent is, for example, a small molecule, it may be present in the pharmaceutical composition in the form of a physiologically acceptable ester or salt, for example in combination with a physiologically acceptable cation or anion as is well known in the art.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the pharmaceutical arts. In general, such methods of preparation include the steps of bringing into association the active ingredient with the carrier or one or more other accessory ingredients and then, if necessary or desired, shaping or packaging the product into the desired dosage unit or units.

Pharmaceutical compositions useful in the methods of the invention may be prepared, packaged or sold in formulations suitable for oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal, buccal, ophthalmic or another route of administration. Other contemplated formulations include sprayed nanoparticles, liposome preparations, re-encapsulated red blood cells containing active ingredients, and immune-based formulations.

The pharmaceutical compositions of the present invention may be prepared, packaged or sold in bulk, as a single unit dose, or as multiple single unit doses. As used herein, a "unit dose" is a discrete amount of a pharmaceutical composition that contains a predetermined amount of an active ingredient. The amount of active ingredient is generally equal to the dose of active ingredient to be administered to the individual or a suitable fraction of such a dose, e.g., 1/2 or 1/3 of such a dose.

The relative amounts of the active ingredients, pharmaceutically acceptable carriers, and any additional ingredients in the pharmaceutical compositions of the invention will vary depending on the nature, size, and condition of the individual being treated, and further depending on the route of administration of the composition. For example, the composition may comprise between 0.1% and 100% (w/w) of active ingredient.

In addition to the active ingredient, the pharmaceutical composition of the present invention may further comprise one or more additional pharmaceutically active therapeutic agents. Additional therapeutic agents of particular interest include antiemetics, antidiarrheals, chemotherapeutic agents, cytokines, and the like.

Controlled or sustained release formulations of the pharmaceutical compositions of the present invention can be prepared using conventional techniques.

Formulations of the pharmaceutical compositions of the present invention suitable for oral administration may be prepared, packaged, or sold in the form of discrete solid dosage units, including but not limited to tablets, hard or soft capsules, cachets, troches, or lozenges, each containing a predetermined amount of the active ingredient. Other formulations suitable for oral administration include, but are not limited to, powder or granule formulations, aqueous or oily suspensions, aqueous or oily solutions, or emulsions.

As used herein, an "oily" liquid is a liquid that comprises carbon-containing liquid molecules and exhibits less polar character than water.

For example, tablets containing the active ingredient may be prepared by compressing or molding the active ingredient, optionally in the presence of one or more additional ingredients. Compressed tablets may be prepared by compressing in a suitable apparatus the active ingredient in a free-flowing form such as a powder or granules optionally mixed with one or more binders, lubricants, excipients, surfactants and dispersants. Tablets may be formed by forming in a suitable apparatus a mixture of the active ingredient, the pharmaceutically acceptable carrier and at least enough liquid to wet the mixture.

Pharmaceutically acceptable excipients used in the manufacture of tablets include, but are not limited to, inert diluents, granulating and disintegrating agents, binding agents, and lubricating agents. Known dispersing agents include, but are not limited to, potato starch and sodium starch glycolate. Known surfactants include, but are not limited to, sodium lauryl sulfate. Known diluents include, but are not limited to, calcium carbonate, sodium carbonate, lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogen phosphate and sodium phosphate. Known granulating and disintegrating agents include, but are not limited to, corn starch and alginic acid. Known binders include, but are not limited to, gelatin, acacia, pregelatinized corn starch, polyvinylpyrrolidone, and hydroxypropyl methylcellulose. Known lubricants include, but are not limited to, magnesium stearate, stearic acid, silica, and talc.

Tablets may be uncoated or they may be coated by known methods to achieve delayed disintegration in the gastrointestinal tract of the individual, thereby providing sustained release and absorption of the active ingredient. For example, materials such as glycerol monostearate or glycerol distearate may be employed to coat the tablets. For further example, U.S. Pat. nos. 4,256,108; 4,160,452, respectively; and 4,265,874 to form osmotic controlled release tablets. The tablets may further contain sweetening agents, flavoring agents, coloring agents, preserving agents or some combination of these to provide pharmaceutically elegant and palatable preparations.

Hard capsules comprising the active ingredient can be prepared using physiologically degradable compositions such as gelatin. Such hard capsules comprise the active ingredient and may further comprise additional ingredients including, for example, inert solid diluents such as calcium carbonate, calcium phosphate or kaolin.

Soft gelatin capsules containing the active ingredient may be prepared using physiologically degradable compositions such as gelatin. Such soft capsules comprise the active ingredient, which may be mixed with water or an oil medium, such as peanut oil, liquid paraffin, or olive oil.

Liquid formulations of the pharmaceutical compositions of the present invention suitable for oral administration may be prepared, packaged and sold in liquid form or as a dry product intended to be reconstituted with water or another suitable vehicle prior to use.

Liquid suspensions may be prepared by conventional means to obtain a suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water and isotonic saline. Oily vehicles include, for example, almond oil; an oily ester; ethanol; vegetable oils, such as peanut oil, olive oil, sesame oil or coconut oil; fractionating the vegetable oil; and mineral oils such as liquid paraffin. Liquid suspensions may further contain one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavoring agents, coloring agents, and sweetening agents. The oily suspension may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives, such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose. Known dispersing or wetting agents include, but are not limited to, naturally occurring phospholipids, such as lecithin; condensation products of alkylene oxides with fatty acids, with long-chain aliphatic alcohols, with partial esters derived from fatty acids and hexitol, or with partial esters derived from fatty acids and hexitol anhydrides (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin and acacia gum. Known preservatives include, but are not limited to, methyl, ethyl or n-propyl p-hydroxybenzoic acid, ascorbic acid and sorbic acid. Known sweetening agents include, for example, glycerin, propylene glycol, sorbitol, sucrose, and saccharin. Thickening agents known for use in oily suspensions include, for example, beeswax, hard paraffin and cetyl alcohol.

Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the main difference being that the active ingredient is dissolved rather than suspended in the solvent. Liquid solutions of the pharmaceutical compositions of the present invention may contain each of the components described with respect to the liquid suspension, it being understood that the suspending agent does not necessarily aid in the dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water and isotonic saline. Oily solvents include, for example, almond oil; an oily ester; ethanol; vegetable oils, such as peanut oil, olive oil, sesame oil or coconut oil; fractionating the vegetable oil; and mineral oils such as liquid paraffin.

Powder and granule formulations of the pharmaceutical preparations of the invention can be prepared using known methods. Such formulations may be administered directly to a subject, for example, for forming tablets, filling capsules, or making aqueous or oily suspensions or solutions by adding aqueous or oily vehicles. Each of these formulations may further comprise one or more dispersing or wetting agents, suspending agents and preservatives. Additional excipients, such as fillers and sweetening, flavoring or coloring agents may also be included in the formulations.

The pharmaceutical compositions of the present invention may also be prepared, packaged or sold in the form of oil-in-water emulsions or water-in-oil emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil; mineral oils, such as liquid paraffin; or a combination of these. Such compositions may further comprise one or more emulsifiers, for example naturally occurring gums, such as gum arabic or gum tragacanth; naturally occurring phospholipids, such as soy or lecithin phospholipids; esters or partial esters derived from combinations of fatty acids and hexitol anhydrides, such as sorbitan monooleate; and condensation products of such partial esters with ethylene oxide, such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweeteners or flavoring agents.

The pharmaceutical compositions of the present invention may be prepared, packaged or sold in formulations suitable for rectal administration. For example, such compositions may be in the form of suppositories, retention enema preparations and solutions for rectal or colonic irrigation.

Suppository formulations may be prepared by combining the active ingredient with a non-irritating pharmaceutically acceptable excipient which is solid at ordinary room temperature (i.e., about 20 ℃) and liquid at the rectal temperature of the individual (i.e., about 37 ℃ in a healthy human). Suitable pharmaceutically acceptable excipients include, but are not limited to, cocoa butter, polyethylene glycols and various glycerides. Suppository formulations may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.

Retention enema preparations or solutions for rectal or colonic lavage can be prepared by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, the enema preparation can be administered using a delivery device adapted to the rectal anatomy of the subject, and the enema preparation can be packaged within a delivery device adapted to the rectal anatomy of the subject. The enema preparation may further comprise various additional ingredients including, but not limited to, antioxidants and preservatives.

The pharmaceutical compositions of the present invention may be prepared, packaged or sold in formulations suitable for vaginal administration. For example, such compositions may be suppositories, impregnated or coated vaginal-insertable substances such as tampons, perfusate preparations, or gels or creams or solutions for vaginal douches.

Methods of impregnating or coating a substance with a chemical composition are known in the art and include, but are not limited to, methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of the substance during synthesis of the substance (i.e., with a physiologically degradable substance, for example), and methods of absorbing an aqueous or oily solution or suspension into an absorbent substance, with or without subsequent drying.

Perfusate preparations for vaginal douches can be prepared by combining the active ingredient with a pharmaceutically acceptable liquid carrier. As is well known in the art, the perfusate preparation can be administered using a delivery device adapted to the vaginal anatomy of the individual, and the perfusate preparation can be packaged within the delivery device adapted to the vaginal anatomy of the individual. The perfusate formulation may further comprise various additional ingredients including, but not limited to, antioxidants, antibiotics, antifungal agents, and preservatives.

As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical penetration of a tissue of an individual and administration of the pharmaceutical composition through a breach in the tissue. Thus parenteral administration includes, but is not limited to, administration of the pharmaceutical composition by injection of the composition, administration of the composition through a surgical incision, administration of the composition through a non-surgical tissue penetrating wound, and the like. In particular, parenteral administration is understood to include, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intrasternal injection, and kidney dialysis infusion techniques.

Formulations of pharmaceutical compositions suitable for parenteral administration comprise the active ingredient in combination with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged or sold in a form suitable for bolus administration or for continuous administration. Injectable preparations may be prepared, packaged or sold in unit dosage form, e.g., in ampoules or in multi-dose containers containing a preservative. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained release or biodegradable formulations as discussed below. Such formulations may further comprise one or more additional ingredients, including but not limited to suspending, stabilizing or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granules) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The compositions of the present invention may be administered by various methods known in the art. The route and/or mode of administration will vary depending on the desired result. The active compounds can be formulated with carriers that protect the compound from rapid release, such as controlled release formulations, including implants, transdermal patches, and microencapsulated delivery systems. Biodegradable biocompatible polymers such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid may be used. For example, Robinson, ed., Sustainated and controlledRelease Drug Delivery Systems, Marcel Dekker, Inc., New York, (1978) describe a number of methods for preparing such formulations. The pharmaceutical composition is preferably manufactured under GMP conditions.

Pharmaceutical compositions may be prepared, packaged or sold in the form of sterile injectable aqueous or oleaginous suspensions or solutions. Such suspensions or solutions may be formulated according to known techniques and may contain, in addition to the active ingredient, additional ingredients such as dispersing, wetting or suspending agents as described herein. Such sterile injectable preparations may be prepared using non-toxic parenterally-acceptable diluents or solvents, for example, water or 1, 3-butanediol. Other acceptable diluents and solvents include, but are not limited to, ringer's solution, isotonic sodium chloride solution, and fixed oils, such as synthetic mono-or diglycerides. Other useful parenterally administrable formulations include those comprising the active ingredient in microcrystalline form, in liposomal preparations, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic substances, such as emulsions, ion exchange resins, poorly soluble polymers or poorly soluble salts.

Formulations suitable for topical (topical) administration include, but are not limited to, liquid or semi-liquid preparations, such as liniments, lotions, oil-in-water or water-in-oil emulsions, such as creams, ointments or pastes, and solutions or suspensions. For example, a topically administrable formulation may contain from about 1% to about 10% (w/w) active ingredient, although the concentration of the active ingredient may be as high as the solubility limit of the active ingredient in the solvent. Formulations for topical administration may further comprise one or more additional ingredients as described herein.

The pharmaceutical compositions of the present invention may be prepared, packaged, or sold in a formulation suitable for oral pulmonary administration. Such formulations may comprise dry particles comprising the active ingredient and having a diameter in the range of from about 0.5 to about 7 nanometers, preferably from about 1 to about 6 nanometers. Such compositions are conveniently in the form of a dry powder for administration using a device comprising a reservoir of dry powder which may direct a stream of propellant to disperse the powder or a self-propelled solvent/powder dispensing container, such as a device comprising an active ingredient dissolved or suspended in a low boiling point propellant in a sealed container. Preferably, such a powder comprises particles, wherein at least 98 wt% of the particles have a diameter larger than 0.5 nm and at least 95% by number of the particles have a diameter smaller than 7 nm. More preferably, at least 95% by weight of the particles have a diameter greater than 1 nanometer and at least 90% by number of the particles have a diameter less than 6 nanometers. The dry powder composition preferably includes a solid fine powder diluent, such as a sugar, and is conveniently provided in a unit dosage form.

Low boiling point propellants generally include liquid propellants having a boiling point below 65 ° f at atmospheric pressure. Typically the propellant may constitute from 50 to 99.9% (w/w) of the composition and the active ingredient may constitute from 0.1 to 20% (w/w) of the composition. The propellant may further comprise additional ingredients such as liquid non-ionic or solid anionic surfactants or solid diluents (preferably having a particle size of the same order as the particles comprising the active ingredient).

Pharmaceutical compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension. Such formulations may be prepared, packaged or sold as aqueous or diluted alcoholic solutions or suspensions, which are optionally sterile, contain the active ingredient and may be conveniently administered using any atomising or powdering device. Such formulations may further comprise one or more additional ingredients including, but not limited to, flavoring agents such as sodium saccharin, volatile oils, buffering agents, surfactants, or preservatives such as methylparaben. The droplets provided by such a route of administration preferably have an average diameter in the range of about 0.1 to about 200 nanometers.

Formulations described herein that facilitate pulmonary delivery also facilitate intranasal delivery of the pharmaceutical compositions of the present invention.

