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WO2006133486A1 - Structures cristallines et modeles de complexes fc/recepteurs fc et utilisations de ces derniers - Google Patents

Structures cristallines et modeles de complexes fc/recepteurs fc et utilisations de ces derniers Download PDF

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Publication number
WO2006133486A1
WO2006133486A1 PCT/AU2006/000813 AU2006000813W WO2006133486A1 WO 2006133486 A1 WO2006133486 A1 WO 2006133486A1 AU 2006000813 W AU2006000813 W AU 2006000813W WO 2006133486 A1 WO2006133486 A1 WO 2006133486A1
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WIPO (PCT)
Prior art keywords
atom
dimensional structure
agent
ligand
target site
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PCT/AU2006/000813
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English (en)
Inventor
Phillip Mark Hogarth
Paul Allen Ramsland
Tessa Margaret Bradford
William Farrugia
Geoffrey Allan Pietersz
Caroline Tan Sardjono
Bruce David Wines
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The Macfarlane Burnet Institute For Medical Research And Public Health Limited
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Priority claimed from AU2005903095A external-priority patent/AU2005903095A0/en
Application filed by The Macfarlane Burnet Institute For Medical Research And Public Health Limited filed Critical The Macfarlane Burnet Institute For Medical Research And Public Health Limited
Publication of WO2006133486A1 publication Critical patent/WO2006133486A1/fr

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/06—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies from serum
    • 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
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • 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
    • C07K2317/71—Decreased effector function due to an Fc-modification
    • 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
    • C07K2317/72—Increased effector function due to an Fc-modification

Definitions

  • the present invention relates to the determination by X-ray crystallography of the three-dimensional structure of an Fc receptor protein in complex with the Fc fragment of an Fc receptor ligand, namely, human IgGl, and the use of said structure in identifying and modifying agents for modulating the biological activity of Fc receptors.
  • Fc receptors Interactions between the various classes of antibodies and Fc receptors (FcR) initiate a wide range of immunological responses. These include antibody-specific antigen uptake for presentation of MHC bound peptides to T cells, degranulation of mast cells in allergy, and immune complex mediated hypersensitivity and inflammation.
  • the FcR have also been shown to function as recognition molecules for viral infections in measles and Dengue fever. In humans, the most prevalent and abundant Fc receptor for immunoglobulin G (IgG FcR) is designated as Fc ⁇ RIIa or CD32.
  • Human Fc ⁇ RIIa exists as two predominant alleles classified as the low responder (LR) and the high responder (HR) wild-type polymorphisms. At the level of protein sequence the difference is that the LR receptor has a histidine (H) while the HR receptor has an arginine (R) residue at position 134 (often designated in the literature as position 131) in the amino acid sequence (Warmerdam et al, 1990).
  • the differences between the LR and HR Fc ⁇ RIIa alleles relate to their different abilities to bind mouse IgGl and human IgG2 (Sautes et al, 1991; Parren et al, 1992).
  • the signalling ITAM is located within the cytoplasmic tail of Fc ⁇ RIIa.
  • Other activating FcR molecules associate with ITAM-containing accessory molecules, which mediate the intracellular aspects of the signalling event (Hogarth, 2002).
  • the crystal structure of the LR allele of the Fc ⁇ RIIa glycoprotein was reported to have a major crystallographic dimer formed around a twofold axis in the P2i2i2 crystals (Maxwell et al, 1999). Such an arrangement brings two ITAM-containing cytoplasmic tails of Fc ⁇ RIIa into close proximity.
  • this novel dimeric form is intrinsically involved in the signalling complex of Fc ⁇ RIIa and, therefore, is of use in elucidating the biology and modulation of this receptor and other cell membrane- associated protein receptors. More particularly, the novel dimeric form of HRsss identified in PCT/ AU2005/ 000176 is of use in identifying and modifying agents for modulating the biological activity of Fc receptors.
  • the process of rational or structure-based drug design requires no explanation or teaching for the person skilled in the art, but a brief description is given here of computational design for the lay reader.
  • the person skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with a target molecule. For example, the screening process may begin by visual inspection of the target molecule, or a portion thereof, on a computer screen, generated from a machine-readable storage medium. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within identified or possible binding pockets (i.e. target sites).
  • Docking may be accomplished using software such as Quanta (Accelrys, Inc, Burlington, Mass, USA) and Sybyl (Tripos Associates, St Louis, Mo, USA) followed by energy minimisation and molecular dynamics with standard molecular mechanics force fields, such as CHARMM (Accelrys, Inc, Burlington, Mass, USA) and AMBER (Weiner et al, 1984; Kollman, PA, University of California, San Francisco, Ca, USA).
  • Quanta Quanta
  • Sybyl Tripos Associates, St Louis, Mo, USA
  • energy minimisation and molecular dynamics with standard molecular mechanics force fields such as CHARMM (Accelrys, Inc, Burlington, Mass, USA) and AMBER (Weiner et al, 1984; Kollman, PA, University of California, San Francisco, Ca, USA).
  • Specialised computer programs may also assist in the process of selecting fragments or chemical entities. These include:
  • GRID Goodford, 1985. GRID is available from Oxford University, Oxford, UK. 2. MCSS (Miranker, 1991). MCSS is available from Accelrys, Inc, Burlington, Mass., USA.
  • DOCK (Kuntz, 1982). DOCK is available from University of Calif ornia, San Francisco, Ca, USA.
  • CAVEAT Bartlett et al 1989. CAVEAT is available from the University of California, Berkeley, Ca, USA;
  • 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, Ca, USA). This area is reviewed in Martin, 1992; and
  • inhibitory or other target-binding compounds may be designed as a whole or de novo. Methods for achieving such include:
  • LUDI (Bohm, 1992). LUDI is available from Accelrys, Inc, Burlington, Mass, USA; 2. LEGEND (Nishibata, 1991). LEGEND is available from Accelrys, Inc, Burlington, Mass, USA; and
  • an effective entity will preferably demonstrate a relatively small difference in energy between its bound and free states (i.e. a small deformation energy of binding).
  • the most efficient entities should preferably be designed with a deformation energy of binding of not greater than about 10 kcal/mole, and preferably, not greater than 7 kcal/mole.
  • some entities may interact with the target site in more than one conformation that is similar in overall binding energy. In those cases, the deformation energy of binding is taken to be the difference between the energy of the free entity and the average energy of the conformations observed when the entity binds to the target site.
  • a compound or chemical complex designed or selected so as to bind to a target site may be further computationally optimised so that in its bound state it would preferably lack repulsive electrostatic interaction with the target protein.
  • Such non-complementary (e.g. electrostatic) interactions include repulsive charge-charge, dipole-dipole and charge-dipole interactions.
