US20120178703A1 - Multimeric polypeptides of hla-g including at least two alpha3 domains and pharmaceutical uses thereof - Google Patents

Multimeric polypeptides of hla-g including at least two alpha3 domains and pharmaceutical uses thereof Download PDF

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US20120178703A1
US20120178703A1 US13/379,525 US201013379525A US2012178703A1 US 20120178703 A1 US20120178703 A1 US 20120178703A1 US 201013379525 A US201013379525 A US 201013379525A US 2012178703 A1 US2012178703 A1 US 2012178703A1
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hla
alpha3
multimer
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Joël Le Maoult
Edgardo Delfino Carosella
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Commissariat a lEnergie Atomique et aux Energies Alternatives CEA
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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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/001—Preparations to induce tolerance to non-self, e.g. prior to transplantation
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection

Definitions

  • the present invention relates to multimeric polypeptides and pharmaceutical uses thereof.
  • the invention more specifically relates to multimers comprising alpha3 domains of an HLA-G antigen.
  • the invention also relates to methods of producing such multimers, pharmaceutical compositions comprising the same, as well as their uses for treating various diseases including organ/tissue rejection.
  • MHC antigens are divided up into three main classes, namely class I antigens, class II antigens (HLA-DP, HLA-DQ and HLA-DR), and class III antigens.
  • Class I antigens comprise classical antigens, HLA-A, HLA-B and HLA-C, which exhibit 3 globular domains ( ⁇ 1, ⁇ 2 and ⁇ 3) associated with bet ⁇ 2 microglobulin, as well as non classical antigens HLA-E, HLA-F, and HLA-G.
  • HLA-G is a non-classic HLA Class I molecule expressed by extra-villous trophoblasts of normal human placenta, thymic epithelial cells and cornea.
  • HLA-G antigens are essentially expressed by the cytotrophoblastic cells of the placenta and function as immunomodulatory agents protecting the foetus from the maternal immune system (absence of rejection by the mother).
  • HLA-G gene has been described [1,2] and comprises 4396 base pairs.
  • This gene is composed of 8 exons, 7 introns and a 3′ untranslated end, corresponding respectively to the following domains: exon 1: signal sequence, exon 2: alpha1 extracellular domain, exon 3: alpha2, extracellular domain, exon 4: alpha3 extracellular domain, exon 5: transmembrane region, exon 6: cytoplasmic domain I, exon 7: cytoplasmic domain II (untranslated), exon 8: cytoplasmic domain III (untranslated) and 3′ untranslated region.
  • HLA-G Seven isoforms of HLA-G (SEQ ID NO:6) have been identified, among which 4 are membrane bound (HLA-G1, HLA-G2, HLA-G3 and HLA-G4) and 3 are soluble (HLA-G5, HLA-G6 and HLA-G7) (see [3] for review).
  • the mature HLA-G1 protein isoform comprises the three external domains ( ⁇ 1, ⁇ 2 and ⁇ 3), the transmembrane region and the cytoplasmic domain.
  • the HLA-G2 protein isoform does not comprise the ⁇ 2 domain, i.e., the ⁇ 1 and ⁇ 3 domains are directly linked, followed by the transmembrane domain and the cytoplasmic domain.
  • the HLA-G3 protein isoform lacks both the ⁇ 2 and ⁇ 3 domains, i.e., it comprises the ⁇ 1 domain directly linked to the transmembrane domain and the cytoplasmic domain.
  • the HLA-G4 protein isoform lacks the ⁇ 3 domain, i.e., it comprises the ⁇ 1 domain, the ⁇ 2 domain, the transmembrane domain and the cytoplasmic domain.
