CN101184505A - Soluble immunoregulatory factor - Google Patents

Abstract

The invention provides soluble derivatives of the vOX2 protein encoded by Kaposi's sarcoma-associated herpesvirus (KSHV), and their use for the treatment of inflammatory disorders such as autoimmune diseases, allergy and graft rejection.

Description

Solubility vOX2 albumen as immunoregulatory factor

Technical field

The present invention relates to immune modulatory molecules, particularly the solubility vOX2 protein derivatives of encoding by kaposi sarcoma-associate herpesvirus (KSHV).

Background technology

The physiological function of immune system is to make the host defense infection by microorganisms.This defence is by the delayed response mediation of the early reaction and the adaptive immunity of innate immunity.Activated immune system antiviral component the earliest is an inflammatory response, it is characterized in that discharging the chemotactic mediator, so that leukocyte (mainly being cellular component---neutrophil cell the abundantest in the immune system) is raised site to inflammatory reaction by peripheral blood by damaged cell (comprising endotheliocyte).Neutrophil cell is removed them by digestion microorganism and infected cell in the phagocytosis process, thereby brings into play pivotal role in innate immune responses.This process is accompanied by the generation of engulfing intravital effector " respiratory burst (respiratory burst) ", and the unexpected raising that it comprises oxidative metabolism causes producing virose reactive oxygen intermediate metabolite (ROI; As O ₂, OH, H ₂O ₂).Though the generation of ROI is in order to remove pathogen in cell, they can pass cell membrane inevitably and be diffused into the inflammation site, and they can be at described inflammation site damaged tissue, thereby has increased immune morbidity.

In the inflammatory response between most important short inflammatory cell mediator be chemotactic factor, interleukin 8 (IL-8) and macrophage chemoattractant albumen-1 (MCP-1) etc.More and more evidences shows, IL-8 and MCP-1 are respectively the main mediator (Akahoshi etc. of acute (neutrophil cell) and chronic (monocytes/macrophages) inflammation, 1994, Frangogiannis etc., 2002, Hatano etc., 1999, Smith etc., 1997, Villiger etc., 1992).CXC-chemotactic factor IL-8 is leukocytic short existence (pro-survival) signal; Many reports show, comprise that mononuclear cell, T lymphocyte, neutrophil cell, fibroblast, endotheliocyte and epithelial various cell express IL-8mRNA and synthetic IL-8 albumen apace.Yet it is mainly synthetic by the cell of monocyte/macrophage pedigree, and mainly raises neutrophil cell (summarizing in (Baggiolini, 2001, Mukaida etc., 1998)).MCP-1 is with the motion of cell and stimulates relevant short scorching chemotactic factor, mainly acts on lymphocyte, mononuclear cell, mastocyte and eosinophilic granulocyte (Mukaida etc., 1998).This chemotactic factor is as different stimulated such as IL-1, tumor necrosis factor (TNF) and immunoglobulin (Ig) replying of G are expressed in the various kinds of cell type, comprise mononuclear cell, smooth muscle cell and human vascular endothelial (HUVEC) (Rollins etc., 1990, Sica etc., 1990, Yang etc., 2004).TNF-α and IgG complex also are the known stimulating factors (Satriano etc., 1993) that produces ROI.

Virus is evolved with its host, and they also and then evolved out and escaped the strategy of immunne response.Kaposi sarcoma-associate herpesvirus (KSHV) is the up-to-date infection mankind's that identify herpesvirus (Chang etc., 1994), it is the cause of disease of Kaposi sarcoma (KS), and may be the cause of disease (summary is consulted (Sarid etc., 1999)) of lymphoma primary effusion and multicentricity castleman's disease.In the genome that comprises about 90 KSHV opening code-reading frames (ORF), at least 16 protein (Russo etc., 1996) that are defined as the scalable host immune response.Can think that inflammatory response is the immunoregulatory crucial target that enhanced virus infects and scatters.In fact, KSHV coding three kinds of CXC chemotactic factor regulatory factors (Boshoff etc., 1997).

Synthetic another kind of protein is by the opening code-reading frame coded (Jenner etc., 2001) of K14 by name in the lytic cycle of KSHV.This protein demonstrates and the mammal OX2 albumen homology of (also being called CD200), the latter belongs to the Ig superfamily of film associated glycoprotein, be expressed on the surface of many cell types, comprise endotheliocyte, B cell, T cell, neurocyte and tonsil vesicle (tonsil follicle) (Morris﹠amp; Beech, 1987, Wright etc., 2001).

The effect of OX2 believes it is to regulate the active a member of myeloid cell in the cell, may be as auxiliary signal (Wright etc., 2000).The rising that OX2 expresses in mice and rat has improved the survival (Gorczynski etc., 1998, Gorczynski etc., 1999) of kidney and skin allograft, and prompting OX2 transmits tolerance signal (summarizing in (Gorczynski, 2001)).The signal transmission of OX2 may be by realizing that with combining of OX2 part (receptor) expression of this part (receptor) is confined to myeloid cell (Gorczynski etc., 2000, Wright etc., 2003).

Since this homology, the so-called vOX2 of this virus protein.Yet this is call easily just.These two kinds of albumen are in only have an appointment 40% sequence homogeneity of amino acid levels, so can not suppose that this virus protein can the proteic any activity of analog cell.And this virus protein also has homology with nerve cell adhesion molecule (NCAM), Thy-1 and L1, so vOX2 also is known as vNCAM and v-adh.

Summary of the invention

Result of study for the vOX2 function is mutual contradiction up to now.Chung etc. (2002) report, the vOX2 albumen that is expressed as soluble g ST fusion rotein can stimulate former generation mononuclear cell, macrophage and dendritic cell, and induces their to produce inflammatory cytokine.In this research, the vOX2 that is expressed in the bone-marrow-derived lymphocyte surface also shows can stimulate mononuclear cell to produce inflammatory cytokine.The author proposes, and this short scorching activity can make LR to the KSHV sites of infection, and this provides the cells that are used for viral infection more, and the system that therefore promotes virus is spread to infected host everywhere.On the contrary, Foster-Cuevas etc. (2004) reach a conclusion, and the vOX2 of film expression has reduced the activation of macrophage.They propose, and the possibility of result of Chung is because due to the false folding of its solubility recombiant protein, but this can not explain in two researchs the contradictory outcome that the protein with cell surface expression obtains.Therefore, the biological activity of vOX2 is still unknown so far.

The inventor has been found that solubility vOX2 can bring into play anti-inflammatory effect in innate immune system.Therefore, in prevailing form, the invention provides the purposes of solubility vOX2 as antiinflammatory.

In aspect first, the invention provides the purposes that solubility vOX2 is used to suppress the inflammatory immunne response, comprise the immune system cell group is contacted with solubility vOX2.All these purposes of carrying out in external, stripped or body all comprise within the scope of the invention.

Described cell mass generally includes the cell of innate immune system, phagocyte especially, and for example neutrophil cell, mononuclear cell and/or macrophage randomly with immune multiple other cell combination, comprise that the T lymphocyte (comprises CD4 ⁺And CD8 ⁺The T lymphocyte) and bone-marrow-derived lymphocyte.

Without wishing to be held to any specifically theoretically, think that solubility vOX2 is to phagocyte such as mononuclear cell (as macrophage) and neutrophil cell performance anti-inflammatory effect.For example, it can reduce the macrophage of handling through interferon gamma specifically is that MCP-1 and IL-8 among the U937 produces, and can also suppress the oxidative burst of neutrophil cell.

Therefore, this method can comprise the step of mensuration to the inhibition degree of inflammatory immunne response.This can be by measuring inflammatory cytokine or chemotactic factor generation especially the generation (for example MCP-1 and/or IL-8) in the macrophage and/or reactive oxygen intermediate (as O ₂Or OH group or H ₂O ₂) generation realize.Certainly, when implementing this method in vivo, can use any suitable inflammatory symptom to monitor the proteic effect of vOX2, for example the tissue infiltration of inflammatory cell, swelling, rubescent etc.