  • the sum of all electrostatic interactions between the entity or other entity and the target site, when the entity is bound to the target site preferably make a neutral or favourable contribution to the enthalpy of binding.
  • substitutions may be made in some of its atoms or side groups. Generally, initial substitutions of this kind will be conservative, that is the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. It should, of course, be understood that components known in the art to alter conformation should be avoided. Such substituted chemical compounds may then be analysed for efficiency of fit to a specific target site by the same computer methods described in detail above.
  • Another approach is the computational screening of small molecule databases for compounds or chemical complexes that can interact in whole, or in part, to a target site.
  • the quality of fit of such entities to the target site may be judged either by shape complementarity or by estimated interaction energy (see, for example, Meng et al, 1992).
  • the applicant has crystallised the glycosylated form HR of Fc ⁇ RIIa in complex with the Fc fragment of an Fc receptor ligand, namely human IgGl, and has determined the three-dimensional structure of the complex by X-ray crystallography.
  • the elucidation of the interactions between the Fc receptor and the Fc ligand is of use in identifying and modifying agents for modulating the biological activity of Fc receptors.
  • the present invention provides a method for identifying an agent for modulating the biological activity of an Fc receptor protein (FcR), said method comprising the steps of:
  • the present invention provides a method for screening compounds and/ or chemical complexes for a candidate agent for modulating the biological activity of an Fc receptor protein (FcR), said method comprising the steps of:
  • the present invention provides a method for modifying a candidate agent for modulating the biological activity of an Fc receptor protein (FcR), said method comprising the steps of:
  • the present invention provides a method for identifying an agent for modulating the biological activity of an Fc ligand, said method comprising the steps of:
  • the present invention provides a method for screening compounds and/ or chemical complexes for a candidate agent for modulating the biological activity of an Fc ligand, said method comprising the steps of:
  • the present invention provides a method for modifying a candidate agent for modulating the biological activity of an Fc ligand, said method comprising the steps of:
  • the methods of the first to sixth aspects of the invention are preferably in silico methods.
  • the present invention provides a method of designing a variant of high responder Fc ⁇ RIIa (HRsss), a variant of a low responder Fc ⁇ RIIa (LRsss) or a variant of an Fc ligand with altered biological activity, said method comprising the steps of:
  • the present invention provides a computer for producing a three- dimensional structure model of a complex of an Fc receptor protein (FcR) with an Fc ligand or a portion of said complex, said structure model comprising the three- dimensional structure of a target site to which an agent may interact and thereby modulate the activity of an Fc receptor, wherein said computer comprises:
  • a machine-readable data storage medium e.g. a magnetic or optical storage medium such as a hard drive, floppy disc or a CD-ROM
  • a machine-readable data storage medium comprising the atomic coordinate data of Table 4;
  • a working memory for storing instructions for processing said atomic coordinate data contained on the machine-readable data storage medium;
  • a central processing unit coupled to said working memory and to said machine- readable data storage medium for processing said atomic coordinate data to generate said three-dimensional structure model;
  • a display coupled to said central processing unit for displaying a representation of said three-dimensional structure model.
  • the present invention provides a machine-readable data storage medium comprising the atomic coordinate data of Table 4 or a portion thereof.
  • the present invention provides a candidate agent identified in accordance with the method of the first, second, fourth or fifth aspects, an agent produced in accordance with the third or sixth aspects, or a variant of HRsss, a variant of LRs88 or a variant of an Fc ligand designed in accordance with the seventh aspect.
  • the present invention provides the use of the candidate agent, agent or variant of the tenth aspect in the preparation of a medicament for modulating the biological activity of FcR (particularly, Fc ⁇ RIIa) or an Fc ligand in a subject.
  • the present invention provides a method of modulating the biological activity of FcR (particularly, Fc ⁇ RIIa) or an Fc ligand in a subject, said method comprising administering a medicament comprising a candidate agent, agent or variant of the tenth aspect.
  • the present invention provides a method of producing a medicament, wherein said method comprises:
  • the present invention provides a method of treating an Fc receptor-mediated disease or condition in a subject, said method comprising administering to said subject a pharmaceutically-eff ective amount of an agent or a variant of an Fc ligand which binds to a surface on an Fc receptor (FcR) selected from:
  • the present invention provides a method of treating an Fc receptor- mediated disease or condition in a subject, said method comprising administering to said subject a pharmaceutically-eff ective amount of an agent which binds to a surface of an Fc ligand selected from:
  • Figure 1 is reproduced from PCT/ AU2005/000176 and provides a diagrammatic representation of the structure of the predominant twofold dimer of HRsss found in the crystalline state, (a) The HRsss dimer in an orientation suitable for assembly in the membrane of a cell with the IgG binding surfaces of each protein monomer at the top and the terminal polypeptide residues facing toward the plasma membrane (bottom of page), (b) The HRsss dimer has been rotated by 90° to generate this side-on view of the receptor assembly.
  • Figure 2 provides a molecular model of the outside-to-inside signalling or activation complex of Fc ⁇ RIIa.
  • the activation complex was originally predicted using the
  • the dimeric form of HRsss found in the crystal lattice is shown by the modelled signalling complex to be capable of binding simultaneously to two Fc (or antibody) ligand molecules.
  • the amino- (N) and carboxyl- (C) termini of the proteins are indicated.
  • the antigen binding portions of the antibodies (Fab) emerge from the N-termini (hinge regions) of the two bound Fc molecules.
  • Figure 3 provides a representation (C ⁇ -trace) of the crystal structure of the complex of HRs88-Fc ⁇ RIIa and the Fc portion of human IgGl (Fc ⁇ l).
  • Panel a - shows how the FcR binds between the CH2 domains and hinge regions of the two heavy chains of the Fc ligand. Carbohydrate residues are space-filling (CPK) representations.
  • Panel b - shows a close-up view of the interaction between FcR and Fc. The F-G loop on FcR and hinge regions on Fc are indicated.
  • Figure 4 is a schematic model of Fc receptor clustering based on available crystallographic information. The model shows the possible states of receptor in equilibrium between active and inactive states.
  • an equilibrium is predicted from a knowledge of the crystallographic information describing what we predict is an inactive dimer (Maxwell et al 1999), the active dimer PCT/ AU2005/ 000176 and in the description of the crystal structure of the complex of HRsss-Fc ⁇ RIIa and human IgGl described herein.
  • An inactive dimer state shown at left is an equilibrium with a monomeric interacting with IgG shown centrally which in turn can form an active dimer state (at right) with IgG bound to each monomer of the Fc receptor dimer forming an active dimeric state which is able to initiate the ITAM dependant signal transduction cascade.
  • Figure 5 provides solvent-accessible surface views of the of HRsss-Fc ⁇ RIIa complex with Fc ⁇ l. Two views are provided related by an approximately 90° rotation around the long axis of the complex. N-linked carbohydrate on the Fc and FcR are also displayed.
