METHODS OF MAKING HYPER-SIALYLATED IMMUNOGLOBULIN
20230374062 · 2023-11-23
Inventors
Cpc classification
C07K2317/41
CHEMISTRY; METALLURGY
C12Y204/01038
CHEMISTRY; METALLURGY
International classification
Abstract
Disclosed herein are methods galatosylating IgG antibodies, methods of preparing hypersialylated (hsIgG), e.g., using immobilized β1,4-Galactosyltransferase I (β4GalT1), as well as polypeptides comprising β1,4-Galactosyltransferase I (β4GalT1) bound to a solid support and compositions comprising the same.
Claims
1. A method of galatosylating IgG antibodies, the method comprising: (a) providing a mixture of IgG antibodies; and (b) incubating the mixture of IgG antibodies in a reaction mixture comprising: a polypeptide comprising an enzymatically active portion of human β1,4-Galactosyltransferase I (β4GalT1) bound to a solid support; and UDP-Gal, thereby producing galactosylated IgG antibodies.
2. A method of preparing hypersialylated (hsIgG), the method comprising: (a) providing galactosylated IgG antibodies produced by the method of claim 1; and (b) incubating the galactosylated IgG antibodies in a reaction mixture comprising: a polypeptide comprising human ST6Gal1 or enzymatically active portion thereof; and CMP-NANA, thereby producing hsIgG.
3. The method of claim 2, further comprising: (c) isolating the polypeptide comprising an enzymatically active portion of human β1,4-Galactosyltransferase I (β4GalT1) bound to a solid support from the reaction mixture, thereby producing recycled β4GalT1; and repeating steps (a)-(b), wherein the β4GalT1 in the reaction mixture is the β4GalT1 isolated in step (c).
4. A method of preparing hypersialylated (hsIgG), the method comprising (a) providing a mixture of IgG antibodies, (b) incubating the mixture of IgG antibodies in a reaction mixture comprising: a polypeptide comprising an enzymatically active portion of human β1,4-Galactosyltransferase I (β4GalT1) bound to a solid support; and UDP-Gal, thereby producing galactosylated IgG antibodies; and (c) incubating the galactosylated IgG antibodies in a reaction mixture comprising: a polypeptide comprising human ST6Gal1 or enzymatically active portion thereof; and CMP-NANA, thereby producing hsIgG.
5. The method of claim 4, further comprising: (d) isolating the polypeptide comprising an enzymatically active portion of human β1,4-Galactosyltransferase I (β4GalT1) bound to a solid support from the reaction mixture, thereby producing recycled β4GalT1; and repeating steps (a)-(c), wherein the β4GalT1 in the reaction mixture is the β4GalT1 isolated in step (d).
6. The method of claim 1, wherein the human β1,4-Galactosyltransferase I (β4GalT1) bound to a solid support is separated from the galactosylated IgG antibodies after step (b).
7. The method of claim 1, wherein the enzymatically active portion of human β4GalT1 comprises SEQ ID NO:8.
8. The method of claim 7, wherein the polypeptide comprising the enzymatically active portion of human β4GalT1 is at least 85% identical SEQ ID NO: 37, 38, or 39, or a variant thereof having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, additions, or subtractions.
9. The method of claim 2, wherein the human ST6Gal1 or enzymatically active portion thereof comprises SEQ ID NO:14.
10. The method of claim 1, wherein the polypeptide comprising an enzymatically active portion of human β4GalT1 further comprises an affinity tag, wherein the affinity tag is attached to the solid support.
11. The method of one of claim 10, wherein the affinity tag is C-terminal.
12. The method of claim 10, wherein the at least one tag is selected from the group comprising polyhistidine, chitin binding protein (CBP), glutathione S-transferase (GST), maltose-binding protein (MBP), hemagglutinin (HA), Myc, streptavidin-binding peptide (SBP), calmodulin-tag, Spot-tag, a streptavidin tag, FLAG-tag, biotin, and combinations thereof.
13. The method of claim 12, wherein the polyhistidine tag comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 histidines.
14. The method of claim 13, wherein the polyhistidine tag comprises 7 or 8 histidines.
15. The method of claim 1, wherein the solid support is a magnetic bead.
16. The method of claim 1, wherein the IgG antibodies comprise IgG antibodies isolated from at least 1000 donors.
17. The method of claim 1, wherein at least 70% w/w of the IgG antibodies are IgG1 antibodies.
18. The method of claim 1, wherein at least 90% of the donor subjects have been exposed to a virus.
19. The method of claim 2, wherein about 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the IgG antibodies in the hsIgG preparation have a sialic acid on both the α1,3 branch and the α1,6 branch.
20. The method of claim 1, wherein at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain of the IgG antibodies in the hsIgG preparation have a sialic acid on both the α 1,3 arm and the α 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage; and at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fc domain of the IgG antibodies in the hsIgG preparation have a sialic acid on both the α 1,3 arm and the α 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage.
21. A polypeptide comprising: an enzymatically active portion of human β1,4-Galactosyltransferase I (β4GalT1); and an affinity tag, wherein the polypeptide is bound to a solid support.
22. The polypeptide of claim 12, wherein the enzymatically active portion of β4GalT1 comprises SEQ ID NO:8.
23. The polypeptide of claim 21, wherein the affinity tag comprises a poly-histidine tag selected from the group consisting of HHHH (SEQ ID NO:26), HHHHH (SEQ ID NO:27), HHHHHH, (SEQ ID NO:28), HHHHHHH (SEQ ID NO:29), HHHHHHHH (SEQ ID NO:30), HHHHHHHHH (SEQ ID NO:31), and HHHHHHHHHH (SEQ ID NO:32).
24. The polypeptide of claim 21, wherein the solid support is an agarose magnetic bead.
25. A composition comprising: the polypeptide of claim 21; a ST6Gal1; UDP-Gal; CMP-NANA; and IgG antibodies.
Description
DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION
[0073] Antibodies are glycosylated at conserved positions in the constant regions of their heavy chain and on the Fab domain. For example, human IgG antibodies have a single N-linked glycosylation site at Asn297 of the CH2 domain. Each antibody isotype has a distinct variety of N-linked carbohydrate structures in the constant regions. For human IgG, the core oligosaccharide normally consists of GlcNAc.sub.2Man.sub.3GlcNAc, with differing numbers of outer residues. Variation among individual IgG's can occur via attachment of galactose and/or galactose-sialic acid at one or both terminal GlcNAc or via attachment of a third GlcNAc arm (bisecting GlcNAc).
[0074] The present disclosure encompasses, in part, methods for preparing immunoglobulins (e.g., human IgG) having an Fc region having particular levels of branched glycans that are sialylated on both of the arms of the branched glycan (e.g., with a NeuAc-α 2,6-Gal terminal linkage). The levels can be measured on an individual Fc region (e.g., the number of branched glycans that are sialylated on an α1,3 arm, an α1,6 arm, or both, of the branched glycans in the Fc region), or on the overall composition of a preparation of polypeptides (e.g., the number or percentage of branched glycans that are sialylated on an α1,3 arm, an α1,6 arm, or both, of the branched glycans in the Fc region in a preparation of polypeptides).