Another formulation suitable for intranasal administration is a coarse powder containing the active ingredient and having an average particle size of about 0.2-500 microns. The formulations are administered in the form of snuff that is rapidly inhaled from a container of powder near the nostrils through the nasal passages.

For example, formulations suitable for nasal administration may comprise as little as about 0.1% (w/w) and as much as 100% (w/w) of the active ingredient, and may further comprise one or more additional ingredients as described herein.

The pharmaceutical compositions of the present invention may be prepared, packaged or sold in formulations suitable for buccal administration. Such formulations may be in the form of tablets or lozenges, for example prepared using conventional methods, and may comprise, for example, 0.1-20% (w/w) of the active ingredient, with the remainder comprising the orally dissolvable or degradable composition and optionally one or more additional ingredients described herein. Alternatively, formulations suitable for buccal administration may comprise a powder or an aerosolized or powdered solution or suspension comprising the active ingredient. When dispersed, such powder, aerosolized or aerosolized formulations preferably have an average particle or droplet size in the range of from about 0.1 to about 200 nanometers, and may further comprise one or more additional ingredients described herein.

The pharmaceutical compositions of the present invention may be prepared, packaged or sold in formulations suitable for ophthalmic administration. Such formulations may be, for example, in the form of eye drops, comprising, for example, a 0.1-1.0% (w/w) solution or suspension of the active ingredient in an aqueous or oily liquid carrier. Such drops may further comprise a buffer, salt or one or more other additional ingredients as described herein. Other useful formulations for ocular administration include those comprising the active ingredient in microcrystalline form or in a liposomal preparation.

As used herein, "additional ingredients" include, but are not limited to, one or more of the following: an excipient; a surfactant; a dispersant; an inert diluent; granulating and disintegrating agents; a binder; a lubricant; a sweetener; a flavoring agent; a colorant; a preservative; physiologically degradable compositions, such as gelatin; an aqueous vehicle and a solvent; oily vehicles and solvents; a suspending agent; dispersing or wetting agents; emulsifiers, demulcents; a buffer solution; salt; a thickener; a filler; an emulsifier; an antioxidant; (ii) an antibiotic; an antifungal agent; a stabilizer; and pharmaceutically acceptable polymeric or hydrophobic substances. Other "additional ingredients" that may be included in the pharmaceutical compositions of the present invention are known in the art and are described, for example, in Remington's pharmaceutical Sciences, Genaro, ed., Mack Publishing Co., Easton, Pa (1985), which is incorporated herein by reference.

In one embodiment of the invention, the composition comprising a therapeutic protein comprises a sterile solution comprising 20mM histidine buffer, pH 5.5, 84mg/ml anhydrotrehalose, 0.2mg/ml polysorbate 80 and 0.1mg/ml disodium EDTA hydrate. In one aspect, the protein is packaged in a clear glass vial with a rubber stopper and an aluminum seal. In another aspect, the vial contains about 20mg/ml of the protein with a nominal fill of about 400mg per vial.

The therapeutic protein compositions of the invention can be administered to an animal, preferably a human. When the composition comprises a combination of a therapeutic protein and a second therapeutic agent, the precise dosage administered for each active ingredient will vary depending on any number of factors including, but not limited to, the type of animal and the type of disease state being treated, the age of the animal, and the route of administration.

The therapeutic protein can be administered to the animal at a frequency of several times per day, or can be administered less frequently, such as once per day, once per week, once every two weeks, once per month, or even less frequently, such as once every few months or even once per year or less. The frequency of administration will be apparent to the skilled artisan and will depend on any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, and the like.

The therapeutic protein can be administered to the animal at a frequency of several times per day, or can be administered less frequently, such as once per day, once per week, once every two weeks, once per month, or even less frequently, such as once every few months or even once per year or less. The frequency of administration will be apparent to the skilled artisan and will depend on any number of factors, such as, but not limited to, the type and severity of the disease being treated, the type and age of the animal, and the like.

The therapeutic protein and the second therapeutic agent (e.g., the second protein) can be co-administered, where they can be administered separately, on different days or at different times of day, and simultaneously or on the same day. Co-administration thus encompasses any temporal combination of administration of the protein and the second therapeutic agent, such that administration of both substances mediates a therapeutic benefit to the patient that is detectably greater than administration of either substance in the absence of the other.

The combination of the therapeutic protein of the invention and the second therapeutic agent may be co-administered with a number of other compounds (other anti-hormonal therapeutic agents, cytokines, chemotherapeutics and/or antivirals, etc.). Alternatively, the compound, or any permutation thereof, may be administered one hour, one day, one week, one month, or even earlier prior to the protein-therapeutic combination. In addition, the compound, or any permutation thereof, may be administered one hour, one day, one week, or even later after the administration of radiation, stem cell transplantation, or the administration of any therapeutic agent (e.g., cytokine, chemotherapeutic compound, etc.). The frequency and administration regimen will be apparent to the skilled artisan and will depend on any number of factors such as, but not limited to, the type and severity of the disease being treated, the age and health of the animal, the nature of the compound or compounds being administered, the route of administration of the various compounds, and the like.

Dosage regimen

The dosage regimen may be adjusted to provide the best desired response. For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the urgency of the treatment situation. It is particularly advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suitable as unitary dosages for the individual mammals to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect, in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention depends on and directly depends on (a) the unique characteristics of the therapeutic protein and the particular therapeutic or prophylactic effect to be achieved, and (b) limitations inherent in the art of synthesizing such active compounds for sensitive therapy in an individual.

An exemplary, non-limiting range of therapeutically effective amounts of therapeutic proteins administered according to the present invention is at least about 1mg/kg, at least about 5mg/kg, at least about 10mg/kg, over about 10mg/kg, or at least about 15mg/kg, such as about 1-30mg/kg, or such as about 1-25mg/kg, or such as about 1-20mg/kg, or such as about 5-20mg/kg, or such as about 10-20mg/kg, or such as about 15 mg/kg. It should be noted that dosage values may vary with the type and severity of the condition being alleviated, and may include single or multiple doses. It is further understood that for any particular individual, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the dosage ranges set forth herein are exemplary only and are not intended to limit the scope or application of the claimed compositions. Determining an appropriate dose and regimen for administering a therapeutic protein is well known in the relevant art, and it is understood that once the teachings disclosed herein are provided, the skilled artisan will determine an appropriate dose and regimen for administering a therapeutic protein.

In one embodiment, the anti-IgE antibody, or antigen-binding portion thereof, of the invention is administered as an intravenous formulation comprising, as a sterile aqueous solution, about 5-20mg/ml of the antibody in a suitable buffer system.

In one embodiment, for low dose administration, a partial dose is administered by intravenous bolus injection, while the remainder is infused by the antibody formulation. For example, 0.01mg/kg of the intravenously injected antibody can be administered as a bolus injection, and the remaining predetermined antibody dose can be administered by intravenous injection. In another embodiment, the entire low dose is administered as a single bolus. For higher doses, e.g., 3mg/kg, the antibody is not administered as a bolus, but the entire amount is administered by infusion. For example, a predetermined dose of the antibody may be administered over a period of about one hour and half an hour to about five hours.

In one embodiment of the invention, the therapeutic protein (e.g., sRAGE-Fc fusion protein, an antibody such as an anti-IgE antibody, etc.) is administered about every 3 weeks, more preferably for about four cycles, after which the therapeutic protein is administered every 3 months. In one aspect of this embodiment, the protein is administered at about 10 mg/kg.

The skilled artisan will appreciate that a therapeutic protein of the invention may be administered concurrently with a second therapeutic protein or therapeutic agent in combination, or that a protein of the invention may be administered at different times with a second therapeutic protein or therapeutic agent. For example, in one embodiment, the therapeutic protein is administered as a single injection and/or infusion, and once per day administration of a second therapeutic agent (e.g., a non-proteinaceous compound) is initiated before, during, or after administration of the protein. However, the present invention is not limited to any particular dosage or administration regimen of the therapeutic agent. Rather, the optimal dosage, route and regimen for administering the antibody and therapeutic agent can be readily determined by one of ordinary skill in the relevant art using well known methods.

For example, a single dose or multiple doses of a therapeutic protein may be administered. Alternatively, at least 1 dose, or at least 3, 6 or 12 doses may be administered. For example, the dose may be administered every 2 weeks, every 3 weeks, every month, every 20 days, every 25 days, every 28 days, every 30 days, every 40 days, every 6 weeks, every 50 days, every 2 months, every 70 days, every 80 days, every 3 months, every 6 months, or annually. In one aspect, the therapeutic protein is administered once every 3 weeks, preferably for 4 cycles, followed by administration of the therapeutic protein every 3 months. Further, the second therapeutic agent or protein may be administered several times daily or once daily, weekly, every 2 weeks, every 3 weeks, every 4 weeks, every month, every 3 months, every 6 months, once a year, or any other period that provides a therapeutic benefit to the patient as determined by the skilled artisan.

Use of the protein

The therapeutic proteins of the present invention may be used as affinity purifiers. In this method, the protein is immobilized on a solid phase using methods well known in the art, such as Sephadex^TMResin or filter paper. The immobilized protein is contacted with a sample containing the binding partner to be purified, and the support is then washed with a suitable solvent that will remove substantially all of the material in the sample except for the binding partner to be purified that binds to the immobilized protein. Finally, the support is washed with another suitable solvent, such as glycine buffer, which releases the binding partner from the immobilized protein.

The proteins may also be used in diagnostic assays, e.g., to detect the expression of a binding partner of interest in a particular cell, tissue or serum. For diagnostic applications, the proteins of the invention will typically be labeled with a detectable moiety. A number of labels are available, including techniques for quantifying the change in fluorescence when the protein is labeled with a fluorophore. The chemiluminescent substrate becomes electronically excited by a chemical reaction and can then emit light, which can be measured (e.g., using a chemiluminescent analyzer) or supplied with energy to a fluorescent acceptor. Examples of enzymatic labels include luciferases (e.g., firefly luciferase and bacterial luciferases; U.S. Pat. No. 4,737,456), luciferin, 2, 3-dihydrophthalazinediones, malate dehydrogenase, urease, peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase, beta-galactosidase, glucoamylase, lysozyme, carbohydrate oxidase (e.g., glucose oxidase, galactose oxidase and glucose-6-phosphate dehydrogenase), heterocyclic oxidases (e.g., urate oxidase and xanthine oxidase), lactoperoxidase, microperoxidase, and the like. Techniques for conjugating enzymes to proteins, including antibodies or fragments thereof, are described in, for example, O' Sullivan et al, Methods for the Preparation of Enzyme-Antibody Conjugates for Use in Enzyme Immunoassay, in: methods in Enzym. (ed.j. langone & h. vanvunakis), Academic press, New York, 73: 147, 166 (1981).

Sometimes, the label is indirectly conjugated to the protein. The skilled person will be aware of various techniques for achieving this. For example, a protein such as an antibody or antigen-binding portion thereof can be conjugated to biotin, and any of the three broad classes of labels mentioned above can be conjugated to avidin, or vice versa. Biotin binds selectively to avidin, and thus the label can be conjugated to the protein in this indirect manner. Optionally, to achieve indirect conjugation of the label to the protein, the protein is conjugated to a small hapten (e.g., digoxin) and one of the different types of labels mentioned above is conjugated to an anti-hapten antibody (e.g., anti-digoxin antibody). Thus, indirect conjugation of the label to the protein may be achieved.

In another embodiment of the invention, the protein need not be labeled, and its presence can be detected using a labeled antibody that binds to the protein.

The proteins of the invention, including antibodies, may be used in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays. Zola, MonoclonalAntibodies: a Manual of Techniques, pp.147-158(CRC Press, Inc.1987).

Competitive binding assays rely on the ability of a labeled standard to compete with a test sample for binding to a limited amount of protein. The amount of target in the test sample is inversely proportional to the amount of standard bound to the protein. To facilitate determination of the amount of bound standard, the protein is generally insoluble before or after competition. As a result, standards and test samples bound to the protein can be conveniently separated from standards and test samples that remain unbound.

Sandwich assays involve the use of two binding proteins, each capable of binding to a different binding domain of the protein to be detected, or in the case of antibodies, each capable of binding to a different epitope of the protein to be detected. In a sandwich assay, a first antibody immobilized on a solid support binds to a test sample to be analyzed, after which a second binding protein, including a second antibody, binds to the test sample, thus forming an insoluble three-part complex. See, for example, U.S. Pat. No. 4,376,110. The second antibody itself may be labeled with a detectable moiety (direct sandwich assay) or may be measured using an anti-immunoglobulin antibody labeled with a detectable moiety (indirect sandwich assay). For example, one type of sandwich assay is an ELISA assay, in which case the detectable moiety is an enzyme.

For immunohistochemistry, the biological sample may be fresh or frozen, or may be embedded in paraffin and fixed with a preservative such as formalin.

The proteins may also be used in vivo diagnostic assays. Typically, with radionuclides (e.g. of the type¹¹¹In、⁹⁹Tc、¹⁴C、¹³¹I、³H、³²P or³⁵S) labels the protein, so that the tissue to which the protein binds to the binding partner, or in the case of antibodies, the tissue to which the antibody binds to its antigen, can be located using immunoimaging.For example, the anti-IgE antibodies of the invention may be used to detect the amount of IgE present in, for example, the lungs of asthma patients. Alternatively, the RAGE fusion proteins of the invention may be used to detect the amount of ligand present in a patient.

The proteins of the invention may be provided in a kit, i.e., a packaged combination of predetermined amounts of reagents with instructions for performing a diagnostic assay. When the protein is labeled with an enzyme, the kit may include substrates and cofactors required by the enzyme (e.g., substrate precursors that provide a detectable chromophore or fluorophore). In addition, other additives may be included, such as stabilizers, buffers (e.g., blocking buffers or lysis buffers), and the like. The relative amounts of the various reagents may be varied widely to provide solution concentrations of the reagents that substantially optimize assay sensitivity. In particular, the reagents may be provided as a dry powder, which is typically lyophilized, including excipients, which, when dissolved, will provide a reagent solution having the appropriate concentration.

