CN101070540B - New dendritic cell co-stimulatory molecules - Google Patents

Abstract

A novel costimulatory protein molecule, B7-DC, which is a member of the B7 family, is described as is DNA coding therefor and expression vectors comprising this DNA. B7-DC protein, fragments, fusion polypeptides/proteins and other functional derivatives, and transformed cells expressing B7-DC are useful in vaccine compositions and methods. Compositions and methods are disclosed for inducing potent T cell mediated responses that can be harnessed for anti-tumor and anti-viral immunity.

Description

New dendritic cell co-stimulatory molecules

The application is international application no is PCT/US01/13430, and international filing date is April 27 calendar year 2001, and Chinese application number is 01812016.4, and denomination of invention is the divisional application of " new dendritic cell co-stimulatory molecules " patent application.

Background of invention

The description of background technology

The generation that T lymphocyte immunity reacts is a complicated process, it relates to the interaction of cell and cell and the generation of soluble mediators (cytokine or lymphokine), this reaction is by several T cell surface molecular as " acceptor ", comprise the adjustment of φt cell receptor (TCR) complex body and other " assisting " surface molecular, described " assisting " surface molecular is much the cell surface " differentiation antigen " defined first by monoclonal antibody (" CD molecule ").

Make all lymphocytes reach optimal activation state and need two kinds of signals: a kind of antigen-specific or the signal of clone and a kind of second signal (Janeway of antigen-non-specific, C., Cold Spring Harbor Symp.Quant.Biol.54:1-14 (1989)).Were it not for the common stimulation being called costimulatory molecules (B7 as described below), when lymphocyte runs into separately antigen, clonal anergy can be produced, also referred to as " anergy " (Schwartz, R.Science 248:1349 (1990)) or apoptosis (apoptosis); If provide costimulatory signal, the clone being specific to stimulator antigen will be produced and expand.Do not stimulate altogether, would not occur significantly for the immune response of given antigen increase (June etc. (Immunology Today 15:321-331,1994); Chen etc. (Immunology Today 14:483486); Townsend, SE and Allison, JP (1993) Science 259:368-370).

Immunoreactive character and potential depend on the kind of antigen presenting cell (APC) to a great extent, this cell process by antigen presentation to T cell.T cell combine and activation time, for the density of the peptide antigen/MHC ligand in conjunction with TCR with provide solvable by APC and/or membrane-bound costimulatory signal is important.Due to these reasons, Immunotherapy Strategy has started to concentrate on provides the target antigen of (a) appropriate APC types and (b) suitable costimulatory molecules to strengthen T cell activation.

The APC of the signal required for activating T cell is provided to comprise monocyte/macrophage, bone-marrow-derived lymphocyte and most important dendritic cell (DC).In the past, think that the scavenger cell of activation is the APC of the key causing t cell immune response in vivo.This concept effectively can engulf antigen based on this cell and by antigen at surface display and presenting.Particularly nearest, people forward attention to DC, it can be used as the major initiator that body endoantigen Specific T cell immunity reacts.DC has the different phenotype of scavenger cell with activation, and can be divided into and can cause different immunoreactive different subtype.The functional characteristics of DC is approximately large than scavenger cell 100 times of the ability of their activating naive T cells in vitro.Up to now, be the quantity variance based on the known molecule very important for antigen presentation to the explanation of this potential.The present invention is based on a kind of important quantity variance of discovery.

First signal of antigen presentation is caused by TCR and reacting to each other of antigen, antigen is wherein present in (Allen, Immunol.Today 8:270 (1987)) in the scope of II type major histocompatibility complex (MHC) molecule on APC.Costimulatory signal derives from other molecule, and best what characterize is B7 family (i.e. B7.1, B7.2 with or B7.3), and they are also present on APC.

The part of the best sign of costimulatory molecules such as B7 or reverse acceptor at two kinds of protein of T cell surface expression.CD28 is homodimer sugar-protein (Aruffo and Seed of immunoglobulin (Ig) superfamily, Proc.Natl.Acad.Sci.84:8573-8577 (1987)), it is present on most of ripe human T-cell of working in T cell activation.CD28 is constitutive expression on Resting T cells, and increases after activation.Signal via after φt cell receptor, the ligation inducing T cell proliferation of CD28, and secrete IL-2 (Linsley, PS, etc. (1991) J Exp.Med.173,721-730; Gimmi, CD, etc. (1991) Proc.Natl.Acad.Sci.USA.88,6575-6579; Thompson, C.B., etc. (1989) Proc.Natl.Acad.Sci.USA.86,1333-1337; June, C.H., etc. (1990) Immunol.Today.11,211-6; Harding, F.A., etc. (1992) Nature.356,607-609.).The contact (" intercellular adhesion ") of CD28 mediate cell-cell, the necessary non-antigen dependent form cell-cell interaction of a kind of immune response (Springer etc., Ann.Rev.Immunol.5:223-252 (1987)).

CTLA4 is T cell surface molecular that is a kind of and CD28 very high homology, but it does not express on Resting T cells, occur after T cell activation (Brunet, J.F., etc., (1987) Nature328,267-270).CTLA-4 is identified by the differential screening of the cytolytic T cell cDNA library of mouse, Brunet etc. see above.Linsley etc. discuss the effect of CTLA-4 as the Co receptor of B7 in (1991) J.Exp.Med.174:561-569, and disclose B7 to CTLA4 than to CD28, there is higher affinity.Freeman etc. (1993) Science 262:907-909 discusses the CTLA-4 in B7 deficient mice.The part of CTLA-4 is at Lenschow etc. there is description in (1993) Proc.Nat ' l.Acad.Sci.90:11054-11058.

Cells secrete growth and differentiation-inducing cytokine such as IL-2, IL-4 and IL-6 may be present in Th-B cells contacting district with the form of assembling, and which ensure that and only to activate antigen presentation to the B cell of Th cell, avoid the B cell activating other.

CD28 and CTLA-4 and costimulatory molecules interact, and costimulatory molecules is generally B7.B7 is described to a kind of B cell active antigen at first, called after B7/BB-1, because it finds (Linsley etc., Proc.Natl.Acad.Sci.USA 87:5031-5035 (1990) in B cell.After this, this molecule is called as B7, B7-1 or B7.1).B7 and the B7 homologue particularly described recently are also a member in Ig superfamily, compare with CD28 with CTLA-4, B7 contains the outer Ig structural domain of two born of the same parents: a N-terminal can change (V) structural domain, is next constant region sample (C) structural domain.

B7 family member generally expresses on APC, and as mentioned, they are very important for the activation of Resting T cells.These family members comprise B7-1 (=B7 also claims CD80) and B7-2 (also claiming CD86).About the reference of the description of B7-1 comprises Schwartz, R.H.Cell 71:1065-1068,1992; The .Cell 71:1093-1102 such as Chen, L., 1992; The J Immunol 143:2714-2722 such as Freeman, G.J., 1989; And the J Exp.Med.174:625-631 such as Freeman, G.J., 1991)).About the reference of the description of B7-2 comprises (the Science 262:909-911 813-960 such as Freeman, G.J., 1993).Up to now, mouse B7-1 and B7-2 and people B7-1 and B7-2 be described (Freeman etc., 1989, see above; 1991, see above; With 1993, see above).The human B lymphocyte of activation expresses CTLA4/CD28 in conjunction with counter receptor B7-2 and B7-3, both by CD28 or CTLA4, costimulatory signal is delivered to T cell.

After stimulating 24 hours altogether with the mAb of anti-Ig or anti-II type MHC, B cell expresses B7-2.B7-2 induces detectable IL-2 to secrete and T cell propagation.About 48-72 hour after activation, B cell expresses B7-1 and the third CTLA4 counter receptor with mAbBB-1 qualification, (Yokochi, T, etc. (1982) J Immunol.128,823-827), called after B7-3.B7-3 also can express in the B cell that B7-is negative activated, and can stimulate the propagation of T cell altogether and not produce detectable IL-2, and this shows that B7-1 and B7-3 molecule is different.B7-3 expresses on various cell widely, comprises the B cell of activation, the monocyte of activation, dendritic cell, Langerhans cell and keratinocyte.Latter 72 hours of B cell activation, the expression of B7-1 and B7-3 started to reduce.In conjunction with counter receptor, these CTLA4/CD28 existed on the bone-marrow-derived lymphocyte surface of activation show that the common stimulation of T cell is modulated, partly activated the adjustment of these molecule transient expressions rear by B cell.

B7:CD28/CTLA4 stimulates the importance of approach to be confirmed in vitro and in vivo altogether.Have between the increase that the increase of T cell activity and B7 express direct contact (Razi-Wolf etc., Proc.Natl.Acad.Sci.USA, 89:4210-4214 (1992)).When T cell lack in conjunction with on the cell of the common stimulation part of CD28 with peptide antigen contact time, can anergy be become.This stimulate the blocking-up of approach to cause will producing antigen specific tolerance in mouse and robot system altogether development (Harding etc., see above; Lenschow, D.J. etc. (1992) Science.257,789-792; Turka, LA etc. (1992) Proc.Natl.Acad.Sci.USA.89,11102-11105; Gimmi, CD etc. (1993) Proc.Natl.Acad.Sci USA 90,6586-6590; Boussiotis, V. etc. (1993) J Exp.Med.178,1753-1763).Contrary, B7 negative murine tumor cells expresses the specific immune response of B7 inducing T cell mediation, along with tumor rejection and the permanent protection to tumor challenge.(Chen, L, etc. (1992) Cell 71:1093-1102; Townsend etc., see above; Baskar, S, etc. (1993) Proc.Natl.Acad.Sci.90,5687-5690.).Therefore, the regulation and control of B7:CD28/CTLA4 approach can produce very large potentiality to stimulate or to suppress the immune response in human body.

Identify react to each other (Linsley etc., J.Exp.Med.173:721-730 (1991)) between CD28 and B7 by the extracellular segment of B7 or CD28 and the genetic fusion of Ig C γ 1 chain.When B7 Ig fused protein is fixed, or when B7 is expressed on the Chinese hamster ovary celI surface of cell such as transfection, they stimulate T cell to breed altogether.The stimulation also specific raising stimulating IL-2 transcriptional level of B7+CHO cell-T cell.

U.S.5,521,288 methods describing the reaction of a kind of immunity moderation, the fragment of part DNA encoding by coding B7 contacts with CD28 positive T cell by the method, and the fragment of wherein said part DNA encoding is mainly corresponding to the extracellular domain (ECD) of B7.Also immunity moderation reaction can be carried out with the derivative of B7, derivative is wherein fused protein construct, it comprises B7 ECD and another protein at least partially, if the solubility of change B7, binding affinity and/or valent people Ig C γ 1 structural domain.Such as, the DNA of coding B7 ECD 1-125 amino acids residue is connected with the DNA of coding corresponding to the hinge of people Ig C γ 1, the amino-acid residue of CH2 with CH3 region sequence, forms the DNA fusion product of a coding B7 Ig fused protein.This article also disclose a kind of by administration B7 or B7Ig fused protein with the method by treating the disease of immune system that T cell mediates in conjunction with CD28 acceptor and t cell responses.By CD28+T cell and B7 antigen or the qualitative response of B7 Ig fusion rotein, the T cell in graft versus host disease is inhibit to breed in conjunction with a kind of immunosuppressor.

United States Patent (USP) 5,861,310 tumour cells disclosing modification, this cell can express one or more T cell costimulatory moleculeses, comprises B7-2 and B7-3.A specific embodiments also comprises expresses B7.Described modification can be the nucleic acid transfection with coding B7-2, B7-3 or B7 protein.Tumour cell also can in vivo by genetic modification.The tumour cell of this kind of modification it is said that to treatment tumour patient be useful, to prevent or to suppress the recurrence of transitivity diffusion or Tumor suppression.This article discloses a kind of method of CD4+T cell response of antineoplastic specificity induction.

United States Patent (USP) 5,942,607 nucleic acid disclosing separation, the CTLA4/CD28 part of its new common stimulation T cell activation of encoding.In a specific embodiment, the nucleic acid encoding B7-2 of this separation.This article also discloses a kind of nucleic acid containing disclosed total length B7-2 sequence at least partially.According to this article, this nucleotide sequence can be integrated in different expression vector, and carrier wherein comprises the synthesis instructing corresponding protein or peptide in Mammals and insect cell at multiple host cell.This article also discloses the host cell of conversion, and this cells produce is by the protein of these nucleic acid sequence encodings or peptide and the protein be separated containing B7-2 sequence at least partially and peptide.

Dong H etc., Nat Med 1999 5:1365-1399 describes the 3rd member of B7 family, is named as B7-H1, and it does not combine with CD28, CTLA4 or ICOS (derivable stimulator altogether).The ligation of B7-H1 stimulates T cell to react to polyclone stimulator and isoantigen altogether, preferably stimulates the generation of interleukin 10.It is required for being total to effect of stimulation with the IL-2 produced on a small quantity to B7-H1.This research has identified a kind of costimulatory molecules of previous the unknown, and this molecule may be relevant to cell-mediated immunoreactive negative regulator.Same laboratory (Wang S etc., Blood.2000; 96:2808-2813) describe a kind of new people B7 sample gene, called after B7-H2, its expression is detected on the immature DC surface of cells of monocytic origin.The fused protein of solvable B7-H2 and Ig, B7-H2Ig can and activate T cell but be not Resting T cells combine.Above-mentioned combination is suppressed by the ICOS of soluble form (ICOSIg), and is not suppressed by CTLA4Ig.The Chinese hamster ovary celI of B7-H2 gene transfection can dye by ICOSIg.The CD3 be cross-linked with suboptimal, as stimulator, finds that the common stimulation that B7-H2Ig breeds T cell is dose-dependent, and relevant with the secretion of IL-2, and the CD3 ligation of the best preferably stimulates the generation of IL-10.Author thinks that B7-H2 is the possible part of ICOS T cell molecule.

Swallow MM etc., Immunity, 1999, the 11:423-432 clones reporting a new gene b7h, it is the homologue that the B7 molecule of expressing on APC is close.B7h stimulates the propagation of the T cell of purifying altogether by acting on the acceptor being different from CD28 or CTLA-4.Surprisingly, although B7h expresses in the B cell do not stimulated, at non-lymphoidocyte (the 3T3 cell with TNF α process; Embryo fibroblast) in, this expression also can be induced, and this expression, with LPS, is raised in a kind of non-lymphoid tissue of mouse of effective TNFa Treatment with activating agent.These researchs define a kind of new T cell and stimulate part altogether, indicate and utilize TNF α directly can increase Urine scent in inflammatory process to the induction of B7h.

Yoshinaga SK etc., Nature, 1999,402:827-832 describe new mouse costimulatory receptor-part pair altogether.Acceptor is wherein relevant to CD28, is the mouse homologue of human protein ICOS, and expresses in the T cell and resting memory T cells of activation.Part wherein, itself and B7 molecule homologous, be named as B7 related protein-1 (B7RP-1).B7RP-1 is 1 type transmembrane protein, has the amino acid identities of 20% and 19% respectively with mouse B7.1 (CD80) and B7.2 (CD86).Because B7.1 and B7.2 only has the amino acid identities of 27%, this homologue be effective (Freeman, GJ etc., J.Exp.Med.178:2185-2192 (1993)).This homologue contains for the very important halfcystine of the formation of Ig ring at conserved positions (from the methionine(Met) started, residue 62,138,185 and 242).The total length of B7RP-1 and the relevant position of trans-membrane region and B7 molecular mimicry (Greenfield, EA etc., Crit.Rev.Immunol.18:389-418 (1998)).B7RP-1 expresses in B cell and scavenger cell.ICOS and B7RP-1 not with the protein interaction in CD28-B7 approach, and B7RP-1 stimulates T cell altogether independent of CD28.Lymphocytic hyperplasia is there is in the transgenic mouse of expressing the fused protein (" B7-RPl-Fc ") of the Fc fragment of B7RP-1 and Ig in spleen, lymphoglandula and aggregate lymphatic nodule.When antigen is attacked, strengthen the common stimulating activity confirming B7RP-1 in vivo with delayed hypersensitivity in the antigen pre-sensitizing mouse of B7RP-1-Fc process.Author thinks that ICOS and B7RP-1 is a kind of new receptor-ligand pair of uniqueness, and its structure is relevant to CD28-B7, participates in adaptive immunity reaction.

Yoshinaga SK etc., Int Immunol, 2000,12:1439-1447 was reported and was stimulated altogether by people B7RP-1 and the ICOS human T-cell carried out that interacts October.The KD value of this ligand-receptor Thermodynamic parameters is approximately 33nM, t _(1/2)the dissociation rate (off-rate) of > 10min.TNF α differentially regulates the expression of the people B7RP-1 on B cell, monocyte and DC.TNF α enhancing B cell and monokaryon carefully go up the expression of B7RP-1, but suppress the expression of B7RP-1 on DC.A kind of people B7RP-1-Fc protein or the cell of expressing membrane-bound B7RP-1 stimulate the propagation of T cell in vitro altogether.Special cytokine, as IFN γ and IL-10 by B7RP-1 stimulate altogether induce.Although the level of IL-2 does not significantly increase, the common stimulation of B7RP-1 induction depends on IL-2.These researchs identify people's orthologous gene (human ortholog) of mouse B7RP-1, and characterize the interaction of itself and people ICOS.

PD-1 is the immunosuppression acceptor of being expressed by T, B of activating and medullary cell.The mouse of PD-1 defect shows the autoimmunization of various ways due to the forfeiture of its peripheral tolerance.Freeman, GJ etc., J.Exp.Med.192:1027-1034 (2000) reports the part (PD-L1) of PD-1, and this part is a member of B7 gene family.PD-1 and PD-L1 combines the lymphocytic activation (secretion of propagation, cytokine) that result in and suppress TCR mediation.In addition, the common stimulation of the CD28 mediation of PD-1 signal suppressing suboptimal level.PD-L1 is expressed (person monocytic cell stimulated with TNF γ, the people DC of activation) by APCs.In addition, PD-L1 also expresses in heart and lung.Author infers that the relative size of the PD-L1 signal suppressed on APC and the B7-1/B7-2 signal stimulated altogether can determine the threshold value between the degree of T cell activation and tolerance and autoimmunity.In non-lymphoid tissue, the existence of PD-L1 can to the immunoreactive magnitude of inflammation sites.

Above-cited document does not also mean that above-mentioned any document is all and relevant prior art herein.The statement on all about date of these documents and about the description of content be based on the application can information, but not certainly about the date of these documents or the exactness of content.

In prior art, the example of the report of success " transgenosis " comprises: plasmid DNA is injected directly in mouse muscle tissue by (a), to cause the expression (Wolff at uncertain time limit internal labeling gene, J.A. etc., Science 247:1465 (1990); Acsadi, G. etc., The New Biologist 3:71 (1991)); (b) retroviral vector in body and original position infect vascular tissue be effective; (c) antiretroviral agents trans-portal vein injection and being injected directly in liver is affected vivo gene transfer and expression (Horzaglou, M. etc., JBiol.Chem.265:17285 (1990); Koleko, M. etc., Human Gene Therapy 2:27 (1991); Ferry, N. etc., Proc.Natl.Acad.Sci.USA 88:8387 (1991)); (d) recombinant adenovirus tracheal strips is injected in lung tissue for transfer and long-term expression in the body of foreign gene in pulmonary respiration epithelium be effectively (Rosenfeld, M.A. etc., Science 252:431 (1991)); (e) herpes simplex virus vector can in vivo by transgenosis to (Ahmad in cerebral tissue, F. etc., compile, Miami Short Reports-Advances inGene Technology: The Molecular Biology of Human Genetic Disease, volume 1, BoerringerManneheim Biochemicals, USA, 1991).

The human body therapy of retrovirus-mediated method uses retroviral systems (Temin, H.M., Human Gene Therapy 1:111 (1990) of two preferendum, replication defect type; Temin etc., United States Patent (USP) 4,980,289; Temin etc., United States Patent (USP) 4,650,764; Temin etc., U.S. Patent number 5,124,263; Wills, J.W. United States Patent (USP) 5,175,099; Miller, A.D., U.S. Patent number 4,861,719).This kind of carrier has been used to be imported to by functional DNA in people's cell or tissue, such as, adenosine deaminase gene is imported in lymphocyte, the channel genes of NPT-II gene and encoding tumor necrosis factor is infiltrated in (infiltrating) lymphocyte to tumour.The transgenosis of retrovirus-mediated method generally need target cell proliferation for transgenosis (Miller, D.G. etc., Mol.Cell.Biol.10:4239 (1990)).By be sure oing the preferred target cell of DNA molecular of the present invention by importing and the tumour cell of active growth, and this condition is met.By any method with plasmid transfection and the gene therapy of cystic fibrosis carried out with retroviral vector transfection by Collins etc., United States Patent (USP) 5,240,846 is open.

The DNA molecular of coding B7-DC sequence can be packaged in retroviral vector with package cell line, package cell line wherein produces replication defect type retrovirus, as in the art known (see, such as, Cone, R.D. etc., Proc.Natl.Acad.Sci.USA 81:6349-6353 (1984); Mann, R.F. etc., Cell 33:153-159 (1983); Miller, A.D. etc., Molec.Cell.Biol.5:431-437 (1985); Sorge, J., etc., Molec.Cell.Biol.4:1730-1737 (1984); Hock, R.A. etc., Nature320:257 (1986); Miller, A.D. etc., Molec.Cell.Biol.6:2895-2902 (1986)).The package cell line that can carry out the renewal of transgenosis safely and effectively is also disclosed (Bank etc., the U.S. 5,278,056).

This method can be used to be transferred to by retroviral vector by the tissue selected or organ in site-specific mode.Therefore, such as can use catheter delivery system (Nabel, EG etc., Science 244:1342 (1989)).Use these class methods of retroviral vector or liposome vectors for being effective especially in the blood circulation nucleic acid of expression being transferred to vessel wall or tumour.

Also can use other virus vector, comprise neuronal specificity transfer and stop recombinant adenovirus (Horowitz, M.S., In:Virology, Fields, BN etc., compile, Raven Press, New York, 1990,1679 pages; Berkner, K.L., Biotechniques 6:616 9191988), Strauss, S.E., In:The Adenoviruses, Ginsberg, HS, compile, Plenum Press, New York, 1984, Chapter 11), hsv (HSV).The benefit utilizing adenovirus carrier to carry out people's gene treatment comprises seldom recombinates, do not have to find the human malignant lesion relevant with this viroid, the genome of adenovirus is double-stranded DNA, it can be processed the foreign gene accepting to arrive greatly 7.5kb size, and the adenovirus of living is safe people's vaccine organisms.According to the present invention, adeno associated virus be also used to people treatment (Samulski, R.J. etc., EMBO is (1991) J.10:3941).

Can express DNA molecular of the present invention, and in treatment of the present invention, be particularly vaccinia virus to another kind of carrier useful in the treatment of people, this virus can be made into non-replicating (United States Patent (USP) 5,225,336; 5,204,243; 5,155,020; 4,769,330; Sutter, G etc., Proc.Natl.Acad.Sci USA (1992) 89:10847-10851; Fuerst, T.R. etc., Proc.Natl.Acad.Sci.USA (1989) 86:2549-2553; Falkner F.G. etc.; Nucl.Acids Res (1987) 15:7192; Chakrabarti, S etc., Molec.Cell.Biol. (1985) 5:3403-3409).Descriptions of recombinant vaccinia virus and other description containing the virus of allogeneic dna sequence DNA and the application in immunity and DNA treatment thereof are shown in: Moss, B., Curr.Opin.Genet.Dev. (1993) 3:86-90; Moss, B.Biotechnology (1992) 20:345-362; Moss, B., Curr Top MicrobiolImmunol (1992) 158:25-38; Moss, B., Science (1991) 252:1662-1667; Piccini, A etc., Adv.Virus Res. (1988) 34:43-64; Moss, B. etc., Gene Amplif Anal (1983) 3:201-213.

