CN1435433A

CN1435433A - Long-acting broad-spectrum chemotactic factor receptor inhibiting matter

Info

Publication number: CN1435433A
Application number: CN 02129301
Authority: CN
Inventors: 龚疆红
Original assignee: Individual
Current assignee: Individual
Priority date: 2002-08-30
Filing date: 2002-08-30
Publication date: 2003-08-13

Abstract

A chimeric protein compound for durable and efficient suppression to different chemotactic factor receptors, its nucleic acid sequence, and the process for preparing and testing its products are disclosed. It can be used to prevent and cure HIV infection, tumor transfer, tissue transplant rejection and self immunopathy. Its advantages are high durability, broad spectrum, and high selectivity.

Description

The chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum

One, technical field

The present invention relates to the chimeric protein compound, relate in particular to a kind of chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum.

Two, background technology

Discover, protein of cell chemotactic factor (Chemokine) and acceptor thereof play an important role to the pathologic process of numerous disease: HIV (human immunodeficiency virus) is to utilize the acceptor of chemokine to infect human body, so, if can seal these acceptors, just can stop the infection of HIV (human immunodeficiency virus); Some tumour cell also is to finish by the Chemokine Receptors that tumor cell surface has to the transfer of some specific organs in addition; When various non-special inflammatory diseases take place, the phenomenon that all has chemokine mediated white corpuscle to infiltrate to focus.Thereby the acceptor of chemokine inhibiting just can suppress cell and moves effectively, reaches the effect of treatment and prevention nonspecific inflammation disease and metastases.

Research finds that also numerous disease all relates to multiple different Chemokine Receptors.It is human to utilize CCR5 and CXCR4 Chemokine Receptors to infect as the main HIV-1 virus of acquired immune deficiency syndrome (AIDS), but also has many HIV-1 viruses can utilize CCR1, and acceptors such as CCR2, CCR3 infect; The breast cancer cell surface has CXCR4 and two kinds of acceptors of CCR7, thereby has determined these cancer metastasis to marrow, lymphoglandula and lung; It is monocyte (by the CCR2 acceptor) and neutrophil leucocyte (by CXCR1, the CXCR2 acceptor) etc. that ulcerative colitis causes the principal element of pathological lesion.The drug screening principle all is that a kind of medicine is only at a kind of acceptor at present.These medicines can suppress this a kind of acceptor, but can not suppress disease.By way of example, polypeptide compound SDF-1P2G is proved to be efficient special inhibition CXCR4 acceptor, the virus that can suppress the full-blown AIDS people, however the HIV-1 virus of early stage patient and many other hypotypes but can not be suppressed, because other acceptors that these viruses are mentioned above utilizing.And the biggest problem that the acquired immune deficiency syndrome (AIDS) problem solves be exactly HIV-1 virus itself can be along with the course of disease constantly produces sudden change, thereby infect used acceptor also in continuous change.Can not suppress all acceptors and just can not accomplish really to suppress virus infection, also just not have clinical value widely.Just because of this reason, up to the present, also have no idea to prevent the infection of HIV-1 in the world.Again such as breast cancer cell, if only suppress a kind of among its two kinds of acceptor, cancer cells still can shift (with reference to illustration 7) by another acceptor, and similarly situation also has many other diseases.Thereby, in order to prevent and treat this class disease, need research and development can suppress many targets property medicine of multiple different Chemokine Receptors simultaneously.Save substantial contribution and time when in addition, another advantage of the medicine of many targets property is developed exactly.Because develop a combination drug of forming by several different compounds, its clinical trial requires and must do earlier clinical trial respectively to every single medicine, remake comprehensive clinical trial, time and expense all expend huge, infeasible, and the exploitation of the medicine of target more than only needs a clinical trial.

On the other hand, effective inhibition of Chemokine Receptors depends on keeping of medicine effective concentration, because Chemokine Receptors is to rely on the G protein receptor, being characterized in can be by recirculation and biosynthesizing regeneration after being suppressed, need to suppress again (document 8), only keep inhibitor effective concentration in vivo and just can accomplish acceptor is suppressed completely, thereby stop the infection of HIV-1 virus and the transfer of tumour effectively, so another important factor that intravital long drug effect also is this type of medicine.

Three, summary of the invention

Main purpose of the present invention is in view of the compound of the present chemokine inhibiting acceptor of developing or too single-minded not wide spectrum, problem of unstable too in vivo, the several of discovery derive from many targets property protein inhibitor of virus owing to be a structure fixed molecule, both can't select the scope that suppresses, (because it is a viral protein) can cause allergic reaction again, be not suitable for the clinical present situation of using repeatedly, and provide a kind of chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum, it is long-acting in vivo, wide spectrum, efficiently, being free from side effects (having specificity and diversity simultaneously), is a kind of new drug that can prolonged and repeated use.

The objective of the invention is to realize by following technical scheme.

The chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum of the present invention is characterized in that, chimeric protein that can two or more Chemokine Receptors of antagonism, and it includes following protein fragments:

A, human immunoglobulin constant region;

B, two kinds of different pair cell Chemokine Receptors have the protein fragments of antagonistic action;

E, wherein the C-end of each receptor antagonist by its molecule links to each other with the N-of human immunoglobulin constant region is terminal, formation fusion rotein monomer;

D, these fusion rotein monomers that contain the antagonist of isoacceptor not link to each other with covalency each other again, can amphiploids, and tetraploid, or polymeric form output.

The chemotactic factor receptor inhibiting matter of the aforesaid long-acting, broad-spectrum of stating is characterized in that, the monomeric human immunoglobulin constant region of described fusion rotein partly is meant human immunoglobulin's the constant region of heavy chain and/or the constant region fragment of light chain; The monomeric receptor antagonist of described fusion rotein partly comprise following any two at the protein of isoacceptor not:

A.XR4 antagonist 1, promptly SDF-1P2G (sees Appendix 1, protein sequence 1.1; 1.2; And genes encoding ND.1);

B.XR4 antagonist 2, promptly SDF-1 (2-67) (sees Appendix 1, protein sequence 2.1; 2.2, and genes encoding ND.2);

C. two amino acid of N-terminal head produce through chemically modified, have the SDF-1 derivative that suppresses the receptor CXCR 4 effect;

D.R5 antagonist 1, i.e. RANTES (9-68) (see protein sequence 3.1,3.2,4.1,4.2 and genes encoding ND.4);

E.R5 antagonist 2, i.e. RANTES (8-68) (seeing protein sequence 4.1,4.2 and genes encoding ND.3);

F.XR3 antagonist 1, i.e. ITAC (4-73) (seeing protein sequence 5.1,5.2 and genes encoding ND.5);

G.XR3 antagonist 2, i.e. ITAC (3-73) (seeing protein sequence 6.1,6.2 and genes encoding ND.6);

H. 3 amino acid of N-terminal head obtain through chemically modified, have the ITAC derivative that suppresses acceptor CXCR3 effect;

I.R7 antagonist 1, i.e. MIP-3 β (8-77) (seeing protein sequence 7.1,7.2 and genes encoding ND.7);

J. N-terminal head two 7 amino acid obtain through chemically modified, have the MIP-3 β derivative that suppresses acceptor CCR7 effect;

K.R7 antagonist 2, i.e. 6CKine (8-79) (seeing protein sequence 8.1,8.2 and genes encoding ND.8);

L. 7 amino acid of N-terminal head obtain through chemically modified, have the pulsating derivative of 6CKine (1-79) that suppresses acceptor CCR7 effect;

M.XR1 antagonist 1, i.e. IL-8 (6-72) (seeing protein sequence 9.1,9.2 and genes encoding ND.9);

N.R2 antagonist 1, i.e. MCP-3 (9-76) (seeing protein sequence 10.1,10.2 and genes encoding ND.11);

O.R2 antagonist 2, i.e. MCP-3 (8-76) (seeing protein sequence 11.1,11.2 and genes encoding ND.10);

P. N-terminal head nine amino acid obtains through chemically modified, has the MCP-3 derivative that suppresses acceptor CCR2 effect;

Q.R2 antagonist 3, i.e. MCP-1 (9-76) (seeing protein sequence 12.1,12.2 and genes encoding ND.13);

R.R2 antagonist 4, i.e. MCP-1 (8-76) (seeing protein sequence 13.1,13.2 and genes encoding ND.12);

S. 9 amino acid of N-terminal head obtain through chemically modified, have the derivative of the MCP-1 that suppresses acceptor CCR2 effect;

T.XR2 antagonist 1, i.e. GROa (8-73) (seeing protein sequence 14.1,14.2 and genes encoding ND.14);

U. 7 amino acid of N-terminal head obtain through chemically modified, have the derivative of the GRO α that suppresses acceptor CXCR2 effect; The mode that described two kinds of different fusion rotein monomers are connected each other preferably by disulphide bridges, also can be passed through covalent linkage such as polypeptide chain, or polysaccharide molecule; Described two kinds of continuous different fusion rotein monomers comprise between two kinds of different fusion rotein monomers that contain heavy chain, or contain heavy chain and contain between the fusion rotein monomer of light chain.

The chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum, wherein human immunoglobulin's CH includes hinge region and CH3 fragment; Described human immunoglobulin's CH includes hinge region and CH1, CH2 and CH3 fragment; Human immunoglobulin's CH comprises the CH of IgG γ 1 and IgG γ 4; The constant region fragment of described light chain comprises the constant region of the κ and the λ of human immunoglobulin's light chain.

The purposes of the chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum, wherein the chimeric protein of two or more Chemokine Receptors of antagonism can be used for treating repulsion and graft versus host disease (GVH disease), treatment HIV-1 virus infection and the acquired immune deficiency syndrome (AIDS) that autoimmune disease and nonspecific inflammation disease, treatment tumour relative disease, treatment/prevention of organ transplant cause; The chimeric protein of described two or more Chemokine Receptors of antagonism and other method or medicine are done combination therapy.

The purposes of the chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum, wherein the using dosage during treatment/prevention is 3-300 milligram/kg body weight scope; Intramuscular injection dosage should be the several times of this dosage.

The production method of the chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum, the method of chimeric protein that wherein can two or more Chemokine Receptors of antagonism comprises: utilize gene recombination technology to make up and include the not monomeric carrier of chimeric protein of Chemokine Receptors of the same race of antagonism; Utilize above-mentioned different carrier, set up to express and the cell strain or the bacterial strain of the chimeric protein that secretion can two or more Chemokine Receptors of antagonism; Cultivate above-mentioned cell or bacterial strain, collect and the purifying secreted chimeric protein that goes out.

The production method of the chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum wherein can the antagonism chemokine receptor CCR 5, CCR1, CCR2, CCR3, the chimeric protein XR4/R5/R1 of CXCR4, its method comprises: utilize gene recombination technology to make up according to money 1 indication and include nucleotide sequence ND.1, ND.2, ND.3, ND.4 or expressing protein are equivalent to sequence (1), (2), (3), the monomeric carrier of the chimeric protein of (4); Utilize above-mentioned different carrier, set up to express and secretion can the antagonism chemokine receptor CCR 5, CCR1, CCR2, CCR3, the cell strain of the chimeric protein of CXCR4; Cultivate above-mentioned cell, collect and the purifying secreted chimeric protein that goes out.

The purposes of the chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum, wherein the chimeric protein XR4/R5/R1 autoimmune disease that is used for the treatment of/prevents HIV virus infection, acquired immune deficiency syndrome (AIDS) and relate to above-mentioned acceptor; Described chimeric protein XR4/R5/R1 is used for doing combination therapy with other method or medicine; Described chimeric protein XR4/R5/R1 and nucleic acid encoding thereof are used for the vaccine therapy method.

The purposes of the chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum, chimeric protein XR4/R5/R1 wherein is used for the treatment of/and using dosage when preventing is 3-300 milligram/kg body weight; Intramuscular injection dosage is the several times of above-mentioned dosage; Use formulation to comprise injection type and mucous membrane local application formulation; The gene therapy method of described chimeric protein XR4/R5/R1 nucleic acid encoding and dosage are 0.4-4 milligram/kg body weight.

The production method of the chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum, wherein can antagonism Chemokine Receptors CCR1, CCR5, the method of the chimeric protein XR3/R5/R1 of CXCR3 comprises: utilize gene recombination technology to make up and include nucleotide sequence ND.3, ND.4 ND.5, ND.6, or their corresponding proteins sequences (3), (4), (5), the monomeric carrier of the chimeric protein of (6); Utilize above-mentioned different carrier, set up to express and secretion can the antagonism chemokine receptor CCR 5, CCR1, the cell strain of the chimeric protein of CXCR3; Cultivate above-mentioned cell, collect and the purifying secreted chimeric protein that goes out.

The purposes of the chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum, wherein chimeric protein XR3/R5/R1 is used for the treatment of/repulsion that prevention of organ transplant causes, graft versus host disease (GVH disease) and the autoimmune disease that relates to above-mentioned acceptor; Described chimeric protein XR3/R5/R1 is used for doing combination therapy with other method or medicine; The vaccine therapy method of chimeric protein XR3/R5/R1 and nucleic acid encoding thereof.

