CN1377279A - Use of soluble cosmetimulatory molecules to enhance immune responses - Google Patents

Abstract

Methods of enhancing immune responses in which soluble forms of costimulatory molecules, e.g., B7 molecules, are administered to augment immune responses to antigens, e.g., to tumor cells and infectious agents are provided. The subject methods are useful for both prophylactic and therapeutic immunization of subjects.

Description

The purposes of soluble cosmetimulatory molecules to enhance immune responses

Related application

The application requires the priority of No. the 60/132nd, 944, the U.S. Provisional Application submitted on May 6th, 1999, and its content quotes in full at this as a reference.

Background of invention

The T cell is in order to respond extraneous protein, and antigen-presenting cell (APC) must provide two kinds of signals (Jenkins, M. and Schwartz, R. (1987) " The Journal of Experimental Medicine " 165,302-319 to tranquillization T lymphocyte; Mueller, and D.L. etc. (1990) " IMMUNOLOGY KEY WORDS INDEX 144,3701-3709).First kind of signal given immunne response with specificity, and it is to transduce via TXi Baoshouti (TCR) after the identification of presenting the exotic antigen peptide in main histocompatibility complex (MHC).Second kind of signal is called common stimulation, inducing T cell propagation and become functional (Lenschow etc. 1996 " immunity is commented academic year " 14:233).The common stimulation neither antigenic specificity, neither MHC restrictive, it is believed that it is (Jenkins, M.K. etc. 1988 " IMMUNOLOGY KEY WORDS INDEX 140, the 3324-3330 that different cell surface molecule provided that is expressed by APC by one or more; Linsley, P.S. etc. 1991 " The Journal of Experimental Medicine " 173,721-730; Gimmi, C.D. etc. 1991 " institute of NAS newspaper " 88,6575-6579; Young, J.W. etc. 1992 " Journal of Clinical Investigation " 90,229-237; Koulova, L. etc. 1991 " The Journal of Experimental Medicine " 173,759-762; Reiser, H. etc. 1992 " institute of NAS newspaper " 89,271-275; Van-Seventer, G.A. etc. (1990) " IMMUNOLOGY KEY WORDS INDEX 144,4579-4586; LaSalle, J.M. etc. 1991 " IMMUNOLOGY KEY WORDS INDEX 147,774-80; Dustin, M.I. etc. 1989 " The Journal of Experimental Medicine " 169,503; Armitage, R.J. etc. 1992 " nature " 357,80-82; Liu, Y. etc. 1992 " The Journal of Experimental Medicine " 175,437-445).If the T cell only by TXi Baoshouti by being stimulated, do not receive other common stimulus signal, they can become unresponsiveness, anergy so, and are perhaps dead, lead the downward modulation of immunne response.

The CD80 that expresses on APC (B7-1) is conclusive co stimulatory molecule (Freeman etc. 1991 " The Journal of Experimental Medicine " 174:625 with CD86 (B7-2) albumen; 1989 " IMMUNOLOGY KEY WORDS INDEX 143:2714 such as Freeman; Azuma etc. 1993 " nature " 366:76; Freeman etc. 1993 " science " 262:909).As if B7-2 play a leading role during primary immune response, and B7-1 was raised in the immunne response process later stage, it may be important (Bluestone, 1995 " immunity " 2:555) to prolonging first t cell response or stimulating once more t cell response jointly.

A kind of and B7-1 and the bonded part of B7-2 are CD28, and it is constitutive expression on tranquillization T cell, express increase after activation.After the TXi Baoshouti signalling, the transduction inducing T cell propagation of the coupled reaction of CD28 and common stimulus signal and secretion IL-2 (Linsley, P.S. etc. 1991 " The Journal of Experimental Medicine " 173,721-730; Gimmi, C.D. etc. 1991 " institute of NAS newspaper " 88,6575-6579; June, C.H. etc. 1990 " immunology today " 11,211-6; Harding, F.A. etc. 1992 " nature " 356,607-609).Second kind of part is called CTLA4 (CD152), and it and CD28 are homologous, but do not express on tranquillization T cell, and appear at after the T cell activation (Brunet, J.F. etc. 1987 " nature " 328,267-270).As if opposite with CD28, CTLA4 is conclusive (Waterhouse etc. 1995 " science " 270:985) to the negative adjusting of t cell response.The blocking-up of CTLA4 has been found that and can remove the signal that disinthibites, and the gathering of CTLA4 has been found that the inhibition signal (Allison and Krummel, 1995 " science " 270:932) that the downward modulation t cell response can be provided.

The exploitation enhance immunity method of replying existed always press for.For example, utilize art-recognized method also to be difficult to increase the immunne response of and tumor cell viral up to now to some.Be used for general enhance immunity and reply, particularly strengthen replying tumor antigen and infectant (for example virus, antibacterial and/or parasite antigen) with of great advantage.

Summary of the invention

The invention provides by handling the method that common stimulation approach enhance immunity is replied.These methods are to increasing tumor antigen and being effective especially from antigenic the replying of infectant.The present invention at least on the part degree based on such discovery, the soluble form of co stimulatory molecule can be preventative and the therapeutic enhance immunity reply.Although solubility co stimulatory molecule of the present invention is in administration on the solid phase (for example not administration on cell), and be do not have cross-linking agent in the presence of administration, also seen this potentiation.These discoveries are surprising especially, because according to existing instruction, the soluble form of B7-1 and B7-2 molecule can not produce common stimulation responses (Hayden etc. 1996 " tissue antigen " 48:242; U.S. patent 5,580, and 756).

Therefore, the present invention provides preventative enhancing curee the method to antigenic immunne response on the one hand,, will comprise the soluble composition administration of the extracellular domain of co stimulatory molecule, so that strengthen this curee to this antigenic immunne response that is.

The present invention provides therapeutic to strengthen the method for curee to antigenic immunne response on the other hand,, will comprise the soluble composition administration of the extracellular domain of co stimulatory molecule, so that strengthen this curee to this antigenic immunne response that is.

In one embodiment, this co stimulatory molecule is selected from the group of being made up of B7-1 and B7-2.

On the other hand, the invention provides and strengthen the method for curee the restricted antigenic CD8+T cell response of I class, promptly, to comprise the restricted antigen of I class or its segmental first kind of reagent and the soluble composition administration that comprises the extracellular domain of B7 molecule, so that in case this curee's administration is promptly strengthened the restricted antigenic CD8+T cell response of I class.

