Enzymes for sialylation of glycans

Abstract

Described herein are fusion proteins, e.g., fusion proteins comprising an enzymatically active portion of ST6Gal1 or B4GalT1, as well as methods for producing them, nucleic acid molecules encoding the fusion proteins, vectors comprising the nucleic acid molecules, and host cells comprising the vectors. Also described herein are methods for sialyating immunoglobulin G (IgG) antibodies.

Description

[Technical Field]

[0001] (Priority Claim) This application claims the benefit of U.S. Provisional Application No. 62 / 981,293, filed February 25, 2020, and U.S. Provisional Application No. 63 / 026,927, filed May 19, 2020, the entire contents of which are incorporated herein by reference.

[0002] (Sequence Listing) This application contains a Sequence Listing that has been submitted electronically in ASCII format, which is incorporated herein by reference in its entirety. The ASCII copy was created on April 5, 2021, is named 14131-0227WO1_SL.txt, and is 54,833 bytes in size.

[0003] FIELD OF THE INVENTION The present disclosure relates to fusion proteins, e.g., fusion proteins comprising an enzymatically active portion of ST6Gal1 or B4GalT1, as well as methods for producing them, nucleic acid molecules encoding the fusion proteins, vectors comprising the nucleic acid molecules, and host cells comprising the vectors. Also described herein are methods for sialyating immunoglobulin G (IgG) antibodies. [Background technology]

[0004] Intravenous immunoglobulin (IVIg), prepared from pooled human donor plasma (e.g., plasma pooled from at least 1,000 donors), is used to treat a variety of inflammatory disorders. However, IVIg preparations have obvious limitations, including variable efficacy, clinical risks, high cost, and finite supply. Although different IVIg preparations are frequently treated as clinically interchangeable, significant differences exist among product preparations that can affect tolerability and activity in selected clinical applications. Current maximum dosing regimens often result in only partial and inadequately sustained responses. Additionally, the long infusion times (4–6 h) associated with high-dose IVIg treatment consume significant resources at infusion centers and negatively impact patient-reported outcomes, such as convenience and quality of life.

[0005] The identification of the important anti-inflammatory role of Fc domain sialylation presents an opportunity to develop more potent immunoglobulin therapies. Commercially available IVIg preparations generally exhibit low levels of sialylation on the Fc domain of the present antibodies. Specifically, they exhibit low levels of disialylation of branched glycans on the Fc region.

[0006] Washburn et al. (Proceedings of the National Academy of Sciences, USA 112:E1297-E1306 (2015)) described a controlled sialylation process to generate highly tetra-Fc-sialylated IVIg and showed that this process resulted in a product with consistent and enhanced anti-inflammatory activity. Summary of the Invention [Means for solving the problem]

[0007] Described herein is a method for preparing immunoglobulin G (IgG) with very high levels of Fc sialylation. The method described herein can provide hypersialylated IgG (hsIgG), in which more than 70% of the branched glycans on the Fc domain are sialylated on both branches (i.e., on the α1,3 branch and the α1,6 branch). HsIgG contains a diverse mixture of IgG antibody subtypes, with IgG1 antibodies being the most common, followed by IgG2. Because the starting material is IgG antibodies pooled from hundreds or thousands of donors, antibody diversity is extremely high. The IgG antibodies used to prepare hsIgG can be obtained, for example, from pooled human plasma (e.g., plasma pooled from at least 1,000 to 30,000 donors). Alternatively, IVIg, including commercially available IVIg, can be used to prepare hsIgG. HsIgG has a much higher level of sialic acid on the branched glycans on the Fc region than IVIg. This results in a composition that differs from IVIg in both structure and activity. HsIgG can be prepared as described in WO 2014 / 179601 or Washburn et al. (Proceedings of the National Academy of Sciences, USA 112:E1297-E1306 (2015)), both of which are incorporated herein by reference.

[0008] Described herein is an improved method for preparing hsIgG.

[0009] In highly sialylated IgG, at least 60% (e.g., 65%, 70%, 75%, 80%, 82%, 85%, 87%, 90%, 92%, 94%, 95%, 97%, 98%, including up to 100%) of the branched glycans on the Fc region are disialylated via NeuAc-α2,6-Gal terminal linkages (i.e., on both the α1,3 branches and the α1,6 arms). In some embodiments, less than 50% (e.g., less than 40%, 30%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%) of the branched glycans on the Fc region are monosialylated via NeuAc-α2,6-Gal terminal linkages (e.g., sialylated only on the α1,3 arms or only on the α1,6 arms).

[0010] In some embodiments, the polypeptide is derived from plasma, e.g., human plasma. In certain embodiments, the polypeptide is predominantly an IgG polypeptide (e.g., IgG1, IgG2, IgG3, or IgG4, or a mixture thereof), although minor amounts of other, containing minor amounts of other immunoglobulin subclasses, may be present.

[0011] As used herein, the term "antibody" refers to a polypeptide that includes an amino acid sequence providing at least one immunoglobulin variable region, e.g., an immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody may include a heavy (H) chain variable region (referred to herein as V H ) and a light (L) chain variable region (abbreviated as V L In another example, an antibody comprises two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab, F(ab')2, Fd, Fv, and dAb fragments) as well as whole antibodies, e.g., intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (and subtypes thereof). The light chains of the immunoglobulin may be of type kappa or lambda.

[0012] As used herein, the term "constant region" refers to a polypeptide corresponding to or derived from one or more constant region immunoglobulin domains of an antibody. A constant region may include any or all of the following immunoglobulin domains: C, C, C-terminal domains ... H 1 domain, hinge region, C H 2 domains, C H 3 domains (derived from IgA, IgD, IgG, IgE, or IgM), and C H It may contain four domains (derived from IgE or IgM).

[0013] As used herein, the term "Fc region" refers to a dimer of two "Fc polypeptides," each of which comprises the constant region of an antibody excluding the first constant region immunoglobulin domain. In some embodiments, an "Fc region" comprises two Fc polypeptides linked by one or more disulfide bonds, chemical linkers, or peptide linkers. "Fc polypeptide" refers to the last two constant region immunoglobulin domains of IgA, IgD, and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and may also include some or all of the flexible hinge N-terminal to these domains. In the case of IgG, "Fc polypeptide" includes immunoglobulin domains C gamma 2 (Cγ2) and C gamma 3 (Cγ3), as well as the lower portion of the hinge between C gamma 1 (Cγ1) and Cγ2. Although the boundaries of the Fc polypeptide might vary, the human IgG heavy chain Fc polypeptide is usually defined to include residues starting from T223, or C226, or P230, through the carboxyl terminus, where this numbering follows the EU index in Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Services, Springfield, VA). For IgA, the Fc polypeptide includes immunoglobulin domains C alpha 2 (Cα2) and C alpha 3 (Cα3), as well as the lower portion of the hinge between C alpha 1 (Cα1) and Cα2. The Fc region may be synthetic, recombinant, or produced from natural sources, such as IVIg.

[0014] As used herein, a "glycan" is a sugar, which may be a monomer or polymer of at least three sugar residues, such as sugars, and may be linear or branched. A "glycan" may contain natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylneuraminic acid, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, phosphomannose, 6' sulfo-N-acetylglucosamine, etc.). The term "glycan" includes homo- and heteropolymers of sugar residues. The term "glycan" also encompasses glycan components of glycoconjugates (e.g., polypeptides, glycolipids, proteoglycans, etc.). The term also encompasses free glycans, including glycans that have been cleaved or otherwise released from glycoconjugates.

[0015] As used herein, the term "glycoprotein" refers to a protein containing a peptide backbone covalently attached to one or more sugar moieties (i.e., glycans). The sugar moieties may be in the form of monosaccharides, disaccharides, oligosaccharides, and / or polysaccharides. The sugar moieties may comprise a single unbranched sugar residue or may comprise one or more branched chains. A glycoprotein may contain O-linked and / or N-linked sugar moieties.

[0016] As used herein, "IVIg" refers to a preparation of pooled multivalent IgG containing all four IgG subgroups extracted from the plasma of at least 1,000 human donors. IVIg is approved as a plasma protein replacement therapy for immunocompromised patients. The level of IVIg Fc glycan sialylation varies between IVIg preparations but is generally less than 20%. The level of disialylation is generally much lower. As used herein, the term "derived from IVIg" refers to a polypeptide resulting from the manipulation of IVIg, such as a polypeptide purified from IVIg (e.g., enriched for sialylated IgG) or modified IVIg (e.g., enzymatically sialylated IVIg IgG).