In vivo uses of the antibodies

It is contemplated that the proteins of the invention may be used to treat mammals. For example, in one embodiment, the protein is administered to a non-human mammal for the purpose of obtaining preclinical data. Exemplary non-human mammals to be treated include non-human primates, dogs, cats, rodents, and other mammals in which preclinical studies are conducted. Such mammals may be established animal models of the disease to be treated with the protein, or may be used to study the toxicity or pharmacokinetics of the protein of interest. In each of these embodiments, a dose escalation study can be conducted on a mammal.

The protein is administered by any suitable method, including parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, as well as intralesional administration if local immunosuppressive therapy is desired. Parenteral infusion includes intramuscular, intravenous, intraarterial, intraperitoneal or subcutaneous administration. Furthermore, it is suitable to administer the protein, in particular a decreasing dose of the protein, by pulsed infusion. Administration by injection is preferred, and intravenous or subcutaneous injection is more preferred, depending in part on whether the administration is transient or chronic.

For the prevention or treatment of disease, the appropriate dosage of the antibody or protein will depend on the type of disease to be treated, the severity and course of the disease, whether the antibody variant is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the antibody variant, and the discretion of the attending physician. In certain embodiments, an anti-human IgE antibody of the invention may be suitably administered to a patient at one time or over a series of treatments. Similarly, in other embodiments, the sRAGE-Ig fusion proteins of the invention are administered to a patient at one time or over a series of treatments.

Depending on the type and severity of the disease, about 0.1mg/kg to 150mg/kg (e.g., 0.1 to 20mg/kg) of protein is an initial candidate dose for administration to the patient, e.g., by one or more separate administrations, or by continuous infusion. Typical daily dosages may range from about 1mg/kg to 100mg/kg or more, depending on the factors mentioned above. Depending on the disease condition, for repeated administrations over several days or longer, treatment is continued until the desired suppression of disease symptoms occurs. However, other dosage regimens may be useful. The course of such treatment is readily monitored by conventional techniques and assays. Exemplary dosage regimens for anti-LFA-1 or anti-ICAM-1 antibodies are disclosed in WO 94/04188.

The use of sRAGE-Ig fusion proteins of the invention for treating various diseases or disorders is described in WO 2004/016229; WO 2006/017643; WO 2006/017647; WO 2007/094926; WO 2007/130302; and WO 2008/100670.

The protein composition is formulated, dosed and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular condition being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the condition, the site of delivery of the substance, the method of administration, the timing of administration and other factors known to the physician. A "therapeutically effective amount" of a protein to be administered will be governed by such considerations and is the minimum amount necessary to prevent, ameliorate, or treat a disease or condition. The protein need not, but is optionally formulated with one or more substances currently used for the prevention or treatment of the disease of interest. The effective amount of such other substances will depend on the amount of protein present in the formulation, the type of disorder or treatment, and other factors discussed above. These formulations are generally used at about 1-99% of the same dosage and route of administration as used above, or previously employed dosages.

The anti-IgE antibodies of the present invention that recognize IgE as their target may be used to treat "IgE-mediated disorders". These conditions include diseases such as asthma, allergic rhinitis & conjunctivitis hay fever), eczema, urticaria, atopic dermatitis and food allergies. Serious physiological conditions such as bee stings, snake bites, food or drug induced anaphylactic shock are also included within the scope of the present invention.

The sRAGE-Ig fusion proteins of the present invention that recognize as their targets advanced glycosylation end products (AGEs), amyloid beta (A β), Serum Amyloid A (SAA), S100, Carboxymethyllysine (CML), amphoterin, and CD11b/CD18 may be used to treat any disease, disease condition, or disorder mediated by or associated with the binding of RAGE to its ligands, i.e., "RAGE-mediated disorders". RAGE is implicated in various disease states, and such RAGE-mediated disorders include, but are not limited to, diabetic symptoms or diabetic late complications symptoms, e.g., diabetic nephropathy, diabetic retinopathy, diabetic foot ulcers, cardiovascular complications, or diabetic neuropathy; amyloidosis, alzheimer's disease, cancer, renal failure, or inflammation associated with autoimmunity, inflammatory bowel disease, rheumatoid arthritis, psoriasis, multiple sclerosis, hypoxia, stroke, heart attack, hemorrhagic shock, sepsis, organ transplantation, or impaired wound healing; osteoporosis, renal failure, transplant rejection, inflammation and/or rejection associated with transplantation, and the like.

The present invention is described in further detail with reference to the following experimental examples. These examples are provided for the purpose of illustration only and are not intended to be limiting unless otherwise specified. Thus, the present invention should not be construed as limited in any way to the following examples, but rather should be construed to include any and all variations that become apparent as a result of the teachings provided herein.

Examples

Example 1:

control of glycosylation: reduced glycan site occupancy by tunicamycin glycosylation inhibitors to affect RAGE ligand binding

Receptor for advanced glycation end products (RAGE) is involved in the pathology of many chronic disorders and is therefore actively pursued as a target for therapeutic intervention. Recent studies have demonstrated that RAGE is a glycoprotein whose glycosylation state can influence binding to its ligand. In this study, we expressed recombinant human sRAGE-Ig fusion protein and examined its glycosylation and proteolytic processing. The sRAGE-Ig fusion protein has three N-linked glycosylation sites, two in the sRAGE domain and one in the Fc region, and is about 70% fully occupied when expressed in CHO cells.

Tunicamycin treated cell cultures were used to express site-occupying variants of the protein. The site-occupying variants were isolated and the effect of glycan occupancy on binding to RAGE ligand S100b was evaluated. The non-glycosylated variant showed a 10-fold increase in binding to S100b compared to the fully occupied form, while the double occupied variant showed moderate binding between the two. Furthermore, we found that about 20% of the CHO-expressed sRAGE-Ig fusion proteins were cleaved by the subtilisin-like protease furin, resulting in a deletion of the RAGE domain. These studies provide insight into the post-translational requirements for optimal ligand binding activity of recombinant RAGE molecules and highlight potential challenges associated with expression of full-length proteins in CHO cells.

Advanced glycation end product Receptors (RAGE) are members of the immunoglobulin superfamily, the expression of which is upregulated in a number of human diseases including diabetes, chronic inflammation, and Alzheimer's disease (Ramasamy et al, 2005, Glycobiology 15: 16R-28R). Ligands for RAGE include advanced glycation end products (AGEs), which are heterostructures resulting from exposure of lipids and proteins to reducing sugars followed by irreversible complex molecular rearrangements (Brownlee et al, 1988, New eng.j.med.318: 1315-1320; schmidt et al, 1994, Arterioscler.Thromb.14: 1521-; chavakis et al, 2004, Microbes infection.6: 1219-; kokkola et al, 2005, Scand.J. Immunol.61: 1-9; schmidt et al, 2001, J.Clin.invest.108: 949-; rocken et al, 2003, am.J.Pathol.162: 1213-.

Various disease conditions involve activation of RAGE by binding to its ligand in different tissues (Hofmann et al, 1999, Cell 97: 889-.

Ligand binding to the RAGE receptor is responsible for its upregulation (Schmidt et al, 2000, Biochim. Biophys. acta 1498: 99-111). Increased receptor expression leads to cell signaling and activation, leading to the pathology of chronic diseases (Schmidt et al, 1999, Circ. Res.84: 489-497). One approach to down-regulate RAGE and interrupt ligand coordination cycles is to generate "decoys" or soluble RAGE (srage) lacking a transmembrane domain. Treatment with exogenous sRAGE in a diabetic mouse model was shown to inhibit the acceleration of diabetic atherosclerosis (Park et al, 1998, Nature Medicine 4: 1025-.

The sRAGE-Ig fusion proteins described in this study are recombinant molecules designed as potential therapeutic decoys. The protein contains a C linked to a human IgG1 heavy chain molecule_H2 and C_HThe ligand of the 3-domain human RAGE binds to the V-like domain and the constant type-like C1 domain (fig. 1). While sRAGE has been isolated as a monomer from mice (Handord et al, 2004, J.biol.chem.279: 50019-50024) and has been expressed as a monomer in E.coli (Wilton et al, 2006, protein expression purify.47: 25-35) and yeast (Ostendorp et al, 2006, biochem.Biophys.Res.Commun.347: 4-11), we report that human sRAGE-Ig fusion proteins are expressed as dimers. We describe the proteolytic cleavage and glycosylation pattern of sRAGE-Ig fusion proteins when expressed in Chinese Hamster Ovary (CHO) cells. To evaluate the functional effect of N-linked glycosylation of human RAGE on binding to its ligand, we used tunicamycin to generate N-linked site occupying variants of sRAGE-Ig fusion proteins and measured the binding of the glycosylated variants to RAGE ligand S100 b.

Experimental methods

Production of host cell lines expressing sRAGE-Ig fusion proteins: a CHO cell line expressing the sRAGE-Ig fusion protein was generated by electroporation using a linearized expression plasmid containing sRAGE-Ig cDNA encoding the amino acid sequence shown in FIG. 2 (SEQ ID NO: 1). The nucleic acid encoding the sRAGE-Ig fusion protein comprises a sequence encoding the human RAGE signal sequence (the first 23 amino acids of SEQ ID NO: 1). Following transfection, cells were selected in methylsulfoximine (msx) and screened for protein production by ELISA from conditioned medium ("CM") in the survival pools. Cells producing the fusion protein were transferred from static culture to suspension culture and scaled up to a non-clonal pool. Pool capillary clones with acceptable growth rate and titer in shake flask assays were cloned. Clones were scaled up to suspension culture and modified based on their growth and expression levels in shake flask assays. Conditioned media was collected from non-clonal pools and clone candidates grown in shake flasks and purified by small MabSelect columns (ge healthcare, Piscataway, NJ) for product quality analysis. The cloned CHO cell line expressing the sRAGE-Ig fusion protein used in this study was selected based on growth, titer, and product quality characteristics,

And the clonal CHO cell line is representative of the total number of cell lines evaluated.

Treating tunicamycin: the clonal CHO cell line expressing the sRAGE-Ig fusion protein was cultured to mid-log phase in 50mL of medium in shake flasks. Cells were treated with 5. mu.g/mL of tunicamycin (Sigma-Aldrich) dissolved in DMSO and incubated for 3 days at 37 ℃ in a shaker. After 3 days the conditioned medium was collected, filtered and purified. Purified proteins were analyzed for glycan site occupancy by RP-HPLC and peak fractions were collected for S100b binding assay.

Purification of sRAGE-Ig protein from conditioned Medium: laboratory scale protein purification was performed on a 5mL HiTrap MabSelect column from GE Healthcare (Piscataway, NJ) using an AKTA FPLC chromatographic slide from GE Healthcare. The column was equilibrated with 5 Column Volumes (CV) of 50mM phosphate, 1M NaCl, pH 7.0. The clarified CM was then applied to the column and washed with 10 CVs of 50mM phosphate, 1M NaCl, pH 7.0, followed by 5 CVs of acetate, pH 5.5. The protein was then eluted from the column using 25mM acetate, pH 3.5, and the elution pool was collected as a single fraction based on absorbance at 280 nm. The column was run at 5mL/min throughout the run. The purified protein pool was then dialyzed into stabilization buffer using a 10K dialysis cassette from Pierce (Rockford, IL). The concentration of the purified protein was determined on a spectrophotometer using absorbance at 280 nm.

SDS-PAGE analysis of sRAGE-Ig, PNGaseF, and furin digestions: purified sRAGE-Ig fusion protein (178. mu.g) was digested with 7.5mU of peptide-N-glycosidase F (PNGase F) (Cat. No. GKE-5006, Prozyme, San Leandro, Calif.) in a total volume of 250. mu.L buffer for 30 hours at 37 ℃. Mu.g of purified sRAGE-Ig fusion protein was digested with 24U of furin (Cat No. P8077S, New England Biolabs, Ipswich, Mass.) in a total volume of 100. mu.L buffer at 37 ℃ for 15 hours. Samples of undigested and digested fusion proteins were prepared in reducing SDS-PAGE sample buffer, 2.7ug total protein per lane and run on a 4-12% gradient NuPage bis-tris gel (Cat. No. NP0322, Invitrogen, Carlsbad, Calif.).

Analysis of sRAGE-Ig by Size Exclusion Chromatography (SEC): purified sRAGE-Ig was diluted to 3mg/mL in mobile phase (350mM citrate, pH 6.0) and analyzed by SEC using an Alliance 2695 liquid chromatograph (Waters, Millford, Mass.). Superdex 20010/300GL column was used for separation (Cat. No.17-5175-01, GE Healthcare, Piscataway, NJ) at flow rate of 0.75mL/min for 40min at ambient temperature. The detection used is the absorbance at 280 nm.

Glycosylation site occupancy was determined by RP-HPLC and peaks were identified by liquid chromatography/mass spectrometry (LC/MS): the glycosylation site occupancy of sRAGE-Ig fusion proteins was determined under reverse phase HPLC conditions. An Agilent 1100 liquid chromatograph (Agilent technologies, Palo Alto, CA) with four pumps and a UV detector was used for HPLC separation. Data acquisition and integration is accomplished using the chemstation (agilent) or empower (waters) software packages. Briefly, 20. mu.L of a 1.0mg/mL protein solution was separated on a Zorbax300SB-C18 column (Agilent) using a shallow gradient of HPLC grade water and acetonitrile (Sigma Aldrich, St. Louis, Mo.) at a flow rate of 0.3 mL/min. The initial gradient at time zero was set at 67% mobile phase a (water with 0.1% trifluoroacetic acid (ACS grade, Pierce, Rockford, IL) and 33% mobile phase B (acetonitrile with 0.085% trifluoroacetic acid)). Mobile phase B was increased to 34% from t 0 to 2 minutes, further to 36% from t 2 to 10 minutes, and then equilibrated at 67% mobile phase a and 33% mobile phase B for 10 minutes. The absorbance at 214nm was measured.

