Cells for glycosylation engineering and methods of use

Abstract

The present disclosure relates to glycosylation engineering, including cells and methods for glycosylation engineering to engineer recombinant glycoproteins, whereby the glycoproteins produced bind to desired glycans.

Description

Cross Reference to Related Applications

[0001] This application claims priority to U.S. Provisional Patent Application No. 63 / 639,286, filed April 26, 2024. The entire contents of the foregoing application are incorporated herein by reference.

[0002] SEQUENCE LISTING The instant application contains a Sequence Listing, which is filed electronically in.xml format and incorporated herein by reference in its entirety. The.xml copy, created on June 17, 2024, is named “A1000-01700PCT_20240617_SeqListing.xml” and is 77 kilobytes in size. TECHNICAL FIELD

[0003] The present disclosure relates to compositions and methods for glycoprotein engineering and production of glycosylation-engineered therapeutic antibodies. BACKGROUND

[0004] Protein therapeutics have gained increasing attention in almost every medical field over the past few years. A large portion of these protein therapeutics are glycoproteins, and it has been demonstrated that the glycans on the glycoproteins are necessary and determinative for therapeutic efficacy. Therefore, post-translational modifications of these biologies, especially the post-translational modification of their glycosylation status, have drawn increasing attention from the academic and industry fields. With the trend of consistently producing protein therapeutics in mammalian expression platforms, there is a continuing need for platforms that provide the desired glycan profile that favors improved efficacy. SUMMARY

[0005] In one aspect, the present disclosure provides a cell for expressing a sialylated glycoprotein, wherein the cell constitutively and / or controllably expresses an exogenous sialyltransferase catalytic peptide and an exogenous galactosyltransferase catalytic peptide, wherein the exogenous sialyltransferase catalytic peptide and the exogenous galactosyltransferase catalytic peptide are translated in close proximity.

[0006] In one aspect, the present disclosure provides a method for glycosylation engineering of a recombinant glycoprotein, comprising: delivering an expression vector into a cell according to one embodiment of the present disclosure, wherein the expression vector comprises a payload nucleic acid configured to encode the recombinant glycoprotein; and expressing the payload nucleic acid in the cell, thereby obtaining a plurality of recombinant glycoproteins, wherein at least one of the plurality of recombinant glycoproteins is bound to a sialylated glycan.

[0007] In one aspect, the present disclosure provides a plurality of enriched recombinant glycoproteins, wherein at least 50% of the plurality of recombinant glycoproteins are configured to bear a sialylated glycan.

[0008] In one aspect, the present disclosure provides a cell for expressing GlcNAc glycoproteins, which lacks N-acetylglucosaminyltransferase I (GnTI) activity and constitutively or controllably expresses an exogenous endoglycosidase.

[0009] In one aspect, the present disclosure provides a method for glycoengineering recombinant glycoproteins, comprising: delivering an expression vector into a cell according to one embodiment of the present disclosure, wherein the expression vector comprises a payload nucleic acid configured to encode a recombinant glycoprotein; and expressing the payload nucleic acid in the cell, thereby obtaining a plurality of recombinant glycoproteins, wherein at least one of the plurality of recombinant glycoproteins is bound to a GlcNAc glycan.

[0010] In one aspect, the present disclosure provides a plurality of enriched recombinant glycoproteins, wherein at least 50% of the plurality of recombinant glycoproteins are configured to bear a GlcNAc glycan. BRIEF DESCRIPTION OF DRAWINGS

[0011] Figure 1 is a graphical representation of a flow cytometry assay showing exemplary results for SNA staining assays performed using exemplary Adalimumab produced by exemplary CHO cells of the present disclosure (SWG-006). Comparative cells transfected with B4GALT1 and ST6GAL1, configured to be expressed separately, and untransfected cells (i.e., parental cells, as a negative control). In this particular exemplary embodiment, the Adalimumab antibody produced by exemplary cells of the present disclosure (SWG-006) had a 5.44% SNA-positive population. The Adalimumab antibody produced by comparative cells had a 4.58% SNA-positive population. The parental cells expressed 0.022% SNA-positive Adalimumab antibody.

[0012] Figure 2 is a graphical representation of a flow cytometry assay showing results for SNA staining assays performed using Adalimumab produced by exemplary SWG-006 CHO cells of the present disclosure, exemplary SWG-015 cells of the present disclosure, and untransfected cells (i.e., parental cells, as a negative control). The Adalimumab antibody produced by SWG-006 cells of the present disclosure had a 39.8% SNA-positive population. The Adalimumab antibody produced by SWG-015 cells of the present disclosure had a 28.5% SNA-positive population. The parental cells expressed 0.25% SNA-positive Adalimumab antibody.

[0013] Figure 3Ais a graphical representation of LS / MS-MS spectra performed to reveal the glycoforms of the adalimumab antibody produced by the exemplary stable clone SAI-G1 of SWG-006 CHO cells according to the present disclosure. Sialylated glycans are marked with solid arrows. Diamonds represent sialic acid; open circles represent galactose; squares represent GlcNAc; solid circles represent mannose; triangles represent fucose.

[0014] Figure 3B is a graphical representation of LS / MS-MS spectra performed to reveal the glycoforms of the exemplary adalimumab antibody produced by the exemplary stable clone SAI-G1 of SWG-006 CHO cells according to the present disclosure. Sialylated glycans are marked with solid arrows. Diamonds represent sialic acid; open circles represent galactose; squares represent GlcNAc; solid circles represent mannose; triangles represent fucose.

[0015] Figure 3C is a graphical representation of LS / MS-MS spectra performed to reveal the glycoforms of the exemplary adalimumab antibody produced by the exemplary stable clone SAI-D4 of SWG-006 CHO cells according to the present disclosure. Sialylated glycans are marked with solid arrows. Diamonds represent sialic acid; open circles represent galactose; squares represent GlcNAc; solid circles represent mannose; triangles represent fucose.

[0016] Figure 3D is a graphical representation of LS / MS-MS spectra performed to reveal the glycoforms of the exemplary adalimumab antibody produced by the exemplary stable clone SAI-F12 of CHO SWG-006 cells according to the present disclosure. Sialylated glycans are marked with solid arrows. Diamonds represent sialic acid; open circles represent galactose; squares represent GlcNAc; solid circles represent mannose; triangles represent fucose.

[0017] Figure 3E is a graphical representation of LS / MS-MS spectra performed to reveal the glycoforms of the exemplary adalimumab antibody produced by the exemplary stable clone SAI-F12 of CHO SWG-006 cells according to the present disclosure. Sialylated glycans are marked with solid arrows. Diamonds represent sialic acid; open circles represent galactose; squares represent GlcNAc; solid circles represent mannose; triangles represent fucose.

[0018] Figure 4A is a graphical representation of LS / MS-MS spectra performed to reveal the glycoforms of the exemplary adalimumab antibody produced by the exemplary stable clone SAI-F12 of CHO SWG-006 cells according to the present disclosure. Sialylated glycans are marked with solid arrows. Diamonds represent sialic acid; open circles represent galactose; squares represent GlcNAc; solid circles represent mannose; triangles represent fucose. ®Figure 1 is a graph of data from an LS / MS-MS spectrum performed to profile the glycoforms of an adalimumab antibody produced by parental CHO cells. No sialylated glycans were observed. Diamonds represent sialic acid; open circles represent galactose; squares represent GlcNAc; filled circles represent mannose; triangles represent fucose.

[0019] Figure 4B Figure 2 is a graph of data from an LS / MS-MS spectrum performed to profile the glycoforms of an adalimumab antibody produced by the exemplary stable clone SAII-A3 of SWG-006 CHO cells according to the present disclosure. Sialylated glycans are labeled with filled arrows. Diamonds represent sialic acid; open circles represent galactose; squares represent GlcNAc; filled circles represent mannose; triangles represent fucose.

[0020] Figure 4C Figure 3 is a graph of data from an LS / MS-MS spectrum performed to profile the glycoforms of an adalimumab antibody produced by the exemplary stable clone SAII-A3 of SWG-006 CHO cells according to the present disclosure. Sialylated glycans are labeled with filled arrows. Diamonds represent sialic acid; open circles represent galactose; squares represent GlcNAc; filled circles represent mannose; triangles represent fucose.

[0021] Figure 5A Figure 4 is a graph of data from an intact protein mass (IPM) analysis performed to profile the glycoforms of a chMC81370 antibody produced by parental HEK293T cells. No sialylated glycans were observed. Diamonds represent sialic acid; open circles represent galactose; squares represent GlcNAc; filled circles represent mannose; triangles represent fucose.

[0022] Figure 5B Figure 5 is a graph of data from an intact protein mass (IPM) analysis performed to profile the glycoforms of a chMC81370 antibody produced by the exemplary HEK293 cells according to the present disclosure. Sialylated glycans are labeled with filled arrows. Diamonds represent sialic acid; open circles represent galactose; squares represent GlcNAc; filled circles represent mannose; triangles represent fucose.

[0023] Figure 6 Figure 6 is a graph of data from a glycoform analysis performed to study a chMC81370 antibody produced by wild-type cells (i.e., parental cells) and the exemplary HEK293 cells according to the present disclosure (“engineered” cells). Left: percentage of fucosylated glycans; middle: percentage of galactosylated glycans; right: percentage of sialylated glycans.

[0024] Figure 7This is a diagram illustrating data from a complete protein quality (IPM) analysis performed to demonstrate the glycoform of the chMC81370 antibody, produced in exemplary HEK293 cells according to this disclosure in the following manner: untreated (top), treated with Streptococcus pneumoniae α2-3 neuraminic acid sucrase (middle), or treated with Clostridium perfringens neuraminic acid sucrase (bottom). Sialidized glycans are labeled with solid arrows. Rhombuses represent sialic acid; hollow circles represent galactose; squares represent GlcNAc; solid circles represent mannose; and triangles represent fucose.

[0025] Figure 8 This is a graphic representation of complete protein quality (IPM) analysis data performed to visualize the glycoform of the chMC81370 antibody, produced using comparative cells transfected with the hB4GalT1 gene instead of the hST6GAL1 gene. The data show increased galactosylation of the antibody produced by cells with only hB4GalT1 gene knock-in, but no terminal sialylation was observed. Rhombuses represent sialic acid; hollow circles represent galactose; squares represent GlcNAc; solid circles represent mannose; and triangles represent fucose.

[0026] Figure 9A This is a diagram illustrating data from a complete protein quality (IPM) analysis performed to study the changes in glycoform of the chMC81370 antibody produced by exemplary HEK293 cells according to this disclosure over five days. Top: Day 0 to Day 3 (samples collected on Day 3); Bottom: Day 4 to Day 5 (samples collected on Day 5). Sialidized glycans are labeled with solid arrows. Rhombuses represent sialic acid; hollow circles represent galactose; squares represent GlcNAc; solid circles represent mannose; triangles represent fucose.

[0027] Figure 9B This is a graph illustrating data from a complete protein quality (IPM) analysis performed to study the changes in the glycoform of the chMC81370 antibody produced by comparative FUT8 gene knockout cells over five days. Top: Day 0 to Day 3 (samples collected on Day 3); Bottom: Day 4 to Day 5 (samples collected on Day 5). Rhombuses represent sialic acid; hollow circles represent galactose; squares represent GlcNAc; solid circles represent mannose; triangles represent fucose.

[0028] Figure 10This is a graphic representation of data from a complete protein quality (IPM) analysis performed to study the changes in glycoform of the chMC81370 antibody produced by exemplary HEK293 cells according to this disclosure over three days. Samples were collected on day 1 (top), day 2 (middle), and day 3 (bottom). Rhombuses represent sialic acid; hollow circles represent galactose; squares represent GlcNAc; solid circles represent mannose; and triangles represent fucose.

[0029] Figure 11 This is a graphic representation of ELISA data, showing the binding affinity of antibodies produced by wild-type cells (i.e., parental cells) and exemplary HEK293 cells (“SCT-enriched” cells) according to this disclosure for the FcγIIA receptor (left), FcγIIB receptor (middle), and FcγIIIA receptor (right). Antibody binding was measured using anti-human IgG Fc antibodies that bind to HRP.

[0030] Figure 12A The results of protein electrophoresis show that the molecular weights of the chMC18370 antibody expressed by exemplary HEK293 cells (Endo H KI "+") and parental GnTI-KO EXPI293 cells (Endo H LI "-") are different due to observed band shifting.

[0031] Figure 12B The results of the transglycosylation assay are presented, showing that the chMC18370 antibody expressed in exemplary Endo H gene knock-in cells according to this disclosure has high transglycosylation efficiency. Transglycosylation was observed within 15 minutes.

[0032] Figure 12C This is a graphic representation of data from a complete protein quality (IPM) analysis performed to study the glycoform of the chMC81370 antibody produced from parental cells (GnTI-KO EXPI293 cells). Squares represent GlcNAc, and solid circles represent mannose.

[0033] Figure 12D This is a diagram illustrating data from a complete protein quality (IPM) analysis performed to study the glycoform of the chMC81370 antibody generated by exemplary Endo H gene knock-in cells according to this disclosure. Squares represent GlcNAc, and solid circles represent mannose.

[0034] Figure 12EThis is a diagram illustrating data from glycoform analysis performed using the chMC81370 antibody generated below: GnTI-KO EXPI293 cells, exemplary Endo H gene knock-in cells according to this disclosure, and exemplary Endo S2 gene knock-in cells according to this disclosure. Left: Percentage of mannose-type glycans; Right: Percentage of mono-GlcNAc glycans.

[0035] Figure 13A The results of protein electrophoresis show that the molecular weights of the chMC18370 antibody expressed by exemplary HEK293 cells (Endo S2 KI "+") and parental GnTI-KO EXPI293 cells (Endo S2 KI "-") are different due to observed band shifts.

[0036] Figure 13B The results of the transglycosylation assay are presented, showing that the chMC18370 antibody expressed in exemplary Endo S2 gene knock-in cells according to this disclosure has high transglycosylation efficiency. Transglycosylation was observed within 15 minutes.

