WO2003095487A2 - Complexes immunogenes - Google Patents

Complexes immunogenes Download PDF

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Publication number
WO2003095487A2
WO2003095487A2 PCT/GB2003/002069 GB0302069W WO03095487A2 WO 2003095487 A2 WO2003095487 A2 WO 2003095487A2 GB 0302069 W GB0302069 W GB 0302069W WO 03095487 A2 WO03095487 A2 WO 03095487A2
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Prior art keywords
atom
cdl
ligand complex
leu
ligand
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PCT/GB2003/002069
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English (en)
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WO2003095487A3 (fr
Inventor
Vincenzo Cerundolo
Stephan Gadola
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Isis Innovation Limited
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Priority to AU2003227935A priority Critical patent/AU2003227935A1/en
Publication of WO2003095487A2 publication Critical patent/WO2003095487A2/fr
Publication of WO2003095487A3 publication Critical patent/WO2003095487A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70539—MHC-molecules, e.g. HLA-molecules
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Definitions

  • the present invention relates to immunogenic complexes. Particularly, but not exclusively, the present invention relates to the production and use of type II glycoproteins displaying glycolipid and phospholipid antigens .
  • the GDI gene cluster on human chromosome lq22-23 encodes a family of five type II glycoproteins which are expressed on the cell surface in association with beta-2- microglobulin ( ⁇ 2m). According to their amino acid sequence homology the five GDI isoforms segregate into group 1, containing GDla,” b, c, and CDle, and group 2, containing CDld. While GDI group 1 molecules are not present in mice and rat, CDld is conserved in all mammalian species studied to date.
  • CD1 molecules are non-polymorphic. It was probably for this reason, that their role as antigen presenting molecules remained unrecognised for more than a decade after their initial description. The lack of polymorphism indicates that, as opposed to HLA class I and class II genes, the CD1 genes are subjected to very weak evolutionary pressure. Two features of CD1 molecules offer an explanation for this phenomenon. Firstly, GDI molecules do not present peptides as HLA class I and HLA class II molecules do, but they present glycolipids and phospholipids to T lymphocytes. Immune escape of microbes from HLA class I or class II-mediated responses can simply be achieved with functionally irrelevant changes in protein sequences abrogating binding and/or presentation of immunogenic peptides.
  • lipid antigens cannot be easily mutated because lipids are end products of highly complex biosynthetic pathways, and because their physicochemical properties, which are essential to the organism, rely on their correct structure.
  • a second characteristic ' of GDI molecules which could also lower the ligand-induced evolutionary pressure on CD1, is their apparent high degree of ligand binding adaptability.
  • human CDlb can present the carbohydrate epitope of either a mycobacterial derived eighty carbon containing (C80) glucomonomycolate (GMM) or a shorter thirty-two carbon (C32) synthetic GMM to the same T cell receptor, suggesting either adaptive conformational changes within the GDI antigen binding groove or simply protrusion of the longer alkyl chain of the ligand at one end of the groove.
  • the crystal structure of mouse CDld which was determined in the absence of ligand, revealed two electrostatically neutral voluminous pockets A' and F' , suited to bind the alkyl chains of CDl lipid ligands .
  • WO 01/94949 relates to compositions and methods for identifying GDI-antigens and CDl-restricted T cells.
  • the compositions include soluble CDl molecules.
  • a method for identifying a CDl-restricted T cell comprising contacting a CDl-presented antigen complex with a putatative CDl- restricted T cell is disclosed.
  • WO 96/12190 relates to the presentation of hydrophobic antigens to T-cells by CDl molecules and discloses the identification of human CDla, CDlb, CDlc, CDld and CDle .
  • WO 95/00163 relates to a method for isolating CDl- presented antigens from a sample.
  • the inventors subsequently obtained two crystal structures of human CDlb, one with phosphatidylinositol (PI) at 2.26A and another with ganglioside GM2 at 2.8 A.
  • the mode of ligand binding was almost identical for both structures.
  • the C16 alkyl chain detergent used in the inventor's refolding protocol acted as an additional ligand in both structures by binding to those areas of the CDlb-binding groove that were not occupied by the glycolipid or phospholipid ligands.
  • the inventors generated fluorescent tetramers of human CDld molecules loaded with the synthetic glycolipid alpha-galactosylceramide (aGC) . These tetramers were shown by the inventors to specifically stain a human immunoregulatory T-lymphocyte population, i.e. invariant NKT cells.
  • aGC glycolipid alpha-galactosylceramide
  • the invention provides a method for producing a CDl/ligand complex; methods of diagnosing or treating patients using said CDl/ligand complex; and screening methods for determining new therapeutic targets on the CDl complex, following determination of its crystal structure.
  • a method of producing correctly folded recombinant CDl molecules around various lipid ligands such as gangliosides, phospholipids and glycosylceramides .
  • the resulting CDl/ligand complex is biologically active, i.e. the ligand is displayed correctly by the CDl molecule so that it can be presented to immune components, e.g. T lymphocytes .
  • CDla and CDlb have been successfully refolded using immobilised enzymes.
  • immobilised enzymes there was no evidence of ligand being present in the refolded molecules.
  • the inventors have devised a novel approach using single chain detergents. This approach is inexpensive and can easily be scaled up for industrial purposes.
  • the inventors have further provided direct evidence that the lipid ligand is bound to the hydrophobic groove of CDl molecules.
  • CDl/ligand complex said method comprising the steps of: a) obtaining a denatured CDl protein; b) contacting said denatured CDl protein with ligand in an environment comprising detergent; and c) isolating said CDl/ligand complex.
  • the CDl protein is fully denatured and reduced to ensure that the protein is unfolded when initially contacted with ligand.
  • the presence of native disulfide bridges would not prevent the protein from refolding with ligand.
  • the ligand may be any lipid, but most preferably, it is a glycolipid (e.g.
  • the CDl molecule is capable of displaying lipids of various sizes.
  • the molecule is preferably anything up to 60 carbons in length, e.g. between 5, 10 or 15 and 30, 40 or 50 carbons in length.
