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WO2006077152A2 - Molecules de fusion hla et utilisations associees - Google Patents

Molecules de fusion hla et utilisations associees Download PDF

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Publication number
WO2006077152A2
WO2006077152A2 PCT/EP2006/000560 EP2006000560W WO2006077152A2 WO 2006077152 A2 WO2006077152 A2 WO 2006077152A2 EP 2006000560 W EP2006000560 W EP 2006000560W WO 2006077152 A2 WO2006077152 A2 WO 2006077152A2
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Prior art keywords
fusion molecule
polynucleotide
hla
alpha
domain
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PCT/EP2006/000560
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English (en)
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WO2006077152A3 (fr
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Nalân UTKU
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Utku Nalan
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Priority to EP06706364A priority Critical patent/EP1919949A2/fr
Priority to CA002595462A priority patent/CA2595462A1/fr
Publication of WO2006077152A2 publication Critical patent/WO2006077152A2/fr
Publication of WO2006077152A3 publication Critical patent/WO2006077152A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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

Definitions

  • the present invention generally relates to the technical field of immunology. More specifically, the present invention relates to a fusion molecule derived from MHC-class II proteins, especially from the membrane proximal domain of the HLA-DR alpha chain (HLA- DR alpha 2 protein), wherein said fusion molecule is involved in the signal transduction of T- cell activation and/or proliferation.
  • the fusion molecule of the present invention provides immunomodulatory signals which in turn are involved in cytokine expression and/or secretion of activated T-cells.
  • the present invention relates to compositions comprising said fusion molecule and to methods of modulating MHC II mediated immune responses, and treating immune response related diseases.
  • T-cell activation is a serial process involving multiple signaling pathways and sequential changes in gene expression resulting in differentiation of T-cells into distinct subpopulations, i.e. ThI and Th2, which are distinguishable by their pattern of cytokine production and characterize the mode of cellular immune response.
  • the T-cell response is initiated by the interaction of the antigen-specific T-cell receptor (TCR) with peptides presented by major histocompatibility complex (MHC) molecules on the surface of antigen presenting cells (APCs).
  • TCR antigen-specific T-cell receptor
  • MHC major histocompatibility complex
  • Additional signals are provided by a network of receptor-ligand interactions mediated by a number of membrane proteins such as CD28/CTLA4 and B7, CD40/CD40L, LFA-I and ICAM-I (Lenschow, Science 257 (1992), 789-792; Linsley, Annu. Rev. Immunol. 11 (1993), 191-212; Xu, Immunity 1 (1994), 423-431; Bachmann, Immunity 7 (1997), 549- 557; Schwartz, Cell 71 (1992), 1065-1068) collectively called costimulatory signals (Perez, Immunity 6 (1997), 411).
  • membrane proteins such as CD28/CTLA4 and B7, CD40/CD40L, LFA-I and ICAM-I (Lenschow, Science 257 (1992), 789-792; Linsley, Annu. Rev. Immunol. 11 (1993), 191-212; Xu, Immunity 1 (1994), 423-431; Bachmann, Immunity 7 (1997), 549- 557; Schwartz,
  • the technical problem of the present invention is to provide means and methods for modulation of the immune response in a subject.
  • the solution to said technical problem is achieved by providing the embodiments characterized in the claims, and described further below.
  • the present invention is directed to a fusion molecule comprising a first domain comprising the HLA-DR alpha 2 domain or at least a fragment thereof and a second, preferably functional domain, hi particular, said second domain of the fusion molecule of the present invention comprises at least a fragment of an immunoglobulin or derivative thereof. Furthermore, the invention is directed to a polynucleotide encoding the fusion molecule of the invention, a vector comprising said polynucleotide and to a host cell comprising said vector for manufacturing said fusion molecule. The present invention further relates to a pharmaceutical composition comprising the mentioned fusion molecule, which is used in cell or organ transplantation and for the treatment of autoimmune, allergic or infectious diseases, or for the treatment of tumors. Furthermore, the present invention is directed to a diagnostic agent, comprising said fusion molecule.
  • Fig. IA Complete HLA-DR alpha chain protein sequence in which the extracellular alpha 2 domain which was used for generating the HLA-DR-V5 fusion protein is underlined.
  • Fig. IB Purified HLA-DR alpha 2-V5 fusion protein was detected by silver staining and showed the expected size of the fusion protein at 7,5 kDa.