[0037] Figure 13C This is a diagram illustrating data from a complete protein quality (IPM) analysis performed to study the glycoform of the chMC81370 antibody generated from exemplary Endo S2 gene knock-in cells according to this disclosure. The squares represent GlcNAc.

[0038] Figure 14A This is a graphic representation of ELISA data showing the binding affinity of antibodies generated by exemplary HEK293 cells (“SCT enrichment”) according to this disclosure and exemplary Endo H gene knock-in cells (“SCT homogenization”) that have undergone transglycosylation according to this disclosure.

[0039] Figure 14B This is a graph of data from an ADCC reporter assay, showing a comparison between antibodies generated from: wild-type cells (i.e., parental cells), exemplary HEK293 cells according to this disclosure (“SCT enriched”), and exemplary transglycosylated Endo H knock-in cells according to this disclosure (“SCT homogenized”). Detailed Implementation

[0040] Glycosylation is a post-translational or co-translational modification found on most cellular proteins, especially surface proteins. The influence of the glycan form (i.e., glycoform) of glycoproteins on therapeutic efficacy is recognized. Taking therapeutic antibodies as an example, the binding between antibodies and target cells or pathogens triggers various downstream immune functions, including phagocytosis, cytotoxicity, vaccination, and complement activation. These immune cell-based responses require the binding of the antibody's Fc domain to specific Fc receptors on immune cells, where the antibody's glycoform is considered crucial. For instance, IgG N-glycosylation at N297 on the constant region of the heavy chain is considered critical for the binding of IgG to the FcγⅢA receptor on NK cells, which induces antibody-dependent cytotoxicity (ADCC) activation. Therefore, engineering and obtaining optimized glycoforms to improve the efficacy and safety of therapeutic glycoproteins is important.

[0041] Conventional mammalian cell lines used for glycoprotein production typically produce a mixture of glycoforms and core-fucosylated biantennary complex glycans. These glycoforms are not optimal for binding to Fcγ receptors due to the inhibitory function of core fucosylation or off-target delivery caused by terminal galactosylation. In contrast, α2-6 linked sialic acid complex (SCT) glycans provide enhanced binding to FcγIIIA and FcγIIA receptors, which are associated with ADCC, antibody-dependent phagocytosis (ADCP), and vaccine activity. Furthermore, mono-GlcNAc (N-acetylglucosamine) and GlcNAc-Fuc glycoforms are good receptor substrates for endoglycosidase-mediated transglycosylation and are therefore beneficial for engineering the desired glycan chains. Therefore, efforts must be made to modify the glycoforms.

[0042] cell

[0043] Cells used to express sialylated glycoproteins

[0044] One aspect of this disclosure provides cells for expressing sialylated glycoproteins, said cells constitutively and / or controllably expressing exogenous sialyltransferase catalytic peptides and exogenous galactosyltransferase catalytic peptides, wherein the exogenous sialyltransferase catalytic peptides and exogenous galactosyltransferase catalytic peptides are translated in close proximity. As used herein, "translated in close proximity" describes translation events of the exogenous sialyltransferase catalytic peptide and the exogenous galactosyltransferase catalytic peptide occurring close to each other both temporally and spatially.

[0045] In some embodiments, the exogenous sialyltransferase catalytic peptide and the exogenous galactosyltransferase catalytic peptide are expressed in a single transcript. As used herein, "in a single transcript" means that the transcription of the exogenous sialyltransferase catalytic peptide and the transcription of the exogenous galactosyltransferase catalytic peptide occur transcribed under the same promoter, or that the DNA encoding the exogenous sialyltransferase catalytic peptide and the DNA encoding the exogenous galactosyltransferase catalytic peptide are transcribed into a single mRNA molecule.

[0046] This disclosure covers the translation of an exogenous sialyltransferase-catalyzed peptide and an exogenous galactosyltransferase-catalyzed peptide adjacent to each other or the expression of both peptides in a single transcription, thereby providing higher sialylation for glycoproteins. It is not intended to be bound by any theory, because the catalytic reaction exerted by galactosyltransferase produces galactosylated glycans (which are substrates of sialyltransferase), so translating the two enzymes adjacently or expressing them in a single transcription ensures that the two catalytic reactions occur closely or almost simultaneously, thereby improving sialylation efficiency.

[0047] Ribosome translocation method. In some embodiments, the cell of this disclosure comprises a first nucleic acid configured to express an exogenous sialyltransferase catalytic peptide; and a second nucleic acid configured to express an exogenous galactosyltransferase catalytic peptide, wherein the first and second nucleic acids are controlled under the same promoter. In some embodiments, the first and second nucleic acids are linked to each other by a linker nucleic acid configured to encode a ribosome translocation peptide. The ribosome translocation peptide is configured to exhibit ribosome skipping, wherein the ribosome translocation peptide prevents ribosome covalently linking of newly inserted amino acids while continuing translation, thereby polymerizing proteins for co-translational cleavage into independent peptides / proteins. In some embodiments, the ribosome translocation peptide comprises the amino acid sequence DxExNPGP, where x represents any amino acid, D represents aspartic acid, E represents glutamic acid, N represents asparagine, P represents proline, and G represents glycine. In some embodiments, the ribosome translocation peptide comprises the amino acid sequence set forth in SEQ ID NO: 06, SEQ ID NO: 07, SEQ ID NO: 08, or SEQ ID NO: 09. In some embodiments, the ligation nucleic acid comprises the sequence set forth in SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21 or SEQ ID NO: 22.

[0048] Fusion Protein Approach. Alternatively, in some embodiments, exogenous sialyltransferase catalytic peptides and exogenous galactosyltransferase catalytic peptides are configured to be expressed as fusion proteins. In some embodiments, the fusion protein comprises a first portion having a sialyltransferase catalytic peptide and a second portion having a galactosyltransferase catalytic peptide, wherein the first and second portions are linked to each other by a cleavable linker, and the cleavable linker is configured to be cleavable post-translationally of the fusion protein, thereby releasing the sialyltransferase catalytic peptide and the galactosyltransferase catalytic peptide as separate proteins upon cleavage.

[0049] In some embodiments, the sialyltransferase catalytic peptide is α-2,6-sialyltransferase. In some embodiments, the sialyltransferase is β-galactosyl α-2,6-sialyltransferase 1. In some embodiments, the sialyltransferase catalytic peptide comprises the product of the ST6Ga11 gene or the PspST gene. In some embodiments, the sialyltransferase catalytic peptide comprises the amino acid sequence as set forth in SEQ ID NO: 01 or SEQ ID NO: 02. However, in some embodiments, the sialyltransferase catalytic peptide comprises the amino acid sequence set forth in SEQ ID NO: 03 or SEQ ID NO: 04.

[0050] In some embodiments, the galactosyltransferase catalytic peptide is β-1,4-galactosyltransferase 1. In some embodiments, the galactosyltransferase catalytic peptide comprises the product of the B4GALT1 gene. In some embodiments, the galactosyltransferase catalytic peptide comprises the amino acid sequence set forth in SEQ ID NO: 05.

[0051] In some embodiments, the cell contains a first nucleic acid configured to express an exogenous sialyltransferase catalytic peptide, wherein the first nucleic acid is derived from the ST6Ga11 gene or the PspST gene. As used herein, “derived from” describes that the first nucleic acid contains a nucleotide sequence identical to that of the reference gene; for example, the first nucleic acid may contain the nucleotide sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11. In some other cases, “derived from” describes that the first nucleic acid contains a nucleotide sequence modified according to the reference gene, provided that the product of the modified nucleotide sequence performs the same or similar catalytic function as the product of the reference gene. Modifications may be made to suit codon usage or to optimize transcription / translation efficiency or accuracy in the host cell. In other instances, a signal peptide is added to the modification, which guides the gene product to a location inside or outside the cell. For example, the first nucleic acid may be derived from the PspST gene, wherein the first nucleic acid contains a nucleotide sequence encoding the product of the PspST gene and a nucleotide sequence encoding a signal peptide that guides the gene product to the glycosylated Golgi body. In some embodiments, the signal peptide may be the signal peptide of the ST6Ga11 gene or the B4GALT1 gene. In the embodiments described, the first nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13.

[0052] In some embodiments, the cell contains a second nucleic acid configured to express an exogenous galactosyltransferase catalytic peptide, wherein the second nucleic acid is derived from the B4GALT1 gene. In some embodiments, the second nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 14.

[0053] In some embodiments, the first and second nucleic acids are transcribed under the same promoter, which can be a constituent promoter or an activatable promoter. Constituent promoters can be, but are not limited to, CMV promoters, T7 promoters, human elongation factor 1α (EF1α) promoters, chicken β-actin (CAG) promoters, or SV40 promoters. Activatable promoters provide controlled expression of exogenous sialyltransferases and exogenous galactosyltransferases. Activatable promoters can be, but are not limited to, tetracycline-inducible promoters (which can be activated by deoxycycline) or dihydrofolate reductase (DHFR) gene promoters (for selecting and amplifying gene expression).

[0054] Parental Cells. In some embodiments, the cells of this disclosure are derived from parental cells (e.g., wild-type cells), which may be mammalian cells. In some embodiments, the parental cells do not possess endogenous sialyltransferases and / or endogenous galactosyltransferases. In some other embodiments, the parental cells may possess endogenous sialyltransferases and / or endogenous galactosyltransferases. However, this disclosure has unexpectedly found that overexpression of endogenous sialyltransferases and / or endogenous galactosyltransferases does not increase sialylation as much as expression of exogenous sialyltransferases and / or exogenous galactosyltransferases. In some embodiments, the parental cells and / or the cells of this disclosure lack fucosyltransferase 8 activity. In some embodiments, the parental cells and / or the cells of this disclosure lack the FUT8 gene encoding the product of fucosyltransferase activity. In some embodiments, the parental cells and / or the cells disclosed herein are or are derived from Chinese hamster ovary (CHO) cells or HEK293 cells. CHO cells may be, but are not limited to, ExpiCHO, CHO-K1, CHO-C, CHOZN, and CHOK1Q. In some embodiments, the term "cell" as used herein may be used interchangeably with "cell line."

[0055] Cells for expressing recombinant glycoproteins. In some embodiments, the cells also contain a payload nucleic acid configured to encode the recombinant glycoprotein, and the expression of the payload nucleic acid is transcribed under the control of a constitutive or activatable promoter. Constitutive and activatable promoters are as described above and herein. In some embodiments, the recombinant glycoprotein expressed or produced by the cells is bound to a glycan. In some embodiments, the glycan may be an N-linked glycan (i.e., an N-glycan) or an O-linked glycan (i.e., an O-glycan).

[0056] Glycans and glycoproteins. The cells of this disclosure are capable of expressing sialylated glycoproteins. In some embodiments, the sialylated glycoproteins comprise sialyl complex (SCT) glycans that can bind to proteins via lysine residues (i.e., O-glycans) or asparagine residues (i.e., N-glycans). In some embodiments, the cells of this disclosure are configured to express SCT-enriched proteins. As used herein, “SCT-enriched” describes a higher percentage of SCT glycans in proteins expressed by the cells of this disclosure compared to the same proteins expressed by parental cells used to generate the cells of this disclosure. In some embodiments, the percentage of SCT glycans is 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200%, 300%, 400%, 500%, or any range defined by the foregoing endpoints, such as 10% to 500%, 10% to 400%, 10% to 300%, 10% to 200%, 10% to 100%, 1 0% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 30% to 500%, 30% to 400%, 30% to 300%, 30% to 200%, 30% to 100%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, or 30% to 40%. In some embodiments, the SCT glycan percentage is enriched to 1.2, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times the SCT glycan percentage of the protein expressed by the parent cell, or any range defined by the aforementioned endpoints, such as 1.2 to 10 times, 1.2 to 9 times, 1.2 to 8 times, 1.2 to 7 times, 1.2 to 6 times, 1.2 to 5 times, 1.2 to 4 times, 1.2 to 3 times, 1.2 to 2 times, 2 to 10 times, 2 to 9 times, 2 to 8 times, 2 to 7 times, 2 to 6 times, 2 to 5 times, 2 to 4 times, 2 to 3 times, 5 to 10 times, 5 to 9 times, 5 to 8 times, 5 to 7 times, or 5 to 6 times. In some embodiments, the glycan is a single-antennae or double-antennae α2-6 sialic acid complex (SCT) glycan. In some embodiments, the glycan is a galactose-rich SCT glycan with or without a core fucose. In some other embodiments, the glycan is a fully galactosylated SCT glycan with or without a core fucose.

[0057] In some embodiments, the glycoprotein (e.g., a recombinant glycoprotein) is an antibody or its antigen-binding fragment. The basic antibody unit is a heterotetraglycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. Each L chain is connected to the H chain by at least one (and usually one) covalent disulfide bond, while the two H chains are connected to each other by one or more disulfide bonds, depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VN) at its N-terminus. H ), followed by the three constant fields (C) of each α-chain and γ-chain. H ) and the four Cs of μ-isotype and ε-isotype. H Domain. Each L-chain has a variable domain (V) at its N-end. L ), followed by the constant domain (C) at its other end. L V L With V H Align and C L With the first constant field of the heavy chain (C) H1 Alignment. It is assumed that specific amino acid residues form an interface between the light chain variable domain and the heavy chain variable domain. V H With V L They pair up to form a single antigen-binding site.

[0058] L-chains from any vertebrate species can be based on their constant domain (C L Based on the amino acid sequence, they are classified into one of two distinct types (called Kappa (κ) and Lambda (λ)). The heavy chain of immunoglobulins (C...) H Immunoglobulins can be classified into different classes or isotypes, depending on the amino acid sequence of their constant domains. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, each with a heavy chain named α (alpha), δ (delta), ε (epsilon), γ (gamma), and μ (mu), respectively. Based on relatively minor differences in CH sequence and function, the γ and α classes are further subdivided into subclasses, for example, humans express the following subclasses: IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. It should be understood that mammals encoding multiple Ig isotypes will be able to perform isotype switching.