  • Soluble HLA-G isoforms all lack the transmembrane and cyto-plasmic domains. More specifically:
  • HLA-G proteins are able to inhibit allogeneic responses such as proliferative T lymphocyte cell response, cytotoxic T lymphocytes mediated cytolysis, and NK cells mediated cytolysis [7,8,9]. More recent studies have also shown that HLA-G is capable of inducing the differentiation of regulatory T cells, which can then inhibit allogeneic responses themselves, and are known to participate in the tolerance of allografts [10,11]. Because of this broad inhibitory function, it has been shown that the expression of HLA-G correlates with a better acceptance of allogeneic transplants, whether HLA-G is expressed by the graft or is detected in the plasma of patients, as a soluble molecule [12,13,14].
  • HLA-G-based procedures have been proposed for treating graft rejection in allogeneic or xenogenic organ/tissue transplantation.
  • HLA-G proteins have also been proposed for the treatment of cancers (EP 1 054 688), inflammatory disorders (EP 1 189 627) and, more generally, immune related diseases. It has also been proposed to fuse HLA-G proteins to specific ligands in order to target HLA-G to particular cells or tissues (WO 2007/091078). It should be noted, however, that no results or experimental data have been provided to show that such targeting fusions are active.
  • HLA-G has been shown to bind three main receptors: ILT2/LILRB1/CD85j, ILT4/LILRB2/CD85d and KIR2DL4.
  • ILT2 is mainly expressed by T cells, B cells, NK cells, monocytes, and dendritic cells.
  • ILT4 is expressed only by myeloid cells, i.e. mainly monocytes and dendritic cells.
  • KIR2DL4 is mainly expressed by decidual NK cells and by a small subset of peripheral NK cells.
  • HLA-G may exert its tolerogenic function on all the effectors of immune responses that are responsible for anti-viral immunity, auto-immune reactions, anti-tumor immunity, inflammatory diseases, and rejection of transplants.
  • KIR2DL4 is a specific receptor for HLA-G.
  • KIR2DL4 docks on the alpha1 domain of HLA-G, and more specifically on residues Met 76 and Gln 79 which are characteristic to HLA-G [15]. It was further shown that these two residues are crucial to the inhibitory function of HLA-G through KIR2DL4, and that mutating them prevented the inhibition of cytolytic activity of KIR2DL4-expressing NK cells by HLA-G in vitro.
  • KIR2DL4 is not likely to play a significant role in HLA-G inhibitory function except in the context of pregnancy, mainly because of its expression that is restricted to decidual NK cells, and because in vitro and in vivo, it was shown that ILTs played the key role through interaction with HLA-G alpha3 domain. It is possible that the alpha1 domain of HLA-G plays a direct role in the function of HLA-G, through KIR2DL4 or another, as yet unknown receptor, but the evidence available to date points to a tolerogenic function of HLA-G that is mediated mainly if not entirely by the interaction of its alpha3 domain with ILT2 and ILT4 molecules.
  • ILT2 and ILT4 are not specific receptors for HLA-G, and it was shown they can bind other HLA Class I molecules through their alpha3 domain [16,17,18]. The capability of the HLA-Class I domain to bind to ILT molecules is well described. ILT2, in particular, has been reported to bind “most if not all” HLA Class I molecules.
  • HLA-G is the ligand of highest affinity for ILT2 and ILT4, as illustrated in Table 1 of Shiroishi et al [19].
  • ILT2 and ILT4 bind more strongly to HLA-G than to classical HLA class 1 molecules. (see [20,21]).
  • HLA-G This stronger ILT-binding capacity of HLA-G compared to other HLA Class I molecules is particularly well illustrated by the fact that HLA-G at the surface of tumor cells, but not classical HLA class I molecules are capable to engage the ILT2 and/or ILT4 receptors of cytolytic effectors with sufficient strength to block the function of these effectors and thus protect the tumor cells from immune destruc-tion [22].
  • ILT2 and ILT4 do not bind the same HLA-G structures [21]. Indeed, ILT2 recognizes only ⁇ 2 microglobulin ( ⁇ 2m)-associated HLA-G structures, whereas ILT4 has the capability to recognize both ⁇ 2m-associated and ⁇ 2m-free HLA-G heavy chains [21,23]. Yet, ILT4 clearly binds ⁇ 2m-free HLA-G heavy chains better that ⁇ 2m-associated ones.