To characterize the mass spectra of glycosylation site occupying variants, samples were analyzed by LC/MS using an Agilent 1100 liquid chromatograph attached to a Micromass time-of-flight (Q-TOF) mass spectrometer (Q-TOF MS) (Waters, Milford, MA). The eluates from the reverse phase separation were introduced 1: 1 separately into the mass spectrometer source. Mass spectra were averaged over each chromatographic peak and resolved using MaxEnt algorithm provided by Masslynx software.

Released glycans were analyzed by normal phase hplc (nplc) with fluorescence detection: the samples were diluted to 0.25mg and desalted using a Nanosep (P/N OD010C34, Pall Life Sciences, East Hills, NY) membrane filter with a molecular weight cut-off (MWCO 10K) of 10 kilodaltons. N-glycans were released from glycoproteins by incubation with PNGaseF (Cat. No. GKE-5006, Prozyme, San Leandro, Calif.) overnight at 37 ℃. The enzymatic reaction was carried out in the presence of beta-mercaptoethanol (GKE-5006, Prozyme, San Leandro, CA) and SDS (Prozyme, San Leandro, CA). After release, proteins were removed from the released glycans using a Nanosep membrane filter (MWCO 10K). The sample containing the glycan moiety was evaporated to dryness prior to the labeling reaction. The glycans were then labeled with 2-aminobenzamide (2-AB) (GKK-404, Prozyme, San Leandro, Calif.) at 65 ℃ for 3-4 hours. Excess labeling reagent was removed using a GlycoCleanS cartridge (GKK-4726, Prozyme, San Leandro, Calif.). Finally, the mixture of labeled glycans was analyzed by NPLC on an Agilent 1100 liquid chromatograph equipped with a fluorescence detector (Agilent Technologies, PaloAlto, CA). The excitation (Ex) wavelength was 330nm and the emission (Em) wavelength was 420 nm. A TSK-GEL amide-80 column (250mmx4.6mm, 5 μm particle size) maintained at 30 ℃ was used for separation (Tosoh Bioscience, South San Francisco, Calif.). A gradient separation was used, with a flow rate of 0.4mL/min, starting with 70% a and 30% B, and increasing to 40% B within 90 min. Mobile phase B was then increased to 95% in 50min, and to 100% in 5min, then decreased to 30% for re-equilibration. The mobile phases used were as follows: mobile phase a was acetonitrile and mobile phase B was 250mM ammonium formate pH 4.4. An injection volume of 10 μ L was used for the analysis.

Charge characterization by anion exchange chromatography: the samples were diluted to 0.5mg and prepared for Normal Phase Liquid Chromatography (NPLC) as described above. The mixture of released and labeled glycans was analyzed by anion exchange High Performance Liquid Chromatography (HPLC) on an Agilent 1100 liquid chromatograph equipped with a fluorescence detector (Agilent Technologies, Palo Alto, CA). The excitation wavelength was 330nm and the emission wavelength was 420 nm. A GlycoSpC column (100mmx7.5mm, 10 μm particle size) at ambient temperature (GKI-4721, Prozyme, San Leandro, Calif.) was used for the separation. A gradient separation was used, flow rate 0.5mL/min, starting with 100% a and 0% B for 5 minutes and increasing to 50% B in 45 minutes. Mobile phase B was then increased to 100% over 5 minutes and maintained for 5 minutes, then returned to 0% for re-equilibration. The mobile phases used were as follows: mobile phase a was 20% acetonitrile, 80% water (v/v) and mobile phase B was 20% acetonitrile, 80% 250mM ammonium acetate (v/v) ph 4.5. An injection volume of 100 μ L was used for analysis.

Site-specific analysis by trypsin mapping LC/MS: the fusion protein was diluted to 4mg/mL in about 100mM Tris pH 7.5 and 25% acetonitrile to a final volume of 100. mu.L. This solution was added to 40. mu.g of trypsin (V511A, Promega, Madison, Wis.) and incubated overnight at 37 ℃. The digestion was stopped by 1: 10 dilution with 0.1% trifluoroacetic acid (TFA). The digest was analyzed by LC/MS on an Agilent 1100 liquid chromatograph connected to a Q-TofMicromass spectrometer (Waters, Millford, Mass.), using a linear gradient of 0% to 50% acetonitrile: water with 0.1% TFA over 50 min. Zorbax 300-SB columns (4.6X150mm, Agilent, Palo Alto, Calif.) were used for separation and mass analysis. The flow from the LC was 1: 2 split before introduction to the mass spectrometer source.

Peak fractions were collected for S100b binding assay: the sRAGE-Ig fusion protein samples were concentrated to 4.0mg/mL using a 10K Amicon ultrafiltration column (Millipore, Bedford, Mass.), and then separated under reverse phase conditions for glycan site occupancy as described above. Three fractions representing the site-occupying variants of sRAGE-Ig were pooled, trisaccharide (fraction 1, F1), bisaccharide (fraction 2, F2) and non-glycosylated (fraction 3, F3). The collected samples were concentrated and exchanged into stabilization buffer. Protein concentrations of the fraction samples were determined with a DU 650 spectrophotometer (Beckman Coulter, Fullerton, CA) before subjecting the proteins to the S100b ELISA binding assay.

S100b binding to ELISA: coating buffer (Tris buffered saline [ TBS ] 100. mu.L/well]pH8.0, containing 5mM CaCl₂) 20. mu.g/mL S100b protein (Cat. No.559290, Calbiochem, Gibbstown, NJ) was coated onto ELISA assay plates overnight at 4 ℃. Then blocking buffer (TBS pH8.0, containing 5mM CaCl)₂0.05% Tween 20 and 1% bovine serum albumin [ BSA ]]) The plate was washed 3 times and blocked with blocking buffer for 1-1.5 hr. sRAGE-Ig fusion samples were prepared in dilution plates alone, with a maximum concentration of 10. mu.g/mL in blocking buffer, and 1: 4 serial dilutions down to 2.4ng/mL in blocking buffer. After blocking, the assay plate was washed with blocking buffer and 100 μ Ι _ of sample was transferred from the dilution plate to the assay plate. The assay plates were incubated at room temperature for 2.5 hr. Assay plates were then washed 3 times with blocking buffer with 1min soak time between washes. Horseradish peroxidase-conjugated goat anti-human IgG (Cat. No. A2290, Sigma, St. Louis, Mo.) diluted 1: 2500 in blocking buffer was added to the plate at 100. mu.L/well. The plate was incubated at room temperature for 1 hr. The assay plate was then washed 3 more times with blocking buffer, 100 μ L of 3, 3 ', 5, 5' -tetramethylbenzidine (TMB; Cat. No.34022, Pierce, Rockford, Ill.) was added to each well, and the plate was incubated for 8 minutes at room temperature. Then 100. mu.L of 1M H ₂SO₄Add each well, shake the plate, and read on a microplate reader at 450 nm.

Results

Expressing the sRAGE-Ig fusion protein in a host cell: SDS-PAGE analysis of purified fusion protein from CHO cell conditioned media in non-reducing and reducing gels showed that the fusion protein migrated as a monomer, indicating that the dimers were linked by non-covalent association. It should be noted that because the purification of the fusion protein employs protein a, it is expected that only Fc-containing fragments that retain the portion that binds to protein a are observed. FIG. 3 shows a photograph of a reduced SDS-PAGE gel and shows that the Full Length Monomer (FLM) migrating at about 55kDa is the major form of the fusion protein (lanes 1 and 6). The properties of fragments a and B (fig. 3, lanes 1 and 6) were confirmed by mass spectrometry in separate analyses. Fragment A represents amino acids 55-438 of the FLM migrating at about 46kDa (see FIG. 2), and fragment B represents about amino acids 199-438 of the FLM migrating at about 30 kDa. The amino acid numbering is based on the sequence shown in FIG. 2 (SEQ ID NO: 1). Treatment of the fusion protein with PNGaseF (fig. 3, lane 3) showed a shift in migration of all three bands, indicating that FLM plus fragments a and B were N-glycosylated. Complete digestion of the fusion protein with furin (FIG. 3, lane 5) resulted in the two major bands shown. The upper band in lane 5 co-migrates with the N-glycosylated form of fragment B seen in lane 6. The lower band in lane 5 co-migrates with the non-glycosylated form of fragment B seen in lane 3. This result, together with mass spectrometry data showing that fragment B contains the sequence immediately following the furin consensus cleavage site (amino acids 199-.

Analysis of full-length sRAGE-Ig fusion by size exclusion chromatography: the results of SEC analysis of purified fusion proteins from CHO conditioned media are shown in figure 4 and show 3 main peaks: peak 1 at 16.94 min; peak 2 at 18.64 min; and peak 3 at 21.63 minutes. The approximate molecular weight associated with peak 1 was about 96kDa, with a ± 10% error, as determined by laser scattering in combination with SEC. This result is consistent with the assignment of peak 1 as sRAGE-Ig homodimer, since the mass range calculated for the homodimer is about 97-107kDa, depending on glycosylation. Based on this result, it is possible that peak 2 on the SEC spectrum shown in fig. 4 represents a heterodimer (one full-length molecule associated with a furin-cleaved Fc fragment), and peak 3 may represent an Fc dimer or possibly a full-length monomer. A compositional summary of these peaks, also shown in figure 4, shows that the sRAGE-Ig fusion protein is composed primarily of homodimers (about 78%) and heterodimers (about 19%), with a small amount of Fc dimers or monomers (about 3%) and high molecular weight species (HMMS 2)/aggregates (less than 1%).

Fractionation of glycosylation site occupancy and site occupancy variants of sRAGE-Ig fusion proteins: the glycosylation site occupancy spectrum of the sRAGE-Ig fusion protein FLM is shown as sample 1(S1) in the data shown in FIG. 5. The spectrum shows that sample 1 contains predominantly 3-glycan type (67%) designated sample 1 fraction 1(S1F1) and 2-glycan type (22%) designated sample 1 fraction 2 (S1F 2) with very small amounts of 1-glycan and 0-glycan type (designated sample 1 fraction 3[ S1F3 ]). The purity of the fractions was confirmed by re-injection using reverse phase separation. Results demonstrating the nature of the 3-glycan and 2-glycan peaks of the S1 sample shown in figure 5 using LC/MS as described below.

Glycosylation site occupancy variants were analyzed by LC/MS: mass spectra of the peaks designated S1F1 and S1F2 as shown in fig. 5 were generated and the results are shown in fig. 6, comprising the upper panel (fig. 6A) and the lower panel (fig. 6B). This spectrum confirms the presence of several types within each sample 1 fraction, consistent with the glycosylation heterogeneity expected from the 3 potential N-linked glycan sites present in the molecule. FIG. 6 (top panel, FIG. 6A) shows that the earliest eluting peak, S1F1, is consistent with the pattern in which all 3 sites are occupied by N-linked glycans (52, 999-. The molecular weight of the unoccupied (0-glycan site occupied) species predicted based on the amino acid sequence is 48,594Da, a mass difference of about 4000-6000 Da. Fig. 6 (lower panel, fig. 6B) shows the mass spectrum of the resolved peak S1F 2. The mass spectrum showed a mass consistent with the type of bis-occupied (2-glycan site occupancy) (51,785-52,400 Da). Overall, LC/MS analysis showed a heterogeneous population of the types present in S1, with varying amounts of glycosylation. As shown by relative UV absorbance, the major types are fully occupied glycoproteins with lower levels of the diglycosylated type, and the presumed mono-and non-glycosylated types. The relative occupancy of 3N-linked sites was assessed using proteolytic mapping and a more detailed analysis of the glycan type present at each site was performed as described below.

Size and charge characterization of N-glycans released from sRAGE-Ig fusions: a graph showing normal phase HPLC chromatogram of N-linked glycans released from the fusion protein by incubation with PNGaseF is shown in fig. 7. Peaks were identified by comparison to commercially available glycan standards. The data show the apparent presence of sialylated glycan structures, as well as core fucosylated biantennary glycans typical of IgG molecules. Oligomannose structures were also observed (Man5, Man 6). The relative amounts of each oligosaccharide are shown in table 1, along with the corresponding proposed structures.

TABLE 1

Relative amount of N-glycan (%)

Legend: ■ -N-acetylglucosamine (GlcNac) tangle-solidup-fucose (Fuc) ● -mannose (Man) · o-galactose (Gal) · sialic acid ═ galactose (Gal) ·

The term "G0" refers to a biantennary structure in which there is no terminal sialic acid (NeuAc) or Gal, "G1" refers to a biantennary structure with one Gal and no NeuAc, and "G2" refers to a biantennary structure with two terminal gals and no NeuAc.

Analysis of the released glycans by anion exchange chromatography was used to quantify the relative amounts of neutral, single-charge, and double-charge types present. The results summarized in table 2 fit well with the glycan characterization data, showing that about 25% of the species present are sialylated.

TABLE 2

Relative amount of charged glycan

Neutral polysaccharide (%)	Monosialylated glycan (%)	Disialylated glycan (%)
			74.7	20.0	5.3

Sialylated glycans were not specifically identified in the chromatogram. The peaks marked "sialylation" in figure 7 were identified by treatment with sialidase and their corresponding shift in residence time. Acid hydrolysis was used and the residence time on reverse phase separation was then labelled and compared with the sialic acid reference figure, showing that the sialic acid type present on the molecule is N-acetylneuraminic acid. Mass spectrometry analysis of trypsin digestion confirmed these configurations.

