WO2012158932A2 - Élimination d'épitopes α-gal de glycoprotéines thérapeutiques afin d'éviter toute interaction avec l'anticorps anti-gal - Google Patents

Élimination d'épitopes α-gal de glycoprotéines thérapeutiques afin d'éviter toute interaction avec l'anticorps anti-gal Download PDF

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WO2012158932A2
WO2012158932A2 PCT/US2012/038371 US2012038371W WO2012158932A2 WO 2012158932 A2 WO2012158932 A2 WO 2012158932A2 US 2012038371 W US2012038371 W US 2012038371W WO 2012158932 A2 WO2012158932 A2 WO 2012158932A2
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gal
galactosidase
glycoproteins
epitopes
column
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WO2012158932A3 (fr
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Uri Galili
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University Of Massachusetts
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders

Definitions

  • the present invention is related to the field of therapeutic glycoproteins.
  • the present invention provides compositions and methods for avoiding the binding of the natural anti-Gal antibody to monoclonal antibodies, and to natural or recombinant glycoproteins or to other molecules that have cc-gal epitopes (carbohydrate chains with terminal Galal-3Gal) and which are injected or infused for therapeutic purpose into humans or other vertebrates producing the anti-Gal antibody.
  • the prevention of this binding eliminates the risk of complement activation, allergic reactions and accelerated removal of the injected glycoproteins, which otherwise may occur if the anti-Gal antibody binds to a-gal epitopes on said glycoproteins.
  • Therapeutic glycoproteins which are introduced into the blood stream or into various tissues by injection or infusion, range from a variety of monoclonal antibodies, to recombinant enzymes, hormones and various glycoproteins with biological activities.
  • the asparagines (Asn or N)-linked carbohydrate chains of these glycoproteins are synthesized on asparagines (N) which are part of an amino acids sequence N-X-S/T (glycosylation sites with the sequence of asparagine- any amino acid- serine or threonine).
  • Anti-Gal is produced in humans as -1% of IgG (Galili et al, J Exp Med 160: 1519, 1984), >1% of IgM, and is readily detected in various body secretions as an IgA antibody (Hamadeh et al., Clin Diagnos Lab Immunol 2: 125, 1995). Recently, anti-Gal was shown to be produced in a proportion of the population as IgE antibody that mediates allergic reactions (Chung et al., New Engl J Med 358: 1109, 2008).
  • anti-Gal is naturally produced in humans, apes and Old World monkeys (monkeys of Asia and Africa) (Galili et al, Proc Natl Acad Sci (USA), 84:1369, 1987)
  • the a-gal epitopes is synthesized by cells in nonprimate mammals, prosimians and New World monkeys (monkeys of Central and South America), since the latter group of mammals has active al,3galactosyltranferase ( l,3GT), the enzyme that synthesizes within the Golgi apparatus a-gal epitopes (Galili et al., Proc Natl Acad Sci (USA), 84: 1369, 1987; Galili et al., J Biol Chem, 263: 17755, 1988).
  • Anti-Gal readily interacts in vivo with a-gal epitopes as indicated in xenotransplantation studies. Transplantation of pig organs or cells (i.e. cells expressing a-gal epitopes) into Old World monkeys or humans results in anti-Gal binding to these epitopes and activation of complement which lyses the xenograft cells, resulting in rapid (hyperacute) rejection (Galili, Immunol Today, 14:480, 1993; and Collins et al., J Immunol, 154:5500, 1995). A similar interaction may occur between anti-Gal and a-gal epitopes on therapeutic glycoproteins injected or infused into humans. This interaction may lead to at least three apparent detrimental outcomes: 1.
  • complement cleavage factors such as C5a and C3a which, if produced in high enough concentration, may cause systemic inflammatory response syndrome (SIRS) that has symptoms similar to sepsis.
  • SIRS systemic inflammatory response syndrome
  • the treated patient has anti-Gal IgE antibodies bound to mast cells, the binding of the a-gal epitopes on the therapeutic glycoprotein to these IgE antibodies will result in cross-linking of the IgE molecules on the mast cells, thus inducing the activation of the mast cells and induction of an allergic reaction, which may ultimately cause an anaphylactic shock.
  • the present invention is related to the field of therapeutic glycoproteins.
  • the present invention provides compositions and methods for processing glycoprotein molecules with linked a-gal epitopes for elimination of the a-gal epitopes from these molecules thereby preventing the in vivo binding of the natural anti-Gal antibody to the a-gal epitopes.
  • the present invention provides methods for prevention of allergic reactions, prevention of complement activation and prevention of accelerated removal of the injected therapeutic glycoprotein, by avoiding in vivo anti-Gal binding to these therapeutic glycoproteins. This is achieved by the use of an enzyme that cleaves (hydrolyses) the terminal galactose from the a-gal epitope thereby it destroys a-gal epitopes.
