Fusion proteins for reshaping antibody glycoforms

Abstract

The present disclosure provides a fusion protein comprising a fucosidase or a truncated fragment or mutant thereof fused to the N-terminus or C-terminus of an endoglycosidase or a truncated fragment or mutant thereof. The present disclosure also provides a nucleic acid molecule expressing the fusion protein and a method for reshaping the polysaccharide of an antibody Fc region.

Description

Technical Field

[0001] The present invention relates to glycoform remodeling; in particular, to fusion proteins for remodeling antibody glycoforms. Background Art

[0002] Antibodies (also known as immunoglobulins (Ig)) are glycoproteins that play a major role in immune responses. IgG antibodies and fragments of IgG antibodies have been developed as important biotherapeutics for treating a wide variety of diseases. The constant (Fc) region of natural antibodies is usually N-glycosylated. Specific glycosylation (i.e., glycotype) significantly affects Fc effector function and the stability of the antibody. Therefore, one way to improve the therapeutic efficacy of antibodies is to modify the glycotype in the Fc region.

[0003] Fc glycoforms often include core fucose residues. In the context of cancer therapy, anti-tumor antibodies with glycoforms in the Fc region and with lower core fucose content have been reported to display higher binding affinity for CD16 (FcγRIIIA) on NK cells. The higher affinity results in improved induction of antibody-dependent cellular cytotoxicity against tumor cells.

[0004] To produce antibodies with low levels of core fucose, various strategies have been developed. For example, inhibitors of fucosyltransferases required for core fucose addition can be added to the culture medium used to grow antibody-producing cells. In another example, antibody-producing cells can be genetically engineered to lack fucosyltransferase activity.

[0005] In a different strategy, purified enzymes can be used in vitro to directly improve antibody glycotypes in a predictable manner. One advantage of this strategy is that antibodies can be produced and then improved to have a well-defined low core fucose Fc glycotype and desired biological properties. This will facilitate the use of high-yield non-mammalian expression systems to produce antibodies instead of mammalian expression systems that are typically used to ensure proper core glycosylation. US 20190185898 provides fucosidase mutants that are used as fucose ligases for core fucosylation of a range of biological glycopeptides and glycoproteins, including intact therapeutic antibodies.

[0006] However, existing fucosidases are inefficient in cleaving core fucose from the Fc region. There is a need to develop enzymes that can efficiently and predictably reshape antibody glycoforms. Summary of the invention

[0007] Fusion proteins are provided in the present disclosure. The assembly of multiple enzymes in fusion proteins promotes the synergistic action of the enzymes and exhibits the beneficial effect of predictably reshaping the antibody glycoform.

[0008] In one aspect, the present disclosure provides a fusion protein comprising a fucosidase or a truncated fragment or mutant thereof fused to the N-terminus or C-terminus of an endoglycosidase or a truncated fragment or mutant thereof; wherein the fusion protein exhibits both fucosidase activity and endoglycosidase activity.

[0009] Some examples of fucosidases include, but are not limited to, Lactobacillus casei α-L-fucosidase C (Alfc), Bacteroides fragilis fucosidase (BF3242), Bacteroides thetaiotamicron α-L-fucosidase (BT2970), Emticicia oligotrophica α-L-fucosidase (EO0918), and Elizabethkingia miricola α-(1-6) fucosidase (Emfuc3), or fragments thereof or mutants thereof. In some embodiments, the fucosidase is Lactobacillus casein α-L fucosidase C, Elizabeth miller α-(1-6) fucosidase (Emfuc3), or a truncated fragment or mutant thereof.

[0010] Some embodiments of endoglycosidases include, but are not limited to, Streptococcus pyogenes endoglycosidase S, Streptococcus pyogenes endoglycosidase S2, or fragments thereof, or mutants thereof. Specifically, the endoglycosidase is Streptococcus pyogenes endoglycosidase S2, or a truncated fragment or mutant thereof.

[0011] In one embodiment, the fucosidase or endoglycosidase is a truncated fragment. The truncated fragment of the endoglycosidase is its IgG binding domain. In some embodiments, the truncated fragment of the endoglycosidase is the IgG binding domain of Streptococcus pyogenes endoglycosidase S or Streptococcus pyogenes endoglycosidase S2.

[0012] In one embodiment, the fucosidase or endoglycosidase is a mutant polypeptide. Specifically, the endoglycosidase mutant is a Streptococcus pyogenes endoglycosidase S having a mutation at amino acid position -D233, preferably -D233Q. In some embodiments, the endoglycosidase mutant is a Streptococcus pyogenes endoglycosidase S2 having a mutation at amino acid position T138, D182, D184, D186, D226 or T227; preferably having T138E, T138M, T138Q, T138R, T138L, T138H, T138N, T138K, D182Q, D184M, D184Q, D184T, D184L, D184F, D184S, D184V, D184K, D184W, E186A, D226Q or T227Q.

[0013] Certain embodiments of fusion proteins include, but are not limited to, Lactobacillus casei α-L fucosidase C fused to: Streptococcus pyogenes endoglycosidase S, Streptococcus pyogenes endoglycosidase S2, the IgG binding domain of Streptococcus pyogenes endoglycosidase S, the IgG binding domain of Streptococcus pyogenes endoglycosidase S2, Streptococcus pyogenes endoglycosidase S with a mutation at amino acid position D233, Streptococcus pyogenes endoglycosidase S with a mutation at amino acid position T138, Streptococcus pyogenes endoglycosidase S2, Streptococcus pyogenes endoglycosidase S2 with a mutation at amino acid position D182, Streptococcus pyogenes endoglycosidase S2 with a mutation at amino acid position D184, Streptococcus pyogenes endoglycosidase S2 with a mutation at amino acid position D186, Streptococcus pyogenes endoglycosidase S2 with a mutation at amino acid position D226, or Streptococcus pyogenes endoglycosidase S2 with a mutation at amino acid position T227.

[0014] Specifically, the fusion protein comprises an amino acid sequence selected from the group consisting of: SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125 8, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, and 204 or a substantially similar sequence thereof. Preferably, the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202 and 204 or a substantially similar sequence thereof.

[0015] In another aspect, the disclosure provides nucleic acid molecules that express the fusion proteins described herein. Some examples of nucleic acids include, but are not limited to, SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117 , 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201 and 203 or substantially the same sequence thereof. In some other embodiments, the nucleic acid has a nucleotide sequence selected from the group consisting of SEQ ID NO: 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, and 203, or a sequence substantially identical thereto.

[0016] In another aspect, the disclosure provides a vector comprising a nucleic acid of the invention. The disclosure also provides a host cell comprising a vector.

[0017] In another aspect, the present disclosure provides a method for remodeling the polysaccharide of the Fc region of an antibody, comprising the following steps: obtaining an antibody having heterogeneous polysaccharides in the Fc region of the antibody and contacting the antibody with a fusion protein as described herein. Specifically, the method comprises providing an antibody having heterogeneous polysaccharides in its Fc region, a fusion protein as described herein, and a target polysaccharide oxazoline, wherein the polysaccharide of the Fc region of the antibody comprises an N-acetylglucosamine (GlcNAc) residue; and contacting the antibody with the fusion protein and linking the target polysaccharide oxazoline to the Fc region of the antibody; thereby, a remodeled polysaccharide of the Fc region of the antibody can be obtained.

