WO2011074965A1 - Méthodes et moyens pour contrer une activité d'un domaine fc - Google Patents

Méthodes et moyens pour contrer une activité d'un domaine fc Download PDF

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WO2011074965A1
WO2011074965A1 PCT/NL2010/050864 NL2010050864W WO2011074965A1 WO 2011074965 A1 WO2011074965 A1 WO 2011074965A1 NL 2010050864 W NL2010050864 W NL 2010050864W WO 2011074965 A1 WO2011074965 A1 WO 2011074965A1
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domain
immunoglobulin
isolated
recombinant
disulfide bonds
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PCT/NL2010/050864
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English (en)
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Theo Rispens
Robertus Carolus Aalberse
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Stichting Sanquin Bloedvoorziening
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39591—Stabilisation, fragmentation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54393—Improving reaction conditions or stability, e.g. by coating or irradiation of surface, by reduction of non-specific binding, by promotion of specific binding
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6854—Immunoglobulins
    • G01N33/6857—Antibody fragments
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • the invention relates to the fields of biology and medicine
  • Therapeutic antibodies form one of the fastest growing segments of the pharmaceutical market.
  • Specific therapeutic antibodies make up 25% of all therapeutic biologicals used, and this percentage steadily increases.
  • Nonspecific antibody preparations such as intravenous immunoglobulins (IVIG) have been used as therapeutic proteins since the early 1980's.
  • IVIG intravenous immunoglobulins
  • IVIG is a blood product used as therapeutic proteins.
  • IVIG is prepared from serum of at least 1000 donors per batch in order to obtain a wide range of antibody specificities. Preparations mainly contain
  • immunoglobulin G (IgG, approximately 97-98%) with a distribution of subclasses similar to normal human plasma.
  • IVIG was first used as replacement therapy in patients with primary antibody deficiencies such as hypogammaglobulinemia to reduce or prevent infections. It is used in a wide variety of specialties including neurology, haematology, immunology, nephrology, rheumatology and dermatology. Specific indications for the use of IVIG are secondary antibody deficiencies (chronic lymphocytic leukemia, multiple myeloma) and
  • autoimmune disease such as autoimmune thrombocytopenic purpura
  • IVIG is used at low dose for replacement therapy and mainly at high dose (up to 2 g/kg) in the treatment of autoimmune disease.
  • Therapeutic antibodies are used in the treatment of several major diseases including autoimmune, cardiovascular and infectious diseases, cancer and inflammation.
  • Examples include infliximab and adalimumab for the treatment of reumato ' id arthritis, bevacizumab for the treatment of colorectal cancer, natalizumab for the treatment of multiple sclerosis, rituximab for the treatment of non-Hodgkin lymphoma.
  • IVIG preparations and/or therapeutic antibodies include complement activation, neutrophil activation and platelet activation and are a result of undesired activity of Fc domains, often as a result of aggregation of immunoglobulins.
  • the regions of an immunoglobulin responsible for a therapeutic effect are located in the antigen binding Fab domain of an immunoglobulin.
  • Unfolded or partly unfolded IgG in IVIG is more susceptible to aggregation and (partly) unfolded IgG is also more susceptible to digestion by pepsin. Hence aggregation of IgG is at least partly prevented by addition of pepsin to IVIG.
  • Disadvantages of pepsin treatment include variation in concentration of Ig's, and since removal of pepsin is difficult, a substantial amount of added (non-human) pepsin is administered to patients, especially given the high doses of IVIG often used.
  • the present invention provides the insight that administration of an isolated or recombinant fragment of an Ig, which fragment at least comprises a CH3 domain and at most an Fc domain excluding the hinge region, diminishes adverse side effects of IVIG preparations or therapeutic antibodies.
  • the invention provides a method for counteracting an activity of an Fc domain, comprising administering to said Fc domain an isolated or recombinant part of an immunoglobulin, said part comprising at least a CH3 domain and at most an Fc domain, and allowing said part to bind said Fc domain.
  • the invention also provides a method for counteracting an activity of an Fc domain comprising administering to said Fc domain an isolated or recombinant immunoglobulin without the disulfide bonds connecting the two heavy chains, or a part thereof, said part comprising at least a CH3 domain, and allowing said isolated or recombinant
  • an activity of an Fc domain preferably is aggregation of antibodies.
  • the invention thus provides a method for counteracting aggregation of an antibody, comprising administering to said antibody an isolated or recombinant part of an immunoglobulin comprising at least a CH3 domain and at most an Fc domain, or an isolated or recombinant immunoglobulin without the disulfide bonds connecting the two heavy chains, or a part thereof, said part comprising at least a CH3 domain.
  • said part or said immunoglobulin is allowed to bind an Fc domain of said antibody.
  • the invention also provides the use of an isolated or recombinant part of an immunoglobulin comprising at least a CH3 domain and at most an Fc domain, and/or an isolated or recombinant immunoglobulin without the disulfide bonds connecting the two heavy chains, or a part thereof, said part comprising at least a CH3 domain, for counteracting aggregation of antibodies.
  • a part according to the invention, or immunoglobulin according to the invention is used for counteracting aggregation of antibodies in vitro.
  • IgG4 binds to intact IgGl and intact IgG4, both coupled to a solid phase, whereas intact IgGl does not bind to solid phase-bound intact IgG4 (Rispens et al. The Journal of Immunology, 2009, 182:4275-4281).
  • intact IgG is meant an IgG consisting of two heavy chains and two light chains connected via disulfide bonds. It was hypothesized that conformational changes in solid phase-bound IgGl trigger IgG4 binding. Therefore, soluble intact IgGl is not able to bind intact IgG4 coupled to a solid phase.
  • IgG4 Fc fragment does not bind to IgG4 in solution, because both IgG4 and the Fc fragment of IgG4 are > 99% monomeric in solution.
  • pFc'/(CH3)2 fragments isolated from Nanogam which are mainly derived from IgGl and IgG2, bind to the Fc domain of IgGl and to antibody components of IVIG in solution. Binding of the pFc'/(CH3)2 fragments to IgGl prevents aggregation induced by acid treatment (figure 7) and binding of pFc'/(CH3)2 fragments to antibody components of IVIG reduces neutrophil activation induced by IVIG (figure 9). Binding of Fc fragments to
  • an IgG4 Fc fragment is able to bind immunoglobulins other than IgG4, because immunoglobulins other than IgG4 are not mainly present as monomers in solution.
  • a part of an immunoglobulin according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains or part thereof is derived from, or comprises, IgG4, these are preferably used to counteract aggregation or binding of the Fc domain of antibodies which are not derived from IgG4, or do not comprise, IgG4.
  • a part of an immunoglobulin according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains or part thereof which is derived from, or comprises, IgG4 can be used to counteract binding of for instance IgGl, IgG2, IgA, IgM, IgD and/or IgE.
  • a part of an immunoglobulin according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains or part thereof which is derived from, or comprises, IgG4 can be used to counteract binding of for instance IgGl, IgG2, IgA, IgM, IgD and/or IgE.
  • immunoglobulin according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains or part thereof which is not derived from, or which does not comprise, IgG4 can be used to counteract binding of any immunoglobulin, including IgGl, IgG2, IgG4, IgA, IgM, IgD and/or IgE.
  • an isolated or recombinant part of an immunoglobulin comprising at least a CH3 domain and at most an Fc domain is further referred to as "a part according to the invention".
  • the term “an isolated or recombinant immunoglobulin without the disulfide bonds connecting the two heavy chains, or a part thereof, said part comprising at least a CH3 domain” is further referred to as "an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains”.