  • the molecule may contain anything between 18 carbons to 100 carbons, e.g. between 10, 20, 30 or 40 and 70, 80, or 90 carbons in length.
  • the detergent is a single chain detergent, such as acyclic single alkyl chain detergents with chain length C2-C60; sphingosines; ceramides with truncated alkyl chains; diacylglycerol-type lipids with truncated alkyl chains; and triacylglycerol-type lipids with truncated alkyl chains.
  • CTAB cetyltrimethylammonium bromide
  • the method preferably further comprises the step of removing excess detergent from the environment prior to isolation of the CDl/ligand complex.
  • the excess detergent which has not stably incorporated into the protein structure may be removed by adding methylated or unmethylated beta-cyclodextrin in molar excess over detergent.
  • the inventors prefer to use at least 12 molar excess of ethyl-beta-cyclodextrin.
  • other methods will be known to the person skilled in the art. For example, other cyclodextrins (alpha and gamma) which vary in the size of their hydrophobic cavity.
  • the inventors preferred methyl-beta-cyclodextrin but other cyclodextrins will work in accordance with the invention.
  • Other methods for stripping off the excess detergent include the use of resins or dialysis.
  • the method according to the first aspect of the invention may be carried out using any CDl molecules. However, preferably, the method is carried out on CDld, CDlc and most preferably on CDlb.
  • an aqueous buffer comprising Urea, L-Arginine, a buffer (e.g. Tris), and a redox- system (e.g. oxidized and reduced glutathione) .
  • the buffer constituents can be varied: L-Arginine can be used in various concentrations from lOOmM, preferably at a concentration between lOOmM to IM, but may not even be essential to get a certain yield of refolded CDl.
  • Urea which can certainly be used in concentrations ranging from OM to 4M. At the higher concentrations a dialysis step may be necessary.
  • glutathione another redox system such as cysteamine- cystin, could be used and the molarities of glutathione can be varied.
  • the CDl protein may be engineered to contain one or more biotinylation sites providing a means for complexing CDl proteins using avidin to obtainmultimeric, e.g. dimer, trimer, tetramer, forms of CDl which may be useful in amplifying output signals in methods and assays for identifying ligand specific T-cells or antibodies.
  • the CDl protein may be complexed with the Fc portion of a selected immunoglobulin.
  • the complex may be formed by using binding partners (biotin - avidin) or by chemical means (covalent, di-sulfide, H-bonds) .
  • the method of the first aspect may further comprise the step of labelling the CDl protein with a chemical marker, more preferably a fluorescent compound, e.g. RPE or FITC.
  • a chemical marker more preferably a fluorescent compound, e.g. RPE or FITC.
  • the labelled CDl protein useful in identification of specific T-cell populations and for diagnosis of specific disease states.
  • mulitmers, e.g dimers, trimers or tetramers, of labelled CDl/ligand complex may be used.
  • the method may further comprise the step of incorporating said CDl/ligand complex into a pharmaceutical composition.
  • the pharmaceutical composition may comprise, in addition to one of the above substances, a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art.
  • Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient.
  • the precise nature of the carrier or other material may depend on the rate of administration, e.g. oral, intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
  • the pharmaceutical composition may be used as a vaccine to boost the immune response in an individual.
  • the composition further comprises a suitable adjuvant.
  • the pharmaceutical composition according to the present invention may be used to elicit cellular immune responses including lipid-specific CD4 + CD8 " , CD4 " CD8 + , and CD4 " CD8 ⁇ T cell responses.
  • antibody responses to the pharmaceutical composition would also be of therapeutic value.
  • the present invention provides a pharmaceutical composition comprising a CDl/ligand complex for treating diseases such as infectious diseases caused by parasites, mycobacteria, fungi, and bacteria; tumours and autoimmune diseases such as multiple sclerosis.
  • the ligand will be a lipid that is capable of inducing an immune response, against substances (e.g. tumour cells, bacteria, myobacteria etc.) associated with the disease.
  • ligands may be a) Mycobacterial cell wall lipids: Glycosyl-esters of mycolic acid (glucomonomycolate, mannose-monomycolate, etc.) Phosphatidylinositomannosides (PIM2 to PIM6) , as well as synthetic lipids modelled after mycobacterial cell wall lipids (synthetic glucomonomycolate, etc.) b) ganglioside lipids such as GM1 or GM2 or GM3, etc.; Also diacylglycerol-type bacterial cell wall lipids; c) sulfatide; d) trypanosomal phospholipids; malarial cell wall lipids .
  • ligands may be a) Mycobacterial cell wall lipid
  • embodiments of the present invention provide a CDl/ligand complex for use in the preparation of a medicament for treating infectious diseases caused by parasites, mycobacteria, fungi, and bacteria; solid tumours; and autoimmune diseases.
  • a CDl/ligand complex produced in accordance with the first aspect of the invention and a method of medical treatment comprising administering the CDl/ligand complex in therapeutically effective amounts are provided.
  • a method of inducing or boosting an immune response in an individual to a lipid antigen comprising administering a CDl/ligand complex to said individual wherein the ligand in the CDl/ligand complex is said lipid antigen.
  • the CDl/ligand complex may be produced according to the first aspect of the invention.
  • the method may further comprise identifying the lipid antigen associated with a disease of the individual, i.e. cancer, infectious disease or autoimmune disease.
  • the method may comprise identifying a lipid antigen over-expressed on the surface of tumour cells present in the individual.
  • the identified lipid antigen may then be folded into a CDl molecule in accordance with the first aspect of the invention thereby producing a CDl/ligand complex which may be administered as a vaccine to said individual to raise an immune response against said tumour.
  • the CDl ligand complex may further comprise a toxin which is capable of disrupting an immune response raised against the lipid antigen.
  • a toxin- conjugated CDl/ligand complex may be used to eliminate specific T lymphocytes which interacted with the complex. Autoimmune T lymphocytes represent a therapeutic target for such toxin-conjugated CDl/ligand complexes.
