WO2024220732A1 - Mutants d'endoglycosidase s2 (endos2) et leurs utilisations - Google Patents

Mutants d'endoglycosidase s2 (endos2) et leurs utilisations Download PDF

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WO2024220732A1
WO2024220732A1 PCT/US2024/025296 US2024025296W WO2024220732A1 WO 2024220732 A1 WO2024220732 A1 WO 2024220732A1 US 2024025296 W US2024025296 W US 2024025296W WO 2024220732 A1 WO2024220732 A1 WO 2024220732A1
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endos2
mutation
recombinant
amino acid
enzyme
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Eric Sundberg
Jonathan DU
Tala AZZAM
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Emory University
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Emory University
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    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/52—Genes encoding for enzymes or proenzymes
    • 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
    • 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
    • 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)
    • C12Y302/01096—Mannosyl-glycoprotein endo-beta-N-acetylglucosaminidase (3.2.1.96)
    • 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

Definitions

  • Antibodies play an integral role in immunity by neutralizing pathogens and by stimulating other immune responses against them.
  • the latter known as antibody- mediated effector functions, occurs when antibodies bind to Fc gamma receptors (FcgRs) on other immune cells triggering activating or inhibitory signals.
  • FcgRs Fc gamma receptors
  • Groups of sugars (glycans) are often attached to the asparagine (N) amino acid position 297 (also referred to as N297) on the Fc region of IgG antibodies.
  • N297-linked (N- linked) glycosylation of IgGs provide an important point of regulation that can be used do direct antibody function, particularly when it comes to monoclonal antibody (mAb) therapeutics.
  • N-linked glycans can be efficiently and specifically removed from IgGs with an endo-beta- A-acetylglucosaminidase (ENGase) enzyme also referred to an EndoS2.
  • EndoS2 catalyzes the hydrolysis of the beta- 1,4 linkage between the first and second N-acetylglucosamine (GlcNAc) residues of N-linked glycans.
  • GlcNAc N-acetylglucosamine
  • EndoS2 can be made into a glycosynthase by mutating one of the aspartic acid (D) residues in the EndoS2 catalytic domain, DI 84, to a methionine (M), referred to as D184M.
  • D aspartic acid
  • DI 84 a methionine
  • EndoS2 D184M can transfer an oxazoline- linked glycan onto IgGs.
  • this glycosynthase still retains some hydrolytic activity.
  • glycosynthases that catalyze transglycosylation of IgG antibodies at position Asn297.
  • the glycosynthases are EndoS2 double mutants having high efficiency and reduced residual hydrolysis for use in engineering IgG antibodies with improved properties.
  • this disclosure relates to uses of EndoS2 mutants disclosed herein for transferring a glycan onto the Fc of immunoglobulins.
  • the EndoS2 mutants can transfer an oxazoline-linked glycan onto IgG.
  • the EndoS2 mutants have improved glycosynthase function, e.g., when compared to the single EndoS2 mutation D184M, by increasing transglycosylation and/or reducing residual hydrolysis.
  • this disclosure relates to a non-naturally occurring recombinant endoglycosidase S2 (EndoS2) enzyme comprising a first mutation and a second mutation in the sequence of a wild-type EndoS2.
  • the mutant of EndoS2 has a hydrolyzing activity lower than that of the EndoS2 D184M mutant and/or has a transglycosylation activity higher than that of the wild-type EndoS2 and/or DI 84M mutant.
  • the first mutation in the EndoS2 is a D184M wherein the first mutation is in reference to positions in amino acid sequence GLDIDIE (SEQ ID NO: 1) (segment of Streptococcus pyogenes endo-beta-N-acetylglucosaminidase (EndoS2) [Streptococcus pyogenes M49 591] Chain A, Secreted Endo-beta-N-acetylglucosaminidase (EndoS), World Protein Data Bank PDB: 6E58 A), wherein the N-terminal amino acid glycine (G) is position 180.
  • GLDIDIE SEQ ID NO: 1
  • the second mutation in EndoS2 is II 85 A wherein the mutation is in reference to positions in amino acid sequence GLDIDIE (SEQ ID NO: 1) wherein the N- terminal amino acid glycine (G) is position 180.
  • the recombinant EndoS2 enzyme has an amino acid sequence comprising SEQ ID NO: 2.
  • the second mutation in EndoS2 is I185F wherein the mutation is in reference to positions in amino acid sequence GLDIDIE (SEQ ID NO: 1) wherein the N- terminal amino acid glycine (G) is position 180.