[0017] As used herein, "N-glycosylation site of an Fc polypeptide" refers to an amino acid residue within an Fc polypeptide to which a glycan is N-linked. In some embodiments, an Fc region contains a dimer of Fc polypeptides, and the Fc region comprises two N-glycosylation sites, one on each Fc polypeptide.

[0018] As used herein, "percent (%) branched glycans" refers to the number of moles of glycan X relative to the total moles of glycans present, where X represents the glycan of interest.

[0019] The term "pharmaceutically effective amount" or "therapeutically effective amount" refers to an amount (e.g., dosage) effective to treat a patient having a disorder or condition described herein. It is also understood that, as used herein, a "pharmaceutically effective amount" may be interpreted as an amount that provides the desired therapeutic effect, either alone or in combination with other therapeutic agents, taken at a single time, or taken in any dosage or route.

[0020] "Pharmaceutical formulations" and "pharmaceutical products" may be included in kits containing the formulation or product and instructions for use.

[0021] A "pharmaceutical formulation" and a "pharmaceutical product" generally refer to a composition that achieves a final, predetermined level of sialylation and is free of process impurities. As such, a "pharmaceutical formulation" and a "pharmaceutical product" are substantially free of ST6Gal sialyltransferase and / or sialic acid donors (e.g., cytidine 5'-monophosphonic-N-acetylneuraminic acid) or their by-products (e.g., cytidine 5'-monophosphate).

[0022] A "pharmaceutical preparation" and a "pharmaceutical product" generally are substantially free of the cells (eg, endoplasmic or cytoplasmic proteins and RNA) in which the glycoprotein, if recombinant, is produced.

[0023] "Purified" (or "isolated") refers to a polynucleotide or polypeptide that has been removed or separated from other components present in its natural environment. For example, an isolated polypeptide is one that is separated from other components of the cell in which it is produced (e.g., endoplasmic reticulum or cytoplasmic proteins and RNA). An isolated polynucleotide is one that is separated from other nuclear components (e.g., histones) and / or upstream or downstream nucleic acids. An isolated polynucleotide or polypeptide may be 60%, or at least 75%, or at least 90%, or at least 95% free from other components present in the natural environment of the referenced polynucleotide or polypeptide.

[0024] As used herein, the term "sialylated" refers to a glycan having a terminal sialic acid. The term "monosialylated" refers to a branched glycan having one terminal sialic acid, for example, on the α1,3 arm or the α1,6 arm. The term "disialylated" refers to a branched glycan having terminal sialic acids on two arms, for example, on both the α1,3 arm and the α1,6 arm.

[0025] Provided herein is a fusion protein comprising an N-terminal signal sequence and an enzymatically active portion of human α-2,6-sialyltransferase 1 (ST6Gal1).

[0026] In some embodiments, the enzymatically active portion of ST6Gal1 comprises SEQ ID NO: 4. In some embodiments, the enzymatically active portion of ST6Gal1 consists of SEQ ID NO: 4.

[0027] In some embodiments, the signal sequence is an N-terminal azurocidin signal sequence. In some embodiments, the azurocidin signal sequence comprises MTRLTVLALLAGLLASSRA (SEQ ID NO: 30). In some embodiments, the azurocidin signal sequence consists of MTRLTVLALLAGLLASSRA (SEQ ID NO: 30).

[0028] In some embodiments, the fusion protein further comprises an affinity tag.

[0029] In some embodiments, the affinity tag is selected from the group consisting of polyhistidine, glutathione S-transferase (GST), maltose-binding protein (MBP), chitin-binding protein, streptavidin tag (e.g., Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 31)), FLAG tag (e.g., DYKDDDDK (SEQ ID NO: 32)), biotin tag, and combinations thereof.

[0030] In some embodiments, the polyhistidine tag is selected from the group consisting of HHHH (SEQ ID NO: 11), HHHHH (SEQ ID NO: 12), HHHHHH (SEQ ID NO: 13), HHHHHHH (SEQ ID NO: 14), HHHHHHHHH (SEQ ID NO: 15), HHHHHHHHHH (SEQ ID NO: 16), and HHHHHHHHHHH (SEQ ID NO: 17).

[0031] In some embodiments, the affinity tag is located toward the N-terminus of the enzymatically active portion of ST6Gal1.

[0032] In some embodiments, the N-terminal signal sequence comprises MTRLTVLALLAGLLASSRA (SEQ ID NO:30) and the enzymatically active portion of ST6Gal1 comprises SEQ ID NO:4.

[0033] In some embodiments, the fusion protein contains a hexa-histidine tag. (SEQ ID NO: 13) In some embodiments, the nucleic acid further comprises a hexa-histidine tag. (SEQ ID NO: 13) is between the N-terminal signal sequence and the enzymatically active portion of ST6Gal1. In some embodiments, the fusion protein consists of SEQ ID NO:6.

[0034] Also provided herein are nucleic acid molecules encoding the fusion proteins, vectors comprising the nucleic acid molecules, and host cells, preferably stably transformed with the vectors.

[0035] In some embodiments, the vector further comprises a promoter operably linked to the nucleic acid encoding the fusion protein, hi some embodiments, the promoter is a cytomegalovirus (CMV) promoter.

[0036] In some embodiments, the host cell is a human embryonic kidney (HEK) cell or a derivative thereof. In some embodiments, the host cell is the HEK derivative HEK293.

[0037] Also provided herein is a method for producing a polypeptide, comprising culturing a host cell described herein in a medium under conditions permissive for expression of the fusion protein, and isolating the fusion protein from the medium.

[0038] Also provided herein is a fusion protein comprising an N-terminal signal sequence and an enzymatically active portion of human β-1,4-galactosyltransferase (B4GalT1).

[0039] In some embodiments, the enzymatically active portion of B4GalT1 comprises SEQ ID NO: 43. In some embodiments, the enzymatically active portion of B4GalT1 consists of SEQ ID NO:43.

[0040] In some embodiments, the signal sequence is an N-terminal azurocidin signal sequence. In some embodiments, the azurocidin signal sequence comprises MTRLTVLALLAGLLASSRA (SEQ ID NO: 30). In some embodiments, the azurocidin signal sequence consists of MTRLTVLALLAGLLASSRA (SEQ ID NO: 30).

[0041] In some embodiments, the fusion protein further comprises an affinity tag.

[0042] In some embodiments, the affinity tag is selected from the group consisting of polyhistidine, glutathione S-transferase (GST), maltose binding protein (MBP), chitin binding protein, streptavidin tag (e.g., Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 31)), FLAG tag (e.g., DYKDDDDK (SEQ ID NO: 32)), biotin tag, and combinations thereof.

[0043] In some embodiments, the polyhistidine tag is selected from the group consisting of HHHH (SEQ ID NO: 11), HHHHH (SEQ ID NO: 12), HHHHHH (SEQ ID NO: 13), HHHHHHH (SEQ ID NO: 14), HHHHHHHH (SEQ ID NO: 15), HHHHHHHHH (SEQ ID NO: 16), and HHHHHHHHHHH (SEQ ID NO: 17).

[0044] In some embodiments, the affinity tag is located toward the C-terminus of the enzymatically active portion of B4GalT1.

[0045] In some embodiments, the N-terminal signal sequence comprises MTRLTVLALLAGLLASSRA (SEQ ID NO:30) and the enzymatically active portion of B4GalT1 comprises SEQ ID NO:43.

[0046] In some embodiments, the fusion protein comprises a septahistidine tag. (SEQ ID NO: 14) In some embodiments, the septahistidine tag (SEQ ID NO: 14) is the C-terminus.

[0047] In some embodiments, the fusion protein consists of SEQ ID NO:45.

[0048] Also provided herein are nucleic acid molecules encoding the fusion proteins, vectors comprising the nucleic acid molecules, and host cells, preferably stably transformed with the vectors.

[0049] In some embodiments, the vector further comprises a promoter operably linked to the nucleic acid encoding the fusion protein, hi some embodiments, the promoter is a cytomegalovirus (CMV) promoter.

[0050] In some embodiments, the host cell is a human embryonic kidney (HEK) cell or a derivative thereof, hi some embodiments, the host cell is the HEK derivative HEK293.

[0051] Also provided herein is a method for producing a polypeptide, comprising culturing a host cell described herein in a medium under conditions permissive for expression of the fusion protein, and isolating the fusion protein from the medium.