Except naked DNA or RNA or virus vector, engineering bacteria also can be used as carrier.A large amount of bacterial isolateses comprises salmonella (Salmonella), BCG and Listeria monocytogenes (Listeriamonocytogenes)-(LM) (Hoiseth & Stocker, Nature 291,238-239 (1981); The .J.Exp.Med.168 such as Poirier, TP, 25-32 (1988); (Sadoff, J.C., etc., Science 240,336-338 (1988); Stover, C.K., etc., Nature 351,456-460 (1991); Aldovini, A. etc., Nature 351,479-482 (1991); Schafer, R., etc., J.Immunol.149,53-59 (1992); Ikonomidis, G. etc., J.Exp.Med.180,2209-2218 (1994)).These organisms are used as vaccine carrier and show two promising characteristics: (1) intestines approach is injected, and provides the possibility of oral vaccine delivery; (2) unicellular/scavenger cell is infected and by Antigen Location on special APC.

Except virus-mediated vivo gene transfer, physical method well known in the art also can be used for direct transfer DNA, comprise administration plasmid DNA (Wolff etc., 1990, see above) and particle bombardment mediation transgenosis (Yang, N.-S., etc., Proc.Natl.Acad.Sci.USA 87:9568 (1990); Williams, R.S. etc., Proc.Natl.Acad.Sci.USA 88:2726 (1991); Zelenin, A.V. etc., FEBS Lett.280:94 (1991); Zelenin, A.V. etc., FEBS Lett.244:65 (1989); Johnston, S.A. etc., In VitroCell.Dev.Biol.27:11 (1991)).In addition, electroporation, a kind of known in vitro by transgenosis to the method in cell, DNA of the present invention also can be used for transfer to (Titomirov in in-vivo tissue, A.V. etc., Biochim.Biophys.Acta 1088:131 (1991)).

" carrier mediated transgenosis " also had description (Wu, C.H. etc., J.Biol.Chem.264:16985 (1989); Wu, G.Y. etc., J Biol.Chem.263:14621 (1988); Soriano, P. etc., Proc.Natl.Acad.Sci.USA 80:7128 (1983); Wang, C-Y. etc., Proc.Natl.Acad.Sci.USA 84:7851 (1982); Wilson, J.M. etc., J.Biol.Chem.267:963 (1992)).Preferred carrier be target liposomes (Nicolau, C. etc., Proc.Natl.Acad.Sci.USA 80:1068 (1983); Soriano etc., see above), if the mAb of acidylate is attached to immunoliposome in lipid bilayer (Wang etc., see above).Also polycation can be used, as asialoglycoprotein/polylysine (Wu etc., 1989, see above), binding substances wherein comprises the molecule (asialoorosomucoid in such as liver) that identifies target tissue and a kind of compound in conjunction with DNA with in conjunction with DNA to be transfected.Polylysine is the example of a DNA binding molecule, and it can not damage it in conjunction with DNA.This binding substances is combined for transfer with plasmid DNA of the present invention subsequently.

Plasmid DNA for transfection or microinjection can be prepared by method well known in the art, as utilized the method (Quiagen) of Quiagen, then carrying out DNA by known method and purifying, the method for lifting as set forth herein.

In addition, mentioned by above-mentioned, the B7-DC molecule of transduction is utilized not need stable expression according to the present invention.On the contrary, the transient expression of polypeptide is enough to make transducer cell exercise its immunogenicity and/or common stimulatory function.

Done general explanation to the present invention above, will deepen the understanding of the present invention by reference to embodiment hereafter, embodiments of the invention provide by way of example, if undeclared, these embodiments have no intention to limit the present invention.

Invention field

The invention belongs to biological chemistry and medical field, relate to the new protein surface of dendritic cells selective expression, this albumen mass-energy be used as cell surface molecule or in vaccine composition kind with solvable form to stimulate generation immune response.

Summary of the invention

In order to identify the encoding gene of new dendritic cell (DC) specific costimulatory molecules for T cell activation, the present inventor has screened the deduction cDNA library between DC and the scavenger cell of activation.This cDNA subtraction method can be determined to be expressed by DC but can't help the gene of Expression of Macrophages that activates.Profit has had been found that several new DC specific gene in this way, and these genes are useful strengthening in the potential depending on the vaccine of T cell activation.The application relates to a kind of such gene.

Based on being present in the fact not being present in the macrophage library of activation in DC library, identify a new encoding sequence, called after " B7-DC ".B7-DC gene is a member of the B7 family gene of coding costimulatory molecules.B7-DC is first B7 family member with the specific expressed and different receptor-specific of DC.The product of this gene has vital role in the unique ability of mediation DC stimulation T cell.Functional selection shows that B7-DC has higher activity than B7-1 in the IFN γ stimulating T cell to produce.Therefore, in the composition that B7-DC DNA and polypeptide are used to improve cell and molecular vaccine compositions usefulness and method, and antigen-specific is not considered.

In a specific embodiment, the invention provides a kind of nucleic acid molecule of separation, the mammalian proteins matter of a kind of called after B7-DC of this nucleic acid molecule encoding, this protein in dendritic cell selective expression and not activation scavenger cell on express.This nucleic acid molecule preferably comprises the nucleotide sequence that is selected from SEQ ID NO:1 (people source) or SEQ ID NO:5 (mouse source).The invention still further relates to a kind of under strict hybridization conditions with the isolating nucleic acid of above-mentioned making nucleic acid molecular hybridization.Preferred stringent condition comprises: in 45 DEG C of insulations in 6X sodium chloride/sodium citrate (SSC), then rinse with about 0.2X SSC at about 50 DEG C of temperature.Preferably, above-mentioned nucleic acid molecule contains nucleotide sequence SEQ ID NO:1.As mentioned above preferred nucleic acid molecule encoding a kind of there is the aminoacid sequence being selected from SEQ ID NO:2 and SEQ ID NO:4 protein or the bioactive fragment of this protein of encoding, homologue or other functional derivatives.Preferably, this nucleic acid molecule encoding has the bioactive fragment of the protein of sequence SEQ ID NO:2 (B7-DC in people source) or coding SEQ ID NO:2., homologue or other functional derivatives.

In a preferred embodiment, the extracellular domain of this nucleic acid molecule encoding B7-DC protein, this extracellular domain comprises residue 26-221, and its of encoding it stimulates homologue, fragment or other functional derivatives altogether.

In another embodiment, the above-mentioned nucleic acid molecule of coding B7-DC fused protein comprises:

A () is encoded the first nucleotide sequence of the first polypeptide, this first polypeptide is complete B7-DC protein or a part wherein (being preferably SEQ ID NO:2 or SEQ ID NO:4);

B first nucleotide sequence optionally, merges with the linker nucleic acid sequence of a joint peptide of encoding by () in frame; With

C the second nucleotide sequence that () is connected with the first nucleotide sequence or is connected with linker nucleic acid sequence in frame, this second nucleic acid sequence encoding second polypeptide.

Second polypeptide preferably contains one or more Ig heavy-chain constant domains, the C-structure territory of two of preferred human IgG, preferred IgG1.

Present invention also offers a kind of expression vector, it contains arbitrary above-mentioned nucleic acid molecule, nucleic acid molecule wherein operationally with (a) promotor and (b) optionally, in eukaryotic cell, regulate the Additional regulatory sequences of this expression of nucleic acid to be connected.

Above-mentioned expression vector can be plasmid or virus vector.The RNA viruses particle that these carriers contain rna replicon (DNA-start or RNA) of self-replacation, suicide type RNA carrier, DNA virus (as adenovirus, vaccinia virus etc.) and grow in package cell line.

This carrier DNA or RNA can with gold grain complexing, to be imported in host with particle gun, also can with another kind of polymer complex, such as, in the delivery formulations controlled, this enhancing by polymer is sent to desirable target cell and tissue.

The present invention also comprises a kind of carrier compositions, and it comprises:

(a) first recombinant expression vector, in the sequence of this carrier, incorporate the nucleotide sequence of a coding object antigen, object antigen induction wherein produces immune response; With

(b) second recombinant expression vector, the nucleotide sequence that one or more coding stimulates polypeptide is altogether incorporated in the nucleotide sequence of this carrier, a polypeptide is wherein had at least to be B7-DC, or its bioactive fragment, homologue or other functional derivatives.

Wherein expression vector energy coinfection or a kind of host cell of cotransfection, produce antigen and stimulate polypeptide altogether, the coexpression of polypeptide fragment, homologue or derivative.

In an improvement of above-mentioned embodiment, the invention provides the 3rd nucleotide sequence of coding target protein matter, its (i) promotes that expression product (antigen) is at iuntercellular, diffusion preferably between APC, (ii) improve the displaying of antigen on the APC of express nucleic acid, and/or (iii) promotes to have imported presenting (intersect and cause (cross-priming)) again and showing of the antigen in the host APC of carrier.The nucleic acid of coding target protein matter can merge mutually with both nucleic acid of coding for antigens or costimulatory molecules or more.First or Second support (acid the firstor the second vector) comprise nucleic acid.In one embodiment, the nucleic acid (being preferably B7-DC) of the nucleic acid of coding for antigens, coding costimulatory molecules and the nucleic acid of coding " target protein matter " are incorporated in the construct of a single fusion by this carrier compositions.

The present invention comprises the cell with above-mentioned any nucleic acid molecule or expression vector conversion or transfection.This cell is preferably eukaryotic cell, more preferably mammalian cell, and optimum is chosen cell, and this cell can be dendritic cell or its precursor (progenitors) cell.In another embodiment, this cell is tumour cell, and more preferably this tumour cell carries an antigen, this antigen consistent with a kind of antigen in host's tumour or with its generation cross reaction, thus cause immune response.

Preferred embodiment is the mammalian tumor cell be separated with exogenous nucleic acid molecule or its bioactive fragment, homologue or other functional derivatives transfection of encoding mammalian B7-DC protein (preferred SEQ ID NO:2 or SEQ ID NO:4), when the protein described in above-mentioned tumor cells expression, its fragment, homologue or derivative and this tumour cell contacts with T cell time

I () B7-DC protein, its fragment, homologue or derivative are combined with T cell; With

(ii) tumour cell stimulates T cell to make it breed altogether and/or produces and secrete cytokines.

The present invention also relates to selective expression in dendritic cell and the polypeptide of not expressing on the scavenger cell of activation, this polypeptide has following character:

(a) and the binding partner binds in T cell; With

B () stimulates T cell make it breed and/or produce and secrete cytokines altogether.

The present invention also comprises the bioactive fragment of this polypeptide, homologue or other functional derivatives.

This polypeptide, fragment, homologue or functional derivatives are preferably by the nucleic acid molecule with sequence SEQ ID NO:1 or SEQID NO:5, or the fragment of this nucleic acid molecule, homologue or they encode.Preferred polypeptide has aminoacid sequence SEQ ID NO:2 or SEQ ID NO:4.

This polypeptide or its bioactive fragment, homologue or other functional derivatives can be by the recombinant expressed generation of one of above-mentioned nucleic acid.

Preferred polypeptide contains the extracellular domain of B7-DC protein, is preferably

The amino-acid residue 26-221 of (a) SEQ ID NO:2 (people), or

The amino-acid residue 26-221 of (b) SEQ ID NO:4 (mouse).

Aforementioned polypeptides can form primarily of the extracellular domain of B7-CD.

Present invention also offers a B7-DC fusion polypeptide containing the first fusion partner, this fusion partner comprises all or part of B7-DC protein, this protein

(ii) direct and the second peptide fusion, or

(ii) optionally, merge with the linker peptide sequence merged mutually with the second polypeptide.

Above-mentioned B7-DC fused protein also can with the second polypeptide, preferred one or more Ig heavy-chain constant domains, preferably has corresponding to the hinge area of human normal immunoglobulin matter C γ 1 chain, C _h2 and C _hthe aminoacid sequence in 3rd district merges mutually.

In an embodiment of above-mentioned fused protein, the first fusion partner is the extracellular domain of B7-DC protein, the full length sequence of SEQ IDNO:2 or SEQ ID NO:4.

Binding partners in this fused protein preferred combination T cell, and stimulate T cell altogether when the adequate stimulus that there is φt cell receptor.

Present invention also offers a kind of dimerization or trimerization fused protein, it is dimer or the tripolymer of above-mentioned fused protein.Preferably, each chain by disulfide linkage or other interchain covalent bonds interconnection.

In preferred dimeric fusion protein matter, dimer is by the C of two Ig heavy chains _hthe covalent linkage of the cysteine residues in district is formed by connecting, and cysteine residues is wherein identical with the cysteine residues connected with disulfide linkage in the normal IgH chain of dimerization.

Fused protein of the present invention can comprise the polymer of the first fusion partner of two or more repetition, and the first fusion partner wherein joins end to end each other, or is connected with the joint sequence between one or more monomer.

Present invention also offers an a kind of epi-position to B7-DC protein and have specific antibody, epi-position wherein is not present among the known member of B7 family protein.This epi-position can be wire or the conformational epitope of SEQ ID NO:2 or SEQ ID NO:4 polypeptide.Antibody is preferably monoclonal antibody, more preferably the monoclonal antibody of people or humanization (passing through genetically engineered).

Present invention also offers a kind of utilize above-mentioned antibody to identify or in quantitating cell populations in the method for the cell of its surface expression B7-DC polypeptide, the method comprises

(a) by the cell in cell mass and above-mentioned antibody contacts, to make antibody and to express the Cell binding of epi-position;

B () detects the existence of cell or the quantity of detection by quantitative antibodies cell of antibodies.

Present invention also offers the another kind of method being separated in the cell of its surface expression B7-DC polypeptide from cell mass, the method comprises:

(a) by cell mass and above-mentioned antibody contacts, to make antibody and to express the Cell binding of epi-position;

B () is just being screened with the cell of antibodies or is being born the cell screening and do not have with antibodies.

Present invention also offers a kind of B7-DC of detection in the sample to which polypeptide, its fragment or the existence of homologue or the method for quantitative B7-DC polypeptide, its fragment or homologue, the method comprises following steps:

A (), by the antibody contacts in sample and claim 43, is combined with any polypeptide containing epi-position or fragment to make antibody;

The polypeptide of (b) detection and the polypeptide of antibodies or the existence of fragment or detection by quantitative and antibodies or fragment.

The invention still further relates to the expression that a kind of induction or enhancement antigen are the B7-DC polypeptide in delivery cell or its precursor cell, to strengthen when there is adequate stimulus to φt cell receptor this cell in vitro or stimulate the method for ability of T cell in body altogether, it is delivery cell or precursor cell that the method comprises with expression vector conversion as described above or transfect antigen, thus on cell, induce or strengthen the expression of B7-DC polypeptide.This antigen presenting cell is preferably dendritic cell, and precursor is dendritic cell precursor.

The invention provides and be total to cell co-stimulatory composition and polypeptide the method that stimulator carrys out immune response stimulating.A kind ofly comprise to strengthen the method for mammalian subject to the t cell response of antigenic stimulation the above-mentioned cell using significant quantity to experimenter, preferred tumour cell, with antigenic stimulation thing, cell wherein can strengthen the t cell response of experimenter to antigenic stimulation effectively.Aforesaid method preferred injections of antigens and common stimulating composition simultaneously.

A kind of tumor associated antigen that utilizes comprises to strengthen the method for mammalian subject to the t cell response of antigenic stimulation the above-mentioned tumour cell using significant quantity to experimenter, antigen described in tumor cells expression wherein, the tumour cell used is effective for strengthening experimenter to the t cell response that tumour antigen stimulates.

A kind ofly strengthen the method for mammalian subject to the t cell response of antigenic stimulation, it comprises uses the polypeptide described above of significant quantity, fragment, homologue or functional derivatives to experimenter, or above-mentioned fusion polypeptide or protein, with antigenic stimulation thing, the polypeptide wherein used is effective for the t cell response of enhancing experimenter to antigenic stimulation.

Present invention also offers a kind of mammalian subject that suppresses to the method for the t cell response of antigenic stimulation, it comprises the above-mentioned antibody using significant quantity to experimenter, the antibody wherein used for blocking t cell stimulation or to eliminate antigen reactivity T cell be effective, thus inhibit t cell response.These methods can be used in particular for the experimenter of treated tissue or organ transplantation to suppress transplant rejection and/or to promote to transplant, and for autoantigen, the method can block or reduce autoimmune response and their pathologic sequelae.

The invention provides the method that utilization is undertaken by the T cell of composition internal stimulus of the present invention treating.A kind of immunoreactive method of mammalian subject to antigenic stimulation that strengthen comprises:

A () obtains T cell from experimenter, from the immunology compatible donor of described experimenter or from the acceptable culturing cell system of immunology;

B (), by the above-mentioned cells contacting of T cell ex vivo and significant quantity, wherein this contact is effective for the response of raising T cell to antigenic stimulation; With

(c) to the T cell of experimenter's step of applying (b),

Thus enhance the immune response of experimenter.

In another embodiment, strengthen the immunoreactive method of mammalian subject to antigenic stimulation to comprise:

A () obtains T cell from experimenter, from the immunology compatible donor of described experimenter or from the acceptable culturing cell system of immunology;

B () is by (i) aforementioned polypeptides of T cell ex vivo and significant quantity, fragment, homologue or functional derivatives, or (ii) above-mentioned fusion polypeptide contact, wherein this contact is effective for the response of raising T cell to antigenic stimulation; With

(c) to the T cell of experimenter's step of applying (b),

Thus strengthen (or generation) immune response of experimenter.

Present invention also offers a kind of vaccine composition, comprise

A () (i) expresses the cell of B7-DC construct as described above, (ii) B7-DC polypeptide, fragment, homologue or functional derivatives, (iii) B7-DC fusion polypeptide or protein

B () is usual, the antigen in another kind of source, although the immune response for this antigen be expect-in based on the vaccine of cell, do not need this antigen, cell wherein self can express this antigen (tumour cell as containing tumour antigen);

(c) optionally, a kind of immunologic stimulant of routine or assistant agent; With

(d) for (a), (b) and (c) one pharmaceutically with acceptable vehicle or carrier in immunology.

Induction or enhancing mammalian subject are to an immunoreactive method for antigen, and the method comprises the above-mentioned vaccine composition using significant quantity to experimenter.

Present invention also offers a kind of common stimulating composition used together with antigen or vaccine, said composition comprises:

(a) B7-DC polypeptide (preferred SEQ ID NO:2 or SEQ ID NO:4), its fragment, homologue or functional derivatives or B7-DC fusion polypeptide, and

(b) one pharmaceutically with acceptable vehicle or carrier in immunology.

Strengthen the immunoreactive method of mammalian subject to antigen or vaccine, the method comprises to the co-administered above-mentioned stimulating composition altogether of experimenter and antigen or vaccine.

Stimulate the method that experimenter reacts the systemic immunity of tumour, the method comprises uses genetically engineered tumour cell to experimenter, this cell

A () derives from the tumour in experimenter, and

B () carries out hereditary change by the B7-DC nucleic acid imported in body as above, described nucleic acid is expressed and provided a kind of costimulatory signal in experimenter,

Wherein, rear generation is used directly for the stimulation that the systemic immunity of tumour reacts.

This tumour cell is preferably processed, preferably by radiation treatment, grows after being applied to prevent it.

Before using above-mentioned therapeutic composition, chemotherapeutic tumor regression process, irradiation or surgical discectomy can be carried out to experimenter.

Present invention also offers a kind of method of inducing antitumor reaction in the Mammals containing antigen positive (antigen-positive) tumour, the method comprises:

A () provides the cell of tumour or tumor cell line, this cell

I () expresses the antigen total with mammal tumor;

(ii) with the nucleic acid carrier transfection of above-mentioned coding B7-DC, when expressing, B7-DC molecule causes cell co-stimulatory to the t cell response of tumour antigen;

(ii) optionally, irradiated before (b) step;

(b) to the cell of administration significant quantity, this cell expressing antigen and B7-DC molecule;

Thus inducing antitumor response.

In the above-mentioned methods, this antitumor response feature is:

(A) sum of the maximum perpendicular diameter product of all measurable infringements at least reduces 50%;

(B) sign producing new infringement is not had, and

(C) any original infringement does not all worsen.

Present invention also offers a kind of method that primary growth of induced tumor in the Mammals with tumour or regeneration are disappeared or decayed, the method comprises:

A () provides the cell of tumour or tumor cell line, this cell

I () expresses the antigen total with mammal tumor;

(ii) with the nucleic acid carrier transfection of above-mentioned coding B7-DC, when expressing, B7-DC molecule makes cell co-stimulatory T cell produce reaction to tumour antigen;

(iii) optionally, irradiated before (b) step;

(b) to the cell of administration significant quantity, this cell expressing antigen and B7-DC molecule; Thus induction is reacted the systemic immunity of Melanoma Tumor antigen-specific, thus induction is disappeared or is decayed.

Suppress a method for antigen positive tumor recurrence growth in Mammals, the method comprises:

A () provides the cell of tumour or tumor cell line, this cell

I () expresses the antigen total with mammal tumor;

(ii) with the nucleic acid carrier transfection of above-mentioned coding B7-DC, when expressing, cell co-stimulatory T cell is made to produce reaction to tumour antigen;

(iii) optionally, irradiated before (b) step;

(b) to the cell of administration significant quantity, this cell expressing antigen and B7-DC molecule;

Thus in Mammals, induce the systemic immunity reaction special to tumour antigen, the recurrence growth of this immune response inhibition tumor cell.

Another embodiment relate to a kind of locate to use near (locally-administered) antigen in mammalian subject a kind of costimulatory signal is provided, to promote that Inflammatory response and immunoreactive location occur, thus the method for the former systemic immunity that creates antagonism, the method comprises to experimenter's spot application

A () above-mentioned expression stimulates the cell of the B7-DC polypeptide of significant quantity, fragment, homologue or functional derivatives altogether, and

(b) antigen

Make to stimulate the adjacent regions of health to impel the fixed point of reaction to produce altogether with antigen, produce systemic immunity.

In above method, antigen is preferably tumour antigen, and in (b), this antigen is applied with the form of tumour cell or subeellular antigenic material.This tumour cell also can be the cell of expression B7-DC polypeptide, fragment, homologue or derivative in (a).

Accompanying drawing is sketched

Fig. 1 shows the hB7-DC collection of illustrative plates be positioned on human chromosome 9p24.BAC clones the hB7-DC collection of illustrative plates of RPCI-11.2.

Fig. 2 shows B7-DC between DC and scavenger cell by differential expression.Carry out effective Northern engram analysis by 0.5 μ g/ road DNA of Purified in electrophoresis on the sepharose of 1%, detect the distribution of B7-DC mRNA in marrow DC, spleen DC, scavenger cell, macrophage system and tissue.G3PDH is with comparing.J774A1, Raw264.7, Pu5-1.8 and WEHI cell is macrophage system.BM: marrow.