  • Fig. 1C Purified HLA-DR alpha 2-V5 fusion protein was detected by Western blot analysis and showed the expected size of the fusion protein at 7,5 kDa.
  • Fig. ID Complete HLA-DR alpha chain protein sequence in which the domain used for generating the HLA-DR alpha 2-Fc fusion protein is underlined.
  • Fig. IE COS7 cells were transiently transfected with an expression vector for HLA-
  • Fig. IF Western blot analysis of HLA-DR-Fc fusion protein.
  • SDS acrylamid gels were blotted to nitrocellulose to detect protein using anti-human Fc antibody coupled to alkaline phosphatase (AP). Bands were detected using BCIP/NBT and revealed the expected size of 45 kDa protein.
  • Fig. IG IFN- ⁇ and IL-IO levels in the supernatant were measured by quantitative sandwich ELISA which revealed a significant inhibition of interferon gamma expression, whereas no inhibition for IL-IO expression was observed as soluble HLA-DR alpha 2 protein inhibited the interferon gamma expression by 50% at a concentration of 100 ⁇ g/ml, compared with the controls.
  • the results show the summary of three independent experiments.
  • PBMC of human healthy donors were isolated according to the Ficoll-Paque density centrifugation protocol.
  • Fig. 2 Intracellular IFN- ⁇ levels in splenocytes of Balb/C mice (10-14 weeks old), treated either with HLA-DR alpha 2-Fc (200 ⁇ g in PBS) or human Fc (50 ⁇ g in PBS) following 50 ⁇ g LPS induction, were measured on FACS. The results of the human Fc treated control group was set to 100 %. Splenocytes of the HLA-DR alpha 2-Fc treated mice revealed a significant inhibition of intracellular IFN- ⁇ expression of about 40 %.
  • the present invention relates to a fusion molecule comprising a first domain and a second domain, wherein said first domain comprises the HLA-DR alpha 2 domain or a fragment thereof.
  • Said fusion molecule is capable of modulating the T-cell mediated immune response.
  • the present invention is based on investigations that the alpha 2 domain of the HLA-DR alpha molecule interacts with its natural receptor located on activated lymphocytes, especially T-cells. hi particular, it could be shown in accordance with the present invention the inhibition of activated T-cell IFN- ⁇ production by soluble HLA-DR alpha 2 fusion protein.
  • fusion molecules derived from HLA-DR alpha 2 and described herein are capable of modulating the signaling of MHC class II restricted T-cells hi cell-mediated immunity, for example by interfering with the interaction with its natural receptor.
  • fusion molecule interfering with the interaction of HLA-DR alpha 2 with its corresponding receptor means in accordance with the present invention an agent capable of inhibiting and/or modulating the interaction of HLA-DR alpha 2 with its corresponding receptor. Since the interaction of HLA-DR alpha 2 with its receptor(s) interferes with events which are valuable in course of immune responses, such inhibitor should also be capable of modulating immune responses.
  • said fusion molecule preferably interacts with its receptor(s), for example by specific binding.
  • Specific binding means “specifically interacting with”, whereby said interaction may be, inter alia, covalently, non-covalently and/or hydrophobic.
  • Said fusion molecules include molecules which bind to, interfere with and/or occupy relevant sites on said receptor. Examples of such molecules include (poly-) peptides or peptide-like molecules.
  • treatment means obtaining a desired pharmacological and/or physiological effect.
  • the effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease.
  • treatment covers any treatment of a disease in a mammal, particularly a human, and includes either preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it, inhibiting the disease, i.e. arresting its development or relieving the disease, i.e. causing regression of the disease.
  • the term "subject” as employed herein relates to mammals in need of amelioration, treatment and/or prevention of immunological diseases as disclosed herein.
  • the term “mammal” means any member of the higher vertebrate animals included in the class Mammalia, as defined in Webster's Medical Desk Dictionary 407 (1986), and includes but is not limited to humans, other primates, pigs, dogs, and rodents (such as immune-suppressed mice). In the preferred embodiment of this invention, the mammal is a human.
  • the present invention relates to a fusion molecule comprising a first domain comprising the HLA-DR alpha 2 domain or at least a fragment thereof, and a second domain.
  • Said first domain of said fusion molecule is HLA (Human Leukocyte associated Antigen) class II alpha chain, also referred to as HLA-DR alpha 2 domain, or a fragment thereof. Specialized forms thereof are shown in the Figures and described in Examples 1 to 4.
