CN101804193B

CN101804193B - CTLA4lg and derivatives thereof used for resisting viruses

Info

Publication number: CN101804193B
Application number: CN200910004904A
Authority: CN
Inventors: 吴军; 王晓娟; 胡婕; 罗高兴; 贺伟峰
Original assignee: CHONGQING XI'NAN HOSPITAL
Current assignee: CHONGQING XI'NAN HOSPITAL
Priority date: 2009-02-16
Filing date: 2009-02-16
Publication date: 2012-09-26
Anticipated expiration: 2029-02-16
Also published as: CN101804193A

Abstract

The invention discloses new application of CTLA4lg or derivatives of the CTLA4lg in the preparation of antiviral drug. The invention discovers the remarkable activation of the CTLA4lg or the derivatives of the CTLA4lg to the cytotoxicity of NK cells (Natural Killer Cells) for the first time and demonstrates that molecular mechanism of the CTLA4lg or the derivatives of the CTLA4lg for improving the cytotoxicity of the NK cells is to remarkably improve the expressions of NKG2D, NKp44 and other activated receptors on the surfaces of NK cell membranes. Proved by experiments, by improving the cytotoxicity of organic NK cells, the CTLA4lg or the derivatives of the CTLA4lg can be used in antiviral treatment.

Description

CTLA4Ig and derivant thereof are used for antiviral

Technical field

The new drug that the present invention relates to known substance is used.Particularly, the present invention relates to application and the new application in the preparation antiviral drugs in treatment virus of CTLA4Ig or CTLA4Ig derivant.

Background technology

CTLA4Ig

The activated T cell plays crucial effect in numerous disease and pathological process.Research shows, the T cell must activation fully under first signal (TCR/MHC-Ag) and costimulating signal combined effect ( ^1,2,3,4, see after and attach the list of references tabulation.As follows).The former is the basis of T lymphocyte activation, and has determined the specificity of t cell responses; The latter is the essential condition of T cell activation, and both are indispensable.Lack or the blocking-up costimulating signal, will cause that anergy (anergy) or the apoptosis (apoptosis) of T cell and clone lose (deletion) ^(2,3,4,5)Found multiple costimulating signal system till now, like B7/CD28-CTLA4 system, CD40/CD40L system, CD95 (Fas)/Fas L, CD2/LFA3, ICAM1/LFA1, VCAM1/VLA4 system etc. ^(6,7,8,9)The latter is a T cell membrane upper surface molecule, and the former is the respective ligand that is expressed on the APC film, and the two-way interaction brings into play the secondary stimulus effect.Discover that in all costimulating signal approach, the B7/CD28-CTLA4 system is considered to most important costimulating signal system (6).

(Cytotoxic Lymphocyte Antigen 4 CTLA4) is the glycoprotein of the 4th species-specific antigen gene code found in the cytotoxic T lymphocyte to CTLA4.Find its encoding gene when the screening mice CTL cell cDNA subtracted library by Brunt etc. the earliest ⁽¹⁰⁾The mankind, its gene is positioned at long-armed 33 bands (2q33) of No. 2 chromosomes (CD28 gene also coexist this site), and single copy is about 4Kb, contains 4 exons and 3 introns.Exons 1 contains 108bp, the one section leading strand that contains hydrophobic amino acid of encoding, and exon 2 contains 348bp; 116 aminoacid in coding CTLA4V district, exon 3 contains 111bp, 37 aminoacid of encoding; Comprise the outer a part of aminoacid of born of the same parents, stride that functional areas begin several amino acid in film district hydrophobic amino acid and the endochylema, exon 4 has 102bp, coding CTLA4 endochylema section Most amino-acids; Be the 1150bp of 3 ' noncoding region, contain 60 the AT repetitive sequence rollers of having an appointment thereafter, maybe be relevant with the stability of control mRNA ⁽¹¹⁾CTLA4 is a kind of transmembrane glycoprotein, and except that signal peptide and termination signal, human CTLA 4 is made up of 187 aminoacid, and the 109th to 111 is glycosylation site.By 166 aminoacid of film outskirt, stride film district 37 aminoacid and 34 aminoacid of cytoplasmic domain are formed.Mainly be expressed on the activating T cell film, and find, it is expressed in to stimulate and reached the peak in back about 48 hours ⁽¹²⁾CTLA4 can combine with B7 molecule on the APC film with CD28 molecule competitive inhibition, thus the B7-CD28 auxiliary signal approach in the blocking-up T lymphocyte activation, and then the activation of blocking t cell.On the other hand, when T cell CTLA4 molecule with after the B7 molecule combines, cause CTLA4 born of the same parents' inner segment phosphorylation, produce negative tonal signal, thereby blocking-up T lymphocyte activation and function thereby think that CTLA4 is a kind of negativity regulatory factor of T cell activation ⁽¹³⁾

The major function of CTLA4 extracellular fragment is to combine with its associated ligands B7 molecule, has found the B7 molecule more than three kinds now, respectively called after B71 (CD80), B72 (CD86), B73 etc. ^(14,15)But the CTLA4 extracellular region is bigger 20 to 100 times than CD28 with the adhesion of B7 molecule ⁽¹⁶⁾Striding the film district is that extracellular region is passed in cell by the bonded signal of B7 molecule.The CTLA4 intracellular region is that extraneous signal is transformed into the negative tonal signal of cell, and reduces the function of T cell.So CTLA4 is bringing into play the effect of reducing the T cell function through two approach at least ^(17,18)The one, combine through CTLA4 extracellular region and CD28 are competitive, and the B7 molecule that suppresses on the antigen presenting cell combines with CD28 molecule on the T cell membrane, thereby make the necessary B7-CD28 auxiliary signal of blocking t cell activation approach, cause the T cell function to be reduced.On the other hand, when CTLA4 with after the B7 molecule combines, directly produce negative tonal signal, reduce the T cell function equally.

As noted earlier; Chinese scholars application of ct scan LA4 and B7 molecule high affinity characteristic; Just endeavouring to develop the CTLA4 extracellular fragment preparation of blocking-up B7-CD28 approach; With the B7/CD28 approach in the blocking t cell activation, and then the complete activation of blocking t cell, finally reach the diseases related purpose of treatment T cell transition activation.From the consideration of aspects such as purification process, linesly etc. at first merge CTLA4 extracellular fragment and part IgG Fc section, and with its called after CTLA4Ig ⁽¹⁹⁾They are with the CH2 of human CTLA 4 extracellular region the 1st to 125 aminoacid and human IgG Fc section, and the fusion of the corresponding gene group in CH3 and γ district forms.Owing to wherein contain IgG Fc section, can use the protein A chromatography purification more conveniently ⁽¹⁹⁾In addition, because of CTLA4 is the immunoglobulin superfamily member, after its extracellular fragment and the fusion of IgG Fc section, can also keeps the integrity and the space conformation of molecule preferably, thereby guarantee good with functions such as the B7 molecule combine.