In one embodiment, these methods further comprise the restricted antigen of II class this curee's administration.In another embodiment, these methods further comprise adjuvant this curee's administration.

In one embodiment, this B7 molecule is the B7-1 molecule.In another embodiment, this B7 molecule is the B7-2 molecule.

In one embodiment, this co stimulatory molecule is a monospecific.In another embodiment, this co stimulatory molecule is dimer and bivalence.In one embodiment, this solubility co stimulatory molecule be monospecific with dimer and bivalence.

In another embodiment of the present invention, the extracellular part of B7 molecule is to merge with second kind of protein that comprises an immunoglobulin molecules part or polypeptide.In one embodiment, this partial immunity globulin molecule comprises cysteine residues.In one embodiment, this partial immunity globulin molecule comprises hinge region, CH2 and the CH3 district of human normal immunoglobulin's molecule.In another embodiment, this partial immunity globulin molecule comprises hinge region, CH1, CH2 and the CH3 district of human normal immunoglobulin's molecule.In one embodiment, this immunoglobulin molecules through modifying, has reduced complement fixation and/or Fc receptors bind.

In one embodiment, this antigen is tumor-cell antigen.

In another embodiment, this curee suffers from the cancer of the set type of being selected from down: colon cancer, breast carcinoma, carcinoma of prostate, renal cell carcinoma, leukemia, lymphoma, melanoma, mastocytoma, sarcoma and bladder cancer.

In one embodiment, this antigen is selected from the group of being made up of bacterial antigens, virus antigen and parasite antigen.

In one embodiment, this immunne response is a cellullar immunologic response.In another embodiment, this immunne response is a humoral immunoresponse(HI).

Marginal data

Fig. 1 shows with the antigenic specificity propagation of II class restriction peptide immune mouse cell and replys, and is with or without the co-administered of B7-2Ig (100 μ g) simultaneously.

The mouse cell of accepting single B7-2Ig treatment when Fig. 2 is presented at initial immunity has bigger propagation in immunity back once more than the mice of never accepting B7-2Ig and replys.Data are from parallel laboratory test.

Fig. 3 shows that the CTL that the co-administered of B7-2Ig strengthens the immunity of I class restriction peptide replys.

Fig. 4 is presented under the existence that is with or without II class restriction peptide and B7-2Ig treatment, and the CTL that limits the peptide immune mouse with the I class replys.

Fig. 5 shows that B7-IgG provides common stimulus signal for the in-vitro multiplication and the lymphokine secretion of splenocyte.

Fig. 6 shows that B7Ig is effective adjuvant in preventative tumor vaccine model.

Fig. 7 shows that inoculating the mice induced tumor with the radiation treatment P815 tumor cell therapeutic that is mixed with B7-1-or B7-2-IgG disappears, and prolongs survival period.

Fig. 8 shows that be effective as the therapeutic radiation treatment tumour-cell vaccine immune mouse of adjuvant to some different mouse tumor models in order to B7-IgG.

Fig. 9 shows that the therapeutic administration of independent B7-IgG is suitable with its effect as vaccine adjuvant to the antitumor action of mice.

Figure 10 shows that T or B cell are that the anti-tumor activity of B7-IgG-mediation is required.

Figure 11 shows CD8+ but not the CD4+T cell is that mediation B7-IgG anti-tumor activity is required.

Figure 12 shows that the B7-IgG therapy and the host IFN-γ that have set up tumor are irrelevant.

Describe in detail

The invention provides improving one's methods of enhancing immune response, that is, and with the solubility co stimulatory molecule (for example extracellular domain of B7 molecule or B7 fusion) administration should thereby strengthen immunity Answer. The solubility co stimulatory molecule does not have crosslinking agent when administration, still shockingly stimulate the T cell Reply. In fact, the applicant has been found that this method than the Co stituation branch of presenting from the teeth outwards Son has increased the Co stituation level, for example the co stimulatory molecule on the cell surface.

The invention further specify before, for convenience's sake, collected here be used in specification, Some term in embodiment and attached claims.

I, definition

Term used herein " preventative " comprise co stimulatory molecule the contact antigen before or with This simultaneously administration forms, increases and/or strengthen immune response for this antigen.

Term used herein " therapeutic " comprises that the co stimulatory molecule drug treatment is existing or carries out In infection or disease (for example cancer or virus or bacterium infect), control with co stimulatory molecule Treatment will be useful to this infection or disease. About therapeutic treatment, that co stimulatory molecule is anti-in contact Put sometime administration after former, form, increase and/or strengthen immune response for this antigen. No Say and explain, also may have with the immune response of co stimulatory molecule therapeutic treatment to the curee Other beneficial effects for example are not that this specific antigen is peculiar.

That term used herein " immunocyte " comprises hematopoietic origin and in immune response, play the part of The cell of personation. Immunocyte comprises lymphocyte, for example B cell and T cell; Naturally kill Hinder cell; Bone marrow cell, for example monocyte, macrophage, eosinocyte, mast cell, Basocyte and granulocyte.

Term used herein " immune response " comprises T and/or B cell response, just cell And/or HI. In one embodiment, method required for protection can be used for subtracting Few t helper cell is replied. In another embodiment, method required for protection can be used for Reducing cytotoxic T cell replys. Method required for protection can be used for reduce first with again Immune response. Curee's immune response for example can be by measuring antibody generation, immunocyte The release of propagation, cell factor, the expression of cell surface marker thing, cytotoxicity etc. are measured.

Term about activating immune cell used herein " Co stituation " comprises the Co stituation branch Son provides the ability of the receptor-mediated signal of second disactivation (" Co stituation signal "), this letter Number induce propagation or effector functions. For example, the Co stituation signal can for example be accepted T Cause cytokine secretion in the T cell of cell receptor-mediated signal. Term used herein " altogether Same stimulation molecule " comprise the molecule that is present on the antigen presenting cell (for example B7-1, B7-2, B7RP-1 (Yoshinaga etc. 1999 " nature " 402:827), (Swallow etc. 1999 for B7h " immunity " 11:423) and/or relevant molecule (for example homologue)), on they and the T cell The Co stituation acceptor (such as CD28, CTLA4, ICOS (Hutloff etc. 1999 " nature " 397:263), B7h part (Swallow etc. 1999 " immunity " 11:423) and/or relevant branch Son). This paper also is generically and collectively referred to as these molecules " B7 molecule ".