  • the CDl/ligand complex according to the present invention may also be used to diagnose a disease in an individual, by identifying the presence or absence of an immune response, i.e. CD/1 lipid-specific T-lymphocytes or antibodies, to a particular lipid antigen.
  • an immune response i.e. CD/1 lipid-specific T-lymphocytes or antibodies
  • a sample may be obtained from an individual, and contacted with a CDl/ligand complex of the invention. If the CDl/ligand complex was being used to identify the presence of an autoimmune disease, the complex would display a lipid antigen associated with this disease. If the sample comprised T-lymphocytes or antibodies already primed to the lipid antigen, these will be detected by the CDl/ligand complex contacted with the sample. Standard labelling techniques may be used to identify any binding between the CDl/ligand complex and the immune components in the sample.
  • the CDl/ligand complex may be provided as a monomer, di er, trimer, tetramer etc. Complexes may be joined by standard means known in the art, e.g. using binding partners (biotin - avidin) or chemical means (co-valent, di-sulfide, hydrogen bonds), so that the ligand is displayed and could be recognised by the T cell receptor.
  • binding partners biotin - avidin
  • chemical means co-valent, di-sulfide, hydrogen bonds
  • the inventors have prepared a crystal of CDl/ligand complex and determined the crystal structure of CDl and CDl/ligand complex. This provides for the first time methods of identifying or obtaining substances (e.g. agonists or antagonists) for modulating the activity of CDl or CDl/ligand complex. Crystal structure information presented herein is useful in designing potential inhibitors and modelling them or their potential interaction with a CDl or CDl/ligand complex binding cavity.
  • substances e.g. agonists or antagonists
  • Potential modulating substances may be brought into contact with CDl or CDl/ligand complex to test for ability to interact with the CDl binding cavity.
  • Actual substances may be identified from among potential substances synthesized following design and model work performed in silico .
  • a substance identified using the present invention may be formulated into a composition, for instance a composition comprising a pharmaceutically acceptable excipient, and may be used in the manufacture of a medicament for use in a method of treatment.
  • the ligand is a lipid, more preferably a glycolipid or a phospholipid.
  • a 87.5 A ⁇ 0.2%
  • the crystal structure of CDl and CDl/ligand complex has the three dimensional atomic co-ordinates of Table 1.
  • the ligand is phosphatidylinositol (PI) or ganglioside GM2 and the CDl molecule is CDlb, CDlc or CDld, preferably CDlb.
  • a method for growing the crystal of the fourth aspect by sitting drop crystallisation using a precipitant comprising 0.2M Lithium nitrate and 20% w/v Polyethylene Glycol is also provided.
  • the coordinates of Table 1 provide a measure of atomic location in Angstroms (A), to a third decimal place.
  • the coordinates are a relative set of positions that define a shape in three dimensions, but the skilled person would understand that an entirely different set of coordinates having a different origin and/or axes could define a similar or identical shape.
  • the skilled person would understand that varying the relative atomic positions of the atoms of the structure so that the root mean square deviation of the residue backbone atoms (i.e.
  • the nitrogen-carbon-carbon backbone atoms of the protein amino acid residues is less than 1.5 A (preferably less than 1.0 A and more preferably less than 0.5 A) when superimposed on the coordinates provided in Table 1 for the residue backbone atoms, will generally result in a structure which is substantially the same as the structure of Table 1 in terms of both its structural characteristics and potency for structure-based design of CDl inhibitors.
  • Table 1 in terms of both its structural characteristics and potency for structure-based design of CDl inhibitors.
  • changing the number and/or positions of the water molecules and/or substrate molecules of Table 1 will not generally affect the potency of the structure for structure-based design of CDl inhibitors.
  • the Table 1 coordinates are transposed to a different origin and/or axes; the relative atomic positions of the atoms of the structure are varied so that the root mean square deviation of residue backbone atoms is less than 1.5 A (preferably less than 1.0 A and more preferably less than 0.5 A) when superimposed on the coordinates provided in Table 1 for the residue backbone atoms; and/or the number and/or positions of water molecules and/or substrate molecules is varied.
  • Reference herein to the coordinate data of Table 1 thus includes the coordinate data in which one or more individual values of the Table are varied in this way.
  • root mean square deviation we mean the square root of the arithmetic mean of the squares of the deviations from the mean.
  • varying the atomic positions of the atoms of the structure by up to about 0.2 A in any direction will result in a structure which is substantially the same as the structure of Table 1 in terms of both its structural characteristics and utility e.g. for structure-based drug design.
  • the provision of the high resolution structure of Table 1 provides those of skill in the art with a detailed insight into the mechanisms of action of CDl or CDl/ligand complex. This insight provides a means to design new substances which have the potential to modulate, e.g. inhibit or enhance the process by which CDl presents ligand to the immune system.
  • CDl/ligand complex allows a novel approach for drug discovery for modulators of this enzyme. Accordingly, in a fifth aspect of the invention a computer-based method of rational drug design is provided comprising the steps of: providing the structure of the CDl or CDl/ligand complex as defined by the coordinates of Table 1; providing the structure of a candidate modulator molecule; and fitting the structure of the candidate modulator molecule to the structure of the CDl or CDl/ligand complex of Table 1.
  • the method of the invention may utilise the coordinates of atoms of interest of the CDl which are in the vicinity of a putative ligand pocket in order to model the pocket in which the ligand fits.
  • the invention provides a computer-based method of rational drug design which comprises: providing the coordinates of at least two atoms of the CDl of Table 1 ("selected coordinates") ; providing the structure of a candidate modulator molecule; and fitting the structure of the candidate modulator molecule to the selected coordinates of the CDl.
  • the invention also relates to fragment linking or fragment growing approaches to rational drug design.
  • the step of providing the structure of a candidate modulator molecule may be performed by providing the structures of a plurality of molecular fragments and linking the molecular fragments to form a candidate modulator molecule.
