CHEMIOENZYMATIC GLYCOENGINEERING OF ANTIBODIES AND FC FRAGMENTS OF THE SAME

专利摘要:
chemoenzymatic glycoengineering of antibodies and fc fragments thereof. the present invention provides recombinant endo-s mutants that exhibit reduced hydrolysis activity and increased transglycosylation activity for glycoprotein synthesis, wherein a desired sialylated oxazoline or synthetic oligosaccharide oxazoline is added to a fucosylated or non-fucosylated core protein g1cnac-acceptor . such recombinant endo-s mutants are useful for efficient glycosylation remodeling of the igg1-fc domain to provide different antibody glycoforms that structurally carry well-defined fc n-glycans.
公开号:BR112014019825B1
申请号:R112014019825-0
申请日:2013-02-11
公开日:2021-08-24
发明作者:Wang Lai-Xi；Huang Wei
申请人:University Of Maryland, Baltimore；
IPC主号:

专利说明:

GOVERNMENT RIGHTS IN THE INVENTION IÕQ
[001] This invention was made with government support under grant numbers GM080374 and GM096973 granted by the National Institutes of Health. The government has certain rights in the invention. CROSS REFERENCE TO RELATED'S REFERENCE
[002] The application claims priority to US Interim Application No. 61/597,468 filed February 10, 2012, the contents of which are incorporated herein by reference for all purposes. BACKGROUND OF THE INVENTION
[003] Field of Invention
The invention relates to the synthesis of glycoproteins, and more particularly to the use of a recombinant mutant Endo S, an endo-PN^acetylglucosaminyldase from Strepcococcus pyogenest which has transglycosylation activity and limited hydrolysis activity of mode allowing efficient glycosylation remodeling of the antibody-Fc domain.
[005] Description of Related Technique
[006] Monoclonal antibodies [mAb) of the IgG type, are an important class of therapeutic proteins used for the treatment of cancer, autoimmune diseases and infectious diseases. (,1-A) IgG antibodies are composed of two heavy chains and two light chains that are associated to form three distinct domains of the protein, including two variable Fab domains and a coustanre Fc (crystallizable) domain connected by a flexible hinge region. The Fab' domains are responsible for antigen binding, while the Fc domain is involved in Fc receptor-mediated efferent functions such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). (2/4) The Fc domain is a hotnodimer that carries two N-glycans at conserved N-glycosylation sites (N297). The linked E>1igosaccharides are of the biantennary complex type with. considerable structural heterogeneity, in which the M-linked heptasaccharide core can be differentially decorated with nuclear fucose (Fuc), M-acetylglucosamine (GldNAc) bisector, terminal galactose (Gal), and terminal sialic acid (Sia) as shown in Figure 1 , (5-1) Crystallographic X-ray and NMR structural studies indicate that Fc glycans are placed between the two CH2/CH3 subdomains and have multiple non-covalent interactions with the Fc domains. (8-14) These studies have shown that the binding of different Fc glycans can have a distinct impact on the conformations of the Fc domain, implying an important role for gl. isolation in the maintenance of one. adequate Fc domain structure for interactions with respective Fc receptors associated with antibody effector functions (8-14) .
[007] It has further been shown that the fine structures of N-glioans Fc are important determinants of the pro- and anti-inflammatory activities of antibodies. (2, 15) , for example, the lack of core fucose as well. as the binding of a bisector GlcNAc moiety, it dramatically increases the antibody's affinity for the Fc-yllla receptor (FcRIIIa), which is responsible for antibody-dependent cellular cytotoxicity (ADCC). (11, 16-18) Thus, mAbs with low fucose content are. sought after, for the improvement of anticoadreneral efficacy in vivo. (19, 20) On the other hand, terminal α-2, 6-sialylated glycerin FC, a minor component of intravenous immunoglobulin (IVTG) collected from the sera of thousands of healthy blood donors, has recently been identified as the active species for the anti-inflammatory activity of IViG in a mouse model of xeumatoid arthritis (ÃR). (21-23) However, commercially available IgGs, including monoclonal antibodies and IVIG, typically exist as mixtures of glycoforms that are not ideal for their respective therapeutic activities. For example, the largest Fc glycoforms of monoclonal antibodies currently used for cancer treatment are fucosylated core that have a xelatively low affinity for the FαyRIIIa activating receptor, demonstrating little efficacy particularly for those patients with the low EdyRHIa-FlSS allelic polymorphism affinity. £2, 19, 20)
[008] The impact of glycosylation on biological functions and- therapeutic outcome of antibodies. IgG has stimulated great interest in the development of methods to control the glycosylation of antibodies. One approach is to control glycosylation profiles during production by engineering the glycan biosynthetic pathway into various expression systems, including mammalian, plant and yeast host cells (24-30). resulted in the production of low-fuCQs and non-fucosylated monoclonal antibodies with improved ADCC activity.However, the glycoforms that can be generated by this approach have been limited, and in most cases, complete control to a defined homogeneous glycoform it's difficult.
[009] A recent analysis of the various therapeutic glycoprotein drugs available on the market, including monoclonal antibody rituximab, indicated significant changes in the glycosylation profiles of different batches produced at different times. [311 This analysis poses the challenge of maintaining a consistent production of glycoprotein-based drugs and also raises regulatory concerns, as alterations in Fc glycosylation would be more likely to impact therapeutic efficacy.
An alternative approach to dealing with the inconsistency and heterogeneity in glycoprotein glycosylation is to perform glycosylation remodeling by trimming heterogeneous N-glycans and extending sugar chains by enzymatic glycosylation. (32, 33) Such enzymatic glycosylation has recently been described using a guimioenzymatic method for remodeling Fc glycosylation that takes advantage of the transglycosylation activity of various endoglycosidases and their glycosynthase mutants using glycan oxazolines as their substrates (34-36) This remodeling approach consists of in two steps: trimming all heterogeneous N-glycans by an endoglycosidase to leave only the first GlcNAc in the glycosylation site(s) and then re-add a well-defined block N-glycan structure by via an endoglycosidase catalyzed transglycosylation reaction. (32)
Recent work has demonstrated that XgG-Fc domain glycosylation engineering can be achieved by a combination of the expression in CHO or yeast cells of the Fc domain and its subsequent chemoenzymatic remodeling via an enzymatic deglycasylation/reglycoslation approach. (34-36) The endo-PN-acetylglucosamidase from Arthrocysterprotophorifiiae, endoA, has been shown to be highly efficient to glycosylate the Fc domain containing GlcNAc using various synthetic d.and N-glycan core oxazolines as substrates. (34, 35) However, the limitations of the current state of the method are evident: (a) neither endoA nor endoM (another endoglycoside from Mucor hiemalis) was able to transform I.gG-Fc Eucosylated-nucleus domain., (35 ) the main glycoforms of recombinant mAbs and IV1G; (B) EndpD mutants were able to attach a ManuGlcNAc core to a fucosylated GlcNAc-Fc domain, (36) but nentium of endoD, endoA, endoM, and their mutants £36-39) were able to transfer N-glycan-like intact complex to GlcNAc-Fc domain either fucosylated or non-fucosylated; and (c) glycosylation remodeling of intact full-length IgG antibodies with complex-type N-glycans is yet to be achieved.
[0012] In an attempt to develop an efficient glycosylation/enzymatic glycosylation system for glycosylation remodeling of glycoprorein, attention has turned to endoS, an endo-β-N-acetylglucosaminidase (ENGaseJ from Streptococcus pyogenes, which is capable of hydrolyzing the Fc N-glycans of intact IgG antibodies by cleaving the (31,4-glycosidic bond in the chitobiose core of M-glycans. (40^-42) Endo-S has transglycosylation activity, such as that capable of utilizing MansGlcNAc oxazoline as a donor substrate for glycosllax a GlcNAc acceptor. However, wild-type Endo-S also possesses highly active Itidrolithium activity, so the glycosylated IgG product is also subject to rapid hydrolysis if wild-type Endo-S is used for the synthesis and remodeling of glycosylation.
[0013] In light of the above known activities of Endo S, it would be advantageous to provide a mutant of Endo-S that exhibits a transglycosylar activity with reduced hydrolyzing activity. SUMMARY OF THE INVENTION
[0D14] The present invention provides selected mutants and recombinant Endo-S thereof, which exhibit reduced hydrolysis activity and increased transglycosylation activity for the synthesis of IgG antibodies to Fc fragments thereof, in which a desired sugar chain is added to a Fucosylated or non-fucosylated core GicNAc-IgG acceptor. As such, the present invention allows for a. synthesis and remodeling of therapeutic antibodies and Fc fragments thereof to provide certain biological activities, such as, prolonged in vivo half-life, lower immunogenicity, improved in vivo activity, increased targeting ability, and/bu capacity of delivering a therapeutic agent.
[0015] In one aspect, the present invention provides the transglycasylation activity of an endo-PN-acetylglucosamidase from Streptococcus pyogenes ISEQ ID NO: 1) and its mutants, wherein the mutants have at least homology thereto and exhibit transglycosylation activity in both fucosylated and non-fucosylated GlcNc-IgG acceptors where endoglieosidases allow the transfer of an oligosaccharide (in the form of an activated sugar oxazoline) in block to a fucosylated or non-fucosylated (or non-fucosylated) GicNAc-IgG. an Fc fragment thereof) in order to form a new IgG glycoform (or an Fc fragment thereof).
[0016] In another aspect, the present invention provides Endo-S mutants that show somewhat increased transglycoylation efficacy or decreased or abolished product hydrolytic activity. Mutants preferably include site-specific mutations, including an Asp-233 mutation. Matters include, but are not limited to, D233Q (SEQ ID MO: 2} and D233A (SEQ ID MQ: 3)
[0017] In another one. In this aspect, the present invention provides a chemoendymatic method for preparing homogeneous fucosylated or non-fucosylated core glycoforms of antibodies. IgG, which comprises:a. providing an acceptor selected from the group consisting of a fucosylated core GlcNAc-IqG, non-fucosylated core GlcNAc-IgG or corresponding IgG-Ec fragments; and b. reacting the D acceptor with a donor substrate, including an activated oligosaccharide moiety, in the presence of Endo-S-Asp 233 mutants of Streptococcus pyogenes to transfer the activated α-ligosaccharide moiety to the acceptor and produce the homogeneous Eucosylated or non-fucosliated glycoptotein.
In yet another aspect, the present invention provides a method for preparing a fucosylated core IgG or IgG-Fc fragment having a predetermined oligosaccharide portion, comprising: a. providing a fucosylated core IgG acceptor comprising one. asparagine-linked fucosylated core N-acecylglucosamine residue (GlcNAc); and b. enzymatically reacting the fucosylated core IgG acceptor with an activated oligosaccharide donor in the presence of S-Etidoglycosidase mutant D233Q [. activated αligαsaccharide donor carries an oligosaccharide portion comprising one. predetermined number and type of sugar residues, wherein the portion of the oligosaccharide is covalently linked to the fucosylated core IgG: acceptor, thus preparing the fucosylated core IgG or IgG-Fc fragment having the predetermined oligosaccharide portion.
In yet another aspect, the present invention provides an activated oligosaccharide moiety such as a glycan or the oxazoline-saccharide oligo.s, glycosyl fluoride, glycosyl azaldehyde or an arlla glycoside, such as a substrate donor for the synthesis of homogeneous fucosylated core glycoproteins or non-fucosylated glycoproteins. Preferably, the activated oligosaccharide moiety is an oxazoline oligosaccharide.
[0020] In another aspect, the present invention relates to a chemoenzyme method for the preparation of an antibody of homogeneous fucasylated or non-fucosylated monomers or an Ec fragment thereof, said method comprising: providing an apeptof selected from the GlcNAc - fucosylated or non-fucosylated core antibody or an Fc fragment thereof; and reacting the acceptor with a donor substrate in the presence of an Endo-S Asp-233 mutant of Straptcoccus pyogenes, wherein the donor substrate comprises a predetermined oligosaccharide component with a defined number and type of sugar residues, and specific binding types, thus providing the homogeneous fucosylated or non-fucosylated monomer antibody or an Fc fragment thereof. In one embodiment, a protein-containing fucosylated GlcNAc is an alpha-1-6-fucosyl-GlcNAc-a protein.
[0021] In another aspect, the present invention relates to a method of remodeling an antibody or Fc fragment thereof with an aligos.sugar ideo having a predetermined aligos saccharide component with a defined number and type of sugar residues and types binding specifics, comprising the method: a. provide a fucosylated core antibody or Phedo fragment even if it comprises Fc N-glycans; b. treating the fucosylated core antibody or Fc fragment with an enzymatic hydrolysis to produce an Asn-linked GlcNAc moiety; and c. joining the oligosaccharide to the Asn-linked GlcNAc portion in the presence of an Endo-S mutant having an amino acid sequence selected from the group consisting of S.EQ ID NO: 2 and SEQ ID NO: 3, thus adding the oligosaccharide component predetermined.
[0022] In another aspect, the present invention relates to a method of remodeling a fucosylated or non-fucosylated core IgG or IgG-Fc fragment with an oligosaccharide having a predetermined oligosaccharide component with a defined number and type of sugar residues and with the specific binding types, the method comprising: a. provide a fucosylated or non-fucosylated core IgG or fragment. IgG-Fc obtained from natural or recombinant sources that carry N-glycanoshet and roganes; B. treat the natural or recombinant IgG or IgG-Fc fragment with a wild-type endo-enzyme or a mutant endoglycoside with efficient hydtolytic activity to hydrolyze the bond between the two GlcNAc residues positioned closest to the domain peptide thereby forming a deglycosylated protein carrying a fucosylated or non-fucosylated core GlcNAc acceptor; and c. joining the predetermined oligosaccharide component to the GlcNAc acceptor to reconstitute the beta-1,4-glycosidic bond through transglycoslation with an Endo-S Asp-233 mutant of Streptococcus pyogenes, thus adding a predetermined oligosaccharide component to remodel the fucosylated or non-fucosylated core IgG or IgG-Ec fragment.
Applicable oxazoline oligosaccharides include, but are not limited to, high mannose type, hybrid type, oxazoline sialoglycan and complex type N-glycan, as well as their selectively modified derivatives, such as those with specific tags. Preferably, di, tri-, tetra-, penta-, hexyl-, hepta-, octyl-, nona-, deca-2 or undecaasaccharide oxazolines are used as donor substrates for the highly efficient chemoenzymatic synthesis of IgG core antibodies homogeneous fuco.silated or non-fucosylated and IgG-Fc fragments,
[0024] In yet another aspect, the present invention relates to a method for synthesizing an antibody or modified fragment of. himself, understanding the method; a. providing a naturally existing IgG antibody, a recombinant antibody, or an Fc domain that carries Fc N-glycans as precursors; B. Deglycosylate Fc using an epdogylcosidase such as wild-type Endo-S to deglycosylate the Fc domain to form one. GlcNAc acceptor; wherein the GlcNAc acceptor is positioned in the Fc region of the antibody and o. GlcNAc acceptor is either fucosylated or non-fucosylated nucleus; ec. transglycosylate the GlcNAc acceptor on the naturally existing IgG antibody, recombinant antibody or Fc domain with an oxatoline oligosaccharide or a sialoglycan oxazoline having a predetermined number of sugar residues under catalysis of an enzyme selected from the group. consists of Endo-S mutants including SEQ ID NO: 2 and SEQ ID NO: 3, to form the modified antibody with a predetermined number of sugar residues r.
In yet another aspect, the present invention provides a method of remodeling an intravenous immunoglobulin [IVTGJ exhibiting sialylated Fc-glycoforms, the method comprising: a. provide an IVIG bearing Fc N-glycans; b. Fc deglycosylate the. Fc N-glycans using an endoglycosidases including wild-type Endo-S to form GlcNAc acceptors; wherein the GlcNAc acceptors are positioned in the Fc region of IVIG and the GlcNAc acceptors are either fucosylated or non-fucosylated; and c. transglycosylate the GlcNAc acceptors with a sialoglycan oxazoin having a predetermined number of sugar residues under catalysis of an enzyme selected from the group consisting of Enda-S inutants including SEQ EQ NO; 2 and SEQ ED NO: 3 to form an allylated IVIG.
Another aspect of the present invention provides a composition containing an IVIG preparation comprising by 901 yen of homogeneous sialylated F'c glycoforms to enhance anti-inflammatory activity, wherein the sialylated Fc glycoforms are synthesized using a mutant of Streptococcus pyogenes Endo-£ Asp-233 was combined with a GlcNAc portion positioned in the Fc region of a degliosylated IVIG and sialoglycerin oxazoline lime having a predetermined number of sugar residues.
In yet another aspect, the present invention relates to a method for the synthesis of homogeneous fucosylated or non-fucosylated core IgG antibodies or IgG-Fc fragments, the method comprising: a. providing a natural or recombinant IgG antibody or IgG-Fc fragment, wherein the recombinant IgG or IgG-Fc is produced from a typical protein expression system, including but not limited to yeast, insects, plants, and any other protein system. mammalian expression;bi remove the N-glycans of an enzyme selected from the group consisting of Endo-H, Endo-A, Endo-S, and/or endo-F3 to form a fucosylated or non-core GlcNAc-containing protein fucosylated; c. providing a sugar oxazoline or sialoglycan oxazoline with a desired oligosaccharide component comprising a number and type of sugar residues in the defined chain; ed. enzymatically transglycosylate the fucosylated or non-fucosylated GlcNAc-containing protein with a sugar oxazolin yielding a desired number of sugar residues or sialoglycan oxazoline having a desired number of sugar and sialic acid residues with an endoglycosidase selected from the group consisting of mutants from Streptococcus pyogenes Endo-S Asp -233, thus forming a homogeneous fucosylated or non-fucosylated core IgG antibody or IgG-Fc fragment having an extension of the desired number of sugar and/or sialic acid residues.
It is envisioned that the oxazoline polysaccharide or sialoglycan oxazoline having a predetermined oligosaccharide component with a defined number and type of sugar residues may further comprise one. additional label or portion, including, a therapeutic agent or drug, such as for the treatment of: cancer, HIV or other viruses, substances that activate receptors on the plasma membrane of cells, agents that affect intracellular chemistry, agents that affect cell physics , genes, gene analogues, RNA, RNA analogues, DNA, DNA analogues, amino acid sequences of surface receptors such as CCR5 or CD4, antigenic structure which has affinity for a specific antibody.; amino acid sequences of receptor ligands such as gp120, gp41 or gplGO, receptor antagonists, receptor blockers, enzymes, enzyme substrates, enzyme inhibitors, enzyme modulators, therapeutic proteins, protein analogues, metabolites, metabolite analogues , oligonucleotides, oligonucleotide analogues, antigens, antigen analogues, antibodies or fragments thereof, antibody analogues, an antibody other than the modified antibody, which is reactive to other recipient bacteria, viruses, inorganic ions, metal ions, metal aggregates, polymers, fluorescent compounds and any combinations thereof.
[0029] As such, the present invention further provides a delivery device for administering a drug or therapeutic agent having a biological activity to treat a condition, the delivery device comprising: an IgG-Fc or remodeled IgG fragment having a predetermined sugar chain or sialoglycan and a therapeutic agent or drug attached to the terminal sugar residue or sialic acid.
The present invention envisions the modification of HIV-related monoclonal antibodies, including, but not limited to, 17b, 48d, A32, CU, 2G12, F24Q, IgGlbl2, 19e, X5, TNX-355 and F91, all .03 which are commercially available.
[0031] Other antibodies related to cancer or other diseases can also be remodeled to an individual fit to certain receptors, thus increasing biological activity, monoclonal antibodies can include, but are not limited to, cetuximab, rituximab, muromonab-CD3, abciximab, daclizumab, basiliximab, palivizumab, infliximab, trastuzumab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimmnab, omalizumab, tositumαmab, I-131 tositumomab, efalizumab, efalizumab, efalizumab, vacitum Biogen Idee and PDL BioPharm), Anti-CDSO mAb (Biogen Idee), Anti-CD23 mAb (Biogen Idel), CAT-3088 (Cambridge Antibody Technology):, C-DP-791 (Imcioné), eraptuzumab {ImmunomedicsJ, MDX- 010 (Medaεex and BMS), MDX-06O (Medarex), MDX^070 (Medarex), matuzumab (Merck), CP-675.2Q6 (Pfizer), CAL (Roche), SGN-30 (Seattle Genetics), zanolimumab ( Seronα and Genmab), adecaturauraab (Serene), oεegovomab (United The rapeutics), nimotαzumab (YM Bioscience), ABT-^74 (Abbott Laboratories), denosumab (.Amgen), AM 108 (Amgen), AMG 714 (Amgen), fontolizαmab (Biogen Idee arid PDL BioPharm) , daclizuroiab (Biogent Idee and PDL BioPharm), goliumab (Centocor and Schering-Plough), CNTO 1275 (Centocor], ocrelizumab (Genetech and Roche], HuMax-CD20 (Genmab), belimumab (PGS and G5K), epratuzumab (Immunomedics), MLN1202 (Millenmedics) , visllizumab (PDL BioPharm], tocilizuinab (Roche), ocrerlizurnab (Roche), certolizumab pegol [UC0, formerly Celltech), eculiztunab (Alexion Pharmaceuticals), pexelizumab (Alexion Pharmaceuticals and Procter & Gamble), abeiximab (Centocor), ranibizim ), mepolizurnab (GSK), TNX-355 (TanoX), αu MYO-Q2S (Wyeth),.
Yet another aspect of the invention relates to a method of remodeling an antibody which initially includes a heterogeneous sugar chain, the method comprising: a. removing the antibody's heterogeneous sugar chain with an endoglycosidase to leave a single fucosylated or non-fucosylated GlcNAc moiety attached to an original glycosylation site; and b. transferring a sialoglycan oligosaccharide or oxazoline core with at least one tag to the fucosylated or non-fucosylated GlcNAc moiety by an endoglycosidase catalyzed transglycosylation to obtain a labeled antibody, wherein the endoglycosidase is selected from if selected from from the group consisting of Endo-S mutants including SEQ ID NO: 2 and SEQ ID NO: 3.
The tag portion may include, but is not limited to, antigens, therapeutic drugs such as for cancer or HIV, toxins, fluorescent probes, biotin, PEG species, lipids or nucleotides.
[0034] In another aspect, the present invention provides a composition comprising at least one mutant of Streptococcus pyogenes Endo-S Asp-233 selected from the group consisting of D233Q (SEQ ID NO:2) and D233A (SEQ ID NO:2). : 3) .
In yet another aspect, the present invention provides a substantially homogeneous preparation of a fucosylated or non-fucosylated core antibody or an Ec fragment thereof having a predetermined oligosaccharide portion, wherein the substantially homogeneous preparation is produced by either method. mentioned above, Compositions comprising such homogeneous preparations are also provided.
In yet another aspect, the present invention provides a method of treatment utilizing a remodeled antibody having a desired glycosylation state and/or sialylated form in an amount sufficient to modulate the biological activity of the treated individual.
[0037] Other aspects, features and embodiments of the invention will be more apparent from the following disclosure and appended claims. BRIEF DESCRIPTION OF THE DRAWINGS
[0038] Figure 1 shows the structures of an IgG antibody and typical of Fc N-glycans. a) Structure of the human IgG Alpha backbone, showing functional regions (modeled based on the PDB code 1H3HJ : b) The structure of a full length complex-like bi-antennaiy of N-glycans linked to Asn-297 in the domain