Wording used herein " B7 " or " B7 molecule " comprise naturally occurring B7-1 molecule, B7-2 molecule, B7RP-1 molecule (Yoshinaga etc. 1999 " nature " 402:827), B7h Relevant molecule, these branches on molecule (Swallow etc. 1999 " immunity " 11:423), the structure Fragment and/or its function equivalent of son. Term " equivalent " attempts to comprise that encoding function is suitable The amino acid sequence of co stimulatory molecule, they have the activity of B7 molecule, and are for example thin with immunity The ability of natural B 7 ligand bindings on the born of the same parents, for example CTLA4, the ICOS on the T cell and/or CD28, and/or the ability of modulation immunocyte Co stituation.

" polypeptide " used herein refers to and comprises arbitrarily two or more amino acid whose peptides or albumen Matter, these amino acid are connected to each other by peptide bond or modification peptide bond. " polypeptide " refers to short chain, Be commonly referred to peptide, oligopeptides and oligomer, and long-chain, be commonly referred to as protein. Polypeptide can contain Amino acid except 20 kinds of gene coding amino acids. " polypeptide " not only comprises through natural mistake Those that journey is modified, for example processing is translated rear modification with other, and comprises through the chemical modification skill Those that art is modified. Such modification fully is described in basic article and more detailed monograph and length In the piece of writing Research Literature, they are well-known to those skilled in the art. It will be appreciated that, giving In the fixed polypeptide, the modification of same type can be present in some positions in identical or different degree On. And given polypeptide can contain polytype modification. Modification can occur in polypeptide In arbitrarily everywhere, comprise polypeptide backbone, amino acid side chain and amino or carboxyl terminal. Modify example As comprise the covalently bound, blood red of acetylation, acidylate, ADP-ribosylation, amidatioon, flavine Covalently bound, lipid or the lipid of covalently bound, the nucleotides of plain part or nucleotide derivative spread out Covalently bound, crosslinked, cyclisation, the disulfide bond of biological covalently bound, phosphatidylinositols generate, The generation of demethylation, covalent cross-linking, the generation of cysteine, the generation of pyroglutamate, first Acidylate, γ-carboxylated, glycosylation, the generation of GPI deadman, hydroxylating, iodate, methylate, Myristoylation, oxidation, proteolysis processing, phosphorylation, isopentene group, racemization, γ-the carboxylated of lipid connection, sulphation, glutaminic acid residue, hydroxylating and ADP-ribosylation, The amino acid of selenic acid, transfer RNA mediation add become protein (for example arginyl) and time At albumen. For example referring to " protein---structure and molecular property " the 2nd edition, T.E. Creighton, W.H.Freeman and Company, New York (1993); Wold, F. " translates After protein modification: viewpoint and prospect ", " protein translate rear covalent modification " pgs.1-12, B.C.Johnson, Ed., Academic Press, New York (1983); " the zymetology side such as Seifter Method " 182:626-646 (1990); Rattan etc. " protein is synthetic: translate rear modification with aging " " Ann.N.Y.Acad.Sci. " 663:48-62 (1992). Polypeptide can be side chain or the band Have or without the ring-type of branch. Ring-type, side chain and can be from translating with the ring type polypeptide of branch After natural process obtain, also can utilize the synthetic method preparation fully.

" polypeptide of separation " used herein or " protein of separation " refer to such polypeptide Or protein, when after from cell, separating or with recombinant DNA technology, producing, basically not Contain other polypeptide, protein, cell material and culture medium, perhaps when synthetic with chemical method, Be substantially free of precursor or other chemicals. " separation " or " purifying " polypeptide or its Biologically-active moiety is substantially free of the tissue-derived cell from this cell or the B7 polypeptide of deriving Material or other contaminative polypeptide, perhaps when synthetic with chemical method, be substantially free of chemistry before Body or other chemicals. Wording " is substantially free of cell material " and comprises that wherein this polypeptide is from this The B7 polypeptide formulations that separates in the cellular component of cell is from this cell separation or use recombination method Produce this polypeptide. In one embodiment, wording " is substantially free of cell material " and comprises non-B7 polypeptide (this paper is also referred to as " contaminative polypeptide ") is less than the B7 of about 30% (with dry weight basis) Polypeptide formulations, more preferably non-B7 polypeptide is less than approximately 20%, and then more preferably non-B7 polypeptide is few In about 10%, most preferably be non-B7 polypeptide and be less than about 5%. When B7 polypeptide or its biologically active section Divide be with recombination method produce the time, it also preferably is substantially free of culture medium, that is to say, Cultivate fiduciary point polypeptide formulations volume less than about 20%, more preferably be less than approximately 10%, most preferably be Be less than about 5%.

Wording " is substantially free of precursor or other chemicals " and comprises that wherein this polypeptide is from ginseng With the B7 polypeptide formulations that separates in the synthetic precursor of this polypeptide or other chemicals. A kind of In the embodiment, wording " is substantially free of precursor or other chemicals " and comprises precursor Or non-B7 chemicals is less than the B7 polypeptide formulations of about 30% (with dry weight basis), more preferably chemistry It is about 20% that precursor or non-B7 chemicals are less than, and then more preferably precursor or non-B7 chemicals Be less than approximately 10%, most preferably be precursor or non-B7 chemicals is less than about 5%.

Preferred B7 nucleic acid molecules and polypeptide are " naturally occurring ". Used herein " natural Existing " molecule refers to the (natural B of for example encoding more than 7 that has naturally occurring nucleotide sequence Peptide) B7 molecule. In addition, also have the natural or non-natural of these polypeptide and nucleic acid molecules to exist Variant, they have kept identical functional activity, for example modulate the adaptation to stress reaction And/or the ability of microorganism virulence. Such variant can prepare like this, for example utilizes this area ripe The technology of knowing is suddenlyd change. Select as an alternative, variant can be synthetic with chemical method.

Term used herein " variant " comprises such nucleic acid molecules or polypeptide, and they are different from sequence with reference to nucleic acid molecules or polypeptide, but keeps its essential attributes.The variation of variant nucleotide sequence may change, also may not change the amino acid sequence of polypeptide that is referenced nucleic acid molecule encoding.The variation of nucleotide can cause amino acid whose replacement, addition taking place in being referenced the polypeptide of sequential coding, leaving out, merge and block, and discusses as follows.Typical polypeptide variants is different from another kind of with reference to polypeptide on aminoacid sequence.Difference generally is limited, so that generally speaking is very similar with reference to polypeptide to the sequence of variant, and is same in a lot of zones.Variant and with reference to polypeptide can be by combination in any on aminoacid sequence one or more replacements, addition and/or leave out and distinguished.Nucleic acid molecules or variant polypeptides can be naturally occurring, allelic variant for example, and perhaps it can be a whether naturally occurring variant of the unknown.The nucleic acid molecules that non-natural exists and polypeptide variants can be by induced-mutation techniques known to the skilled, directly synthesize and other recombination methods are prepared.