  • HLA class II is a heterodimer of two transmembrane glycoproteins, the alpha and beta chains.
  • both chains comprise two extracellular domains, each of 90 - 100 amino acids, connected to a short cytoplasmic tail by a hydrophobic amino acid sequence that makes a single pass through the cell membrane.
  • the membrane distal domain is known as alpha 1 and the membrane proximal domain as alpha 2.
  • the membrane distal domain is known as beta 1 and the membrane proximal domain as beta 2.
  • Both membrane proximal domains possess structural characteristics of Cl -type immune globulin domains.
  • the alpha 1 and beta 1 domains are polymorph and occupied with presentations of peptides (12 - 24mers) to the T- cell receptor during the course of T-cell activation. Studies using site-specific mutants have mapped the site of CD4 binding to the membrane proximal beta 2 domain of the HLA class II molecule.
  • HLA-DR alpha is conserved in humans and has not been recognized as a ligand for other molecules.
  • the CD4 molecule interacts with the beta 2 domain of HLA class II (Marsh et al., The HLA Facts Book (2000)).
  • the T-cell receptor binds to polymorphic beta 1 domains of HLA-DR with associated peptide antigen (Parham, Immunological Reviews 171 (1999), 1).
  • Other molecules which bind to HLA-DR include super antigens such as staphylococcus enterotoxin B (SEB) (Li et al., Ann. Rev. Immunol.
  • HLA class II molecules and their polymorphism are strongly associated with a number of diseases such as insulin-dependent diabetes mellitus, Goodpasture syndrome, Pemphigus vulgaris, Systemic lupus erythramatosus, Multiple sclerosis, Grave's disease, Rheumatoid arthritis and Myastenia gravis.
  • a person's immune system develops tolerance to the self HLA class I and II allotypes expressed on the surface of that same person's cell.
  • a person's immune system is not tolerant of the many hundreds of non-self HLA allotypes expressed by other human beings such as after organ transplantation. Therefore, once a person receives a transplant, hyperacute or acute rejection of the transplanted organ is likely to occur if the recipient and donor are not compatible in their HLA antigen types expressed on the cell surface.
  • said first domain and/or second domain, and preferably the entire fusion molecule is a protein. Most preferably, said fusion molecule is in a soluble form.
  • the second domain of said fusion molecule comprises an immunoglobulin molecule or a fragment thereof.
  • said immunoglobulin is a human immunoglobulin or a fragment thereof.
  • said immunoglobulin fragment of the immunoglobulin is the Fc portion of the immunoglobulin, wherein in a particularly preferred embodiment said Fc portion is the Fc portion of immunoglobulin Gl (IgGl).
  • IgGl immunoglobulin Gl
  • immunomodulating fusion proteins examples include IL-IO-Fc, wherein IL-IO, an anti- inflammatory and antirejection agent has been fused to murine Fc[gamma]2a (Zheng et al., The Journal of Immunology 154 (1995), 5590-5600), tumor necrosis factor receptor linked with the Fc protein of human IgG 1 (Fisher et al., N. Engl. J. Med. 334 (1996), 1697-1702; Van Zee et al., The Journal of Immunology 156 (1996), 2221-2230), or fusion of Fc with CD4 receptor (Capon et al., Nature, 337 (1989), 525-531).
  • HLA-DR alpha 2 domain normally located on the cell surface of antigen-presenting cells (APC)
  • APC antigen-presenting cells
  • immunoglobulin heavy chain constant regions can be used for said second domain of the fusion molecule of the present invention derived from any of the human IgG antibody subclasses referred to in the art as IgGl, IgG2, IgG3, and IgG4.
  • Immunoglobulin heavy chain constant region domains have cross-homology among the immunoglobulin classes as described, for example, in US2004/082039.
  • nucleic acid sequences encoding, and amino acid sequences defining a human immunoglobulin Fc region are used in the practice such as disclosed in WO00/40615, WO00/69913, WO00/24782 or in the Genbank and/or EMBL databases, for example, AF045536.1 (Macaca fuscicularis), AF045537.1 (Macaca mulatta), AB016710 (Felix catus), K00752 (Oryctolagus cuniculus), U03780 (Sus scrofa), 248947 (Camelus dromedarius), X62916 (Bos taurus), L07789 (Mustela viso ⁇ ), X69797 (Ovis aries), U17166 (Cricetulus migratorius), X07189 (Rattus rattus), AF5761
  • the fusion molecule of the present invention preferably comprises further the immunoglobulin molecules and fragments thereof, preferably the Fc portion of murine IgG subclasses known by the person skilled in the art as IgGl, IgG2a, IgG2b, IgG3.