  • the recombinant EndoS2 enzyme has an amino acid sequence comprising SEQ ID NO: 3.
  • the second mutation in EndoS2 is second mutation in EndoS2 is I185C, wherein the mutation is in reference to positions in amino acid sequence GLDIDIE (SEQ ID NO: 1) wherein the N-terminal amino acid glycine (G) is position 180.
  • the recombinant EndoS2 enzyme has an amino acid sequence comprising SEQ ID NO: 4.
  • the second mutation in EndoS2 is second mutation in EndoS2 is Il 85V wherein the mutation is in reference to positions in amino acid sequence GLDIDIE (SEQ ID NO: 1) wherein the N-terminal amino acid glycine (G) is position 180.
  • the recombinant EndoS2 enzyme has an amino acid sequence comprising SEQ ID NO: 5.
  • the second mutation in EndoS2 is second mutation in EndoS2 is I185Y wherein the mutation is in reference to positions in amino acid sequence GLDIDIE (SEQ ID NO: 1) wherein the N-terminal amino acid glycine (G) is position 180.
  • the recombinant EndoS2 enzyme has an amino acid sequence comprising SEQ ID NO: 6.
  • any of the recombinant EndoS2 enzymes reported herein further comprise D233A and/or D233Q mutation, wherein the mutation is in reference to positions in amino acid sequence NYIDASQ (SEQ ID NO: 7) wherein the N-terminal amino acid asparagine (N) is position 230.
  • this disclosure relates to recombinant nucleic acids encoding a recombinant EndoS2 enzyme as reported herein in operable combination with a heterologous promoter.
  • this disclosure relates to a vector comprising the recombinant nucleic acid encoding a recombinant EndoS2 enzyme as reported herein in operable combination with a heterologous promoter.
  • this disclosure relates to a cell or cell-free protein expression system comprising a recombinant nucleic acid or vector as reported herein.
  • this disclosure relates to a somatic cell comprising a recombinant nucleic acid or vector as reported herein.
  • this disclosure relates to methods for preparing an engineered glycoprotein using the mutant of EndoS2 as reported herein, comprising contacting an activated oligosaccharide with a glycoprotein acceptor providing an extended glycoprotein conjugated to the glycoprotein acceptor.
  • the activated oligosaccharide is a glycan oxazoline.
  • the glycoprotein acceptor contains a N-acetylglucosamine (GlcNAc) monosaccharide.
  • the glycoprotein acceptor is a non-fucosylated GlcNAc-acceptor.
  • the glycoprotein acceptor is a glycopeptide, a glycoprotein, an antibody, or a fragment thereof.
  • the glycoprotein acceptor is a core fucosylated or non-fucosylated GlcNAc-IgG acceptor or a fragment thereof.
  • the GlcNAc-IgG acceptor is derived from a therapeutic monoclonal antibody.
  • composition comprising the glycoprotein reported herein and a pharmaceutically acceptable carrier or excipient.
  • this disclosure relates to methods of treating a disease or condition comprising administering an effective amount of a glycoprotein made by processes disclosed herein to a subject in need thereof.
  • the disease or condition is a cancer, autoimmune disease, or inflammatory disease.
  • Figure 1A shows data on transglycosylation activity of EndoS2 D184M in which the enzyme transfers the glycan onto the deglycosylated IgG in a stepwise manner to form monoglycosylated IgG and subsequently diglycosylated IgG.
  • Figure IB shows data on transglycosylation activity of EndoS2 D184SeMet in which the enzyme transfers the glycan onto the deglycosylated IgG in a stepwise manner to form monoglycosylated IgG and subsequently diglycosylated IgG.
  • Figure 2 shows data from glycosynthase screens of EndoS2 D184M and the EndoS2 D184M 1185 mutants in which 0.5 pg of EndoS2 D184M and 1185 was incubated with 100 pg of deglycosylated IgG at a concentration of 34.5 pM and an excess of oxazolinedinked glycan (ox- S2G2). The reaction was sampled after 10, 15, 30, 50, and 100 minutes. EndoS2 D184M and Il 85 A, C, V, F, and Y have similar or improved transglycosylation when compared to EndoS2 D184M. EndoS2 D184M and I185L, M, T have lower transglycosylation, and other mutants tested had little to no glycosynthase activity.
  • Figure 3 shows data from hydrolysis screens of EndoS2 D184M, EndoS2 D184M and I185A, EndoS2 D184M and I185C, EndoS2 D184M and I185F, EndoS2 D184M and I185V, EndoS2 D184M and I185Y.