For example, self-evident is that B7 polypeptide as herein described also comprises its equivalent.Such variant for example can prepare like this, utilizes methods known in the art to suddenly change.Select as an alternative, variant can be synthetic with chemical method.For example, utilize technology well known in the art can prepare the mutant form of B7 polypeptide, they be of equal value on function (for example having and CTLA4 and/or the bonded ability of CD28).Sudden change for example can comprise at least a dispersive point mutation, and the latter can cause replacement, perhaps comprises at least aly leaving out or inserting.For example, can use random mutagenesis.Sudden change can also or utilize cassette mutagenesis to finish by random mutagenesis.About the former, operation is selected or screened to the complete coding region of molecule by one of Several Methods (chemistry, PCR, doping oligonucleotide synthesize) mutation to the elements collection of random mutation.About the latter, carry out saturated or half random mutagenesis to the polypeptide dispersion area that is equivalent to defined structure or function determiner, again these boxes by mutation are reintroduced in other wild-type allele structures.In one embodiment, can use PCR mutation.For example, can use large primer PCR (O.H.Landt, 1990 " gene " 96:125-128).

Term used herein " enhance immunity is replied " comprises and utilizes method required for protection treatment curee, increases T and/or B cell response, i.e. cell and/or humoral immunoresponse(HI).In one embodiment, method required for protection can be used to strengthen t helper cell and replys.In another embodiment, method required for protection can be used to strengthen cytotoxic T cell and replys.Method required for protection can be used for strengthening first and secondary immune response.Preferably, when with untreated curee or do not compare with the curee of method required for protection treatment, method required for protection increases curee's immunne response.In case the increase of immunne response for example can show as with after the method required for protection treatment, curee's immunocyte is replied increase to antigenic.Curee's immunne response for example can utilize in the various external or bodies activated immune cell effect to be measured, and for example measures the expression, cytotoxicity of release, the cell surface marker thing of antibody generation, immune cell propagation, cytokine etc.

Term used herein " solubility " comprises not and the associating molecule of cell, for example co stimulatory molecule.Kept the former function from the deutero-solubility co stimulatory molecule of cell associated molecule, that is to say that they can combine with the connection part on the T cell, via CD28 on the T cell and/or CTLA4 mediation signal transduction, but, they are solubilities, just not with membrane-bound.Preferably, soluble composition comprises the extracellular domain of B7 molecule.

Term used herein " extracellular domain of co stimulatory molecule " comprises the part of co stimulatory molecule, and it is extracellular in the cell association form of co stimulatory molecule.Preferably, the extracellular domain of co stimulatory molecule comprises the extracellular domain of B7 molecule.The B7 extracellular domain comprises the bonded part of co stimulatory molecule mediation and CD28 and/or CTLA4.For example, people B7-1 extracellular domain comprises about amino acid/11 of adult form B7-1 to about aminoacid 208 (SEQ ID NO:1), and people B7-2 extracellular domain comprises about aminoacid 24 of adult form B7-2 to about aminoacid 245 (SEQ ID NO:2).In one embodiment, the solubility co stimulatory molecule comprises the extracellular domain of B7 molecule, further comprises signal sequence.

Term used herein " I class limited antigen " comprises such antigen, and it combines with the main I of histocompatibility complex (MHC) class ditch, and is in MHC I quasi-molecule structure and passs T cell.I class limited antigen mainly stimulates the CD8+T cell.Term used herein " II class limited antigen " comprises such antigen, and it combines with MHC II class ditch, and is in MHC II quasi-molecule structure and passs T cell.II class limited antigen mainly stimulates the CD4+T cell.

Term used herein " adjuvant " comprises the reagent of reinforcement to antigenic immunne response.Adjuvant can with common stimulation molecule administering drug combinations, with other increase immunne response.

Term used herein " monospecific " comprises only having a species specific solubility co stimulatory molecule, that is to say, they combine with connection part on the T cell, for example CD28 or CTLA4.Such monospecific reagent does not also comprise other specificity, thereby does not combine with other cell surface molecules with oriented approach.Term used herein " few specificity " comprises having more than one specific solubility co stimulatory molecules, for example the molecule except that the B7 part had other specificity, the for example specificity of pair cell surface molecular, for example tumor-cell antigen or TXi Baoshouti.Term used herein " bivalence " comprises that each solubility co stimulatory molecule has two binding sites, is used for interacting with its connection part, for example CD28 and/or CTLA4.Term used herein " dimer " comprises the soluble form that exists with homodimer, the unit that forms by two identical subunits just, and the two for example links together by disulfide bond.Term used herein " poly " comprises the soluble form with two above subunits.

II, solubility co stimulatory molecule

B7 antigen is at the antigen-presenting cell (for example mononuclear cell, arborescent cell, langerhans' cells) of bone-marrow-derived lymphocyte, specialty and a class co stimulatory molecule of finding on cell (for example keratinocyte, endotheliocyte, spider cell, fibroblast, the oligodendrocyte) surface of immunocyte antigen-presenting.Other known or still indefinite receptors bind on CTLA4, CD28 on these co stimulatory molecules and the T cell surface and/or ICOS or the immunocyte.The member of this class co stimulatory molecule can provide common stimulation to the activated T cell, thus inducing T cell propagation and/or cytokine secretion.

People have established the purification technique of B7 molecule, in addition from many species clonings B7 gene (cDNA), comprise people and mice (for example referring to Freeman, G.J. etc. (1993) " science " 262:909-911; Azuma, M. etc. (1993) " nature " 366:76-79; Freeman, G.J. etc. (1993) " The Journal of Experimental Medicine " 178:2185-2192).

The nucleotide sequence of co stimulatory molecule is known in the art, can find in document or data base, for example GenBank.For example referring to B7-2 (Freeman, G.J. etc. 1993 " science " 262:909 or GenBank go into Tibetan P42081 or A48754), B7-1 (" The Journal of Experimental Medicine " 1991.174:625 such as Freeman or GenBank go into Tibetan P33681 or A45803), CTLA4 (for example going into Tibetan P16410 or 291929), CD28 (Aruffo and Seed " institute of NAS newspaper " 84:8573 or GenBank go into to hide number 180091), ICOS (Hutloff etc. 1999 " nature " 397:263 referring to 1985 " science " 228:1401 such as Ginsberg or GenBank; WO 98/38216) with relevant sequence.