The purposes of the chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum, wherein chimeric protein XR3/R5/R1 be used for the treatment of/using dosage when preventing is 3-300 milligram/kg body weight scope, intramuscular injection, dosage should be the several times of this dosage; Described chimeric protein XR3/R5/R1 is used for the treatment of/use formulation when preventing, comprise injection type; The gene therapy method of the nucleic acid encoding of described various chimeric proteins and dosage are 0.4-4 milligram/kg body weight.

The production method of the chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum, wherein can antagonism Chemokine Receptors CCR7, the method of the chimeric protein R7/XR4 of CXCR4 comprises: utilize gene recombination technology to make up and include nucleotide sequence ND.1, ND.2, ND.7, ND.8, or their corresponding proteins sequences (1), (2), (7), the monomeric carrier of the chimeric protein of (8); . utilize above-mentioned different carrier, set up to express and secretion can antagonism Chemokine Receptors CCR7, the cell strain of the chimeric protein of CXCR4; . cultivate above-mentioned cell, collect and the purifying secreted chimeric protein that goes out.

The purposes of the chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum, wherein chimeric protein R7/XR4 is used for the treatment of tumour and treatment and prophylaxis of tumours transfer, and the autoimmune disease that relates to above-mentioned acceptor; Described chimeric protein R7/XR4 is used for doing combination therapy with other method or medicine; The vaccine therapy method of described chimeric protein R7/XR4 and nucleic acid encoding thereof.

The purposes of the chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum, wherein chimeric protein R7/XR4 be used for the treatment of/using dosage when preventing is 3-300 milligram/kg body weight; Intramuscular injection dosage is the several times of this dosage; Described chimeric protein R7/XR4 is used for the treatment of/and use formulation when preventing comprises injection type; The gene therapy method of the nucleic acid encoding of described various chimeric proteins and dosage are 0.4-4 milligram/kg body weight.

The production method of the chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum wherein can antagonism Chemokine Receptors CCR2, CXCR1, the method of the chimeric protein XR1/R2 of CXCR2 comprises: utilize gene recombination technology to make up and include nucleotide sequence ND.9, ND.10, ND.11, ND.12, ND.13, No.14 or their corresponding proteins sequences (9), (10), (11), the monomeric carrier of chimeric protein of (12) (13) (14); Utilize above-mentioned different carrier, set up to express and secretion can antagonism Chemokine Receptors CCR2, the cell strain of the chimeric protein of CXCR1/CXCR2 or bacterial strain; Cultivate above-mentioned cell or bacterial strain, collect and the purifying secreted chimeric protein that goes out.

The purposes of the chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum, wherein chimeric protein XR1/R2 is used for the treatment of autoimmune disorder and non-special inflammation; Described chimeric protein XR1/R21 is used for doing combination therapy with other method or medicine; The vaccine therapy method of described chimeric protein R2/XR1 and nucleic acid encoding thereof.

The purposes of the chemotactic factor receptor inhibiting matter of aforesaid long-acting, broad-spectrum, wherein chimeric protein XR1/R2 be used for the treatment of/using dosage when preventing is 3-300 milligram/kg body weight scope; Intramuscular injection dosage is the several times of this dosage; Described chimeric protein XR1/R2 is used for the treatment of/use formulation when preventing, comprise intestinal absorption formulation, injection type; The gene therapy method of the nucleic acid encoding of described various chimeric proteins and dosage are 0.4-4 milligram/kg body weight.

Four, description of drawings

Figure 1A to Fig. 1 E is the composition mode synoptic diagram of chimeric protein compound of the present invention.

Fig. 2 is that the present invention secretes with anti-people source immune globulin antibody and combines the titration curve diagrammatic sketch.

Fig. 3 A is the chemokine quantity per-cent diagrammatic sketch of the present invention and receptors bind.

Fig. 3 B is the chemokine per-cent diagrammatic sketch of the present invention and receptors bind.

Fig. 4 is the per-cent diagrammatic sketch that inducing cell of the present invention moves quantity.

Fig. 5 is the chemokine quantity per-cent diagrammatic sketch of the present invention and receptors bind.

Fig. 6 A is the per-cent diagrammatic sketch that chemokine of the present invention is induced mobile cell quantity.

Fig. 6 B is the per-cent diagrammatic sketch that inducing tumor cell of the present invention moves.

Fig. 7 is the chemokine quantity per-cent diagrammatic sketch of the present invention and receptors bind.

Fig. 8 is the per-cent diagrammatic sketch that chemokine of the present invention is induced mobile cell quantity.

Fig. 9 is the chemokine quantity per-cent diagrammatic sketch of the present invention and receptors bind.

Figure 10 is the per-cent diagrammatic sketch that chemokine of the present invention is induced mobile cell quantity.

Five, embodiment

The present invention design and preparation be that some contain the chimeric protein compound at the multiple different efficient antagonist of Chemokine Receptors.These contain at the fused protein monomer of different Chemokine Receptors antagonists each other covalency link to each other, can amphiploid, tetraploid, or polymeric form output.Figure 1A-one E has illustrated the composition mode of these chimeric protein compounds.

Specifically, above-mentioned Chemokine Receptors antagonist partly includes, but is not limited to the ITAC that N-terminal is modified, the SDF-1 that N-terminal is modified, the RANTES that N-terminal is modified, the MCP-1 that N-terminal is modified, the MCP-3 that N-terminal is modified, the MIP-3 β that N-terminal is modified, a GRO and a friendly fragment of 6Ckine that N-terminal is modified, Deng the polypeptide compound of the no chemokine function of suddenling change, and their derivative or fragment.Relevant sequence sees Appendix 1.Analogy, the antagonist of chimeric protein XR4/R5/R1 partly comprises protein sequence 1 (or sequence 2) and sequence 3 (or 4); Chimeric protein XR3/R5/R1 antagonist partly comprises protein sequence 3 (or 4) and protein sequence 5 (or 6); Chimeric protein R7/XR4 antagonist partly includes protein sequence 1 (or 2) and sequence 7 (or 8); Chimeric protein XR1/R2 comprises protein sequence 9 (or sequence 14) and sequence 10 (or 11 or 12 or 13)

It is the constant zone of the heavy chain and the light chain of people source immunoglobulin (Ig) that above-mentioned and receptor antagonist merge part mutually.As required, merging different receptor antagonist and CH, mainly is IgG1, IgG4, the constant region of IgG2 (its sequence sees Appendix 1), or in the constant region of light chain any one.In addition, also can utilize the constant region sequence (seeing world's gene pool and EMBL database) of other immunoglobulin (Ig).Since can conjugated complement with the chimeric compound of IgG1, cause the ADCC reaction, thereby can kill and wound and its bonded target cell.On the contrary, the compound debond complement chimeric with IgG4 can not cause the ADCC reaction.Fusion is carried out on gene level, contains at linking to each other with covalency between the fusion rotein monomer of different receptor antagonists.Mode of connection comprises disulphide bridges, polypeptide, structures such as organic compound.Here can be to link to each other between two heavy chains, also link to each other between heavy chain and the light chain.Product can be an amphiploid, tetraploid, or polymeric form.

Functional character of the present invention is the proteinic fragment that chimeric protein all adopts the people source, can not cause tangible anaphylaxis in vivo.Every kind of chimeric protein all has significant inhibitory effect to two kinds or above different Chemokine Receptors.The acceptor here includes, but is not limited to Chemokine Receptors CCR1, CCR2, CCR3, CCR5, CCR7, CXCR1, CXCR2, CXCR3, CXCR4.Significant inhibitory effect refers to these chimeric proteins itself and does not have activity, but can combine closely (having high-affinity) activity of chemokine inhibiting by occupying acceptor with corresponding acceptor separately.The activity of chemokine refers to the blood leucocyte that they can induce human body herein, mainly comprises the T-lymphocyte, the B-lymphocyte, and monocyte, eosinophilic granulocyte, neutrophil leucocytes etc., or some tumour cell are such as moving of breast cancer cell.In addition, the stimulation of chemokine also has the intracellular calcium concentration of making to be increased, and/or the functions such as release of some intracellular enzymes.Different chemokines is induced the moving of T-cell such as IP-10 by the CXCR3 acceptor of T-lymphocytic cell surface by inducing the target cell generation of carrying acceptor to move with specific receptors bind and stimulation.For example, chimeric protein XR4/R5/R1 suppresses CCR5, CCR1, CCR3, CCR2 and CXCR4 acceptor; Chimeric protein XR3/R5/R1 suppresses CXCR3, CCR1 and CCR5 acceptor; Chimeric protein XR4/R7 suppresses CCR7 and CXCR4 acceptor, and chimeric protein XR1/R2 suppresses CCR2 and CXCR1, CXCR2 acceptor.If with the binding ability that the receptor antagonist of the good chimeric protein of purifying and equal gram molecular weight comes comparison and acceptor, then chimeric protein XR4 R5/R1 is than the high about 1.5-3 of binding ability times of not chimeric antagonist.

Another feature of above-mentioned chimeric protein is all obviously to improve than receptor antagonist itself in animal body half life, and the multiple of raising can reach more than hundred times.Illustration 12 shows chimeric protein XR4/R5/R1 through intravenous injection herein, and the half life in rabbit blood plasma is about 45 hours, and half life (0.25 hour) is grown nearly 180 times in vivo than not chimeric receptor antagonist.The half life of IgG1 antibody in rabbit blood plasma of known person, be about 100 hours, but intravital half life reach 21 days the people (because blood of human body recycle ratio rabbit slowly many).Thereby chimeric protein of the present invention should be near the half life (people's IgG antibody 1 is 21 days intravital half life the people) of people's IgG antibody 1 in people intravital half life.

The preparation method of the chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum of the present invention is that above-mentioned many targets property CFI chimeric protein can utilize gene recombination method to make up.Because above-mentioned receptor antagonist all is the non-functional chemokine derivative of transgenation, can utilize the RT-PCR method gene fragment of the various chemokines of clone earlier by the nucleic acid library of human body according to the paper of having delivered, utilize clone technology and transgenation technology that the gene order of these chemokines is transformed again.

About the heavy chain of the people source immunoglobulin (Ig) that wherein merges mutually with above-mentioned receptor antagonist and the constant area part of light chain, its gene order can obtain by the RT-PCR method from the nucleic acid library of human body according to the academic paper of having delivered.The gene fragment 5 of this heavy chain or light chain ' is held with above-mentioned receptor antagonist gene fragment 3 ' end branch and is connected to form fused protein.

In addition, the invention provides to expressing the necessary expression vector of above-mentioned chimeric protein.Expression vector can utilize plasmid vector, shown in embodiment 1, and also virus vector, or other general carrier.Have promotor (as the CMV promotor) in the carrier, and enhanser (as the EU enhanser) or the like important gene regulation and control fragment.The certain carrier that also can pack into and use as gene therapy, in order to screen conveniently, carrier also can be put into the impedance gene at different antibiotic, as the anti-Neomycin of utilization herein and the gene of anti-Puromycin.

The present invention also provides to expressing the necessary host of above-mentioned chimeric protein gene.The expression vector that above-mentioned two or three that build contain different antigen-4 fusion protein genes is imported into simultaneously in the same appropriate host by electric shock or other method and expresses.The host here preferably uses eukaryotic cell, and the Chinese hamster ovary celI of use is selected in analogy herein.Its strong point is that 1. to utilize CHO express to produce protein formulation be the most ripe and welcome in the world at present way.Big industrialized producing technology is ripe; 2.CHO can growing in serum-free medium, cell goes down to posterity.This makes whole process of production need not to use any animal preparation, thereby has avoided virus pollution; 3. produce protein technology with Chinese hamster ovary celI and obtained the U.S. FDA approval.The some kinds of protein formulations of producing with this method reach clinical criterion of acceptability fully.In addition, HEK293 cell in addition commonly used, the COS cell, NS/O or HELA cell are gone back insect cell, as the Rf9 cell, or yeast cell or the like; Prokaryotic cell prokaryocyte such as intestinal bacteria E.Coli etc. also can be used as these genes of host expresses.Different is that the chimeric protein that the latter produces can not be by saccharification.The cell strain of stably express obtains by the antibiotic impedance gene of screening vector.

The present invention also provides the way that how to improve product production.The way that improves MTX herein such as the utilization of using filters out the high expressing cell strain, uses for producing.In addition, GK enzyme gene can be received in the identical carrier of chimeric protein gene, output be improved by improving enzyme substrates.

The invention provides and how above-mentioned many targets property inhibitors of chemokine receptors chimeric protein is produced the method for expressing with the secretory protein form, both the N-terminal at protein gene added leader, and expressed products is secreted in the supernatant liquor of culturing cell.Leader is cut out in the protein secreting process.Annex 1 has provided the example of some leaders.This method benefit is that downstream egg white matter product purification is relatively easy.In addition, also can the endogenous protein formal representation.

Listed examples of the present invention shows that producing oozy chimeric protein output can reach 15 mg/litre supernatant/10 ⁹Cell every day.Also can further improve output by the screening cell clone.The present invention further provides the method that how will produce the separation and purification of excretory product albumen matter.What adopt is the partition method of affinity chromatography herein.Affinity chromatography can use a-protein, and it is coupled on carrier support, the immunoglobulin fragment specific combination of it and chimeric protein; Perhaps use the antibody of anti-chimeric protein receptor antagonist part, all can reach ideal separation and purification result.