  • the enzyme referred to as a-galactosidase
  • a-galactosidase is used as a solid phase in a column of porous beads through which the solution of the therapeutic glycoprotein is passed for the destruction of the a-gal epitopes.
  • the enzyme a-galactosidase may be isolated from a natural source or in a recombinant form.
  • the enzyme is used in a soluble form which is mixed with the therapeutic glycoprotein.
  • the enzyme may be removed by its selective binding to a matrix such as, but not limited to, an agarose column with a ligand that specifically removes the enzyme from the solution mixture.
  • the invention provides a method, comprising applying a therapeutic glycoprotein preparation having one or more a-gal epitopes having a terminal Galal-3Gal or terminal a-galactosyls to a column having covalently or non-covalently coupled ⁇ -galactosidase in porous beads or on particulate material in order to destroy the a-gal epitope by enzymatic removal of the terminal galactose by the ⁇ -galactosidase.
  • the therapeutic glycoproteins are monoclonal antibodies.
  • the therapeutic glycoproteins are recombinant glycoproteins.
  • the therapeutic glycoproteins are natural glycoproteins.
  • the ⁇ -galactosidase is obtain from a natural source including, but not limited to microbial source, fungal source, or plant source.
  • the ⁇ -galactosidase is a recombinant a-galactosidase produced in a prokaryotic gene expression system or in a eukaryotic gene expression system or in a transgenic animal.
  • the invention also provides a method, comprising destruction of one or more a-gal epitopes having a terminal Galal-3Gal or terminal a-galactosyls on therapeutic glycoproteins by mixing the glycoproteins with tagged ⁇ -galactosidase in order to destroy the a-gal epitope by enzymatic removal of the terminal galactose and to subsequently remove the ⁇ -galactosidase.
  • the tagged ⁇ -galactosidase is a recombinant ⁇ -galactosidase produced in a prokaryotic gene expression system or an eukaryotic gene expression system or in a transgenic animal and the enzyme is tagged with an oligopeptide, polypeptide, protein or other tag that enables the removal of the ⁇ -galactosidase from the reaction mixture by passing the mixture through a column of porous beads or with particulate material carrying linked ligand to the tag.
  • the ⁇ -galactosidase is tagged with an oligopeptide of 6 histidines (Hise), or tagged with fusion proteins as protein A, Fc portion of an immunoglobulin molecule, glutathione S-transferase, or tagged with biotin, and the corresponding columns with matrix for removal of the tagged ⁇ -galactosidase including nickel-sepharose column, IgG coupled column, protein A coupled column, glutathione coupled column, or avidin coupled column, respectively.
  • the ⁇ -galactosidase is not tagged.
  • Figure 1 illustrates schematically a-gal epitopes on N-linked carbohydrate chains of therapeutic glycoproteins and the destruction of these epitopes by ⁇ -galactosidase.
  • the a-gal epitopes are marked with rectangles of broken lines.
  • anti- Gal binds to a-gal epitopes (left glycoprotein).
  • the a-gal epitopes can be destroyed by the enzyme ⁇ -galactosidase. This enzyme cleaves (i.e.
  • Figure 2 shows a schematic illustration of a porous bead within the column of a- galactosidase attached to a matrix of agarose beads (called also Sepharose beads).
  • the column is also referred to as a-galactosidase column, a-galactosidase in linked to the matrix either covalently as in agarose beads activated with cyanogens bromide, or linked non-covalently.
  • a non-covalently linked enzyme is recombinant ⁇ -galactosidase with a tag of a number of histidines (His 6 tag) that links the enzyme to nickel-agarose.
  • the therapeutic glycoprotein passing through the column is subjected to the enzymatic activity of a- galactosidase linked to the column, resulting in the removal of terminal galactose from the carbohydrate chains of the glycoproteins and thus, destruction of the a-gal epitopes.
  • Figure 3 demonstrates the in vitro binding of the monoclonal anti-Gal antibody M86 (Galili et al., Transplantation 65: 1129, 1998) to a-gal epitopes of various glycoproteins prior ( Figure 3A), and post treatment with recombinant a-galactosidase (10 Units/ml) ( Figure 3B).
  • the glycoproteins served as solid-phase antigens in an enzyme-linked immunosorbent assay (ELISA).
  • ELISA wells were coated with the ⁇ g/ml glycoproteins in carbonate buffer pH 9.5 by standard methods known to those skilled in the art.