Except the natural existence form of co stimulatory molecule, term " co stimulatory molecule " also comprises the form that non-natural exists, the for example variant of co stimulatory molecule or mutant forms, they remain with the function of co stimulatory molecule, for example with the bonded ability of connection counter receptor.For example, under the condition of avoiding with non-co stimulatory molecule gene recombination (for example being equivalent under the condition of 65 ℃ of 5 * SSC (1 * SSC=150mM NaCl/0.15M sodium citrate)), can be used to prepare anti-B7 antibody with the DNA sequence of the DNA hybridization of coding B7 molecule.Select as an alternative, can be used for producing in the DNA sequence of the regional reservation queue homogeneity of nucleic acid molecule encoding protein domain and can be used as immunogenic common stimulatory protein(SP), these protein domains are important to the function of co stimulatory molecule, for example combine with other co stimulatory molecules.Preferably, the co stimulatory molecule of non-natural existence and the aminoacid sequence of naturally occurring co stimulatory molecule extracellular domain have significant aminoacid homogeneity (for example greater than 70%, being preferably more than 80%, more preferably greater than 90-95%).

May to combine with its counter receptor co stimulatory molecule be the amino acid residue of important co stimulatory molecule in order to measure, can contrast the aminoacid sequence of the extracellular domain of the co stimulatory molecule that comprises different plant species, for example mice and people, (for example same) residue that record is conservative.For example this can utilize the program of standard contrast arbitrarily to carry out, for example MegAlign (DNASTAR).Such contrast program is described in detail as follows.Conservative or the same residue of this class may be necessary concerning correct combination of co stimulatory molecule and its receptor, thereby there is no fear of change.

For example, the function of mediation and CD28 and CTLA4 being interacted is that important B7-1 molecular domains is differentiated by sudden change.Two hydrophobic residue in the B7-1V-spline structure territory, be included in from all B7-1 of various species clonings and B7-2 molecule all to be the Y87 residue of guarding, to be found to be conclusive (Fargeas etc. 1995 " The Journal of Experimental Medicine " 182:667).Utilize these or similar technology, can measure the amino acid residue of the extracellular domain of decisive co stimulatory molecule, they are conclusive, therefore can not change.

Use B7 cDNA molecule, have the active peptide of B7 and can utilize standard technique to be produced.For the transfected host cell of expression of peptides can be prokaryotic cell or eukaryotic cell arbitrarily.For example, having the active peptide of B7 can be expressed in following cell: bacterial cell, for example escherichia coli, insect cell (baculovirus), yeast, or mammalian cell, for example Chinese hamster ovary cell (CHO) and NS0 cell.Other host cell and expression vectors that are fit to can be referring to Goeddel, and (1990), on seeing, or well known by persons skilled in the art.Expression vector example in the saccharomyces cerevisiae comprises pYepSecl (Baldari etc. (1987) " EMBO's magazine " 6:229-234), pMFa (Kurjan and Herskowitz, (1982) " cell " 30:933-943), pJRY88 (Schultz etc. (1987) " gene " 54:113-123) and pYES2 (Invitrogen Corporation, San Diego, CA).The baculovirus vector that can be used for protein expression in the insect cell of being cultivated (SF9 cell) comprises pAc series (Smith etc. (1983) " molecular cytobiology " 3:2156-2165) and pVL series (Lucklow, V.A. and Summers, M.D., (1989) " virusology " 170:31-39).Usually, COS cell (Gluzman, Y., (1981) " cell " 23:175-182) with such as carriers such as pCDM8 (Seed, B., (1987) " nature " 329:840) unite the of short duration amplification/expression that is used in the mammalian cell, and CHO (Chinese hamster ovary) cell and the stable amplification/expression that is used for such as carriers such as pMT2PC (Kaufman etc. (1987) " EMBO's magazine " 6:187-195) in the mammalian cell.The cell line that is preferred for recombinant protein production is the NS0 myeloma cell line, can obtain (catalogue #85110503) from ECACC, is described in Galfre, G. and Milstein, ((1981) " Enzymology method " 73 (13): 3-46 among the C.; " MONOCLONAL ANTIBODIES SPECIFIC FOR: strategy and operation " AcademicPress, N.Y., N.Y.).Carrier DNA can be incorporated in the mammalian cell via routine techniques, transfection, lipofection reagent or the electroporation of for example calcium phosphate or calcium chloride co-precipitation, the mediation of DEAE-glucosan.The method that is suitable for transformed host cell can be referring to (" molecular cloning laboratory manual " the 2nd edition, Cold Spring Harbor Laboratory press (1989)) and other laboratory textbooks such as Sambrook.In the time of in being used in mammalian cell, the control function of expression vector is often provided by viral material.For example, promoter commonly used is deutero-from polyoma, adenovirus 2, cytomegalovirus, and modal is simian virus 40.

That is expressed in mammalian cell or other cells has the active peptide of B7 and can carry out purification according to the standard operation of this area, comprise ammonium sulfate precipitation, fractionation column chromatograph (for example ion exchange, gel filtration, electrophoresis, affinity chromatography etc.), comprise crystallization (generally referring to " purification of enzyme and correlation technique " " Enzymology method " 22:233-577 (1971)) at last.

The B7 molecule that is used to prepare the used solubility B7 molecule of this method can derive from any mammalian species, preferably is the people.The nucleotide sequence of several sources B7 molecule is known in the art.The GenBank of the global DNA sequence of people B7-1 (CD80) go into to hide and number to be L25259, is published in " science " 1993 such as Freeman, and " natures " 1993 such as 262:9090 or Azuma (are seen the sequence of WO 96/40915 about B7-1 and B7-2) among the 366:76 in addition.The nucleotide sequence of people B7-1 and people B7-2 is also shown in SEQ ID Nos 1 and 2 (B7-1) and SEQ ID Nos 3 and 4 (B7-2).Select as an alternative, a kind of like this sequence can be measured like this, based on sequence and ability known, for example people B7 sequence hybridization, separates the B7 nucleic acid molecules from required source.For example, can detect the B7 molecule has the making nucleic acid molecular hybridization of the active peptide of B7 with known coded under high or low stringent condition ability.The stringent condition of suitable promotion DNA hybridization is the 6.0 * sodium chloride/sodium citrate (SSC) under about 45 ℃ for example, wash with 2.0 * SSC down at 50 ℃ then, these conditions are well known by persons skilled in the art, perhaps can be referring to " the popular scheme of molecular biology " JohnWiley ﹠amp; Sons, N.Y. (1989), 6.3.1-6.3.6.For example, the salinity in the washing step can be selected from the high stringent condition of 50 ℃ of the low stringency conditions to 0.2 of 50 ℃ of about 2.0 * SSC * SSC.In addition, the temperature in the washing step can be increased to about 65 ℃ high stringent condition from the about 22 ℃ low stringency condition of room temperature.In preferred embodiment, the B7 molecule is a human B 7-1 molecule.