[0018] In one embodiment of the present invention, the method further comprises purifying the antibody having the remodeled glycan in the Fc region.

[0019] The present disclosure is described in detail in the following sections. Other features, objectives and advantages of the present disclosure can be found in the detailed description and claims.

[0020] Simple diagram description

[0021] Figure 1 Shown are bar graphs showing the percentage of fucose hydrolyzed by the indicated enzymes (ie, fucosidase and fucosidase / EndoS2 fusion protein), expressed as a percentage of the starting fucose removed. The enzymes are identified in Tables 1 to 5 below.

[0022] Figure 2 Shown are bar graphs showing the percentage of fucose hydrolyzed by Alfc fucosidase or Alfc / EndoS2 fusion protein expressed as a percentage of the starting fucose removed. The fusion proteins are identified in Tables 1 to 5 below.

[0023] Figure 3 Shown are bar graphs showing the percentage of fucose hydrolyzed by Alfc fucosidase or five fusion proteins with truncated EndoS2, expressed as a percentage of the starting fucose removed. The fusion proteins are identified in Tables 1 to 5 below.

[0024] Figure 4 Shown are bar graphs showing the percentage of fucose hydrolyzed by Alfc or Emfuc3 fucosidase or Alfc or Emfuc3 / EndoS2 fusion proteins expressed as a percentage of the starting fucose removed. The fusion proteins are identified in Tables 1 to 5 below.

[0025] Figure 5 Shown are bar graphs showing the percentage of fucose hydrolyzed by Alfc or Emfuc3 fucosidase, Alfc / EndoS or EndoS or EndoS2 / Emfuc3 fusion proteins expressed as a percentage of the starting fucose removed. The fusion proteins are identified in Tables 1 to 5 below.

[0026] Figure 6 Shown are bar graphs showing the percentage of fucose hydrolyzed by Alfc, Emfuc3 or BF3242 fucosidase, EndoS or EndoS2 / Emfuc3 or EndoS / BF3242 fusion proteins expressed as a percentage of the starting fucose removed. The fusion proteins are identified in Tables 1 to 5 below.

[0027] Figure 7Shown are bar graphs showing the percentage of fucose hydrolyzed by BF3242 fucosidase, EndoS or EndoS2 / BF3242 fusion protein expressed as a percentage of the starting fucose removed. The fusion proteins are identified in Tables 1 to 5 below.

[0028] Fig. 8A SDS-PAGE analysis showing the time course of trastuzumab (TRZ) glycan remodeling by C-C401. 2 (α2-6)Gal 2 GlcNAc 2 Man 3 GlcNAc 2 Lane 2: TRZ standard. Lane 3: TRZ-N / N (TRZ containing two GlcNAcs) standard. Lane 4: TRZ treated with C-C401 for 20 hours to obtain TRZ-N / N. Lanes 5-7: TRZ-N / N glycosylated with 95%, 98% and 98% by C-C401 using SCT-oxazoline for 10, 20 and 30 minutes, respectively. Lane 8: Purified glycoengineered TRZ (TRZ-G2S2 / G2S2).

[0029] Figure 8B SDS-PAGE analysis showing remodeling of TRZ by C-C401 glycans. 2 GlcNAc 2 Man 3 GlcNAc 2 (G2) replacement. Lane 2: Trastuzumab standard. Lane 3: TRZ treated with C-C401 for 16 hours to obtain TRZ-N / N. Lanes 4-7: 98% glycosylated TRZ-N / N by C-C401 using CT-oxazoline for 15, 30, 45 and 60 minutes. Lane 8: Purified glycoengineered TRZ (TRZ-G2 / G2). Lane 9: TRZ-G2S2 / G2S2 standard.

[0030] Fig. 9SDS-PAGE analysis showing glycan remodeling of rituximab (RTX) by C-C401. Lane 2: Standard of trastuzumab. Lane 3: Standard of TRZ-N / N. Lane 4: Standard of rituximab. Lane 5: RTX treated with C-C401 for 20 hours to obtain RTX-N / N. Lanes 6-8: 95%, 98%, and 98% glycosylated RTX-N / N by C-C401 using SCT-oxazoline. Lane 9: Purified glycoengineered RTX (RTX-G2S2 / G2S2).

[0031] Fig.10 SDS-PAGE analysis showing the glycan remodeling of TRZ by C-C406. Lane 2: Trastuzumab standard. Lane 3: TRZ-N / N standard. Lane 4: TRZ treated with C-C406 for 16 hours to obtain TRZ-N / N. Lanes 5-8: TRZ-N / N glycosylated at 90%, 95%, 98% and 98% by C-C406 with SCT-oxazoline for 30, 60, 90 and 120 minutes, respectively. Lane 9: Purified glycoengineered TRZ (TRZ-G2S2 / G2S2).

[0032] Fig.11 SDS-PAGE analysis showing glycan remodeling of TRZ by C-C407. Lane 2: Standard of trastuzumab. Lane 3: Standard of TRZ-N / N. Lanes 4-7: 80%, 95%, 98% and 98% glycosylated TRZ-N / N by C-C407 using SCT-oxazoline for 30, 60, 90 and 120 minutes, respectively. Lane 8: TRZ treated with C-C407 for 12 hours to obtain TRZ-N / N. Lane 9: Purified glycoengineered TRZ (TRZ-G2S2 / G2S2).

[0033] Fig.12 SDS-PAGE analysis showing the glycan remodeling of TRZ by C-C204. Lane 2: Trastuzumab standard. Lane 3: TRZ treated with C-C204 for 16 hours to obtain TRZ-N / N. Lanes 4-9: TRZ-N / N glycosylated with 85%, 97%, 98%, 98%, 97% and 90% by C-C204 using SCT-oxazoline for 15, 30, 45, 60, 90, 120 and 240 minutes, respectively. Lane 10: Purified glycoengineered TRZ (TRZ-G2S2 / G2S2). DETAILED DESCRIPTION

[0034] The Fc domains of all IgGs have conserved N-glycosylation sites to which biantennary complex-type glycans are usually attached, but terminal and core modifications result in significant structural heterogeneity. Certain glycoforms of human antibodies exhibit improved therapeutic effects, while other glycoforms have undesirable properties. Therefore, the ability to control the glycosylation process plays a vital role in obtaining antibodies containing desired glycoforms to improve their therapeutic efficacy. The present disclosure provides fusion proteins comprising a plurality of enzymes for remodeling antibody glycoforms, which can then generate glycopeptides with glycosylation patterns suitable for therapeutic use in mammals.

[0035] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, recombinant DNA, and immunology, which are well known to those skilled in the art and are fully explained in the literature. See, e.g., Molecular Cloning A Laboratory Manual, 2nd edition, edited by Sambrook, Fritsch and Maniatis (Cold Spring Harbor Laboratory Press, 1989); DNA Cloning, Volumes I and II (D. N. Glover, ed., 1985); Culture Of Animal Cells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide To Molecular Cloning (1984); treatise Methods In Enzymology (Academic Press, Inc., NY); Gene Transfer Vectors For Mammalian Cells (J. H. Miller and M. P. Calos, eds., 1987, Cold Spring Harbor Laboratory); Methods In Enzymology, Volumes 154 and 155 (Wu et al., eds.), Immunochemical Methods In Cell And Molecular Biology (Mayer and Walker, eds., Academic Press, London, 1987); Antibodies: A Laboratory Manual, Harlow and Lanes (Cold Spring Harbor Laboratory Press, 1988); and Handbook Of Experimental Immunology, Volumes I-IV (DM Weir and CC Blackwell, eds., 1986).

