EP4677079A2 - Nouvelles enzymes fucosidases pour applications thérapeutiques - Google Patents

Nouvelles enzymes fucosidases pour applications thérapeutiques

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Publication number
EP4677079A2
EP4677079A2 EP24709098.8A EP24709098A EP4677079A2 EP 4677079 A2 EP4677079 A2 EP 4677079A2 EP 24709098 A EP24709098 A EP 24709098A EP 4677079 A2 EP4677079 A2 EP 4677079A2
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EP
European Patent Office
Prior art keywords
composition
fucosidase
protein
enzyme
glycosylated
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
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Application number
EP24709098.8A
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German (de)
English (en)
Inventor
Nico Callewaert
Berre VAN MOER
Wim NERINCKX
Hendrik GROOTAERT
Jasper ZUALLAERT
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Universiteit Gent
Vlaams Instituut voor Biotechnologie VIB
Original Assignee
Universiteit Gent
Vlaams Instituut voor Biotechnologie VIB
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Application filed by Universiteit Gent, Vlaams Instituut voor Biotechnologie VIB filed Critical Universiteit Gent
Publication of EP4677079A2 publication Critical patent/EP4677079A2/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/005—Glycopeptides, glycoproteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01051—Alpha-L-fucosidase (3.2.1.51)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01052—Beta-N-acetylhexosaminidase (3.2.1.52)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Definitions

  • the present invention relates to the field of glyco-engineering of antibodies, more specifically the field of fucosidases capable of removing core fucose from N-glycan substrates present on antibody Fc regions.
  • the present invention in particular relates to a composition for defucosylation of an N-glycosylated Fc- containing protein, said composition comprising the Capnocytophaga canimorsus Glycosyl Hydrolase family 29 (GH29) ⁇ -fucosidase or a variant thereof, and a pretreatment enzyme.
  • said composition comprises a pretreatment enzyme for trimming the N-glycosylated Fc-containing protein sample to trimannosyl-glycan substrates on said Fc-containing proteins, for subsequent defucosylation by the Capnocytophaga canimorsus GH29 enzyme.
  • the invention further specifies methods and uses of said enzymes and compositions for production of defucosylated Fc-containing proteins and their therapeutic utility.
  • BACKGROUND Antibodies binding to their target antigen elicit effector functions, performed by immune cells.
  • One of said effector functions concerns antibody dependent cellular cytotoxicity (ADCC), wherein natural killer cells induce cell death in aberrant cells, which are marked by opsonization with antibodies binding to a cell surface antigen (Johnson, & Glennie.
  • Immune cells specifically recruit to those sites through their Fcgamma receptors (Fc ⁇ Rs) which bind to the Fc domain of antibodies, specifically Fc ⁇ RIIIa (CD16a) on natural killer cells (Sulica, et al. 2001. Int Rev Immunol.2001 Jun;20(3-4):371-414.).
  • Fc ⁇ Rs Fcgamma receptors
  • CD16a Fc ⁇ RIIIa
  • Antibody therapies are suboptimal as anti-tumor treatments when their N297 N-glycan is core-fucosylated, since the a1-6 linked saccharide drastically reduces the affinity between antibodies and FcgRIIIa, due to steric hindrance to the N162 N-glycan of the receptor (Shields, et al.2002; J. Biol. Chem.277, 26733–26740; Shinkawa, et al.2003; J. Biol. Chem.278, 3466– 3473).
  • CHO cells the main production hosts for commercial antibodies, commonly incorporate core-fucose in IgG (Mori et al. 2007; NiCa/GH29/800 Cytotechnology 55(2-3):109-14), making clinically relevant monoclonal antibodies expressed in CHO less efficient as cancer treatments.
  • core-fucose in IgG
  • NiCa/GH29/800 Cytotechnology 55(2-3):109-14 core-fucose
  • monoclonal antibodies expressed in CHO less efficient as cancer treatments In order to reduce the level of fucosylation in CHO cells, manipulations during protein productions have been carried out. The most straightforward, genetic solution to remove fucosylation is implemented in the POTELLIGENT technology in which the responsible fucosyltransferase gene (FUT8) is knocked out in CHO cells (Yamane-Ohnuki, et al., 2004; Biotechnology and Bioengin.87(5) 614-622).
  • FUT8 responsible fucosyltransferase gene
  • FUT8 is only responsible for core ⁇ 1-6 fucosylation, other fucosylation products such as the Lewis antigens are preserved.
  • Alternative approaches for obtaining defucosylated antibodies in hosts involve changes in the fucose metabolism (e.g. blocking de novo synthesis through knocking out or mutating GDP-L-fucose synthase and/or GDP-mannose 4,6 dehydratase), mutation of the N- glycosylation pathway (e.g. removing GnT-I required for GlcNac required for core-fucosylation), applying fucosylation inhibitors during manufacturing (e.g.
  • non- fucosylated antibodies can also be generated by using host cells that intrinsically have a reduced fucosylation activity (e.g. CHO Lec13 cells which have a deficient GMDS activity).
  • host cells that intrinsically have a reduced fucosylation activity
  • most non- mammalian expression systems differ in fucosylation capacity. For instance, yeast glycoproteins are not incorporated with fucose, but only consist of GlcNAc and terminally positioned mannose residues.
  • enzymatic alternatives have been disclosed as well, which can be implemented after antibody production.
  • Enzymes capable of hydrolyzing fucose residues from N-glycans belong to the glycosyl hydrolase (GH) families GH29 and GH95 (Grootaert, et al. 2020; Glycobiology, 30, 9, 735–745; Lombard, et al. 2014; Nucleic Acids Res. 42, D490–D495). While the GH95 family mainly houses ⁇ 1-2 fucosidases, the GH29 family contains fucosidases with a wider substrate specificity: ⁇ 1-2/3/4/6 (McCarter and Withers, 1994; Curr.
  • GH glycosyl hydrolase
  • fragilis fucosidase BfFucH removes core-fucose from IgG, however only after using endoglucosaminidases (ENGases) such as Endo S to trim the N-glycan into a fucosylated GlcNAc residue (Huang et al., 2012; Am. Chem. Soc. 134, 29, 12308–12318; Li et al., 2016; J Biol Chem.291(32): 16508–16518; Tsai et al., 2017; ACS Chem. Biol.2017, 12, 1, 63–72).