In one embodiment, the solubility co stimulatory molecule is deutero-from naturally occurring B7-1 or B7-2 molecule.Have B7 molecular activity as described herein and also can be expressed, also belong to scope of the present invention with soluble form because of the degeneracy of genetic code has the polypeptide of sequence that is different from the natural B7 of existence molecule.Be equivalent to the polypeptide (for example having the active polypeptide of B7) of B7 on such nucleic acid coding function, but on sequence, be different from B7-1 known in the art or B7-2.For example, a large amount of aminoacid are by an above triplet name.Because the degeneracy of genetic code may exist the codon or the synonymous codon (for example CAU and CAC are the synonymous codon of histidine) of specifying same amino acid.As an example, the dna sequence polymorphism of (especially in the 3rd base of codon) may cause " silence " sudden change among the DNA in the B7 molecular core nucleotide sequence, and this does not influence coded aminoacid.But, be expected at and have the dna sequence polymorphism that causes that really B7 antigen aminoacid sequence changes in a certain population.Those skilled in the art will recognize that, since natural allelic variation, these variations (up to about 3-4% of nucleotide) in one or more nucleotide of the nucleic acid of the peptide that may existing in a certain population individuality encodes has novel bone-marrow-derived lymphocyte antigen active.Such nucleotide diversity and due to amino acid polymorphism also belong to scope of the present invention.In addition, also may there be one or more isotypes or relevant cross reactivity B7 molecule.

Except naturally occurring allele variant, the B7 molecule in the scope of the invention can utilize art-recognized technology to be prepared.In another embodiment, the solubility co stimulatory molecule is the modified forms of B7-1 or B7-2, and it has kept the function of B7 co stimulatory molecule, just is identical on function.The antigenic DNA sequence of bone-marrow-derived lymphocyte is modified through genetic technique, can produce altered protein of aminoacid sequence or polypeptide.Such sequence is considered as belonging to scope of the present invention, wherein can be combined with CTLA4 and/or CD28 by polypeptide expressed, and modulation T cell-mediated immune responses and immunologic function.

For example, in one embodiment, can in dna molecular, introduce sudden change according to any one of big metering method, comprise be used to produce simply leave out or insertion, systematicly leave out, the insertion of alkali base cluster or the replacement of replacement or single base, generate the variant of bone-marrow-derived lymphocyte antigen dna or modify after equivalent.For example, the change of B7-1 or B7-2 cDNA sequence, for example amino acid whose replacement or leave out preferably obtains by site directed mutagenesis.The site directed mutagenesis system is well known in the art.Scheme and reagent commercial can be from Amersham International PLC, Amersham, U.K. obtains.

Have the B7 molecular activity, just have with polypeptide after the native ligand of B7 molecule combines and modulate the modification of ability of t cell mediated immune response and be considered as belonging to scope of the present invention, this ability is for example produced by the cytokine of the T cell that receives elementary activation signals and/or the T cel l proliferation is confirmed.

Another kind modification example with peptide of B7 molecular activity is that cysteine residues is preferably replaced by alanine, serine, threonine, leucine or glutaminic acid residue, to reduce the dimerization via disulfide bond.In addition, the amino acid side chain with the active peptide of B7 can be modified with chemical method.The another kind of modification is the cyclisation of peptide.

For enhanced stability and/or reactivity, have the active polypeptide of B7 through modifying, can in the antigen aminoacid sequence, mix one or more polymorphisms, this is caused by natural arbitrarily allelic variation.In addition, D-aminoacid, alpha-non-natural amino acid or non-amino acid analogue be through replacing or addition, can produce the protein after the modification that belongs to the scope of the invention.In addition, according to A.Sehon and partner's method (Wie etc., the same), peptide can use Polyethylene Glycol (PEG) to modify, and produces the peptide with the PEG conjugated.In addition, can during the chemosynthesis of peptide, add PEG.Other modifications of peptide comprise reduction/alkylation (Tarr: " the little characterization method of protein " J.E.Silver ed., Humana Press, Clifton NJ 155-194 (1986)); Acidylate (Tarr, the same); Chemical coupling (Mishell and Shiigi with suitable carrier, eds " cellular immunology method for concentrating " WH Freeman, San Francisco, CA (1980), United States Patent (USP) 4,939,239) or rare formalin handle (Marsh (1971) " international allergy and applied immunology document " 41:199-215).

Preferred B7 polypeptide has the B7 activity, and has homogeneity at least about 60% with naturally occurring B7 aminoacid sequence, is preferably the homogeneity at least about 70%, more preferably at least about 80% homogeneity.Have the B7 activity and have at least about 90% homogeneity with naturally occurring B7 molecule, be preferably at least about 95%, more preferably the polypeptide at least about 98-99% homogeneity also belongs to scope of the present invention.Term refers to when contrasting the aminoacid sequence of two kinds of peptides at the aminoacid on the given position " homogeneity ", has identical aminoacid on correspondence position.If a certain position is occupied by identical aminoacid in the sequence that is compared, then molecule is same on this position.The degree of homogeneity (or percentage rate) is the coupling shared by these sequences or the function of same position quantity between the sequence.

