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WO2003011909A1 - Agents liants a activite differentielle - Google Patents

Agents liants a activite differentielle Download PDF

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Publication number
WO2003011909A1
WO2003011909A1 PCT/GB2002/003455 GB0203455W WO03011909A1 WO 2003011909 A1 WO2003011909 A1 WO 2003011909A1 GB 0203455 W GB0203455 W GB 0203455W WO 03011909 A1 WO03011909 A1 WO 03011909A1
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WO
WIPO (PCT)
Prior art keywords
binding agent
binding
agent according
biological target
target
Prior art date
Application number
PCT/GB2002/003455
Other languages
English (en)
Inventor
George Telford Stevenson
Original Assignee
University Of Southampton
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GBGB0118662.6A external-priority patent/GB0118662D0/en
Priority claimed from GB0123260A external-priority patent/GB0123260D0/en
Application filed by University Of Southampton filed Critical University Of Southampton
Priority to EP02749075A priority Critical patent/EP1412389A1/fr
Priority to US10/484,638 priority patent/US20050031626A1/en
Priority to JP2003517099A priority patent/JP2005509595A/ja
Publication of WO2003011909A1 publication Critical patent/WO2003011909A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/283—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against Fc-receptors, e.g. CD16, CD32, CD64
    • 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
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
    • 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
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/40—Immunoglobulins specific features characterized by post-translational modification
    • C07K2317/41—Glycosylation, sialylation, or fucosylation