[0075] Naturally derived polypeptides that can be used to prepare hypersialylated IgG include, for example, IgG in human serum (particular human serum pooled from more than 1,000 donors), intravenous immunoglobulin (IVIg) and polypeptides derived from IVIg (e.g., polypeptides purified from IVIg (e.g., enriched for sialylated IgGs) or modified IVIg (e.g., IVIg IgGs enzymatically sialylated).
[0076] N-linked oligosaccharide chains are added to a protein in the lumen of the endoplasmic reticulum. Specifically, an initial oligosaccharide (typically 14-sugar) is added to the amino group on the side chain of an asparagine residue contained within the target consensus sequence of Asn-X-Ser/Thr, where X may be any amino acid except proline. The structure of this initial oligosaccharide 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, resulting in a short, branched core oligosaccharide composed of two N-acetylglucosamine and three mannose residues. One of the branches is referred to in the art as the “α 1,3 arm,” and the second branch is referred to as the “α 1,6 arm,” as shown in
[0077] N-glycans can be subdivided into three distinct groups called “high mannose type,” “hybrid type,” and “complex type,” with a common pentasaccharide core (Man (α 1,6)-(Man(α 1,3))-Man(β 1,4)-GlcpNAc(β 1,4)-GlcpNAc(β 1,N)-Asn) occurring in all three groups.
[0078] The more common Fc glycans present in IVIg are shown in
[0079] Additionally or alternatively, one or more monosaccharides units of N-acetylglucosamine may be added to the core mannose subunits to form a “complex glycan.” Galactose may be added to the N-acetylglucosamine subunits, and sialic acid subunits may be added to the galactose subunits, resulting in chains that terminate with any of a sialic acid, a galactose or an N-acetylglucosamine residue. Additionally, a fucose residue may be added to an N-acetylglucosamine residue of the core oligosaccharide. Each of these additions is catalyzed by specific glycosyl transferases.
[0080] “Hybrid glycans” comprise characteristics of both high-mannose and complex glycans. For example, one branch of a hybrid glycan may comprise primarily or exclusively mannose residues, while another branch may comprise N-acetylglucosamine, sialic acid, galactose, and/or fucose sugars.
[0081] Sialic acids are a family of 9-carbon monosaccharides with heterocyclic ring structures. They bear a negative charge via a 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 sialyl residues found in polypeptides produced in mammalian expression systems are N-acetyl-neuraminic acid (NeuAc) and N-glycolylneuraminic acid (NeuGc). These usually occur as terminal structures attached to galactose (Gal) residues at the non-reducing termini of both N- and O-linked glycans. The glycosidic linkage configurations for these sialyl groups can be either α 2,3 or α 2,6.
[0082] Fc regions are glycosylated at conserved, N-linked glycosylation sites. For example, each heavy chain of an IgG antibody has a single N-linked glycosylation site at Asn297 of the CH2 domain. IgA antibodies have N-linked glycosylation sites within the CH2 and CH3 domains, IgE antibodies have N-linked glycosylation sites within the CH3 domain, and IgM antibodies have N-linked glycosylation sites within the CH1, CH2, CH3, and CH4 domains.
[0083] Each antibody isotype has a distinct variety of N-linked carbohydrate structures in the constant regions. For example, IgG has a single N-linked biantennary carbohydrate at Asn297 of the C.sub.H2 domain in each Fc polypeptide of the Fc region, which also contains the binding sites for C1q and FcγR. For human IgG, the core oligosaccharide normally consists of GlcNAc2Man3GlcNAc, with differing numbers of outer residues. Variation among individual IgG can occur via attachment of galactose and/or galactose-sialic acid at one or both terminal GlcNAc or via attachment of a third GlcNAc arm (bisecting GlcNAc).
[0084] Immunoglobulins, e.g., IgG antibodies, can be sialylated by carrying out a galactosylation step followed by a sialylation step. Beta-1,4-galactosyltransferase 1 (B4GalT) is a Type II Golgi membrane-bound glycoprotein that transfers galactose from uridine 5′-diphosphosegalactose ([[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2R,3R,4S,5R,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl] hydrogen phosphate; UDP-Gal) to GlcNAc as 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-Nacetylneuraminic 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 as an α-2,6 linkage. Schematically, the reactions proceed shown in
[0085] Glycans of polypeptides can be evaluated using any methods known in the art. For example, sialylation of glycan compositions (e.g., level of branched glycans that are sialylated on an α1,3 branch and/or an α1,6 branch) can be characterized using methods described in WO2014/179601.
[0086] In some embodiments of the 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 a sialic acid on both the α 1,3 arm and the α 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage. In addition, in some embodiments, at least 40%, 50%, 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain have a sialic acid on both the α 1,3 arm and the α 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage. Overall, in some embodiments, at least 60%, 65%, 70%, 75%, 80%, 85%, or 90% of the branched glycans have a sialic acid on both the α 1,3 arm and the α 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage.
Enzymes
[0087] Beta-1,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galt1, as well as orthologs, mutants, and variants thereof, including enzymatically active portions of beta-1,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galt1, as well as orthologs, mutants, and variants thereof, along with fusion proteins and polypeptides comprising the same are suitable for use in the methods described herein. B4Galt1 is one of seven beta-1,4-galactosyltransferase (beta4GalT) genes that each encode type II membrane-bound glycoproteins that appear to have exclusive specificity for the donor substrate UDP-galactose; all transfer galactose in a beta1,4 linkage to similar acceptor sugars: GlcNAc, Glc, and Xyl. B4Galt1 adds galactose to N-acetylglucosamine residues that are either monosaccharides or the nonreducing ends of glycoprotein carbohydrate chains. B4GalT1 is also called GGTB2. Four alternative transcripts encoding four isoforms of B4GALT1 (NCBI Gene ID 2683) are described in Table 1.