[0036] definition

[0037] The present disclosure may be more readily understood by reference to the following detailed description, examples, and drawings and tables of various embodiments of the present disclosure and their associated descriptions. It should be understood that, unless otherwise expressly specified by the claims, the present disclosure is not limited to a particular method of preparation, carrier, or formulation, or a particular manner of formulating the extracts of the present disclosure into a product or composition intended for topical, oral, or parenteral administration, as will be apparent to those skilled in the art, such matters may of course vary. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only and are not intended to be limiting.

[0038] As used in accordance with the present disclosure, the following terms shall be understood to have the following meanings, unless otherwise indicated.

[0039] It must be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly indicates otherwise. Thus, unless the context requires otherwise, singular terms shall include pluralities and plural terms shall include the singular.

[0040] As used herein, the term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes examples where the event or circumstance occurs and examples where the event or circumstance does not occur. For example, the phrase "optionally includes an agent" means that the agent may or may not be present.

[0041] As used herein, the term "glycan" refers to a polysaccharide or an oligosaccharide. Glycans are also used herein to refer to the carbohydrate portion of a glycoconjugate (e.g., a glycoprotein, a glycolipid, a glycopeptide, a glycoprotein body, a peptidoglycan, a lipopolysaccharide, or a proteoglycan). Glycans are generally composed only of O-glycosidic linkages between monosaccharides. For example, cellulose is a glycan (or more specifically, a glucan) composed of β-1,4-linked D-glucose, and chitin is a glycan composed of β-1,4-linked N-acetyl-D-glucosamine. Glycans can be homopolymers or heteropolymers of monosaccharide residues and can be straight or branched. Glycans can be found attached to proteins, such as in glycoproteins and proteoglycans. They are generally found on the outside surface of cells. O- and N-linked glycans are very common in eukaryotic cells, but can also be found (although less commonly) in prokaryotes. N-linked glycans are found attached to the R-group nitrogen (N) of asparagine in the sequon. The sequon is an Asn-X-Ser or Asn-X-Thr sequence, where X is any amino acid except proline.

[0042] As used herein, the term "engineered" describes a polypeptide whose amino acid sequence has been designed by man and / or whose existence and production requires human intervention and / or activity.

[0043] As used herein, the terms "polynucleotide" or "nucleic acid" are used interchangeably herein and refer to polymers of nucleotides of any length and include DNA and RNA. Nucleotides may be deoxyribonucleotides, ribonucleotides, modified nucleotides or bases and / or their analogs or any substrate that can be incorporated into a polymer by DNA or RNA polymerase or by a synthetic reaction. Polynucleotides may include modified nucleotides, such as methylated nucleotides and their analogs. If present, modification of the nucleotide structure may be performed before or after assembly of the polymer. The nucleotide sequence may be mixed with non-nucleotide components. Polynucleotides may be further modified after synthesis, for example, by coupling with a label. Other types of modifications include, for example, "caps", substitution of one or more of the natural nucleotides with an analog; internucleotide modifications, such as those with uncharged linkages (e.g., methylphosphonates, phosphotriesters, phosphoamidates, carbamates, etc.) and those with charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), those containing side moieties, such as proteins (e.g., nucleases, toxins, antibodies, signal peptides, poly-L-lysine, etc.), those with intercalators (e.g., acridine, psoralen, etc.), those containing chelators (e.g., metals, radioactive metals, boron, oxidative metals, etc.), those containing alkylating agents, those with modified linkages (e.g., alpha mutameric nucleic acids, etc.), as well as unmodified forms of polynucleotides. In addition, any of the hydroxyl groups typically present in the sugar can be replaced by, for example, a phosphonate group, a phosphate group, protected by a standard protecting group, or activated to prepare additional linkages with additional nucleotides, or can be coupled to a solid or semisolid support. The 5' and 3' terminal OH can be phosphorylated or partially substituted with an amine or an organic capping group of 1 to 20 carbon atoms. Other hydroxyl groups can also be derivatized to standard protecting groups. Polynucleotides can also contain similar forms of ribose or deoxyribose sugars commonly known in the industry, including, for example, 2'-O-methyl-ribose, 2'-O-allyl-ribose, 2'-fluoro-ribose or 2'-azido-ribose, carbocyclic sugar analogs, α-mutanosaccharides, epimeric sugars (e.g., arabinose, xylose or lyxose), pyranose, furanose, sedoheptulose, acyclic analogs, and abasic nucleoside analogs (e.g., methyl nucleosides). One or more phosphodiester linkages can be replaced by alternative linking groups. The alternative linking groups include, but are not limited to, the following embodiments: wherein the phosphate ester is P(O)S ("thioester"), P(S)S ("dithioester"), (O)NR 2 ("amid"), P(O)R, P(O)OR', CO or CH 2("methyl acetal") substitution, wherein each R or R' is independently H or optionally a substituted or unsubstituted alkyl (1-20 C), aryl, alkenyl, cycloalkyl, cycloalkenyl or aralkyl containing an ether (--O--) linkage. All linkages in a polynucleotide need not be identical. The foregoing description applies to all polynucleotides mentioned herein, including RNA and DNA.

[0044] As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.

[0045] As used herein, the term "host" refers to a system (e.g., a cell, an organism, etc.) in which a polypeptide of interest is present. In some embodiments, a host is a system susceptible to infection by a particular infectious agent. In some embodiments, a host is a system that expresses a particular polypeptide of interest.

[0046] As used herein, the term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to natural amino acids. Natural amino acids are those encoded by the genetic code and those amino acids that are subsequently modified, for example, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as natural amino acids (i.e., the α carbon is linked to hydrogen, carboxyl, amine, and R groups), for example, homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. The analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as natural amino acids. Amino acid mimetics refer to chemical compounds that have a structure different from the general chemical structure of amino acids but function in a manner similar to natural amino acids.

[0047] As used herein, the terms "polypeptide", "peptide" and "protein" are used interchangeably herein and can be used to refer to polymers in which monomers are amino acids and are joined together by amide bonds, alternatively referred to as peptides. In addition, non-natural amino acids are also included, such as β-alanine, phenylglycine and homoarginine. Amino acids that are not encoded by nucleic acids can also be used in the present invention. In addition, amino acids that have been modified to include reactive groups, glycosylation sites, polymers, therapeutic moieties, biomolecules and the like can also be used in the present invention. All amino acids used in the present invention can be their D- or L-isomers. The L-isomer is generally preferred. In addition, other peptide mimetics can also be used in the present invention. As used herein, "peptide" is an oligopeptide, polypeptide, peptide, protein or glycoprotein. As used herein, "polypeptide" refers to glycosylated and unglycosylated peptides. Peptides that are incompletely glycosylated by a system that expresses peptides are also included.

[0048] As used herein, the term "fusion protein" (also known as "chimeric protein") refers to a protein translated from the joining of two or more genes that originally encoded separate proteins.