  • the first domain of said fusion molecule comprises the amino acid sequence depicted in SEQ ID NO: 1 (Fig. IA and D) or a fragment thereof. Most preferably said fragment comprises amino acid sequence position 128 to 198 of SEQ ID NO: 1 as described in Example 2.
  • the fusion molecule of the present invention may comprise, e.g., linker molecules.
  • the linker could be made up of amino acids linked together by peptide bonds as described in WO/02066514.
  • the present invention comprises chemical cross-linking of the above mentioned first and second domain, as described in Yang et al., Biochemistry 42 (2003), 3527-3535. Principally, two domains could be fused without using a linker molecule as described above. Therefore, the Fc portion can be fused to a target protein or peptide via its C- or N-terminus using the N- and C-terminus of the protein, respectively.
  • a chimera of Fc and TNF and EPO is disclosed in EP 0 464 533, wherein the N-terminus of Fc was coupled to the C-terminus of the protein (X-Fc).
  • the identical conjunction was selected for leptin-Fc chimeras as disclosed in WO97/00319 and WO97/24440.
  • Fc-protein chimeras such as Fc-(IL-2), Fc-EPO, Fc-PSMA, Fc-(IL-12), Fc-TNFa, Fc-(GM-CSF), Fc-TNFR, Fc-endostatin, Fc, angiostatin, Fc-g ⁇ l20, Fc- leptin, Fc-IFNa, Fc-(G-CSF) are described for example in WO96/08570, WO98/28427, WO99/02709 and WO99/58662.
  • WO00/24782 discloses a huge number of possible Fc-X conjugates, wherein the linkage between the two partners may be Fc-X or X-Fc.
  • An extensive development of Fc-X molecules was realized by Lexigen/Merck KgaA as disclosed in US-A-5,541,087, WO99/43713, WO99/29732, WO99/52562, WO99/53958, WOOO/11033, WO01/07081, WO01/36489.
  • X-Fc and Fc-X molecules which have "lost" their antigen binding sites, as well as molecules, wherein the binding sites und thus their antigen-specific targeting functions are conserved, are of great interest as promising therapeutic proteins, and there exists a further need to develop analogue compositions for different clinical applications.
  • Non-natural therapeutic proteins are often particularly immunogenic.
  • Enbrel is a fusion protein consisting of an extracellular domain of a Tumor Necrosis Factor Receptor (TNF-R) fused to an Fc region of an antibody. All of those Fc fusion techniques can be applied in order to produce a fusion molecule of the present invention.
  • TNF-R Tumor Necrosis Factor Receptor
  • the present invention relates to a fusion molecule, which is encoded by a polynucleotide.
  • said fusion molecule comprises in a first functional domain the HLA-DR alpha 2 chain, which is normally exposed on the cell surface of APC, forming a part of the MHC class II complex.
  • the most important advantage of the present invention is to provide an experimental system which allows the recombinant expression and purification of said fusion molecule in a soluble and stable form as shown in Fig. 1 and 2 and described in the appended Examples 1 and 2.
  • the recombinant fusion molecule simplifies the elucidation of the T-cell signaling pathway and therefore gives basis for lots of experimental purposes in questions concerning in the T-cell mediated immune response.
  • the present invention relates to a polynucleotide encoding the fusion molecule of the present invention.
  • the polynucleotide of the invention encoding the above- described fusion molecule may be, e.g., DNA, cDNA, RNA or synthetically produced DNA or RNA or a recombinantly produced chimeric nucleic acid molecule comprising any of those polynucleotides, either alone or in combination.
  • said polynucleotide is part of a vector, wherein said polynucleotide is operatively linked to expression control sequences allowing expression in prokaryotic or eukaryotic cells.
  • Such vectors may comprise further genes such as marker genes which allow for the selection of said vector in a suitable host cell and under suitable conditions.
  • Expression of said polynucleotide comprises transcription of the polynucleotide into a translatable mRNA.