  • Binding of the monoclonal anti-Gal M86 in Figures 3A and 3B was determined by the subsequent binding of goat anti-mouse IgM coupled to horseradish peroxidase (HRP) and color development with O-phenylene diamine (OPD). Binding of anti-Gal in human serum to untreated glycoproteins in ELISA is presented in Figure 3C, whereas binding of anti-Gal in human serum to glycoproteins treated with a- galactosidase is presented in Figure 3D. Binding of anti-Gal in human serum to a-gal epitopes was determined by the subsequent binding of rabbit anti-human IgG coupled to HRP and color development with OPD.
  • Figure 3A provides a graph of the binding of monoclonal anti-Gal M86 to synthetic a-gal epitopes on bovine serum albumin (a-gal BSA) ( ⁇ ), bovine thyroglobulin ( ⁇ ), a-gal fetuin (A) or mouse laminin ( ⁇ ), human thyroglobulin ( ⁇ ), human laminin (0), or original fetuin lacking a-gal epitopes ( ⁇ ).
  • a-gal BSA bovine serum albumin
  • ⁇ bovine thyroglobulin
  • ⁇ a-gal fetuin
  • A mouse laminin
  • ⁇ human thyroglobulin
  • ⁇ human laminin (0)
  • original fetuin lacking a-gal epitopes ⁇
  • Figure 3C provides a graph of the binding of anti-Gal in human serum to the various glycoproteins with symbols as in Figure 3A.
  • Figure 3D the binding anti-Gal in human serum was evaluated after the assayed glycoproteins were treated with ⁇ -galactosidase. Note that monoclonal anti-Gal and human serum anti-Gal readily bind to the a-gal epitopes on a-gal BSA, bovine thyroglobulin, a-gal fetuin or mouse laminin.
  • Figure 4 illustrates the activation of complement in human serum, by human anti-Gal binding to a-gal epitopes on glycoproteins, and prevention of this activation by a-galactosidase mediated destruction of a-gal epitopes on the glycoproteins.
  • Figure 4A is a schematic of the readout system for complement activity involving the cytolysis of the anti-Gal producing hybridoma cells M86 (Galili et al, Transplantation 65: 1129, 1995). These cells have anti-Gal bound to their cell membranes because, in addition to production of a monoclonal anti-Gal, these cells express a-gal epitopes.
  • complement Upon addition of complement, as in the addition of human serum to M86 cells, the complement is activated by the monoclonal anti-Gal bound to the a-gal epitopes on the M86 hybridoma cell membrane, resulting in complement mediated lysis (cytolysis) of the M86 cells.
  • cytolysis complement mediated lysis
  • anti-Gal in the serum binds to a-gal epitopes on glycoproteins and activates serum complement as a result of this binding, the complement in the serum is consumed. If consumed, the complement is not available for subsequent cytolysis of M86 cells upon addition of the cells to the serum containing the glycoproteins.
  • Figure 4B provides a graph showing the cytolysis of M86 cells by complement after incubation at 37°C for lh with human serum at various dilutions of the serum ( ⁇ ).
  • heat inactivation of serum results in inactivation of the complement within the human serum and hence no cytolysis of the M86 cells is observed at any serum dilution (O).
  • the cytolysis of M86 cells by human serum is dependent only on complement activity. This is indicated by a similar cytolysis observed with normal rabbit serum which completely lacks anti- Gal antibodies (since rabbits, like all nonprimate mammals have a-gal epitopes on their cells) ( ⁇ ).
  • Figure 4C provides a graph showing that the interaction of anti-Gal in human serum with a- gal epitopes on glycoproteins results in complement activation and consumption, as measured by a loss of serum cytolytic activity.
  • Human serum was incubated for 2h at 37°C at serial two-fold dilutions with lOmg/ml a-gal BSA (O) that has 21 a-gal epitopes per molecule or with 5mg/ml a-gal fetuin ( ⁇ ) that has 9 a-gal epitopes per molecule.
  • Figure 5 illustrates the destruction of a-gal epitopes on a glycoprotein with a-gal epitopes passed through an ⁇ -galactosidase column.
  • the glycoprotein was collected in 2ml saline and plated in ELISA wells at serial two-fold dilutions in carbonate buffer pH 9.5 starting at 10 ⁇ g/ml.
  • the original glycoprotein solution of a-gal BSA (O) was plated at similar two-fold dilutions, starting at 10 ⁇ g/ml. After 2h incubation at 37°C and overnight incubation at 4°C, the ELISA plate was blocked with 1% BSA in PBS. The presence of a-gal epitopes on the glycoprotein attached to the wells was determined by incubation for 2h of the monoclonal anti-Gal antibody M86, followed by washes, addition of FfRP coupled goat anti- mouse IgM and color development with OPD.
  • Figure 6 illustrates human complement activation by a-gal BSA passed through an a- galactosidase column.
  • Human serum was incubated for 2h at 37°C at serial two-fold dilutions with 100 ⁇ 1 ⁇ -gal BSA (O) or with 100 ⁇ g ml a-gal BSA that was passed through an a- galactosidase column ( ⁇ ) and subsequently cytolysis of M86 cells was measured. Cytolysis of M86 cells by human serum in the absence of a-gal BSA is described as ( ⁇ ).