[0059] An "antibody fragment" or "antigen-binding fragment of an antibody" is a polypeptide or polypeptide comprising a portion of a complete antibody, preferably the antigen-binding region or variable region of the complete antibody. Examples of antibody fragments include F(ab')2 fragments, Fv fragments, single-chain Fv (ScFv) antibodies, bifunctional antibodies, microantibodies, and nanobodies (V... HH) and linear antibodies (see U.S. Patent No. 5,641,870; Zapata et al., Protein Engineering 8(10): 1057-1062

[1995] ); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0060] Antibody digestion with papain produces two identical antigen-binding fragments, called the "Fab" fragment and the residual "Fe" fragment, names reflecting their ease of crystallization. The Fab fragment consists of the intact L chain and the variable region (V) of the H chain. H ) and the first constant field of a heavy chain (C H1 The F(ab')2 antibody fragment is composed of two disulfide-linked Fab fragments, each of which is monovalent in terms of antigen binding, i.e., it has a single antigen-binding site. Treatment of the antibody with pepsin produces a single large F(ab')2 fragment, which roughly corresponds to two disulfide-linked Fab fragments with bivalent antigen-binding activity and is still capable of cross-linking the antigen. Both Fab and F(ab')2 are examples of "antigen-binding fragments". The Fab' fragment differs from the Fab fragment in that it has a few additional residues at the carboxyl terminus of the CH1 domain, which includes one or more cysteine ​​residues from the hinge region of the antibody. Fab'-SH is the name given in this paper to Fab' fragments with free thiol groups on the cysteine ​​residues of the constant domain. The F(ab')2 antibody fragment is initially produced as a pair of Fab' fragments with hinge cysteine ​​residues between them. Other chemical couplings of antibody fragments are also known.

[0061] The “Fc” fragment contains the carboxyl terminus of two H chains held together by disulfide bonds (i.e., the C of IgG). H2 and C H3 The effector function of an antibody is determined by the sequence in the Fc region. The Fc domain is a portion of the antibody recognized by a cellular receptor (e.g., FcR) and binds to the complement-activating protein C1q. As discussed herein, the Fc domain can be modified (e.g., by amino acid substitution) to modify (e.g., improve, reduce, or eliminate) one or more functions of an Fc-containing polypeptide (e.g., the antibody of this disclosure).

[0062] "Fv" is the smallest antibody fragment containing both a complete antigen recognition and binding site. This fragment consists of a dimer of a tightly bound, non-covalently bound heavy chain variable region and a light chain variable region. Folding from these two domains produces six hypervariable rings (three from the H chain and three from the L chain), which provide the amino acid residues for antigen binding and give the antibody antigen-binding specificity. However, even a single variable domain (or half of an Fv containing only the three CDRs specific to the antigen) can recognize and bind to the antigen, but typically with lower affinity than the entire binding site.

[0063] "Single-chain Fv" is also abbreviated as "sFv" or "scFv," and is an antibody fragment containing VH and VL antibody domains linked to a single polypeptide chain. Preferably, the sFv polypeptide also contains a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, The Pharmacology of Monoclonal Antibodies, Vol. 113, edited by Rosenburg and Moore, Springer-Verlag, New York, pp. 269-315 (1994); Borrebaeck 1995, see below.

[0064] The term "bifunctional antibody" refers to a small antibody fragment prepared by means of: constructing a V H sFv fragments (see previous paragraph) with short linkers (approximately 5-10 residues) between the VL domain and the sFv domain enable interchain rather than intrachain pairing of the V domain, resulting in bivalent fragments, i.e., fragments with two antigen-binding sites. Bispecific bifunctional antibodies are heterodimers of two “crossover” sFv fragments, where the V domains of the two antibodies are linked together. H and V L Domains exist on different polypeptide chains. Bifunctional antibodies are more fully described, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al., Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci. USA), 90:6444-6448 (1993). Other antibody fragments and molecules containing them include, for example, linear antibodies, tandem scFv, scFv-Fc, tandem scFv-Fc, scFv dimers, scFv-zippers, bifunctional antibody-Fc, and bifunctional antibody-C. H3 The following are also covered in this article: sc bifunctional antibody, sc bifunctional antibody-Fc, sc bifunctional antibody-CH3, nanobody, TandAbs, micro antibody, small antibody, trifunctional antibody, tetrafunctional antibody, scFab, Fab-scFv, Fab-scFv-Fc, scFv-CH-CL-scFv and F(ab')2-scFv2.

[0065] In embodiments where the glycoprotein is an antibody or its antigen-binding fragment, the glycan may be located on the heavy chain or the Fc region. It is noteworthy that the glycosylation sites of antibodies that regulate antibody function can vary depending on the subtype. For example, the preBCR complex is crucial for B cell development and is tightly regulated by N-glycan at N46 on the µHC. N-glycan at N402 on the µHC is associated with antibody oligomerization and complement activation. Furthermore, IgG N-glycosylation at N297 on the γHC plays a key role in complement activation and Fcγ receptor activation, thereby producing various effector functions. Therefore, in some embodiments, the glycan may be located at N46, N402, and / or N297 of the heavy chain.

[0066] In some embodiments, the glycoprotein is adalimumab (Humira). ® ), Adalimumab-atto (Amjevita) ® ), Rituximab ® ), Rituximab-atto (Truximab) ® Cetuximab (Erbitux) ® ), Bevacizumab (Avastin) ® ), Infliximab (Remicade ® Trastuzumab (Herceptin) ® Pembrolizumab (Keytruda) ® Etanercept (Enbrel) ® Ipilimumab (Yervoy) ® Ofatumumab (Arzerra), Golimumab (Simponi) ® Atezolizumab (Tecentriq) ® Ocrelizumab (OCREVUS) ® Durvalumab ® Avelumab (Bavencio) ® Nivolumab (Opdivo) ®Pertuzumab (Perjeta) ® Obinutuzumab (Gazyva) ® ), Gazyvaro Infliximab (Remicade ® ) or trastuzumab emtansine (Kadcyla) ® ).

[0067] In some embodiments, the glycoprotein is an immunogenic protein, such as a viral envelope protein or a viral spike protein. For example, an immunogenic protein may be, but is not limited to, influenza hemagglutinin or the SARS-CoV-2 spike protein.

[0068] Cells used to express GlcNAc glycoprotein

[0069] One aspect of this disclosure provides cells for expressing GlcNAc glycoproteins, wherein the cells lack N-acetylglucosamine aminotransferase I (GnTI) activity and constitutively or controllably express exogenous endoglucosidases. As described herein, “GlcNAc glycoprotein” describes a glycan bound to a glycoprotein that is primarily composed of GlcNAc. In some embodiments, the GlcNAc glycan may be a mono-GlcNAc glycan or a GlcNAc-Fuc glycan. In some embodiments, the glycoprotein is as described above and herein.

[0070] The exogenous endonuclease may be, but is not limited to, endonuclease H (Endo H) or endonuclease S2 (Endo S2). In some embodiments, the exogenous endonuclease comprises the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16. In some embodiments, the cell comprises a nucleic acid configured to encode the exogenous endonuclease, wherein the nucleic acid may comprise the nucleotide sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18. The nucleic acid may be controlled under a constitutive or activatable promoter. A constitutive promoter may be, but is not limited to, the CMV promoter, the T7 promoter, the EF1A promoter, the CAG promoter, or the SV40 promoter. An activatable promoter provides controlled expression of exogenous sialyltransferase and exogenous galactosyltransferase. An activatable promoter may be, but is not limited to, a tetracycline-inducible promoter (which can be activated by deoxyhydroxytetracycline) or a dihydrofolate reductase (DHFR) gene promoter (for selecting and amplifying gene expression).

[0071] As described in this article, "lack of N-acetylglucosamine transferase I (GnTI) activity" means that the cell lacks functional N-acetylglucosamine transferase I, which can be caused by gene knockout of the endogenous gene encoding N-acetylglucosamine transferase I or mutations at genes that result in missense, nonsense, or frameshift silencing. Without being bound by any theory, cells lacking GnTI activity produce mannose-rich glycans, which are preferentially cleaved by endoglycosidases, thereby producing GlcNAc glycoproteins.

[0072] Parental Cells. In some embodiments, the cells of this disclosure are derived from parental cells, which may be mammalian cells. In some embodiments, the parental cells lack N-acetylglucosamine aminotransferase I (GnTI) activity. In some other embodiments, the parental cells do not lack acetylglucosamine aminotransferase I (GnTI) activity, while the cells of this disclosure are genetically engineered to lack GnTI activity. In some embodiments, the parental cells and / or the cells of this disclosure lack fucosyltransferase 8 activity. In some embodiments, the parental cells and / or the cells of this disclosure lack the FUT8 gene encoding the product of fucosyltransferase activity. In some embodiments, the parental cells and / or the cells of this disclosure are or are derived from Chinese hamster ovary (CHO) cells or HEK293 cells. CHO cells may be, but are not limited to, ExpiCHO, CHO-K1, CHO-C, CHOZN, and CHOK1Q, and HEK293 cells may be Expi293F™ GnTI KO.

[0073] Cells for expressing recombinant glycoproteins. In some embodiments, the cells further comprise a payload nucleic acid configured to encode the recombinant glycoprotein, and the expression of the payload nucleic acid is controlled by a constituent or activatable promoter. Constituent and activatable promoters are as described above and herein. In some embodiments, the recombinant glycoprotein expressed or produced by the cells is bound to a glycan. In some embodiments, the glycan may be an N-linked glycan.

[0074] The production of cells disclosed in this paper

[0075] The generation of cells according to one embodiment of this disclosure requires engineering parental cells to express exogenous enzymes. In some embodiments, the generation of cells requires engineering parental cells to insert and / or knock out relevant genes. In some embodiments, the engineering can be performed using transfection or gene editing methods, which can be stable or temporary.

[0076] transfection

[0077] Transfection can be performed using any conventional method, taking into account high transfection efficiency, minimal cytotoxicity, low or no significant impact on normal physiology, and ease of operation.

[0078] In some embodiments, transfection can be performed using a virus-mediated method. Virus-mediated methods use viral vectors to introduce nucleic acids configured to encode the desired gene product into host cells. Some commonly used viral vectors include (but are not limited to) adenovirus, adeno-associated virus, retroviral murine leukemia virus, herpes simplex virus, vaccinia virus, and Sindbe virus. Virus-mediated methods are highly efficient due to the infectious nature of viral particles. However, a disadvantage of these methods lies in the issues of immunogenicity and cytotoxicity. However, various virus-mediated methods have been thoroughly studied and developed to mitigate these problems. Furthermore, in some embodiments, the cells of this disclosure are used to produce the desired glycoprotein and are not directly transfected into patients or human individuals during their use. Therefore, immunogenicity and cytotoxicity issues are less important to this disclosure.

[0079] In some embodiments, transfection can be performed using chemical methods, such as cationic polymers, calcium phosphate, cationic lipids, or cationic amino acids. Specific examples of chemical methods include, but are not limited to, the use of DEAE-polydextrose, polyethyleneimine, dendritic polymers, agglutinating amines, calcium phosphate, liposomes, DOTAP, lipody amines, CTAB / DOPE, or DOTMA. The underlying principle of these methods is the preparation of nucleic acid / chemical complexes using positively charged chemicals with negatively charged nucleic acids. It is assumed that the nucleic acid / chemical complexes are attracted to the negatively charged cell membrane and eventually cross the cell membrane via endocytosis, phagocytosis, or both. The efficiency of these chemical methods can be affected by several factors, including the nucleic acid / chemical ratio, the pH of the environment, and cell membrane conditions.

[0080] In some embodiments, transfection can be performed using physical methods, such as direct microinjection, gene gun particle delivery, electroporation, laser-based transfection, ultrasound transfection, and magnetic transfection. These methods may be applicable, but are generally technically demanding and labor-intensive.

[0081] Genome editing

[0082] In some embodiments, engineering modifications can be performed using genome editing. Genome editing methods can be non-targeted gene editing, such as Sleeping Beauty transposons or I-SceI-mediated transposons; or targeted gene editing, such as using transcription activator-like effector nucleases (TALENs) or clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-related proteins. Gene editing integrates exogenous nucleic acids into the genome of the host cell (parent cell).

[0083] Targeted gene editing methods induce DNA double-strand breaks (DSBs) at target loci, allowing for the alteration of the function of the relevant gene (insertion of a foreign gene or silencing of an endogenous gene). The TALEN method requires the use of chimeric molecules consisting of a transcription activator-like effector (TALE) domain and a FokI nuclease catalytic domain. The TALE domain contains a DNA-binding motif that can be programmed to target specific loci in the host cell's genome. Typically, a pair of chimeric molecules, each targeting the forward and reverse directions respectively, are required. Once the TALE domain binds to the target loci in both directions, the FokI nuclease catalytic domains of the two chimeric molecule pairs dimerize, thereby activating the nuclease's catalytic activity to cleave DNA and produce DSBs. The CRISPR method involves using a complex of a Cas protein and a guide RNA. The guide RNA contains a backbone sequence for Cas binding and a spacer (approximately 20 nucleotides) with a complementary sequence to a specific locus in the host cell's genome. Typically, the spacer must have a unique sequence specific to the target locus to prevent off-target binding, and the target locus is followed by a protospacer adjacent motif (PAM) site necessary for Cas protein recognition. Once the guide RNA / Cas protein complex binds to the target site, Cas nuclease activity is activated, and DNA is cleaved to produce DCB.

[0084] Induced DSB can be repaired through three different repair mechanisms, including homology-directed repair (HDR), non-homologous end joining (NHEJ), and microhomologous end joining (MMEJ). During the repair mechanism, deletion or frameshift can lead to gene silencing, or by using donor vectors containing microhomologous sequences homologous to the target locus, the repair mechanism can effectively induce the targeted integration of exogenous genes into the genome.