  • Enzymes 0.5 pg were incubated of with 100 pg of deglycosylated IgG at a concentration of 34.5 pM. Relative fractions of (Right) deglycosylated, (Center) monoglycosylated, and (Left) di glycosylated IgG after 100 minutes are provided.
  • Figure 4 shows data on transglycosylation activity of EndoS2 D184M and II 85 A in which the enzyme transfers the glycan onto the deglycosylated IgG (circles) in a stepwise manner to form monoglycosylated IgG (square) and subsequently diglycosylated IgG (triangles).
  • an “embodiment” of this disclosure refers to an example and infers that the example is not necessarily limited to the example.
  • Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of medicine, organic chemistry, biochemistry, molecular biology, pharmacology, and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
  • Patent law in that when applied to methods and compositions encompassed by the present disclosure refers to the idea of excluding certain prior art element(s) as an inventive feature of a claim, but which may contain additional composition components or method steps, etc., that do not materially affect the basic and novel characteristic(s) of the compositions or methods, compared to those of the corresponding compositions or methods disclosed herein.
  • an “oxazoline” refers a chemical group with a 5-(hydroxymethyl)-2- methyl-3a,6,7,7a-tetrahydro-5H-pyrano[3,2-d]oxazole-6,7-diol structure optionally substituted with a glycol, saccharides, or polysaccharide, which can be prepared by the procedures set out in Noguchi et al. J. Org. Chem. 2009, 74, 2210-2212. Reactions with glycols, saccharides and polysaccharide results in N-acetyl-2-amino sugars when catalyzed by the EndoS2 D 184M enzyme.
  • an “antibody” refers to a protein-based molecule that is naturally produced by animals in response to the presence of a protein or other molecule or that is not recognized by the animal’s immune system to be a “self’ molecule, i.e., recognized by the animal to be a foreign molecule, i.e., an antigen to the antibody.
  • the immune system of the animal will create an antibody to specifically bind the antigen, and thereby targeting the antigen for degradation or elimination, or any cell or organism attached to the antigen. It is well recognized by skilled artisans that the molecular structure of a natural antibody can be synthesized and altered by laboratory techniques.
  • antibody is intended to include natural antibodies, monoclonal antibody, or non-naturally produced synthetic antibodies, such as specific binding single chain antibodies, bispecific antibodies, or fragments thereof. These antibodies may have chemical modifications.
  • monoclonal antibodies refers to a collection of antibodies encoded by the same nucleic acid molecule that are optionally produced by a single hybridoma (or clone thereof) or other cell line, or by a transgenic mammal such that each monoclonal antibody will typically recognize the same antigen.
  • the term “monoclonal” is not limited to any particular method for making the antibody, nor is the term limited to antibodies produced in a particular species, e.g., mouse, rat, etc.
  • an antibody is a combination of proteins: two heavy chain proteins and two light chain proteins.
  • the heavy chains are longer than the light chains.
  • the two heavy chains typically have the same amino acid sequence; however, embodiments of this disclosure are directed to antibodies that have two heavy chains that are dissimilar sequences or have alternative mutation patterns.
  • the two light chains typically have the same amino acid sequence; however, light chains with dissimilar sequences are contemplated.
  • each of the heavy and light chains contain a variable segment that contains amino acid sequences which participate in binding to the antigen.
  • the variable segments of the heavy chain do not have the same amino acid sequences as the light chains.
  • the variable segments are often referred to as the antigen binding domains.
  • the antigen and the variable regions of the antibody may physically interact with each other at specific smaller segments of an antigen often referred to as the "epitope.”
  • Epitopes usually consist of surface groupings of molecules, for example, amino acids or carbohydrates.
  • the terms “variable region,” “antigen binding domain,” and “antigen binding region” refer to that portion of the antibody molecule which contains the amino acid residues that interact with an antigen and confer on the antibody its specificity and affinity for the antigen. Small binding regions within the antigen-binding domain that typically interact with the epitope are also commonly alternatively referred to as the "complementarity-determining regions, or CDRs.”
  • a "chimeric antibody” is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules such that the entire molecule is not naturally occurring.
  • Examples of chimeric antibodies include those having a variable region derived from a non-human antibody and a human immunoglobulin constant region.
  • the term is also intended to include antibodies having a variable region derived from one human antibody grafted to an immunoglobulin constant region of a predetermined sequences or the constant region from another human for which there are allotypic differences residing in the constant regions of any naturally occurring antibody having the variable regions, e.g., CDRs 1, 2, and 3 of the light and heavy chain.
  • Human heavy chain genes exhibit structural polymorphism (allotypes) that are inherited as a haplotype.