Trypsin mapping analysis of sRAGE-Ig fusion proteins: the UV chromatogram of the tryptic digestion of the sRAGE-Ig fusion protein is shown in FIG. 8. Tryptic peptide fragments containing a common N-junction site were identified based on mass and were labeled T1, T10, and T31 in the chromatogram. The amino acid sequences of the trypsin digestion peptide fragments are as follows: t1 comprises pENITAR (SEQ ID NO: 5); t10 comprises VLPNGSFLPAVGIQDEGIFR (SEQ ID NO: 6); and T31 comprises EEQYNSTYR (SEQ ID NO: 7). T1 contained a first N-linked glycosylation site in the RAGE domain at Asn2(N2), T10 contained a second at Asn58(N58), and T31 contained an Fc domain N-linked glycosylation site present at Asn288(N288) of the fusion protein (but in other cases Asn297 of human IgG 1). The tryptic peptide fragments predicted to contain glycosylation sites are shown in FIG. 9. The T1 peptide has an N-terminal glutamine (Gln, Q) that is cyclized to form a pyroglutamic acid (pE) type, shown as T1 pE. The corresponding glycopeptides of these peptides were also tested. The inset in fig. 8 shows a magnified view of the region where T1 and T31 glycopeptides elute. Because of the co-eluting peaks, highly accurate quantitative data regarding the level of site occupancy may not be obtained. However, an estimate of the level at each site can be made. That is, the T1 peptide containing the first RAGE glycosylation site at Asn2 was approximately 90% occupied based on UV absorbance of the tryptic peptide fragments with and without glycan present. The glycopeptide of T1 co-eluted with glycopeptides from T31, resulting in this estimate. The same is true for the T31 peptide containing an IgG glycosylation site at Asn 288. However, very little unoccupied peptide was observed, and therefore it can be estimated that this site is at least 95% occupied. Finally, the T10 peptide containing the second RAGE glycosylation site at Asn58 was estimated to be approximately 85% occupied. Overall, these measurements agree well with the values obtained by a complete type of reverse phase analysis as described previously herein.

A mass spectrum of chromatographic peaks of the glycopeptides was generated to determine the nature of the glycans present at each site. Assigning glycan properties based on the mass of the glycopeptide measured from mass spectrometry in combination with properties determined from normal phase chromatographic analysis of released glycans summarized in table 1. The nature of the glycans present at each of the 3 glycosylation sites was also determined. The data show that most sialylation occurs at the Asn2 site and exists as G2 glycan, G2+2NANA, and G2+ NANA along with G1+ NANA glycan with one and two terminal N-acetylneuraminic acids, respectively. The Asn58 site mostly contains the oligomannose structure, mainly Man 5. Several small structures also exist at this site. Asn288 shows the predominant presence of G0 and G1 structures, typical of human IgG glycans. A summary of the glycans present at each site is shown in table 3, along with an estimate of their relative abundance.

TABLE 3

Properties of glycans present at each of 3 sites in the sRAGE-Ig fusion protein

Site of the body	Polysaccharides	Relative abundance
			Asn2(T1pE)	G2+2NANA	++
	G2+NANA	++
				G1+NANA	++
			Asn58(T10)	Man5	++
	Man6	+
				Man4	+
			Asn288(T31)	G0	+++
	G1	++

Tunicamycin treatment

The antibiotic tunicamycin is a glycosylation inhibitor that inhibits the glycosyltransferase that transfers a phosphate-N-acetylglucosamine (P-GlcNAc) from Uridine Diphosphate (UDP) -GlcNAc to form dolichol phosphate (Dol-P) -GlcNAc. Tunicamycin is used herein to demonstrate that inhibition of glycosylation can reduce the glycan site occupancy of a protein and, more preferably, can affect the binding characteristics of the protein to its cognate ligand binding.

Site occupancy of tunicamycin treated samples. The glycosylation site occupancy profile of the full-length sRAGE-Ig fusion protein expressed from CHO cells treated with tunicamycin is shown in sample 2(S2) in FIG. 10. The spectrum shows that the sample contains mainly 3-glycan type (42%), 2-glycan type (16%) and 0-glycan type (31%), and no 1-glycan type was detected. The nature of the 3-glycan, 2-glycan and 0-glycan peaks of the S2 sample in fig. 10 was confirmed using mass spectrometry before fractionation and collection of the S2 fractions 1(S2F1) (3-glycan), S2F2 (2-glycan) and S2F3 (0-glycan) for binding analysis (fig. 11). The 0-glycan type is consistent with the theoretical mass of sRAGE-Ig fusions with pyroglutamic acid (pE) at the N-terminus, and with or without a mixture of variants of C-terminal lysines, typical of IgG heavy chain molecules, confirming the heterogeneous C-terminus due to partial proteolytic processing during expression (i.e., lysine cleavage).

The assay site occupying variant binds to S100 b. S100b is a known RAGE ligand whose interaction with RAGE has been mapped to the VC1 domain. Binding of S100b to RAGE extracellular stimulates elevated RAGE levels, which leads to increased cellular responses and establishment of chronic inflammation and disease. Mixtures and fractions of site-occupying variants isolated from sRAGE-Ig fusion proteins as described above were therefore tested for their ability to bind to S100b, and the results are shown in table 4. The results are reported as IC50 values and relative binding to S100b (%) compared to S1 control (untreated sRAGE-Ig produced by CHO cells grown without tunicamycin).

TABLE 4

S100b binding ELISA

When sRAGE-Ig fusion protein was expressed in Chlamydine-treated CHO cells (sample S2), the glycan occupancy profile shifted from a mixture of 67% 3-glycan + 22% 2-glycan (untreated/control sample S1) to 42% 3-glycan + 16% 2-glycan + 31% 0-glycan (treated with Chlamydine/sample S2). Sample S2 bound to S100b (295%) about 3 times more than sample S1 (100%), indicating that the lower glycan occupancy of the mixture resulted in better binding of sRAGE-Ig to S100 b. Additional binding studies with isolated glycan fractions confirmed this finding. The 2-glycan fraction (S1F2) isolated from the S1 control showed increased binding to S100b (173%) compared to the 3-glycan fraction (46%) also isolated from the S1 control (S1F 1). The highest binding to S100b was observed in the 0-glycan fraction (S2F3) isolated from tunicamycin treated sample S2. The non-glycosylated variant (S2F3) showed a 10-fold increase (529%) when bound to S100b compared to the fully occupied 3-glycan form of sample S1F1 (46%) and a 5-fold increase when compared to the unfractionated, untreated S1 control (100%).

Discussion of the related Art

Human sRAGE-Ig fusion proteins were expressed and characterized in this study. In addition, the effect of glycosylation on the binding of the protein to RAGE ligand S100b was examined. While fusion proteins have traditionally been produced to increase the serum half-life of the fusion partner, it is rarely known how linking a portion of RAGE or a portion of sRAGE to an immunoglobulin domain or dimerization can affect the folding and glycosylation of sRAGE or its ability to attract ligands.

The data disclosed herein indicate that sRAGE-Ig fusion proteins expressed in CHO cells exist primarily as non-covalent homodimers, with the remainder as heterodimers formed by furin cleavage of the homodimers missing a single RAGE V-like domain. Because of the furin consensus sequence in the C1 domain of RAGE, it is unclear why the homodimer population is not cleaved more efficiently. Without wishing to be bound by any particular theory, the incomplete cleavage of FLM by furin, which results in a heterogeneous population of cleaved and uncleaved dimers, may be due to the fact that CHO and mammalian cells typically produce very small amounts of endogenous furin (Ayoubi et al, 1996, Molecular Biology reports.23: 87-95).

Also of interest is the biological purpose of the furin cleavage site in the RAGE molecule and whether furin cleavage has any functional significance for the biological function of endogenous RAGE molecules. Furin cleavage was shown to release soluble forms of apolipoprotein E receptor 2(ApoER2) (Koch et al, 2002, EMBO J.21: 5996-. Thus, and without wishing to be bound by any particular theory, furin may be involved in the production of soluble RAGE receptor fragments in vivo, which may be used to modulate or inhibit ligand interaction with its cellular targets or to control RAGE signaling. Although it has been shown that the novel splice variant of human RAGE mRNA is one of the mechanisms responsible for the generation of soluble forms of RAGE receptors (Yonekura et al, 2003, biochem. J.370: 1097-1109), less is known for the proteolytic process by which endogenous soluble RAGE can be generated. Interestingly, in mice, soluble RAGE was produced by carboxy-terminal truncation, in contrast to the alternative splicing mechanisms reported in humans (Hanaford et al, 2004, J.biol.chem.279: 50019-50024). Thus, without wishing to be bound by any particular theory, the furin cleavage observed in sRAGE-Ig fusion proteins may be indicative of endogenous cleavage of RAGE and may play a role in the biological function of the molecule in vivo.

The two potential sites of native RAGE in the V-like domain, N2 and N58, are N-glycosylated. Many studies have shown that glycosylation of RAGE may affect its affinity and/or specificity for RAGE ligands, and that this binding may vary depending on the ligand under consideration. Wilton et al (2006, protein expression Purif.47: 25-35) report that soluble RAGE (monomeric, non-glycosylated) expressed in bacteria binds to several RAGE ligands, including advanced glycation end product (AGE) -BSA, immunoglobulin light chain amyloid fibers, and glycosaminoglycans. However, sRAGE derived from e.coli, which is not a fusion protein, does not bind AGE Carboxymethyllysine (CML) -BSA. Dattilo et al (2007, Biochemistry 46: 6957-one 6970) also demonstrated that in addition to binding to S100b and amphoterin, soluble RAGE expressed by bacteria binds to AGE-BSA. In contrast, Srikrishna et al (2002, J. neurohem.80: 998-1008) reported that native, full-length glycosylated RAGE receptors isolated from bovine lung exhibited a significant reduction in binding to amphoterin when the receptor was deglycosylated. Osawa et al (2007, Biochimica et Biophysica Acta 1770: 1468-1474) also showed that deglycosylated mutants of soluble RAGE expressed in mammalian cells exhibit reduced binding to AGEs compared to the glycosylated wild-type. Thus, it is unclear what the effect of glycosylation or its lack on RAGE or sRAGE ligand binding is. Furthermore, none of the previous studies examined the effect of glycosylation on sRAGE binding to its ligand when it is fused to an Ig domain.

The data disclosed herein demonstrate for the first time that linking an immunoglobulin region to sRAGE does not appear to interfere with glycosylation of the V-like domain of RAGE, as the expressed fusion protein shows that about 90% and 85% of the N-attachment sites at Asn2 and Asn58, respectively, are occupied in the fusion protein. Without wishing to be bound by any particular theory, it is possible that because this fusion protein exists as a dimer, dimerization of sRAGE may have an effect on its affinity for ligands. Studies by Ostendorp et al (2007, EMBO J.26: 3868-3878) have shown that dimerization plays a role in ligand binding, and they have demonstrated that tetrameric S100b binds optimally to dimer sRAGE. This study demonstrated that sRAGE-Ig fusion proteins (3-glycan type) with N-glycosylation in 2 of all 3 site-V like domains and 1 of the Ig regions showed little binding to S100b compared to the non-glycosylated form (e.g., the form with no more than 2 occupied N-linked glycosylation sites). When 1N-junction site in the protein was not occupied (2-glycan type), sRAGE-Ig fusion protein bound to S100b increased, and showed the highest binding to S100b when all 3 sites in the protein were unoccupied (0-glycan type). Because it is not possible to distinguish between occupied N-attachment sites in RAGE V-like domains and occupied sites in Ig regions, the 2-glycan type may represent that both sites in RAGE V-like domains (N2 and N58) are occupied with no glycans at N288, or the 2-glycan type may represent glycan occupancy at N2 and N288 or at N58 and N288. However, because trypsin mapping data shows that the N-glycan site (Asn288) in the Ig region is most highly occupied in the 3 (95% compared to 90% for Asn2 and 85% for Asn 58), this data suggests that at least the 2-glycan type of 2/3 will contain a site occupied at Asn288, and thus the 2-glycan type of 2/3 should have only a single occupied N-attachment site, Asn2 or Asn58, in a RAGE V-like domain. The data disclosed herein show that the 2-glycan type of sRAGE-Ig fusion protein binds to S100b in increased compared to the 3-glycan type, and thus, without wishing to be bound by any particular theory, the increased binding may be the result of loss of glycosylation in the RAGE V-like domain. This trend is confirmed by the data disclosed herein, which show that the 0-glycan type of sRAGE-Ig fusion protein binds the highest to S100b compared to the 2-glycan or 3-glycan types. These data further show that increased ligand binding is the result of reduced glycosylation in the RAGE V-like domain, which is most pronounced for 0-glycan types that lack glycosylation entirely in the RAGE V-like domain.

In general, the data disclosed herein demonstrate for the first time that non-glycosylated variants of sRAGE-Ig fusion proteins provide optimal binding to the RAGE ligand S100 b. Since the binding site on RAGE for S100b has been identified as the VC1 domain (daillo et al, 2007, Biochemistry 46: 6957-6970), and without wishing to be bound by any particular theory, these data suggest that N-glycosylation at positions Asn2 and Asn58 in soluble RAGE may physically inhibit S100b, or may alter the recognition of the ligand binding site. In addition to studying the binding of RAGE ligand S100b, sRAGE-Ig fusion proteins and glycosylated variants thereof were tested for binding to A- β peptide and similar results were found, as demonstrated below.

Example 2:

increased A β binding of RAGE comprising reduced glycan site occupancy

sRAGE-Ig was expressed in untreated CHO cells (control; sample 1 ═ S1) and Tunicamycin (TMYCN) -treated CHO cells (sample 2 ═ S2) as described elsewhere herein previously. The sample is then fractionated as previously described to obtain a fraction comprising the fusion protein having the following glycan site occupancy: sample 1-fraction 1(S1F1) ═ untreated cells, 3-glycan occupied; sample 1-fraction 2(S1F2) ═ untreated cells, 2-glycan occupied; sample 2-fraction 1(S2F1) ═ TMYCN treated cells, 3-glycan occupied; sample 2-fraction 2(S2F2) ═ TMCYN treated cells, 2-glycan occupied; sample 2-fraction 3(S2F3) ═ TMYCN treated cells, 0-glycan occupied. Each fraction was compared to binding of S1 (control, untreated cells) for binding to a β, and the results are summarized below in table 5.