Definitions

  • the present invention relates to binding agents, especially to binding agents useful in targeting cancer cells or other biological targets.
  • the invention relates to antibodies and antibody constructs, especially bispecific antibody constructs.
  • Antibodies and derivatives thereof have been used to target biological targets, including cancer cells, for many years.
  • the predominant class of antibody is the IgG class, which has 3 globular modules, 2 Fab and one Fc, joined by an extended and flexible hinge.
  • the Fab modules each display an integral antigen-binding site, while the Fc is responsible (a) for recruiting the molecular and cellular effectors needed to destroy an antibody-coated target cell and (b) for directing certain trafficking and metabolic characteristics of each antibody class.
  • the cells recruited by the Fc module display molecules called Fc-receptors (FcR), which dock at sites on the surface of the Fc.
  • FcR Fc-receptors
  • FIG. 1 A A mouse IgGl molecule is shown in Figures 1 A and IB.
  • Figure 1 A illustrates the disposition of chains and interchain disulfide (SS) bonds. Chains (2 light, 2 heavy, N termini at the top, C termini at the bottom) are represented by black ribbons. Sets of interchain noncovalent bonds are depicted by hatched rectangles, and the two antibody sites by dashed arcs. Human IgG differs only in having 2 rather than 3 inter-heavy chain SS bonds.
  • SS interchain disulfide
  • Figure IB shows a 2-dimensional diagrammatic representation of the overall protein conformation.
  • Antibody sites are represented by triangular indentations, and noncovalent interactions between the chains by dashed lines.
  • the chains are seen to be organized into 3 globular regions joined by a hinge comprising an extended sequence of each heavy chain.
  • the Fc region displays sequences for recruitment of effector molecules (a set known as complement) and effector cells (chiefly macrophages and NK lymphocytes).
  • the Fc contains a further set of sequences which prolong the metabolic life of the IgG molecule by sequestering it away from lysosomal enzymes.
  • the mouse IgG molecule is seen as foreign by the human immune system, which after about 10 days often produces antibodies against it, frequently against its Fc zone, thus further shortening the survival of the mouse antibody in the human host.
  • FIGS. 2A and 2B The two most common designs for bispecific antibodies are shown in Figures 2A and 2B. Each has been the subject of clinical trials, but with only limited success.
  • the construct shown in Figure 2A binds only weakly to tumour cells and has a limited half-life of only about 24 hours.
  • the construct shown in Figure 2B also binds only weakly to tumour cells. Additionally, if attached to effector cells only, it can cause damage due to recruitment of effectors by the Fc portion of the construct.
  • a binding agent comprising: (a) a first part that comprises one or more of the biological activities of an antibody Fc region when the binding agent is bound to a biological target; (b) a second part that is capable of binding to the biological target with a valency of two or more; and (c) a third part that is capable of monovalent binding to an effector cell so that the effector cell can act upon the biological target when the second part is bound to the biological target.
  • the effector cell is capable of destroying, damaging, altering or removing the biological target.
  • the biological target is deleterious to a human or non-human animal.
  • the biological target is a cancer cell or a part thereof.
  • At least one of the biological activities of the first part is modulated when the binding agent is bound to the biological target in comparison with when the binding agent is bound to the effector cell only.
  • At least one of the biological activities of the first part is at least ten times higher when the binding agent is bound to the biological target in comparison with when the binding agent is bound to the effector cell only.
  • the binding agent may be such that, in the absence of binding of the second part to the biological target, the binding agent is configured so that at least one biological activity of the first part is prevented or reduced due to steric hindrance, and in which the steric hindrance is removed or reduced when the second part binds to the biological target.
  • the first part comprises an FcRn docking site that is not sterically hindered in the absence of binding of the second part to the biological target.
  • the first part comprises one or more of the following biological activities when the binding agent is bound to a biological target: (a) complement activation; and (b) binding to the neonatal or Brambell Fc-receptor (FcRn).
  • the binding agent is modified to reduce activation of an effector cell in the absence of binding of the binding agent to a biological target.
  • it is modified to reduce binding to an FcRI, FcRII and/or an FcRIII receptor in the absence of binding of the binding agent to a biological target.
  • the first part comprises an Fc region which lacks one or more glycans, preferably two glycans, normally associated with a natural Fc molecule.
  • the glycans are those linked to asparagine corresponding to position 297 of the IgG heavy chain.
  • the first part comprises an Fc region which is enzymatically deglycosylated, preferably with glycoamidase PNGaseF.
  • the first part comprises a recombinant Fc region in which the asparagine residue corresponding to position 297 of the IgG heavy chain is replaced with a non-gylcosylatable amino acid residue.
  • the first part further comprises one or more of the following biological activities when the binding agent is bound to a biological target: (c) induction or stimulation of phagocytosis by phagocytic cells; and (d) antibody-dependent cellular cytotoxicity (ADCC).
  • the at least one biological activity may include binding with FcRI, FcRII and/or FcRIII receptors.
  • endosomal binding to the first part so as to reduce lysosomal degradation of the binding agent in vivo is not prevented.
  • the second part is capable of binding to a plurality of different biological targets or to a plurality of different parts of the same biological target.
  • the binding agent comprises one or more Fab, Fab' or F(ab') 2 regions or parts thereof. Preferably, it comprises one or more Fc regions, or parts thereof. Preferably, it comprises at least one anti-target Fab, Fab' or F(ab') 2 regions or parts thereof, at least one anti-effector cell Fab, or Fab' regions or parts thereof, and at least one Fc region or a part thereof. In preferred embodiments, the binding agent comprises at least two anti-target Fab, Fab' or F(ab') 2 regions or parts thereof.
  • any one or more of the first, second and third parts of the binding agent are derived from an IgG molecule. Any one or more of the first, second and third parts may be covalently linked to each other.
  • the binding agent may comprise one or more tandem thioether links that interconnect cysteine residues.
  • the second part binds specifically to the biological target.
  • the second part comprises anti-CD 20 and/or anti CD-37 binding activity.
  • the third part binds specifically to the effector cell.
  • the third part comprises anti-CD 16 binding activity.
  • the binding agent may comprise a modular structure, in which one module is capable of binding to a biological target, one module is capable of binding to an effector cell and another module comprises one or more of the biological activities of an antibody Fc region when the binding agent is bound to a biological target.
  • it comprises two modules capable of binding to the same biological target.
  • a part, component or module of a binding agent for use in the manufacture of a binding agent as described.
  • a method of providing a binding agent comprising providing a plurality of modules and connecting them via tandem thioether linkages between cysteine residues.
  • a method of providing a binding agent comprising the steps of: (a) providing a first part comprising one or more of the biological activities of an antibody Fc region when the binding agent is bound to a biological target; (b) providing a second part capable of binding to the biological target with a valency of two or more; (c) providing a third part capable of monovalent binding to an effector cell so that the effector cell can act upon the biological target when the second part is bound to the biological target; and covalently joining the first, second and third parts.
  • the modules or parts of the binding agent are as set out in any preceding aspect of the invention.
  • the modules or parts are linked via a maleimide linker (e.g. o-phenylenedimaleimide (PDM).
  • PDM o-phenylenedimaleimide
  • a binding agent according to any preceding aspect of the invention for use in medicine.
  • the disease or disorder is selected from the group consisting of: cancer, including a lymphoma (e.g. a B-cell lymphoma), an infectious disease or disorder and an autoimmune disease or disorder.
  • a lymphoma e.g. a B-cell lymphoma
  • an infectious disease or disorder e.g. a B-cell lymphoma
  • composition comprising a binding agent as described; the composition optionally comprising a pharmaceutically acceptable carrier, diluent or excipient.
  • an image or model preferably a computer generated image or model, of a binding agent as described.
  • a data carrier that comprises data for such an image or model.
  • a computer that comprises data for such an image or model, and/or that comprises such a data carrier.
  • a fourteenth aspect of the invention there is provided a method comprising providing an image or model as described, a data carrier as described, or a computer as described and using it to predict the structure and/or function of potential new therapeutic binding agents.
  • the method comprises making one or more changes to the image or model and, optionally, predicting or analysing an effect of those changes.
  • a drug development program that uses a binding agent as described, an image or model as described, a data carrier as described, a computer as described, or a method as described.