  • the step of fitting the structure of the candidate modulator molecule may be performed before the molecular fragments are linked together, by separately fitting the structure of each molecular fragment, or after the molecular fragments are linked together.
  • the computer-based method of rational drug design may comprise: providing the coordinates of at least two atoms of the CDl or CDl/ligand complex of Table 1; providing the structures of a plurality of molecular fragments; fitting the structure of each of the molecular fragments to the selected coordinates of the CDl or CDl/ligand complex; and assembling the molecular fragments into a single molecule to form a candidate modulator molecule.
  • the computer-based method may further comprise the steps of: obtaining or synthesising the candidate modulator molecule; contacting the candidate modulator molecule with CDl; and determining the ability of the candidate modulator molecule to interact with CDl.
  • the computer-based method may further comprise the steps of: obtaining or synthesising the candidate modulator molecule; forming a complex of CDl and said candidate modulator molecule; and analysing said complex by X-ray crystallography to determine the ability of said candidate modulator molecule to interact with CDl.
  • a further aspect of the invention provides a compound having a chemical structure selected using the method of any one of the previous aspects, said compound being a modulator of the activity of CDl, e.g. an inhibitor or enhancer of CDl ligand presentation.
  • the step of providing the structure of a candidate modulator may involve selecting the compound by computationally screening a database of compounds for interaction with the active site. For example, a 3-D descriptor for the potential modulator may be derived, the descriptor including geometric and functional constraints derived from the architecture and chemical nature of the active site. The descriptor may then be used to interrogate the compound database, a potential modulator being a compound that has a good match to the features of the descriptor. In effect, the descriptor is a type of virtual pharmacophore.
  • the determination of the three-dimensional structure of CDl and CDl/ligand complex provides a basis for the design of new and specific ligands for CDl or modulators of CDl activity.
  • computer modelling programs may be used to design different molecules expected to interact with possible or confirmed active sites, such as binding sites or other structural or functional features of CDl.
  • a potential modulator of CDl/ligand complex activity can be examined through the use of computer modelling using a docking program such as GRAM, DOCK, or AUTODOCK (see Walters et al., Drug Discovery Today, Vol.3, No .4 , (1998), 160-178, and Dunbrack et al., Folding and Design, 2, (1997), 27-42).
  • a docking program such as GRAM, DOCK, or AUTODOCK
  • the present invention provides a machine readable data storage medium comprising a data storage material encoded with machine readable data, wherein the data is defined by all or a portion of the structure coordinates of CDl/ligand complex according to Table 1.
  • the invention further includes use of the machine readable data storage medium to design modulators of the CDl/ligand complex.
  • a computer system intended to generate structures and/or perform rational drug design for CDl/ligand complex, or complexes of CDl/ligand with a potential modulator, the system containing machine readable data comprising:
  • atomic coordinate data of Table 1 said data defining the three dimensional structure of CDl/ligand complex, or at least one sub-domain of the three-dimensional structure of CDl/ligand complex, or the coordinates of at least two atoms of CDl/ligand complex;
  • the method preferably further comprises the steps of: obtaining or synthesising the candidate modulator; and contacting the candidate modulator with CDl/ligand complex to determine the ability of the candidate modulator to interact with CDl/ligand complex. More preferably, in the latter step the candidate modulator is contacted with CDl/ligand complex under conditions to determine its function.
  • a modulator e.g. an enhancer or inhibitor
  • it may be manufactured and/or used in the preparation, i.e. manufacture or formulation, of a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals.
  • the present invention provides a method for identifying a candidate modulator (e.g. potential enhancer) of CDl/ligand complex comprising the steps of: providing the three-dimensional structure of CDl/ligand complex, or at least one sub-domain thereof, to characterise at least one active site of CDl, the three-dimensional structure being defined by atomic coordinate data according to Table 1; and identifying a candidate modulator molecule for interaction with the active site.
  • the candidate modulator molecule is identified by designing or selecting the molecule to interact with the active site.
  • the modulator may be formed by linking the respective compounds into a larger compound which maintains the relative positions and orientations of the respective compounds at the active sites .
  • the larger compound may be formed as a real molecule or by computer modelling.
  • high throughput screening of compounds to select compounds with binding activity may be undertaken, those compounds showing binding activity being selected as possible candidate modulators, and further crystallized with CDl (e.g. by co-crystallization or by soaking) for X-ray analysis.
  • the resulting X-ray structure may be compared with that of Table 1 for a variety of purposes. For example, where the contacts made by such compounds interact with a plurality of active sites, e.g. where the contacts overlap with those made by lipid antigen, novel molecules comprising residues contacting both lipid antigen and the bound compound may be obtained.
  • Identified modulators e.g. an enhancers or inhibitors
  • a composition such as a medicament, pharmaceutical composition or drug. These may be administered to individuals.
  • a tenth aspect of the present invention provides a method of assessing the ability of a candidate modulator molecule to interact with CDl or CDl/ligand complex comprising the steps of: obtaining or synthesising said candidate modulator molecule; forming a crystallised composite of CDl or
  • CDl/ligand complex and said candidate modulator; and analysing the composite by X-ray crystallography to determine the ability of the candidate modulator to interact with CDl or CDl/ligand complex.
  • the composite diffracts X-rays for the determination of atomic coordinates of the composite to a resolution of better than 3A, more preferably better than 2A.
  • the crystallised composite may be formed by crystal soaking or co-crystallisation.
  • the step of analysing the composite may involve analysing the intensities and/or positions of X-ray diffraction spots from the composite to determine the ability of the candidate modulator molecule to interact with CDl or CDl/ligand complex.
  • the invention relates to a method of determining three dimensional structures of CDl/ligand complex homologues or analogues of unknown structure by utilising the structural coordinates of Table 1.
  • CDl homologue or analogue of unknown structure For example, if X-ray crystallographic or NMR spectroscopic data is provided for a CDl homologue or analogue of unknown structure, the structure of CDl as defined by Table 1 may be used to interpret that data to provide a likely structure for the CDl homologue or analogue by techniques which are well known in the art, e.g. phase modelling in the case of X-ray crystallography.