  • Figure 6 shows the binding site on FcR for its Fc ligand as determined by crystallography of the HRsss-Fc ⁇ RIIa: Fc ⁇ l complex.
  • Front and back views of the FcR are provided with the contact residues with Fc shaded on the solvent-accessible surface.
  • the N-linked glycans at positions 64 and 145 are also included, but these do not participate directly in the interaction with Fc.
  • Insert shows a close-up of the FcR residues involved in the interaction with Fc. These residues are considered to form the binding site of Fc ⁇ RIIa for human Fc ⁇ l.
  • Figure 7 shows the residues on Fc ⁇ l contacted by Fc ⁇ RIIa as identified by crystallography of the HRsss-Fc ⁇ RIIaiFc ⁇ l complex.
  • a solvent-accessible view of the Fc ligand is provided with the residues in contact with FcR shaded.
  • Insert shows a close- up of the Fc residues in contact with FcR (note that Fuc designates the residue 802 on chain B).
  • These residues can be considered to form the Fc ⁇ l contact site recognized by Fc ⁇ RIIa. Numbering for the Fc residues is according to the Eu Index (Kabat et al 1991).
  • Figure 8 depicts the locations of the epitopes on Fc ⁇ RIIa that interact with inhibitory monoclonal antibodies.
  • Panel a - shows the epitope (region 1) defined for the IV.3 monoclonal antibody.
  • Panel b - shows the epitope (regions 1 and 2) defined for the 8.7 and 7.30 monoclonal antibodies.
  • Region 1 is defined by FcR residues 132-137 (FSRLDP; SEQ ID NO:1). Note that position 134 is H in LR-Fc ⁇ RIIa.
  • Regions 2 and 3 are defined by FcR residues 112-119 (SWKDKPLV; SEQ ID NO:2) and 157-162 (NIGYTL; SEQ ID NO:3), respectively.
  • Figure 9 depicts MAbs 8.7, 7.30 and IV.3 binding to the Fc ⁇ RIIa domain 2.
  • the Fc ⁇ RIIa, Fc ⁇ RI, and ⁇ and ⁇ chimeric receptors are shown schematically.
  • the mAbs binding by FACS analysis is indicated with (+) positive or (-) negative.
  • Fc ⁇ RIIa domains are represented in grey; Fc ⁇ RI in black.
  • Levels of expression of the chimeric receptor were determined using niAb 47 (murine anti-hFc ⁇ RI domain 2) when there was lacked binding of 7.30, 8.7 and IV.3 mAbs.
  • Figure 10 depicts elucidation of the 8.7 and 7.30 epitopes using Fc ⁇ RIIa/Fc ⁇ RI chimeric receptors binding assay.
  • Dl domain 1;
  • D2 domain 2; (+) positive binding; (-) negative binding; amino acid number in parenthesis indicate the sequences changed. Regions originating from Fc ⁇ RIIa were represented in grey and from Fc ⁇ RI in black. In the absence of binding by 8.7 and 7.30 mAbs, IV.3 mAb was used to confirm the levels of expression of the chimeric receptors. * predicted epitope amino acid residues
  • Figure 11 depicts elucidation of the IV.3 mAb epitope using Fc ⁇ RIIa/Fc ⁇ RI chimeric receptors binding assay.
  • Dl domain 1;
  • D2 domain 2; (+) positive binding; (-) negative binding; amino acid number in parenthesis indicate the changes designation. Regions originated from Fc ⁇ RIIa were represented in grey and from Fc ⁇ RI in black. In the lack of binding by 8.7 and 7.30 mAbs, IV.3 mAb was used to determine the levels of expression of the chimeric receptors. * predicted epitope amino acid residues.
  • the present applicant has determined the structure of the glycosylated form HR of Fc ⁇ RIIa (HRsss-Fc ⁇ RIIa) and found that it forms an active dimer in unliganded crystals. This dimeric form was not mentioned in the analysis of Sonderman et al. (2001) of the non-glycosylated HR Fc ⁇ RIIa crystal structure, in which the authors identified only a monomeric receptor.
  • the applicant has crystallised the glycosylated form HR of Fc ⁇ RIIa in complex with an Fc ligand, namely, human IgGl, and has determined the three- dimensional structure of the complex by X-ray crystallography. The elucidation of the interactions between the Fc receptor and the Fc ligand is of use in, for example, identifying and modifying agents for modulating the biological activity of Fc receptors.
  • the three-dimensional structural information provided by the present invention has further enabled the applicant to model a relationship between the receptor dimer observed in PCT/ AU2005/ 000176 and two molecules of IgGl.
  • This model has shown that two Fc portions of human IgGl are easily accommodated in the dimer of glycosylated HR Fc ⁇ RIIa observed in the C222i crystals of unliganded HR Fc ⁇ RIIa.
  • the model allows for the prediction of the sites of interaction between two molecules of IgGl when bound to a receptor dimer. Identification of the sites of liganddigand interaction is of further use in identifying and modifying agents for modulating the biological activity of Fc receptors.
  • the model has enabled the applicant to conceive of a schematic model for the mechanism of Fc receptor clustering (Figure 4).
  • Fc receptors which can modulate the biological activity of Fc receptors would be of invaluable benefit for the therapy of immunological disorders such as antibody- mediated inflammation, autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis, cancer, infection etc.
  • immunological disorders such as antibody- mediated inflammation, autoimmune diseases such as systemic lupus erythematosus (SLE) and rheumatoid arthritis, cancer, infection etc.