  • ENGases endoglucosaminidases
  • the structure of the Fc domain is however similarly collapsed as non-glycosylated IgG, thereby reducing effector functionality (Allhorn et al., 2008; PLoS One. ; 3(1): e1413; Sjögren, et al., 2015; Glycobiology, 25, Issue 10, 1053–1063).
  • Restoring the structure NiCa/GH29/800 to the level of naturally N-glycosylated antibodies can be done through transglycosylation of the GlcNAc stump with chemically activated sugar donors. However, this final step would make the overall strategy very expensive.
  • the NEB fucosidase O enzyme from the Omnitrophica bacterium OLB16 is the only GH29 enzyme that indicated to be active in (partially) removing core-fucose on N-glycosylated IgG, with its optimal activity window at pH 5.5 and at 50°C. Complete fucose removal was demonstrated for RFMS-labeled forms of hIgGs, though not for non-trimmed or non-labelled hIgGs (Vainauskas et al. 2018; Sci Rep. 8(1):9504; Taron et al. US10260056B2).
  • this CC-H2 enzyme is positioned in a totally different branch of the GH29 family phylogenetic tree as compared to Fucosidase O, its substrate specificity could thus not be predicted based on the activity observed for the phylogenetically distant Fuc O.
  • the newly identified enzyme with activity on N-glycans of Fc- containing protein has a pH optimum close to the physiological range whereas Fucosidase O operates optimally at acidic pH (4.5-5.5).
  • NiCa/GH29/800 The C.
  • canimorsus Fuc H2 enzyme activity described herein was further optimized in view of increasing its efficiency towards defucosylation of N-glycosylated Fc-containing proteins, partially trimmed, to provide for the first fucosidase composition with optimal productivity at physiological conditions.
  • the invention herein describes a novel solution for defucosylation of antibodies carrying the larger GlcNAc2Man3GlcNAc2Fuc N-glycan, as this is the main glycoform incorporated on CHO-produced antibodies, by providing a composition combining the Capnocytophaga canimorsus (‘H2’) ⁇ -fucosidase with a pretreating enzyme, preferably a hexosaminidase, for pre-trimming the GlcNAc 2 Man 3 GlcNAc 2 Fuc, as to provide Man 3 GlcNAc 2 Fuc N-glycan substrate for CC-H2 fucosidase.
  • H2 Capnocytophaga canimorsus
  • a pretreating enzyme preferably a hexosaminidase
  • Jack Bean hexosaminidase digest of intact VHH-Fcs removed most terminal GlcNAc residues. Overnight Jack Bean hexosaminidase (Agilent) or mock digest of intact ExpiCHO-produced VHH-Fc, aiming to remove GlcNAc residues from GlcNAc2Man 3 GlcNAc 2 Fuc glycans to generate Man 3 GlcNAc 2 Fuc. After the digest, N-glycans were released by PNGaseF treatment and detected via capillary electrophoresis. Figure 3. Identification of three fucosidases active on VHH-Fc N-glycan, labeled with APTS.
  • VHH- Fc + Hex hexosaminidase from Jack Bean. This is indicated as (VHH- Fc + Hex) next to the panels.
  • fucosidases selected from our GH29 collection were incubated overnight with the APTS-labeled N-glycans and subsequently ran on CE.
  • Figure 4 Fucosidase H2 and Fuc O partially remove core-fucose from intact VHH-Fc.
  • VHH-Fc pretreated with hexosaminidase (Jack’s Bean), was incubated overnight at 37 C with fucosidases (H2, fuc O and A8) in 5 mM CaCl2 and 50 mM NaAcO- at pH 5.5.
  • fucosidases H2, fuc O and A8
  • NiCa/GH29/800 Figure 5 Phylogenetic tree of the in house GH29 family collection. The collection was pruned to include the maximal diversity with the fewest sequences. Additionally, sequences for fucosidase O and five fucosidases from GH95 (‘Extra’ enzymes indicated in brown in panel C) were added.
  • the fucosidases included in the screening for defucosylation of VHH-Fc are indicated in bold. Fucosidase O and AlfC from L. casei BL23 are indicated in green in panel B, while H2 (Capnocytophaga canimorsus) is indicated in blue in panel A.
  • VHH- Fc was treated with Endo E GH20 in PBS for 24h at 37 C.
  • VHH-Fc was purified by protein A chromatography and subjected to DSA FACE for glycan analysis.
  • Figure 7. Activity screening for fucosidases selected from the GH29 family collection. Fucosidases were incubated for 30 minutes with 1 mM chloro-nitrophenol-fucose (CNP-fucose) in 50 mM sodium phosphate pH 7. Units were determined based on comparison with a dilution series of H2 #1.
  • Figure 8. Fucosidases H2 and O are capable of removing core-fucose from VHH-Fc, in a pH dependent manner. VHH-Fc was pretreated with Endo E GH20 and purified by protein A chromatography.
  • Fucosidase H2 is most active on VHH-Fc carrying Man 3 GlcNAc 2 Fuc at physiological pH.
  • the activity of fucosidase H2 was compared in a range of pH conditions in different buffers.
  • the substrate of interest was VHH-Fc pretreated with Endo E GH20 to carry some level of Man 3 GlcNAc 2 Fuc.
  • Fucosidase H2 was incubated overnight at 37C in one of the following buffer: MclIvaine buffer (mix of 0.1M citric acid and 0.2M Na2HPO4), 40 mM NH4AcO-, 50 mM Na2HPO4 (NaPi), 50 mM Tris.
  • N-glycans were analyzed by DSA FACE and the area for the non-fucosylated Man3GlcNAc2 peak plotted for each condition.
  • NiCa/GH29/800 Figure 11 Defucosylation of VHH-Fc, carrying Man 3 GlcNAc 2 Fuc is not influenced by urea, GlcNAc or mannose residues, but is reduced in DMSO.
  • VHH-Fc pretreated with Endo E GH20 was incubated overnight at 37 C with fucosidase H2 at pH 7 in the presence of varying amounts of urea, DMSO, GlcNAc or mannose. The control condition with no additives is shown left with a black bar.
  • N-glycans were analyzed by DSA FACE and the area for the non-fucosylated Man3GlcNAc2 peak plotted for each condition.
  • Figure 12. Defucosylation of VHH-Fc, carrying Man 3 GlcNAc 2 Fuc, can be boosted by repeatedly adding the same amount of fucosidase H2. Two batches H2#1 and H2#2 were tested for their defucosylatoin activity on VHH-Fc pretreated with Endo E GH20, incubated at 37° C with fucosidase H2 at pH 7. The number of enzyme additions varied between the samples.