TABLE-US-00001 TABLE 1 Human B4GALT1 isoforms SEQ Length Transcript Length (nt) Protein ID NO: (aa) Isoform NM_001497.4 4176 NP_001488.2 1 398 1 NM_001378495.1 3999 NP_001365424.1 2 385 2 NM_001378496.1 4053 NP_001365425.1 3 357 3 NM_001378497.1 1520 NP_001365426.1 4 225 4
TABLE-US-00002 >NP_001488.2 B4GALT1 [organism = Homo sapiens] [GeneID = 2683] [isoform = 1] (SEQ ID NO: 1) MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLS RLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRP GGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIE FNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLK YWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYD YTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGG VSALSKQQFLTINGFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVG RCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRY PLYTQITVDIGTPS >NP_001365424.1 B4GALT1 [organism = Homo sapiens] [GeneID = 2683] [isoform = 2] (SEQ ID NO: 2) MPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLSRLPQLVGVSTPLQ GGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRPGGDSSPVVDSGPG PASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQ NPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQ QLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIP MNDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTIN GFPNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKN EPNPQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTP S >NP_001365425.1 B4GALT1 [organism = Homo sapiens] [GeneID = 2683] [isoform = 3] (SEQ ID NO: 3) MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLS RLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRP GGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIE FNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLK YWLYYLHPVLQRQQLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYD YTCFVFSDVDLIPMNDHNAYRCFSQPRHISVAMDKFGFRLVFRGMSIS RPNAVVGRCRMIRHSRDKKNEPNPQRFDRIAHTKETMLSDGLNSLTYQ VLDVQRYPLYTQITVDIGTPS >NP_001365426.1 B4GALT1 [organism = Homo sapiens] [GeneID = 2683] [isoform = 4] (SEQ ID NO: 4) MRLREPLLSGSAAMPGASLQRACRLLVAVCALHLGVTLVYYLAGRDLS RLPQLVGVSTPLQGGSNSAAAIGQSSGELRTGGARPPPPLGASSQPRP GGDSSPVVDSGPGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIE FNMPVDLELVAKQNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLK YWLYYLHPVLQRQQLDYGIYVINQYEKIRRLLW
TABLE-US-00003 TABLE 2 Topology of B4GALT1 isoform 1 (SEQ ID NO: 1) Feature AAs Description Length Sequence SEQ ID NO: Topological 1-24 Cytoplasmic 9 MRLREPLLSGSAAMPGASLQR 5 domain ACR Transmembrane 25-44 Helical; 17 LLVAVCALHLGVTLVYYLAG 6 Signal- anchor for type II membrane protein Topological 45-398 Lumenal 380 RDLSRLPQLVGVSTPLQGGSN 7 domain SAAAIGQSSGELRTGGARPPP PLGASSQPRPGGDSSPVVDSG PGPASNLTSVPVPHTTALSLP ACPEESPLLVGPMLIEFNMPV DLELVAKQNPNVKMGGRYAPR DCVSPHKVAIIIPFRNRQEHL KYWLYYLHPVLQRQQLDYGIY VINQAGDTIFNRAKLLNVGFQ EALKDYDYTCFVFSDVDLIPM NDHNAYRCFSQPRHISVAMDK FGFSLPYVQYFGGVSALSKQQ FLTINGFPNNYWGWGGEDDDI FNRLVFRGMSISRPNAVVGRC RMIRHSRDKKNEPNPQRFDRI AHTKETMLSDGLNSLTYQVLD VQRYPLYTQITVDIGTPS
TABLE-US-00004 TABLE 3 Binding sites of B4GALT1 isoform 1 (SEQ ID NO:1) Position(s) Description Reference(s) 250 Metal binding; Manganese 310 Binding site; “Structural snapshots of beta-1,4- UDP-alpha-D- galactosyltransferase-I along the kinetic pathway.” galactose Ramakrishnan B., Ramasamy V., Qasba P. K. J. Mol. Biol. 357:1619-1633(2006) 343 Metal binding; Manganese; via tele nitrogen 355 Binding site; N- “Oligosaccharide preferences of beta1,4- acetyl-D- galactosyltransferase-I: crystal structures of glucosamine Met340His mutant of human beta1,4- galactosyltransferase-I with a pentasaccharide and trisaccharides of the N-glycan moiety.” Ramasamy V., Ramakrishnan B., Boeggeman E., Ratner D. M., Seeberger P. H., Qasba P. K. J. Mol. Biol. 353:53-67(2005) “Deoxygenated disaccharide analogs as specific inhibitors of beta1-4-galactosyltransferase 1 and selectin-mediated tumor metastasis.” Brown J. R., Yang F., Sinha A., Ramakrishnan B., Tor Y., Qasba P. K., Esko J. D. J. Biol. Chem. 284:4952-4959(2009)
TABLE-US-00005 TABLE 4 Post Translational Amino Acid Modifications of B4GALT1 isoform 1 (SEQ ID NO: 1) Feature key Position(s) Description Reference(s) Glycosylation 113 N-linked (GlcNAc . . .) asparagine Disulfide 130 .Math. 172 “Oligosaccharide preferences of beta1,4- bond galactosyltransferase-I: crystal structures of Met340His mutant of human beta1,4- Disulfide 243 .Math. 262 galactosyltransferase-I with a bond pentasaccharide and trisaccharides of the N- glycan moiety.” Ramasamy V., Ramakrishnan B., Boeggeman E., Ratner D. M., Seeberger P.H., Qasba P. K. J. Mol. Biol. 353:53-67(2005) “Structural snapshots of beta-1,4- galactosyltransferase-I along the kinetic pathway.” Ramakrishnan B., Ramasamy V., Qasba P. K. J. Mol. Biol. 357:1619-1633(2006)
[0088] The soluble form of B4GalT1 derives from the membrane form by proteolytic processing. The cleavage site is at positions 77-78 of B4GALT1 isoform 1 (SEQ ID NO:1).
[0089] In some embodiments, one or more of the amino acids of the B4GalT1 corresponding to amino acids 113, 130, 172, 243, 250, 262, 310, 343, or 355 of B4GALT1 isoform 1 (SEQ ID NO:1) is conserved as compared to (SEQ ID NO:1).
[0090] In some embodiments, the enzyme is an enzymatically active portion of, e.g., B4GalT1. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 1 (SEQ ID NO:1), or an ortholog, mutant, or variant of SEQ ID NO:1. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 2 (SEQ ID NO:2), or an ortholog, mutant, or variant of SEQ ID NO:2. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 3 (SEQ ID NO:3), or an ortholog, mutant, or variant of SEQ ID NO:3. In some embodiments, the enzyme is an enzymatically active portion of B4GALT1 isoform 4 (SEQ ID NO:4), or an ortholog, mutant, or variant of SEQ ID NO:4.
[0091] In some embodiments, the enzymatically active portion of B4GalT1 does not comprise a cytoplasmic domain, e.g., SEQ ID NO:5. In some embodiments, the enzymatically active portion of B4GalT1 does not comprise a transmembrane domain, e.g., SEQ ID NO:6. In some embodiments, the enzymatically active portion of B4GalT1 does not comprise a cytoplasmic domain, e.g., SEQ ID NO:5 or a transmembrane domain, e.g., SEQ ID NO:6.
[0092] In some embodiments, the enzymatically active portion of B4GalT1 comprises all or a portion of a luminal domain, e.g., SEQ ID NO:7, or an ortholog, mutants, or variants thereof.
[0093] In some embodiments, the enzymatically active portion of B4GalT1 comprises amino acids 109-398 of SEQ ID NO:1, or an ortholog, mutants, or variants thereof. In some embodiments, the enzymatically active portion of B4GalT1 consists of SEQ ID NO:1, or an ortholog, mutant, or variant of SEQ ID NO:1.
[0094] A suitable functional portion of an B4GalT1 can comprise or consist of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO:8.
TABLE-US-00006 SEQ ID NO: 8 GPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQ NPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQ LDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMN DHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFP NNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNP QRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPS
[0095] ST6Gal1, e.g., human ST6Gal1, as well as orthologs, mutants, and variants thereof, including enzymatically active portions of ST6Gal1, e.g., human ST6Gal1, as well as orthologs, mutants, and variants thereof, along with fusion proteins and polypeptides 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 the Galβ1.fwdarw.4GlcNAc structure on glycoproteins, such as asialofetuin and asialo-a1-acid glycoprotein. ST6Gal1 is also called as ST6N or SIAT1. Four alternative transcripts encoding two isoforms of ST6GAL1 (NCBI Gene ID 6480) are described in Table 1.