Discover now; CTLA4Ig is not only to the various autoimmune property disease relevant with the activation of T cell transition and transplant the back immunological rejection certain treatment and preventive effect are arranged, and it is also alleviating the shock damage, prolonging in the gene therapy aspect such as transgene expression and bringing into play important effect.For example, CTLA4Ig has therapeutical effect to ischemical reperfusion injury, can be applicable to can prolong the allograft survival period in the treatment of autoimmune disease, can promote transgene expression etc.

The derivant of CTLA4Ig

CTLA4Ig can be combined its part CD80 and CD86 molecule by extensive proof, and blocks CD80/CD86-CD28 thus and stimulate path to cause the apoptosis (apoptosis) or the no response (anergy) of T cell altogether.But CTLA4Ig combines the ability of CD80 and CD86 molecule that difference is arranged; That is: the affinity of CTLA4Ig and CD80 is stronger; It is 100 times of [Linsley PS of blocking-up CD86-CD28 path that its blocking-up CD80-CD28 stimulates the usefulness of path altogether; Greene JL, Brady W, BajorathJ; Ledbetter JA and Peach R.Human B7-1 (CD80) bind with similar aviditiesbut distinct kinetics to CD28 and CTLA4 receptors.Immunity 1994,1:793-801].Exactly because also stimulate path altogether owing to CTLA4Ig can not effectively block CD86-CD28; So animal model shows CTLA4Ig blocking t cell activation [Ronchese F fully; Hausmann B; Hubele S; Lane P.Mice transgenic for a soluble form of murineCTLA4 show enhanced expansion of antigen-specific CD4+T cells anddefective antibody production in vivo.J.Exp.Med.1994,179:809-817.Sayegh MH, Akalin E; Hancock WW; Et al.CD28-B7 de afteralloantigenic challenge in vivo bits Th1 cytokines but spares Th2.J.Exp.Med.1995,181:1869-1874.Khoury SJ, Akalin E; Chandraker A; Et al.CD28-B7 costimulatory blockade by CTLA4Ig prevents ely inducedexperimental autoimmune encephalomyelitis and inhibits Th1 but spares Th2cytokines in the central nervous system.J.Immunol.1995,155:4521-4524.Griggs ND, Agersborg SS; Noelle RJ; Ledbetter JA, Linsley PS and Tung KS.The relative contribution of the CD28 and gp39 costimulatory pathways in theclonal expansion and pathogenic acquisition of self-reactive T cells.J.Exp.Med.1997,185:393-403.].

In order effectively to block CD80-CD28 and the CD86-CD28 path biological function with enhanced CT LA4Ig simultaneously, Bristol-Myers Squibb company is parent molecule with CTLA4Ig, selects 20 aminoacid to carry out point mutation (site-directed mutagenesis), has screened 2300 filial generation CTLA4Ig fusion rotein; Discovery substitutes the dissociation rate (comparing with wild type CTLA4Ig) that glutamic acid can significantly reduce (2 times) CTLA4Ig mutant and CD86 at 104 with leucine, and biological effectiveness is more excellent, and this albumen is named L104E [Larsen CP; Pearson TC, Adams AB, Tso P; Shirasugi N, Strobert E, Anderson D; Cowan S, Price K, Naemura J; EmswilerJ, Greene J, Turk LA; Bajorath J, Townsend R, Hagerty D; Linsley PS andPeach RJ.Rational development of LEA29Y (balatacept), a high-affinityvariant of CTLA4-Ig with potent immunosuppressive properties.American JTransplant.2005,5:443-453].Be the PCR masterplate again with L104E-Ig, find that the albumen of A29 sudden change descends 4 times for molecule CTLA4Ig with the dissociation rate ratio of CD86 is female, and be named LEA29Y, but the A29 that suddenlys change separately then do not have influence to dissociation rate.Experiment showed, that LEA29Y has stronger and affinity CD80 and CD86 than CTLA4Ig, and its ability that suppresses the T cell proliferation of CD86 regulation and control is 10 times of CTLA4Ig.LEA29Y shows that in experiment made on the living it can effectively suppress humoral immunization, separately or associating routine immunization inhibitor can the significant prolongation animal time-to-live of allosome renal transplantation thing.This research also prove the 24th and 104 of the CTLA4Ig aminoacid sequence to most important [Larsen CP, Pearson TC, Adams AB, the Tso P of combining of part; Shirasugi N, Strobert E, Anderson D; Cowan S, Price K, Naemura J; EmswilerJ, Greene J, Turk LA; Bajorath J, Townsend R, Hagerty D; Linsley PS andPeach RJ.Rational development of LEA29Y (balatacept), a high-affinityvariant of CTLA4-Ig with potent immunosuppressive properties.American JTransplant.2005,5:443-453; Bristol-Myers Squibb Co.:WO0202638.Engineering high avidity CTLA4Ig for therapy of rheumatic diseases.ExpertOpin.Ther.Patents 2002,12:1455-1457].

In addition; Researcher also partly suddenlys change to the Fc of CTLA4Ig, that is: substitute the 130th, 136 and 139 cysteine (cysteine) of IgG constant region with serine (serine); 148 proline (proline) substitutes with serine, and is named Abatacept.Experiment shows that Abatacept no longer has the ability that combines CD16 and CD32, and only has the ability of more weak combination CD69.These characteristics make Abatacept no longer include ADCC and CDC effect [PM.Davis; R.Abraham; L.Xu; ST Nadler and SJ Suchard.Abatacept binds to the Fc receptorCD64 but does not mediate complement-dependent cytotoxicity orantibody-dependent cellular cytotoxicity.J Rheumatology 2007,34:2204-10].

In sum, up to now, CTLA4Ig and all functions derivant thereof; Include but not limited to Abatacept, Belatacept, LEA29Y etc.; All be through combining, thereby blocking-up CD80-CD28 stimulate path altogether with CD86-CD28, reaches the effect of controlling immunne response with CD80 and CD86 molecule.Therefore, have reason to think the every CTLA4Ig and functional deriv thereof that can combine CD80 and CD86 molecule; Include but not limited to Abatacept; Belatacept, LEA29Y etc. have same or analogous biological function; The treatment of diseases that can be used to be correlated with, and can reach same or analogous curative effect.

Abatacept

Abatacept is the clinical CTLA4Ig that is used to treat rheumatoid arthritis, by CTLA4 extracellular region and human IgG1's CH2, the fusion rotein that the CH3 district forms, CD80/CD86 molecule affinity is higher than CTLA4 itself.And it is not engaged with cell surface CD16/CD32, thereby avoid the generation of ADCC effect to the modification of Fc section.In zoopery, the large usage quantity of CTLA4Ig.Therefore no matter Abatacept considers or the consideration of the characteristics of its effect from economic angle, is undoubtedly the succedaneum of best CTLA4Ig.

NK cell (natural killer cell, natural killer cell)

The NK cell is and T, B cell the 3rd monoid lymphocyte arranged side by side.The NK cell quantity is less, in peripheral blood, accounts for 15% of TLC, in spleen, has 3%～4% approximately, also can appear at lungs, liver and intestinal mucosa, but rare in thymus, lymph node and thoracic duct.