To the change and the modification of the chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum of the present invention, following various to the patent thing change and modify and should be included within the present invention.

Basically is if be to strengthen treatment or prophylactic effect in order to reach to the change of above-mentioned patent thing and modification; Or in order to improve product at external stability (to the defensive ability/resistance ability of body endoproteinase); Or the modification that patent protein is done in the synthetic back of pirate recordings, as changing saccharification or phosphorylation composition,, both should be included within the present invention as long as the product that is derived by these variations remains with the principal character of this patent thing.

This modification comprises to be passed through to replace to the amino acid of patent thing or their nucleotide sequence, and increase or brachymemma come change, but the product that changes still keeps the principal character of patent thing.Can be replaced by Isoleucine or a word used in person's names propylhomoserin such as leucine, (or between basic aminoacids, between the neutral amino acids, between the polare Aminosaeren or between the nonpolar amino acid, between the aromatic amino acid) or the like can replace mutually between the acidic amino acid.It is generally acknowledged as long as product and segmental aminoterminal 40 amino acid of various proteic sequence, especially chemokine receptor anagonists of the present invention after changing, have more than 70% identically, just should belong within the scope of the invention.

In addition, patent thing protein or their nucleotide sequence also can add other compound and keep the principal character of patent thing, as alkali, thereby increase its solubility on its pendant chemical groups; Or isotropic substance, as cobalt 60, iodine 125, sulphur 35 or the like, or fluorescence reaching in the effect (biological missile) that kills and wounds target cell or the body tracer action to target cell (as tumour cell), thereby heighten the effect of a treatment or are applied to in-vivo diagnostic.In addition, patent thing protein or their nucleotide sequence also can with the protein or the chimeric hybridization of their nucleotide sequence of other function, as the toxin fragment, to reach the effect that kills and wounds target cell.These all are the methods that the reservation patent owner of widespread wants feature.

At last, as required, fusion can be different receptor antagonist respectively with following any one: the human igg heavy chain constant region mainly is (but being not limited to) IgG1, IgG4, the constant region of IgG2, or in the constant region of light chain.Analogy CCR2 receptor antagonist can be chimeric with the IgG1 constant region, or chimeric with the IgG4 constant region, or merge with the constant region of light chain.Here CH refers to and includes hinge region (hingeregion) and can add CH1, CH2 and CH3 fragment, hinge region adds the fragment of CH1 and CH2, or hinge region adds the fragment of CH2 and CH3.The κ that comprises human immunoglobulin's light chain of the constant region fragment indication of light chain or the constant region of λ.

The testing method of the chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum of the present invention.

Many targets property Chemokine Receptors of the present invention suppresses chimeric protein can be identified by following experimental technique: measure its Chemokine Receptors binding ability different with two or more and select the target cell that has acceptor to be measured (for example people's monocyte strain has the CCR1 acceptor, can be used as the target cell of CCR1; Also have the CCR2 acceptor, thereby also can be used as the target cell of CCR2).With the part chemokine (analogy is MIP-1 α at the part chemokine of CCR1 acceptor, and the part chemokine of the acceptor of CCR2 is MCP-1, and details see Table 1) that will measure acceptor with isotropic substance (such as iodine 125) mark in addition.Tagged ligand chemokine with fixed amount is incubated with being subjected to somatic target cell together with the chimeric protein to be measured of different amounts on the other hand.If chimeric protein and this receptors bind, then isotope-labeled part chemokine is subjected to the competition of chimeric protein, must reduce with the receptors bind amount.So identify by mensuration and different target cell bonded amounts whether chimeric protein to be measured competes bind receptor with their part chemokine, and ability (avidity) of competition.

Measuring chimeric protein can be with following any one way to the restraining effect of two or more different Chemokine Receptors: the part chemokine can induce it moved towards the chemokine direction by somatic target cell.By measuring the function that effect that pair cell moves can detect chimeric protein.Method is to get a saturating film test panel, and chemokine and chimeric protein to be measured are added in the hole below the test panel, then only adds in the upper surface hole to be subjected to somatic target cell.Behind the insulation certain hour, the film that induced by chemokine moved through between two holes by somatic target cell enters down in the face.If chimeric protein to be measured suppresses the function of acceptor, then part chemokine inducing action lowers, and the target cell quantity that causes moving reduces.

In addition, the part chemokine can also induce it improved by the interior calcium ion concn of somatic target cell.Method is be incubated with specific fluorescent reagent by somatic target cell, makes it enter cell.The stimulation of part chemokine can cause the activation of acceptor, thereby causes intracellular free calcium level to improve, and the moment that shows as the fluorescent absorption degree raises.Acceptor inhibitor itself is because can not activated receptor, thus there is not above-mentioned effect, but can suppress this effect of part chemokine.

Measure half life in the body.Measure chimeric protein to be measured and be expelled in the animal body after (or other method administration), the amount in blood is reduced to half needed time of injection volume.Details are seen embodiment 12.

The application of the chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum of the present invention:

Present invention includes these chimeric proteins or their gene fragment (DNA, application RNA).A). in clinical treatment, the protein of the lot of research proof chemokine of having delivered and acceptor thereof all play important effect to the pathologic process of numerous disease as medicinal application.Research finds that also many diseases all relate to multiple different Chemokine Receptors.The analogy HIV (human immunodeficiency virus) infects allosome organ transplant rejection, the transfer of some tumour cell, various autoimmune diseases, nonspecific inflammation disease (on seeing).Also have other numerous disease not enumerate one by one at this.Here Fa Ming chimeric protein owing to can suppress multiple different Chemokine Receptors efficiently, has overcome the diversity complicacy of disease pathology.Simultaneously,, can keep medicine effective concentration, so fundamentally overcome Chemokine Receptors constantly regeneration and the halfway puzzlement of inhibition that caused because chimeric protein half life in vivo is very long.More than two advantages obviously improved the clinical value of chimeric protein widely, be applicable to this class treatment of diseases and prevention.Concrete (but being not limited to) for example is as follows: it is human that the HIV-1 virus of acquired immune deficiency syndrome (AIDS) mainly utilizes CCR5 and CXCR4 Chemokine Receptors to infect, and in addition, many HIV-1 viruses also can be utilized CCR1, and acceptors such as CCR2, CCR3 infect.Thereby chimeric protein XR4/R5/R1 of the present invention can efficiently suppress above-mentioned all acceptors (embodiment 4, embodiment 5) because of it, thereby can suppress various dissimilar HIV-1 virus infectiones.In addition, the performance that chimeric protein XR4/R5/R1 is stable long-lived in vivo guarantees the keeping for a long time of intravital effective concentration (embodiment 10), really stops the infection of HIV-1 virus effectively.These characteristics of chimeric protein XR4/R5/R1 make it must become one of the most effective in the world at present prevention HIV-1 medicine for treating viral infections just.Can be used for the treatment and the prevention of acquired immune deficiency syndrome (AIDS), especially should be applicable to the woman that prevents AIDS the infection of fetus, or with HIV virus carrier's sexual intercourse after protect from infection remedy rapidly.

And for example, the breast cancer cell surface almost all has CXCR4 and two kinds of acceptors of CCR7, thereby has determined these cancer metastasis to marrow, lymphoglandula and lung, and be fixed up there, increase rapidly.Chimeric protein XR4/R7 of the present invention can suppress above-mentioned two kinds of acceptors (embodiment 6, embodiment 7) longer, so can suppress the transfer of breast cancer cell to these organs effectively.This uses the transfer (embodiment 6) that also is applicable to some other tumour cell.

And for example, the rejection of allosome tissue's organ transplantation resembles the repulsion of representative heart transplantation, and animal experiment shows it is owing to have CXCR3, CCR5, and the white corpuscle of CCR1 acceptor is in action.Chimeric protein XR3/R5/R1 of the present invention (embodiment 8 and embodiment 9) can be efficient, suppresses above-mentioned three kinds of acceptors (embodiment 10) longer, thus can be used for preventing being ostracised of histoorgan, and graft versus host disease (GVH disease).

And for example autoimmune disorder also has nonspecific inflammation, discovers that the principal element that causes pathological lesion is monocyte and neutrophil leucocyte (respectively by CCR2, CXCR1 and CXCR2 acceptor).Chimeric protein XR2/R2 of the present invention can efficiently suppress above-mentioned three kinds of acceptors (illustration 10 and 11), and drug effect is long in the body, so can suppress this two kinds of cells, mitigation symptoms effectively.B). as the medicine integral part

The present invention includes above-mentioned patent thing is prepared to different formulations and is used for the treatment of.Separately or with other composition, can be drug for injection as the formulation of medicine, oral drug or local application (comprise rectal administration, or the formulation of spray delivery such as nose spraying or mouthful inhale spraying etc.) various formulations.Illustration 12 is the examples as the solution intravenously administrable.

The present invention also comprises the effective dose that above-mentioned patent thing is used for the treatment of.Such as the chimeric protein intravenously administrable, according to the situation of disease, dosage is in 3-300 milligram/kg body weight scope; As intramuscular injection, dosage should be above-mentioned several times; Do gene therapy in this way, the dosage of plasmid vector is in 0.4-4 milligram/kg body weight; Or the like.C). be applied to gene therapy

Nucleotide sequence among the present invention can be used as gene therapy.These sequences can be installed in the different Vectors in Gene Therapy that is used for, such as known nacked dna vector or virus vector (such as adenovirus carrier or retroviral vector).Gene is imported in the body, reaches result of treatment by expressing voluntarily in vivo.The disease of treatment includes, but is not limited to acquired immune deficiency syndrome (AIDS), tumour and the organ rejection of allosome tissue, various autoimmune disorders.D). be applied to combination therapy with other method or medicine

Here Fa Ming chimeric protein also can be used for the combination therapy with other method or medicine.Analogy (but being not limited to) chimeric protein XR4/R7 can use with chemotherapy and radiotherapy, reaches and not only suppresses metastases, goes back the effect of kill tumor cell; Chimeric protein XR4/R5/R1 can unite use with drug cocktail therapy (treatment), suppresses the rate of increase of HIV-1 virus by multiple different effect channel, to reach better result of treatment; Chimeric protein XR3/R5/R1 can share with a small amount of steroid hormone, and better result of treatment is played in the repulsion of transplanting allosome tissue's organ; Chimeric protein XR1/R2 can share with a small amount of steroid hormone or other immunosuppressor, tackles stubbornness and serious autoimmune disorder etc. jointly.E). be applied to in-vivo diagnostic

Here Fa Ming chimeric protein also can be used for diagnosis.The analogy (but being not limited to) with chimeric protein through be used for the tracking that breast cancer cell shifts after the isotopic labeling in the patient body.

For the ease of understanding, below list some relevant specific embodiments of the invention to this invention.The gene recombination of embodiment 1 chimeric protein XR4/R5/R1

The cDNA of chemokine SDF-1 and RANTES (comprising leader) obtains from the RNA of people's tissue by the RT-PCR method.CDNA is cloned in the PCR2.1 carrier.Utilize of the genetic modification of transgenation method, form other nucleotide sequence chemokine.On the other hand, utilizing the cDNA of the CH of people source Immunoglobulin IgG4, reported sequence obtains from the cDNA storehouse of people's spleen, and this fragment is received (that usefulness is the pCR3.1 carrier pHC that transforms in the carrier of expression here, utilize CMV to be promotor, EU is an enhanser, and the neomycin insensitive gene is the screening sign).3 ' end of the sequence fragment of the RANTES sequence fragment that said gene is engineered and the SDF-1 of transformation is held with 5 ' of the CH1 section of the CH (IgG γ 4) of IgG4 respectively by the Restriction Enzyme point of contact and is connected.On the other hand, the cDNA fragment of the people source light chain immunoglobulin κ constant region of utilizing reported sequence to obtain from the cDNA storehouse of people's spleen is received (that usefulness is the pCR3.1 carrier pLC that transforms, and the Puromycin insensitive gene is the screening sign) in the expression vector here.

The direction that merges is 3 ' terminally being connected with 5 ' end of constant region for immunoglobulin of gene fragment (correspondingly, its expressed protein product must be that the N-terminal peptide bond in the constant zone of the heavy chain of C-terminal and immunoglobulin (Ig) by the receptor antagonist molecule and light chain is connected) (Fig. 3 B) of antagonist.