The anti-inflammatory property that solubility vOX2 showed makes it be applicable to treatment inflammatory diseases or other disease, and wherein unfavorable or unwanted immunne response causes patient's symptom and/or disease incidence, comprises autoimmune disease, allergy and transplant rejection.Such disease comprises rheumatoid arthritis, psoriasis, inflammatory bowel, multiple sclerosis, asthma, chronic obstructive pulmonary disease (COPD), contact and allergic dermatosis, inflammatory eye disease, transplant rejection, vascular inflammation (heart disease) and inflammatory nervous syndrome.

Therapeutic Method described in this description can comprise any appropriate method that solubility vOX2 is delivered to the inflammation site.For example, solubility vOX2 albumen directly can be applied to the patient who has this to need.Perhaps the nucleic acid of coding solubility vOX2 can be applied to the patient, thereby the cell of this albumen by this patient self synthesizes and secretes.Or, the cell that can express and secrete solubility vOX2 can be applied to the patient.

Therefore, the cell of solubility vOX2 maybe can be expressed and secrete to the nucleic acid that the invention provides the solubility vOX2 albumen that is used for therapy, the solubility of encoding vOX2.

The cell of solubility vOX2 maybe can be expressed and secrete to the nucleic acid of solubility vOX2 albumen in the medicine that the present invention also is provided for preparing prevention or treating inflammatory diseases, coding solubility vOX2.

The present invention also provides the method for prevention or treatment inflammatory diseases, comprises that the nucleic acid that the patient that these needs are arranged is used the solubility vOX2 albumen of effective dose, the solubility of encoding vOX2 maybe can express and secrete the cell of solubility vOX2.

The present invention also provides pharmaceutical composition, and it comprises with the solubility vOX2 albumen of pharmaceutically suitable carrier combination, the nucleic acid of coding solubility vOX2 maybe can express and secrete the cell of solubility vOX2.

The vOX2 albumen that uses in the method for the present invention generally obtains by recombinant expressed and secretion, preferably from eukaryotic cell.

This vOX2 albumen can be polyvalent.That is to say that it comprises two or more vOX2 parts in complex bivalence, tervalent, quaternary or more high price.Without wishing to be held to any particular theory ground, therefore polyvalent vOX2 may can more effectively bring into play anti-inflammatory activity than monovalence albumen than the lip-deep receptor of the more effectively crosslinked target cell of monovalence vOX2.

Single vOX2 part can be with covalently or non-covalently mode combination in the vOX2 complex.This combination can be directly (promptly between the vOX2 part) or indirect (by the allos component that partly links to each other with vOX2).

For example, vOX2 part chemical crosslinking each other.

Perhaps, vOX2 partly can be expressed as the fusion rotein that comprises two (or more) vOX2 parts in single polypeptide chain, and it randomly by suitable peptide linker separately.

Perhaps, vOX2 part can be connected with allos (being non-vOX2's) component, causes combination thereby described allos component is interact with each other.Therefore, this complex can comprise the first and second vOX2 parts that link to each other with the first and second allos components respectively, and the wherein said first and second allos components are bonded to each other.The described first and second allos components can be identical or different, and can be bonded to each other by covalently or non-covalently interacting.

Preferably, each vOX2 part is expressed as fusion rotein with its allos component separately.The example of allos component comprises the oligomerization domain of transcription factor, as the leucine zipper motif that is bonded to each other by hydrophobic interaction.Yet particularly preferred allos component example is the antibody Fc district.The vOX2-Fc fusion rotein of native conformation normally comprises the dimer of two polypeptide chains, and wherein every chain partly is made up of a Fc part and a vOX2, and described polypeptide chain is by the disulfide bond covalent bond between the Fc part.

Described allos component can be regulated the pharmacokinetic properties of vOX2 molecule.For example, it can regulate bioavailability, dissolubility, stability, interior half-life of body etc.Compare with independent effector molecule, the fusion rotein that comprises the effector molecule that is connected with antibody Fc district (also being immunoadhesin) has enhanced dissolubility, stability and half-life usually.Though (expectation improves these characteristics of vOX2 part usually, should be appreciated that, needs to reduce some or all in these characteristics sometimes.Can correspondingly select suitable allos component.)

Also may expect the vOX2 part is connected with the allos component, described allos component is regulated its pharmacokinetic properties in the mode of expectation, partly is not incorporated in the multivalence complex but do not mediate vOX2.

In another aspect, the invention provides the solubility antiinflammatory, it comprises the complex of at least two vOX2 parts.

As mentioned above, this complex can comprise the first and second vOX2 parts that link to each other with the first and second allos components respectively, and the wherein said first and second allos components mutually combine.

When described allos component was protein or peptide, it preferably was expressed as fusion rotein with its vOX2 part separately.Therefore, the invention provides the nucleic acid of the such fusion rotein of coding, this fusion rotein preferably can be by secretory host cell.The host cell that comprises these expression of nucleic acids carriers and comprise these expression vectors also is provided.

When described allos component identical (the identical allos component that promptly mutually combines), usually might be by only from a kind of such nucleic acid construct expressing protein product, thus form the multivalence complex.

When described allos component difference (promptly an allos component combines with another dissimilar allos component), then must be from the expression of nucleic acid protein product of the every type of fusion rotein of encoding.

Therefore, the invention provides compositions, it comprises first nucleic acid and second nucleic acid, described first nucleic acid coding contains the fusion rotein of the vOX2 part and the first allos component, described second nucleic acid coding contains the fusion rotein of the 2nd vOX2 part and the second allos component, wherein when being expressed as protein, the described first and second allos components mutually combine.Described fusion rotein can be secreted by appropriate host cell usually.Described first and second nucleic acid can be on expression vector that separates or same expression vector.The present invention also provides the host cell that comprises described first and second nucleic acid.

In preferred embodiments, described allos component is the antibody Fc district.Therefore, the invention provides solubility vOX2-Fc fusion rotein.

The present invention also provides nucleic acid, and its coding can be by the excretory solubility vOX2-Fc fusion rotein of appropriate host cell.Therefore, the signal peptide of the common encoding fusion protein N of described nucleic acid end, thus make this fusion rotein can be by the secretory host cell of expressing it.

The present invention also provides the expression of nucleic acids carrier that comprises coding solubility vOX2-Fc fusion rotein.Described carrier generally comprises with the sequence of encoding fusion protein effectively is connected suitable transcribes and translates the adjusting sequence, thereby makes host cell transcribe and translate described protein.

The present invention also provides the host cell that contains above-mentioned expression vector.Described host cell is preferably eukaryotic host cell, as mammalian cell, insect cell or yeast cells.In certain embodiments, preferred mammal cell (as Chinese hamster ovary celI or people's cell).

The present invention also provides pharmaceutical composition, and it contains fusion rotein, nucleic acid, expression vector or host cell described in the second aspect present invention that makes up with pharmaceutically suitable carrier.

Description of drawings

The generation of Fig. 1: vOX2-Fc and purification.A. in the Chinese hamster ovary celI of stable transfection, produce vOX2-Fc.The supernatant through transforming Chinese hamster ovary celI to results carries out the Western engram analysis, uses anti-human IgG-HRP antibody to analyze, and uses ECL trace reagent (Amersham) to develop.Swimming lane 1 is without the normal Chinese hamster ovary celI of transfection; Swimming lane 2, with the Chinese hamster ovary celI of reorganization pDR2 Δ EF1 α plasmid derivative thing transfection, it expresses irrelevant Fc-fusion rotein, KSHV complement control albumen (KCP:Fc); Swimming lane 3, stable transfection the Chinese hamster ovary celI system of pvOX2-Fc (CHO-15 " 69).The position of molecular size labelling is marked in the left side of trace.B. protein purification and refining (polishing): the reorganization vOX2-Fc through affinity purification and " making with extra care " is carried out the Western engram analysis.Swimming lane 1 is without the normal Chinese hamster ovary celI of transfection; Swimming lane 2, the transfection of purification self-stabilization the reorganization vOX2-Fc of the Chinese hamster ovary celI system of pvOX2-Fc (CHO-15 " 69).Carry out protein purification by affinity chromatograph (Hi Trap A albumen post) and size exclusion chromatography (Superdex 200).Use anti-human IgG-HRP antibody that purified vOX2-Fc albumen is carried out the Western engram analysis, and with ECL trace reagent develop (Amersham).