The NK cell is bigger, contains cytoplasmic granule, so claim large granular lymphocyte.The NK cell can non-special direct killing target cell, and this NK activity neither needs also not need antibody to participate in advance by antigen sensibilization, and does not have the MHC restriction.

The target cell of NK cell killing mainly is organ, tissue of tumor cell, virus infected cell, bigger pathogen (like antibacterial, fungus and parasite), allograft etc., but does not injure normal cell.

NK cell surface receptor (NKR) can be discerned by the polysaccharide molecule of the cell surface expression of viral infection.The lethal effect of NK cell is by the toxicity molecule mediation that discharges after its activation, like perforin, granzyme and TNF α (tumor necrosis factor) etc.

NK cytosis mechanism

NK cell antineoplastic mechanism maybe be through three steps: (1) NK cell combines with target cell, and the NK cell surface has the receptor of identification target cell, and the target cell surface has by the determinant of receptor identification; (2) target cell stimulates the NK cell to discharge the NK CF; (3) the NK CF combines with target cell, impels some enzyme to change, and causes the target cell dissolving.

In addition, the NK cell plays an important role in body anti-virus infection, immune surveillance through the cytotoxicity of NKT and ADCC performance.(1) viral infection resisting: NK optionally kills and wounds the target cell of viral infection.The IFN that is produced by accesory cell or NK cell can work in coordination with the antivirus action of NK, and normal cell is had protective effect.On the other hand, the virus antigen on virus infected cell surface makes its killer cell effect to NK become responsive more with other surface molecular.External, the target cell of NK solubilized herpesvirus, vaccinia virus, Measles virus, mumps virus, cytomegalovirus and influenza infection.In vivo test shows that NK low activity mouse species is responsive more to some viral infection; The anti-Asialo GM1 antibody that injection suppresses the NK cell can increase the weight of mice influenza property pneumonia.In addition, the NK cell also can kill and wound antibacterial, fungus, protozoon etc. external, and possibly discharging some with the NK cell, to kill and wound medium relevant.(2) the NK cell possibly have more important role than T cell at the tumor cell of immune surveillance, kill mutation.Some disease such as Chediak-Higashi or X property couplet lymphocytic hyperplasia syndrome patient are because the NK functional defect is to the special susceptible of malignant lymphatic cell breeding disease.(3) participate in graft leukemia effect after the bone marrow transplantation (graft-versus-leukemia effect, GVL): can kill and wound some lymph appearance and myeloid leukemia cell at external NK cell.After the bone marrow transplantation in several weeks, in PBL, account for quite high ratio from the NK cell of donor.In addition, the NK cell also can kill and wound some immature cell such as bone marrow stem cell, thymocyte cell subgroup etc. in vivo.

The NK cell surface receptor

The NK cell is closely related to the part on the receptor of the killing activity of target cell and its cell surface and target cell surface.NKG2D is the activation receptor of NK cell, is expressed in all NK cell surfaces, is the main activation receptor of mediation NK cell recognition and lytic virus cell.The part of NKG2D be MHCI class chain related gene product (MICA, MICB) and ULBPS (ULBP3), the part of NKG2D is expressed at multiple virus infected cell for the proteic conjugated protein ULBP1 of human cytomegalic inclusion disease virus UL16, ULBP2.NKG2D influences the killing activity of NK cell to target cell, and the expression that improves NKG2D might improve the antiviral activity of NK cell.

The NK cell receptor also comprises NKp46, NKp44 and NKp30 etc., and wherein NKp46 and NKp30 are expressed in all static and activatory NK cells, and NKp44 then optionally is expressed in activatory NK cell.Ligand molecular on the target cell causes the cracking and a large amount of production of cytokines of target cell with the activation that combines (crosslinked) to cause the NK cell of NK cell receptor.At present confirmed that NKp46 and NKp44 can discern the ligand molecular hemagglutinin on the virus, the acetylsulfuric acid polysaccharide of NKp46 on then can recognizing cells.

Summary of the invention

The inventor is through further investigation; Be surprised to find that CTLA4Ig or CTLA4Ig derivant (include but not limited to Abatacept; Belatacept, LEA29Y etc., preferred Abatacept and Balatacept) cytotoxicity of NK cell (natural killer cell) is had significant activation; And confirm that it is activated receptor such as the NKG2D that has significantly improved the NK surface of cell membrane that CTLA4Ig improves the Cytotoxic molecular mechanism of NK, the expression of NKp44 etc.Experiment showed, that CTLA4Ig can be used for antiviral therapy through improving the cytotoxicity of body NK cell.

Particularly, the present invention provides the following:

1.CTLA4Ig or the application of CTLA4Ig derivant in the preparation antiviral drugs.

2. according to above 1 application, wherein said CTLA4Ig derivant has the activity of cytotoxicity function of the NK cell of activation organism (human body or animal body).

3. according to above 2 application; Wherein preferred said CTLA4Ig derivant is the CTLA4Ig derivant that keeps the ability that combines CD80 and CD86, and more preferably said CTLA4Ig derivant is Abatacept and the Balatacept that keeps and strengthen the ability that combines CD80 and CD86.Said derivant comprises that also wherein the Fc fragment of CTLA4Ig is modified the CTLA4Ig derivant with the ADCC effect of avoiding the mediation of Fc section.CTLA4Ig derivant of the present invention can be in the ability that keep to combine CD80 and CD86 and modified to avoid the ADCC effect of Fc section mediation in the Fc of CTLA4Ig fragment, like Abatacept.

4. according to above 3 application, wherein said CTLA4Ig derivant is Abatacept and Balatacept.

5. according to any one application in above 1 to 4, wherein said virus is selected from the group of being made up of following: herpesvirus, vaccinia virus, Measles virus, mumps virus, cytomegalovirus and influenza virus.

6.CTLA4Ig or the CTLA4Ig derivant is used for activating the application of reagent of cytotoxicity function of the NK cell of organism (human body or animal body) in preparation; Said CTLA4Ig derivant has the activity of the cytotoxicity function that activates the NK cell; Preferred said CTLA4Ig derivant is the CTLA4Ig derivant that keeps the ability that combines CD80 and CD86, and more preferably said CTLA4Ig derivant is Abatacept and the Balatacept that keeps and strengthen the ability that combines CD80 and CD86.Said derivant comprises that also wherein the Fc fragment of CTLA4Ig is modified the CTLA4Ig derivant with the ADCC effect of avoiding the mediation of Fc section.CTLA4Ig derivant of the present invention can be in the ability that keep to combine CD80 and CD86 and modified to avoid the ADCC effect of Fc section mediation in the Fc of CTLA4Ig fragment, like Abatacept.