[0049] As used herein, the term "antibody" also includes antigen-binding fragments of intact antibody molecules. Antigen-binding fragments of antibodies can be derived from, for example, intact antibody molecules using any suitable standard technique, such as proteolysis or recombinant genetic engineering techniques involving manipulation and expression of DNA encoding antibody variable domains and, if appropriate, constant domains. The DNA is known and / or readily available from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. The DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable and / or constant domains into a suitable configuration, or to introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0050] As used herein, the term "antibody Fc region" refers to the crystallizable region fragment, which is the tail region of the antibody that interacts with cell surface receptors called Fc receptors and some proteins of the complement system. Glycosylation of the Fc fragment is required for Fc receptor-mediated activity.

[0051] As used herein, the term "glycotype" refers to an antibody comprising a specific glycosylation state. The term "glycosylation state" refers to an antibody having a specific or desired glycosylation pattern.

[0052] As used herein, the term "remodeling the glycans of the Fc region of an antibody" refers to changing or modifying the type, position, number, form or linkage of the residues in the glycans of the Fc region of an antibody. In one embodiment, the remodeling reduces the amount or length of the core fucose.

[0053] As used herein, the term "target polysaccharide oxazoline" refers to a polysaccharide oxazoline whose glycoform is to be reshaped.

[0054] Fusion Protein

[0055] Glycosylation pathway engineering has been developed to improve biological functions and reduce the heterogeneity of therapeutic antibodies. An approach to obtain homogeneous glycoproteins is based on a strategy of glycoprotein remodeling. Fucosidases are able to catalyze the hydrolysis of α-L-fucoside. For example, the hydrolysis of α-L-fucoside involves a chemical reaction that cleaves the 1,2-linkage, 1,3-linkage, or 1,6-linkage between α-L-fucose and N-acetylglucosamine residues. On the other hand, endoglycosidases catalyze the release of oligosaccharides from glycoproteins or glycoesters. For example, the release of oligosaccharides from glycoproteins is carried out by hydrolyzing the β-1,4-di-N-acetylcitrobioside core of asparagine-linked glycans or hydrolyzing the endo-β-N-acetylglucosaminides on all N-linked glycans and diantennary and sialylated glycans of AGP (α1-acid glycoprotein). The combination of fucosidase and endoglycosidase to process antibody glycoforms can obtain homogeneous antibodies with well-defined glycans at the Fc region.

[0056] The present disclosure provides a fusion protein comprising a fucosidase or a truncated fragment or mutant thereof fused to the N-terminus or C-terminus of an endoglycosidase or a truncated fragment or mutant thereof; wherein the fusion protein exhibits both fucosidase activity and endoglycosidase activity.

[0057] Fucosidase or its truncated fragment or mutant and endoglycosidase or its truncated fragment or mutant are fused together directly or via a linker. In one embodiment, fucosidase or its truncated fragment or mutant is directly or indirectly linked to the N-terminus of the truncated fragment of endoglycosidase or its mutant. In another embodiment, fucosidase or its truncated fragment or mutant is directly or indirectly linked to the C-terminus of the truncated fragment of endoglycosidase or its mutant.

[0058] The fucosidase may be in a wild-type form or in a modified form compared to its wild-type form, such as a truncated fragment or mutant thereof. Examples of fucosidases include, but are not limited to, Lactobacillus casei α-L-fucosidase C (Alfc), Bacteroides fragilis fucosidase (BF3242), Bacteroides polymorpha α-L-fucosidase (BT2970), Oligotrophic bacteria Hosokobacterium α-L-fucosidase (EO0918) and Elizabeth miller α-(1-6) fucosidase (Emfuc3), or fragments thereof or mutants thereof. In some embodiments, the fucosidase is Lactobacillus casei α-L-fucosidase C and Elizabeth miller α-(1-6) fucosidase (Emfuc3) and truncated fragments or mutants thereof.

[0059] The endoglycosidase may be in wild-type form or in a modified form compared to its wild-type form, such as a truncated fragment or mutant of the endoglycosidase. Examples of endoglycosidases include, but are not limited to, Streptococcus pyogenes endoglycosidase S, Streptococcus pyogenes endoglycosidase S2, or a fragment or mutant thereof. In some embodiments, the endoglycosidase is Streptococcus pyogenes endoglycosidase S2, or a truncated fragment or mutant thereof.

[0060] Truncated fragments of fucosidase or endoglycosidase can also be used in the present disclosure. Truncated fragments of fucosidase or endoglycosidase have been truncated at the N-terminus of the peptide, the C-terminus of the peptide, or both ends or the internal region of the peptide. In some embodiments, the truncated fragment of fucosidase or endoglycosidase is a truncated fragment of its C-terminus or N-terminus. In one embodiment, the truncated fragment of endoglycosidase is its IgG binding domain. In some other embodiments, the truncated fragment of endoglycosidase is the IgG binding domain of Streptococcus pyogenes endoglycosidase S or Streptococcus pyogenes endoglycosidase S2.

[0061] The fucosidase or endoglycosidase may be in a mutant form thereof. In one embodiment, the endoglycosidase mutant is a Streptococcus pyogenes endoglycosidase S having a mutation at amino acid position -D233, preferably having a mutation at amino acid position D233Q. In some embodiments, the endoglycosidase mutant is a Streptococcus pyogenes endoglycosidase S2 having a mutation at the following amino acid positions: T138, D182, D184, D186, D226 or T227; preferably having T138E, T138M, T138Q, T138R, T138L, T138H, T138N, T138K, D182Q, D184M, D184Q, D184T, D184L, D184F, D184S, D184V, D184K, D184W, E186A, D226Q or T227Q.

[0062] In some embodiments, the fusion protein comprises Lactobacillus casein α-L fucosidase C fused to: Streptococcus pyogenes endoglycosidase S, Streptococcus pyogenes endoglycosidase S2, the IgG binding domain of Streptococcus pyogenes endoglycosidase S, the IgG binding domain of Streptococcus pyogenes endoglycosidase S2, Streptococcus pyogenes endoglycosidase S with a mutation at amino acid position D233, Streptococcus pyogenes endoglycosidase S with a mutation at amino acid position T138, pyogenes endoglycosidase S2, a Streptococcus pyogenes endoglycosidase S2 having a mutation at amino acid position D182, a Streptococcus pyogenes endoglycosidase S2 having a mutation at amino acid position D184, a Streptococcus pyogenes endoglycosidase S2 having a mutation at amino acid position D186, a Streptococcus pyogenes endoglycosidase S2 having a mutation at amino acid position D226, or a Streptococcus pyogenes endoglycosidase S2 having a mutation at amino acid position T227.

[0063] In some embodiments, the fusion protein comprises a-(1-6)fucosidase from Elizabeth Millipore fused to: Streptococcus pyogenes endoglycosidase S, Streptococcus pyogenes endoglycosidase S2, the IgG binding domain of Streptococcus pyogenes endoglycosidase S, the IgG binding domain of Streptococcus pyogenes endoglycosidase S2, a Streptococcus pyogenes endoglycosidase S with a mutation at amino acid position D233, a Streptococcus pyogenes endoglycosidase S with a mutation at amino acid position T138, or a Streptococcus pyogenes endoglycosidase S with a mutation at amino acid position T239. Streptococcus pyogenes endoglycosidase S2, Streptococcus pyogenes endoglycosidase S2 with a mutation at amino acid position D182, Streptococcus pyogenes endoglycosidase S2 with a mutation at amino acid position D184, Streptococcus pyogenes endoglycosidase S2 with a mutation at amino acid position D186, Streptococcus pyogenes endoglycosidase S2 with a mutation at amino acid position D226, or Streptococcus pyogenes endoglycosidase S2 with a mutation at amino acid position T227.