  • human serum incubated with a-gal BSA displays decreased cytolysis of M86 cells because of complement activation and consumption as a result of anti-Gal binding to oc-gal epitopes of oc- gal BSA.
  • a-gal BSA was passed through an a-galactosidase column, consumption of complement was much lower, because a large proportion of the a-gal epitopes on a-gal BSA were destroyed by ⁇ -galactosidase within the column.
  • therapeutic glycoproteins refers to any protein molecule that carries carbohydrate chains and is used for clinical treatment by infusion, injection intravenously or to any tissue such as intramuscular or intradermal.
  • Therapeutic glycoproteins may originate from blood, tissue extracts or secretion of various animals, or may be produced in natural or recombinant form from various cells grown in cultures.
  • Some non-limiting examples of therapeutic glycoproteins are: monoclonal antibodies, recombinant or natural hormones, recombinant growth factors and cytokines.
  • a-gal epitope refers to any molecule, or part of a molecule, with a terminal structure comprising Galal-3Gaipi -4GlcNAc-R, Galal -3Galpl-3GlcNAc-R, or any carbohydrate chain with terminal Galal-3Gal, or terminal a-galactosyl at the non-reducing end, and which are capable of binding natural or elicited anti-Gal antibodies.
  • glycoproteins with a-gal epitopes refers to any protein with at least one carbohydrate chain which has one or more a-gal epitopes, or any oligopeptide or polypeptide and which has one or more a-gal epitopes.
  • N-acetyllactosamine refers to any molecule, or part of a molecule, with a terminal structure comprising Gai i-4GlcNAc-R, or Gal l-3GlcNAc-R.
  • anti-Gal antibodies also called anti- -gal-antibodies, or anti-a-galactosyl- antibodies or anti-galactose al-3galactose antibodies or anti-Galotl-3Gal antibodies and refers to naturally occurring or elicited antibodies that bind to a-gal epitopes.
  • a-galactosidase refer to a glycosidase enzyme that cleaves (i.e. hydrolyzes) terminal galactose units on carbohydrate chains that are linked in an a-anomeric linkage to the penultimate carbohydrate unit that may be galactose or any other carbohydrate.
  • a natural source of a-galactosidase may include, but not limited to, green coffee beans, soy beans, or fungi.
  • Recombinant ⁇ -galactosidase is produced by cloning an ⁇ -galactosidase gene from any of the natural sources, insertion of the cloned gene into an expression system of microbial or eukaryotic cell or transgenic animal and production of the enzyme in the expression system cells.
  • the recombinant ⁇ -galactosidase may be produced in its native form (i.e. without any tag), or linked to a tag such as, but not limited to, (His)6, protein A, Fc portion of an immunoglobulin molecule, or glutathione S-transferase.
  • ⁇ -galactosidase column refers to a column of agarose beads, Sepharose beads Sephacryl beads, or any other matrix to which any ⁇ -galactosidase enzyme in linked covalently or non-covalently.
  • al,3-galactosyltransferase refers to an enzyme synthesizing a-gal epitopes. The enzyme is naturally expressed in most mammals with the exception of humans, apes and Old World monkeys. This enzyme is also referred to as "al,3GT”.
  • the carbohydrate structure produced by the enzyme is immunogenic in man and most healthy people have high titer natural anti a-gal antibodies, also referred to as anti-galactosea 1-3 galactose antibodies or anti-Gal antibodies.
  • the term "al,3GT” refers to a common marmoset gene (e.g., Callithrix jacchus - GENBANK Accession No.
  • al,3GT refers to mouse al,3GT (e.g., Mus musculus - nucleotides 445 to 1560 of GENBANK Accession No. NM_010283), bovine al,3GT (e.g., Bos taurus - GENBANK Accession No. NM_177511), feline al,3GT (e.g., Felis catus - GENBANK Accession No.
  • NM_001009308 ovine al,3GT (e.g., Ovis aries - GENBANK Accession No. NM_001009764), rat al,3GT (e.g., Rattus norvegicus - GENBANK Accession No. NM_145674) and porcine al,3GT (e.g., S s scrofa - GENBANK Accession No. NM_213810).
  • the a,l,3GT gene is also referred to as Ggtal gene.
  • a-gal fetuin refers to the bovine fetal serum glycoprotein fetuin on which the terminal carbohydrate epitope SA(sialic acid)-Gaipi-4GlcNAc-R was enzymatically replaced with a-gal epitopes (Gal l-3Galpl-4GlcNAc-R) (Chen et al., Glycobiology 11:577, 2001).