7.CTLA4Ig or the CTLA4Ig derivant is used for improving the application of reagent of expression of activation receptor of the NK cell of organism (human body or animal body) in preparation; Said CTLA4Ig derivant has the activity of the expression of the activation receptor that improves the NK cell; Preferred said CTLA4Ig derivant is the CTLA4Ig derivant that keeps the ability that combines CD80 and CD86, and more preferably said CTLA4Ig derivant is Abatacept and the Balatacept that keeps and strengthen the ability that combines CD80 and CD86.Said derivant comprises that also wherein the Fc fragment of CTLA4Ig is modified the CTLA4Ig derivant with the ADCC effect of avoiding the mediation of Fc section.CTLA4Ig derivant of the present invention can be in the ability that keep to combine CD80 and CD86 and modified to avoid the ADCC effect of Fc section mediation in the Fc of CTLA4Ig fragment, like Abatacept.

8. according to above 7 application, the activation receptor of wherein said NK cell is NKG2D and/or NKp44.

9. anti-viral pharmaceutical compositions; It comprises CTLA4Ig or CTLA4Ig derivant and medicinal adjuvant; Said CTLA4Ig derivant has the activity of cytotoxicity function of the NK cell of activation organism (human body or animal body); Preferred said CTLA4Ig derivant is the CTLA4Ig derivant that keeps the ability that combines CD80 and CD86, and more preferably said CTLA4Ig derivant is Abatacept and the Balatacept that keeps and strengthen the ability that combines CD80 and CD86.Said derivant comprises that also wherein the Fc fragment of CTLA4Ig is modified the CTLA4Ig derivant with the ADCC effect of avoiding the mediation of Fc section.CTLA4Ig derivant of the present invention can be in the ability that keep to combine CD80 and CD86 and modified to avoid the ADCC effect of Fc section mediation in the Fc of CTLA4Ig fragment, like Abatacept.

The preparation of pharmaceutical composition is known in those skilled in the art, for example referring to Remington ' sPharmaceutical Sciences, and Mack Publishing Co. (A.R.Gennaro version 1985).

10. according to above 9 pharmaceutical composition, it also contains other antiviral activity composition.

11. comprising to said experimenter, a method of treating experimenter's viral infection, this method use CTLA4Ig or above-mentioned CTLA4Ig derivant or according to the step of above 9 or 10 pharmaceutical composition.Preferred said experimenter is the human or animal.

Those skilled in the art can confirm the dosage level of CTLA4Ig and pharmaceutical composition respectively through routine test.Should be appreciated that; Concrete dosage level about any concrete experimenter depends on multiple factor, comprises the seriousness etc. of the disease specific of experimenter's age, body weight, general health state, sex, diet, time of application, route of administration and excretion rate, medication combined and experience treatment.

12. the method according to above 11, wherein said experimenter also accepts another kind of antiviral therapy simultaneously.

13. comprising to said experimenter, a method that activates the cytotoxicity function of NK cell among the experimenter, said method use CTLA4Ig or above-mentioned CTLA4Ig derivant or according to the step of above 9 or 10 pharmaceutical composition.Preferred said experimenter is the human or animal.

14. comprising to said experimenter, a method that improves the expression of the activation receptor of NK cell among the experimenter, said method use CTLA4Ig or above-mentioned CTLA4Ig derivant or according to the step of above 9 or 10 pharmaceutical composition.Preferred said activation receptor is NKG2D or NKp44.Preferred said experimenter is the human or animal.

Should be appreciated that the combination in any that this invention is intended to comprise above-mentioned each item.

Description of drawings

Fig. 1 .CTLA4Ig is to human peripheral blood single nucleus cell (PBMC) toxicity regulating action.A: the CTLA4Ig concentration of adding is to the Cytotoxic influence of PBMC; The activity ratio of the regulating action of B:CTLA4Ig (in this paper and accompanying drawing, also being expressed as CTLA-4Ig or CTLA4-Ig sometimes);

Fig. 2 .CTLA4Ig promotes NK cells of human beings to kill and wound the ability of responsive target cell (>=1 μ g/ml) effectively;

Fig. 3 .CTLA4Ig promotes NK cells of human beings to kill and wound the ability of responsive target cell (＜1 μ g/ml) effectively;

Fig. 4 A:CTLA4Ig is to the influence of NK cell surface NKG2D expression of receptor;

Fig. 4 B:CTLA4Ig is to the influence of NK cell surface NKP44 expression of receptor;

Fig. 5. the cytotoxic effect (ADCC) that antibody relies on activates possible effect in the NK cell function at CTLA4Ig.But the cytotoxicity of A:CTLA4Ig enhanced NK 92; It partly is the non-dependence of ADCC that B:CTLA4Ig strengthens NK cells of human beings toxicity;

Fig. 6 .CTLA4Ig and IL-2 are for the toxic influence of NK cell killing;

Promote NK cell killing toxicity in Fig. 7 .CTLA-4Ig body;

Promote NK cell killing toxicity in the derivant Abatacept body of Fig. 8 .CTLA-4Ig;

The effect of Fig. 9 .CTLA4Ig in the viral infection model;

The CTLA4 zone of Figure 10 .Abatacept and the aminoacid sequence in Fc zone;

The CTLA4 zone of Figure 11 .CTLA4Ig and the aminoacid sequence in Fc zone.

The specific embodiment

Hereinafter is described the reference implementation example in detail the present invention, and said embodiment only is intended as illustrative explanation the present invention, rather than intention restriction scope of the present invention.Scope of the present invention is specifically limited accompanying Claim.

Embodiment 1 CTLA4Ig is to the research of human peripheral blood single nucleus cell (PBMC) toxicity regulating action

1. reagent and material:

Human PBMC: derive from the Xinan Hospital, Chongqing Blood Transfusion Department, the manual White Blood Cells Concentrate that divides;

CTLA4Ig, Recombinant Human CTLA-4/Fc Chimera, Catalog Number:325-CT is purchased the R&D system company from the U.S.;

Human lymphocyte separating medium 200ml, Catalog Number:LTS1077 is purchased the Hao ocean biological product Co., Ltds (TBD) from Tianjin;

CFSE (CF 5(6)-Carboxyfluorescein diacetate succinimide ester), Catalog Number:ALX-610-030 is purchased the biochemicals from Alexis;

Propidium Iodide/ propidium iodide, Catalog Number:537059 is purchased from Calbiochem, San Diego, CA;

K562 (human chronic myelogenous leukemia's cell line): available from Chinese Academy of Sciences's Shanghai cell bank;

Flow cytometer, U.S. Beckman Ku Erte stream type cell analyzer (Beckman co μ ltercell) model: be purchased SC from Cell Lab Quanta.