  • Regulatory elements ensuring expression in eukaryotic cells, preferably mammalian cells, are well-known to those skilled in the art. They usually comprise regulatory sequences ensuring initiation of transcription and optionally poly-A signals ensuring termination of transcription and stabilization of the transcript. Additional regulatory elements may include transcriptional as well as translational enhancers, and/or naturally associated or heterologous promoter regions.
  • Possible regulatory elements permitting expression in prokaryotic host cells comprise, e.g., the PL, lac, trp or tac promoter in E. coli, and examples for regulatory elements permitting expression in eukaryotic host cells are the AOXl or GALl promoter in yeast or the CMV-, SV40- , RSV-promoter (Rous sarcoma virus), CMV-enhancer, SV40-enhancer or a globin intron in mammalian and other animal cells.
  • Beside elements which are responsible for the initiation of transcription such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site, downstream of the polynucleotide.
  • transcription termination signals such as the SV40-poly-A site or the tk-poly-A site
  • leader sequences capable of directing the polypeptide to a cellular compartment or secreting it into the medium may be added to the coding sequence of the polynucleotide of the invention and are well-known in the art.
  • the leader sequence(s) is (are) assembled in appropriate phase with translation, initiation and termination sequences, and preferably, a leader sequence capable of directing secretion of translated protein, or a portion thereof, into the periplasmic space or extracellular medium.
  • the heterologous sequence can encode a fusion protein including a C- or N-terminal identification peptide imparting desired characteristics, e.g., stabilization or simplified purification of expressed recombinant products.
  • suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDVl (Pharmacia), pCDM8, pRc/CMV, pcDNAl, ⁇ cDNA3 (Invitrogen), or pSPORTl (GIBCO BRL).
  • the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells, but control sequences for prokaryotic hosts may also be used.
  • the vector Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level expression of the nucleotide sequences, and, as desired, the collection and purification of the fusion protein may follow; see, Beychok, Cells of Immunoglobulin Synthesis, Academic Press, N. Y., (1979).
  • the present invention relates to vectors, particularly plasmids, cosmids, viruses and bacteriophages used conventionally in genetic engineering that comprise the polynucleotide encoding the fusion molecule of the invention.
  • the polynucleotide of said vector is operably linked to regulatory sequences allowing the transcription and optionally expression of said polynucleotide.
  • said vector is an expression vector and/or a gene transfer or targeting vector.
  • Expression vectors derived from viruses such as retroviruses, vaccinia virus, adeno-associated virus, herpes viruses, or bovine papilloma virus, may be used for delivery of the polynucleotides or vector of the invention into targeted cell population.
  • the polynucleotides and vectors of the invention can be reconstituted into liposomes for delivery to target cells.
  • the vectors containing the polynucleotides of the invention can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host.
  • the present invention furthermore relates to a host cell transformed with a polynucleotide under the control of a heterologous promoter encoding said fusion protein or a vector of the invention.
  • Said host cell may be a prokaryotic or eukaryotic cell.
  • the polynucleotide or vector of the invention which is present in the host cell may either be integrated into the genome of the host cell or it may be maintained extrachromosomally.
  • the host cell can be any prokaryotic or eukaryotic cell, such as a bacterial, insect, fungal, plant, animal or human cell.
  • Preferred fungal cells are, for example, those of the genus Saccharomyces, in particular those of the species & cerevisiae.
  • the term "prokaryotic” is meant to include all bacteria which can be transformed or transfected with DNA or RNA molecules for the expression of a fusion protein of the invention.
  • Prokaryotic hosts may include gram-negative as well as gram- positive bacteria such as, for example, E. coli, S. typhimurium, Serratia marcescens and Bacillus subtilis.
  • eukaryotic is meant to include yeast, higher plant, insect and preferably mammalian cells, most preferably NSO and CHO cells.
  • the fusion protein encoded by the polynucleotide of the present invention may be glycosylated or may be non-glycosylated.
  • the fusion protein of the invention may also include an initial methionine amino acid residue.
  • a polynucleotide of the invention can be used to transform or transfect the host using any of the techniques commonly known to those of ordinary skill in the art.
  • Suitable source cells for the DNA sequences and host cells for immunoglobulin expression and secretion can be obtained from a number of sources, such as the American Type Culture Collection ("Catalogue of Cell Lines and Hybridomas," Fifth edition (1985) Rockville, Maryland, U.S.A., which is incorporated herein by reference).