  • Said immunoglobulin without the disulfide bonds connecting the two heavy chains preferably is a reduced immunoglobulin.
  • isolated also encompasses a purified part or immunoglobulin with a purity of at least 70%, more preferably with a purity of at least 80%, more preferably with a purity of at least 90% and most preferably with a purity of at least 95%.
  • Antibodies are composed of two heavy chains and two light chains. Each light chain consists of a constant (CL) and a variable domain (VL), of which the variable domain is important for antigen binding.
  • the isotype of an antibody is determined by its heavy chain. Heavy chains consist of one variable domain (VH), important for antigen binding, and 3 or 4 constant domains (CHl-4).
  • the amino terminal side of an Ig is called F(ab')2 fragment.
  • the Fc region fragment chrystallizable region
  • the different heavy and light chains are connected via disulfide bonds in several cysteine residues. Between the CHI and CH2 domains of Ig heavy chains is a so called hinge region containing several disulfide bonds.
  • a pFc' domain usually consists of a noncovalent dimer of two peptides, each consisting of a CH3 domain and seven amino acids of the CH2 domain. CH3 domains form dimers similar to pFc'.
  • immunoglobulin without the disulfide bonds connecting the two heavy chains consists of one Ig heavy chain and one Ig light chain, disulfide bonds in the hinge region connecting two heavy chains are absent.
  • An isolated or recombinant part of an immunoglobulin according to the present invention preferably comprises at least a CH3 domain. Said part is allowed to comprise one amino acid more than a CH3 domain or to comprise two or more amino acids more than a CH3 domain.
  • An isolated or recombinant part of an immunoglobulin according to the invention comprises at most a CH3 domain bound to a CH2 domain, or an Fc domain.
  • a part according to the invention comprises at most an Fc domain.
  • Said part preferably consists of a CH3 domain optionally including at most 10 additional amino acids, as such a fragment by itself is unable to bind to most known Fc gamma receptors, if any, nor complement factor. Hence adverse effects of the fragment itself are at least in part avoided.
  • a part according to the invention comprises a truncated form of a CH3 domain which is still capable of folding Said truncated CH3 preferably comprises at least 50 amino acids, more preferably at least 60 amino acids, most preferably at least 70 amino acids.
  • a human CH3 domain typically has a length of between 90 and 120 amino acids.
  • a CH3 domain according to the invention preferably has at least 70% sequence identity with a contiguous stretch of at least 90 amino acids of the sequence
  • a part of an immunoglobulin according to the invention or an immunoglobulin according to the invention comprises a sequence which has at least 70% sequence identity to a contiguous stretch of at least 90 amino acids of the sequence
  • an immunoglobulin according to the invention or an immunoglobulin according to the invention comprises a sequence which has at least 80% sequence identity to a contiguous stretch of at least 90 amino acids of the sequence
  • said part or immunoglobulin without the disulfide bonds connecting the two heavy chains comprises a sequence of at least 90 amino acids which has at least 90% sequence identity to the sequence GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSL SLSPGK (IgGl), or
  • said part or immunoglobulin without the disulfide bonds connecting the two heavy chains comprises a sequence which has at least 95% sequence identity to a contiguous stretch of at least 90 amino acids of the sequence
  • a truncated CH3 domain comprising at least 70 amino acid residues is also suitable.
  • said part comprises an amino acid sequence with a length of at least 70 amino acids, which has at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% sequence identity with a contiguous stretch of at least 70 amino acids of any of the above mentioned amino acid sequences.
  • a part of an immunoglobulin according to the invention or immunoglobulin (without the disulfide bonds connecting the two heavy chains) according to the invention is preferably an isolated part of an immunoglobulin or an isolated immunoglobulin without the disulfide bonds connecting the two heavy chains, for example isolated from IVIG or plasma. More preferably, however, said part or immunoglobulin is a recombinant part of an immunoglobulin according to the invention.
  • immunoglobulin or a recombinant immunoglobulin without the disulfide bonds connecting the two heavy chains can be produced by methods well known in the art, such as for instance production in a eukaryotic cell such as for instance HEK293 cells, and need no further explanation.
  • An immunoglobulin without the disulfide bonds connecting the two heavy chains is for instance a reduced immunoglobulin, preferably IgGl or IgG2.
  • immunoglobulin can be isolated from for example IVIG or plasma by methods well known in the art, such as for example affinity chromatography.
  • a reduced immunoglobulin or part thereof can also be obtained by isolating or
  • a reduced immunoglobulin thus comprises at most one heavy chain and one light chain.
  • a reduced immunoglobulin consists of a heavy chain, as heavy chain-light chain disulfide bonds are often reduced as well under similar conditions.
  • a part of an immunoglobulin or immunoglobulin without the disulfide bonds connecting the two heavy chains, according to the invention is for instance of human or non-human, preferably murine, origin.
  • an immunoglobulin is preferably composed of a single chain domain or Fc fragment, because such single chain domain or fragment is particularly suitable for binding to the Fc domain of antibodies. Therefore, an immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention and a part according to the invention preferably consist of a single chain.
  • the term "isolated or recombinant immunoglobulin without the disulfide bonds connecting the two heavy chains, or part thereof, said part comprising at least a CH3 domain” encompasses a reduced immunoglobulin or part thereof as well as an immunoglobulin or part thereof in which cysteines that are normally able to form disulfide bonds between the heavy chains are mutated. Such a mutation is for instance replacement by any other amino acid or deletion. Thus the disulfide bonds are absent for instance as a result of reduction, or enzymatic cleavage, or modification, deletion or masking of cysteine residues that are normally able to form said disulfide bonds.
  • adverse side effects of an Ig molecule or a collection of Ig molecules are diminished by administering to said Ig molecule or collection of Ig molecules an isolated or recombinant part of an immunoglobulin comprising at least a CH3 domain and at most an Fc domain.
  • the invention also provides a method for diminishing adverse side effects of an Ig molecule or a collection of Ig molecules by administering to said Ig molecule or collection of Ig molecules an isolated or recombinant
  • immunoglobulin without the disulfide bonds connecting the two heavy chains, or a part thereof, said part comprising at least a CH3 domain.
  • Said Ig molecules or a collection of Ig molecules preferably comprise IVIG and/or therapeutic antibodies and/or functional equivalents thereof.
  • An isolated or recombinant part of an immunoglobulin according to the invention or immunoglobulin (without the disulfide bonds connecting the two heavy chains) according to the invention is for instance administered to an individual at the same time that an Ig molecule or a collection of Ig molecules is administered to said individual to reduce undesired activity of an Fc domain in vivo.
  • an isolated or recombinant part of an immunoglobulin or immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains is administered to an Ig molecule or collection of Ig molecules before administration of said Ig molecule or collection of Ig molecules to an individual.
  • IVIG is treated with a part according to the invention or immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention in vitro, after which the resulting IVIG is particularly suitable for use in vivo as a result of reduced side effects.
  • a collection of Ig molecules such as IVIG does not need to be treated with pepsin and disadvantages of pepsin treatment such as variation in concentration of Ig's, and addition of (non-human) pepsin to the product, such as for instance an immune reaction against the non-human pepsin, are prevented. Therefore, in a preferred embodiment, in a method according to the invention for diminishing adverse side effects of IVIG and/or therapeutic antibodies and/or functional equivalents thereof, a part according to the invention , or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains, are administered to said IVIG and/or therapeutic antibodies and/or functional equivalents thereof which have not been treated with a protease.
  • said IVIG and/or therapeutic antibodies and/or functional equivalents thereof have not been treated with pepsin. Then, the disadvantages of treatment with a protease and/or pepsin, such as an immune reaction against non-human protease and/or pepsin are prevented.
  • immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains, added to an Ig molecule or collection of Ig molecules is preferably between 0.1 and 5.0% (weight percentage) of said Ig molecule or collection of Ig molecules.
  • An Ig molecule or collection of Ig molecules is preferably IVIG and/or therapeutic antibodies and/or functional equivalents thereof. More preferably the amount of a part according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains added to an Ig molecule or collection of Ig molecules is between 0.2 and 2.5% (weight percentage) of said Ig molecule or collection of Ig molecules.
  • the amount of a part according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains added to an Ig molecule or collection of Ig molecules is between 0.5 and 1.0% (weight percentage) of said Ig molecule or collection of Ig molecules.
  • the amount of a part according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains added is between 0.5 and 1.0 % of IVIG and/or therapeutic antibodies and/or functional equivalents thereof.
  • Therapeutic antibodies treated with a method according to the invention are preferably murine, chimeric, humanized or human antibodies. They can be monoclonal or polyclonal therapeutic antibodies.
  • a functional equivalent of an antibody is defined herein as a compound which has at least one same property as said antibody in kind, not necessarily in amount. Said functional equivalent is capable of binding the same antigen as said antibody, albeit not necessarily to the same extent.
  • a functional equivalent of an antibody preferably comprises a single domain antibody, a single chain antibody, a nanobody, a unibody or a single chain variable fragment (scFv). Nanobodies are the smallest fragments of naturally occurring heavy-chain antibodies that have evolved to be fully functional in the absence of a light chain. A unibody is a stable, smaller antibody format with an anticipated longer therapeutic window.
  • a functional equivalent of an antibody is also for instance produced by altering an antibody such that at least one property - preferably an antigen- binding property - of the resulting compound is essentially the same in kind, not necessarily in amount. This is done in many ways, for instance through conservative amino acid substitution, whereby an amino acid residue is substituted by another residue with generally similar properties (size, hydrophobicity, etc), such that the overall functioning is likely not to be seriously affected.
  • Adverse side-effects of a therapeutic Ig molecule or a collection of Ig molecules (such as IVIG) that are preferably counteracted by a method according to the invention comprise aggregation, a binding activity, a complement activation activity, a neutrophil activation activity and/or a platelet activation activity. Aggregation of Ig's for instance occurs when Fc domains of immunoglobulins are misfolded. Aggregated immunoglobulins are particularly potent in activating complement and neutrophils, and therefore aggregation of immunoglobulins is preferably prevented. As described above, aggregation of immunoglobulins is currently counteracted by pepsin treatment, however according to the present invention pepsin treatment is no longer necessary. Now, an isolated or recombinant part according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains can be used to diminish adverse side-effects.
  • Binding activity of an antibody component of IVIG and/or a therapeutic antibody and/or functional equivalent thereof for instance comprises binding of the Fc domain of said antibody component, therapeutic antibody or functional equivalent to Fc domains of other immunoglobulins, for instance derived from IVIG and/or therapeutic antibodies and/or functional equivalents thereof or immunoglobulins present in an individual. This binding will interfere with normal functioning of an immunoglobulin, e.g. binding to Fc receptors on immune cells with their Fc domain. Binding of an Fc domain of an immunoglobulin to Fc domains of other immunoglobulins is therefore preferably prevented using a part according to the invention or an
  • a part according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains preferably comprises a sequence which has at least 70%, more preferably at least 80%, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% sequence identity to a contiguous stretch of at least 90 amino acids of the sequence
  • an isolated or recombinant immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention preferably is a reduced immunoglobulin.
  • the Fc region of antigen-bound immunoglobulins is able to bind and activate the first component (Cl) of the complement system.
  • the complement system is a cascade of related proteins that is an effector of the immune system causing for instance lysis of cells, bacteria and viruses or formation of many biologically active complement fragments that act as anaphylatoxins, opsonins or chemotactic factors.
  • Activation of the complement system by antigen-bound immunoglobulins is known as the classical complement activation pathway.
  • the classical activation pathway can be activated by IgG and IgM antibodies, but not IgA, IgD and IgE.
  • Complement activation after administration of IVIG and/or therapeutic antibodies and/or functional equivalents thereof is disadvantageous because it can contribute to disease progression when the complement system is activated by antibodies bound to self-antigens or antibodies bound to a host's own cells or tissue.
  • Immunoglobulins have also been demonstrated to be able to regulate complement activation by binding activated complement fragments.
  • unwanted activation of the complement system after administration of IVIG and/or therapeutic antibodies and/or equivalents thereof to an individual is counteracted, because a part, or immunoglobulin without the disulfide bonds connecting the two heavy chains, according to the invention binds to an Fc domain and thus diminishes, delays or prevents its complement activating ability.
  • US patent US 7,049,282 describes a method for blocking the biochemical function of the classical complement pathway.
  • small peptides derived from the CH3 domain (10 and 17 amino acids long) are used to block the complement pathway by preventing binding of complement components to their targets after
  • complement activation has occurred.
  • the present invention discloses a method wherein complement activation is counteracted. Hence, complement activation does not occur, or occurs to a significantly lesser extent as compared to US 7,049,282.
  • a part according to the invention or immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention comprising at least 70 amino acids, it is possible to diminish or prevent activation of complement components by binding to an Ig Fc domain, leaving this domain unable to bind to complement factors. Therefore, the present invention provides a method which is preferred over US 7,049,282. It is more preferred to prevent or diminish activation of complement components, than to block the complement pathway after activation has already taken place. With a method according to the invention, counteracting unwanted effects of complement activation is more effective, because intervention occurs at an earlier stage in the process of complement aggregation.
  • pepsin treatment of IVIG was not known to prevent other activities of an immunoglobulin Fc domain, such as neutrophil activation or platelet activation.
  • an immunoglobulin Fc domain such as neutrophil activation or platelet activation.
  • the present inventors have identified the mechanism of action of pepsin treatment, and found that a part according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains is capable of counteracting aggregation of immunoglobulins and complement activation by binding to an Fc domain, it is now also possible to counteract such other activities of an Fc domain with a method according to the invention.
  • Activation of neutrophils can be triggered by binding of
  • immunoglobulins in particular IgG, to Fc receptors.
  • Neutrophils constitutively express FcyRIIa (CD32) and FcyRIIIb (CD16) on their cell membrane. Binding of immunoglobulins to neutrophils results in engulfment of the foreign agent or cell bound to the Ig.
  • ligation of unbound immunoglobulins can result in release of intracellular components and contribute to disease progression in inflammatory or auto-immune disorders.
  • unwanted activation of neutrophils after administration of IVIG and/or therapeutic antibodies and/or equivalents thereof to an individual is therefore preferably counteracted because a part according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains is capable of binding an Fc domain, thereby at least in part preventing binding of said Fc domain to a neutrophil Fc receptor.
  • Binding of the Fc domain of IgG to Fc receptors expressed on platelets leads to platelet activation and subsequent platelet adhesion and aggregation, resulting in an increased risk of for instance thrombosis.
  • Intravascular thrombosis has been reported to be a side-effect of IVIG treatment.
  • unwanted activation of platelets and subsequent increased risk of thrombosis after administration of IVIG and/or therapeutic antibodies and/or equivalents thereof to an individual is therefore also preferably counteracted because a part according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains is capable of binding an Fc domain thereby at least in part preventing binding of said Fc domain to a platelet Fc receptor.
  • the invention provides a method for counteracting an activity of an Fc domain, comprising administering to said Fc domain an isolated or recombinant part of an immunoglobulin comprising at least a CH3 domain and at most an Fc domain wherein said activity of said Fc domain comprises aggregation, a binding activity, a complement activation activity, a neutrophil activation activity and/or an anti platelet activation activity.