  • a method comprising providing a binding agent as described, or a drug or drug candidate as described and testing in vivo or in vitro the activity and/or binding of the binding agent, drug, or drug candidate against a biological target.
  • a method comprising providing a binding agent as described, or a drug or drug candidate as described and testing in vivo or in vitro the toxicity of the binding agent, drug, or drug candidate.
  • a binding agent as described or a drug or drug candidate as described, when in immobilised form.
  • an array comprising a binding agent as described, or a drug or drug candidate as described .
  • a method comprising the steps of: (a) exposing a Fc-containing polypeptide to a matrix; (b) allowing the Fc-containing polypeptide to bind to the matrix by a hydrophobic interaction; (c) removing the Fc-containing polypeptide from the matrix by disrupting the hydrophobic interaction.
  • a method of separating an Fc-containing polypeptide from other components in a sample comprising: (a) exposing the sample to a matrix; (b) allowing the Fc-containing polypeptide to bind to the matrix by a hydrophobic interaction; and optionally removing one or more components of the sample by washing the matrix; and (c) removing the Fc- containing polypeptide from the matrix by disrupting the hydrophobic interaction.
  • the matrix comprises Toyopearl TSK-butyl-650.
  • the effector cell is capable of destroying, damaging, altering or removing the target.
  • the effector cell may do this directly and/or indirectly (e.g. via the recruitment of other moieties, such as cytokines or other cells).
  • an "effector cell” may thus be any cell capable of giving rise to or promoting a desired biological effect.
  • Preferred cells include cytotoxic T-cells, natural killer (NK) cells, monocytes and dendritic cells.
  • the effector cell is advantageously protected from the antibody Fc biological activities mediated by the first part of the molecule.
  • the activity of the Fc module is hindered in the absence of target binding, for example by steric means, such that the antibody Fc activity of the binding agent-effector cell complex is relatively low in the absence of target cell binding.
  • at least one antibody Fc activity is increased by 10 times on target binding, preferably by 20 times, 40 times, 60 times, 100 times or more. In a further preferred aspect more than one antibody Fc activity may be so increased, for example 2, 3, 4 or more such activities.
  • the binding agent is constructed by covalently linking together antibody fragments.
  • the first part comprises an antibody Fc region
  • the second and third parts are selected from antibody binding fragments, such as Fv, scFv, Fab, F(ab') 2 and Fab'.
  • the fragments are linked together advantageously via a maleimide linker, such as o-phenylenedimaleimide.
  • compositions described here are variously applicable to medicine, as described below, including veterinary medicine and diagnostics.
  • the molecules as described here may moreover be represented in silico for use in molecular modelling and drug design.
  • a structural model of the binding agent is generated using molecular modelling techniques.
  • these are computer implemented modelling techniques.
  • Suitable programs include grid-based techniques and/or multiple copy simultaneous search (MCSS) methods. These will be familiar to those skilled in the art.
  • MCSS multiple copy simultaneous search
  • visual inspection of a computer model of a binding agent can be used, in association with manual docking of models of functional groups into its binding pockets.
  • targets and/or effector groups to simulate or modulate target binding may for example be identified by one or more of the following techniques: de novo compound design, by defining a pharmacophore as herein defined, and/or by using automated docking algorithms as herein described.
  • the first aspect (de novo compound design) may be performed using suitable computer software.
  • the software is selected from the group consisting of: QUANTA, SYBYL, HOOK and CAVEAT.
  • linking the functional groups may be performed manually.
  • Suitable in silico libraries for use in the methods and compositions described here will be familiar to those skilled in the art, and includes the Available Chemical Directory (MDL Inc), the Derwent World Drug Index (WDI), BioByteMasterFile, the National Cancer Institute database (NCI), and the Maybridge catalogue.
  • MDL Inc Available Chemical Directory
  • WDI Derwent World Drug Index
  • NCI National Cancer Institute database
  • Maybridge catalogue a compound identifiable using one or more of the methods as described here
  • a computer readable medium for a computer characterised in that the medium contains the atomic-co-ordinates of a binding agent as described herein.
  • compositions described here may employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, microbiology, recombinant DNA and immunology, which are within the capabilities of a person of ordinary skill in the art. Such techniques are explained in the literature. See, for example, J. Sambrook, E. F. Fritsch, and T. Maniatis, 1989, Molecular Cloning: A Laboratory
  • Figures 1A and IB show schematic representations of a mouse IgGl molecule.
  • Figure 1A illustrates the disposition of chains and interchain disulfide (SS) bonds.
  • Figure IB shows a 2-dimensional diagrammatic representation of overall protein conformation.
  • Figure 2 shows two types of bispecific antibody construct.
  • Figure 2 A shows a bis-
  • FIG. 2B shows an IgG bispecific antibody, usually prepared by hybridoma technology.
  • Figure 3 shows a schematic representation of an embodiment of a differentially activated bispecific antibody.
  • Figure 4 illustrates methods of joining polypeptide chains using linkers.
  • a binding agent as described here comprises three parts.
  • a first part of the binding agent has one or more of the biological activities of an antibody Fc region, when the binding agent is bound to a biological target.
  • the first part of the binding agent desirably has one or more of the following biological activities when the binding agent is bound to a biological target: (a) complement activation (which is achieved in mammals by binding to Clq); and (b) induction or stimulation of phagocytosis.
  • the first part of the binding agent further comprises any one or more of the following biological activities when the binding agent is bound to a biological target: (c) antibody-dependent cellular cytotoxicity (ADCC); (d) binding to the neonatal or Brambell Fc receptor (FcRn).
  • ADCC antibody-dependent cellular cytotoxicity
  • FcRn neonatal or Brambell Fc receptor
  • the binding agent When the binding agent is not bound to the target, it is not essential that any or all of the biological activities of the first part are present. However, it is preferred that the ability to bind to the neonatal receptor FcRn be present in a binding agent when it is not bound to the target.
  • one or more of these activities are not present, or are present at only a relatively low level.
  • a relatively low level may, for example, be considered to be less than 50%, less than 10%), or less than 1% of the level of activity present when the binding agent is bound to the target.
  • IgG natural antibody molecules
  • the activation of biological activities is dependent on aggregate binding to form arrays of Fc portions; binding to effector cells may also upregulate such activities.
  • activities (a) to (c) above are hindered in the event that only the effector cell is bound, and are dependent on target binding.
  • the cell lysis activity of the binding agent when it is not bound to the target is relatively lower than when it is bound to the target.
  • the cell lysis activity of the first part is at least ten times higher when the agent is bound to the target in comparison with when the agent is bound to the effector cell only.
  • the cell lysis activity is at least 10 times higher.
  • the titre of the binding agent which induces 50%> of plateau cell lysis is at least 5 times less, preferably 10 times less, more preferably 50 times or 100 times less, when it is bound to the target compared to when it is bound to the effector cell only.
  • the methods and compositions described here allows for "differential activity", whereby certain activities of the first part of the binding agent are reduced or prevented until binding of the second part to the target occurs.
  • the property of differential activity is useful when the effector cell is bound to the binding agent but the target is not bound, because the effector cell can be protected from significant Fc mediated cell damage that might otherwise occur (e.g. due to premature activation of complement and/or ADCC).
  • relatively high activity against a target can still be provided when the target is bound to the binding agent.
  • binding agents with such differential activity. Indeed prior art binding agents that were designed to bind both effector cells and cancer cell targets often suffered from the disadvantage that complement activation and/or ADCC occurred at an early stage so that effector cells were destroyed or damaged before being brought into the proximity of target cells. The provision of the binding agents as described here therefore represents a major advance in the field of immunotherapy.
  • differential activity is achieved due to steric hindrance of one or more sites present on the first part of the binding agent when the binding agent is bound to an effector cell but not to the target; Removal of or reduction in steric hindrance is thought to occur when the second part of the binding agent binds to the target. More specifically, it is believed that the second part of the binding agent may, in its unbound state, cause steric hindrance of one or more functional sites present on the first part of the binding agent and that a conformational change occurs on binding to the target so as to remove or reduce steric hindrance of said one or more sites of the first part. Furthermore, in embodiments where the second part has a valency of two or more, the differential valency in the molecule for target and effector cell epitopes may also contribute to the differential activity.
