WO2009060159A1 - Molécules ilt mutées - Google Patents

Molécules ilt mutées Download PDF

Info

Publication number
WO2009060159A1
WO2009060159A1 PCT/GB2007/004257 GB2007004257W WO2009060159A1 WO 2009060159 A1 WO2009060159 A1 WO 2009060159A1 GB 2007004257 W GB2007004257 W GB 2007004257W WO 2009060159 A1 WO2009060159 A1 WO 2009060159A1
Authority
WO
WIPO (PCT)
Prior art keywords
dimer
polypeptide
molecule
mutated human
ilt
Prior art date
Application number
PCT/GB2007/004257
Other languages
English (en)
Inventor
Bent Karsten Jakobsen
Yi Li
Ruth Karen Moysey
Original Assignee
Medigene Limited
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Medigene Limited filed Critical Medigene Limited
Priority to PCT/GB2007/004257 priority Critical patent/WO2009060159A1/fr
Publication of WO2009060159A1 publication Critical patent/WO2009060159A1/fr

Links

Classifications

    • 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
    • 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
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to mutated human ILT molecules comprising amino acids 4-197 of SEQ ID NO: 6. Also provided are monomeric and dimeric polypeptide fusions comprising said mutated human ILT molecules and immunoglobulin Fc segments, and methods for using these molecules and polypeptide fusions.
  • Immunoglobulin-like transcripts are also known as Leukocyte Immunoglobulin-like receptors (LIRs), monocyte/macrophage immunoglobulin-like receptors (MIRs) and CD85. This family of immunoreceptors form part of the immunoglobulin superfamily. The identification of ILT molecules was first published in March 1997 in a study (Samaridis et al, (1997) Eur J Immunol 27 660-665) which detailed the sequence of LIR-I (ILT-2), noted their similarity to bovine FC ⁇ 2R, human killer cell inhibitory receptors (KIRs), human Fc ⁇ R, and mouse gp49. This study also noted that LIR-I, unlike KIRs, is predominately expressed on monocytic and B lymphoid cells.
  • LIRs Leukocyte Immunoglobulin-like receptors
  • MIRs monocyte/macrophage immunoglobulin-like receptors
  • CD85 This family of immunoreceptors form part of the
  • the ILT family of immunoreceptors are expressed on the surface of lymphoid and myeloid cells.
  • the ILT molecules share 63-84% homology in their extracellular regions and all except the soluble LIR-4 are type I transmembrane proteins. All the currently identified ILT molecules have either two or four immunoglobulin superfamily domains in their extracellular regions. (Willcox et al, (2003) 4 (9) 913- 919)
  • Individual ILT molecules may also be expressed as a number of distinct variants / isoforms. (Colonna et al, (1997) J Exp Med 186 (11) 1809-1818) and (Cosman et al., (1997) Immunity 7 273-282)
  • WO9848017 discloses the genetic sequences encoding ILT family members and their deduced amino acid sequences. This application classified LIR molecules into three groups. The first group containing polypeptides with a transmembrane region including a positively charged residue and a short cytoplasmic tail. The second group comprising polypeptides having a non-polar transmembrane region and a long cytoplasmic tail. And finally a third group containing a polypeptide expressed as a soluble polypeptide having no transmembrane region or cytoplasmic tail. Also disclosed were processes for producing polypeptides of the LIR family, and antagonistic antibodies to LIR family members.
  • LIR family members to treat autoimmune diseases and disease states associated with suppressed immune function.
  • the use of soluble forms of an LIR family member is advantageous for certain applications. These advantages included the ease of purifying soluble forms of ILTs/LIRs from recombinant host cells, that they are suitable for intravenous administration and their potential use to block the interaction of cell surface LIR family members with their ligands in order to mediate a desirable immune function.
  • the possible utility of soluble LIR fragments that retain a desired biological activity, such as binding to ligands including MHC class I molecules was also noted.
  • ILT-4 binds to HLAs-A, B and G, but not HLA-Cw3 or HLA-Cw5.
  • WO03041650 discloses a method of treating Rheumatoid Arthritis (RA) using modulators of LIR-2 and/or LIR-3/ LIR-7 activity.
  • the modulators disclosed include both agonists and antagonists of LIR activity.
  • WO2006033811 discloses the use of ILT-3 polypeptides and fusions thereof as therapeutic agents for the inhibition of graft rejection.
  • Fc fusion-based therapeutics are on the market including Abatacept®, a CTLA4-Fc fusion polypeptide.
  • WO 98/48017 describes the production of soluble two domain (D1D2) analogues of wild-type ILT, and Fc fusion polypeptides comprising these soluble analogues of ILTs.
  • Mutated human ILT molecules and polypeptide monomers and dimers such as Fc fusions with the pMHC binding characteristics of such ILT mutated human molecules and multivalent complexes thereof provide a means of blocking the CD8 binding site on pMHC molecules, for example for the purpose of inhibiting CD8 + T cell-mediated autoimmune disease. For that purpose it is desirable for these mutated human ILT molecules to have an even higher affinity and/or an even slower off-rate for the target pMHC molecules than the class of mutated ILT-like polypeptides disclosed in WO 2006/125963.
  • the present invention relates to mutated human ILT molecules comprising amino acids 4-197 of SEQ ID NO: 6. Also provided are monomeric and dimeric polypeptide fusions comprising said mutated human ILT molecules and immunoglobulin Fc segments, and methods for using these molecules and polypeptide fusions.
  • the mutated human ILT2 molecules of the present invention represent an improvement relative to those disclosed by WO 2006/125963 in that they have both a higher affinity and a slower off rate that the mutant ILT molecules known from WO 2006/125963. Furthermore, when presented as Fc fusion polypeptides, the molecules of the present invention also demonstrate enhanced suppression of T cell activation and the T cell-mediated killing of antigen-presenting cells relative to Fc fusions comprising the closest analogous mutated human ILT molecule known from WO 2006/125963, namely homodimer Fc fusions comprising amino acids 2-198 of mutated human ILT molecule of SEQ ID NO: 23 using the numbering of SEQ ID NO: 3.
  • Such Fc fusions have the advantage over the non-fused ILT-like monomers and dimers, for example in terms of improved pharmacokinetic properties such as longer plasma half-life.
  • the monomers and dimers of the invention may be associated with therapeutic agents, may be assembled into multivalent complexes, and may be used in the treatment of autoimmune diseases.
  • ILT molecules are also known as LIRs, MIRs and CD85.
  • ILT as used herein is understood to encompass any polypeptide within this family of immunoreceptors.
  • This invention provides additional human mutated ILT molecules having higher affinity and/or a slower off rate for the interaction between these molecules and Class I MHC complexes that the affinity and/or off-rate for the interation between wild-type human ILT2 and Class I pMHC complexes within the scope of, but not disclosed in, our co-pending application WO 2006/125963.
  • the present invention provides a mutated human ILT2 molecule comprising amino acids 4-197 of SEQ ID NO: 6.
  • affinity and/or off-rate of these mutated human ILT molecules for pMHC complexes can be determined.
  • said affinity (K D ) and/or off-rate (k o f f ) may be determined by Surface Plasmon Resonance.
  • Example 4 herein provides a Biacore- based assay suitable for carrying out such determinations
  • mutated human ILT molecules comprising:
  • amino acids 3-197 of SEQ ID NO: 6; amino acids 2-197 of SEQ ID NO: 6; or amino acids 1-197 of SEQ ID NO: 6 are embodiments of the present invention.
  • the present invention also provides a mutated human ILT2 molecule selected from the group consisting of:
  • mutated human ILT molecule amino acid sequences herein are expressed in bacteria, they will have an N-terminal methionine (Met or M) residue. As will be known to those skilled in the art this residue may be removed during the production of recombinant proteins, for example this methionine would not normally be present in mutated human ILT molecules expressed by eukaryotic cells.
  • methionine would not normally be present in mutated human ILT molecules expressed by eukaryotic cells.
  • naturally occurring ILT polypeptides have either two or four immunoglobulin superfamily domains in their extracellular regions.
  • the high affinity ILT-like polypeptides of the invention may be expressed in forms having four, three or two of said domains.
  • the currently preferred embodiments of the invention have two immunoglobulin superfamily domains corresponding to the two N-terminal domains of human ILT-2 containing one or more mutation(s) which confer high affinity for Class I pMHC. These N-terminal domains are domains one and two using the notation of Cosman et al, (1999) Immunol Revs 168: 177-185. ILT-like polypeptides having those two N-terminal domains generally have a sequence corresponding to amino acids 1-198 of SEQ ID NO: 3.
  • the mutations in the amino acid sequence of the mutated human ILT molecules of the invention may be one or more of substitution(s), deletion(s) or insertion(s). These mutations can be carried out using any appropriate method including, but not limited to, those based on polymerase chain reaction (PCR), restriction enzyme-based cloning, or ligation independent cloning (LIC) procedures. These methods are detailed in many of the standard molecular biology texts.
  • PCR polymerase chain reaction
  • LIC ligation independent cloning
  • polypeptide of the invention comprising amino acids corresponding to at least amino acids 2-195 of SEQ ID No: 3.
  • polypeptides are two-domain embodiments comprising domains corresponding to the two N- terminal immunoglobulin superfamily domains of human ILT-2
  • sequences present in the mutated ILT molecules that are not directly involved in contacts with the peptide-MHC complex to which the mutated ILT molecules bind they may be shorter than, or may contain substitutions or deletions relative to the sequence of native ILT2.
  • a further embodiment is provided by mutated human ILT2 molecules of the invention which are soluble.
  • the mutated human ILT2 molecules of the invention may be used as soluble therapeutics. In such instances is desirable to increase the solubility of these polypeptides.
  • the invention encompasses polypeptides which comprise one or more mutation(s) which increase the solubility of the polypeptide relative to a corresponding polypeptide lacking said mutations.