  • the present invention relates to a method for the production of a fusion molecule, or a biologically active fragment thereof, which comprises either culturing the host cell capable of expressing the fusion molecule of the present invention under conditions allowing for the expression of the fusion molecule, the in vitro translation of the polynucleotide encoding said fusion molecule or the crosslinking the domains and recovering the fusion molecule produced as described above.
  • the present invention relates to a fusion molecule which is obtainable by the above-mentioned method.
  • the present invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the above-described fusion molecules, polynucleotides, vectors or host cells expressing said fusion molecule, and optionally to a pharmaceutically acceptable carrier.
  • the pharmaceutical composition of the present invention can include pharmaceutically acceptable salts of the components therein.
  • Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide) that are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, tartaric, mandelic and the like.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine, procaine and the like.
  • inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2- ethylamino ethanol, histidine, procaine and the like.
  • Particularly preferred is the HCl salt when used in the preparation of cyclic polypeptide [alpha]v antagonists.
  • Physiologically tolerable carriers are well-known in the art.
  • said pharmaceutical composition is in a soluble form.
  • liquid carriers are sterile aqueous solutions that contain no materials in addition to the active ingredients and water, or contain a buffer such as sodium phosphate at physiological pH value, physiological saline or both, such as phosphate-buffered saline. Still further, aqueous carriers can contain more than one buffer salt, as well as salts such as sodium and potassium chlorides, dextrose, polyethylene glycol and other solutes. Liquid compositions can also contain liquid phases in addition to and to the exclusion of water. Exemplary of such additional liquid phases are glycerin, vegetable oils such as cottonseed oil, and water-oil emulsions.
  • the present invention relates to a pharmaceutical composition for use in a broad variety of therapeutic needs, which concern preferably cell or organ transplantation, wound healing, the treatment of autoimmune, cardiovascular, allergic or infectious diseases, or the treatment of tumors.
  • an immune system disorder such as inflammation, actinic keratosis, acquired immunodeficiency syndrome (AIDS), Addison's disease, adult respiratory distress syndrome, allergies, ankylosing spondylitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, bronchitis, bursitis, cholecystitis, cirrhosis, contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis, diabetes mellitus, emphysema, erythroblastosis fetalis, erythema nodosum, atrophic gastriti
  • an immune system disorder such as inflammation, actin
  • said pharmaceutical composition is designed to be administered to a subject.
  • said subject is a mammal.
  • said pharmaceutical composition is adapted in a form to be administered orally, intravenously, subcutaneously, intramuscular or by inhalation.
  • the appropriate concentration of the therapeutic agent might be dependent on the particular agent.
  • the therapeutically effective dose has to be compared with the toxic concentrations; the clearance rate as well as the metabolic products play a role as do the solubility and the formulation.
  • Therapeutic efficacy and toxicity of compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • the present invention relates to a diagnostic composition
  • a diagnostic composition comprising the above-described fusion molecule, a polynucleotide or a vector encoding, or a host cell expressing said fusion molecule, and optionally suitable means for detection.
  • a variety of immunological methods as well as molecular biological methods like nucleic acid hybridization assays, PCR assays or DNA Enzyme Immunoassays (Mantero et al., Clinical Chemistry 37 (1991), 422-429) have been developed and are well known in the art.
  • ligand fusion molecules such as HLA class II alpha 2 chain nucleic acid molecules may also comprise PNAs, modified DNA analogs containing amide backbone linkages. Such PNAs are useful, inter alia, as probes for DNA/RNA hybridization.
  • the above-described diagnostic composition may be used for methods for detecting expression of HLA class II alpha 2 chain polynucleotide by detecting the presence of mRNA coding for a HLA class II alpha 2 chain (polypeptide which comprises, for example, obtaining mRNA from cells of a subject and contacting the mRNA so obtained with a probe/primer comprising a nucleic acid molecule capable of specifically hybridizing with a HLA class II alpha 2 chain polynucleotide under suitable hybridization conditions, and detecting the presence of mRNA hybridized to the probe/primer.