  • anti-Gal binding epitope refers to any molecule or part of molecule that is capable of binding the anti-Gal antibody.
  • tag refers here to an oligopeptide, polypeptide, protein, or any molecule that can be attached to a natural or recombinant a-galactosidase and which can bind to a corresponding molecule (ligand) on a column, thereby enabling the specific removal of a- galactosidase from solution.
  • patient and “subject” refer to a mammal or an animal that is a candidate for receiving medical treatment.
  • saline refers to physiologic salt solution of 0.9% NaCl in distilled water.
  • the present invention relates to the field of therapeutic glycoproteins and describes methods and compositions for preventing adverse effects of therapeutic glycoproteins that have a-gal epitopes on their carbohydrate chains and thus, can interact in vivo with the natural anti- Gal antibody (left carbohydrate chain in Figure 1).
  • Anti-Gal is an abundant natural antibody in humans constituting -1% of serum immunoglobulins (Galili et al., J Exp Med, 160:1519, 1984). This antibody interacts specifically with the a-gal epitope (Galal-3Gai i-4GlcNAc-R or Galal-3Gaip i-3GlcNAc-R) on glycolipids and glycoproteins (Galili, Springer Semin Immunopathol, 15:155, 1993). Anti-Gal is produced throughout life as a result of antigenic stimulation by bacteria of the gastrointestinal tract (Galili et al., Infect Immun, 56:1730, 1988).
  • the a-gal epitope is synthesized by the glycosylation enzyme al,3galactosyltransferase (al,3GT) and expressed in very large amounts on cells of non-primate mammals, prosimians and in New World monkeys (Galili et al., J Biol Chem, 263: 17755, 1988).
  • al,3GT gene was inactivated in ancestral Old World primates.
  • humans, apes, and Old World monkeys lack a-gal epitopes and produce high titer anti-Gal antibodies (Galili, Springer Semin Immunopathol, 15: 155, 1993).
  • Anti-Gal antibodies bind avidly in vivo to -gal epitopes when administered to humans or Old World monkeys. This is particularly evident in the context of xenotransplantation, where the in vivo binding of anti-Gal to a-gal epitopes on transplanted pig heart or kidney is the main cause for the rapid rejection of such grafts in humans and Old World monkeys (Galili, Immunol Today, 14:480, 1993; Collins et al., J Immunol, 154:5500, 1995).
  • One of the main mechanisms mediating xenograft rejection is the activation of the complement cascade due to anti-Gal binding to a-gal epitopes on the endothelial cells of the xenograft. This results in the destruction of these endothelial cells by the activated complement molecules, causing collapse of the vascular bed and xenograft ischemia followed by rapid rejection of the xenograft (Collins et al., J Immunol, 154:5500, 1995).
  • This in situ interaction of anti-Gal with a-gal epitopes may also occur when therapeutic glycoproteins carrying these epitopes are injected into patients.
  • the in situ interaction of anti-Gal with a-gal epitopes on therapeutic glycoproteins may result in a number of adverse effects, some of which are detailed below.
  • a-gal epitopes are synthesized on carbohydrate chains of glycoproteins and glycolipids in the Golgi apparatus of cells containing al,3GT by the following reaction:
  • the number of a-gal epitopes differs between various glycoproteins and depends on the number of carbohydrate chains per protein that can be subjected to the enzymatic activity of l,3GT in the trans-Golgi apparatus in cells, and on the activity of al,3GT in comparison to other competing glycosyltransferases within the trans-Golgi compartment where al,3GT is active, such as sialyltransferase (Smith et al, J Biol chem. 265:6225, 1990).
  • the various glycosyltransferases in the trans-Golgi apparatus compete with each other for "capping" the N- acetyllactosamine of the carbohydrate chains with the carbohydrates they transfer to the chain.
  • the monoclonal antibody produced by such hybridoma cells will have a-gal epitopes (Borrebaeck et al., Immunol Today 14:477, 1993). This is because the al,3GT encoded by the al,3GT gene of the myeloma fusion partner will synthesize a-gal epitopes on a proportion of the carbohydrate chains present on all monoclonal antibodies.
  • recombinant glycoproteins produced in cells that contain active al,3GT may have a-gal epitopes because the recombinant glycoprotein passing through the trans-Golgi compartment will be subjected to the activity of al,3GT.
  • Non-limiting examples are recombinant human Factor VIII produced in baby hamster kidney cells (Hironaka et al., J Biol Chem 267:8012, 1992) and recombinant human Interferon ⁇ produced in mouse epithelial cells (Kagawa et al., J Biol Chem 263 : 17508, 1988). Analysis of the carbohydrate chains on these recombinant glycoprotein demonstrated presence of a-gal epitopes on their carbohydrate chains.
  • any therapeutic glycoprotein produced in mammalian cells that contain active l,3GT may have a-gal epitopes.