[0052] Also, a) providing a composition comprising an IgG antibody; Also provided herein is a method for sialyating immunoglobulin G (IgG), comprising: b) exposing the composition to an enzymatically active portion of ST6Gal1 comprising β1,4-galactosyltransferase 1 and SEQ ID NO: 4 in the presence of UDP-Gal and CMP-NANA, thereby producing a composition comprising sialyated IgG (sIgG).

[0053] Also provided herein is a method for sialyating immunoglobulin G (IgG), comprising: a) providing a composition comprising an IgG antibody; b) exposing the IgG antibody to β1,4-galactosyltransferase 1 in the presence of UDP-Gal, thereby producing a composition comprising a galactosylated IgG; and c) exposing the composition comprising the galactosylated IgG antibody to an enzymatically active portion of ST6Gal1 comprising SEQ ID NO: 4 in the presence of CMP-NANA, thereby producing a composition comprising a sialyated IgG (sIgG).

[0054] In some embodiments, the composition comprising the galactosylated IgG antibody is not purified prior to step (c).

[0055] In some embodiments, the method further comprises supplementing one or more of the compositions with CMP-NANA.

[0056] In some embodiments, the mixture of IgG antibodies is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and combinations thereof.

[0057] In some embodiments, at least 60% of the branched glycans on the Fc region of the antibodies in the composition comprising sIgG are disialylated.

[0058] In some embodiments, less than 50% of the branched glycans on the Fc region of the antibodies in a composition comprising sIgG are monosialylated.

[0059] Provided herein are human embryonic kidney (HEK) cells stably transformed with a nucleic acid molecule comprising a nucleic acid sequence encoding a fusion protein comprising an azurocidin signal sequence and a portion of human ST6 sialyltransferase consisting of SEQ ID NO:4.

[0060] In some embodiments, the fusion protein comprises a sequence selected from HHHHH (SEQ ID NO:12), HHHHHH (SEQ ID NO:13), HHHHHHH (SEQ ID NO:14), HHHHHHHH (SEQ ID NO:15), HHHHHHHHH (SEQ ID NO:16), HHHHHHHHHH (SEQ ID NO:17), HHHHHM (SEQ ID NO:18), HHHHHHM (SEQ ID NO:19), HHHHHHM (SEQ ID NO:20), HHHHHHHHM (SEQ ID NO:21), HHHHHHHHHM (SEQ ID NO:22), and HHHHHHHHHHM (SEQ ID NO:23), located between the azaricidin signal sequence and the portion of human ST6 sialyltransferase consisting of SEQ ID NO:4.

[0061] In some embodiments, the fusion protein lacks the human ST6 sialyltransferase amino terminal to SEQ ID NO:4.

[0062] In some embodiments, the fusion protein comprises SEQ ID NO:4 but lacks the human ST6 sialyltransferase amino terminal to SEQ ID NO:4.

[0063] In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 6. In some embodiments, the fusion protein comprises the amino acid sequence of SEQ ID NO: 3.

[0064] In some embodiments, the nucleic acid molecule comprises a promoter operably linked to the nucleic acid encoding the fusion protein, hi some embodiments, the promoter is a cytomegalovirus promoter.

[0065] Also provided herein is a method for preparing a polypeptide comprising SEQ ID NO:3, comprising culturing HEK cells in a medium under conditions permissive for expression of the fusion protein, and isolating the polypeptide comprising SEQ ID NO:3 from the medium.

[0066] In some embodiments, the method further comprises purifying the isolated polypeptide to at least 95% w / w.

[0067] Also provided herein are polypeptides comprising SEQ ID NO:3 or SEQ ID NO:6.

[0068] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials for use in the present invention are described herein; other suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and are not intended to be limiting. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will govern.

[0069] Other features and advantages of the invention will become apparent from the following detailed description and claims. [Brief explanation of the drawings]

[0070] [Figure 1] A short, branched core oligosaccharide containing two N-acetylglucosamine and three mannose residues is shown. One of the branches is referred to in the art as the "α1,3 arm," and the second branch as the "α1,6 arm." Squares: N-acetylglucosamine; dark gray circles: mannose; light gray circles: galactose; diamonds: N-acetylneuraminic acid; triangles: fucose. [Figure 2] Figure 1 shows common Fc glycans present in IVIg. Squares: N-acetylglucosamine, dark grey circles: mannose, light grey circles: galactose, diamonds: N-acetylneuraminic acid, triangles: fucose. The figure discloses SEQ ID NO: 7. [Figure 3] 1 shows how immunoglobulins, such as IgG antibodies, can be sialylated by performing a galactosylation step followed by a sialylation step: squares: N-acetylglucosamine, dark grey circles: mannose, light grey circles: galactose, diamonds: N-acetylneuraminic acid, triangles: fucose. [Figure 4]Representative example reaction products of IgG-Fc glycan profiles for reactions starting with IVIg are shown. The left panel is a schematic of the enzymatic sialylation reaction for converting IgG to hsIgG. The right panel is the IgG Fc glycan profile of the starting IVIg and hsIgG. The bars correspond, from left to right, to IgG1, IgG2 / 3, and IgG3 / 4, respectively. DETAILED DESCRIPTION OF THE INVENTION

[0071] Antibodies are glycosylated at conserved positions within the constant regions of their heavy chains and within the Fab. For example, within the Fc domain, human IgG antibodies have a single N-linked glycosylation site at Asn297 in the CH2 domain. Each antibody isotype has a variety of different N-linked carbohydrate structures in the constant region. For human IgG, the core oligosaccharide usually consists of GlcNAc2Man3 with different numbers of outer residues. Variation between individual IgGs can occur through the attachment of galactose and / or galactose-sialic acid to one or both of the terminal GlcNAc residues, or the attachment of a third GlcNAc arm (bisected GlcNAc).

[0072] The present disclosure encompasses, in part, methods for preparing immunoglobulins (e.g., human IgG) having Fc regions with particular levels of branched glycans that are sialylated on both the α1,3 arm, the α1,6 arm, or both of the branched glycans (e.g., with NeuAc-α2,6-Gal terminal linkages). The levels can be measured on individual Fc regions (e.g., the number of branched glycans that are sialylated on the α1,3 arm, the α1,6 arm, or both of the branched glycans within an Fc region) or on the overall composition of a polypeptide preparation (e.g., the number or percentage of branched glycans that are sialylated on the α1,3 arm, the α1,6 arm, or both of the branched glycans within an Fc region in a polypeptide preparation).

[0073] Naturally occurring polypeptides that can be used to prepare hypersialylated IgG include, for example, IgG in human serum (particular human serum pooled from over 1,000 donors), intravenous immunoglobulin (IVIg), and polypeptides derived from IVIg, such as polypeptides purified from IVIg (e.g., enriched for sialylated IgG) or modified IVIg (e.g., enzymatically sialylated IVIg IgG).

[0074] N-linked oligosaccharide chains are added to proteins within the lumen of the endoplasmic reticulum. Specifically, an initial oligosaccharide (typically 14 saccharides) is added to the 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 except proline. This initial oligosaccharide structure is common to most eukaryotes and contains three glucose, nine mannose, and two N-acetylglucosamine residues. This initial oligosaccharide chain can be trimmed by specific glycosidase enzymes in the endoplasmic reticulum to yield a short, branched core oligosaccharide consisting of two N-acetylglucosamine and three mannose residues. One of the branches is referred to in the art as the "α1,3 arm," and the second branch is referred to as the "α1,6 arm," as shown in Figure 1.

[0075] N-glycans can be subdivided into three distinct groups, termed "high mannose," "hybrid," and "complex," with a common pentasaccharide core (Man(α1,6)-(Man(α1,3))-Man(β1,4)-GlcpNAc(β1,4)-GlcpNAc(β1,N)-Asn) occurring in all three groups.

[0076] The more common Fc glycans present in IVIg are shown in FIG.

[0077] Additionally or alternatively, one or more monosaccharide units of N-acetylglucosamine may be added to the core mannose subunit to form a "complex glycan." Galactose may be added to the N-acetylglucosamine subunit, and sialic acid subunits may be added to the galactose subunit, resulting in chains terminating in either sialic acid, galactose, or N-acetylglucosamine residues. Furthermore, fucose residues may be added to the N-acetylglucosamine residue of the core oligosaccharide. Each of these additions is catalyzed by a specific glycosyltransferase.

[0078] A "hybrid glycan" contains characteristics of both high-mannose and complex glycans. For example, one branch of a hybrid glycan may contain primarily or exclusively mannose residues, while another branch may contain N-acetylglucosamine, sialic acid, galactose, and / or fucose sugars.