Fig. 2: suppress the generation of inflammatory chemokine in the U937 mononuclear cell by the vOX-2:Fc albumen of reorganization.The level of A.MCP-1.The level of B.IL-8.Measure by Luminex culture fluid carried out cytokine quantitatively before, under the situation that has or do not exist reorganization IFN-γ (5ng) and vOX-2:Fc (10 μ g/ml) with 1 * 10 ⁶Density cultivated the U937 cell 48 hours.

Fig. 3: the vOX-2:Fc albumen by reorganization suppresses the oxidative burst in people's peripheral blood neutrophil cell of former generation rather than swallows (engulfment).A. oxidative burst.B. swallow.Measure (OPREGEN Pharma by commercially available Bursttest and Phagotest; BD Biosciences) with the component of these activate the phagocytic capacity in the flow cytometry measurement whole blood, granulocyte is set up gate (gating), and obtain 5000 incidents.Before measuring, with the whole blood of heparinization with the human IgG (10 μ g/ml) of reorganization vOX2-Fc (10 μ g/ml) or purification hatch (90 minutes, 37 ℃, 5%CO ₂), perhaps keep not handling.When collecting, each blood sample is carried out differential blood count, this makes and is determined at escherichia coli: the ratio of neutrophil cell is to carry out in 6: 1 o'clock, and each experimenter's data are carried out normalization to average fluorescent strength (MFI).The cumulative data that has shown 13 whole blood samples researchs of 11 health volunteers in three independent experiments.

Fig. 4: suppress the inductive acute inflammation of carrageenan in vivo by reorganization vOX-2:Fc and reply.Induce the generation acute inflammation of BALB/c mouse foot pad by a rear solid end that the Sargassum extract carrageenan is applied to each animal.Using carrageenan preceding 30 minutes, 4 groups of mices are accepted reorganization vOX2-Fc (the 80 μ g/ mices of intraperitoneal (IP) injection; The ■ group), human IgG (the 80 μ g/ mices of purification; ▲ group), dexamethasone (500 μ g/ mices; ● group) or PBS (◆ group), after using carrageenan, carry out once again after 6,12 and 24 hours then.Assess the inflammation degree by the thickness of measuring foot pad, the metapedes pad that is calculated as the inoculation carrageenan and same animal are without the difference in thickness between the metapedes pad of inoculating.Data merge from three single blind experiments independently, represent every group meansigma methods+/-SE (it is 24 mices that PBS and vOX2-Fc handle every group, and 8 mices are accepted the human IgG of purification, handles with dexamethasone for 16).Comparing data between the group is shown in the table.

Fig. 5 and Fig. 6: vOX2-Fc is to the arthritic generation collagen-induced in the DBA mice and the influence of seriousness.In every width of cloth figure, figure A shows the average arthritis score of every group of 10 mices.Figure B shows arthritic percentage ratio incidence rate in each group.Fig. 5 shows that the disease of as many as in the time of the 37th day takes place.Fig. 6 shows the result of as many as identical experiment in the time of the 42nd day.

Fig. 7: the aminoacid sequence of the vOX2-Fc fusion rotein of using in this research.The vOX2 sequence is shown as normal font and underscore is arranged.This represents the 78-307 amino acids in the full length sequence that Russo etc. submits to.Sequence A ADPI (normal font, no underscore) is a connector area.Immunoglobulin γ 1Fc sequence is shown as italic.The hinge region of immunoglobulin (PKSCDKPHTCP) is shown as italic and underscore is arranged.

Detailed Description Of The Invention

vOX2

VOX2 is the opening code-reading frame encoded protein matter (Jenner etc., 2001) by called after K14 in the KSHV virus.The vOX2 albumen exemplary sequence of taking from (PNAS 93:14862 (1996)) such as Russo is as follows:

1

MIHTFFDCPGRRVVEGGVISSYFLIGAPGRTAIKTEEGVSALQN

78

LPPTVLPVAGTGSVVRPVVCAPPWTTPSASRGSMSSLFISLPWVAFIWLALLGAVGGA

RVQGPMRGSAALTCAITPRADIVSVTWQKRQLPGPVNVATYSHSYGVVVQTQYRHKAN

ITCPGLWNSTLVIHNLAVDDEGCYLCIFNSFGGRQVSCTACLEVTSPPTGHVQVNSTE

DADTVTCLATGRPPPNVTWAAPWNNASSTQEQFTDSDGLTVAWRTVRLPRGDNTTPSE

3D7 TM

GICLITWGNESISIPASIQGPLAHDLPAAQ GTLAGVAITLVGLFGIFALHHCRRKQGG

ASPTSDDMDPLSTQ

This virus protein is an embrane-associated protein, comprises N end ectodomain, membrane spaning domain (having underscore) and C end born of the same parents intracellular domain.Described opening code-reading frame contains two possible initial methionines, at 1 and 78 amino acids places.It has been generally acknowledged that 78 methionines most possibly are used as intravital start codon.

VOX2 function in vivo is controversial.Therefore yet the inventor has now found that solubility vOX2 demonstrates anti-inflammatory property, and can be in vivo be used to suppress inflammatory response with external.

The solubility vOX2 albumen that uses in the inventive method can be polyvalent.That is to say that they can comprise two or more vOX2 parts.Multivalence vOX2 may be more effectively more crosslinked with the lip-deep receptor of target cell than monovalence vOX2.

VOX2 part can covalently or non-covalently mutually combine, although for stability and also have the former of active aspect thereby preferably covalently combination possibly.

Two vOX2 parts can coexpression be fusion rotein.In order to produce fusion rotein, made up the nucleic acid expression vector that in a continuous opening code-reading frame, comprises the coded sequence of each part, thereby these two parts can be translated as the part of same polypeptide chain.

Usually, between two structures, comprise flexible peptide linker, to allow these two components freely to interact and not have sterically hindered.The technical staff can design suitable joint fully.Routinely, such joint length is between 12 to 20 aminoacid, and the little ratio with hydrophilic amino acid residue (for example glycine and serine) is very high, so that required flexibility to be provided, and does not destroy the water solublity of this molecule.

Perhaps, can transform the vOX2 part to improve its mutual affinity.This can be accomplished in several ways.For example, cysteine can be introduced so that these two parts form disulfide bond each other.

Or, can promote two interactions between the vOX2 part by each vOX2 part is connected with the allos component, wherein said two allos components can mutually combine.When this allos component was polypeptide, it can partly be expressed as fusion rotein with described vOX2.

Preferred allos component is to comprise the antibody Fc sequence, be preferably one or more antibody Fc domains (for example the CH2 of IgG, IgM, CH3 and/or CH4 domain (suitably time) etc.) polypeptide.Preferably, also comprise the hinge sequence that is usually located between CH1 and the CH2 domain.Hinge region contains cysteine residues, and it forms disulfide bond between the heavy chain of complete natural antibody.Therefore, if having hinge region in the vOX2-Fc molecule as herein described, then will form similar key to stablize the interaction between the chain.The human IgG1 is preferred fusion partner.

The technical staff is clear can be used for strengthening or stable vOX2 part between interactional substituting allos component.They comprise the leucine zipper polypeptide, and it is by the hydrophobic interaction dimerization.

Although preferred recombination method, vOX2 part also can be covalently bound by chemical means.Be suitable for making peptide molecule to put together each other or crosslinked difunctional and multifunctional chemical linkers is well-known for the skilled person.

No matter be multivalence or monovalence, each vOX2 part preferably comprises the proteic ectodomain of vOX2 (promptly 78 sequences to 309 amino acids comprise or do not comprise signal peptide as required) or it is enough to show the fragment of anti-inflammatory effect in the solubility vOX2 albumen described herein.When anti-inflammatory effect refers to use the arbitrary mensuration described in the embodiment to assess the IL-8 of U937 cell or MCP-1 produce, oxidative burst, the inflammation in the carrageenan model of neutrophil cell or use disease in the collagen-induced arthritis model of DBA mice to take place or seriousness in one or more significance,statistical reduce.

Preferably, described vOX2 partly comprises at least 50 aminoacid that above show in the ECD sequence, and can comprise at least 100,150 or 200 aminoacid that above show in the ECD sequence.

Yet the technical staff will appreciate that, can carry out some change to this aminoacid sequence and keeps the anti-inflammatory activity of this soluble protein basically.Therefore, described vOX2 albumen or part can comprise at least 50,100,150 or 200 amino acid whose sequences, this sequence and above-mentioned sequence have at least 80% homogeneity relevant on overlapping, preferably has at least 85% sequence homogeneity with this sequence, more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% homogeneity needs only it and shows required anti-inflammatory activity.