[0064] Examples of amino acid sequences of fusion proteins include, but are not limited to, SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125 8, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, and 204 or a substantially similar sequence thereof. Preferably, the fusion protein has an amino acid sequence selected from the group consisting of SEQ ID NO: 34, 36, 38, 40, 42, 44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, 198, 200, 202, and 204, or a substantially similar sequence thereof.

[0065] When applied to polypeptides, the term "substantially similar" or "substantially similar" means that two peptide sequences have at least 95% sequence identity, even better at least 98% or 99% sequence identity when optimally aligned (e.g., by using the programs GAP or BESTFIT with preset gap weights). Preferably, the inconsistent residue positions differ by conservative amino acid substitutions. "Conservative amino acid substitutions" are those in which an amino acid residue is replaced by another amino acid residue having a side chain (R group) of similar chemical properties (e.g., charge or hydrophobicity). In general, conservative amino acid substitutions will not substantially change the functional properties of the protein. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percentage of sequence identity or similarity may be adjusted upward to correct for the conservative nature of the substitution. The manner in which this adjustment is made is well known to those skilled in the art. Examples of groups of amino acids having side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine, and isoleucine; (2) aliphatic-hydroxy side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains: cysteine ​​and methionine. Preferred conservative amino acid substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine.

[0066] Mutation can be the replacement, deletion or insertion of one or more codons encoding an antibody or polypeptide, which results in a change in the amino acid sequence compared to the native sequence of the antibody or polypeptide. Standard techniques known to those skilled in the art can be used to induce mutations in the nucleotide sequence encoding the molecules provided herein, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. Insertion or deletion can be within the range of about 1 to 5 amino acids as appropriate. In certain embodiments, replacement, deletion or insertion includes less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions or less than 2 amino acid substitutions compared to the original molecule. Allowed variation can be determined by systematically making amino acid insertions, deletions or substitutions in the sequence and testing the resulting variants for the activity displayed by the full-length or mature native sequence.

[0067] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions (ranging in length from one residue to polypeptides containing 100 or more residues), as well as intrasequence insertions of single or multiple amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue. Other insertion variants of the antibody molecule include fusion of the N- or C-terminus of the antibody to an enzyme (e.g., for antibody-directed proenzyme therapy) or a polypeptide that increases the serum half-life of the antibody.

[0068] Conservative amino acid substitutions are those in which an amino acid residue is replaced by an amino acid residue with a side chain of similar charge. Families of amino acid residues with side chains of similar charge have been defined in the industry. The families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Alternatively, mutations can be introduced randomly along all or part of the coding sequence, for example, by saturation mutagenesis, and the resulting mutants can be screened for biological activity to identify mutants that retain activity. After mutagenesis, the encoded protein can be expressed and the activity of the protein can be determined.

[0069] Substantial modification of the biological properties of the antibody is achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the size of the side chain.

[0070] Non-conservative substitutions entail exchanging a member of one of the classes for another class. The substituted residues may also be introduced into conservative substitution sites or into remaining (non-conservative) sites.

[0071] Mutations can be made using methods known in the art, such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (e.g., see Carter, 1986, Biochem J. 237: 1-7; and Zoller et al., 1982, Nucl. Acids Res. 10: 6487-500), cassette mutagenesis (e.g., see Wells et al., 1985, Gene 34: 315-23) or other known techniques can be performed on cloned DNA to generate variant DNA.

[0072] Nucleic acid molecules encoding fusion proteins disclosed herein and expression systems for producing fusion proteins disclosed herein

[0073] The disclosure also provides nucleic acid molecules that express the fusion proteins described herein.

[0074] In some embodiments of the present disclosure, the nucleic acid molecule has a nucleotide sequence selected from the group consisting of: SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117 , 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, and 203, or substantially the same sequence thereof. In some other embodiments, the nucleic acid has a nucleotide sequence selected from the group consisting of SEQ ID NO: 33, 35, 37, 39, 41, 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, 199, 201, and 203, or a sequence substantially identical thereto.

[0075] The term "substantial identity" or "substantially identical" when referring to a nucleic acid or a fragment thereof indicates that when appropriate nucleotide insertions or deletions are optimally aligned with another nucleic acid (or its complementary strand), at least 95%, and more preferably at least 96%, 97%, 98% or 99% of the nucleotide bases exist in nucleotide sequence identity with the entire sequence of the reference nucleic acid sequence, as measured by any well-known algorithm for sequence identity (e.g., FASTA, BLAST or Gap), as discussed below. In certain cases, a nucleic acid molecule having substantial identity to a reference nucleic acid molecule can encode a polypeptide having the same or substantially similar amino acid sequence as a polypeptide encoded by the reference nucleic acid molecule.

[0076] Fusion proteins can be produced using various expression systems, including prokaryotic and eukaryotic expression systems. Many such systems are widely available from commercial suppliers. In one embodiment, the fusion protein can be expressed using a vector, wherein the polynucleotide encoding the fusion protein is operably linked to a promoter sequence. In one embodiment, the promoter is a constitutive promoter. In another embodiment, the promoter is an inducible promoter.

[0077] In one embodiment, the polynucleotide or vector is contained in a virus. In another embodiment, the virus is selected from the group consisting of retrovirus, lentivirus, adenovirus and adeno-associated virus. In a preferred embodiment of the present disclosure, the polynucleotide or vector is contained in an adeno-associated virus (AAV) shuttle plasmid.

[0078] Application of the Fusion Protein of the Disclosure

[0079] The present disclosure provides a method for remodeling the glycans of an antibody Fc region, comprising the steps of obtaining an antibody having heterogeneous glycans in the antibody Fc region, and contacting the antibody with a fusion protein described herein.

[0080] In one embodiment of the present disclosure, the method comprises:

[0081] Provided are antibodies having heterogeneous glycans in their Fc regions, fusion proteins of the present disclosure, and target glycan oxazolines, wherein the glycans in the Fc region of the antibody comprise an N-acetylglucosamine (GlcNAc) residue linked to an Asn residue in the Fc region; preferably, the glycans in the Fc region of the antibody comprise an N-acetylglucosamine (GlcNAc) residue linked to Asn-297 in the Fc region; and

[0082] contacting the antibody with the fusion protein and linking the target polysaccharide oxazoline to the antibody Fc region;

[0083] This can be used to obtain remodeled glycans in the Fc region of antibodies.

[0084] In one embodiment, the target polysaccharide oxazoline is linked to the antibody Fc region instead of a portion of the polysaccharide in the Fc region. It is believed that, although not wishing to be bound by any theory, the fusion protein of the present disclosure removes the N-linked GlcNAc in the polysaccharide in the antibody Fc region, and then glycosylation occurs to add the target polysaccharide oxazoline to the antibody Fc region. Thus, a remodeled polysaccharide in the antibody Fc region can be obtained.