[0079] Sialic acids are a family of 9-carbon monosaccharides with heterocyclic ring structures. They carry a negative charge via the carboxylic acid group attached to the ring, as well as other chemical decorations, including N-acetyl and N-glycolyl groups. The two main types of sialic acid residues found in polypeptides produced in mammalian expression systems are N-acetylneuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). They typically occur as terminal structures attached to galactose (Gal) residues at the non-reducing ends of both N- and O-linked glycans. The glycosidic bond configuration for these sialic groups can be either α2,3 or α2,6.

[0080] The Fc region is glycosylated at conserved N-linked glycosylation sites. For example, each heavy chain of an IgG antibody contains C H It has a single N-linked glycosylation site at Asn297 in the C2 domain. H 2 and C H3 domain has N-linked glycosylation sites, and IgE antibodies H 3 domain, and IgM antibodies have N-linked glycosylation sites within the C H 1. C H 2. C H 3, and C H 4 has an N-linked glycosylation site.

[0081] Each antibody isotype has a variety of different N-linked carbohydrate structures in the constant region. For example, IgG has C1q and C2q in each Fc polypeptide of the Fc region, which also contains binding sites for C1q and FcγR. H The core oligosaccharide of human IgG has a single N-linked biantennary carbohydrate at Asn297 of the 2 domain. For human IgG, the core oligosaccharide usually consists of GlcNAc2Man3 with different numbers of outer residues. Variation between individual IgGs can occur through the attachment of galactose and / or galactose-sialic acid at one or both terminal GlcNAc, or through the attachment of a third GlcNAc arm (bisected GlcNAc).

[0082] Immunoglobulins, such as IgG antibodies, can be sialylated by performing a galactosylation step followed by a sialylation step. β-1,4-galactosyltransferase 1 (B4GalT) is a type II Golgi membrane-bound glycoprotein that transfers galactose from uridine 5'-diphosphoseglucactose ([[(2)R,3S,4R,5R)-5-(2,4-dioxapyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl][(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]hydrogenphosphate, UDP-Gal) to GlcNAc via a β-1,4-linkage. Alpha-2,6-sialyltransferase 1 (ST6) is a type II Golgi membrane-bound glycoprotein that transfers sialic acid from cytidine 5'-monophospho-N-acetylneuraminic acid (((2R,4S,5R,6R)-5-acetamido-2-[(2R,3S,4R,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-4-hydroxy-6-(1,2,3-trihydroxypropyl)oxane-2-carboxylic acid, CMP-NANA, or CMP-sialic acid) to Gal via an α-2,6 linkage. The reaction proceeds as shown in Figure 3.

[0083] The glycans of a glycoprotein can be evaluated using any method known in the art. For example, the sialylation of the glycan composition (e.g., the level of branched glycans sialylated on the α1,3 arm and / or the α1,6 arm) can be characterized using the methods described in WO 2014 / 179601.

[0084] In some embodiments of hsIgG compositions prepared by the methods described herein, at least 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the branched glycans on the Fc domain have sialic acid on both the α1,3 and α1,6 arms connected via a NeuAc-α2,6-Gal terminal linkage. Additionally, in some embodiments, at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain have sialic acid on both the α1,3 and α1,6 arms connected via a NeuAc-α2,6-Gal terminal linkage. Overall, in some embodiments, at least 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the branched glycans have sialic acid on both the α1,3 and α1,6 arms connected via a NeuAc-α2,6-Gal terminal linkage.

[0085] enzyme β-1,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galt1, and its orthologs, mutants, and variants, including enzymatically active portions of β-1,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galt1, and its orthologs, mutants, and variants, and fusion proteins containing the same, are suitable for use in the methods described herein. B4Galt1 is one of seven β-1,4-galactosyltransferase (β4GalT) genes, each encoding a type II membrane-bound glycoprotein that appears to have exclusive specificity for the donor substrate UDP-galactose, with all incoming galactose in a β1,4 linkage to the similar acceptor sugars: GlcNAc, Glc, and Xyl. B4Galt1 adds galactose to N-acetylglucosamine residues that are either monosaccharides or the non-reducing terminus of glycoprotein carbohydrate chains. B4GalT1 is also known as GGTB2. Four alternative transcripts (NCBI gene ID 2683) encoding four isoforms of B4GALT1 are listed in Table 1.

[0086] [Table 1]

[0087] [Table 2]

[0088] [Table 3]

[0089] [Table 4]

[0090] [Table 5]

[0091] [Table 6]

[0092] [Table 7]

[0093] [Table 8]

[0094] The soluble form of B4GalT1 is derived from the membrane form by proteolytic processing, with the cleavage site located at positions 77-78 of B4GALT1 isoform 1 (SEQ ID NO: 37).

[0095] In some embodiments, one or more of the amino acids of B4GalT1 corresponding to amino acids 113, 130, 172, 243, 250, 262, 310, 343, or 355 of B4GALT1 isoform 1 (SEQ ID NO: 37) are conserved compared to (SEQ ID NO: 37).

[0096] For example, provided herein are enzymatically active portions of B4GalT1. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 1 (SEQ ID NO: 37), or an ortholog, mutant, or variant of SEQ ID NO: 37. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 2 (SEQ ID NO: 38), or an ortholog, mutant, or variant of SEQ ID NO: 38. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 3 (SEQ ID NO: 39), or an ortholog, mutant, or variant of SEQ ID NO: 39. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 4 (SEQ ID NO: 40), or an ortholog, mutant, or variant of SEQ ID NO: 40.

[0097] In some embodiments, an enzymatically active portion of B4GalT1 does not include a cytoplasmic domain, e.g., SEQ ID NO: 41. In some embodiments, an enzymatically active portion of B4GalT1 does not include a transmembrane domain, e.g., SEQ ID NO: 42. In some embodiments, an enzymatically active portion of B4GalT1 does not include a cytoplasmic domain, e.g., SEQ ID NO: 41, or a transmembrane domain, e.g., SEQ ID NO: 42.

[0098] In some embodiments, the enzymatically active portion of B4GalT1 is the luminal domain, e.g., SEQ ID NO: 63 , or an ortholog, mutant, or variant thereof.

[0099] In some embodiments, an enzymatically active portion of B4GalT1 comprises amino acids 109-398 of SEQ ID NO: 37, or an ortholog, mutant, or variant thereof. In some embodiments, an enzymatically active portion of B4GalT1 consists of SEQ ID NO: 37, or an ortholog, mutant, or variant of SEQ ID NO: 37.

[0100] A suitable functional portion of B4GalT1 may comprise or consist of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98%, or 100%) identical to SEQ ID NO:43.

[0101] [Table 9]

[0102] ST6Gal1, e.g., human ST6Gal1, and its orthologs, mutants, and variants comprising enzymatically active portions of ST6Gal1, e.g., human ST6Gal1, and its orthologs, mutants, and variants, as well as fusion proteins comprising the same, are suitable for use in the methods described herein. ST6GAL1, β-galactoside α-2,6-sialyltransferase 1, transfers sialic acid from CMP-sialic acid to a Galβ1→4GlcNAc structure on glycoproteins such as asialofetuin and asialo-α1-acid glycoprotein. ST6Gal1 is also referred to as ST6N or SIAT1. Four alternative transcripts encoding two isoforms of ST6GAL1 (NCBI gene ID 6480) are listed in Table 1.

[0103] [Table 10]

[0104] >NP_001340845.1 (NP_003023.1, NP_775323.1) ST6GAL1 [organism=Homo sapiens][Gene ID=6480][Isoform=a] (SEQ ID NO:28) [Table 11]

[0105] >NP_775324.1 ST6GAL1 [organism=Homo sapiens][Gene ID=6480][Isoform=b] (SEQ ID NO:29) [Table 12]

[0106] [Table 13]

[0107] [Table 14]

[0108] [Table 15]

[0109] The soluble form of ST6Gal1 is derived from the membrane form by proteolytic processing.

[0110] In some embodiments, one or more of the amino acids of ST6Gal1 corresponding to amino acids 142, 149, 161, 184, 189, 212, 233, 335, 353, 354, 364, 365, 369, 370, 376, or 406 of ST6Gal1 isoform a (SEQ ID NO: 28) teeth , conserved compared to SEQ ID NO:28.

[0111] Also provided herein, for example, are enzymatically active portions of ST6Gal1. In some embodiments, the enzyme is an enzymatically active portion of ST6Gal1 isoform a (SEQ ID NO: 28), or an ortholog, mutant, or variant of SEQ ID NO: 28. In some embodiments, the enzyme is an enzymatically active portion of STG6Gal1 isoform b (SEQ ID NO: 29), or an ortholog, mutant, or variant of SEQ ID NO: 29.