  • a method of determining three dimensional structures of CDl or CDl/ligand complex homologues of unknown structure comprising the steps of: aligning a representation of an amino acid sequence of a CDl or CDl/ligand complex homologue or analogue of unknown structure with the amino acid sequence of CDl or CDl/ligand complex to match homologous regions of amino acid sequences; modelling the structure of the matched homologous regions of the homologue or analogue of unknown structure on the structure as defined in Table 1 of the corresponding regions of CDl or CDl/ligand complex; and determining a conformation for the homologue or analogue of unknown structure which substantially preserves the structure of said matched homologous regions .
  • said homologues have an amino acid sequence having at least 50% homology with said CDl, more preferably at least 60%, 70%, 80% or 90% homology.
  • a method for determining the structure of a protein comprising the steps of: providing the coordinates of Table 1; and either (a) positioning said coordinates in the crystal unit cell of said protein so as to provide a structure for said protein, or (b) assigning NMR spectra peaks of said coordinates.
  • a method for determining the structure of a compound bound to CDl/ligand complex comprising the steps of: providing a crystal of CDl/ligand complex; and soaking the crystal with the compound to form a complex; and determining the structure of the complex by employing the data of Table 1.
  • Figure 1 Structure of the human CDlb complex and of its ligands.
  • a CDlb structure ( ⁇ l- ⁇ 3 domains in light shading, ⁇ 2m in dark shading with left to right hatching) with bound PI (alkyl chains in grey shading with right to left hatching (C ) and dark shading (A' ) , inositol dot filled in light grey and adjacent phosphate dot filled in black) and detergent molecules (dark shading (F' ) and dot filled in black (T' ) ) shown as Van Der Waals spheres.
  • the internal hydrophobic cavity of ⁇ l ⁇ 2 is drawn as a transparent surface, and ligand binding channels are indicated as A' , C , F' and T' .
  • b Chemical structures of lipid and detergent ligands used in the refolding of CDlb complexes .
  • Figure 2 Binding and presentation of alkyl chain ligands by CDlb. All panels depict the CDlb/PI complex excepting b and c which show CDlb/GM2. a, Overview of the ⁇ l ⁇ 2 domain of CDlb with bound ligands. Close up views of the framed regions of the groove are shown in panels b-g. b and c, Positioning of the first glucosyl- and the phosphoinositol head groups in the CDlb-GM2 and CDlb-PI structures, respectively. Carbon atoms of the lipid ligand are grey shaded with right to left hatching, oxygens are black spheres, phosphate is hashed, and nitrogen dot filled black.
  • Hydrogen bonds are shown as black dotted line, d, The F' channel with bound monoalkyl detergent, e, A single detergent molecule bound to tunnel T' traverses a path, unobstructed by bulky side chains, between channels A' and F' .
  • f A portal in the C channel, stabilised by a unique disulfide bond Cysl31-Cysl45 (square dot infill) , allows egress of the lipid from the interior of the protein, g, The bottom of the A' channel contains a hydrophobic pole formed by Vall2 and Phe70, which could guide the lipid ligand from the A' channel into the T' tunnel.
  • the F 0 ⁇ F C omit map electron density (thin line mesh, contoured at 2.5 ⁇ ) was calculated after a simulated annealing during which the glycolipid (plus any residues and detergent within a distance of 3.5A) were omitted, in panels d-g, the 2 F 0 -F c
  • ⁇ ca i c electron density of the protein structure is shown as a white surface, contoured at I ⁇ .
  • Figure 3 Differences between CDlb and other CDl isoforms. a, b, Structural comparison of the antigen binding cavity for hCDlb and mCDld. The hydrophobic groove and key side chains of CDlb and CDld are shaded. The ligands present in the CDlb structures were superimposed onto the mCDld structure for direct comparison, c, Sequence alignment of the ⁇ l ⁇ 2 domains of human CDla-e and mCDld. Secondary structure elements of CDlb are shown above the protein sequence.
  • a shaded background indicates those residues which confer the lipid binding properties of CDlb and which are conserved at equivalent positions in other CDl isoforms.
  • Hydrophobic residues guiding the lipid ligands in CDlb at the bottom of channel A' (see Fig. 2g) and between channels C and F' are indicated by triangles, respectively. Cysteine residues are boxed.
  • Figure 4 Models for binding of mycolic acid and triacylglycerol to CDlb.
  • a Chemical structures of mycolic acid from M. tuberculosis and of the triacylglycerol trilaurin.
  • b Mycolic acid modeled into the CDlb structure.
  • the C60 long meromycolate chain (hash infill) could be fully contained within channels A' , T' and F' , thus forming a superchannel of some 70A length.
  • CDld/aGC complexes are specific for human invariant NKT cells.
  • Use of fluorescent CDld/aGC tetramers as a diagnostic composition in identifying human invariant NKT cells by FACS analysis a, FSC-H/SSC- H plot used for gating of lymphocyte population, b, Propidium iodide staining gates out dead cells.
  • c Staining of human invariant NKT cells with anti-T-cell receptor (anti-TCR) Valpha24 antibody, d, e, CDld/aGC tetramers stain human invariant NKT cells.
  • in vitro folding of proteins is generally much less effective.
  • Mammalian cells possess a range of specialised helper proteins, e.g. chaperonins, disulfidases, proline isomerases, etc., which enhance the yield of correctly folded molecules.
  • Standard in vitro refolding protocols which were successful for HLA class I molecules were completely unsuccessful for refolding CDl molecules.
  • CDl molecules are significantly more hydrophobic than HLA class I molecules, the inventors thought that the difficulties with standard protocols were due to hydrophobic interactions between partially folded CDl molecules, causing the proteins to aggregate and precipitate.
  • the second stage of the process is to strip off the detergent from these micelles so as to allow the molecules to completely fold.
  • the detergent is stripped using, for example, soluble cyclodextrins .
  • the inventors have been able to crystallise CDlb molecules generated by this method and have obtained high resolution structure of CDlb with bound ligand.