  • polypeptides which comprise one or more mutation(s) which increase the solubility of the polypeptide relative to a corresponding polypeptide lacking said mutations.
  • mutate amino acids which are solvent exposed. These solvent exposed amino acids can be identified by reference to the crystal structure of ILT-2. (See Chapman et al, (2000) Immunity 12 727-736)
  • the invention encompasses polypeptides wherein one or more solvent-exposed amino acid(s) are mutated.
  • polypeptides of the invention comprising at least one mutation wherein a solvent exposed hydrophobic amino acid is substituted by a charged amino acid.
  • solubility enhancing mutations are in within the C-terminal 6 amino acids of the polypeptides of the invention.
  • the inclusion of one or both of mutations corresponding to 195D and/or 197D using the numbering of SEQ ID NO: 3 provide preferred means of increasing the solubility of the high affinity ILT-like polypeptides of the invention relative to the corresponding polypeptides lacking said mutation(s).
  • the exemplary polypeptide of the invention provided in Figure 4(SEQ ID NO: 6) incorporates both the 195L ⁇ D and 197L ⁇ D mutations.
  • mutated human ILT2 molecules of the invention in which amino acid 195D and/or 197D using the numbering of SEQ ID NO: 3 are substituted for 195L and/or 197L respectively.
  • a further embodiment is provided by a mutated human ILT2 molecule of the invention which comprises a transmembrane domain, such as the transmembrane domain of human ILT which is underlined in SEQ ID NO: 1.
  • the amino acid sequence a mutated human ILT molecule of the invention incorporating such a transmembrane domain is provided by SEQ ID NO: 22. This is the amino acid sequence of a "full- length" mutated human ILT molecule of the present invention in which amino acid 195D and 197D using the numbering of SEQ ID NO: 3 are substituted for 195L and 197L respectively.
  • Polypeptide monomers and dimers comprising high affinity ILT -like polypeptides and Fc-like portions
  • the present invention also provides monomelic polypeptides comprising an ILT-like segment which is a soluble mutated human ILT molecule of the invention and an Fc- like segment wherein either
  • the ILT-like segment is the N-terminal segment of the polypeptide;
  • the Fc-like segment is the C-terminal segment of the polypeptide and comprises a portion of the constant domain of one of the heavy chains of an immunoglobulin having at least 70% identity and/or 80% similarity to the corresponding portion of SEQ ID NO: 9; or
  • the Fc-like segment is the N-terminal segment of the polypeptide and comprises a portion of the constant domain of one of the heavy chains of an immunoglobulin having at least 70% identity and/or 80% similarity to the corresponding portion of SEQ ID NO: 9; and the ILT-like segment is the C-terminal segment of the polypeptide.
  • the present invention also provides polypeptide dimers comprising a first polypeptide and a second polypeptide, in which dimer
  • the first and/or the second polypeptide comprises an ILT-like segment which is a soluble mutated human ILT molecule of the invention
  • each of the first and second polypeptides comprises an Fc-like segment comprising a portion of the constant domain of one of the heavy chains of an immunoglobulin having at least 70% identity and/or 80% similarity to the corresponding portion of SEQ ID NO: 9;
  • the ILT-like segment(s) is/are the N-terminal segment(s) of the first and/or second polypeptides
  • the Fc-like segments are the C-terminal segments of the first and second polypeptides
  • the Fc-like segments are the N-terminal segments of the first and/or second polypeptides
  • the ILT-like segment(s) is/are the C-terminal segment(s) of the first and second polypeptides.
  • polypeptide dimers of the invention wherein the ILT- like segment(s) is/are the N-terminal segment(s) of the first and/or second polypeptides, and the Fc-like segments are the C-terminal segments of the first and second polypeptides
  • Polypeptide monomers and dimers which meet the above homology and Class I pMHC-binding criteria may be regarded as polypeptide monomers comprising high affinity soluble human ILT-like portions and Fc-like portions and may be referred to herein as such.
  • FC-like segments of the polypeptide monomers and dimers of the invention comprise at least one inter-chain covalent link between a residue in one of the said Fc-like segments and a residue in the other said Fc-like residue.
  • inter-chain covalent links may correspond to links present between cysteine residues in the heavy chain constant domains of native immunoglobulins and/or non-native interchain links may be introduced.
  • polypeptide monomers or dimers of the invention having the property of binding to an Fc receptor via the said Fc-like segments.
  • the ability of the polypeptide dimers of the invention to bind to a given Fc receptor can be may be assessed by any suitable means.
  • Example 8 herein provides a Fluorescence Activated Cell Sorting (FACS) based competitive binding assay for assessing this ability.
  • FACS Fluorescence Activated Cell Sorting
  • Polypeptide monomers or dimers of the invention wherein the Fc-like segment or segments comprise respectively one or both of the chains of the Fc portion of an immunoglobulin provide another aspect of the invention.
  • Such Fc portions can be comprised of the CH2 and CH3 domains of an immunoglobulin and optionally the hinge region of the immunoglobulin.
  • the Fc fragment can be of an IgG, an IgA, an IgM, an IgD, or an IgE.
  • the said immunoglobulin is an IgG immunoglobulin.
  • the said immunoglobulin may an IgGl immunoglobulin, such as human IgGl immunoglobulin.
  • the Fc-like segment or segments comprise respectively one or two of amino acid sequence SEQ ID NO: 9.
  • polypeptide monomers or dimers of the invention wherein the Fc -like segment or segments comprise respectively one or both of the chains of a mutated Fc portion of an immunoglobulin.
  • mutation(s) in these Fc portion amino acid sequences may be one or more of substitution(s), deletion(s) or insertion(s).
  • mutations can be carried out using any appropriate method including, but not limited to, those based on polymerase chain reaction (PCR), restriction enzyme-based cloning, or ligation independent cloning (LIC) procedures. These methods are detailed in many of the standard molecular biology texts.
  • Such mutations may be introduced for a number of reasons. For example it may de desirable to introduce mutations to the said Fc-like segment(s) which impact one or more of disulfide bond formation, expression levels achievable in a selected host cell, N-terminal heterogeneity upon expression in a selected host cell, Fc portion glycosylation or the level of antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cellular cytotoxicity (CDCC) responses to the polypeptide monomers and dimers of the invention.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cellular cytotoxicity
  • polypeptide monomers or dimer of the invention mutated so as to reduce antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cellular cytotoxicity (CDCC) responses thereto wherein the said Fc-like segment or segments has or have a sequence or sequences corresponding to SEQ ID NO: 9 in which one or more of amino acids corresponding to amino acids 13 E, 14L, 15 L, 16G, 107 A, 11OA or 11 IP of SEQ ID NO: 9 is/are mutated.
  • ADCC antibody-dependent cellular cytotoxicity
  • CDC complement-dependent cellular cytotoxicity
  • the said Fc-like segment or segments has or have a sequence or sequences corresponding to SEQ ID NO: 9 having one or more of the following mutations 13E ⁇ P, 14L— *W, 15L ⁇ A, deletion of 16G, 107A ⁇ G, 1 lOA ⁇ S or 11 lP ⁇ S using the numbering of SEQ ID NO: 9.
  • polypeptide monomers or dimers of the invention wherein the said Fc-like segment or segments is/are mutated so as so as to increase the plasma half-life of the monomer or dimer.
  • polypeptide monomers or dimer of the invention wherein the said Fc-like segment or segments has or have a sequence or sequences corresponding to SEQ ID NO: 9 in which one or both of amino acids corresponding to amino acids 3OT and 208M is/are mutated.
  • the said Fc-like segment or segments has or have a sequence or sequences corresponding to SEQ ID NO: 9 having one or more of the following mutations 30T- ⁇ Q or 208M— »L using the numbering of SEQ ID NO: 9.
  • IgGl antibodies containing these Fc mutations have been shown to have serum half-lives in rhesus monkeys than the corresponding wild-type antibodies.
  • the increased serum half-lives of antibodies incorporating these mutations is believed to be due to their increased affinity for the human neo-natal FC receptor (FcRn) which, in turn, is believed to allow these mutated antibodies to avoid lysosomal degradation and to be returned into the circulation.. (Hinton et al, (2005) J. Immunol. 176: 346-356)
  • the Fc-like segment(s) of the polypeptide monomers and dimers of the invention are CHARACTERISED IN THAT said segment(s) have at least 80% identity and/or 90% similarity to SEQ ID NO: 9.
  • the Fc-like segment(s) of the polypeptide monomers and dimers of the invention are CHARACTERISED IN THAT said segment(s) have at least a 90% identity and/or 95% similarity to SEQ ID NO: 9.
  • the Fc-like segment(s) of the polypeptide monomers and dimers of the invention are CHARACTERISED IN THAT said segment(s) have at least a 95% identity and/or 98% similarity to SEQ ID NO: 9.
  • Sequence identity means identical amino acids at corresponding positions in the two sequences which are being compared. Similarity in this context includes amino acids which are identical and those which are similar (functionally equivalent). For example a single substitution of one hydrophobic amino acid present at a given position in a polypeptide with a different hydrophobic amino acid would result in the formation of a polypeptide which was considered similar to the original polypeptide but not identical).
  • the parameters "similarity” and “identity” as used herein to characterise polypeptides of the invention are determined by use of the FASTA algorithm as implemented in the FASTA programme suite available from William R. Pearson, Department of Biological Chemistry, Box 440, Jordan Hall, Charlottesville, Virginia. The settings used for determination of those parameters via the FASTA programme suite are as specified in Example 6 herein.
  • polypeptide monomers or dimers of the invention wherein the Fc-like segment, or both Fc-like segments, comprise an amino acid sequence selected from the group consisting of SEQ ID NOs: 14 to 21. (See Figures 8a - 8d respectively for the amino acid sequences of these polypeptides)
  • the ILT-like segment(s) of the polypeptide monomers or dimers of the invention are either high affinity soluble mutated human ILT-like molecules, or are functional equivalents thereof.