Especially, the conservative replacement in the vOX2 sequence (comparing with reference sequences) especially can better tolerate, and not appreciable impact function.

Conservative is replaced and can be defined as the replacement within the aminoacid classification and/or be the replacement of positive score value in the BLOSUM62 matrix.

According to a kind of classification, the aminoacid classification be tart, alkaline, uncharged polar with nonpolar, wherein acidic amino acid is Asp and Glu; Basic amino acid is Arg, Lys and His; Uncharged polar amino acid is Asn, Gln, Ser, Thr and Tyr; Nonpolar amino acid is Ala, Gly, Val, Leu, Ile, Pro, Phe, Met, Trp and Cys.

According to another kind classification, the aminoacid classification is little hydrophilic, acid/amide/hydrophilic, alkaline, little hydrophobic and aromatics, and its medium and small hydrophilic amino acid is Ser, Thr, Pro, Ala and Gly; Acid/amide/hydrophilic amino acid is Asn, Asp, Glu and Gln; Basic amino acid is His, Arg and Lys; Little hydrophobic amino acid is Met, Ile, Leu and Val; Aromatic amino acid is Phe, Tyr and Trp.

As follows for the replacement of just marking in the BLOSUM62 matrix:

Original residue	C	?S	?T	?P	?A	?G	?N	?D	?E	?Q	?H	?R	?K	?M	?I	?L	?V	?F	?Y	?W
																					Replace	-	?T?A?N	?S	?-	?S	?-	?S?D?H	?N?E	?D?Q?K	?E?R?K	?N?Y	?Q?K	?E?Q?R	?I?L?V	?M?L?V	?M?I?V	?M?I?L	?Y?W	?H?F?W	?F?Y

Be defined as with the amino acid sequence identity percentage ratio (%) of reference sequences: carrying out sequence alignment and introducing breach where necessary with after realizing highest serial homogeneity percentage ratio, the percentage ratio of the amino acid residue identical with amino acid residue in the reference sequences in the candidate sequence, any conservative are replaced and are not thought and constitute sequence homogeneity.Can pass through the numerical value that WU-BLAST-2 (Altschul etc., Methods in Enzymology, 266:460-480 (1996)) determines homogeneity percentage ratio.WU-BLAST-2 uses the several retrieval parameter, and wherein great majority use as default.Adjustable parameter is set to following value: overlapping span=1, and overlapping mark (overlap fraction)=0.125, speech threshold value (word threshold) is (T)=11.The identical residue number of the coupling of determining with WU-BLAST-2 multiply by 100 again, thereby determines % amino acid sequence identity value divided by the residue sum of reference sequences (ignore by what WU-BLAST-2 introduced in reference sequences and make the maximized breach of comparison scoring).

Amino acid similarity percentage ratio (%) defines in the mode identical with homogeneity, just calculates the residue for just marking in the BLOSUM62 matrix.Therefore, inequality but residue that have a similar characteristic (for example replaced by conservative and obtain) also counts.

Similarly, be defined as with the nucleotide sequence homogeneity percentage ratio (%) of reference nucleic acid: in the candidate sequence with reference nucleic acid sequence in the percentage ratio of the identical nucleotide residue of nucleotide residue.Homogeneity value used herein can be produced by the BLASTN module that is set to default parameters among the WU-BLAST-2, and wherein overlapping span and overlapping mark are set to 1 and 0.125 respectively.

Described in this manual solubility vOX2 albumen preferred expression is from eukaryotic cell, as yeast cells, insect cell (for example Sf9 cell) and mammalian cell (for example CHO (Chinese hamster ovary) cell).

Preferably, therefore described albumen is correctly folding also can combine with CD200 receptor (CD200R).The list of references that suitable mensuration is described in (2004) such as Foster-Cuevas and quotes.Preferably, in the vOX2 protein Preparation thing at least 20% vOX2 part can with the CD200 receptors bind; More preferably, in the vOX2 protein Preparation thing at least 30%, 40%, 50%, 60%, 70%, 80% or 90% vOX2 part can with the CD200 receptors bind.Use the affinity purification technology of CD200 receptor to be used in to realize ideal in the solubility vOX2 protein Preparation thing in conjunction with activity level.

The proteic therapeutic use of solubility vOX2

The inventor shows that solubility vOX2 has anti-inflammatory property, especially can reduce the reaction of the congenital part of immune system (as neutrophil cell and macrophage) mediation.It is that MCP-1 and IL-8 among the U937 produces that this albumen can reduce the macrophage of handling through interferon gamma specifically, can also suppress the oxidative burst of neutrophil cell.It also can reduce in the mouse model by the inductive inflammation of carrageenan, importantly, in its appreciable impact DBA mice by collagen-induced arthritic generation and seriousness.This result is astonishing especially.

Therefore, solubility vOX2 can be used for treating inflammation, inflammatory diseases or other disease, the reason that the incorrect or undesirable immunne response that relates to innate immune system in these diseases is this disease symptoms and/or disease incidence.Such disease comprises autoimmune disease, allergy and transplant rejection.Therefore, for example, solubility vOX2 can be used for treating rheumatoid arthritis, psoriasis, inflammatory bowel, multiple sclerosis, asthma, chronic obstructive pulmonary disease (COPD), contact and allergic dermatosis, inflammatory eye disease, transplant rejection, vascular inflammation (heart disease) and inflammatory nervous syndrome.

Solubility vOX2 albumen directly can be applied to the experimenter with pharmaceutical composition.

Perhaps, can use coding solubility vOX2 proteic nucleic acid to the experimenter, thereby by experimenter's self cellular expression justacrine solubility vOX2 albumen.Described nucleic acid is the part of one or more expression vectors normally, can be used as that naked nucleic acid is used or uses in delivery vector such as viral vector (for example adenovirus, slow virus or retrovirus).

Perhaps, can use through transforming proteic cell the experimenter with secretion solubility vOX2.Preferably, described cell and experimenter are homologous or tissue compatible.For example, cell can be taken from the experimenter, after one or more appropriate carriers transfections, is applied to this experimenter again.As a supplement or substitute, described secretory cell can incapsulate, and for example in the biologically inert polymer, interacts between transplanted cells and the host immune system preventing.

The technical staff can be designed for the therapeutic use suitable nucleic acid expression vector of (and being used for other purposes described in this description).Described carrier will generally contain suitable adjusting sequence, comprise promoter sequence, terminator fragment, enhancer sequence, marker gene and other sequence, and this depends on the proteic concrete form of solubility vOX2 to be administered (seeing above).This carrier can be intended to be integrated into the host cell gene group, or can be used as episome and be independent of host genome and exist and duplicate.

The preferred experimenter who handles by the inventive method is a mammal.Preferred experimenter is primates (comprising the people), Rodents (comprising mice and rat), and other common laboratory animal, domestic animal and agricultural animal, includes but are not limited to rabbit, Canis familiaris L., cat, horse, cattle, pig, sheep, goat etc.

Complex as herein described, polypeptide, nucleic acid and cell can be mixed with pharmaceutical composition.Except one of above-mentioned substance, these compositionss can comprise pharmaceutically acceptable excipient, carrier, buffer agent, stabilizing agent or other material well-known to those skilled in the art.These materials should be nontoxic, and should not disturb the effectiveness of described active component.The definite character of described carrier or other material can be depending on route of administration, for example oral, intravenous, skin or subcutaneous, per nasal, intramuscular and intraperitoneal approach.

Be used for pharmaceutical composition for oral administration and can be the form of tablet, capsule, powder or liquid.Tablet can comprise solid carrier such as gelatin or adjuvant.Composition of liquid medicine generally includes liquid-carrier, as water, oil, animal or plant oil, mineral oil or artificial oil.Can comprise normal saline, glucose or other sugar juice or dihydroxylic alcohols such as ethylene glycol, propylene glycol or Polyethylene Glycol.

With regard to intravenous, skin or subcutaneous injection or ill site injection, active component will be for can be used for parenteral aqueous solution form, and it does not contain pyrogen, and has suitable pH, isotonicity and stability.This area person skilled can expertly prepare suitable solution, uses for example to wait to ooze carrier (as sodium chloride injection, ringer's injection, lactated Ringer's injection).When needing, can comprise antiseptic, stabilizing agent, buffer agent, antioxidant and/or other additive.