  • the first part comprises at least a portion of a Fc region of an immunoglobulin, preferably, an Fc region of an IgG antibody.
  • the Fc region displays sequences for recruitment of effector molecules (a set known as complement) and effector cells (chiefly macrophages and NK lymphocytes).
  • the Fc contains a further set of sequences which prolong the metabolic life of the IgG molecule by sequestering it away from lysosomal enzymes.
  • the space between the 2 heavy chains at the hinge-proximal region is filled by carbohydrate in the form of 2 oligosaccharide chains, depicted in Figure 1 by rectangles, attached to asparagine 297 on each heavy chain.
  • FIG. 1 Docking sites for Fc interactions are indicated by hatched areas in Figure 1. (A mirror-image second set of sites, reflecting the symmetry of the molecule, is not shown.) Effector cells such as macrophages display 3 classes of receptor for the Fc of IgG: FcRI, II and III. These receptors compete for a common site involving and adjacent to the lower hinge, a cup-like area kept patent by the carbohydrate chains. The site for complement is also seen to be near the lower hinge in Figure 1. The receptor for prolonging metabolic survival, the neonatal Fc-receptor (FcRn) or Brambell receptor, found on a variety of cells throughout the body, has a docking site further towards the C-terminus of the heavy chain.
  • FcRn neonatal Fc-receptor
  • Brambell receptor found on a variety of cells throughout the body, has a docking site further towards the C-terminus of the heavy chain.
  • the docking site for FcRI, II and III has been visualized by X-ray crystallography (P Sondermann et al. The 3.2-A crystal structure of the human IgGl Fc fragment-Fc ⁇ RIII complex. Nature 406:267-273, 2000), as has the site for FcRn (WL Martin et al. Crystal structure at 2.8 A of an FcRn/heterodimeric Fc complex: mechanism of pH-dependent binding. Molecular Cell 7:876-77, 2001).
  • the complement site has been deduced from site-directed mutagenesis, with the details subject to some ambiguity (AR Duncan & G Winter. The binding site for Clq on IgG. Nature 332:738-740, 1988; MH Tao et al. Structural features of human immunoglobulin-G that determine isotype-specific differences in complement activation. J. Exp. Med. 178:661-667, 1993).
  • FcRn docking site is distinct from the shared site for the FcRI, FcRII and FcRIII families on effector cells ( Figure 1).
  • FcRn is present on a variety of cells and is called the "neonatal Fc-receptor", because it is involved in the transport of maternal IgG across the placenta and across the neonatal gut.
  • FcRn is responsible for the prolonged metabolic survival of the IgG molecule. In man IgG is seen to survive in the body with a half-life of about 20 days, compared with 3-4 days for the other antibody classes - a point of great therapeutic importance.
  • FcRn is displayed on endosomes of the cells which internalize and break down plasma proteins. Most of the IgG taken up is bound to this FcRn, which returns the IgG to the bloodstream instead of letting it progress with other proteins to destruction in the cell's lysosomes.
  • One site that is believed to be sterically hindered when the binding agent binds to an effector cell (prior to binding to the target) is present on the Fc region of IgG and is involved in the induction of complement mediated cell damage when the binding agent is bound to the target. In humans this site is the Clq binding site.
  • Another site that is believed to be sterically hindered is the site that binds to FcRI, FcRII and FcRIII receptors.
  • the FcRn binding site (involved in binding to an endosomal receptor so as to prevent or reduce lysosomal degradation) is believed not to be substantially sterically hindered.
  • the absence of substantial steric hindrance at this site is believed to be of great advantage in increasing the half-life of the binding agents when in use.
  • the half- life in humans is at least 1 day. More preferably, it is at least 2, at least 4, or at least 8 days. Most preferably, it is at least 16 days.
  • Preferred binding agents comprise a target-binding part with a higher binding valency than the effector cell-binding part.
  • the target-binding part may bind to two, three or four epitopes (which may be the same or different) and the effector cell- binding part may bind to fewer epitopes (e.g. to a single epitope).
  • the target-binding part binds to two epitopes and the effector cell-binding part binds to one epitope.
  • preferred embodiments comprise target-binding parts with a valency of two or more, univalent binding agents are also envisaged. Such a construct may comprise one instead of two Fab(anti-target) modules, and may be useful in certain situations.
  • Desirably binding is specific so that the effector and target-binding parts do not cross-react with undesired moieties when binding the effector and target respectively.
  • the effector-binding part may have specific anti-CD 16 binding activity and the target-binding part may have specific anti-CD20 and/or anti-CD37 binding activity.
  • the binding agent may be provided in a form in which it is already bound to an effector cell. Alternatively it may be provided in unbound form and may bind an effector cell in vivo.
  • the first part of the binding agent is modified such that its binding to Fc receptors is reduced or impaired.
  • the first part does not comprise Fc receptor binding activity.
  • the Fc receptor is selected from one or more of FcRI, FcRII and FcRIII receptor families.
  • Reduction or abolition of Fc binding activity is preferably accomplished by removal of one or more oligosaccharides or glycans normally associated with a natural IgG Fc region.
  • a glycan covalently linked to the Fc region is removed.
  • the glycan comprises an asparagine linked glycan, more preferably, a glycan attached to an asparagine residue corresponding to residue number 297 on the IgG heavy chain.
  • Deglycosylation may be accomplished enzymatically, e.g., with a suitable oligosaccharide cleaving enzyme such as an amidase or glycoamidase.
  • glycoamidase any suitable glycoamidase may be used, a preferred enzyme is peptide-N ⁇ N-acetyl- ⁇ - glucosaminyl)asparagine amidaseP ⁇ Gase F (EC 3.5.1.52), described in detail in Tarentino and Plummer (1994, Meth Enz 230, 44-57).
  • the binding agent may be reacted with the enzyme before during or after its component parts are put together, i.e., the glycan may be removed from the Fc region before or after linkage to any of the Fab components.
  • the Fc component comprising the first part of the binding agent is treated with enzyme prior to being assembled.
  • glycan removal may be effected by genetic means.
  • a mutation may be introduced into a coding sequence for the Fc region, or a heavy chain encoding sequence.
  • a suitable mutation replaces the asparagine residue in the encoded amino acid with a non-glycosylatable residue, such as a residue other than serine or threonine, preferably an alanine residue.
  • Preferred binding agents are derived from IgG molecules or parts thereof, although other immunoglobulins or parts thereof may be used, if desired.
  • IgG molecules may be obtained as monoclonal or polyclonal antibodies.
  • polyclonal antibodies can be obtained by purifying them from a human or animal host after the host has been exposed to an immunogen.
  • an adjuvant may also be administered to the host to aid in immunostimulation.
  • Well- known adjuvants include Freund's adjuvant (complete or incomplete) and aluminium hydroxide.
  • Monoclonal antibodies can be produced from hybridomas. These can be formed by fusing together myeloma cells and spleen cells that produce a desired antibody in order to form an immortal cell line. Thus the well-known Kohler & Milstein technique (Nature 256 (1975)), or subsequent variations upon this technique can be used.
  • Antibodies can be purified by various techniques, including adsorption to staphylococcal protein A.
  • the staphylococcal protein will usually be coupled to a solid support, such as Sepharose beads. This can be done via cyanogen bromide coupling.
  • Antibodies bind to protein A chiefly by hydrophobic interactions, which can be disrupted when desired so as to elute the antibodies (e.g. via transient exposure to low pH).
  • Antibodies binding to known epitopes can be purified by elution using the epitopes in immobilised form to select the antibodies, followed by elution with an appropriate buffer
  • antibodies can be provided in forms not occurring in nature - i.e. in synthetic form.
  • humanised (or primatised) antibodies may be provided.
  • An example of a humanised antibody is an antibody having human framework regions, but rodent hypervariable regions.
  • Binding agents can be obtained by providing parts of antibodies and linking them together covalently.
  • the parts may be obtained via enzymatic cleavage (although other techniques including chemical synthesis and genetic engineering may be used).
  • papain cleavage can be used to provide two Fab fragments and an Fc fragment from an IgG molecule.
  • pepsin cleavage can be used to provide an F(ab') 2 fragment and a pFc' fragment from such a molecule. Chemical reduction of the F(ab') 2 fragment can then be used to provide two Fab' fragments.
  • Roitt et al describes immunoglobulin structure and function in detail, including the provision of the aforesaid fragments.
  • a binding agent of preferably comprises a plurality of Fab, Fab' or F(ab') 2 regions or parts thereof. Desirably it also comprises one or more Fc regions, or parts thereof.
  • the binding agent may comprise at least two anti-target antigen binding regions or parts thereof (e.g. at least two anti-target Fab ⁇ ' fragments); at least one anti-effector cell antigen binding region or part thereof (e.g. at least one anti-effector Fab ⁇ ' fragment); and at least one Fc ⁇ fragment or part thereof.