[0039] Figure 2 shows the alignment of the eridoscopy sequence (SEQ ID NO: 4) and EπdoF3 (SEQ ID NO: 5].
Figures 3 A and B show the scheme for the glycosylation remodeling of rituximab to natural and selectively modified homogeneous glycoforms.


[0041] Figure 4 shows the SDS-PAGE and ESI-MS analysis of rituximab glycosylation remodeling, (a) SDS-PAGE analysis: lane 0, protein markers; Lane lz rituximab commercial; Lane 2, EndoS de-glycosylated rituximab (1); Lane 3, transglycosylation product (31 from the Endos-D233A catalyzed reaction between (1) and slaloglycanα oxazoline (2); Lane 4, transglycosylation product from the Endos-D233Q catalyzed reaction of (1) and (2); lane 5, the transglycosylation product 15) from the endos-D233Q catalyzed reaction between degliαosylated rituximab (1) and Man3GlcNAc oxazoline (4); lane 6r the transglycosylation product (7) from the endoS-D233Q catalyzed reaction between the deglycosylated rituximab (1) and N3Man3GlcNA6 oxazoline (6). (b) ESI-MS (after deconvolution) of commercial rituximab heavy chain, (e) ESI-MS of de-gliosylated rituximab (1). (d) ESI-MS of the transglycation product (3). (e) ESI-MS of the transglycosylation product (5). [f) ESI-MS of the transglycosylation product (7).
[0042] Figures 5 A and B show the enzymatic remodeling to homogeneous non-tucosylated glycoform of derituximab
Sia) .
[0043] Figure 6 shows the glycoengineered SDS-PAGE and ESI-MS analysis of rituximab for the non-fucosylated G2 glycoform. (a) SDS-PAGE analysis: lane 0, protein markers; Lane 1, commercial rituximab; Lane 2, endoS de-glycosylated rituximab (1); Lane 3, the. product def’jcosiladα (8); Lane 4, G2 glycoform modified by g Li ceengenharia. (b) ESI-MS (after deconvolution) of defucosylated rituximab heavy chain (8). (c) G2 glycoengineering modified rituximab heavy chain ESI-MS I ID).
Figure 7 shows the site-specific Fc glycpengen aria of human IVIG.

Figure 8 shows the HPLC fluorescence profiles of 2AB labeled N-glycans from IVIG Fab and Fc. a) from Fc of native IVIG; bj from gllcoengineered-modified IVIG Fc; Cl from Fab of IVIG Tjactive; d) from 1 Glycoengineered-modified IVIG Fab.: The glycan structures. include the following components:


Figures 9A-C show typical SPR binding sansograms of G2-rituximab and commercial rituximab, with respective Fey receptors: FcYRHIa-V15.8 [A], FcyRIIIa-F158 (iB), and EeyRIIb (C). Antibodies were immobilized by Protein A capture and binding was analyzed by injecting the respective Fcy receptors in a 2-fold dilution series starting at 40 pg/ml (1.33 mM).
[0047] Figure 10 shows the MALDI-TOF MS of the Fc N-glycans released by treatment with PNGase F with the same symbols, as defined in Figure 1.
[0048] Figure 11 shows the MALDI-TOF MS of the Fc N-glycans released by treatment with, EndoS with the same symbols., as defined in Figure 1.
Figures 12 A-C show the LC-MS analysis of rituximab; a) the reduced rituximab LC profile'; b) light chain ESI-MS; c) decorevolved light chain MS; d) heavy chain ESIMS; and j deconvoluted heavy chain MS.
[0050] Figure 13 shows the fluorescent HPLC profile of 2-AB-labeled N-glycans released from commercial and glycoengineered rituximab samples by treatment with PNGase F, a) from commercial rituximab; b) from sialylated rituximab (3); c) from non-fucosylated rituximab (10) .