Character regardless of the activating agent in individuality to be administered (for example cell of the present invention, polypeptide, nucleic acid molecules, other medicinal agents), all preferably to be enough to show that " prevention effective dose " or " treatment effective dose " (under the situation that might occur, the preventing also can think treatment although be) to the benefit of individuality use.Actual dosage and the speed of administration and character and the seriousness that time-histories will depend on treated individual.The prescription (for example dosage determine etc.) of treatment in omni-doctor and other doctor's the Limitation on Liability, consider usually disease to be treated, individual patient situation, send the known other factors of site, medication and practitioner.The example of above-mentioned technology and scheme is found in Remington ' s Pharmaceutical Sciences, 20th Edition, 2000, pub.Lippincott, Williams﹠amp; Wilkins.

Perhaps, by using targeted system such as antibody or cell specific ligand, can use targeted therapy that activating agent is delivered to some cell type more specifically.May be because multiple former thereby need targeted therapy, for example, agents useful for same has unacceptable toxicity, perhaps otherwise will need too high dosage, perhaps otherwise can not enter target cell.

Compositions can be used separately, perhaps simultaneously or use with other therapeutic combination continuously, depends on situation to be treated.

Material and method

Clone vOX2-Fc fusion rotein

With aminoterminal domain and the human IgG of KSHV vOX2 albumen as fusion rotein ₁The C end fragment in Fc district is expressed in the frame together.Based on our research (data not shown) and previous disclosed analysis (Chung etc., 2002, Neipel etc., 1997, Russo etc., 1996), select in two possible methionine residues (residue 1 or residue 78) second as start codon.By pcr amplification ORF K14, and TA clones into pCR2.1-TOPO carrier (Invitrogen).Described gene amplification is used following PCR primer from the lymphoma primary effusion cell line (Cesarman etc., 1995) that BC-1 KSHV infects: justice is arranged, 5 '-GCT CTAGAT GTC TAG CCT CTT CAT TTC ATT AC-3 '; Antisense, 5 '-TAT GCG GCCGCG GCC GCG GGA AGG TCA TGG GC-3 '.These amplimers comprise restriction site (Not I and Xba I), make this gene sub-clone to be advanced expression vector.K14 is inserted segmental two chains check order, confirm that PCR does not introduce mistake, and this gene sub-clone is advanced pDR2 Δ EF1 alpha expression carrier (consulting (Spiller etc., 2003)) and generation pvOX2-Fc.Sub-clone comprises that using Xba I and Not I that K14 is inserted fragment digests from pCR2.1-TOPO, and is connected in the pDR2 Δ EF1 α of SpeI and Not I digestion.Insert the site at K14 the pvOX2-Fc plasmid is checked order, produced virus and IgG to guarantee to connect ₁The frame endomixis of gene.The aminoacid sequence of the vOX2-Fc fusion rotein that obtains is shown in Fig. 7.Cell and cell culture

At 37 ℃, 5%CO ₂Down, cultivation people monoblast is U937 in RPMI 1640 culture medium (BioWhittaker) of having replenished 10% heat-inactivated fetal bovine serum (FBS), L-glutaminate (2mM), penicillin-streptomycin (1%), non essential amino acid (0.1mM).In order to make the U937 cell differentiation is the macrophage-like phenotype, with it at recombinant interferon (IFN)-γ (5ng/ml; R﹠amp; D Systems) there are cultivation down 48 hours.

Obtain the informed consent postscript, collect the venous blood of heparinization from the adult volunteer of health.Use Sysmex SE, (Sysmex Corp. Japan) carries out the white blood count and differential counting to 9500 blood analysers.In some are measured, by the hypotonic cracking of glucosan sedimentation and erythrocyte, carry out difference density gradient centrifugation by Ficoll (Sigma) subsequently and come from whole blood, to separate polymorphonuclear cell.

In order to obtain producing the proteic cell line of vOX2-Fc, Chinese hamster ovary celI is advanced in the pvOX2-Fc transfection.In the culture medium that contains HYG (500 μ g/ml), obtain stable cell lines, and use anti-human IgG-FITC (Sigma) or anti-human IgG-HRP antibody to filter out the stable cell lines that produces recombiant protein by immunofluorescence and Westen trace respectively.VOX2-Fc protein is produced maximum cell line called after CHO-15 " 69.

Albumen produces and purification

Collect CHO-15 " 69 cell conditioned medium liquid, and (Hi Trap Amersham) upward carries out affinity purification to vOX2-Fc at A albumen post to use AKTA protein purification system (Amersham).For vOX:Fc and the Fc protein fragments of removing truncate, then by on Superdex200 size-exclusion column (Amersham), carrying out this albumen of gel filtration " making with extra care ".Evaluating protein purity by the following method: polyacrylamide gel electrophoresis is also utilized coomassie brilliant blue R250 (Bio-Rad Laboratories) or gel is analyzed in Sypro Ruby (Analgene) dyeing, uses anti-human IgG-HRP and anti-cattle Ig-HRP (Sigma) to carry out the western trace and detects.Downcutting all protein bands also identifies by mass spectrum (MS) or (MALDI-TOF) mass spectrum of substance assistant laser desorpted ionized-flight time.In some experiments, utilize same scheme purification human IgG purification as negative control.

Oxidative burst is measured

Utilization is measured (OPREGEN Pharma based on the commercialization Bursttest of flow cytometry; BD Biosciences, Oxford UK), measures the oxidative burst component of phagocytosis in whole blood periphery polymorphonuclear mononuclear cell.This is measured and allows the leukocytic activity of quantitative assay, and need not purifying cells in advance.Bursttest measures and to depend on unlabelled through conditioning escherichia coli (E.coli) and as the dihydro rhodamine (DHR) 123 of oxidation activity fluorogenic substrate as the microgranule stimulus object.In brief, with the whole blood of heparinization with the human IgG (10 μ g/ml) of reorganization vOX2-Fc (10 μ g/ml) or purification hatch (90 minutes, 37 ℃, 5%CO ₂), again with through the conditioning Bacillus coli cells (6 cell/leukocyte) hatch (7 minutes, 37 ℃, 5%CO ₂).The sample of non-stimulated thing is as negative (background) contrast.Add then DHR123 and incubated cell (10 minutes, 37 ℃, 5%CO ₂).Add the lysis buffer cessation reaction, this buffer section is leukocyte and splitting erythrocyte fixedly.At last, in order to get rid of antibacterial or hematoblastic gathering illusion, cell DNA is dyeed before carrying out flow cytometry facing.Utilize flow cytometer (FACSCalibur, BD Biosciences) pair cell to analyze, obtain 5000 granulocyte incidents obtaining producing the cell percentage ratio and the number of reactive oxygen group, and its enzymatic activity degree of weighing with average fluorescent strength.

Engulf mensuration

Utilization is based on the commercialization Phagotest test kit (OPREGENPharma of flow cytometry; BD Biosciences), in whole blood periphery polymorphonuclear mononuclear cell, measure aspect phagocytotic the swallowing.Similar with Bursttest, this mensuration system allows the leukocytic activity of swallowing of quantitative assay, and need not purification in advance.In brief, with the whole blood of heparinization with the human IgG (10 μ g/ml) of reorganization vOX2-Fc (10 μ g/ml) or purification hatch (90 minutes, 37 ℃, 5%CO ₂), again with through the conditioning FITC labelling Bacillus coli cells (6 cell/leukocyte) hatch (10 minutes, 37 ℃, 5%CO ₂).Hatch and add cancellation solution by 4 ℃ and stop picked-up.This solution makes the bonded FITC fluorescent quenching of bacterium surface and the particulate fluorescence of internalization is constant, thereby allows to utilize flow cytometry to differentiate that adhere to and antibacterial internalization.Behind the washing step, remove erythrocyte by hypotonic cracking.At last, described in Bursttest mensuration, cell DNA is dyeed.Utilize flow cytometry (FACSCalibur, BD Biosciences) pair cell to analyze, and obtain 5000 granulocyte incidents.