  • SLE systemic lupus erythematosus
  • the present invention provides a method for identifying an agent for modulating the biological activity of an Fc receptor protein (FcR), said method comprising the steps of:
  • the method is for identifying a candidate agent for modulation of the interaction between the monomers of a dimer of HRsss or LRsss / said method comprising the steps of:
  • the method is for identifying a candidate agent for modulation of the interaction between the monomers of a dimer comprising a monomer of HRsss and a monomer of LRsss, said method comprising the steps of:
  • the method is for identifying a candidate agent for modulation of the interaction between an Fc receptor protein (FcR) and an Fc ligand, said method comprising the steps of:
  • said structure model comprises the three-dimensional structure of a target site with which an agent may interact and thereby modulate the biological activity of the receptor, wherein said structure model has been generated from a three- dimensional structure model of a complex of the FcR and an Fc ligand by subsequently removing or discounting the Fc ligand structure;
  • the present invention provides a method for screening compounds and/ or chemical complexes for a candidate agent for modulating the biological activity of an Fc receptor protein (FcR), said method comprising the steps of:
  • a method for screening compounds and/ or chemical complexes for a candidate agent for modulation of the interaction between the monomers of a dimer of HRsss or LRsss comprising the steps of: (i) generating a three-dimensional structure model of a dimer of HRsss or LRsss or a portion thereof in which portions of each monomer are represented, wherein said structure model comprises the three-dimensional structure of a target site with which an agent may interact and thereby modulate the interaction between the monomers; and
  • a method for screening compounds and/ or chemical complexes for a candidate agent for modulation of the interaction between the monomers of a dimer comprising a monomer of HRsss and a monomer of LRsss/ said method comprising the steps of:
  • a method for screening compounds and/ or chemical complexes for a candidate agent for modulation of the interaction between a Fc receptor protein (FcR) and an Fc ligand comprising the steps of:
  • said structure model comprises the three-dimensional structure of a target site with which an agent may interact and thereby modulate the biological activity of the receptor, wherein said structure model has been generated from a three- dimensional structure model of a complex of the FcR and an Fc ligand by subsequently removing or discounting the Fc ligand structure;
  • the present invention provides a method for modifying a candidate agent for modulating the biological activity of an Fc receptor protein (FcR) 7 said method comprising the steps of:
  • a method for modifying a candidate agent for modulation of the interaction between the monomers of a dimer of HRsss or LRsss to provide an agent with improved activity comprising the steps of:
  • a method for modifying a candidate agent for modulation of the interaction between a Fc receptor protein (FcR) and an Fc ligand to provide an agent with improved activity comprising the steps of:
  • said structure model comprises the three-dimensional structure of a target site with which an agent may interact and thereby modulate the biological activity of the receptor, wherein said structure model has been generated from a three- dimensional structure model of a complex of the FcR and an Fc ligand by subsequently removing or discounting the Fc ligand structure;
  • the crystal structure disclosed herein also provides a three-dimensional structure model of the Fc portion of IgGl which is an Fc ligand.
  • This three-dimensional structure model of the Fc portion of IgGl can be used in the selection or design of compounds that can modulate the activity of IgGl and other Fc ligands. Accordingly, in a fourth aspect, the present invention provides a method for identifying an agent for modulating the biological activity of an Fc ligand, said method comprising the steps of:
  • the present invention provides a method for screening compounds and/ or chemical complexes for a candidate agent for modulating the biological activity of an Fc ligand, said method comprising the steps of:
  • the present invention provides a method for modifying a candidate agent for modulating the biological activity of an Fc ligand, said method comprising the steps of:
  • the methods of the first to sixth aspects of the invention are preferably in silico methods.
  • the three-dimensional structure model generated in step (i) of each of the methods of the first to third aspects comprises, at least, the three-dimensional structure of a target site to which a candidate agent or a developed agent (i.e. modified candidate agent) may interact (e.g. bind) with, preferably HRsss or a portion thereof or, otherwise, a complex of HRsss or a portion thereof with an Fc ligand or a portion thereof.
  • the atomic coordinate data for the amino acids within the three-dimensional structure model of a complex of HRsss with an Fc ligand is provided in Table 4 hereinafter.
  • the structure model is preferably generated from a three-dimensional structure model of a complex of the FcR and an Fc ligand by subsequently removing or discounting the Fc ligand structure to reveal the target site.
  • a convenient means for generating such a structure model is to utilise only the atomic coordinate data for the amino acids of the Fc receptor protein provided in Table 4 (i.e. atomic coordinate data for atoms 3415- 4745), or only the atomic coordinate data for the amino acids and linked carbohydrate of the Fc receptor protein provided in Table 4 (i.e. atomic coordinate data for atoms 3415-4745 and 5037-5145).
  • the method of the fourth to sixth aspects comprises the three-dimensional structure model of a target site to which a candidate agent or a developed agent (i.e. modified candidate agent) may interact (e.g. bind) with an Fc ligand or a portion thereof, preferably, the Fc portion of IgGl or a portion thereof.
  • a convenient means for generating such a structure model is to utilise only the atomic coordinate data for the amino acids of the Fc ligand provided in Table 4 (i.e. atomic coordinate data for atoms 1-3414), or only the atomic coordinate data for the amino acids and linked carbohydrate of the Fc ligand provided in Table 4 (i.e. atomic coordinate data for atoms 1-3414 and 4746-5036).
  • the structure model is generated from only the atomic coordinate data for the amino acids and the linked f ucose carbohydrate (residue 802) of the Fc ligand provided in Table 4 (i.e. atomic coordinate data for atoms 1-3414 and 4760-4769); the f ucose residue 802 interacts with the Fc receptor and it has been reported that fucosylated Fc ligands may preferentially bind to certain isof orms of Fc receptors (e.g. the HRsss Fc ⁇ RIIa).
  • the present applicant has recognised that the structural data of the Fc receptor:Fc complex which has been disclosed herein, can be used in combination with the structural data of the unliganded dimer disclosed in PCT/ AU2005/ 000176 in order to provide new insights into the structure of an Fc receptor dimer bound to two Fc ligands. Accordingly, where the method comprises generating a three-dimensional structure model of an FcR or a portion thereof (with or without bound Fc ligand(s)), it is preferable that the structure model comprises a dimer of said FcR or portion thereof.
  • the step of identifying a candidate agent may be achieved by methods described above for designing and selecting compounds or chemical complexes with three-dimensional structures that fit and interact with a target site.
  • the method of the first and fourth aspects may further comprise a step of assessing the deformation of energy of the candidate agent when brought from the free state to the target site-interacting state (e.g. bound state).
  • the deformation of energy is not greater than 10 kcal/mole and, more preferably, not greater than 7 kcal/mole.
  • the method of the first aspect may comprise a step of assessing the enthalpy of the interaction (e.g. binding) of the candidate agent with the target site.
  • the candidate agent shall make a neutral or favourable contribution to the enthalpy of the interaction.
  • the step of screening compounds and/ or chemical complexes to identify any compound(s) or chemical complex(es) with a three- dimensional structure enabling interaction with the target site may be achieved by methods described above.
  • the screened compounds and/ or chemical complexes may belong to a library or database of suitable compounds and/ or chemical complexes (e.g. ACD-SC (Available Chemicals Directory Screening Compounds), MDL Inc, San Leandro, Ca, USA).
  • step of modifying a candidate agent may be achieved by methods described above such as substituting one or more groups (e.g. functional groups) on compounds.
  • the candidate agent and agent is preferably selected from small chemical entities (SCE), peptides or peptido-mimetics, and monoclonal antibodies (and fragments thereof such as Fab and F(ab')2 fragments and scFv).
  • SCE small chemical entities
  • peptides or peptido-mimetics and monoclonal antibodies (and fragments thereof such as Fab and F(ab')2 fragments and scFv).