  • the serologically defined allotypes differ within and between population groups. See Jefferis et al. mAb, 1 (2009), pp. 332-338.
  • the antibody, antibody heavy chain, antigen binding fragment, the light chain, or the heavy chain comprises a non-naturally occurring chimeric amino acid sequence such that there is at least one mutation that is not present in naturally occurring antibodies.
  • hmAbs antigen-specific chimeric human monoclonal antibodies
  • ASCs antibody-secreting cells
  • the antibody genes of the ASCs are then amplified by RT-PCR and nested PCR, cloned into expression vectors, and transfected into a human cell line.
  • Meijer et al. report methods for isolation of human antibody repertoires with preservation of the natural heavy and light chain pairing. J Mol Biol, 2006, 358(3):764-72.
  • Wrammert et al. report using immunoglobulin variable regions isolated from sorted single ASCs to produce human monoclonal antibodies (mAbs). Nature, 2008, 453(7195): 667-671.
  • Chimeric antibodies comprising one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule can be produced using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; International Publication No. WO 91/09967; and U.S.
  • EndoS2 D184M mutant demonstrates transglycosylation activity with residual hydrolysis activity. J Biol Chem, 2016, 291(32): 16508-18.
  • the EndoS2 D184M mutant is in reference to the sequence GLDIDIE (SEQ ID NO: 1) where the N-terminal G is position 180 of EndoS2, an endoglycosidase from S. pyogenes of serotype M49 (NCBI Reference Sequence: WP 012560921.1).
  • Polypeptides sequences of EndoS2 mutants as disclosed herein can be produced by any commonly used method. Typical examples include the recombinant expression in suitable host, cell, or cell-free systems, e.g., cell, mammalian cell, bacteria, or yeast.
  • the EndoS2 mutants may be produced by living host cells that have been genetically engineered to produce the EndoS2 mutant polypeptides. Methods of genetically engineering cells to produce proteins are well known in the art. See e.g., Ausubel et al., eds. (1990), Current Protocols in Molecular Biology (Wiley, New York). Such methods include introducing nucleic acids that encode and allow expression of the EndoS2 mutant polypeptide in host cells.
  • host cells can be bacterial cells, fungal cells, or animal cells grown in culture.
  • EndoS2 mutants are produced in mammalian cells.
  • Typical mammalian host cells for expressing the peptide include Chinese Hamster Ovary (CHO cells), lymphocytic cell lines, e.g., NSO myeloma cells, SP2 cells, COS cells.
  • the recombinant expression vectors may carry additional sequences, such as sequences that regulate replication of the vector in host cells (e.g., origins of replication) and selectable marker genes.
  • the selectable marker gene facilitates selection of host cells into which the vector has been introduced (see e.g., U.S. Pat. Nos. 4,399,216; 4,634,665; and 5,179,017).
  • the selectable marker gene confers resistance to drugs, such as G418, hygromycin, or methotrexate, on a host cell into which the vector has been introduced.
  • Standard molecular biology techniques can be used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells and recover the EndoS2 mutant peptides or cells coated with the peptide from the culture medium.
  • the EndoS2 mutant peptides or cells can be isolated by affinity chromatography.
  • this disclosure relates to nucleotide sequences or nucleic acids that encode the EndoS2 mutants as disclosed herein, genetic constructs that include nucleotide sequences or nucleic acids and one or more elements for genetic constructs known per se. In certain embodiments, this disclosure relates to hosts or host cells that contain such nucleotide sequences or nucleic acids, and/or that express (or are capable of expressing) EndoS2 mutant peptides disclosed herein.
  • this disclosure relates to methods for preparing EndoS2 mutants or cells expressing the EndoS2 mutants using constructs disclosed herein, which method comprises cultivating or maintaining a host cell under conditions such that said host cell produces or expresses the EndoS2 mutants.
  • a “mutation,” “mutant,” or the like of a peptide sequence refers to the expression of a variant amino acid(s) within a heavy chain antibody defined by positions compared to base amino acids.
  • the mutants may be constructed by building peptide sequences synthetically or, more typically, constructed using recombinant nucleic acid techniques, e.g., expression of the peptide in a cell from a template nucleic acid. Due to three codon translation of amino acids from nucleic acid, several three nucleotide codons may express the same amino acid variant. Sometimes the variant is due to a single nucleotide change, and sometimes the variant is due to more than one nucleotide change. Thus, reference to a “mutation,” “mutant,” or the like of a peptide sequence are not necessarily limited to solely single nucleotide changes.