[0112] In some embodiments, an enzymatically active portion of ST6Gal1 does not include a cytoplasmic domain, e.g., SEQ ID NO: 34. In some embodiments, an enzymatically active portion of ST6Gal1 does not include a transmembrane domain, e.g., SEQ ID NO: 35. In some embodiments, an enzymatically active portion of ST6Gal1 does not include a cytoplasmic domain, e.g., SEQ ID NO: 34, or a transmembrane domain, e.g., SEQ ID NO: 35.

[0113] In some embodiments, the enzymatically active portion of ST6Gal1 comprises all or a portion of the luminal domain, eg, SEQ ID NO: 36, or an ortholog, mutant, or variant thereof.

[0114] In some embodiments, an enzymatically active portion of ST6Gal1 comprises amino acids 87-406 of SEQ ID NO:28 (SEQ ID NO:4), or an ortholog, mutant, or variant thereof. In some embodiments, an enzymatically active portion of ST6Gal1 consists of SEQ ID NO:4, or an ortholog, mutant, or variant of SEQ ID NO:4.

[0115] A suitable functional portion of ST6Gal1 may comprise or consist of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98%, or 100%) identical to SEQ ID NO:3 or SEQ ID NO:4.

[0116] [Table 16]

[0117] variant In some embodiments, the enzymes described herein are at least 80%, e.g., at least 85%, 90%, 95%, 98%, or 100% identical to the amino acid sequence of an exemplary sequence (e.g., as provided herein), and include, e.g., mutations described herein substituted with conservative mutations, or in addition, have differences of, e.g., up to 1%, 2%, 5%, 10%, 15%, or 20% of the residues of the exemplary sequence. In preferred embodiments, the variant retains the desired activity of the parent, e.g., β-galactoside α-2,6-sialyltransferase activity or β-1,4-galactosyltransferase activity.

[0118] To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences for optimal alignment, and non-homologous sequences can be ignored for comparison purposes). The length of the reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments, at least 90% or 100%. Nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, nucleic acid "identity" is equivalent to nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced to optimally align the two sequences.

[0119] The percent identity between a subject polypeptide or nucleic acid sequence (i.e., query) and a second polypeptide or nucleic acid sequence (i.e., target) can be determined using, for example, Smith Waterman Alignment (Smith, T.F. and M.S. Waterman (1981) J. Mol. Biol. 147:195-7), "BestFit" as implemented in GeneMatcher Plus™ (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)), Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, MO, Ed., pp. 353-358, or the BLAST program (Basic Local Alignment Search Tool, (Altschul, S.F., W. Gish, et al. (1990) J. Mol. Biol. 215:403-10), BLAST-2, BLAST-P, BLAST-N, BLAST-X, WU-BLAST-2, ALIGN, ALIGN-2, CLUSTAL, or Megalign (DNASTAR) software, and other publicly available computer software. In addition, those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the length of the sequences being compared. Generally, for target proteins or nucleic acids, the comparison length can be any length (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%), up to and including the full length of the target. For purposes of this disclosure, percent identity is relative to the full length of the query sequence.

[0120] For purposes of this disclosure, comparison of sequences and determination of percent identity between two sequences may be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5.

[0121] Conservative substitutions typically include the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.

[0122] Fusion proteins Also provided herein are fusion proteins comprising the enzymes described herein or portions thereof.

[0123] In one embodiment, the fusion protein comprises a signal sequence. In some embodiments, the signal sequence is about 15 to about 20 amino acids in length, e.g., about 15, 16, 17, 18, 19, or 20 amino acids in length. In some embodiments, the signal sequence comprises a hydrophobic core region (h region) flanked by an n region and a c region. In some embodiments, the c region comprises a signal peptidase consensus cleavage site.

[0124] In some embodiments, the signal sequence is an N-terminal signal sequence.

[0125] In some embodiments, the signal sequence is an azurocidin signal sequence. In some embodiments, the azurocidin signal sequence comprises or consists of MTRLTVLALLAGLLASSRA (SEQ ID NO: 30). In some embodiments, the signal sequence is a serum albumin signal sequence. In some embodiments, the serum albumin signal sequence comprises or consists of MKWVTFISLLFLFSSAYS (SEQ ID NO: 46). In some embodiments, the signal sequence is an immunoglobulin heavy chain signal sequence. In some embodiments, the immunoglobulin heavy chain signal sequence comprises or consists of MDWTWRVFCLLAVTPGAHP (SEQ ID NO: 47). In some embodiments, the signal sequence is an immunoglobulin light chain signal sequence. In some embodiments, the immunoglobulin light chain signal sequence comprises or consists of MDWTWRVFCLLAVTPGAHP (SEQ ID NO: 48).

[0126] In some embodiments, the signal sequence is a cystatin S signal sequence. In some embodiments, the cystatin-S signal sequence comprises or consists of MARPLCTLLLLMATLAGALA (SEQ ID NO:49). In some embodiments, the signal sequence is an IgKappa signal sequence. In some embodiments, the IgKappa signal sequence comprises or consists of MDMRAPAGIFGFLLVLFPGYRS (SEQ ID NO:50). In some embodiments, the signal sequence is a trypsonogen 2 signal sequence. In some embodiments, the trypsonogen 2 signal sequence comprises or consists of MRSLVFVLLIGAAFA (SEQ ID NO:51). In some embodiments, the signal sequence is a potassium channel blocking signal sequence. In some embodiments, the potassium channel blocking sequence comprises or consists of MSRLFVFILIALFLSAIIDVMS (SEQ ID NO:52).

[0127] In some embodiments, the signal sequence is the alpha-conotoxin Ip1.3 signal sequence. In some embodiments, the alpha-conotoxin Ip1.3 signal sequence comprises or consists of MGMRMMFIMFMLVVLATTVVS (SEQ ID NO: 53). In some embodiments, the signal sequence is an α-galactosidase signal sequence. In some embodiments, the α-galactosidase signal sequence comprises or consists of MRAFLFLTACISLPGVFG (SEQ ID NO: 54). In some embodiments, the signal sequence is a cellulase signal sequence. In some embodiments, the cellulase signal sequence comprises or consists of MKFQSTLLLAAAAGSALA (SEQ ID NO: 55). In some embodiments, the signal sequence is an aspartic proteinase nepenthesin-1 signal sequence. In some embodiments, the aspartic proteinase nepenthesin-1 signal sequence comprises or consists of MASSLYSFLLALSIVYIFVAPTHS (SEQ ID NO: 56). In some embodiments, the signal sequence is an acid chitinase signal sequence. In some embodiments, the acid chitinase signal sequence comprises or consists of MKTHYSSAILPILTLFVFLSINPSHG (SEQ ID NO: 57). In some embodiments, the signal sequence is a K28 preprotoxin signal sequence. In some embodiments, the K28 preprotoxin signal sequence comprises or consists of MESVSSLFNIFSTIMVNYKSLVLALLSVSNLKYARG (SEQ ID NO: 58). In some embodiments, the signal sequence is a killer toxin digocin precursor signal sequence. In some embodiments, the killer toxin digocin precursor signal sequence comprises or consists of MKAAQILTASIVSLLPIYTSA (SEQ ID NO: 59). In some embodiments, the signal sequence is a cholera toxin signal sequence. In some embodiments, the cholera toxin signal sequence comprises or consists of MIKLKFGVFFTVLLSSAYA (SEQ ID NO: 60). In some embodiments, the signal sequence is a human growth hormone signal sequence.In some embodiments, the human growth hormone signal sequence comprises or consists of MATGSRTSLLLAFGLLCLPWLQEGSA (SEQ ID NO: 61).

[0128] In some embodiments, the fusion protein comprises one or more affinity tags. In some embodiments, the purification tag is selected from the group consisting of polyhistidine, glutathione S-transferase (GST), maltose binding protein (MBP), chitin binding protein, streptavidin tag (e.g., Strep- t ag®, e.g., Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 31), FLAG tag (e.g., DYKDDDDK (SEQ ID NO: 32)), biotin tag (e.g., AviTag™), and combinations thereof.

[0129] In some embodiments, the affinity tag is located towards the N-terminus of the enzyme or portion thereof, hi some embodiments, the affinity tag is N-terminal.

[0130] In some embodiments, the affinity tag is located towards the C-terminus of the enzyme or portion thereof, hi some embodiments, the affinity tag is C-terminal.