  • the agents may modulate biological activity by, for example, binding to or mimicking the action of an FcR, mimicking the action of an Fc ligand (e.g. a mimick of an Fc ligand may comprise a peptide fragment of an Fc ligand or a peptido-mimetic of an Fc ligand), disrupting cellular signal transduction through an FcR by, for example, preventing dimerisation of two FcR proteins, or enhancing cellular signal transduction or binding to an FcR by, for example, enhancing dimerisation of two FcR proteins.
  • an Fc ligand e.g. a mimick of an Fc ligand may comprise a peptide fragment of an Fc ligand or a peptido-mimetic of an Fc ligand
  • the agent may also be selected from Fc ligands which may have been modified by one or more random amino acid modifications (e.g. substitution, addition and/ or deletion) to the region of the Fc ligand that forms the surface that interacts with the immunoglobulin-binding site of an FcR.
  • the method may be used to identify a candidate agent by designing or selecting an Fc ligand with one or more random amino acid modifications to the region of the Fc ligand forming the surface that interacts with the immunoglobulin- binding site of an FcR.
  • the agent may also be selected from monoclonal antibodies and fragments thereof such as Fab and F(ab')2 fragments and scFv. Such monoclonal antibodies and fragments thereof may inhibit binding between FcR and an Fc ligand (e.g. by binding to the immunoglobulin-binding site).
  • the agents may modulate biological activity by, for example, binding to an Fc ligand; preventing or enhancing complexation of an Fc ligand to an FcR, or preventing or enhancing interactions between Fc ligands when, for example, two Fc ligands are bound to a dimer of an FcR.
  • the target site is preferably a surface on the HRs88 or LRs88 selected from:
  • site A the surface forming a large groove between two FcR monomers of a dimerised receptor (hereinafter referred to as site A);
  • site B the surface forming a cavity, channel and two identical pockets adjacent to the dimerisation interface between two FcR monomers of a dimerised receptor (hereinafter referred to as site B).
  • two FcR monomers of a dimerised receptor includes: (a) two HRsss monomers, (b) two LRsss monomers, and (c) a HRsss monomer and an LRsss monomer.
  • interface refers to the group of atoms and residues from separate polypeptide chains (e.g. monomer 1 and monomer 2 of Fc ⁇ RIIa) that are in direct contact (i.e. hydrophobic, van der Waals or electrostatic contact) and nearby residues, not necessarily in direct contact, which may be reasonably regarded as contributing to the protein:protein interaction.
  • the surface comprises a structure defined by the conformation of, at least, the amino acid residues 90, 113, 117, 119, 120, 128, 129, 132, 134, 135 and 160. More preferably, the surface comprises a structure defined by the conformation of the amino acid residues 90, 113- 117, 119, 120, 128, 129, 131-135, 155, 156 and 158-160.
  • the surface comprises a structure defined by the conformation of amino acid residues 26, 33, 54-56, 58, 102, 103, 105, 142 and 143 of one monomer of the FcR dimer and the equivalent residues of the other monomer of the dimer.
  • the surface comprises a structure defined by the conformation of amino acid residues 22-24, 60, 107, 109, 110, 112, 114-118, 131, 133-138, 140 and 160 of one monomer of the FcR dimer and the equivalent residues of the other monomer of the dimer.
  • the surface comprises a structure defined by the conformation of amino acid residues 12-16, 26, 96, 100 and 105 of one monomer of the FcR dimer and the equivalent residues of the other monomer of the dimer.
  • Agents which interact may modulate the biological activity of an FcR protein, particularly Fc ⁇ RIIa, by inhibiting or enhancing cellular signal transduction by the receptor or through inhibiting or enhancing binding of the receptor to the Fc portion of an immunoglobulin protein (e.g. IgG) or fragment thereof.
  • an FcR protein particularly Fc ⁇ RIIa
  • an immunoglobulin protein e.g. IgG
  • the target site is preferably a surface of an Fc ligand selected from:
  • the surface which, when the Fc ligand is bound to an Fc receptor, interacts with another Fc ligand similarly bound to an Fc receptor e.g. where the Fc receptor is dimerised.
  • the target site is the surface of the Fc ligand that interacts with the immunoglobulin binding site of an Fc receptor
  • the surface comprises a structure defined by, in the case where the Fc ligand is human IgGl, the conformation of amino acid residues 234-237, 239, 265-267, 297 and 327-329 or corresponding amino acid residues from other Fc ligands. Numbering for Fc residues is according to the Eu Index and corresponding residues of other IgG subclasses can be inferred using this convention and by reference to standard sequence alignments such as (Kabat et al 1991).
  • the target site is the surface which, when the Fc ligand is bound to an Fc receptor, interacts with another Fc ligand similarly bound to an Fc receptor (e.g. where the Fc receptor is dimerised)
  • the surface comprises a structure defined by, in the case where the Fc ligand is human IgGl, by the conformation of amino acid residues 231-233 and 326 or corresponding amino acid residues from other Fc ligands.
  • the Eu index can be used to determine similar residues from other Fc ligands.
  • the present invention provides a method of designing a variant of high responder Fc ⁇ RIIa (HRsss), a variant of low responder Fc ⁇ RIIa (LRsss) or a variant of an Fc ligand, preferably human IgGl, with altered biological activity, said method comprising the steps of:
  • variable we refer to any molecule that differs from HRsss / LRsss or the particular Fc ligand, but which retains similarity in biological activity or, in certain embodiments, has substantially modified biological activity.
  • a variant may therefore have substantial overall structural similarity with HRsss, LRsss or a particular Fc ligand or only structural similarity with one or more regions of HRsss, LRsss or a particular Fc ligand (e.g. a soluble HRsss variant may only have structural similarity to the extracellular region of HRsss).
  • a variant of HRsss, LRsss or a particular Fc ligand will be provided by, or be the result of, one or more amino acid modification such as the addition of one or more amino acids to the amino acid sequence of HRsss / LRsss or the particular Fc ligand, deletion of one or more amino acids from the amino acid sequence of HRsss or LRsss or the particular Fc ligand, and/ or substitution of one or more amino acids of the amino acid sequence of HRsss or LRsss or the particular Fc ligand.
  • Inversion of amino acids and other mutational changes that result in the alteration of the amino acid sequence are also encompassed.
  • substitution of an amino acid may involve a conservative or non-conservative amino acid substitution.
  • conservative amino acid substitution it is meant that an amino acid residue is replaced with another amino acid having similar characteristics and which does not substantially alter the biological function of the polypeptide.
  • Exemplary conservative amino acid substitutions are provided in Table 1 below. Particular conservative substitutions envisaged are: G, A, V, I, L, M; D, E, N, Q; S, C, T; K, R, H: and P, N ⁇ -alkylamino acids.
