Invented by Lai-Xi Wang, Wei Huang, University of Maryland at Baltimore
The University of Maryland at Baltimore invention works as followsThe invention allows for recombinant Endos-S mutants with reduced hydrolysis activity and higher transglycosylation activities for the synthesis glycoproteins. In this case, a sialylated or synthetic oligosaccharide or oxazoline is combined to a core fucosylated GlcNAc protein acceptor. These recombinant Endos mutants can be used to efficiently glycosylate remodel IgG1Fc domains and provide different antibody glycoforms with structurally defined Fc N-glycans.
Background for Chemoenzymatic glycoengineering antibodies and FC fragments
Field of Invention
The invention relates to glycoprotein synthesis, and more particularly, to the use of a recombinant and mutant Endo S, an Endo-?-N-acetylglucosaminidase from Streptococcus pyogenes, that possesses transglycosylation activity and limited hydrolyzing activity thereby providing for efficient glycosylation remodeling of antibody-Fc domain.
Description of Related Art
Monoclonal antibody (mAbs), of the IgG class, are an important class therapeutic proteins that can be used to treat cancer, autoimmune and infectious diseases. IgG antibodies consist of two heavy chains and two lighter chains. These chains are linked to form three distinct protein domains. The Fab domains are responsible to antigen binding. While the Fc domain engages in Fc receptor-mediated effectsor functions such as complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity. (2, 4) Fc is a homodimer that contains two N-glycans at N297 (conserved N-glycosylation locations). Attached oligosaccharides can be decorated with biantennary complex types with significant structural heterogeneity. The N-linked Heptasaccharide core may be differentially decorated by core fucose or core galactose (Gal), bisecting N acetylglucosamines (GlcNAc), terminal sialic acid, (Sia), as shown in FIG. 1. NMR structural and X-ray crystallographic studies (5-7) show that Fc glycans can be found sandwiched between two CH2/CH3 subdomains. They also have multiple noncovalent interactions and interactions with Fc domains. (8-14) These studies show that different Fc glycocans can have different effects on Fc domain conformations. This implicates the important role of glycosylation for maintaining appropriate Fc domain structures to allow interactions with Fc receptors involved in antibody’s effector functions. (8-14)
It was further shown that the fine structures in Fc N-glycans play an important role in the pro- and/or anti-inflammatory properties of antibodies. (2, 15). For example, the absence of core fucose and the attachment of a bisecting GlcNAc moiety dramatically increase the affinity of antibody to the Fc.IIIa receptor (Fc.RIIIa), responsible for antibody-dependent cell cytotoxicity (ADCC). For improved anticancer efficacy in vivo, mAbs with low fucose levels are desired. (19, 20). On the other side, the terminal?-2.6-sialylated Fc Glyform, a minor component intravenous immunoglobulin, was recently identified to be the active species for anti-inflammatory activity in IVIG in a mouse model rheumatoid (RA) model. (21-23) But, IgGs commercially available, including monoclonal antibodies, and IVIG, often exist in mixtures of glycoforms that do not suit their therapeutic functions. The major Fc glycoforms for monoclonal antibody treatment for cancer are core-fucosylated, which have a low affinity for Fc.RIIIa. This results in low efficacy, especially for patients with low-affinity Fc.RIIIa-F158 polymorphism. (2, 19, 20)
The impact of glycosylation in the therapeutic outcomes and biological functions of IgG antibodies has sparked a lot of interest in developing ways to regulate antibody’s glycosylation. One way to manage the glycosylation profiles of IgG antibodies during production is through glycan biosynthetic pathway engineering in various expression systems including yeast, mammalian and plant host cells. (24-30). This control of glycosylation led to the production of monoclonal monoclonal antibodies that are low-fucose and nonfucosylated with enhanced ADCC activities. However, this method can only produce a limited number of glycoforms and it is not possible to control the entire glycoform.
An analysis of therapeutic glycoprotein drugs, including monoclonal antibody Rituximab, revealed significant changes in the glycosylation profiles of different batches made at different times. This analysis highlights the difficulty in maintaining consistent production of glycoprotein drugs. It also raises regulatory concerns as changes to the Fc glycosylation could most likely affect the therapeutic efficacy.