  • the anti anti-target binding regions may be derived from a different species from the species for which the binding agent is to be used in treatment. It is however preferred that part or all of the Fc regions is derived from the same species as that for which the binding agent is to be used in treatment.
  • the binding agent may be chimeric and may be humanised.
  • amino acids have similar characteristics and that one or more amino acids can often be substituted by one or more other such amino acids without eliminating a desired property of a polypeptide. Substitutions of this nature are often referred to as “conservative” or “semi-conservative” substitutions.
  • the amino acids glycine, alanine, valine, leucine and/or isoleucine can often be substituted for one another (amino acids having aliphatic side chains).
  • glycine and alanine are used to substitute for one another (because they have relatively short side chains) and that valine, leucine and isoleucine are used to substitute for one another (because they have larger aliphatic side chains which are hydrophobic).
  • amino acids having aromatic side chains include phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and/or cysteine and methionine (amino acids having sulphur-containing side chains).
  • amino acids having aromatic side chains include phenylalanine, tyrosine and tryptophan (amino acids having aromatic side chains); lysine, arginine and histidine (amino acids having basic side chains); aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains); and/or cysteine and methionine (amino acids having sulphur-containing side chains).
  • Another possibility is to provide one or more deletions. This can be advantageous because the overall length and the molecular weight of a polypeptide can be reduced, whilst still retaining a desired property. Thus, if desired, non-essential or non-important sequences may be removed from a given polypeptide.
  • Amino acid insertions can also be made. This may be done to alter the nature of the polypeptide (e.g. to assist in identification, purification or expression.)
  • sequence identity for the appropriate part of a binding agent as described with the sequence of a naturally occurring Fab, Fab', F(ab') 2 , Fc, or Fc' region. More preferably, the degree of sequence identity for said part with said region is at least 50%, at least 60%, at least 70%, or at least 80%. High sequence identities of at least 90%, at least 95%, or at least 99% are most preferred.
  • the percentage sequence identity between two amino acid sequences can be determined in a number of ways. For example:
  • S 100 X (M N); where M is the number of nucleotides or amino acids in the given sequence that are identical with nucleotides or amino acids at the corresponding positions in the reference sequence and N is the total number of amino acids or nucleotides in the reference sequence.
  • gaps may be penalised in a simple manner simply by considering the gaps to represent amino acid or nucleotide mismatches over the full length of any gaps present. Two gaps of 5 and 10 amino acids would therefore be considered to represent mismatches totalling 15 amino acids and the proportion of matches would be reduced (relative to a system in which gaps were not penalised). More sophisticated systems for penalising gaps are also known and may involve separate penalties for gap lengths and for the numbers of gaps present.
  • Gapped BLAST can be utilised as described in Altschul et al., 1997 Nucleic Acids Research 25(17):3389-3402.
  • BLAST and gapped BLAST programs it is preferred that the default parameters of the respective programs (e.g., XBLAST and NBLAST) are used (see e.g. http://www.ncbi.nlm.nih.gov/BLAST)
  • the FASTA program can also be used. This is based upon a modified version of the Wilbur and Lipman algorithm (see e.g. http://www2.ebi.ac.uk/fasta 3).
  • sequence identity is determined by using Gapped BLAST (version 2.0), using the default parameters provided.
  • thioether links are used to link cysteine residues, so as to link covalently different parts at or proximal to a hinge structure. Details of the provision of thioether links and hinge structures are provided by Stevenson (Antibody Engineering, Chem. Immunol., Basel, Karger, 1997, 65, 57-72).
  • the linking together of parts is preferably used to provide a binding agent having a tetramodular structure, whereby two modules are capable of binding to a biological target (or to two different biological targets), one module is capable of binding to an effector cell and another module has one or more of the biological activities of an antibody Fc region when the binding agent is bound to the biological target.
  • Binding agents may be provided in substantially pure form if desired.
  • the term "substantially pure form" is used to indicate that a given component is present at a high level.
  • the component is desirably the predominant protein component present in a composition. Preferably it is present at a level of more than 75%, of more than 90%, or even of more than 95%o, said level being determined on a dry weight / dry weight basis with respect to the total protein composition under consideration. At very high levels (e.g. at levels of more than 90%, of more than 95% or of more than 99%) the component may be regarded as being in "isolated form".
  • inorganic buffer salt components may be present, and if taken into account their mass may exceed that of the protein fraction and thus substantially reduce the percentage figures set forth above.
  • a method comprising the steps of: (a) exposing a Fc- containing polypeptide to a matrix; (b) allowing the Fc-containing polypeptide to bind to the matrix by a hydrophobic interaction; (c) removing the Fc-containing polypeptide from the matrix by disrupting the hydrophobic interaction.
  • a method of separating an Fc-containing polypeptide from other components in a sample comprising: (a) exposing the sample to a matrix; (b) allowing the Fc-containing polypeptide to bind to the matrix by a hydrophobic interaction; and optionally removing one or more components of the sample by washing the matrix; and (c) removing the Fc- containing polypeptide from the matrix by disrupting the hydrophobic interaction.
  • the matrix comprises one or more hydrophobic groups, for example, phenyl or butyl groups. More preferably, the matrix comprises polyhydroxymethacrylate gel bonded with phenyl or butyl group.
  • the matrix is in the form of a column,
  • step (b) is carried out in the presence of a higher salt concentration than in step (c). More preferably, step (c) is carried out by elution of the matrix with dilute aqueous salt solution, preferably in the presence of an organic solvent.
  • a suitable organic solvent comprises 10% dimethylformamide.
  • a composition containing a Fc-containing polypeptide is exposed to a suitable hydrophobic column under high salt conditions.
  • the bound protein is then eluted with a suitable solvent (such as a decreasing salt gradient).
  • a suitable solvent such as a decreasing salt gradient.
  • Suitable columns for hydrophobic interaction chromatography are known in the art, and are available for example from Amersham Biosciences (e.g., HiTrap HIC Selection Kit) and Agilent Technologies (e.g., TSK Phenyl-5PW, TSK Ether-5PW or SynChropak H-Propyl).
  • the matrix comprises Toyopearl TSK-butyl-650.
  • Hydrophobic interaction chromatography is a technique for the purification and separation of biomolecules based on differences in their surface hydrophobicity. HIC techniques have been used as apart of protein purification strategies in combination with other chromatographic techniques such as GF and IEX, as well as an analytical tool for the detection of protein conformational changes. Many biomolecules, generally considered to be hydrophilic, also have sufficient numbers of hydrophobic groups allowing interaction with hydrophobic ligands coupled to the chromatographic matrix. While HIC and RPC are closely related techniques, adsorbents for RPC are more highly substituted with hydrophobic ligands than HIC adsorbents. This feature allows the use of mild elution conditions to help maintain the biological activity of the sample.
  • Binding agents as described in this document are particularly useful in medicine. They can be used in the preparation of a medicament for treating a disease or disorder caused by or involving a biological target.
  • Treatments may benefit a human or non-human animal.
  • human and veterinary treatments are within the scope of the methods and compositions described here.
  • the treatment of mammals is particularly preferred.
  • a treatment may be in respect of an existing condition or it may be prophylactic. It may be of an adult, a juvenile, an infant, a foetus, a cell, tissue, or organ, or a part of any of the aforesaid.
  • Binding agents as described are useful in treating cancer (e.g. lymphomas). However they can be used to treat other diseases or disorders where the target is deleterious to a human or non-human animal. Examples of such other diseases or disorders include pathogenic or autoimmune diseases or disorders, and infectious diseases wherein infected cells may be detected - such as viral infections.
  • the medicament will usually be supplied as part of a pharmaceutical composition.
  • the pharmaceutical composition will desirably be provided in sterile form. It may be provided in unit dosage form and may also be provided in a sealed container. A plurality of unit dosage forms may be provided.
  • compositions may include one or more of the following: pharmaceutically carriers, preserving agents, solubilising agents, stabilising agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts, buffers, coating agents, antioxidants, adjuvants, excipients and diluents. They may also contain other therapeutically active agents in addition to binding agents. Where two or more therapeutic agents are used they may be administered separately (e.g. at different times and/or via different routes) and therefore do not always need to be present in a single composition. Thus combination therapy is within the scope of the methods and compositions described here.