Determination of a β peptide binding activity: briefly, two human Α β peptides were tested: abeta 1-40 (product number # A-1153-2, Rceptide), and Abeta 1-28 (product number #24232, AnaSpec). With 1% NH₄Plates were coated overnight at 4 ℃ with 20. mu.g/mL peptide in OH. Then blocking buffer (TBS pH8.0, 0.05% Tween)20 and 1% BSA) wash plates and block for 1hr at room temperature. All other steps are performed essentially as previously described herein for the S100b binding assay.

TABLE 5

Effect of glycan site occupancy on sRAGE-Ig binding to A β

As previously disclosed herein with respect to binding of the fusion protein to S100b, when sRAGE-Ig fusion protein was expressed in Chlamydin-treated CHO cells (sample S2), the glycan occupancy profile shifted from a mixture comprising about 67% 3-glycan + 22% 2-glycan (untreated/control sample S1) to a mixture comprising about 42% 3-glycan + 16% 2-glycan + 31% 0-glycan (treated with tunicamycin/sample S2).

The relative binding of sample S2 to a β (151%) was about one and one half times that of sample S1 (100%), indicating that the lower glycan occupancy of the S2 mixture resulted in better binding of sRAGE-Ig to a β compared to the more glycosylated S1 sample. Additional binding studies with isolated glycan fractions confirmed this finding. Similar to the previously disclosed results for binding of S100b, the 2-glycan fraction isolated from the S1 control (S1F2) showed increased binding to S100b (170%) compared to the 3-glycan fraction also isolated from the S1 control (102%) (S1F 1). The highest binding to a β was observed for the 0-glycan fraction (S2F3) isolated from tunicamycin treated sample S2. The non-glycosylated variant (S2F3) showed a 6-fold increase (295%) in binding to a β compared to the fully occupied 3-glycan form (49%) of sample S2F1 and a 3-fold increase compared to the unfractionated, untreated S1 control (100%).

In summary, decreased glycan site occupancy mediates increased binding of sRAGE-Ig fusion proteins to a β. Thus, the data disclosed herein demonstrate that control of glycosylation can be used to improve the characteristics of a protein binding to its ligand.

Example 3:

glycosylation was controlled using 2-deoxy-D-glucose: reduced glycan site occupancy affects RAGE ligand binding

The RAGE-Fc fusion protein used in this study contains the ligand binding site for RAGE, which comprises a moiety C linked to a human IgG1 molecule_H2 domain and C_H3 domains of human RAGE V-like and C-like C1 domains. The fusion protein contains 3N-linked glycosylation sites, 2 in the RAGE domain at N2 and N58, and 1 in the Fc region (at amino acid residue 288 of the fusion peptide), which is about 70% fully occupied when expressed in CHO cells. As disclosed elsewhere previously herein, tunicamycin is used in cell culture to express the fusion protein and produce site-occupying variants. The glycosylation variants were isolated and the effect of glycan occupancy on binding to RAGE ligands was determined. As previously disclosed, the non-glycosylated 0-glycan site-occupying variant showed a 10-fold increase (529%) in binding to S100b compared to the fully occupied 3-glycan form (46%), while the double occupied 2-glycan variant showed moderate binding between the two (180%). Similarly, with respect to binding to a β, the non-glycosylated 0-glycan variant showed a 6-fold increase in binding to a β (295% compared to the unfractionated control glycan mixture) relative to the fully occupied 3-glycan form (49% compared to the unfractionated control glycan mixture), and the 2-glycan site occupied variant (238% relative to the unfractionated control glycan mixture) showed a moderate increase in binding (238% compared to the unfractionated control mixture), very similar to that of the 0-glycan variant. In summary, the data disclosed elsewhere herein using tunicamycin as a glycan site occupancy inhibitor demonstrates for the first time that the reduced site occupancy increases the affinity of RAGE for binding to its ligand, and more specifically, that RAGE-Ig fusion protein binding affinity can be controlled by inhibiting glycan site occupancy. Thus, these data demonstrate that the glycosylation pattern of a recombinant protein can be manipulated intracellularly to increase binding of the protein to its ligand. These data suggest the development of the use of inhibitors of N-glycan site occupancy for making a drug with increased knots Biological methods of synthesizing the active molecules are useful.

The less costly and non-toxic N-linked glycan site occupying inhibitor 2-deoxy-D-glucose is used to develop such a novel biological approach. 2-deoxy-D-glucose functions as a glycolytic antagonist by competitively inhibiting hexokinase and glucose phosphate isomerase (phosphorisomerase), and competes with mannose during N-linked glycosylation by incorporating dolichol-pyrophosphate linked oligosaccharides. The effect of using 2-deoxy-D-glucose on cell cultures expressing the sRAGE-Fc fusion proteins described previously using a controlled fed-batch bioreactor process was evaluated. The data disclosed herein demonstrate the effect of this inhibitor on N-junction site occupancy and on the activity of subsequent binding of the fusion protein to the S100b ligand. The results disclosed herein demonstrate that the yield of non-glycosylated variants of the fusion protein is increased relative to more glycosylated variants due to the use of the inhibitor. More importantly, the data disclosed herein demonstrate that fusion proteins produced using biological methods containing 2-deoxy-D-glucose demonstrate increased affinity for the S100b ligand.

Thus, the data disclosed herein demonstrate that 2-deoxy-D-glucose can be effectively used in a bioreactor process to control N-linked glycan site occupancy of recombinant molecules expressed in mammalian cells. Likewise, this inhibitor may be used during the manufacturing process to manipulate preferred characteristics of the molecule, such as optimal target/ligand/tissue binding activity, solubility, half-life/clearance from serum, etc.

Materials and methods

Cell line maintenance and bioreactor inoculum acclimatization

CHO cells expressing RAGE-Fc fusion proteins were maintained and expanded as suspension cultures in 125, 250, 500, 1000 or 2000ml aerated shake flasks (Corning) shaken at 140rpm, 1 inch swing and 36.5 ℃ on a shaker platform incubator.

0.5L bioreactor operation-DASGIP cell culture system

Having a maximum of 8 pH and O₂A modular system DASGIP Cellferm-pro culture system (Julich, Germany) set up for parallel culture of mammalian cells was controlled according to the manufacturer's instructions for the 0.5L bioreactor operation.

1L bioreactor System

For 1L bioreactor operation, with submerged O₂/CO₂An Applikon culture system (Cat No. v3mb010012, Applikon inc., Foster City, CA) inflated with air and a constant working volume of 1L stirred glass container was overlaid into the headspace controlled by an Applikon 1030ADI controller connected to an Applikon flow control console and a P100ADI1032 stirrer controller.

For substrate dosing (dosing), a Watson-Marlow 101U/R pump (Cat No.010.4202.00A, Watson-Marlow Bredel Inc., Wilmington, Mass.) was used.

Control Medium

Commercially available production medium CD CHO medium is a protein-free chemically defined medium for CHO growth and monoclonal antibody production (Cat No.10743.029, Invitrogen, Carlsbad, CA). Inoculum training medium was supplemented with 250. mu.l (25. mu.M) of 100mM L-methionine sulfoximine (L-methionine sulfoximine) (Cat No. GSS-1015-F Millipore). The production medium was preloaded with additional amino acids, 0.77g/L isoleucine, 1.08g/L leucine, 0.91g/L tyrosine 2Na, 0.38g/L valine, 0.405g/L cysteine, and 0.07g L tryptophan.

Experimental culture medium

Commercially available CD Opti-CHO media are protein-free chemically defined media (RefNo.12681-01, Invitrogen). Inoculum acclimatization medium was supplemented with 250. mu.l (25. mu.M) of 100mM L-Methionine Sulfoximine (MSX) (Cat No. GSS-1015-F, Millipore, Bedford, MA).

Opti-CHO without lipids is a commercially available medium with a known chemical composition without proteins (Invitrogen, Sku. ME080016). Inoculum acclimatization medium was supplemented with 250. mu.l (25. mu.M) of 100mM L-Methionine Sulfoximine (MSX) (Millipore, Cat No. GSS-1015-F).

Ex-Cell 325 medium is a protein-free commercially available medium (Cat No.14340C, Sigma). Inoculum training medium was supplemented with 250. mu.l (25. mu.M) of 100mM L-methionine sulfoximine (Millipore, Cat No. GSS-1015-F).

Nutritional supplements and supplements: the specialized feeding cdfv6.2 nutrient feeding solution is a chemically-defined powder of a powder formulation manufactured by Invitrogen (Carlsbad, CA). CDFv6.2 was formulated as a lipid-free nutrient feed solution with no subclasses of fatty acid precursors. Recombination of these feeds involved dissolving the feed in WFI and adjusting the pH to 7.0.

Glucose feed solution: the feed was a 300g/L (30%) glucose (Cat No. G-8920, Sigma) solution.

2-deoxy-D-glucose treatment: 2-deoxy-D-glucose (Cat No. D8375-100G or Cat No. D3179, Sigma) was dissolved in about 40ml of water for injection (WFI) and boluses were made based on the dosing strategy for each reactor as seen in Table 6 below.

ViCell cell counter: an automated cell viability analyzer manufactured by Beckman Coulter (model ViCell XR) performs the trypan blue exclusion method by video imaging of the flow-through cells.

Cedex cell counter: an automatic cell counter (model CEDEX AS20, innovatis AG, Bielefeld, Germany) designed to be equal to a hemocytometer detects and distinguishes live and dead cells by the trypan blue exclusion method of image analysis of microscopic images of CEDEX flow cells.

NOVA BioProfile 400 and Flex analyzer: an automated metabolite analyzer for mammalian cell suspensions (model BioProfile 400 and model BioProfile Flex, novabimultimedia, Waltham, MA) was used. BioProfile 400 analyses cell suspensions for nutrients/metabolites (glutamine, glucose, lactate, glutamate), acid/base status (pH, PCO2, PO2), electrolytes (sodium, potassium) and parameters calculated by specific enzymatic or non-enzymatic sensors (osmolality, air and CO2 saturation, HCO)₃). BioProfile Flex combines multiple analyzers in a single modular instrument that analyzes cell suspensions, module 1: gluc, Lac, Gln, Glu, NH4+, pH, PCO₂、PO₂Na +, K +, Ca + +, by electrochemistry; and (3) module 2: osmolality, reduced by freezing point; and a module 3: cell density/cell viability by digital imaging.

Osmometer: an automatic, single sample osmometer Advanced micro-osmometer (model 3320) (Advanced Instruments, inc., Norwood, MA) designed to handle 20- μ l of sample was used. Model 3320 freezing point depression was used by cooling the sample before analyzing the osmolality of the sample.

Agilent HPLC analysis: the sRAGE-Ig Fc fusion Protein production titers of unpurified cell culture broth samples were analyzed using the antibody affinity protocol POROS6B.M (Lincoln PGMSOP "Protein A HPLC, HB-0004-01") and Agilent 1100 series instruments (model G1316A, SN DE11122116 and model G1329A, SN DE 91603800). The procedure included the following chromatographic conditions: balance mobile phase: 1 XPBS (Invitrogen without MgCl)₂And CaCl₂) (ii) a Elution of mobile phase: 12mM HCl, 150ml NaCl, pH 2.25; washing buffer solution: WFI water; washing buffer solution: 6M guanidine and 70% ethanol; column: applied BioSystems A protein immunoassay sensor cartridge (Cat. No.2-1001-00, SN 6940, 7318); injection volume: 20 mu L of the solution; and a detector: UV absorbance lambda is 280 nm.

Results and discussion

Tunicamycin treatment-determination of binding of site occupying variants to S100b

The antibiotic tunicamycin is a glycosylation inhibitor that inhibits the glycosyltransferase that transfers a phosphate-N-acetylglucosamine (P-GlcNAc) from Uridine Diphosphate (UDP) -GlcNAc to form dolichol phosphate (Dol-P) -GlcNAc. Data disclosed elsewhere herein previously demonstrate that tunicamycin can be used to reduce the glycan site occupancy of a protein, more preferably, to affect binding of the protein to its ligand. However, tunicamycin is not a desirable inhibitor for use in bioreactor processes because it is toxic and expensive. Thus, the additional glycan site occupancy inhibitor 2-deoxy-D-glucose is used to control glycan site occupancy in RAGE-Ig fusion protein assays.

As noted elsewhere previously herein, S100b is a known RAGE ligand whose interaction with RAGE has been mapped to the ligand binding site of the V/C1 domain. The ability of the mixture and fractions of site-occupying variants isolated from tunicamycin-treated RAGE-Fc fusion protein to bind to S100b as described above was tested and the results are shown in table 5 above. The results are reported as IC50 values and relative binding to S100b (%) compared to S1 control (unfractionated isolated RAGE-Fc produced by untreated cells). When RAGE-Fc fusion protein was expressed in CHO cells treated with tunicamycin (sample S2), the glycan occupancy profile shifted from a mixture of about 67% 3-glycan + 22% 2-glycan (untreated/control sample S1) to a mixture containing about 42% 3-glycan + 16% 2-glycan + 31% 0-glycan (treated with tunicamycin/sample S2). Sample S2 bound to S100b (295%) approximately 3 times more than sample S1 (100%), indicating that the lower glycan occupancy of the mixture results in better binding of RAGE-Fc to S100 b. Additional binding studies with isolated glycan fractions confirmed this finding. The 2-glycan fraction isolated from the S1 control (S1F2) exhibited increased binding to S100b (173%) compared to the 3-glycan fraction also isolated from the S1 control (46%) (S1F 1). The highest binding to S100b was observed in the 0-glycan fraction (S2F3) isolated from tunicamycin treated sample S2. The non-glycosylated variant (S2F3) showed a 10-fold increase (529%) when bound to S100b compared to the fully occupied 3-glycan form of sample S1F1 (46%) and a 5-fold increase compared to the untreated S1 control (100%).