  • recombinant glycoproteins produced in vivo such as in the cells of mammary glands having active al,3GT and secreted in the milk of mammalian species including, but not limited to, cow, sheep or goat, may have a-gal epitopes synthesized by al,3GT in cells of the mammary glands.
  • a-gal epitopes were demonstrated as free oligosaccharides in ovine milk (Urashima et al., Biochim Biophys Acta 992:375, 1989), or bovine milk (Urashima et al., Biochim Biophys Acta 1073:225, 1991).
  • glycoproteins that have a-gal epitopes When injected into treated individuals, therapeutic glycoproteins that have a-gal epitopes, can bind via their a-gal epitopes to anti-Gal IgE antibodies on mast cells, induce degranulation of these cells resulting in an allergic response. This was demonstrated in patients treated with the monoclonal antibody to epidermal growth factor receptor named "cetuximab” (Chung et al. New Engl J Med 358: 1109, 2008). Glycoproteins with a-gal epitopes binding in vivo anti-Gal may also induce complement activation in the blood.
  • the complement cleavage peptides C3a and C5a can induce a systemic response called “systemic inflammatory response syndrome” (“SIRS”) which has adverse effects similar to septic shock, including disseminated intravascular coagulopathy “DIC” (Guo and Ward Recent Patents Anti-Infect Drug Disc 1:57, 2006; Ward, Focus on Complement 5:2, 2007).
  • SIRS systemic inflammatory response syndrome
  • Anti- Gal bound to cc-gal epitopes on glycoproteins can further decrease the half life of the glycoprotein in the circulation because of accelerated removal of the immune complexes with anti-Gal by the reticuloendothelial system.
  • This invention teaches compositions and methods for elimination of oc-gal epitopes on therapeutic glycoproteins by the use of the enzyme ⁇ -galactosidase. As shown in Figure 1, this enzyme cleaves the terminal galactose by hydrolysis of the a 1,3 linkage between the terminal galactose of the a-gal epitope and the penultimate galactose in the following reaction.
  • the carbohydrate chains of therapeutic glycoproteins will have the terminal structure Gaipi-4GlcNAc-R, or Gaipi-3GlcNAc-R, which is referred to as N- acetyllactosamine (Right structure in Figure 1).
  • ⁇ -galactosidase may be isolated from natural sources such as, but not limited to, green coffee beans, soy beans, or fungi, ⁇ -galactosidase has been obtained also in a recombinant form.
  • ⁇ -galactosidase that of the coffee bean ⁇ -galactosidase gene that was cloned and inserted into the genome of the yeast Pichia pastoris (Zhu et al., Arch Biochem Biophys 324:65, 1995).
  • This yeast produces the recombinant ⁇ -galactosidase and secretes it into the yeast growth medium.
  • the enzyme can subsequently purified from the growth medium by using on an ion exchange chromatography column as known to those skilled in the art.
  • the cloned a-galactosidase gene may be inserted into genomes of eukaryotic or prokaryotic cells comprising various gene expression systems and the recombinant ⁇ -galactosidase may be produced in its native form (i.e. without any tag), or linked to a tag such as, but not limited to,
  • the recombinant a-galactosidase may be subsequently produced in vitro and isolated from the growth medium, or in vivo in transgenic animals containing the ⁇ -galactosidase transgene and producing it in various cells.
  • a non-limiting example is production of this recombinant enzyme by the cells of the mammary glands of animals transgenic for the ⁇ -galactosidase gene and isolation of the recombinant enzyme from the milk of such transgenic animals.
  • the present invention provides methods for elimination of a-gal epitopes from therapeutic glycoproteins by incubating them with the enzyme ⁇ -galactosidase as illustrated in Figure 1.
  • a-gal epitopes on therapeutic glycoproteins may be destroyed by a-galactosidase
  • these glycoproteins have to be injected into treated individuals in a pure form, i.e. without contaminating ⁇ -galactosidase.
  • the present invention provides methods and compositions for the prevention of contamination of therapeutic glycoprotein solution by ⁇ -galactosidase used for the destruction of a-gal epitopes. Two non-limiting methods are as follows:
  • Solid-Phase ⁇ -galactosidase This invention teaches a method for the destruction of a-gal epitopes on therapeutic glycoproteins by ⁇ -galactosidase that is linked to a matrix (i.e. solid-phase ⁇ -galactosidase) such as, but not limited to, porous agarose beads, Sepharose beads or Sephacryl beads.
  • a matrix i.e. solid-phase ⁇ -galactosidase
  • natural or recombinant ⁇ -galactosidase is covalently or non-covalently linked to the porous beads or any other matrix by a variety of methods known to those skilled in the art.