[0085] Examples of remodeled glycans include, but are not limited to, Sia 2 (α2-6)Gal 2 GlcNAc 2 Man 3 GlcNAc 2 、Sia 2(α2-6)Gal 2 GlcNAc 3 Man 3 GlcNAc 2 、Sia 2 (α2-3)Gal 2 GlcNAc 2 Man 3 GlcNAc 2 、Sia 2 (α2-3)Gal 2 GlcNAc 3 Man 3 GlcNAc 2 、Sia 2 (α2-3 / α2-6)Gal 2 GlcNAc 2 Man 3 GlcNAc 2 、Sia 2 (α2-6 / α2-3)Gal 2 GlcNAc 2 Man 3 GlcNAc 2 、Sia 2 (α2-3 / α2-6)Gal 2 GlcNAc 3 Man 3 GlcNAc 2 、Sia 2 (α2-6 / α2-3)Gal 2 GlcNAc 3 Man 3 GlcNAc 2 、Sia(α2-6)Gal 2 GlcNAc 2 Man 3 GlcNAc 2 、Sia(α2-3)Gal 2 GlcNAc 2 Man 3 GlcNAc 2 、Sia(α2-6)Gal 2 GlcNAc 3 Man 3 GlcNAc 2 、Sia(α2-3)Gal 2 GlcNAc 3 Man 3 GlcNAc 2 、Sia(α2-6)GalGlcNAc2 Man 3 GlcNAc 2 、Sia(α2-3)GalGlcNAc 2 Man 3 GlcNAc 2 、Sia(α2-6)GalGlcNAc 3 Man 3 GlcNAc 2 、Sia(α2-3)GalGlcNAc 3 Man 3 GlcNAc 2 , Gal 2 GlcNAc 2 Man 3 GlcNAc 2 、GalGlcNAcMan 3 GlcNAc 2 , Gal 2 GlcNAc 3 Man 3 GlcNAc 2 , GalGlcNAc 2 Man 3 GlcNAc 2 , GalGlcNAc 3 Man 3 GlcNAc 2 , GlcNAc 3 Man 3 GlcNAc 2 , GlcNAc 2 Man 3 GlcNAc 2 , GlcNAcMan 3 GlcNAc 2 or Man 3 GlcNAc 2 .

[0086] Preferably, the method further comprises purifying antibodies whose Fc regions have remodeled glycans.

[0087] The aspects of the present invention are further described by means of the following illustrative non-limiting examples that provide a better understanding of the embodiments of the present invention and its many advantages. The following examples are intended to show preferred embodiments of the present invention. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques that are used in the present invention to function well in the practice of the present invention, and therefore can be considered to constitute preferred modes for its practice. However, it should be understood by those skilled in the art with the aid of this disclosure that many changes can be made to the disclosed specific embodiments without departing from the spirit and scope of the present invention and still obtaining the same or similar results.

[0088] Examples

[0089] Fusion Protein

[0090] Example 1: Production of fusion protein

[0091] Fusion proteins containing full-length or truncated (IgG binding motif only) EndoS or EndoS2 fused to the fucosidases BF3242 (Table 1), Alfc (Table 2), BT2970 (Table 3), EO0918 (Table 4), and Emfuc3 (Table 5) were generated as shown in Tables 1 to 5 below.

[0092] Table 1. BF3242 fusion proteins

[0093]

[0094]

[0095] a Only the IgG binding domain of endoS or endoS2

[0096] b Wild-type endoS or endoS2

[0097] Table 2. Alfc fusion proteins

[0098]

[0099]

[0100] Table 3. BT2970 fusion proteins

[0101]

[0102]

[0103] Table 4. EO0918 fusion proteins

[0104]

[0105] Table 5. Emfuc3 fusion proteins

[0106]

[0107] Remodeling the glycans in the Fc region of antibodies.

[0108] Example 2: Fucosidase activity analysis

[0109] The test antibody (ie, trastuzumab (TRZ)) was treated with endoS2 in Tris-HCl (pH 7.0) at 37°C for 1 hour. The treated antibody was purified by adsorption onto a protein A affinity chromatography resin (MabSelect TM ), washed with Tris-HCl (pH 7.4) and purified by elution with citric acid buffer (pH 3.0). Most of the residual polysaccharides in the Fc region of the obtained antibody are N-acetylglucosamine-fucose (GlcNAc-Fuc). The purified antibody was treated with fusion protein or native fucosidase in Tris-HCl buffer (pH 7.0) at 37°C for 16h, followed by heating at 55°C for 20min to inactivate the enzyme activity. The reaction mixture was filtered by a 0.22μm membrane and the treated antibody was purified by affinity chromatography.

[0110] The content of residual fucose was determined by analyzing the mass of intact protein. The ratios of TRZ-N / N (TRZ containing two GlcNAc in the Fc region), TRZ-N / NF (TRZ containing one GlcNAc and one GlcNAc-Fuc in the Fc region), and TRZ-NF / NF (TRZ containing two GlcNAc-Fuc in the Fc region) were identified by the intensity of each mass peak. The results are shown in Tables 6 and Figure 1 middle.

[0111] Table 6. Percentage of Fucose Removed from Test Antibody TRZ

[0112]

[0113] The results showed that the fucosidase activity of B. fragilis fucosidase BF3242 was not improved by fusion to S. pyogenes endoS2 D184M mutant. BF3242 (18.22% fucose removal) was compared to E-C401 (14.35%) and E-N401 (7.11%).

[0114] Surprisingly, the fucosidase activity of Alfc was significantly increased by fusing this enzyme to the N-terminus or C-terminus of the D184 mutant endoS2 enzyme. For example, Alfc removed 20.72% of fucose from the test antibody, while Alfc fused to the N-terminus of the EndoS2 D184M mutant removed 40.19% of fucose. Alfc was compared to C-N401. Even more unexpectedly, Alfc fused to the C-terminus of EndoS2 D184M (C-C401) removed up to 86.37% of fucose from the test antibody. In particular, all tested Alfc fusions at the C-terminus of EndoS2 D184 mutants (ie, D184M, D184Q, D184T, D184L, and D184F) showed unexpectedly high fucosidase activity compared to Alfc fusions at the N-terminus of EndoS2 mutants.

[0115] Example 3: Fucosidase activity of additional Alfc fusion proteins

[0116] Additional fusion proteins with Alfc fused to the C-terminus of native or site-specific mutant EndoS2 enzymes were tested for fucosidase activity against TRZ as described above in Example 2. The results are shown in Tables 7 and Figure 2 middle.

[0117] Table 7. Percentage of Fucose Removed from Test Antibody TRZ

[0118]

[0119]

[0120] The results showed that all Alfc / EndoS2 fusion proteins tested removed significantly more fucose from TRZ compared to Alfc alone.

[0121] Additional fusion proteins of five fucosidases with truncated EndoS2 were tested for fucosidase activity against TRZ as described above in Example 2. The results are shown in Tables 8 and Figure 3 middle.

[0122] Table 8 Percentage of Fucose Removed from Test Antibody TRZ

[0123]

[0124] The defucosylating ability of Alfc was significantly improved. The fusion proteins of BT2970 and EO0918 could not remove the core-fucosyl of the antibody.

[0125] Example 4 Additional Fusion Proteins with Alfc or Emfuc3 Fused to Truncated EndoS2 or Site-Specific Mutant EndoS2 Enzymes

[0126] Additional fusion proteins with Alfc or Emfuc3 fused to truncated EndoS2 or site-specific mutant EndoS2 enzymes were tested for fucosidase activity against TRZ as described in Example 2 above. The results are shown in Tables 9 and Figure 4 middle.