  • a monomeric or protomer is defined as a single polypeptide chain from amino-terminal end (also referred to herein as N-term or N-terminus or N-terminal end) to carboxy-terminal end (also referred to herein as C-term or C-terminus or C-terminal end).
  • a “protein subunit” as used herein refers to a monomer or protomer, which may form part of a multimeric protein complex or assembly.
  • the terms "chimeric polypeptide”, “chimeric protein”, “chimer”, “fusion polypeptide”, “fusion protein”, are used interchangeably herein and refer to a protein that comprises at least two separate and distinct polypeptide components that may or preferably may not originate from the same protein.
  • the term also refers to a non-naturally occurring molecule which means that it is man-made.
  • the term “fused to”, and other grammatical equivalents, such as “covalently linked”, “connected”, “attached”, “ligated”, “conjugated”, and as specifically used herein ‘inserted in’ when referring to a chimeric or fusion polypeptide (as defined herein) refers to any chemical or recombinant mechanism for linking two or more polypeptide components.
  • the fusion of the two or more polypeptide components may be a direct NiCa/GH29/800 fusion of the sequences or it may be an indirect fusion, e.g. with intervening amino acid sequences or linker sequences, or chemical linkers.
  • the fusion of amino acid residues or (poly)peptides to an Ena protein or insertion into an Ena protein sequence, or to another protein of interest as described herein, may be a covalent peptide bond, or also refer to a fusion obtained by chemical linking.
  • the term “fused to”, as used herein, and interchangeably used herein as “connected to”, “conjugated to”, “ligated to” refers, in particular, to “genetic fusion”, e.g., by recombinant DNA technology, as well as to “chemical and/or enzymatic conjugation” resulting in a stable covalent link.
  • recombinant polypeptide is meant a polypeptide made using recombinant techniques, i.e., through the expression of a recombinant or synthetic polynucleotide, which may be obtained in vitro and/or in a cellular context.
  • a recombinant or synthetic polynucleotide which may be obtained in vitro and/or in a cellular context.
  • the chimeric polypeptide or fusion polypeptide or biologically active (i.e. functional) portion thereof is recombinantly produced, it is also preferably enriched, purified or substantially free of culture medium, i.e., the impurities represent less than about 20 %, more preferably less than about 10 %, and most preferably less than about 5 % of the volume of the protein preparation.
  • “isolated” or “purified” is meant material that is substantially or essentially free from components that normally accompany it in its native state.
  • “Homologue”, “Homologues” of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having amino acid substitutions, deletions and/or insertions relative to the unmodified or wild-type protein in question and having similar biological and functional activity as the unmodified protein from which they are derived.
  • amino acid identity refers to the extent that sequences are identical on an amino acid-by-amino acid basis over a window of comparison.
  • a "percentage of sequence identity” is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical amino acid residue (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met, also indicated in one-letter code herein) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity.
  • the identical amino acid residue e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met, also indicated in one-letter code herein
  • substitution results from the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively as compared to an amino acid sequence or nucleotide sequence of a parental protein or a fragment thereof. It is understood that a protein or a fragment thereof may have conservative amino acid substitutions which have substantially no effect on the protein's activity.
  • the percentage of amino acid identity as provided herein is preferably in view of a window of comparison corresponding to the total length of the native or natural wild-type protein, or of the specific amino acid sequence referred to.
  • wild-type refers to a gene or gene product isolated from a naturally occurring source, or included in a cell, cell line or organism.
  • a wild-type gene or gene product is that which is most frequently observed in a population and is thus arbitrarily designed the “normal” or “wild-type” form of the gene or gene product a observed in nature.
  • the term “modified”, “engineered”, “mutant” or “variant” refers to a gene or gene product that displays modifications in sequence, post-translational modifications and/or functional properties (i.e., altered characteristics) when compared to the wild-type or naturally-occurring gene or gene product.
  • a knock-out refers to a modified or mutant or deleted gene as to provide for non-functional gene product and/or function.
  • mutants or variants may be isolated; these are identified by the fact that they have altered characteristics when compared to the wild-type gene or gene product, and a different sequence as compared to the reference gene or protein.
  • antibody refers to an immunoglobulin (Ig) molecule or a molecule comprising an immunoglobulin (Ig) domain, which specifically binds with an antigen.
  • Antibodies can further be intact immunoglobulins derived from natural sources or from recombinant sources and can be immunoreactive portions of intact immunoglobulins.
  • IgG comprises the majority, representing 60 % of total serum Igs in humans.
  • the human IgG molecule is composed of two identical fragment antigen binding (Fab) domains and one fragment crystallizable (Fc) domain that make it multivalent and multifunctional.
  • the two Fab fragments each consist of a heterodimer of a light chain and the N-terminal part of the heavy chain, whereas the C-terminal half of the two heavy chains dimerizes to form the Fc fragment of the IgG antibody.
  • the N-terminal domains of the Fab fragment are the variable domains (V L and V H ) that are responsible for antigen recognition, whereas the C-terminal part of the heavy chains compose the Fc fragment that is responsible for humoral and cellular effector functions.
  • the two Fabs and the Fc are connected by the hinge region, which facilitates the spatial alignment of the three moieties for binding to antigens and effector ligands.
  • said Fc domain is thus responsible for antibody function
  • antibody Fc engineering stands for engineering functions of antibodies, which are effector functions, such as antibody-dependent cellular cytotoxicity (ADCC) and antibody-dependent cellular phagocytosis (ADCP), and controlling serum half-life.
  • Engineered Fc domains may therefore be present in the form of mutants or variants containing amino acid substitutions, insertions or deletions as to allow different modifications of the Fc in post-translational modifications, dimerization behavior, effector function, serum half life, among others.
  • active antibody fragment refers to a portion of any antibody or antibody-like structure that by itself has high affinity for an antigenic determinant, or epitope, and contains one or more complementarity determining regions (CDRs) accounting for such specificity, typically at least 3 CDRs, or in conventional antibodies, defined by 6 CDRs.
  • CDRs complementarity determining regions
  • Non-limiting examples of active antibody fragments include immunoglobulin domains, Fab, F(ab)'2, scFv, heavy-light chain dimers, immunoglobulin single variable domains (ISVDs), Nanobodies (or VHH antibodies), domain antibodies, and single chain structures, such as a complete light chain or complete heavy chain.