  • a column is constructed from the beads containing solid-phase ⁇ -galactosidase. In order to remove a-gal epitopes from therapeutic glycoproteins, preparations of such glycoproteins in solutions are passed through the solid-phase ⁇ -galactosidase column. The flow rate through the column may vary according to the type of glycoprotein and its concentration.
  • the ⁇ -galactosidase molecules bound to the matrix of the column cleave the terminal galactose units from the a-gal epitopes, resulting in glycoproteins with N-acetyllactosamines as terminal structures on their carbohydrate chains, instead of a-gal epitopes as illustrated in Figure 2.
  • Tagging recombinant ⁇ -galactosidase with a His- His-His-His-His-His tag i.e. an oligopeptide of 6 histidines (His)e
  • the a-galactosidase-(His)6 recombinant enzyme is added to the therapeutic glycoprotein solution for the destruction of a-gal epitopes.
  • the solution is passed through a nickel-sepharose which specifically binds the enzyme through the -(His) 6 tag. This results in elimination of a-galactosidase. 2.
  • Tagging recombinant ⁇ -galactosidase with glutathione S- transferase enables subsequent removal of the enzyme with columns containing glutathione. 3.
  • Tagging recombinant ⁇ -galactosidase with the protein A enables the subsequent removal of the enzyme in columns of IgG bound to beads.
  • a-galactosidase originally cloned from the coffee beans cc-galactosidase gene (Zhu et al., Arch Biochem Biophys 324:65, 1995) is very effective in destruction of the a- gal epitopes on glycoproteins. This can be demonstrated by ELISA with various glycoproteins carrying a-gal epitopes, used as solid-phase antigens in ELISA wells.
  • glycoproteins included mouse laminin with 50 a-gal epitopes/molecule (Arumugham et al., Biochim Biophys Acta 883: 112, 1986), bovine thyroglobulin with 12 a-gal epitopes/molecule (Spiro and Bhoyroo J Biol Chem 259:9858, 1984), a-gal fetuin originating from the bovine serum fetuin, on which 9 a-gal epitopes were synthesized by the use of recombinant al,3GT (Chen et al., Glycobiology 11 :577, 2001), and a-gal BSA which is a bovine serum albumin molecule carrying 21 synthetic a-gal epitopes, each linked via a 14 carbon spacer to the protein molecule (Dextra, Reading, UK).
  • Anti-Gal IgG within human serum also readily bound to bovine thyroglobulin, mouse laminin, and to a-gal fetuin, but not to the glycoproteins lacking a-gal epitopes, including human thyroglobulin, human laminin, or fetuin ( Figure 3C).
  • Incubation with 10 Units/ml a- galactosidase resulted in the subsequent inability of anti-Gal in human serum to bind to these glycoproteins because of the destruction of a-gal epitopes on these glycoproteins by this enzyme (Figure 3D).
  • EXAMPLE 2 PREVENTION OF COMPLEMENT ACTIVATION BY a- GALACTOSIDASE MEDIATED ELIMINATION OF a-GAL EPITOPES ON GLYCOPROTEINS
  • the cloned anti-Gal hybridoma cells produce monoclonal anti-Gal IgM antibody, as well as maintain al,3GT activity synthesizing a-gal epitopes (contributed by the myeloma fusion partner).
  • a portion of anti-Gal produced by the M86 cells binds to the a-gal epitopes expressed on these anti-Gal secreting cells ( Figure 4A).
  • M86 cells are added to human serum, they are readily lysed by the complement cascade that is activated by the monoclonal anti-Gal IgM molecules bound to its antigen, (i.e. ligand) a-gal epitopes on these cells.
  • EXAMPLE 3 a-GALACTOSIDASE COLUMNS DESTROY a-GAL EPITOPES ON a- GAL BSA
  • the present invention teaches how to destroy a-gal epitopes on glycoproteins with a-gal epitopes by passing them through a column of porous beads, or of any other matrix to which the enzyme ⁇ -galactosidase is coupled covalently or bound non-covalently.
  • the present example describes a column of ⁇ -galactosidase coupled to cyanogen bromide activated agarose beads, as schematically illustrated in Figure 2, and the efficacy of such a column in destroying a-gal epitopes on glycoproteins is demonstrated.
  • Cyanogen bromide activated agarose beads also called Sepharose beads (Sigma Co. St Louis, MO) were placed in a small 1 ml column attached to a 10ml reservoir above it. The column was equilibrated with carbonate buffer, pH 8.3. Subsequently, 50ml of recombinant a- galactosidase (Zhu et al, Arch Biochim Biophys 324:65, 1995) at a concentration of 100 Units/ml in carbonate buffer, pH 8.3, were passed through the 1ml column of the cyanogens bromide activated beads. The enzyme solution was passed twice through the column to enable binding of recombinant a-galactosidase to the matrix of the beads.