[0085] Methods for glycosylation engineering of recombinant glycoproteins and the recombinant glycoproteins obtained therefrom

[0086] One aspect of this disclosure provides a method for glycosylation engineering of recombinant glycoproteins. The method includes delivering an expression vector into a cell according to an embodiment of this disclosure, wherein the expression vector comprises a payload nucleic acid configured to encode a recombinant glycoprotein; and expressing the payload nucleic acid in the cell to obtain a plurality of recombinant glycoproteins, wherein at least one of the plurality of recombinant glycoproteins binds to a desired glycan.

[0087] Glycoproteins can be the glycoproteins described herein. In some embodiments, the glycoprotein is a therapeutic antibody or an antigen-binding fragment thereof. In embodiments where the glycoprotein is an antibody or an antigen-binding fragment thereof, the glycan may be located on the heavy chain or the Fc region. In some embodiments, the glycan is located at a glycosylation site of the antibody. In some embodiments, the glycan may be located at N46, N402, and / or N297 of the heavy chain.

[0088] As used herein, “glycosylation engineering” refers to the use of cells to produce recombinant glycoproteins, whereby the recombinant glycoproteins carry or bind to a desired glycoform or glycan. In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of the recombinant glycoprotein carries the desired glycoform (e.g., sialylated glycans or GlcNAc glycans), or an integer ranging from any of the two numbers above, or any range defined by the foregoing endpoints, such as 50 to 100%, 50 to 99%, 50 to 95%, 50 to 90%, 50 to 85%, 50 to 80%, 50 to 75%, 50 to 70%, 50 to 65%. %, 50 to 60%, 50 to 55%, 60 to 100%, 60 to 99%, 60 to 95%, 60 to 90%, 60 to 85%, 60 to 80%, 60 to 75%, 60 to 70%, 60 to 65%, 70 to 100%, 70 to 99%, 70 to 95%, 70 to 90%, 70 to 85%, 70 to 80%, 70 to 75%, 80 to 100%, 80 to 99%, 80 to 95%, 80 to 90%, 80 to 85%, 90 to 100%, 90 to 99%, or 90 to 95%.

[0089] In some embodiments, multiple recombinant glycoproteins are homogeneously bound to a desired glycoform (e.g., sialylated glycans or GlcNAc glycans) with the following properties: at least 1%, 2%, 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%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 42%, 43%, 44%, 45%, 46%, 4 7%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range defined by the aforementioned endpoints.For example, 1% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 1% to 10%, 5% to 100%, 5% to 95%, 5% to 85%, 5% to 75%, 5% to 65%, 5% to 55%, 5% to 45%, 5% to 35%, 5% to 25%, 5% to 10%, 10% to 100%, 10% to 99%, 10% to 95%, 10% to 90%, 10% to 85%, 10% to 80%, 10% to 75%, 10% to 70%, 10% to 65%, 10% to 60%, 10% to 55%, 10 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, 10% to 15%, 20% to 100%, 20% to 99%, 20% to 95%, 20% to 90%, 20% to 85%, 20% to 80%, 20% to 75%, 20% to 70%, 20% to 65%, 20% to 60%, 20% to 55%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, 20% to 25%, 40% to 100%, 40% to 99%, 40% to 95%, 40% to 90%, 40% to 85%, 40% to 80%, 40% to 75%, 40% to 70%, 40% to 65%, 40% to 60%, 40% to 55%, 40% to 50%, 60% to 100%, 60% to 99%, 60% to 95%, 60% to 90%, 60% to 85%, 60% to 80%, 60% to 75%, 50% to 100%, 50% to 99%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, 55% to 100%, 55% to 99%, 55% to 98%, 55% to 97%, 55% to 96%, 55% to 95%, 55% to 94%, 55% to 93%, 55% to 92%, 55% to 91%, 55% to 90%, 55% to 85%, 55% to 80%, 55% to 75%, 55% to 70%, 55% to 65%, 55% to 60%, 70% to 100%, 70% to 99%, 70% to 95%, 70% to 90%, 70% to 85%, 70% to 80%, 70% to 75%, 80% to 100%, 80% to 99%, 80% to 95%, 80% to 90%, 80% to 85%, 90% to 100%, 90% to 99%, or 90% to 95%, including or excluding any of the foregoing figures. All figures may be modified by “about” as defined herein.

[0090] In some embodiments, the cells are cells according to the present disclosure described above for expressing sialylated glycoproteins. In some embodiments, the sialylated glycoprotein comprises sialyl complex (SCT) glycans. In some embodiments, the methods of the present disclosure are configured to express SCT-enriched proteins as defined herein. In some other embodiments, the cells are cells according to the present disclosure described above for expressing GlcNAc glycoproteins, as defined herein.

[0091] In some embodiments, the method further includes collecting recombinant glycoproteins within 200, 180, 150, 120, 100, 80, 50, 30, 20, 10, 5, or 1 hour after the start of expression of the payload nucleic acid, or an integer ranging between any two of the aforementioned numbers, or any range defined by the aforementioned endpoints, such as 1 to 200 hours, 1 to 180 hours, 1 to 150 hours, 1 to 120 hours, 1 to 100 hours, 1 to 80 hours, 1 to 50 hours, 1 to 30 hours, 1 to 20 hours, 1 to 10 hours, 1 to 5 hours, 5 to 200 hours, 5 to 180 hours, 5 to 150 hours, 5 to 120 hours, 5 to 100 hours, 5 to 80 hours, 5 to 50 hours, 5 to 30 hours, 5 to 20 hours, or 5 to 10 hours. In some embodiments, the recombinant glycoprotein is collected within 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 day after the start of expression of the payload nucleic acid, or an integer ranging from any two of the aforementioned numbers, or any range defined by the aforementioned endpoints, such as 1 to 10 days, 1 to 9 days, 1 to 8 days, 1 to 7 days, 1 to 6 days, 1 to 5 days, 1 to 4 days, 1 to 3 days, or 1 to 2 days. As described herein, “after the start of expression of the payload nucleic acid” means the time point at which the expression vector is delivered to the cell, or the time point at which the promoter controlling the expression of the payload nucleic acid is activated after the expression vector is delivered to the cell.

[0092] In some embodiments, the expression of the payload nucleic acid is constitutive or controllable. In some embodiments, the expression of the payload nucleic acid is controlled by a constitutive promoter or an activatable promoter. Constitutive promoters may be, but are not limited to, CMV promoters, T7 promoters, EF1A promoters, CAG promoters, or SV40 promoters. Activatable promoters provide controllable expression of exogenous sialyltransferases and exogenous galactosyltransferases. Activatable promoters may be, but are not limited to, tetracycline-inducible promoters (which can be activated by deoxyhydroxytetracycline) or dihydrofolate reductase (DHFR) gene promoters (for selecting and amplifying gene expression).

[0093] In some embodiments, the expression vector can be delivered by genetic engineering, which can be performed using transfection or gene editing methods, and can be stable or transient. The transfection and gene editing methods can be those described herein with respect to the cells that produce this disclosure.

[0094] Another aspect of this disclosure provides a variety of recombinant glycoproteins, wherein at least 50% of the various recombinant glycoproteins are configured to carry a desired glycan, which may be a sialylated glycan or a GlcNAc glycan. Recombinant glycoproteins may be described herein. In some embodiments, the various recombinant glycoproteins are obtained by using methods for glycosylation engineering of the recombinant glycoproteins of this disclosure.

[0095] In some embodiments, the multiple recombinant glycoproteins are multiple enriched recombinant glycoproteins. The term "enriched" describes a higher or more concentrated percentage of desired glycans in the multiple recombinant glycoproteins of this disclosure compared to the same protein expressed by parental cells or wild-type cells used to produce the cells of this disclosure, or a higher or more concentrated percentage of desired glycans in the multiple recombinant glycoproteins of this disclosure compared to the desired percentage of desired glycans in a variety of naturally occurring proteins having the same or similar amino acid sequences. In some embodiments, the desired glycan percentage is enriched to at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 250%, 300%, 350%, 400%, 450%, or 500%, or an integer ranging between any two of the foregoing numbers, or any range defined by the foregoing endpoints, such as 10% to 500%, 10% to 400%, 10% to 300%, or 10%. Up to 200%, 10% to 100%, 10% to 90%, 10% to 80%, 10% to 70%, 10% to 60%, 10% to 50%, 10% to 40%, 10% to 30%, 10% to 20%, 30% to 500%, 30% to 400%, 30% to 300%, 30% to 200%, 30% to 100%, 30% to 90%, 30% to 80%, 30% to 70%, 30% to 60%, 30% to 50%, or 30% to 40%, including or excluding any of the foregoing figures. All figures may be modified by “about” as defined herein. In some embodiments, the desired glycan percentage is enriched to 1.2, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 times the percentage of SCT glycans of the protein expressed by the parent cell, or an integer ranging between any two of the aforementioned numbers, or any range defined by the aforementioned endpoints, such as 1.2 to 10 times, 1.2 to 9 times, 1.2 to 8 times, 1.2 to 7 times, 1.2 to 6 times, 1.2 to 5 times, 1.2 to 4 times, 1.2 to 3 times, 1.2 to 2 times, 2 to 10 times, 2 to 9 times, 2 to 8 times, 2 to 7 times, 2 to 6 times, 2 to 5 times, 2 to 4 times, 2 to 3 times, 5 to 10 times, 5 to 9 times, 5 to 8 times, 5 to 7 times, or 5 to 6 times, including or excluding any of the aforementioned numbers. All numbers may be modified by “about” as defined in this article.

[0096] In some embodiments, the desired percentage of glycan enrichment is at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95%, or an integer between any two of the aforementioned numbers, or any range defined by the aforementioned endpoints, such as 51% to 95%, 51% to 94%, 5 ... 1% to 93%, 51% to 92%, 51% to 91%, 51% to 90%, 51% to 85%, 51% to 80%, 51% to 75%, 51% to 70%, 51% to 65%, 51% to 60%, 55% to 95%, 55% to 90%, 55% to 85%, 55% to 80%, 55% to 75%, 55% to 70%, 55% to 65%, 55% to 60%, 60% to 95%, 60% to 90%, 60% to 85%, 60% to 80%, 60% to 75%, 60% to 70%, 60% to 65%, 70% to 95%, 70% to 90%, 70% to 85%, 70% to 80%, or 70% to 75%, including or excluding any of the foregoing figures. All figures may be modified by “about” as defined herein.

[0097] In some embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99%, or 100% of a plurality of recombinant glycoproteins carry the desired glycotype (e.g., sialylated glycans or GlcNAc glycans), or an integer ranging from any of the two numbers above, or any range defined by the aforementioned endpoints, such as 50 to 100%, 50 to 99%, 50 to 95%, 50 to 90%, 50 to 85%, 50 to 80%, 50 to 75%, 50 to 70%, 50 to 6 ... 5%, 50 to 60%, 50 to 55%, 60 to 100%, 60 to 99%, 60 to 95%, 60 to 90%, 60 to 85%, 60 to 80%, 60 to 75%, 60 to 70%, 60 to 65%, 70 to 100%, 70 to 99%, 70 to 95%, 70 to 90%, 70 to 85%, 70 to 80%, 70 to 75%, 80 to 100%, 80 to 99%, 80 to 95%, 80 to 90%, 80 to 85%, 90 to 100%, 90 to 99%, or 90 to 95%. All numbers may be modified by “about” as defined herein.

[0098] In some embodiments, multiple recombinant glycoproteins are homogeneously bound to sialylated glycans in the following proportions: at least 1%, 2%, 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%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 4 9%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range defined by the aforementioned endpoints.For example, 1% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 1% to 10%, 5% to 100%, 5% to 95%, 5% to 85%, 5% to 75%, 5% to 65%, 5% to 55%, 5% to 45%, 5% to 35%, 5% to 25%, 5% to 10%. 10% to 100%, 10% to 99%, 10% to 95%, 10% to 90%, 10% to 85%, 10% to 80%, 10% to 75%, 10% to 70%, 10% to 65%, 10% to 60%, 10% to 55%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20% 10% to 15%, 20% to 100%, 20% to 99%, 20% to 95%, 20% to 90%, 20% to 85%, 20% to 80%, 20% to 75%, 20% to 70%, 20% to 65%, 20% to 60%, 20% to 55%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, 20% to 25% 40% to 100%, 40% to 99%, 40% to 95%, 40% to 90%, 40% to 85%, 40% to 80%, 40% to 75%, 40% to 70%, 40% to 65%, 40% to 60%, 40% to 55%, 40% to 50%, 60% to 100%, 60% to 99%, 60% to 95%, 60% to 90%, 60% to 85%, 60% to 80%, 60% to 75% 50% to 100%, 50% to 99%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, 55% to 100%, 55% to 99%, 55% to 98%, 55% to 97%, 55% to 96%, 55% to 95%, 55% to 94%, 55% to 93%, 55% to 92%, 55% to 91%, 55% to 90%, 55% to 85% 55% to 80%, 55% to 75%, 55% to 70%, 55% to 65%, 55% to 60%, 70% to 100%, 70% to 99%, 70% to 95%, 70% to 90%, 70% to 85%, 70% to 80%, 70% to 75%, 80% to 100%, 80% to 99%, 80% to 95%, 80% to 90%, 80% to 85%, 90% to 100%, 90% to 99%, or 90% to 95%, including or excluding any of the foregoing figures. All figures may be modified by “about” as defined herein.

[0099] In embodiments where the cells are those of the present disclosure for expressing GlcNAc glycoproteins as described above, the method of the present disclosure may further include a transglycosylation step. The transglycosylation step may include collecting multiple recombinant glycoproteins expressed in the foregoing steps of the method and culturing the multiple recombinant glycoproteins together with an endoglucosidase and a donor glycan, thereby binding the donor glycan to at least one of the multiple recombinant glycoproteins (i.e., the transglycosylation reaction).

[0100] In some embodiments, the donor glycan comprises an oxazoline moiety configured to react with an endoglycosidase, thereby binding the donor glycan to at least one of a plurality of recombinant glycoproteins. In some embodiments, the donor glycan is a sialyl complex (SCT) glycan. In some embodiments, the endoglycosidase is endoglycosidase H (Endo H), endoglycosidase S2 (Endo S2), or a derivative thereof. In some embodiments, the derivative of Endo S2 comprises an Endo S2 D184 mutant, including but not limited to the D184M mutant or the D184Q mutant.