  • antibody fragment and “active antibody fragment” or “functional variant” as used herein refers to a protein comprising an immunoglobulin domain or an antigen-binding domain capable of specifically binding the antigen.
  • Antibodies are typically tetramers of immunoglobulin molecules.
  • immunoglobulin (Ig) domain or more specifically “immunoglobulin variable domain” (abbreviated as “IVD”) means an immunoglobulin domain essentially consisting of four “framework regions” which are referred to in the art and herein below as “framework region 1” or “FR1”; as “framework region 2” or “FR2”; as “framework region 3” or “FR3”; and as “framework region 4” or “FR4”, respectively; which framework regions are interrupted by three “complementarity determining regions” or “CDRs”, which are referred to in the art and herein below as “complementarity determining region 1” or “CDR1”; as “complementarity determining region 2” or “CDR2”; and as “complementarity determining region 3” or “CDR3”, respectively.
  • an immunoglobulin variable domain can be indicated as follows: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4. It is the immunoglobulin variable domain(s) (IVDs) that confer specificity to an antibody for the antigen by carrying the antigen-binding site.
  • IVDs immunoglobulin variable domain(s)
  • a heavy chain variable domain (VH) and a light chain variable domain (VL) interact to form an antigen binding site.
  • VH heavy chain variable domain
  • VL light chain variable domain
  • the complementarity determining regions (CDRs) of both VH and VL will contribute to the antigen binding site, i.e. a total of 6 CDRs will be involved in antigen binding site formation.
  • the antigen- binding domain of a conventional 4-chain antibody (such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art) or of a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody, binds to the respective epitope of an antigen by a pair of (associated) immunoglobulin domains such as light and heavy chain variable domains, i.e., by a VH-VL pair of immunoglobulin domains, which jointly bind NiCa/GH29/800 to an epitope of the respective antigen.
  • a conventional 4-chain antibody such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art
  • a Fab fragment such as an IgG, IgM, IgA, IgD
  • immunoglobulin single variable domain refers to a protein with an amino acid sequence comprising 4 Framework regions (FR) and 3 complementary determining regions (CDR) according to the format of FR1-CDR1-FR2-CDR2-FR3-CDR3- FR4.
  • An “immunoglobulin domain” refers to “immunoglobulin single variable domains” (abbreviated as "ISVD"), equivalent to the term “single variable domains”, and defines molecules wherein the antigen binding site is present on, and formed by, a single immunoglobulin domain.
  • the immunoglobulin single variable domain may be a (single) domain antibody (or an amino acid sequence that is suitable for use as a (single) domain antibody), a "dAb” or dAb (or an amino acid sequence that is suitable for use as a dAb) or a Nanobody (as defined herein, and including but not limited to a VHH); other single variable domains, or any suitable fragment of any one thereof.
  • VHH domain has been chosen to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “VL domains”).
  • antibody or ‘Fc-fusion’ or ‘VHH-Fc’’ as used herein further refers to the genetic linking or fusion of antigen-binding fragments or antigen-binding domains with an Fc constant domain as to obtain dimers forming or resembling an antibody structure when expressed in a recombinant host.
  • antibody fragments, or single domain antibodies such as ISVDs may be C-terminally fused to the N- terminus of an Fc domain, preferably via a linker or hinge region.
  • antibody fragments, or single domain antibodies such as ISVDs may be fused at the N-terminus to the C-terminal end of an Fc domain, preferably via a linker or hinge region.
  • Said single domain antibody or ISVD fused to said Fc may comprise one or more VHHs or Nbs.
  • a “therapeutically active agent” or “therapeutically active composition” means any molecule or composition of molecules that has or may have a therapeutic effect (i.e. curative or prophylactic effect) in the context of treatment of a disease.
  • a therapeutically active agent is a disease-modifying agent, which can be a cytotoxic agent, such as a toxin, or a cytotoxic drug, or an enzyme capable of converting a prodrug into a cytotoxic drug, or a radionuclide, or a cytotoxic cell, or which can be a non- cytotoxic agent.
  • a therapeutically active agent has a curative effect on the disease.
  • the composition, or pharmaceutical composition of the invention may act as a therapeutically active agent, when beneficial in treating patients in need for antibody-therapy requiring defucosylated antibodies.
  • the term ”subject”, “individual” or “patient”, used interchangeably herein, relates to any organism such as a vertebrate, particularly any mammal, including both a human and another mammal, for whom diagnosis, therapy or prophylaxis is desired, e.g., an animal such as a rodent, a rabbit, a cow, a sheep, a horse, a dog, a cat, a lama, a pig, or a non-human primate (e.g., a monkey).
  • the rodent may be a mouse, rat, hamster, guinea pig, or chinchilla.
  • the subject is a human, a rat or a non-human primate.
  • treatment can be used in a medical context and is thus defined by a therapeutic intervention that slows, interrupts, arrests, controls, stops, reduces, or reverts the progression or severity of a sign, symptom, disorder, condition, or disease, but does not necessarily involve a total elimination of all disease-related signs, symptoms, conditions, or disorders.
  • Therapeutic treatment is thus designed to treat an illness or to improve a person's health, rather than to prevent an illness.
  • Treatment may also refer to a prophylactic treatment which relates to a medication or a treatment designed and used to prevent a disease from occurring, herein referred to as “prevention”.
  • composition relates to a combination of one or more active molecules, and may further include buffered solutions and/or solutes such as pH buffering substances, water, saline, physiological salt solutions, glycerol, preservatives, etc. for which a person skilled in the art is aware of the suitability to obtain optimal performance.
  • buffered solutions and/or solutes such as pH buffering substances, water, saline, physiological salt solutions, glycerol, preservatives, etc. for which a person skilled in the art is aware of the suitability to obtain optimal performance.
  • Suitable conditions as used herein could also refer to suitable binding conditions.
  • the present invention is based on the finding that a fucosidase enzyme (named ‘H2’ or ‘CC-H2’ further herein) removes core-fucosylation from Fc domains, at least if the N-glycan at N297 is trimmed down to NiCa/GH29/800 the Man 3 core glycoform, and is optimally active at physiological pH.