[0131] In some embodiments, the affinity tag is a polyhistidine tag. In some embodiments, the polyhistidine tag is selected from the group consisting of HHHH (SEQ ID NO: 11), HHHHH (SEQ ID NO: 12), HHHHHH (SEQ ID NO: 13), HHHHHHH (SEQ ID NO: 14), HHHHHHHHH (SEQ ID NO: 15), HHHHHHHHHH (SEQ ID NO: 16), and HHHHHHHHHHH (SEQ ID NO: 17). In some embodiments, the polyhistidine tag is a hexahistidine tag (e.g., HHHHHH (SEQ ID NO: 13)).

[0132] In some embodiments, the fusion protein comprises or consists of SEQ ID NO:43, SEQ ID NO:44, or SEQ ID NO:45.

[0133] [Table 17]

[0134] [Table 18]

[0135] In some embodiments, the fusion protein comprises or consists of SEQ ID NO:3 or SEQ ID NO:5.

[0136] [Table 19]

[0137] [Table 20]

[0138] Expression system To use the enzymes and / or fusion proteins described herein, it may be desirable to express them from the nucleic acids encoding them. This can be done in a variety of ways. For example, the nucleic acids encoding the enzymes and / or fusion proteins can be cloned into intermediate vectors for transformation into prokaryotic or eukaryotic cells for replication and / or expression. The intermediate vectors are typically prokaryotic vectors, such as plasmids, or shuttle vectors, or insect vectors, for storage or manipulation of the nucleic acids encoding the enzymes and / or fusion proteins. The nucleic acids encoding the enzymes and / or fusion proteins can also be cloned into expression vectors for administration to plant cells, animal cells, preferably mammalian or human cells, fungal cells, bacterial cells, or protozoan cells.

[0139] To achieve expression, sequences encoding enzymes and / or fusion proteins are typically subcloned into expression vectors containing a promoter to direct transcription. Suitable bacterial and eukaryotic promoters are well known in the art and are described, for example, in Sambrook et al., Molecular Cloning, A Laboratory Manual (3rd ed. 2001), Kriegler, Gene Transfer and Expression: A Laboratory Manual (1990), and Current Protocols in Molecular Biology (Ausubel et al., eds., 2010). Bacterial expression systems for expressing engineered proteins are available, for example, for E. coli, Bacillus sp., and Salmonella (Palva et al., 1983, Gene 22:229-235). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available.

[0140] The promoter used to direct expression of a nucleic acid will depend on the particular application, for example, strong constitutive promoters are typically used for expression and purification of fusion proteins.

[0141] In some embodiments, the promoter is selected from the group consisting of human cytomegalovirus (CMV), EF-1 alpha (EF1A), elongation factor 1 alpha short (EFS), CMV enhancer chicken β-actin promoter (CAG) and rabbit β-globin splice acceptor site (CAG), hybrid CBA (CBh), spleen focus-forming virus (SFFV), murine stem cell virus (MSCV), simian virus 40 (SV40), mouse phosphoglycerate kinase 1 (mPGK), human phosphoglycerate kinase 1 (hPGK), and ubiquitin C (UBC) promoter. In some embodiments, the promoter is a human cytomegalovirus (CMV) promoter.

[0142] In addition to the promoter, an expression vector contains a transcription unit, or expression cassette, which contains all the additional elements required for expression of a nucleic acid in a host cell, either prokaryotic or eukaryotic. Thus, a typical expression cassette contains, for example, a promoter operably linked to a nucleic acid sequence encoding an enzyme and / or a fusion protein, and any signals required, for example, for efficient polyadenylation of the transcript, transcription termination, ribosome binding sites, or translation termination. Additional elements of the cassette may include, for example, enhancers and heterologous spliced ​​intron signals.

[0143] In some embodiments, the expression vector comprises a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE). See, e.g., Zufferey et al., "Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element Enhances Expression of Transgenes Delivered by Retroviral Vectors," Journal of Virology 73(4):2886-92 (1999).

[0144] The particular expression vector used to transport the genetic information into the cell is selected with regard to the intended use of the enzyme and / or fusion protein, e.g., expression in plants, animals, bacteria, fungi, protozoa, etc.

[0145] Standard transfection methods are used to produce bacterial, mammalian, yeast, or insect cell lines that express large amounts of protein, which are then purified using standard techniques (see, e.g., Colley et al., 1989, J. Biol. Chem., 264:17619-22; Guide to Protein Purification, in Methods in Enzymology, vol. 182 (Deutscher, ed., 1990)). Transformation of eukaryotic and prokaryotic cells is carried out according to standard techniques (see, e.g., Morrison, 1977, J. Bacteriol. 132:349-351; Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (Wu et al., eds, 1983)).

[0146] Any of the known procedures for introducing foreign nucleotide sequences into host cells can be used. These include calcium phosphate transfection, polybrene, protoplast fusion, electroporation, nucleofection, liposomes, microinjection, naked DNA, both episomal and integrative plasmid vectors, and any of the other well-known methods for introducing cloned genomic DNA, cDNA, synthetic DNA, or other foreign genetic material into host cells (see, e.g., Sambrook et al., supra). The particular genetic engineering procedure used need only be capable of successfully introducing at least one gene into the host cell capable of expressing the enzyme and / or fusion protein.

[0147] In some embodiments, the host cell is stably transformed.

[0148] In some embodiments, the host cells are grown under non-hypoxic conditions.

[0149] The enzymes and / or fusion proteins described herein can be produced by any protein production system known in the art, such as a host cell-based expression system, a synthetic biology platform, or a cell-free protein production platform. In some embodiments, the protein production system is capable of post-translational modifications, including, but not limited to, one or more of glycosylation, e.g., N-glycosylation of proteins, disulfide bond formation, and tyrosine phosphorylation. See, e.g., Boh and Ng, "Impact of Host Cell Line Choice on Glycan Profile," Critical Reviews in Biotechnology 38(6):851-67 (2018).

[0150] In some embodiments, the host cell is a mammalian host cell, hi some embodiments, the mammalian cell is selected from the group consisting of Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells, NS0 myeloma cells, Sp2 / 0 hybridoma mouse cells, human embryonic kidney (HEK) cells, HT-1080 human cells, and derivatives thereof.

[0151] In some embodiments, the host cell is a non-human mammalian host cell, hi some embodiments, the non-human mammalian host cell is selected from CHO cells, BHK-21 cells, mouse NS0 myeloma cells, Sp2 / 0 hybridoma cells, and derivatives thereof.

[0152] In some embodiments, the host cell is a human mammalian host cell, hi some embodiments, the human cell is selected from the group consisting of HEK, PER.C6, CEVEC's amniocyte production (CAP), AGE1.HM, HKB-11, HT-1080 cells, and derivatives thereof.

[0153] In some embodiments, the host cell is a human embryonic kidney cell (HEK, ATCC® CRL-1573™) or a derivative thereof.

[0154] In some embodiments, HEK cells express a temperature-sensitive allele of the SV40 T antigen. In some embodiments, HEK cells are resistant to ricin toxin after ethyl methanesulfonate (EMS) mutagenesis and lack N-acetylglucosaminyltransferase I activity, e.g., encoded by the MGAT1 gene. In some embodiments, HEK cells modify glycoproteins primarily with Man5GlcNAc2 N-glycans. In some embodiments, HEK cells express the tetR repressor, allowing tetracycline-inducible protein expression.

[0155] In some embodiments, the HEK derivative is selected from the group consisting of HEK293, HEK293T (293tsA1609neo, ATCC® CRL-3216™), HEK293T / 17 (ATCC® CRL-11268™), HEK293T / 17 SF (ATCC® ACS-4500™), HEK293S, HEK293SG, HEK293F™, HEK293SGGD, HEK293F™, HEK293E, and HKB-11.

[0156] Synthetic biology platforms such as those described in Kightlinger et al., "Synthetic Glycobiology: Parts, Systems, and Applications," ACS Synth. Biol. 9:1534-62 (2020) are also suitable for producing the enzymes and / or fusion proteins described herein.

[0157] Also provided herein are cells, including vectors and cells, and kits, including the proteins and nucleic acids described herein, for example, for use in the methods described herein. [Example]

[0158] The present invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0159] Example 1: Preparation of hypersialylated IgG IgG in which greater than 60% of the overall branched glycans are disialylated can be prepared as follows.