The gene recombination of embodiment 2 chimeric protein XR4/R7

The gene of chemokine MIP-3 β obtains from the cDNA storehouse of people's tissue by PCR method, and is cloned in the PCR2.1 carrier.Utilize the transgenation method that it is transformed into new nucleotide sequence.On the other hand, cDNA (utilizing, the reported sequence obtains) fragment of the CH of people source Immunoglobulin IgG1 is received (that usefulness is carrier pHC in the carrier of expression here from the cDNA storehouse of people's spleen, utilize CMV to be promotor, EU is an enhanser, and the neomycin insensitive gene is the screening sign).The sequence fragment of genetic engineering modified MIP-3 β and the sequence fragment 3 ' end of SDF-1 are connected by the enzyme point of contact with the 5 ' end of IgG γ 1 respectively.In addition, producer source light chain immunoglobulin κ constant region fragment expression carrier is with embodiment 1.Embodiment 3 expression of chimeric protein XR4/R5/R1 gene in eukaryotic cell CHO

To have above-mentioned CCR5 antagonist-IgG γ 4 sequences and have CXCR4 antagonist-IgG γ 4 sequences, and the DNA of three kinds of different expression vectors of constant region of light chain κ, import in the Chinese hamster ovary celI simultaneously after the linearizing and expressed.Process G418 (1 mg/ml), and the screening in Purimycin (3 mcg/ml) 3 weeks obtain stable expression cell strain.In order to obtain high yielding cell sarain, utilize the way that improves MTX to filter out the high expressing cell strain.The cell strain of screening is cultivated the culture supernatant of collecting cell after a day.

Identify by the ELISA method whether genetically modified cell synthesizes the justacrine chimeric protein.Particularly, the culture supernatant of collecting is combined test with the mouse-anti human IgG antibody who prepares.Fig. 2 shows that cloned genes has been inserted into Chinese hamster ovary celI gene and successful expression, and the synthetic chimeric protein is secreted in the nutrient solution, thereby can be the dose-dependent competition that combines with anti-human body IgG antibody.

By to different Chemokine Receptors in conjunction with the test (seeing embodiment 4) prove, wherein about 20% clone secrete chimeric protein respectively with multiple receptors bind such as CXCR4 and CCR5, illustrate that these chimeric proteins contain two kinds of different monomers.Enlarge and the several clones of freezing preservation.

The secretion supernatant is measured through separation and purification, and output is that per 106 cells are secreted 15-25 mcg/ml chimeric protein every day.The about 75kDa size of its main molecules adds that express cell is all insensitive to Puromycin and Neomycin, and the tetraploid that chimeric protein is made up of heavy chain and light chain chimeric protein is described.

Above-mentioned experimental result shows, no matter be form between two heavy chain chimeric proteins amphiploid, the still tetraploid of forming by heavy chain and light chain chimeric protein, both as the described structure of Figure 1A, but successful expression all.

In order to identify whether chimeric protein XR4/R5 has the function of acceptor inhibitor, done the evaluation of the chimeric protein XR4/R5/R1 feature that 4 couples of following two experiment: embodiment production expresses-altogether to the combination of different Chemokine Receptors.

Measure at first that the said gene engineering is made and excretory chimeric protein R5/XR4 whether can with CCR5, CCR1, CCR2, CCR3 and the ground combination of CXCR4 acceptor high affinity.Done the receptors bind competitive assay for this reason.People's monocyte surface has CCR1, CCR2, and the CCR5 acceptor, people's eosinophilic granulocyte surface has the CCR3 acceptor, and people's T-lymphocyte strain has the CXCR4 acceptor.These three cell strains are as the target cell of these acceptors.To contain the culture supernatant of chimeric protein XR4/R5/R1 and the isotropic substance I of fixed dosage ¹²⁵The part chemokine of mark mixes, and combines with these receptor competitions: wherein, MIP-1 α is the part chemokine of CCR1, MCP-1 is the part of CCR2, Eotaxin is the part of CCR3, and RANTES is the part of CCR5, and SDF-1 α then is the part of CXCR4 acceptor.Fig. 3 shows chimeric protein XR4/R5/R1 and the very strong bonded ability (50% cell conditioned medium can suppress combining of most chemokines) of above-mentioned acceptor performance.Utilize a-protein to do affinity chromatography, with the secretory protein separation and purification in the supernatant.Afterwards, with identical molconcentration relatively, chimeric protein XR4/R4 is high than the avidity of the antagonist that does not merge.The evaluation of the chimeric protein XR4/R5/R1 feature that 5 couples of embodiment production is expressed-to the inhibition of different chemokine receptor functions.

Utilize the cell migration inhibition test to detect the restraining effect of the chimeric protein XR4/R5/R1 of gene engineering making to acceptor.The part chemokine can induce the target cell that has their acceptors directed mobile.With people's monocyte, these three cell strains of people's eosinophilic granulocyte and T-lymphocyte strain are as the target cell of these acceptors.As shown in Figure 4, various part chemokines induce separately target cell to move (be decided to be mobile 100%) respectively.The adding that contains the chimeric protein XR4/R5/R1 culture supernatant of different concns is dosage and relies on shape ground and suppress moving of cell: by suppressing CCR1, and CCR2 and CCR5 acceptor and suppress α, MCP-1 and RANTES inductive THP-1 cell mobile by MIP-1; Suppress moving of Eotaxin inductive eosinophilic granulocyte by suppressing the CCR3 acceptor; Suppress moving of SDF-1 α inductive CEM-M3 cell by inhibition to the CXCR4 acceptor.Inhibition degree and chimeric protein XR4/R5/R1 match to the binding ability of acceptor.The evaluation of the chimeric protein XR4/R7 feature that 6 couples of embodiment production is expressed-to the combination of different Chemokine Receptors.Measure gene engineering making and excretory chimeric protein XR4/R7 whether can with CCR7, the combination of CXCR4 acceptor high affinity.Done the receptors bind competitive assay for this reason.People's T-lymphocyte strain (CXCR4 acceptor) and people's the T cell strain that has the CCR7 acceptor is as the target cell of acceptor.The culture supernatant that will contain chimeric protein XR4/R7 respectively with isotropic substance I ¹²⁵The part chemokine SDF-1 α of mark and 6Ckine or MIP-3 β mix, and combine with these acceptors do competitions.Fig. 5 shows the ability (50% cell conditioned medium can suppress combining of most chemokines) that chimeric protein XR4/R7 and above-mentioned acceptor are combined closely.If with identical molconcentration relatively, chimeric protein XR4/R7 is obviously higher than the avidity of the antagonist that does not merge.Embodiment 7 chimeric protein XR4/R7 suppress moving of breast carcinoma cell strain

Utilize two people's breast carcinoma cell strain T-47D and MDA-7 as target cell, tested the restraining effect that chimeric protein XR4/R7 moves breast cancer cell.As shown in Figure 6, the part chemokine 6CKine of CCR7 acceptor or MIP-3 β, and the part SDF-1 of CXCR4 induces move (result of T-47D only is shown) of these two breast carcinoma cell strains herein.The adding of culture supernatant that contains the chimeric protein XR4/R7 of different concns is dosage and relies on shape ground and suppress the mobile of SDF-1 α and 6CKine (or MIP-3 β) inductive breast cancer cell.Inhibition degree and chimeric protein XR4/R7 match to the binding ability of acceptor.The evaluation of the chimeric protein XR3/R5/R1 feature that 8 couples of embodiment production is expressed-to the combination of different Chemokine Receptors.Measure gene engineering making and excretory chimeric protein XR3/R5/R1 whether can with CCR1, CCR5 and the combination of CXCR3 acceptor high affinity.With the T cell of the activated human peripheral target cell as CXCR3 and CCR5, monocyte is as the target cell of CCR1 acceptor.The T cell that separation is obtained is incubated with cytokine interleukin element-2, makes the T cell obtain activating.The culture supernatant of activated T cells and monocyte and chimeric protein XR3/R5/R1, and respectively with isotropic substance I ¹²⁵The part chemokine IP-10 or the RANTES of mark, or MIP-1 α is mixed, and does competition with these acceptors and combining.Fig. 7 shows the ability (50% cell conditioned medium can suppress combining of most chemokines) that chimeric protein XR3/R5/R1 and above-mentioned acceptor are combined closely.The evaluation of the chimeric protein XR3/R5/R1 feature that 9 pairs of productions of embodiment are expressed-suppress target cell by inhibition to move to different Chemokine Receptors.

Utilize the target cell of the T cell of activated human peripheral as CXCR3 and CCR5, monocyte is as the target cell of CCR1 acceptor.The preparation of cell such as illustration 8.As shown in Figure 8, the part chemokine MIP-1 α of CCR1 acceptor, and the part RANTES of the ligand i P-10 of CXCR3 and CCR5 induces moving of above-mentioned target cell.The adding of culture supernatant that contains the chimeric protein XR3/R5/R1 of different concns is dosage and relies on shape ground and suppress IP-10, MIP-1 α or RANTES inductive target cell mobile.Inhibition degree and chimeric protein XR3/R5/R1 match to the binding ability of acceptor.The evaluation of the chimeric protein XR1/R2 feature that 10 couples of embodiment production is expressed-to the combination of different Chemokine Receptors.

Measure gene engineering making and excretory chimeric protein XR2/R2 whether can with CCR2, CXCR1 and the combination of CXCR2 acceptor high affinity.With the neutrophil leucocyte of the human peripheral target cell as CXCR1 and CXCR2, monocyte is as the target cell of CCR2 acceptor.Neutrophil leucocyte that separation is obtained and monocyte immediately with the culture supernatant of chimeric protein XR2/R2, and respectively with isotropic substance I ¹²⁵The part chemokine IL-8 of mark or GRO-α, or MCP-1 is mixed, and does competition with these acceptors and combining.Fig. 9 shows the ability (50% cell conditioned medium can suppress combining of most chemokines) that chimeric protein XR2/R2 and above-mentioned acceptor are combined closely.The evaluation of the chimeric protein XR1/R2 feature that 11 pairs of productions of embodiment are expressed-suppress target cell by inhibition to move to different Chemokine Receptors.Utilize the target cell of the neutrophil leucocyte of human peripheral blood as CXCR1 and CXCR2 acceptor, monocyte has been tested chimeric protein XR1/R2 to CCR2 and CXCR1 as the target cell of CCR2 acceptor, the restraining effect that the receptor-mediated cell of CXCR2 moves.As shown in figure 10, the part chemokine MCP-1 of CCR2 acceptor, and the part GRO α of the ligand i L-8 of CXCR1 and CXCR2 induces moving of above-mentioned target cell.The adding of culture supernatant that contains the chimeric protein XR1/R2 of different concns is dosage and relies on shape ground and suppress IL-8, GRO α or MCP-1 inductive target cell mobile.Inhibition degree and chimeric protein XR1/R2 match to the binding ability of acceptor.Embodiment 12 chimeric protein R5/XR4 medicine in animal body is for dynamic experiment

50 microgram purifying are also used I ¹²⁵The chimeric protein XR4/R5/R1 (diploid) of mark, chimeric protein XR4/R5/R1 (tetraploid) or the antagonist protein matter that does not merge are squeezed into respectively in the rabbit body of 2 kilograms of body weight by left ear vein.Got 5-10 milliliter blood every 10 minutes from the auris dextra vein of rabbit.By measuring the content of people's heavy chain immunoglobulin in the isotope detection blood.Because it is very not fast that the antagonist protein matter that merges disappears, and chimeric protein is stable at blood level, since the and day, change into taking out from the latter and get sample one time every 1-2 days.Shown in following table 2, I ¹²⁵The half life of non-fusion antagonist protein in the rabbit peripheral blood of mark, only have about 15 minutes, and I ¹²⁵The half life of the chimeric protein XR4/R5/R1 (diploid) of mark, be approximately 45 hours, I ¹²⁵The half life of the chimeric protein XR4/R5/R1 (tetraploid) of mark, be approximately 110 hours, be the antagonist protein matter that do not merge about 180-400 doubly.

The definition that relates among the present invention

1. chemokine: an extended familys protein that exists naturally in the body, they induce various white corpuscles directed mobile specifically.Some of them participate in inflammatory reaction, and other are responsible for white corpuscle and locate in vivo.Each chemokine all has own corresponding acceptor.Particular case sees Table 1.

2. Chemokine Receptors antagonist (abbreviation receptor antagonist): can combine with Chemokine Receptors, itself does not have function, but the but compound of chemokine inhibiting function.What here, receptor antagonist referred to all is the non-functional chemokine derivative of some sudden changes.

3. people source immunoglobulin (Ig): be divided into and be IgG, IgA, IgM, IgD, the big class of IgE5.Wherein IgG is divided into IgG1 again, IgG2, and IgG3, the IgG4 subclass, (being made up of heavy chain and light chain), their heavy chain is by called after IgG γ 1 correspondingly, and IgG γ 2, IgG γ 3, IgG γ 4, light chain then have two kinds of κ or λ.

4. many targets property CFI: the compound that can suppress at least two different Chemokine Receptors.

5. fusion rotein: the gene segment of two different proteins is connected, and its expression product is a single chain protein matter that is connected with peptide bond each other by two different proteins.

6. chimeric protein compound (abbreviation chimeric protein): two different proteinic gene fragments are expressed in same host, and what produce is two different protein monomers, covalently bound more each other product.