  • HLA-G antigen appears to adopt a dimer conformation in vivo as a result of the formation of an intermolecular disulfide bridge between cysteine residue 42 of the ⁇ 1 domains of two HLA-G molecules [20,23 and 25; WO2007/011044].
  • HLA-G The dimeric structure of HLA-G has been described in Shiroishi et al. [20]. Two molecules of wild-type HLA-G exist in an asymmetric unit; each monomer is covalently attached with the symmetrical partner via the Cys42-Cys42 disulfide bridge along with 2-fold crystallographic axis.
  • the full-length HLA-G1 protein is composed of H chain, associated ⁇ 2-microglobulin ( ⁇ 2m) and a nonameric peptide similar to the classical MHC class I structure. It has been proposed that receptor binding sites of HLA-G dimers are more accessible than those of corresponding monomers, so that dimers would have a higher affinity and slower dissociation rate than monomers. However, it is not clear what conformation is the most active for pharmaceutical purpose, which isoform is the most efficient, or how appropriate HLA-G dimers or oligomers may be produced.
  • Dimerization of HLA-G occurs via the creation of disulfide bridges between the cysteines in position 42 (alpha 1 domain) of two HLA-G molecules (see FIG. 8 of Gonen-Gross et al. [24]) and is crucial to HLA-G function. Indeed, it was shown that mutant HLA-G molecules that lack the cysteine in position 42 and do not make dimers also lack inhibitory function [24].
  • FIG. 4 of Shiroishi et al [20] provides the disulfide linked HLA-G dimer structure.
  • HLA-G inhibitory function it goes mainly through the binding of HLA-G dimers to ILT molecules through their unique alpha3 domain.
  • ILT4/HLA-G complex structure [21] reveals that ILT4 shows remarkably distinct major histocompatibility class I (MHCI) binding recognition compared to ILT2, binding more to the ⁇ 3 domain than to ⁇ 2m.
  • MHCI major histocompatibility class I
  • HLA-G1/G5 molecule is a trimolecular complex of a HLA-G complete heavy chain ( ⁇ 1, ⁇ 2 and ⁇ 3 domains) non-covalently associated with ⁇ 2m and a nonapeptide.
  • the function of such a construct is well established, but due to the complexity of its structure, its production is difficult, its purification risky, and its stability is poor.
  • the present invention relates to multimers or multimeric polypeptides, characterized in that they comprise at least two monomers, each of said monomers being selected in the group consisting of a peptide P1 (named also here after alpha3 monomer) of formula X1 ⁇ X2, wherein X1 represents a flexible peptidic linker including a cysteine amino acid and X2 represents an alpha3 domain (SEQ ID NO:1) (or alpha3 peptide) of HLA-G.
  • a peptide P1 named also here after alpha3 monomer
  • X1 represents a flexible peptidic linker including a cysteine amino acid
  • X2 represents an alpha3 domain (SEQ ID NO:1) (or alpha3 peptide) of HLA-G.
  • multimers of the invention include dimers of formula P1 ⁇ P1 and multimers of formula (P1)n, comprising exclusively alpha3 monomers (SEQ ID NO:3 or SEQ ID NO:5).
  • the multimers according to the invention thus include dimers, as well as molecules comprising 3, 4, 5, 6, 7 or even more P1 monomers.
  • Multimers according to the instant invention may comprise up to 100, 500, 1000 or even more P1 monomers.
  • alpha3 monomers SEQ ID NO:3 or SEQ ID NO:5
  • alpha3 polypeptides also named alpha3 polypeptides
  • HLA-G antigens function as immunomodulatory agents protecting the foetus from the maternal immune system.