Those of ordinary skills can contrast two seed amino acid sequences easily, to provide the justice contrast are arranged biology.The percentile mensuration of homogeneity can also utilize mathematical algorithm to finish between the comparison of sequence and the two kinds of sequences.In one embodiment, homogeneity percentage rate between the two seed amino acid sequences utilizes Needleman and Wunsch (" molecular biology magazine " (48): 444-453 (1970)) algorithm to measure, this algorithm has been combined in the GAP program the GCG software kit (can from http://www.gcg.com obtain), adopt Blossom 62 matrixes or PAM250 matrix, the spacing weight is 16,14,12,10,8,6,5 or 4, and the length weight is 1,2,3,4,5 or 6.In another embodiment, homogeneity percentage rate between two kinds of nucleotide sequences is to utilize GAP program determination the GCG software kit (can obtain from http://www.gcg.com), adopt the NWSgapdna.CMP matrix, the spacing weight is 40,50,60,70 or 80, and the length weight is 1,2,3,4,5 or 6.In another embodiment, homogeneity percentage rate between two seed amino acids or the nucleotide sequence is to utilize E.Meyers and W.Miller algorithm (CABIOS, 4:11-17 (1989)) measures, this algorithm has been combined in the ALIGN program (2.0 editions), adopt PAM120 weight residue table, notch length is compensated for as 12, and breach is compensated for as 4.

Nucleic acid of the present invention and protein sequence can further be used as " search sequence ", for example other family members or relevant sequence are retrieved in common data base.Such retrieval can utilize NBLAST and the XBLAST program (2.0 editions) of (1990) " molecular biology magazine " 215:403-10 such as Altschul to carry out.The retrieval of BLAST nucleotide can utilize NBLAST program, score=100, word length=12 to carry out, to obtain and the homologous nucleotide sequence of nucleic acid molecules of the present invention.The retrieval of BLAST protein can utilize XBLAST program, score=50, word length=3 to carry out, to obtain and the homologous aminoacid sequence of B7 protein molecule of the present invention.For the contrast that obtains having breach in order to relatively, can adopt Gapped BLAST, as (1997) " nucleic acids research " 25 (17) such as Altschul: as described in the 3389-3402.When adopting BLAST and Gapped blast program, can use the default parameters of each program (for example XBLAST and NBLAST).See http://www.ncbi.nlm.nih.gov.

" at least a portion extracellular domain of B7 molecule " is defined as a kind of like this aminoacid sequence, it comprises intact cell external structure territory sequence or its part of B7, this sequential coding have activity (just with immunocyte on the bonded ability of natural B 7 parts, for example combine with CTLA4 and/or CD28 on the T cell) polypeptide.Has the active peptide of B7 in conjunction with CTLA4 and/or CD28, modulation T cell-mediated immune responses, this for example obtains combining with these parts or the inducing cell factor produces and/or received the confirmation of the T cel l proliferation of elementary activation signals, shown in attached embodiment.In preferred embodiment, " at least a portion extracellular domain of B7 molecule " comprises such peptide species, it comprises the outer part of the antigenic intact cell of people B7 (for example about 1-208 amino acid residue of B7-1 sequence or about 24-245 amino acid residue of B7-2 sequence), and this part can be used in conjunction with CTLA4 and/or CD28.

Except only comprising naturally occurring amino acid whose B7 polypeptide, also provide the B7 peptide mimics.Peptide analogues is commonly used for non-peptide medicine in pharmaceutical industries, have the character that is similar to the template peptide.The non-peptide compound of these types is called " analogies of peptide " or " peptide mimics " (Fauchere, J. (1986) " drug research progress " 15:29; Veber and Freidinger (1985) " TINS " are p.392; Evans etc. (1987) " medical chemistry magazine " 30:1229 quotes at this as a reference), under development usually by the modeling of computer molecule.

The analogies of structurally similar to the peptide with therapeutic value peptide can be used to produce treatment or preventive effect of equal value.Usually, peptide mimics structurally with polypeptide example (polypeptide that just has biology or pharmacologically active), for example B7 is similar, but wherein one or more peptide bond is selected from down the key replacement of group :-CH2NH-alternatively,-CH2S-,-CH2-CH2-,-CH=CH-(cis and trans),-COCH2-,-CH (OH) CH2-and-CH2SO-, method is known in the art, further describe in following list of references: Spatola, A.F. " aminoacid, peptide and proteinic chemistry and biochemistry " B.Weinstein, eds., Marcel Dekker, New York, p.267 (1983); Spatola, A.F. " Vega Data " (1983.3.), 1 the volume, 3 phases, " peptide backbone modifications " (summary); Morley, J.S. " pharmaceutical science trend " (1980) pp.463-468 (summary); Hudson, and D. etc. " international peptide and protein research magazine " (1979) 14:177-185 (CH2NH-, CH2CH2-); Spatola, A.F. etc. " life sciences " (1986) 38:1243-1249 is (CH2-S); Hann, M.M. " the will Charles Bell gold transactions I of Englishize association " (1982) 307-314 (CH-CH-, cis and trans); Almquist, R.G. etc. " medical chemistry magazine " (1980) 23:1392-1398 is (COCH2-); Jennings-White, C. etc. " tetrahedron wall bulletin " (1982) 23:2533 is (COCH2-); Szelke, M. etc., European patent application EP 45665 (1982) CA:97:39405 (1982) (CH (OH) CH2-); Holladay, M.W. etc. " tetrahedron wall bulletin " (1983) 24:4401-4404 (C (OH) CH2-); Hruby, (CH2-S-), they quote at this as a reference respectively V.J. " life sciences " (1982) 31:189-199.Particularly preferred non-peptide bond is-CH2NH-.

The analogies of this class peptide can have the advantage that significantly is better than the polypeptide embodiment, for example comprise: produce more economical, chemical stability is higher, pharmacological properties strengthens that (half-life, absorption, effectiveness, effect etc.), specificity change (for example broad-spectrum biological activity), antigenicity reduces and other or the like.The labelling of peptide mimics is usually directed to one or more labels directly or by the non-interfering position covalent bond on spacer groups (for example amide groups) and the peptide mimics, predict by D-M (Determiner-Measure) construction-activity data and/or molecule modeling these positions.Such non-interfering position generally is such position, and they do not form directly with macromole and contact, and peptide mimics combines the generation therapeutic effect with these macromole.Derive (for example labelling) of peptide mimics should not disturb the required biology or the pharmacologically active of peptide mimics basically.

One or more aminoacid of B7 aminoacid sequence are replaced (for example D-lysine replaces L-lysine) methodically by the D-aminoacid of same type, can be used to generate more stable molecule.In addition, can generate the constraint peptide (Rizo and Gierasch (1992) " bioid academic year comments " 61:387 quotes at this as a reference) that comprises B7 aminoacid sequence or same basically sequence variations according to methods known in the art; For example, add intrinsic cysteine residues, these residues can form the intramolecular disulfide bridge, make the peptide cyclisation.