[0101] In some embodiments, the transglycosylation step produces a variety of recombinant glycoproteins that bind to donor glycans in the following homogeneous manner: at least 1%, 2%, 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%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 42%, 43%, 44%, 45%, 46%, 47%, 4 8%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, or any range defined by the aforementioned endpoints.For example, 1% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 1% to 10%, 5% to 100%, 5% to 95%, 5% to 85%, 5% to 75%, 5% to 65%, 5% to 55%, 5% to 45%, 5% to 35%, 5% to 25%, 5% to 10%, 10% to 100%, 10% to 99%, 10% to 95%, 10% to 90%, 10% to 85%, 10% to 80%, 10% to 75%, 10% to 70%, 10% to 65%, 10% to 60%, 10% to 55%, 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30 ... 0%, 10% to 25%, 10% to 20%, 10% to 15%, 20% to 100%, 20% to 99%, 20% to 95%, 20% to 90%, 20% to 85%, 20% to 80%, 20% to 75%, 20% to 70%, 20% to 65%, 20% to 60%, 20% to 55%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 20% to 30%, 20% to 25%, 40% to 100%, 40% to 99%, 40% to 95%, 40% to 90%, 40% to 85%, 40% to 80%, 40% to 75%, 40% Up to 70%, 40% to 65%, 40% to 60%, 40% to 55%, 40% to 50%, 60% to 100%, 60% to 99%, 60% to 95%, 60% to 90%, 60% to 85%, 60% to 80%, 60% to 75%, 50% to 100%, 50% to 99%, 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, 55% to 100%, 55% to 99%, 55% to 98%, 55% to 97%, 55% to 96%, 55% to 95%. 55% to 94%, 55% to 93%, 55% to 92%, 55% to 91%, 55% to 90%, 55% to 85%, 55% to 80%, 55% to 75%, 55% to 70%, 55% to 65%, 55% to 60%, 70% to 100%, 70% to 99%, 70% to 95%, 70% to 90%, 70% to 85%, 70% to 80%, 70% to 75%, 80% to 100%, 80% to 99%, 80% to 95%, 80% to 90%, 80% to 85%, 90% to 100%, 90% to 99%, or 90% to 95%, including or excluding any of the foregoing figures. All figures may be modified by “about” as defined herein.

[0102] definition

[0103] Unless otherwise expressly defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Unless otherwise stated, the techniques employed or covered herein are standard methods well-known to one of ordinary skill in the art. Unless otherwise instructed, the practice of this disclosure will employ conventional techniques of microbiology, tissue culture, molecular biology, chemistry, biochemistry, and recombinant DNA technology, within the skill of the art. Materials, methods, and examples are illustrative and non-limiting only. The following is presented in an illustrative manner and is not intended to limit the scope of this disclosure.

[0104] The figures used to describe and claim certain embodiments of this disclosure, representing the amount and characteristics of components (e.g., homogeneity, enrichment, molecular weight, reaction conditions, and results, etc.), should be understood to be modified by the term "about" in some cases. Those skilled in the art will understand the meaning of the term "about" in its defined value. The numerical values ​​presented in some embodiments of this disclosure may contain some error arising from the standard deviation in their corresponding test measurements. For example, as used herein, the term "about" refers to a measurable value, such as an amount, duration, etc., and is intended to cover variations of ±5%, ±1%, or ±0.1% from a specified value, provided such variations are appropriate.

[0105] As used herein, “substantially” means sufficient to achieve the intended purpose. Therefore, the term “substantially” allows for small, insignificant variations in absolute or perfect states, dimensions, measurements, results, etc., that would be expected by someone skilled in the art in a manner that would not significantly affect overall performance. When used with respect to numerical values ​​or parameters or characteristics expressed as numerical values, “substantially” means within ten percent.

[0106] As used herein, “treat,” “treatment,” and “treating” refer to a method for achieving a beneficial or desired outcome, such as a clinical outcome. For the purposes of this disclosure, a beneficial or desired outcome may include inhibiting or suppressing the onset or progression of an infection or disease; alleviating symptoms of an infection or disease or reducing its development; or a combination thereof.

[0107] As used herein, “preventing” and “prevention” are used interchangeably with “prophylaxis” and can mean complete prevention of infection or prevention of the development of symptoms of said infection, delaying the onset of disease or its symptoms; or reducing the severity of subsequently developed infection or its symptoms.

[0108] As used herein, the term "polysaccharide" refers to a polysaccharide, oligosaccharide, or monosaccharide. Polysaccharides can be monomers or polymers of sugar residues and can be linear or branched. Polysaccharides can include natural sugar residues (e.g., glucose, N-acetylglucosamine, N-acetylglucosamine, galactose, mannose, fucose, hexose, arabinose, ribose, xylose, etc.) and / or modified sugars (e.g., 2'-fluororibose, 2'-deoxyribose, mannose phosphate, 6'-sulfonate N-acetylglucosamine, etc.).

[0109] As used herein, “recombinant glycoprotein” or “recombinant protein” is a protein produced by introducing engineered nucleic acids into a host organism (such as bacteria, yeast, or mammalian cells) using laboratory or industrial methods, and in some cases, a protein isolated or purified for clinical or industrial use.

[0110] Example

[0111] Example 1: Exemplary CHO cells of this disclosure for expressing sialylated glycoproteins

[0112] This experiment used parental CHO cells to create exemplary cells according to one embodiment of this disclosure. ExpiCHO cells (Thermo Fisher Scientific) were purchased and ExpiCHO™ expression medium was maintained according to the product manual. Subsequently, the cells were transferred using the ExpiFectamine™ CHO transfection kit with the SWG-006 plasmid (i.e., the expression vector) according to the manufacturer's instructions. The SWG-006 plasmid (SEQ ID NO: 23) was prepared from pcDNA™ 3.1 plasmid and constructed using the CMV enhancer, CMV promoter, ST6GAL1 gene (SEQ ID NO: 10), P2A sequence (SEQ ID NO: 19), and B4GALT1 gene (SEQ ID NO: 14). Alternatively, cells were co-transfected with ST6GAL1 plasmid (SEQ ID NO: 25) and B4GALT1 plasmid (SEQ ID NO: 26) to construct cells that express the ST6GAL1 gene and the B4GALT1 gene respectively under the CMV promoter.

[0113] Subsequently, cells were transfected with the adalimumab plasmid (pcDNA2 TADA, SEQ ID NO: 27) to express adalimumab (Humira) using the ExpiFectamine™ CHO transfection kit. Three days post-transfection, transfected cells (transient clones) were washed with staining buffer (EDTA 1 mM, NaN 30.02%, BSA 1%, in PBS) and stained with biotinylated SNA (VectorLabs), which was designed to bind sialic acid linked to N-acetylgalactosamine or galactose. After washing, cells were stained with Streptoavidin-Alexa647 (BioLegend) and analyzed by flow cytometry (BD LSRFortessa).

[0114] result( Figure 1 The results showed that transfection with the SWG-006 plasmid or co-transfection with the ST6GAL1 and B4GALT1 plasmids promoted α2,6-sialylation. In contrast, parental CHO cells (ExpiCHO cells) lack endogenous α2,6-sialyl transferases, and therefore no signal was detected from untransfected cells. Most importantly, the results also showed that transfection with the SWG-006 plasmid provided significantly higher α2,6-sialylation than co-transfection with the ST6GAL1 and B4GALT1 plasmids.

[0115] Similarly, another exemplary cell type according to one embodiment of this disclosure was obtained by transfecting ExpiCHO cells (Thermo Fisher Scientific) with the SWG-015 plasmid (SEQ ID NO: 24) using the ExpiFectamine™ CHO transfection kit. The SWG-015 vector was prepared from the pcDNA™ 3.1 plasmid and constructed with the CMV enhancer, CMV promoter, modified PspST gene (SEQ ID NO: 12), P2A sequence (SEQ ID NO: 19), and B4GALT1 gene (SEQ ID NO: 14). Cells were also transfected with the adalimumab plasmid (pcDNA2 TADA, SEQ ID NO: 27) to express adalimumab (Humira). Subsequently, sialylation of adalimumab produced by the cells (which are also transient clones) was observed using SNA staining as described above. Cells transfected with the SWG-006 vector were also examined for comparison with cells transfected with the SWG-015 vector. The results showed that cells transfected with SWG-015 provided similar levels of sialylation as cells transfected with SWG-006 vector. Figure 2 ).

[0116] Example 2: Stabilization of exemplary cells of this disclosure

[0117] Plasmid SWG-006 was linearized by digestion with PvuI followed by purification. Linearized SWG-006 was transfected into EXPICHO cells using the ExpiFectamine™ CHO Transfection Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. On day 2 post-transfection, G418 was added at 400 μg / ml for drug selection. After recovery to 90% viability, cells were stained with biotinylated SNA followed by streptavidin-BV421, where SNA binds to endogenous membrane proteins produced by the cells. Cells were then sorted for high SNA binding into single cells cultured in 96-well plates using a WOLF G2 cell sorter (Nanocellect Biomedical, Inc.). After a period of culture, single clones were stained with biotinylated SNA followed by streptavidin-APC and analyzed by flow cytometry to select clones with high surface α2,6-sialylation. The selected cells (>99% SNA positive) were then transfected with the adalimumab expression plasmid (SEQ ID NO: 27) to overexpress adalimumab. The expressed adalimumab was purified using protein A beads and analyzed using SNA hybridization blot to select clones expressing highly α2,6-sialylated adalimumab. The glycoform of adalimumab was further analyzed by LC / MS-MS.

[0118] LC / MS-MS data showed that all four stable clones tested in this case—SAII-A3, SAI-GI, SAI-D4, and SAI-F12—expressed a high percentage of sialylation for adalimumab. The stable clone SAII-A3 showed 70.9% (approximately 70% to 72%) sialylation of its expressed adalimumab. Figure 3A Stable clone SAI-G1 showed 55.39% (approximately 55% to 57%) sialylation of adalimumab expression. Figure 3B Stable clone SAI-D4 showed 16.8% (approximately 16% to 18%) sialylation of its expressed adalimumab. Figure 3C Furthermore, the stable clone SAI-F12 showed 36.14% (approximately 36% to 38%) sialylation of its expressed adalimumab. Figure 3D In contrast, adalimumab expressed by parental cells did not show significant sialylation. Figure 3E ).

[0119] Example 3: Glycosylation engineering of adalimumab using exemplary stable cells according to this disclosure

[0120] In this experiment, according to the manufacturer's instructions, the stable clone (SAII-A3) of SWG-006 cells obtained as described in Example 2 above was transfected with the ExpiFectamine™ CHO transfection kit (Thermo Fisher Scientific) using the adalimumab expression vector (SEQ ID NO: 27) to express adalimumab, and its glycoform was observed and compared with the same antibody expressed by parental cells (ExpiCHO cells).

[0121] Simply put, dilute the cells to 5 × 10⁶ cells in a flask preheated to 37°C with 15 mL of fresh ExpiCHO™ expression medium. 6 A final density of viable cells / mL was achieved. The flask was then gently swirled. Cells were then maintained in a humidified atmosphere at 37°C with air containing 8% CO2 on a 125 rpm orbital shaker prior to transfection. The plasmid DNA (i.e., the expression vector of SEQ ID NO: 27, 12 μg) was diluted with 0.6 mL of cold OptiPRO™ medium, and the ExpiFectamine™ CHO reagent (48 μL) was diluted with 552 μL of cold OptiPRO™ medium. The expanded DNA was then mixed with the diluted ExpiFectamine™ CHO reagent to obtain the ExpiFectamine™ CHO / plasmid DNA complex. The ExpiFectamine™ CHO / plasmid DNA complex was added to cell cultures in a humidified atmosphere at 37°C with air containing 8% CO2 on a 125 rpm orbital shaker. After transfection, 90 μl of ExpiFectamine™ CHO enhancer and 3.6 mL of ExpiCHO™ feed volume were added to the cells. The cells in the flasks were then kept in a humidified atmosphere at 37°C with 8% CO2 while shaking. Eight to ten days post-transfection, the supernatant from the cell culture was collected for protein purification.

[0122] Subsequently, antibodies produced by SAII-A3 cells and parental EXPICHO cells were isolated using Protein A beads (GE HealthCare). Simply put, 15 mL of supernatant was passed through a 200 μL Protein A column. The column was washed with 10 column volumes (CV) of PBS. The bound antibody was eluted with 15 CV of 0.1 M glycine at pH=3. Antibodies were dialyzed against 10 mM ammonium acetate. The antibody was then reduced with 25 mM DTT and analyzed by LC / MS-MS. Commercial adalimumab (Humira) was purchased. ®And it was used as a control for analysis using LC / MS-MS.

[0123] LC / MS-MS data showed that adalimumab produced via exemplary cells of this disclosure possesses a variety of sialylated glycans (see [link to LC / MS data]). Figure 4C Sialized glycans, labeled with solid arrows, include G2S2F, G2S1F, G1S1F, N3H6F1S1, N3H4S1, N3H5F1S1, N4H6F1S1, and N4H6F1S2. The sialylation percentage of the total glycans is 70.9%, and the sialylation percentage of the biantennary complex glycans is 79.8%. In comparison, although commercial products ( Figure 4A ) and adalimumab produced via parental ExpiCHO cells ( Figure 4B It also carries glycans, but no sialylation was observed.

[0124] Example 4: Demonstrative HEK293 cells of this disclosure for expressing sialylated glycoproteins

[0125] This experiment used CRISPR technology to create exemplary cells according to one embodiment of this disclosure from parental HEK293T cells. The results were remarkably similar to those described in Example 1 for CHO cells using the ExpiFectamine™ CHO transfection kit.