  • Various HLA-G isoforms have been reported, which are either membrane-bound or soluble. These isoforms contain distinct functional domains, selected from extracellular globular domains, designated ⁇ 1 (SEQ ID NO:2), ⁇ 2 and ⁇ 3 (SEQ ID NO:1), a trans-membrane domain and a cytoplasmic domain. While the biological activity and mechanism of action of certain HLA-G isoforms (such as mature HLA-G1) have been documented, the relative contribution of each domain to the immunoregulatory activity, especially in soluble form, has not been studied in detail.
  • HLA-G antigen is mediated by binding to ILT inhibitory receptors ILT2 or ILT4. More specifically, it has been proposed that such binding occurs through the alpha3 domain of HLA-G (Shiroishi et al., [21]).
  • alpha3 polypeptides comprising only alpha3 domains of HLA-G (SEQ ID NO:1) are able to protect graft rejection in vivo or inhibition of alloproliferation in vitro.
  • results obtained show that the multimers according to the instant invention exhibit high immunoregulatory activity in vivo and therefore represent efficient drugs for treating immune-related disorders, particularly for reducing unwanted or deleterious immune responses in a subject.
  • results obtained more specifically show that multimers of this invention can induce a 100% or even more increase in graft survival in vivo compared to placebo or ⁇ 1- ⁇ 3 multimers comprising at least two monomers, each of said monomers consisting of a peptide comprising from the N-terminal end to the C-terminal end an alpha1 domain of HLA-G and an alpha3 domain of HLA-G (named indifferently hereafter alpha3 ⁇ alpha1 monomer or alpha1 ⁇ alpha3 monomer although the alpha1 domain is always at the N-terminal end).
  • alpha3 multimers of the invention are significantly more efficient compared to soluble HLA-G6 isoform, alpha3 multimers comprising a non flexible peptidic linker or alpha1 ⁇ alpha3 multimers.
  • the functional fragment contains at least 60 consecutive amino acids of the alpha3 domain.
  • the functionality of the fragment may be verified as disclosed in the experimental section.
  • the functionality may be verified by preparing a multimer of the fragments, administering the multimer to an animal model prior to organ/tissue transplantation, and verifying the graft survival rate. Where the multimer extends the duration of graft survival by 50%, as compared to placebo, the fragment may be considered as functional.
  • HLA-G5, HLA-G6 and HLA-G7 are also available from U.S. Pat. No. 5,856,442, U.S. Pat. No. 6,291,659, FR2,810,047, or Paul et al., Hum. Immunol 2000; 61: 1138, from which the sequence of the alpha1 and alpha3 domain can be obtained directly.
  • the alpha3 peptide consists essentially of amino acids 183-274 of a mature HLA-G antigen, or a functional fragment thereof.
  • the alpha 1 peptide consists essentially of amino acids 1-90 of a mature HLA-G antigen, or a functional fragment thereof.
  • sequence of a preferred alpha3 peptide is provided in SEQ ID NO:1.
  • alpha1 peptide The sequence of alpha1 peptide is provided in SEQ ID NO:2.
  • the sequence of a preferred alpha3 monomer is provided in SEQ ID NO:3 (alpha3 ⁇ L1).
  • the linker L1 corresponds to positions 1-12 (SEQ ID NO:7) and contains a cysteine in position 2; positions 13 and 14 correspond to two amino acids of the alpha2 domain (see SEQ ID NO:6 corresponding to HLA-G and in which alpha2 corresponds to positions 115-206).
  • Positions 15-106 correspond to the alpha3 domain and positions 107-108 correspond to two amino acids of the transmembrane domain; all the hydrophilic tail of HLA G may be inserted.
  • Main contact residues with ILT molecules are in positions 27 and 29 of said SEQ ID NO:3.
  • SEQ ID NO:5 Another sequence of an alpha3 monomer is provided in SEQ ID NO:5.
  • the linker L2 corresponds to positions 1-18 (SEQ ID NO:8) and contains a cysteine in position 1; positions 19 and 20 correspond to two amino acids of the alpha2 domain (see SEQ ID NO:6 corresponding to HLA-G and in which alpha2 corresponds to positions 115-206).