2. experimental technique

The human PBMC separates: will divide White Blood Cells Concentrate slowly to add on the equal-volume lymphocyte separation medium face by hand, room temperature be centrifugal, 3000rpm/min; 20min, tunica albuginea layer in the middle of getting, a large amount of PBS washed cells; Count subsequent use (list of references: Ulmer; A.J., W.Scholz, M.Ernst; E.Brandt, andH.D.Flad.1984.Isolation and subfractionation of human peripheral bloodmononuclear cells (PBMC) by density gradient centrifugation on Percoll.Immunobiology 166:238-250);

Cytotoxicity detection method (list of references: Marcusson-Stahl; M., and K.Cederbrant.2003.A flow-cytometric NK-cytotoxicity assay adapted for use in rat repeateddose toxicity studies.Toxicology 193:269-279):

With the responsive target cell K562 of CFSE labelling NK cells of human beings: become the storage liquid of 5mmol/L with DMSO dissolving living cells green fluorescence dyestuff (CFSE), keep in Dark Place in-20 ℃.During use, with serum-free RPMI 1640 culture medium (contain L-glutaminate, #C11875500BT, it is subsequent use Invitrogen) to be diluted to the working solution of 5 μ mol/L.Make K562 cell suspension 2-5 * 10 ⁶Individual/ml, add equal-volume CFSE working solution, hatch 10min in 37 ℃, with the cold calf serum of 1640+10% volume end mark immediately.Behind twice of the centrifuge washing, with an amount of RPMI1640 (+10% hyclone) re-suspended cell.

Effector lymphocyte, target cell are cultivated altogether: 10 ⁶Individual PBMC+2 * 10 ⁴The K562 of individual CFSE labelling adds 96 orifice plates, cultivates altogether 2.5 hours.Do not add the natural death contrast of the K562 of PBMC as target cell.

PI (propidium iodide) labelling dead cell detects with flow cytometer; Be made into PI storage liquid 250mg/ml with PBS, working solution 50 μ g/ml.Take out the cell in the culture plate, centrifugal back is with 100 μ lPI working solution labelling dead cells.The cell of band CFSE/PI double labelling is dead K562 cell in the flow cytometer.The killing-efficiency of PBMC can be calculated with formula:

Cytotoxicity (cytotoxicity) %=100 * (the K562 mortality rate-natural mortality rate that adds PBMC)/(100-natural mortality rate)

In co-culture system, add variable concentrations CTLA4Ig (final concentration is respectively 0.1,0.5,2,5,10 and 20 μ g/ml), detect it the toxic influence of PBMC.

Statistical method: one factor analysis of variance, significant difference has been confirmed as in P＜0.05.Used software is spss 13.0.

3. experimental result (seeing Fig. 1 and table 1):

Table 1

	0	0.1	0.5	2	5	10	20
								0		0.119	0.03	0.019	0.003	0.004	0.005
0.1	0.119		0.957	0.855	0.196	0.276	0.408
								0.5	0.030	0.957		1	0.588	0.729	0.880
2	0.019	0.855	1		0.763	0.880	0.969
								5	0.003	0.196	0.588	0.763		1	0.996
10	0.004	0.276	0.729	0.880	1		1
								20	0.005	0.408	0.880	0.969	0.996	1

Annotate: numerical value is P value (as follows) in each table.

Conclusion:

Can find out that from Fig. 1 and table 1 CTLA4Ig promotes the human PBMC to kill and wound the responsive target cell of NK cell, its MEC is 0.5 μ g/ml, above after this concentration, and activation capability and dosage indifference.

Embodiment 2 CTLA4Ig promote NK cells of human beings to kill and wound the ability of responsive target cell (>=1 μ g/ml) effectively

1. reagent and material:

NK cells of human beings separating kit (NK Cell Isolation Kit human), Catalog Number:130-092-657 is purchased from miltenyi biotec company (Germany).

LD?columns，Catalog?Number：130-042-901，miltenyi?biotec

All the other materials are with embodiment 1.

2. experimental technique:

The human PBMC separates with embodiment 1;

NK cells of human beings separates, and the strict test kit description of pressing is operated;

The cytotoxicity detection method:

The method of the responsive target cell K562 of CFSE labelling NK cells of human beings is with embodiment 1.

Effector lymphocyte, target cell are cultivated altogether: 10 ⁵Individual NK cells of human beings+2 * 10 ³The K562 of individual CFSE labelling adds 96 orifice plates, cultivates altogether 2.5 hours.The K562 that does not add PBMC contrasts as the target cell natural death.

The computational methods of the killing-efficiency of PI labelling dead cell, flow cytometer detection and NK cell are with embodiment 1.

In co-culture system, add the CTLA4Ig (being respectively 1,5,10 and 20 μ g/ml) of variable concentrations, detect it the Cytotoxic influence of NK.

Statistical method: one factor analysis of variance, there is significant difference p＜0.05.

3. experimental result (referring to table 2 and Fig. 2):

Table 2

	0	1	5	10	20
						0		0.002	0.001	0.002	0.002
1	0.002		0.474	0.965	1.000
						5	0.001	0.474		0.777	0.436
10	0.002	0.965	0.777		0.946
						20	0.002	1.000	0.436	0.946

Conclusion:

Can find out obviously that from table 2 and Fig. 2 CTLA4Ig promotes NK cells of human beings to kill and wound the ability of responsive target cell effectively.During greater than 1 μ g/ml, CTLA4Ig promotes that the ability of NK cell killing target cell is the dose dependency in concentration.

Embodiment 3 CTLA4Ig effectively promote NK cells of human beings to kill and wound the ability of responsive target cell (＜1 μ g/ml)

1. reagent and material:

With embodiment 2.

2. experimental technique:

The human PBMC separates with embodiment 1;

The cytotoxicity detection method:

Effector lymphocyte, target cell are cultivated altogether: 10 ⁵Individual NK cells of human beings+2 * 10 ³The K562 of individual CFSE labelling adds 96 orifice plates, cultivates altogether 2.5 hours.The K562 that does not add PBMC contrasts as the target cell natural death.The K562 that adds IL-2 is as positive control.

In co-culture system, add the CTLA4Ig (being respectively 0.01,0.1,0.5 and 1 μ g/ml) of variable concentrations, detect it the Cytotoxic influence of NK.

3. experimental result (referring to table 3 and Fig. 3):

Table 3

	0	0.01	0.1	0.5	1	IL-2
							0		0.839	0.090	0.000	0.000	0.000
0.01	0.839		0.652	0.001	0.002	0.000
							0.1	0.090	0.652		0.005	0.007	0.000
0.5	0.000	0.001	0.005		1.000	0.121
							1	0.000	0.002	0.007	1.000		0.091
IL-2	0.000	0.000	0.000	0.121	0.091

Conclusion:

Can find out obviously that from table 3 and Fig. 3 the MEC that CTLA4Ig promotes NK cells of human beings to kill and wound the responsive target cell of NK cell is 0.1 μ g/ml, and activation capability strengthens with concentration, above behind the 0.5 μ g/ml, activation capability and dosage indifference.

IL-2 is the strong activator of NK cell of generally acknowledging; In document through being usually used in doing the positive control of NK cell-stimulating; Be mainly used in the clear and definite CTLA4Ig degree that effect is compared with IL-2 to the NK cell-stimulating here; Finally draw the CTLA4Ig conclusion suitable with IL-2, in the research of back, mention specially NK cell-stimulating ability.