  • mRNA coding for a HLA class II alpha 2 chain polypeptide which comprises, for example, obtaining mRNA from cells of a subject and contacting the mRNA so obtained with a probe/primer comprising a nucleic acid molecule capable of specifically hybridizing with a HLA class II alpha 2 chain polynucleotide under suitable hybridization conditions, and detecting the presence of mRNA hybridized to
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • CGH comparative genome hybridization
  • RDA representative difference analysis
  • the invention comprises methods of detecting the presence of a HLA class II alpha 2 chain protein in a sample, for example, a cell sample, which comprises obtaining a cell sample from a subject, contacting said sample with one of the aforementioned antibodies under conditions permitting binding of the antibody to the HLA class II alpha 2 chain protein, and detecting the presence of the antibody so bound, for example, using immuno assay techniques such as radioimmunoassay or enzymeimmunoassay.
  • one skilled in the art may specifically detect and distinguish polypeptides which are functional HLA class II alpha 2 chain proteins from mutated forms which have lost or altered their HLA class II alpha 2 chain activity by using an antibody which either specifically recognizes a (polypeptide which has HLA class II alpha 2 chain activity but does not recognize an inactive form thereof or which specifically recognizes an inactive form but not the corresponding polypeptide having HLA class II alpha 2 chain activity.
  • the above described diagnostic compositions and methods can preferably be used to diagnose a disease or disorder as mentioned herein before.
  • Example 1 Cloning, expression and purification of a HLA-DR alpha 2 fusion molecule
  • HLA-DR alpha 2 The ability of HLA-DR alpha 2 to interact with its receptor(s) can be analyzed hi binding studies utilizing a soluble HLA-DR alpha 2 fusion protein.
  • the HLA-DR alpha 2 domain (Fig. IA) was cloned into an insect expression vector containing the V5- protein cDNA and expressed as a fusion protein in insect cells to establish DES-expression system for HLA-DR alpha 2-V5 fusion protein expression, HLA-DR alpha 2-V5 fusion protein was generated using the DES-Drosophila expression system (Invitrogen).
  • HLA-DR alpha 2 extracellular domain (amino acids 132-188) was cloned into the pMTBiP/V5-His A, a vector containing the V5 -epitope and a His-tag for purification.
  • HLA-DR alpha 2-V5 fusion protein expression vector was stably transfected into Schneider 2 (S2) cells with calcium phosphate precipitation and selected by co-transfection with a blasticidin selection vector. Protein secretion was induced by addition of copper sulfate and medium supernatant was taken for protein purification. Fusion protein was purified using Ni 2+ -columns. For silver staining, HLA-DR alpha 2-V5 fusion proteins were purified from cell culture supernatant.
  • HLA-DR alpha 2-Fc fusion protein a mammalian expression vector was developed in which the IGCl domain of HLA-DR alpha 2 chain is expressed in its whole integrity (amino acids 128-198) fused to the human IgGl-Fc fragment.
  • leader sequence the IG kappa leader was chosen, that is recommended for the secretion of recombinant antibodies.
  • HLA-DR alpha 2-Fc fusion protein For expression of HLA-DR alpha 2-Fc fusion protein, COS7 cells were transiently transfected with the mammalian expression vector for HLA-DR alpha 2-Fc fusion protein using Fugene ⁇ Transfection Reagent (Roche). One day after transfection media was exchanged to OptiMEM I (Gibco). After an additional 96 h HLA-DR alpha 2-Fc fusion protein was purified by Sepharose A column. Previous attempts to generate a fusion protein utilizing the HLA-DR-V5 expression system resulted in a very low yield of the expressed protein in the supernatants secreted by the transfected cells.
  • a new fusion protein was designed, containing the HLA-DR alpha 2 domain in its entirety coupled with a human IgGl Fc protein previously proven to be efficiently expressed in a mammalian expression system (Fig. ID).
  • This expression vector was transfected into COS7 cells and supernatants were subjected to Western blot using an anti-Fc-protein specific antibody.
  • This construct revealed a band on Western blot which matches the theoretical 45 kDa weight of the HLA-DR - Fc fusion protein (Fig. IE and IF).
  • Example 3 Biological activity of HLA-DR-alpha 2 fusion molecule
  • HLA-DR alpha 2 fusion molecules To examine the biological activity of the HLA-DR alpha 2 fusion molecules the effect of soluble HLA-DR alpha 2-Fc fusion protein on cytokine expression was studied in PHA activated human T-cells.
  • PBMC of human healthy donors were isolated according to the Ficoll-Paque density centrifugation protocol. 5 x 10 4 PBMC/well were incubated with 1 ⁇ g/ml PHA (Sigma) at 5% CO 2 , 37°C for 48 h in presence of HLA-DR peptides, control peptide or HLA-DR-Fc and Fc control respectively, in a total volume of 100 ⁇ l/well.