TABLE-US-00007 TABLE 1 Human ST6GAL1 isoforms SEQ Length ID Length Transcript (nt) Protein NO: (aa) Isoform NM_173216.2 4604 NP_775323.1 9 406 a NM_173217.2 3947 NP_775324.1 10 175 b NM_003032.3 4303 NP_003023.1 9 406 a NM_001353916.2 4177 NP_001340845.1 9 406 a
TABLE-US-00008 >NP_001340845.1 (NP_003023.1, NP_775323.1) ST6GAL1 [organism = Homo sapiens] [GeneID = 6480] [isoform = a] (SEQ ID NO : 9) MIHTNLKKKFSCCVLVFLLFAVICVWKEKKKGSYYDSFKLQTKEFQVLKS LGKLAMGSDSQSVSSSSTQDPHRGRQTLGSLRGLAKAKPEASFQVWNKDS SSKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNV SMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGR EIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYN EGILIVWDPSVYHSDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQM PWELWDILQEISPEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKT DVCYYYQKFFDSACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPG FRTIHC >NP_775324.1 ST6GAL1 [organism = Homo sapiens] [GeneID = 6480] [isoform = b] (SEQ ID NO: 10) MNSQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNY KTYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIM MTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVKH LNQGTDEDIYLLGKATLPGFRTIHC
TABLE-US-00009 TABLE 2 Topology of ST6Gal1 isoform a (SEQ ID NO: 28) Feature AAs Description Length Sequence SEQ ID NO: Topological 1-9 Cytoplasmic 9 MIHTNLKKK 11 domain Transmembrane 10-26 Helical; 17 FSCCVLVFLLFAVICVW 12 Signal- anchor for type II membrane protein Topological 27-406 Lumenal 380 KEKKKGSYYDSFKLQTKEFQVLKS 13 domain LGKLAMGSDSQSVSSSSTQDPHRG RQTLGSLRGLAKAKPEASFQVWNK DSSSKNLIPRLQKIWKNYLSMNKY KVSYKGPGPGIKFSAEALRCHLRD HVNVSMVEVTDFPFNTSEWEGYLP KESIRTKAGPWGRCAVVSSAGSLK SSQLGREIDDHDAVLRFNGAPTAN FQQDVGTKTTIRLMNSQLVTTEKR FLKDSLYNEGILIVWDPSVYHSDI PKWYQNPDYNFFNNYKTYRKLHPN QPFYILKPQMPWELWDILQEISPE EIQPNPPSSGMLGIIIMMTLCDQV DIYEFLPSKRKTDVCYYYQKFFDS ACTMGAYHPLLYEKNLVKHLNQGT DEDIYLLGKATLPGFRTIHC
TABLE-US-00010 TABLE 3 Binding sites of ST6Gal1 isoform a (SEQ ID NO: 28) Position(s) Description Reference(s) 189 Substrate; via “The structure of human alpha-2,6-sialyltransferase amide nitrogen reveals the binding mode of complex glycans.” 212 Substrate Kuhn B., Benz J., Greif M., Engel A. M., Sobek H., 233 Substrate Rudolph M.G. Acta Crystallogr. D 69:1826- 353 Substrate; via 1838(2013) carbonyl oxygen 354 Substrate 365 Substrate 369 Substrate 370 Substrate “The structure of human alpha-2,6-sialyltransferase reveals the binding mode of complex glycans.” 376 Substrate Kuhn B., Benz J., Greif M., Engel A. M., Sobek H., Rudolph M. G. Acta Crystallogr. D 69:1826- 1838(2013)
TABLE-US-00011 TABLE 4 Post Translational Amino Acid Modifications of ST6Gal1 isoform a (SEQ ID NO: 28) Feature key Position(s) Description Reference(s) Disulfide 142 .Math. 406 “The structure of human alpha-2,6- bond sialyltransferase reveals the binding mode of complex glycans.” Kuhn B., Benz J., Greif M., Engel A.M., Sobek H., Rudolph M. G. Acta Crystallogr. D 69:1826- 1838(2013) Glycosylation 149 N-linked “Glycoproteomics analysis of human (GlcNAc . . .) liver tissue by combination of multiple asparagine enzyme digestion and hydrazide chemistry.” Chen R., Jiang X., Sun D., Han G., Wang F., Ye M., Wang L., Zou H. J. Proteome Res. 8:651-661(2009); and “The structure of human alpha-2,6- sialyltransferase reveals the binding mode of complex glycans.” Kuhn B., Benz J., Greif M., Engel A. M., Sobek H., Rudolph M. G. Acta Crystallogr. D 69:1826- 1838(2013) Glycosylation 161 N-linked “Glycoproteomics analysis of human (GlcNAc . . .) liver tissue by combination of multiple asparagine enzyme digestion and hydrazide chemistry.” Chen R., Jiang X., Sun D., Han G., Wang F., Ye M., Wang L., Zou H. J. Proteome Res. 8:651-661(2009) Disulfide 184 .Math. 335 “The structure of human alpha-2,6- bond sialyltransferase reveals the binding mode of complex glycans.” Kuhn B., Benz J., Greif M., Engel A. M., Sobek H., Rudolph M. G. Acta Crystallogr. D 69:1826- 1838(2013) Disulfide 353 .Math. 364 “The structure of human alpha-2,6- bond sialyltransferase reveals the binding mode of complex glycans.” Kuhn B., Benz J., Greif M., Engel A. M., Sobek H., Rudolph M. G. Acta Crystallogr. D 69:1826- 1838(2013) Modified 369 Phosphotyrosine “Quantitative phosphoproteomic residue analysis of T cell receptor signaling reveals system-wide modulation of protein-protein interactions.” Mayya V., Lundgren D. H., Hwang S.-I., Rezaul K., Wu L., Eng J. K., Rodionov V., Han D. K. Sci. Signal. 2:RA46-RA46(2009)
[0096] The soluble form of ST6Gal1 derives from the membrane form by proteolytic processing.
[0097] In some embodiments, one or more of the amino acids of the 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:9) is conserved as compared to SEQ ID NO:9.
[0098] Also provided herein is an enzymatically active portion of, e.g., ST6Gal1. In some embodiments, the enzyme is an enzymatically active portion of STG6Gal1 isoform a (SEQ ID NO:9), or an ortholog, mutant, or variant of SEQ ID NO:9. In some embodiments, the enzyme is an enzymatically active portion of STG6Gal1 isoform b (SEQ ID NO:10), or an ortholog, mutant, or variant of SEQ ID NO:10.
[0099] In some embodiments, the enzymatically active portion of ST6Gal1 does not comprise a cytoplasmic domain, e.g., SEQ ID NO:11. In some embodiments, the enzymatically active portion of ST6Gal1 does not comprise a transmembrane domain, e.g., SEQ ID NO:12. In some embodiments, the enzymatically active portion of ST6Gal1 does not comprise a cytoplasmic domain, e.g., SEQ ID NO:11 or a transmembrane domain, e.g., SEQ ID NO:12.