The influence that embodiment 4 CTLA4Ig express NK cell surface activated receptor

1. reagent and material:

Zenapax (the anti-Tac monoclonal antibody of anti-Tac monoclonal antibody injection (Zenapax) Daclizumab, Shanghai Luo Shi (containing hIgG1); (Shanghai Luo Shi produces the medicine be used to prevent acute rejection after the renal transplantation, and Zenapax is a trade name, and Daclizumab is the medicine name, wherein contains the contrast that the hIgG1 fragment can be used as this test)

Fluorescein isothiocyanate (FITC) anti-human CD56 (N-CAM, NCAM), Catalog Number:11-0569-71/25tests is purchased from eBioscience (U.S.);

PE anti-human CD56 (NCAM), Catalog Number:318305/25 tests, Biolegend (U.S.)

PE?anti-human?CD336(NKp44)，Catalog?Number：325107/25?tests，Biolegend

Fluorescein?isothiocyanate(FITC)antihuman?NKG2D/CD314(activating)，Catalog?Number：11-5878/25?tests，eBioscience

All the other reagent and material are with embodiment 2.

2. experimental technique:

The human PBMC separates with embodiment 1;

After separating the NK cell, add 96 orifice plates immediately, 10 ⁵Individual/hole, and add CTLA4Ig2.5 μ g/ml respectively, (the Fc section possibly combine with NK cell surface CD16 equimolecular hIgG1 among the CTLA4Ig of the purification that uses in the in vitro tests; Thereby mediation ADCC effect increases the NK cytoactive, and the purpose that adds hIgG1 is in order to get rid of the influence of ADCC effect) 2.5 μ g/ml, IL-2 (injection recombination human interleukin-2; Be purchased two aigret Pharmaceuticaies from Beijing, bibliographical information thinks that IL-2 can act on the NK cell) 1000U/ml (list of references: Lin, S.J.; R.L.Roberts; B.J.Ank, Q.H.Nguyen, E.K.Thomas; And E.R.Stiehm.1997.Human immunodeficiency virus (HIV) type-1 GP120-specificcell-mediated cytotoxicity (CMC) and natural killer (NK) activity in HIV-infected (HIV+) subjects:enhancement with interleukin-2 (IL-2); IL-12, and IL-15.Clinical immunology andimmunopathology 82:163-173), interact after 16 hours; Take out cell and press document (Dasgupta; S., M.Bhattacharya-Chatterjee, B.W.O ' Malley; Jr., and S.K.Chatterjee.2005.Inhibition ofNK cell activity through TGF-beta 1 by down-regulation of NKG2D in a murine model ofhead and neck cancer.J Immunol 175:5541-5550; And Mavoungou; E.; M.K.Bouyou-Akotet; And P.G.Kremsner.2005.Effects of prolactin and cortisol on natural killer (NK) cell surfaceexpression and function of human natural cytotoxicity receptors (NKp46; NKp44 andNKp30) .Clinical and experimental immunology 139:287-296) method labeled cell surface receptor detects with flow cytometer behind the PBS flush away antibody.

3. experimental result:

A:CTLA4Ig is to the influence (referring to Fig. 4 A and table 4) of the expression of the activation receptor NKG2D of NK cell surface

Table 4

	NK	NK+CTLA4-Ig	NK+IL-2	NK+hIgG1
					NK		0.000	0.000	0.784
NK+CTLA4-Ig	0.000		0.667	0.000
					NK+IL-2	0.000	0.667		0.000
NK+hIgG1	0.784	0.000	0.000

Conclusion:

CTLA4 can raise the expression of NK cell surface NKG2D.

Should be pointed out that the Fc section possibly combine with NK cell surface CD16 equimolecular among the CTLA4Ig of the purification that uses in the in vitro tests, thus the cytotoxicity function of activation NK cell.Because the Fc section of CTLA4Ig is the same with the Fc section of hIgG1, be in order to prove that CTLA4Ig is not the effect of Fc section to the effect of NK so add the hIgG1 purpose, but the effect of CTLA4.

B:CTLA4Ig is to the influence (referring to Fig. 4 B and table 5) of the expression of the activation receptor NKP44 of NK cell surface

Table 5

	NK	NK+CTLA4-Ig	NK+IL-2	NK+hIgG1
					NK		0.000	0.000	0.223
NK+CTLA4-Ig	0.000		0.046	0.000
					NK+IL-2	0.000	0.046		0.000
NK+hIgG1	0.223	0.000	0.000

Conclusion:

CTLA4 can raise the expression of NK cell surface NKp44.

The cytotoxic effect (ADCC) that embodiment 5 antibody rely on activates possible effect in the NK cell function at CTLA4Ig

But A.CTLA4Ig enhanced NK 92 cytotoxicities

1. reagent and material:

CTLA4Ig，Recombinant?Human?CTLA-4/Fc?Chimera，Catalog?Number：325-CT，R&D?system；

Zenapax (the anti-Tac monoclonal antibody of anti-Tac monoclonal antibody injection (Zenapax) Daclizumab, Shanghai Luo Shi (containing hIgG1);

NK92 cell strain: available from Chinese Academy of Sciences's Shanghai cell bank;

CFSE (CF 5(6)-Carboxyfluorescein diacetate succinimide ester), Catalog Number:ALX-610-030, Alexis biochemicals;

Propidium Iodide/ propidium iodide, Catalog Number:537059, Calbiochem, SanDiego, CA;

Flow cytometer U.S. Beckman Ku Erte stream type cell analyzer (Beckman co μ ltercell) model: Cell Lab Quanta SC;

2. experimental technique:

The cytotoxicity detection method:

The responsive target cell K562 of CFSE labelling NK cells of human beings: the storage liquid with DMSO is dissolved into 5mmol/L, keep in Dark Place in-20 ℃.During use, the working solution that is diluted to 5 μ mol/L with the serum-free RPMI-1640 is subsequent use.Make K562 cell suspension 2-5 * 10 ⁶Individual/ml, add equal-volume CFSE working solution, hatch 10min in 37 ℃, with the cold calf serum of 1640+10% volume end mark immediately.Behind twice of the centrifuge washing, with an amount of complete culture solution re-suspended cell.

Effector lymphocyte, target cell are cultivated altogether: 10 ⁶Individual NK92 cell+2 * 10 ⁴The K562 of individual CFSE labelling adds 96 orifice plates, cultivates 4h altogether.The K562 that does not add NK92 contrasts as the target cell natural death.

PI labelling dead cell detects with flow cytometer; Be made into PI storage liquid 250mg./ml with PBS, working solution 50 μ g/ml.Take out the cell in the culture plate, centrifugal back is with 100 μ l PI working solution labelling dead cells.The two mark of CFSE/PI cell is dead K562 cell in the flow cytometer.Then the killing-efficiency of NK cell can be calculated with formula:

Cytotoxicity %=100 * (the K562 mortality rate-natural mortality rate that adds NK92)/(100-natural mortality rate)

In co-culture system, add CTLA4Ig respectively, hIgG1 2.5 μ g/ml detect it to the Cytotoxic influence of NK92.