[0160] Briefly, a mixture of IgG antibodies is subjected to sequential enzymatic reactions using the enzymes β1,4-galactosyltransferase 1 (B4-GalT) and α2,6-sialyltransferase (ST6-Gal1). B4-GalT does not need to be removed from the reaction prior to the addition of ST6-Gal1, and no partial or complete purification of the product is required between enzymatic reactions.

[0161] Galactosyltransferase enzymes selectively add galactose residues to existing asparagine-linked glycans. The resulting galactosylated glycans serve as substrates for sialyltransferases, which selectively add sialic acid residues to cap the attached asparagine-linked glycan structures. Thus, the overall sialylation reaction employs two sugar nucleotides: uridine 5'-diphosphogalactose (UDPGal) and cytidine-5'-monophosphoro-N-acetylneuraminic acid (CMP-NANA). The latter is periodically replenished to increase the proportion of disialylated products relative to monosialylated products. The reaction involves the cofactor manganese chloride.

[0162] A representative example of the IgG-Fc glycan profile of such a reaction starting with IVIg and the reaction product is shown in Figure 4. In Figure 4, on the left is a schematic diagram of the enzymatic sialylation reaction for converting IgG to hsIgG, and on the right are the IgG Fc glycan profiles of the starting IVIg and hsIgG. In this study, the glycan profiles of different IgG subclasses were derived via glycopeptide mass spectrometry. The peptide sequences used to quantify glycopeptides for the different IgG subclasses were IgG1 = EEQYNSTYR (SEQ ID NO: 7), IgG2 / 3 = EEQFNSTFR (SEQ ID NO: 8), IgG3 / 4 = EEQYNSTFR (SEQ ID NO: 9), and EEQFNSTYR (SEQ ID NO: 10).

[0163] Glycan data are presented per IgG subclass. Glycans from IgG3 and IgG4 subclasses cannot be quantified separately. As shown, for IVIg, the sum of all nonsialylated glycans is greater than 80%, and the sum of all sialylated glycans is <20%. For the reaction product, the sum of all nonsialylated glycans is <20%, and the sum of all sialylated glycans is greater than 80%. The nomenclature of the different glycans listed in the glycoprofile uses the Oxford notation for N-linked glycans.

[0164] Example 2: Alternative sialylation conditions For example, alternative suitable reaction conditions for galactosylation and sialylation to generate hsIgG in 50 mM BIS-TRIS (pH 6.9) involve galactosylation of an IgG antibody (e.g., pooled IgG antibody, pooled immunoglobulin, or IVIg) as follows: 7.4 mM MnCl, 38 μmol UDP-Gal / g IgG antibody, and incubation at 37° C. for 16-24 hours, followed by sialylation with 7.5 units B4GalT / g IgG antibody in 7.4 mM MnCl, 220 μmol CMP-NANA / g IgG antibody (added twice, i.e., once at the beginning of the reaction and again 9-10 hours later), and 15 units ST6-Gal1 / g IgG antibody with incubation at 37° C. for 30-33 hours. The reaction can be carried out by adding ST6-Gal1 and CMP-NANA to the galactosylation reaction. Alternatively, all of the reactants can be combined at the beginning and supplemented with CMP-NANA.

[0165] Example 3: Production of ST6Gal A fusion protein containing the enzymatically active portion of ST6Gal was designed for high-level expression in HEK cells and ease of purification. SEQ ID NO: 6 contains a portion of human ST6Gal (SEQ ID NO: 4), a 6HIS tag, and (SEQ ID NO: 13) , azurocidin-derived signal sequence ( MTRLTVLALL AGLLASSRA GSSPLLD(SEQ ID NO: 62), 19 aa are underlined signals), and the amino acids resulting from the cloning process. SEQ ID NO: 3 is the secreted form, and SEQ ID NO: 5 is the 6HIS tag. (SEQ ID NO: 13) and the ST6GalT portion.

[0166] [Table 21]

[0167] [Table 22]

[0168] [Table 23]

[0169] [Table 24]

[0170] Other embodiments While the present invention has been described in connection with its detailed description, it is understood that the foregoing description is illustrative of the scope of the invention and is not intended to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. The inventions described in the original claims of this application are listed below. [Invention 1] A fusion protein comprising: an N-terminal signal sequence; and an enzymatically active portion of human α-2,6-sialyltransferase 1 (ST6Gal1). [Invention 2] The fusion protein according to claim 1, wherein the enzymatically active portion of ST6Gal1 comprises SEQ ID NO:4. [Invention 3] 3. The fusion protein according to claim 2, wherein the enzymatically active portion of ST6Gal1 consists of SEQ ID NO:4. [Invention 4] 4. The fusion protein according to any one of Inventions 1 to 3, wherein the signal sequence is an N-terminal azurocidin signal sequence. [Invention 5] 5. The fusion protein according to claim 4, wherein the azurocidin signal sequence comprises MTRLTVLALLAGLLASSRA (SEQ ID NO: 30). [Invention 6] 5. The fusion protein according to claim 4, wherein the azurocidin signal sequence consists of MTRLTVLALLAGLLASSRA (SEQ ID NO: 30). [Invention 7] 7. The fusion protein according to any one of Inventions 1 to 6, further comprising an affinity tag. [Invention 8] 8. The fusion protein according to claim 7, wherein the affinity tag is selected from the group consisting of polyhistidine, glutathione S-transferase (GST), maltose binding protein (MBP), chitin binding protein, streptavidin tag (e.g., Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 31)), FLAG tag (e.g., DYKDDDDK (SEQ ID NO: 32)), biotin tag, and combinations thereof. [Invention 9] The fusion protein according to invention 8, wherein the polyhistidine tag is selected from the group consisting of HHHH (SEQ ID NO: 11), HHHHH (SEQ ID NO: 12), HHHHHH (SEQ ID NO: 13), HHHHHHH (SEQ ID NO: 14), HHHHHHHH (SEQ ID NO: 15), HHHHHHHHH (SEQ ID NO: 16), and HHHHHHHHHHH (SEQ ID NO: 17). [Invention 10] 10. The fusion protein according to any one of Inventions 7 to 9, wherein the affinity tag is located towards the N-terminus of the enzymatically active portion of ST6Gal1. [Invention 11] 2. The fusion protein according to claim 1, wherein the N-terminal signal sequence comprises MTRLTVLALLAGLLASSRA (SEQ ID NO: 30) and the enzymatically active portion of ST6Gal1 comprises SEQ ID NO: 4. [Invention 12] 12. The fusion protein according to claim 11, further comprising a hexahistidine tag. [Invention 13] 13. The fusion protein according to claim 12, wherein the hexahistidine tag is located between the N-terminal signal sequence and the enzymatically active portion of ST6Gal1. [Invention 14] 14. The fusion protein according to invention 13, consisting of SEQ ID NO: 6. [Invention 15] A nucleic acid molecule encoding the fusion protein according to any one of inventions 1 to 14. [Invention 16] A vector comprising the nucleic acid molecule according to invention 15. [Invention 17] 17. The vector according to claim 16, further comprising a promoter operably linked to said nucleic acid encoding said fusion protein. [Invention 18] 17. The vector according to claim 16, wherein the promoter is a cytomegalovirus (CMV) promoter. [Invention 19] A host cell stably transformed with the vector according to invention 16. [Invention 20] 18. The host cell according to invention 17, wherein the cell is a human embryonic kidney (HEK) cell or a derivative thereof. [Invention 21] 21. The host cell according to claim 20, wherein the cell is an HEK derivative HEK293. [Invention 22] 1. A method for producing a polypeptide, comprising: Culturing the host cell according to any one of claims 19 to 21 in a medium under conditions that allow expression of the fusion protein; and isolating the fusion protein from the medium. [Invention 23] A fusion protein comprising: an N-terminal signal sequence; and an enzymatically active portion of human β-1,4-galactosyltransferase (B4GalT1). [Invention 24] 24. The fusion protein according to claim 23, wherein the enzymatically active portion of B4GalT1 comprises SEQ ID NO: 43. [Invention 25] 24. The fusion protein according to claim 23, wherein the enzymatically active portion of B4GalT1 consists of SEQ ID NO: 43. [Invention 26] 26. The fusion protein according to any one of Inventions 23 to 25, wherein the signal sequence is an N-terminal azurocidin signal sequence. [Invention 27] 24. The fusion protein according to claim 23, wherein the azurocidin signal sequence comprises MTRLTVLALLAGLLASSRA (SEQ ID NO: 30). [Invention 28] 24. The fusion protein according to claim 23, wherein the azurocidin signal sequence consists of MTRLTVLALLAGLLASSRA (SEQ ID NO: 30). [Invention 29] 29. The fusion protein according to any one of inventions 23 to 28, further comprising an affinity tag. [Invention 30] 30. The fusion protein of claim 29, wherein the affinity tag is selected from the group consisting of polyhistidine, glutathione S-transferase (GST), maltose binding protein (MBP), chitin binding protein, streptavidin tag (e.g., Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO: 31)), FLAG tag (e.g., DYKDDDDK (SEQ ID NO: 32)), biotin tag, and combinations thereof. [Invention 31] The fusion protein of invention 30, wherein the polyhistidine tag is selected from the group consisting of HHHH (SEQ ID NO: 11), HHHHH (SEQ ID NO: 12), HHHHHH (SEQ ID NO: 13), HHHHHHH (SEQ ID NO: 14), HHHHHHHH (SEQ ID NO: 15), HHHHHHHHH (SEQ ID NO: 16), and HHHHHHHHHHH (SEQ ID NO: 17). [Invention 32] 32. The fusion protein according to any one of inventions 23 to 31, wherein the affinity tag is located towards the C-terminus of the enzymatically active portion of the B4GalT1. [Invention 33] 24. The fusion protein according to claim 23, wherein the N-terminal signal sequence comprises MTRLTVLALLAGLLASSRA (SEQ ID NO: 30) and the enzymatically active portion of B4GalT1 comprises SEQ ID NO: 43. [Invention 34] 34. The fusion protein according to claim 33, further comprising a septahistidine tag. [Invention 35] 35. The fusion protein according to claim 34, wherein the septahistidine tag is at the C-terminus. [Invention 36] 36. A fusion protein according to invention 35, consisting of SEQ ID NO: 45. [Invention 37] A nucleic acid molecule encoding the fusion protein according to any one of inventions 23 to 36. [Invention 38] A vector comprising the nucleic acid molecule according to invention 37. [Invention 39] 39. The vector according to invention 38, further comprising a promoter operably linked to said nucleic acid encoding said fusion protein. [Invention 40] 40. The vector according to claim 39, wherein the promoter is a cytomegalovirus (CMV) promoter. [Invention 41] A host cell stably transformed with the vector according to invention 16. [Invention 42] 42. The host cell according to invention 41, wherein said cell is a human embryonic kidney (HEK) cell or a derivative thereof. [Invention 43] 43. The host cell according to claim 42, wherein the cell is an HEK derivative HEK293. [Invention 44] 1. A method for producing a polypeptide, comprising: Culturing the host cell according to any one of inventions 41 to 43 in a medium under conditions that allow expression of the fusion protein; and isolating the fusion protein from the medium. [Invention 45] 1. A method for sialyating an immunoglobulin G (IgG) antibody, comprising: a) providing a composition comprising an IgG antibody; b) exposing the composition to an enzymatically active portion of ST6Gal1 comprising β1,4-galactosyltransferase 1 and SEQ ID NO: 4 in the presence of UDP-Gal and CMP-NANA, thereby producing a composition comprising sialyated IgG (sIgG). [Invention 46] 1. A method for sialyating an immunoglobulin G (IgG) antibody, comprising: a) providing a composition comprising an IgG antibody; b) exposing the IgG antibody to β1,4-galactosyltransferase 1 in the presence of UDP-Gal, thereby producing a composition comprising a galactosylated IgG antibody; c) exposing a composition comprising the galactosylated IgG antibody to an enzymatically active portion of ST6Gal1 comprising SEQ ID NO: 4 in the presence of CMP-NANA, thereby producing a composition comprising sialyated IgG (sIgG). [Invention 47] 47. The method of claim 46, wherein the composition comprising the galactosylated IgG antibody is not purified prior to step (c). [Invention 48] 48. The method of any one of claims 45 to 47, further comprising supplementing one or more of said compositions with CMP-NANA. [Invention 49] 49. The method according to any one of Inventions 45 to 48, wherein the mixture of IgG antibodies is selected from the group consisting of IgG1, IgG2, IgG3, IgG4, and combinations thereof. [Invention 50] 47. The method of claim 45 or 46, wherein at least 60% of the branched glycans on the Fc region of the antibodies in the composition comprising the sIgG are disialylated. [Invention 51] 47. The method of claim 45 or 46, wherein less than 50% of the branched glycans on the Fc region of the antibodies in the composition comprising the sIgG are monosialylated.