  • polypeptide monomers or dimers of the invention wherein the N-terminal segment(s) of the first and/or second polypeptide(s) consist(s) of or include(s) amino acids 4-197 of SEQ ID No: 6. Further embodiments are provided by polypeptide monomers or dimers of the invention comprising an amino acid sequence selected from the group consisting of SEQ ID Nos: 14 to 21.
  • polypeptide dimers of the invention which are homodimers.
  • a homodimer consisting of two polypeptides having SEQ ID No: 20.
  • the inhibitory activities of the polypeptide of SEQ ID NO: 6 and any given ILT-like segment(s) contained within polypeptide monomers or dimers of the invention may be determined by any conventional assay from which the read-out is related to the binding affinity of CD8 for the given pMHC. In general the read-out will be an IC 50 value.
  • the CD8 binding inhibition provided by the test ILT-like segment(s) and that of SEQ ID NO: 6 will be assessed at comparable concentrations and their respective IC 50 's determined by reference to the inhibition curves plotted from the individual results.
  • a suitable assay is that described in Example 5 herein.
  • mutated human ILT molecules and polypeptide monomers and dimers of the invention may be used in multimeric forms or in association with other moieties. In this regard it is desirable to produce mutated human ILT molecules and polypeptide monomers and dimers thereof which comprising a means of attaching other moieties thereto.
  • mutated human ILT molecules and polpeptide monomers and dimers thereof which comprises a C-terminal reactive site for covalent attachment of a desired moiety.
  • This reactive site may be a cysteine residue.
  • reactive chemistries which are suitable for this purpose. These include, but are not limited to, cysteine residues, hexahistidine peptides, biotin and chemically reactive groups. The presence of such reactive chemistries may also facilitate purification of the molecules
  • mutated human ILT molecules and polypeptide monomers and dimers of the invention are associated with at least one polyalkylene glycol chain(s). This association may be caused in a number of ways known to those skilled in the art.
  • the polyalkylene chain(s) is/are covalently linked to the mutated human ILT molecules or polypeptide monomers and dimers thereof.
  • the polyethylene glycol chains of the present aspect of the invention comprise at least two polyethylene repeating units.
  • One aspect of the invention provides a multivalent complex comprising at least two mutated human ILT molecules or polypeptide monomers and dimers of the invention.
  • At least two mutated human ILT molecules or polypeptide monomers and dimers thereof of the invention are linked via linker moieties to form multivalent complexes.
  • the mutated human ILT molecules or polypeptide monomers and dimers thereof of the invention are linked by a non-peptidic polymer chain or a peptidic linker sequence.
  • the multivalent complexes are water soluble, so the linker moiety should be selected accordingly.
  • the linker moiety should be capable of attachment to defined positions on the mutated human ILT molecules or polypeptide monomers and dimers thereof, so that the structural diversity of the complexes formed is minimised.
  • One embodiment of the present aspect is provided by a multivalent complex of the invention wherein the polymer chain or peptidic linker sequence extends between amino acid residues of each mutated human ILT molecule or polypeptide monomer and dimer thereof which are not located in the Class I pMHC binding domain of thereof.
  • the linker moieties should be chosen with due regard to their pharmaceutical suitability, for example their immunogenicity.
  • linker moieties which fulfil the above desirable criteria are known in the art, for example the art of linking antibody fragments.
  • linker There are two classes of linker that are preferred for use in the production of multivalent complexes of the present invention.
  • a multivalent complex of the invention in which the polypeptides are linked by a polyalkylene glycol chain or a peptidic linker derived from a human multimerisation domain provide certain embodiments of the invention.
  • Suitable hydrophilic polymers include, but are not limited to, polyalkylene glycols.
  • the most commonly used polymers of this class are based on polyethylene glycol or PEG, the structure of which is shown below.
  • n is greater than two.
  • suitable, optionally substituted, polyalkylene glycols include polypropylene glycol, and copolymers of ethylene glycol and propylene glycol.
  • Such polymers may be used to treat or conjugate therapeutic agents, particularly polypeptide or protein therapeutics, to achieve beneficial changes to the pharmacokinetic (PK) profile of the therapeutic, for example reduced renal clearance, improved plasma half-life, reduced immunogenicity, and improved solubility.
  • PK pharmacokinetic
  • Such improvements in the PK profile of the PEG-therapeutic conjugate are believe to result from the PEG molecule or molecules forming a 'shell' around the therapeutic which sterically hinders the reaction with the immune system and reduces proteolytic degradation.
  • the size of the hydrophilic polymer used may in particular be selected on the basis of the intended therapeutic use of the mutated human ILT molecules or polypeptide monomers and dimers thereof.
  • the polymer used can have a linear or branched conformation.
  • Branched PEG molecules, or derivatives thereof, can be induced by the addition of branching moieties including glycerol and glycerol oligomers, pentaerythritol, sorbitol and lysine.
  • the polymer will have a chemically reactive group or groups in its structure, for example at one or both termini, and/or on branches from the backbone, to enable the polymer to link to target sites in the mutated human ILT molecules or polypeptide monomers and dimers thereof.
  • This chemically reactive group or groups may be attached directly to the hydrophilic polymer, or there may be a spacer group/moiety between the hydrophilic polymer and the reactive chemistry as shown below: Reactive chemistry-Hydrophilic polymer-Reactive chemistry
  • spacer used in the formation of constructs of the type outlined above may be any organic moiety that is a non-reactive, chemically stable, chain, Such spacers include, by are not limited to the following:
  • a multivalent complex of the invention in which a divalent alkylene spacer radical is located between the polyalkylene glycol chain and its point of attachment to a mutated human ILT molecule or polypeptide monomer and dimer of the complex provides a further embodiment of the present aspect.
  • a multivalent complex of the invention in which the polyalkylene glycol chain comprises at least two polyethylene glycol repeating units provides a further embodiment of the present aspect.
  • hydrophilic polymers linked, directly or via a spacer, to reactive chemistries that may be of use in the present invention. These suppliers include Nektar Therapeutics (CA, USA), NOF Corporation (Japan), Sunbio (South Korea) and Enzon Pharmaceuticals (NJ, USA).
  • hydrophilic polymers linked, directly or via a spacer, to reactive chemistries that may be of use in the present invention include, but are not limited to, the following:
  • coupling chemistries can be used to couple polymer molecules to protein and peptide therapeutics.
  • the choice of the most appropriate coupling chemistry is largely dependant on the desired coupling site.
  • the following coupling chemistries have been used attached to one or more of the termini of PEG molecules (Source: Nektar Molecular Engineering Catalogue 2003):
  • non-PEG based polymers also provide suitable linkers for multimerising the mutated human ILT molecules or polypeptide monomers and dimers thereof of the present invention.
  • linkers for multimerising the mutated human ILT molecules or polypeptide monomers and dimers thereof of the present invention.
  • moieties containing maleimide termini linked by aliphatic chains such as BMH and BMOE (Pierce, products Nos. 22330 and 22323) can be used.
  • Peptidic linkers are the other class of multivalent complex linkers. These linkers are comprised of chains of amino acids, and function to produce simple linkers or multimerisation domains onto which the polypeptides of the present invention can be attached.
  • the biotin / streptavidin system has previously been used to produce tetramers of TCRs and pMHC molecules (see WO 99/60119) for in- vitro binding studies.
  • streptavidin is a microbially-derived polypeptide and as such not ideally suited to use in a therapeutic.
  • Multivalent complexes of the invention in which the mutated human ILT molecules or polypeptide monomers and dimers thereof are linked by a peptidic linker derived from a human multimerisation domain provide one embodiment of the present aspect.
  • human proteins that contain a multimerisation domain that could be used in the production of multivalent mutated human ILT molecules or polypeptide monomers and dimers thereof complexes.
  • the tetramerisation domain of p53 has been utilised to produce tetramers of scFv antibody fragments which exhibited increased serum persistence and significantly reduced off-rate compared to the monomeric scFv fragment.
  • Haemoglobin also has a tetramerisation domain that could potentially be used for this kind of application.
  • the multivalent complexes of the invention may be dimers or tetramers.
  • Examples 11 and 12 herein provide detailed methodologies for the production of dimeric and tetrameric PEG-linked mutated human ILT molecule complexes of the invention respectively.
  • a multivalent complex of the invention comprising at least two mutated human ILT molecules or polypetide monomers and dimers thereof of the invention, wherein at least one of said mutated human ILT molecules or polypeptide monomers and dimers thereof is associated with a therapeutic agent provides a further embodiment of this aspect.
  • a further aspect is provided by a polypeptide monomer or a polypeptide dimer of the invention, or a multivalent complex thereof which is soluble.
  • the mutated human ILT molecules, or polypeptide monomers or dimers or multivalent complexes thereof of the invention may be labelled with an imaging compound, for example a label that is suitable for diagnostic purposes.
  • an imaging compound for example a label that is suitable for diagnostic purposes.
  • Such labelled mutated human ILT molecules, or polypeptide monomers or dimers or multivalent complexes thereof of the invention are useful in a method for detecting target pMHC molecules which method comprises contacting the pMHC with mutated human ILT molecules, or polypeptide monomers or dimers or multivalent complexes thereof of the invention which bind to the pMHC; and detecting said binding.