Figure 14 shows SDS-PAGE analysis of transglycosylation by wild-type BndoS. Lane 0, .protein markers; Lane 1, commercial rituximab; Lane 2, EndoS de-=glycosylated rituximab (11; Lane 3' to lane 7, monitoring the transglycosylation reaction between de-glycosylated rituximab (1) and sialoglycan oxaroline (2): Lane 3.15 minutes; 4.30 min; Lane 5, 1 h; Lane 6, 2 h; Lane 7.4 h.
[0052] Figures 15. A and B show the monitoring of L.C-MS for defucosylation of Fuc (of2,6) GlcNAc-rituximab (1) with bovine kidney α-fucoaidase. The ES1-MS deconvoluted profiles of rituximab heavy chain were shown (FG-Rx, Fuc heavy chain («2,.6) GlcNAc-rituximab; G-Bx, GlcNAc-rituximab heavy chain) - al incubation with a- fucosidase, for 2 days; b) incubation with ct-fucasidase for 7 days; c) incubation with o-fucosidase for 14 days; and dj incubation with o-fucosidase for 20 days.
[0053] Figure 16 shows the 3DS-PAGE analysis of IVIG glycoengineering. Lane 0, protein marker; Lane 1, commercial IVIG; Lane 2f IVIG (11) after deglycosylation by EndoS; Lane 3, IVIG (12) after Endos-D233Q catalyzed transglycosylation with oxazoline sialoglione,
Figure 13 A and B show the amino acid residues of Streptococcus pyogenes Eπdo-S-Asp 233 mutants D233Q [SEQ ID NO: 2) and D233A (SEQ ID NO: 31, respectively). DETAILED DESCRIPTION OF THE INVENTION
[0055] The present invention provides novel Endos Asp 2.33 glycosynthase mutants that. exhibit a remarkable transglycosylation efficiency capable of transferring complex-type N-glycans of activated glycan oxazolines to degliosylate intact antibodies without product hydrolysis. We found here that the Endos A.sp 233 glycosynthase mutants acted efficiently in both the fucosylated and non-fucosylated core GlcNAc-Fc domain of intact antibodies to provide several defined IgG glycoforms. In addition, intravenous antibodies and immunoglobulins were transformed into Ec glycoforms fully sialylated with increased anti-inflammatory activity. In addition, the present invention provides a homogeneous glycoform having increased ADCC activity with enhanced FCyTXla receptor binding activity and azide-tagged glycoforms, which can be further transformed into others glycoforms.
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of immunology, molecular biology, microbiology, cell biology and recombinant DNA, which are within the skill of the art. See, for example, Sambrook, et al, MOLECULAR CLONING CLONING: A Laboratory Manual, 2nd edition (1969); Current Protocols in MOLECULAR BIOLOGY (FM Ausubel, et al θds, (1987)..); the METHODS IM ENZYMOLOGY series (Academic Press, Inc.J: PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. 11995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE IR. I. Freshney, ed, (1987)).
Aspects of the present invention described herein are understood to include "compound" and/or "consisting essentially of" aspects.
[0058] Definitions
As used herein in the specification, "a" or "an" may mean one or more. As used herein in the claims, when used in conjunction with the word "comprises", the words ''a1' or "one" may mean one or more than one. As used herein "other" may mean at least one second or more.
[0060] As used herein, "biological activity" refers to pharmacodynamic and pharmacokinetic properties, including, for example, the resulting molecular affinity or biochemical or physiological effects, affinity for the receptor or the resulting physiological or biochemical effect, non-receptor affinity or biochemical or physiological effect, efficacy, bioavailability, absorption, distribution, metabolism or elimination.
As used herein, "sugar" refers to a molecule containing an oxidized or unoxidized carbohydrate, including, but not limited to, an oligosaccharide or polysaccharide. galactose, Placetilneureminco acid (sialic acid), glucose, fructose, fucose, sorbose/rhamnose, Mannoeptulse, N-acetylgalactosarnin, dihydroxyacetone, xylose, xyullose, arabinose, glyceraldehyde, sucrose, any combination, maloepulse, Lactose fact that the L-or D-isotnera. Sugar also refers that these molecules produced .ma Lura Imente, recombinant®, synthetically, and/or semi-synthetically
As used herein, "homogeneous" refers to fucosylated core glycoproteins or non-fucosylated glycoproteins wherein the oligosaccharide component comprises at least 75%, more preferably at least 80*, at least 85 B OR at least minus 90%, and more preferably at least 95% of the same number and types of sugar residues.
[0063] As used herein, "protein" or "glycoprotein" is interchangeable with the terms peptide and glycopeptides,
As used herein, "homology" refers to the amino acid sequence with substantial identity or similarity between two polypeptides being at least 90%, and more preferably at least 95% similar to a polypeptide. ideo of the reference. For polypeptides, the length of comparison to obtain the above-described percent homologies between sequences will generally be at least 25 amino acids, to 1 therm at least 50 amino acids, more likely, at least 100 amino acids, and most likely 200 amino acids or more. Substantially identical or homologous polypeptides include additions, deletions, internal truncations with insertions, conservative and non-conservative substitutions, or other modifications located at positions in the amino acid sequence that do not destroy the endogliuosidase function. Those skilled in the art will recognize the numerous amino acids that can be modified or substituted by other chemically similar residues without substantially altering activity.
As used herein, "modulate" refers to an increase or a decrease in "biological activity" as defined above when compared to a glycosylation engineering antibody of the present invention to a non-antibody. modified by glycosylation engineering,
As used herein, "immunoglobulin molecule" or "antibodies" refers to molecules that contain an antigen binding site that specifically binds to an antigen or an Fc region that binds to receptors cell phones. Structurally, the simplest naturally occurring antibody (e.g., IgG) comprises four polypeptide chains, two heavy (H) and two inter-chain light (L) chains linked by disulfide bonds. Natural immunoglobulins constitute a large family of molecules that includes several types of molecules, such as IgD, IgG, IgA, IgM and IgE. The term also encompasses hybrid antibodies, or altered antibodies, and fragments thereof, including Fc ffagtnent(s),
The antibodies can be fragmented using conventional techniques as described herein and the fragments screened for utility in the same fashion as described for the whole antibodies. A Fab fragment of an immunoglobulin molecule is a multimeric protein that consists of the portion of an immunoglobulin molecule that contains the immunologically active portions of an immunoglobulin heavy chain and an immunoglobulin light chain covalently coupled together and is covalently coupled together. capable of specifically combining with an antigen. Fab and Fc fragments can be prepared by proteolytic digestion of substantially intact immunoglobulin molecules with papain, using methods that are well known in the art. However, a Fab or Fc fragment can also be prepared by expressing in a suitable host cell the desired portions of immunoglobulin heavy chain and immunoglobulin light chain using methods known in the art.
[0068] As used herein, with respect to antibodies, "substantially pure" means separated from those contaminants that accompany it in its natural state from the contaminants produced or used in the process of obtaining the antibody. This term further includes the desired product with a single glycosylation state, whether or not that state includes glycosylation at a single location or multiple locations. Typically, the antibody is substantially pure when it constitutes at least 6.0% by weight of the antibody in the formulation. For example, the antibody in the preparation is at least about 736 em. in certain embodiments at least about 80%, in certain embodiments about 85%, in certain embodiments at least about 90%, in certain embodiments at least about 95%, and more effectively, at least about of 99% by weight of the desired antibody. A substantially pure antibody includes an antibody produced not to an Intent, either recombinantly or synthetically.
As used herein, "therapeutically effective amount" refers to an amount that results in an amelioration or reduction in the symptoms of the disease or condition.
Antigens useful for attachment as a tag to a modified fucosylated or non-fucosylated core glycoprotein of the present invention and more preferably an antibody or fragment thereof may be a foreign antigen, an endogenous antigen, its fragments or variants having the same functional activity.
[0D71] As used herein, "endogenous antigen" refers to a protein or part thereof that is naturally present in the animal recipient cell or tissue, such as a cellular protein, an immunoregulatory agent, or a therapeutic agent.
As used herein, "foreign antigen" refers to a protein, or a fragment thereof, which is foreign to the recipient animal cell or tissue, including, but not limited to, a vital protein, a protein. of the parasite, an immunoregulatory agent or a therapeutic agent.
The foreign antigen may be a protein, an antigenic fragment, or antigenic fragments thereof that originate from viral and parasitic pathogens.
Alternatively, the foreign antigen may be encoded by a synthetic gene and may be constructed using standard recombinant DNA methods; the synthetic gene can express antigens or parts thereof that originate from viral and parasite patαgenαs. These paragens can be infectious in human hosts, domestic animals and wild animals.
The foreign antigen may be any molecule that is expressed by any viral pathogen or parasite, prior to or during entry, colonization, or replication within the host animal.
Viral pathogens from which viral antigens are derived include, but are not limited to, orthomyxoviruses such as influenza virus (Taxonomy ID: 59*771); retroviruses such as R5V, HTLV-1 (Taxonomy ID: 39015) and HTLV-II (Taxonomy ID: 11909); Herpes virus, such as EBV (Taxonomy ID: 10295), CMV CID Taxonomy: 10359) or herpes simplex virus (ATCC No.: VR-1487); Lentiviruses, such as HIV-1 (Taxonomy ID: 12721] and HIV-2 (Taxonomy ID: 11709); Rabdoviruses, such as rabies; Picotnoviruses, such as polioviruses (Taxonomy ID: 12080); Poxviruses, such as vaccinia (Taxonomy ID: 10245); Rotavirus (Taxonomy ID: 10912); is Parvoviruses, such as adeno-associated virus 1 (Taxonomy ID: 85106).
Examples of viral antigens include, but are not limited to, the human immunodeficiency virus Nef antigens (National Institute of Allergy and Infectious Disease HIV, Deposit Cat. No. 1'83; Nα GeπBaπk AF238278), Gag , Eπv (National Institute of Allergy and Infectious Disease HIV, Deposit Cat. No. 2433; No. GenBank. Ü39362), Tar (National Institute of Allergy and Infectious Disease HIV, Deposit Cat. No. 827; ° GeπBank ML3137), Rev (National Institute of Allergy and Infectious Disease HIV, Deposit Cat. No. 2088; M° GeπBank L14572)y Pol (National Institute of Allergy and Infectious Disease HIV, Deposit Cat. No. 238 GenBank No. AJ237568) and T and B cell epitopes of gp120i the hepatitis B sdperfiαi-e antigen (GeπBank No. AE043578); rotavirus antigens such as VP4 (GenBank No. AJ293721) and VP7 (GenBank No. AY003871); influenza virus antigens such as hemagglutinin (GenBank No. AJ404627); ucleop.ro tel na (ND GenBank AJ289B72); and herpes simplex virus antigens such as thymidine kinase (GenBank Accession No. AB0473781.
Bacterial pathogens from which bacterial antigens are derived include, but are not limited to, Hycαbactorium spp., Helicobacter pylori, Salmonella spp., Shigella spp., E. poly, Rickettsia spp., Listeria spp. Legionella pneumoniae, Pseudomonas spp., Vibrio spp. and Bore-Ilia burgdorferi.
Examples of protective antigens from bacterial pathogens include the enterotoxigenic E. coli somatic antigens, such as the fimbrial CFA/I antigen and the non-toxic B subunit of the heat-labile toxin; pertactin from Bordetella pertussis, adenylate cyelase-hemolysin from B. pertussis, a fragment C of tetanus toxin from Clostridium tetani, OspA from Horellia burgdorferi, paxacrystalline proteins on the surface of the protective layer of Rickettsia properektypi and Rickettsia . , listeriolysin (also known as "blo '*e" f-ily *'l and/or the superoxide dismutase (also known as ''SOD "e" p60 ") from Listeria monocytogenes, urease from Helicobacter pylori, and the domain: of binding to the lethal thorin receptor and/or the protective antigen of Bacillus anthrak.
Examples of biological weapons antigens or pathogens include, but are not limited to, smallpox, anthrax, tularemia, plague, Listeria, brucellosis, hepatitis, vaccinia, mycobacteria, coxsackie, tuberculosis, malaria, ehrlichosis and bacterial meningitis.
The pathogenic parasites from which the parasite antigens are derived include, but are not limited to, Plasmodium spp., such as Plasmodium falciparum (ATCC nD:- 50145},* Trypanosome spp., such as Trypanosoma cruzi (ATCC no.: 50797]; Glarelia .spp, such as Giardia intestinalis [ATCC no.: 30888D); Boophilus spp., Babesia spp., such as Babesia microti (ATCC no.: 30221); spp., such as .Entamoeba histolytica (ATCC no: 30015); Eimeria spp., such as Èimerià maxima (ATCC no. 40357).; Leishmania spp., (Taxonqmia ID: 38568); Schistosoma spp., such as Schistosoma mansoni (GenBank No. AZ301495); Brugia spp. such as Brugia malayi (GenBank No. BE352806]; Pascida spp. such as Fasciola hepatica (ND GenBank AF2B6903); Dirofilaria spp. such as Dixofliaria immitis (GenBank AF No. AF2B6903); '008300).; Wuchereria spp., such as Vochereria bancrofti IN* GenBank AF250'996); and Onchocerea spp; as Onchocerca volvulus (GenBank No. BE588251], .
Examples of parasite antigens include, but are not limited to, pre-erythroocyte stage antigens of Plasmodium spp. , such as the circumsporozoite antigen of Plasmodium falciparum GenBank.M22982) P. vívaA* (GenBank Accession No. M20670); Plasmodium sppt liver stage antigens such as liver stage 1 antigen (as referred to as LSA-1; GenBank No. AF0B6802); antigens of the merotoite stage of Plasmodium spp; such as merozoite surface antigen 1 (also referred to αomα MSA-1 or MSP-1; Na GenBank AF19941D); surface antigens from Entamoeba histolytica, such as galactose-specific ILlectin (GenBank Accession No. M59850] or the serine rich protein from Entamoeba histolytica; surface proteins from Leishmania spp, such as 63 kDa glycoprotein (qp63j from Leishmanis major (GenBaπk No. Y00647} or 46 kDa glycoprotein (gp46) from Leishmania major; paramyosia from Erugia malayi (bJ° GenBaπk. U77590], triose-phosphate isomerase from Schistosoma mansoni (GenBaπk No. W.06781], a). secreted globing-like protein from Triohosrrangy Ius colubriformis (Nα GenBank M63263); the glutathione-S-transfers from Fasciola hepatica (GenBank No. M77682); Schistosoma bovis IN"3 GenBank M77682J; S. japonic.um (GenBank No. M77682); U58012.) and KLH from Schistosoma japαnicum and S. bovis (Bashir, et al, supra)..
[0083] Examples of tumor specific antigens include prostate specific antigen (PSA), TAG-72 and CEA; human cyrosinase (ND GenBank M27160); tyrosiase-related protein (also referred to as TRP;GenBank All3293.3); and tumor-specific peptide antigens.
Examples of transplant antigens include the CD3 molecule on T cells and histocrapatibility antigens such as HLA A, HLA-B, HLA-C, BLA DR and HLA.
[0085] Examples of autoimmune antigens include the IAS β chain, which is useful in therapeutic vaccines against autoimmune encephalomyelitis (Na GenBank DB8762); glatamic acid decarboxylase, which is useful in therapeutic vaccines against insulin-dependent type 1 diabetes (No GenBank NMDI3445).; thyrottophine receptor (TSHR), which is useful in therapeutic vaccines against Grave's disease (Na GenBank NM0D0369) and tyrosinase-related protein 1, which is useful in therapeutic vaccines against vitiligo CN° GenBank MMOÚ'0550) .
Anti-HIV drugs that can be used to construct the antibodies or labeled fragments thereof include, but are not limited to, anti-viral agents, such as F.T nucleoside inhibitors, inhibitors/antagonists
CCR5 inhibitors/antagonists such as SCH-C, SCH-D, PRO 140, TAK 779, TAK-220, RANTES analogues, AK602, UK-427, 8.57, monoclonal antibodies; and viral entry inhibitors such as Fuzeon (T-2D) (enifuvirtide), NB-2, NB-64, T-649, T-1249, C-SCH, SCH-D, PRO 140, TAK 779, TAK - 220, analogues of RANTES, AK602, UK-427, 857; and functional analogues or their equivalents.
It is believed that many different fucosylated core glycoproteins and non-fucosylated glycoproteins can be modified in accordance with the methods of the present invention, or used as a therapeutic agent for conjugation to a terminal sugar, including but not limited to a, adrenocorticotropic hormone (ACTH); adrenocorticotropic hormone derivatives (eg, ebitatide); angiotensins; of angiotensins II; asparaginase; atrial natriuretic psiprids; atrial sodium diuretic peptides; bacitracin; beta-endorphins; blood coagulation factors VII, VIII and IX; blood thymic factor (FTS); blood thymic factor derivatives; bombesin; bone morphogenic factor (BMP); bone morphogenetic protein; bradykinin; cerulein; calcitonin gene polypeptide (CGRP); calcitonins; CCK-8; cell growth factors (e.g., EGF; TGF-alpha; TGF-beta; FDGF; acidic FGF; basic FGF); cerulein; chemokines; cholecystokinin; cholecystokinin-8; cholecystokinin-pancreozymine (CCK-FZ); colistin; colony stimulating factors (eg CSF; GCSF; GMCSF; MCSF); corticotropin releasing factor (CRF); cytokines; desmopressins; dynorphin; dipeptide; dismutase; dynorphin; eledoisin; endorphins; endothelin; endothelin-antiagonist peptides; endoterins; enkephalins; enkephalin derivatives; epidermal growth factor (EGF); erythropoietin (EPO); follicle-stimulating hormone (FSH); galanin; gastric inhibitory polypeptide; Gastrin Releasing Polypeptide (GRP); gastrins; G-CSF; glucagon; glutathione peroxidase; glutathione peroxidase; gonadotropins (for example, human chorionic gon.adotrotin and alpha and beta, subunits thereof); gramicidin; gramiedines; growth factor (EGF); growth factor releasing hormone [GRD; growth hormones; hormone releasing hormone (LH.RH) ; human arterial natriuretic polypeptide (h-ANP); human placental lactogen; insulin; insulin-like growth factors (IGF-I; IG'F-11); interferon; interferons (for example, alpha-beta and gamma-interferons); interleukins (for example 1, 2, 3, 4, 5, 6, 7, 8, 9, ID, 11 and 12); intestinal polypeptide (VIP); kalicrein; chyotorphyria; luliberin; luteinizing hormone (LH); lαteiπizaπte hormone-releasing hormone (LH-P.H) ; lysozyme chloride; melanocyte-stimulating hormone (MSH); stimulating hormone melanophore; mellitin; matiline; muramyllic; mutamildipeptide; nerve growth factor (NGF) ; nerve nutritional factors (eg, NT-3; NT-4; CNTF; GDWF; BDNF); neuropeptides Y; heurotensin; oxytocin; pancreastatin; pancreatic polypeptide; pancreozymine; parathyroid hormone (PTB); pentagastrin; polypeptide YY; pituitary cyclase activating polypeptides (adenyl PACAPs); growth factor-derived platelets; polymyxin B; prolactin; polypeptide that stimulates protein synthesis; PTH-related protein; relaxin; renin; secretin; serum thymus factor; somatomedins; somatostatins derivatives; superoxide dismutase; taftsin; tetragastrin; thrombopoietin (TPO); thymic humoral factor (THF); thymopoietin; thymosin; thymostimulin; thyroid hormone releasing hormone; thyroid stimulating hormone (T.SH) ; thiotrophin-releasing hormone (TRH1 ; trypsin; tuft-sin; tumor growth factor (TG.F-alpha) ; tumor necrosis factor (TNF); thyrocidin; urogastrone; urokinase; vasoactive intestinal polypeptide; and vasopressin.
Fucosylated and non-fucosylated core glycoproteins are important classes of biomolecules that play a crucial role in many biological events, such as cell adhesion, tumor metastasis, pathogenic infections, and immune response. As indicated hereinbefore, one of the main problems in structural and functional studies of fucosylated or non-fucosylated glycoproteins is their structural micro-titerogeneity. Natural and recombinant fucosylated or non-fucosylated glycoproteins are generally produced as a mixture of glycoforms that differ only in structure from the pendant oligosaccharides.
Remodeled glycoproins such as antibodies can be subjected to other structural modifications that are necessary or desired, including, without limitation, glycosyl transfer, and selective binding (eg, click chemistry, Staudinger reaction, etc.) to introduce additional functional groups or labels. Functional groups can be of any suitable type, including, without limitation, toxins, specific antigens (such as alpha-Gal), radioactive species, photoactive species, PEGS, etc. The glycoprotein can be catalytically tagged in a cycloaddition reaction. of "click chemistry" of the azide functionality of the glycoprotein with an alkyne having the functional portion of interest. The azide and alkyne functional groups can be linked into the respective binding components, and the glycoprotein can be functionalized with an alkynyl functionality and reacted with an azide-functionalized compound, including the portion of interest. It will also be appreciated that other bonding pairs can be designed to make the chemical reaction click.
Fucosylated core antibodies or non-fucoslylated antibodies or fragments thereof, produced according to the methods described herein, can be used for diagnosis and therapy. About two-thirds of therapeutic proteins. such as the monoclonal antibodies used on the market and/or currently in clinical trials are glycoproteins. However, structural heterogeneity in different glycoforms of natural and recombinant glycoproteins presents an important barrier in the development of glycoprotein-based drugs, as different glycoforms can have different biological activities and controlling the glycosylation of a homogeneous glycoform is extremely difficult during expression . The earlier discovery of the transglycosylation activity of a class of endoglycosidases represents a major advance in the field of glycosylation engineering to improve the therapeutic and diagnostic potentials of glycoproteins and the Endo-S mutants of the present invention are capable of transglycosylate fucosylated core glycoproteins and non-physical linked natural and recombinants without the negative aspects of hydrolysis.
[0092] The features and advantages of the present invention are more fully illustrated through the following, non-limiting examples.
[0093] Generation die Mutants EndoS Glycosyntase and its use for Remodeling of Intact Monoclonal Antibody Rituiximab Glycosylation