7. part chemokine: be called as the part chemokine (table 1) of this acceptor with the chemokine that certain receptor-specific combined and produced biological effect

Part chemokine that table 1 is relevant with this paper and corresponding acceptor

The name of the international novel receptor title of the habitual chemokine of chemokine name

MCP-1????????????????CCL2????????????????????????CCR2

MCP-3????????????????CCL7????????????????????????CCR2

MIP-1α??????????????????????????????CCL3????????????????????????CCR1

RANTES???????????????CCL5????????????????????????CCR5

TP-10????????????????CXCL10??????????????????????CXCR3

I-TAC?????????????????CXCL11???????????????CXCR3

Eotaxin???????????????CCL11????????????????CCR3

MIP-3β????????????????????????????????CCL19????????????????CCR7

6CKine????????????????CCL21????????????????CCR7

SDF-1?????????????????CXCL12???????????????CXCR4

IL-8??????????????????CXCL8????????????????CXCR1/CXCR2

GROα????????????????????????????????????CXCL1????????????????CXCR2

Table 2

Some feature of chimeric protein

Title	The structure of unit	Half life in rabbit plasma (hour)	Combine with complement	Combine with a-protein
Title	The structure of unit	Half life in rabbit plasma (hour)	Combine with complement	Combine with a-protein	Chemokine protein matter	Non-chimeric protein	????0.25	Debond	Debond
The chimeric protein amphiploid	Heavy chain ₂	????45	Debond	In conjunction with	Chemokine protein matter	Non-chimeric protein	????0.25	Debond	Debond
The chimeric protein amphiploid	Heavy chain ₂	????45	Debond	In conjunction with	The chimeric protein tetraploid	Heavy chain ₂Light chain ₂	????110	In conjunction with	In conjunction with

The feature chemical structure characteristic of the chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum of the present invention

Part above-mentioned and that receptor antagonist merges mutually is the constant zone of the heavy chain and the light chain of people source immunoglobulin (Ig).As required, fusion can be different receptor antagonist and CH, mainly is IgG1, IgG4, the constant region of IgG2 (its sequence sees Appendix 1), or in the constant region of light chain any one.In addition, also can utilize the constant region sequence (seeing world's gene pool and EMBL database) of other immunoglobulin (Ig).Since can conjugated complement with the chimeric compound of IgG1, cause the ADCC reaction, thereby can kill and wound and its bonded target cell.On the contrary, with the chimeric compound of IgG4 debond complement then, can not cause the ADCC reaction.Fusion is carried out on gene level.Contain at linking to each other with covalency between the fusion rotein monomer of different receptor antagonists.Mode of connection comprises disulphide bridges, polypeptide, structures such as organic compound.Here can be to link to each other between two heavy chains, also link to each other between heavy chain and the light chain.Product can be an amphiploid, tetraploid, or polymeric form.Functional character

Because chimeric protein all adopts people's source protein matter fragment, can not cause tangible anaphylaxis in vivo.

Every kind of chimeric protein all has significant inhibitory effect to two kinds or above different Chemokine Receptors.The acceptor here includes, but is not limited to Chemokine Receptors CCR1, CCR2, CCR3, CCR5, CCR7, CXCR1, CXCR2, CXCR3, CXCR4.Significant inhibitory effect refers to these chimeric proteins itself and does not have activity, but can combine closely (having high-affinity) activity of chemokine inhibiting by occupying acceptor with corresponding acceptor separately.The activity of chemokine refers to the blood leucocyte that they can induce human body herein, mainly comprises the T-lymphocyte, the B-lymphocyte, and monocyte, eosinophilic granulocyte, neutrophil leucocytes etc., or some tumour cell are such as moving of breast cancer cell.In addition, the stimulation of chemokine also has the intracellular calcium concentration of making to be increased, and/or the functions such as release of some intracellular enzymes.Different chemokines is induced the moving of T-cell such as IP-10 by the CXCR3 acceptor of T-lymphocytic cell surface by inducing the target cell generation of carrying acceptor to move with specific receptors bind and stimulation.For example, chimeric protein XR4/R5/R1 suppresses CCR5, CCR1, CCR3, CCR2 and CXCR4 acceptor; Chimeric protein XR3/R5/R1 suppresses CXCR3, CCR1 and CCR5 acceptor; Chimeric protein XR4/R7 suppresses CCR7 and CXCR4 acceptor, and chimeric protein XR1/R2 suppresses CCR2 and CXCR1, CXCR2 acceptor.

If with the binding ability that the receptor antagonist of the good chimeric protein of purifying and equal gram molecular weight comes comparison and acceptor, then chimeric protein XR4 R5/R1 is than the high about 1.5-3 of binding ability times of not chimeric antagonist.

The sequence of the chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum of the present invention (annex 1, illustration is not limited only to this)

Sequence Table <110> National Food Research Institute <120> DNA level rice palatability evaluation methods, and by half a unhulled / No Unpolished rice analytical methods to choose tasty rice <130> P131218K <160> 92 <210> 1 <211> 30 <212> DNA <213> Artificial Sequence <400> 1 ccagctgaac gcctgtacta caagaattaa 30 <210> 2 <211> 30 <212> DNA <213> Artificial Sequence <400> 2 ccagctgtac gtcttcccca gcgccggcgg 30 <210> 3 <211> 21 <212> DNA <213> Artificial Sequence <400> 3 ccagctgtac gcctgtacta c 21 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <400> 4 ccagctgtac gtcttcccca gc 22 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <400> 5 tgcctcgcac cagaaatagt ataatcccaa 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <400> 6 tgcctcgcac catgaggtgt ggccgagtac 30 <210> 7 <211> 19 <212> DNA <213> Artificial Sequence <400> 7 tgcctcgcac cagaaatag 19 <210> 8 <211> 16 <212> DNA <213> Artificial Sequence <400> 8 tgcctcgcac catgag 16 <210> 9 <211> 30 <212> DNA <213> Artificial Sequence <400> 9 gtttcgctcc tacagtaatt aaggggctat 30 <210> 10 <211> 30 <212> DNA <213> Artificial Sequence <400> 10 gtttcgctcc catgcaatct gcaaaagttt 30 <210> 11 <211> 25 <212> DNA <213> Artificial Sequence <400> 11 gtttcgctcc tacagtaatt aaggg 25 <210> 12 <211> 20 <212> DNA <213> Artificial Sequence <400> 12 gtttcgctcc catgcaatct 20 <210> 13 <211> 30 <212> DNA <213> Artificial Sequence <400> 13 caggtgtggg ttacaaggat gacccttggg 30 <210> 14 <211> 30 <212> DNA <213> Artificial Sequence <400> 14 caggtgtggg ttcacggcct tgattaataa 30 <210> 15 <211> 22 <212> DNA <213> Artificial Sequence <400> 15 caggtgtggg ttacaaggat ga 22 <210> 16 <211> 17 <212> DNA <213> Artificial Sequence <400> 16 caggtgtggg ttcacgg 17 <210> 17 <211> 30 <212> DNA <213> Artificial Sequence <400> 17 ccacagcagt gcttcatgtc atgtagaata 30 <210> 18 <211> 30 <212> DNA <213> Artificial Sequence <400> 18 ccacagcagt tcaaatacac caggaatttc 30 <210> 19 <211> 15 <212> DNA <213> Artificial Sequence <400> 19 ccacagcagt gcttc 15 <210> 20 <211> 21 <212> DNA <213> Artificial Sequence <400> 20 ccacagcagt gcttcatgtc a 21 <210> 21 <211> 30 <212> DNA <213> Artificial Sequence <400> 21 tggccggcat gactcacata cccaacatat 30 <210> 22 <211> 30 <212> DNA <213> Artificial Sequence <400> 22 actggccggc atcaagacca accaatttgg 30 <210> 23 <211> 17 <212> DNA <213> Artificial Sequence <400> 23 tggccggcat gactcac 17 <210> 24 <211> 18 <212> DNA <213> Artificial Sequence <400> 24 actggccggc atcaagac 18 <210> 25 <211> 30 <212> DNA <213> Artificial Sequence <400> 25 ggaatggaac cgaagtggag ctattccctg 30 <210> 26 <211> 30 <212> DNA <213> Artificial Sequence <400> 26 ggaatggaac cgccgtaaac ttgaatgcta 30 <210> 27 <211> 20 <212> DNA <213> Artificial Sequence <400> 27 ggaatggaac cgaagtggag 20 <210> 28 <211> 21 <212> DNA <213> Artificial Sequence <400> 28 ggaatggaac cgccgtaaac t 21 <210> 29 <211> 30 <212> DNA <213> Artificial Sequence <400> 29 tacctggttg atgtatacag atctggttat 30 <210> 30 <211> 30 <212> DNA <213> Artificial Sequence <400> 30 atccctcgat ccctctagca ttatatcctc 30 <210> 31 <211> 28 <212> DNA <213> Artificial Sequence <400> 31 tacctggttg atgtatacag atctggtt 28 <210> 32 <211> 24 <212> DNA <213> Artificial Sequence <400> 32 atccctcgat ccctctagca ttat 24 <210> 33 <211> 30 <212> DNA <213> Artificial Sequence <400> 33 accactccat atatatcatc caaagttcta 30 <210> 34 <211> 30 <212> DNA <213> Artificial Sequence <400> 34 accactccat atcaccacaa ggcgtttagg 30 <210> 35 <211> 25 <212> DNA <213> Artificial Sequence <400> 35 accactccat atatatcatc caaag 25 <210> 36 <211> 22 <212> DNA <213> Artificial Sequence <400> 36 accactccat atcaccacaa gg 22 <210> 37 <211> 30 <212> DNA <213> Artificial Sequence <400> 37 gagaccgata tgcgattcgc ggcattggac 30 <210> 38 <211> 30 <212> DNA <213> Artificial Sequence <400> 38 gtggtgttta gatccagaga cttaacttta 30 <210> 39 <211> 18 <212> DNA <213> Artificial Sequence <400> 39 gagaccgata tgcgattc 18 <210> 40 <211> 24 <212> DNA <213> Artificial Sequence <400> 40 gtggtgttta gatccagaga ctta 24 <210> 41 <211> 30 <212> DNA <213> Artificial Sequence <400> 41 ctcactcaaa tttacagtgc attttcttgt 30 <210> 42 <211> 30 <212> DNA <213> Artificial Sequence <400> 42 agggccatga tacaagactc tgttctgtag 30 <210> 43 <211> 27 <212> DNA <213> Artificial Sequence <400> 43 ctcactcaaa tttacagtgc attttct 27 <210> 44 <211> 23 <212> DNA <213> Artificial Sequence <400> 44 agggccatga tacaagactc tgt 23 <210> 45 <211> 30 <212> DNA <213> Artificial Sequence <400> 45 ggccgtcgtt ctgcgatggt ctccaagaat 30 <210> 46 <211> 30 <212> DNA <213> Artificial Sequence <400> 46 ggagaatccc acagtaagtt tttctttgtt 30 <210> 47 <211> 17 <212> DNA <213> Artificial Sequence <400> 47 ggcggtcgtt ctgcgat 17 <210> 48 <211> 28 <212> DNA <213> Artificial Sequence <400> 48 ggagaatccc acagtaagtt tttctttg 28 <210> 49 <211> 30 <212> DNA <213> Artificial Sequence <400> 49 gtcggagtgg tcagaccggg ctagcttttg 30 <210> 50 <211> 30 <212> DNA <213> Artificial Sequence <400> 50 gtcggagtgg atggagtagc ggtgggtgtg 30 <210> 51 <211> 18 <212> DNA <213> Artificial Sequence <400> 51 gtcggagtgg tcagaccg 18 <210> 52 <211> 20 <212> DNA <213> Artificial Sequence <400> 52 gtcggagtgg atggagtagc 20 <210> 53 <211> 30 <212> DNA <213> Artificial Sequence <400> 53 acaacgcctc cgatgatcga accatatctt 30 <210> 54 <211> 30 <212> DNA <213> Artificial Sequence <400> 54 acaacgcctc cgacaacaag attttctcct 30 <210> 55 <211> 16 <212> DNA <213> Artificial Sequence <400> 55 acaacgcctc cgatga 16 <210> 56 <211> 15 <212> DNA <213> Artificial Sequence <400> 56 acaacgcctc cgaca 15 <210> 57 <211> 30 <212> DNA <213> Artificial Sequence <400> 57 gtccactgtg accacaacat ttcttccagc 30 <210> 58 <211> 30 <212> DNA <213> Artificial Sequence <400> 58 gtccactgtg gggattgttc cataaaagat 30 <210> 59 <211> 20 <212> DNA <213> Artificial Sequence <400> 59 gtccactgtg accacaacat 20 <210> 60 <211> 20 <212> DNA <213> Artificial Sequence <400> 60 gtccactgtg gggattgttt 20 <210> 61 <211> 30 <212> DNA <213> Artificial Sequence <400> 61 aacgggccaa aaacggaggt cgtatggagc 30 <210> 62 <211> 30 <212> DNA <213> Artificial Sequence <400> 62 aacgggccaa cgcagccatt aaagagaaat 30 <210> 63 <211> 20 <212> DNA <213> Artificial Sequence <400> 63 aacgggccaa aaacggaggt 20 <210> 64 <211> 15 <212> DNA <213> Artificial Sequence <400> 64 aacgggccaa cgcag 15 <210> 65 <211> 30 <212> DNA <213> Artificial Sequence <400> 65 acaacggtcc gtccttgctt aggaaaaggc 30 <210> 66 <211> 30 <212> DNA <213> Artificial Sequence <400> 66 acaacggtcc aacagatact tttgaaaaac 30 <210> 67 <211> 20 <212> DNA <213> Artificial Sequence <400> 67 acaacggtcc gtccttgctt 20 <210> 68 <211> 25 <212> DNA <213> Artificial Sequence <400> 68 acaacggtcc aacagatact tttga 25 <210> 69 <211> 30 <212> DNA <213> Artificial Sequence <400> 69 agtgcagcca ttatatagga ctaacaagga 30 <210> 70 <211> 30 <212> DNA <213> Artificial Sequence <400> 70 agtgcagcca aaccagaaga aagccatgtt 30 <210> 71 <211> 25 <212> DNA <213> Artificial Sequence <400> 71 agtgcagcca ttatatagga ctaac 25 <210> 72 <211> 20 <212> DNA <213> Artificial Sequence <400> 72 agtgcagcca aaccagaaga 20 <210> 73 <211> 30 <212> DNA <213> Artificial Sequence <400> 73 gtcgttcctg tggttaggac agggtcgcaa 30 <210> 74 <211> 30 <212> DNA <213> Artificial Sequence <400> 74 gtcgttcctg ctggtgtctc agatcgttcg 30 <210> 75 <211> 25 <212> DNA <213> Artificial Sequence <400> 75 gtcgttcctg tggttaggac agggt 25 <210> 76 <211> 24 <212> DNA <213> Artificial Sequence <400> 76 gtcgttcctg ctggtgtctc agat 24 <210> 77 <211> 30 <212> DNA <213> Artificial Sequence <400> 77 aacggcgaca taaaataagt tgttacatgt 30 <210> 78 <211> 30 <212> DNA <213> Artificial Sequence <400> 78 aacggcgaca gtggcatgct cgatgacgac 30 <210> 79 <211> 22 <212> DNA <213> Artificial Sequence <400> 79 aacggcgaca taaaataagt tg 22 <210> 80 <211> 25 <212> DNA <213> Artificial Sequence <400> 80 aacggcgaca gtggcatgct cgatg 25 <210> 81 <211> 30 <212> DNA <213> Artificial Sequence <400> 81 ggtgaacgct gtagttggaa tataagtata 30 <210> 82 <211> 30 <212> DNA <213> Artificial Sequence <400> 82 ggtgaacgct cagatttaaa tataattagt 30 <210> 83 <211> 24 <212> DNA <213> Artificial Sequence <400> 83 ggtgaacgct gtagttggaa tata 24 <210> 84 <211> 26 <212> DNA <213> Artificial Sequence <400> 84 ggtgaacgct cagatttaaa tataat 26 <210> 85 <211> 30 <212> DNA <213> Artificial Sequence <400> 85 ccgcagttag atgcaccatt agaattgctt 30 <210> 86 <211> 30 <212> DNA <213> Artificial Sequence <400> 86 ccgcagttag atcaagtggc aaggttccat 30 <210> 87 <211> 20 <212> DNA <213> Artificial Sequence <400> 87 ccgcagttag atgcaccatt 20 <210> 88 <211> 20 <212> DNA <213> Artificial Sequence <400> 88 ccgcagttag atcaagtggc 20 <210> 89 <211> 40 <212> DNA <213> Artificial Sequence <400> 89 gtcgttcctg tggttaggac agggtcgcaa attcagtctt 40 <210> 90 <211> 40 <212> DNA <213> Artificial Sequence <400> 90 gtcgttcctg ctggtgtctc agatcgttcg ggggcttgcg 40 <210> 91 <211> 12 <212> DNA <213> Artificial Sequence <400> 91 gtcgttcctg tg 12 <210> 92 <211> 12 <212> DNA <213> Artificial Sequence <400> 92 gtcgttcctg ct 12 ...

MKVSAAALAVILIATALCAPASAPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVF VTRKNRQVCANPEKKWVREYINSLEMS3.2 secreted protein sequence

PCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYINS LEMS (4) R5 antagonist 2 (RANTES (8-68)) 4.1 band leaders

MKVSAAALAVILIATALCAPASATPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVV FVTRKNRQVCANPEKKWVREYINSLEMS4.2 secreted protein sequence TPCCFAYIARPLPRAHIKEYFYTSGKCSNPAVVFVTRKNRQVCANPEKKWVREYIN SLEMS (5) XR3 antagonist 1 (ITAC (4-73)) 5.1 band leaders

MSVKGMAIALAVILCATVVQGFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDK IEVIITLKENKGQRCLNPKSK QARLIIKKVE RKNF5.2 secreted protein sequence

FKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKENKGQRCLNPKSK QARLIIKKVERKNF (6) XR3 antagonist 2 (ITAC (3-73)) 6.1 band leaders

MSVKGMAIALAVILCATVVQGMFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCD KIEVIITLKENKGQRCLNPKSK QARLIIKKVE RKNF6.2 secreted protein sequence

MFKRGRCLCIGPGVKAVKVADIEKASIMYPSNNCDKIEVIITLKENKGQRCLNPKS KQARLIIKKVERKNF (7) .R7 antagonist 1 (MIP-3 β (8-77)) 7.1 band leader MALLLALSLLVLWTSPAPTLSCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFT TLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSS7.2 secreted protein sequences

CCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQ RLQRTSAKMKRRSS (8) R7 antagonist (6CKine (8-79)) 8.1 band leader MAQSLALSLLILVLAFGIPRTQGCCLKYSQRKIPAKVVRSYRKQEPSLGCSIPAIL FLPRKRSQAELCADPKELWVQQLMQHLDKTPSPQKPAQG8.2 secreted protein sequence CCLKYSQRKIPAKVVRSYRKQEPSLGCSIPAILFLPRKRSQAELCADPKELWVQQL MQHLDKTPSPQKPAQG

(9) XR1 antagonist 1 (IL-8 (6-72)) 9.1 band leaders

MTSKLAVALLAAFLISAALCEGAVLPR---RCQCIKTYSKPFHPKFIKELRVIESG PHCANTEIIVKLSDGRELCLDPKENWVQRVVEKFLKRAENS " 9.2 secreted protein sequences, RCQCIKTYSKPFHPKFIKELRVIESGPHCANTEIIVKLSDGRELCLDPKENWVQRV VEKFLKRAENS ", (, 10, ), R2 antagonist 1, (, MCP-3, (, 9-76, ), ), 10.1 band leader MKASAALLCLLLTAAAFSPQGLA---TTCCYRFINKKIPKQRLESYRRTTSSHCPR EAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKL10.2 secreted protein sequence TTCCYRFINKKIPKQRLESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDFM KHLDKKTQTPKL, (, 11, ), R2 antagonist 2, (, MCP-3, (, 8-76, ), ), 11.1 band leader MKASAALLCLLLTAAAFSPQGLA---STTCCYRFINKKIPKQRLESYRRTTSSHCP REAVIFKTKLDKEICADPTQKWVQDFMKHLDKKTQTPKL11.2 secreted protein sequence STTCCYRFINKKIPKQRLESYRRTTSSHCPREAVIFKTKLDKEICADPTQKWVQDF MKHL, (, 12, ), the R2 antagonist, (, MCP-1, (, 9-76, ), ), 12.1 band leader MKVSAALLCLLLIAATFIPQGLA--VTCCYNFTNRKISVQRLASYRRITSSKCPKE AVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT12.2 secreted protein sequence

VTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDSM DHLDKQTQTPKT (13) R2 antagonist 4 (MCP-1 (8-76)) 13.1 band leader MKVSAALLCLLLIAATFIPQGLA--PVTCCYNFTNRKISVQRLASYRRITSSKCPK EAVIFKTIVAKEICADPKQKWVQDSMDHLDKQTQTPKT13.2 secreted protein sequences

PVTCCYNFTNRKISVQRLASYRRITSSKCPKEAVIFKTIVAKEICADPKQKWVQDS MDHLDKQTQTPKT (14) XR2 antagonist 1 (GRO α (8-73)) 14.1 band leader MARAALSAAPSNPRLLRVALLLLLLVAAGRRAAGASVATELRCQCLQTLQGIHPKN IQSVNVKSPGPHCAQTEVIATLKNG

RKACLNPASPIVKKIIEKMLN SDKSN14.2 secreted protein sequence

The chimeric protein sequence of ASVATELRCQCLQTLQGIHPKNIQSVNVKSPGPHCAQTEVIATLKNGRKACLNPAS PIVKKIIEKMLN SDKSN (example, but be not limited to): 15. people source Immunoglobulin IgG1s decide the district:

ASFKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPC PAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVH NAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPV LDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK16. people source Immunoglobulin IgG2 determine district:

ASFKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAP PVAGPSVFLFPPKPKDTLMISRTPEVTWVVVDVSHEDPEVQFNWYVDGVEVHNAKT KPREEQFNSTFCVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKTKGQPRE PQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSD GSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK17. people source Immunoglobulin IgG4 determine district:

ASFKGPSVFPLVPCSRSTSESTAALGCLVKDYFPEPVTVSWNSCALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPSCPAP EFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAK TKPREEQFNSTYRVVRVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEDNYKTTPPVLDS DGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSPGK " 18. people source light chain immunoglobulin κ determine district:

TAAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTY SLSSTLTL SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC Chemokine receptor antagonist nucleic acid sequence (eg, but not limited to): (ND.1) XR4 antagonist 1 (SDF-1P2G) atgaacgcca aggtcgtggt cgtgctggtc ctcgtgctga ccgcgctctg cctcagcgac gggaagGGcg tcagcctgag ctacagatgc ccatgccgat tcttcgaaag ccatgttgcc agagccaacg tcaagcatct caaaattctc aacactccaa actgtgccct tcagattgta gcccggctga agaacaacaa cagacaagtg tgcattgacc cgaagctaaa gtggattcag gagtacctgg agaaagcttt aaacaagagg ttcaagatgtga (ND.2) XR4 antagonist 2 (SDF_1 (2-73) atgaacgcca aggtcgtggt cgtgctggtc ctcgtgctga ccgcgctctg cctcagcgac gggcccg tcagcctgag ctacagatgc ccatgccgat tcttcgaaag ccatgttgcc agagccaacg tcaagcatct caaaattctc aacactccaa actgtgccct tcagattgta gcccggctga agaacaacaa cagacaagtg tgcattgacc cgaagctaaa gtggattcag gagtacctgg agaaagcttt aaacaagagg ttcaagatgtga (ND.3) R5 antagonist 1 RANTES (8-68) atgaaggtc tccgcggcag ccctcgctgt catcctcatt gctactgccc tctgcgctcc tgcatctgcc acaccctgc tgctttgcct acattgcccg cccactgccc cgtgcccaca tcaaggagta tttctacacc agtggcaagt gctccaaccc agcagtcgtc tttgtcaccc gaaagaaccg ccaagtgtgt gccaacccag agaagaaatg ggttcgggag tacatcaact ctttggagat gagctag (ND.4) R5 antagonist 2 (RANTES (9-68)) atgaaggtc tccgcggcag ccctcgctgt catcctcatt gctactgccc tctgcgc ccc tgcatctgcc tcctgc tgctttgcct acattgcccg cccactgccc cgtgcccaca tcaaggagta tttctacacc agtggcaagt gctccaaccc agcagtcgtc tttgtcaccc gaaagaaccg ccaagtgtgt gccaacccag agaagaaatg ggttcgggag tacatcaact ctttggagat gagctag (ND.5) XR3 antagonist 1 (ITAC (4-73)) atgag tgtgaagggc atggctatag ccttggctgt gatattgtgt gctacagttg ttcaaggc --- ttc aaaagaggac gctgtctttg cataggccct ggggtaaaag cagtgaaagt ggcagatatt gagaaagcct ccataatgta cccaagtaac aactgtgaca aaatagaagt gattattacc ctgaaagaaa ataaaggaca acgatgccta aatcccaaat cgaagcaagc aaggcttata atcaaaaaag ttgaaagaaa gaatttttaa (ND.6) XR3 antagonist 2 (ITAC (3-73)) atgag tgtgaagggc atggctatag ccttggctgt gatattgtgt gctacagttg ttcaaggc --- Atgttc aaaagaggac gctgtctttg cataggccct ggggtaaaag cagtgaaagt ggcagatatt gagaaagcct ccataatgta cccaagtaac aactgtgaca aaatagaagt gattattacc ctgaaagaaa ataaaggaca acgatgccta aatcccaaat cgaagcaagc aaggcttata atcaaaaaag ttgaaagaaa gaatttttaa (ND.7) R7 antagonist 1 (MIP-3β (8-77)) at ggccctgcta ctggccctca gcctgctggt tctctggact tccccagccc caactctgag t__tgctgcct gtctgtgacc cagaaaccca tccctgggta catcgtgagg aacttccact accttctcat caaggatggc tgcagggtgc ctgctgtagt gttcaccaca ctgaggggcc gccagctctg tgcaccccca gaccagccct gggtagaacg catcatccag agactgcaga ggacctcagc caagatgaag cgccgcagca gttaa (ND.8) R7 antagonist 2 (6CKine (8-79)) atggctcagtca ctggctctga gcctccttat cctggttctg gcctttggca tccccaggac ccaaggctg ttgcctcaag tacagccaaa ggaagattcc cgccaaggtt gtccgcagct accggaagca ggaaccaagc ttaggctgct ccatcccagc tatcctgttc ttgccccgca agcgctctca ggcagagcta tgtgcagacc caaaggagct ctgggtgcag cagctgatgc agcatctgga caagacacca tccccacaga aaccagccca gggc (ND.9) XR1 antagonist 1 (IL-8 (6-72)) atgacttcca agctggccgt ggctctcttg gcagccttcc tgatttctgc agctctgtgt gaaggtgcag ttttgccaag g --- agat gtcagtgcat aaagacatac tccaaacctt tccaccccaa atttatcaaa gaactgagag tgattgagag tggaccacac tgcgccaaca cagaaattat tgtaaagctt tctgatggaa gagagctctg tctggacccc aaggaaaact gggtgcagag ggttgtggag aagtttttga agagggctga gaattcataa (ND.10) R2 antagonist 1 (MCP-3 (8-76)) atgaaagcct ctgcagcact tctgtgtctg ctgctcacag cagctgcttt cagcccccag gggcttgct --- tcaactacct gctgctacag atttatcaat aagaaaatcc ctaagcagag gctggagagc tacagaagga ccaccagtag ccactgtccc cgggaagctg taatcttcaa gaccaaactg gacaaggaga tctgtgctga ccccacacag aagtgggtcc aggactttat gaagcacctg gacaagaaaa cccaaactcc aaagctttga (ND.11) R2 antagonist 2 (MCP-3 (9-76)) atgaaagcct ctgcagcact tctgtgtctg ctgctcacag cagctgcttt cagcccccag gggcttgct --- actacct gctgctacag atttatcaat aagaaaatcc ctaagcagag gctggagagc tacagaagga ccaccagtag ccactgtccc cgggaagctg taatcttcaa gaccaaactg gacaaggaga tctgtgctga ccccacacag aagtgggtcc aggactttat gaagcacctg gacaagaaaa cccaaactcc aaagctttga (ND.12) R2 antagonist 3 (Mcp-1 (8-76)) atgaaag tctctgccgc ccttctgtgc ctgctgctca tagcagccac cttcattccc caagggctcg ct ---- ccagtca cctgctgtta taacttcacc aataggaaga tctcagtgca gaggctcgcg agctatagaa gaatcaccag cagcaagtgt cccaaagaag ctgtgatctt caagaccatt gtggccaagg agatctgtgc tgaccccaag cagaagtggg ttcaggattc catggaccac ctggacaagc aaacccaaac tccgaagact tgaa (ND.13) R2 antagonist 4 (Mcp-1 (9-76)) atgaaag tctctgccgc ccttctgtgc ctgctgctca tagcagccac cttcattccc caagggctcg ct ---- gtca cctgctgtta taacttcacc aataggaaga tctcagtgca gaggctcgcg agctatagaa gaatcaccag cagcaagtgt cccaaagaag ctgtgatctt caagaccatt gtggccaagg agatctgtgc tgaccccaag cagaagtggg ttcaggattc catggaccac ctggacaagc aaacccaaac tccgaagact tgaa (ND.14) XR2 antagonist 1 GROα (8-73) a tggcccgcgc tgctctctcc gccgccccca gcaatccccg gctcctgcga gtggcactgc tgctcctgct cctggtagcc gctggccggc gcgcagcagg a-cgctgcca gtgcttgcag accctgcagg gaattcaccc caagaacatc caaagtgtga acgtgaagtc ccccggaccc cactgcgccc aaaccgaagt catagccaca ctcaagaatg ggcggaaagc ttgcctcaat cctgcatccc ccatagttaa gaaaatcatc gaaaagatgc tgaacagtga caaatccaac tga Chimeric nucleic acid sequences (eg, but not limited to): ND15. Human immunoglobulin IgG1 fixed zone: 1 gcaagcttca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 61 ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 121 tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 181 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 241 tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 301 aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 361 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 421 gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 481 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 541 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 601 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 661 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 721 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 781 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 841 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 901 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 961 cagaagagcc tctccctgtc tccgggtaaa ND16. Humanized immunoglobulin IgG2 fixed zone: 1 gcaagcttca agggcccatc ggtcttcccc ctggcgccct gctccaggag cacctccgag 61 agcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 121 tggaactcag gcgctctgac cagcggcgtg cacaccttcc cagctgtcct acagtcctca 181 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcaacttcgg cacccagacc 241 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagac agttgagcgc 301 aaatgttgtg tcgagtgccc accgtgccca gcaccacctg tggcaggacc gtcagtcttc 361 ctcttccccc caaaacccaa ggacaccctc atgatctccc ggacccctga ggtcacgtgg 421 gtggtggtgg acgtgagcca cgaagacccc gaggtccagt tcaactggta cgtggacggc 481 gtggaggtgc ataatgccaa gacaaagcca cgggaggagc agttcaacag cacgttctgt 541 gtggtcagcg tcctcaccgt tgtgcaccag gactggctga acggcaagga gtacaagtgc 601 aaggtctcca acaaaggcct cccagccccc atcgagaaaa ccatctccaa aaccaaaggg 661 cagccccgag aaccacaggt gtacaccctg cccccatccc gggaggagat gaccaagaac 721 caggtcagcc tgacctgcct ggtcaaaggc ttctacccca gcgacatcgc cgtggagtgg 781 gagagcaatg ggcagccgga gaacaactac aagaccacac ctcccatgct ggactccgac 841 ggctccttct tcctctacag caagctcacc gtggacaaga gcaggtggca gcaggggaac 901 gtcttctcat gctccgtgat gcatgaggct ctgcacaacc actacacgca gaagagcctc 961 tccctgtctc cgggtaaa ND 17. Humanized immunoglobulin IgG4 fixed zone: 1 gcaagcttca agggcccatc ggtcttcccc ctggtgccct gctccaggag cacctccgag 61 agcacagccg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 121 tggaactcat gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 181 ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacgaagacc 241 tacacctgca acgtagatca caagcccagc aacaccaagg tggacaagag agttgagtcc 301 aaatatggtc ccccatgccc atcatgccca gcacctgagt tcctgggggg accatcagtc 361 ttcctgttcc ccccaaaacc caaggacact ctcatgatct cccggacccc tgaggtcacg 421 tgcgtggtgg tggacgtgag ccaggaagac cccgaggtcc agttcaactg gtacgtggat 481 ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagttcaa cagcacgtac 541 cgtgtggtca gggtcctcac cgtcctgcac caggactggc tgaacggtaa ggagtacaag 601 tgcaaggtct ccaacaaagg cctcccgtcc tccatcgaga aaaccatctc caaagccaaa 661 gggcagcccc gagagccaca ggtgtacaccc tgcccccat cccaggagga gatgaccaag 721 aaccaggtca gcctgacctg cctggtcaaa ggcttctacc ccagcgacat cgccgtggag 781 tgggagagca atgggcagcc ggaggacaac tacaagacca cgcctcccgt gctggactcc 841 gacggctcct tcttcctcta cagcaggcta accgtggaca agagcaggtg gcaggagggg 901 aatgtcttct catgctccgt gatgcatgag gctctgcaca accactacac acagaagagc 961 ctctccctgt ctccgggtaa actctccctgtctctgggtaaa ND 18. Humanized immunoglobulin light chain κ fixed zone: 1 actgcggccg caccatctgt cttcatcttc ccgccatctg atgagcagtt gaaatctgga 61 actgcctctg ttgtgtgcct gctgaataac ttctatccca gagaggccaa agtacagtgg 121 aaggtggata acgccctcca atcgggtaac tcccaggaga gtgtcacaga gcaggacagc 181 aaggacagca cctacagcct cagcagcacc ctgacgctga gcaaagcaga ctacgagaaa 241 cacaaagtct acgcctgcga agtcacccat cagggcctga gctcgcccgt cacaaagagc 301 ttcaacaggg gagagtgttga Information about major chart illustrates the invention ...

Figure 1A-C is the synoptic diagram of the present invention's some chimeric protein of designing and making.The antagonist 1,2,3 here can be represented the included any four kinds of different antagonists of the present invention respectively.Similarly, the heavy chain here can be that people source immunoglobulin (Ig) comprises IgG1, IgG4, any one heavy chain of IgG2; The light chain here can be any among κ or the λ.The CH1 here, CH2, CH3 are meant three fragments of the constant region of heavy chain; What CL represented is the constant region of light chain; Here connection between the chimeric protein monomer or disulphide bridges representative be the covalently bound thing not of the same race that comprises disulfide bridge bond.Details are seen detailed description of the invention.

Fig. 2 with the ELISA method do anti-human body IgG antibody in conjunction with competition experiments.

The result shows that clone's chimeric protein XR4/R5/R1 gene has been inserted into Chinese hamster ovary celI gene and successful expression, and the synthetic chimeric protein is secreted in the nutrient solution, thereby can be the dose-dependent competition that combines with anti-human body IgG antibody.

The competition experiments that combines of the different chemokines of Fig. 3 A and its acceptor.

People's monocyte surface has CCR1, CCR2, and the CCR5 acceptor, people's eosinophilic granulocyte surface has the CCR3 acceptor, and people's T-lymphocyte has the CXCR4 acceptor.These three cell strains are as the target cell of these acceptors.To contain the culture supernatant of chimeric protein XR4/R5/R1 and the isotropic substance I of fixed dosage ¹²⁵The part chemokine of mark mixes, and combines with these receptor competitions: wherein, MIP-1 α is the part chemokine of CCR1, MCP-1 is the part of CCR2, Eotaxin is the part of CCR3, and RANTES is the part of CCR5, and SDF-1 α then is the part of CXCR4 acceptor.The result shows chimeric protein XR4/R5/R1 and the very strong bonded ability (50% cell conditioned medium can suppress combining of most chemokines) of above-mentioned acceptor performance.

The albumen of the different fusions of Fig. 3 B and the test that combines of acceptor

5 ' terminal 5 ' the end that is connected the protein (C-N merges, ring marker) that merges and nucleotide sequence ND.1 of 3 ' terminal (being equivalent to the proteic C-terminal of SDF-1P2G) that to contain nucleotide sequence ND.1 and nucleotide sequence ND.14 is connected iodine 125 marks of the secreted supernatant of transgenic cell of protein (trilateral sign) of fusion and fixed amount with the end of nucleotide sequence ND.14 chemokine SDF-1 does the receptors bind competition experiments.The result shows that the C-N fusion rotein has receptor binding capacity.

The migration inhibition test of Fig. 4 cell.

With people's monocyte, these three cell strains of people's eosinophilic granulocyte and T-lymphocyte strain are as the target cell of these acceptors.As shown in the figure, various part chemokines induce separately target cell to move (be decided to be mobile 100%) respectively.The adding that contains the chimeric protein XR4/R5/R1 culture supernatant of different concns is dosage and relies on shape ground and suppress moving of cell: by suppressing CCR1, and CCR2 and CCR5 acceptor and suppress α, MCP-1 and RANTES inductive THP-1 cell mobile by MIP-1; Suppress moving of Eotaxin inductive eosinophilic granulocyte by suppressing the CCR3 acceptor; Suppress moving of SDF-1 α inductive CEM-M3 cell by inhibition to the CXCR4 acceptor.

Fig. 5 receptors bind competitive assay.

People's T-lymphocyte strain that has the CXCR4 acceptor and people's the cell strain () that has the CCR7 acceptor are as the target cell of acceptor.The culture supernatant that will contain chimeric protein XR4/R7 respectively with isotropic substance I ¹²⁵The part chemokine SDF-1 α of mark and 6Ckine or MIP-3 β mix, and combine with these acceptors do competitions.As show the ability (50% cell conditioned medium can suppress combining of most chemokines) that chimeric protein XR4/R7 and above-mentioned acceptor are combined closely.

Fig. 6 chimeric protein XR4/R7 suppresses moving of breast carcinoma cell strain

The breast carcinoma cell strain T-47D that utilizes two people has tested the restraining effect that chimeric protein XR4/R7 moves breast cancer cell as target cell.As shown in the figure, the part chemokine 6CKine of CCR7 acceptor or MIP-3 β, and the part SDF-1 of CXCR4 induces moving of these two breast carcinoma cell strains.Fig. 6 A: the adding of culture supernatant that contains the chimeric protein XR4/R7 of different concns is dosage and relies on shape ground and suppress the mobile of SDF-1 α and 6CKine (or MIP-3 β) inductive breast cancer cell.Fig. 6 B:CXCR4 receptor antagonist SDF-1P2G can only suppress SDF-1 inductive T47 breast cancer cell by inhibition CXCR4 acceptor and move, and but can not suppress 6Ckine (by the CCR7 acceptor) inductive breast cancer cell and move.Illustrate that two acceptors that have only as breast cancer cell T47D all are suppressed, could stop moving of cancer cells.

Fig. 7 measure gene engineering making and excretory chimeric protein XR3/R5/R1 whether can with CCR5, CCR1 and the combination of CXCR3 acceptor high affinity.