  • fluorescent streptavidin can be used to provide a detectable label.
  • a fluorescently-labelled tetramer is suitable for use in FACS analysis, for example to detect antigen presenting cells.
  • a polypeptide mutated human ILT molecules, or polypeptide monomers or dimers or multivalent complexes thereof of the present invention may alternatively or additionally be associated with (e.g. covalently or otherwise linked to) a therapeutic agent or detectable label.
  • said mutated human ILT molecules, or polypeptide monomers or dimers or multivalent complexes thereof of the invention may be covalently linked to a therapeutic agent or detectable label.
  • the therapeutic agent may be linked to an Fc-like segment of said polypeptide monomer or dimer.
  • said therapeutic agent is an immune effector molecule.
  • the said immune effector molecule may be a cytokine.
  • Mutated human ILT2 molecules, or polypeptide monomers or dimers or multivalent complexes thereof of the invention are preferred embodiments of the invention.
  • Mutated human ILT2 molecules, or polypeptide monomers or dimers or multivalent complexes thereof of the invention associated with IL-4, IL-IO or IL- 13 or a phentoypically silent variant or fragment of these cytokines provide specific embodiments of the present invention.
  • a multivalent complex of the invention may have enhanced binding capability for a given pMHC compared to the corresponding non-multimerised mutated human ILT molecule, polypeptide monomer or polypeptide dimer of the invention.
  • the multivalent complexes according to the invention are particularly useful for tracking or targeting cells presenting particular antigens in vitro or in vivo, and are also useful as intermediates for the production of further multivalent complexes having such uses.
  • compositions comprising a mutated human ILT2 molecule, or polypeptide monomer or dimer or multivalent complex thereof, optionally associated with a therapeutic agent, or a plurality of cells or particles presenting a mutated human ILT molecule of the invention together with a pharmaceutically acceptable carrier provide a further aspect of the invention.
  • a related embodiment is provided by the therapeutic use of a mutated human ILT2 molecule, or polypeptide monomer or dimer or multivalent complex thereof, optionally associated with a therapeutic agent, or a plurality of cells or particles presenting a mutated human ILT molecule of the invention.
  • a mutated human ILT2 molecule or polypeptide monomer or dimer or multivalent complex thereof, optionally associated with a therapeutic agent, or a plurality of cells or particles presenting a mutated human ILT molecule of the invention in the manufacture of a medicament for the treatment of autoimmune disease or Asthma, Eczema, Allograft rejection, Graft- versus Host Disease, Hepatitis and Cerebral malaria.
  • said medicament may be adapted for parenteral administration. Suitable parenteral routes of administration include subcutaneous, intradermal or intramuscular routes.
  • Autoimmune diseases which may be amenable to treatment by the compositions of the present invention include, but are not limited to, diseases such as Diabetes, Goodpasture's syndrome, Multiple sclerosis, Psoriasis, Rheumatoid arthritis, Myositis, Ankylosing spondylitis, Artery aneurysms in acute Kawasaki disease, Hashimoto's disease and Crohn's disease.
  • compositions of the present invention include, but are not limited to, Asthma, Eczema, Allograft rejection, Graft-versus Host Disease, Hepatitis and Cerebral malaria.
  • Soluble mutated human ILT2 molecules, or polypeptide monomers or dimers or multivalent complexes thereof of the invention may be linked to an enzyme capable of converting a prodrug to a drug. This allows the prodrug to be converted to the drug only at the site where it is required (i.e. targeted by the said mutated human IL T2 molecule, or polypeptide monomer or dimer or multivalent complex thereof).
  • mutated human ILT2 molecules or polypeptide monomers or dimers or multivalent complexes thereof, optionally associated with a therapeutic agent, or a plurality of cells or particles presenting a mutated human ILT molecule of the invention disclosed herein may be used in methods for the diagnosis and treatment of autoimmune disease or Asthma, Eczema, Allograft rejection, Graft- versus Host Disease, Hepatitis and Cerebral malaria.
  • the invention also provides a method of treatment of autoimmune disease or Asthma, Eczema, Allograft rejection, Graft- versus Host Disease, Hepatitis and Cerebral malaria comprising administering to a subject suffering such autoimmune disease an effective amount of a mutated human ILT2 molecule, or polypeptide monomer or dimer or multivalent complex thereof, optionally associated with a therapeutic agent, or a plurality of cells or particles presenting a mutated human ILT molecule of the invention.
  • the invention provides for the use of a mutated human ILT2 molecule, or polypeptide monomer or dimer or multivalent complex thereof, optionally associated with a therapeutic agent, or a plurality of cells or particles presenting a mutated human ILT molecule of the invention in the preparation of a composition for the treatment of autoimmune disease.
  • a mutated human ILT2 molecule or polypeptide monomer or dimer or multivalent complex thereof, optionally associated with a therapeutic agent, or a plurality of cells or particles presenting a mutated human ILT molecule of the invention in the preparation of a composition for the treatment of autoimmune disease.
  • Examples 9 and 10 herein describes in-vitro methods suitable for assessing the ability composition odf the inventions, such as the preferred polypeptide homodimer of the invention comprising two monomers of SEQ ID NO: 20 to inhibit cytotoxic T cell activation and killing respectively.
  • Therapeutic or imaging mutated human ILT2 molecules, or polypeptide monomers or dimers or multivalent complexes thereof, optionally associated with a therapeutic agent, or a plurality of cells or particles presenting a mutated human ILT in accordance with the invention will usually be supplied as part of a sterile, pharmaceutical composition which will normally include a pharmaceutically acceptable carrier.
  • This pharmaceutical composition may be in any suitable form, (depending upon the desired method of administering it to a patient). It may be provided in unit dosage form, will generally be provided in a sealed container and may be provided as part of a kit. Such a kit would normally (although not necessarily) include instructions for use. It may include a plurality of said unit dosage forms.
  • the pharmaceutical composition may be adapted for administration by any appropriate route, for example parenteral, transdermal or via inhalation, preferably a parenteral (including subcutaneous, intramuscular, or, most preferably intravenous) route.
  • a parenteral route for example parenteral, transdermal or via inhalation, preferably a parenteral (including subcutaneous, intramuscular, or, most preferably intravenous) route.
  • Such compositions may be prepared by any method known in the art of pharmacy, for example by mixing the active ingredient with the carrier(s) or excipient(s) under sterile conditions.
  • Dosages of the substances of the present invention can vary between wide limits, depending upon the disease or disorder to be treated, the age and condition of the individual to be treated, etc. and a physician will ultimately determine appropriate dosages to be used.
  • a mutated human ILT2 molecule, or polypeptide monomer or dimer or multivalent complex thereof of the invention of the present invention may be provided in substantially pure form, or as a purified or isolated preparation. For example, it may be provided in a form which is substantially free of other proteins.
  • nucleic acid or nucleic acids encoding mutated human ILT2 molecule, or polypeptide monomer or dimer of the invention
  • a nucleic acid or nucleic acids encoding two different mutated human ILT2 molecules, or polypeptide monomers or dimers of the invention.
  • Said nucleic acid or nucleic acids may be adapted for high level expression in a host cell.
  • nucleic acid optimisation as a service
  • Related embodiments include expression vectors incorporating said nucleic acid or nucleic acids and cells containing said vectors.
  • a related embodiment is provided by isolated cells or particles presenting at least one mutated human ILT2 molecule of the invention. As will be known to those skilled in the art there are a number of particles which are suitable for coating with the mutated human ILT molecules of the invention, for example Dynabeads® (Invitrogen).
  • a further aspect of the invention is provided by a method of producing a soluble mutated human ILT2 molecule or a soluble polypeptide monomer or dimer of the invention comprising:
  • the host cells are selected from Chinese Hamster Ovary (CHO) cells, E.coli cells or yeast cells, for example Pichia pastoris cells.
  • Example 7 herein provides a method for the production of polypeptide Fc fusion dimers of the invention in CHO cells.
  • a final aspect of the invention provided by a method of producing a cell presenting a mutated human ILT molecule of the invention comprising: (i) transforming a host cell with a vector incorporating a nucleic acid or nucleic acids encoding a mutated human ILT molecule of thie invention; and
  • the host cells are human T cells or human haematopoietic stem cells.
  • Figure Ia is the full amino acid sequence of a wild type human ILT-2 (SEQ ID No: 1)
  • the highlighted amino acids show residues of this polypeptide which differ from the corresponding residues of isoform 1 of Wild-type human ILT-2.
  • the amino acids of the transmembrane domain are underlined.
  • Figure Ib is the full DNA sequence of a wild type human ILT-2 (SEQ ID No: 2) which encodes the amino acid sequence of Figure Ia.
  • the DNA sequence corresponds to that given NCIMB Nucleotide accession NO: NM 006669.
  • Figures 2a and 2b respectively are the amino acid and DNA sequence of a soluble two domain form of the wild-type ILT-2 sequences provided in figures Ia and Ib. These truncated sequences contain / encode for only extracellular domains Dl and D2 of ILT-2. (SEQ ID No: 3 and SEQ ID NO: 4 respectively)
  • Figure 3 is the full DNA sequence inserted into the pGMT7-based vector in order to express the soluble two domain form of the wild-type ILT-2 polypeptide of Figure 2a.
  • the HindIII and Ndel restriction enzyme recognition sequences are underlined.