  • the invention also provides a method for counteracting an activity of an Fc domain, comprising administering to said Fc domain an isolated or recombinant immunoglobulin without the disulfide bonds connecting the two heavy chains, or a part thereof, said part comprising at least a CH3 domain wherein said activity of said Fc domain comprises aggregation, a binding activity, a complement activation activity, a neutrophil activation activity and/or an anti platelet activation activity.
  • said activity of said Fc domain comprises an interaction of said Fc domain with the Fc domain of an antibody or with a complement factor or with an immune cell or with a platelet.
  • a part according to the invention or an immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention is capable of binding to an Fc domain of a therapeutic antibody or antibody component of an IVIG preparation and thus diminishes, delays or prevents aggregation of Ig's, or binding of said Fc domain to a complement factor, an immune cell or a platelet.
  • a complement factor is defined as a protein of the complement system.
  • Complement factors are constituents of the antibody- dependent classical activation pathway.
  • Cl is a complex of three distinct proteins Clq, Clr and Cls.
  • Subtypes as for example C3a are fragments formed as a result of proteolytic cleavage of complement factor and are also encompassed by the term "complement factor”.
  • Activation of the classical complement pathway starts with activation of the Cl complex.
  • the end product of the complement cascade responsible for lysis of cells, bacteria or viruses is a complex consisting of complement factors C5b, C6, C7, C8 and C9.
  • An immune cell is defined as a cell of the immune system and is for instance a neutrophil, a basophil, an eosinophil, a monocyte, a macrophage, a dendritic cell, a T lymphocyte or a B lymphocyte.
  • a part according to the invention for diminishing adverse side effects of an Ig molecule or a collection of Ig molecules, preferably for diminishing adverse side effects of IVIG and/or therapeutic antibodies and/or functional equivalents thereof.
  • the invention also provides a use of an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains, for diminishing adverse side effects of an Ig molecule or a collection of Ig molecules, preferably for diminishing adverse side effects of IVIG and/or therapeutic antibodies and/or functional equivalents thereof.
  • Said adverse side- effects preferably comprise aggregation, a binding activity, a complement activation activity, a neutrophil activation activity and/or an anti platelet activation activity, as described above.
  • immunoglobulin according to the invention without the disulfide bond connecting the two heavy chains is used for diminishing adverse side effects of IVIG and/or therapeutic antibodies and/or functional equivalents thereof in vitro.
  • immunoglobulin according to the invention without the disulfide bond connecting the two heavy chains is used for diminishing adverse side effects of IVIG and/or therapeutic antibodies and/or functional equivalents thereof, whereby said part or immunoglobulin is added to said IVIG and/or therapeutic antibodies and/or functional equivalents thereof before administration to an individual.
  • a part according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains can be derived from any class or isotype; IgA (IgAl or IgA2), IgD, IgE, IgG (IgGl, IgG2, IgG3 or IgG4) or IgM.
  • said part according to the invention is derived from IgG.
  • said immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention comprises IgG.
  • immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains, originating from IgG has a stronger binding capacity for an Fc domain than IgA, IgD, IgE or IgM.
  • a part according to the invention or immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains is preferably derived from IgG, more preferably IgGl, IgG2 or IgG4.
  • the invention further provides the insight that a part according to the invention originating from IgG4 has the strongest binding capacity for an Fc domain. Therefore, the invention most preferably provides a method for counteracting aggregation of antibodies, comprising administering to said antibodies an isolated or recombinant part of an IgG4 immunoglobulin comprising at least a CH3 domain and at most an Fc domain.
  • part of IgG4 is usually present in the form of non- covalent dimers, of which each monomer consists of one heavy chain and one light chain, whereas IgGl and IgG2 are present in the form of a covalent dimer, consisting of two heavy chains and two light chains.
  • part of IgG4 is in solution usually present as an immunoglobulin without the disulfide bonds connecting the two heavy chains. Counteracting aggregation of antibodies in solution is thus preferably carried out using an immunoglobulin without the disulfide bonds connecting the two heavy chains which comprises IgGl or IgG2, since part of IgG4 is already present in the from of an
  • an immunoglobulin without the disulfide bonds connecting the two heavy chains preferably comprises IgGl or IgG2.
  • the invention also provides a method for counteracting an activity of an Fc domain, preferably aggregation of antibodies, comprising administering to said Fc domain an isolated or recombinant IgGl or IgG2 immunoglobulin without the disulfide bonds connecting the two heavy chains, or a part of an immunoglobulin without the disulfide bonds connecting the two heavy chains derived from IgG4, IgGl or IgG2, said part comprising at least a CH3 domain.
  • Such part of IgG4, IgGl or IgG2 or IgGl or IgG2 without the disulfide bonds connecting the two heavy chains is particularly suitable for diminishing side effects of therapeutic antibodies.
  • a method comprising diminishing adverse side effects of an Ig molecule or a collection of Ig molecules, preferably IVIG and/or therapeutic antibodies and/or functional equivalents thereof, by administering to said Ig molecule or collection of Ig molecules an isolated or recombinant part of an IgG4, IgGl or IgG2 immunoglobulin comprising at least a CH3 domain and at most an Fc domain.
  • Also provided is a method comprising diminishing adverse side effects of an Ig molecule or a collection of Ig molecules, preferably IVIG and/or therapeutic antibodies and/or functional equivalents thereof, by administering to said Ig molecule or collection of Ig molecules an isolated or recombinant IgGl or IgG2 immunoglobulin without the disulfide bonds connecting the two heavy chains, or a part thereof, said part comprising at least a CH3 domain.
  • said part or IgG without the disulfide bonds connecting the two heavy chains is administered to said therapeutic antibodies in vitro.
  • the present invention further provides a use of an IgG4, IgGl or IgG2 part according to the invention for diminishing adverse side effects of an Ig molecule or a collection of Ig molecules, preferably for diminishing adverse side effects of IVIG and/or therapeutic antibodies and/or functional equivalents thereof. Also provided is a use of an IgGl or IgG2 immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains for diminishing adverse side effects of an Ig molecule or a collection of Ig molecules, preferably for diminishing side effects of IVIG and/or therapeutic antibodies and/or functional equivalents thereof.
  • the invention further provides a method for diminishing adverse side effects of an Ig molecule or a collection of Ig molecules, preferably IVIG and/or therapeutic antibodies and/or functional equivalents thereof, comprising administering said Ig molecule or collection of Ig molecules to an individual, and administering a part according to the invention and/or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains to said individual.
  • a part according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains is capable of binding Fc domains of therapeutic antibodies in vivo, thereby reducing unwanted binding activity of said Fc domains, and thus diminishing adverse side effects of an Ig molecule or a collection of Ig molecules.
  • a part of an immunoglobulin or immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention is for instance administered to an Ig molecule or collection of Ig molecules at most 1 hour before administration of said Ig molecule or collection of molecules to an individual.
  • a part according to the invention or an immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention is administered to an individual at the same time that an Ig molecule or a collection of Ig molecules is administered to said individual.
  • a part according to the invention or an immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention is administered to an individual at most 1 hour after administration of an Ig molecule or collection of Ig molecules to said individual.
  • Administration of said Ig molecule or collection of molecules and a part according to the invention or immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains to an individual can be performed via any administration route known in the art, preferably via parenteral administration, more preferably via intravenous injection or infusion.
  • therapeutic antibodies including IVIG
  • a part according to the invention or a part according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains according to the invention is administered to an individual and is capable of binding therapeutic antibodies in vivo.