[0127] Table 9. Percentage of Fucose Removed from Test Antibody TRZ

[0128]

[0129] When fused to the C-terminus of the intact EndoS2 mutation (C-C204), the defucosylation capacity of Alfc was increased more than that of fused to truncated EndoS2 (IgG binding domain), A-C202, and A-N202. When fused to the N-terminus of truncated EndoS2 (IgG binding domain), A-N205, the defucosylation capacity of Emfuc3 was improved, but not when fused to the C-terminus of truncated EndoS2 (IgG binding domain), A-C205.

[0130] Example 5

[0131] Additional fusion proteins with Alfc or Emfuc3e fused to the N-terminus of truncated or site-specific mutant EndoS or EndoS enzymes were tested for fucosidase activity against TRZ as described in Example 2 above. The results are shown in Tables 10 and 10 below. Figure 5 middle.

[0132] Table 10. Percentage of Fucose Removed from Test Antibody TRZ

[0133]

[0134] The activity of Alfc was dramatically improved after fusion with EndoS2, but not with EndoS. In contrast, the activity of Emfuc3 was more significantly improved after fusion with EndoS but not EndoS2.

[0135] Example 6 Additional Fusion Proteins with EndoS or EndoS2 / Emfuc3, EndoS / BF3242, Alfc, Emfuc3 or BF3242 Fucosidase

[0136] Additional fusion proteins with EndoS or EndoS2 / Emfuc3, EndoS / BF3242, Alfc, Emfuc3 or BF3242 fucosidase enzymes were tested for fucosidase activity against TRZ as described in Example 2 above. The results are shown in Tables 11 and 11 below. Figure 6 middle.

[0137] Table 11. Percentage of Fucose Removed from Test Antibody TRZ

[0138]

[0139]

[0140] Compared to Example 5, when Emfuc3 was fused to the N-terminus of EndoS-mutation and EndoS2-mutation, more significant improvement was observed than when fused to the C-terminus of the endoglycosidase.

[0141] Example 7 Additional Fusion Proteins with BF3242 Fused to Truncated or Site-Specific Mutant EndoS2 or EndoS Enzymes

[0142] Additional fusion proteins with BF3242 fused to truncated or site-specific mutant EndoS2 or EndoS enzymes were tested for fucosidase activity against TRZ as described above in Example 2. The results are shown in Tables 12 and Figure 7 middle.

[0143] Table 12. Percentage of Fucose Removed from Test Antibody TRZ

[0144]

[0145] The core-fucose removal ability of BF3242 was not improved by fusion with endoglycosidase.

[0146] Example 8 Glycoengineering of TRZ via Fusion Protein C-C401

[0147] The test antibody TRZ was treated with the fusion protein C-C401 (i.e., Alfc fused to the C-terminus of endoS2 D184M) in Tris-HCl (pH 7.0) at 37°C for 16 to 20 h. After adjusting the reaction to 30°C, sialylated polysaccharide-oxazoline (i.e., Sia 2 (α2-6)Gal 2 GlcNAc 2 Man 3 GlcNAc-oxazoline) or the unsialylated version (Gal 2 GlcNAc 2 Man 3GlcNAc-oxazoline) and the reaction was incubated for 30 min to 1 h. The glycoengineered TRZ was purified as described in Example 2 above. The resulting glycoengineered TRZ contained two Sia 2 (α2-6)Gal 2 GlcNAc 2 Man 3 GlcNAc 2 (G2S2) or two Gal 2 GlcNAc 2 Man 3 GlcNAc 2 (G2). Samples of the reaction were taken out at different time intervals and analyzed by SDS-PAGE. The results are presented graphically in 8A and 8B.

[0148] like Fig. 8A As shown in , TRZ (lane 1) was treated with C-C401 for 20 h to remove all glycans except N-linked GlcNAc (lane 4) to obtain TRZ-N / N (GlcNAc / GlcNac; lane 3). 2 (α2-6)Gal 2 GlcNAc 2 Man 3 The amount of TRZ-N / N glycosylated after GlcNAc-oxazoline was 95%, 98% and 98% after 10 min., 20 min. and 30 min., respectively. See lanes 5, 6 and 7. The purified glycoengineered TRZ-G2S2 / G2S2 is shown in lane 8.

[0149] Steering Figure 8B , which shows that after 16 h of TRZ deglycosylation (lane 3), after adjusting the temperature and adding Gal 2 GlcNAc 2 Man 3 The amount of TRZ-N / N glycosylated after GlcNAc-oxazoline was 98% at all time points. See lanes 4-7. Purified glycoengineered TRZ-G2 / G2 is shown in lane 8.

[0150] Example 9 Glycoengineering of Rituximab (RTX) via Fusion Protein C-C401

[0151] The test antibody RTX was treated with the fusion protein C-C401 in Tris-HCl. After adjusting the temperature to 30°C, Sia 2 (α2-6)Gal 2 GlcNAc 2 Man 3GlcNAc-oxazoline and the reaction was incubated for 1 h. The polysaccharide remodeled RTX was purified as described in Example 2 above. The resulting RTX contained two G2S2 in the Fc region. Samples of the reaction were taken at different time intervals and analyzed by SDS-PAGE. The results are shown in Fig. 9 middle.

[0152] RTX (lane 4) was treated with C-C401 for 20 h to remove glycans except N-linked GlcNAc to obtain RTX-N / N (GlcNAc / GlcNac; lane 5). 2 (α2-6)Gal 2 GlcNAc 2 Man 3 The amount of RTX-N / N glycosylated after GlcNAc-oxazoline was 95%, 98% and 98% after 10 min., 20 min. and 30 min. respectively. See lanes 6, 7 and 8. The purified glycoengineered RTX-G2S2 / G2S2 is shown in lane 9.

[0153] Example 10 Glycoengineering of TRZ via Fusion Protein C-C406

[0154] The test antibody TRZ was treated with the fusion protein C-C406 (i.e., Alfc fused to the C-terminus of endoS2 D184S) in Tris-HCl (pH 7.0) at 37°C for 16 h. After adjusting the temperature to 30°C, Sia 2 (α2-6)Gal 2 GlcNAc 2 Man 3 GlcNAc-oxazoline was added and the reaction was incubated for 1 h. The polysaccharide remodeled TRZ was purified as described in Example 2 above. The resulting TRZ contained two G2S2 in the Fc region. Samples of the reaction were taken out at different time intervals and analyzed by SDS-PAGE. The results are shown in Fig.10 middle.

[0155] TRZ (lane 1) was treated with C-C406 for 16 h to remove all glycans except N-linked GlcNAc to form TRZ-N / N (GlcNAc / GlcNac; lane 4). 2 (α2-6)Gal 2 GlcNAc 2 Man 3The amount of TRZ-N / N glycosylated after GlcNAc-oxazoline was 90%, 95%, 98% and 98% after 30 min., 60 min., 90 min. and 120 min. respectively. See lanes 5, 6, 7 and 8. Purified glycoengineered TRZ-G2S2 / G2S2 is shown in lane 9.