  • Example 4 Reduction of IFN-gamma positive cells after LPS induced sepsis and HLA-DR alpha 2-Fc treatment
  • mice were intraperitoneally induced with 50 ⁇ g LPS on day 0.
  • 4 mice were intraperitoneally treated with HLA-DR alpha 2-Fc (200 ⁇ g in PBS) or as control group with human Fc (50 ⁇ g in PBS).
  • HLA-DR alpha 2-Fc 200 ⁇ g in PBS
  • human Fc 50 ⁇ g in PBS
  • the splenocytes were isolated with a cell strainer. The cells without clumps were transferred in 15 ml tubes and centrifuged for 6 min at 1100 rpm. The erythrocytes were lysed with a red-blood lysis buffer from Sigma. Afterwards 5x10 6 cells were taken for intracellular FACS staining. The cells were washed with PBS/3 % FCS and then incubated with Fc-block (1 ⁇ g/lxl ⁇ 6 cells) for 20 min. After a further washing step the cells were fixed and permeabilized with CellFix/Perm-solution 2 (1:10 in PBS) for 10 minutes. The cells were washed twice with PBS/3 % FCS.

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Abstract

La présente invention se rapporte à une molécule de fusion comportant un premier domaine et un second domaine, ledit premier domaine comportant le domaine HLA-DR alpha 2 ou un fragment de ce domaine. Ladite molécule de fusion a la capacité de moduler la réponse immunitaire médiée par les lymphocytes T. En outre, l'invention se rapporte à des compositions pharmaceutiques comportant ces molécules de fusion et destinées au traitement chez un sujet de diverses maladies du type réaction du greffon contre l'hôte, maladies auto-immunes, maladies allergiques, maladies infectieuses et tumeurs.
PCT/EP2006/000560 2005-01-21 2006-01-23 Molecules de fusion hla et utilisations associees WO2006077152A2 (fr)

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CN103382224A (zh) * 2013-06-28 2013-11-06 英科隆生物技术（杭州）有限公司 含有四聚体形式的异源交联抗体的异源交联物及其应用
WO2013169922A1 (fr) 2012-05-08 2013-11-14 Western University Of Health Sciences Plateformes ex vivo standardisées pour l'expansion antigène-spécifique de populations de lymphocytes t cd4+

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WO1999009064A1 (fr) * 1997-08-19 1999-02-25 Mount Sinai School Of Medicine Of New York University ; Element de la classe ii du complexe majeur d'histocompatibilite portant un epitope/molecules chimeres d'immunoglobuline
EP1054984A1 (fr) * 1998-02-19 2000-11-29 President And Fellows Of Harvard College Proteines hybrides monovalentes, multivalentes et multimeres caracterisees par un domaine de liaison cmh (complexe majeur d'histocompatibilite), conjugues de ces proteines, et utilisations correspondantes
IL136511A0 (en) * 2000-06-01 2001-06-14 Gavish Galilee Bio Appl Ltd Genetically engineered mhc molecules
EP1430084A2 (fr) * 2001-09-17 2004-06-23 GenPat77 Pharmacogenetics AG Peptides capables de moduler une reponse immunitaire

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2013169922A1 (fr) 2012-05-08 2013-11-14 Western University Of Health Sciences Plateformes ex vivo standardisées pour l'expansion antigène-spécifique de populations de lymphocytes t cd4+
JP2015516164A (ja) * 2012-05-08 2015-06-11 ウエスタン ユニバーシティ オブ ヘルス サイエンシズ ＣＤ４＋Ｔ細胞集団の抗原特異的な拡大のために標準化されたｅｘｖｉｖｏプラットフォーム
US9943578B2 (en) 2012-05-08 2018-04-17 Western University Of Health Sciences Standardized ex vivo platforms for the antigen-specific expansion of CD4+ T cell populations
EP2847322B1 (fr) * 2012-05-08 2019-06-26 Western University Of Health Sciences Plateformes ex vivo standardisées pour l'expansion antigène-spécifique de populations de lymphocytes t cd4+
CN103382224A (zh) * 2013-06-28 2013-11-06 英科隆生物技术（杭州）有限公司 含有四聚体形式的异源交联抗体的异源交联物及其应用

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