  • a kit of parts comprising a part according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains, and an Ig molecule or a collection of Ig molecules, preferably IVIG, is also herewith provided, as well as a kit of parts comprising a part according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains, and a therapeutic antibody or functional equivalent thereof.
  • IVIG and/or therapeutic antibodies and/or equivalents thereof are for instance treated with an isolated or recombinant part or an immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention.
  • said part or immunoglobulin without the disulfide bonds connecting the two heavy chains preferably a reduced immunoglobulin, according to the invention comprises (part of) IgG
  • said immunoglobulin without the disulfide bonds connecting the two heavy chains comprises IgGl or IgG2 and said part comprises part of IgG4, IgGl or IgG2.
  • a part according to the invention or an immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention is used in order to improve an assay wherein antibodies are used.
  • antibody-based assays exist for the detection of protein in a sample; these assays for instance include enzyme-linked immunosorbent assays (ELISA), radio-immuno assays (RIA), Western Blot assays and immunohistochemical staining assays. These assays are well known in the art. Shortly, with an ELISA a protein (antigen) can be detected or quantified in a sample.
  • the technique comprises applying a sample on a solid support, for example a 96-well plate, and immobilizing the protein to be detected or quantified on the surface of the solid support.
  • a capture antibody is fixated on the surface of a solid support after which a sample containing the protein to be detected or quantified is applied to the immobilized capture antibody allowing the protein of interest to bind.
  • Non- binding proteins are than washed away. Consequently a specific antibody conjugated to an enzyme or a primary antibody followed by a secondary antibody conjugated to an enzyme is added to the solid support. Finally the enzyme's substrate is added and converted to a detectable signal, for example a colored substance or fluorescence.
  • RIA is a competition assay. A known quantity of protein (antigen) is radioactively labeled. Subsequently, a known quantity of antibody is added to the antigen mixture. A sample of interest is added to the mixture, which competes with the radioactively labeled antigen. Finally antigen bound to antibody is separated from free antigen after which radioactivity can be measured. The radioactivity detected is a measure (reversely correlated) for the amount of protein in the sample.
  • proteins are separated based on size using gel electrophoresis. Proteins are transferred to a membrane, for example nitrocellulose or PVDF. Proteins on the membrane can be detected by adding specific antibody conjugated to an enzyme or a primary antibody followed by a secondary antibody conjugated to an enzyme and subsequently the enzyme's substrate which is converted to a detectable signal.
  • a membrane for example nitrocellulose or PVDF. Proteins on the membrane can be detected by adding specific antibody conjugated to an enzyme or a primary antibody followed by a secondary antibody conjugated to an enzyme and subsequently the enzyme's substrate which is converted to a detectable signal.
  • immunohistochemical staining assay proteins are detected in fixed paraffin embedded tissue sections or cyrosections. Detection of proteins is performed as described in the above mentioned assays via specific antibody conjugated to an enzyme or a primary antibody followed by a secondary antibody conjugated to an enzyme.
  • ELISA enzyme-linked immunosorbent assay
  • RIA Western blot assay
  • immunohistochemical staining assay are known in the art.
  • Non-specific background signaling is for instance caused by binding of enzyme conjugated antibodies to proteins or materials other than the protein for which said antibody has specificity.
  • this non-specific binding is reduced by counteracting binding via an Fc domain of (enzyme conjugated) antibodies to proteins or materials other than the protein for which said antibody has specificity.
  • the invention thus provides a use of an isolated or recombinant part of an immunoglobulin comprising at least a CH3 domain and at most an Fc domain, for reducing non-specific background signaling in an assay which assay comprises the use of antibodies.
  • Said immunoglobulin without the disulfide bonds connecting the two heavy chains preferably is a reduced immunoglobulin.
  • the invention further provides a method for reducing non-specific background signaling in an assay which assay comprises the use of antibodies, comprising administering to said antibodies before or during the performance of said assay an isolated or recombinant part of an in immunoglobulin comprising at least a CH3 domain and at most an Fc domain.
  • a secondary labeled antibody is used for detection of binding of a primary antibody to an antigen
  • a part according to the invention or an immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains is preferably added to said secondary antibody before said secondary antibody is added to said primary antibody- antigen complex.
  • an assay comprising the use of antibodies, further comprises the use of an antigen and a part according to the invention immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains or part thereof is administered to an antibody before said antibody is brought into contact with an antigen or an antibody-antigen complex.
  • said assay comprises an enzyme-linked immunosorbent assay (ELISA) or a radio- immuno assay (RIA) or a Western Blot assay or an immunohistochemical staining assay.
  • IgG4 Fc binding to other Ig's limits the possibilities of detecting Ag- specific IgG4.
  • specific and non-specific binding of IgG4 is not easily distinguished.
  • IgG4 Fc binding to immunoglobulins is now counteracted using parts according to the invention or immunoglobulins without the disulfide bonds connecting the two heavy chains according to the invention, reducing false positive results.
  • a part according to the invention or an immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention preferably contains a single chain CH3 domain, optionally comprising additional amino acids, or a single Fc fragment in order to facilitate binding to the Fc domain of a therapeutic antibody or antibody component of an IVIG preparation. Therefore, an immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention preferably comprises part of an immunoglobulin without the disulfide bonds connecting two heavy chains.
  • a part according to the invention or an immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention comprises a single chain part of IgG
  • said immunoglobulin without the disulfide bonds connecting the two heavy chains comprises a single chain part of IgGl or IgG2
  • said part of an immunoglobulin without the disulfide bonds connecting the two heavy chains comprises a single chain part of IgGl or IgG2
  • immunoglobulin without the disulfide bonds connecting the two heavy chains comprises a single chain part of IgG4, IgGl or IgG2, said part comprising at least a CH3 domain.
  • a method according to the invention is used for prolonging the storage life or increasing the maximum storage temperature of an Ig molecule or a collection of Ig molecules, preferably IVIG and/or therapeutic antibodies and/or equivalents thereof. Therefore, the invention provides a method for prolonging the storage life or increasing the maximum storage temperature of an Ig molecule or a collection of Ig molecules, preferably IVIG and/or therapeutic antibodies or equivalents thereof, comprising administering to said Ig molecule or collection of Ig molecules an isolated or recombinant part of an immunoglobulin comprising at least a CH3 domain and at most an Fc domain, or an isolated or recombinant
  • immunoglobulin without the disulfide bonds connecting the two heavy chains or a part thereof, said part comprising at least a CH3 domain, the method further comprising storing said Ig molecule or collection of Ig molecules.
  • Said immunoglobulin without the disulfide bonds connecting the two heavy chains preferably is a reduced immunoglobulin.
  • Aggregation of immunoglobulins limits the storage life of preparations containing immunoglobulins. During storage unfolding or misfolding of immunoglobulins occurs followed by aggregation of said immunoglobulins. Aggregation increases with time when immunoglobulins are stored. In addition, aggregation of immunoglobulins depends on storage temperature. Both Fab domains and Fc domains are able to undergo aggregation. Aggregation of Fc domains is often caused by low pH. A low pH ( ⁇ 3) is for instance used during affinity purification of antibodies, and various IVIG preparations are produced at a pH of around 4. Thus, aggregation of Fc domains during or after production of therapeutic antibodies or IVIG is potentially a substantial problem.
  • the storage life of a collection of Ig molecules is prolonged and/or the maximum storage temperature of a collection of Ig molecules is increased.