  • conservative amino acid substitutions will be selected on the basis that they do not have any substantial effect on (a) the structure of the peptide backbone in the region of the substitution, (b) the charge or hydrophobicity of the polypeptide at the site of substitution, and/ or (c) the bulk of the side chain at the site of substitution.
  • the variant may also include an amino acid or amino acids not encoded by the genetic code, such as ⁇ -carboxyglutamic acid and hydroxyproline.
  • D-amino acids rather than L-amino acids may be included.
  • the variant is a mimetic of HRsss such as a peptido- mimetic. Table 1 Exemplary conservative amino acid substitutions
  • Particularly preferred variants which may be designed in accordance with the method of the seventh aspect are soluble HRsss or LRsss variants comprising the extracellular region of FcR, wherein the extracellular region includes one or more amino acid modification such as those mentioned above, such that the HRsss or LRsss variants have enhanced binding for an Fc ligand (i.e. relative to the unmodified extracellular region of the HRsss or LRsss).
  • Such soluble HRsss or LRsss variants with enhanced Fc ligand binding may be particularly useful in the treatment of an FcR-mediated disease or ⁇ condition.
  • variants of monoclonal antibodies wherein the Fc portion includes one or more amino acid modification such as those mentioned above, such that the monoclonal antibody has diminished or enhanced binding of FcR.
  • a method of designing a variant of a dimer of HRsss or LRsss with altered biological activity comprising the steps of:
  • a method of designing a variant of a dimer comprising a monomer of HRsss and a monomer of LRsss with altered biological activity comprising the steps of:
  • the method of the seventh aspect of the invention is preferably an in silico method.
  • the method of the seventh aspect therefore provides a means for designing variants of HRs88 or LRs88 or an Fc ligand which may have substantially modified biological activity by analysing the structure and interactions between individual amino acids of the FcR or Fc ligand, and thereafter introducing one or more amino acid modifications to modify those interactions.
  • variants of HRsss or LRsss can be designed with enhanced binding to immunoglobulin proteins or immune complexes of immunoglobulin proteins for use as therapeutic compounds to prevent immune complex binding to cells or enhance biological responses such as cellular signal transduction upon binding of FcR to immunoglobulin proteins or complexes thereof.
  • recombinant soluble FcR engineered to embody certain improvements can be produced on the basis of the knowledge of the three-dimensional structure of FcR and their interaction with Fc ligands.
  • recombinant soluble Fc ligands e.g. recombinant soluble human IgGl
  • engineered to embody certain improvements can be produced on the basis of the knowledge of the three-dimensional structure of Fc ligands and their interaction with the FcR.
  • an Fc ligand which may be designed in accordance with method of the seventh aspect, is a monoclonal antibody wherein the Fc portion includes one or more amino acid modification such that the monoclonal antibody has diminished or enhanced binding of FcR.
  • PCT/US2003/030249 Publication No WO 2004/029207), the entire disclosure of which is to be regarded as incorporated herein by reference, methods of generating optimised Fc ligand variants are proposed.
  • the present invention provides structural information which can be used to direct and focus the preparation of optimised Fc variants using the methods described in PCT/ US2003/ 030249 (i.e.
  • the structural information described herein may be used to identify and select Fc ligands such as the Fc portions or regions, as they are referred to in PCT/ US2003/ 030249, of immunoglobulins, or groups of Fc ligands for further screening, which may show diminished or enhanced binding of FcR, and it is to be understood that such use of the structural information is encompassed by the present invention).
  • FcR binding activity of an Fc portion of a monoclonal antibody may be enhanced such that recruitment of Fc ⁇ RIIa-carrying macrophages to a cell carrying the antigen to which the monoclonal antibody binds is encouraged. This can have important therapeutic implications.
  • the effectiveness of such antibodies might be improved by enhancing their ability to recruit macrophages (i.e. through enhancing binding of the Fc portion to Fc ⁇ RIIa) to the target tumour cells.
  • the binding activity of the Fc portion of a therapeutic antibody may be diminished or even removed; this might find application in, for example, the therapeutic use of anti-TNF- ⁇ monoclonal antibodies.
  • Fc ligands wherein the FcR binding activity has been removed may also provide a useful carrier protein in the production of fusion proteins.
  • the three-dimensional structure model generated in step (i) of the method of the seventh aspect is generated using at least a portion of the atomic coordinate data of Table 4.
  • a recombinant protein according to a variant of HRsss, or a dimer thereof (or LRsss or dimer thereof), or a dimer of HRsss and LRsss, or an Fc ligand (e.g. human IgGl), may be prepared by any of the methods well known to the person skilled in the art.
  • the recombinant protein may be prepared by firstly generating a DNA molecule encoding the variant protein by site- directed mutagenesis of a DNA molecule encoding the Fc receptor (e.g. HRsss), and thereafter expressing the DNA molecule in a suitable host cell.
  • a DNA molecule encoding Fc ⁇ RIIa and methods for expressing DNA molecules encoding Fc ⁇ RIIa and variants thereof (including soluble variants), are disclosed in International patent application no PCT/ AU87/00159 (Publication no WO 87/07277) and International patent application no PCT/ AU95/ 00606 (Publication no WO 96/08512). The disclosures of these two International patent applications are to be regarded as incorporated herein by reference.
  • the present invention provides a computer for producing a three- dimensional structure model of a complex of an Fc receptor protein (FcR) with an Fc ligand or a portion of said complex (particularly, a portion comprising FcR or, alternatively, a portion comprising the Fc ligand), said structure model comprising the three-dimensional structure of a target site to which an agent may interact and thereby modulate the activity of an Fc receptor, wherein said computer comprises:
  • a machine-readable data storage medium e.g. a magnetic or optical storage medium such as a hard drive, floppy disc or a CD-ROM
  • a machine-readable data storage medium comprising the atomic coordinate data of Table 4;
  • the computer may further comprise:
  • the atomic coordinate data for the range of chemical components and substituents and the atomic coordinate data for the range of compounds and/ or chemical complexes can be obtained from suitable databases.
  • the present invention provides a machine-readable data storage medium comprising the atomic coordinate data of Table 4 or a portion thereof (e.g. the atomic coordinate data for the amino acids of the Fc receptor protein only, or the atomic coordinate data for the amino acids of the Fc ligand only).
  • the present invention provides a candidate agent identified in accordance with the method of the first, second, fourth or fifth aspects, an agent produced in accordance with the third or sixth aspects or a variant of HRsss, a variant of LRs88 or a variant of an Fc ligand (e.g. human IgGl) designed in accordance with the seventh aspect.
  • the candidate agent, agent or variant of the tenth aspect may be used to prepare a medicament to modulate the biological activity of FcR (in particular, an FcR selected from Fc ⁇ R, Fc ⁇ R, Fc ⁇ R such as Fc ⁇ RIIa, Fc ⁇ RIIb and Fc ⁇ RIIc, and mixtures thereof) or an Fc ligand in a subject.