Experimental result (referring to Fig. 5 A and table 6):

Table 6

	Nk92	Nk92+hIgG1	Nk92+CTLA4Ig
				Nk92		0.720	0.032
Nk92+hIgG1	0.720		0.046
				Nk92+CTLA4Ig	0.032	0.046

Conclusion:

Because there is not CD16/CD32/CD64 (Maki in the NK92 cell surface; G.; H.G.Klingemann, J.A.Martinson, and Y.K.Tam.2001.Factors regulating the cytotoxic activity of the humannatural killer cell line; NK-92.Journal of hematotherapy & stem cell research 10:369-383), can think that it does not possess the ADCC effect.So CTLA4Ig mediates NK92 cytotoxicity potentiation right and wrong ADCC.

It partly is the non-dependence of ADCC that B.CTLA4Ig strengthens NK cells of human beings toxicity

1. reagent and material:

With above-mentioned embodiment.

2. experimental technique:

The human PBMC separates with embodiment 1;

The cytotoxicity detection method:

HIgG1 encapsulates 96 orifice plates: be made into 2.5 μ g/ml among the 10 μ g/ml hIgG1 adding PBS; 200 μ l add 96 orifice plates, 3 parallel holes, 4 ℃; 12 hours (Dubois; S., H.J.Patel, M.Zhang; T.A.Waldmann, and J.R.Muller.2008.Preassociation of IL-15 with IL-15Ralpha-IgG1-Fc enhances its activity on proliferation of NK andCD8+/CD44high T cells and its antitumor action.J Immunol 180:2099-2106).

In co-culture system, add CTLA4Ig respectively, hIgG1 2.5 μ g/ml add cell mixing simultaneously in encapsulating the hole, detect CTLA4Ig to the toxic influence of NK cells of human beings.

Experimental result (referring to Fig. 5 B and table 7):

Table 7

	NK	NK+CTLA4Ig	NK+Soluble?Ig	Nk+bound-Ig
					NK		0.000	0.012	0.02
NK+CTLA4Ig	0.000		0.000	0.000
					NK+Soluble?Ig	0.012	0.000		0.680
Nk+bound-Ig	0.02	0.000	0.680

(Soluble Ig is meant the hIgG1 that in cultivating system, adds solubility; Liberation state in the analog cell microenvironment, bound-Ig then are at the state that test utilized bibliographical information method analogue membrane to express in preceding 12 hours hIgG1 to be fixed on the Tissue Culture Plate)

3. conclusion:

HIgG1 can significantly strengthen NK cells of human beings toxicity through the ADCC effect, and CTLA4Ig has compared significant difference to NK cytotoxicity activation with the ADCC effect that hIgG1 is mediated, and promptly it relies on NK cytotoxicity activation part right and wrong ADCC.

Thus, CTLA4Ig mainly is to be its CTLA4 part to the active part of NK cytotoxicity activation.Therefore, can the Fc of CTLA4Ig partly be modified, avoided the CTLA4Ig derivant of the ADCC effect of Fc section mediation with acquisition.

Embodiment 6 CTLA4Ig and IL-2 are for the toxic influence of NK cell killing

1. reagent and material:

The manual White Blood Cells Concentrate that divides of human PBMC southwest hospital Blood Transfusion Department

CTLA4Ig，Recombinant?Human?CTLA-4/Fc?Chimera，Catalog?Number：325-CT，R&D?system

Human lymphocyte separating medium 200ml, Catalog Number:LTS1077, TBDNK Cell Isolation Kit human, Catalog Number:130-092-657, miltenyibiotec

LD?columns，Catalog?Number：130-042-901，miltenyi?biotec

Injection recombination human interleukin-2 50 ten thousand IU/ bottle, the two aigret Pharmaceuticaies in Beijing

CFSE (CF 5(6)-Carboxyfluorescein diacetate succinimide ester), Catalog Number:ALX-610-030, Alexis biochemicals

Propidium Iodide/ propidium iodide, Catalog Number:537059, Calbiochem, SanDiego, CA

K562 (human chronic myelogenous leukemia's cell line) is available from Chinese Academy of Sciences's Shanghai cell bank

Flow cytometer U.S. Beckman Ku Erte stream type cell analyzer (Beckman co μ ltercell) model: Cell Lab Quanta SC

2. experimental technique:

1, the human PBMC separates: the manual White Blood Cells Concentrate that divides slowly adds on the equal-volume lymphocyte separation medium face, 3000rpm/min, and room temperature, 20min, tunica albuginea layer in the middle of getting, a large amount of PBS washed cells are counted subsequent use;

2, NK cells of human beings separates, and the strict test kit description of pressing is operated

3, the cytotoxicity detection method:

(1) the responsive target cell K562 of CFSE labelling NK cells of human beings: the storage liquid with DMSO is dissolved into 5mmol/L, keep in Dark Place in-20 ℃.During use, the working solution that is diluted to 5umol/L with the serum-free RPMI-1640 is subsequent use.Make K562 cell suspension 2-5 * 106/ml, add equal-volume CFSE working solution, hatch 10min in 37 ℃, with the cold calf serum of 1640+10% volume end mark immediately.Behind twice of the centrifuge washing, with an amount of complete culture solution re-suspended cell.

(2) effector lymphocyte, target cell are cultivated altogether: 10 ⁵Individual NK cells of human beings+2 * 10 ³The K562 of individual CFSE labelling adds 96 orifice plates, cultivates 2.5h altogether.The K562 that does not add PBMC contrasts as the target cell natural death.

PI labelling dead cell detects with flow cytometer; Be made into PI storage liquid 250mg./ml with PBS, working solution 50 μ g/ml.Take out the cell in the culture plate, centrifugal back is with 100 μ l PI working solution labelling dead cells.The two mark of CFSE/PI cell is dead K562 cell in the flow cytometer.Then the killing-efficiency of NK cell can be calculated with formula: cytotoxicity%=100 * (the K562 mortality rate-natural mortality rate that adds NK cells of human beings)/(100-natural mortality rate)

4, in co-culture system, add CTLA4Ig 2.5 μ g/ml and IL-21000U/ml (3) respectively, relatively CTLA4Ig and IL-2 are to the toxic influence of NK cells of human beings.

Experimental result (referring to Fig. 6 and table 8):

Table 8

	NK	NK+hIgG1	NK+CTLA4Ig	NK+IL-2
					NK		0.012	0.000	0.000
NK+hIgG1	0.012		0.000	0.000
					NK+CTLA4Ig	0.000	0.000		0.337
NK+IL-2	0.000	0.000	0.337

(hIgG1 is contrast, is used to get rid of the influence of ADCC effect)

Conclusion:

CTLA4Ig is suitable to the ability and the IL-2 of NK cytotoxicity function activation.