  • the crystal structure revealed that the large groove of Cdlb is not only occupied by ligand but also by the detergent molecule .
  • the ligand is loaded in a way that allows optimal presentation of the antigenic epitope to the T cell receptor, while two detergent molecules per CDl molecule fill the rest of the large groove.
  • the loaded CDl molecule (CDl/ligand complex) is considered biologically active.
  • the inventors have refolded the extracellular region of human CDlb (hCDlb; heavy chain residues 1-283 plus beta-2- microglobulin, ⁇ 2m) in vitro from completely denatured and reduced E. coli-derived proteins.
  • Single alkyl chain detergents of sixteen carbon length (C16) were used as refolding assistants to protect exposed hydrophobic surfaces during early refolding stages, thereby reducing hydrophobic protein aggregation and precipitation.
  • This protocol yielded stable soluble CDlb/ ⁇ 2m-complexes, which mass spectrometry confirmed to be loaded with the specified lipid ligands plus detergent (see methods) .
  • Soluble CDlb/ ⁇ 2m-proteins loaded either with phosphatidylinositol (PI) or ganglioside GM2 (GM2) 8 were crystallised, their structures determined by molecular replacement and refined using data to 2.26A and 2.8A, respectively (see Fig. 1, 2, methods and supplementary information) .
  • PI phosphatidylinositol
  • GM2 ganglioside GM2
  • the CDlb heavy chain and ⁇ 2m structures are identical in the two complexes and unless otherwise stated the following analyses are based on the higher resolution CDlb/PI structure.
  • the structures reveal a defined network of hydrophobic channels at the core of the c.l ⁇ 2 domain which are precisely tailored for acyl binding and are saturated by four hydrocarbon chains of 11 to 22 carbon atoms in length.
  • This binding groove architecture is radically different to that of classical MHC class I and II molecules.
  • the total volume (2200A 3 ) of the network is essentially filled by the hydrocarbon chains. No buried water molecules are present in either structure.
  • the three CDlb binding channels which connect directly to the surface are denoted as A' , C and F' , with the fourth, a unique tunnel, designated T' (Fig. 1, 2).
  • T' a unique tunnel
  • Channels A', C , and F' interconnect via T' .
  • the sequential connection of A' , T' and F' provides the potential to accommodate up to 60 carbon atoms of a single acyl chain which could enter and exit between the ⁇ l and ⁇ 2 helices (along A' and F' ) .
  • Channel C remains separate, leading from the T cell receptor (TCR) recognition surface between the ⁇ l and ⁇ 2 helices to a portal in the side of the molecule beneath the ⁇ 2 helix (Fig 1, 2d) .
  • TCR T cell receptor
  • PI and GM2 ligands occupy channels A' and C , whilst two detergent molecules fill channels F' and T' (Fig. 1, 2, 3) .
  • CDlb shows the glycolipid head groups to be presented by CDlb in a position analogous to that of the P4 residue in a peptide presented by MHC class I.
  • the surface presented by the CDlb/glycolipid complexes appears compatible with standard TCR recognition, a conclusion borne out by mutagenesis studies mapping TCR binding to CDlb 9 .
  • key features of the ⁇ 3 domain required for MHC class I binding to CD8 are not conserved in CDlb 10 .
  • CDld is closed off in CDld by Phel28 and Trpl33, being residues with bulky side chains, replacing Vall26 and Cysl31 respectively in CDlb.
  • CDl alleles other than CDlb the absence of a disulphide bridge (Cys 131 - Cysl45, Fig 2f) pulls the ⁇ 2 helix main chain more closely to the CDl backbone.
  • a disulphide bridge Cys 131 - Cysl45, Fig 2f
  • channel F' is partially occluded in mCDld by the side chain of Leu84 the side chain conformation selected by the equivalent residue (Phe84) in the current CDlb structure frees up sufficient space for F' to accommodate a detergent molecule (Fig. 2d, 3).
  • Phe84 may be expected to adopt an alternate conformation (analogous to that of Leu84 in mCDld) to pack against Phel44, Phe88 and Met90. This suggests a mechanism whereby the hydrophobic binding capacity of the channels may be tailored to ligand requirements, a phenomenon previously observed for binding of non- nucleoside inhibitors to HIV Reverse Transcriptase 11 .
  • CDlb appears to be unique among CDl molecules .
  • the arrangement of the combined detergent and lipid ligands in the CDlb/PI and CDlb/GM2 structures provide a general model for describing the interaction of the CDlb- binding groove with alkyl chain containing ligands.
  • Mycobacterial mycolates (Fig. 4a) , which play a crucial role in the adaptation of mycobacteria to intracellular growth and survival 12 , were the first fully characterised ligands of human CDlb 2 . It has long been speculated about how lipid ligands of such large size could bind to CDlb.
  • Modelling of mycolic acid into the current CDlb structures demonstrates how the long C50-C56 meromycolate chain could be fully contained within a super channel consisting of the interconnected A', T' and F' channels, with the shorter C22-C26 alkyl chain binding to C (Fig. 4b) .
  • the super channel has a maximum length of some 70A providing binding capacity for a single fatty acid chain of up to 60 carbons.
  • the F' channel could be closed off by selection of an alternative side chain conformation for Phe84 (see previously and Fig. 2d) .
  • CDlb can present either the large C80 mycobacterial glucomonomycolate or the shorter C32 synthetic glucomonomycolate to the same T cell line 13 .
  • the superchannel also allows for the option of stably accommodating long chain monoalkyl ligands, which can therefore serve as T cell antigens.
  • Short chain fatty acids of the endoplasmic reticulum could also act as ligands for CDlb in a similar manner to the C16 chain detergents used in the current refolding protocol (see methods).
  • endogenous short chain fatty acids could exert a chaperone-like function before binding of higher affinity ligands. Consistent with this hypothesis is the fact that, despite a 500 fold molar excess of detergent over lipid in the refolding buffer, detergent molecules were bound only to the T' tunnel and F' channel, while both the A' and C channels were occupied by the two alkyl chains of the lipid ligand. This also suggests that surface CDlb molecules may still retain endogenous chaperones to account for any excess binding capacity, in particular in tunnel T' .