Fig. 3 shows effective Northern engram analysis that B7-DC expresses on people DC.Road 1 shows the people DC cultivated with GM-CSF+Flt-3L, and road 2 shows Human plactnta and road 3 shows the people DC cultivated with GM-CSF+IL4.From people B7-DC 5 ' and 3 ' UTR oligonucleotide be used as PCR DNA probe to carry out effective Northern engram analysis to people DC total serum IgE.Beta-actin matter is with comparing the quality determining mRNA.

Fig. 4 represents the flow cytometry of the surface expression of display B7-DC on ripe BM-DC.The mouse BM-DCs growing 9 days is closed by Fc, and dyes with control antibodies or B7-DC antiserum(antisera).By adding the specificity that B7-DC-Ig confirms the combination on DC surface to combine to compete anti-B7-DC.

Fig. 5 shows B7-DC and PD-1 and combines, but is not combined with CTLA-4 or CD28.With pCAGGS-B7.1o pCAGGS-B7-DC transient transfection 293T cell.With PD-l-Ig, 28-Ig and CTLA-4-Ig fusion molecule dyeing transfectant, then with the second antibody dyeing of PE mark.The dyeing of PCAGGS (empty carrier) transfectant is negative (not shown).

The common stimulation that Fig. 6 (left figure and right figure) display AntiCD3 McAb and B7-DC-Ig breed T cell.Zuo Tu: the T cell (CD4+CD8) of culture purified in hole, this hole is in advance with fixing B7.1-Ig (◆), B7-DC-Ig (●) or Isotype control (▲) the bag quilt of the AntiCD3 McAb (mAb2C11) and fixed concentration (0.1 μ g/ml) that increase concentration.Result describes a typical test in three.Culturing cell 72h, and mark with 3H-thymidine CPM, count per minute.Right figure: the cd8 t cell of culture purified in hole, this hole is in advance with fixing B7.1-Ig (◆), B7-DC-Ig (●) or Isotype control (▲) the bag quilt of the AntiCD3 McAb and fixed concentration (0.1 μ g/ml) that increase concentration.Result is a typical test in the two.Culturing cell 72h, and use ³h-thymidine CPM marks, count per minute.

Fig. 7 shows the common stimulation of T cells with antigenic specificity proliferative response.With IFN γ process RENCA cell 72 hours, to induce the expression of II type MHC, this cell is cultivated together with the HA110-120 polypeptide of 12.5 μ g/ml.The HA+I-Ed specific transgenic T cells of purifying is added in the B7.1-Ig (◆) of the increase concentration of soluble form, B7-DC-Ig (●) or Isotype control (▲).Culturing cell 48h, and use ³h-thymidine CPM marks, count per minute.Result is a typical test in three.

The secretion of the cytokine of the T cell that Fig. 8 display B7-DC stimulates altogether.

Top: the T cell of culture purified in hole, fixing B7.1-Ig (◆), B7-DC-Ig (●) or Isotype control (▲) the bag quilt of AntiCD3 McAb (0.12 μ g/ml) and 0.1 μ g/ml is used in advance as Fig. 6 (left side) in this hole.Result is a typical test in three.

Bottom: with the HA+I-E of purifying ^dspecific transgenic T cells and solvable B7.1-Ig, the B7-DC-Ig of 2 μ g/ml or Isotype control (marking the same) cultivate the RENCA cell that the γ-IFN with 12.5 μ g/ml HA (110-120) polypeptide processed.Result is a typical test in both.Collect supernatant liquor after cultivating 24h and 48h, and detect the lymphokine of instruction with ELISA.

After Fig. 9 display stimulates in vivo altogether, B7-DC-Ig greatly strengthen Antigen specific proliferation.In suitable transferase 12 .5 × 10 ⁶after the TCR transgenic cell that individual HA is special, after mouse, in palmula, be used alone HA polypeptide (110-120), incomplete Freund's adjuvant (IFA) or be combined IFA and B7-DC-Ig+IFA or a kind of isotype control Ab immunity three groups of mouse.The lymphoglandula of discharging is collected at the 7th day.By 1.5 × 10 ⁵lN cell and HA peptide cultivate 48h, with 1 μ Ci [ ³h] thymidine pulse, and after 12h, detect the radioactivity of combination.

The description of preferred embodiment

The present invention has identified new protein and nucleic acid, and this protein and nucleic acid can as the bases of improving immunotherapeutic composition and method.The common stimulating protein being named as the people of B7-DC and the new of mouse form has been found and has been disclosed in this.

The DNA of encoding human B7-DC has nucleotide sequence SEQ ID NO:1, shows below:

1 atgatcttcctcctgctaatgttgagcctggaattgcagcttcaccagatagcagcttta

61 ttcacagtgacagtccctaaggaactgtacataatagagcatggcagcaatgtgaccctg

121 gaatgcaactttgacactggaagtcatgtgaaccttggagcaataacagccagtttgcaa

181 aaggtggaaaatgatacatccccacaccgtgaaagagccactttgctggaggagcagctg

241 cccctagggaaggcctcgttccacatacctcaagtccaagtgagggacgaaggacagtac

301 caatgcataatcatctatggggtcgcctgggactacaagtacctgactctgaaagtcaaa

361 gcttcctacaggaaaataaacactcacatcctaaaggttccagaaacagatgaggtagag

421 ctcacctgccaggctacaggttatcctctggcagaagtatcctggccaaacgtcagcgtt

481 cctgccaacaccagccactccaggacccctgaaggcctctaccaggtcaccagtgttctg

541 cgcctaaagccaccccctggcagaaacttcagctgtgtgttctggaatactcacgtgagg

601 gaacttactttggccagcattgaccttcaaagtcagatggaacccaggacccatccaact

661 tggctgcttcacattttcatcccctcctgcatcattgctttcattttcatagccacagtg

721 atagccctaagaaaacaactctgtcaaaagctgtattcttcaaaagacacaacaaaaaga

781 cctgtcaccacaacaaagagggaagtgaacagtgctatc 819

People B7-DC protein has aminoacid sequence SEQ ID NO:2, shows below (annotation with leader sequence, membrane spaning domain and cytoplasmic tail):

The ectodomain of this protein is from residue P ²⁶to residue W ²²¹.

The DNA clone comprising the encoding sequence of coding mouse B7-DC has nucleotide sequence SEQ ID NO:3, shows below.This encoding sequence (is drawn and is had underscore, represent with the form of triplet) start from the Methionine codon atg (runic) at Nucleotide 210 place, terminate in tag terminator codon (runic) (Nucleotide 951-953).

gaattcggcacgaggtcaaatgtggcatatctttgttgtctccttctgtctcccaactag 60

agagaacacacttacggctcctgtcccgggcaggtttggttgtcggtgtgattggcttcc 120

agggaacctgatacaaggagcaactgtgtgctgccttttctgtgtctttgcttgaggagc 180

tgtgctgggtgctgatattgacacagacc 209

atg ctg ctc ctg ctg ccg ata ctg aac ctg agc tta caa ctt cat cct257

gta gca gct tta ttc acc gtg aca gcc cct aaa gaa gtg tac acc gta305

gac gtc ggc agc agt gtg agc ctg gag tgc gat ttt gac cgc aga gaa353

tgc act gaa ctg gaa ggg ata aga gcc agt ttg cag aag gta gaa aat401

gat acg tct ctg caa agt gaa aga gcc acc ctg ctg gag gag cag ctg449

ccc ctg gga aag gct ttg ttc cac atc cct agt gtc caa gtg aga gat497

tcc ggg cag tac cgt tgc ctg gtc atc tgc ggg gcc gcc tgg gac tac545

aag tac ctg acg gtg aaa gtc aaa gct tct tac atg agg ata gac act593

agg atc ctg gag gtt cca ggt aca ggg gag gtg cag ctt acc tgc cag641

gct aga ggt tat ccc cta gca gaa gtg tcc tgg caa aat gtc agt gtt689

cct gcc aac acc agc cac atc agg acc ccc gaa ggc ctc tac cag gtc737

acc agt gtt ctg cgc ctc aag cct cag cct agc aga aac ttc agc tgc785

atg ttc tgg aat gct cac atg aag gag ctg act tca gcc atc att gac833

cct ctg agt cgg atg gaa ccc aaa gtc ccc aga acg tgg cca ctt cat881

gtt ttc atc ccg gcc tgc acc atc gct ttg atc ttc ctg gcc ata gtg929

ata atc cag aga aag agg atc tag953

gggaagctgtattacggaagaagtggtctcttcttcccagatctggacctgcggtcttgg 1013

gagttggaaggatctgatgggaaaccctcaagagacttctggactcaaagtgagaatctt 1073

gcaggacctgccatttgcacttttgaaccctttggacggtgacccagggctccgaagagg 1133

agcttgtaagactgacaatcttccctctgtctcaagactctctgaacagcaagaccccaa 1193

tggcactttagacttacccctgggatcctggaccccagtgagggcctaaggctcctaatg 1253

actttcagggtgagaacaaaaggaattgctctccgccccacccccacctcctgctttccg 1313

cagggagacatggaaattcccagttactaaaatagattgtcaatagagttatttatagcc 1373

ctcatttcctccggggacttggaagcttcagacagggtttttcataaacaaagtcataac 1433

tgatgtgttttacagcatcctagaatcctggcagcctctgaagttctaattaactggaag 1493

catttaagcaacacgtcaagtgcccctgctgtggtatttgtttctacttttctgttttta 1553

aagtgtgagtcacaaggtaattgttgtaacctgtgatatcactgtttcttgtgtctcttc 1613

tttcaactacatcttttaaaacaaaaaaaaaaaaaaaaaaaa 1655

SEQ ID NO:5 is the coding sequence portion of SEQ ID NO:3.

By SEQ ID NO:3, the mouse B7-DC protein of the coding region encodes of (such as by SEQ ID NO:5) has aminoacid sequence SEQ ID NO:4, shows below (annotation with leader sequence, membrane spaning domain and cytoplasmic tail):

The ectodomain of this protein is from residue P ²⁶to residue W ²²¹.

The DNA sequence dna (initial called after " butyrophilin matter sample protein " or " Btdc ") of complete mouse B7-DC has GenBank Accession AF142780.2.

basic molecular method

The method is in an embodiment by for a more detailed description.The present inventor utilizes PCR back-and-forth method (PCR Select), and the method has two important features.The first, the initial p CR reaction before hybridization step only needs a small amount of RNA.This technology allows to use highly purified ripe DC, DC wherein substantially to eliminate the scavenger cell of pollution, precursor cell or other potential contamination of cells.Known highly purified like this DC is difficult to a large amount of acquisition.The second, PCR select procedure can clone the gene of low copy number, differential expression.

In order to identify by corresponding to its cell counterpart, the gene that the gene of the DC differential expression of the scavenger cell of activation is relevant to DC exceptional function with qualification, the present inventor applies cDNA substractive process (cDNA subtractionapproach).They employ the PCR-based of improvement, in conjunction with suppression PCR (PCR Select ^tM) " typical differences display analysis (RDA) " (Diatchenko, L. etc., Proc.Natl.Acad.Sci USA 93:66025-6030 (1996)).

conventional recombinant DNA method

The version on basis discloses molecular biological ordinary method, all these are by reference to being incorporated into this, comprise: Sambrook, J etc., Molecular Cloning:A Laboratory Manual, the second edition, ColdSpring Harbor Press, Cold Spring Harbor, New York, 1989; The .CurrentProtocols in Molecular Biology such as Ausubel, FM, Vol.2, Wiley-Interscience, New York, (current version); Kriegler, Gene Transfer and Expression:A Laboratory Manual (1990); Glover, DM, ed, DNACloning:A Practical Approach, volume .I & II, IRL Press, 1985; Albers, B. etc., MolecularBiology of the Cell, the second edition, Garland Publishing, Inc., New York, NY (1989); Watson, JD etc., Recombinant DNA, the second edition, Scientific American Books, New York, 1992; And Old, RW etc., Principles of Gene Manipulation:An Introduction to Genetic Engineering, the second edition, press of University of California (University of California Press), Berkeley, California (1981).

Except as otherwise noted, concrete nucleotide sequence tends to comprise its conservative varient (replacement of such as degenerate codon) of replacing and complementary sequence.Term " nucleic acid " and " polynucleotide " are synonyms, tend to comprise a gene, cDNA molecule, mRNA molecule and their fragment such as oligonucleotide, further also comprise its Equivalent (hereafter will do to explain more fully).The size of nucleic acid represents with kilobase (kb) or base pair (bp).This is according to agarose or polyacrylamide gels (PAGE) and determined by user or disclosed nucleotide sequence measures.The size of the protein molecular weight of kilodalton (kDa) or length (total number of atnino acid) represent.The size of protein measures according to PAGE, order-checking, the aminoacid sequence derived based on the nucleotide sequence of coding or known aminoacid sequence.

Particularly, the cDNA molecule of the aminoacid sequence corresponding to B7-DC or its fragment or derivative of encoding can use the primer deriving from protein sequence disclosed herein to synthesize (see such as U.S.4 by polymerase chain reaction (PCR), 683,202).Then these cDNA sequence are transferred into eucaryon or prokaryotic expression carrier, and the carrier of acquisition, at suitable host cell, such as, can be used to guide the synthesis of B7-DC or its fragment or derivative in COS or Chinese hamster ovary celI.

The present invention comprises the nucleic acid of separation, and this nucleic acid has the nucleotide sequence of coding new B7-DC, its fragment or its Equivalent.Term nucleic acid used herein tends to comprise this kind of fragment or Equivalent.Nucleotide sequence of the present invention can be DNA or RNA.Preferred nucleic acid is that coding has the people B7-DC of sequence SEQ ID NO:1 or the cDNA of its Equivalent.

Preferably, nucleic acid of the present invention is the cDNA molecule of B7-DC at least partially of encoding.This cDNA can be prepared by mRNA, and mRNA wherein extracts from the ripe DC or other cell of this protein of natural expression.The nucleotide sequence of coding B7-DC can obtain from the genomic dna of DC.Therefore, the DNA of coding B7-DC can clone from cDNA or genomic library according to known technology.

The cDNA nucleotide sequence of coding B7-DC can by being separated total mRNA and obtaining from suitable clone.Double-strand cDNA is prepared from total mRNA.The known technology of any one can be utilized to be inserted in suitable plasmid, phage or virus vector by this cDNA.

About nucleotide sequence, term " Equivalent " tends to the encoding sequence comprising analog and/or function equivalence protein.Such as, the proof that can suddenly change as " silence " not changing aminoacid sequence of the natural polymorphism (particularly the 3rd base of codon) of B7-DC nucleotide sequence.But, the polymorphism relating to the aminoacid sequence change in B7-DC may reside in people (or other Mammals) group, those ordinarily skilled in the art can be understood, owing to there is Natural allelic variants, therefore probably can find that there is one or more Nucleotide and change these Alielic variants of (3-4% accounting at most whole encoding sequence) in crowd.In the DNA of coding B7-DC, all this Alielic variants and the nucleic acid of generation and the polymorphism of polypeptide are included in scope of the present invention.

Further, can also be the family member that the one or more naturally occurring isomer of B7-DC protein described herein or relevant, immunology can occur in cross reaction.This kind of isomer or family member are defined as the protein of intimate aminoacid sequence with B7-DC, even if they are by the genes encoding of different genes seat.

nucleic acid fragment

Nucleic acid sequence fragments is defined as the nucleotide sequence with the Nucleotide more less than the nucleotide sequence of encoding full leng B7-DC protein.The present invention includes the nucleic acid fragment of this kind of coded polypeptide, this polypeptide remains the immune response (measuring by the cytokine product of T cell and/or T cell propagation that receive primary activation signal) of the T cell mediation that (1) B7-DC activates in conjunction with the ability of its native ligand in T cell and (2) enhancer or inhibitor (depending on them how to be presented).

Such as, nucleic acid fragment encodes B7-DC polypeptide herein, this polypeptide remains with in conjunction with T cell surface receptor, and transmits costimulatory signal to the lymphocytic ability of T, and acceptor does not wherein have identified (but not being obviously CD28 or CTLA-4).According to another standard, nucleic acid fragment of the present invention is the nucleic acid fragment with the nucleic acid hybridization coming from another kind of animal species, and therefore it can be used to screening assay to detect the new protein that cross reaction occurs with B7-DC.

Usually, the nucleotide sequence of coding B7-DC polypeptide fragment contains the Nucleotide of the encoding sequence from mature protein.But, in some instances, all or part of leader sequence of nucleic acid also can be contained.Nucleotide sequence of the present invention also can contain joint sequence, restriction endonuclease sites that is natural or that modify and to about clone, express and the protein of coding or useful other sequence of the purification process of fragment.These and other nucleotide sequence modified describes or well known in the art herein.

In one embodiment, utilize PCR, by coding corresponding to the aminoacid sequence of the ECD of B7-DC DNA with coding corresponding to people Ig C γ 1 hinge area, C _h2 and C _hthe DNA of the aminoacid sequence in 3rd district connects, thus forms the construct of expressing B7-DC-Ig fused protein, and wherein the ECD of B7-DC contains the amino acid of about site 26-221.

A kind of similar DNA molecule of the B7-Ig of coding fused protein is disclosed in U.S.5,521, in 288, and be preserved in the American type culture collection (American Type Culture Collection) of Rockville.Md, preserving number is 68627.

Technology such as the synthesis, PCR, transformant, carrier construction, expression system etc. of oligonucleotide of resetting and express the DNA of coding B7-DC and solvable B7-DC fused protein are very ripe in this area, and those of ordinary skill in the art is familiar for the material of the standard resource of actual conditions and step.

In another embodiment, by a structural domain of B7-DC or the DNA of fragment and the another kind of B7 family protein of encoding of encoding, the nucleic acid fusion of such as B7.1, B7.2 or B7.3 major part or whole remainder.The DNA sequence dna of complete people B7.1 (CD80) has gene pool (Genbank) registration number X60958; The registration number of mouse sequence is X60958; The registration number of rabbit sequence is U05593.The complete cDNA sequence of people B7.2 (CD86) has GenBank Accession L25259; The registration number of mouse sequence is L25606.

expression vector and host cell

The present invention includes the expression vector of the nucleotide sequence containing coding B7-DC polypeptide, nucleotide sequence wherein and at least one regulate sequence to be operatively connected." be operatively connected " refer to encoding sequence with allow the mode of the expression of encoding sequence with adjustment sequence be connected.Known adjustment sequence is selected to the expression instructing target protein matter in suitable host cell.Therefore, term " adjustment sequence " comprises promotor, enhanser and other expression regulation element.This kind of regulating and controlling sequence is at such as Goeddel, Gene Expression Technology.Methods in Enzymology, volume .185, academic press (Academic Press), San Diego, California. be described in (1990).

Those of ordinary skill in the art can understand, and concrete structure of expression vector of the present invention needs to consider the kind such as the host cell of transfection and/or the protein of expression.

Expression vector of the present invention contains B7-DC in the various embodiment of coding: full length protein and its functional derivatives (definition of this place), comprise the total length nucleic acid molecule of polypeptide fragment, varient, fused protein etc.Therefore, in one embodiment, this expression vector comprises independent or has merged another kind of protein, the nucleic acid of the B7-DC protein such as ECD at least partially of encoding.

This kind of expression vector is used to transfection host cell comprises fused protein or peptide coded protein with expressible dna and production.Be appreciated that the genetically modified cell of expressing B7-DC polypeptide can being enough to make described cell for instant expression of exogenous DNA in for some time of set object.Therefore, if cell as in vivo with the external immunogen all with the common stimulation ability of increase, required for so expressing or the time span of cell survival is its immunogenicity of cells play and/or stimulatory function necessary time altogether.Such as, for the tumour cell of transduction of expression B7-DC of the present invention, the expression of B7-DC can be only 6 hours, preferred 24h, more preferably at least 2-4 days.Certainly, expressing also can be stable (such as, for viable cell).(e.g., (as derivable or constitutive promoter) can the stability required for expression be selected for hereafter discussed suitable expression vector and controlling element.

The invention provides the method for producing B7-DC protein, fragment and derivative.Such as, with the nucleic acid carrier transfection host cell of the B7-DC protein at least partially of encoding, cultivate this host cell under suitable conditions and make it express B7-DC polypeptide.

Host cell also can with one or more expression vector transfection, this expression vector can comprise B7-DC protein DNA and to encode the second protein DNA at least partially at least partially of encoding either individually or in combinations, thus the fusion polypeptide making host cell produce two portions all to contain.

When recombinant vectors contains the DNA of the DNA of a part of B7-DC that encodes and coding another kind of protein such as people IgC γ 1, the fused protein of generation may have the solvability of change, binding affinity and/or valency.Such as, therefore B7-DC Ig fused protein preferably secreted by host cell transfected in culture, and can be separated from substratum.Alternatively, if protein is retained in tenuigenin, then results also lysing cell, isolated protein from lysate.

Culture typically comprises host cell, suitable growth medium and other by product.Suitable substratum is well-known in the art.Utilize conventional protein purification can be separated B7-DC protein from substratum or cell lysate with the technology of peptide, described technology comprises ammonium sulfate precipitation, fractional column chromatography is (as ion-exchange, gel-filtration, affinity chromatography etc.) and/or electrophoresis (usually see " Enzyme Purificationand Related Techniques ", Methods in Enzymology, 22:233-577 (1971)).Once purifying, part or homogeneous, restructuring B7-DC protein of the present invention just can be used for herein by pharmaceutical composition for a more detailed description.

Be converted or transfection to express the eucaryon of B7-DC or its homologue or functional derivatives or prokaryotic host cell all within the scope of the present invention.Such as, B7-DC can bacterial cell such as intestinal bacteria (E.coli), and insect cell (baculovirus), yeast or mammalian cell be Chinese hamster ovary cell (CHO) or people's cells such as.Other suitable host cell can at Goeddel, finds or known to a person of ordinary skill in the art in (1990) (seeing above).

Glycosylation partially or completely can be caused at eukaryotic expression and/or formed in the corresponding chain of recombinant protein or interchain disulfide bond.

The example carrying out the carrier of expressing in cereuisiae fermentum (S.cerevisiae) comprise pYepSecl (Baldari etc., (1987) EMBO J6:229-234), pMFa (Kurjan etc. (1982) Cell 30:933-943), pJRY88 (Schultz etc., (1987) Gene 54:113-123), with pYES2 (Invitrogen Corporation, San Diego, California).For the baculovirus vector of marking protein in the insect cell (SF 9 cell) cultivated comprise pAc series (Smith etc., (1983) Mol.Cell Biol.3:2156-2165) and pVL series (Lucklow, V.A., and Summers, M.D., (1989) Virology 170:31-39).Usually, by COS cell (Gluzman, Y., (1981) Cell 23:175-182) and this kind of carrier such as pCDM 8 (Aruffo A. and Seed, B., see above) combine with transient amplification/expression in mammalian cell, and CHO (dhfr-negative CHO) cell and carrier such as pMT2PC (Kaufman etc. (1987), EMBOJ.6:187-195) use and increase to stablize in mammalian cell/express.NS0 myeloma cell line (a kind of NADPH-linked glutamate synthase expression system) is purchased from Celltech company limited.