  • the 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 nucleic acid base (e.g., A, T, C, G, U, or I) 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 nucleic acid base e.g., A, T, C, G, U, or I
  • sequence “identity” refers to the number of exactly matching amino acids (expressed as a percentage) in a sequence alignment between two sequences of the alignment calculated using the number of identical positions divided by the greater of the shortest sequence or the number of equivalent positions excluding overhangs wherein internal gaps are counted as an equivalent position. Numerous online resources use this as a default method of calculating percentage of “sequence identity” as calculated by comparing two optimally aligned sequences using default values, see e.g., National Library of Medicine, Basic Local Alignment Search Tool (BLAST®).
  • any recitation of sequence identity expressed herein may be substituted for sequence similarity.
  • Percent “similarity” is used to quantify the similarity between two sequences of the alignment. This method is identical to determining the identity except that certain amino acids do not have to be identical to have a match. Amino acids are classified as matches if they are among a group with similar properties according to the following amino acid groups: Aromatic - F Y W; hydrophobic-A V I L; Charged positive: R K H; Charged negative - D E; Polar - S T N Q. The amino acid groups are also considered conserved substitutions.
  • polypeptide polypeptide
  • peptide protein
  • polymers of amino acids of any length can comprise modified amino acids.
  • the terms also encompass an amino acid polymer that has been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component.
  • nucleic acid refers to a polymer of nucleotides, or a polynucleotide, e.g., RNA, DNA, or a combination thereof. The term is used to designate a single molecule, or a collection of molecules, e.g., one or more nucleic acids. Nucleic acids may be single stranded or double stranded and may include coding regions and regions of various control elements.
  • a "heterologous" nucleic acid sequence or peptide sequence refers to a nucleic acid sequence or a peptide sequence that does not naturally occur, e.g., because the whole sequence contains a segment from other plants, bacteria, viruses, other organisms, or joinder of two sequences that occur the same organism but are joined together in a manner that does not naturally occur in the same organism or any natural state.
  • nucleic acid molecule when made in reference to a nucleic acid molecule refers to a nucleic acid molecule which is comprised of segments of nucleic acid joined together by means of molecular biological techniques provided that the entire nucleic acid sequence does not occurring in nature, i.e., there is at least one mutation in the overall sequence such that the entire sequence is not naturally occurring even though separately segments may occur in nature. The segments may be joined in an altered arrangement such that the entire nucleic acid sequence from start to finish does not naturally occur.
  • recombinant when made in reference to a protein or a peptide refers to a protein molecule that is expressed using a recombinant nucleic acid molecule.
  • vector refers to a recombinant nucleic acid containing a desired coding sequence and appropriate nucleic acid sequences necessary for the expression of the operably/functionally linked coding sequence in a particular host organism or expression system, e.g., cellular or cell-free expression system.
  • Nucleic acid sequences necessary for expression in prokaryotes usually include a promoter, an operator (optional), and a ribosome binding site, often along with other sequences.
  • Eukaryotic cells are known to utilize promoters, enhancers, and termination and polyadenylation signals.
  • a promoter is functionally linked to a nucleic acid (e.g., coding) sequence if it is able to control or modulate transcription of the linked nucleic acid sequence in the cis position.
  • a functionally linked nucleic acid sequences are close together.
  • a functionally linked promoter is generally located upstream of the coding sequence it does not necessarily have to be close to it. Enhancers need not be close either, provided that they assist the transcription of the nucleic acid sequence.
  • a polyadenylation site is functionally linked to a gene sequence if it is positioned at the 3' end of the gene sequence in such a way that the transcription progresses via the coding sequence to the polyadenylation signal.
  • this disclosure contemplates a vector encoding EndoS2 mutants as disclosed herein in operable combination with a heterologous promoter, enhancer, and/or polyadenylation signal.
  • the term "cell” refers to a biological compartment containing a lipid membrane and cytosol which may contain a nucleus containing genetic material, mitochondria, and other organelles. In certain embodiments, the cell is a somatic cell.
  • label refers to a detectable moiety that is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule.
  • label include fluorescent tags, enzymatic linkages, and radioactive isotopes.
  • a label includes the incorporation of a radiolabeled amino acid or the covalent attachment of biotinyl moi eties to a peptide that can be detected by marked avidin (for example, streptavidin containing a fluorescent marker or enzymatic activity that can be detected by optical or colorimetric methods).