  • Positions 21-111 correspond to the alpha3 domain and positions 112-113 correspond to two amino acids of the transmembrane domain.
  • alpha1 ⁇ alpha3 monomer The sequence of alpha1 ⁇ alpha3 monomer is provided in SEQ ID NO:4. Positions 1-90 of SEQ ID NO:4 correspond to alpha1 domain; positions 91-182 of SEQ ID NO:4 correspond to alpha3 domain and positions 183-184 correspond to two amino acids of the transmembrane domain; all the hydrophilic tail of HLA G may be inserted. Cys42 is used for dimerization. Main contact residues with ILT molecules are in positions 103 and 105.
  • the various monomers may be linked together in different manner such as, without limitation, through disulfide bridging (especially for a dimer), or through a spacer group and/or a carrier.
  • the alpha3 monomers, as defined here above are linked covalently or through an affinity interaction.
  • a particular example of a multimer of the invention is a P1 ⁇ P1 dimer.
  • the invention relates to an alpha3 dimer, having two monomers P1 of SEQ ID NO:3 associated together through a disulfide bridge. More specifically, the two alpha3 peptides are linked through a disulfide bridge between cysteine residues present at the N terminus of the linker X1.
  • the alpha3 monomers are linked through a spacer or a carrier.
  • monomers are linked to a carrier, thereby producing a multimer.
  • the carrier can be of different nature. It is preferably biocompatible, and most preferably biologically inert.
  • the carrier may be a molecule, such as a protein, e.g., albumin (e.g., human serum albumin), or an inert solid carrier.
  • the monomers may be linked to the carrier through different types of coupling reactions, such as affinity interaction or the use of functional groups. Affinity interaction may be obtained by coating the carrier with ligands that bind alpha3 or alpha1 peptides (e.g., antibodies or fragments thereof).
  • Affinity interaction may also be obtained by adding to the alpha3 monomers and to the carrier, respectively a member of a binding pair (e.g., avidin and biotin). Coupling may also be obtained through bi-functional groups such as maleimide, etc.
  • multimers may contain monomers linked to a carrier and further engaged in inter-molecular disulfide bridging.
  • a multimer of the instant invention is a molecule comprising two or more alpha3 monomers (SEQ ID NO:3 or SEQ ID NO:5) linked to a carrier.
  • the multimers of this invention can be produced by various techniques. As discussed above, the monomers may be coupled together through different coupling techniques, such as covalent linkage (e.g., disulfide bridge, bifunctional group, etc) or affinity reaction.
  • covalent linkage e.g., disulfide bridge, bifunctional group, etc
  • affinity reaction e.g., affinity reaction
  • Alpha1-alpha3 multimers may be obtained in the same conditions, from alpha1 ⁇ alpha3 monomers obtained by chemical synthesis.
  • the monomers are typically incubated in the presence of the carrier under conditions allowing attachment of the monomers on the carrier and, preferably, the multimer is separated.
  • the carrier may be e.g., a solid carrier.
  • the carrier may also be a protein, such as serum-albumin.
  • the carrier may be functionalized to contain reactive groups able to interact with the monomers.
  • the carrier may be coated with a ligand of alpha1 or alpha3 peptides (SEQ ID NO:1 and SEQ ID NO:2), such as antibodies or fragments thereof (e.g., Fab fragments, CDR fragments, ScFv, etc) or a chemical coupling reagent (e.g., maleimide).
  • the carrier may be functionalized by a reactant able to bind a ligand of the alpha1 polypeptides.
  • the carrier may be coated with an anti-human IgG Fc fragment, and the ligand may be a human polyclonal IgG directed against an HLA-G1 antigen. In such a case, the monomers, carrier and ligand may be incubated together, in order to allow proper association of the monomers to the beads.