Usually, in fusion expression vector, introduce a proteolytic cleavage site in the junction of reporter group and target protein matter, to make after purified fusion protein matter, target protein mass-energy is enough to be separated with reporter group.The protease and the recognition sequence thereof that are suitable for this cutting comprise Xa factor, zymoplasm and enteropeptidase.

Typical fusion expression vector comprises pGEX (Amrad company, Melbourne, Australia), pMAL (New England Biolabs, Beverly, and pRIT5 (Pharmacia Mass.), Piscataway, New Jersey), these carriers are respectively by glutathione S-transferase, maltose E binding protein, a-protein merges mutually with object recombinant protein.

Derivable nonfusion expression vector comprises pTrc (Amann etc., (1988) Gene 69:301-315) and pET 11d (Studier etc., Gene Expression Technology:Methods in Enzymology 185, academic press, San Diego, California. (1990) 60-89).What the expression of goal gene depended on that host RNA polymerase carries out from the hybrid trp-lac fusion promotor in pTrc transcribes, and the expression being inserted into the goal gene in pET 11d depends on transcribing by carrying out from T7gn10-lacO promoter, fusion of mediating of the viral rna polymerase of coexpression (T7gn1).This varial polymerases is provided by Host Strains BL21 (DE3) or HMS174 (DE3), and described Host Strains is from the stop gamma bacteriophage with the T7gn1 be positioned under the control of lacUV 5 promoter transcription.

One embodiment of the invention are the transfectional cells of again expressing new B7-DC.For the cell such as ripe DC that have expressed B7-DC, the cell of transfection is in the B7-DC protein of its surface expression increasing amount or its fragment.

Such as, expression vector transfection tumor cell that following tumor cell surface instructs B7-DC to express is used in as sarcoma, melanoma, leukemia, lymphoma, cancer or neuroblastoma.The tumour cell of this kind of transfection can be used as immunogen to induce therapeutic anti-tumor immunity described herein.

the structure of carrier

Building suitable encoding containing object uses standard well known in the art to be connected and restriction technologies with the carrier of regulating and controlling sequence.By the form cutting on demand of the oligonucleotide of the plasmid of separation, DNA sequence dna or synthesis, tailing be connected again.

The DNA sequence dna forming carrier can have many sources.Backbone Vector and regulator control system are generally present in " host " carrier available, be used as sequence container in construct.For relevant encoding sequence, initial construct can be and be often a kind of probably suitable sequence again obtained from cDNA or genome dna library.But, once this sequence is disclosed, just whole gene orders can be synthesized in vitro from single nucleotide derivative.Whole gene order of the gene of suitable length such as 500-1000bp by synthesizing independent overlapping complementary oligonucleotide, and can be prepared by the Non-overlapping Domain of archaeal dna polymerase filling strand under deoxynucleotide triphosphoric acid existent condition.The method has successfully constructed the gene of several known array.Such as, see Edge, M.D., Nature (1981) 292:756; Nambair, K.P., etc., Science (1984) 223:1299; And Jay, E., J Biol Chem (1984) 259:6311.

The phosphotriester method described by above-mentioned reference or Beaucage, S.L. can be utilized, and Caruthers, M.H., Tet Lett (1981) 22:1859; And Matteucci, M.D., and the oligonucleotide that the phosphoramidite method preparation described by Caruthers, M.H., J Am Chem Soc (1981) 103:3185 is synthesized, also can prepare with commercially available oligonucleotide automatic DNA synthesizer DNA.At 50mM Tris, H 7.6,10mM MgCl ₂, 5mM dithiothreitol (DTT), 1-2mMATP, 1.7pmoles γ- ³²p-ATP (2.9mCi/mmole), 0.1mM spermidine, 0.1mM EDTA deposits in case, realizes annealing with the substrate of the polynucleotide kinase process 1nmol of excessive such as about 10 units or carries out the kinases process marking front strand.

Once thereby is achieved each several part of required carrier, just can cut it with the restriction of standard and linker and be connected.The cutting of site-specific DNA under the generally well-known condition in this area, can come with the process of suitable restriction enzyme (or enzyme) under the condition particularly illustrated by the manufacturers of these commercially available restriction enzymes.See, as New England Biolabs, Product Catalog.In general, in the buffered soln of about 20ml, the plasmid of 1mg or the DNA sequence dna enzyme of 1 unit cut; In embodiment herein, typically, use excessive restriction enzyme to guarantee the complete digestion of DNA substrate.Although change allows, it is generally incubation 1 to 2 hours at about 37 DEG C.After each incubation, with phenol/chloroform extrct deproteinize, then can use ether extracting, by with alcohol settling, from aqueous phase, reclaim nucleic acid.If desired, standard technique can be utilized to be separated the cutting fragment of different size by polyacrylamide gel or agarose gel electrophoresis.The general description that clip size is separated is shown in Methods in Enzymology (1980) 65:499-560.

When existence four kinds deoxynucleotide triphosphoric acid (dNTPs), by 50mM Tris pH 7.6,50mM NaCl, 6mM MgCl ₂, in 6mM DTT and 0.1-1.0mM dNTP, make it become flat end with the large fragment (Klenow) of E.coli DNA polymerase i incubation 15-25 minute process restriction cutting fragment at 20-25 DEG C.Even if there are four kinds of dNTPs, this Klenow fragment is filled with 5 ' single-stranded overhang, and has cut away 3 ' outstanding strand.If need, in the limited field that the character by overhang determines, can by provide only one or optionally provide dNTP to carry out Selective repair.After Klenow process, use alcohol settling with phenol/chloroform extracting mixture.Under suitable conditions with S1 nuclease or BAL-31 process, any single stranded portion is hydrolyzed.

In 15-50ml volume, under following standard conditions and temperature, carry out typical ligation: such as, 20mM Tris-HCl pH 7.5,10mM MgCl ₂10mM DTT, 33 μ g/ml BSA, 10-50mMNaCl, with the T4 DNA ligase of 40 μMs of ATP, 0.01-0.02 (Weiss) units, 0 DEG C (" sticky end " connects) or 1mM ATP, the T4 DNA ligase of 0.3-0.6 (Weiss) unit, 14 DEG C (" flat end " connects).Carrying out the DNA total concn that intermolecular " sticky end " connect is 33-100 μ g/ml (final total concn is 5-100nM).Carrying out the final total concn of DNA that intermolecular " flat end " connect is 1mM.

Employ in vector construction " carrier segments ", this fragment self is connected with preventing to remove 5 ' phosphoric acid with bacterial alkaline phosphatase (BAP) or calf intestine alkaline phosphatase (CIAP) process usually.In about 10mMTris-HCl, 1mM EDTA, pH 8, with the amount BAP of about 1 unit/mg carrier or CLAP 60 DEG C of digestion about 1 hour.With this product of phenol/chloroform extracting, use alcohol settling.Alternatively, can prevent from connecting again in by the carrier of other restriction enzyme double digestion, and be separated unnecessary fragment.

Can introduce sudden change to produce varient of the present invention in encoding sequence by any method, these sudden changes comprise simple disappearance or insertion, systematic absence, insertion or the replacement of bunch base or the replacement of single base.

Such as, site-directed mutagenesis is utilized to modify B7-DC DNA sequence dna (cDNA or genomic dna), site-directed mutagenesis is known technology, its scheme and reagent are commercially available (Zoller, MJ etc., Nucleic AcidsRes (1982) 10:6487-6500 and Adelman, JP etc., DNA (1983) 2:183-193)).Such as, by with connect mixture first Transformed E .coli bacterial strain MC1061 (Casadaban, M., etc., J Mol Biol (1980) 138:179-207) or other suitable host determine the exact connect ion of plasmid construction.Utilizing conventional method, according to the mode of plasmid construction, screening transformant based on there is penbritin one, tsiklomitsin one or other antibiotics resistance gene (or other selection markers).Prepare plasmid from the transformant with optional chloramphenicol amplification, optionally then carry out paraxin amplification (Clewell, DB etc., Proc Natl Acad Sci USA (1969) 62:1159; Clewell, D.B., J Bacteriol (1972) 110:667).Several micro-DNA fragmentation (preps) of usual use, see, as " Holmes, DS, etc., Anal Biochem (1981) 114:193-197; Birmboim, HC etc., Nucleic Acids Res (1979) 7:1513-1523.By limiting and/or checking order by the dideoxyribonucleoside method (Proc Natl Acad Sci USA (1977) 74:5463) of Sanger or carry out the DNA of analytical separation by the method for .Methods in Enzymology (1980) 65:499 such as Maxam, dideoxyribonucleoside acid system is wherein at Messing, Deng., do further description in Nucleic Acids Res (1981) 9:309.

By the technology of routine, as carrier DNA imports in mammalian cell by the transfection of calcium phosphate or calcium chloride coprecipitation, the mediation of DEAE-dextran, fat transfection or electroporation.The suitable method of transformed host cell can at Sambrook etc. and see above, and find in other article.

Usually, in fusion expression vector, imported a proteolytic cleavage site in the junction of reporter group and target protein matter, after purified fusion protein matter, made target protein mass-energy enough be separated with reporter group like this.The protease and the recognition sequence thereof that are suitable for this cutting comprise Xa factor, zymoplasm and enteropeptidase.

Typical fusion expression vector comprise pGEX (Amrad company., Melbourne, Australia), pMAL (New England Biolabs, Beverly, and pRIT5 (Pharmacia Mass.), Pi Sitawei, New Jersey), these carriers are respectively by glutathione S-transferase, maltose E binding protein, a-protein merges mutually with object recombinant protein.

Derivable nonfusion expression vector comprise pTrc (Amann etc., (1988) Gene 69:301-315) and pET 11d (Studier etc., Gene Expression Technology:Methods in Enzymology 185, academic press, San Diego, California. (1990) 60-89).What the expression of goal gene depended on that host RNA polymerase carries out from the hybrid trp-lac fusion promotor in pTrc transcribes, and the expression being inserted into the goal gene in pET 11d depends on transcribing by carrying out from T7gn10-lacO promoter, fusion of mediating of the viral rna polymerase of coexpression (T7gn1).This varial polymerases is provided by Host Strains BL21 (DE3) or HMS174 (DE3), and described Host Strains is from the stop gamma bacteriophage with the T7gn1 be positioned under the control of lacUV 5 promoter transcription.

promotor and enhanser

Transcribing of the nucleotide sequence of " being operatively connected ", in conjunction with RNA polymerase, is started in the promoter region of DNA or RNA molecule.As using herein, " promoter sequence " refers to the nucleotide sequence of promotor, and this promotor is positioned on DNA or RNA chain, by rna polymerase transcribe.When two sequences of nucleic acid molecule, as promotor and encoding sequence be interconnected in some way time, they " are operatively connected ", and described mode allows all transcribed rna transcription body in same rna transcription body or in a permission sequence of these two sequences to start to be extended in second sequence.Therefore, if promoter sequence initial transcribe the encoding sequence rna transcription body that generation one is operatively connected, then the encoding sequence of two sequences such as promoter sequence and DNA or RNA is operatively connected.In order to realize " being operatively connected ", do not need two sequences are closed on immediately each other in linear order.

The preferred promoter sequence of the present invention must be exercisable in mammalian cell, can be eucaryon or viral promotors.Suitable promotor can be derivable, quenchable or composing type.The example of the promotor of composing type is viral promotors MSV-LTR, this promotor in various types of cell all effectively and have activity, and, compared with other promotor of great majority, this promotor stagnate and growth cell in there is the same activity strengthened.Other preferred viral promotors comprises and is present in CMV-LTR (from cytomegalovirus) (Bashart, M. etc., Cell 41:521 (1985)) or RSV-LTR (from Rous sarcoma virus) (Gorman, C.M., Proc.Natl.Acad.Sci.USA 79:6777 (1982)) in promotor.Other useful promotor be mouse metallothionein I gene promotor (Hamer, D., etc., J.Mol.Appl.Gen.1:273-288 (1982)); The TK promotor (McKnight, S., Cell 31:355-365 (1982)) of simplexvirus; SV40 early promoter (Benoist, C., Deng., Nature 290:304-310 (1981)) and yeast gal4 gene promoter (Johnston, S.A., Deng., Proc.Natl.Acad.Sci. (USA) 79:6971-6975 (1982); Silver, P.A., etc., Proc.Natl.Acad.Sci (USA) 81:5951-5955 (1984)).The transcription factor relevant with promoter region and respective activity describe with other illustrative in conjunction with the DNA of transcription factor and comprise: Keegan etc., Nature (1986) 231:699; Fields etc., Nature (1989) 340:245; Jones, Cell (1990) 61:9; Lewin, Cell (1990) 61:1161; Ptashne etc., Nature (1990) 346:329; Adams etc., Cell (1993) 72:306.The relevant disclosure of above-mentioned listed all reference is by reference to being incorporated into this.

Described promoter region can also comprise eight aggressiveness districts further, this eight aggressiveness district by with certain protein interaction in particular organization, play a role as tissue-specific enhancer.The enhancing subarea of DNA construct of the present invention has specificity to transfected target cell or by the enhancing subarea of the cytokine high level activation of this kind of target cell.The example of carrier (plasmid or retrovirus) is disclosed in (Roy-Burman etc., U.S. Patent number 5,112,767) in.About the generality discussion of the enhanser in transcribing and activity thereof is see Lewin, B.M., Genes IV, Oxford University Press (Oxford University Press), Oxford, (1990), 552-576 page.Useful especially enhanser is retrovirus enhanser (such as viral LTR).Enhanser is preferably placed at the upstream of promotor, influences each other to express with stimulated gene with promotor.If use retrovirus vector, endogenous contaminating virus LTR is to be used as weak enhanser, and can the enhancer sequence needed for other replace, this required enhancer sequence gives tissue specificity or the character needed for other, as the transcriptional efficiency of the DNA molecular to coding B7-DC of the present invention.

Nucleotide sequence of the present invention also can use standard technique chemosynthesis.The various methods of chemosynthesis polydeoxyribonucleotide are known, comprise solid phase synthesis, as peptide symthesis, utilize commercially available DNA synthesizer its can fully automated (see, e.g., Itakura etc. U.S. Patent number 4,598,049; Caruthers etc. U.S. Patent number 4,458,066; With Itakura U.S. Patent number 4,401,796 and 4,373,071, by reference to being incorporated into this).

proteins and peptides

The present invention comprises a kind of " separation " B7-DC protein with sequence SEQ ID NO:2 or SEQ ID NO:4.Specification sheets of the present invention for the people of total length and mouse B7-DC protein (and DNA), but is to be understood that the homologue of B7-DC that have feature disclosed herein, that come from other mammalian species and its mutant also belong to scope of the present invention.

The present invention also comprises " functional derivatives " of B7-DC, and " functional derivatives " refers to the varient that amino acid is replaced, and " fragment " or " chemical derivative " of B7-DC, term " fragment " and " chemical derivative " are explained hereinafter.A functional derivatives remains with detectable B7-DC activity, and preferably T cell can be stimulated active in conjunction with the acceptor in T cell and altogether, this activity allows it according to effectiveness of the present invention." functional derivatives " comprises " varient " and " fragment ", no matter and they are used simultaneously in the present invention or are used alone.

A kind of Functional homologue must have above-mentioned biochemistry and biological activity.Consider its functional character, the homologous proteins B7-DC coming from other species used, comprise the protein still do not found, if these protein have sequence similarity and the biological chemistry enumerated and biological activity, so these protein all within the scope of the present invention.

In order to determine the identity per-cent of two aminoacid sequences or two nucleotide sequences, in order to the object contrast sequence (such as all importing breach to carry out the best contrast in one of the first and second amino acid or nucleotide sequence or both, removing nonhomologous sequence to compare) that the best compares.In a preferred control methods, compared for cysteine residues.

In a preferred embodiment, the sequence length compared at least accounts for 30% of reference sequences length, is preferably at least 40%, is more preferably at least 50%, is even more preferably at least 60%, and is even more preferably at least 70%, 80% or 90%.Such as, when second sequence contrasts with the people B7-DC protein amino acid sequence (SEQ ID NO:2) containing 276 amino-acid residues, at least 83, be preferably at least 110, be more preferably at least 138, even be more preferably at least 166, and be even more preferably at least 193,221 or 248 amino-acid residues are contrasted).Then the amino-acid residue (or Nucleotide) of more corresponding amino acid sites (or nucleotide site).If a position in First ray and the corresponding position in the second sequence have identical amino-acid residue (or Nucleotide), so these two molecules are consistent (amino acid used herein or nucleic acid " identity " are equal to amino acid or nucleic acid " homology ") in this position.Consider the needs of the best contrast in order to carry out two sequences and the quantity of breach that imports and the length of each breach, the identity per-cent of two sequences is the parameters of the same number of sites that a characterised sequences has.

Gene comparision between two sequences and identity percentage test can utilize mathematical algorithm.In a preferred embodiment, the identity per-cent of two aminoacid sequences uses Needleman and Wunsch (J.MoL.BioL.48:444-453 (1970)) operational method, use Blossom 62 matrix and PAM250 matrix and 16,14,12,10,8, the breach value (gap weight) of 6 or 4 and the length value (length weight) of 1,2,3,4,5 or 6 determine, this operational method be contained in GAP program in GCG software package ( http:// www.gcg.comcan obtain) in.In another preferred embodiment, identity per-cent between two nucleotide sequences use GAP program in GCG software package ( http:// www.gcg.comcan obtain), use NWSgapdna, CMP matrix and 40,50,60, the gap weight and 1 of 70 or 80,2,3,4, the length weight of 5 or 6 measures.In another embodiment, the identity per-cent of two amino acid or nucleotide sequence is with E.Meyers and W.Miller operational method (CABIOS, 4:11-17 (1989)), use PAM120 weight residue table (weight residue table), gap LENGTH PENALTY is 12, gap point penalty is 4 to measure, and operational method wherein has been contained in ALIGN program (2.0 version).

Nucleic acid of the present invention and protein are also used as " search sequence " to carry out the retrieval of public database, such as, identify other family member or correlated series.This retrieval can with Altschul etc. NBLAST and the XBLAST program (2.0 version) of (1990) J.Mol.Biol.215:403-10 is carried out.NBLAST is utilized to carry out BLAST nucleotide search, score value (score)=100, wordlength=12, to obtain the nucleotide sequence with people or mouse B7-DC nucleic acid molecule homologous.Also BLAST protein retrieval can be carried out by XBLAST program, score value (score)=50, wordlength=3, to obtain the aminoacid sequence with people of the present invention or mouse B7-DC protein molecule homology.In order to obtain gapped alignments (alignment) to compare, can as Altschul etc. the description of (1997) Nucleic Acids Res.25:3389-3402 utilizes GappedBLAST.When utilizing BALST and Gapped blast program, use the default parameters in each program (such as XBLAST and NBLAST).See http://www.ncbi.nlm.nih.gov.

Therefore, the homologue of B7-DC protein as described above has the functionally active of following character (a) natural B 7-DC, (b) when measuring with aforesaid method, 30% (amino acid levels) is at least about with the sequence similarity of natural B 7-DC protein (such as SEQ ID NO:2 or SEQ ID NO:4), be preferably at least about 50%, be more preferably at least about 70%, be even more preferably at least about 90%.

Based on published B7-DC sequence, utilize DNA probe to obtain and express the common practise that this protein is this area.Then by art-recognized method method as described herein, such as standard T cell proliferation or cytokine secretion measure the biochemistry and the biological activity that detect this protein.Whether display homologue is had required activity and as " functional " homologue by the Biological Detection that T cell stimulates altogether.

The functional performance of preferred test determination B7-DC is as stimulated the T cell synthetic cell factor, and this depends on the combination of TCR or the transmission of crosslinked (" primary activation signal ") and costimulatory signal.B7-DC is combined with its native ligand and sends one and cause cytokine in T cell, and as the signal that IL-2 increment is produced, it stimulates proliferation conversely, and described propagation can measure by ordinary method.

" varient " of B6-DC refers to and its full length protein or the substantially consistent molecule of its fragment, and in this molecule, one or several amino-acid residue is substituted (replacement varient) or one or several residue deletions (Deletion variants) or increases (increase varient)." fragment " of B6-DC refers to any hypotype of this molecule, the molecule preferably containing ECD, the i.e. shorter polypeptide of this full length protein.

There is a lot of method can be used for the fragment of DNA sequence dna of preparative separation, mutant and varient.The little subprovince of nucleic acid of coding B7-DC protein or fragment, such as length is 1-30 base, can prepare by the chemical synthesis of standard.Antisense oligonucleotide and primer can be used for preparing larger synthesis fragment.

A kind of preferred functional derivatives is a kind of fused protein, a kind of polypeptide containing B7-DC functional fragment.Such as, a kind of useful B7 derivative is B7-DC-Ig fused protein, and this fused protein contains the C district of polypeptide corresponding to the ECD of B7-DC and a kind of Ig.

The existence of fusion partner can change the solvability of B7-DC protein, avidity and/or valency (be defined as each molecule can the number of binding site) here.A solvable B7-DC fused protein, can produce the biological effect different with the natural protein of expressing on APC when itself and receptors bind in T cell, namely by competitive binding instead of stimulate the stimulation carrying out suppressor T cell altogether.

The extracellular domain (ECD) of B7-DC used herein refers to and to identify and in conjunction with the whole extracellular part of this protein of other acceptor on PD-1 or T cell or its any fragment, the acceptor in T cell is not wherein CD28 or CTLA-4.Preferably, the ECD of B7-DC be by the site 26 of SEQ ID NO:2 or SEQ IDNO:4 to site 221 amino-acid residue coded by part.

" solubility B7-DC " refers to the B7-DC of cell-free form, and it can be deviate from (shed), secretion or other extracting from the cell produced.The B7-DC of solubility include, but are not limited to the fused protein of solubility as the free ECD of B7-DC-Ig, B7-DC or the B7-DC ECD merging (genetic method or chemical process) with bioactive molecules.

As mentioned before, the present invention also comprises the hybrid fusion protein matter formed by a structural domain of a B7-DC structural domain and another kind of B7 family protein or fragment, preferably expresses with the form of common stimulation at cell surface.

One group of preferred B7-DC varient has an amino-acid residue at least, preferably the only varient that replaced by different residue of a residue.About the detailed description of protein chemistry and structure, see Schulz, GE etc., Principles of Protein Structure, Springer Verlag, New York, 1978, and Creighton, T.E., Proteins: Structure and Molecular Properties, W.H.Freeman & Co., San Francisco, 1983, above-mentioned reference is by reference to being incorporated into this.The analytical results of the frequency that the type of the replacement that can occur in protein molecule can change based on the amino acid between the homologous protein of different plant species, as Fig. 3-9 of table 1-2 and Creighton (seeing above) of (seeing above) such as Schulz.Based on this analysis, conservative a group of replacing in following 5 groups of definition exchanges by the present invention:

1	little aliphatics, nonpolarity and have micropolar residue	l-Ala, Serine, Threonine (proline(Pro), glycine);
			2	polarity, electronegative residue and acid amides thereof	aspartic acid, l-asparagine, L-glutamic acid, glutamine;
3	polarity, positively charged residue	histidine, arginine, Methionin;
			4	large aliphatics, nonpolarity residue	methionine(Met), leucine, Isoleucine, α-amino-isovaleric acid (halfcystine)
5	large aromatic residue	phenylalanine, tyrosine, tryptophane.