  • the medicament can be used for, for example, reducing IgG- mediated tissue damage; stimulating an IgG humoral immune response in an animal; and improving the therapeutic effects of an antibody that is administered to an animal to treat, by opsonisation or Fc ⁇ R-dependent effector functions (e.g. antibody-dependent Fc ⁇ R-mediated cytotoxicity, phagocytosis or release of cellular mediators), a particular disease, including, but not limited to, inflammatory diseases, autoimmune diseases, cancer or infectious disease (e.g. oral infections such as HIV, herpes, bacterial infections, yeast infections or parasite infections).
  • a particular disease including, but not limited to, inflammatory diseases, autoimmune diseases, cancer or infectious disease (e.g. oral infections such as HIV, herpes, bacterial infections, yeast infections or parasite infections).
  • the agent of the tenth aspect is selected from small chemical entities (SCE), peptides and peptide-mimetics, and monoclonal antibodies or fragments thereof such as Fab and F(ab')2 fragments and scFv.
  • SCE small chemical entities
  • peptides and peptide-mimetics and monoclonal antibodies or fragments thereof such as Fab and F(ab')2 fragments and scFv.
  • the present invention provides the use of the candidate agent, agent or variant of the tenth aspect in the preparation of a medicament for modulating the biological activity of FcR (particularly, Fc ⁇ RIIa) or an Fc ligand (particularly, human IgG) in a subject.
  • the present invention provides a method of modulating the biological activity of FcR (particularly, Fc ⁇ RIIa) or an Fc ligand (particularly, human IgG) in a subject, said method comprising administering a medicament comprising a candidate agent, agent or variant of the tenth aspect.
  • the subject referred to in the eleventh and twelfth aspects may be a human or other animal (e.g. companion animals and livestock).
  • the candidate agent, agent or variant of the tenth aspect may be formulated with any pharmaceutically-acceptable delivery vehicle or adjuvant for administration to the subject.
  • Administration may be by any suitable mode including, for example, intramuscular injection, intravenous administration, nasal administration via an aerosol spray, and oral administration.
  • the amount of the candidate agent, agent or variant of the tenth aspect that may be administered to a subject may vary upon a number of factors including the immune status of the subject and the severity of any disease or condition being treated.
  • an agent according to the seventh aspect may be administered to a subject at a dose of about 0.001 to 10 mg /kg body weight, preferably from 0.1 to 1 mg/kg body weight.
  • the present invention provides a method of producing a medicament, wherein said method comprises:
  • the agent is a small chemical entity, peptide or peptido-mimetic
  • the agent can be synthesised by any suitable method known to the person skilled in the art of chemical synthesis.
  • the agent is a peptide or protein (e.g. a monoclonal antibody)
  • the agent can be prepared by any suitable method known to the person skilled in the art including recombinant DNA technology.
  • the present invention provides a method of treating an Fc receptor-mediated disease or condition in a subject, said method comprising administering to said subject a pharmaceutically-eff ective amount of an agent or an Fc ligand which binds to a surface on an Fc receptor (FcR) selected from:
  • two FcR monomers of a dimerised receptor includes: (a) two HRs88 monomers, (b) two LRsss monomers, and (c) a HRsss monomer and an LRsss monomer.
  • the agent or Fc ligand in binding to one of said surfaces on FcR, causes inhibition of binding of immunoglobulin to FcR.
  • the FcR referred to in the fourteenth aspect is selected from the group consisting of Fc ⁇ R, Fc ⁇ R, Fc ⁇ R (e.g. Fc ⁇ RIIa, Fc ⁇ RIIb and Fc ⁇ RIIc) and mixtures thereof.
  • the said FcR is Fc ⁇ RIIa.
  • the present invention provides a method of treating an Fc receptor- mediated disease or condition in a subject, said method comprising administering to said subject a pharmaceutically-effective amount of an agent which binds to a surface of an Fc ligand selected from: (a) the surface that interacts with the immunoglobulin binding site of an Fc receptor; and
  • the Fc ligand referred to in the fifteenth aspect is a human IgG (e.g. IgGl, IgG2, IgG3 and IgG4).
  • the FcR-mediated disease or condition which may be treated by the method of the fourteenth or fifteenth aspects may be selected from the group consisting of; IgG- mediated tissue damage, IgE-mediated diseases or conditions, inflammation, an autoimmune disease (e.g. rheumatoid arthritis, systemic lupus erythematosus, immune thrombocytopenia, neutropenia, and haemolytic anaemias).
  • an autoimmune disease e.g. rheumatoid arthritis, systemic lupus erythematosus, immune thrombocytopenia, neutropenia, and haemolytic anaemias.
  • the method of the fourteenth and fifteenth aspects may also be used to treat an FcR- mediated disease or condition wherein aggregates of antibodies are produced or where immune complexes are produced by contact of antibody with intrinsic or extrinsic antigen.
  • diseases include immune complex diseases, autoimmune diseases, cancers or infectious diseases (e.g. Dengue virus-dengue hemorrhagic fever and measles virus infection) and vasculitities (e.g. polyarteritis nodosa, and systemic vasculitis).
  • Table 1 provides a list of conservative amino acid substitutions.
  • Table 2 provides a summary of statistics for the X-ray data and crystallographic refinements used for structure determination of the HRs88Fc ⁇ RIIa:Fc complex.
  • Data from the HRsss Fc ⁇ RIIa:Fc co-crystal were obtained using a MicroMax007/R-Axis IV ++ rotating anode X-ray generator system operated at 40 kV and 20 mA.
  • Data was reduced and scaled using the DENZO and Scalepack programs from the HKL suite version 1.97 (HKL Research Inc, USA).
  • the crystal structure was solved and refined using the CNS program package version 1.0 (Brunger et al, 1998);
  • Table 3 provides interatomic distances less than 4 A defining the interface between the HRsss-Fc ⁇ RIIa and its authentic IgG (Fc) ligand.
  • the chains are designated as follows: (Chain A) the first heavy chain component of the Fc ligand; (Chain B) the second heavy chain component of the Fc ligand; and, (Chain C) the extracellular domains of the HRs88-Fc ⁇ RIIa.
  • the distances were calculated from the attached coordinates for the refined molecular replacement solution for the HRsss-Fc ⁇ RIIa :Fc complex (Table 4).
  • Table 4 provides the atomic coordinates for the crystal structure of the HRs88-Fc ⁇ RIIa:Fc complex.
  • Table 5 provides results of in vitro biological activity results for two compounds selected in accordance with the present invention.
  • Table 6 provides the nomenclature for receptor proteins and chimeras mentioned in Example 4.
  • HRsss-Fc ⁇ RIIa was prepared by the methods set out in PCT/ AU2005/00176.