[0126] Cells, Plasmids, and Transfection. Parental HEK293T cells (ATCC) were cultured in a cell culture incubator at 37°C and 5% CO2 in DMEM (Thermo Fisher Scientific) supplied with 10% FBS (Thermo Fisher Scientific). Plasmids for CRISPR / Cas9 expression were constructed as recommended in the kit manual (Thermo Fisher Scientific). In short, an empirically proven sgRNA sequence targeting the glycosyltransferase was synthesized (Integrated DNA Technologies) and cloned into a vector for Cas9 and sgRNA expression. A synthetic codon-optimized gene insert (Integrated DNA Technologies) flanked by the homologous arm of the target gene at the sgRNA target site was cloned into an empty vector as a donor plasmid. Transfection of 293T cells was mediated via TransIT-293 (Mirus Bio) following the kit manual. The plasmid used to express the antibody chMC81370 (a humanized antibody targeting stage-specific embryonic antigen 4 (SSEA4)) was transfected into cells, which were then cultured. At harvest, the culture medium was collected and subsequently subjected to a protein A agarose bead (GE Healthcare) column to purify the antibody.

[0127] Analysis. The purified antibody was analyzed using intact protein quality (IPM) analysis. Simply put, samples were diluted to concentrations between 5 and 10 µM with LCMS-grade water and analyzed by 6230 TOF LC / MS using dual AJS ESI ion sources (Agilent Technologies) and a PLRP-S 1000 Å 5 µm column (Agilent Technologies). Solvent A was H₂O containing 0.1% formic acid, and solvent B was CAN containing 0.1% formic acid. Results showed that the antibody produced by parental HEK293T cells (… Figure 5A Compared to ), galactosylated and sialylated antibody glycoforms were significantly increased. Figure 5B The results of IPM glycoform analysis are presented in a bar chart, showing a significant increase in antibody galactosylation and sialylation, but a lack of core fucosylation. Figure 6 ).

[0128] Characterizing sialylation. To confirm that antibody sialylation is mediated by hST6Gal1, antibodies were treated overnight at 37°C with two different sialicases, namely Streptococcus pneumoniae α2-3 neuraminidase and Clostridium perfringens neuraminidase, followed by purification or gel electrophoresis. Intact protein analysis did not show any significant difference in the distribution of asialylated and sialylated antibodies between the untreated group and the α2-3 neuraminidase-treated group. Figure 7 The upper and middle sections; marked with solid arrows for sialylated glycoforms), indicate that the sialylation of the antibody is not attributed to endogenous α2-3 sialyltransferases. On the other hand, treatment with Clostridium perfringens neuraminidase, which removes all sialylation, largely eliminates terminal sialyl ( Figure 7 (Bottom). Furthermore, antibodies with increased galactosylation produced from cells with only hB4GalT1 gene knock-in did not show terminal sialylation ( Figure 8 These results confirm that antibody sialylation is caused by hST6Gal1, and that increased hB4Gal1 expression actually enhances antibody galactosylation (Figure 5).

[0129] Sialyltransferase hST6Gal1 is known to be the only human glycosyltransferase mediating α2-6 sialylation, and it preferentially uses the α1-3 branch arms of biantennary glycans as substrates. Some in vitro studies have shown that glycosylation engineering of antibody glycans via hST6Gal1 is time-consuming and difficult to produce fully sialylated antibody glycans. Furthermore, this could explain why only antibodies with at least one fully galactosylated glycan are sialylated. If the galactose on the G1 glycan is not located on the α1-3 branch arm, then hST6Gal1 may not sialyze it. Therefore, in this case, the expression level of the glycosyltransferase may not be the bottleneck. Instead, substrate preference and enzyme activity are the main factors. Using α2-6 sialyltransferases from other organisms can yield similar or better activities than hST6Gal1, as demonstrated by other examples using the PspST gene described in this paper.

[0130] Example 5: Time factor in glycosylation engineering of exemplary cells using the present disclosure

[0131] This experiment tested whether antibody expression levels and timing in the demonstrative cells used in this disclosure affected glycosylation. The culture time was extended to 5 days, and culture media were collected on days 3 and 5, respectively. The produced antibodies were examined using intact protein quality (IPM) analysis. Results showed that sialylation was relatively strong in the first collection (first three days). Figure 9A (D0-3), and subsequently sialylation decreased in the second collection (last two days), accompanied by an increase in terminal galactosylation ( Figure 9A (D4-5) indicates that proteins involved in glycosylation may not be sufficient to effectively generate Fc-SCT antibodies in the later stages.

[0132] Because hST6Gal1 and hB4GalT1, which are involved in the production of FC-SCT antibodies, are almost artificially and constitutively expressed in cells, we also examined whether endogenous glycosyltransferases faced the same problem of reduced glycosylation. By expressing the antibody in FUT8 KO cells, the antibody was almost entirely terminally glycosylated with at least one or more galactoses in the first collection. Figure 9B (D0-3), but the proportion of galactosylated antibodies decreased, and even antibody glycans without any galactose became the dominant glycoform in the last two days ( Figure 9B (D4-5). The results showed that dynamic glycosylation levels after antibody expression could also be observed on endogenous glycosylated products.

[0133] The aforementioned findings indicate that antibody production at different time points can affect glycosylation in different ways. Therefore, in the next experiment, antibodies were purified daily after plasmid transfection, and the dynamic changes in antibody glycans from multiple engineered cells were analyzed. Antibodies with complete sialylation could be detected by intact protein quality analysis on day one, and subsequently decreased gradually over time. Figure 10 Regarding galactosylation, the antibody-glycan is capped by at least two types of galactose (a total of 2 to 4 types) on the first day, but this capping begins on the second day, as evidenced by the presence of a single galactose on the antibody-glycan. Figure 10 These observations suggest that antibody glycosylation is almost complete at the onset of protein expression and gradually decreases over time.

[0134] Example 6: Binding between sialylated antibodies and Fcγ receptors

[0135] FcγRIIA (Fc gamma RIIA) and FcγRIIB (Fc gamma RIIB) are typically expressed simultaneously by antigen-presenting cells such as dendritic cells and macrophages, and these two receptors work together to regulate immune responses. FcγRIIA is crucial for NK cell-mediated ADCC3. This experiment tested whether Fc-SCT-enriched antibodies generated using exemplary cells of this disclosure exhibited better binding affinity than broad-spectrum antibodies (i.e., antibodies generated by parental cells). Fc-SCT-enriched antibodies were generated and purified as described in Example 4. Binding affinity was examined using an ELISA assay.

[0136] ELISA. Recombinant soluble FcγIIA, FcγIIB, and FcγRIIIA (Fc gamma RIIIA, R&D) were coated at 50 ng / well in bicarbonate / carbonate coating buffer (50 mM, pH 10) overnight at 4 °C, followed by blocking the wells overnight at 4 °C with TPBS containing 5% BSA (0.05% Tween 20 in PBS). Antibodies were added to the wells, starting at a final concentration of 100 μg / ml, and serially diluted five times and incubated at room temperature for 1 hour, followed by incubation at room temperature for another 1 hour with HRP-bound goat anti-human IgG antibody (Jackson ImmunoResearch). Finally, TMB substrate (Bethyl Laboratories) was added to react with HRP at room temperature, and the reaction was stopped by H2SO4. The absorbance at 450 nm was measured using a SpectraMax M5 spectrometer (MolecularDevice). The wells were washed 3–5 times with TPBS between each step.

[0137] The results showed that in all three tested Fc receptors, the binding of Fc-SCT-enriched antibodies to FcR was increased. Figure 11 Among these receptors, the increased binding of FcγRIIA and FcγRIIIA is consistent with previous studies. Interestingly, WT antibodies showed extremely weak binding to the inhibitory receptor FcγRIIB. Although the improvement was not as significant as that for FcγRIIA and FcγRIIIA, the cellular glycosylation engineering of this disclosure improved antibody binding to receptor FcγRIIB. Although the increased binding appears to conflict with the binding of the two relatively functional receptors FcγRIIA and FcγRIIB, which are usually co-expressed on antigen-presenting cells (APCs), the final immune response was determined by the expression levels and signaling strength of FcγRIIA and FcγRIIB on effector cells. Antibodies enriched in Fc-SCT also exhibited stronger FcγRIIIA binding capacity than antibodies produced from wild-type cells. Figure 11 Not wanting to be bound by theory, this enhancement was also facilitated by removing core fucose 11 and adding galactose.

[0138] Example 7: Exemplary HEK293 cells for expressing GlcNAc glycoprotein of this disclosure

[0139] This experiment used CRISPR technology to create an exemplary cell according to an embodiment of this disclosure from parental GnT1 KO ExpiHEK293F cells. The parental cells were prepared as described in Example 4. The resulting cell line expressed a high-mannose N-glycan that was preferentially cleaved by Endo H to produce an Fc-GlcNAc antibody. Therefore, this experiment aimed to knock-in Endo H (UniProtKB / Swiss-Prot: P04067.1, see SEQ ID NO: 15 and SEQ ID NO: 17) into this cell line via CRISPR-Cas9. Plasmids for CRISPR technology were prepared as described below.

[0140] Plasmids and Transfection. Plasmids for CRISPR / Cas9 expression were constructed as recommended in the kit manual (Thermo Fisher Scientific). Simply put, an empirically proven sgRNA sequence targeting the glycosyltransferase (Edtec) was synthesized and cloned into a vector for Cas9 and sgRNA expression. A synthetic codon-optimized gene insert (Edtec) was side-linked with the homologous arm of the target gene to the sgRNA target site and cloned into an empty vector as a donor plasmid. Transfection of 293T cells was mediated using the Expi293Fectamine™ Transfection Kit (Thermo Fisher Scientific), following the kit manual. The plasmid for expressing the antibody chMC81370 was transfected into the cells, followed by incubation. At harvest, the culture medium was collected and subsequently treated with a Protein A Agarose Beads (GE Healthcare) column to purify the antibody.

[0141] Analysis. Antibodies expressed by the cells were analyzed by electrophoresis. Proteins were heated at 95°C for 5 min in LDS sample buffer supplied with 2-mercaptoethanol and then subjected to gel electrophoresis with 12% SDS-PAGE. Proteins on the gel were stained with Coomassie blue buffer (ApexBio). The results showed that the antibody heavy chain (AbHC) expressed by these cells had a faster migration rate than AbHC from its parent cells (GnT1 KO), and most of the AbHC migrated downwards. Figure 12A ).

[0142] The antibody was also subjected to a transglycosylation assay. The antibody, along with SCT glycan-oxazoline and the Endo S2 mutant, was incubated in Tris buffer at 37°C for a specified time, followed by collection for purification or gel electrophoresis. Transglycosylation yielded an antibody with homogeneous glycan (in this experiment, homogeneous SCT glycan). The transglycosylation efficiency was quite good. Figure 12B The glycans on these antibodies were then analyzed by intact protein quality (IPM). The results showed that the majority of the antibodies (over 95%) contained GlcNAc only on each HC. Figure 12C , Figure 12D and Figure 12E ).

[0143] Instead of embedding Endo H, an endoglucosidase S2 from *Streptococcus pyogene* (Endo S2; UniProtKB / Swiss-Prot: T1WGN1.1, see SEQ ID NO: 16 and SEQ ID NO: 18) was embedded, as this enzyme can hydrolyze both high-mannose and complex N-glycans into Fc-GlcNAc. The donor vector and CRISPR-Cas9 plasmid were delivered to GnT1 KO cells. The antibody-glycans produced by these cells were then examined as previously described. Protein gels showed a significant downward shift of the AbHC band compared to its parent cells (GNT1 KO). Figure 13A In addition, polysaccharides can be added to AbHC via enzymatic transglycosylation. Figure 13B Furthermore, complete protein quality analysis showed a clear signal from the Fc-GlcNAc antibody. Figure 13C Most antibodies (over 90%) contain GlcNAc only on each HC. Figure 12E ).

[0144] Based on these results, it is concluded that both Endo H and Endo S2 can be introduced into cells via CRISPR-Cas9 to process N-glycans into Fc-GlcNAc antibodies for in vitro glycosylation. Furthermore, this cell-based approach can be used for other glycoproteins, such as influenza hemagglutinin and the SARS-CoV-2 spike protein, to generate single-GlcNAc modified glycoforms for vaccines, thereby inducing a broad-spectrum protective immune response.

[0145] Next, since the Fc-SCT-enriched antibody (i.e., the antibody obtained in Example 4) exhibits increased binding to the FcγIIIA receptor, the binding affinity of the homogeneous Fc-SCT antibody obtained above via transglycosylation to the FcγIIIA receptor was compared with that of the Fc-SCT-enriched antibody. The results showed that the Fc-SCT-enriched antibody exhibited similar binding affinity to the homogeneous SCT antibody. Figure 14A ).

[0146] In addition, ADCC reporter assays were performed. Simply put, the assays were performed according to the kit manual. SK-OV3 target cells expressing SSEA4 were seeded in 96-well plates, followed by the addition of antibody at a final concentration of 1 g / ml, serially diluted five times. Subsequently, effector cells expressing FcγRIIIA (effector:target cell ratio, 6:1) were added and incubated with the target cells at 37°C for 6 hours. The plates were then placed at room temperature for 15 minutes, followed by the addition of luciferase substrate. After 5 minutes of incubation, cold light was measured using a SpectraMax M5 spectrometer. The fold induction was calculated using RLU (inducing background) / RLU (antibody-free control background). It was observed that at the lowest antibody concentration used for induction, the WT antibody did not induce any significant cell activation, but homogenized Fc-SCT and Fc-SCT-enriched antibodies induced cell activation, reaching a maximum rapidly at the next lower concentration. Figure 14B Even at higher concentrations, WT antibodies can achieve maximum induction, but their activity is still far lower than that of homogeneous Fc-SCT or Fc-SCT-enriched antibodies. Figure 14B ).

[0147] Exemplary Examples

[0148] Example 1: A cell for expressing sialylated glycoproteins, wherein the cell constitutively and / or controllably expresses an exogenous sialyltransferase catalytic peptide and an exogenous galactosyltransferase catalytic peptide, wherein the exogenous sialyltransferase catalytic peptide and the exogenous galactosyltransferase catalytic peptide are translated very closely.