  • labels for peptides include, but are not limited to, the following: radioisotopes or radionucleotides (such as 35 S, 18 F, or 131 I), fluorescent labels (such as fluorescein isothiocyanate (FITC), rhodamine, lanthanide phosphors), enzymatic labels (such as horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent markers, biotinyl groups, predetermined peptide epitopes recognized by a secondary reporter (such as a leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags), or magnetic agents, such as gadolinium chelates.
  • labels are attached by spacer arms (linking groups) of various lengths to reduce potential steric hindrance.
  • the disclosure relates to recombinant EndoS2 mutants comprising sequences disclosed herein or variants or fusions thereof wherein the interior amino acid sequence, the amino terminal end, or the carbon terminal end of the amino acid sequence are optionally attached to a heterologous amino acid sequence, label, or reporter molecule.
  • the disclosure relates to the recombinant vectors comprising a nucleic acid encoding an EndoS2 mutant peptide as disclosed herein.
  • the recombinant vector optionally comprises a mammalian, human, insect, viral, bacterial, bacterial plasmid, yeast associated origin of replication or gene such as a gene or retroviral gene or lentiviral LTR, TAR, RRE, PE, SLIP, CRS, and INS nucleotide segment or gene selected from tat, rev, nef, vif, vpr, vpu, and vpx or structural genes selected from gag, pol, and env.
  • a mammalian, human, insect, viral, bacterial, bacterial plasmid, yeast associated origin of replication or gene such as a gene or retroviral gene or lentiviral LTR, TAR, RRE, PE, SLIP, CRS, and INS nucleotide segment or gene selected from tat, rev, nef, vif, vpr, vpu, and vpx or structural genes selected from gag, pol, and env.
  • the recombinant vector optionally comprises a gene vector element (nucleic acid) such as a selectable marker region, lac operon, a CMV promoter, a hybrid chicken B-actin/CMV enhancer (CAG) promoter, tac promoter, T7 RNA polymerase promoter, SP6 RNA polymerase promoter, SV40 promoter, internal ribosome entry site (IRES) sequence, cis-acting woodchuck post regulatory element (WPRE), scaffold-attachment region (SAR), inverted terminal repeats (ITR), c-myc tag coding region, metal affinity tag coding region, streptavidin binding peptide tag coding region, polyHis tag coding region, HA tag coding region, MBP tag coding region, GST tag coding region, polyadenylation coding region, SV40 polyadenylation signal, SV40 origin of replication, Col El origin of replication, fl origin, pBR322 origin, or pUC origin, TEV prote
  • this disclosure contemplates antibody heavy chain(s) or antibodies containing the same, wherein a desired sugar chain is added to a core fucosylated or nonfucosylated GlcNAc-acceptor, including fucosylated or nonfucosylated GlcNAc-IgG acceptor using EndoS2 mutants disclosed herein.
  • the present disclosure allows for the synthesis and remodeling of therapeutic antibodies, or Fc fragments thereof, to provide for certain biological activities, such as, prolonged half-life time in vivo, less immunogenicity, enhanced in vivo activity, increased targeting ability, and/or ability to deliver a therapeutic agent.
  • this disclosure relates to a non-naturally occurring recombinant endoglycosidase S2 (EndoS2) enzyme, comprising a first mutation and a second mutation in the sequence of a wild-type EndoS2, wherein the mutant of EndoS2 has a hydrolyzing activity lower than that of the EndoS2 the D184M mutant and/or has transglycosylation activity higher than that of the wild-type EndoS2 and/or DI 84M mutant.
  • EndoS2 endoglycosidase S2
  • the first mutation in EndoS2 is aD184M wherein the first mutation is in reference to positions in amino acid sequence GLDIDIE (SEQ ID NO: 1) (segment of Streptococcus pyogenes endo-beta-N-acetylglucosaminidase (EndoS2) [Streptococcus pyogenes M49 591] Chain A, Secreted Endo-beta-N-acetylglucosaminidase (EndoS), World Protein Data Bank PDB: 6E58_A), wherein the N-terminal amino acid glycine (G) is position 180.
  • GLDIDIE SEQ ID NO: 1
  • second mutation in EndoS2 is I185A wherein the mutation is in reference to positions in amino acid sequence GLDIDIE (SEQ ID NO: 1) wherein the N-terminal amino acid glycine (G) is position 180.
  • the recombinant EndoS2 enzyme comprises the amino acid sequence of SEQ ID NO: 2,
  • the variants have greater than 90, 95, 96, 97, 98, or 99% sequence identity. In certain embodiments, variants are conserved variants. In certain embodiments, there are one, two or three variants. In certain embodiments, the variants are within the first 50 or 100 amino acids on the N-terminus or within the last 50 or 100 amino acids of the C-terminus.