Three amino-acid residues in upper table parenthesis have special effect in the structure of protein.Glycine is unique a kind of residue not having side chain, thus makes chain have elasticity.Proline(Pro), due to the Geometry that it is unusual, and fetters chain tightly.Halfcystine can participate in the formation of disulfide linkage, disulfide linkage protein folding in be important.

(or immunologic) attribute aspect that is biochemical, function the more change of essence is replaced by selectivity and produces, and it is lower that this selectivity replaces conservative property, as between above-mentioned 5 groups, instead of the replacement in 5 groups.This change is more obviously different from the structure that they are keeping (a) to replace the peptide main chain in district, such as sheet or helicoidal structure, b () is in the electric charge of target site molecule or hydrophobicity, or the effect of the aspect of the volume of (c) side chain.The example of this replacement is that (i) sweet acid acid and/or proline(Pro) are by another kind of aminoacid replacement or disappearance or insert glycine or proline(Pro); (ii) hydrophilic residue such as Serine or Threonine replaces hydrophobic residue as leucine, Isoleucine, phenylalanine, α-amino-isovaleric acid or L-Ala, (being replaced by this hydrophobic residue); (iii) cysteine residues replaces other residue (or being replaced by other residue); (iv) residue of electropositivity side chain such as Methionin, arginine or Histidine is with to replace the electronegative residue of band as L-glutamic acid or aspartic acid (or being replaced by the electronegative residue of band); Or (v) residue with bulky side chain such as phenylalanine replaces residue containing this side chain as glycine (or the residue not containing this side chain replaces).

Being those according to the most receptible disappearance in the present invention, insertion and replacement does not produce disappearance that essence changes, insertion and replacement to the characteristic of B7-DC protein in its T cell altogether stimulating activity.But, carrying out replacing, lack, insert before when being difficult to accurately foretell that it affects, those of ordinary skill in the art can understand can by conventional detection method, method as described herein, and does not need excessive test to assess its impact.

For the present invention, comparatively short chain varient can be prepared by chemosynthesis, it is preferably the site-specific mutagenesis of the nucleic acid by coding B7-DC polypeptide compared with long-chain varient, expression variance body nucleic acid in cell culture, optionally, from cell culture, purified polypeptide such as carries out immune affinity chromatographic purified polypeptide with the specific antibody be fixed on post (with by adsorbing varient in conjunction with at least one epi-position) and typically prepares.

the chemical derivative of B7-DC

" chemical derivative " of B7-DC is containing the other chemical part not usually being protein part.The covalent modification of polypeptide is also contained within scope of the present invention.This kind of derivative part can improve solvability, adsorption, biological halflife etc.The part receiving this kind of effect is disclosed in such as Remington ' sPharmaceutical Sciences, the 16th edition., Mack Publishing Co., Easton, PA (1980).

By the targeted amino acid residues of polypeptide and organic derivatization reagent are reacted, this modification is imported in molecule, the side chain that derivatization reagent wherein can and be selected or terminal residue reaction.Another kind of modification is the cyclisation of protein.

The example of the chemical derivative of polypeptide is as follows.

Derive lysine residue and n terminal residue with succinyl oxide or other carboxylic acid anhydride, there is with the derivative that cyclic carboxylic acids acid anhydride produces the effect changing lysine residue electric charge.Other suitable derivative reagent containing amino residue comprises imido-ester as methyl picolinimidate (methyl picolinimidate); Pyridoxal phosphate; Pyridoxal; Chloroborohydride; Trinitro-benzene-sulfonic acid; O-methyl-isourea; 2,4-diacetylmethane; With reaction that is transaminase-catalyzed and oxoethanoic acid.

By (2-morpholinyl-(4-ethyl) carbon imide or 1-ethyl-3-(4-nitrogen-4,4-dimethyl amyl group) carbon imide react and come selective modification Carboxyl side groups, aspartyl or glutamyl 1-cyclohexyl-3-with carbon imide (R-N=C=N-R ').And, by aspartyl and glutamyl being converted to asparaginyl and Glutaminyl with ammonia react.

Other modification comprises the hydroxylation of proline(Pro) and Methionin, Serine or the phosphorylation of hydroxyl of the threonine residues, (Creighton that methylates of the amino of Methionin; see above, 79-86 page), the acetylize of N-terminal amino and the amidation of C-terminal carboxyl.

Also comprise the amino acid whose polypeptide of the one or more L-of wherein one or more D-aminoacid replacement.

polypeptide (Multimeric Peptides)

The present invention also comprises longer polypeptide, and the basic peptide sequence obtained from B7-DC sequence in this polypeptide is repeated about 2-about 100 times, and with or without interleaves interval and joint.Clearly this polymer can thus place definition any peptide varient build.And a peptide multimer can contain the various combination of peptide monomer and its disclosed varient replaced.This oligomerization or polypeptide can be prepared by chemosynthesis described herein or recombinant DNA technology.When chemically producing, the basic peptide sequence that this oligomer preferably repeats containing 2-8.When producing with recombination method, this polymer can allow many repetitions containing expression system, such as 2-about 100 repetition.

In the tandem multimers of B7-DC peptide or polypeptide, be preferably dimer and tripolymer, by intrachain disulfide bond or other " artificial " covalent bonds between each chain, therefore, each chain is " shoulder to shoulder " instead of " joining end to end ".Preferred dimer and tripolymer are the fused proteins having B7-DC, the dimer formed between B7-DC-Ig as described herein and tripolymer.

the antibodies specific of B7-DC peptide

In the following description, the known various methodology of the those of ordinary skill in immunology, Celluar and Molecular Biology field will be incorporated by reference.The full content of the publication and other material of illustrating these known methods is all by reference to being incorporated into this.The general reference of illustrating immunology ultimate principle comprises A.K.Abbas etc., Cellular and Molecular Immunology (the 4th edition), W.B.Saunders Co., Philadelphia, 2000; C.A.Janeway etc., immunobiology .The Immune System in Health and Disease, the 4th edition., Garland Publishing Co., New York, 1999; Roitt, I. etc., Immunology, (current ed.) C.V Mosby Co., St.Louis, MO (1999); Klein, J., Immunology, Blackwell ScientificPublications, Inc., Cambridge, MA, (1990).

Monoclonal antibody (mAbs) and its preparation are described in Kohler and Milstein, Nature 256:495-497 (1975) with using method; U.S. Patent number 4,376,110; Hartlow, E. etc., Antibodies:A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988); Monoclonal Antibodies and Hybridomas:A New Dimension in Biological Analyses, Plenum Press, New York, New York (1980); H.Zola etc., in Monoclonal Hybridoma Antibodies:Techniques and Applications, CRC Press, 1982)).

The volumes such as method of immunity is also described in Coligan, J.E.., Current Protocols in Immunology, Wiley-Interscience, New York 1991 (or current version); Butt, W.R. (volume) PracticalImmunoassay:The State of the Art, Dekker, New York, 1984; Bizollon, Ch.A., compile., Monoclonal Antibodies and New Trends in Immunoassays, Elsevier, New York, 1984; Butler, J.E., ELISA (the 29th chapter), In:van Oss, C.J. etc., (volume), IMMUNOCHEMISTRY, Marcel Dekker, Inc., New York, 1994,759-803 page; Butler, J.E. (volume), Immunochemistry ofSolid-Phase Immunoassay, CRC Press, Boca Raton, 1991; Weintraub, B., Principles ofRadioimmunoassays, Seventh Training Course on Radioligand Assay Techniques, TheEndocrine Society, March, 1986; Work, T.S. etc., Laboratory Techniques andBiochemistry in Molecular Biology, North Holland Publishing Company, New York, (1978) (chapter write by Chard.T, " An Introduction to Radioimmune Assay and RelatedTechniques ").

Antiidiotypic antibody is also described in, such as, and Idiotypy in Biology and Medicine, academic press, New York, 1984; Immunological Reviews 79 volume, 1984; Immunological Reviews 90 volume, 1986, Curr.Top.Microbiol., Immunol.119 roll up, and 1985; Bona, C. etc., CRC Crit.Rev.Immunol., 33-81 page (1981); Jerne, NK, Ann.Immunol.125C:373-389 (1974); Jerne, NK, In:Idiotypes-Antigens on the Inside, Westen-Schnurr, I., compile., Editiones Roche, Basel, 1982, Urbain, J etc., Ann.Immunol.133D:179-(1982); Rajewsky, K etc., Ann.Rev.Immunol.1:569-607 (1983).

The invention provides antibody, comprise monoclonal and polyclonal antibody, in this antibody capable and known B7 family protein, the new epi-position of non-existent B7-DC reacts.This antibody can be allos, allotypic, isogenic or its modified forms, as humanized or chimeric antibody.Also the idiotype comprising antagonism B7-DC antibody has specific antiidiotypic antibody.The implication of term " antibody " also comprises entire molecule and contains antigen binding site and the fragment of this molecule that can be combined with the epi-position of B7-DC.These comprise Fab and F (ab ') of the Fc fragment lacking complete antibody ₂fragment, it removes more quickly than complete antibody from circulation, and has less nonspecific tissue combination (Wahl etc., J.Nucl.Med.24:316-325 (1983)).Also comprise Fv fragment (Hochman, J. etc. (1973) Biochemistry 12:1130-1135; Sharon, J. etc. (1976) Biochemistry 15:1591-1594) .).These different fragments can with routine techniques as proteolytic enzyme cutting or chemical chop (see, e.g., Rousseaux etc., Meth.Enzymol., 121:663-69 (1986)) prepare.

From being obtained the polyclonal antibody of serum form the animal of immunity such as rabbit, goat, rodent class etc., this antibody can not need process further directly to use, or also can carry out conventional concentrated or purification process as ammonium sulfate precipitation, ion-exchange chromatography and affinity chromatography (see Zola etc., see above).

This immunogen can contain complete B7-D6 protein, or its fragment or derivative.Preferred immunogen contains the ECD (amino-acid residue 26-221) of all or part of people B7-DC, natural B 7-DC finds these residues include posttranslational modification as glycosylation.By the immunogen containing extracellular domain that the existing various method in this area is produced, as the clone gene of expressing with the recombination method of routine, from initiating cell, express the middle separation such as the cell mass of high-level B7-DC.

Can by conventional hybridoma technology, as the method introduced with Kohler and Milstein (Nature, 256:495-97 (1975)), and its method (see above-mentioned reference) improved produces mAbs.With a kind of animal of above-mentioned a kind of immunogen immune, be preferably mouse, to produce required antibody in by the animal of immunity.

Usually, when exist merge promotor as polyoxyethylene glycol (PEG), will bone-marrow-derived lymphocyte and the myeloma cell fusion of the lymphoglandula of immunized animal, spleen or peripheral blood be derived from.Any cell of rat bone marrow tumour cell system can be used for merging: P3-NS1/1-Ag4-1, P3-x63-k0Ag8.653, Sp2/0-Ag14, or HL1-653 myeloma cell line (can obtain from ATCC, Rockville, MD).Following step be included in selective medium the parent myeloma cell that grows to make not occur to merge and donor lymphocyte completely dead, and only have hybridoma cells survive.Clone and cultivate these hybridomas, and screening has required specific antibody from supernatant liquor, as the immunoassay screening by using B7-DC-Ig fused protein.Positive colony, by subclone, such as, by limiting dilution assay subclone, and is separated mAbs.

With technology well known in the art can (in ascites fluid) breeding is produced according to these methods in vitro or in body hybridoma (usually see Fink etc., Prog.Clin.Pathol., 9:121-33 (1984)).In general, breed individual cells system in the medium, contained the substratum of high density list mAb by decant, filtration or collected by centrifugation.

The antibody produced can be single-chain antibody or scFv, instead of normal multimeric structure.This single-chain antibody comprises the hypervariable region of object Ig, and when single-chain antibody is complete Ig a part of, reproduce natural Ig antigen binding site (Skerra, A. etc. (1988) Science, 240:1038-1041; Pluckthun, A. etc. (1989) Methods Enzymol.178:497-515; Winter, G. etc. (1991) Nature, 349:293-299); Bird etc., (1988) Science 242:423; Huston etc. (1988) Proc.Natl.Acad.Sci.USA 85:5879; Jost CR etc. .J.Biol Chem.1994 269:26267-26273; U.S. Patent number 4,704,692,4,853,871,4,94,6778,5,260,203,5,455,0Kn contacts with the solution of the traget antibody (it is as a kind of " reporter molecule ") containing unknown quantity.Second time makes the antibody of mark be combined with antigen after cultivating, and this antigen is combined with solid support by unlabelled antibody, again rinses solid support to remove unreacted traget antibody.It can be that a kind of simple " Yes/No " measures that such forward sandwich (forward sandwich) measures, to identify whether antigen exists, also quantitative assay can be carried out by comparing traget antibody with the amount of the antibody obtained from the standard model of the antigen containing known quantity.

In " sandwich " of another kind of type measures, use so-called " synchronously " and " oppositely " assay method.Synchronous detection relates to a culturing step, because be all simultaneously applied in tested sample in conjunction with the antibody of solid support and the antibody of mark.After cultivation terminates, rinse solid support to remove residual liquid sample and unconjugated traget antibody.Then the existence of the traget antibody be combined with solid support is detected as " forward " sandwich detection of routine.

" oppositely " in detection, in liquid sample, first progressively adding the solution of traget antibody, after hatching the suitable time, then adding the unmarked antibody be attached on solid support.After second time is hatched, rinse solid phase in a conventional manner to remove detected residual sample and the unreacted antibody-solutions be labeled.Then detect as " synchronously " and " forward " and detect the traget antibody be combined with solid support.

Above-mentioned antibody is useful in suppressor T cell stimulates the disease relevant to unwanted T cell activation with treatment as transplant rejection and autoimmune method.The method experimenter related to this treatment of needs uses the antibody of significant quantity, is preferably mAb, is more preferably the special people of the common stimulation epi-position of B7-DC or humanized mAb.The antibody used must be effective in the stimulation of blocking t cell or elimination antigen-reactive T cells, thus suppresses directed t cell response.Relevant dosage range will describe hereinafter.

the application of the nucleic acid of coding B7-DC protein

By detecting the expression of B7-DC in biological sample cell or detecting the impact of a kind of reagent on the expression of B7-DC, nucleic acid of the present invention can be used for detecting the process of disease in diagnosis.This realizes preferably by the mRNA level in-site detecting cell.In order to use in this kind of diagnostic method; this nucleotide sequence is marked by detectability; such as; utilize radioactivity or fluorescent mark or biotin labeling, and for the Dot blot of routine or Northern hybridization step to detect the preparation that mRNA molecule is present in total kor poly (A+) RNA of biological example sample.

therapeutic composition and administration thereof

B6-DC polypeptide or the cell such as DC or tumour cell that express this polypeptide are applied to mammalian subject, preferred people.The immunity of the lymphocytic reactivity of T and generation is improved with Cell binding, polypeptide that is fixing or other aggregate form.B6-DC-Ig fused protein forms a dimer also, as shown in the Examples, stimulates T cell altogether.The B6-DC polypeptide of soluble and monomeric form in conjunction with the acceptor in T cell, and can not produce stimulating activity, is thus considered to by the competitive inhibitor stimulating the T cell of the molecule of form generation to stimulate altogether or antagonist.The combination of this B6-DC antagonist can suppress ongoing T cell activity maybe can disturb by endogenous B6-DC or the effect of costimulatory signal of even being presented by its acceptor (as CD28 or CTLA-4) by other B7 family member.

The composition with B7-DC activity described in the invention carrys out administration with biologic effective dose or treatment significant quantity in pharmaceutical carrier.This B7-DC polypeptide (or expressing the cell of this polypeptide) can individually dosed or with other oroteins or polypeptide as there is the active protein of another member of B7 family or polypeptide or another kind of molecules of immunization stimulus in conjunction with administration.Treatment can comprise a kind of adjuvant of administration, and this adjuvant comprises any nonspecific immune-stimulating compound in a broad sense, as Interferon, rabbit.The adjuvant that the present invention considers comprises Resorcinol, and the tensio-active agent of non-ionic type is as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether.

Here is the dosage to snibject's antibody of the present invention.

Treatment significant quantity refers to and will reach required immunology or the dosage of clinical effectiveness when administration one section of validity period.

The therapeutic activity amount with the polypeptide (or antibody of a kind of anti-B7-DC) of B7-DC activity can change according to factor, and described factor such as the body weight of the state of disease, age, sex and individuality and peptide cause the ability of required reaction in individual body.Dose therapies can be adjusted to provide best therapeutic response.Such as, can daily several dosage of separating or as suitably reduced dosage by treatment situation in the urgent need to indicated.Treatment significant quantity, can determine according to protein or cell equivalents with the protein of cell associated form.

Therefore, the significant quantity of the recipient of each kg body weight is that about 1ng-is about 1g, is more preferably about 1 μ g-100mg/kg, is more preferably about 100 μ g-and is about 100mg/kg.Be applicable to the activeconstituents of dosage form preferably containing the about 0.1mg-500mg of (dosage range for the latter) per unit of inner administration.Based on the gross weight of said composition, this activeconstituents can change in 0.5-95% weight range.Alternatively, express the cell of B7-DC, the effective dose of so preferred transducer cell as the tumour cell of DC or non-activity is each object about 10 ⁴-10 ⁹individual cell, more preferably from about 10 ⁶-10 ⁸individual cell, is preferably the dosage form be separated.The those of ordinary skill of immunotherapy field can adjust these dosage and not need too much test.

This active compound (as B6-DC polypeptide or the cell of transduceing with B6-DC DNA) can in a conventional manner such as by conventional and effective approach drug administration by injection.Preferred approach comprises subcutaneous, intradermal, intravenously and intramuscular route.Other possible approach comprises oral administration, in sheath, suck, transdermal administration or rectal administration.In order to treat the tumour of not excising completely, also can direct intra-tumoral injection.

Depend on the approach of administration, active compound can be coated in a kind of material to prevent this compound by the natural condition effect of enzyme, acid and other this compound of deactivation.Therefore, for by intestines administration, there is the polypeptide of B7-DC activity or the mode of peptide, must by composition with a kind of prevent the material bag of its inactivation by or by composition and a kind of material co-administered preventing its inactivation.Such as, with suitable carrier, thinner or adjuvant form to individual administration peptide, with enzyme inhibitors (insulin inhibitor, diisopropylfluorophosphate (or ester) (DEP) and Trypsin inhibitor,Trasylol as pancreas) administration altogether, or with suitable carrier as liposome (comprise water-in-oil-in-water (water-in-oil-in-water) milk sap and conventional liposome (Strejan etc., (1984) JNeuroimmunol 7:27)) form administration.

The present invention's " pharmaceutical carrier " used comprises any and all solvents, dispersion agent, coating, antiseptic-germicide and anti-mycotic agent, isotonic with absorption delay agent etc.The active substance that these media and reagent are used as in pharmacy is well known in the art.Except at any conventional media or reagent except active compound is incompatible, their application in therapeutic composition can be considered.Auxiliary active compound also may be used for composition.

Preferred medicinal diluent comprises salt solution and water-containing buffering liquid.The pharmaceutical composition being suitable for injecting comprises the aqueous solution (water soluble) of sterilizing or the sterile powder of the Injectable solution of dispersion and sterilizing or the extemporaneous preparation of dispersion.Isotonic agent is as sugar, and polyvalent alcohol is as N.F,USP MANNITOL, Sorbitol Powder, and sodium-chlor can be included among pharmaceutical composition.Under any circumstance, composition should sterilizing and should be fluid., should be stable under manufacture and storage conditions, must containing sanitas to prevent microorganism as bacterium and fungal contamination.Dispersion also can be prepared in glycerine, liquid polyethylene glycol and composition thereof and in oil.Under general preservation and working conditions, these preparations can containing sanitas to prevent microorganism growth.

Carrier can be a kind of solvent or dispersion agent, and it comprises as water, ethanol, polyvalent alcohol (polyoxyethylene glycol etc. as glycerol, propylene glycol and liquid state) and their suitable mixture.Suitable mobility can be kept, such as, by using a kind of coating as Yelkin TTS, by keeping the granular size needed for dispersion and being realized by use tensio-active agent.

Utilize various antiseptic-germicide and anti-mycotic agent, as parabens, butylene-chlorohydrin, phenol, xitix, thiomersal(ate) etc. can suppress the effect of microorganism.

By comprising a kind of preparation postponing to adsorb in the composition, as aluminum monostearate and gelatin can realize the prolongation absorption of Injectable composition.

Preferably prepare parenteral composi with the form of dose unit, be beneficial to administration and dosage is homogeneous.Dosage unit form refers to and is suitable for as single dose the individual on the health of mammalian subject administration; Active compound and the required pharmaceutical carrier of the result for the treatment of of each unit needed for the calculated generation of predetermined volume form.To the explanation of dose unit of the present invention according to or directly depend in (a) active compound uniqueness characteristic and to reach concrete result for the treatment of, and (b) treats limitation intrinsic in the field of individual sensitivity mixing this active compound.

For lung instillation, can use the solution of atomization, in sprayable aerosol preparations, active protein can be combined with solid-state or liquid inert carrier material.Its also can be packaged in squeeze bottle or and pressurized volatile, normally propellant gases mixing.This aerosol preparations, except containing except protein of the present invention, can contain solvent, damping fluid, tensio-active agent and antioxidant.

For topical application, protein of the present invention can in conjunction with the vehicle of topical application as ointment or ointment, and described vehicle has also can directly be administered into infected region by activeconstituents to the smoothing effect of skin.

Carrier for activeconstituents both can be sprayable form also can be not sprayable form.Not sprayable form can be semi-solid or solid form, and it contains one and is suitable for the carrier of topical application and has the dynamic viscosity being preferably greater than water.Suitable preparation include, but are not limited to solution, suspension, milk sap, emulsifiable paste, ointment, powder, liniment, ointment etc.If needed, these can by sterilizing or with auxiliary agent as sanitas, stablizer, wetting Agent for Printing Inks, buffer reagent or the salt etc. that affects osmotic pressure mix.Example for the preferred vehicle of not sprayable topical formulations comprises ointment base, as PEG-6000 (PEG-1000), conventional emulsifiable paste as HEB emulsifiable paste, gel and Vaseline gel etc.

Other pharmaceutical carrier for B7-DC polypeptide of the present invention has liposome, the pharmaceutical composition containing active protein, and described composition is dispersion or exists with different particle form, and particle is wherein made up of the water layer be combined with lipid layer.Active protein to be preferably present in water layer and in lipid layer, inner outside or any position, or in the non-homogeneous system of known Liposomal suspensions.Hydrophobic layer, or oil layer, usually but not exclusively containing phosphatide, as Yelkin TTS and sphingophospholipid, steroid, as cholesterol, ionic surface active agent more or less, as dicetylphosphate, stearylamine or phosphatidic acid and/or other hydrophobic nature material.

the modification of tumour cell is to express B7-DC and multiple costimulatory molecules

Another aspect of the present invention is a kind of cell, preferred tumour cell, and it is modified to express multiple costimulatory molecules.In the B cell of activation, the transient expression of costimulatory molecules B7, B7-2 and B7-3 is different.Such as, the early expression that B7-2 activates in B cell, and B7-3 expresses late.Therefore costimulatory moleculeses different in immunoreaction process has different functions.An effective t cell response needs T cell acceptance from the costimulatory signal of multiple costimulatory molecules.

Therefore, the present invention contains tumour cell that is genetically modified or that express more than one costimulatory molecules, such as, can modify tumour cell to express B7-DC and one or more B7, B7-2 and B7-3.