[0094] Glycosynthases have previously been made from various GH85 endoglycosidases (ENGases), including endoA, endoM, and endoD> by site-directed mutagenesis of a key asparagine (Asn) residue responsible for the promotion of intermediate oxazolinium ion formation during hydrolysis (36.-39, 43) is an endoS endoglycosidase belonging to the family of glycoside hydrolases 18 (GHIL, (40/41), which is of the same GH family as EndoF1, EndoF2, and EndoF'3 which have been recently shown which have transglycosylation activity (44), Based on the assumption that EndoS catalyzed hydrolysis also undergoes a substrate-assisted mechanism involving the formation of an oxazolinium ion intermediate, as demonstrated by other GH18 endoglycosidases, such as EndoF3, ( 45) potential endoS glycosynthases were created through the identification and mutation of the residue responsible for promoting the formation of oxazolinium ions. Structural and previous mutagenesis studies res on EndoFl demonstrated that an aspartic acid residue at position 165 (D165), instead of an as.paragine residue as in the GH85 family enzymes, is responsible for promoting the formation of oxaulin and that the residue El67 is the general acid/base for catalytic hydrolysis [45J,. Alignment of EndoS sequences with EndoF3 (Figure 2) led to the identification of two key residues in EndoS for catalysis: the residue D233 (corresponding to D165 in EndoF3) responsible for promoting the formation of oxazolinium ions and the residue E235 ( equivalent to E167 of RndoFJ) as the general acid/base residue in the glycan hydrolysis as shown in Figure 2 . Functionally, residue D2 33 must also be equivalent to N171, N175f, and N322 nas, endoglycosides GU.B5, éndoÃ, endoM, and EndoD, respectively. Thus, following the approach, for the creation of endoA/endoM, and EndoD g 1 icosynases that proceed in a substrate-assisted mechanism via an io.n oxazolinium intermediate, (36-39) two specific murants, D233A (SEQ ID NO: 2) and D233Q (SEQ ID NO: 3), as shown in Figure 17, were generated by EndoS site-directed mutagenesis (SEQ ID NO: 1). These mutants, as well as wild-type EndoS, were expressed in Escherichia coll in high yield (30-40 mg/L) as a GST fusion protein and purified by means of glutathione affinity chromatography.
Rituximab, a therapeutic monoclonal antibody, was used as a mAb model to examine the deglycosylation activity and potential transglycosylation activity of the enzymes. The major commercial Fc die rituximab glycans are complex-type biantenar fucosylated core oligosaccharides carrying 0-2 portions of galactose, named glycophorites G0F, GIF and G2F, respectively, as revealed by laser-assisted desorption and ionization mass spectrometry analysis by time-of-flight matrix (MALDI-TOF MS.) of the N-glycans released by PNGase F, as shown in Figure 10. Treatment of rituximab with the GST-EndoS fusion protein (herein referred to as wild-type EndoS or EndoS) resulted in a rapid deglycosylation to obtain the corresponding Fc N-glycans (with only one GlcNAc at the reducing end), as shown in Figure 11, and the deglycoinated rituximab which carries the fucosylated GlcNAc disaccharide moiety (Fucal, GGlcNAc) at the glycosylation sites (N297). These results confirm the remarkable Fc glycan hydrolysis activity of wild-type endoS to intact IgG, implying its usefulness in the first step for remodeling the glycosylation of mAbs. The transglycosylation potential of EndoS and its mutants then was examined using deglycosylated rituximab as the acceptor and various synthetic glycan oxazolines as the donor substrates, as depicted in Figures 3 A and B. The glycosylation remodeling process was monitored by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-^ PAGE) and liquid chromatography mass spectrometry (LC-MS), as shown in Figure 4. Rituximab heavy chain and light chain appeared at about 50 kDa and about 25 kPa, respectively, under reducing conditions (a, lane 1, in Figure 4AJ After deglycosylation with wild-type EndoS, the heavy chain appeared as a single band at approximately 48 kDa, suggesting that removal of the two N-g lycans (each from a heavy chain) of rituximab (a, lane 2, in Figure 4A). Incubation of the deglycoβylated rituximab (1) and the synthetic sialoglycan oxazoline (2) (see Figure 3A for structures) (donor/acceptor molar ratio, 50:1) with the En.dos-D233A mutant gave a transglycosylation product (3) , whose heavy bitch appeared as a single band that was about 2 kDa larger than the do. deglycosylated rituximab (1) (a, blink 3, Figure 4A). This result suggests that a new N-glycan was linked to each of the Fc heavy chains. Incubation of (1) and (2) Endas-D.233Q cam gave the same transglycosylation product (a, flash 4, Figure 4A ) . Interestingly, an essentially quantitative transglycosylation for the Fc domain of the intact antibody was achieved within 1 h of incubation. It was noted that an incubation time (10 h) does not lead to hydrolysis of the transglycosylation product. These results indicate that the two EndoS mutants are effective new glycosynthases that allow glycosylation of deglycosylated intact IgG with the complex-type N-glycan, without product hydrolysis.
Transglycosylation was further characterized by LC-MS analysis. Rituximab heavy chain and light chain were separated under an LCHMâ condition, as shown in Figure 12. Deconvolution of the Light chain MS data gave a mass of 23,044, which was consistent with the calculated mass of rituximab light chain (M = 23,042 Da} (47). , as shown in □ graph B, in Figure 4 A, which were in good agreement with the theoretical mass of heavy chain glycoforms; GQF, M = 50 515 Da; GIF, M = 50 677 Da; and G2F, M = 50 839 Da ; respectively. (47) Deconvoluted electron spεay ionization mass spectrometry (ESI-MS) of deglycosylated rituximab heavy chain (1) showed a single species of 491 420, as shown in graph C in Figure 4B, which matches well with a heavy chain carrying a Fucαl,βGlcNAc disaccharide moiety (calculated, M ~ 49 420 Da). In glycosylation remodeling, a single peak at 51426 was observed from the heavy chain of the transglycosylation product (3) with an addition of 2.006 Da to the deglycosylated rituximab heavy chain, as shown in graph d in Figure 4B. This result indicates the attachment of an oxazedin sialoglycan corresponding to the sugar (2) to the heavy chain, there is a single band on SDS-PAGE and pure MS spectra of the transglycosylation product suggest <21 clearly that the transglycosylation was essentially quantitative over the two sites of glycosylation of the Fc domain in rituximab (incomplete glycosylation of either of the two Fc homodimer sites would result in the observation of Fucol heavy chain, 6Glc'NAc after reduction, M - 49 420 DaJ . To further confirm that the W-glycan was . specifically linked to the GlcNAc of the Fc domain, the set of M-glycans were released from the glycα-remodeledα rituximab (3) by txatam.enta cam PNGase F, which specifically hydrolyzes the amide bond between the Asn-glycan bond. Released N-glycans were labeled by 2-aminobenzaldehyde (2-AB) fluorescent labeling and were subjected to high performance liquid fluorescence chromatography (HPLCJ and MS analysis. LC-MS analysis revealed c. Clearly, the released N-glycan was expected to be the N-glycan type, biantennary complex carrying fucose core and the terminal ialic acids, which consisted of about 92t of disialylated N-glycans and about about monosialylated N-glycans, as shown in Figure 13b. The composition of N-glycans was very consistent with the relationship found in the oxazoline N-q.lycan corresponding to C2) used for the tian.sgliaosuation. This result confirms that the transfer of N-glycans was specifically linked to the GlcNAc primer in deglycosylated rituximab.
[0097] The results presented in this document represent the first glycosylation remodeling report of an intact IgG monoclonal antibody, with an en-block transfer of a full-length complex-type natural N-glycan to the Fc domain through a protocol, degl Highly efficient glycosylation-reglycosylation enabled by the combined use of EndoS and EndoS-based glycosynthase. After completion of transglycosylation, the product was purified by means of a simple protein A affinity chromatography, giving the well-defined homogeneous glycoform. It should be noted that commercial rituximab contains only traces of stalllated glycoform, as shown in Figure 13a. Since sialylated Fc and IqG have been proposed to have anti-inflammatory activity, glycoengineered modified rituximab carrying fully sialylated Fc N-glycans may have an anti-inflammatory function, thus potentially expanding its therapeutic coverage of cancer treatment to the treatment of autoimmune diseases. (31, 22)
In addition to the sialylated complex-type oxazoline N-glycans (2), the Endcis mutants were equally efficient in using the core oxasolin of Ma u 3 Glc 5'Ac (4) (48] and the N3Pian3GlcNAc-tagged oxazoline with azide (6) (49) to glycoengineer rituximab, leading to the formation of the corresponding homogeneous glycoforms, (5) and (7), respectively, as shown in Figure 3A. transglycosylation [5J showed a single species of 50 112, as shown in the Lco graticum and in Figure 4C, which corresponded well with the calculated molecular mass (M - 50 109 Da) of the heavy chain of rituximab carrying a Man3GlcNAe2 glycan. The deconvoluted ESI-MS of the transglycosylation product heavy chain (7) showed a single species of 50 143, as shown in graph f in Figure 4C, which was in good agreement with the calculated molecular mass (M = 50 134 Da) gives rituximab heavy chain carrying an M3Man3GlcNAc2 glycan. Indeed, these results indicate that transglycosylation. it is essentially quantitative. It should be noted that decreasing the donor/acceptor molar ratio of 25:1 still resulted in efficient transformation, implying the remarkable transglycosylation efficiency of the endoS glycosynthase mutants. In particular, selective introduction of azidan functionality into the Fc core of N-glycans in intact monoclonal antibodies will allow for more site-specific modifications of antibodies through clique chemistry, (50, 51) which can be used for labeling and of targeting, cu to expand the diversity of antibody glycoforms for structure-activity relationship studies.
[0099] EndoS from. wild type was also tested for transglycosylation of rituximab deglycosylate (1) with the oxazolines glycan (2 and 4) under the same conditions as the EndoS mutants and it was observed that only transient formation of the corresponding transglycosylation products was found as monitored by LC-MS, probably due to the rapid in situ hydrolysis of the products by the wild-type enzyme. Recently, Scanlan, Davis, and colleagues reported an independent study on the substrate specificity of EndoS and demonstrated that wild-type EndoS could use Man3GlcNAc oxazoline for efficient transglycosylation of desqlicosylated IgG. (42) To address this apparent discrepancy in observations, the transglycosylation efficiency of wild-type EndoS was re-evaluated at a lower temperature (4°C) using a much smaller amount of enzyme, following the. recent report (42] Using this modified condition, transglycosylated Significant was observed for desglycosylated rituximab (1) with the oxazoline sugar complex (2) by wild-type endoS in the initial incubation period, but the product was gradually hydrolyzed when a, incubation continued, as shown in Figure 14. Thus, the reaction condition must be carefully controlled in order to trap the transglycosylation product when wild-type EndoS is used. For practical application, EndoS glycosynthase mutants should be the choice for efficient and complete transglycosylation, since they are devoid of hydrolytic activity product.
[00100] Rituximab glycoengineering to provide Non-fucosylated and galactosylated G2 glycoform
[00101] For anticancer therapy, non-fucosylated IgG glycoforms are desirable, since it was previously shown that mAbs with low fucose content of FC N-glycans showed improved ADCC activity in vitro and greater anticancer efficacy in vivo, especially for those patients carrying the low affinity F158 allele of the FcYIIIa receptor. (16-19, 52) in the available efficient method to efficiently transform an existing fucosylated mAb (the major glycoform of recombinant mAbs produced in mammalian cells) to a non-fucosylated mAb. To solve this problem, a number of commercially available cr-fucosidases have been tested, but none can remove the cri,õ-fuccse in intact rituximab, see schematic in Figures 5 A and B. These results imply that the «1,6 portion β-fucose can be protected by the Fc domain and/or the complex N-glycan, making them inaccessible to α-fucosidases. It has been theorized that, after deglycosylation, the Fuc(al,6}GlcNAc glycoform resulting from rituximab might be more accessible to ct-fuc.osidases. Therefore, the activity of several commercially available or-phycosidases was tested in deglycosylated rituximab (1 ). ) which carries only the Fuc(al,6) GlcNAc moiety. An unspecific bovine kidney α-fucosidase was found to have moderate activity and was able to remove the fucose residue from the deglycosylated rituximab (1) to give the rituximab containing GlcNAc (B) (see Figures 15 A and B) Although a relatively large amount of a-fucosidase and a prolonged reaction time were required to achieve complete defuosylation of rituximab EndioS-deglycylated due to moderate activity than α-fucosidase, the discovery of this α-fucosidase activity provides an alternative way to obtain the defucosylated precursor rituximab (â) for further glycoengineering.
[00102] Next, it is determined that EndQS-D2 33A and Endq5-D233Q glycosynthases were also efficient to recognize the non-fucosylated GlcNAc in (8) for transglycosylation with a sialylated oxazoline N-glycan (9) to provide the glycoform Homogeneous, non-fucosylated G2 (10) in an essentially quantitative conversion, Figure 5. The product was purified by protein A affinity chromatography. , as shown in Figure 6. Defuαosylated rituximab (8) showed a single species of 49,224 (Figure 6b), which confirms the removal of fucose (calculated for GlcNAc-rituximab heavy chain, M = <9 9 274 Gives) . The deconvoluted ESI-MS of the transglycosylation product heavy chain (10) appeared as a single species of 50,695 (Figure Sc), which corresponded well with the calculated molecular mass (M = 50,693 Da] of rituximab heavy chain carrying a biantennary asialylated complex type N-glycan, Gal2.GlcNAc2Man3GlcNAc2. In a comparative study, it was also found that although the D233A and D233Q mutants recognized both fucosylated GlcNAc-rituximab (1) and non-fucosylated GlcNAc-rituximab ( 3) as acceptors for transglycosylation, the two glycosynthase mutants preferred the fucosylated GlcNAc-GlcNAc-rituximab (1) as the acceptor, with a faster transglycosylation reaction than the non-fucosylated acceptor (8) (data not shown). Taken together, these experimental results revealed a combined enzymatic approach to make the homogeneous non-fucosylated and fully galactosylated glycoform from commercially available monoclonal antibodies. The resulting ado and galactosylated would have improved AD.CC and CDC effector functions, as suggested in earlier studies. (2, 16-20, 52)
[00103] IVIG site-specific Fc glycoengineering to provide sialylated all-Fc IVIG glycoforms
[00104] The successful glycosylation remodeling of rituximab has led to the examination of the chemoenzymatic method for glycoengineering of IVIG with the aim of increasing its anti-inflammatory activity. IVIG are pooled IgG fractions purified from the plasma of thousands of healthy donors. Recent studies have suggested that a smaller, α2,6-sialylated Fc glycoform is the active species in IVIG that confers anti-inflammatory activity, as demonstrated in a rat model of rheumatoid arthritis. (21, 22, 53, 54] Since sialylated Fc glycoforms are minor components in IVIG, (55), the dependence of anti-inflammatory activity of IVIG on terminal Fc sialylation may explain in part why a high dose ( 1-2 g/kg) of IVIG infusion is required to provide protection Direct sialylation of Fc and IVIG has been attempted using human ofl,,6-sialyltransferase (ST6Gal-I), but efficiency was low, and mostly Of these cases, only monosialylated glycoforms were obtained as major products.(22, 56) In addition, about 30 µl of the FAB domains in IVIG are N-glycosylated and the enrichment of the sialylated Fc glycoforms of IVIG would be less effective when FAB glycans are sialylated.(2, 57) Therefore, it would be highly desirable if Fc-specific glycoengineering with sialylated N-glycans could be achieved without altering FAB glycosylation.
endoS was found to be kappa.Z: to selectively deglycosylate the Fc domain of IVIG without hydrolyzing the N-glycans to the FAB domains under mild conditions. In addition, the Fc domain of deglycosylated IVIG (11) could be selectively glycosylated with a s la log 11 oxazoline (2) by the EndoS-D233.Q mutant to give the fully sialylated IVIG Fc 112), as shown in Figure 7 Glycoengineering was first monitored by SDS-E'AGE analysis. Deglycosylation and reglycosylation of IVIG were apparent as shown in the change in heavy chain band size, as shown in Figure 16. To further characterize the site selectivity of the glycoengineering of IVIG, the Fab and Fc domains were disconnected by papain digestion. (58) The Fc domain was isolated by protein A affinity chromatography and the FAB domains left in the flow-through were isolated by size exclusion chromatography in the fast protein liquid chromatography (FPLC) system. Then, the N-glycans of Fc and FAB were released separately by treatment with PNGase F, labeled with 2-aminobenzamide (2-AB), (59) and analyzed by HPLC (fluarescent detection and quantification) and characterization by MS. The FAB and Fc N-glycan profiles before and after glycoengineering of IVIG were shown in Figure 8. Fc glycosylation patterns of 1V1G were found to be more complex than monoclonal antibody Fc glycosylation with rituximab. In addition to the GO.F, G1F and G2F glycophoxmas as. the major components, there was a significant amount of monosialylated glycoforms (peaks 2 and 7) (approximately 10%) and glycoforms containing bissectional GlcNAc (peaks 13-15) (51) (Figure 8a). Fc glycosylation after glycoengineering (through EndoS-deglycosylation and further transglycosylation with sialoglycan oxazoline (21 by EndoSl-D233Q) showed the fully sialylated glycans (peaks 1 and 6) as the major glycoforms (>90ij (Figure 8b). Interestingly, FAB glycosylation patterns were similar before and after the glycoengineering process (compare with figure â, c and d) except for the generation of a small amount of the fully allylylated glycoform (peak 6).These results indicate the glycosylation remodeling process. EndoS-based is highly selective for Fc N-glycans of intact IgG antibodies, even in the presence of FAB glycosylation. Remarkable selectivity and high efficiency of current Fc glycoengineering approach provide a new pathway to transform commercial IVIG in a fully Fc-sialylated IVIG preparation that is expected to exhibit increased anti-inflammatory activity, as demonstrated in previous studies using a mould. mouse delo. (21, 22, 53, 54)
[00106] Binding of Glycoengineered Modified Rituximab to Stimulatory Fcy Receptor (FcYRIIIa) and Inhibitory Fcy Receptor (FcyRIlb)
The affinity of the remodeled rituximab glycoforms for the respective receptors (FcYRHla-F158, FcRIITa-V158, and FcRIlb) was examined by surface plasmon resonance (SFR) analysis. The glyc-O forms of rituximab were site-specific immobilized on protein chips and Fey receptors at various concentrations were injected as analytes, following recently reported procedures (35). As rough, the non-fucosylated G2 glycoform showed significant improvement in affinity for both the high affinity and low affinity Fcylfla receptors, FcRIlIa-F15B and FcRIlIa-Vl58. when compared to the commercially available rituximab as shown in Figure 9. The Kd values for G2 glycoform binding (10) for FcRIIla-F158 and FcRIIIa-V15B were 123 t 11 and 12 i 2 nM, respectively, which were obtained by fitting the binding data with a 1:1 steady state model using the BIAcore TIDO evaluation software. On the other hand, the. KD values for binding of commercial rituximab to FcyRIIIa-F'158 and FcyRIIIa-V15B were estimated to be 1042 1 155 and 252 i 18 nM, respectively. Thus, the affinity of the glycoengineered-modified G2 glycoform for low-affinity and high-affinity Fc receptors (FcRIIa-Fl58 or FcyRllIa-Vl58) was about 9-fold and 20-fold greater than commercial rituximab, respectively. . On the other hand, the G2 glycoform and commercial rituximab demonstrated comparable affinity for the Fcy receptor FcyRIIb inhibitor with KD values of 2.3 ± 0.5 and 2.0 ± 0.7 μM, respectively. These results reveal a clear gain of beneficial functions for the glycoengineered modified rituximab. It should be noted that an efficient preparation of high-affinity binding FcYRIIa glycoforms is clinically significant to address the issue of 'Fcy receptor polymorphism found in cancer patients that are at least or not responsive to treatment with common monoclonal antibodies. the FcyBIHa-Fl&S allele has a low affinity for therapeutic mA.bs', such as rituximab, compared to the high affinity receptor, the FcyRIIIa-V158 allele. (52, 60, 61), Fcy receptor-mediated effector functions have also been suggested to be an important mechanism for achieving protective immunity from neutralizing HIV antibodies, (62) Thus, the glycoengineering approach described in the present document may find wide applications in the production of various defined glycoforms of monoclonal antibodies valuable for functional studies as well. as for biomedical applications,
[00108] An efficient approach to chemoenzymatic glycoengineering of intact IgG antibodies is described herein. The two novel EndoS-based glycosynthes generated by the site-directed mutagenesis demonstrate broad substrate specificity capable of transferring sialylated and asialylated and complex-type N-glycans, as well as core N-glycans selectively modified from oxazoline glycans corresponding to intact Fc antibodies -deglycosylated. Furthermore, the deglycosylation/reglycosylation approach is effective for both fucosylated and non-fucosylated core IgG antibodies when an α-fucosidase is properly combined. These discovered notes significantly increase the scope of the chemoenzymatic method and make possible an efficient transformation of the intact monoclonal antibodies into a number of well-defined glycoforms which are hitherto difficult to obtain by existing methods. It is hoped that this glycoengineering approach can facilitate the development of biosimilar and/or bioimproved biological products that have therapeutic efficacy and/or obtain new functions. Materials and methods
Rituximab monoclonal antibody (rituxan, Genentech Inc., South San Francisco, CA) and IVIG were purchased from Premium Health Services Inc. (Columbia, MD). S-ialoglycan oxaline (2) and complex asialoglycan-type die oxalolin (5J were synthesized following procedure reported ant ericr nienLee.(38, 46) Bovine kidney α1-fucosidase was purchased from Sigma (St. Louis , MO) and Prozyme (Hayward, CA ) Arthrobacter protophonniae endo-PN-acetylglucosaminidase (endoA) and Mu cor hiemalis endo-PN-acetylglucosaminidase (endoM) and their mutants were overproduced in E. coli following reported procedures (38) PNGase F was purchased from New England Biolabs (Ipswich, MA).
[00110] Liquid Chromatography of Mass Spectrometry (LC-M5)
[00111] The LC-MS was performed on an LXQ system (Thermo Scientific) with a Hypersil GOLD column (1.9 mμ, 50 x 2.1 mm,). IgG samples. were treated with 0.5% β-mercaptoethanol and heated at .60 °C for 15 min, then subjected to LC-MS measurement. The analysis was carried out at 60°C, eluting with a linear gradient of 1040% MeCN containing 0.1% formic acid in 10 min at a flow rate of 0.25 ml/min.
[00112] Electron Spray Ionization Mass Spectrometry (E5I-MS) and Time of Flight Matrix Assisted Laser Desorption and Ionization Mass Spectrometry (MALDI-TOP MS)