[0149] Example 2: Cells according to Example 1, wherein the exogenous sialyltransferase catalytic peptide and the exogenous galactosyltransferase catalytic peptide are expressed in a single transcript.

[0150] Example 3: A cell according to Example 1 or Example 2, comprising a first nucleic acid configured to express the exogenous sialyltransferase catalytic peptide and a second nucleic acid configured to express the exogenous galactosyltransferase catalytic peptide, wherein the first nucleic acid and the second nucleic acid are transcribed under the same promoter.

[0151] Example 4: The cell according to Example 3, wherein the first nucleic acid and the second nucleic acid are linked to each other by a linker nucleic acid configured to encode a ribosomal transpeptide.

[0152] Example 5: The cell according to Example 4, wherein the ribosome translocation peptide comprises the amino acid sequence DxExNPGP, where x represents any amino acid, D represents aspartic acid, E represents glutamic acid, N represents asparagine, P represents proline, and G represents glycine.

[0153] Example 6: The cell according to Example 4 or Example 5, wherein the ribosome translocation peptide comprises the amino acid sequence set forth in SEQ ID NO: 06, SEQ ID NO: 07, SEQ ID NO: 08 or SEQ ID NO: 09.

[0154] Example 7: The cell according to Example 3, wherein the exogenous sialyltransferase catalytic peptide and the exogenous galactosyltransferase catalytic peptide are co-configured to be expressed as a fusion protein.

[0155] Example 8: The cell according to Example 7, wherein the fusion protein comprises a first portion having a sialyltransferase catalytic peptide domain and a second portion having a galactosyltransferase catalytic peptide domain, wherein the first portion and the second portion are connected to each other by a cleavable linker, wherein the cleavable linker is configured to be cleavable after translation of the fusion protein, thereby releasing the sialyltransferase catalytic peptide and the galactosyltransferase catalytic peptide as separate proteins upon cleavage.

[0156] Example 9: Cells according to any one of Examples 3 to 8, wherein the first nucleic acid is derived from the ST6Ga11 gene or the PspST gene.

[0157] Example 10: A cell according to any one of Examples 3 to 9, wherein the first nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 10 or SEQ ID NO: 11.

[0158] Example 11: The cell according to Example 10, wherein the first nucleic acid comprises the nucleotide sequence described in SEQ ID NO: 12 or SEQ ID NO: 13.

[0159] Example 12: A cell according to any one of Examples 3 to 11, wherein the second nucleic acid is derived from the B4GALT1 gene.

[0160] Example 13: A cell according to any one of Examples 3 to 12, wherein the second nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 14.

[0161] Example 14: A cell according to any one of Examples 3 to 13, wherein the promoter is a constituent promoter or an activatable promoter.

[0162] Example 15: The cell according to Example 14, wherein the constituent promoter is the CMV promoter, T7 promoter, human elongation factor 1α (EF1α) promoter, chicken β-actin (CAG) promoter or SV40 promoter.

[0163] Example 16: The cell according to Example 14, wherein the activatable promoter is a tetracycline-inducible promoter (which can be activated by deoxytetracycline) or a dihydrofolate reductase (DHFR) gene promoter.

[0164] Example 17: Cells according to any one of Examples 1 to 16, wherein the sialyltransferase catalytic peptide is α-2,6-sialyltransferase.

[0165] Example 18: Cells according to Example 17, wherein the sialyl transferase is β-galactoside α-2,6-sialyl transferase 1.

[0166] Example 19: A cell according to any one of Examples 1 to 18, wherein the sialyltransferase catalytic peptide comprises the amino acid sequence set forth in SEQ ID NO: 01 or SEQ ID NO: 02.

[0167] Example 20: The cell according to Example 19, wherein the sialyl transferase catalytic peptide comprises the amino acid sequence set forth in SEQ ID NO: 03 or SEQ ID NO: 04.

[0168] Example 21: Cells according to any one of Examples 1 to 20, wherein the galactosyltransferase catalyzing peptide is β-1,4-galactosyltransferase 1.

[0169] Example 22: The cell according to Example 21, wherein the galactosyltransferase catalytic peptide comprises the amino acid sequence described in SEQ ID NO: 05.

[0170] Example 23: Cells according to any one of Examples 1 to 22, wherein the cells lack fucosylation activity.

[0171] Example 24: Cells according to Example 23, wherein the cells lack fucosyltransferase 8 activity.

[0172] Example 25: Cells according to Example 23 or Example 24, wherein the cells lack the FUT8 gene encoding the product of the fucosylation activity.

[0173] Example 26: A cell according to any one of Examples 1 to 25, wherein the cell is a mammalian cell.

[0174] Example 27: Cells according to Example 26, wherein the cells are derived from Chinese hamster ovary cells or HEK293 cells.

[0175] Example 28: A cell according to any one of Examples 1 to 27, wherein the cell further comprises a payload nucleic acid configured to encode a recombinant glycoprotein, and the expression of the payload nucleic acid is transcribed under the control of a constituent promoter or an activatable promoter.

[0176] Example 29: Cells according to Example 28, wherein the recombinant glycoprotein is an antibody or its antigen-binding fragment.

[0177] Example 30: Cells according to Example 29, wherein the antibody is a therapeutic antibody.

[0178] Example 31: A method for glycosylation engineering of recombinant glycoproteins, comprising: delivering an expression vector into a cell according to any one of Examples 1 to 27, wherein the expression vector comprises a payload nucleic acid configured to encode the recombinant glycoprotein; and expressing the payload nucleic acid in the cell to obtain a plurality of recombinant glycoproteins, wherein at least one of the plurality of recombinant glycoproteins is bound to sialylated glycans.

[0179] Example 32: The method according to Example 31, wherein the sialylated polysaccharide is an α2-6 sialyl complex (SCT) polysaccharide.

[0180] Example 33: The method according to Example 32, wherein the α2-6 sialic acid complex (SCT) polysaccharide is a single-antennae or a double-antennae.

[0181] Example 34: The method according to Example 31 or Example 32, wherein the sialylated polysaccharide does not contain core fucose.

[0182] Example 35: The method according to any one of Examples 31 to 34, wherein the expression of the payload nucleic acid is constitutive or controllable.

[0183] Example 36: The method according to any one of Examples 31 to 35, wherein at least 50% of the plurality of recombinant glycoproteins are bound to the sialylated glycan.

[0184] Example 37: The method according to any one of Examples 31 to 36, wherein the plurality of recombinant glycoproteins are bound to the sialylated glycan with the following enrichment homogeneity: at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 99%, 100%, or an integer ranging from any two of the above numbers.

[0185] Example 38: The method according to any one of Examples 31 to 37 further includes collecting the various recombinant glycoproteins within 200 hours after the initiation of expression of the payload nucleic acid in the cells.

[0186] Example 39: According to the method of Example 38, the samples were collected within 100 hours after the initiation of expression of the payload nucleic acid in the cells by the plurality of recombinant glycoproteins.

[0187] Example 40: The method according to any one of Examples 31 to 39, wherein the recombinant glycoprotein is an antibody or its antigen-binding fragment.

[0188] Example 41: The method according to Example 40, wherein the glycan is located on the constant region of the antibody.

[0189] Example 42: The method according to Example 41, wherein the polysaccharide is located on the heavy chain of the antibody.

[0190] Example 43: The method according to any one of Examples 40 to 42, wherein the glycan is located at the glycosylation site of the antibody.

[0191] Example 44: The method according to Example 43, wherein the glycan is located at sites N46, N402 and / or N297 of the antibody.

[0192] Example 45: The method according to any one of Examples 40 to 44, wherein the antibody is a therapeutic antibody.

[0193] Example 46: The method according to Example 45, wherein the therapeutic antibody is adalimumab (Humira). ® ), Adalimumab-atto (Amjevita) ® ), Rituxan ® ), Rituximab-atto (Truximab) ® ), cetuximab (Erbitux) ® Avastin bevacizumab ® ), Infliximab (Remicade ® trastuzumab (Herceptin) ® ), Pembrolizumab (Keytruda) ® Enbrel ® ), Yervoy ® Arzerra (or simponi), and Simponi (or simponi) ®Atezolizumab (Tecentriq) ® OCREVUS (Orizumab) ® Durvalumab ® ), Bavencio ® Nivolumab (Opdivo) ® Pertuzumab (Perjeta) ® ), Gazyva ® ), Remicade ® ) or trastuzumab entathiacin (Kadcyla) ® ).

[0194] Example 47: Multiple enriched recombinant glycoproteins, wherein at least 50% of the multiple recombinant glycoproteins are configured to carry sialylated glycans.

[0195] Example 48: A plurality of enriched recombinant glycoproteins according to Example 47, wherein at least 70% of the plurality of enriched recombinant glycoproteins are configured to carry the sialylated glycan.

[0196] Example 49: A variety of enriched recombinant glycoproteins according to Example 47 or Example 48, wherein the variety of enriched recombinant glycoproteins are homogeneously bound to the sialylated glycan as follows: at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100%.

[0197] Example 50: A variety of enriched recombinant glycoproteins according to Example 47 or Example 48, wherein the various enriched recombinant glycoproteins are homogeneously bound to the sialylated glycan in the following proportions: 1% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 1% to 10%, 5% to 100%, 5% to 95%, 5% to 85%, 5% to 75%, 5% to 65%, 5% to 55%, 5% to 45%, 5% to 35%, 5% to 25%, 5% to 10%, 10% to 100%, 10% to 99%, 10% to 95%, 10% to 90%, 10% to 85%, 10% to 80%, 10% to 75%, 10% to 70%, 10% to 65%, 10% to 60%, 10% to 55%. 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, 10% to 15%, 20% to 100%, 20% to 99%, 20% to 95%, 20% to 90%, 20% to 85%, 20% to 80%, 20% to 75%, 20% to 70%, 20% to 65%, 20% to 60%, 20% to 55%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 2 0% to 30%, 20% to 25%, 40% to 100%, 40% to 99%, 40% to 95%, 40% to 90%, 40% to 85%, 40% to 80%, 40% to 75%, 40% to 70%, 40% to 65%, 40% to 60%, 40% to 55%, 40% to 50%, 60% to 100%, 60% to 99%, 60% to 95%, 60% to 90%, 60% to 85%, 60% to 80%, 60% to 75%, 50% to 100%, 50% to 99% 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, 70% to 100%, 70% to 99%, 70% to 95%, 70% to 90%, 70% to 85%, 70% to 80%, 70% to 75%, 80% to 100%, 80% to 99%, 80% to 95%, 80% to 90%, 80% to 85%, 90% to 100%, 90% to 99%, or 90% to 95%.

[0198] Example 51: A variety of enriched recombinant glycoproteins according to any one of Examples 47 to 50, wherein the sialylated glycan is an α2-6 sialyl complex (SCT) glycan.

[0199] Example 52: Various enriched recombinant glycoproteins according to Example 51, wherein the α2-6 sialic acid complex (SCT) glycan is a single-antennae or a double-antennae.

[0200] Example 53: Various enriched recombinant glycoproteins according to Example 51 or Example 52, wherein the sialylated glycans do not contain core fucose.

[0201] Example 54: A variety of enriched recombinant glycoproteins according to any one of Examples 47 to 53, wherein the recombinant glycoprotein is an antibody or its antigen-binding fragment.

[0202] Example 55: Multiple enriched recombinant glycoproteins according to Example 54, wherein the glycan is located on the constant region of the antibody.

[0203] Example 56: Multiple enriched recombinant glycoproteins according to Example 55, wherein the glycan is located on the heavy chain of the antibody.

[0204] Example 57: A variety of enriched recombinant glycoproteins according to any one of Examples 54 to 56, wherein the glycan is located at the glycosylation site of the antibody.

[0205] Example 58: Multiple enriched recombinant glycoproteins according to Example 57, wherein the glycans are located at N46, N402 and / or N297 sites of the antibody.

[0206] Example 59: A variety of enriched recombinant glycoproteins according to any one of Examples 54 to 58, wherein the antibody is a therapeutic antibody.

[0207] Example 60: A variety of enriched recombinant glycoproteins according to any one of Examples 47 to 59, which are obtained by using the method for glycosylation engineering of recombinant glycoproteins according to any one of Examples 31 to 46.

[0208] Example 61: A cell for expressing GlcNAc glycoprotein, which lacks N-acetylglucosamine aminotransferase I (GnTI) activity and constitutively or controllably expresses exogenous endoglucosidase.

[0209] Example 62: Cells according to Example 61, wherein the exogenous endonuclease is endonuclease H (Endo H) or endonuclease S2 (Endo S2).

[0210] Example 63: The cell according to Example 62, wherein the exogenous endonuclease comprises the amino acid sequence set forth in SEQ ID NO: 15 or SEQ ID NO: 16.

[0211] Example 64: A cell according to any one of Examples 61 to 63, wherein the cell contains a nucleic acid configured to encode the exogenous endonuclease.

[0212] Example 65: The cell according to Example 64, wherein the nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO: 17 or SEQ ID NO: 18.

[0213] Example 66: A cell according to any one of Examples 61 to 65, wherein the cell is a mammalian cell.

[0214] Example 67: Cells according to Example 66, wherein the cells are derived from Chinese hamster ovary cells or HEK293 cells.

[0215] Example 68: Cells according to any one of Examples 61 to 67, wherein the cells are derived from Expi293F™ GnTI KO cells.

[0216] Example 69: A cell according to any one of Examples 61 to 68, wherein the cell further comprises a payload nucleic acid configured to encode a recombinant glycoprotein, and the expression of the payload nucleic acid is controlled by a constituent promoter or an activatable promoter.

[0217] Example 70: Cells according to Example 69, wherein the recombinant glycoprotein is an antibody or its antigen-binding fragment.

[0218] Example 71: Cells according to Example 70, wherein the antibody is a therapeutic antibody.