  • channels A' , C and F' strongly suggests that lipids containing three alkyl chains, such as endogenous triacylglycerols or mycobacterial triacyl trehalose, may bind to human CDlb (Fig.4c).
  • Triacylglycerols which are synthesised on membranes of the endoplasmic reticulum, are independent risk factors for coronary heart disease 14 ' 15 .
  • Activated memory T lymphocytes accumulate in atherosclerotic plaques 16, 17 , and oxidized lipoproteins, which contain triacylglycerols, can act as immunogens for T cell 18 ' 19 .
  • CDlb is highly expressed on macrophages in atherosclerotic lesions, but not on normal tissue macrophages 20 . It is therefore interesting to speculate on the role of CDlb- mediated presentation of triacylglycerols to T lymphocytes in atherosclerosis.
  • CDlb The topology of CDlb is MHC class I-like (Fig. la) with close structural similarity to murine CDld.
  • Tubes of electron density delineate binding channels, buried between the ⁇ l and ⁇ 2 helices, occupied by four acyl chains. These hydrocarbon chains are up to 80 carbon atoms in length.
  • two of the acyl chains (in A' and C ) are assigned to the tails of the glycolipid ligand and the remaining two (in F' and T' ) are accounted for by detergent molecules.
  • Electron density at the CDlb surface linking chains A' and C' provide partial information for the position of the glycolipid head-group structures, which are relatively mobile as judged from crystallographic temperature factors.
  • the detergents used in the refolding of the single lipid complexes have supplemented GM2 and PI to saturate the acyl binding capacity of CDlb.
  • the structure of murine CDld 7 does not include a specific bound ligand but the ⁇ l ⁇ 2 domain contains cavities designated as binding pockets A' and F' . Comparison with the CDlb complexes indicates broad equivalence of pocket A' to channels A' and of pocket F' to channel F' .
  • CDld cavities have a reduction in volume relative to the total CDlb network.
  • the main chain topology of CDlb differs markedly from MHC class I in the peak height attained by the kink in the ⁇ 2 helix (residues 150-157 and 149-152 in CDlb and MHC class I respectively) .
  • Direct substitution of CDlb into a MHC class I/TCR complex therefore results in significant steric clashes between the TCR and this portion of the CDlb ⁇ 2 helix.
  • the upward displacement of the TCR necessary to provide a sterically acceptable dock onto the CDlb structure may facilitate incorporation at the recognition interface of the increased size of the glycolipid head group compared to an amino acid side chain.
  • the surface presented by the CDlb/glycolipid complexes appears compatible with standard TCR recognition, a conclusion borne out by mutagenesis studies mapping TCR binding to CDl.
  • Superposition of CDlb into the HLA A2/CD8 ⁇ crystal structure 10 again reveals substantial steric clashes.
  • Given the highly conserved nature of the MHC class I/CD8 interaction these changes imply relative abolition of any such interaction. This is again consistent with the functional recognition of CDl by CD8 ⁇ T cells 5 .
  • CDld-tetramers generated using detergent refolding specifically detected human invariant natural killer (NK)T cells which recognise glycolipid presented by CDld and show specificity for alpha-galactosylceramide (aGC) , ( Figure 5) .
  • Human NKT cells expressing an invariant TCR Valpha24 chain are highly specific for CDld/aGC complex.
  • Biotinylated human CDld molecules loaded with the synthetic glycolipid aGC were generated from completely denatured and reduced CDld protein and complexed with fluorecent streptavidin.
  • Fluorescent CDld/aGC tetramers specifically stained human NKT cells and demonstrated the use of CDl/ligand complexes for in vivo and in vitro T-cell identification and disease state diagnosis ( Figure 5d, e) .
  • a unique network of channels allows CDlb to accommodate glycolipids with two very long acyl tails.
  • CDlb the absence of side chains at Gly98 and Glyll ⁇ opens channel T' (Fig 3c sequence alignments) .
  • T' Fig 3c sequence alignments
  • certain of the CDl family may have the capability to bind three acyl chain lipids .
  • Such ligands would be expected to show strong binding consistent with enhanced avidity.
  • single acyl chain lipids would be penalized by reduced avidity.
  • hCDlb human CDlb
  • hCDlb-pET23d plasmid hCDlb-pET23d
  • the hCDlb coding sequence was amplified by PCR from monocyte- derived dendritic cell cDNA. Oligonucleotides used for PCR amplification (5' -3' and 3' -5') introduced 5'Ncol and 3' BamHI restriction sites, which allowed cloning into the bacterial expression vector pET23d (Invitrogen) .
  • the correct sequence of the hCDlb coding sequence was confirmed using automated sequencing. Plasmid hb2m-pET23d encoding extracellular human beta2-microglobulin ( ⁇ 2m) has been described previously. Both proteins were expressed separately in E.coli BL21 (Invitrogen) and purified from inclusion bodies as described below.
  • the extracellular ⁇ l- ⁇ 3 domains of human CDlb (SWISS- PROT: P29016) and ⁇ 2m were synthesised using a prokaryotic expression system (pET23d; Novagen, Milwaukee, WI) . Both proteins were purified from E.coli (strain BL21) inclusion bodies, which were subsequently solubilised using 6M guanidine buffer containing lOmM DTT.
  • Refolding of the fully denatured and reduced proteins was carried out at room temperature by dilution into buffer 1 (IM Urea, 300mM L-Arginine, 50mM Tris pH 7.5, 2mM EDTA, 5mM reduced glutathione, 0.5mM oxidized glutathione) supplemented with 500 ⁇ M hexadecyltrimethyl- ammoniumbromide (SIGMA, Illinois, USA) and 1 ⁇ M of either synthetic GM2 8 ligand or soy bean purified PI ligand (AVANTILIPIDS, USA) .