The low dose of CTLA-4Ig of embodiment 7 intravenous injections can promote NK cell killing toxicity in vivo

Reagent and animal:

CTLA-4Ig，Recombinant?Human?CTLA-4/Fc?Chimera，Catalog?Number：325-CT，R&D?system

Erythrocyte cracked liquid preparation: at first prepare storage liquid, i.e. 0.16M ammonium chloride: 8.3g/L, 0.17M Tris: 20.6g Tris is dissolved in the distilled water of 900ml, behind the adjustment pH value to 7.65, complements to 1000ml.Working solution: 90ml 0.16M ammonium chloride and 10ml 0.17M Tris (pH 7.65) are mixed, adjustment pH value to 7.2,0.22 micron filtration sterilization is subsequent use.

YAC-1 (Mus T lymphoma cell line) is available from Chinese Academy of Sciences's Shanghai cell bank

Flow cytometer: U.S. Beckman Ku Erte stream type cell analyzer (Beckman co μ ltercell) model: Cell Lab Quanta SC

The Balb/c mice is available from animal institute of Third Military Medical University

Zenapax (the anti-Tac monoclonal antibody of anti-Tac monoclonal antibody injection (Zenapax) Daclizumab, Shanghai Luo Shi (containing the human IgG1)

Experimental technique:

Mouse tail vein injection CTLA4Ig; Behind the alcohol disinfecting mouse tail vein, 200 μ l (containing 10 μ g CTLA4Ig) along mouse tail vein, less than 30 ° of inserting needles, are injected in the mice body with the 1ml syringe.

Behind the 24h, kill mice, get spleen, add calf serum and grind and to process 350g behind the cell suspension, centrifugal 6min.Abandon the erythrocyte cracked liquid that supernatant adds the above preparation of 10ml, leave standstill behind the 2-4min centrifugal, abandon supernatant after counting subsequent use.

The cytotoxicity detection method:

The responsive target cell YAC-1 of CFSE labelling NK cells in mice: the storage liquid with DMSO is dissolved into 5mmol/L, keep in Dark Place in-20 ℃.During use, the working solution that is diluted to 5 μ mol/L with serum-free RPMI 1640 culture medium is subsequent use.Make YAC-1 suspension 2-5 * 10 ⁶Individual cell/ml adds equal-volume CFSE working solution, hatches 10min in 37 ℃, with the cold calf serum of RPMI 1640+10% volume end mark immediately.Behind twice of the centrifuge washing, with an amount of complete culture solution re-suspended cell.

Effector lymphocyte, target cell are cultivated altogether: with 10 ⁶Individual mouse spleen lymphocyte+2 * 10 ⁴The YAC-1 of individual CFSE labelling adds 96 orifice plates, cultivates 3h altogether.The YAC-1 that does not add the effector lymphocyte contrasts as the target cell natural death.

PI labelling dead cell, last machine testing; Be made into PI storage liquid 250mg./ml with PBS, working solution 50 μ g/ml.Take out the cell in the culture plate, centrifugal back is with 100 μ l PI working solution labelling dead cells.The two mark of CFSE/PI cell is dead YAC-1 in the flow cytometer.Then the killing-efficiency of NK cell can be calculated with formula: cytotoxicity%=100 * (the YAC-1 mortality rate-natural mortality rate that adds mouse spleen lymphocyte)/(100-natural mortality rate).

Do not classify inject CTLA4Ig as matched group as with the mouse spleen lymphocyte of injection people Ig, detecting spleen lymphocyte (being the NK cell) toxicity has zero difference.

Experimental result (referring to Fig. 7 and table 9):

Table 9

	Normal group (normal)	HIg injection groups (hIg injected)	CTLA4Ig injection groups (CTLA4Ig injected)
				Normal group		0.918	0.004
The hIg injection groups	0.918		0.007
				The CTLA4Ig injection groups	0.004	0.007

Conclusion: the low dose of CTLA4Ig of mouse tail vein injection can significantly improve the kill and wound toxicity of its spleen NK cell for target cell YAC-1.

The derivant Abatacept of embodiment 8 intravenous injection CTLA4Ig can promote the toxicity of killing and wounding of NK cell in vivo

Except (trade name is Orencia with Abatacept; Available from U.S. Shi Guibao company (Bristol-Myers-Squibb)) beyond the replacement CTLA4Ig, all the other reagent that use in the present embodiment and animal, experimental technique (detect injection Abatacept (300 μ g/) after 48 hours Balb/c spleen cell to the toxicity of killing and wounding of target cell), statistical method are all identical with embodiment 7.

Experimental result is seen Fig. 8.

Conclusion: the derivant Abatacept of mouse tail vein injection CTLA4Ig can significantly improve the kill and wound toxicity of its spleen NK cell for target cell YAC-1.

The effect of embodiment 9 CTLA4Ig in the viral infection model

Animal, cell and reagent:

SCID (severe combined immunodeficiency mice) mice: be available commercially from animal institute of Third Military Medical University;

MCMV CMV (murine cytomegalovirus (MCMV); Smith strain MCMV; Bukowski, J.F., J.F.Warner; G.Dennert, and R.M.Welsh.1985.Adoptive transfer studies demonstrating the antiviral effect of natural killercells in vivo.The Journal of experimental medicine 161:40-52);

CTLA4Ig：Recombinant?Human?CTLA-4/Fc?Chimera?Catalog?Number：325-CT?R&D?system

Zenapax (the anti-Tac monoclonal antibody of anti-Tac monoclonal antibody injection (Zenapax) Daclizumab, Shanghai Luo Shi (containing hIgG1)

Experimental technique:

Divide into groups: CMV+CTLA4Ig injection groups, CMV+hIgG1 (human IgG1) injection groups, CMV+PBS injection groups

The SCID caudal vein is injected 200 μ l CTLA4Ig (1.5 μ g/ μ l) respectively, hIgG1 10 μ g/ and 200 μ l PBS, each 10; 24h pneumoretroperitoneum injection 8 * 10 ³(experimental procedure is referring to Bukowski for PFU (0.1ml) MCMV CMV; J.F.; J.F.Warner, G.Dennert, and R.M.Welsh.1985.Adoptive transfer studies demonstrating the antiviral effect ofnatural killer cells in vivo.The Journal of experimental medicine 161:40-52); Every other day inject CTLA-4Ig later on one time; HIgG1 and PBS according to the mice life span, draw survival curve.

Experimental result is seen Fig. 9, and wherein

is the CMV+PBS group;

is the CMV+hIgG1 group;

is CMV+CTLA4Ig group (rightmost broken line).

Conclusion:

CTLA4Ig passes through the activation to NK cytotoxicity function, the life span that the ability significant prolongation infects the mice of CMV.

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Claims

1.CTLA4Ig the application in preparation anti-cytomegalovirus medicine.

2. anti-cytomegalovirus pharmaceutical composition, it comprises CTLA4Ig and medicinal adjuvant.

3. according to the pharmaceutical composition of claim 2, it also contains other antiviral activity composition.