[0100] In some embodiments, the enzymatically active portion of ST6Gal1 comprises all or a portion of a luminal domain, e.g., SEQ ID NO:13, or an ortholog, mutants, or variants thereof.
[0101] In some embodiments, the enzymatically active portion of ST6Gal1 comprises amino acids 87-406 of SEQ ID NO:9 (SEQ ID NO:14), or an ortholog, mutants, or variants thereof. In some embodiments, the enzymatically active portion of ST6Gal1 consists of SEQ ID NO:4, or an ortholog, mutant, or variant of SEQ ID NO:4.
[0102] A suitable functional portion of an ST6Gal1 can comprise or consist of an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO:14.
TABLE-US-00012 SEQ ID NO: 14 AKPEASFQVWNKDSSSKNLIPRLQKIWKNYLSMNKYKVSYKGPGPGIKF SAEALRCHLRDHVNVSMVEVTDFPFNTSEWEGYLPKESIRTKAGPWGRC AVVSSAGSLKSSQLGREIDDHDAVLRFNGAPTANFQQDVGTKTTIRLMN SQLVTTEKRFLKDSLYNEGILIVWDPSVYHSDIPKWYQNPDYNFFNNYK TYRKLHPNQPFYILKPQMPWELWDILQEISPEEIQPNPPSSGMLGIIIM MTLCDQVDIYEFLPSKRKTDVCYYYQKFFDSACTMGAYHPLLYEKNLVK HLNQGTDEDIYLLGKATLPGFRTIHC
[0103] In some embodiments, the ST6Gal1 comprises or consists of SEQ ID NO:14, the portion of SEQ ID NO:14 from amino acid 4 to 320, or the portion of SEQ ID NO:14 from amino acid 5 to 320.
[0104] Also suitable for use in the methods described herein is an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO:15.
TABLE-US-00013 SEQ ID NO: 15 gssplldmlehhhhhhhhmAKPEASFQVWNKDSSSKNLIPRLQKIWKNY LSMNKYKVSYKGPGPGIKFSAEALRCHLRDHVNVSMVEVTDFPFNTSEW EGYLPKESIRTKAGPWGRCAVVSSAGSLKSSQLGREIDDHDAVLRFNGA PTANFQQDVGTKTTIRLMNSQLVTTEKRFLKDSLYNEGILIVWDPSVYH SDIPKWYQNPDYNFFNNYKTYRKLHPNQPFYILKPQMPWELWDILQEIS PEEIQPNPPSSGMLGIIIMMTLCDQVDIYEFLPSKRKTDVCYYYQKFFD SACTMGAYHPLLYEKNLVKHLNQGTDEDIYLLGKATLPGFRTIHC
Variants
[0105] In some embodiments, the enzyme(s) 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), e.g., have differences at up to 1%, 2%, 5%, 10%, 15%, or 20% of the residues of the exemplary sequence replaced, e.g., with conservative mutations, e.g., including or in addition to the mutations described herein. In preferred embodiments, the variant retains desired activity of the parent, e.g., β-galactoside α-2,6-sialyltransferase activity or β-1,4-galactosyltransferase activity.
[0106] To determine the percent identity of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90% or 100%. The 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, then the molecules are identical at that position (as used herein nucleic acid “identity” is equivalent to nucleic acid “homology”). The percent identity between the 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, which need to be introduced for optimal alignment of the two sequences.
[0107] Percent identity between a subject polypeptide or nucleic acid sequence (i.e. a query) and a second polypeptide or nucleic acid sequence (i.e. target) is determined in various ways that are within the skill in the art, for instance, using publicly available computer software such as Smith Waterman Alignment (Smith, T. F. and M. S. Waterman (1981) J Mol Biol 147:195-7); “BestFit” (Smith and Waterman, Advances in Applied Mathematics, 482-489 (1981)) as incorporated into GeneMatcher Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M. O., Ed, pp 353-358; 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. 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. In general, for target proteins or nucleic acids, the length of comparison can be any length, up to and including full length of the target (e.g., 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%). For the purposes of the present disclosure, percent identity is relative to the full length of the query sequence.
[0108] For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.
[0109] Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
B4GalT Immobilization
[0110] In some embodiments, the protein(s) comprising enzyme(s) or portions thereof as described herein are immobilized on a surface, e.g., a solid support.
[0111] Methods for protein immobilization, including both covalent and non-covalent approaches, are known and described in the art.
[0112] Covalent approaches, such as enzymatic approaches (e.g., sortase A), enzyme self-labeling (e.g., SNAP-tag, HaloTag, and CLIP-tag) chemical approaches (e.g., oxime ligation, Cu(I)-catalyzed axide-alkyne cycoloaddition (CuAAC) reaction, strain-promoted azide-alkyne cycloaddition (SPAAC) reaction, strain-promoted alkyne-nitrone cycloaddition (SPANC) reaction, and inverse electron-demand Diels-Alder reaction (IEDDA) reaction). See, e.g., Meldal and Schoffelen, “Recent Advances in Covalent, Site-Specific Protein Immobilization,” F1000Research 216, 5(F1000 Faculty Rev):2303.
[0113] In some embodiments, the protein is immobilized via a non-covalent approach (affinity-mediated mobilization) such as the use of protein A or G for binding of antibodies, peptide tags such as polyhistidine, protein tags such as maltose-binding protein and glutathione-S-transferase, DNA-directed immobilization, or the biotin-streptavidin interaction pair. See e.g., Steen et al., “Protein engineering for directed immobilization,” Bioconjug Chem. 2013; 24(11):1761-77; Liu et al., “Oriented immobilization of proteins on solid supports for use in biosensors and biochips: a review,” Microchim Acta 2016; 183:1-19; Sapsford et al., “Functionalizing nanoparticles with biological molecules: developing chemistries that facilitate nanotechnology,” Chem Rev. 2013; 113(3):1904-2074; Benes̆ová et al., “Affinity Interactions as a Tool for Protein Immobilization,” In: Magdeldin S, editor. Affinity Chromatography: InTech. 2012; 29-46; Trilling et al., “Antibody orientation on biosensor surfaces: a minireview,” Analyst 2013; 138(6):1619-27; and Meyer et al., “Advances in DNA-directed immobilization,” Curr Opin Chem Biol. 2014; 18:8-15.
Polypeptides
[0114] Thus, also provided herein are polypeptides comprising: i) a B4GalT enzyme (e.g., a Beta-1,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galt1, or an ortholog, mutants, or variants of Beta-1,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galt1, including enzymatically active portions of beta-1,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galt1, as well as orthologs, mutants, and variants of an enzymatically active portions of beta-1,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galt1); and ii) at least one affinity tag.
[0115] In some embodiments, the at least one tag is at the N terminus, C terminus, or at both the N terminus and the C terminus.