  • Alpha3 multimers of the invention may be produced by techniques known per se in the art, such as recombinant techniques, enzymatic techniques or artificial synthesis, preferably by artificial synthesis, such as the Merrifield synthesis.
  • the alpha3 peptides X2 (SEQ ID NO:1) and the alpha3 monomers P1 (SEQ ID NO:3 or SEQ ID NO:5) are produced by artificial synthesis using known chemistry and synthesisers.
  • the alpha3 multimers may comprise either natural amino acids, or non-natural or modified amino acid residues. They may be in L and/or D conformation.
  • the peptides may comprise either amine linkages and/or modified peptidomimetic linkages. Also, the peptides may be terminally protected and/or modified, e.g., through chemical or physical alteration of lateral functions, for instance.
  • the carrier and monomers may be modified to contain cross-reactive groups (e.g., avidin and biotin). In such a case, incubation of the carrier and monomers will cause multimerisation on the carrier.
  • cross-reactive groups e.g., avidin and biotin
  • the multimer formed (i.e., the complex between the carrier and the alpha3 monomer) can be isolated using various techniques known per se in the art, including centrifugation, sedimentation, electromagnetic separation, etc.
  • these multimers are able to promote graft tolerance in vivo.
  • the dimers of alpha3 monomer of SEQ ID NO:3 also represent specific objects of the invention.
  • the invention indeed shows that said dimers have substantial in vivo activity for treating graft rejection and may be used to prepare very active multimers.
  • the invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a multimer as defined above or obtainable by a method as disclosed above and, preferably, at least a pharmaceutically acceptable vehicle or carrier.
  • a further object of this invention is a pharmaceutical composition
  • a pharmaceutical composition comprising an alpha3 dimer having two monomers of SEQ ID NO:3 and, preferably, at least a pharmaceutically acceptable vehicle or carrier.
  • Suitable vehicles or carriers include any pharmaceutically acceptable vehicle such as buffering agents, stabilizing agents, diluents, salts, preservatives, emulsifying agents, sweeteners, etc.
  • vehicle typically comprises an isotonic aqueous or non aqueous solution, which may be prepared according to known techniques. Suitable solutions include buffered solutes, such as phosphate buffered solution, chloride solutions, Ringer's solution, and the like.
  • the pharmaceutical preparation is typically in the form of an injectable composition, preferably a liquid injectable composition, although other forms may be contemplated as well, such as tablets, capsules, syrups, etc.
  • compositions according to the invention may be administered by a number of different routes, such as by systemic, parenteral, oral, rectal, nasal or vaginal route. They are preferably administered by injection, such as intravenous, intraarterial, intramuscular, intraperitoneal, or subcutaneous injection. Transdermal administration is also contemplated. The specific dosage can be adjusted by the skilled artisan, depending on the pathological condition, the subject, the duration of treatment, the presence of other active ingredients, etc. Typically, the compositions comprise unit doses of between 10 ng and 100 mg of multimer, more preferably between 1 ⁇ g and 50 mg, even more preferably between 100 ⁇ g and 50 mg.
  • compositions of the present invention are preferably administered in effective amounts, i.e., in amounts which are, over time, sufficient to at least reduce or prevent disease progression.
  • compositions of this invention are preferably used in amounts which allow the reduction of a deleterious or unwanted immune response in a subject.
  • Said multimeric polypeptides can be used as tolerogenic agents capable of mimicking HLA-G full function.
  • the prime therapeutic uses of these compounds would be transplantation, in order to induce and maintain tolerance to allografts, but may also be auto-immune diseases, or inflammatory diseases, in order to stop auto-immune responses and inflammation, and possibly re-establish auto-tolerance.
  • polypeptides production/purification protocols comparatively easier, cheaper, more controlled, safer than classical production methods that involve prokaryotic or eukaryotic organisms and significantly more active than HLA-G6 isoform or control peptides as defined in the examples [(alpha3-L2) ⁇ 2 of SEQ ID NO:5 and (alpha1 ⁇ alpha3) ⁇ 2] of SEQ ID NO:4.