Before modifying, cell, can not express any costimulatory molecules as tumour cell and maybe can express certain costimulatory molecules and the costimulatory molecules of not expressing other.As described in the present invention, by the independent nucleic acid transfection with coding B7-DC or tumour cell can be modified with different costimulatory molecules transfections.Such as, can also further with the nucleic acid transfection of coding B7 with the tumour cell of the nucleic acid transfection of coding B7-DC.The sequence of the cDNA molecule of encoding human or mouse B7-DC protein is the encoding part of SEQ ID NO:1 and SEQ ID NO:3 respectively.Alternatively, can use more than a kind of modification.Such as, can stimulate by the tumour cell of the nucleic acid transfection of coding B7-DC with the reagent that induction B7-1, B7-2 or B7-3 express.

the antigen relevant to pathogenic agent

Main application of the present invention is the application of composition of the present invention in therapeutic vaccine, causes falling ill and dead major chronic viral infections in this vaccine energy Therapeutic cancer and world wide.Design this vaccine to eliminate infected cell, this needs T cell to react when antibody loses efficacy.Except the epitope of self, vaccine of the present invention also comprises:

(a) carrier, the rna replicon as naked DNA, naked RNA, self-replacation is sub and viral, and described virus comprises vaccinia virus, adenovirus, adeno associated virus (AAV), slow virus genus and RNA alphavirus;

(b) Antigen Location (targeting) or processing signal, if the domain II, herpes simplex VP22 target protein etc. of the extracellular domain of HSP70, calretinin, Flt-3 part, ETA (ETA) is (see the U.S. Patent application 09//421,608 of common transfer; 09/501,097; 09/693,450; 60/222,9002; 60 ,/222,985; 60/268,575 and Chang, W-F etc., J.Virol.75:2368-2376 (2001), whole full text is by reference to being incorporated into this); With

C () costimulatory signal, is preferably B7-DC protein of the present invention or its fused protein, fragment or functional derivatives (separately or with other known stimulating protein altogether as combinations such as B7.1, B7.2, solvable CD40).

With the host cell of an a kind of nuclear transformation of antigen of coding, transfection or other mode transduced tumors cell or other type, comprise APC, antigen wherein can cause immune response.The epi-position of this antigen preferably pathogenic microorganism, host cell is replied by effector T cell, comprises that cytotoxic T lymphocyte (CTL) and delayed hypersensitivity obtain to resist pathogenic microorganism protecting.These pathogenic microorganisms typically comprise virus, cytozoon as plasmodium (malaria) and on the bacterium of Intracellular growth as mycobacterium (mycobacteria) and Listeria (listeria).Therefore, the type being included in the antigen in vaccine of the present invention combination is any form relevant with these pathogenic bacterias (certainly also comprising tumour specific antigen).It should be noted that some virus antigens are also tumour antigens when virus is the paathogenic factor of cancer.

In fact, two kinds of cancers the most general in the world: hepatocellular carcinoma and cervical cancer are all relevant with virus infection.Hepatitis B virus (HBV) (Beasley, R.P. etc., Lancet 2,1129-1133 (1981)) be used as the pathogenic agent of hepatocellular carcinoma.The cervical cancer of 80-90% expresses E6 and HPV-16 E7, this antigen is from four kinds of " high-risk (high risk) " human papillomavirus types: the one (Gissmann in HPV-16, HPV-18, HPV-31 and HPV-45, L. etc., Ciba Found Symp.120,190-207 (1986); Beaudenon, S., wait .Nature 321,246-249 (1986)).Because it generally expresses in cervical cancer, HPV E6 and HPV-16 E7 are most possibly the target antigens of cancer relevant to virus in immunocompetent individuals.Except their importance as the target antigen of therapeutic cancer vaccine, the tumour antigen that virus is relevant is also the ideal candidate of preventative vaccine.In fact, in Asia, the introducing of preventative HBV vaccine has decreased the sickness rate (Chang, M.H., wait .New Engl.J.Med.336,1855-1859 (1997)) of liver cancer, and this creates very large impact to cancer prevention.

The most important virus in chronic human viral infections aspect is human papillomavirus (HPV), hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), Epstein-Barr virus (EBV) and hsv (HSV).

Except for except human cancer and infectious diseases, the present invention also can treat Animal diseases in veterinary applications.Therefore, the method that the present invention describes can by those of ordinary skill in the art ready-made be used for treat the herpesvirus infection of livestock, comprise the marek's disease virus in EHV, bovine herpes virus, chicken and other poultry; Animal retroviral diseases, pseudorabies and rabies etc.

Following reference gives basic virology, the principle in medical virology and veterinary virology field and current information, all by reference to being incorporated into this: Fields Virology, Fields, BN etc., compile., LippincottWilliams & Wilkins, New York, 1996; Principles of Virology:Molecular Biology, Pathogenesis, and Control, Flint, S.J. etc., compile. and, Amer Society for Microbiology, Washington, 1999; Principles and Practice of Clinical Virology, the 4th edition, Zuckerman.A.J. etc., compile, John Wiley & Sons, New York, 1999; The Hepatitis C Viruses, by Hagedom, CH etc., compile, Springer Verlag, 1999; Hepatitis B Virus:Molecular Mechanisms inDisease and Novel Strategies for Therapy, Koshy, R. etc., compile, World Scientific Pub Co, 1998; Veterinary Virology, Murphy, F.A. etc., compile. and, Academic Press, New York, 1999; AvianViruses:Function and Control, Ritchie, B.W., Iowa State University Press, Ames, 2000; Virus Taxonomy:Classification and Nomenclature of Viruses:Seventh Report of theInternational Committee on Taxonomy of Viruses, by M.H.V Van Regenmortel, MHV etc., compile, Academic Press; New York, 2000.

target molecule

The multiple proteins of being permitted with different binding mode is used for being combined with antigen by as " target " molecule, preferably as fusion polypeptide, to make Antigen Location in cell and subcellular compartment, thus promote that antigen presentation is in T cell in more effective and useful mode.

Antigen and heat shock protein (HSP) chain represents a kind of method of the potential validity for improving nucleic acid (with other) vaccine.HSP is obviously the natural biologic adjuvants in cancer and virus vaccines.Reside in gp96 HSP in endoplasmic reticulum (ER) and tenuigenin Hsp70 can as immunological adjuvant (Srivastava, PK etc., Semin.Immunol.3,57-64 (1991); Udono, H etc., Proc.Natl.Acad.Sci.USA 91,3077-3081 (1994)).These HSP or chaperonins are in conjunction with a large amount of peptides (Lammert, E., wait .Eur.J Immunol.27,923-927 (1997)).Hsp70 be a kind of can by relevant protein positioning to the chaperonins matter on the cell protein combined enzyme agent of proteoplast-main, cell protein combined enzyme agent wherein can produce peptide to be combined with MHC I quasi-molecule.Therefore, the antigen that direct and Hsp70 links can more effectively be presented (guide and produce, especially CTL response) by I class MHC.There are two feature of course relevant to the adjuvanticity of HSP: (1) in vitro, antigen imports in I class MHC process approach by Toplink effectively that be combined with gp96; (2) secretion in conjunction with energy inducible proinflammatory cytokine of gp96 and scavenger cell, so expand the function of the fixed cell of peptide antigen target.

With can induce from tumour or the HSP complex body immunity that is separated from virus infected cell effective antitumour immunity (Srivastava, PK etc., Int J Cancer.33:417-22,1984; Srivastava, PK etc., ProcNatl Acad Sci USA.83:3407-11,1986; Udono, H etc., J Immunol.152:5398-5403,1994; Blachere, NE etc., J Immunother.14:352-6,1993; Udono, H etc., see above; Tamura, Y etc., Science.278:117-20,1997; Janetzki, S etc., J Immunother.21:269-76,1998)) or antiviral immunity (Heikema, A etc., Immunol Lett.57:69-74,1997; Suto, R etc., Science.269:1585-8,1995).In vitro peptide is mixed with HSP produce immunogenic HSP-peptide complex body (Ciupitu, AM etc., J Exp Med.187:685-91,1998; Blachere, NE etc., J Exp Med.186:1315-22,1997).Some protein vaccines based on HSP relate to antigen and HSP fusion (Suzue, K etc., J Immunol.156:873-9,1996; Suzue, K. etc., Proc Natl Acad Sci USA 94:13146-51,1997).Recently, the present inventor and its colleague (as Chen, C-H etc., Canc.Res.60:1035-1042 (2000)) in the DNA or the sub-vaccine of rna replicon of chimeric versions thereof, employ HSP.They use HPV-16E7 as antigen, merge with Mycobacterium tuberculosis (Mycobacterium tuberculosis) HSP70, show the amplification of E7 specific C D8+T cell and the enhancing of activation, this causes the effective antitumor immunity (Lin to existing tumour, K.-Y. etc., Cancer Res.56:21-26., 1996).

Another kind of useful target molecule is the translocation domain of ETA (ETA), the domain II (dII) (comprising residue 253-364) of such as ETA.Translocation domain is the polypeptide in the tenuigenin of a kind of energy induced protein or polypeptide transporte to cells.Such as, the similar polypeptide be suitable for derives from diphtheria, Clostridium (Clostridia) (Clostridium botulinum (botulinum), tetani), anthrax, Yersinia (Yersinia), vibrio cholerae (Vibrio cholerae) or Bordetella pertussis toxin.In the preparation of this composition, the DNA of the toxin domain of this element element of encoding preferably is suddenlyd change or is lacked.

Calprotectin (CRT) is the protein of an abundant 46kDa, be positioned in endoplasmic reticulum (ER) chamber, this protein shows lectin activity, known and nascent sugar-protein folding and assemble relevant (Nash (1994) Mol.Cell.Biochem.135:71-78; Hebert (1997) J Cell Biol.139:613-623; Vassilakos (1998) Biochemistry 37:3480-3490; Spiro (1996) J Biol.Chem.271:11588-11594.Be combined with CRT by the peptide that translocator is transported in ER, this translocator is processed relevant to antigen, as TAP-1 and TAP-2 (Spee (1997) Eur.J. Immunol.27:2441-2449).CRT forms complex body with peptide in vitro.When these complex bodys are delivered medicine to mouse, cause peptide specific CD8+T cell response (Basu (1999) J Exp.Med.189:797-802; Nair (1999) J.Immunol.162:6426-6432).From mouse tumour, the CRT of purifying produces the tumour to being used as CRT source, but not the specific immune of the tumour of antigen uniqueness (Basu sees above).Stimulate DC further in vitro with the CRT of binding peptide, again presented in the DC I type molecule of this peptide above, and the specific CTL of stimulator polypeptide (Nair sees above).

Flt-3 part stimulates the growth of DC precursor in vivo, and can promote a large amount of DC generation (Maraskovsky, E. etc., J Exp Med.184:1953-62,1996; Shurin, MR. etc., Cell Immunol.179:174-84,1997).Flt3, and a kind of mouse tyrosine kinase receptor (Rosnet, O. etc., Oncogene 6:1641-50,1991) be the member (see Lyman, SD, Curr Opin Hematol.5:192-6,1998) of type III receptor kinase family.In hemopoietic tissue, the expression of Flt3 is only limited to CD34 positive precursor.Flt3 is used to qualification and the corresponding part of subclone, and Flt3-part (Lyman, SD etc., Cell 75:1157-67,1993; Hannum, C etc., Nature 368:643-8,1994).The Flt-3 part of the principal mode of synthesis, as transmembrane protein, therefrom prepares intimate solvable ECD (Lyman etc. see above) with proteolytic cleavage.These protein bound also activate unique tyrosine kinase receptor.In hematopoietic cell, the expression of Flt-3 acceptor is mainly limited to the most original precursor cell, comprises DC precursor.The ECD of Flt-3 part to several mouse model tumors, comprise fibrosarcoma, mastocarcinoma, liver cancer, lung cancer, melanoma and lymphoma produce strong antitumor action (Lynch, DH etc., Nat Med.3:625-631,1997; Chen, K etc., Cancer Res.57:3511-3516,1997; Braun, SE etc., Hum Gene Ther.10:2141-2151,1999; Peron, JM etc., J Immunol.161:6164-6170,1998; Chakravarty, PK etc., Cancer Res.59:6028-6032,1999; Esche, C etc., Cancer Res.58:380-383,1998.) (19).The DNA of coding HPV Ek7 albumen is connected with the DNA of coding Flt-3 part ECD by the colleague of the present inventor.With this construct immunity, improve amplification and the activity of HPV-16 E7 specific C D8+T cell significantly, effective antitumour immunity is created to the metastatic tumo(u)r of existing expression E7.

Can use, HSV-1 protein VP22 is a kind of prototype protein, and owing to having transfer performance in outstanding cell, this albumen mass-energy, especially improves the diffusion (Elliott, G., and P.O ' Hare.1997.Cell88:223-33) of antigen.Such as, with p53 (Phelan, A. etc., 1998, Nat Biotechnol 16:440-443) or thymidine kinase (Dilber, MS etc., 1999, Gene Ther 6:12-21) VP22 that combines, the short nearly proteins diffusion connected is in the cell of surrounding and Therapeutic mode tumour in vitro.In DNA vaccination above, the VP22 be connected with HPV-16 HPV-16 E7 causes increasing considerably (about 50 times) by the E7 specific C D8+T cell precursors quantity in the mouse of immunity, makes the DNA vaccination of poor efficiency be transformed into the vaccine of the tumour significant effective to expression E7.The VP22 mutant of non-diffusing can not strengthen effect of vaccine.VP22 and the protein with similar action pattern strengthen effect of vaccine in several ways: (1) promotes that antigen is from the diffusion of the cell of transfection APC towards periphery, thus increases the quantity by the APC of I class MHC approach antigen-presenting; (2) more effectively antigen-presenting in the cell of transfection; (3) " intersect and cause (cross-priming) " is carried out thus release VP22/ antigen coalescence protein matter, cause the absorption of DC (or other APC) and process thus present (Huang by MHC-I restrictive approach, AY etc., 1994, Science 264:961-965).

Those those of ordinary skill in the art understand appropriate epitope how to identify from the associated protein of pathogenic agent as CTL epi-position, to apply according to the present invention.

b7-DC DNA is to the transfer of cell and animal

DNA shifts, such as, realize usually known " gene therapy ", relate to and import in cell by " external source " DNA, finally import in animal alive.The gene therapy technology of several routine is studied and repeated (Yang, N-S., Crit.Rev.Biotechnol.12:335-356 (1992) by a large amount of; Anderson, W.F., Science 256:808-813 (1992); Miller, A.S., Nature 357:455-460 (1992); Crystal, R.G., Amer.J Med 92 (suppl 6A): 44S-52S (1992); Zwiebel, J.A. etc., Ann.N.Y.Acad.Sci.618:394-404 (1991); McLachlin, J.R. etc., Prog Nucl.Acid Res.Molec.Biol.38:91-135 (1990); Kohn, D.B. etc., Cancer Invest.7:179-192 (1989), these reference are incorporated into this by ginseng card in full).

A method comprises to be transferred in the primary cell of cultivation by nucleic acid, then by the cell autotransplantation be converted ex vivo in host, carrying out homology transfer transfers in host by the cell of conversion, both can transfer to whole body, and also can be transferred in concrete organ or tissue.

In order to realize object of the present invention, realize exonuclease treatment by being transferred directly in vivo in mammiferous body tissue or organ by functionally active DNA.The transfer of DNA can be realized by many methods described below.Whether whether successful expression is to screen the clone of the transfection of expressible dna to detect these systems in vitro by selected marker (as G418 resistance), then exist (after the process of derivable system inductor) with the expression product that the antibody of product detects B7-DC by suitable immunoassay.The validity of the method, comprises the absorption of DNA, the stability of the integration of plasmid and integrated plasmid can by being improved as " carrier " cotransfection with known method linearization plasmid with the mammalian DNA of high molecular.

Example I

Materials and methods

the preparation of cell and cultivation

6-12 week age female BAl BIc/c mouse buy from NCI, for the preparation of DC and scavenger cell.

As previously mentioned (26), cultivate in RPMI-1640 (Gibco BRL) substratum and derive from the DC of marrow, in this substratum, be supplemented with 5% foetal calf serum (FCS) (Hyclone), penicillin/streptomycin (the restructuring mouse GM-CSF (Immunex) of (JRHBiosciences), gentamicin (Sigma), non-essential amino acid (JRH Biosciences), Pidolidone (salt) (JRH Biosciences), Sodium.alpha.-ketopropionate (Sigma), 2 mercapto ethanol (Sigma) and 1000 units/ml.The DC deriving from marrow growing 8 days is dyeed by conventional method monoclonal antibody.The monoclonal antibody of the anti-II class MHC of purifying from the supernatant liquor of hybridoma, 14-4-4s.William doctor Baldwin, Johns Hopkins University (Johns Hopkins University) provides CTLA4-Ig fusion molecule with open arms.The antibody (28-14-8) of I class MHC, the antibody (Cl.A3-1) of F4/80, the antibody (1G10) of B7.1, the antibody (GL1) of B7.2, Fc _γthe antibody (2.4G2) of RII/III and the antibody (M1/70) of Mac-1 are purchased from PharMingen.In order to prepare detection cDNA, tumor center of Johns Hopkins University (Johns Hopkins University Oncology Center) 14-4-4s and CTLA4-Ig has purified the growth cell of 8 days by Cell Sorter.II after sorting ^hiclass MHC and B7 ^hithe purity of population is 93-98%.

The scavenger cell deriving from marrow is cultivated in RPMI-1640 substratum, the restructuring mouse M-CSF of the FCS of 10%, penicillin/streptomycin, non-essential amino acid, Sodium.alpha.-ketopropionate, Pidolidone (salt), 2 mercapto ethanol and 250 units/ml is supplemented with in this substratum, as previously mentioned (27), with the γ-IFN (Pharmingen) of 500 units/ml and LPS (Sigma) process of 5 μ g/ml.After stimulation, cultivate the cell surface expression with fluidic cell metering (cytometric) analysis confirmation II class MHC and B7 after 10 days.

Macrophage system WEHI-3, RAW264.7, J774.A.1, PU5-1.8 are by NIAID, and Joshua doctor Farber of healthy national institute (National Institutes of Health) provides.By the culture medium culturing that ATCC recommends.

the lymphocyte reaction of allos mixing

Grow 8 days, be characterised in that II ^hiclass MHC and B7 ^hithe DC in BM source be detected its ability at MLC moderate stimulation allogenic T cells.By the BALB/c irritation cell of increasing amount is added to 3 × 10 ⁵in individual allos C57BL/6 lymphocyte, in 96 hole flat bottom microtiter plate, carry out MLC reaction.Cultivate after 3 days, to each Kong Zhongzai add 1 μ Ci [ ³h]-methyl-thymidine (Amersham), cultivate 18h, the propagation of evaluation T cell.Then collecting cell, measures radioactive combination with β counter (Packard 96).

cDNA subtractive hybridization

The total serum IgE of the DC of sorting and the scavenger cell of activation is come from TRIZOL (Gibco BRL) extracting.With Oligotex mRNA purification kit (Qiagen) purifying messenger RNA(mRNA).We use the SMART cDNA synthesis system (Clonetech) of PCR-based to increase cDNA, and the subtraction system PCR then carrying out PCR-based selects (Clonetech).Subtrahend is carried out according to the scheme of manufacturers.The last time after subtrahend PCR, DNA fragmentation is connected in plasmid vector pCR2.1 (Invitrogen) or pCR Blunt (Invitrogen).After conversion, cultivate each clone to carry out plasmid DNA amplification and to prepare DNA in a small amount, then digest with alleged occurrence Insert Fragment with EcoRI.Then Plasmid Dot Blot is carried out to confirm that this cDNA clone is specific for dendritic cells.Gone up and SMART cDNA probe hybridization to Hybond N+ film (Amersham) by the DNA point sample of the micropreparation of alkaline denaturation, this probe derives from the DC of sorting or the scavenger cell of activation.With random primering (Stratagene Prime-It II), use ³²p marks these cDNA probes, and (28) carry out hybridizing and rinsing as mentioned previously.By film exposure in film (Amersham) 1-2 days, and develop.

plasmid Dot Blot Analysis

By the DNA sample point sample of alkaline denaturation micropreparation on Hybond N+ film (Amersham), with the SMART of the scavenger cell of the DC or activation that derive from sorting cDNA probe hybridization.With random primering (Stratagene Prime-It II), 32P is used to mark these cDNA probes.(cites as mentioned previously?) carry out hybridizing and rinsing, by film exposure in film (Amersham) 1-2 days, and develop.

the clone of the structure of cDNA library and screening---B7-DC

Without sorting, the DC of derived from bone marrow is collected in growth for 8 days afterwards.These cell high level expressions II class MHC and B7 of about 20%-40%.Carry out the extracting of total serum IgE and the purifying of polyA RNA as mentioned above.For the DC library construction that few dT guides, we use γ ZAP to express cDNA synthesis system (Stratagene).The PCR DNA fragmentation mark of B7-DC is used for screening as probe., the scheme according to Stratagene carries out film transfer, sex change, renaturation.As above the radio-labeled of probe, hybridization, flushing and radioautograph is carried out.Be separated positive colony, carry out postsearch screening.After postsearch screening, excise plasmid by excision in body, detected by Dot blot and order-checking.Sequencing has been carried out in Univ Johns Hopkins Med (JohnsHopkins University School of Medicine) by Core Facility.In Genbank (NCBI), the homology search of nucleotide sequence is carried out to determine the similarity of itself and the previous gene reported with blast program.From DC cDNA library, isolate total length B7-DC cDNA clone.5 '-RACE is carried out with SMART RACE cDNA amplification kit (Clontech).To check order in 5 '-RACE product cloning to pCR2.1 carrier.Obtain by RT-PCR the B7-DC that two exceed total length, compare their sequence to avoid sequence errors.