2-deoxy-D-glucose treatment-determination of binding of site occupying variants to S100b

It has been reported that treatment of cultures with 2-deoxy-d-glucose interferes with specific steps in the N-linked glycosylation pathway or functions as a substrate antagonist. 2-deoxy-D-glucose functions as a glycolytic antagonist by competitively inhibiting hexokinase and glucose phosphate isomerase. Furthermore, this carbohydrate analog was reported to compete with mannose during N-linked glycosylation by incorporating dolichol-pyrophosphate linked oligosaccharides.

Reactor run 39(R39) sampled at day 10 as listed in Table 6 represents the baseline glycan site occupancy profile and S100b binding activity of the RAGE-Fc fusion protein, purified from conditioned media harvested at day 10, using the CHO control protocol in a 0.5L DASGIP bioreactor. The CHO control method included a temperature shift from 34 ℃ to 31 ℃ on day 7. As shown in table 7, the results on day 10 and day 12 were essentially the same for this reactor. The RAGE-Fc fusion protein in this control approach is predominantly hyperglycosylated, with the majority of the molecules being in the form of 3-glycans (about 46%) and 2 glycans (about 28%). These samples showed minimal binding to S100 b.

Table 7 shows the results of the reactor R53 is the same reactor as R39, the same method of operation but adding in the method of the 9 th and 11 th day of the application of two 1mg/mL (based on the volume of the reactor of 6mM) bolus of 2-deoxy-D-glucose. The glycan site occupancy profile and S100b binding activity of RAGE-Fc fusion protein purified from conditioned medium of this reactor harvested at day 10 and day 12 are also shown in Table 2 and FIG. 1. The data disclosed herein demonstrate a 100-1521% increase in binding activity in this reactor at day 12 compared to that of baseline bioreactor R39. This increase in binding activity is associated with an increase in the type of non-glycosylation as well as a decrease in the higher glycosylation pattern.

A correlation between binding activity and site occupancy was observed in different media formulations and administration strategies. Commercially available CHO media (i.e., Ex Cell 325PF CHO and OptiCHO) were formulated with proprietary concentrations of media components such as amino acids, glucose, vitamins, salts, and trace elements. To confirm the effect of the addition of medium in combination with 2-deoxy-D-glucose on the glycosylation profile in the bioreactor, RAGE-Fc fusion protein-expressing CHO cells were directly adapted to CD OptiCHO (Invitrogen) medium and Ex Cell 325PF CHO (Sigma). Chemically defined, animal component and protein free medium CD OptiCHO was formulated as different batches: catalog formulations as well as lipid-free custom formulations to correspond to lipid-free formulations of Invitrogen CD CHO catalog media. Bioreactor R66 utilized 1L CHO Applikon control method minus amino acid preload, CD OptiCHO medium without lipid precursors, and cdfv6.2 nutrient feed without fatty acid precursor subclasses. A single 1mg/ml (6 mM based on 1L reactor volume) bolus of 2-deoxy-d-glucose was administered to the bioreactor on day 9. Media samples were harvested at day 12 and day 14, conditioned media samples were purified and tested for glycan site occupancy and S100b binding activity, with the results listed in table 7 and figure 12. The results show that at harvest, the fully glycosylated forms decreased and the non-glycosylated forms increased relative to the baseline "control" bioreactor R39 (3-glycan: day 12R 3945.8 vs. day 12R 6632.9%; 0-glycan: day 12R 390.3 vs. day 12R 6612.2%). This shift in glycoform distribution correlates with a 32-fold increase in S100b binding activity (100% baseline vs. day 10R 39. day 12R 663246%) and a 32-fold increase in S100b binding activity when compared to the day 12 baseline bioreactor R39 sample. In addition, the results shown in table 6 and fig. 12 indicate that the glycan occupancy profile and S100b binding activity increased with continued culture.

Protein-free medium formulated without glutamine Ex-Cell325PF CHO medium was studied in a 1L Applikon bioreactor run R79 using the CHO control method minus 6 amino acid medium preload. Two 1mg/mL (6 mM based on 1L bioreactor volume) bolus doses of 2-deoxy-d-glucose were administered to the bioreactor on days 7 and 9. Media samples were harvested at day 12 and day 14, conditioned media was purified and glycan site occupancy and S100b binding activity were determined. Data for these bioreactor conditions are shown in table 7 and R79 in fig. 12. The data disclosed herein show an increase in the type of complete glycosylation (3-glycans: R7968.0% vs. 12 day R3945.8%) and an increase in the 0-glycan form (0.8% R79vs. 12 day 0.3% R39 on day 12). This change in glycoform distribution correlates with a 5-fold increase in S100b binding activity at day 12 compared to baseline bioreactor R39 (day 10) (100% at baseline R39 compared to 527% at baseline R79, day 10 control R39 binding). Furthermore, the data disclosed herein, e.g., table 7 and fig. 12, indicate that glycan occupancy profiles and S100b binding activity increase with continued culture.

Similar increases in binding of the non-glycosylated variant to S100b were obtained when 2-deoxy-d-glucose bolus injection was performed using CD CHO medium. Reactor R80 in Table 7 was identical to reactor R79 and was run in the same manner, but using CD-CHO instead of Ex Cell325PF CHO medium. The data for this bioreactor are shown in table 7 and R80 in fig. 12. The data disclosed herein show that similar to R79, treatment with 2-deoxy-D-glucose results in an overall reduction in the type of complete glycosylation, and also mediates an increase in the 0-glycan form. Notably, the fully glycosylated form was reduced (45.7-21.7%) and the non-glycosylated form was increased (0.3-31.8%) relative to the baseline bioreactor (day 10R 39). The shift in glycan occupancy significantly increased S100b binding activity (100-. The S100b binding activity of the day 14 sample from the R80 reactor was the highest obtained and was significantly higher than that associated with the sample from the baseline reactor R39-surprisingly, the bioreactor run R80 was about 8077% or about 80-fold higher than the control bioreactor R39 on day 10.

Overall, 2-deoxy-D-glucose supplementation produces an increase in RAGE-Ig non-glycosylated (0-glycan site occupancy) variants that also exhibit increased binding to S100b ligands. The surprising increase in binding affinity with the non-glycosylated variant is the result of a change in the glycosylation profile pattern following the 2-deoxy-D-glucose dosing strategy. The increased production of non-glycosylated variants of the RAGE-Ig fusion protein was demonstrated in a bioreactor using the CHO control method in combination with two 1g/L administrations of 2-deoxy-D-glucose. The increased production of non-glycosylated variants in turn mediated a surprising increase in S100b binding activity compared to an untreated reference bioreactor with a greater amount of fully glycosylated (3-glycan sites occupied) variants. The data disclosed herein demonstrate that inhibitors of glycan site occupancy, such as, but not limited to, 2-deoxy-D-glucose, can be used in manufacturing processes to control the N-linked site occupancy of recombinant proteins in order to manipulate preferred characteristics of the proteins affected by glycosylation, such as optimal target/ligand/tissue binding activity, solubility, half-life, clearance from serum, and the like. One skilled in the art will appreciate that once the disclosure provided herein is provided, which utilizes glycan site occupancy inhibitors to control glycan site occupancy in recombinant proteins without the need to introduce amino acid mutations to delete glycosylation sites, provides an important new approach for rapidly controlling protein characteristics without affecting protein conformation, loss of activity, increased immunogenicity, etc., which are potentially associated with such mutations.

Example 4:

glycosylation was controlled using 2-deoxy-D-glucose: reduced glycan site occupancy by antibodies

Antibodies that specifically bind human immunoglobulin E for use in determining that the novel cell culture methods of the invention can be used to produce glycoproteins that comprise reduced glycosylation as compared to the level of glycosylation of the same protein produced under otherwise identical cell culture conditions but in the absence of a glycosylation inhibitor.

As shown in fig. 13A to 13D, the fully human anti-IgE antibody was designated antibody 5.948.1 (also referred to herein as "IgE mAb" or "mAb 5.948.1"). Antibody 5.948.1 contains potential N-linked glycosylation sites, i.e., [ nts ], at amino acid residues 73-75 of the heavy chain polypeptide V domain, and at amino acid residues 301-303 of the constant domain, i.e., [ NST ] (see FIG. 13A). See also international patent application No. PCT/US2008/004286, filed on 1/4/2008, and now published as WO 2008/123999 on 16/10/2008 (incorporated herein by reference in its entirety). FIG. 13A shows the amino acid sequence of the heavy chain (SEQ ID NO: 8), with variable domain residues in lower case and CDRs underlined. The V domain glycosylation site (N2) is capitalized, and the constant domain glycosylation site (N301) is underlined. FIG. 13B shows the nucleic acid sequence (SEQ ID NO: 9) encoding the heavy chain amino acid sequence (SEQ ID NO: 8). FIG. 13C shows the amino acid sequence of the light chain (SEQ ID NO: 10), with variable domain residues in lower case and CDRs underlined. FIG. 13D shows a nucleic acid sequence (SEQ ID NO: 11) encoding a light chain amino acid sequence (SEQ ID NO: 10).

Thus, mAb 5.948.1 is a fully human IgG comprising a heavy chain₂The heavy chain comprises two glycosylation sites: asn73 in framework region 3(FR3) of the V domain and a typical Fc heavy chain glycosylation site Asn 301. Figure 14 provides a graph showing the 4 possible glycan-occupying site variants of antibody 5.948.1. That is, a 0-glycan occupying site variant that does not comprise a glycan, a 1-glycan occupying site variant that comprises a glycan at Asn301, a 1-glycan occupying site variant that comprises a glycan at Asn73, and a 2-glycan occupying variant that comprises a glycan at both Asn73 and Asn 301. Because each antibody comprises two heavy chains, various permutations are possible, including various combinations of each of the above glycan occupying variants with two light chains.

Cell cultures expressing mAb 5.948.1 were grown in the presence and absence of 2-deoxy-D-glucose. Briefly, a clonal population of CHO cells expressing mAb 5.948.1 was designated 0.3X10⁶One cell/mL was inoculated into a 6x500mL aerated shake flask, 100mL medium per flask. Shake flasks (S5, S6, S7, S9, S10, S11, see Table 9) containing cells were placed in a non-addition flaskOn a wet shaker at 36.5 ℃ with 5% CO₂And 140rpm oscillation with a 1 inch swing. Cell counts and viability were sampled daily in 14 days of culture. Beginning on day 3 of culture, all flasks were fed with nutrients (16 mL/L/day) and a bolus of glucose (2.5 mL/day 100g/L glucose stock). On days 7 and 9 of culture 1g/L of 2-deoxy-d-glucose was added to the test flasks (S10 and S11, respectively), whereas no 2-deoxy-d-glucose inhibitor was added to the control flasks (S6 and S7). On day 7 of culture, shake flasks S5 and S9 were harvested for titer and glycan site occupancy analysis by HPLC. S9 was harvested before the addition of 2-deoxy-d-glucose. On day 12 of culture, shake flasks S6 (without inhibitor) and S10 (with inhibitor) were harvested for titer determination and glycan site occupancy analysis. On day 14 of culture, shake flasks S7 (without inhibitor) and S11 (with inhibitor) were harvested for titer determination and glycan site occupancy analysis.

Protein titers were measured for each sample and the results are provided in table 8. Protein titers increased with culture time and there was no significant difference in protein titers between cultures without 2-deoxy-D-glucose and cultures containing glycosylation inhibitors. Thus, the presence of 2-deoxy-D-glucose does not appear to affect protein expression in cells grown in the presence of the inhibitor.

TABLE 8

Sample (I)	Sample ID	2-deoxy-D-glucose	Titer (mg/ml)
				S5	Day 7 of S5	Is free of	3.77
S6	Day 12 of S6	Is free of	4.37
				S7	Day 14 of S7	Is free of	4.92
S9	Day 7 of S9	Is free of	3.79
				S10	Day 12 of S10	Is provided with	4.43
S11	Day 14 of S11	Is provided with	4.61

The effect of the addition of 2-deoxy-D-glucose on glycan site occupancy was compared to the glycan site occupancy of mAb 5.948.1 produced by the cells in the absence of the inhibitor. The glycan profile of each sample was determined and the chromatogram is shown in figures 16 to 24. The amount of each glycan site occupying variant in each sample was determined and the results are shown in table 9 below.

TABLE 9

More specifically, the glycan profile of the control sample of mAb 5.948.1 produced under conditions that provided the greatest glycosylation is shown in figure 15. In addition, the control demonstrated about 98% 2-glycans and about 2% 1-glycans, with no detectable 0-glycans present. Interestingly, the majority of the 1-glycan variants present (about 75%) were glycans containing a sugar at Asn301, the Fc glycosylation site present in the constant region, and the remaining 25% of the 1-glycan fraction contained Asn73 glycan, which contained N-linked glycans in the variable region (antigen binding fragment; Fab).

The glycan profiles of both culture samples (S5 and S9) without inhibitor taken on day 7 were similar to the control. That is, on day 7, the glycans comprise about 98% 2-glycans and about 2% 1-glycans, with no detectable 0-glycan variants. In addition, most of the 1-glycan forms are glycosylated at the Fc glycosylation site (Asn301), and the remaining 1-glycans are glycosylated in the variable region.

The glycan profile of the antibody produced in the control culture grown in the absence of the inhibitor demonstrated a reduction in the percentage of 2-glycans present over time, with the percentage of 2-glycans decreasing from about 98% to about 95.5% at day 14. In addition, the percentage of glycosylated 1-glycans in the Fc region increases from about 1.2% to about 4.1%. However, no 0-glycan was detected at any time in the control culture and the amount of glycosylated 1-glycan in Fab did not change over time.