  • a fucosidase enzyme named ‘H2’ or ‘CC-H2’ further herein
  • the invention relates to a composition or assembly of components for removing the core-fucose present at the first GlcNac of a N-glycosylated Fc-containing protein, said composition or mixture combining a functional pretreatment enzyme and a GH29 fucosidase enzyme, wherein said fucosidase comprises the CC-H2 sequence, SEQ ID NO: 1, or a functional variant or homologue with at least 85 % identity thereof, more preferably at least 95% identity thereof.
  • a functional variant or homologue of the GH29 CC-H2 fucosidase is meant herein that the function of being enzymatically active on the same substrate as the wild type CC-H2 fucosidase is maintained in the functional variant or homologous protein, wherein enzymatically active refers to the observation of providing a substrate convergence that is in the same order of magnitude as the wild type enzyme.
  • the functional variant or homologue of the CC-H2 enzyme as provided herein may envisage mutant variants wherein one or more amino acid residues are substituted or mutated to result in a functional variant or homologue with at least 85% amino acid identity to the wild type CC-H2 mature protein form of SEQ ID NO:1, and thus with retained enzymatic activity on (trimmed) Fc-containing proteins such as N-glycan antibody substrates or N-glycan Fc-based protein substrates.
  • Said substrates may comprise alternative antibody type proteins such as Fc-fusions with antibody fragments or antigen-binding domains, or VHH- Fc fusions, or IgG-type proteins, as described above herein.
  • said functional variant or homologue of the CC-H2 fucosidase comprises or consists or an amino acid sequence, in its mature form (i.e. the protein after cleavage of the signal peptide), with at least 50 % identity to CC-H2 SEQ ID NO:1, or at least 60 % identity to CC-H2 SEQ ID NO:1, or at least 70 % identity to CC-H2 SEQ ID NO:1, or at least 75% identity to CC-H2 SEQ ID NO:1, or at least 80 % identity to CC-H2 SEQ ID NO:1, or at least 85 % identity to CC-H2 SEQ ID NO:1, or at least 90 % identity to CC-H2 SEQ ID NO:1, or at least 95 % identity to CC-H2 SEQ ID NO:1, or at least 98 % identity to CC-H2 SEQ ID NO:1.
  • CC-GH29 (of which the mature protein form is presented in SEQ ID NO:1) is phylogenetically very different from further fucosidase enzymes with the potential to defucosylate N-glycan antibody substrates, such as Fuc O ( Figure 5).
  • the closest homologue of CC-H2 of the collection taken up in our analysis was shown to be Chitinophaga pinensis GH29 ( Figure 5A), with an amino acid sequence (of its mature protein form) identity of only 48 % to CC- H2 SEQ ID NO:1.
  • a BlastP of the CC-H2 mature protein shows that the naturally-appearing variants within the same species show at least 85 % identity to the amino acid sequence of SEQ ID NO:1.
  • One embodiment relates to said composition in which the pretreatment requires the trimming of naturally intact N-glycans on antibodies to trimannosyl structures, for instance obtained using hexosaminidase thereby providing Man3GlcNAc2Fuc-glycans on said Fc-containing proteins as substrate NiCa/GH29/800 for core-fucosylation by CC-H2.
  • An alternative embodiment relates to said composition in which the pretreatment requires that the CC-H2 is brought in proximity to the N-glycosylated Fc branch, which may be provided by the presence of an ‘N-glycosylated Fc-containing protein scavenger enzyme’, such as catalytically non-active endoglucosaminidases, or domains thereof.
  • an ‘N-glycosylated Fc-containing protein scavenger enzyme’ such as catalytically non-active endoglucosaminidases, or domains thereof.
  • N-glycosylated Fc- containing protein scavenger enzyme as used herein is referred to those proteins, in particular inactivated forms of enzymes, which bind the N-glycan structures present on Fc-containing proteins produced in eukaryotic cells, such as endoglucosaminidase enzyme exemplified herein, which are functioning herein as scavengers of the N-glycan antibody substrate for the GH29 fucosidase enzyme, thereby bringing the fucosidase in proximity with the N-glycan substrates.
  • these scavenger protein domains or enzymes are present in the composition as a fusion with the CC-H2 enzyme or variant of the present invention.
  • a preferred embodiment relates to said composition, wherein said hexosaminidase comprises the GH20 domain of Endo E (SEQ ID NO: 10 or a homologue with at least 95 % identity thereof), and/or said scavenger enzyme comprising an inactive mutant of the GH18 domain of Endo E (SEQ ID NO: 12), or an inactive mutant of EndoS, or EndoS2 (SEQ ID NO: 14, 16, resp.) or a truncated EndoS or EndoS2 enzyme (e.g. as present in SEQ ID NO: 17), or a functional variant or homologue with at least 95 % identity thereof.
  • the combination of the enzymatic activity for defucosylation of N-glycosylated Fc-containing proteins i.e. the enzymes provided by the GH29 CC-H2 enzyme or functional variant thereof, and the enzyme or enzyme domain or protein domain of one or more pretreatment enzymes, is present as a fusion protein, preferably obtained by recombinant production of a genetic fusion of said enzymes, directly connected, fused by a linker, or designed as swapped or replaced domains of known enzymes.
  • examples of fusions as disclosed herein are provide the fusions of the CC- H2 fucosidase and the GH20 hexosaminidase domain, as exemplified in SEQ ID NOs: 18-19, or a homologue with at least 95 % identity of any one thereof, or a fusion of the GH29 CC-H2 and the EndoS or EndoS2 domains, e.g. as in SEQ ID Nos: 20-21.
  • the invention provides for derived products of said composition , including but not limited to the provision of enzymes or proteins on a solid support structure, such as a resin, column, membrane or coated on a surface, for immobilization; as well as the provision of nucleic acid molecules, vectors, or hosts for recombinantly expressing and producing said composition.
  • a solid support structure such as a resin, column, membrane or coated on a surface, for immobilization
  • nucleic acid molecules, vectors, or hosts for recombinantly expressing and producing said composition.
  • Another aspect of the disclosure relates to a method to produce at least partially defucosylated N- glycosylated Fc-containing proteins, in particular antibodies, comprising the steps of: mixing the protein sample comprising a N-glycosylated Fc-containing protein with said composition described herein, or NiCa/GH29/800 with the solid support comprising the components of the composition; and isolating the (at least partially) defucosylated N-glycosylated Fc-containing protein from the mixture.