An alternative method to address the inconsistence or heterogeneity of glycosylation is glycosylation remodeling. This involves trimming the heterogeneous N glycans and extending sugar chains through enzymatic glycosylation. 32, 33. This enzymatic glycosylation was recently described using a chemoenzymatic method of Fc glycosylation remodeling. It takes advantage of the transglycosylation activities of several endoglycosidase and their glycosynthase mutations using glycan-oxazolines to their substrates. (34-36) This remodeling approach consists of two steps: trimming off all the heterogeneous N-glycans by an endoglycosidase to leave only the first GlcNAc at the glycosylation site(s) and then adding back a well-defined N-glycan en bloc via an endoglycosidase-catalyzed transglycosylation reaction. (32)
Recent work has demonstrated that IgG-Fc domain glycosylation engineering can be achieved by a combination of yeast or CHO cell expression of the Fc domain and its subsequent chemoenzymatic remodeling through an enzymatic deglycosylation/reglycosylation approach. (34-36) It has been shown that the endo-?-N-acetylglucosaminidase from Arthrobacter protophormiae, EndoA, is highly efficient to glycosylate the GlcNAc-containing Fc domain by using various synthetic N-glycan core oxazolines as substrates. However, there are limitations to the method. (34, 35). EndoA and EndoM (another endoglycosidase derived from Mucor hiemalis), were unable to transform the core-fucosylated IgG domain (35); (b) EndoD mutants were able attach a Man3GlcNAc Core to a fucosylated GlcNAc?Fc domain (36); however, EndoD, EndoA, EndoA and their mutants (36-39) and (36-fucosylated) to the fucosylated) and complex type N-glycans; (c) glycosylation of full-length Iglycans (36, (36, 35, 35, but not yet to the fucosylated) to the complex type N-glycans; (c) to the complex type N-glycans; (36, 36-fucosylated or fucosylated) to the fucosylated Gl-glycans; (39) to the complex type N-glycans; glycans; syl-glycansylation of the glycosylated N-glycans; ) to glycans; glycans; a; glycans; syl-glycans; glycans; c) to glycans to glycans to a; c to glycansylated glycans to glycans to glycans to glycans to glycans to glycansylated glycans to glycansylated glycans to glycansylated oAcNAcNAcNAcNAcNAcNAcNAcNAcNAcNAcNAcNAcNAcNAcNAc to cNAcNAcNAcNAcNAcNAcNAcNAcNAcNAcNAcNAcNAcNAcN-fucosyl to glycans to glycans to g
In an attempt to develop efficient enzymatic deglycosylation/glycosylation system for glycoprotein glycosylation remodeling, attention has been turned to EndoS, an endo-?-N-acetylglucosaminidase (ENGase) from Streptococcus pyogenes that is capable of hydrolyzing the Fc N-glycans of intact IgG antibodies by cleaving the ?-1,4-glycosidic bond in the chitobiose core of the N-glycans.(40-42). Endo-S has transglycosylation ability, such that it can use Man3GlcNAc Oxazoline as a donor substrate to glycosylate an GlcNAc accepting protein. Wild type EndoS has a high level of hydrolytic activity. Therefore, the glycosylated IgG product can also be subject to rapid hydrolysis when wild type EndoS is used in synthesis or glycosylation remodeling.
Considering the above-mentioned activities of Endo S it would be beneficial to create a mutant Endo-S with transglycosylating and reduced hydrolyzing activity.
The invention allows for recombinant Endos and selected mutants thereof to exhibit reduced hydrolysis activity and increased transglycosylation activities for the synthesis IgG antibodies. The sugar chain is then added to a core fucosylated GlcNAcIgG acceptor. The present invention provides for the synthesis and remodeling therapeutic antibodies and Fc fragments thereof. This allows for certain biological activities such as longer half-life times in vivo and less immunogenicity. It also increases in vivo activity and targets more easily.
In one aspect, the present invention provides for transglycosylation activity of an endo-?-N-acetylglucosamindase of Streptococcus pyogenes (SEQ ID NO: 1) and mutants thereof, wherein the mutants have at least 95% homology thereto and exhibit transglycosylation activity on both core fucosylated and nonfucosylated GlcNAc-IgG acceptors, wherein the endoglycosidases enable the transfer of an oligosaccharide (in the form of an activated sugar oxazoline) en bloc to a fucosylated or nonfucosylated GlcNAc-IgG (or an Fc fragment thereof) to form a new glycoform of IgG (or an Fc fragment thereof).
Another aspect of the invention is Endo-S mutants, which exhibit remarkable enhanced transglycosylation efficiency while exhibiting diminished or abrogated hydrolytic activity. Site-specific mutations, including one at Asp-233, are preferred in mutants. These mutants include, among others, D233Q (SEQID NO: 2), and D233A, (SEQID NO: 3).
In another aspect, the invention provides a chemoenzymatic process for the preparation homogeneous core fucosylated and nonfucosylated glycoforms IgG antibodies. It includes:
a. providing an acceptable acceptor from the group consisting either a core fucosylated GlcNAcIgG, nonfucosylated GlcNAcIgG or corresponding IgG?Fc fragments;
b. Reacting with the acceptor using a donor substrate that includes an activated Oligosaccharide Moiety in Streptococcus Endo-S Asp233 mutants to transfer activated Oligosaccharide to the acceptor, and yielding homogeneous fucosylated/nonfucosylated glycoprotein.
In an even further aspect, the present invention provides a method of preparing a corefucosylated IgG/IgG-Fc fragment with a predetermined moiety of oligosaccharides. It includes:
a. providing an IgG core-fucosylated acceptor consisting of an asparagine linked core-fucosylated GlcNAc residue; and
b. Enzymatically reacting core-fucosylated IgG accepting with an activated Oligosaccharide donors in the presence Endoglycosidase S D233Q (SEQID NO: 2) or D233A (SEQID NO: 3), wherein activated Oligharide donors carries an oligosaccharide miety consisting of a predetermined type and number of sugar residues. The oligosaccharide molecule is covalently linked with the IgG-fucosylated IgG/Fc fragment with the predetermined oligosaccharide oligosaccharide oligosaccharide oligosaccharide oligosaccharide osaccharide oligosaccharide oligosaccharide oligosaccharide oligos.
In another aspect, the invention provides an activated moiety of oligosaccharides, such as glycosyl fluoride or glycan, to be used as donor substrates for the synthesis homogeneous corefucosylated or nonfucosylated oligosaccharides. The preferred activated oligosaccharide moiety of the invention is an oligosaccharide-oxazoline.Click here to view the patent on Google Patents.