  • buffer 1 IM Urea, 300mM L-Arginine, 50mM Tris pH 7.5, 2mM EDTA, 5mM reduced glutathione, 0.5mM oxidized glutathione
  • SIGMA hexadecyltrimethyl- ammoniumbromide
  • AVANTILIPIDS synthetic GM2 8 ligand or soy bean purified PI ligand
  • Crystals were flash frozen at 100K in mother liquor containing 20% glycerol. Diffraction data from one
  • CDlb/GM2 crystal were collected at beamline ID-14 EH2 (ESRF, Grenoble France) with 0.933A radiation, recorded on an ADSC Q4 CCD detector.
  • Diffraction data from two CDlb/PI crystals were collected at beam line ID-29 (ESRF, Grenoble, France) with 0.977A radiation, recorded on an ADSC Q210 detector.
  • the molecular replacement solutions for both the CDlb/GM2 and CDlb/PI crystals were identified with the program AmoRe 23 , using respectively, as models, the 2.8A resolution structure of mCDld 7 (PDB accession code 1CD1) and the protein component of a partially refined structure of CDlb/GM2.
  • the CNS program suite was used for refinement 24 . Approximately 3% of reflections were set aside for the Rfr ee calculations (535 and 751 observations respectively for the GM2-Cdlb and PI-CDlb structures) .
  • the final refined structures showed good stereochemistry as assessed with the program PROCHECK 28 .
  • VOLUMES Esnouf, unpublished program identified cavities as surfaces accessible to methyls (1.7A radius), but not to large probes (6A radius). The figures have been prepared using Bobscript 9 and Raster3D 30 .
  • Biotinylated human CDld molecules loaded with the synthetic glycolipid alpha-galactosylceramide (aGC) were generated from completely denatured and reduced inclusion body protein (see Methods - Protein expression, refolding and crystallization) .
  • the refolded CDld molecules were used to generate CDld/aGC-tetramers by binding the biotinylated CDld/aGC-complexes to fluorescent streptavidin.
  • the resulting fluorescent CDld/aGC- tetramers were tested for use as diagnostic compositions in the identification of human invariant NKT cells in vi tro by fluorescent cell sorter analysis (FACS) .
  • Plasma triglyceride level is a risk factor for cardiovascular disease independent of high-density lipoprotein cholesterol level: a meta-analysis of population-based prospective studies. J Cardiovasc Risk 3, 213-9. (1996) .
  • T lymphocytes in human atherosclerotic plaques are memory cells expressing CD45RO and the integrin VLA-1. Arterioscler Thromb 12, 206-11. (1992).
  • ATOM 79 C ILEA 13 53.720 18.870 -0.116 1.0023.25
  • ATOM 253 CA GLN A 36 46.631 25.485 -6.019 1.00 19.24 A
  • ATOM 262 CA ILE A 37 48.571 25.657 -9.289 1.00 21.88 A
  • ATOM 342 CA ILEA 48 49.566 18.219-13.237 1.0026.08 A
  • ATOM 379 CA PROA 52 48.021 27.571 -18.395 1.0028.53 A
  • ATOM 454 CD LYSA 61 67.333 21.112-21.061 1.0052.41 A
  • ATOM 516 CA ILEA 69 64.275 15.379 -8.886 1.0027.87 A
  • ATOM 553 CA TYR A 73 63.112 12.039 -3.928 1.00 33.09 A
  • ATOM 713 CA TYR A 92 53.123 2.149 12.419 1.00 48.10 A
  • ATOM 778 C ILE A 99 57.384 23.003 2.940 1.00 28.54 A
  • ATOM 833 CA GLY A 108 62.716 43.038 -3.052 1.00 39.17 A

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Abstract

L'invention concerne des procédés de production d'un complexe CD1/ligand comprenant les étapes suivantes : (a) obtenir une protéine CD1 dénaturée ; (b) mettre en contact ladite protéine CD1 dénaturée avec le ligand, dans un environnement comprenant du détergent ; et (c) isoler le complexe CD1/ligand. L'invention concerne, de plus, l'utilisation du complexe CD1/ligand, obtenu selon le procédé, sa structure cristalline, des procédés et des systèmes informatiques permettant l'élaboration rationnelle des médicaments, l'évaluation des molécules modulatrices candidates et des procédés de détermination de structures de protéines homologues ou analogues.
PCT/GB2003/002069 2002-05-14 2003-05-14 Complexes immunogenes WO2003095487A2 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005066867A2 (fr) * 2004-01-09 2005-07-21 Isis Innovation Limited Modulateurs de recepteurs

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
ALTAMIRANO MM ET AL.: "Ligand-independent assembly of recombinant human CD1 by using oxidative refolding chromatography" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE USA, vol. 98, no. 6, 13 March 2001 (2001-03-13), pages 3288-3293, XP002263519 *
GADOLA SD ET AL.: "Structure of human CD1b with bound ligands at 2.3 A, a maze for alkyl chains" NATURE IMMUNOLOGY, vol. 3, no. 8, August 2002 (2002-08), pages 721-726, XP002263522 *
KARADIMITRIS A ET AL.: "Human CD1d-glycolipid tetramers generated by in vitro oxidative refolding chromatography" PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCE, vol. 98, no. 6, 13 March 2001 (2001-03-13), pages 3294-3298, XP002263520 *
ZENG Z-H: "Crystal structure of Mouse CD1: An MHC-Like Fold with a Large Hydrophobic Binding Groove" SCIENCE, vol. 277, 18 July 1997 (1997-07-18), pages 339-345, XP002263521 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005066867A2 (fr) * 2004-01-09 2005-07-21 Isis Innovation Limited Modulateurs de recepteurs
WO2005066867A3 (fr) * 2004-01-09 2006-01-05 Isis Innovation Modulateurs de recepteurs
US7851598B2 (en) 2004-01-09 2010-12-14 Isis Innovation Limited Receptor modulators
US8945561B2 (en) 2004-01-09 2015-02-03 Isis Innovation Limited Receptor modulators

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