  • a preferred embodiment discloses the method to produce (at least partially) defucosylated N- glycosylated Fc-containing proteins, comprising the steps of: mixing a protein sample comprising a N- glycosylated Fc-containing protein with the enzymatic composition described herein, or with the solid support comprising the components of the composition described herein, wherein said incubation is performed in physiological conditions, referring to at least in an environment with a neutral pH and at around 37°C; and isolating the defucosylated N-glycosylated Fc-containing protein from the mixture.
  • the incubation step a.
  • the N-glycosylated Fc-containing protein sample is initially incubated with an amount of said composition, and after a defined time, additional composition and/or GH29 fucosidase is added to the sample of step a. for further subjecting said N-glycans on said Fc-containing proteins to the enzymatic activity using freshly added enzyme.
  • the Fc-containing protein sample may be repeatedly incubated as for step a.
  • Glycosyl hydrolase 29 family of alpha-fucosidase enzymes In the present application, the result is described from a search for a fucosidase that could remove the ⁇ 1-6 linked core-fucose residue from antibodies at their N297 N-glycan, positioned in the Fc domain. Glycosyl hydrolases with an ⁇ 1-6 core-fucose specificity all cluster together in the GH29 family.
  • C. canimorsus has been observed to evade the immune system, orchestrated by macrophages, by breaking down their membrane, downregulating production of cytokines such as TNF- ⁇ and IL-8, and reducing TLR4 activation. Furthermore, it also evades killing by granulocytes and has NiCa/GH29/800 been observed to deglycosylate host glycans (Shin et al.2009. Infection and Immunity 77: 2262-2271). In this last context, a fucosidase comes into play.
  • a pre- screening step could be introduced to remove any mutant without the desired activity increase, to subsequently test the remaining variants more thoroughly.
  • Such qualitative screening in which “test- worthy” variants perform better than the current fucosidase, could be achieved by employing agonists that recognize non-fucosylated IgG specifically over fucosylated. Two interactions could allow for such selection. Firstly, we could take advantage of CD16a and its increased binding for non-fucosylated IgG. Yet, with its narrow increase in binding with a factor of 30 between fucosylated and non-fucosylated, fewer variants will be excluded than with a more discriminating binding event.
  • VHHs selected to specifically recognize non-fucosylated glycoforms of IgG (Kao et al., 2022; PNAS 119 (48) e2212658119).
  • VHH libraries the authors screened against antibodies with NiCa/GH29/800 homogeneous N-glycosylation, obtained chemoenzymatically.
  • affinity maturation several candidates were disclosed with selective binding to non-fucosylated IgG.
  • Employing one of these VHHs in a pre-screening method could allow to prioritize fucosidase mutants for more in depth analysis of their activities.
  • an ELISA type pre-screening would require 20 – 30x as little VHH-Fc material than our current DSA FACE screen.
  • Enhancing the GH29 CC-H2 enzymatic activity on N-glycan antibody substrates As supported by the Examples described herein, C. canimorsus wild type fucosidase, called herein ‘H2’,by itself, or in combination with a hexosaminidase, is capable to defucosylate VHH-Fcs, which is quite unique, though not in a very efficient manner.
  • fucosidase H2 enzyme may also be fused with EndoS or Endo S2, two ENGases known to specifically hydrolyze IgG N-glycans located at N297. Swapping their 35 kDa GH18 domains with fucosidase H2 may lead to a properly expressed fusion with improved Fc defucosylation, first of all since full length Endo S2 expresses at 250 mg/L in the currently applied expression systems herein, and since swapped fusions using fucosidases have been reported, for instance by Fan et al. (2023; Biochemical and Biophysical Research Communications, 645,p. 40-46) to provide for a synergistic activity.
  • fucosidase H2 for increased activity may be initiated based on the alignment of the fucosidase models and available Fc protein structures, as to design for fucosidase H2 mutant variants, comprising one or more amino acid substitutions for which the activity on VHH-Fc may be tested.
  • One option to provide for an improved activity is to do multiple treatments in smaller amounts of fucosidase H2, so alternatively, an increased stability of the H2 enzyme may be aimed for through engineering.
  • the relevance of the trimmed trimannosyl version is arguable, as it is known that this results in increased clearance rates because of interactions of the terminal mannose residue with mannose receptors on macrophages and liver cells. Additionally, the level of ADCC capacity with antibodies carrying this shorter Man3GlcNAc2 N-glycan needs to be verified. By means of chemoenzymatic workflows, antibodies with homogeneous non-fucosylated glycoforms have been created (Kurogochi et al. 2015; PLoS ONE 10(7): e0132848).
  • the resulting glycoforms ranged from the largest complex type (Sia2Gal2GlcNAc2Man3GlcNAc2) to the Man3GlcNAc2.
  • Kurogochi et al. show that the affinity for CD16a of trastuzumab-Man 3 GlcNAc 2 is the lowest among the chemoenzymatically modified variants, at the same level of fucosylated CHO produced IgG.
  • Man 3 GlcNAc 2 outperformed fucosylated IgG, making its activity more similar to GlcNAc2Man3GlcNAc2.
  • the method for producing defucosylated N-glycosylated Immunoglobulin G1 (IgG1) antibodies comprises the steps of: a. Incubating a protein sample in vitro with the composition described herein, wherein said protein sample may be a sample obtained from a patient or a component or fraction thereof, containing human IgG1s, even more preferably a sample obtained by plasmapheresis; b. Isolating the IgGs from said sample incubated with said composition, preferably using IgG capturing methods known in the art, or alternatively, by collecting the flow-through when the enzyme composition is provided on a solid support.
  • a protein sample which comprises recombinantly produced N-glycosylated antibodies, preferably produced in an eukaryotic host, such as a mammal or human, with the composition or solid structure as described herein, and b. Isolating or purifying the (at least partially) defucosylated N-glycan IgGs or antibodies from said mixture.
  • the sample subjected in step a. of said method is the resulting product of a manufactured therapeutic antibody recombinantly produced in a mammalian host, such as a CHO cell culture, containing antibodies with an N-glycan structure with a core fucose.
  • said fucose increases the potency of said therapeutic antibodies in several applications, such as anti-tumor treatment where an increase in ADCC effector function is expected, potentiating recombinant antibodies, more specifically monoclonal antibodies against cancer.
  • said (at least partially) defucosylated antibodies obtained from the enzyme treatment or incubation with the composition described herein may be used for treating a patient, wherein said initial antibodies subjected to defucosylation are patient-derived antibodies, thus resulting in the treatment of said patient with its own antibodies, in a-fucosylated form. Indeed, one ultimate goal is thus to defucosylate patient-derived antibody samples.