[0219] Example 72: A method for glycosylation engineering of recombinant glycoproteins, comprising: delivering an expression vector into a cell according to any one of Examples 61 to 68, wherein the expression vector comprises a payload nucleic acid configured to encode a recombinant glycoprotein; and expressing the payload nucleic acid in the cell to obtain a plurality of recombinant glycoproteins, wherein at least one of the plurality of recombinant glycoproteins is bound to GlcNAc glycan.

[0220] Example 73: The method according to Example 72, wherein the GlcNAc polysaccharide is a mono-GlcNAc polysaccharide or a GlcNAc-Fuc polysaccharide.

[0221] Example 74: The method according to Example 72 or Example 73, wherein the expression of the payload nucleic acid is constitutive or controllable.

[0222] Example 75: The method according to any one of Examples 72 to 74, wherein at least 50% of the plurality of recombinant glycoproteins are bound to the GlcNAc glycan.

[0223] Example 76: According to the method of any one of Examples 72 to 75, the plurality of recombinant glycoproteins are bound to the GlcNAc glycan with the following homogeneity: at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 45%. Approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100%.

[0224] Example 77: The method according to any one of Examples 72 to 76 further includes collecting the various recombinant glycoproteins within 200 hours after the initiation of expression of the payload nucleic acid in the cells.

[0225] Example 78: The method described in Example 77, wherein the samples are collected within 100 hours after the initiation of expression of the payload nucleic acid in the cells by the plurality of recombinant glycoproteins.

[0226] Example 79: The method according to any one of Examples 72 to 78 further comprises: collecting the plurality of recombinant glycoproteins; and culturing the recombinant glycoproteins together with an endoglucosidase and a donor glycan.

[0227] Example 80: The method according to Example 79, wherein the donor polysaccharide comprises an oxazoline moiety.

[0228] Example 81: The method according to Example 79 or Example 80, wherein the donor glycan is a sialic acid complex (SCT) glycan.

[0229] Example 82: The method according to any one of Examples 79 to 81, wherein the endoglycosidase is endoglycosidase H (Endo H) or endoglycosidase S2 (Endo S2).

[0230] Example 83: The method according to any one of Examples 72 to 82, wherein the recombinant glycoprotein is an antibody or its antigen-binding fragment.

[0231] Example 84: The method according to Example 83, wherein the glycan is located on the constant region of the antibody.

[0232] Example 85: The method according to Example 84, wherein the polysaccharide is located on the heavy chain of the antibody.

[0233] Example 86: The method according to any one of Examples 83 to 85, wherein the glycan is located at the glycosylation site of the antibody.

[0234] Example 87: The method according to Example 86, wherein the glycan is located at the N46, N402 and / or N297 sites of the antibody.

[0235] Example 88: The method according to any one of Examples 83 to 87, wherein the antibody is a therapeutic antibody.

[0236] Example 89: The method according to Example 88, wherein the therapeutic antibody is adalimumab (Humira). ® ), Adalimumab-atto (Amjevita) ® ), Rituxan ® ), Rituximab-atto (Truximab) ® ), cetuximab (Erbitux) ® Avastin bevacizumab ® ), Infliximab (Remicade ® trastuzumab (Herceptin) ® ), Pembrolizumab (Keytruda) ® Enbrel ® ), Yervoy ® Arzerra (or simponi), and Simponi (or simponi) ® Atezolizumab (Tecentriq)® OCREVUS (Orizumab) ® Durvalumab ® ), Bavencio ® Nivolumab (Opdivo) ® Pertuzumab (Perjeta) ® ), Gazyva ® ), Remicade ® ) or trastuzumab entathiacin (Kadcyla) ® ).

[0237] Example 90: A variety of enriched recombinant glycoproteins, wherein at least 50% of the variety of recombinant glycoproteins are configured to carry GlcNAc glycans.

[0238] Example 91: A variety of enriched recombinant glycoproteins according to Example 90, wherein at least 70% of the variety of recombinant glycoproteins are configured to carry the GlcNAc glycan.

[0239] Example 92: A variety of enriched recombinant glycoproteins according to Example 90 or Example 91, wherein the variety of recombinant glycoproteins are homogeneously bound to the GlcNAc glycan as follows: at least about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 31%, about 32%, about 33%, about 34%, about 35%, about 36%, about 37%, about 38%, about 39%, about 40%, about 41%, about 42%, about 43%, about 44%, about 4 5%, approximately 46%, approximately 47%, approximately 48%, approximately 49%, approximately 50%, approximately 51%, approximately 52%, approximately 53%, approximately 54%, approximately 55%, approximately 56%, approximately 57%, approximately 58%, approximately 59%, approximately 60%, approximately 61%, approximately 62%, approximately 63%, approximately 64%, approximately 65%, approximately 66%, approximately 67%, approximately 68%, approximately 69%, approximately 70%, approximately 71%, approximately 72%, approximately 73%, approximately 74%, approximately 75%, approximately 76%, approximately 77%, approximately 78%, approximately 79%, approximately 80%, approximately 81%, approximately 82%, approximately 83%, approximately 84%, approximately 85%, approximately 86%, approximately 87%, approximately 88%, approximately 89%, approximately 90%, approximately 91%, approximately 92%, approximately 93%, approximately 94%, approximately 95%, approximately 96%, approximately 97%, approximately 98%, approximately 99%, or approximately 100%.

[0240] Example 93: A variety of enriched recombinant glycoproteins according to Example 90 or Example 91, wherein the various recombinant glycoproteins are bound to the GlcNAc glycan with the following homogeneity: 1% to 100%, 1% to 90%, 1% to 80%, 1% to 70%, 1% to 60%, 1% to 50%, 1% to 40%, 1% to 30%, 1% to 20%, 1% to 10%, 5% to 100%, 5% to 95%, 5% to 85%, 5% to 75%, 5% to 65%, 5% to 55%, 5% to 45%, 5% to 35%, 5% to 25%, 5% to 10%, 10% to 100%, 10% to 99%, 10% to 95%, 10% to 90%, 10% to 85%, 10% to 80%, 10% to 75%, 10% to 70%, 10% to 65%, 10% to 60%, 10% to 55%. 10% to 50%, 10% to 45%, 10% to 40%, 10% to 35%, 10% to 30%, 10% to 25%, 10% to 20%, 10% to 15%, 20% to 100%, 20% to 99%, 20% to 95%, 20% to 90%, 20% to 85%, 20% to 80%, 20% to 75%, 20% to 70%, 20% to 65%, 20% to 60%, 20% to 55%, 20% to 50%, 20% to 45%, 20% to 40%, 20% to 35%, 2 0% to 30%, 20% to 25%, 40% to 100%, 40% to 99%, 40% to 95%, 40% to 90%, 40% to 85%, 40% to 80%, 40% to 75%, 40% to 70%, 40% to 65%, 40% to 60%, 40% to 55%, 40% to 50%, 60% to 100%, 60% to 99%, 60% to 95%, 60% to 90%, 60% to 85%, 60% to 80%, 60% to 75%, 50% to 100%, 50% to 99% 50% to 95%, 50% to 90%, 50% to 85%, 50% to 80%, 50% to 75%, 50% to 70%, 50% to 65%, 50% to 60%, 70% to 100%, 70% to 99%, 70% to 95%, 70% to 90%, 70% to 85%, 70% to 80%, 70% to 75%, 80% to 100%, 80% to 99%, 80% to 95%, 80% to 90%, 80% to 85%, 90% to 100%, 90% to 99%, or 90% to 95%.

[0241] Example 94: A variety of enriched recombinant glycoproteins according to any one of Examples 90 to 93, wherein the GlcNAc glycan is a mono-GlcNAc glycan or a GlcNAc-Fuc glycan.

[0242] Example 95: A variety of enriched recombinant glycoproteins according to any one of Examples 90 to 94, wherein the recombinant glycoprotein is an antibody or its antigen-binding fragment.

[0243] Example 96: Multiple enriched recombinant glycoproteins according to Example 95, wherein the glycan is located on the constant region of the antibody.

[0244] Example 97: Multiple enriched recombinant glycoproteins according to Example 96, wherein the glycan is located on the heavy chain of the antibody.

[0245] Example 98: A variety of enriched recombinant glycoproteins according to any one of Examples 95 to 97, wherein the glycan is located at the glycosylation site of the antibody.

[0246] Example 99: Multiple enriched recombinant glycoproteins according to Example 98, wherein the glycans are located at N46, N402 and / or N297 sites of the antibody.

[0247] Example 100: A variety of enriched recombinant glycoproteins according to any one of Examples 95 to 99, wherein the antibody is a therapeutic antibody.

[0248] Example 101: A variety of enriched recombinant glycoproteins according to any one of Examples 90 to 100, which are obtained by using a method for glycosylation engineering of recombinant glycoproteins according to any one of Examples 72 to 89.

[0249] sequence

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

[0272]

[0273]

[0274]

[0275]

[0276]

[0277]

[0278]

[0279]

Claims

1. A cell for expressing a sialylated glycoprotein, wherein the cell constitutively and / or controllably expresses an exogenous sialyltransferase catalytic peptide and an exogenous galactosyltransferase catalytic peptide, wherein the exogenous sialyltransferase catalytic peptide and the exogenous galactosyltransferase catalytic peptide are translated in close proximity.

2. The cell according to claim 1, wherein the exogenous sialyltransferase catalytic peptide and the exogenous galactosyltransferase catalytic peptide are expressed in a single transcript.

3. The cell of claim 1, comprising a first nucleic acid configured to express the exogenous sialyltransferase catalytic peptide and a second nucleic acid configured to express the exogenous galactosyltransferase catalytic peptide, wherein the first nucleic acid and the second nucleic acid are transcribed under the same promoter.

4. The cell of claim 3, wherein the first nucleic acid and the second nucleic acid are linked to each other by a linker nucleic acid configured to encode a ribosomal shifting peptide.

5. The cell of claim 3, wherein the exogenous sialyltransferase catalytic peptide and the exogenous galactosyltransferase catalytic peptide are co-configured to be expressed as a fusion protein.

6. The cell of claim 5, wherein the fusion protein comprises a first portion having a sialyltransferase catalytic peptide domain and a second portion having a galactosyltransferase catalytic peptide domain, wherein the first portion and the second portion are connected to each other by a cleavable linker, wherein the cleavable linker is configured to be cleavable after translation of the fusion protein, thereby releasing the sialyltransferase catalytic peptide and the galactosyltransferase catalytic peptide as separate proteins upon cleavage.

7. The cell according to claim 3, wherein the first nucleic acid is derived from the ST6Ga11 gene or the PspST gene.

8. The cell according to claim 3, wherein the second nucleic acid is derived from the B4GALT1 gene.

9. The cell of claim 3, wherein the promoter is a constituent promoter or an activatable promoter.

10. The cell according to claim 1, wherein the sialyl transferase is β-galactosyl α-2,6-sialyl transferase 1.

11. The cell according to claim 1, wherein the galactosyltransferase catalyzing peptide is β-1,4-galactosyltransferase 1.

12. The cell according to claim 1, wherein the cell lacks fucosylation activity.

13. A method for glycosylation engineering recombinant glycoproteins, comprising: Delivery of an expression vector into a cell according to any one of claims 1 to 12, wherein the expression vector comprises a payload nucleic acid configured to encode the recombinant glycoprotein; and The payload nucleic acid is expressed in the cells to obtain a variety of recombinant glycoproteins, wherein at least one of the recombinant glycoproteins is bound to sialylated glycan.

14. The method according to claim 13, wherein the sialylated polysaccharide is an α2-6 sialyl complex (SCT) polysaccharide.

15. The method of claim 13, wherein the sialylated glycan lacks a core fucose.

16. The method of claim 13, wherein at least 50% of the plurality of recombinant glycoproteins are bound to the sialylated glycan.

17. The method of claim 13, further comprising collecting the plurality of recombinant glycoproteins in the cells within 200 hours of the expression of the payload nucleic acid.

18. The method of claim 13, wherein the recombinant glycoprotein is an antibody or an antigen-binding fragment thereof.

19. The method of claim 18, wherein the glycan is located at sites N46, N402 and / or N297 of the antibody.

20. The method of claim 18, wherein the antibody is adalimumab (Humira). ® ), Adalimumab-atto (Amjevita) ® ), Rituximab ® ), Rituximab-atto (Truximab) ® Cetuximab (Erbitux) ® Bevacizumab (Avastin) ® ), Infliximab (Remicade ® Trastuzumab (Herceptin) ® Pembrolizumab (Keytruda) ® Etanercept (Enbrel) ® Ipilimumab (Yervoy) ® Ofatumumab (Arzerra), Golimumab (Simponi) ® Atezolizumab (Tecentriq) ® Ocrelizumab (OCREVUS) ® Durvalumab ® Avelumab (Bavencio) ® Nivolumab (Opdivo) ® Pertuzumab (Perjeta) ® Obinutuzumab (Gazyva) ® ), Gazyvaro Infliximab (Remicade ® ), or trastuzumab emtansine (Kadcyla) ® ).

21. A plurality of enriched recombinant glycoproteins, wherein at least 50% of the plurality of recombinant glycoproteins are configured to have sialylated glycans.

22. The plurality of enriched recombinant glycoproteins according to claim 21, wherein at least 70% of the plurality of enriched recombinant glycoproteins are configured to have the sialylated glycan.

23. The plurality of enriched recombinant glycoproteins according to claim 21, wherein the plurality of enriched recombinant glycoproteins are bound to the sialylated glycan with 40% to 99% homogeneity.

24. The various enriched recombinant glycoproteins according to claim 21, wherein the sialylated glycan is an α2-6 sialyl complex (SCT) glycan.

25. The various enriched recombinant glycoproteins according to claim 21, wherein the recombinant glycoprotein is an antibody or an antigen-binding fragment thereof.

26. The multiple enriched recombinant glycoproteins according to claim 25, wherein the glycan is located at sites N46, N402 and / or N297 of the antibody.

27. The various enriched recombinant glycoproteins according to claim 21, which are obtained by using the method for glycosylation engineering recombinant glycoproteins according to claim 13.

Images