  • compositions may be provided in controlled release form. This can be achieved by providing a pharmaceutically active agent in association with a substance that degrades under physiological conditions in a predetermined manner. Degradation may be enzymatic or may be pH-dependent.
  • compositions may be designed to pass across the blood brain barrier (BBB).
  • BBB blood brain barrier
  • a carrier such as a fatty acid, inositol or cholesterol may be selected that is able to penetrate the BBB.
  • the carrier may be a substance that enters the brain through a specific transport system in brain endothelial cells, such as insulin-like growth factor I or II.
  • the carrier may be coupled to the active agent or may contain/be in admixture with the active agent.
  • Liposomes can be used to cross the BBB.
  • WO91/04014 describes a liposome delivery system in which an active agent can be encapsulated/embedded and in which molecules that are normally transported across the BBB (e.g. insulin or insulin-like growth factor I or II) are present on the liposome outer surface. Liposome delivery systems are also discussed in US Patent No. 4704355.
  • a pharmaceutical composition may be adapted for administration by any appropriate route.
  • it may be administered by the oral (including buccal or sublingual), rectal, nasal, topical (including buccal, sublingual or transdermal), vaginal or parenteral (including subcutaneous, intramuscular, intravenous or intradermal) routes.
  • Such a composition may be prepared by any method known in the art of pharmacy - by admixing one or more active ingredients with a suitable carrier, for example.
  • compositions adapted for parenteral administration include aqueous and non-aqueous sterile injectable solutions or suspensions. These may contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially isotonic with the blood of an intended recipient. Other components that may be present in such compositions include water, alcohols, polyols, glycerine and vegetable oils, for example.
  • Compositions adapted for parenteral administration may be presented in unit- dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of a sterile liquid carrier, e.g. sterile water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets.
  • compositions suitable for injectable use include sterile buffered aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene gloycol, and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation.
  • dispersions are prepared by incorporating the sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof.
  • Dosages of binding agents can vary between wide limits, depending upon the nature of the treatment, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used. However, without being bound by any particular dosages, a daily dosage of from l ⁇ g to lmg/kg body weight may be suitable. The dosage may be repeated as often as appropriate. If side effects develop, the amount and/or frequency of the dosage can be reduced, in accordance with good clinical practice.
  • the principal active ingredients are compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in unit dosage form. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
  • the optimum dose may be determined empirically in a clinical trial by administering escalating doses of antibody until the maximum tolerated dose is determined.
  • the MTD is defined as the highest dose preceding that at which 50% of patients experience dose-limiting toxicity.
  • a further aspect is the provision of an image or model of a binding agent as described here. This may be computer-generated or may be physical. The image or model is preferably two or three-dimensional.
  • It may be an X-ray crystallographic image or model and may comprise a plurality of co-ordinates. It may show one or more: binding sites, hydrophilic or hydrophobic regions, bonds, etc.
  • binding sites e.g. by rotation, by zooming in or out, etc.
  • a data carrier that comprises data for such an image or model is also within the scope of the methods and compositions described here, as is a computer that comprises said data or data carrier.
  • the computer is set up to display the image or model.
  • the image or model is useful in predicting the structure and/or function of potential new therapeutic agents. For example, one or more changes may be made to the image or model and the effect(s) of those changes may be predicted or analysed.
  • a binding agent, image or model, data carrier, or computer as aforesaid is useful in a drug development program.
  • a drug or drug candidate obtained via a drug development program in which the binding agent, image or model, data carrier, or computer is used is also within the scope of the methods and compositions described here.
  • Drugs or drug candidates are tested for activity and/or activity.
  • a method comprising providing a binding agent, drug or drug candidate, as aforesaid, and testing the activity and/or toxicity of the binding agent, drug, or drug candidate in vivo or in vitro.
  • an immobilised binding agent may be provided as part of an organised array (e.g. an array having a grid-like structure). This can aid in identification when screening and is also useful in high throughput screening.
  • the array may comprise or consist of a plurality of binding agents, each as described. It may be provided upon a generally planar surface.
  • DAB differentially activated bispecific
  • Two Fab modules target the abnormal cells. Effectors are recruited by a human Fc module and also by an Fab module directed at effector cells.
  • the construct is differentially active in the sense that initial binding to an effector cell leads to much less implementation of Fc-recruited effector function than does binding to the targeted cell. In this way damage to effector cells, and undesired symptoms due to cytokine release from effector cells in blood, are minimised
  • FIG. 4 illustrates fundamentals of engineering of a DAB antibody.
  • Each module has its hinge-region interchain SS bonds reduced, and sometimes further manipulated by disulfide interchange, to leave one or more SH groups.
  • One module (Fc in this example) is then exposed to a surplus of a bismaleimide linker, so that one end of the linker reacts with an SH group to form a thioether bond while the other end continues to display a reactive maleimide group. Unreacted linker is removed and the maleimide-displaying module is allowed to react with a second SH-displaying module (Fab in this case) to form a bimodular or higher order construct.
  • the linking unit of about 9A length, consists of thioether bonds on either side of an o-phenylenedisuccinimidyl group. If disulfide interchange has left some nascent SH groups, further modular additions are possible.
  • an Fc ⁇ module is deglycosylated enzymaticalt before being used as a building block.
  • the molecular target on the neoplastic B cells is the CD20 molecule and the molecule used for recruiting effector cells with Fab is CD 16.
  • the starting modules are:
  • the method of synthesis is as follows: (a) Fab ' ⁇ - maleimide (anti-CO2 >)
  • F(ab' ⁇ ) 2 ex 1F5 is reduced by lmM dithiothreitol (DTT) at pH 8.0 and the resulting Fab' ⁇ (-SH) 5 separated by gel chromatography. It then reacts with ImM o- phenylenedimaleimide (PDM) at pH 5.0 to yield Fab' ⁇ -maleimide, which is separated and concentrated by ion-exchange chromatography.
  • DTT dithiothreitol
  • PDM ImM o- phenylenedimaleimide
  • F(ab' ⁇ ) 2 ex 3G8 is reduced by ImM DTT at pH 8.0 and the resulting Fab' ⁇ (-SH) 5 separated by gel chromatography.
  • Fc ⁇ l is reduced by ImM DTT at pH 8.0 and the resulting Fc ⁇ (-SH) 4 separated by gel chromatography.
  • F(ab' ⁇ ) 3 - maleimide and Fc ⁇ (-SH) 4 are mixed at a mass ratio of 2:1 and allowed to react at pH 5.0. Finally the reaction mixture undergoes SS -interchange with 0.5 mM cysteine at pH 8.4 in order to close the Fc hinge, and the Fab 3 Fc product is separated by gel chromatography.
  • FcRIII chiefly NK cells and macrophages. Multiple anchoring will hold the effector cell tightly and the effective crosslinking of the FcRIII molecule will activate the cell for cytotoxicity. Crosslinking will also lead to cytokine release, enhancing the cytotoxicity and leading to inflammation in the vicinity of the targeted cells. If these events are widespread throughout the body some symptoms of toxicity will be inevitable, but the physician should be forewarned by the known mass and distribution of the targeted cells.
  • FcRIII-mediated cytotoxicity will be enhanced by the disablement of the Fc site for FcRI, II and III, because among the subtypes of FcRII is the inhibitory receptor FcRIIb whose engagement can considerably downgrade the activation of macrophages. Isolated disablement of FcRII in mice enhances the ability of antibody to destroy tumour (RA Clynes et al. Inhibitory Fc receptors modulate in vivo cytotoxicity against tumor targets. Nature Medicine 6:443-446, 2000).
  • FcRn is present on many body cells, including the vascular endothelial cells thought to be a major site for catabolism of IgG. At the pH of extracellular fluid (7.4) Fc ⁇ has no significant affinity for FcRn. On being endocytosed - a preliminary to catabolism - IgG is in endosomes of decreasing pH, histidines in the docking site for FcRn become protonated, and the IgG combines with FcRn on the endosomal wall. This diverts the molecule away from the path to destructive endolysosomes: instead it is trafficked back onto the cell surface where the higher pH leads to its release back into extracellular fluid.
  • a deglycosylated Differentially Activated Bispecific Antibody (termed Fab 3 Fcd) is made in a similar manner described above in Example 3, except that steps (d) and (e) are modified.
  • the starting materials are identical, but the synthesis involves 2 major stages, the synthesis of Fab 3 and the attachment to it of deglycosylated Fc.
  • F(ab' ⁇ ) 2 ex 1F5 is reduced by 1 mM dithiothreitol (DTT) at pH 8.0 and the resulting Fab(-SH) 5 separated by gel chromatography. It then reacts with 1 mM o- phenylenedimaleimide (PDM) at pH 5.0 to yield Fab-maleimide, which is separated and concentrated by ion-exchange chromatography.