[0171] Other embodiments While the present invention has been described in connection with its detailed description, it is understood that the foregoing description is illustrative of the scope of the invention and is not intended to limit the scope of the invention, which is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A method for ex vivo sialylation of plasma-derived immunoglobulin G (pdIgG) antibodies, comprising: The method comprises: a1) exposing a composition comprising pdIgG to a soluble form of recombinant β1,4-galactosyltransferase 1 (B4GalT1) and a soluble form of recombinant ST6Gal1 in the presence of UDP-Gal and CMP-NANA, thereby producing a composition comprising sialylated pdIgG; or Or, b1) exposing a composition comprising pdIgG to a soluble form of recombinant β1,4-galactosyltransferase 1 (B4GalT1) in the presence of UDP-Gal, thereby producing a composition comprising galactosylated pdIgG antibodies; b2) exposing the composition comprising the galactosylated pdIgG antibody to a soluble form of recombinant ST6Gal1 in the presence of CMP-NANA, thereby producing a composition comprising sialylated pdIgG; Optionally, the composition comprising the galactosylated pdIgG antibody is not purified prior to step (b2); The soluble form of the recombinant B4GalT1 is expressed as a fusion protein with an N-terminal signal sequence containing a signal peptidase cleavage site, and the soluble form of the recombinant ST6Gal1 is expressed as a fusion protein with an N-terminal signal sequence containing a signal peptidase cleavage site. The method.

2. The method of claim 1, further comprising supplementing one or more of the compositions with CMP-NANA.

3. The method described in claim 1, wherein the pdIgG antibody is selected from the group consisting of IgG1, IgG2, IgG3, and IgG4, and combinations thereof.

4. The method described in claim 1, wherein at least 60% of the branched glycans on the Fc region of the sialylated pdIgG are disialylated.

5. The method described in claim 1, wherein less than 50% of the branched glycans on the Fc region of the sialylated pdIgG are monosialylated.

6. The method of claim 1, wherein the soluble form of the recombinant B4GalT1 is expressed as a fusion protein comprising an N-terminal azurocidin signal sequence comprising MTRLTVLALLAGLLASSRA (SEQ ID NO: 30) and the enzymatic active site of B4GalT1 comprising the sequence of SEQ ID NO:

43.

7. The method described in claim 6, wherein the fusion protein further comprises an affinity tag.

8. The method described in claim 7, wherein the affinity tag is selected from the group consisting of polyhistidine, glutathione S-transferase (GST), maltose binding protein (MBP), chitin binding protein, streptavidin tag, FLAG tag, biotin tag, and combinations thereof.

9. The method of claim 8, wherein the polyhistidine tag is selected from the group consisting of HHHH (SEQ ID NO: 11), HHHHH (SEQ ID NO: 12), HHHHHH (SEQ ID NO: 13), HHHHHHH (SEQ ID NO: 14), HHHHHHHH (SEQ ID NO: 15), HHHHHHHHH (SEQ ID NO: 16), and HHHHHHHHHHH (SEQ ID NO: 17).

10. The method of claim 1, wherein the soluble form of the recombinant ST6Gal1 is expressed as a fusion protein comprising an N-terminal azurocidin signal sequence comprising MTRLTVLALLAGLLASSRA (SEQ ID NO: 30) and an enzymatic active site of ST6Gal1 comprising the sequence of SEQ ID NO:

4.

11. The method described in claim 10, wherein the fusion protein further comprises an affinity tag.

12. The method described in claim 11, wherein the affinity tag is selected from the group consisting of polyhistidine, glutathione S-transferase (GST), maltose binding protein (MBP), chitin binding protein, streptavidin tag, FLAG tag, biotin tag, and combinations thereof.

13. The method of claim 12, wherein the polyhistidine tag is selected from the group consisting of HHHH (SEQ ID NO: 11), HHHHH (SEQ ID NO: 12), HHHHHH (SEQ ID NO: 13), HHHHHHH (SEQ ID NO: 14), HHHHHHHH (SEQ ID NO: 15), HHHHHHHHH (SEQ ID NO: 16), and HHHHHHHHHHH (SEQ ID NO: 17).

14. The method of claim 8 or 12, wherein the streptavidin tag is Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (sequence number 31) and / or the FLAG tag is DYKDDDDK (sequence number 32).

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