[0116] In some embodiments, the affinity tag is selected from the group consisting of polyhistidine, chitin binding protein (CBP) (e.g., KRRWKKNFIAVSAANRFKKISSSGAL, SEQ ID NO:16), glutathione S-transferase (GST) (e.g., MSPILGYWKIKGLVQPTRLLLEYLEEKYEEHLYERDEGDKWRNKKFELGLEF PNLPYYIDGDVKLTQSMAIIRYIADKHNMLGGCPKERAEISMLEGAVLDIRYG VSRIAYSKDFETLKVDFLSKLPEMLKMFEDRLCHKTYLNGDHVTHPDFMLYD ALDVVLYMDPMCLDAFPKLVCFKKRIEAIPQIDKYLKSSKYIAWPLQGWQAT FGGGDHPPKSD, SEQ ID NO:17), maltose-binding protein (MBP) (e.g., MGSSHHHHHHSSGLVPRGSHMGSMKIEEGKLVIWINGDKGYNGLAEVGKKF EKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEIT PDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIP ALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVD NAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNI DTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDE GLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAF WYAVRTAVINAASGRQTVDEALKDAQTNSSSLGIEGR, SEQ ID NO:18), hemagglutinin (HA) (e.g., YPYDVPDYA, SEQ ID NO:19), Myc (e.g., EQKLISEEDL, SEQ ID NO:20), streptavidin-binding peptide (SBP) (e.g., MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP, SEQ ID NO:21), calmodulin-tag (e.g., MADQLTEEQIAEFKEAFSLFDKDGDGTITTKELGTVMRSLGQNPTEAELQDMI NEVDADGNGTIDFPEFLTMMARKMKDTDSEEEIREAFRVFDKDGNGYISAAE LRHVMTNLGEKLTDEEVDEMIREADIDGDGQVNYEEFVQMMTAKGSMGWD LTVKMLAGNEFQVSLSSSMSVSELKAQITQKIGVHAFQQRLAVHPSGVALQD RVPLASQGLGPGSTVLLVVDKCDEPLNILVRNNKGRSSTYEVRLTQTVAHLK QQVSGLEGVQDDLFWLTFEGKPLEDQLPLGEYGLKPLSTVFMNLRLRGG, SEQ ID NO:22), Spot-tag (e.g., PDRVRAVSHWSS, SEQ ID NO:23), a streptavidin tag (e.g., Strep-Tag®, e.g., Trp-Ser-His-Pro-Gln-Phe-Glu-Lys (SEQ ID NO:24)), FLAG-tag (e.g., DYKDDDDK (SEQ ID NO:25) biotin, as well as variants thereof and combinations of all of the foregoing.
[0117] 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:26), HHHHH (SEQ ID NO:27), HHHHHH, (SEQ ID NO:28), HHHHHHH (SEQ ID NO:29), HHHHHHHH (SEQ ID NO:30), HHHHHHHHH (SEQ ID NO:31), and HHHHHHHHHH (SEQ ID NO:32). In some embodiments, the at least one tag comprises 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 histidines (SEQ ID NO: 44).
[0118] In some embodiments, the affinity tag is situated towards the N-terminal side of the enzyme. In some embodiments, the affinity tag is situated towards the C-terminal side of the enzyme.
[0119] Additional tags are known in the art and can be used for the purpose of immobilizing the β4GalT1 to a solid support (e.g. resin, column, array, etc.). In some embodiments, these additional tags may be paired with known binding agents attached to the solid support such that the tagged β4GalT1 binds to the solid support.
[0120] In some embodiments, the polypeptide further comprises a cleavage sequence or spacer sequence between the enzyme and the affinity tag (e.g., situated towards the C-terminal side of the enzyme and towards the N-terminal side of the affinity tag). In some embodiments, the spacer sequence is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids long. In some embodiments, the spacer sequence is at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids long. In some embodiments, the cleavage or spacer sequence is at least 3 amino acids long.
[0121] In some embodiments, the spacer sequence comprises or consists of PRD (SEQ ID NO:33). In some embodiments spacer sequence comprises PGG (SEQ ID NO:34).
[0122] A suitable B4GalT with a C-terminal spacer sequence that is suitable for use in the methods described herein, therefore, can comprise an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO:35 or SEQ ID NO:36
TABLE-US-00014 SEQ ID NO: 35 GPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQ NPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQ LDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMN DHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFP NNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNP QRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPSPRD SEQ ID NO: 36 GPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAKQ NPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQQ LDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPMN DHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGFP NNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPNP QRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPSPGG
[0123] A his-tagged human Beta-1,4-galactosyltransferase 1 (B4GalT) is suitable for use in the methods described herein. A suitable B4GalT can comprise an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO:37 or SEQ ID NO:38 (a schematic of which is shown in
TABLE-US-00015 SEQ ID NO: 37 MGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAK QNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQ QLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPM NDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGF PNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPN PQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPSPGG HHHHHHHH SEQ ID NO: 38 MGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAK QNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQ QLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPM NDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGF PNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPN PQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPSPRD HHHHHHH
[0124] A visual map of a portion of SEQ ID NO:38 is shown in
[0125] A biotin-tagged human Beta-1,4-galactosyltransferase 1 (B4GalT) is suitable for use in the methods described herein. A suitable B4GalT can comprise an amino acid sequence that is at least 80% (85%, 90%, 95%, 98% or 100%) identical to SEQ ID NO:39. In some embodiments, the biotin tag is a variant of biotin.
TABLE-US-00016 SEQ ID NO: 39 MGPASNLTSVPVPHTTALSLPACPEESPLLVGPMLIEFNMPVDLELVAK QNPNVKMGGRYAPRDCVSPHKVAIIIPFRNRQEHLKYWLYYLHPVLQRQ QLDYGIYVINQAGDTIFNRAKLLNVGFQEALKDYDYTCFVFSDVDLIPM NDHNAYRCFSQPRHISVAMDKFGFSLPYVQYFGGVSALSKQQFLTINGF PNNYWGWGGEDDDIFNRLVFRGMSISRPNAVVGRCRMIRHSRDKKNEPN PQRFDRIAHTKETMLSDGLNSLTYQVLDVQRYPLYTQITVDIGTPSPG G-Biotin
Supports
[0126] In some embodiments, the support, e.g., solid support, e.g., porous solid support, is a resin, column, array, microarray, solid phase.
[0127] In some embodiments, the support material can comprise a membrane, a bead, a gel, a cassette, a column, a chip, a slide, a plate, an array, a microarray, or a monolith. In some embodiments, the support material may comprise a hydrophilic compound, a hydrophobic compound, an oleophobic compound, an oleophilic compound, or any combination thereof. In some embodiments, the support material may comprise a polymer or a copolymer.
[0128] Examples of suitable support materials, include, but are not limited to polyether sulfone, polyamide, e.g., agarose, cellulose, a polysaccharide, polytetrafluoroethylene, polysulfone, polyester, polyvinylidene fluoride, polypropylene, a fluorocarbon, e.g. poly (tetrafluoroethylene-co-perfluoro(alkyl vinyl ether)), poly carbonate, polyethylene, glass, polycarbonate, polyacrylate, polyacrylamide, poly(azolactone), polystyrene, ceramic, nylon and metal.