[00113] ESI-MS spectra are measured on a single Waters Micromass ZQ-4000 quad mass spectrometer. The MALDI-TOF MS was performed on an Autoflex II MALDI-TOF mass spectrometer (Bruker Daltoni.cs, Billerica, MA). The instrument was calibrated using the ProteoMass Peptide MALDI-MS Calibration Kit (MSCAL2, Sigma / Aldirich). The 2,5-dihydroxybenzoic acid (DH.B) matrix was used for the neutral glycans and the 2’,4',61-trihydroxyαetofenpne (THAPJ) was used for the acidic glycans.
[00114] Over-expression and purification of EndoS and Mutants
[00115] Wild-type EndoS was overproduced in E, call and purified according to the procedures reported previously, (40, 63) using the plasmid pGEX-EndoS which was kindly provided by D_t, M. Collin (University of Lund , Sweden). The two EndoS mutants, D233A and D233Q, were generated using the GeneArt site-directed mutagenesis kit (Invitrogen) according to the manufacturer's instructions. Plasmid pGEX-EndoS was used as template, and LA Taq polymera.se (Takara, Japan) was used for PCR. Mutations were confirmed by DNA sequencing and transformed into BL21(DE3). Transformants were cultured in Luria-Bertani medium containing 100 mg/L carbenicillin and induced with 0.1 mM isopropyl-PD-tlogalactopyranoside for 16 h at 25°C. Cells were harvested by centrifugation at 1700g for 15 min at 4°C. Cell pellet was suspended in phosphate buffered saline (pH 7.4) with lysozyme and PMSF. The lysis mixture was centrifuged at 16000g for 20 min at 4°C. After centrifugation, cell lysis supernatant was applied to 3 ml of 50% glutathione-Sepharose 4B resin (GE Healthcare). Samples were incubated at 25°C for .60 min with gentle shaking. The resin was applied to a 10 ml column (PD-CO from GE Healthcare! and washed five times with PBS. 500 µL of glutathione elution buffer (50 mH Tris-HCl, 10 mW glutations, pH 8.0] was added to the column, incubated at room temperature for 5 min, collected, and then repeated three times. The eluted fractions were pooled and dialyzed against sodium phosphate buffer ( 50 mM, pH 7.0) overnight at 4° C. Protein samples were then concentrated using Amicon 10 kDa ultracentrifuge filters (Mill ipore). Concentrated protein samples were analyzed by SDS-PAGE, and protein concentration was quantified using a 2000c Nano-Urop spectrophotometer.The overproduction yield of wild-type endoS was approximately 40 mg/L, and the yield for mutants was approximately 30 mg/L.
[00116] Deglycosylation of Rituximab by wild-type EndoS to give (Euoal,6) GlcNAc-Rituximab (1)
Commercial Rituximab [20 mg) in Tris-Cl buffer (50 mM, pH 8.0, 2 ml) was In.cub.adó with EndoS (30 µg) at 37°C for 1 h. LC-MS and SDS-PAGE analyzes indicated complete cleavage of the N-glycans in the heavy chain. The reaction mixture was subjected to affinity chromatography on a column of protein A-agarose resin (5 ml) which was pre-equilibrated with a Tris-Cl buffer (20 mM, pH 8.0). The column was washed with Tris-Cl (20 mM, pH 8.0, 25 ml) and glycine-HCl (20 mM, pH 5.0, 20 MJ, successively. Bound IgG was released with glycine-HCl (100 mM) , pH 2.5, 20 ml), and the elution fractions were immediately neutralized with Tris-Cl buffer (1.0 M, pH 8.8) Fractions containing the Fc fragments were combined and concentrated by centrifugal filtration ( centrifugal filter Amicon Ultra, Millipore, Billerica, MA) to give (Fuc cri, 6) GlcNAc-rituximab |li (18 mg) LC-MS: calculated for (Fuc ct1.6) GlcNAc-ritux heavy bit. imab (1), M = 49 420 Da£ (47j found (m/z), 49 420 (deconvolution data).
[00118] Transglycosylation of (Fuçai, 6) GlcNAc-Rituximab Oil with Oxazoline Sialoglycan (2) by EndoS Mutants D.233A or D233Q
A solution of (Fucal,6) GlcNAc^rituximab (1) (.10 mg) and sialoglycan-oxaroline (2) (10 mg) in Tris buffer (50 µM, pH 7.4, 2 ml) was incubated with the EndoS mutant D233A or D233Q (200 pg) at 30°C. Samples were taken at intervals and analyzed by LC-MS. After 2-3 h, LC-MS monitoring indicated complete reaction of (Fuccx1.6) Gl cNAc-rituximab (1) to give the product. of transglycosylation (3) which carries the fully sialylated N-glycans. The reaction mixture was subjected to affinity chromatography on an agarose-protein A column according to the procedure described above. Fractions containing the product were combined and concentrated by ultracentrifugation to give rituximab si to Lillado (3) (11 mg, quantitative). LC-MS: calculated for the heavy chain from (3) bearing the fully sialylated N^glycan, M = 51,421 Da; found (m/z), 51 4 26 (deconvolution data).
[00120] Transglycosylation of (Fuco'1,6) GlcNAc-Ritux imab (1) with ManUGlcNAc oxazoline (4) and azide-labeled Man3GlcNAc oxazoline (6) by endo5-D233Q
[00121] Transglycosylation was performed as described for the preparation of (3J to give the corresponding products. LC-MS analysis of glycoengineered modified rituximab [5 and 7): calculated for the heavy chain of (5) that carries the Msn3 .GlcNAc2 N-fucosylated glycols, M = 50 109 Da; found (rti/z), 50 112 [deconvolution data); calculated for the heavy chain of (7] carrying the azido-Man3GlcMAc2 N-fucosylated glycan, M = 50 134 Pa; found (πi/z), 50 143 (deconvolution data).
[00122] Defucosylation of (Fucα1,6) GlcHAc-Situximab (1) by a bovine kidney fucosidase
[00123] Urea solution of (Fucα1.6l GlcNAc-rituximab (1) (2 mg) in phosphate buffer (50 mM, pH 5.5, 200 μl) containing 0.05 sodium azide was incubated with kidney fucosidase bovine (Frozyme, 5 U) at 37°C. Samples were collected at intervals and analyzed by LC-MS. After 20 days, follow-up LC-MS indicated complete defucosylation of (Fucofl,6l GlcNAc-rituximab (1) for give the product, GlcNAc-rituximab (2). The reaction mixture was subjected to affinity chromatography on a protein A column, following the procedure described above. The fractions containing the product were combined and concentrated by ultracentrifugation to give GlcNAc-rituximab (2) (2 mg, quantitative) LC-MS; calculated for the heavy chain of GlcNAe-rituxlrnab [2] bearing a GlcNAc moiety, M = 49 274 Da; found (m/z), 49 274 [data from deconvolution).
[00124] Transglycosylation of GlcNAc-Rituximab (4) with Asiallated Complex Type Oxazoline Glycan (5) p.or D233Q Mutant
[00125] A solution of Gl.cMAc—rituximab (4) [2 trig) and oxazoline (5) (5 mg) in Tris buffer (50 mM, pH 7.4, 0.5 mlr) was incubated with the endoS-D233Q (200 μg) at 37 "C. Samples were collected at intervals and analyzed by LC-MS. After 2 h, LC-MS monitoring indicated the co-complete reaction of 4 to obtain the corresponding transglycosylation product (6) , A Reaction mixture was subjected to affinity chromatography on a protein A column. Fractions containing the product were combined and concentrated by ultracentrifugation to give the non-fucosylated rituximab glycoform (6) (2 mg, quantitative). calculated for the heavy chain of (6) bearing the non-fucosylated N-glycan, M = 50 693 Da; found (m/z), 50 695 (deconvolution data).
[00126] Site-specific deglycosi on: Fc Domain deIVIG by EndoS
[00127] Commercial IVIG [20 mg) in Tris-Cl buffer (50 mM, pH 8.0, 2 ml) was incubated with EndoS (SEQ ID NO: 1} (30 pg) at 37°C for 1 h. The residue was subjected to affinity chromatography on a protein A column to obtain (Fucal, 6| GlcNAc-1GIV (20 mg, quantitative), in which the Fc N-glycans were removed, leaving the GlcNAc al, 6 -fucosylate. at Locations N297,
[00128] Transglycosylation of (Fucal,6) GlcNAc-IVIG with □xazQ.li.na Sialoglidane (2) by D233Q Mutant
[00129] A solution of ((Fucal,6) GlcNAc-IGIV (3 mg) and siaioglycan-oxazoline (2) (3 mg) in Tris buffer [50 mM, pH 7.4, 2 ml) was incubated with D23 3Q mutant (SEQ ID NO: 2) (60 pg), at 30pC. After 2 h, SDS-PAGE analysis indicated complete reaction of (Fucal>6) GlcNAc-TVIG to give the transglycosylation product. The reaction mixture was subjected to affinity chromatography on a protein A column to provide the glyco-reshaped IVIG (3 mg, quantitative), in which the Fc N-glycans were remodeled to the fully sialylated complex-type N-glycanus .
[00130] Surface plasmon resonance (SPR) binding experiments
The binding between the different IgG: and Fey receptor glycoforms was measured by surface plasmon resonance (SPR) using a Biacore T100 instrument (GE Healthcare, USA). A 5000 RU protein is immobilized on a CM5 biosensor chip (GE Healthcare) using standard primary amine coupling chemistry at pH 4.5 to capture the different IgG glycoforms. A reference flow cell was similarly prepared without protein A injection. Each individual IgG glycoform in HBS-P buffer (10 mM HEPES pH 1.4-, 0.15 M HaCl, 0.05t v/v of P2ÕJ surfactant was injected at 10 pL/min to the surface protein à and reached the capture level of 150 RU. Serial dilution of FcylIIa and Fcyllb receptors was injected at 10 pL/tnin. After each cycle, the surface was regenerated by injecting 10 mM HCl at 10 μL/min for 30 s. Data were fitted in a 1:1 Langmuir binding model using BIAcore TI 00 evaluation software to obtain equilibrium constant (KD) data .
[00133] References
[00134] The contents of all references cited herein are incorporated herein by reference for all purposes.
[00135] 1. Adams, G.P.; Weiner, L.M.y Monoclonal antibody therapy of cancer. Hat. Biotechnol. 2005, 23, 1147-1157.
[00136] 2. Jefferis, R., Glycosylation as a strategy to improve the body-based art of therapeutics. Nat. Rev. Drug Discovery 2009,. 8, 226-234.
[00137] 3. Agigarwal, S., What's fueling the biotechengine-2010 to 2011. Nat. Biotechnol. 2011, 29, 1083-1089.
[00138] 4. Nimmeijahπ, F.; RavBtch, J.V., Fey receptors regulators of immune responses. Nat. Rev. Immunol. 2008, 8, 34-47.
[00139] 5. Takahashi, N.; Nakagawa, H.; Fujikawa, K.; Kawamura, Y.; Tomiya, N., Three-dimensional elution mapping oE pyridylaminated N-liπked neutral and sialyl oligosaccharides. Anal. Biochem. 1995,226, 139-146.
[00140] 6. Wormaid, M.R.; Rudd, P.M.; Harvey, D.J.; Chang, S.C.; Scragg, I.G.; Dwek, R.A., Variations in Oligosaccharide-protein Interactions in Immunoglobulin G Determine the Site-Specific Glycosylation Profiles and Modulate the Dynamic Motion of the Fc Oligosaccharides. Biochemistry 1997, 36, 1370-1380.
[00141] 7. Jefferis, R., Glycosylation of Recombinant Antibody Therapeutics. Biotechnol. Program 2005, 21, 11-16.
[00142] 8. Sondermann, P.; Huber, R.; Oosthuizen, V.; Jacob, U., Ths 3.2-A crystal structure of the human IgG1 Fc fragment-FcyRHI complex. Nature 2000, 406, 267-273.
[00143] 9. Krapp, S.; Mimura, Y.; Jefferis, R.; Huber,R,; Sondermann, P, Structural Analysis of Human IgG-Fc Glycoforms Reveals a Correlation Between Glycosylation and Structural Integrity. J. Mol. Biol, 2003, 325, 979-989.
[00144] 10. Crispin, M.; Bowden, T.A.; Coles, C, H.; Harlos, K.; Aricescu, A.R.; Harvey, D.J.; Stuart, D.I.; Jones, E.Y., Carbohydrate and Domain Architecture of an Immature Antibody Glycoform Exhibiting Enhanced Effector Functions. J. Mol. Biol. 2009, 387, 1061-1066.
[00145] 11. Ferrara, C.; Grade, 5.; Jager, C.; Sondermann, P.; Brunker, P.; Waldhauer, I.; Hennig, M.; Ruf, A.; Rufe.r, A.C, ; Stihle, M.; Umana, P.; Bent, J., Unique carbohydrate-carbohydrate interactions are required for high affinity binding between FcyRIII and antibodies lacking core fucose. Proc. Natl. Academic Know. U.S.A. 2011, 108, 12669-12674.
12. Yamaguchi, Y.; Nishimura, M.; Nagano, M, ;Yagi, H.; Sasakawa, R.; Uchida, H; Shitara, K.; Kata, K., Glycotorm-dependent conformational alteration of the Fc region of human immunoglobulin Gl as revealed by NMR spectroscopy. Biochem. Biophys. Minutes 2006, 1760, 693-700.
[00147] 13. Matsumiya, S.; Yamaguchi, Y.; Saito, J.;Viaganor M.; Sasakawa, H.; Otaki, S.; Satoh, M.; Shitara, K.; Kato, K., Structural Comparison of Fucosylated and Noπfucsylate.d Fc Fragments of Human Immunoglobulin Gl. J. Mol. Biol. 2007, 369, 767-779.
[00149] 14. Barb, A.W.; Prestegard, NMR analysisdemonstrates immunoglobulin G N-glycans are accessible and dynamic. Nat,. Cheiti Biol. 2011, 7, 147-153.
[00149] 15. Nimmeijahn, F.; Ravetch, J.V., Antiinflammatory actions of intravenous immunoglobulin. Year Rev. Immunol. 2008, 26, 513-533.
[00150] 16. Shields, R.L.; Lal, J.; Keck., R.; G'Connell,L. Y.; Hong, K.; Meng, Y.G.; Weibert, S.H.; Presta, L.G., Lack of fucose on human IgG1 N-linked oligosaccharide improves binding to human FcRIHA and antibody-dependent cellular toxicity. J. Biol. Chem. 2002, 277, 26733-26740.
[00151] 17. Shinkawa, T.; Nakamura, K.; Yamane, N.; Shoji-Hosaka, E.; Kanda, Y.; Sakurada, M.; Uchida, K.; Anarawa,:H.; Satoh, M.; Yamasaki, M.; Hanai, N.; Shitara, K., The Absence of Fucose but Not the Presence of Galactose or Bisecting N-Acetylglucosamine of Human IgGl Complex-type Oligosaccharides Shows the Critical Role of Enhancing Antibody-dependent Cellular Cytotoxicity, J. Biol. Chemi. 2003, 2-78, 3466" 3413.
[00152] 18. Niwa, R.; Shoji-Hosaka., E.; Sakurada, M.; Shlαlcawa, T.; Uchida, K.; Nakamura, K.; Matsushima, K.; Ueda, R; Hanai, N.; Shitara, K., Defucosylated Chimeric Anti-CC Chetnokine Receptor 4 IgG1 with Enhanced AntibodyDependent Cellular Cytotoxicity Shows Potent Therapeutic Activity to T-Cell Leukemia and Lymphoma, Cancer Res. 2004* 64,2.127-2133.
[001531 19. Strome, S.E.; Sausville, E.A.; Mann, D., Amechanistic perspective of -monoclonal antibodies in cancer therapy beyond target-related, effects. Oncologist 2007, 12, 1084-1095.
20. Jefferis, R, Glycosylation of antibodytherapeutics: optimization for purpose. Methods Mol. Biol. 2009, 483, 223-239.
[00155] 21. Kaneko, Y.; Ninanexjahn, F.; Ravetch, J.V., Anti-Inflammatory Activity of Immunoglobulin G Resulting from Fc Sialylation. Science 2006, 313, 670-673,
[00156] 22. Anthony, R.M.; Nimmeijahn, F.; Ashline, D.J.; Reinhold, V.N.; Faulson, J. Ç. ; Ravetch, J.V., Recapitulation of IVIG Anti-Inflammatory Activity with a Recombinant IgG Fc. Science 2008, 320, 373-376.
[00157] 23, Anthony, R.M.; Iiermeling, F.; Karlsson, M.C.; Ravetch, J.V., Identification of a receptor required far the anti-inflammatory activity of IVIG. Proc. Natl. Academic Sci. U.S.A. 2008, 105, 19571-19578.
[00158] 24, Umana, ! .; Jean-Mairet, J.; Moudry, R; Amstutz, H.; Bailey, J.E., Engineered, glycαforms of an antineuroblastoma IgGl with, optimized antibody-dependent cellular cytotoxic activity. Nat. Biotechnol. 1999, 17,176-180.
[00159| 25. Yamane-Ohnuki, N.; Kiπoshita, S.; Inoue-Urakubo, M.; Kusuπaki, M.; Lida, S.; Nakano, R.; Wakltani, M.; Niwa, R.; Sakurada, M.; Uchida, K.; Shitara, K.; Batch, M. , Establishment of FUT8 knockout Chinese hamster ovary cells: An ideal host cell line for producing completely defucosylàted antibodies with enhanced antibody-dependent cellular cytotoxicity. Biotechnol, Bioeng. 2004, 87, 614622.
[00160] 26. Stanley, P.; Sundaram, S.; Tang, J.; Shi, S., Molecular analysis of three gain-of-function CRO mutants that add the bisecting GlcNAc to N-glycans. Glycobiology 2005, 15, 43-53.
[00161] 27. Cox, K.M.; Sterling, J.D.; Regan, J.T.; Gasdaska, J.R.; Frantz, K.K.; Peele, C.G.; Black, A.; Passmore, D.; Moldovan-Loomis, 0.; Srinivasan, M.; Cuison, S.; Cardareili, P.M.; Dickey, L.F., Glycan optimization of a human, monoclonal antibody in the aquatic plant Irθmna minor. Nat. Biotechnol. 2006, 24, 1591-1597.
[00162] 28. Strasser, R.; Castilho, A.; Stadlmann, Ji; Kunert, R.; Quendler, R.; Gattinger, P.; Jez, J.; Rademacher, T.; Altmann, F.; Mach, L.; Steinkellner, H.J., Improved Virus Neutralization by Flant-produced Anti-HIV Antibodies with a Homogenecius β1,4- Galactosylated N-Glycan Profile. Biol. Chetti. 2009, 284, 20479-20485.
[00163] 29. Li, H.; Sethuraman, N.; Stadheim, T.A.; Zha,D. ; Prinz, B.; Ballew, N.; Bobrowicz, P.; Choi, B.R.; Cooky W.J.; Cukan, M.; Houston-Cummings, N.R.; Davidson, R.; Gong, B.; Hamilton, S.R.; Hoopes, J.P.; Jiang, X.; Kim, N.; Mansfield, R.; Nett, J.H.; Rivers, 3.; Strawbridge, R.; Wildt, 3.; Gemgross, T.U-, Optimization of humanized IgGs in glycoengineered Pichi-a pastoris. Nat. Biotechnol. 2006, 24, 210-215.
[00164] 30. Zhou, Q.; Shankara, S.; Roy, A.; Qiu, H.; These, S.; McVie-Wylle, A., -Culm-Merdek, K.; Park, A.; Pan, C.; Edmunds, T., Development of a simple and rapid method for producing non-fucosylated oligomannose containing antibodies with increased effector function. Biotechnol. Bioeng. 2008, 99, 652-665.
[00165] 31. Schiestl, M.; Stangler, T.; Toreila, C.; Cepeljnik, T.; Toll, H.; Grade, R., Acceptable changes in quality attributes of glycosylated biopharmaceuticals. Nat. Biotechnol. 2011,29,310-312.
[00166] 32. Wang, L, X.; Lomino, J.V., Emerging Technologies for Making Glycan-Defined Glycoproteins. ACS Chem. Biol. 2012, 7, 110- 122,.
[00167] 33. Wang, L.X., The amazing transglycosylation activity of endo-P-N-acetyIglucosaminidases. Trends Glycosci. Glycotechl. 2011, 23, 33-52.
[00168] 34. Wei, Y.; Li, C.; Huang, W.; Li, B.; Strome, S.; Wang, L.X., Glycoengineering of Human IgG1-Fc through Combined Yeast Expression and in Vitro Chemoenzymatic Glycosylation. Biochemistry 2008, 47, 10294-10304.
[00169] 35. Zou, G.; Ochiai, H.; Huang, W, ; Yang, Q.; Li, C.; Wang, L.X., Chemoenzymatic Synthesis and Fey Receptor Binding of Homogeneous Glycoforms of Antibody Fc Domain. Presence of a Bisecting Sugar Moiety Enhances the Affinity of Fc to Fcyllla Receptor. J. Am. Chem. 3c. 2011, 133, 1B975-LSã91.
[00170] 36. Fan, S, Q.; Huang, W.; Wang, L. X., Remarkable Transglycosylatioπ Activity of Glycosynthase Mutants of Endo-D, an Eπdo-p-N-acetylglucosaiπinid.ase from Streptococcus pneumonia. J. Biol. Chem. 2012, 281, 1127211281.
[00171] 37. Omekawa, M.; Li, C.; Higashiyama, I.; Huang, W.; Ashida, H.; Yamamoto,K. ; Wang, L. X., Efficient mutant glycosynthase derived from Mαcor hiemalis endo-P-N-acetyIglucosaminidase capable of transferring oligosaccharide from both sugars: oxazoline and natural N-glycan. J. Biol. Chera. 2010, 285, 511-521.
[00172] 39. Huang, W.; Li, C.; Li, B.; Umekawa, M.; Yamamoto, K.; Zhang, X.; Wang., 1.. X., Glycosynthases Enable a Highly EfficientChemo.enzymat ic Synthesis of M-Glycoproteins Carrying Intact Natural N-Glycans. J. Am. Ciem. Soc. 2009, 131, 2214-222 3.
[00173] 39. Dmekawa, M.; Huang, W.; Li, B.; Fujita, K.; Ashida, H.; Wang, L.X.; Yamamoto, K. , Mutants of Mucor hiemalis Endo-B-N-acetyIglucosaminidase Show Enhanced Transglycosylation and Glycosynthase-like Activities. J. Biol. Chem. 2008, 293, 4469-4479.
[00174] 40. Collin, M.; Olsen, A., EndoS, a novel secreted protein from Streptococcus pyogenes with endoglycosidase activity on human IgG. EMBQ J. 2001, 20, 3046-3055.
[00175] 41. Allhopi, M.; Olsen, A.; Collin, M., EndoS from Streptococcus pyogenes is hydrolyzed by the cysteine proteinase Spets and requires glutamic acid 235 and tryptophans for IgG glycan-hydrolyzing activity. BMC Microbiol. 2003, 8, 3.
[00176] 42. Goodfellow, J.J.; Baruah, K.; Yamamoto, K.; Bonomelli, C,; Krishna, B.; Harvey, D, J.; Crispin, M.; Scanlan, C.N.; Davis, B.G., An Endoglycosidase with Alternative Glycan Specificity Allows Broadened Glycoprotein Remodelling. J. Am. C-hem. Soo. 2012, 134,B030-8033.
[00177] 43, Umekawa, M.; Higashiyama, T.; Koga, ¥.; Tanaka, T.; Noguchi, M.; Kobayashi, A.; Shoda, S,; Huang, W.; Wang, L.X-; Ashida, H.; Yamamoto, K. , Efficient transfer of sialo-oligosaccharide onto, proteins by combined use of a glycosynthase-like mutant of Mucor hiemalis endoglycosidase and synthetic sialo-cαmplex-type sugar oxazoline. Biochim. ,Biophys. Minutes 2010, 1800, 1203-1209.
[00178] 44, Huang, W.; Li, Wang, L.X., UnusualTransglycαsylation Activity of Flavobacterturn meningosepticum Endoglycosidases Enables ConvergentChemo.enzymati and Synthesis of Core Fucosylated Complex N-Glycopeptides. ChemBioChem 2011, 12, 93.2-941 .
[00179] 45. Waddling, C.A.; Plummer, T.H., Jr.; Tarentino, A.L.; Van P.oey, P., Structural Basis for the Substrate Specificity of Endo-P-W-acatylglucosaminidase F3. Biochemistry 2000, 39, 7878-78.85,
[00180] 46. Huang, W.; Yang, Q.; Umekawa, M.; Yamamoto, K.; Wang, L.X., Arthrαbacter Endo-P-N-AcetylglucosaiTiinidase Show's Transglycosylation Activity on Complex-Type N-Glycan Oxazoliries: One-Pot Conversipπ of Ribonuclease B to Sialylated Ribonuclease C. ChemBioChem 2010, 11, 1350-1355.
[00181] 47, Wan, H.Z.; Kaneshiro, S.; Frenz, J.; Cacia, J. , Rapid method for monitoring galactosylacticn levels during recombinant antibody production by electrospray mass spectrometry with selective-ion monitoring. J. Chromatogr'. , A 3001, 913, 437-446.
[00182] 48. Li, B.; Zeng, Y.; Hauser, S.; Song, H.; Wang, L.X., Highly Efficient Eπdoglycosidase-Catalyzed Synthesis of Glycopeptides Hsing Oligosaccharide Oxazoliiies as Donor Substrates, J. Am. Chem. See. 2005, 127, 9:692-9693.
[001831 49. Ochiai, H.; Huang, W.; Wang, L.X., Expeditious Chemoenzyniatic. ! - Am. Chem. See. 2008, 130, 13790-13903.
50. Sletteπ, E.M. Bertozzi, C.R., From Mechanism to Mouse; A Tale of Two Bioorthogonal Reactions. Ace. Chem. Res. 2011, 44, 666-676.
[00185] 51. Best, M.D., Click Chemistry and Bioorthogonal Reactions: Unpreceded Selectivity in the Labeling of Biological Molecules. Biochemistry 2009, 48, 6571-6564,
[00186] 52. Cartron, G.; Dacheux, L.; Salles, G.; Solal-Celigπy, P,; Bardos, P.,* Colombat, P.; Warier, H, Therapeutic activity of a humanised anti-CD20 monoclonal antibody and polymorphism in IgG Fc receptor FcRIIIa gene. Blood 2002, 99, 754-758.
[00187] 53. Sazinsky, 5, L,; Ott, R.G<; Silver, N.W.; Tidor, B.; Ravetch, J.V.; Wittrup, K.D. , Aglycosylated immunoglobulin G1 variants productively engage activating Fc receptors. Proc. Natl. Academic Know. U.S.A. 2008, 105, 20167-20172.
[00188] 54. Anthony, R.M.; Kobayashi, T.; Wermeliπ.g, F.; Ravetchj J..V., Intravenous gammaglobulin suppresses inflammation through a novel TH2 pathway. Nature 2011, 475, 110’113.
[00189] 55. Huhn, C.; Selman, M.H.; Ruhaak, L.P.; Leelder, A.M.; Wuhrer, M., IgG glycosylation analysis. Proteomics 2009, 9, 882-913,.
[00190] 56. Barb, A.W.; Brady, E.K.; Prestegard, J.H., Branch-Specific Sialysis of IgG-Fc Glycans by ST6Gal-I:. Biochemistry 2009, 48, 9705-9707.
[00191] 57. Guhr, T.; Bloem, J.; Derksen, N.I.; WUhrer,M.; Koendermaπ, A.H.; Aalberse, R.C.; Rispens, T., Enrichment of sialylated IgG by lectin fractionation does not enhance the efficacy of immunoglobulin G in a murine model of immune thrombocytopenia. PloSQne 2011, 6, e2124 6'.
[00192] 58. Raju, T.S.; Scallon, B., Glycosylation in the Fc domain of IgG increases resistance to proteolytic cleavage by papain. J. Biochem. Biophys. Common Res. 2006, 341, 797-803.
[00193] 59. Guile, G.R.,* Rudd, P.M.; Wing, D.R.; Prime, S.B.; Dwek, R. A., A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Anal. Biochem. 1996, 24.0, 210-226.
[00194] 60. Johnson, P., Glennie, M., The mechanisms of action of rituximab in the elimination of tumor cells. Seminar Oncol. 200 3, 30, 3-8.
[00195] 61. Koene, H.R.; Kleijer, M.; Algra, J.; Roos, D.; von dem Borne, A.E.; de Haas, M., FαyRIHa-158V/F Polymorphism influences the binding of IgG by natural killer cell FcyRIIIa, independently of the FcyRHIa-lRL/R./H phenotype. Blood 1997, 90, 1109-1114.
[00196] 62, Hassell, A.J.; Hangartner, L, ; Hunter, M.; Havenith, C.E.; Beurskeπs, F.J.; Bakker, J.M.; Lanigan, C.M.; Landucci, G.; Forthal, D.N.; Parien, P.W.; Marx, P.A.; Burton, D.R., Fc feceptor but not complement binding is important in antibody protection against HIV. Nature 2007, 449, 101-104.
[00197] 63. Collin, M.; Olsen, A., Effect of SpeB and EndαS from Streptococcus pyogenes on human immunoglobulins. Infect, Immun. 2001, 69, 7IB7-7189.