With the T-lymphocyte of human peripheral and monocyte strain target cell as acceptor.The T-cell that separation is obtained activates with cytokine interleukin element-2 earlier.After will contain chimeric protein XR3/R5/R1 culture supernatant respectively with isotropic substance I ¹²⁵The part chemokine IP-10 or the RANTES of mark, or MIP-1 α is mixed, and does competition with these acceptors and combining.The ability (50% cell conditioned medium can suppress combining of most chemokines) that diagram chimeric protein XR3/R5/R1 and above-mentioned acceptor are combined closely.

Fig. 8 T-lymphocyte and monocyte migration inhibition test

Utilize activated human body T-lymphocyte and monocyte strain as target cell, tested chimeric protein XR3/R5/R1, the restraining effect that the receptor-mediated cell of CCR5 and CXCR3 moves CCR1.As shown in the figure, the part chemokine RANTES of CCR5 acceptor, and the ligand i P-10 of CXCR3 induces that activated T-is lymphocytic to be moved, the part chemokine MIP-1 α of CCR1 acceptor induces monocyte to move.The adding of culture supernatant that contains the chimeric protein XR3/R5/R1 of different concns is dosage and relies on shape ground and suppress IP-10, MIP-1 α or RANTES inductive target cell mobile.

Fig. 9 measures gene engineering making and excretory chimeric protein XR1/R2 and CCR2, the combination of CXCR1 and CXCR2 acceptor.

With the neutrophil leucocyte of the human peripheral target cell as CXCR1 and CXCR2, the monocyte strain is as the target cell of CCR2 acceptor.Neutrophil leucocyte that separation is obtained and monocyte immediately with the culture supernatant of chimeric protein XR1/R2, and respectively with isotropic substance I ¹²⁵The part chemokine IL-8 of mark or GRO-α, or MCP-1 is mixed, and does competition with these acceptors and combining.The result shows the ability (50% cell conditioned medium can suppress combining of most chemokines) that chimeric protein XR1/R2 and above-mentioned acceptor are combined closely.

Figure 10 people's neutrophil leucocyte cell and monocyte migration inhibition test

Utilize the target cell of the neutrophil leucocyte of human peripheral blood as CXCR1 and CXCR1 acceptor, the monocyte strain has been tested chimeric protein XR1/R2 to CCR2 and CXCR1 as the target cell of CCR2 acceptor, the restraining effect that the receptor-mediated cell of CXCR2 moves.As shown in the figure, the part chemokine MCP-1 of CCR2 acceptor, and the part GRO α of the ligand i L-8 of CXCR1 and CXCR2 induces moving of above-mentioned target cell.The adding of culture supernatant that contains the chimeric protein XR2/R2 of different concns is dosage and relies on shape ground and suppress IL-8, GRO α or MCP-1 inductive target cell mobile.

Some features of table 2 chimeric protein

The above, it only is preferred embodiment of the present invention, be not that the present invention is done any pro forma restriction, every foundation technical spirit of the present invention all still belongs in the scope of technical solution of the present invention any simple modification, equivalent variations and modification that above embodiment did.

Claims

1, a kind of chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum is characterized in that, chimeric protein that can two or more Chemokine Receptors of antagonism, and it includes following protein fragments:

A, human immunoglobulin constant region;

C, wherein the C-end of each receptor antagonist by its molecule links to each other with the N-of human immunoglobulin constant region is terminal, formation fusion rotein monomer;

2, the chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum according to claim 1, it is characterized in that the monomeric human immunoglobulin constant region of described fusion rotein partly is meant human immunoglobulin's the constant region of heavy chain and/or the constant region fragment of light chain; The monomeric receptor antagonist of described fusion rotein partly comprise following any two at the protein of isoacceptor not: XR4 antagonist 1; XR4 antagonist 2; Two amino acid of N-terminal head produce through chemically modified, have the SDF-1 derivative that suppresses the receptor CXCR 4 effect; R5 antagonist 1; R5 antagonist 2; XR3 antagonist 1; XR3 antagonist 2; 3 amino acid of N-terminal head obtain through chemically modified, have the ITAC derivative that suppresses acceptor CXCR3 effect; R7 antagonist 1; N-terminal head two 7 amino acid obtain through chemically modified, have the MIP-3 β derivative that suppresses acceptor CCR7 effect; R7 antagonist 2; 7 amino acid of N-terminal head obtain through chemically modified, have the pulsating derivative of 6CKine (1-79) that suppresses acceptor CCR7 effect; XR1 antagonist 1; R2 antagonist 1; R2 antagonist 2; N-terminal head nine amino acid obtains through chemically modified, has the MCP-3 derivative that suppresses acceptor CCR2 effect; R2 antagonist 3; R2 antagonist 4; 9 amino acid of N-terminal head obtain through chemically modified, have the derivative of the MCP-1 that suppresses acceptor CCR2 effect; XR2 antagonist 1; 7 amino acid of N-terminal head obtain through chemically modified, have the derivative of the GRO α that suppresses acceptor CXCR2 effect; The mode that described two kinds of different fusion rotein monomers are connected each other preferably by disulphide bridges, also can be passed through covalent linkage such as polypeptide chain, or polysaccharide molecule; Described two kinds of continuous different fusion rotein monomers comprise between two kinds of different fusion rotein monomers that contain heavy chain, or contain heavy chain and contain between the fusion rotein monomer of light chain.

3, the chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum according to claim 1 is characterized in that, described human immunoglobulin's CH includes hinge region and CH3 fragment; Described human immunoglobulin's CH includes hinge region and CH1, CH2 and CH3 fragment; Human immunoglobulin's CH comprises the CH of IgG γ 1 and IgG γ 4; The constant region fragment of described light chain comprises the constant region of the κ and the λ of human immunoglobulin's light chain.

4, a kind of purposes of chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum as claimed in claim 1, it is characterized in that the chimeric protein of described two or more Chemokine Receptors of antagonism can be used for treating repulsion and graft versus host disease (GVH disease), treatment HIV-1 virus infection and the acquired immune deficiency syndrome (AIDS) that autoimmune disease and nonspecific inflammation disease, treatment tumour relative disease, treatment/prevention of organ transplant cause; The chimeric protein of described two or more Chemokine Receptors of antagonism and other method or medicine are done combination therapy.

5, the purposes of the chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum according to claim 4 is characterized in that, the using dosage during described treatment/prevention is 3-300 milligram/kg body weight scope; Intramuscular injection dosage should be the several times of this dosage.

6, a kind of production method of chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum as claimed in claim 1, it is characterized in that, the method of described chimeric protein that can two or more Chemokine Receptors of antagonism comprises: utilize gene recombination technology to make up and include the not monomeric carrier of chimeric protein of Chemokine Receptors of the same race of antagonism; Utilize above-mentioned different carrier, set up to express and the cell strain or the bacterial strain of the chimeric protein that secretion can two or more Chemokine Receptors of antagonism; Cultivate above-mentioned cell or bacterial strain, collect and the purifying secreted chimeric protein that goes out.

7, a kind of production method of chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum as claimed in claim 1 is characterized in that, described can the antagonism chemokine receptor CCR 5, CCR1, CCR2, CCR3, the chimeric protein XR4/R5/R1 of CXCR4, its method comprises: utilize gene recombination technology to make up according to money 1 indication and include nucleotide sequence ND.1, ND.2, ND.3, ND.4 or expressing protein are equivalent to sequence (1), (2), (3), the monomeric carrier of the chimeric protein of (4); Utilize above-mentioned different carrier, set up to express and secretion can the antagonism chemokine receptor CCR 5, CCR1, CCR2, CCR3, the cell strain of the chimeric protein of CXCR4; Cultivate above-mentioned cell, collect and the purifying secreted chimeric protein that goes out.

8, a kind of purposes of chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum as claimed in claim 7 is characterized in that, the autoimmune disease that described chimeric protein XR4/R5/R1 is used for the treatment of/prevents HIV virus infection, acquired immune deficiency syndrome (AIDS) and relates to above-mentioned acceptor; Described chimeric protein XR4/R5/R1 is used for doing combination therapy with other method or medicine; Described chimeric protein XR4/R5/R1 and nucleic acid encoding thereof are used for the vaccine therapy method.

9, the purposes of the chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum according to claim 7 is characterized in that, and described chimeric protein XR4/R5/R1 is used for the treatment of/and using dosage when preventing is 3-300 milligram/kg body weight; Intramuscular injection dosage is the several times of above-mentioned dosage; Use formulation to comprise injection type and mucous membrane local application formulation; The gene therapy method of the nucleic acid encoding of described chimeric protein XR4/R5/R1 and dosage are 0.4-4 milligram/kg body weight.

10, a kind of production method of chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum as claimed in claim 1, it is characterized in that, described can antagonism Chemokine Receptors CCR1, CCR5, the method for the chimeric protein XR3/R5/R1 of CXCR3 comprises: utilize gene recombination technology to make up and include nucleotide sequence ND.3, ND.4 ND.5, ND.6, or their corresponding proteins sequences (3), (4), (5), the monomeric carrier of the chimeric protein of (6); Utilize above-mentioned different carrier, set up to express and secretion can the antagonism chemokine receptor CCR 5, CCR1, the cell strain of the chimeric protein of CXCR3; Cultivate above-mentioned cell, collect and the purifying secreted chimeric protein that goes out.

11, a kind of purposes of chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum as claimed in claim 10, it is characterized in that, described chimeric protein XR3/R5/R1 is used for the treatment of/repulsion that prevention of organ transplant causes, and graft versus host disease (GVH disease) and the autoimmune disease that relates to above-mentioned acceptor; Described chimeric protein XR3/R5/R1 is used for doing combination therapy with other method or medicine; The vaccine therapy method of chimeric protein XR3/R5/R1 and nucleic acid encoding thereof.

12, the purposes of the chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum according to claim 11, it is characterized in that, described chimeric protein XR3/R5/R1 is used for the treatment of/and using dosage when preventing is 3-300 milligram/kg body weight scope, intramuscular injection, dosage should be the several times of this dosage; Described chimeric protein XR3/R5/R1 is used for the treatment of/use formulation when preventing, comprise injection type; The gene therapy method of the nucleic acid encoding of described various chimeric proteins and dosage are 0.4-4 milligram/kg body weight.

13, a kind of production method of chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum as claimed in claim 1, it is characterized in that, described can antagonism Chemokine Receptors CCR7, the method for the chimeric protein R7/XR4 of CXCR4 comprises: utilize gene recombination technology to make up and include nucleotide sequence ND.1, ND.2, ND.7, ND.8, or their corresponding proteins sequences (1), (2), (7), the monomeric carrier of the chimeric protein of (8); . utilize above-mentioned different carrier, set up to express and secretion can antagonism Chemokine Receptors CCR7, the cell strain of the chimeric protein of CXCR4; . cultivate above-mentioned cell, collect and the purifying secreted chimeric protein that goes out.

14, a kind of purposes of chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum as claimed in claim 13 is characterized in that, described chimeric protein R7/XR4 is used for the treatment of tumour and treatment and prophylaxis of tumours to be shifted, and the autoimmune disease that relates to above-mentioned acceptor; Described chimeric protein R7/XR4 is used for doing combination therapy with other method or medicine; The vaccine therapy method of described chimeric protein R7/XR4 and nucleic acid encoding thereof.

15, the purposes of the chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum according to claim 14 is characterized in that, and described chimeric protein R7/XR4 is used for the treatment of/and using dosage when preventing is 3-300 milligram/kg body weight; Intramuscular injection dosage is the several times of this dosage; Described chimeric protein R7/XR4 is used for the treatment of/and use formulation when preventing comprises injection type; The gene therapy method of the nucleic acid encoding of described various chimeric proteins and dosage are 0.4-4 milligram/kg body weight.

16, a kind of production method of chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum as claimed in claim 1 is characterized in that, described can antagonism Chemokine Receptors CCR2, CXCR1, the method of the chimeric protein XR1/R2 of CXCR2 comprises: utilize gene recombination technology to make up and include nucleotide sequence ND.9, ND.10, ND.11, ND.12, ND.13, No.14 or their corresponding proteins sequences (9), (10), (11), the monomeric carrier of chimeric protein of (12) (13) (14); Utilize above-mentioned different carrier, set up to express and secretion can antagonism Chemokine Receptors CCR2, the cell strain of the chimeric protein of CXCR1/CXCR2 or bacterial strain; Cultivate above-mentioned cell or bacterial strain, collect and the purifying secreted chimeric protein that goes out.

17, a kind of purposes of chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum as claimed in claim 16 is characterized in that, described chimeric protein XR1/R2 is used for the treatment of autoimmune disorder and non-special inflammation; Described chimeric protein XR1/R21 is used for doing combination therapy with other method or medicine; The vaccine therapy method of described chimeric protein R2/XR1 and nucleic acid encoding thereof.

18, the purposes of the chemotactic factor receptor inhibiting matter of long-acting, broad-spectrum according to claim 16 is characterized in that, and described chimeric protein XR1/R2 is used for the treatment of/and using dosage when preventing is 3-300 milligram/kg body weight scope; Intramuscular injection dosage is the several times of this dosage; Described chimeric protein XR1/R2 is used for the treatment of/use formulation when preventing, comprise intestinal absorption formulation, injection type; The gene therapy method of the nucleic acid encoding of described various chimeric proteins and dosage are 0.4-4 milligram/kg body weight.