[0156] Example 11 Glycoengineering of TRZ via Fusion Protein C-C407

[0157] The test antibody TRZ was treated with the fusion protein C-C407 (i.e., Alfc fused to the C-terminus of endoS2 D184V) in Tris-HCl (pH 7.0) at 37°C for 12 h. 2 (α2-6)Gal 2 GlcNAc 2 Man 3 GlcNAc-oxazoline and the reaction was incubated for 1 h. The polysaccharide remodeled TRZ was purified as described above. The resulting TRZ contained two G2S2 in the Fc region. Samples of the reaction were taken out at different time intervals and analyzed by SDS-PAGE. The results are shown in Fig.11 middle.

[0158] TRZ (lane 1) was treated with C-C407 for 12 h to remove polysaccharides except N-linked GlcNAc to obtain TRZ-N / N (GlcNAc / GlcNac; lane 8). 2 (α2-6)Gal 2 GlcNAc 2 Man 3 The amount of TRZ-N / N glycosylated after GlcNAc-oxazoline was 80%, 95%, 98% and 98% after 30 min., 60 min., 90 min. and 120 min., respectively. See lanes 4, 5, 6 and 7. Purified glycoengineered TRZ-G2S2 / G2S2 is shown in lane 9.

[0159] Example 12 Glycoengineering of TRZ via Fusion Protein C-C204

[0160] The test antibody TRZ was treated with the fusion protein C-C204 (i.e., Alfc fused to the C-terminus of endoS2 T138M) in Tris-HCl (pH 7.0) at 37°C for 16 h. 2 (a2-6)Gal 2 GlcNAc 2 Man 3GlcNAc-oxazoline was added and the reaction was incubated for 4 h. TRZ remodeled with polysaccharides (50 min) was purified as described above. The resulting TRZ contained two G2S2 in the Fc region. Samples of the reaction were taken at different time intervals and analyzed by SDS-PAGE. The results are shown in Fig.12 middle.

[0161] TRZ (lane 1) was treated with C-C204 to remove polysaccharides other than N-linked GlcNAc to obtain TRZ-N / N (GlcNAc / GlcNac; lane 3). 2 (a2-6)Gal 2 GlcNAc 2 Man 3 The amount of TRZ-N / N glycosylated after GlcNAc-oxazoline was 80%, 95%, 98%, 98%, 95% and 90% after 15min, 30min, 45min, 50min, 120min and 240min, respectively. See lanes 4, 5, 6, 7, 8 and 9. Purified glycoengineered TRZ-G2S2 / G2S2 (50min) is shown in lane 10.

[0162] Although the present disclosure has been described in conjunction with the specific embodiments explained above, many alternatives and modifications and variations thereof will be apparent to those skilled in the art. All such alternatives, modifications and variations are considered to be within the scope of the present disclosure.

Claims

1. A fusion protein consisting of an endoglycosidase or a truncated fragment or mutant thereof and a fucosidase fused to the N-terminus or C-terminus of the endoglycosidase or a truncated fragment or mutant thereof; wherein the fusion protein exhibits both fucosidase activity and endoglycosidase activity; wherein The fucosidase is a casei) α-L-fucosidase C (Alfc), and the endoglycosidase or a truncated fragment or mutant thereof is Streptococcus pyogenes endoglycosidase S2, the IgG binding domain of Streptococcus pyogenes endoglycosidase S2, Streptococcus pyogenes endoglycosidase S with a mutation at amino acid position D233Q, Streptococcus pyogenes endoglycosidase S with a mutation at amino acid position T138E, T138M, T138Q, T138R, T138L, T138H, T138N, T138K, D182Q, D184M, D184Q, D184T, D184L, D184F, D184S, D184V, D184K, D184W, E186A, D226Q or T227Q; or The fucosidase is Elizabethkingia miricola α-(1-6) fucosidase (Emfuc3), and the endoglycosidase or a truncated fragment or mutant thereof is Streptococcus pyogenes endoglycosidase S2, the IgG binding domain of Streptococcus pyogenes endoglycosidase S2, Streptococcus pyogenes endoglycosidase S with a mutation at amino acid position D233Q, Streptococcus pyogenes endoglycosidase S2 with a mutation at amino acid position T138M or D184M.

2. The fusion protein of claim 1, which consists of the following amino acid sequence: SEQ ID NO:44, 46, 48, 50, 52, 54, 56, 58, 60, 62, 64, 66, 68, 70, 72, 74, 76, 78, 84, 86, 88, 100, 102, 104, 106, 108, 186, 188, 202 or 204.

3. A nucleic acid molecule that expresses the fusion protein of claim 1 or 2.

4. The nucleic acid molecule according to claim 3, which consists of the following nucleotide sequence: SEQ ID NO: 43, 45, 47, 49, 51, 53, 55, 57, 59, 61, 63, 65, 67, 69, 71, 73, 75, 77, 83, 85, 87, 99, 101, 103, 105, 107, 185, 187, 201 or 203.

5. A vector comprising the nucleic acid molecule according to claim 3 or 4.

6. A method for reshaping the glycans in the Fc region of an antibody, comprising the following steps: obtaining an antibody having heterogeneous glycans in the Fc region of the antibody and contacting the antibody with the fusion protein of claim 1 or 2.

7. The method of claim 6, comprising: Providing an antibody having a heterogeneous polysaccharide in its Fc region, a fusion protein as claimed in claim 1 or 2, and a target polysaccharide oxazoline, wherein the polysaccharide in the Fc region of the antibody comprises N-acetylglucosamine (GlcNAc) residues; and Contacting the antibody with the fusion protein of claim 1 or 2 and linking the target polysaccharide oxazoline to the Fc region of the antibody; Thus, the remodeled glycans of the antibody Fc region can be obtained.

8. The method of claim 6, further comprising purifying the antibody having the remodeled glycan in the Fc region.

9. The method of claim 6, wherein the remodeled polysaccharide is Sia2(α2-6)Gal2GlcNAc2Man3GlcNAc2, Sia2(α2-6)Gal2GlcNAc3Man3GlcNAc2, Sia2(α2-3)Gal2GlcNAc2Man3GlcNAc2, Sia2(α2-3)Gal2GlcNAc3Man3GlcNAc2, Sia2(α2-3 / α2-6)Gal2GlcNAc2Man3GlcNAc2 lcNAc2, Sia2(α2-6 / α2-3)Gal2GlcNAc2Man3GlcNAc2, Sia2(α2-3 / α2-6)Gal2GlcNAc3Man3GlcNAc2, Sia2(α2-6 / α2-3)Gal2GlcNAc3Man3GlcNAc2, Sia(α2-6)Gal2GlcNAc2Man3GlcNAc2, Sia(α2-3)Gal2GlcNAc2Man3GlcNAc2 , Sia(α2-6)Gal2GlcNAc3Man3GlcNAc2, Sia(α2-3)Gal2GlcNAc3Man3GlcNAc2, Sia(α2-6)GalGlcNAc2Man3GlcN Ac2, Sia(α2-3)GalGlcNAc2Man3GlcNAc2, Sia(α2-6)GalGlcNAc3Man3GlcNAc2, Sia(α2-3)GalGlcNAc3Man3Glc NAc2, Gal2GlcNAc2Man3GlcNAc2, GalGlcNAcMan3GlcNAc2, Gal2GlcNAc3Man3GlcNAc2, GalGlcNAc2Man3GlcNAc 2. GalGlcNAc3Man3GlcNAc2, GlcNAc3Man3GlcNAc2, GlcNAc2Man3GlcNAc2, GlcNAcMan3GlcNAc2 or Man3GlcNAc2.

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