The proteic measurement of MCP-1 and IL-8

When having or not existing reorganization vOX2-Fc albumen (10 μ g/ml), do not handle or with rIFN-γ (5ng/ml) processing U937 cell (1 * 10 ⁶/ ml), and hatch 48 hours (37 ℃, 5%CO ₂).Then, collect this culture fluid, and pass through the concentration of quantitative cytokine of Luminex algoscopy and chemotactic factor.

Research in the body

Test acutely inflamed mouse model with anti-inflammatory effect in the proteic body of assessment reorganization vOX2-Fc.In this model, as described above by bringing out the acute inflammation (Leung etc., 2001) of BALB/c mouse (Jackson Laboratories) foot pad in the rear solid end that the Sargassum extract carrageenan is applied to every animal.In each experiment, four groups of BALB/c mouse were accepted human IgG (80 μ g/ mice), dexamethasone (500 μ g/ mice) or the phosphate-buffered saline (PBS) of intraperitoneal (IP) injection reorganization vOX2-Fc (80 μ g/ mice), purification in 30 minutes before using carrageenan.Determine the inflammation degree by measuring sufficient mat thickness, be calculated as the metapedes pad of inoculation carrageenan and the difference in thickness between the nonvaccinated metapedes pad of same animal.After using carrageenan, carry out these measurements after 6,12 and 24 hours.Carried out independently single blind experiment three times.

Statistical analysis

Check by printenv Mann Whitney and to measure the difference between each processed group in Bursttest, Phagotest and the cytokine assay.If these significant differences are then thought in P≤0.05.For studying in the body, carry out data analysis by the GLM repeated measure, use Tukey as the check of the multiple comparisons between four treatment groups (SPSS) afterwards.In order to analyze the difference between each group of each time point (6,12,24 hours), carried out Mann Whitney check.If significant difference is then thought in P≤0.05.

The result

The proteic clone of reorganization vOX-2:Fc, synthetic and purification

Total length KSHV ORFK14 gene clone is advanced in the pDR2 Δ EF1 α carrier for expression of eukaryon, wherein conduct of vOX2 albumen and human IgG ₁The fusion rotein in Fc C end structure territory produces.VOX2 is expressed as and this IgG ₁The advantage of the fusion rotein of domain comprises: the affinity tag that is used for protein purification (i) is provided and (ii) the half-life of recombiant protein can have been improved nearly 10 times (Harris etc., 2002).Then, at cell line CHO-15 " produce reorganization vOX-2:Fc albumen (Figure 1A) in 69, and utilize the affine and size exclusion chromatography of A albumen from culture fluid, to carry out purification (Figure 1B).Select size exclusion chromatography to be because only can be purified into a plurality of bands (Figure 1B) of reorganization vOX2-Fc by A albumen affinity chromatograph.By MS or MALDI-TOF MS the identity of these bands is defined as vOX2-Fc dimer, monomer and clipped form, and Fc self (data not shown).Behind size exclusion chromatography, obtained the wall scroll band (Figure 1B) of reorganization vOX2-Fc.

By the inflammatory response of vOX-2:Fc albumen at the vitro inhibition myeloid cell

In order to determine whether vOX-2:Fc albumen regulates the inflammatory response of myeloid cell, and the cytokine of the macrophage-like U937 cell that assessment contacts with this recombiant protein produces spectrum.Do not handle cell, perhaps when existing or not having vOX-2:Fc albumen, handle the U937 cell, and measure releasing and activity of inflammatory cytokines with rIFN-γ.VOX2-Fc handles the generation that has significantly reduced MCP-1 (about 30%) (Fig. 2 A) and IL-8 (about 50%) (Fig. 2 B).This effect is specific, because other inflammatory cytokine comprises the generation of eotaxin (eotaxin), MIP-1 α, MIP1 β, RANTES, IL-1 β, IL-6 and TNF α uninfluenced (data not shown).

VOX-2:Fc regulated myeloid cell is active to studies show that further this albumen is significantly reduced the oxidative burst activity (Fig. 3 A) of (at least 25%) former generation people neutrophil cell.This inhibition is specific for phagocytotic this on the one hand, because the swallowing of these cells active uninfluenced (Fig. 3 B).

Reply by suppressing acute inflammation in the reorganization vOX-2:Fc body

Because reorganization vOX2-Fc albumen has anti-inflammatory activity external, so measured its body internal latent heat in this respect.Therefore, for the influence of checking that vOX2-Fc takes place for experimental acute inflammation, with reorganization vOX2-Fc albumen (80 μ g) treatments B ALB/c mice, it used through intraperitoneal bringing out acute inflammation by carrageenan inoculation foot pad in preceding 30 minutes.After 6,12 and 24 hours, measure the inflammation degree (consulting " method ") of foot pad.Replace vOX2-Fc to inject each group (Fig. 4) in three groups of control group mice with the human IgG (80 μ g) of purification, dexamethasone (500 μ g) or PBS.In general, according to analysis of GLM repeated measure and the check of Tukey afterwards, sufficient mat thickness is determined as measuring, and reorganization vOX2-Fc processing has significantly suppressed acute inflammation and replied PBS and vOX2-Fc, P＜0.0001; IgG and vOX2-Fc, P＜0.005; Dexamethasone and vOX2-Fc, P＜0.001.The same with expectation, there is not significant difference between the inflammatory response of PBS and IgG processed group.In order to analyze the inhibition influence between each time point (6,12 and 24 hours) vOX2-Fc group, carried out printenv Mann Whitney check (table 1).These the analysis showed that (24 hours) are compared with the PBS processed group in the research persistent period, and compare with the human IgG processing at least 12 hours, and reorganization vOX2-Fc albumen significantly suppresses the experimental acute inflammation that brings out.

Histopathological analysis to the foot pad has been supported these analyses.Therefore, this data show is compared with untreated mice, and acute inflammation seriousness and inflammatory cell are raised the significance,statistical reduction to the inflammation site.

	The P value of inflammatory response difference between the processed group
				Use the time (h) behind the carrageenan	PBS and vOX2-Fc	IgG and vOX2-Fc	Dex and vOX2-Fc
?6	≤0.0001	≤0.001	≤0.006
				?12	≤0.001	≤0.03	≤0.001
?24	≤0.006	≤0.6	≤0.02

Statistical analysis is carried out in the inhibition of table 1. pair mouse insole carrageenan inflammation that processing is brought out.Utilize the analysis of Mann Whitney printenv that the measurement and the matched group measurement of vOX2-Fc processed group are compared.Experimental technique is referring to the legend of Fig. 4.

Suppress collagen-induced arthritis in the DBA mice

Check that in blind research vOX2-Fc is to arthritic influence collagen-induced in the DBA mice.Use other two kinds of Fc fusion rotein in contrast.They are Fc fusions of Complement Regulatory Protein KCP, and lack the Fc fusions that complement is regulated active KCP albumen three lysine mutation bodies (KCPmut).

At the 0th day, use II Collagen Type VI and Freund's complete adjuvant immune mouse, and attacked with the II Collagen Type VI in the saline at the 21st day.Estimate that first kind of sign of disease took place at the 27th day.Disease widely took place in 35 days in expectation to the.

Each organizes mice (10 every group) was accepted 100 μ g among the PBS at the-1 and the 0th day corresponding Fc fusion rotein.Every group at the 3rd day and accept 50 μ g albumen among the PBS afterwards every three days until the 27th day (comprising the 27th day).Another mice in control group is only accepted PBS.The results are shown in Fig. 5 during to the 37th day.Data are expressed as to put correlation time and go up every group average mark.Result during to the 42nd day (except the KSP:Fc contrast) is shown in Fig. 6.

These data show that vOX2-Fc significantly suppresses arthritic generation and seriousness in this model.

Discuss

KSHV is the human oncogenic virus of describing recently (Chang etc., 1994).It is the paathogenic factor of Kaposi sarcoma (KS), and this disease is the complicated tumor that influences AIDS patient and old people from Mediterranean, and is African most common tumor.The pathogenesis of KS is quite complicated, and nearest evidence points out that KSHV induces infected epithelial cell to transcribe and is programmed to the lymph phenotype again, thereby promotes lymphatic vessel to take place, and promotes that therefore (Hong etc., 2004, Wang etc., 2004) take place tumor.Be responsible for this viral gene of transcribing again tissue and do not determine at last as yet, but may comprise g protein coupled receptor (Bais etc., 2003).However, nearly 20% KSHV genome comprises the gene with immunoregulatory activity.They comprise and are confirmed as suppressing MHC I surface expression (Coscoy﹠amp; Ganem, 2000), regulate T cellular immunization synapse (Coscoy﹠amp; Ganem, 2001), suppress complement activation (Spiller etc., 2003), interrupt the gene of interferon signal cascade (Zhu etc., 2002), chemotactic factor signal (Boshoff etc., 1997) and B-cell receptor signal (Choi etc., 2000).