  • a part according to the invention or an immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention to an Fc domain of immunoglobulins aggregation of these immunoglobulins is inhibited and thus a collection of Ig molecules can be stored for a longer period of time and/or at a higher temperature before unacceptable degree of aggregated Ig molecules is reached.
  • an Ig molecule or a collection of Ig molecules preferably IVIG and/or therapeutic antibodies or equivalents thereof, comprising administering to said Ig molecule or collection of Ig molecules an isolated or recombinant part of an immunoglobulin comprising at least a CH3 domain and at most an Fc domain, the method further comprising storing said Ig molecule or collection of Ig molecules.
  • an immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention for prolonging the storage life or increasing the maximum storage temperature of an Ig molecule or a collection of Ig molecules, preferably IVIG and/or therapeutic antibodies or functional equivalents thereof.
  • a part according to the invention is used for prolonging the storage life or increasing the maximum storage temperature according to the invention of an Ig molecule or a collection of Ig molecules, preferably IVIG and/or therapeutic antibodies or equivalents thereof.
  • a part according to the invention or immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention is preferably composed of a single chain CH3 domain, optionally comprising additional amino acids, or of a single Fc fragment facilitating binding to the Fc domain of a therapeutic antibody or antibody component of an IVIG preparation.
  • a part according to the invention or immunoglobulin according to the invention without the disulfide bonds connecting the two heavy chains comprises (part of) IgG
  • said isolated or recombinant immunoglobulin without the disulfide bonds connecting the two heavy chains comprises IgGl or IgG2
  • said isolated or recombinant part of an immunoglobulin without the disulfide bonds connecting the two heavy chains comprises IgG4, IgGl or IgG2.
  • the invention further provides a part according to the invention or an immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention, for use as a medicament.
  • immunoglobulin without the disulfide bonds connecting the two heavy chains preferably is a reduced immunoglobulin.
  • disorders related to an activity of an Fc domain such as aggregation, a binding activity, a complement activation activity, a neutrophil activation activity and/or a platelet activation activity, are counteracted.
  • said disorder is associated with an interaction of an Fc domain, present within an individual, with the Fc domain of an antibody or with a complement factor or with an immune cell or with a platelet.
  • a part according to the invention or an immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention is capable of binding an Fc domain.
  • an isolated or recombinant part of an immunoglobulin comprising at least a CH3 domain and at most an Fc domain, for the preparation of a medicament for counteracting and/or at least in part preventing a disorder associated with an interaction of an Fc domain, present within an individual, with an Fc domain of an antibody or with a complement factor or with an immune cell or with a platelet.
  • a use of an isolated or recombinant part of an immunoglobulin comprising at least a CH3 domain and at most an Fc domain, for the preparation of a medicament for counteracting and/or at least in part preventing a disorder associated with an interaction of an Fc domain, present within an individual, with an Fc domain of an antibody or with a complement factor or with an immune cell or with a platelet.
  • immunoglobulin without the disulfide bonds connecting the two heavy chains, or a part thereof, said part comprising at least a CH3 domain, for the preparation of a medicament for counteracting and/or at least in part preventing a disorder associated with an interaction of an Fc domain, present within an individual, with the Fc domain of an antibody or with a complement factor or with an immune cell or with a platelet.
  • a method for counteracting and/or at least in part preventing a disorder associated with an Fc domain activity within an individual comprising administering to said individual a therapeutically effective amount of an isolated or recombinant part of an immunoglobulin comprising at least a CH3 domain and at most an Fc domain, or a therapeutically effective amount of an isolated or recombinant
  • said method comprises counteracting and/or at least in part preventing a disorder associated with an interaction of an Fc domain, present within an individual, with an Fc domain of an antibody or with a complement factor or with an immune cell or with a platelet.
  • said individual is firstly diagnosed with a disorder associated with an Fc domain activity.
  • Said disorder associated with an interaction of an Fc domain, present within an individual, with an Fc domain of an antibody or with a complement factor or with an immune cell or with a platelet preferably comprises an autoimmune disease or thrombosis.
  • autoimmune diseases are rheumatoid arthritis, Crohn's disease and Colitis ulcerosa.
  • Rheumatoid arthritis is a chronic inflammatory disease, generally considered an auto-immune disease. It can affect many tissues or organs, but mostly results in destruction of joints. In rheumatoid arthritis immune complexes are formed in affected joints inducing inflammatory responses. In the serum of rheumatoid arthritis patients (auto)antibodies directed against Fc domains of antibodies are found. These are called rheumatoid factors (RF) and are in particular directed against the Fc domain of IgG. RF can be of any immunoglobulin isotype, e.g. IgM, IgA, or IgG. The consequently formed immune complexes contribute to disease progression.
  • binding of IgG Fc region to RF is for instance counteracted.
  • the onset, progression and/or adverse effects of rheumatoid arthritis are diminished.
  • Crohn's disease and Colitis ulcerosa are inflammatory diseases of the intestines, generally considered auto-immune diseases. Crohn's disease may cause abdominal pain, diarrhea, vomiting and weight loss, but may also cause symptoms outside of the gastrointestinal tract. The symptoms of Colitis ulcerosa are similar to those of Crohn's disease. Auto-antibodies are found both in individuals suffering from Crohn's disease and in individuals suffering from Colitis ulcerosa. With a part according to the invention or an immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention unwanted effects of such antibodies are for instance counteracted. As a result, the onset, progression and/or adverse effects of Crohn's disease or Colitis ulcerosa are diminished.
  • Thrombosis is defined as the formation of a blood clot, frequently resulting in vessel occlusion. Thrombus formation usually occurs after vessel injury as a result of aggregation of blood factors, primarily platelets and fibrin with entrapment of cellular elements. With a part according to the invention or an immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention binding of an IgG Fc region to an Fc receptor expressed on a platelet is counteracted, since said part and/or said
  • immunoglobulin without the disulfide bonds connecting the two heavy chains according to the invention compete with an Fc receptor expressed on a platelet for binding to an Fc domain of IgG and therefore platelet activation and aggregation is diminished.
  • a method, a use, a part of an immunoglobulin, and/or an immunoglobulin without the disulfide bonds connecting the two heavy chains, or a part thereof according to the invention wherein said disorder comprises an autoimmune disease or thrombosis.
  • Said autoimmune disease preferably comprises rheumatoid arthritis, Crohn's disease or Colitis ulcerosa.
  • the invention further provides a combination of an Ig molecule or a collection of Ig molecules, preferably IVIG and/or therapeutic antibodies or functional equivalents thereof, and an isolated or recombinant part of an immunoglobulin comprising at least a CH3 domain and at most an Fc domain, for use as a medicament.
  • such combination according to the invention is provided for counteracting and/or at least in part preventing a disorder associated with an interaction of an Fc domain, present within an individual, with an Fc domain of an antibody or with a complement factor or with an immune cell or with a platelet.
  • a combination according to the invention adverse effects of Fc domain interactions, such as aggregation, a binding activity, a complement activation activity, a neutrophil activation activity and/or an anti platelet activation activity, are diminished.
  • unwanted activation of the complement system for instance leading to disease progression in autoimmune disease, unwanted activation of neutrophils, for instance leading to disease progression in inflammatory disorders, or unwanted activation of platelets, for instance leading to thrombosis or increased risk of thrombosis is diminished or even prevented.
  • the invention further provides a use of a combination of an Ig molecule or a collection of Ig molecules and an isolated or recombinant part of an immunoglobulin comprising at least a CH3 domain and at most an Fc domain, for the preparation of a medicament for counteracting and/or at least in part preventing a disorder associated with an interaction of an Fc domain, present within an individual, with an Fc domain of an antibody or with a complement factor or with an immune cell or with a platelet.