  • DTT dithiothreitol
  • PDM o- phenylenedimaleimide
  • F(ab ⁇ ) 2 ex 3G8 is reduced by 1 mM DTT at pH 8.0 and the resulting Fab(-SH) 5 separated by gel chromatography.
  • Fab-maleimide and Fab(-SH) 5 are mixed in a ratio of 2.2:1 and allowed to react at pH 5.0.
  • the resulting Fab 3 is separated by gel chromatography, then reacts with PDM to yield Fab 3 -maleimide.
  • the reaction is stochastic and Fab4 and Fab2 form as byproducts. During gel chromatography the latter is bled off, to be subjected to further reaction with Fab-maleimide.
  • the Fc is first deglycosylated by reaction with the glycoamidase PNGaseF (AL Tarentino & THPlummer. Enzymatic deglycosylation of asparagine-linked glycans: purification, properties, and specificity of oligosaccharide-cleaving enzymes from Flavobacterium meningosepticum. Meth Enzymol 230:44-57, 1994). Any incompletely deglycosylated can be expected to display quite a variety of carbohydrate, so the completely deglycosylated (and therefore quite hydrophobic) Fc is separated and concentrated by hydrophobic interaction chromatography on Toyopearl TSK-butyl-650. The eluted deglycosylated Fc ⁇ l is designated Fed.
  • PNGaseF glycoamidase PNGaseF
  • Fab 3 -maleimide and Fcd(-SH) 4 are mixed at a mass ratio of 2:1 and allowed to react at pH 5.0. Finally the reaction mixture is alkylated with 10 mM iodoacetate at pH 8.0 in order to block surplus SH groups, and the Fab 3 Fcd product is separated by gel chromatography.
  • a binding agent comprising: (a) a first part that has one or more of the biological activities of an antibody Fc region when the binding agent is bound to a biological target (b) a second part that is capable of binding to the biological target with a valency of two or more; and (c) a third part that is capable of monovalent binding to an effector cell so that the effector cell can act upon the target when the second part is bound to the target.
  • Paragraph 2 A binding agent according to Paragraph 1 , wherein the effector cell is capable of destroying, damaging, altering or removing the target.
  • Paragraph 3 A binding agent according to Paragraph 1 or Paragraph 2, wherein the target is deleterious to a human or non-human animal.
  • Paragraph 4 A binding agent according to Paragraph 3, wherein the target is a cancer cell or a part thereof.
  • a binding agent according to any preceding Paragraph wherein the first part has one or more of the following biological activities when the binding agent is bound to a biological target: (a) complement activation; (b) induction or stimulation of phagocytosis by phagocytic cells; (c) antibody-dependent cellular cytotoxicity (ADCC); and (d) binding to the neonatal or Brambell Fc-receptor (FcRn).
  • Paragraph 6 A binding agent according to any preceding Paragraph, wherein at least one of the biological activities of the first part is modulated when the agent is bound to the target cell in comparison with when the agent is bound to the effector cell only.
  • Paragraph 7 A binding agent according to any preceding Paragraph, wherein at least one of the biological activities of the first part is at least ten times higher when the agent is bound to the target cell in comparison with when the agent is bound to the effector cell only.
  • Paragraph 8 A binding agent according to any preceding Paragraph, wherein, in the absence of binding of the second part to the target, the binding agent is configured so that at least one biological activity of the first part is prevented or reduced due to steric hindrance, and wherein said steric hindrance is removed or reduced when the second part binds to the target.
  • Paragraph 9 A binding agent according to Paragraph 8, wherein said at least one biological activity includes complement activation.
  • Paragraph 10 A binding agent according to Paragraph 8 or 9, wherein said at least one biological activity includes binding with FcRI, FcRII and/or FcRIII receptors.
  • Paragraph 11 A binding agent according to Paragraph any of Paragraphs 8 to 10, wherein endosomal binding to the first part so as to reduce lysosomal degradation of the binding agent in vivo is not prevented.
  • Paragraph 12 A binding agent according to Paragraph 11, wherein the first part comprises an FcRn docking site that is not sterically hindered in the absence of binding of the second part to the target.
  • Paragraph 13 A binding agent according to any preceding Paragraph, wherein the second part is capable of binding to a plurality of different targets or to a plurality of different parts of the same target.
  • Paragraph 14 A binding agent according to any preceding Paragraph comprising one or more Fab, Fab' or F(ab') 2 regions or parts thereof.
  • Paragraph 15 A binding agent according to any preceding Paragraph comprising one or more Fc regions, orparts thereof.
  • a binding agent according to any preceding Paragraph comprising at least two anti-target Fab, Fab' or F(ab') 2 regions or parts thereof, at least one anti- effector cell Fab, or Fab' regions or parts thereof, and at least one Fc region or a part thereof.
  • Paragraph 17 A binding agent according to any preceding Paragraph wherein the parts of the binding agent are derived from an IgG molecule.
  • Paragraph 18 A binding agent according to any preceding Paragraph wherein the parts are covalently linked together.
  • Paragraph 19 A binding agent according to any preceding Paragraph comprising one or more tandem thioether links that interconnect cysteine residues
  • Paragraph 20 A binding agent according to any preceding Paragraph wherein the second part binds specifically to the target.
  • Paragraph 21 A binding agent according to any preceding Paragraph wherein the second part has anti-CD 20 and/or anti CD-37 binding activity.
  • Paragraph 22 A binding agent according to any preceding Paragraph wherein the third part binds specifically to the effector cell.
  • Paragraph 23 A binding agent according to any preceding Paragraph, wherein the third part has anti-CD 16 binding activity.
  • Paragraph 24 A binding agent according to any preceding Paragraph having a tetramodular structure, wherein two modules are capable of binding to a biological target, one module is capable of binding to an effector cell and another module has one or more of the biological activities of an antibody Fc region when the binding agent is bound to a biological target.
  • Paragraph 25 A binding agent according to any preceding Paragraph when bound to the effector cell.
  • Paragraph 26 A method of providing a binding agent according to any preceding Paragraph, comprising providing a plurality of modules and connecting them via tandem thioether linkages between cysteine residues.
  • Paragraph 27 A method according to Paragraph 26 wherein modules are linked via a maleimide linker (e.g. o-phenylenedimaleimide (PDM).
  • PDM o-phenylenedimaleimide
  • Paragraph 28 A binding agent according to any of Paragraphs 1 to 25, for use in medicine.
  • Paragraph 29 The use of a binding agent according to any of Paragraphs 1 to 25 in the preparation of a medicament for treating a disease or disorder caused by or involving the biological target.
  • Paragraph 30 The use according to Paragraph 29, wherein the disease or disorder is cancer.
  • Paragraph 31 The use according to Paragraph 30, wherein the disease or disorder is a lymphoma (e.g. a B-cell lymphoma).
  • a lymphoma e.g. a B-cell lymphoma
  • Paragraph 32 The use according to Paragraph 29, wherein the disease or disorder is a pathogenic disease or disorder.
  • Paragraph 33 The use according to Paragraph 29, wherein the disease or disorder is an autoimmune disease or disorder.
  • Paragraph 34 A pharmaceutical composition comprising a binding agent according to any of Paragraphs 1 to 25 ; said composition optionally comprising a pharmaceutically acceptable carrier, diluent or excipient.
  • Paragraph 35 An image or model of a binding agent according to any of Paragraphs 1 to 25.
  • Paragraph 36 An image or model according to any of Paragraphs 1 to 25 that is computer generated.
  • Paragraph 37 A data carrier that comprises data for an image or model according to Paragraph 35.
  • Paragraph 38 A computer that comprises data for an image or model according to Paragraph 35 or 36, and/or that comprises a data carrier according to Paragraph 37.
  • Paragraph 39 A method comprising providing an image or model according to Paragraph 35 or 36, a data carrier according to Paragraph 37, or a computer according to Paragraph 38 and using it to predict the structure and/or function of potential new therapeutic agents.
  • Paragraph 40 A method comprising providing an image or model according to Paragraph 35 or 36 making one or more changes to it and, optionally, predicting or analysing an effect of those changes.
  • Paragraph 41 A drug development program that uses a binding agent according to
  • Paragraph 1 to 25 an image or model according to Paragraph 35 or 36, a data carrier according to Paragraph 37, a computer according to Paragraph 38, or a method according to Paragraph 39 or 40.
  • Paragraph 42 A drug or drug candidate obtained or identified using a drug development program according to Paragraph 41.
  • Paragraph 43 A method comprising providing a binding agent according to any of Paragraphs 1 to 25, or a drug or drug candidate according to Paragraph 42 and testing in vivo or in vitro the activity and/or binding of the binding agent, drug, or drug candidate against a biological target.
  • Paragraph 44 A method comprising providing a binding agent according to any of
  • Paragraphs 1 to 25, or a drug or drug candidate according to Paragraph 42 and testing in vivo or in vitro the toxicity of the binding agent, drug, or drug candidate.
  • Paragraph 45 A binding agent according to any of Paragraphs 1 to 25, or a drug or drug candidate according to Paragraph 42, when in immobilised form.
  • Paragraph 46 An array comprising a binding agent according to any of Paragraphs 1 to 25, or a drug or drug candidate according to Paragraph 42.