  • a number of mutations were introduced into the DNA encoding this polypeptide. These mutations do not alter the amino acid sequence of the expressed polypeptide.
  • Figure 4 (SEQ ID No 6) is the amino acid sequence of a soluble two domain high affinity mutated human ILT molecule ("Clone 138") The residues which have been mutated relative to those of Figure 2a are highlighted
  • Figure 5 is the DNA sequence of a pGMT7-derived vector into which DNA encoding the amino acid sequences of the ILT-like segments can be inserted.
  • Figure 6 is the plasmid map of the pGMT7-derived vector detailed in Figure 5
  • Figure 7 details the amino acid sequence of the Fc monomer of wild-type human IgGl.
  • Figure 8a details the amino acid sequences of a preferred mutated human IgGl Fc monomer.
  • the amino acid residues in these monomers that have been mutated relative to wild-type human IgGl are shaded.
  • the mutations which improve PK are in bold and shaded.
  • the mutations which counter ADCC and/or /CDCC are underlined and shaded.
  • Figure 8b details the amino acid sequences of another preferred mutated human IgGl Fc monomer.
  • the amino acid residues in these monomers that have been mutated relative to wild-type human IgGl are shaded.
  • the mutations which remove native cysteine residues are shaded and itallicised.
  • Figure 8c details the amino acid sequences of a further preferred mutated human IgGl Fc monomer.
  • the amino acid residues in these monomers that have been mutated relative to wild-type human IgGl are shaded.
  • the mutations which counter ADCC and/or /CDCC are underlined and shaded.
  • Figure 8d details the amino acid sequences of a further preferred mutated human IgGl Fc monomer.
  • the amino acid residues in these monomers that have been mutated relative to wild-type human IgGl are shaded. .
  • the mutations which counter ADCC and/or /CDCC are underlined and shaded.
  • Figures 9a to 9h detail the amino acid sequences of preferred high affinity ILT-like Fc fusion polypeptide monomers.
  • the amino acid sequences which are mutated relative to wild-type human ILT-2 and wild-type human IgGl Fc are shaded.
  • the amino acids within the linker sequences between the ILT-like and Fc-like portions of these fusion polypeptides monomers are underlined.
  • Figure 10 is the amino acid sequence of a "full-length" cell-bound form of the mutated human ILT molecule "Clone 138" The residues which have been mutated relative to those of Figure 2a are highlighted and those within the transmembrane domain are underlined.
  • Figure l la (SEQ ID NO: 23) is the amino acid sequence of a soluble mutated human ILT molecule "Clone 64" The residues which have been mutated relative to those of Figure 2a are highlighted.
  • Figure l ib (SEQ ID NO: 24) is the amino acid sequence of an Fc fusion monomer comprising the soluble mutated human ILT molecule "Clone 64" The residues which have been mutated relative to those of Figure 2a are highlighted.
  • Figure 12 is a Biacore trace of the interaction of an Fc fusion of the invention (SEQ ID NO: incorporating a mutated human ILT molecule having amino acids 2-198 of SEQ ID NO: 6 ai an NY-ESO-derived peptide-HLA-A*0201 Class I pMHC.
  • Figure 13 is a graph of the effect of titrating the concentration of two ILT-FC fusion homodimers on the inhibition of T cell activation.
  • Figure 14 is a graph of the effect of titrating the concentration of two ILT-FC fusion homodimers on the inhibition of T cell-mediated cell lysis.
  • Example 1 Production of a soluble wild-type human ILT-2 molecule comprising domains 1 and 2.
  • This examples details the production of a soluble wild-type human ILT-2 molecule comprising domains 1 and 2 (D1D2) thereof.
  • Figure 3 provides the DNA sequence used to express a soluble wild- type ILT-2 containing only domains Dl and D2.
  • This DNA sequence was synthesised de-novo by a contract research companies, GeneArt (Germany). Restriction enzyme recognition sites (Ndel and Hindlll) have been introduced into this DNA sequence in order to facilitate ligation of the DNA sequence into a pGMT7-based expression plasmid, which contains the T7 promoter for high level expression in E.coli strain BL21-DE3(pLysS) (Pan et al, Biotechniques (2000) 29 (6): 1234-8)
  • This DNA sequence is ligated into a pGMT7 vector cut with Ndel and Hindlll. (See Figure 5 for the DNA sequence of this vector and Figure 6 for the plasmid map of this vector).
  • the cut ILT-2 DNA and cut vector are ligated using a rapid DNA ligation kit (Roche) following the manufacturers instructions.
  • Ligated plasmids are transformed into competent E.coli strain XLl -blue cells and plated out on LB/agar plates containing 100mg/ml ampicillin. Following incubation overnight at 37 0 C, single colonies are picked and grown in 10 ml LB containing lOOmg/ml ampicillin overnight at 37 0 C with shaking. Cloned plasmids are purified using a Miniprep kit (Qiagen) and the insert is sequenced using an automated DNA sequencer (Lark Technologies).
  • Figure 2a shows the amino acid sequence of the soluble wild-type ILT-2 polypeptide produced from the DNA sequence of Figure 2b.
  • This polypeptide can be used as the reference polypeptide to compare the pMHC affinity and ability to inhibit CD8/pMHC binding of the high affinity mutated human ILT molecules of the present invention.
  • the methods required to carry out these determinations are detailed in Examples 4 and 5 respectively.
  • the soluble wild-type human ILT-2 polypeptide produced as described in Example 1 can be used as a template from which to produce the polypeptides of the invention which have an increased affinity and/or slower off-rate for class I pMHC molecules.
  • Mutagenesis was carried out using the following conditions : 50ng plasmid template, l ⁇ l of 1OmM dNTP, 5 ⁇ l of 1Ox Pfu DNA polymerase buffer as supplied by the manufacturer, 25 pmol of fwd primer, 25 pmol of rev primer, l ⁇ l pfu DNA polymerase in total volume 50 ⁇ l. After an initial denaturation step of 2 mins at 95C, the reaction was subjected to 25 cycles of denaturation (95C, 10 sees), annealing (55C 10 sees), and elongation (72C, 8 mins). The resulting product was digested with Dpnl restriction enzyme to remove the template plasmid and transformed into E. coli strain XLl -blue. Mutagenesis was verified by sequencing.
  • the expression plasmid containing the soluble human ILT moecules as prepared in Examples 1 or 2 were transformed separately into E.coli strain rosetta DE3pLysS, and single ampicillin / chloramphenicol-resistant colonies were grown at 37°C in TYP (ampicillin lOO ⁇ g/ml, chloramphenicol 15 ⁇ g/ml) medium for 7 hours before inducing protein expression with 0.5mM IPTG. Cells were harvested 15 hours post-induction by centrifugation for 30 minutes at 4000rpm in a Beckman J-6B.
  • Cell pellets were re- suspended in a buffer, re-suspended cells were sonicated in 1 minute bursts for a total of around 10 minutes in a Milsonix XL2020 sonicator using a standard 12mm diameter probe. Inclusion body pellets were recovered by centrifugation for 10 minutes at 4000rpm in a Beckman J2-21 centrifuge. Three detergent washes were then carried out to remove cell debris and membrane components.
  • the inclusion body pellet was homogenised in a Triton buffer (5OmM Tris-HCI, 0.5% Triton-XIOO, 20OmM NaCI, 1OmM NaEDTA, 0.1% (w/v) NaAzide, 2mM DTT, pH 8.0) before being pelleted by centrifugation for 15 minutes at 4000rpm in a Beckman J2-21. Detergent and salt was then removed by a similar wash in the following buffer: 5OmM Tris-HCI, ImM NaEDTA, 0.1% (w/v) NaAzide, 2mM DTT, pH 8.0. Finally, the inclusion bodies were divided into 60mg aliquots and frozen at -70°C. Inclusion body protein yield was quantitated by solubilising with 6M guanidine-HCl and measurement using a UV spectrometer.
  • Triton buffer 5OmM Tris-HCI, 0.5% Triton-XIOO, 20OmM NaCI, 1OmM NaEDTA,
  • guanidine solution (6 M Guanidine-hydrochloride, 1OmM Sodium Acetate, 1OmM EDTA), to ensure complete chain de-naturation.
  • the guanidine solution containing fully reduced and denatured ILT polypeptide was then injected into 1 litre of the following refolding buffer: 10OmM Tris pH 8.5, 40OmM L-Arginine, 2mM EDTA, 5mM reduced Cystaeimine, 0.5mM 2-mercaptoethylamine, 5M urea.
  • the redox couple (2-mercaptoethylamine and cystamine (to final concentrations of 6.6mM and 3.7mM, respectively) were added approximately 5 minutes before addition of the denatured ILT polypeptide. The solution was left for 30 minutes. The refolded soluble human ILT molecules were dialysed in Spectrapor 1 membrane (Spectrum; Product No. 132670) against 10 L 10 mM Tris pH 8.1 at 5°C ⁇ 3°C for 18-20 hours. After this time, the dialysis buffer was changed to fresh 10 mM Tris pH 8.1 (10 L) and dialysis was continued at 5 0 C ⁇ 3 0 C for another 20-22 hours.
  • Soluble human ILT molecules were separated from degradation products and impurities by loading the dialysed refold onto a POROS 50HQ anion exchange column and eluting bound protein with a gradient of 0-50OmM NaCI over 50 column volumes using an Akta purifier (Pharmacia). Peak fractions were stored at 4°C and analysed by Coomassie-stained SDS-PAGE before being pooled and concentrated. Finally, the soluble human ILT molecules were purified and characterised using a Superdex 200HR gel filtration column pre-equilibrated in HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3.5 mM EDTA, 0.05% nonidet p40).
  • a surface plasmon resonance biosensor (Biacore 3000TM) was used to analyse the binding of soluble mutated human ILT molecules of the invention to class I pMHC. This analysis was facilitated by producing soluble biotinylated pMHC (described below) which were immobilised to a streptavidin-coated binding surface in a semi- oriented fashion, allowing efficient testing of the binding of soluble mutated human ILT molecules to up to four different pMHC (immobilised on separate flow cells) simultaneously. Injection of the pMHC allows the precise level of immobilised class I molecules to be manipulated easily.
  • Soluble biotinylated class I HLA-A*0201 loaded with a CEA-derived YLSGANLNL (SEQ ID NO: 7) peptide were refolded in vitro from bacterially-expressed inclusion bodies containing the constituent subunit proteins and synthetic peptide, followed by purification and in vitro enzymatic biotinylation (O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15).
  • MHC-heavy chain was expressed with a C-terminal biotinylation tag which replaces the transmembrane and cytoplasmic domains of the protein in an appropriate construct.
  • Inclusion body expression levels of -75 mg/litre bacterial culture were obtained.
  • the MHC light-chain or ⁇ 2-microglobulin was also expressed as inclusion bodies in E.coli from an appropriate construct, at a level of -500 mg/litre bacterial culture.