权利要求:
Claims (7)
[0001]
1. Method of preparing a fucosylated or non-fucosylated core antibody or an Fc fragment thereof having a predetermined oligosaccharide portion, characterized in that the method comprises: providing an IgG antibody or an IgG-Fc fragment, which comprises an acceptor of fucosylated or non-fucosylated core GlcNAc; eenzymatically reacting the fucosylated or non-fucosylated core GlcNAc acceptor with an activated oligosaccharide donor using a mutant of Streptococcus pyogenes Endoglycosidase-S Asp233, wherein the activated oligosaccharide donor is a synthetic oligosaccharide oxazoline or sialylated oxazoline mutant, wherein -S is selected from a mutant comprising a site-directed mutation D233Q (SEQ ID NO: 2) or D233A (SEQ ID NO: 3), wherein the activated oligosaccharide donor carries an activated oligosaccharide portion comprising a number and predetermined types of sugar residues, in which through an enzymatic reaction, the activated oligosaccharide moiety is covalently linked to the fucosylated or non-fucosylated core GlcNAc acceptor, thus preparing the fucosylated or non-fucosylated antibody or Fc fragment with the predetermined oligosaccharide portion.
[0002]
2. Method according to claim 1, characterized in that the synthetic oligosaccharide oxazoline is a di-, tri-, tetra-, penta-, hexyl-, hepta-, octyl-, nona-, -deca or undecasaccharide oxazoline .
[0003]
3. Method according to claim 1 or 2, characterized in that the fucosylated GlcNAc-acceptor core is an alpha-1-6-fucosyl-GlcNAc containing antibody or Fc fragment.
[0004]
4. Method according to any of the preceding claims, characterized in that the fucosylated or non-fucosylated core antibody is a monoclonal antibody selected from the group consisting of ibalizumab, cetuximab, rituximab, muromonab-CD3, abciximab, daclizumab, basiliximab, palivizumab, infliximab, trastuzumab, gemtuzumab ozogamicin, alemtuzumab, ibritumomab tiuxetan, adalimumab, omalizumab, tositumomab, I-131 tositumomab, efalizumab, bevacizumab, mAb, mAb-umab, anti-tumab-um, anti-natal, anti-natal CD23 mAb, alacizumab, pegol, eraptuzumab, ipilimumab, iratumumab, matuzumab, tremilimumab, zanolimumab, adecatumumab, oregovomab, nimotuzumab, briakinumab, denosumab, fontolizumab, daclizumab, pumalizumab, zanolimumab, pumalizumab, tomumab, benumab, benumab ocrerlizumab, certolizumab pegol, eculizumab, pexelizumab, abciximab, ranibizimumab, mepolizumab, and stamulumab.
[0005]
5. The method according to claim 1, wherein the activated oligosaccharide donor is a synthetic oligosaccharide oxazoline, characterized in that it additionally comprises: (a) providing an IgG antibody with a fucosylated or non-fucosylated core or an IgG- fragment Fc, which comprises heterogeneous or undesirable N-glycans; and(b) removing the heterogeneous or unwanted N-glycans by an enzyme selected from Endo S or Endo-A to form the IgG antibody or IgG-Fc fragment comprising the GlcNAc acceptor with fucosylated or non-fucosylated core.
[0006]
6. Method according to claim 5, characterized in that the enzyme used to remove heterogeneous or unwanted N-glycans is Endo S.
[0007]
7. Composition characterized in that it comprises at least one Streptococcus pyogenes Endo-S Asp-233 mutant selected from the group consisting of D233Q (SEQ ED NO: 2) and D233A (SEQ ID NO: 3).