Following human cloning B7-DC: as previously mentioned (29), cultivate normal peripheral blood mononuclear cells in GM-CSF+IL-4 or GM-CSF+Flt-3L, therefrom obtains people DC.The same extracting RNA.BLAST retrieval determines overlapping EST clone, and GenBank registration number is AK001879, the homology of itself and mouse B7-DC.The samely carry out 5 ' RACE.We have measured the sequence of 5 '-RACE PCR fragment, devise a primer corresponding with the 5 '-UTR of people B7-DC.The following primer of the 5 '-UTR and 3 '-UTR that are arranged in B7-DC is used to the people B7-DC of amplification total length:

5 '-GGAGCTACTGCATGTTGATTGTTTTG-3 ' [SEQ ID NO:6] and

5’-TGCAAACTGAGGCACTGAAAAGTC-3’[SEQ ID NO：7]

The full length cDNA sequence of people and mouse B7-DC cDNA is deposited in EMBL/GenBank/DDBS, and its registration number is AF329193 and AF142780.

screening/the genomic clone in BAC (129SVJ) library and mapping

BAC library screening is carried out according to the scheme (Genome Systems, Inc.) of manufacturers.Primer used is:

5 '-TTGTTGTCTCCTTCTGTCTCCCAAC-3 ' [SEQ ID NO:8] and

5’-ACAGTTGCTCCTTGTATCAGGTTC-3’[SEQ ID NO：9]

Screening BAC library obtains 3 positive colonies.Chromosomal localization collection of illustrative plates is drawn by fluorescence in situ hybridization (Genome Systems Inc.).Altogether analyze 80 medium cells, 79 display specific markers.People B7-DC gene is located by using the blast program of bioinformatics tools commercially, NCBI and international RH mapping agreement (MappingConsortium).In htsg, retrieve this hB7-DC sequence, find that it is positioned two and is positioned in No. 9 chromosomal BAC clone RP11-574F11 (AL162253) and Rp11-635N21 (AL354744).

the Northern trace of virus

The female Balb/c mouse in 4-6 age in week purchased from NCI, and for the preparation of organizing RNA.Carry out RNA extracting as above-mentioned and organize SMART cDNA to synthesize, the scavenger cell of sorting DC and activation.With PCR purification kit (Qiagen) purifying SMART PCR cDNA.DNA leakage of electricity swimming on the sepharose of 1% of the purifying of 0.5 μ g/ swimming lane, transfers on Nytran nylon membrane (Schleier and Schuell).In order to prepare radioactive probe, we with the plasmid DNA deriving from subtracted library for template increases.We utilize the primer pair of the adjacent locations of the cloning site of plasmid DNA by pcr amplified dna, and the pcr dna of the purifying of each clone is used as hybridization probe.The nucleotide sequence of these primers is as follows:

5 '-GTAACGGCCGCCAGTGTGCTG-3 ' [SEQ ID NO:10] and

5’-CGCCAGTGTGATGGATATCTGCA-3’[SEQ ID NO：11]

Also carry out the virtual Northern analysis of the total serum IgE of people DC and contrast placenta.The preparation of probe used and RNA is described above.The radio-labeling of probe, hybridization, flushing and radioautograph are also carried out as mentioned above.

hamster anti-mB7-DC Ab product

Stable B7-DC transfectant in DC2.4, RAW246.7 and RENCA clone is used to immune U.S. hamster.B7-DC is cloned in the pCAGGS carrier (30) of modification.With this hamster of plasmid (Rockland) booster immunization containing B7-DC three times.The anti-B7-DC antibody used in this research from three by the serum of in the hamster of immunity.

cD28-Ig, CTLA4-I and PD-1-Ig binding tests

Utilize fat transfection amine 2000 (Gibco BRL), with B7.1-pCAGGS, B7-DC-pCAGG, PD-1-pCAGGS or independent carrier transfection 293T cell., after 24h, at FACS damping fluid (1 × HBSS, 2% calf serum, 10mM HEPES and 0.1%NaN ₃) middle resuspension cell, at 4 DEG C, 1000rpm rotates 5 minutes.Then Flick out buffer, is added to antibody in test tube, and 4 DEG C of incubations 20 minutes, with FACS wash buffer twice, repeat this program for secondary antibody.FACScan loses shape.B7.1 antibody is carried out the dilution of 1: 5,10 μ l/ samples (Gal-Tag).CD28-Ig, CTLA-4-Ig and PD-1-Ig mosaic of restructuring is used (R & D System, Inc) with the concentration of 2 μ g/ml, 10 μ l/ samples respectively.Sheep F (ab ') ₂anti-human igg-PE uses (Southern Biotechnology Associates, Inc.) after doing the dilution of 1: 20.

the dimeric synthesis of B7-DC-Ig

By merging the amino acid whose sequence of coding B7-DC N-terminal and coding human IgG in pIg-Tail Plus carrier (R & D systems) ₁the amino acid whose sequence of Fc C-terminal prepares B7-DC-Ig construct, and the-terminal amino acid of B7-DC is not wherein containing the membrane spaning domain in reading frame.Utilize fat transfection amine 2000 (Gibco BRL) or GINE JAMMER (Stratagene), with pIg/B7-DC transient transfection COS-7 cell.By saturated ammonium sulphate purifying B7-DC-Ig fused protein from the supernatant liquor of serum-free.SDS-PAGE and silver dye confirm its purity > 90%.

t cell propagation and CYTOKINE ASSAYS

For the common irritant test of AntiCD3 McAb, 96 hole flat undersides (Immulon 4 purchased from Dynex) anti-cd 3 antibodies (2C11, and B7.1-Ig (R & D System), and the 100ng/mlB7-DC-Ig diluted in 1 × PBS (Gibco) pH7.4 or Isotype control (Sigma) at 37 DEG C pre-coated 2 hours Pharmingen).Then use 1 × PBS washing plate 3 times, with the RPMI1640 substratum blocking of plates half an hour being supplemented with 10%FCS, penicillin/streptomycin, non-essential amino acid, Sodium.alpha.-ketopropionate, Pidolidone (salt), 2 mercapto ethanol, then add T cell.Spleen and lymphoglandula is obtained from the BALB/c mouse in 6-10 age in week.By indirect method, with ACK damping fluid cracking RBC, with dynabeads M-280 (Dynex) purified T cell.Rinse this pearl twice with PBS pH 7.4+1%FCS, then add cell, will by anti-IE ^dbe added in cell with the mixtures of antibodies that B220/CD45RO or CD8 α (Pharmingen) forms, be mixed in 4 DEG C of incubations 30 ' along with two-way.Test tube is placed on Dynal MPC 5 ' come isolated cell, 1500rpm centrifugal 5 ', rinses twice to remove unconjugated Ab with 2 × PBS pH7.4+1%FCS.With incubation 15 ', with 2 × 10 ⁵cells/well inoculating cell carrys out the same step of repetition.After incubation 72h, by 10 μ l ³h-thymidine (1 μ Ci/ hole) joins in each hole, incubation 18h.With a Packard Micromate cell harvester cell, reading filtrate on Packard Matrix 96 directly β counter.

For the common irritant test utilizing the RENCA system of presenting HA antigen to carry out, with the RPMI-1640 culture medium culturing RENCA cell being supplemented with 10%FCS, penicillin/streptomycin, non-essential amino acid, Sodium.alpha.-ketopropionate.Express to make II class MHC with IFN-γ (75U/ml) inducing cell 72hr.Then irradiate 13,200 rads, think 2 × 10 ⁴cells/well (96 hole flat underside) is inoculated.Then, Xiang Kongzhong adds the HA110-120 peptide in 2.5 μ g/ holes and the Ig fusion molecule of different concns.Separation transgenosis I-E described above ^d+ HA specific T-cells (H.vonBoehmer is so kind as to give, Harvard University), with 4 × 10 ⁵cells/well is inoculated.After incubation 48hr, by 10 μ l ³h-thymidine (1 μ Ci/ hole) to join in each hole and incubation 18hrs.With a Packard Micromate cell harvester cell, reading filtrate on Packard Matrix 96 directly β counter.

In order to by elisa assay cytokine product, cultivate as described above, collect supernatant liquor in the time of specifying.The concentration of IL-2, IL-10 and IFN-γ is measured with commercially available ELISA kit (Endogen) and IL-4 and IL-6 (R & d system).

stimulate altogether in body

The armpit of the mixing of TCR transgenic mouse system 6.5, inguinal region, uterine cervix and mesenteric mesaraic LN dissociation in RPMI substratum (GIBCOBRL) will be derived from, by the nylon cell strainer of 100 μm, rinse in Hank ' s damping fluid (GIBCO BRL) of sterilizing, transgenic mouse system 6.5 wherein can express one and identify I-E under B10.D2 genetic background ^depi-position ( ¹¹⁰sFERFEIFPKE ¹²⁰[SEQ ID NO:12]) TCR. painted with after the ratio of the cd4 cell measuring clonotypic, containing 2.5 × 10 in 0.2ml sterilizing Hank ' s damping fluid ⁶the cell preparation intravenously (i.v.) of clonotypic cells is expelled in the tail vein of acceptor B10.D2 mouse.Carry out this transfer after 3 days, be expelled in hind paw (hind footpad) by subcutaneous (s.c) and carry out immune animal.Each mouse accepts the two a kind of side injections in three kinds of preparations:

(A) with 1: 1 volume ratio and the HA (each sole) (HA peptide (111-120)) that synthesizes of incomplete Fu Shi adjuvant (IFA) (Sigma) 10 μ g of combining,

(B) the HA-IFA mixture containing 25 μ g B7-DC-Ig, or

(C) the HA-IFA mixture containing 25 μ g isotype control Ab.After 7 days, the LN joint that results are discharged; 1.5 × 10 ⁵individual LN cell is cultivated together with the synthesis HA peptide of prescribed concentration in the tissue culturing plate of round bottom 96 hole.By with 1 μ Ci [ ³h] thymidine pulse culture 48 hours, then cultivate 12 h before harvest, detect the radioactive amount combined and carry out proliferation test.

Example II

the qualification of B7-DC and sign

B7-DC is separated from the subtracted library between DC with activated macrophage.These two cell masses for cDNA subtrahend are the DC that cultivates as the GM-CSF of the derived from bone marrow of " test " group and the M-CSF scavenger cell in adherent bone marrow source activated as the gamma-interferon+LPS of " driving " group.Grow the II of 8 days ^hiclass MHCB7 ^hi" maturation " DC is sorted to the source of purity as test cDNA of > 93%.By flow cytometry, DC is characterized as being the II class MHC level than scavenger cell with approximately high 50 times.These two groups express B7.1 and B7.2, but the level of B7.2 is much higher in DC.The expression level of F4/80 and CD16 in scavenger cell group is higher.Carry out functional comparison confirming DC group in stimulation allogeneic mixed lymphocyte reaction approximately more effective than scavenger cell group 100 times to these two cell masses.

From two groups after extracting RNA, we use the cDNA synthesis system of a PCR-based, then carry out the subtraction procedure of PCR-based, and PCR screens.We are by the called after B7-DC of in the clone of differential expression, the immunoglobulin supergene family member that clones coding wherein is new.The length of mouse B7-DCcDNA is ~ 1.7kb, and the precursor protein matter of the N-terminal signal peptide containing a 23aa of coding one 247 amino acid (aa), predicted molecular mass is ~ 25kd (table 1).The leader sequence of deriving with SOSUI program (31) qualification and membrane spaning domain.In the membrane spaning domain of the 23aa of mB7-DC, find two charged amino acid, show that this place is binding partners.At amino acid levels, mouse mB7-DC and people B7-DC has the homology of 70%, and this shows that they are straight to homology (orthologues) (seeing the following form).HB7-DC is somewhat different than mouse B7-DC, because it has a longer cytoplasmic tail.

Pass through homology search, find B7-DC and the B7-H1 (identity of 34%, the similarity of 48%) (table 2), the butyrophilin (identity of 30% that content is lower, the similarity of 45%) there is very high homology, with B7.1 and B7.2, there is the identity (table 3) of < 20%.Phylogeny research shows, butyrophilin can be reorganized (exon shuffling) (32,33) and contacted with B7 family by exon.They each containing typical IgV-IgC structure and membrane spaning domain.Contrary with other B7 family member, mouse B7-DC has very short cytoplasmic tail (4aa).

Table 1

The aminoacid sequence of mouse B7-DC and people B7-DC compares.On line out mB7-DC derive leader and membrane spaning domain.Utilize Clustalw Gonnet Pam250 matrix to contrast, [*] represents consistent amino acid, and [:] represents conservative replacement, and the very important cysteine residues of the formation for the disulfide linkage in immunoglobulin V district or C district represents by italic.

the leader sequence of deriving

mB7-DC MLLLLPILNLSLQLHPVAALFTVTAPKEVYTVDVGSSVSLECDFDRRECTELEGIRASLQ

hB7-DC MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQ

＊：：＊＊：＊.＊.＊＊＊＊：＊＊＊＊＊＊＊.＊＊＊：＊：：＊＊.＊：＊＊＊：＊＊..：＊.＊＊＊＊＊

mB7-DC KVENDTSLQSERATLLEEQLPLGKALFHIPSVQVRDSGQYRCLVICGAAWDYKYLTVKVK

hB7-DC KVENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVK

＊＊＊＊＊＊＊：＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊.＊＊＊＊＊.＊＊＊：＊：：＊＊.＊＊＊＊＊＊＊＊：＊＊＊

mB7-DC ASYMRIDTRILEVPGTGEVQLTCQARGYPLAEVSWQNVSVPANTSHIRTPEGLYQVTSVL

hB7-DC ASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSVL

＊＊＊：＊：＊：＊＊：＊＊＊.＊＊：＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊＊

the TM structural domain of deriving

mB7-DC RLKPQPSRNFSCMFWNAHMKELTSAIIDPLSRMEPKVPRTWPLHVFIPACTIALIFLAIV

hB7-DC RLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPSCIIAFIFIATV

＊＊＊＊＊.＊＊＊＊＊：＊＊＊：＊：：＊＊＊＊＊＊＊：＊＊＊：.＊＊＊＊：＊＊＊：＊＊＊：＊＊：＊＊

mB7-DC IIQRKRI--------------------------

hB7-DC IALRKQLCQKLYSSKDTTKRPVTTTKREVNSAI

＊＊＊：：

Table 2

The aminoacid sequence of mB7-DC and mB7-H1 compares

mB7-DC MLLLLPILNLSLQLHPVAALFTVTAPKEVYTVDVGSSVSLECDFDRRECTELEGIRASLQ

mB7-H1 -MRIFAGIIFTACCH-LLRAFTITAPKDLYVVEYGSNVTMECRFPVERELDLLALVVYWE

：：：.：：：＊：＊＊：＊＊＊＊：：＊.＊：＊＊.＊：：＊＊＊..：＊.：.：

mB7-DC K----------VENDTSLQSE----RATLLEEQLPLGKALFHIPSVQVRDSGQYRCLVIC

mB7-H1 KEDEQVIQFVAGEEDLKPQHSNFRGRASLPKDQLLKGNAALQITDVKLQDAGVYCCIISY

＊＊：＊.＊.＊＊：＊：：＊＊＊：＊：：＊..＊：：：＊：＊＊＊：：

mB7-DC GAAWDYKYLTVKVKASYMRIDTRILEVPGTGEVQLTCQARGYPLAEVSWQN-----VSVP

mB7-H1 GGA-DYKRITLKVNAPYRKINQRISVDPATSEHELICQAEGYPEAEVIWTNSDHQPVSGK

＊.＊＊＊＊：＊：＊＊：＊.＊：＊：＊＊＊.＊.＊：＊＊＊＊.＊＊＊＊＊＊＊＊＊＊

mB7-DC ANTSHIRTPEGLYQVTSVLRLKPQPSRNFSCMFWNAH--MKELTSAIIDPLSRMEPKVPR

mB7-H1 RSVTTSRTEGMLLNVTSSLRVNATANDVFYCTFWRSQPGQNHTAELIIPELPATHPPQNR

..：＊＊＊：＊＊＊＊＊：：...＊＊＊＊.：：：.：.＊＊＊..＊＊

mB7-DC T-WPLHVFIPACTIALIFLAIVIIQRKRI------------------------

mB7-H1 THWVLLGSILLFLIVVSTVLLFLRKQVRMLDVEKCGVEDTSSKNRNDTQFEET

＊＊＊＊＊.：：：.：：：＊：

Table 3

The aminoacid sequence of B7-DC and B7 family member compares

Retrieve (matrix B LOSUM62) with NCBI blast2 to compare

1. at the amino acid that corresponding site is consistent

2. the amino acid similar in corresponding site-divide into groups as follows: (A, G); (S, T); (E, D); (R, K, H); (Q, N); (V, I, L, M); (Y, F); (W); (C); (P)

3. do not find obvious similarity with matrix B LOSUM62

4.BT=butyrophilin matter

5.＝PDL-1

In order to measure the genome structure of mB7-DC, the present invention by utilize 5 ' and 3 ' UTR probe screen one mixing bacterial artificial chromosome (BAC) library, isolate a genomic clone.Chromosomal localization figure is made with BAC clone.The chromosomal localization of B7-DC is carried out with fluorescence in situ hybridization (FISH).Measure No. 19 karyomit(e)s of 10 specific markers, confirm that mB7-DC is positioned at 47% place of heterochromatin-euchromatic border to No. 19 chromosomal telomere spacings, i.e. the respective regions at band 19C2 and 19C3 interface.Incubation hybridized slides in the anti-digoxigenin antibody of fluorescein, the point then calculated by DAPI is painted carrys out detection specificity hybridization signal.This seat is corresponding to people No. 9 chromosomal regions, and hB7-H1 is located herein.

Find that hB7-DC is positioned on two No. 9 karyomit(e) BAC clones.In addition, find that hB7-DC and hB7-H1 is positioned on single No. 9 karyomit(e) BAC clones, the insertion size (Fig. 1) of this clone with the 164kb that has an appointment.B7-DC and B7-H1 is genomic close to making people associate B7.1/B7.2 couple, in their mutually positioning scopes internal in 1 megabasse.

EXAMPLE III

selective expression B7-DC in dendritic cell

In order to measure the phraseology of B7-DC, carry out virtual Northern analysis with RNA, RNA wherein from various tissue, macrophage system, macrophage culture and derive from marrow and spleen dendritic cell extracting obtain.With B7-DC probe at immature (4,6 days) and ripe (8 days and MHCII through sorting ^hib7 ^hi) strong hybridization detected in the DC of derived from bone marrow and spleen DC, and the BM scavenger cell of 4 kinds of macrophage systems, activation or peritoneal macrophages any one in signal (Fig. 2) all do not detected.Cultivate peripheral blood lymphocytes with the GM-CSF being added with IL-4 or Flt-3L, in the people DC therefrom grown, the strong expression (Fig. 3) of hB7-DC detected.In order to check the cell surface expression of B7-DC protein, anti-m B7-DC antibody was utilized to contaminate DC.Close with solubility B7-DC-Ig, DC finds that there is dyeing (Fig. 4).

b7-DC is not combined with CD28 or CTLA-4, but is combined with PD-1

Although B7-DC has and the structure of B7 family homology and sequence, but it is not containing the CD28/CTLA-4 binding sequence of inferring, SQDXXXELY [SEQ ID NO:13] or XXXYXXRT [SEQ IDNO:14] (34) (wherein any amino acid of X=).In order to directly evaluate combination, have studied the ability of the painted 293T cell through B7-DC or B7.1 transfection of dimer CD28-Ig and CTLA-4-Ig.B7.1 transfectant finds strong combination, and B7-DC transfectant does not combine (Fig. 5).Based on homology (homlogy) and the genomic proximity of B7-DC and B7-H1/PD-L1, carry out the PD-1 testing the binding partners tested as B7-DC candidate.In fact, PD-100IG and B7-DC transfectant combines, but is not combined with B7.1 transfectant.The combination of BPD-1-Ig and B7-DC transfectant than CTL-4-It and CD28-Ig and B7.1 transfectant in conjunction with low, although it is specific.The positive staining that stable B7-DC-GFP transfectant and PD-1-Ig occur further demonstrate that PD-1 can be combined with B7-Dc.Therefore can reach a conclusion, the same as B7-H1 with B7h/B7RP-1, B7-DC is not using CD28 or CTLA-4 as acceptor.On the contrary, the acceptor of PD-1 seemingly B7-DC.

EXAMPLE IV

b7-DC is as the function of T cell costimulatory molecules

Preparation can be added to T cell stimulates the solvable B7-DC-Ig fused protein in detecting whether to have common stimulating activity to be used for detecting B7-DC.When there is B7-DC-Ig, B7.1-Ig or a kind of Isotype control, detect T cell to the propagation response stimulated by the amount increasing fixing AntiCD3 McAb.Fig. 6 (left figure) shows when there is suboptimal AntiCD3 McAb concentration, and B7-DC stimulates the response of T cell propagation larger altogether than B7.1.And the propagation response that B7-DC stimulates altogether in cd4 cell is than higher in cd8 cell (figure six is right).When lacking the stimulator that TCR concentrates, B7-DC can not irritate T cell, and this shows that B7-DC provides a real costimulatory signal.

When MHC peptide complex body produces " signal 1 ", B7-DC also stimulates proliferation response altogether.RENCA cell (analyzed by RT-PCR, this cell does not express endogenous B7.1, B7.2 or B7-DC) is processed to induce the expression of II class MHC with γ-IFN.These cells carry I-E ^drestricted HA110-120 peptide (FERFEIFPKE) (35) [SEQ ID NO:15].Add from I-E ^dthe splenic t-cell of purifying in+HA 110-120 specific TCR transgenic mouse system, measures propagation response when there is B7-DC-Ig, B7.1-Ig or Isotype control.Fig. 7 shows B7-DC and has larger common stimulating activity than B7.1.

b7-DC stimulates the mode producing lymphokine altogether

The t cell response that the best stimulated altogether B7 family molecule characterizes produces lymphokine.These lymphokines are important T cell effect media.Researched and analysed the many different lymphokine that T cell produces, T cell wherein is stimulated by AntiCD3 McAb or HA antigen (Fig. 8) and is stimulated altogether by B7-DC-Ig, B7.1-Ig or Isotype control.The mode that lymphokine stimulates altogether is quite consistent, is no matter as " signal 1 " with AntiCD3 McAb or MHC peptide complex body.Obviously, B7-DC stimulates higher levels of γ-IFN altogether than B7.1.B7-DC also stimulates altogether and produces a large amount of IL-6, and B7.1 stimulates in fact altogether and do not produce completely.When two molecules stimulate generation IL-2 altogether, B7.1 is more effective than B7-DC.Therefore, the mode that B7-DC and B7.1 stimulates altogether is different, and in the pro-inflammatory lymphokine that common stimulation is important, B7-DC is more effective.

EXAMPLE V

b7-DC improves immune response in body

In order to determine whether B7-DC has biological activity in vivo, present inventors studied for peptide vaccine, whether B7-DC-Ig can improve immune response.B7-DC-Ig or one isotype control Ab is added in the immunogenic cocktail of HA 110-120 peptide and IFA.In order to count the HA specific C D4T cell in body, first 3 days of immunity, by 2.5 × 10 ⁶anti-HA 6.5T cell forwards in mouse.Immunity, after 7 days, collects the LN cell of discharging, with the HA110-120 peptide cells stimulated in vitro 2 days of difference amount.Fig. 9 shows the B7-DC-Ig added and has in fact increased substantially and reply the propagation of HA.Relative to isotype antibody controls, the total amount adding group HA specific T-cells in the LN discharged of B7-DC-Ig adds about 2 times.Therefore, can infer that B7-DC has the ability improving antigen-specific reaction, even on each cell base.

Example VI

Discuss and conclusion

The present inventor has been found that and characterizes a kind of new B7 family member, and it is expressed by height limitation in DC, and the T cell with uniqueness stimulates performance altogether.People's straight homologues of B7-DC is also expressed in DC.

Different from previously described B7 family member, this restricted phraseology indicates the immune response that B7-DC participates in being different from known B7.1/2 approach.When a weak B7-DC signal being detected in the scavenger cell activated, the preliminary Real time RT-PCR analysis carried out shows the expression ratio of B7-DC mRNA in DC high > 15 times in the scavenger cell of activation.Equally, very low-level B7-DC is detected with antibody dye technology at the Macrophage Surface of activation.Do not know that whether this is enough for effective T cell activation.

Compare with other B7 family member, B7-DC stimulates altogether and produces the unusual mode of lymphokine and mean a unique biological action.The traditional classification of cytokine is as follows: Th1 cytokine comprises IL-2, γ-IFN and lymphotoxin; Th2 cytokine comprises IL-4, IL-5, IL-6 and IL-13 (36).B7-DC does not induce any class Th1 or Th2 lymphokine distribution (profile).B7-DC induces considerably less IL-4, does not induce IL-10.But IL-6 is considered to a kind of Th2 cytokine.The lower IL-2 stimulated altogether relative to B7.1, B7-DC and the non-compliant Th1 pattern of higher γ-IFN.But high γ-IFN rate schedule understands that B7-DC causes important T cell effector function.