  • the recombinant EndoS2 enzyme has a second mutation in EndoS2 of I185F wherein the mutation is in reference to positions in amino acid sequence GLDIDIE (SEQ ID NO: 1) wherein the N-terminal amino acid glycine (G) is position 180.
  • recombinant EndoS2 enzyme comprises the amino acid sequence of SEQ ID NO: 3
  • the variants have greater than 90, 95, 96, 97, 98, or 99% sequence identity. In certain embodiments, variants are conserved variants. In certain embodiments, there are one, two or three variants. In certain embodiments, the variants are within the first 50 or 100 amino acids on the N-terminus or within the last 50 or 100 amino acids of the C-terminus.
  • the recombinant EndoS2 enzyme has a second mutation in EndoS2 of I185C, wherein the mutation is in reference to positions in amino acid sequence GLDIDIE (SEQ ID NO: 1) wherein the N-terminal amino acid glycine (G) is position 180.
  • the recombinant EndoS2 enzyme has the amino acid sequence of SEQ ID NO: 4
  • the variants have greater than 90, 95, 96, 97, 98, or 99% sequence identity. In certain embodiments, variants are conserved variants. In certain embodiments, there are one, two or three variants. In certain embodiments, the variants are within the first 50 or 100 amino acids on the N-terminus or the C-terminus.
  • the recombinant EndoS2 enzyme has a second mutation in EndoS2 of II 85 V wherein the mutation is in reference to positions in amino acid sequence GLDIDIE (SEQ ID NO: 1) wherein the N-terminal amino acid glycine (G) is position 180.
  • the recombinant EndoS2 enzyme has the amino acid sequence of SEQ ID NO: 5
  • the variants have greater than 90, 95, 96, 97, 98, or 99% sequence identity. In certain embodiments, variants are conserved variants. In certain embodiments, there are one, two or three variants. In certain embodiments, the variants are within the first 50 or 100 amino acids on the N-terminus or within the last 50 or 100 amino acids of the C-terminus.
  • recombinant EndoS2 enzyme has a second mutation in EndoS2 of I185Y wherein the mutation is in reference to positions in amino acid sequence GLDIDIE (SEQ ID NO: 1) wherein the N-terminal amino acid glycine (G) is position 180.
  • recombinant EndoS2 enzyme has the amino acid sequence of SEQ ID NO: 6.
  • the variants have greater than 90, 95, 96, 97, 98, or 99% sequence identity. In certain embodiments, variants are conserved variants. In certain embodiments, there are one, two or three variants. In certain embodiments, the variants are within the first 50 or 100 amino acids on the N-terminus or within the last 50 or 100 amino acids of the C-terminus.
  • the recombinant EndoS2 enzyme further comprises a D233A and/or D233Q mutation, wherein the mutation is in reference to positions in amino acid sequence NYIDASQ (SEQ ID NO: 7) wherein the N-terminal amino acid asparagine (N) is position 230.
  • this disclosure relates to a fusion protein comprising an EndoS2 mutant enzyme as reported herein and heterologous peptide domain.
  • this disclosure relates to recombinant nucleic acids encoding a recombinant enzyme as reported herein with a heterologous nucleic acid sequence, e.g., in operable combination with a heterologous promoter.
  • this disclosure relates to vectors comprising a recombinant nucleic acid encoding an enzyme as reported herein.
  • this disclosure relates to cell or cell-free protein expression systems comprising a recombinant nucleic acid or vector encoding an enzyme as reported herein.
  • the expression system is a somatic cell or cell fee expression system.
  • this disclosure relates to methods for preparing an engineered glycoprotein using the mutant of EndoS2 as reported herein, comprising contacting an activated oligosaccharide with a glycoprotein acceptor providing an extended glycoprotein conjugated to the glycoprotein acceptor.
  • the activated oligosaccharide is a glycan oxazoline.
  • the glycoprotein acceptor contains a GlcNAc monosaccharide.
  • the glycoprotein acceptor is a non-fucosylated GlcNAc-acceptor.
  • the glycoprotein acceptor is a glycopeptide, a glycoprotein, an antibody or a fragment thereof.
  • the glycoprotein acceptor is a core fucosylated or non- fucosylated GlcNAc-IgG acceptor or a fragment thereof.