  • said collection of Ig molecules preferably comprises IVIG and/or therapeutic antibodies or functional equivalents thereof and said disorder preferably comprises an autoimmune disease, most preferably rheumatoid arthritis, Crohn's disease or Colitis ulcerosa, or thrombosis.
  • a combination according to the invention for use as a medicament.
  • Yet another embodiment provides a use of a combination according to the invention for the preparation of a medicament for counteracting and/or at least in part preventing a disorder associated with an interaction of an Fc domain, present within an individual, with an Fc domain of an antibody or with a complement factor or with an immune cell or with a platelet.
  • said disorder preferably comprises an autoimmune disease, such as rheumatoid arthritis, Crohn's disease or Colitis ulcerosa, or thrombosis.
  • Said Ig molecule or collection of Ig molecules preferably comprises IVIG and/or therapeutic antibodies or functional equivalents thereof.
  • Figure 1 Inhibition of binding of 125 I-labeled IgG4 to IgGl Separose by IgG4 (squares), IMIG (circles), and IVIG (triangles). A) inhibition vs ng total IgG, B) inhibition vs ng IgG4. C) Schematic representation of the inhibition assay.
  • Figure 2. A) Inhibition of IgG4 binding to IgGl Sepaharose by IVIG and
  • IMIG before and after protein G affinity chromatography 700 ng/test.
  • Figure 6 Binding of radiolabeled pFc' isolated from Nanogam IVIG to IgG coupled to Sepharose.
  • Figure 7. Acid-induced aggregation of 5 mg/mL IgGl (30 min./pH 2.5 followed by neutralization to pH 7). IgGl (solid line); acid-shocked IgGl (fat line); acid- shocked IgGl in the presence of 0.1 mg/mL (dashed line) or 0.5 mg/mL (dotted line) IVIG pFc7(CH3) 2 .
  • IgG4 Fc binding to IgGl Sepharose by A) IgGl and reduced/alkylated IgGl; B) IgG2 and reduced/alkylated IgG2; C) IgGl Fc, reduced/alkylated IgGl Fc, hingeless IgGl Fc; D) IgG4 Fc, reduced/alkylated IgG4 Fc, hingeless IgG4 Fc.
  • Figure 9 Elastase release after stimulation of neutrophils with IVIG which was not treated with pepsin, IVIG which was not treated with pepsin but which was treated with pFc'/(CH3)2 fragments, and Nanogam, a finalized IVIG product (pepsin- treated).
  • IVIG contains fragments that inhibit IgG4 Fc-Fc binding
  • FIG 1 inhibition of IgG4 binding to IgGl-Sepharose by IgG4 and two polyclonal human IgG products is shown. Without inhibitor, IgG4 or IgG4 Fc binds to IgGl, as shown previously (1).
  • Figure 1A it is shown that adding non-labeled IgG4 inhibits the binding of radiolabeled IgG4 in a dose-dependent manner.
  • IVIG intravenous immunoglobulin
  • IMIG intramuscular immunoglobulin
  • This additional inhibitory component could be separated from IgG using a protein G column ( Figure 2A).
  • IVIG the fraction that does not bind to protein G contains besides 75 and 100 kD fragments also several smaller fragments of between 10 and 25 kD (non-reducing SDS-PAGE, Figure 2E), and inhibits the IgG4 binding efficiently.
  • IMIG nearly all material binds to protein G, and the inhibitory action of IMIG and the purified IMIG are similar.
  • the inhibitory component was identified as a fragment of ca. 25 kD ( Figure 2B).
  • the component could be separated from IVIG also in a single SEC purification step ( Figure 2C).
  • pepsin treatment O/N at 37°C and pH4 removed all inhibitory action of both recombinant IgG4 and IVIG. Reducing the time to about 1 hour resulted in a modest increase in inhibitory potential.
  • adalimumab treated with pepsin similarly inhibits the IgG4 Fc binding, whereas
  • adalimumab itself does not inhibit at all ( Figure 4).
  • the adalimumab pepsin digest was separated on a Superdex200 column and fractions were tested for inhibitory potential. The inhibitory action was centered around the pFc' peak (not shown).
  • Pepsin treatment of adalimumab at 37°C O/N results in no inhibitory action, and SEC shows that besides F(ab')2 only small peptides are present.
  • the difference between the pFc' and (CH3)2 fragments is not significant for IgG4, but pFc' from IgGl appears to inhibit more efficiently as IgGl (CH3)2 fragments.
  • the pFc' fragment from IgG2 is intermediate in inhibitory potential.
  • the pFc' fragment isolated from IVIG inhibits a little better than IgG2 pFc'. In other words, the active fragment in IVIG is most likely mainly IgG2 pFc', perhaps together with a small amount of IgG4 pFc' that was too little to be detected by the nanoLC- MS/MS-based peptide mapping.
  • pFc' and/or CH3 fragments derived from IgG4 or reduced IgG4 are preferred over pFc' and/or CH3 fragments derived from IgGl and IgG2 or reduced IgGl and IgG2.
  • pFc' and/or CH3 fragments derived from IgG2 or reduced IgG2 are preferred over pFc' and/or CH3 fragments derived from IgGl or reduced IgGl.
  • pFc' from IVIG binds to human IgG
  • IgGl (adalimumab) was coupled to CNBr-activated Sepharose (Amersham Biosciences, Uppsala, Sweden) at a density of 2.5 mg/100 mg Sepharose. 250 pL of a 2 mg/mL Sepharose suspension was incubated overnight with 125 I labeled IgG4 Fc together with inhibitor in a total volume of 750 pL. After washing 5 times, binding was measured (expressed as the amount of radioactivity bound relative to the amount of radioactivity added). IgG4 Fc fragments were prepared by papain digestion as described previously (1). IgG4 Fc was labeled by the 125 -I chloramine-T method as described previously (2).
  • IVIG (Nanogam, Sanquin, The Netherlands) or IMIG (Tetaquin, Sanquin, The Netherlands) was layered to a column of 1 mL protein A or G Sepharose followed by PBS to obtain non-bound material. Bound material was eluted with 0.1 M glycine buffer pH 2.5. Fractions were neutralized immediately after collecting with 2 M Tris. Size-exclusion chromatography Samples (100 ⁇ ) were applied to a Superdex 200 HR 10/30 column (Amersham Biosciences, Uppsala, Sweden), which was connected to a HPLC system (AKTAexplorer) from Amersham Biosciences, Uppsala, Sweden. The column was equilibrated in PBS. For estimation of protein size, the column was calibrated with the HMW calibration kit from GE Healthcare. and- CHS affinity chromatography
  • Nanobodies on this matrix bind to an epitope located in the CH3 domain of IgG. Elution of the bound fraction was performed with 0.1 M Glycine pH 3 or 2.5. Fractions were neutralized immediately after collecting with 2 M Tris. Limited pepsin digestion of IgGl, IgG2, and IgG4.
  • adalimumab IgGl, Gml(za)
  • omalizumab IgGl, Gml(f)
  • panitumumab IgG2 or natalizumab (IgG 4) (4 mg/mL in 0.1 M acetate buffer pH 4.2) was digested with pepsin (Sigma) 1:100 w/w at 37 °C. Time was varied between 0 and 1000 minutes; optimal yields of pFc' were obtained after 90 minutes (IgGl), 180 minutes (IgG2) or 20 minutes (IgG4) (based on SDS-PAGE or HP-SEC).
  • pFc' fragments were purified by removal of non-digested material using protein G affinity chromatography (pFc' in flow through), and subsequent removal of pepsin and F(ab)2 using a-CH3 affinity chromatography (Capture Select Human Fc affinity matrix, Nalgene) as described above.