  • the E. coli cells were lysed and inclusion bodies are purified to approximately 80% purity. Protein from inclusion bodies was denatured in 6 M guanidine-HCl, 50 mM Tris pH 8.1, 100 mM NaCl, 10 mM DTT, 10 mM EDTA, and was refolded at a concentration of 30 mg/litre heavy chain, 30 mg/litre ⁇ 2m into 0.4 M L-Arginine-HCl, 100 mM Tris pH 8.1, 3.7 mM cystamine, 6.6 mM ⁇ -cysteamine, 4 mg/ml of the peptide required to be loaded by the MHC, by addition of a single pulse of denatured protein into refold buffer at ⁇ 5 0 C.
  • Refolding was allowed to reach completion at 4 0 C for at least 1 hour.
  • Buffer was exchanged by dialysis in 10 volumes of 10 mM Tris pH 8.1. Two changes of buffer were necessary to reduce the ionic strength of the solution sufficiently.
  • the protein solution was then filtered through a 1.5 ⁇ m cellulose acetate filter and loaded onto a POROS 50HQ anion exchange column (8 ml bed volume). Protein was eluted with a linear 0-500 mM NaCl gradient.
  • the soluble biotinylated HLA-A2-peptide complex eluted at approximately 250 mM NaCl, and peak fractions were collected, a cocktail of protease inhibitors (Calbiochem) was added and the fractions were chilled on ice.
  • Biotinylation tagged pMHC were buffer exchanged into 10 mM Tris pH 8.1, 5 mM NaCl using a Pharmacia fast desalting column equilibrated in the same buffer. Immediately upon elution, the protein-containing fractions were chilled on ice and protease inhibitor cocktail (Calbiochem) was added. Biotinylation reagents were then added: 1 mM biotin, 5 mM ATP (buffered to pH 8), 7.5 mM MgC12, and 5 ⁇ g/ml BirA enzyme (purified according to O'Callaghan et al. (1999) Anal. Biochem. 266: 9- 15). The mixture was then allowed to incubate at room temperature overnight.
  • Biotinylated pMHC were purified using gel filtration chromatography. A Pharmacia Superdex 75 HR 10/30 column was pre-equilibrated with filtered PBS and 1 ml of the biotinylation reaction mixture was loaded and the column was developed with PBS at 0.5 ml/min. Biotinylated pMHC eluted as a single peak at approximately 15 ml. Fractions containing protein were pooled, chilled on ice, and protease inhibitor cocktail was added. Protein concentration was determined using a Coomassie-binding assay (PerBio) and aliquots of biotinylated pMHC were stored frozen at -2O 0 C. Streptavidin was immobilised by standard amine coupling methods.
  • PerBio Coomassie-binding assay
  • Such immobilised pMHC are capable of binding soluble T-cell receptors and the co- receptor CD8 ⁇ , as well as ILT-like molecules, and these interactions can be used to ensure that the immobilised pMHC are correctly refolded.
  • SPR measures changes in refractive index expressed in response units (RU) near a sensor surface within a small flow cell, a principle that can be used to detect receptor ligand interactions and to analyse their affinity and kinetic parameters.
  • the probe flow cells were prepared by immobilising the pMHC complexes in flow cells via biotin-tag binding.
  • the assay was then performed by passing the soluble mutated ILT molecules over the surfaces of the different flow cells at a constant flow rate, measuring the SPR response in doing so.
  • K D was determined by experimentally measuring the dissociation rate constant, kd, and the association rate constant, ka. The equilibrium constant K D was calculated as kd/ka.
  • High affinity soluble mutated ILT molecules were injected over two different cells one coated with -300 RU of CEA-derived YLSGANLNL (SEQ ID NO: 7)-HLA-A*0201 complex, the second was left blank as a control Flow rate was set at 50 ⁇ l/min. Typically 250 ⁇ l of ILT polypeptide at -3 ⁇ M was injected. Buffer was then flowed over until the response had returned to baseline. Kinetic parameters were calculated using Biaevaluation software. The dissociation phase was also fitted to a single exponential decay equation enabling calculation of half-life.
  • the high affinity mutated human ILT molecule of the present invention having the amino acid sequence provided in Figure 4 (SEQ ID No: 6) has a K D for Class I pMHC of 2.56nM, an off-rate (k off ) of 4.56 x 10 "3 S "1 , an on-rate (Ic 0n ) of 1.76 x 10 6 Ms '1 and a half-live of 142 seconds.
  • the closest analogous, and highest affinity, mutated human ILT molecule known from WO 2006/125963 namely the molecule having the sequence of amino acid sequence provided by SEQ ID NO: 23 herein, has a K D for Class I pMHC of 4OnM, an off-rate (k off ) of 4.7 x 10 "2 S "1 , an on-rate (Ic 0n ) of 1.09 x 10 6 Ms '1 and a half- live of 14.7 seconds.
  • Example 5 Biacore surface plasmon resonance analysis of soluble ILT-mediated inhibition of the pMHC/CD8 interaction
  • a surface plasmon resonance biosensor (Biacore 3000TM) is used to analyse the ability of soluble mutated human ILT molecules to mediate inhibition of the class I pMHC/CD8 interaction. This analysis is facilitated by producing soluble pMHC complexes (described below) and biotinylated soluble CD8 ⁇ molecules (also described below).
  • biotinylated soluble CD8 ⁇ molecules are immobilised to a streptavidin-coated binding surface "Biacore chip" in a semi-oriented fashion, allowing efficient testing of the binding of soluble pMHC complexes to the immobilised soluble CD8 ⁇ . Injection of the biotinylated soluble CD8 ⁇ molecules allows the precise level of immobilised CD8 molecules to be manipulated easily.
  • Soluble HLA-A*0201 pMHC loaded with a CEA-derived SEQ ID NO: 7) peptide are produced using the methods substantially as described in (Garboczi et. al, (1992) PNAS USA 89 3429-3433).
  • the soluble pMHC molecules are refolded in vitro from E.coli expressed inclusion bodies containing the constituent subunit proteins and synthetic peptide and then purified.
  • the MHC light-chain or ⁇ 2-microglobulin is also expressed as inclusion bodies in E.coli from an appropriate construct, at a level of -500 mg/litre bacterial culture.
  • E. coli cells are lysed and inclusion bodies are purified, and the over-expressed proteins are refolded and purified using the methods detailed in Example 4 except that the biotinylation steps are omitted.
  • Biotinylated soluble CD8 molecules are produced as described in Examples 1 and 6 of EP 1024822. Briefly, the soluble CD8 ⁇ containing a C-terminal biotinylation tag is expressed as inclusion bodies in E.coli and then purified and refolded to produce CD8 ⁇ homodimers containing a tag sequence that can be enzymatically biotinylated.. (Schatz, (1993) Biotechnology N Y 11. 1138-43). Biotinylation of the tagged CD8 ⁇ molecules is then achieved using the BirA enzyme (O'Callaghan, et al.
  • Biotinylation reagents are : 1 mM biotin, 5 raM ATP (buffered to pH 8), 7.5 mM MgC12, and 5 ⁇ g/ml BirA enzyme (purified according to O'Callaghan et al. (1999) Anal. Biochem. 266: 9-15). The mixture is then allowed to incubate at room temperature overnight.
  • the biotinylated sCD8 ⁇ is immobilised on the surface of a Biacore streptavidin- coated chip producing a change in the refractive index of 1000 response units.
  • Such immobilised CD8 ⁇ molecules are capable of binding soluble pMHC complexes which may be injected in the soluble phase.
  • SPR measures changes in refractive index expressed in response units (RU) near a sensor surface within a small flow cell, a principle that can be used to detect receptor ligand interactions and to analyse their affinity and kinetic parameters.
  • the chips are prepared by immobilising the soluble biotinylated CD8 ⁇ molecules to streptavidin coated chips as described above.
  • Serial dilutions of soluble wild-type ILT-2 (SEQ ID NO: 3) wild-type ILT or high affinity mutated human ILT molecules are prepared and injected at constant flow rate of 5 ⁇ l min "1 over a flow cell coated with 1000 RU of biotinylated CD8 ⁇ in the presence of a suitable concentration of soluble YLSGANLNL (SEQ ID NO: 7) -HLA-A* 0201.
  • the inhibition of the SPR responses for the CD8 ⁇ /pMHC interaction produce a dose response curve which is used to calculate an IC50 value for the polypeptide being assayed for this interaction.
  • Gap -10
  • the wild-type human IgGl Fc amino acid sequence in single letter code as provided in Figure 7 is entered as the first (query) sequence and the amino acid sequence for comparison thereto is entered as the second (library) sequence.
  • the algorithm can then be run and will provide percentage identity and similarity scores for the pair of sequences compared.
  • pFUSE vector DNA (Invivogen; pfuse-hglfc2 or pfc2-hgle3 ) was digested with BgIII and EcoRI restriction enzymes for 4.5h at 37 0 C.
  • These vectors incorporate DNA encoding wild-type (pfuse-hglfc2 vector) and mutated (pfc2-hgle3 vector - SEQ ID NO: 13 (See Figure 8d for amino acid sequence) Fc portions of the human IgGl immunoglobulin respectively and the human IL-2 leader sequence (MYRMQLLSCIALSLALVTNS (SEQ ID NO: 25)) which ensures secretion of the ILT-Fc fusions.
  • Digested vector DNA was purified using commercially available spin-columns.
  • DNA encoding a mutated human ILT Fc fusion of the invention which comprises amino acids 2 -198 of the mutated humam ILT molecule of SEQ ID NO: 6, and a comparison ILT-Fc fusion which comprises amino acids 2 -198 of the mutated humam ILT molecule of SEQ ID NO: 23 were PCRed from template vector DNA using the forward primer SDl 13 (tagged with an EcoRI site) and the reverse primers SDl 14 and SDl 15 (tagged with BgIII sites) which encode two different linkers differing in length by four amino acids.
  • This comparison ILT-Fc fusion comprises amino acids 2 to 198 of the mutated human ILT molecule of Figure 13bd of WO 2006/125963.
  • SEQ ID NO: 20 ( Figure 9g) is the full amino acid sequence of a polypeptide monomer of the polypeptide dimer of the invention.
  • SEQ ID NO: 24 ( Figure 1 Ib) is the full amino acid sequence of a polypeptide monomer of the compariative polypeptide dimer
  • the comparison ILT-Fc fusion contained the mutated human ILT molecule of SEQ ID NO: 23 ( Figure 1 Ia).
  • the full amino acid sequence of this comparison ILT-Fc fusion is provided by SEQ ID NO: 24 ( Figure l ib).
  • the PCR products were digested with EcoRI and BgIII for 3hours at 37 0 C and the digested fragments were gel-purified using a commercially available kit.
  • ILT-linker fragments were ligated into the digested pFUSE vectors and transformed into E.coli strain XL-I Blue. Following selection of transformed clones on solid media containing 100ug/ml zeocin, DNA was isolated for sequencing using a commercially available kit. Clones of the correct sequence were grown in 50ml LB media and Fc-fusion vector DNA isolated for cell transfections using a commercially available kit.