On day 12, the shift in glycan profiles was confirmed by antibodies produced in cultures grown in the presence of the inhibitor (S10). More specifically, the percentage of 2-glycans was reduced to about 94%, and the percentage of 1-glycans was significantly increased, with increased levels of both 1-glycans (1.6%) glycosylated in the Fab region and 1-glycans (3.5%) glycosylated in the Fc region. Even more surprising is that cells grown in the presence of 2-deoxy-D-glucose produced detectable amounts (0.9%) of 0-glycan variants. After 2 days, at day 14, the increase in 1-glycan and 0-glycan variants was even more dramatic, and the decrease in 2-glycan fractions was more pronounced. That is, in S11, the 2-glycan variant comprises about 90% of the antibody produced, and the 1-glycan variant comprises 2.3% glycosylation in the Fab region and 5.2% glycosylation in the Fc region. The percentage of antibody without glycosylation (0-glycans) comprises about 1.7% of the antibody 5.948.1 produced.

These results demonstrate that glycosylation inhibitors can be used to control glycosylation of glycoproteins produced in mammalian cell culture. More specifically, antibodies comprising 2 glycosylation sites in the heavy chain produced in cell culture in the presence of 2-deoxy-D-glucose comprise a reduced amount of 2-glycan variants and increased amounts of 1-glycan and 0-glycan forms. Without wishing to be bound by any particular theory, it appears that glycosylation in the Fc domain of the antibody occurs preferentially compared to glycosylation in the Fab region of the antibody. Also not wishing to be bound by any particular theory, it is possible that the 2-glycan antibodies produced continue to be present in the culture before the inhibitor is added to the culture medium. Thus, the relative decrease in 2-glycan variants present over time and the consequent increase in 1-glycan and eventually 0-glycan variants appear to indicate that only very little, if any, additional 2-glycan variants are produced upon addition of the inhibitor. It can also be shown that once the inhibitor is added, the 1-glycan Fab form is produced while some additional 1-glycan Fc variants continue to be produced. The results also indicate that the addition of the inhibitor mediates the production of 0-glycan non-glycosylated variants. Thus, the data disclosed herein demonstrate for the first time that glycosylation of glycoproteins can be controlled, and even more preferably, that protein titers are not significantly reduced. The results further indicate that additional modifications of culture conditions, including but not limited to increasing the length of culture time, can provide further increases in the production of 0-and 1-glycan forms to about or greater than 30% of the glycoprotein produced.

Example 5:

glycosylation of anti-IgE antibodies with reduction of 2-deoxy-D-glucose: reduced glycan site occupancy and impact on antibody binding to IgE binding

Any effect on antigen binding of mAb 5.948.1 mediated by reduced glycan site occupancy was evaluated using standard methods such as, but not limited to, the methods disclosed in WO 2008/123999 (e.g., IgE cell binding assays using RBL-2H3 cells, degranulation inhibition assays, depletion of free IgE from serum, ELISA IgE binding assays, BIAcore^TMAssays, etc.), and other assays for evaluating antibody binding characteristics are well known in the art.

More specifically, BIAcore is utilized^TM3000 instrument (BIAcore)^TMUppsala, Sweden) by BIAcore^TMCells cultured in the absence (S5, S6, and S7) and presence (S9, S10, S11) of 2-deoxy-D-glucose were evaluated analytically for binding characteristics of mAb 5.948.1 produced. Briefly, antibodies from each sample were covalently immobilized on a CM5 sensor chip (BIAcore) using standard EDC-NHS coupling chemistry and 10mM sodium acetate, ph 5.0 immobilization buffer^TM) The above. The reference flow cells were activated by EDC-NHS and blocked with ethanolamine, but no protein was immobilized thereon.

HBS-EP buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005% surfactant P-20 pH 7.4) at a flow rate of 50 or 100. mu.L/min was used as a buffer for BIAcore^TMSupplied) kinetic measurements were obtained with IgE (Serotec, cat. No. php008x2 or EuropaBioproducts, cat. No. cp1035k) at concentrations ranging from about 0.09 to 600 nM. The injection time for each concentration was about 3.25 minutes followed by 20 minutes of the dissociation phase. The dissociation phase was followed by a regeneration step using a regeneration solution (10mM glycine, pH 1.7). Evaluation software Using BIA 4.1 (BIAcore)^TM) And Scrubber Software version 2.0(BioLogic Software) local fitting sensorgrams (Sensorgram).

The affinity (KD), binding rate (ka, M-1S-1), and dissociation rate (KD) of each sample (S5, S6, and S7) produced in the absence of inhibitor were compared to samples (S9, S10, and S11) produced in the presence of inhibitor to evaluate the effect of reduced glycosylation on antibody binding of mAb 5.948.1 to its antigen human IgE.

The nucleic acid and amino acid sequences described herein and their sequence identifiers are listed in table 10 below.

Watch 10

Sequence of

Each of the publications and each of the patents, patent applications, and publications cited herein are incorporated by reference in their entirety.

While the invention has been disclosed with reference to specific embodiments, it is apparent that other embodiments and variations of the invention may be devised by others skilled in the art without departing from the true spirit and scope of the invention. The appended claims should be construed to include all such embodiments and equivalent variations.

Claims (36)

1. A method for reducing the level of glycosylation of a protein, the method comprising expressing a protein comprising at least one glycosylation site in a host cell grown in culture in the presence of a glycosylation inhibiting amount of a glycosylation inhibitor, wherein the protein comprises a lower level of glycosylation as compared to an otherwise identical protein produced in an otherwise identical host cell grown under otherwise identical conditions in the absence of the inhibitor, thereby controlling the level of glycosylation of the protein.

2. The method of claim 1, wherein the glycosylation site is in a ligand binding site or an antigen binding site.

3. The method of claim 2, wherein the glycosylation level is selected from the group consisting of glycan site occupancy at the glycosylation site and degree of glycosylation at the glycosylation site.

4. The method of claim 3, wherein said glycosylation sites are selected from the group consisting of O-linked glycosylation sites and N-linked glycosylation sites.

5. The method of claim 4, wherein the glycosylation site is an N-linked glycosylation site, and further wherein the glycosylation site comprises the amino acid sequence asparagine-X-serine or asparagine-X-threonine, wherein X is any amino acid except proline.

6. The method of claim 5, wherein the glycosylation inhibitor is at least one selected from the group consisting of: tunicamycin, tunicamycin homologues, streptovirins, brazidomycins, amfomycin, ziromycin, antibiotic 24010, antibiotic MM 19290, bacitracin, corynetoxin, pyrophyllomycin, chlortetracycline, 1-deoxymannose nojirimycin, deoxynojirimycin, N-methyl-1-deoxymannose nojirimycin, brerafenidin A, glucose analogs, mannose analogs, 2-deoxy-D-glucose, 2-deoxyglucose, D- (+) -mannose, D- (+) galactose, 2-deoxy-2-fluoro-D-glucose, 1, 4-dideoxy-1, 4-imino-D-mannitol (DIM), fluoroglucose, fluoromannose, UDP-2-deoxyglucose, deoxynojirimycin, brefeldin A, bredin B, doxycycline A, 2-deoxyglucose, D-, GDP-2-deoxyglucose, mevalonate monoacyl-CoA reductase inhibitors, 25-hydroxycholesterol, swainsonine, actinone, puromycin, actinomycin D, monensin, carbonyl cyanide metachlorobenzohydrazone (CCCP), compactin, polyterpene prolactin-phosphoryl-2-deoxyglucose, N-acetyl-D-glucosamine, hypoxanthine, thymidine, cholesterol, glucosamine, mannosamine, castanospermine, glutamine, bromocyclohexenetetraol, cyclohexenetetraol epoxide, cyclohexenetetraol derivatives, glycosylmethyl p-nitrophenyltriazene, beta-hydroxynorvaline, threo beta-fluoroaspartamide, D- (+) -glucono-delta-lactone, bis (2-ethylhexyl) phosphate, Tributyl phosphate, dodecyl phosphate, 2-dimethylaminoethyl (diphenylmethyl) -phosphate, [2- (diphenylphosphinyloxy) ethyl ] trimethylammonium iodide, iodoacetate and fluoroacetate.

7. The method of claim 6, wherein the glycosylation inhibitor is 2-deoxy-D-glucose.

8. The method of claim 7, wherein the glycosylation inhibiting amount ranges from about 0.5g/L to 3 g/L.

9. The method of claim 8, further comprising maintaining the glucose concentration in the culture in an amount ranging from about 0.05g/L to 10 g/L.

10. The method of claim 8, wherein the host cell is selected from the group consisting of a yeast cell, an insect cell, and a mammalian cell.

11. The method of claim 10, wherein said mammalian host cell is selected from the group consisting of CHO cells, NS0 cells, NS0/1, Sp2/0, human cells, HEK 293, BHK, COS, Hep G2, per.c6, COS-7, TM4, CV1, VERO-76, MDCK, BRL 3A, W138, MMT 060562, TR1, MRC5, and FS 4.

12. The method of claim 11, wherein the host cell is a CHO cell.

13. The method of claim 12, wherein the glycosylation site is at least one glycosylation site comprising an amino acid sequence selected from the group consisting of asparagine-isoleucine-threonine (NIT), asparagine-glycine-serine (NGS), asparagine-serine-threonine (NST), and asparagine-threonine-serine (NTS).

14. A protein produced according to the method of claim 10.

15. A protein produced according to the method of claim 13.

16. The protein of claim 15, wherein the protein is an antibody or antigen-binding portion thereof, and further wherein the antibody is an anti-human IgE antibody.

17. The protein of claim 16, wherein the antibody is a human antibody comprising a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8, and the light chain variable region has the amino acid sequence of SEQ ID NO: 10.

18. The protein of claim 17, wherein said antibody comprises at least one N-linked glycosylation site that is unoccupied and/or comprises at least one sugar moiety less than said otherwise identical antibody produced in the absence of said glycosylation inhibitor, wherein said site is selected from the group consisting of a heavy chain corresponding to SEQ ID NO: 8 at asparagine 73(N73) and asparagine 301 (N301).

19. The protein of claim 18, wherein the antibody comprises an unoccupied N-linked glycosylation site at asparagine 73.

20. The protein of claim 15, wherein the protein comprises a Ligand Binding Site (LBS) for receptor for advanced glycation end products (RAGE) comprising an amino acid sequence selected from the group consisting of SEQ id no: 13. SEQ ID NO: 14 and SEQ ID NO: 15, or a pharmaceutically acceptable salt thereof.

21. The protein of claim 20, wherein the protein is a RAGE fusion protein comprising the amino acid sequence of SEQ ID NO: 1, the signal sequence comprising amino acid residues 1 to number 23, wherein the fusion protein lacks a terminal lysine residue (Lys 438).

22. The protein of claim 21, comprising at least one N-linked glycosylation site that is unoccupied and/or comprises at least one sugar moiety less than an otherwise identical RAGE fusion protein produced in the absence of the glycosylation inhibitor, wherein the site is at least one site selected from the group consisting of asparagine at amino acid residue number 2 (N2), asparagine at amino acid residue number 58 (N58), and asparagine at amino acid residue number 288 (N288), all corresponding to seq id NO: 1.

23. The protein of claim 22, wherein said protein comprises at least two N-linked glycosylation sites that are unoccupied and/or comprise at least one carbohydrate moiety less.

24. The protein of claim 22, wherein the protein comprises three N-linked glycosylation sites, at least one of which is unoccupied and/or comprises at least one carbohydrate moiety less.

25. The protein of claim 18, wherein said protein exhibits increased binding to an antigen as compared to said otherwise identical protein produced in the absence of said inhibitor.

26. The protein of claim 22, wherein said protein exhibits increased binding to a RAGE ligand as compared to the otherwise identical protein produced in the absence of the inhibitor.

27. The method of claim 11, further comprising a temperature transition from a temperature in the range of about 34 ℃ to 39 ℃ to a temperature in the range of about 28 ℃ to 33 ℃.

28. The method of claim 27, comprising a temperature transition from a temperature in the range of about 34 ℃ to 37 ℃ to a temperature in the range of about 30.5 ℃ to 31.5 ℃.

29. A pharmaceutical composition comprising the protein of claim 14 and a pharmaceutically acceptable carrier.

30. A pharmaceutical composition comprising the protein of claim 15 and a pharmaceutically acceptable carrier.

31. A composition comprising an amount of a fusion protein, wherein the fusion protein comprises a RAGE polypeptide linked to an immunoglobulin polypeptide,

a) wherein the RAGE polypeptide comprises human RAGE (SEQ ID NO: 3) wherein the fragment of human RAGE comprises a ligand binding site and at least one amino acid residue that may be glycosylated,

b) Wherein the immunoglobulin polypeptide comprises C of an immunoglobulin_H2 domain or part C_H2 domain and immunoglobulin C_H3 domain, and

c) wherein the N-terminal residue of the immunoglobulin polypeptide is linked to the C-terminal residue of the RAGE polypeptide; and

wherein at least 0.5% of the amount of the fusion protein is non-glycosylated.

32. The composition of claim 31, wherein at least 30% of the total amount of the fusion protein is non-glycosylated.

33. The composition of claim 31, wherein the percentage of the amount of the fusion protein in a fully glycosylated form is less than the percentage of the amount of the fusion protein in all non-fully glycosylated forms.

34. The composition of claim 31, wherein the fusion protein comprises at least three amino acid residues that may be glycosylated, wherein a first potential site of glycosylation is an amino acid residue of the RAGE ligand binding site, a second potential site of glycosylation is an amino acid residue of the RAGE polypeptide, and a third potential site of glycosylation is an amino acid residue of the immunoglobulin polypeptide.

35. A composition comprising a protein comprising at least one potential glycosylation site, wherein the amount of fully glycosylated protein is less than the amount of less than fully glycosylated protein, and further comprising a pharmaceutically acceptable carrier.

36. The composition of claim 35, wherein the protein comprises two potential glycosylation sites, and wherein the amount of fully glycosylated protein is less than the sum of the amount of protein comprising one site that is not glycosylated and/or is less glycosylated and the amount of protein comprising two sites that are not glycosylated and/or are less glycosylated.