[0129] In some embodiments, the support comprises a metal (e.g. metal chelate), Nickel (e.g. Ni2+), Cobalt (e.g. Co2+), chitin, maltose, GSH, an antibody or nanobody, a FLAG-binding antibody or nanobody, a HA-binding antibody or nanobody, a Myc-binding antibody or nanobody, an NE-binding antibody or nanobody, streptavidin, biotin, calmodulin, a Spot-binding antibody or nanobody, variants thereof, and combinations thereof.
[0130] In some embodiments, the support comprises a ligand that binds an affinity tag, e.g., an affinity tag of a polypeptide comprising a B4GalT, e.g., a poly-histidine tag, as described herein. In some embodiments, the support comprises a ligand selected from the group consisting of nickel (e.g., Ni-NTA or Ni-IDA), cobalt, and combinations thereof.
[0131] In some embodiments, the support is a bead, e.g., a magnetic bead. In some embodiments, the support is a magnetic agarose bead. In some embodiments, the magnetic agarose bead is a magnetic sepharose bead. In some embodiments, the support is a resin. In some embodiments, the support is an agarose resin. In some embodiments, the agarose resin is a sepharose resin.
[0132] In some embodiments, the magnetic agarose bead or agarose resin comprises an agarose gel of about 1% to about 10% w/v. In some embodiments, the magnetic agarose bead or agarose resin comprises an agarose gel of about 1% to about 9%, about 1% to about 8%, about 1% to about 7%, about 1% to about 6%, about 1% to about 5%, about 1% to about 4%, about 1% to about 3%, about 1% to about 2%, about 2% to about 10%, about 2% to about 9%, about 2% to about 8%, about 2% to about 7%, about 2% to about 6%, about 2% to about 5%, about 2% to about 4%, about 2% to about 3%, about 3% to about 10%, about 3% to about 9%, about 3% to about 8%, about 3% to about 7%, about 3% to about 6%, about 3% to about 5%, about 3% to about 4%, about 4% to about 10%, about 4% to about 9%, about 4% to about 8%, about 4% to about 7%, about 4% to about 6%, about 4% to about 5%, about 5% to about 10%, about 5% to about 9%, about 5% to about 8%, about 5% to about 7%, to about 6%, about 6% to about 10%, about 6% to about 9%, about 6% to about 8%, about 6% to about 7%, about 7% to about 10%, about 7% to about 9%, about 7% to about 8%, about 8% to about 10%, about 8% to about 9%, or about 9% to about 10% w/v.
[0133] In some embodiments, the pore size range of the support is from about 20 to about 130 nm. In some embodiments, the pore size range of the support is about 20 to about 120, about 20 to about 110, about 20 to about 100, about 20 to about 90, about 20 to about 80, about 20 to about 70, about 20 to about 60, about 20 to about 50, about 20 to about 40, about 20 to about 30, about 30 to about 130, about 30 to about 120, about 30 to about 110, about 30 to about 100, about 30 to about 90, about 30 to about 80, about 30 to about 70, about 30 to about 60, about 30 to about 50, about 30 to about 40, about 40 to about 130, about 40 to about 120, about 40 to about 110, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 130, about 50 to about 120, about 50 to about 110, about 50 to about 100, about 50 to about 90, about 50 to about 80, about 50 to about 70, about 50 to about 60, about 60 to about 130, about 60 to about 120, about 60 to about 110, about 60 to about 100, about 60 to about 90, about 60 to about 80, about 60 to about 70, about 70 to about 130, about 70 to about 120, about 70 to about 110, about 70 to about 100, about 70 to about 90, about 70 to about 80, about 80 to about 130, about 80 to about 120, about 80 to about 110, about 80 to about 100, about 80 to about 90, about 90 to about 130, about 90 to about 120, about 90 to about 110, about 90 to about 100, about 100 to about 130, about 100 to about 120, about 100 to about 110, about 110 to about 130, about 110 to about 120, or about 120 to about 130 nm.
[0134] In some embodiments, the support, e.g., bead or resin, e.g., magnetic bead or magnetic resin, is from about 10 to about 350 μm in size, e.g., in diameter. In some embodiments, the support, e.g., bead, e.g., magnetic bead is from about 10 to about 170, about 10 to about 160, about 10 to about 150, about 10 to about 140, about 10 to about 130, about 10 to about 120, about 10 to about 110, about 10 to about 100, about 10 to about 90, about 10 to about 80, about 10 to about 70, about 10 to about 60, about 10 to about 50, about 10 to about 40, about 10 to about 30, about 10 to about 20, about 20 to about 170, about 20 to about 160, about 20 to about 150, about 20 to about 140, about 20 to about 130, about 20 to about 120, about 20 to about 110, about 20 to about 100, about 20 to about 90, about 20 to about 80, about 20 to about 70, about 20 to about 60, about 20 to about 50, about 20 to about 40, about 20 to about 30, about 30 to about 170, about 30 to about 160, about 30 to about 150, about 30 to about 140, about 30 to about 130, about 30 to about 120, about 30 to about 110, about 30 to about 100, about 30 to about 90, about 30 to about 80, about 30 to about 70, about 30 to about 60, about 30 to about 50, about 30 to about 40, about 40 to about 170, about 40 to about 160, about 40 to about 150, about 40 to about 140, about 40 to about 130, about 40 to about 120, about 40 to about 110, about 40 to about 100, about 40 to about 90, about 40 to about 80, about 40 to about 70, about 40 to about 60, about 40 to about 50, about 50 to about 170, about 50 to about 160, about 50 to about 150, about 50 to about 140, about 50 to about 130, about 50 to about 120, about 50 to about 110, about 50 to about 100, about 50 to about 90, about 50 to about 80, about 50 to about 70, about 50 to about 60, about 60 to about 170, about 60 to about 160, about 60 to about 150, about 60 to about 140, about 60 to about 130, about 60 to about 120, about 60 to about 110, about 60 to about 100, about 60 to about 90, about 60 to about 80, about 60 to about 70, about 70 to about 170, about 70 to about 160, about 70 to about 150, about 70 to about 140, about 70 to about 130, about 70 to about 120, about 70 to about 110, about 70 to about 100, about 70 to about 90, about 70 to about 80, about 80 to about 170, about 80 to about 160, about 80 to about 150, about 80 to about 140, about 80 to about 130, about 80 to about 120, about 80 to about 110, about 80 to about 100, about 80 to about 90, about 90 to about 170, about 90 to about 160, about 90 to about 150, about 90 to about 140, about 90 to about 130, about 90 to about 120, about 90 to about 110, about 90 to about 100, about 100 to about 170, about 100 to about 160, about 100 to about 150, about 100 to about 140, about 100 to about 130, about 100 to about 120, about 100 to about 110, about 110 to about 170, about 110 to about 160, about 110 to about 150, about 110 to about 140, about 110 to about 130, about 110 to about 120, about 120 to about 170, about 120 to about 160, about 120 to about 150, about 120 to about 140, about 120 to about 130, about 130 to about 170, about 130 to about 160, about 130 to about 150 about 130 to about 140, about 140 to about 170, about 140 to about 160, about 140 to about 150, about 150 to about 170, about 150 to about 160, or about 160 to about 170 μm in size, e.g., in diameter. In some embodiments, the support is about 10 to about 40 μm in diameter. In some embodiments, the support is about 10 μm in diameter.