  • Transfections of log-phase CHO-S suspension cells (Invitrogen) growing in serum- free CD-CHO medium (Invitrogen) with the ILT2:pFUSE constructs were performed using Lipofectamine 2000 reagent according to the manufacturers instructions. Transfected cultures were grown under zeocin selection (400ug/ml) for 3-4 weeks to generate stable polyclonal lines. The ILT-Fc-fusion polypeptides secreted from polyclonal lines were purified using Protein A affinity resin according to standard protocols. The isolation of high-expressing discrete clones was performed by FACS seeding single cells into 96-well plates containing 200ul of serum-free medium per well and 400ug/ml zeocin.
  • Example 8 Competitive binding Fluorescence Activated Cell Sorting (FACS) assay or assessing the ability of the polypeptide monomers or dimers of the invention to bind to Fc receptors.
  • FACS Fluorescence Activated Cell Sorting
  • Hinton et ai (2004) J Biol. Chem. 279 (8): 6213-6216 details the methods required to obtain a suitable cell line, and to carry out an appropriate FACS-based assay for assessing the ability of the ILT-FC fusions of the invention to bind to the human neonatal Fc receptor (FcRn).
  • FcRn human neonatal Fc receptor
  • cDNA encoding the human FcRn and human beta-2 microglobulin is cloned by PCR from peripheral blood monucleate cells (PBMCs) and sub-cloned cloned into a vector derived from pVk.
  • PBMCs peripheral blood monucleate cells
  • the NSO mouse myeloma cell line (The European Collection of Animal Cell Cultures, Salisbury, UK) is then transfected with this vector by electroporation to obtain a stably transfected cell line.
  • FACS-based competitive binding assays can then be carried by analysing the ability of the ILT-Fc fusions of the invention to compete against the binding of a range of concentrations of a reference human IgG antibody to FcRn. Any reduction in the observed level of binding of the reference antibody in the presence of the ILT-Fc fusions of the invention would indicate that they were capable of binding to human FcRn.
  • Example 9 - ELISPOT assay for assessing in-vitro inhibition ofcyto-toxic T cell (CTL) activation by the mutated human ILT molecules, polypeptide monomers or polypeptide dimers of the invention or multivalent complexes thereof
  • the following method provides a means of assessing the ability of the soluble mutated human ILT molecules, polypeptide monomers or dimers, or multivalent complexes thereof, to inhibit CD8 co-receptor mediated T cell activation.
  • Assay media 10% FCS (heat-inactivated, Gibco, cat# 10108-165), 88% RPMI 1640 (Gibco, cat# 42401-018), 1% glutamine (Gibco, cat# 25030-024) and 1% penicillin/streptomycin (Gibco, cat# 15070-063).
  • Wash buffer 0.01 M PBS/0.05% Tween 20 (1 sachet of Phosphate buffered saline with Tween 20, pH7.4 from Sigma, Cat. # P-3563 dissolved in 1 litre distilled water gives final composition 0.01 M PBS, 0.138 M NaCl, 0.0027 M KCl, 0.05 % Tween 20).
  • Diaclone EliSpot kit contains all other reagents required i.e. capture and detection antibodies, skimmed milk powder, BSA, streptavidin-alkaline phosphatase, BCIP/NBT solution (Human IFN- ⁇ PVDF Eli-spot 20 x 96 wells with plates (IDS cat# DC-856.051.020, DC-856.000.000.
  • 100 ⁇ l capture antibody was diluted in 10 ml sterile PBS per plate. 100 ⁇ l diluted capture antibody was aliquoted into each well and left overnight at 4 0 C, or for 2 hr at room temperature. The plates wee then washed three times with 450 ⁇ l wash buffer, Ultrawash 96-well plate washer, (Thermo Life Sciences) to remove excess capture antibody. 100 ⁇ l of 2% skimmed milk was then added to each well. (One vial of skimmed milk powder as supplied with the EliSpot,kit is dissolved in 50 ml sterile PBS). The plates were then incubated at room temperature for two hours before washing washed a further three times with 450 ⁇ l wash buffer, Ultrawash 96-well plate washer, (Thermo Life Sciences)
  • a MART-I specific T cell clone (KA/C5) (effector cell line) was harvested by centrifugation (280 x g for 10 min) and resuspended at lxlO 4 /ml in assay media to give 500 cells/ well when 50 ⁇ l was added to the assay plate.
  • a mutated human ILT molecule, polypeptide monomer, dimer or multivalent complex of the invention was diluted in assay media at a 3x concentration to give a Ix final when 50ul is added to the plate in a final volume of 150 ⁇ l. A range of different concentration solutions of this test sample were then prepared for testing.
  • Test samples 50 ⁇ l Mel 624 target cells 50ul of the desired concentration of the mutated human ILT molecule, polypeptide monomer, dimer or multivalent complex.
  • the plates were then incubated overnight at 37°C/5% CO 2 .
  • the plates were then washed six times with wash buffer before tapping out excess buffer.
  • 550 ⁇ l distilled water was then added to each vial of detection antibody supplied with the ELISPOT kit to prepare a diluted solution.
  • 100 ⁇ l of the diluted detection antibody solution was then further diluted in 10 ml PBS/1% BSA per plate and 100 ⁇ l of the diluted detection antibody solution was aliquoted into each well.
  • the plates were then incubated at room temperature for 90 minutes.
  • the plates were then washed thoroughly in tap water and shaken before being taken apart and left to dry on the bench.
  • the number of spots that appeared in each well is proportional to the number of T cells activated. Therefore, any decrease in the number of spots in the wells containing mutated human ILT molecule, polypeptide monomer, dimer or multivalent complex indicates inhibition of CD 8 co-receptor-mediated CTL activation.
  • Figure 13 is a graph of the effect of titrating the concentration of two ILT-FC fusion homodimers on the inhibition of T cell activation.
  • the "cl38 Fc dimer” is an ILT Fc fusion homodimer of the invention.
  • SEQ ID NO: 20 ( Figure 9g) provides the full amino acid sequence of the polypeptide monomer of this homodimer which comprises amino acids 2 -198 of the mutated human ILT molecule of SEQ ID NO: 6.
  • the "c64 Fc dimer” is a comparison ILT Fc fusion homodimer.
  • Example 10 In-vitro cellular assay of T cell -mediated target cell lysis in the presence and absence of the mutated human ILT molecules, polypeptide monomers or polypeptide dimers of the invention or multivalent complexes thereof
  • Target cells (Mel 624 or peptide pulsed T2 cells) were loaded with BATDA reagent for 30min at 37°C/5%CO2 according to package instructions (l-3 ⁇ l BATDA added to 1x10 6 cells in ImI assay media). The target cells were washed three times in assay media containing lOO ⁇ M ⁇ -mercaptoethanol and resuspended at 1x10 5 cells/ml to give 5000 cells/well in 50 ⁇ l. The ILT-Fc fusion polypeptide dimers were added to the wells at varying concentrations (50 ⁇ l of 3X final concentration in assay media) before the addition of effector cells (T cell clones, Melc5 or EBV 176 D5.1).
  • the effector to target ratio was determined for each T cell clone (3:1 Melc5:Mel 624;) and the relevant number of effector cells was added in 50 ⁇ l assay media.
  • Target cells alone spontaneous release
  • target cells + 1% triton maximum release
  • supernatant from the final wash of the targets background
  • the plates were incubated at 37°C/5%CO2 for 2 hours.
  • the plates were centrifuged and 20 ⁇ l of supernatant was transferred to a black plate.
  • 180 ⁇ l europium solution was added to each well and the plates were shaken for 15min before reading in the Wallac Victor II.
  • % Spontaneous release 100 x (spotaneous release-background) / (maximum release- background)
  • Figure 14 is a graph of the effect of titrating the concentration of two ILT-Fc fusion homodimers on the inhibition of T cell-mediated cell lysis.
  • the "cl38 dimer” is an ILT-Fc fusion homodimer of the invention.
  • SEQ ID NO: 20 ( Figure 9g) provides the full amino acid sequence of the polypeptide monomer of this homodimer which comprises amino acids 2 -198 of the mutated human ILT molecule of SEQ ID NO: 6.
  • the "c64 dimer” is a comparison ILT Fc fusion homodimer.
  • Soluble mutated human ILT molecules having amino acids 2 to 198 of SEQ ID NO: 6 and an additional cysteine residue at the C-terminus were prepared using the methods detailed in Examples 1 to 3. These soluble mutated human ILT molecues were cross- linked using non-branched bifunctional maleimide-PEG (MAL-PEG-MAL, MW 3.4KD, NOF Corporation, Japan). The maleimide groups on the termini of this linker confer free thiol binding specificity to the linker. Prior to cross-linking the mutated ILT molecules were pre-treated with a reducing agent, O.lmM DTT (room temperature, overnight), in order to liberate the free cysteine on the soluble mutated human ILT molecules.
  • a reducing agent O.lmM DTT (room temperature, overnight
  • This low concentration of reducing agent is used to selectively reduce the exposed C-terminal cysteine residue.
  • the soluble mutated human ILT molecules was then re-purified by gel-filtration chromatography (Superdex 75) in PBS buffer.
  • the soluble mutated human ILT molecules were then re-concentrated using a 1OkDa cut-off centrifugal membrane concentrator (VivaScience, Satorius).
  • Cross- linking was achieved by the stepwise addition of MAL-PEG-MAL (1OmM in DMF) at an approximately 2:1 (protein to cross-linker) molar ratio and subsequently incubating for 2 hours at room temperature.
  • the product was then purified using Superdex 75 HRl 0/30 gel-filtration column pre-equilibrated in PBS.
  • Soluble mutated human ILT molecules having amino acids 2 to 198 of SEQ ID NO: 6 and an additional cysteine residue at the C-terminus were prepared using the methods detailed in Examples 1 to 3. These soluble mutated human ILT molecues are tetramerised using a tetrameric maleimide-PEG (4arm MAL-PEG, MW 20KD, Shearwater Corporation). The maleimide groups on the termini of this linker confer free thiol binding specificity to the linker. Prior to cross-linking the soluble mutated human ILT molecues are pre-treated with a reducing agent, 0.
  • ImM DTT room temperature, overnight
  • This low concentration of reducing agent is used to selectively reduce the exposed C-terminal cysteine residue.
  • the soluble mutated human ILT molecues are then re-purified by gel-filtration chromatography (Superdex 75) in PBS buffer.
  • the soluble mutated human ILT molecues are then re-concentrated using a 1OkDa cut-off centrifugal membrane concentrator (VivaScience, Satorius).
  • Tetramerisaton is achieved by the stepwise addition of the 4arm MAL-PEG (1OmM in DMF) at an approximately 4:1 (protein to cross-linker) molar ratio and subsequent incubation for 2 hours at room temperature.
  • the product is then purified using Superdex 75 HRl 0/30 gel-filtration column pre-equilibrated in PBS. The eluted fractions are further analysed by SDS-PAGE.
  • Samples from the fractions are pre-treated with standard SDS sample buffer (BioRad) without DTT (non-reducing) or with 15mM DTT (reducing), and are run on a gradient 4-20% PAGE and stained with Coomassie blue stain.