  • the GlcNAc-IgG acceptor is derived from an antibody selected from the group consisting of abagovomab, abciximab, abituzumab, abrezekimab, abrilumab, actoxumab, adalimumab, adecatumumab, aducanumab, afasevikumab, afelimomab, afutuzumab, alacizumab, alemtuzumab, alirocumab, altumomab, amatuximab, anatumomab, andecaliximab, anetumab, anifrolumab, anrukinzumab, apolizumab, aprutumab, arcitumomab, ascrinvacumab, aselizumab, atezolizumab, atinumab, atlizuma
  • compositions comprising the glycoprotein as reported herein and a pharmaceutically acceptable carrier or excipient.
  • this disclosure relates to method of treating a disease or condition comprising administering an effective amount of a glycoprotein as reported herein to a subject in need thereof.
  • the disease or condition is a cancer, autoimmune disease, or inflammatory disease.
  • EndoS2 is an endo-b-N-acetylglucosaminidase (ENGase) that catalyzes the hydrolysis of the beta-1,4 linkage between the first and second N-acetylglucosamine (GlcNAc) residues of N-linked glycans.
  • ENGase endo-b-N-acetylglucosaminidase
  • EndoS2 can be made into a glycosynthase by mutating one of the residues in the catalytic triad, DI 84, to a methionine (D184M).
  • EndoS2 D184M can transfer an oxazoline-linked glycan onto IgGs.
  • this glycosynthase still retains some hydrolytic activity.
  • EndoS2 D184SeMet seleno-methionine
  • EndoS2D184M (0.5pg) or EndoS2D184SeMet (0.5pg) was incubated with 100 pg of deglycosylated IgG at a concentration of 34.5 pM and an excess of oxazoline-linked glycan (ox-S2G2). The reaction was sampled approximately every 4 minutes.
  • the reactions were analyzed by LC-MS using an Agilent 1290 Infinity II LC SystemTM equipped with a 50 mm PLRP-S columnTM from Agilent with 1000 A pore size.
  • the LC system was attached to either an Agilent 6545XTTM quadrupole- time of flight (Q-TOF) or Agilent 6560TM ion mobility QTOF mass spectrometers. Relative amounts of the substrate and hydrolysis products were quantified after deconvolution of the raw data and identification of the corresponding peaks.
  • EndoS2 D184SeMet has a higher transglycosylation rate as compared to EndoS2 D184M ( Figure 1), and kinetic data over a longer timeframe shows that the incorporation of SeMet reduces the residual hydrolysis rate of the enzyme. However, EndoS2 D184SeMet does not reach full transglycosylation and the relative amount of diglycosylated IgG plateaus at about 70%-80%.
  • EndoS2D184SeMet is relatively unstable and prone to oxidation
  • a strategy that involved mutating residues near the active site that are not directly involved in catalysis was taken. These residues contribute to subtle conformational changes that can result in transglycosylation and reduced residual hydrolysis. Specifically, saturation mutagenesis of residue 1185 was performed. Glycosynthase screens of the 19 mutants using the same reaction parameters described earlier, revealed five mutants that had similar or improved transglycosylation compared to EndoS2 D184M: EndoS2 D184M I185A, EndoS2 D184M I185F, EndoS2 D184M I185C, EndoS2 D184M Il 85V, EndoS2 D184M I185Y ( Figure 2).

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Abstract

L'invention concerne des variants de glycosynthases utiles pour modifier des anticorps IgG avec une efficacité élevée et une hydrolyse résiduelle réduite. Dans certains modes de réalisation, la présente invention concerne des compositions et des procédés d'utilisation de mutants d'EndoS2 décrits ici pour transférer un glycane sur le Fc d'immunoglobulines. Dans certains modes de réalisation, les mutants EndoS2 peuvent transférer un glycane à liaison oxazoline sur IgG.
PCT/US2024/025296 2023-04-18 2024-04-18 Mutants d'endoglycosidase s2 (endos2) et leurs utilisations Ceased WO2024220732A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017124084A1 (fr) * 2016-01-15 2017-07-20 University Of Maryland, College Park Utilisation de mutants endo-s2 comme glycosynthases, procédé de fabrication et d'utilisation pour la glycoingénierie de glycoprotéines
US20180298361A1 (en) * 2016-08-24 2018-10-18 CHO Pharma Inc. Endoglycosidase mutants for glycoprotein remodeling and methods of using it

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017124084A1 (fr) * 2016-01-15 2017-07-20 University Of Maryland, College Park Utilisation de mutants endo-s2 comme glycosynthases, procédé de fabrication et d'utilisation pour la glycoingénierie de glycoprotéines
US20180298361A1 (en) * 2016-08-24 2018-10-18 CHO Pharma Inc. Endoglycosidase mutants for glycoprotein remodeling and methods of using it

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