  • Tumor-infiltrating B lymphocytes NiCa/GH29/800 may produce anti-tumor antibodies of the IgG1 subclass (Sharonov, et al.2020; Nat Rev Immunol 20, 294–307). In the assumption that some of these antibodies end up in circulation, activating them by removing their core-fucose is in scope. For instance, if (a fraction of) the total antibody pool could be treated during plasmapheresis, they would re-enter the patient’s body defucosylated, plausibly ADCC- activated, potentially resulting in improved recruitment of NK cells.
  • the GH29 fucosidase family has been established from about 600 sequences about 10 years ago, to a close to a 10.000 genes nowadays, all clustering together based on their amino acid sequences.
  • bacteria with known or annotated fucosidase genes were cultivated to subsequently test their enzymatic activity on simple chromogenic substrates, with only limited activity detectable in few bacterial strains.
  • the strategy shifted to recombinant production of the fucosidases.
  • the GH29 family members were clustered phylogenetically in order to prune the collection based on the length of loops surrounding the conserved catalytic residues. Based on this clustering activity, we selected a collection of 90 sequences, largely from bacterial origin with a few eukaryotic and archaeal genes. Upon cloning and bacterial intracellular expression, lysates and purified fucosidases were tested on a variety of fucosylated substrates. However, none of the enzymes tested showed activity on core-fucosylated antibodies from commercial human sera.
  • AlphaFold2 models were generated for a downsized-GH29 collection to search for enzymes with a minimally constrained catalytic site to accommodate both the Fc backbone and complex N-glycans.
  • the active site was positioned closely to the surface in the generated model.
  • Figure 1 shows a few examples of such conformations (AlfC, fucosidase O, A8 and H2 enzymes, as described NiCa/GH29/800 herein) and examples of enzymes with a more closed structure near the active site (A2 and C9 enzymes).
  • AlfC fucosidase O
  • A8 and H2 enzymes examples of enzymes with a more closed structure near the active site
  • VHH-Fc With smaller N-glycans as substrate, we would increase the likelihood of finding active enzymes in our screening.
  • VHH-Fc carrying terminal mannose residues we treated VHH-Fc with hexosaminidase enzymes to remove the terminal GlcNAc residues, resulting in the shorter Man3GlcNAc2Fuc N-glycan.
  • hexosaminidase enzymes to remove the terminal GlcNAc residues, resulting in the shorter Man3GlcNAc2Fuc N-glycan.
  • the main N-glycan is GlcNAc 2 Man 3 GlcNAc 2 Fuc, with a minimal amount of GlcNAcMan 3 GlcNAc 2 Fuc, while this shifts after digestion to a population consisting mainly of Man 3 GlcNAc 2 Fuc with a limited amount of GlcNAcMan 3 GlcNAc 2 Fuc ( Figure 2).
  • VHH-Fc pretreated overnight with hexosaminidase, as a substrate to test GH29 enzymes with an open active site. These enzymes (as named herein: A8, B6, C1, E1, E7, F11 and H2) were expressed in E.
  • fucosidase H2 generates the most non-fucosylated N- NiCa/GH29/800 glycans in the tested condition: Man 3 GlcNAc 2 takes up 16 % in the total pool.
  • MSA multiple sequence alignment
  • Fucosidases were expressed intracellularly in E. coli BL21-AI cells. The volume for expression ranged from 50 ml to 3 L to ensure that minimally 0.5 mg fucosidase was obtained after purification. After overnight expression, cells were sonicated and the resulting lysates were clarified for loading onto a HisTrap column for IMAC based purification of the His-tagged fucosidases.
  • EndoE a multi-modular glycosyl hydrolase from Enterococcus faecalis, contains a GH20 domain with an activity as exo- ⁇ -1,2-N- acetylglucosaminidase, alongside a GH18 domain that functions as an endo-N-acetylglucosaminidase (ENGase).
  • the wild type or variant H2 fucosidase may be fused to NiCa/GH29/800 other glycosyl hydrolase(GH)-domains, or GH-containing enzymes to boost the activity on antibody core- fucose residues.
  • GH glycosyl hydrolase
  • Endo- ⁇ -N-acetylglucosaminidase E or in short ‘Endo E’ contains two GH domains: GH18, an endoglucosaminidase (ENGase), and GH20, a hexosaminidase.
  • the GH20 domain was already verified to hydrolyze terminal GlcNAc residues of IgG, which is required for activity of its GH18 domain on the resulting deGlcNAcylated N-glycan;
  • EndoS and EndoS2 are two 90 kDa GH18 containing enzymes, with a strict preference of IgG as substrate.
  • GH18 domains they harbor LRR domains and CBM domains, of which the latter is verified to impart the specificity.
  • boosting fucosidase H2’s activity on Fcs may be done by combining the different proteins, to act in a synergistic manner on the N-glycan of the Fc-containing protein, though the ENGase domains should be removed or inactivated by mutations as to avoid trimming to fucosylated GlcNAc residue which would result in a structure of the Fc domain that is similarly collapsed as non-glycosylated IgG, thereby reducing effector functionality.
  • Such catalytically inactivated mutant GH18 Endo E is provided in SEQ ID NO: 12, and inactivated mutant EndoS and EndoS in SEQ ID NO:16 and 17, resp.
  • the proteins of interest are purified from the lysate by IMAC and SEC, and optionally IEC or HIC purification steps may be included prior to sizing, if the protein purity is suboptimal.
  • NiCa/GH29/800 the activity is tested on synthetic substrate (chloro-nitrophenol fucose) and on IgG or VHH-Fc, which are expressed in CHO cells, with GlcNAc 2 Man 3 GlcNAc 2 as main N-glycan.
  • a pretreatment of some of the antibodies with Endo E GH20 is performed to generate GlcNAc1Man3GlcNAc2 and Man3GlcNAc2.
  • E. coli (AI and DE3) and purification Upon synthesizing and cloning of the encoding sequences into E. coli expression plasmids (performed Grootaert et al., 2020 (supra), E. coli BL21 strains were transformed with vectors encoding the fucosidases, either the DE3 strain for IPTG induction or AI strain for arabinose-based induction. After incubation at 37° C to reach exponential growth, the fucosidases were expressed intracellularly for 18h in selective LB broth at 28° C.