Methods of Hypersialylation with Immobilized B4-GalT
[0135] In some embodiments, described herein, inter alia, is a method of preparing hypersialylated (hsIgG), the method comprising: (a) providing a mixture of IgG antibodies, (b) incubating the mixture of IgG antibodies in a reaction mixture comprising β1,4-Galactosyltransferase I (β4GalT1, also called B4GalT) bound to a solid support and UDP-Gal to produce galactosylated IgG antibodies; (c) incubating the galactosylated IgG antibodies in a reaction mixture comprising ST6Gal1 (also called ST6) and CMP-NANA, thereby creating the hsIgG preparation.
[0136] Suitable β4GalT1, e.g., human B4GalT, e.g., human B4Galt1, as well as orthologs, mutants, and variants thereof, including enzymatically active portions of beta-1,4-galactosyltransferase (B4GalT), e.g., human B4GalT, e.g., human B4Galt1, as well as orthologs, mutants, and variants thereof, along with fusion proteins and polypeptides comprising the same, are described herein.
[0137] Suitable ST6Gal1, e.g., human ST6Gal1, as well as orthologs, mutants, and variants thereof, including enzymatically active portions of ST6Gal1, e.g., human ST6Gal1, as well as orthologs, mutants, and variants thereof, along with fusion proteins and polypeptides comprising the same, are described herein.
[0138] In some embodiments, the β4GalT1 is bound to the solid support through at least one affinity tag. Suitable affinity tags and solid supports are described herein.
[0139] In some embodiments, the β1,4-Galactosyltransferase I (β4GalT1) bound to a solid support (e.g. resin, column, array, etc.) is separated from the galactosylated IgG antibodies prior to step (b).
[0140] In some embodiments, GMP-NANA is added 1, 2, 3, or more times during the sialylation reaction.
[0141] In some embodiments, the IgG antibodies comprise IgG antibodies isolated from at least 1000 donors. In some embodiments, at least 70% w/w of the IgG antibodies are IgG1 antibodies. In some embodiments, at least 90% of the donor subjects have been exposed to a virus. In some embodiments, about 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the IgG antibodies in the hsIgG preparation have a sialic acid on both the α1,3 branch and the α1,6 branch. In some embodiments, at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fab domain of the IgG antibodies in the hsIgG preparation have a sialic acid on both the α 1,3 arm and the α 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage; and at least 60%, 65%, 70%, 75%, 80%, or 85% of the branched glycans on the Fc domain of the IgG antibodies in the hsIgG preparation have a sialic acid on both the α 1,3 arm and the α 1,6 arm that is connected through a NeuAc-α 2,6-Gal terminal linkage.
EXAMPLES
Example 1: Hypersialylated IgG Preparation
[0142] IgG in which more than 60% of the overall branched glycans are disialylated can be prepared as follows. An exemplary reaction is shown in
[0143] Briefly, a mixture of IgG antibodies was exposed to a sequential enzymatic reaction using β1,4 galactosyltransferase 1 (B4-GalT or β4GalT1) and α2,6-sialyltransferase (ST6-Gal1) enzymes. The B4-GalT does not need to be removed from the reaction before addition of ST6-Gal1 and no partial or complete purification of the product is needed between the enzymatic reactions. However, multiple purifications steps to remove the enzymes from the hsIgG product typically follow sialylation.
[0144] The galactosyltransferase enzyme selectively adds galactose residues to pre-existing asparagine-linked glycans. The resulting galactosylated glycans serve as substrates to the sialic acid transferase enzyme which selectively adds sialic acid residues to cap the asparagine-linked glycan structures attached to. Thus, the overall sialylation reaction employed two sugar nucleotides (uridine 5′-diphosphogalactose (UDPGal) and cytidine-5′-monophospho-N-acetylneuraminic acid (CMP-NANA)). The latter is replenished periodically to increase disialylated product relative to monosialylated product. The reaction includes the co-factor manganese chloride.
[0145] A representative example of the IgG-Fc glycan profile for such a reaction starting with IVIg and the reaction product is shown in
[0146] The glycan data is shown per IgG subclass. Glycans from IgG3 and IgG4 subclasses cannot be quantified separately. As shown, for IVIg the sum of all the nonsialylated glycans is more than 80% and the sum of all sialylated glycans is <20%. For the reaction product, the sum for all nonsialylated glycans is <20% and the sum for all sialylated glycans is more than 80%. Nomenclature for different glycans listed in the glycoprofile use the Oxford notation for N linked glycans.
Example 2: Hypersialylated IgG Preparation with Immobilized B4-GalT
[0147] IgG in which more than 60% of the overall branched glycans are disialylated can be prepared as follows.
[0148] Briefly, a mixture of IgG antibodies was exposed to a sequential enzymatic reaction using His-tagged β1,4 galactosyltransferase 1 (B4-GalT or β4GalT1) and α2,6-sialyltransferase (ST6-Gal1) enzymes. The B4-GalT was immobilized on a nickel Sepharose resin. No partial or complete purification of the product is needed before the ST6-Gal1 enzymatic reactions.
[0149] Coupling of the His-β4GalT1 to the nickel Sepharose resin occurred by interaction of the chelated nickel molecules by the 8× poly-histidine tag (SEQ ID NO: 30) at the C-terminal end of the β4GalT1. Immobilization was optimized in an aqueous solution suitable for the stability of the enzyme and was shown to be stable for greater than 21 days at 3TC. This stability means that minimal leaching of the enzyme (and therefore Ni) occurred over this time period and that multiple batches of product could be generated from one lot of immobilized enzyme. The amount of immobilized enzyme needed for galactosylation of IVIg was determined by performing protein and enzyme activity assays and quantifying the specific activity, which was 50% to 80% of the soluble enzyme. Galactosylation of IVIg occurred over 72 hours at 37° C. in MOPS buffer at pH 7.4 with UDP-Gal. Constant mixing was carried out using a tube rotator. The immobilized enzyme was filtered away and the extent of galactosylation was characterized and quantified by mass spec methods. The extent of galactosylation was found to be equivalent to that seen for the soluble enzyme and was nearly completely G2F for IgG1, 2, 3, and 4. IgG1 results of 3 separate reactions are shown in
[0150] Thus, about 50%-80% less enzyme used in the galactosylation reaction resulted in similar or more proper glycan structure than soluble enzyme.
Example 2: Immobilization of B4-GalT
[0151] An enzymatically active portion of B4-GalT (SEQ ID NO:38,
Adsorption with Nickel Loaded Beads Achieved Up to 50% Enzyme Activity
[0152] As shown in
[0153] As shown in
Amine Coupling Achieved Up to 17% Enzyme Activity
[0154] As shown in
Epoxy Coupling Reduced 99% Enzyme Activity
[0155] As shown in
OTHER EMBODIMENTS
[0156] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.