类似技术:

---

公开号 | 公开日 | 专利标题

---

BR112014019825B1|2021-08-24|CHEMIOENZYMATIC GLYCOENGINEERING OF ANTIBODIES AND FC FRAGMENTS OF THE SAME

---

US10851174B2|2020-12-01|Core fucosylated glycopeptides and glycoproteins: chemoenzymatic synthesis and uses thereof

---

US9850473B2|2017-12-26|Transglycosylation activity of glycosynthase mutants of an endo-beta-N-acetylglucosaminidase | from Streptococcus pneumoniae

---

US10836815B2|2020-11-17|Generation and comparative kinetic analysis of new glycosynthase mutants from Streptococcus pyogenes endoglycosidases for antibody glycoengineering

---

JP2022023160A|2022-02-07|Endo-S2 mutant as glycosynthase, method for producing glycoprotein and use for glycoprotein manipulation of sugar chains

---

US20180298361A1|2018-10-18|Endoglycosidase mutants for glycoprotein remodeling and methods of using it

同族专利:

---

公开号 | 公开日

---

US9434786B2|2016-09-06|

---

CA2862925A1|2013-08-15|

---

US20210061868A1|2021-03-04|

---

US20190367570A1|2019-12-05|

---

JP2015507925A|2015-03-16|

---

EP2812442A4|2015-12-23|

---

IN2014DN06806A|2015-05-22|

---

IL233779D0|2014-09-30|

---

US20170058040A1|2017-03-02|

---

JP6282232B2|2018-02-21|

---

US10836805B2|2020-11-17|

---

WO2013120066A1|2013-08-15|

---

US20150087814A1|2015-03-26|

---

BR112014019825A2|2017-06-20|

---

IL233779A|2019-09-26|

---

CN104220603B|2017-06-06|

---

US9845360B2|2017-12-19|

---

CN104220603A|2014-12-17|

---

CA2862925C|2020-01-21|

---

US20180186847A1|2018-07-05|

---

EP2812442A1|2014-12-17|

---

US10344063B2|2019-07-09|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

JPH072895A|1993-04-28|1995-01-06|Eli Lilly & Co|Antibody - drug conjugate|

---

JP2005535280A|2001-11-28|2005-11-24|ネオーズテクノロジーズ，インコーポレイテッド|Remodeling of glycoproteins using endoglycanase|