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  • Peptides Or Proteins (AREA)

Abstract

L'invention concerne des agents liants à activité différentielle, certaines activités d'une première partie de l'agent de liaison étant réduites ou empêchées jusqu'à ce que se produise la liaison de l'agent à une cible. Cette propriété est très utile lorsque l'agent de liaison est censé se lier à la fois à une cellule effectrice et à une cible à détruire, la cellule effectrice pouvant alors être protégée de tout dommage cellulaire important qui pourrait se produire autrement (par exemple en raison d'une activation prématurée du complément et/ou de toxicité cellulaire dépendante d'anticorps). De tels agents de liaison sont utiles dans le traitement de cancer, par exemple.
PCT/GB2002/003455 2001-07-31 2002-07-30 Agents liants a activite differentielle WO2003011909A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP02749075A EP1412389A1 (fr) 2001-07-31 2002-07-30 Agents liants a activite differentielle
US10/484,638 US20050031626A1 (en) 2001-07-31 2002-07-30 Binding agents with differential activity
JP2003517099A JP2005509595A (ja) 2001-07-31 2002-07-30 示差的（ｄｉｆｆｅｒｅｎｔｉａｌ）活性を有する結合作用因子

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GBGB0118662.6A GB0118662D0 (en) 2001-07-31 2001-07-31 Binding agents
GB0118662.6 2001-07-31
GB0123260.2 2001-09-27
GB0123260A GB0123260D0 (en) 2001-09-27 2001-09-27 Binding agents

Publications (1)

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WO2003011909A1 true WO2003011909A1 (fr) 2003-02-13

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PCT/GB2002/003455 WO2003011909A1 (fr) 2001-07-31 2002-07-30 Agents liants a activite differentielle

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EP (1) EP1412389A1 (fr)
JP (1) JP2005509595A (fr)
WO (1) WO2003011909A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014134486A3 (fr) * 2013-02-28 2015-03-26 Immunogen, Inc. Conjugués comprenant des agents de liaison cellulaire (cba) et des agents cytotoxiques

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995009917A1 (fr) * 1993-10-07 1995-04-13 The Regents Of The University Of California Anticorps bispecifiques et tetravalents, obtenus par genie genetique
WO2001077342A1 (fr) * 2000-04-11 2001-10-18 Genentech, Inc. Anticorps multivalents et leurs utilisations
WO2002008293A2 (fr) * 2000-07-25 2002-01-31 Immunomedics Inc. Proteine de liaison cible multivalente

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1995009917A1 (fr) * 1993-10-07 1995-04-13 The Regents Of The University Of California Anticorps bispecifiques et tetravalents, obtenus par genie genetique
WO2001077342A1 (fr) * 2000-04-11 2001-10-18 Genentech, Inc. Anticorps multivalents et leurs utilisations
WO2002008293A2 (fr) * 2000-07-25 2002-01-31 Immunomedics Inc. Proteine de liaison cible multivalente

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
ALT M ET AL: "NOVEL TETRAVALENT AND BISPECIFIC IGG-LIKE ANTIBODY MOLECULES COMBINING SINGLE-CHAIN DIABODIES WIHT THE IMMUNOGLOBULIN GAMMA L FC OR CH3 REGION", FEBS LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 454, no. 1/2, 1999, pages 90 - 94, XP000914692, ISSN: 0014-5793 *
KAN K S ET AL: "Thioether-bonded constructs of Fab'gamma and Fc gamma modules utilizing differential reduction of interchain disulfide bonds.", JOURNAL OF IMMUNOLOGY (BALTIMORE, MD.: 1950) UNITED STATES 15 JAN 2001, vol. 166, no. 2, 15 January 2001 (2001-01-15), pages 1320 - 1326, XP002214195, ISSN: 0022-1767 *
PLUCKTHUN ANDREAS ET AL: "New protein engineering approaches to multivalent and bispecific antibody fragments.", IMMUNOTECHNOLOGY (SHANNON), vol. 3, no. 2, 1997, pages 83 - 105, XP002950976, ISSN: 1380-2933 *
RADAEV S ET AL: "Recognition of IgG by Fcgamma receptor. The role of Fc glycosylation and the binding of peptide inhibitors.", THE JOURNAL OF BIOLOGICAL CHEMISTRY. UNITED STATES 11 MAY 2001, vol. 276, no. 19, 11 May 2001 (2001-05-11), pages 16478 - 16483, XP002214196, ISSN: 0021-9258 *
SEGAL D M ET AL: "Bispecific antibodies in cancer therapy", CURRENT OPINION IN IMMUNOLOGY, CURRENT BIOLOGY LTD, XX, vol. 11, no. 5, 1 October 1999 (1999-10-01), pages 558 - 562, XP004257585, ISSN: 0952-7915 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014134486A3 (fr) * 2013-02-28 2015-03-26 Immunogen, Inc. Conjugués comprenant des agents de liaison cellulaire (cba) et des agents cytotoxiques
US9901647B2 (en) 2013-02-28 2018-02-27 Immunogen, Inc. Conjugates comprising cell-binding agents and cytotoxic agents

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EP1412389A1 (fr) 2004-04-28

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