  • the beads in the column were then incubated overnight in a solution of 0.2M glycine in carbonate buffer pH 8.3 in order to block the remaining reactive groups on the beads which did not bind the enzyme. Subsequently, the column was washed with 20ml carbonate buffer followed by 100ml solution of 0.5M NaCl, then with 30ml of saline.
  • a solution of 100 ⁇ g/ml a-gal BSA (bovine serum albumin carrying 21 cc-gal epitopes per molecule) in saline was applied on the ⁇ -galactosidase coupled column at a total volume of 0.5ml (i.e. 50 ⁇ g a-gal BSA) in three 166.6 ⁇ 1 aliquots loaded on the column in 10 min intervals with flow stopping in between the loadings. Subsequently, 1.5ml saline was added to the column. The effluent was collected at a total volume of 2ml. The final concentration of a-gal BSA in the effluent is 25 ⁇ / ⁇ 1.
  • Both glycoproteins were allowed to attach to the ELISA wells for 2h at 37°C followed by 20h at 4°C. After blocking of the wells with 1% BSA in PBS, the wells were washed and the monoclonal anti-Gal antibody M86 was placed for 2h in the wells, at a dilution of 1 :5.
  • This monoclonal antibody binds to a-gal epitopes and thus enables the detection of these epitopes on the enzymatically treated or untreated a-gal BSA in the ELISA wells.

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Abstract

La présente invention concerne des procédés consistant à introduire une préparation à base de glycoprotéines thérapeutiques comportant un ou plusieurs épitopes α-gal comprenant un Gala α1-3Gal terminal ou des α-galactosyles terminaux dans une colonne comportant de l'α-galactosidase couplée par des liaisons covalentes ou non-covalentes à des billes poreuses ou à un matériau particulaire afin de détruire l'épitope α-gal par élimination enzymatique du galactose terminal au moyen de l'α-galactosidase. L'invention concerne, en outre, des procédés comprenant la destruction d'un ou plusieurs épitopes α-gal comportant un Gala α1-3Gal terminal ou des α-galactosyles terminaux sur des glycoprotéines thérapeutiques en mélangeant lesdites glycoprotéines à de l'α-galactosidase marquée afin de détruire l'épitope α-gal par élimination enzymatique du galactose terminal, cela étant suivi de l'élimination de l'α-galactosidase.
PCT/US2012/038371 2011-05-18 2012-05-17 Élimination d'épitopes α-gal de glycoprotéines thérapeutiques afin d'éviter toute interaction avec l'anticorps anti-gal WO2012158932A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015007326A1 (fr) * 2013-07-18 2015-01-22 Institut D'investigació Biomèdica De Bellvitge (Idibell) Agents comprenant une partie alpha-galactosyle terminale destinée à être utilisé dans la prévention et/ou le traitement de maladies inflammatoires
US9758553B2 (en) 2008-05-30 2017-09-12 Merck Sharp & Dohme Corp. Yeast strain for the production of proteins with terminal alpha-1,3-linked galactose

Citations (2)

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Publication number Priority date Publication date Assignee Title
US5770405A (en) * 1993-09-23 1998-06-23 New England Biolabs, Inc. Isolation and composition of novel glycosidases
US20110045496A1 (en) * 2009-01-22 2011-02-24 Carlos Bosques Gal alpha 1-3gal-containing n-glycans in glycoprotein products derived from cho cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5770405A (en) * 1993-09-23 1998-06-23 New England Biolabs, Inc. Isolation and composition of novel glycosidases
US20110045496A1 (en) * 2009-01-22 2011-02-24 Carlos Bosques Gal alpha 1-3gal-containing n-glycans in glycoprotein products derived from cho cells

Non-Patent Citations (2)

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Title
KEVIN R. STONE ET AL.: 'Replacement of human anterior cruciate ligaments with pig ligaments: a model for anti-non-gal antibody response in long-term xenotransplantation' TRANSPLANTATION vol. 83, no. 2, 2007, ISSN 0041-1337 pages 211 - 219 *
SEONGSIK PARK ET AL.: 'Removal of alpha-gal epitopes from porcine aortic valve and pericardium using recombinant human alpha galactosidase A' J. KOREAN MED. SCI. vol. 24, no. 6, 2009, ISSN 1011-8934 pages 1126 - 1131 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9758553B2 (en) 2008-05-30 2017-09-12 Merck Sharp & Dohme Corp. Yeast strain for the production of proteins with terminal alpha-1,3-linked galactose
WO2015007326A1 (fr) * 2013-07-18 2015-01-22 Institut D'investigació Biomèdica De Bellvitge (Idibell) Agents comprenant une partie alpha-galactosyle terminale destinée à être utilisé dans la prévention et/ou le traitement de maladies inflammatoires

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