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Toxicology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Cell Biology (AREA)
  • Peptides Or Proteins (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

La présente invention concerne des molécules ILT humaines mutées comprenant les acides aminés 4 à 197 de la SEQ ID NO: 6. L'invention concerne également des fusions polypeptidiques dimériques et monomériques comprenant lesdites molécules ILT humaines mutées et des segments d'immunoglobuline Fc. De telles compositions sont utiles soit seules, soit associées à un agent thérapeutique, pour cibler des cellules exprimant les molécules pCMHI de classe I.
PCT/GB2007/004257 2007-11-08 2007-11-08 Molécules ilt mutées WO2009060159A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/GB2007/004257 WO2009060159A1 (fr) 2007-11-08 2007-11-08 Molécules ilt mutées

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/GB2007/004257 WO2009060159A1 (fr) 2007-11-08 2007-11-08 Molécules ilt mutées

Publications (1)

Publication Number Publication Date
WO2009060159A1 true WO2009060159A1 (fr) 2009-05-14

Family

ID=39301292

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2007/004257 WO2009060159A1 (fr) 2007-11-08 2007-11-08 Molécules ilt mutées

Country Status (1)

Country Link
WO (1) WO2009060159A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017527616A (ja) * 2014-09-05 2017-09-21 アールエスイーエム，リミティド パートナーシップ 炎症を処置および予防するための組成物および方法
JP2018515072A (ja) * 2015-05-04 2018-06-14 アポジェニックス アーゲー 単鎖ｃｄ４０受容体アゴニストタンパク質

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006125963A2 (fr) * 2005-05-25 2006-11-30 Medigene Limited Polypeptides
EP1772515A1 (fr) * 1999-05-12 2007-04-11 Immunex Corporation Immunorégulateur de la famille nommée 'récepteurs semblables à des récepteurs de leucocytes de type immunoglobuline' (LIR)

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1772515A1 (fr) * 1999-05-12 2007-04-11 Immunex Corporation Immunorégulateur de la famille nommée 'récepteurs semblables à des récepteurs de leucocytes de type immunoglobuline' (LIR)
WO2006125963A2 (fr) * 2005-05-25 2006-11-30 Medigene Limited Polypeptides

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
CHAPMAN TARA L ET AL: "Crystal structure and ligand binding properties of the D1D2 region of the inhibitory receptor LIR-1 (ILT2)", IMMUNITY, CELL PRESS, US, vol. 13, no. 5, November 2000 (2000-11-01), pages 727 - 736, XP002437411, ISSN: 1074-7613 *
DATABASE EMBL ebi; 3 October 2003 (2003-10-03), "(Immunoglobulin-like transcript 2 (ILT-2)", XP002478010, Database accession no. Q8NHL6 *
KUROKI KIMIKO ET AL: "Extensive polymorphisms of LILRB1 (ILT2, LIR1) and their association with HLA-DRB1 shared epitope negative rheumatoid arthritis", HUMAN MOLECULAR GENETICS, OXFORD UNIVERSITY PRESS, SURREY, GB, vol. 14, no. 16, August 2005 (2005-08-01), pages 2469 - 2480, XP002454509, ISSN: 0964-6906 *
SHIROISHI MITSUNORI ET AL: "Human inhibitory receptors Ig-like transcript 2 (ILT2) and ILT4 compete with CD8 for MHC class I binding and bind preferentially to HLA-G", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF USA, NATIONAL ACADEMY OF SCIENCE, WASHINGTON, DC, US, vol. 100, no. 15, 22 July 2003 (2003-07-22), pages 8856 - 8861, XP002437412, ISSN: 0027-8424 *
WILLCOX BENJAMIN E ET AL: "Crystal structure of HLA-A2 bound to LIR-1, a host and viral major histocompatibility complex receptor.", NATURE IMMUNOLOGY, vol. 4, no. 9, September 2003 (2003-09-01), pages 913 - 919, XP002478009, ISSN: 1529-2908 *
WILLCOX BENJAMIN E ET AL: "Crystal structure of LIR-2 (ILT4) at 1.8 A: differences from LIR-1 (ILT2) in regions implicated in the binding of the Human Cytomegalovirus class I MHC homolog UL18", BMC STRUCTURAL BIOLOGY, BIOMED CENTRAL LTD., LONDON, GB, vol. 2, no. 1, 11 October 2002 (2002-10-11), pages 6, XP021017168, ISSN: 1472-6807 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2017527616A (ja) * 2014-09-05 2017-09-21 アールエスイーエム，リミティド パートナーシップ 炎症を処置および予防するための組成物および方法
US10738122B2 (en) 2014-09-05 2020-08-11 Rsem, Limited Partnership Compositions and methods for treating and preventing inflammation
US10766964B2 (en) 2014-09-05 2020-09-08 Rsem, Limited Partnership Compositions and methods for treating and preventing inflammation
US11649292B2 (en) 2014-09-05 2023-05-16 Rsem, Limited Partnership Compositions and methods for treating and preventing inflammation
JP2018515072A (ja) * 2015-05-04 2018-06-14 アポジェニックス アーゲー 単鎖ｃｄ４０受容体アゴニストタンパク質
US10793616B2 (en) 2015-05-04 2020-10-06 Apogenix Ag Single-chain CD40-receptor agonist proteins

Similar Documents

Publication Publication Date Title
US8217144B2 (en) High affinity Melan-A T cell receptors
EP1885754B1 (fr) Récepteurs des lymphocytes t se fixant spécifiquement à vygfvracl-hla-a24
CA2602463C (fr) Recepteurs de lymphocytes t infectes par le vih presentant une affinite elevee
AU730457B2 (en) Soluble monovalent and multivalent MHC class II fusion proteins, and uses therefor
US8017730B2 (en) T cell receptors which specifically bind to VYGFVRACL-HLA-A24
WO2006054096A2 (fr) Proteines bifonctionnelles solubles
US20100135997A1 (en) Polypeptide monomers and dimers containing mutated ilt
EP1885752B1 (fr) Membres modifies de la famille de recepteurs leukocyte du type immunoglobulin (lir's) presentant une affinite accrue pour les molecules de mhc classe i et utilisation de ceux-ci pour modulation d'activation de lymphocytes t
WO2009060159A1 (fr) Molécules ilt mutées
US20110172168A1 (en) Ilt-2 (lir1) variants with increased affinity for mhc class i molecules
AU2013202288B2 (en) High affinity HIV T cell receptors

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 07824490

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 07824490

Country of ref document: EP

Kind code of ref document: A1