  • the cells were pelleted and lysed by sonication in a lysozyme NiCa/GH29/800 containing buffer (25 mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM MgCl2, 100 ⁇ g/ml chicken egg white lysozyme and 20 mM imidazole).
  • a lysozyme NiCa/GH29/800 containing buffer 25 mM Tris-HCl pH 7.5, 200 mM NaCl, 5 mM MgCl2, 100 ⁇ g/ml chicken egg white lysozyme and 20 mM imidazole.
  • IMAC immobilized metal-ion affinity chromatography
  • a nickel-sepharose packed column (HisTrap HP) was equilibrated with equilibration buffer (20 mM sodium phosphate, 20 mM imidazole, 0.5M NaCl, pH 7.2 – 8.0 depending on protein of interest’s pI). After loading of the lysate, the column was washed with equilibration buffer to remove non-specifically bound contaminants. Next, all proteins were eluted with elution buffer (20 mM sodium phosphate, 300 mM imidazole, pH 7.2 – 8.0). Elution fractions were then analyzed by SDS-PAGE and if possible by fucosidase activity assay (see further).
  • NTA beads were loaded with NiSO4. After removing non-chelated nickel ions, the beads were mixed with E. coli lysates for 1h at room temperature. Subsequently, the beads were collected on filter membranes (#7311550, Bio-Rad) and washed with IMAC equilibration buffer. Next, bound proteins were eluted with IMAC elution buffer.
  • Fucosidase activity was quantified spectrophotometrically at 415 nm on an iMarkTM microplate reader (Bio-Rad) after incubation at 37 °C with chromogenic substrate 2-chloro-p-nitrophenol-fucosidase (CNP- fucose, Carbosynth) for 1h.50 ul of (diluted) enzyme or purification fraction was mixed with substrate, to a final concentration of 1 mM CNP-fucose. To buffer the reaction, MES pH 5.0 or sodium phosphate pH 7.0 was added to a final concentration of 50 mM.
  • VHH-Fc production purification and digest with hexosaminidase VHH-Fc was produced in ExpiCHO cells (Thermo Fisher Scientific) by transient transfection. Cultures were incubated at 6x 10 6 cells/ml at 37 °C and 8% CO2. A pcDNA3.4-based expression vector for VHH-Fc was transfected using ExpiFectamine CHO reagent (0.8 ug DNA/ml culture volume).
  • ExpiCHO enhancer and ExpiCHO feed were added, 18 – 22h after transfection, according to the manufacturer’s instructions. Subsequently, cells were incubated at 32 °C and 5% CO 2 . A second feeding was performed 5 days post NiCa/GH29/800 transfection. Recombinant protein expression was stopped when cell viability dropped below 75%, by pelleting the CHO cells and filtering the resulting supernatant. VHH-Fc was purified from the supernatant using the MabSelect SuRe column (GE Healthcare) on ⁇ KTA chromatography systems (Cytiva).
  • MclIvaine buffer phosphate/citrate buffer pH 7.2 (McIlvaine, 1921; Journal of Biological Chemistry 49(1) p183–186)
  • MclIvaine buffer phosphate/citrate buffer pH 7.2
  • McIlvaine, 1921; Journal of Biological Chemistry 49(1) p183–186 bound proteins were eluted in MclIvaine pH 3.
  • the acidic pH was increased to pH 6 with saturated Na 3 PO 4 .
  • a Superdex200 HiLoad 16/600 GE Healthcare
  • equilibrated to storage buffer 25 mM L-histidine, 125 mM NaCl pH 6
  • VHH-Fc was treated with hexosaminidase to remove terminal GlcNAc residues from the N-glycan.
  • VHH-Fc commercially available enzyme extracted from Jack Bean (GKX-5003, Agilent) was incubated with VHH-Fc, 0.3 mU enzyme for 10 ⁇ g VHH-Fc, at 37 °C for 24h in acidic buffer: Agilent buffer (100 mM Na-citrate phosphate pH 5.0) or NEB glycobuffer 1 (5 mM CaCl2 and 50 mM NaAc at pH 5.5).
  • VHH-Fc was treated with Endo E GH20 domain, as non-commercial alternative to hexosaminidase extracted from Jack Bean.
  • Endo E GH20 was produced in house in E. coli BL21-AI and purified as described above.
  • VHH-Fc was mixed with 3.6 mg Endo E GH20 in 25 ml PBS. After 24h incubation at 37 °C, the VHH-Fc was purified identically to described above (protein A and size exclusion chromatography). Defucosylation screening of VHH-Fc Experiments testing fucosidases for activity on Man 3 GlcNAc 3 Fuc on VHH-Fc were performed overnight (16 – 18h) at 37 °C. Unless specified otherwise, 2 ug VHH-Fc was treated with 2 ug fucosidase in 50 mM Na2HPO4 pH 7.0 in a final volume of 40 – 50 ul.
  • DSA FACE The level of defucosylation was subsequently analyzed by DSA FACE, described below.
  • DSA FACE Isolation and analysis of N-glycans was performed as described previously (Laroy et al., 2006; Nature Protocols 1, p.397–405). Briefly, glycoproteins were denatured and immobilized on 96-well PVDF plates (Merck). After reduction and alkylation of disulfide bonds and blocking in 1% PVP, N-glycans were released by employing in-house produced PNGase F (15.4 IUB mU/ul) for 3h at 37 °C.

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Abstract

La présente invention concerne le domaine de la glyco-ingénierie d'anticorps, plus spécifiquement le domaine des fucosidases capables d'éliminer le fucose central de substrats de N-glycane présents sur des régions Fc d'anticorps. La présente invention concerne en particulier une composition pour la défucosylation d'une protéine contenant Fc N-glycosylée, ladite composition comprenant une α-fucosidase de la famille Capnocytophaga canimorsus Glycosyl Hydrolase 29 (GH29) ou un variant de celle-ci, et une enzyme de prétraitement. Plus spécifiquement, ladite composition comprend une enzyme de prétraitement pour rogner l'échantillon de protéine contenant Fc N-glycosylée en substrats de trimannosyl-glycane sur lesdites protéines contenant Fc, pour une défucosylation ultérieure par l'enzyme Capnocytophaga canimorsus GH29. L'invention concerne en outre des procédés et des utilisations desdites enzymes et des compositions pour la production de protéines contenant Fc défucosylées et leur utilité thérapeutique.
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