  • the Fc portion was prepared from plasmin proteolysis of human IgGl. Purified recombinant hu3S193 IgGl at 5 mg/ml was dialyzed against 0.15 M NaCl, 50 mM Tris pH 8.0 and was digested with 1 u/ml plasmin (Roche, Basel, Switzerland) for 48hrs at 21 0 C. The Fc was purified to homogeneity by separation from Fab by protein-A sepharose, followed by removal of residual intact IgGl by protein-L sepharose.
  • Crystals of the HRsss-Fc ⁇ RIIa in complex with the Fc fragment of IgGl were successfully produced by the method of vapour diffusion. Large thin plates with growth habits of single crystals and crystalline clusters were produced in 2 ⁇ l sitting and hanging droplets in 2 to 4 weeks at 18°C.
  • the crystallisation droplets contained 1 ⁇ l of the protein solution (containing 5.5 mg/ml with the HRsss-Fc ⁇ RIIa mixed with purified Fc in a 1:1 molar ratio) was mixed with 1 ⁇ l of the reservoir solution that contained 0.2 M trisodium citrate or tripotassium citrate and 20% PEG 3350.
  • the crystals were flash-cooled in the reservoir solution supplemented with 20-30% (v/v) glycerol in a stream of liquid nitrogen.
  • Datasets were collected by the oscillation method (Otwinowski and Minor, 1997) with a crystal-to-detector distance of between 150 mm and 200 mm with delta-phi slices of 0.5 to 1.5° per diffraction image.
  • the crystal structure was solved by the method of Molecular Replacement using the coordinates for the unliganded form of the HRsss-
  • the crystal structure of HRsss-Fc ⁇ RIIa in complex with an Fc ligand contains the detailed atomic information that relates the footprint of both the receptor and Fc ligand that forms a unique interface ( Figures 3, 5-7 and Tables 3-4) for targeting of agents to modulate the biological activity of an FcR.
  • knowledge of the surface comprising the amino acid residues forming the interface i.e. the immunoglobulin- binding site
  • SCE small chemical entities
  • the dimeric form of FcR disclosed are regarded as representing conformational isomers of the FcR and FcR:Fc complex that can occur on the surface of cells.
  • a scheme based upon the various crystal structures is presented in Figure 4. With reference to that figure, a previously described structure for Fc ⁇ RIIa (see PCT/ IB99/ 00367 (Publication no WO 99/40117)) is now believed to represent a resting or inactive state of the FcR since it is incapable, for steric reasons, of binding an Fc ligand.
  • the crystal structure of the complex of HRsss-Fc ⁇ RIIa and the Fc ligand i.e.
  • human IgGl describes a monomeric form of a ligand-bound state of the FcR as described by the atomic coordinate data provided in Table 4.
  • This monomeric FcR:Fc complex is thought to reassemble in the membrane to form an active dimeric state since it is generally agreed that all ITAM-bearing signalling molecules occur as dimers or higher order biological assemblies.
  • Evidence of such oligomeric states exist can be deduced from publications such as Shields et at which describes mutations to the immunoglobulin G molecule and the effect of such mutations on the interaction with Fc ⁇ RII.
  • mutations in CH3 region notably lysine 414 that reduce binding to Fc ⁇ RII.
  • the CH3 domain and in particular lysine 414 is very distant from the Fc receptor binding site and could not contact nor cause a conformational change within the distant CH2 which contains the residues in contact with Fc ⁇ RII.
  • Table 3 Provides interatomic distances less than 4 A defining the interface between the HRsss-Fc ⁇ RIIa and its authentic IgG (Fc) ligand.
  • ATOM 8 CA PRO A 232 92.691 37.017 30.793 1.00 95.38 A
  • ATOM 369 CA TYR A 278 90.013 37.746 66.801 1.00 2.35 A
  • ATOM 472 CA PRO A 291 78.875 40.278 55.205 1.00 39.74 A
  • ATOM 542 CA THR A 299 86.909 41.163 40.223 1.00 51.12 A
  • ATOM 642 CD GLN A 311 80.228 51.255 75.399 1.00 63.28 A

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Abstract

L'invention porte sur la détermination de la structure tridimensionnelle de complexes récepteurs Fc/ligands Fc, en particulier Fc?RIIa avec l'IgGI Fc, par cristallographie aux rayons X. On utilise la structure pour identifier, modifier ou modéliser des agents permettant de moduler l'activité biologique du récepteur Fc ou du ligand Fc, ou pour concevoir des récepteurs Fc ou des ligands Fc variants présentant une activité biologique modifiée.
PCT/AU2006/000813 2005-06-14 2006-06-14 Structures cristallines et modeles de complexes fc/recepteurs fc et utilisations de ces derniers WO2006133486A1 (fr)

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US10919953B2 (en) 2012-08-24 2021-02-16 Chugai Seiyaku Kabushiki Kaisha FcgammaRIIB-specific Fc region variant
US11142563B2 (en) 2012-06-14 2021-10-12 Chugai Seiyaku Kabushiki Kaisha Antigen-binding molecule containing modified Fc region
US11267868B2 (en) 2013-04-02 2022-03-08 Chugai Seiyaku Kabushiki Kaisha Fc region variant

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EP2078091A4 (fr) * 2007-07-10 2010-09-01 Medimmune Llc Cristaux et structure du variant d'igg fc humain
RU2719132C2 (ru) * 2011-06-30 2020-04-17 Чугаи Сейяку Кабусики Кайся Гетеродимеризованный полипептид
US11142563B2 (en) 2012-06-14 2021-10-12 Chugai Seiyaku Kabushiki Kaisha Antigen-binding molecule containing modified Fc region
US10919953B2 (en) 2012-08-24 2021-02-16 Chugai Seiyaku Kabushiki Kaisha FcgammaRIIB-specific Fc region variant
US10766960B2 (en) 2012-12-27 2020-09-08 Chugai Seiyaku Kabushiki Kaisha Heterodimerized polypeptide
US11267868B2 (en) 2013-04-02 2022-03-08 Chugai Seiyaku Kabushiki Kaisha Fc region variant
CN107249624A (zh) * 2015-02-27 2017-10-13 默沙东公司 抗人类pd‑1单克隆抗体的晶体
EP3261663A4 (fr) * 2015-02-27 2018-10-03 Merck Sharp & Dohme Corp. Cristaux d'anticorps monoclonaux anti-pd-1 humains
US11014986B2 (en) 2015-02-27 2021-05-25 Merck Sharp & Dohme Corp. Crystals of anti-human PD-1 monoclonal antibodies
CN107249624B (zh) * 2015-02-27 2023-01-31 默沙东有限责任公司 抗人类pd-1单克隆抗体的晶体

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