---

US20050064540A1|2002-11-27|2005-03-24|Defrees Shawn Ph.D|Glycoprotein remodeling using endoglycanases|

---

EP1532159A4|2002-06-20|2009-10-21|Univ Maryland Biotech Inst|Scaffolded maleimide clusters for multivalent peptide assembly|

---

WO2004033663A2|2002-10-11|2004-04-22|University Of Maryland Biotechnology Institute Off. Of Research Admin./ Tech. Dev.|Carbohydrate-based synthetic vaccines for hiv|

---

US7604804B2|2004-02-09|2009-10-20|University Of Maryland Biotechnology Institute|Enhancing anti-HIV efficiency through multivalent inhibitors targeting oligomeric gp120|

---

US7728106B2|2005-07-01|2010-06-01|University Of Maryland Biotechnology Institute|HIV-1 glycopeptides and derivatives; preparation and applications thereof|

---

US20100221241A1|2005-07-06|2010-09-02|University Of Maryland Biotechnology Institute|Constrained hiv envelope-based immunogen that simultaneously presents receptor and coreceptor binding sites|

---

CA2663633A1|2005-09-14|2007-03-22|Lai-Xi Wang|Synthetic polyvalent carbohydrates as components of microbicides|

---

EP1957099B1|2005-11-07|2015-03-25|The Rockefeller University|Reagents, methods and systems for selecting a cytotoxic antibody or variant thereof|

---

EP2001358B1|2006-03-27|2016-07-13|University Of Maryland, Baltimore|Glycoprotein synthesis and remodeling by enzymatic transglycosylation|

---

RU2008152435A|2006-06-06|2010-07-20|Дженентек, Инк. |COMPOSITIONS AND METHODS OF REGULATING VESSEL DEVELOPMENT|

---

CA2655246A1|2006-06-09|2007-12-21|University Of Maryland, Baltimore|Glycosylation engineered antibody therapy|

---

GB0624874D0|2006-12-13|2007-01-24|Hansa Medical Ab|Treatment|

---

DK2190984T3|2007-09-14|2013-12-02|Genovis Ab|Methods and kits for purifying and detecting glycosylated IgG|

---

GB0821100D0|2008-11-18|2008-12-24|Hansa Medical Ab|Antibodies|

---

ES2528321T3|2009-10-02|2015-02-06|Roche Glycart Ag|Detection of a-fucosylation in antibodies|

---

CN102770554B|2009-10-29|2016-02-17|詹森生物科技公司|Antibody glycosylation variant|

---

US10087236B2|2009-12-02|2018-10-02|Academia Sinica|Methods for modifying human antibodies by glycan engineering|

---

US10851174B2|2011-03-03|2020-12-01|University Of Maryland, Baltimore|Core fucosylated glycopeptides and glycoproteins: chemoenzymatic synthesis and uses thereof|

---

EP2812442A4|2012-02-10|2015-12-23|Univ Maryland|Chemoenzymatic glycoengineering of antibodies and fc fragments thereof|US7960139B2|2007-03-23|2011-06-14|Academia Sinica|Alkynyl sugar analogs for the labeling and visualization of glycoconjugates in cells|

---

JP5986745B2|2008-07-15|2016-09-06|アカデミア シニカＡｃａｄｅｍｉａ Ｓｉｎｉｃａ|Glycan arrays on PTFE-like aluminum-coated glass slides and related methods|

---

US10087236B2|2009-12-02|2018-10-02|Academia Sinica|Methods for modifying human antibodies by glycan engineering|

---

US10338069B2|2010-04-12|2019-07-02|Academia Sinica|Glycan arrays for high throughput screening of viruses|

---

CA2835489C|2010-05-10|2018-03-06|Chi-Huey Wong|Zanamivir phosphonate congeners with anti-influenza activity and determining oseltamivir susceptibility of influenza viruses|

---

US9175326B2|2011-03-03|2015-11-03|University Of Maryland, Baltimore|Transglycosylation activity of glycosynthase mutants of an endo-beta-N-acetylglucosaminidasefrom streptococcus pneumoniae|

---

EP2812442A4|2012-02-10|2015-12-23|Univ Maryland|Chemoenzymatic glycoengineering of antibodies and fc fragments thereof|

---

US10130714B2|2012-04-14|2018-11-20|Academia Sinica|Enhanced anti-influenza agents conjugated with anti-inflammatory activity|

---

CA2880701A1|2012-08-18|2014-02-27|Academia Sinica|Cell-permeable probes for identification and imaging of sialidases|

---

EP2888238A4|2012-08-21|2016-01-27|Academia Sinica|Benzocyclooctyne compounds and uses thereof|

---

DK2991683T3|2013-05-02|2019-11-04|Glykos Finland Oy|CONJUGATES OF A GLYCOPROTEIN OR A GLYCAN WITH A TOXIC CHARGE|

---

US10086054B2|2013-06-26|2018-10-02|Academia Sinica|RM2 antigens and use thereof|

---

WO2014210564A1|2013-06-27|2014-12-31|Academia Sinica|Glycan conjugates and use thereof|

---

EP3041949B1|2013-09-05|2021-04-28|Vib Vzw|Cells producing fc containing molecules having altered glycosylation patterns and methods and use thereof|

---

JP6486368B2|2013-09-06|2019-03-20|アカデミア シニカＡｃａｄｅｍｉａ Ｓｉｎｉｃａ|Activation of human iNKT cells using glycolipids containing modified glycosyl groups|

---

EP3058084A4|2013-10-16|2017-07-05|Momenta Pharmaceuticals, Inc.|Sialylated glycoproteins|

---

EP3079719B8|2013-12-09|2019-12-04|Allakos Inc.|Anti-siglec-8 antibodies and methods of use thereof|

---

US9689016B2|2013-12-18|2017-06-27|Caliber Biotherapeutics, Llc|Method for in vivo production of deglycosylated recombinant proteins used as substrate for downstream protein glycoremodeling|

---

PL406737A1|2013-12-31|2015-07-06|Michał Lower|Method for industrial production of therapeutic proteins with the desired profile of glycosylation|

---

US10495645B2|2015-01-16|2019-12-03|Academia Sinica|Cancer markers and methods of use thereof|

---

DK3250590T3|2015-01-30|2021-10-18|Academia Sinica|Compositions and Methods relating to universal glycoforms for enhanced anti-SSEA4 antibody efficacy|

---

US10118969B2|2014-05-27|2018-11-06|Academia Sinica|Compositions and methods relating to universal glycoforms for enhanced antibody efficacy|

---

US10150818B2|2014-01-16|2018-12-11|Academia Sinica|Compositions and methods for treatment and detection of cancers|

---

US9975965B2|2015-01-16|2018-05-22|Academia Sinica|Compositions and methods for treatment and detection of cancers|

---

JP2017507118A|2014-01-16|2017-03-16|アカデミア シニカＡｃａｄｅｍｉａ Ｓｉｎｉｃａ|Compositions and methods for the treatment and detection of cancer|

---

TWI682033B|2014-03-17|2020-01-11|泉盛生物科技股份有限公司|Methods for producing recombinant glycoproteins with modified glycosylation|

---

TW202026300A|2014-03-27|2020-07-16|中央研究院|Reactive labelling compounds and uses thereof|

---

JP6750148B2|2014-04-25|2020-09-02|公益財団法人野口研究所|Process for producing sugar chain cleaving antibody and uniform sugar chain antibody|

---

KR20170003720A|2014-05-27|2017-01-09|아카데미아 시니카|Anti-cd20 glycoantibodies and uses thereof|

---

EP3904388A1|2014-05-27|2021-11-03|Academia Sinica|Fucosidase from bacteroides and methods using the same|

---

JP2017523131A|2014-05-27|2017-08-17|アカデミア シニカＡｃａｄｅｍｉａ Ｓｉｎｉｃａ|Anti-HER2 glycoengineered antibody group and use thereof|

---

EP3154582A4|2014-05-28|2018-01-10|Academia Sinica|Anti-tnf-alpha glycoantibodies and uses thereof|

---

WO2016040369A2|2014-09-08|2016-03-17|Academia Sinica|HUMAN iNKT CELL ACTIVATION USING GLYCOLIPIDS|

---

JP6779887B2|2015-01-24|2020-11-04|アカデミア シニカＡｃａｄｅｍｉａ Ｓｉｎｉｃａ|New glycan conjugate and how to use it|

---

JP6830576B2|2015-02-26|2021-02-17|東京化成工業株式会社|Endo M mutant and method for producing N-linked sugar chain-containing compound or N-linked sugar chain-containing protein|

---

JP6744738B2|2015-06-29|2020-08-19|公益財団法人野口研究所|Glycosynthase|

---

JP6752203B2|2015-07-16|2020-09-09|第一三共株式会社|New EndoS mutant enzyme|

---

WO2017018474A1|2015-07-29|2017-02-02|公益財団法人野口研究所|Method for preparing core fucose-containing antibody|

---

KR20180050339A|2015-09-04|2018-05-14|오비아이 파머 인코퍼레이티드|Glycan arrays and how to use them|

---

CN105277718B|2015-09-29|2018-03-20|上海知先生物科技有限公司|For the product of the examination of malignant tumour correlation and assessment, application and method|

---

JP6618176B2|2015-11-30|2019-12-11|公益財団法人野口研究所|Method for producing heptasaccharide oligosaccharide|

---

WO2017124084A1|2016-01-15|2017-07-20|University Of Maryland, College Park|Endo-s2 mutants as glycosynthases, method of making and use for glycoengineering of glycoproteins|

---

EP3426693A4|2016-03-08|2019-11-13|Academia Sinica|Methods for modular synthesis of n-glycans and arrays thereof|

---

US10980894B2|2016-03-29|2021-04-20|Obi Pharma, Inc.|Antibodies, pharmaceutical compositions and methods|

---

BR112018070097A2|2016-03-29|2019-02-12|Obi Pharma, Inc.|antibody, hybridoma, pharmaceutical composition, method for treating cancer in an individual, method for inhibiting cancer cell proliferation, method for diagnosing cancer in an individual, method for treating a human patient, method for imaging an individual, conjugate of antibody-antibodymethod for treating cancer, bispecific antibody and method for preparing a homogeneous antibody population|

---

EP3480211A4|2016-07-01|2019-12-25|Daiichi Sankyo Company, Limited|Hanp-fc-containing molecular conjugate|

---

US11193155B2|2016-08-10|2021-12-07|University Of Maryland, College Park|Designer α 6-fucosidase mutants enable direct core fucosylation of intact N-glycopeptides and N-glycoproteins|

---

EP3500594A4|2016-08-22|2020-03-11|Cho Pharma Inc.|Antibodies, binding fragments, and methods of use|

---

CN107778372B|2016-08-22|2019-11-26|中国科学院上海药物研究所|A kind of oligosaccharides connexon and the antibody-drug conjugates of the fixed point connection using oligosaccharides connexon preparation|

---

CN106596827B|2017-01-23|2019-06-07|上海知先生物科技有限公司|Application of the Gal index in treating autoimmune diseases sensibility and curative effect evaluation|

---

US11203638B2|2017-05-05|2021-12-21|Allakos Inc.|Methods and compositions for treating perennial allergic conjunctivitis and keratoconjunctivitis|

---

US20190002542A1|2017-06-29|2019-01-03|University Of Maryland, College Park|Transglycosylation of endo-s and endo-s mutants for antibody glycosylation remodeling|

---

KR20200061376A|2017-09-29|2020-06-02|다이이찌 산쿄 가부시키가이샤|Antibody-pyrrolobenzodiazepine derivative conjugate|

---

EP3805389A1|2018-05-31|2021-04-14|Daiichi Sankyo Company, Limited|Anti-human tlr7 antibody|

---

US11203645B2|2018-06-27|2021-12-21|Obi Pharma, Inc.|Glycosynthase variants for glycoprotein engineering and methods of use|

---

AU2019315177A1|2018-07-31|2021-02-25|Daiichi Sankyo Company, Limited|Treatment of metastatic brain tumor by administration of antibody-drug conjugate|

---

AU2019320336A1|2018-08-06|2021-03-04|Daiichi Sankyo Company, Limited|Combination of antibody-drug conjugate and tubulin inhibitor|

---

CN112912109A|2018-09-20|2021-06-04|第一三共株式会社|Treatment of HER 3-mutated cancer by administration of anti-HER 3 antibody-drug conjugates|

---

EP3882349A1|2018-11-14|2021-09-22|Daiichi Sankyo Company, Limited|- conjugate|

---

AU2019396895A1|2018-12-11|2021-07-08|Daiichi Sankyo Company, Limited|Combination of antibody-drug conjugate with PARP inhibitor|

---

TW202038957A|2018-12-21|2020-11-01|日商第一三共股份有限公司|Combination of antibody-drug conjugate and kinase inhibitor|

---

TW202102228A|2019-03-25|2021-01-16|日商第一三共股份有限公司|Antibody-pyrrolobenzodiazepine derivative conjugate|

---

EP3949987A1|2019-03-25|2022-02-09|Daiichi Sankyo Company, Limited|Anti-her2 antibody-pyrrolobenzodiazepine derivative conjugate|

---

WO2020196712A1|2019-03-27|2020-10-01|第一三共株式会社|Combination of antibody-pyrrolobenzodiazepine derivative conjugate and parp inhibitor|

---

WO2021140981A1|2020-01-10|2021-07-15|国立研究開発法人理化学研究所|Antibody population uniformly including antibodies having left-right asymmetric sugar chain, and method for producing same|

---

WO2021148955A1|2020-01-21|2021-07-29|Yissum Research Development Company Of The Hebrew University Of Jerusalem Ltd.|Utilization of plant protein homologues in culture media|

---

WO2021260580A1|2020-06-24|2021-12-30|Astrazeneca Uk Limited|Combination of antibody-drug conjugate and atm inhibitor|

---

WO2021260582A1|2020-06-24|2021-12-30|Astrazeneca Uk Limited|Combination of antibody-drug conjugate and aurora b inhibitor|

---

WO2021260578A1|2020-06-24|2021-12-30|Astrazeneca Uk Limited|Combination of antibody-drug conjugate and cdk9 inhibitor|

---

WO2021260579A1|2020-06-24|2021-12-30|Astrazeneca Uk Limited|Combination of antibody-drug conjugate and atr inhibitor|

---

WO2021260583A1|2020-06-24|2021-12-30|Astrazeneca Uk Limited|Combination of antibody-drug conjugate and dna-pk inhibitor|

---

CN112442124B|2020-12-09|2022-01-14|福州迈新生物技术开发有限公司|anti-CD 23 protein monoclonal antibody, cell line, preparation method and application thereof|

法律状态:
2018-01-23| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|

---

2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2020-05-26| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

---

2021-01-26| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

---

2021-05-04| B06I| Publication of requirement cancelled [chapter 6.9 patent gazette]|Free format text: ANULADA A PUBLICACAO CODIGO 6.21 NA RPI NO 2612 DE 26/01/2021 POR TER SIDO INDEVIDA. |

---

2021-06-15| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

---

2021-08-24| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 11/02/2013, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

---

申请号 | 申请日 | 专利标题

---

US201261597468P| true| 2012-02-10|2012-02-10|

---

US61/597,468|2012-02-10|

---

PCT/US2013/025553|WO2013120066A1|2012-02-10|2013-02-11|Chemoenzymatic glycoengineering of antibodies and fc fragments thereof|

---