The function of the product of the vOX2 of one of the immunomodulating KSHV gene of inferring (ORF K14) is controversial, is summed up as macrophage activation (Chung etc., 2002) and suppresses (Foster-Cuevas etc., 2004) activity.

In order to assess the function of vOX2 in this research, it is expressed as has human IgG ₁The N end fusion rotein of FC (Fc) domain C end.We assess the extracorporeal anti-inflammatory characteristic of vOX2-Fc to the influence that phagocyte is swallowed, oxidative burst and proinflammatory cytokine discharge by measuring it in primary cell or cell line, and find that it reduces the generation of oxidative burst in neutrophil cell active and short scorching chemotactic factor IL-8 of downward modulation and MCP-1.Importantly, also in model, assessed the influence of vOX2-Fc, in this model by using the acute inflammation of Sargassum extract carrageenan inducing mouse foot pad.Administered recombinant vOX2-Fc significantly suppresses this inflammatory effector, provides this recombiant protein to suppress definite evidence that acute inflammation is replied (the particularly influx of neutrophilic granulocyte).Our discovery shows that reorganization vOX2-Fc albumen will be born the immunomodulating signal and pass to phagocyte, thus inhibition of innate immune responses.The vOX2-Fc fusion rotein also can suppress arthritic generation collagen-induced in the Muridae model.

VOX2-Fc albumen suppresses the human peripheral neutrophil cell and is subjected to the generation that bacterial cell stimulates back ROI.The ROI stable state is critical to normal cell function, because low-level ROI is most important to many cell signalling incidents.Under physiological condition, there is balance between ROI level that produces in the normal cell metabolism and the endogenous antioxidant level, described endogenous antioxidant is used to prevent that tissue is subjected to oxidative damage (McCord, 1993).VOX2-Fc suppresses oxidative burst in the neutrophil cell and depends on vOX2-Fc and cell preincubate at least 2 hours.The dependency of this pair cell and other immunomodulator preincubate has report (Chen etc., 2004, Lehn etc., 1989) before.Should also be noted that the IgG complex is the known stimulant (Satriano etc., 1999) that ROI produces, it has supported that further the inhibition activity of vOX2-Fc in our the IgG comparative study is not because the Fc component of this molecule.

Oxidative stress and inflammatory reaction are interrelated, even the instantaneous raising of ROI level also is the biological important mediator of blood vessel, influences cell growth, apoptosis, migration, inflammation and secretion and (summarize in (Fattman etc., 2003, Touyz﹠amp; Schiffrin, 2004)).And the excessive generation of ROI is relevant with the pathogenesis of numerous disease, (summarizes in (Bowler﹠amp as cardiovascular disease, sacred disease, renal failure and lung disease; Crapo, 2002, Delanty﹠amp; Dichter, 1998, Fukai etc., 2002)).

ROI also can represent the second messenger system (summarizing in (Droge, 2002)) of gene activation, and it may have importance (DeForge etc. to the generation of chemotactic factor during the tissue injury such as MCP-1 and IL-8,1993, Satriano etc., 1993, Vlahopoulos etc., 1999).In fact, discharging chemotactic factor is the another kind of medullary system function of vOX2-Fc negative regulation.We find to handle and mononuclear cell U937 cell that differentiation takes place produces the ability of MCP-1 and IL-8 (the two all has short scorching effect) and significantly suppressed (Fig. 2) (Akahoshi etc., 1994, Baggiolini through IFN-γ, 2001, Endo etc., 1994, Frangogiannis etc., 2002, Hatano etc., 1999, Poddar etc., 2001, Withanage etc., 2004, Yla-Herttuala etc., 1991).

Because short scorching chemotactic factor and toxicity oxygen metabolism thing have increased the morbidity of cardiovascular, arthritis, tumor and neurodegenerative diseases, so vOX2-Fc helps to develop the new therapy at these diseases.

Although the present invention's combination embodiment above is described, yet after providing the disclosure content, the improvement of many equivalences and change are conspicuous for those skilled in the art.Therefore, disclosed embodiment of the present invention should be thought exemplary and nonrestrictive.Can carry out various changes and not deviate from the spirit and scope of the present invention described embodiment.All lists of references that this paper quoted are all incorporated this paper into by the reference mode clearly.

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Claims (81)

1. suppress the method for inflammatory immunne response, it comprises makes the immune system cell group contact with solubility vOX2.

According to the process of claim 1 wherein described contact in vivo, external or stripped carrying out.

3. according to the method for claim 1 or claim 2, wherein said cell mass comprises the cell of innate immune system.

4. according to the method for claim 3, wherein said cell mass comprises phagocyte, as neutrophilic granulocyte, mononuclear cell and/or macrophage.

5. according to the method for claim 3 or 4, wherein said cell mass also comprises T lymphocyte and/or bone-marrow-derived lymphocyte.

6. according to each method in the claim 1 to 5, also comprise the inhibition degree of mensuration to the inflammatory immunne response.

7. according to the method for claim 6, it comprises the generation of measuring inflammatory cytokine or chemotactic factor.

8. according to the method for claim 7, wherein said inflammatory cytokine or chemotactic factor are MCP-1 and/or IL-8.

9. according to the method for claim 6, it comprises the generation of measuring reactive oxygen intermediate.

10. depend on the method according to claim 6 of claim 1, wherein said method is carried out in vivo, and uses tissue infiltration, swelling, the rubescent inhibition degree of monitoring the inflammatory immunne response that waits of inflammatory symptom such as inflammatory cell.

11. according to each method in the claim 1 to 10, wherein said vOX2 albumen comprises two or more vOX2 parts.

12. according to the method for claim 11, wherein said vOX2 part links to each other with the allos component, the combination that causes the vOX2 part interact with each other of this allos component.

13. according to the method for claim 12, wherein each vOX2 part is as expressing with the fusion rotein of its allos component separately.

14. according to the method for claim 12 or claim 13, wherein said allos component is the antibody Fc district.

15. according to the method for claim 12 or claim 13, the oligomerization domain that wherein said allos component is a transcription factor.

16. according to the method for claim 15, wherein said allos component is the leucine zipper motif.

17. according to each method in the claim 1 to 16, it carries out in vivo, is used for prevention or treatment autoimmune disease, allergy or transplant rejection.

18. method according to claim 17, it carries out in vivo, is used for prevention or treatment rheumatoid arthritis, psoriasis, inflammatory bowel, multiple sclerosis, asthma, chronic obstructive pulmonary disease (COPD), contact or allergic dermatosis, inflammatory eye disease, transplant rejection, vascular inflammation (heart disease) or inflammatory nervous syndrome.

19. according to the method for claim 17 or claim 18, it comprise to the experimenter that these needs are arranged use solubility vOX2 albumen, the proteic cell of solubility vOX2 can be expressed and secrete to coding solubility vOX2 proteic nucleic acid maybe.

20. solubility vOX2 albumen, it is used for the therapeutic treatment method.

21. solubility vOX2 albumen, it is used for prevention or treatment inflammatory diseases.

22. the method for prevention or treatment inflammatory diseases comprises the solubility vOX2 albumen of the patient that these needs are arranged being used effective dose.

23. pharmaceutical composition, it comprises the solubility vOX2 albumen with pharmaceutically suitable carrier combination.

24. according to the solubility vOX2 albumen or the method for claim 21 or claim 22, wherein said inflammatory diseases is autoimmune disease, allergy or transplant rejection.

25. according to the solubility vOX2 albumen or the method for claim 24, wherein said inflammatory diseases is rheumatoid arthritis, psoriasis, inflammatory bowel, multiple sclerosis, asthma, chronic obstructive pulmonary disease (COPD), contact or allergic dermatosis, inflammatory eye disease, transplant rejection, vascular inflammation (heart disease) or inflammatory nervous syndrome.