  • the multimers (SEQ ID NO:3 or SEQ ID NO:5) of the instant invention have strong immune-regulatory activity and may be used to treat a variety of disease conditions associated with abnormal or unwanted immune response. More specifically, the multimers of the invention are suitable for treating immune-related disorders such as, particularly, organ or tissue rejection, inflammatory diseases or auto-immune diseases. They can substantially inhibit allogeneic graft rejection in vivo.
  • the instant invention also relates to a multimer or composition as disclosed above for treating graft rejection.
  • the invention further relates to a method of treating graft rejection in a subject, the method comprising administering to a subject in need thereof an effective amount of a composition as disclosed above.
  • treating designates for instance the promotion of the graft tolerance within the receiving subject.
  • the treatment can be performed prior to, during and/or after the graft, and may be used as an alternative therapy to existing immuno-suppressive agents or, as a combined therapy with actual immunosuppressive agents.
  • the invention is applicable to allogenic, semi-allogenic or even xenogenic transplantation, and may be used for any type of transplanted organs or tissues including, without limitation, solid tissues, liquid tissues or cells, including heart, skin, kidney, liver, lung, liver-kidney, etc.
  • the invention relates also to an improved method for transplanting an organ or tissue in a subject, the improvement comprising administering to the subject, prior to, during and/or after transplantation, an effective amount of a composition as disclosed above.
  • the invention further relates to a method for promoting graft tolerance in a subject, the method comprising administering to the subject, prior to, during and/or after transplantation, an effective amount of a composition as disclosed above.
  • the invention further relates to a method for reducing graft rejection in a subject, the method comprising administering to the subject, prior to, during and/or after transplantation, an effective amount of a composition as disclosed above.
  • the composition is administered at least twice to the subject.
  • the results shown in this application demonstrate that a repeated administration leads to a further increased benefit, e.g., to a further significantly increased graft tolerance in vivo.
  • the amount of the composition actually administered shall be determined and adapted by a physician, in the light of the relevant circumstances including the condition or conditions to be treated, the exact composition administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration. Therefore, the above dosage ranges are intended to provide general guidance and support for the teachings herein, but are not intended to limit the scope of the invention.
  • FIG. 1 3D model of the (alpha3) ⁇ 2 dimer of SEQ ID NO:1.
  • a Model of the dimerized polypeptide Each monomer is in a different grey. The artificially introduced free cysteine is shown by spheres, allowing dimerization.
  • Alpha3 peptide is shown in 3D ribbon rendering. The structures of the alpha3 domain of HLA-G and of the alpha3 peptide are superimposed.
  • FIG. 2 3D model of the binding of the (Alpha3) ⁇ 2 of SEQ ID NO:1 polypeptide to ILT4 molecules. Only one half of the (alpha3) ⁇ 2 dimer is shown.
  • FIG. 3 Binding of different dimers to HLA-G receptors ILT2, ILT4, KIR2DL4: (alpha3 ⁇ L1) ⁇ 2 of SEQ ID NO:3: P1 ⁇ P1 dimer according to the invention) wherein L1 (SEQ ID NO:7) represents a particular linker X1; (alpha3 ⁇ L2) ⁇ 2 of SEQ ID NO:5: control peptide; (alpha3 ⁇ alpha1) ⁇ 2 of SEQ ID NO:4: control peptide.
  • a peak that is situated on the right of the vertical bar indicates recognition by the indicated receptor.
  • FIG. 4 Cell toxicity results.
  • FIG. 5 Effect of different peptides on the alloproliferation of T lymphocytes in vitro: (alpha3 ⁇ L1) ⁇ 2 of SEQ ID NO:3: P1 ⁇ P1 dimer according to the invention) wherein L1 represents a particular linker X1 (SEQ ID NO:7); (alpha3 ⁇ L2) ⁇ 2 of SEQ ID NO:5: control peptide; (alpha1 ⁇ alpha3) ⁇ 2 of SEQ ID NO:4: control peptide.