B7-DC stimulates the ability of IL-6 to merit attention altogether.The strong T cell proliferative response of being induced by B7-DC is that part produces IL-6 because it stimulates altogether consumingly, and this is not when observing with when B7.1.IL-6 is an agent of effectively increasing (being combined with other proliferative stimuli) (37,38) of T cell propagation.IL-6 is a kind of multi-functional cytokine, can not only the function of regulatory T-cell, can also regulate the growth (39,40) of proinflammatory reaction, monocytic differentiation, the differentiation of B cell, thrombosis, bone resorption and some hematopoetic tumor.IL-6 and solubility IL-6 acceptor (sIL-6R) cooperation induced chemokine and leukocyte recruitment (41).It can by the effective anti-apoptosis effect of Stat-3 activation mediation.Report the important channel (42,43) that the IL-6 depending on Stat-3 activation in T cell is the T cell survival of activation, although other report infers that Stat-3 plays a role in Resting T cells.

Although B7-DC is not combined with CD28 or CTLA-4, it is in conjunction with an acceptor (22,47,48) of PD-1, B7-H1/PD-L1.Whether it does not also determine in conjunction with acceptor (23-25,44-46)-ICOS of B7h/B7RP-1.The physical linkage that obvious homology (higher than the homology between B7.1 with B7.2) between B7-DC and B7-H1/PDL-1, hB7-H1/PD-L1 and hB7-DC are similar and they show that they are produced by a relative gene duplication event recently with common receptors bind and correlate.This is similar to the relation of B7.1 with B7.2, B7.1 and B7.2 is positioned at mouse No. 16 karyomit(e)s and the chromosomal megabasse of people No. 3 to (49) in scope.

Understand that the Relative biological effect of B7-DC to B7-H1/PD-L1 is important when PD-1 is receptor-mediated with other deduction.After T cell activation, PD-1 is expressed, and seem suppressor T cell activation.Under stimulating the condition of T cell with the AntiCD3 McAb of high density, PD-1 causes Apoptosis.PD-1 can reject mouse and occur autoimmune syndrome (22), and it is characterized by the clinical manifestations of hypertrophic cardiomyopathy.On the contrary, under Dong etc. (21) are reported in the AntiCD3 McAb of low concentration, B7-H1/PD-L1 stimulates the release of T cell propagation and cytokine altogether.Relation according to CD28/CTLA-4 is analogized, and PD-L1 can be a kind of counter receptor of the active acceptor still do not identified.Although have the performance in conjunction with PD-1, it is different that B7-DC and B7-H1 is total in stimulation mode in their lymphokine; B7-H1 stimulates T cell IL-10 product altogether, and B7-DC can not.The cellular expression patterns of the uniqueness of B7-DC and common hormesis indicate it in immunization, have unique effect.

The reference quoted above and below all adds herein by reference, and no matter whether it is add particularly.

Sufficient description is done to the present invention, those of ordinary skill in the art can understand, when without departing from the spirit and scope of the present invention, in wide synchronization parameters, concentration and a condition and range, also can implement the present invention, and do not need too much test.

Although be described the present invention in conjunction with specific embodiment of the invention scheme, be construed as and can be improved further the present invention.The application tend to contain usually follow fundamental principle of the present invention any change, application or improvement, comprise and this kind ofly to depart from relative to of the present invention, such as known in the field that the invention belongs to or departing from common practical framework, such as can be applied to departing from essential features above, these essential features are illustrated within the scope of accompanying claim.

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<130>2240-171934

<140>PCT/US01/13430

<141>2001-04-17

<160>16

<170>PatentIn version 3.1

<210>1

<211>819

<212>DNA

<213> people

<220>

<221>CDS

<222>(1)..(819)

<223>

<400>1

atg atc ttc ctc ctg cta atg ttg agc ctg gaa ttg cag ctt cac cag 48

Met Ile Phe Leu Leu Leu Met Leu Ser Leu Glu Leu Gln Leu His Gln

1 5 10 15

ata gca gct tta ttc aca gtg aca gtc cct aag gaa ctg tac ata ata 96

Ile Ala Ala Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile

20 25 30

gag cat ggc agc aat gtg acc ctg gaa tgc aac ttt gac act gga agt 144

Glu His Gly Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser

35 40 45

cat gtg aac ctt gga gca ata aca gcc agt ttg caa aag gtg gaa aat 192

His Val Asn Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn

50 55 60

gat aca tcc cca cac cgt gaa aga gcc act ttg ctg gag gag cag ctg 240

Asp Thr Ser Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu

65 70 75 80

ccc cta ggg aag gcc tcg ttc cac ata cct caa gtc caa gtg agg gac 288

Pro Leu Gly Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp

85 90 95

gaa gga cag tac caa tgc ata atc atc tat ggg gtc gcc tgg gac tac 336

Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr

100 105 110

aag tac ctg act ctg aaa gtc aaa gct tcc tac agg aaa ata aac act 384

Lys Tyr Leu Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr

115 120 125

cac atc cta aag gtt cca gaa aca gat gag gta gag ctc acc tgc cag 432

His Ile Leu Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln

130 135 140

gct aca ggt tat cct ctg gca gaa gta tcc tgg cca aac gtc agc gtt 480

Ala Thr Gly Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val

145 150 155 160

cct gcc aac acc agc cac tcc agg acc cct gaa ggc ctc tac cag gtc 528

Pro Ala Asn Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val

165 170 175

acc agt gtt ctg cgc cta aag cca ccc cct ggc aga aac ttc agc tgt 576

Thr Ser Val Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys

180 185 190

gtg ttc tgg aat act cac gtg agg gaa ctt act ttg gcc agc att gac 624

Val Phe Trp Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp

195 200 205

ctt caa agt cag atg gaa ccc agg acc cat cca act tgg ctg ctt cac 672

Leu Gln Ser Gln Met Glu Pro Arg Thr His Pro Thr Trp Leu Leu His

210 215 220

att ttc atc ccc tcc tgc atc att gct ttc att ttc ata gcc aca gtg 720

Ile Phe Ile Pro Ser Cys Ile Ile Ala Phe Ile Phe Ile Ala Thr Val

225 230 235 240

ata gcc cta aga aaa caa ctc tgt caa aag ctg tat tct tca aaa gac 768

Ile Ala Leu Arg Lys Gln Leu Cys Gln Lys Leu Tyr Ser Ser Lys Asp

245 250 255

aca aca aaa aga cct gtc acc aca aca aag agg gaa gtg aac agt gct 816

Thr Thr Lys Arg Pro Val Thr Thr Thr Lys Arg Glu Val Asn Ser Ala

260 265 270

atc 819

Ile

<210>2

<211>273

<212>PRT

<213> people

<400>2

Met Ile Phe Leu Leu Leu Met Leu Ser Leu Glu Leu Gln Leu His Gln

1 5 10 15

Ile Ala Ala Leu Phe Thr Val Thr Val Pro Lys Glu Leu Tyr Ile Ile

20 25 30

Glu His Gly Ser Asn Val Thr Leu Glu Cys Asn Phe Asp Thr Gly Ser

35 40 45

His Val Asn Leu Gly Ala Ile Thr Ala Ser Leu Gln Lys Val Glu Asn

50 55 60

Asp Thr Ser Pro His Arg Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu

65 70 75 80

Pro Leu Gly Lys Ala Ser Phe His Ile Pro Gln Val Gln Val Arg Asp

85 90 95

Glu Gly Gln Tyr Gln Cys Ile Ile Ile Tyr Gly Val Ala Trp Asp Tyr

100 105 110

Lys Tyr Leu Thr Leu Lys Val Lys Ala Ser Tyr Arg Lys Ile Asn Thr

115 120 125

His Ile Leu Lys Val Pro Glu Thr Asp Glu Val Glu Leu Thr Cys Gln

130 135 140

Ala Thr Gly Tyr Pro Leu Ala Glu Val Ser Trp Pro Asn Val Ser Val

145 150 155 160

Pro Ala Asn Thr Ser His Ser Arg Thr Pro Glu Gly Leu Tyr Gln Val

165 170 175

Thr Ser Val Leu Arg Leu Lys Pro Pro Pro Gly Arg Asn Phe Ser Cys

180 185 190

Val Phe Trp Asn Thr His Val Arg Glu Leu Thr Leu Ala Ser Ile Asp

195 200 205

Leu Gln Ser Gln Met Glu Pro Arg Thr His Pro Thr Trp Leu Leu His

210 215 220

Ile Phe Ile Pro Ser Cys Ile Ile Ala Phe Ile Phe Ile Ala Thr Val

225 230 235 240

Ile Ala Leu Arg Lys Gln Leu Cys Gln Lys Leu Tyr Ser Ser Lys Asp

245 250 255

Thr Thr Lys Arg Pro Val Thr Thr Thr Lys Arg Glu Val Asn Ser Ala

260 265 270

Ile

<210>3

<211>1655

<212>DNA

<213>Murinae gen.sp.

<220>

<221>CDS

<222>(210)..(953)

<223>

<400>3

gaattcggca cgaggtcaaa tgtggcatat ctttgttgtc tccttctgtc tcccaactag 60

agagaacaca cttacggctc ctgtcccggg caggtttggt tgtcggtgtg attggcttcc 120

agggaacctg atacaaggag caactgtgtg ctgccttttc tgtgtctttg cttgaggagc 180

tgtgctgggt gctgatattg acacagacc atg ctg ctc ctg ctg ccg ata ctg 233

Met Leu Leu Leu Leu Pro Ile Leu

1 5

aac ctg agc tta caa ctt cat cct gta gca gct tta ttc acc gtg aca 281

Asn Leu Ser Leu Gln Leu His Pro Val Ala Ala Leu Phe Thr Val Thr

10 15 20

gcc cct aaa gaa gtg tac acc gta gac gtc ggc agc agt gtg agc ctg 329

Ala Pro Lys Glu Val Tyr Thr Val Asp Val Gly Ser Ser Val Ser Leu

25 30 35 40

gag tgc gat ttt gac cgc aga gaa tgc act gaa ctg gaa ggg ata aga 377

Glu Cys Asp Phe Asp Arg Arg Glu Cys Thr Glu Leu Glu Gly Ile Arg

45 50 55

gcc agt ttg cag aag gta gaa aat gat acg tct ctg caa agt gaa aga 425

Ala Ser Leu Gln Lys Val Glu Asn Asp Thr Ser Leu Gln Ser Glu Arg

60 65 70

gcc acc ctg ctg gag gag cag ctg ccc ctg gga aag gct ttg ttc cac 473

Ala Thr Leu Leu Glu Glu Gln Leu Pro Leu Gly Lys Ala Leu Phe His

75 80 85

atc cct agt gtc caa gtg aga gat tcc ggg cag tac cgt tgc ctg gtc 521

Ile Pro Ser Val Gln Val Arg Asp Ser Gly Gln Tyr Arg Cys Leu Val

90 95 100

atc tgc ggg gcc gcc tgg gac tac aag tac ctg acg gtg aaa gtc aaa 569

Ile Cys Gly Ala Ala Trp Asp Tyr Lys Tyr Leu Thr Val Lys Val Lys

105 110 115 120

gct tct tac atg agg ata gac act agg atc ctg gag gtt cca ggt aca 617

Ala Ser Tyr Met Arg Ile Asp Thr Arg Ile Leu Glu Val Pro Gly Thr

125 130 135

ggg gag gtg cag ctt acc tgc cag gct aga ggt tat ccc cta gca gaa 665

Gly Glu Val Gln Leu Thr Cys Gln Ala Arg Gly Tyr Pro Leu Ala Glu

140 145 150

gtg tcc tgg caa aat gtc agt gtt cct gcc aac acc agc cac atc agg 713

Val Ser Trp Gln Asn Val Ser Val Pro Ala Asn Thr Ser His Ile Arg

155 160 165

acc ccc gaa ggc ctc tac cag gtc acc agt gtt ctg cgc ctc aag cct 761

Thr Pro Glu Gly Leu Tyr Gln Val Thr Ser Val Leu Arg Leu Lys Pro

170 175 180

cag cct agc aga aac ttc agc tgc atg ttc tgg aat gct cac atg aag 809

Gln Pro Ser Arg Asn Phe Ser Cys Met Phe Trp Asn Ala His Met Lys

185 190 195 200

gag ctg act tca gcc atc att gac cct ctg agt cgg atg gaa ccc aaa 857

Glu Leu Thr Ser Ala Ile Ile Asp Pro Leu Ser Arg Met Glu Pro Lys

205 210 215

gtc ccc aga acg tgg cca ctt cat gtt ttc atc ccg gcc tgc acc atc 905

Val Pro Arg Thr Trp Pro Leu His Val Phe Ile Pro Ala Cys Thr Ile

220 225 230

gct ttg atc ttc ctg gcc ata gtg ata atc cag aga aag agg atc tag 953

Ala Leu Ile Phe Leu Ala Ile Val Ile Ile Gln Arg Lys Arg Ile

235 240 245

gggaagctgt attacggaag aagtggtctc ttcttcccag atctggacct gcggtcttgg 1013

gagttggaag gatctgatgg gaaaccctca agagacttct ggactcaaag tgagaatctt 1073

gcaggacctg ccatttgcac ttttgaaccc tttggacggt gacccagggc tccgaagagg 1133

agcttgtaag actgacaatc ttccctctgt ctcaagactc tctgaacagc aagaccccaa 1193

tggcacttta gacttacccc tgggatcctg gaccccagtg agggcctaag gctcctaatg 1253

actttcaggg tgagaacaaa aggaattgct ctccgcccca cccccacctc ctgctttccg 1313

cagggagaca tggaaattcc cagttactaa aatagattgt caatagagtt atttatagcc 1373

ctcatttcct ccggggactt ggaagcttca gacagggttt ttcataaaca aagtcataac 1433

tgatgtgttt tacagcatcc tagaatcctg gcagcctctg aagttctaat taactggaag 1493

catttaagca acacgtcaag tgcccctgct gtggtatttg tttctacttt tctgttttta 1553

aagtgtgagt cacaaggtaa ttgttgtaac ctgtgatatc actgtttctt gtgtctcttc 1613

tttcaactac atcttttaaa acaaaaaaaa aaaaaaaaaa aa 1655

<210>4

<211>247

<212>PRT

<213>Murinae gen.sp.

<400>4

Met Leu Leu Leu Leu Pro Ile Leu Asn Leu Ser Leu Gln Leu His Pro

1 5 10 15

Val Ala Ala Leu Phe Thr Val Thr Ala Pro Lys Glu Val Tyr Thr Val

20 25 30

Asp Val Gly Ser Ser Val Ser Leu Glu Cys Asp Phe Asp Arg Arg Glu

35 40 45

Cys Thr Glu Leu Glu Gly Ile Arg Ala Ser Leu Gln Lys Val Glu Asn

50 55 60

Asp Thr Ser Leu Gln Ser Glu Arg Ala Thr Leu Leu Glu Glu Gln Leu

65 70 75 80

Pro Leu Gly Lys Ala Leu Phe His Ile Pro Ser Val Gln Val Arg Asp

85 90 95

Ser Gly Gln Tyr Arg Cys Leu Val Ile Cys Gly Ala Ala Trp Asp Tyr

100 105 110

Lys Tyr Leu Thr Val Lys Val Lys Ala Ser Tyr Met Arg Ile Asp Thr

115 120 125

Arg Ile Leu Glu Val Pro Gly Thr Gly Glu Val Gln Leu Thr Cys Gln

130 135 140

Ala Arg Gly Tyr Pro Leu Ala Glu Val Ser Trp Gln Asn Val Ser Val

145 150 155 160

Pro Ala Asn Thr Ser His Ile Arg Thr Pro Glu Gly Leu Tyr Gln Val

165 170 175

Thr Ser Val Leu Arg Leu Lys Pro Gln Pro Ser Arg Asn Phe Ser Cys

180 185 190

Met Phe Trp Asn Ala His Met Lys Glu Leu Thr Ser Ala Ile Ile Asp

195 200 205

Pro Leu Ser Arg Met Glu Pro Lys Val Pro Arg Thr Trp Pro Leu His

210 215 220

Val Phe Ile Pro Ala Cys Thr Ile Ala Leu Ile Phe Leu Ala Ile Val

225 230 235 240

Ile Ile Gln Arg Lys Arg Ile

245

<210>5

<211>744

<212>DNA

<213>Murinae gen.sp.

<400>5

atgctgctcc tgctgccgat actgaacctg agcttacaac ttcatcctgt agcagcttta 60

ttcaccgtga cagcccctaa agaagtgtac accgtagacg tcggcagcag tgtgagcctg 120

gagtgcgatt ttgaccgcag agaatgcact gaactggaag ggataagagc cagtttgcag 180

aaggtagaaa atgatacgtc tctgcaaagt gaaagagcca ccctgctgga ggagcagctg 240

cccctgggaa aggctttgtt ccacatccct agtgtccaag tgagagattc cgggcagtac 300

cgttgcctgg tcatctgcgg ggccgcctgg gactacaagt acctgacggt gaaagtcaaa 360

gcttcttaca tgaggataga cactaggatc ctggaggttc caggtacagg ggaggtgcag 420

cttacctgcc aggctagagg ttatccccta gcagaagtgt cctggcaaaa tgtcagtgtt 480

cctgccaaca ccagccacat caggaccccc gaaggcctct accaggtcac cagtgttctg 540

cgcctcaagc ctcagcctag cagaaacttc agctgcatgt tctggaatgc tcacatgaag 600

gagctgactt cagccatcat tgaccctctg agtcggatgg aacccaaagt ccccagaacg 660

tggccacttc atgttttcat cccggcctgc accatcgctt tgatcttcct ggccatagtg 720

ataatccaga gaaagaggat ctag 744

<210>6

<211>26

<212>DNA

<213> artificial sequence

<220>

<223> primer

<400>6

ggagctactg catgttgatt gttttg 26

<210>7

<211>24

<212>DNA

<213> artificial sequence

<220>

<223> primer

<400>7

tgcaaactga ggcactgaaa agtc 24

<210>8

<211>25

<212>DNA

<213> artificial sequence

<220>

<223> primer

<400>8

ttgttgtctc cttctgtctc ccaac 25

<210>9

<211>24

<212>DNA

<213> artificial sequence

<220>

<223> primer

<400>9

acagttgctc cttgtatcag gttc 24

<210>10

<211>21

<212>DNA

<213> artificial sequence

<220>

<223> primer

<400>10

gtaacggccg ccagtgtgct g 21

<210>11

<211>23

<212>DNA

<213> artificial sequence

<220>

<223> primer

<400>11

cgccagtgtg atggatatct gca 23

<210>12

<211>11

<212>PRT

<213>Murinae gen.sp.

<400>12

Ser Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu

1 5 10

<210>13

<211>9

<212>PRT

<213> is unknown

<220>

<223> binding sequence

<220>

<221>misc_feature

<222>(4)..(6)

The Xaa of <223>4-6 position can be any amino acid

<400>13

Ser Gln Asp Xaa Xaa Xaa Glu Leu Tyr

1 5

<210>14

<211>8

<212>PRT

<213> is unknown

<220>

<223> binding sequence

<220>

<221>misc_feature

<222>(1)..(3)

The Xaa of <223>1-3 position can be any amino acid

<220>

<221>misc_feature

<222>(5)..(6)

The Xaa of <223>5-6 position can be any amino acid

<400>14

Xaa Xaa Xaa Tyr Xaa Xaa Arg Thr

1 5

<210>15

<211>10

<212>PRT

<213>Murinae gen.sp.

<400>15

Phe Glu Arg Phe Glu Ile Phe Pro Lys Glu

1 5 10

<210>16

<211>290

<212>PRT

<213>Murinae gen.sp.

<400>16

Met Arg Ile Phe Ala Gly Ile Ile Phe Thr Ala Cys Cys His Leu Leu

1 5 10 15

Arg Ala Phe Thr Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr

20 25 30

Gly Ser Asn Val Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu

35 40 45

Asp Leu Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val

50 55 60

Ile Gln Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn

65 70 75 80

Phe Arg Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn

85 90 95

Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr

100 105 110

Cys Cys Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu

115 120 125

Lys Val Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp

130 135 140

Pro Ala Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro

145 150 155 160

Glu Ala Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly

165 170 175

Lys Arg Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val

180 185 190

Thr Ser Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys

195 200 205

Thr Phe Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile

210 215 220

Ile Pro Glu Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr His Trp

225 230 235 240

Val Leu Leu Gly Ser Ile Leu Leu Phe Leu Ile Val Val Ser Thr Val

245 250 255

Leu Leu Phe Leu Arg Lys Gln Val Arg Met Leu Asp Val Glu Lys Cys

260 265 270

Gly Val Glu Asp Thr Ser Ser Lys Asn Arg Asn Asp Thr Gln Phe Glu

275 280 285

Glu Thr

290

Claims (11)

1. the nucleic acid molecule be separated, the fusion rotein that its coding first fusion partner and the second polypeptide form, wherein said first fusion partner is made up of the-terminal amino acid of SEQ ID NO:2, and not there is membrane spaning domain, the second wherein said polypeptide contains the structural domain of (a) one or more Ig CH; Two C-structure territories of (b) IgG CH; Or (c) human normal immunoglobulin C _γthe hinge area of 1 chain, C _h2nd district and C _h3rd district, and wherein said fusion rotein is in conjunction with programmed death molecule-1 (PD-1), wherein said-terminal amino acid is made up of the amino acid/11-221 of SEQ ID NO:2.

2. the nucleic acid molecule be separated, the fusion rotein of its coding first fusion partner and the second polypeptide composition, the leader sequence that wherein said first fusion partner is connected by the first fusion partner of the claim 1 with maturation forms, and described second polypeptide contains the structural domain of (a) one or more Ig CH; Two C-structure territories of (b) IgG CH; Or (c) human normal immunoglobulin C _γthe hinge area of 1 chain, C _h2nd district and C _h3rd district.

3. the nucleic acid molecule of any one of claim 1 and 2, it is in expression vector, and is operationally connected with promotor.

4. the nucleic acid molecule of claim 3, it is operationally connected with regulating the additive regulating sequence of described expression of nucleic acid in eukaryotic cell.

5. the nucleic acid molecule of claim 3 or 4, wherein this expression vector is plasmid or virus vector.

6. a host cell, it is transformed or transfection by the nucleic acid molecule of any one of claim 1-5, and wherein said host cell is not embryonic stem cell, or the sexual cell of human or animal.

7. the host cell of claim 6, this cell is a kind of mammalian cell.

8. the host cell of claim 7, wherein said cell is selected from by dendritic cell, the group of its precursor cell and tumour cell composition.

9. the host cell of claim 7, wherein said mammalian cell is Chinese hamster ovary (CHO) cell.

10. the ripe fusion rotein of the separation of the supernatant liquor acquisition of the host cell of an any one of Accessory Right requirement 6-9.

The fusion rotein of 11. claims 10 for the preparation of enhancing mammalian subject to the application in the reagent of the t cell response that antigenicity stimulates, wherein said reagent effectively increases the t cell response that described experimenter stimulates antigenicity.