26. according to each solubility vOX2 albumen, method or pharmaceutical composition in the claim 20 to 25, wherein said vOX2 albumen comprises two or more vOX2 parts.

27. according to solubility vOX2 albumen, method or the pharmaceutical composition of claim 26, wherein said vOX2 part links to each other with the allos component, the combination that causes the vOX2 part interact with each other of this allos component.

28. according to solubility vOX2 albumen, method or the pharmaceutical composition of claim 27, wherein each vOX2 part is as expressing with its allos component fusion rotein separately.

29. according to solubility vOX2 albumen, method or the pharmaceutical composition of claim 27 or claim 28, wherein said allos component is the antibody Fc district.

30. according to solubility vOX2 albumen, method or the pharmaceutical composition of claim 27 or claim 28, the oligomerization domain that wherein said allos component is a transcription factor.

31. according to solubility vOX2 albumen, method or the pharmaceutical composition of claim 30, wherein said allos component is the leucine zipper motif.

32. the nucleic acid of coding solubility vOX2, it is used for the therapeutic treatment method.

33. the nucleic acid of coding solubility vOX2, it is used for prevention or treatment inflammatory diseases.

34. the method for prevention or treatment inflammatory diseases comprises the nucleic acid of the patient that these needs are arranged being used the coding solubility vOX2 of effective dose.

35. pharmaceutical composition, it comprises the nucleic acid of the coding solubility vOX2 that makes up with pharmaceutically suitable carrier.

36. according to the nucleic acid or the method for claim 33 or claim 34, wherein said inflammatory diseases is autoimmune disease, allergy or transplant rejection.

37. according to the nucleic acid or the method for claim 36, wherein said inflammatory diseases is rheumatoid arthritis, psoriasis, inflammatory bowel, multiple sclerosis, asthma, chronic obstructive pulmonary disease (COPD), contact or allergic dermatosis, inflammatory eye disease, transplant rejection, vascular inflammation (heart disease) or inflammatory nervous syndrome.

38. according to each nucleic acid, method or pharmaceutical composition in the claim 32 to 37, wherein said solubility vOX2 albumen can be secreted from express this proteic host cell.

39. according to each nucleic acid, method or pharmaceutical composition in the claim 32 to 38, wherein said nucleic acid coding comprises the fusion rotein of two or more vOX2 components, this vOX2 component is separated by junctional complex alternatively.

40. according to each nucleic acid, method or pharmaceutical composition in the claim 32 to 38, wherein said nucleic acid coding comprises the fusion rotein of the solubility vOX2 component that links to each other with the allos component, thus wherein can the be bonded to each other combination of mediation vOX2 part of two such allos components.

41. according to nucleic acid, method or the pharmaceutical composition of claim 39 or claim 40, wherein said fusion rotein can come out from express this proteic host cell in secretion.

42. according to nucleic acid, method or the pharmaceutical composition of claim 40, wherein said allos component is the antibody Fc district.

43. can express and secrete the cell of solubility vOX2, it is used for the therapeutic treatment method.

44. can express and secrete the cell of solubility vOX2, it is used for prevention or treatment inflammatory diseases.

45. the method for prevention or treatment inflammatory diseases comprises the cell that can express and secrete solubility vOX2 of the patient that these needs are arranged being used effective dose.

46. pharmaceutical composition, it comprises the cell that can express and secrete solubility vOX2 with pharmaceutically suitable carrier combination.

47. according to the cell or the method for claim 44 or claim 45, wherein said inflammatory diseases is autoimmune disease, allergy or transplant rejection.

48. according to the cell or the method for claim 47, wherein said inflammatory diseases is rheumatoid arthritis, psoriasis, inflammatory bowel, multiple sclerosis, asthma, chronic obstructive pulmonary disease (COPD), contact or allergic dermatosis, inflammatory eye disease, transplant rejection, vascular inflammation (heart disease) or inflammatory nervous syndrome.

49. according to each cell, method or pharmaceutical composition in the claim 43 to 48, wherein said solubility vOX2 albumen is each defined solubility vOX2 albumen in the claim 26 to 31.

50. comprise the compositions of first nucleic acid and second nucleic acid, described first nucleic acid coding contains the fusion rotein of the vOX2 part and the first allos component, described second nucleic acid coding contains the fusion rotein of the 2nd vOX2 part and the second allos component, wherein, when being expressed as protein, the described first and second allos components mutually combine.

51. according to the compositions of claim 50, wherein said fusion rotein can come out from express this proteic host cell in secretion.

52. according to the compositions of claim 50 or claim 51, wherein said first and second nucleic acid are on the expression vector that separates.

53. according to the compositions of claim 50 or claim 51, wherein said first and second nucleic acid are on same expression vector.

54. according to each compositions in the claim 50 to 53, the oligomerization domain that wherein said allos component is a transcription factor.

55. according to the compositions of claim 54, wherein said allos component is the leucine zipper motif.

56. according to each compositions in the claim 50 to 55, it is used for the therapeutic treatment method.

57. according to each compositions in the claim 50 to 55, it is used for prevention or treatment inflammatory diseases.

58. the method for prevention or treatment inflammatory diseases comprises the patient that these needs are arranged is used each described first and second nucleic acid in the claim 50 to 55 of effective dose.

59. pharmaceutical composition, it comprises in the claim 50 to 55 with pharmaceutically suitable carrier combination each compositions.

60. according to the compositions or the method for claim 59, wherein said inflammatory diseases is autoimmune disease, allergy or transplant rejection.

61. according to the compositions or the method for claim 60, wherein said inflammatory diseases is rheumatoid arthritis, psoriasis, inflammatory bowel, multiple sclerosis, asthma, chronic obstructive pulmonary disease (COPD), contact or allergic dermatosis, inflammatory eye disease, transplant rejection, vascular inflammation (heart disease) or inflammatory nervous syndrome.

62. test kit, it comprises each described first and second nucleic acid in the claim 50 to 55.

63. according to the test kit of claim 62, wherein every kind of nucleic acid all makes up with pharmaceutically suitable carrier.

64. according to the test kit of claim 62 or claim 63, wherein said first and second nucleic acid are positioned on separately first and second expression vectors.

65. host cell, it comprises each described nucleic acid in the claim 38 to 42, perhaps comprises each described first and second nucleic acid in the claim 50 to 55.

66. according to the cell of claim 65, it is used for the therapeutic treatment method.

67. according to the cell of claim 65, it is used for prevention or treatment inflammatory diseases.

68. the method for prevention or treatment inflammatory diseases comprises the cell according to claim 65 of the patient that these needs are arranged being used effective dose.

69. pharmaceutical composition, it comprises the cell according to claim 65 with pharmaceutically suitable carrier combination.

70. according to the cell or the method for claim 67 or claim 68, wherein said inflammatory diseases is autoimmune disease, allergy or transplant rejection.

71. according to the cell or the method for claim 72, wherein said inflammatory diseases is rheumatoid arthritis, psoriasis, inflammatory bowel, multiple sclerosis, asthma, chronic obstructive pulmonary disease (COPD), contact and allergic dermatosis, inflammatory eye disease, transplant rejection, vascular inflammation (heart disease) or inflammatory nervous syndrome.

72. the solubility antiinflammatory, it comprises the complex of at least two vOX2 parts.

73. the solubility antiinflammatory according to claim 72 wherein comprises fusion rotein, described fusion rotein comprises at least two vOX2 parts of randomly separating by joint.

74. according to the solubility antiinflammatory of claim 72, it comprises the first and second vOX2 parts that link to each other with separately the first and second allos components, the wherein said first and second allos components are connected with each other.

75. according to the solubility antiinflammatory of claim 74, wherein said allos component is and its fusion rotein of vOX2 part separately.

76. according to the solubility antiinflammatory of claim 74 or claim 75, wherein said allos component is the antibody Fc district.

77. according to the solubility antiinflammatory of claim 74 or claim 75, the oligomerization domain that wherein said allos component is a transcription factor.

78. according to the solubility antiinflammatory of claim 77, wherein said allos component is the leucine zipper motif.

79. the nucleic acid of each described fusion rotein in the coding claim 73 or 75 to 78.

80. comprise expression of nucleic acids carrier according to claim 79.

81. host cell, it comprises according to the nucleic acid of claim 79 or 0 expression vector according to Claim 8.

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