  • Bar graph proliferation indexed on controls, SEM are reported as error bars.
  • % inhibition represents the extent of inhibition mediated by the peptides.
  • FIG. 6 General structures of (alpha3 ⁇ L1) ⁇ 2 of SEQ ID NO:3 and (alpha3 ⁇ L2) ⁇ 2 of SEQ ID NO:5 dimers.
  • FIG. 7 Structural scheme of alpha3 ⁇ L1 of SEQ ID NO:3 and alpha3 ⁇ L2 of SEQ ID NO:5 monomers.
  • FIG. 8 General structure of (alpha3 ⁇ alpha1) ⁇ 2 dimers.
  • the peptides of SEQ ID NO:1-5 were synthesised using a peptide synthesiser.
  • FIGS. 6 , 7 and 8 illustrate the general structures of the synthesized peptides.
  • Alpha3 monomers of SEQ ID NO:3 or SEQ ID NO:5 were synthesized chemically. Monomers were first synthesized, then refolded by allowing the generation of disulfide bonds between the two cysteines within the alpha3 domain (cysteines 35 and 91 of SEQ ID NO:3, cysteines 41 and 97 of SEQ ID NO:5). Dimerization was then performed by generating a disulfide bridge between the cysteines within the linker X1 of two monomers (Cysteine 2 of SEQ ID NO:3, cysteine 1 of SEQ ID NO:5). The purity of the synthesized products was verified by mass spectrometry.
  • FIG. 1 A three dimensional model of the dimer of SEQ ID NO:3 is shown in FIG. 1 . Based on computational modelization, this structure is able to bind HLA-G receptor ILT4 (shown in FIG. 2 ; see also FIG. 3 ).
  • Alpha1 ⁇ alpha3 monomers of SEQ ID NO:4 were synthesized chemically. Monomers were first synthesized, then refolded by allowing the generation of disulfide bonds between the two cysteines within the alpha3 domain (cysteines 111 and 167 of SEQ ID NO:4). Dimerization was then performed by generating a disulfide bridge between two cysteines within the alpha1 domain of two monomers (Cysteine 42 of SEQ ID NO:4). The purity of the synthesized products was verified by mass spectrometry.
  • alpha1+alpha3 polypeptide The sequence of the alpha1+alpha3 polypeptide is shown in SEQ ID NO:4.
  • Beads and receptors were then incubated for 90 minutes in the dark on a shaker before wash twice with 200 ⁇ l of 1 ⁇ PBS, 0.05% Tween. Beads were then resuspended in 50 ⁇ l of PBS Luminex assay Buffer containing 2 ⁇ g/ml of Phycoerythrin-conjugated Goat anti Human IgG antibody (Sigma) for 30 minutes in the dark on a rotating shaker. Beads were then washed twice with 200 ⁇ l of 1 ⁇ PBS, 0.05% Tween, and resuspended in 300 ⁇ l of 1 ⁇ PBS.
  • Fluorescence, indicative of peptide recognition by the receptors was evaluated by flow cytometry performed on an Epics XL Cytometer (Beckman Coulter) using EXPO32 software (Beckman Coulter).
  • FIG. 3 illustrates the results and clearly show that all peptides containing alpha3 domain indeed bind specifically to ILT4 receptor.
  • Results show that said dimers are not toxic towards PBMC.
  • MLR Mixed Lymphocytes Reaction
  • PBMC Peripheral blood mononuclear cells
  • FIG. 5 illustrates the results and shows that alpha3 dimers according to the invention [(alpha3 ⁇ L1) ⁇ 2 of SEQ ID NO:3 in said FIG. 5 ] inhibit significantly alloproliferation of T lymphocytes compared to (alpha3 ⁇ L2) ⁇ 2 dimer of SEQ ID NO:5 and (alpha1 ⁇ alpha3) ⁇ 2 dimer of SEQ ID NO:4.

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