  • pFc' fragments from omalizumab and panitumumab this procedure yielded very low yields.
  • pFc'fragments were purified by HP-SEC.
  • the CH3 regions of IgGl and IgG4 were amplified by PCR with primers containing restriction sites Ncol and NotI and subsequently inserted into pUC-HAV20 (4). Sequences of the primers used are 5' - C CATGG CTC AG C C C CGAG AGC C AC AGGTGTAC - 3' (forward, ⁇ 1/4), 5'- GCGGCCGCTCATTTACCCGGGGACAGGGAGAG-3' (reverse, Y l), and 5'- GCGGCCGCTCATTTACCCAGAGACAGGGAGAG-3' (reverse, Y 4).
  • the CH3 domains with leader sequence were cloned into a pcDNA3.1 expression vector (Invivogen), obtaining respectively a pCDNA3.1-CH3(Gl) and a pCDNA3.1- CH3(G4) vector.
  • the CH3 domains were expressed by transfecting pCDNA3.1- CH3(G1) or pCDNA3.1-CH3(G4) expressing vector in HEK-293F cells using 293fectin and FreeStyleTM 293 expression Medium according to the manufactures's instructions (Invitrogen).
  • Protein concentrations were determined either by measuring the absorbance at 280 nm with a NanoDrop ND-1000 spectrophotometer or by using the BCA protein assay (Pierce, Rockford, USA). SDS-PAGE
  • Non-reducing SDS-PAGE was performed according to the instructions of the manufacturer (Invitrogen, Carlsbad, CA). Samples were heated for 10 minutes at 70 °C. Gradient gels of 4 - 12% were used. Gels were stained using the silver- staining kit from Invitrogen.
  • Mass spectroscopy based sequencing was carried out as described in (3).
  • IgGl (adalimumab) was incubated at 5 mg/mL for 30 minutes 2.5 in 100 mM Glycine buffer at a pH of 2.5 with 0. 0.1, or 0.5 mg/mL IVIG CH3, followed by neutralization to pH 7 with 1M Tris. Samples were analyzed by HP-SEC as described above. Reduction and alkylation of IgGl, IgG4 and their Fc fragments
  • IgGl, IgG2, IgGl Fc, and IgG4 Fc were incubated for 0.5 h at room
  • Papain was pre-activated at 4.2 mg/ ml, 0.1 M DTT for 30 min in 37 oC. IgG (10 mg/ml final concentration) together with pre-activated papain (1:70) and 3mM EDTA was incubated for 3 hours at 37 C°. Digested material was separated from undigested material by size-exclusion chromatography
  • Hingeless IgGl/ IgG4 Fc digestion with FabRICATOR 7.5 ⁇ of FabRICATOR (Genovis Ab) was added to 250 ⁇ of IgGl or IgG4 (5 mg/ml in the PBS pH 6.6) Fc fragments were separated from F(ab)2 and undigested material with size exclusion chromatography using a Superdex 200 HR 10/30 column, which was connected to a FPLC system (AKTAexplorer) from Amersham Biosciences, using PBS as running buffer.
  • IVIG which was not treated with pepsin
  • IVIG which was not treated with pepsin but which was treated with
  • Venous blood was obtained from healthy volunteers by venapuncture in citrate- containing tubes. Citrated blood was diluted in a ratio of 1:1 in PBS containing 0.4% human serum albumin (HSA) and 0.38% trisodium citrate, layered on Lymphoprep and centrifuged at 1000 rcf for 25 minutes at room temperature (RT) without brake. The pellet containing erythrocytes and neutrophils was lysed twice in ice-cold isotonic NH4CI solution (155 mmol/1 NH4CI, 10 mmol/1 KHCO3, 0.1 mmol/1 EDTA, pH 7.2).
  • HSA human serum albumin
  • the cell suspension was diluted in culture medium to a final concentration of 2*10 5 cells/ml. To minimize spontaneous degranulation, we equilibrated the cell suspension for 1 hour at RT before further use. Degranulation of neutrophils
  • Isolated neutrophils were incubated with IVIG products (10 mg/ml) and dialyzed against culture medium with or without pFc'/(CH3)2 fragments obtained from Nanogam (0.35 mg/ml).
  • buffer contained poloxamer 207 to a final concentration of 0.05%.
  • the incubation was performed in 96-well microtiter plates using a thermomixer (Eppendorf, Hamburg, Germany) operated at 700 rpm for 1 hour at 37 °C. To harvest the supernatants, the plates were centrifuged at 2000 rpm for 1 minute at RT and the cell free supernatants collected.
  • elastase Released elastase was measured with a sandwich ELISA. Briefly, a microtiter plate coated with 1.5 ⁇ g/ml polyclonal rabbit IgG directed against human elastase kindly provided by the department of Immunopathology (Sanquin, Amsterdam, Netherlands) was incubated with supernatants diluted 1:50 in PBS/0.1% Tween20/0.2% gelatine (PTG) for 1 hour shaking at RT. After subsequent washing with PBS/0.02% Tween20 1 ⁇ g/ml biotinylated rabbit anti-human elastase diluted in PTG was added and the plates were incubated for 1 hour shaking at RT.

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Abstract

La présente invention a pour objet des méthodes et des moyens pour contrer l'agrégation des anticorps par l'administration auxdits anticorps d'une partie isolée ou recombinante d'une immunoglobuline ou d'une immunoglobuline isolée ou recombinante sans les liaisons disulfure reliant les deux chaînes lourdes ou une partie d'entre elles. La présente invention concerne également des méthodes et des moyens pour diminuer les effets secondaires indésirables des IVIG et/ou des anticorps thérapeutiques, par exemple une agrégation, une activité de liaison, une activité d'activation des compléments, une activité d'activation des neutrophiles et/ou une activité d'activation des plaquettes. La présente invention concerne aussi des associations pour le traitement d'un trouble associé à une interaction d'un domaine Fc, une méthode de réduction de la signalisation de fond non spécifique dans une analyse, laquelle analyse comprend l'utilisation d'anticorps et une méthode permettant de prolonger la durée de stockage ou d'augmenter la température de stockage maximale des IVIG ou des anticorps thérapeutiques.
PCT/NL2010/050864 2009-12-17 2010-12-17 Méthodes et moyens pour contrer une activité d'un domaine fc WO2011074965A1 (fr)

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WO2015002528A1 (fr) 2013-07-04 2015-01-08 Isobionics B.V. Procédé de fermentation biphasique pour la production d'un composé organique
EP3009450A1 (fr) * 2011-07-19 2016-04-20 Glaxo Group Limited Formulation liquide contenant adalimumab et un tampon acetate
CN110297093A (zh) * 2019-03-18 2019-10-01 山西瑞豪生物科技有限公司 一种检测人免疫球蛋白g4的方法和试剂盒
US11448651B2 (en) 2018-07-10 2022-09-20 Regeneron Pharmaceuticals, Inc. Modifying binding molecules to minimize pre-exisiting interactions

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3009450A1 (fr) * 2011-07-19 2016-04-20 Glaxo Group Limited Formulation liquide contenant adalimumab et un tampon acetate
WO2015002528A1 (fr) 2013-07-04 2015-01-08 Isobionics B.V. Procédé de fermentation biphasique pour la production d'un composé organique
US11448651B2 (en) 2018-07-10 2022-09-20 Regeneron Pharmaceuticals, Inc. Modifying binding molecules to minimize pre-exisiting interactions
CN110297093A (zh) * 2019-03-18 2019-10-01 山西瑞豪生物科技有限公司 一种检测人免疫球蛋白g4的方法和试剂盒

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