Those skilled in the art need not over-drastic experimental technique can produce the polypeptide that is equivalent to B7 peptide sequence and sequence variants thereof.This class polypeptide can be produced like this, in protokaryon animal or eucaryon animal reservoir cell, expresses the polynucleotide of coding B7 peptide sequence, often the part of the bigger polypeptide of conduct.Select as an alternative, such peptide can be synthetic with chemical method.The expression of xenogenesis polypeptide in recombinant host, the chemosynthesis and the external method of translating of polypeptide are well known in the art, further describe " molecular clonings: laboratory manual " such as Maniatis (1989) the 2nd editions Cold SpringHarbor, N.Y.; Berger and Kimmel " Enzymology method " 152 volumes " molecule clone technology guide " (1987), Academic Press, Inc., San Diego, Calif.; Merrifield, J. (1969) " JACS " 91:501; Chaiken I.M. (1981) " CRC Crit.Rev.Biochem. " 11:255; Kaiser etc. (1989) " science " 243:187; Merrifield, B. (1986) " science " 232:342; Kent, S.B.H. (1988) " bioid academic year comments " 57:957; Offord, R.E. (1980) " semi-synthetic protein " Wiley Publishing quotes at this as a reference.

Peptide for example can be produced by direct chemosynthesis.The peptide that peptide can be made into to modify, wherein non-peptide moiety combines with N-end and/or C-end by covalent bond.Some preferred embodiment in, carboxyl terminal or amino terminal or the two all are through chemical modification.The prevailing modification of terminal amino group and carboxyl is respectively acetylation and amidatioon.Amino terminal is modified for example acidylate (for example acetylation) or alkylation (for example methylating), and carboxyl terminal is modified for example amidatioon, and other are end modified, comprise cyclisation, and these can be embodied in the various embodiment of the present invention.Some amino terminal and/or carboxyl terminal are modified and/or peptide is extended for core sequence, favourable physics, chemistry, biochemistry and pharmacological properties can be provided, and for example: stable enhancing, effectiveness and/or effect increase, tolerance serum albumin enzyme, desirable pharmacokinetics character and other or the like.

The present invention also provides B7 chimeric or fused polypeptide.B7 used herein " chimeric polyeptides " or " fused polypeptide " comprise the B7 polypeptide that is connected with non-B7 polypeptide with operation method." B7 polypeptide " refers to has the polypeptide that is equivalent to the B7 amino acid sequence of polypeptide, and " non-B7 polypeptide " refers to and has the polypeptide that is equivalent to a kind of like this amino acid sequence of polypeptide, the former polypeptide and B7 polypeptide are not homologous basically, for example are different from the B7 polypeptide and derive from the polypeptide of identical or different organism.In the B7 fused polypeptide, the B7 polypeptide can be equivalent to all or part of of B7 polypeptide.In preferred embodiment, the B7 fused polypeptide comprises at least one biologically-active moiety of B7 polypeptide.In fused polypeptide, term " connects with operation method " and is intended to show that B7 polypeptide and non-B7 polypeptide are the framework endomixis each other.Non-B7 polypeptide can merge with the N-end or the C-end of B7 polypeptide.

Preferred nucleic acid fragment code length is at least about the B7 polypeptide of 40 amino acid residues, and preferably length is at least about 80 amino acid residues, and more preferably length is at least about 120 amino acid residues.Particularly preferred fragment is at least about 200 aminoacid on length, for example comprise the intact cell external structure territory of B7 molecule.Big metering method can be used to generate the fragment of DNA isolation sequence.The nucleic acid subprovince or the fragment of coding B7-1 or B7-2 protein, a for example 1-30 base length can be prepared according to the organic chemistry synthesizing mean of standard.This technology also can be used for preparing primer, and primer is used to generate bigger synthetic B7 dna fragmentation.

The bigger subprovince or the fragment of the antigenic gene of coding bone-marrow-derived lymphocyte can be expressed as peptide like this, utilize polymerase chain reaction (PCR) to synthesize relevant DNA fragment (Sambrook, Fritsch and Maniatis " molecular cloning: laboratory manual " the 2nd edition, Cold Spring Harbor, N.Y., (1989)), gained DNA is connected in the suitable expression.Utilize PCR, generated the particular sequence of the double-stranded DNA of being cloned, be cloned in the expression vector, measure CTLA4/CD28 then in conjunction with activity.For example, in order to utilize the proteic secretion of PCR expressing human B7-1 or B7-2 (solvable) form, can synthesize the not DNA that strides film and cytoplasmic region of coded protein.This dna molecular can be connected in the suitable expression, is incorporated in the host cell, and for example CHO synthesizes and secretion B7 protein fragments there.Can from culture medium, obtain the B7 protein fragments easily then.

In one embodiment, at least a portion of coding B7 molecule, for example lack molecule and stride the nucleic acid molecules of the extracellular domain part of membrane portions and be placed in the expression vector, by host cell expression, so that the B7 molecule is not expressed on cell surface.For example, the cDNA in coding B7 molecular cell external structure territory when synthetic, can use from disclosed B7-1 or B7-2 sequence (referring to Freeman etc. " IMMUNOLOGY KEY WORDS INDEX 1989.143:2714 or " science " 1993.262:9090) deutero-primer, utilize polymerase chain reaction (United States Patent (USP) 4,683,202).Gained cDNA sequence can be assembled into eukaryote or prokaryotic expression carrier then, and this carrier can be used for the extracellular domain of the synthetic B7 of directed suitably host cell, for example COS or Chinese hamster ovary celI.

In another embodiment, expression vector comprise the coding have the B7 antigen active peptide DNA and the coding second peptide species DNA.Second peptide species preferably is not deutero-from co stimulatory molecule.Preferably, the chimeric or fusion rotein of B7 of the present invention is to produce with the recombinant DNA technology of standard.For example, the dna fragmentation of the different peptide sequences of coding according to linking together in the routine techniques framework, is utilized and adopts blunt end or staggered end to connect, restriction endonuclease digestion is to provide suitable end, take the circumstances into consideration to insert cohesive end, alkaline phosphatase treatment is to avoid worthless connection, with being connected of enzyme.In another embodiment, fusion gene can be synthetic with routine techniques, comprises automatic dna synthesizer.Select as an alternative, the pcr amplification of genetic fragment can utilize anchor primer to carry out, this primer causes the complementary jag between two consecutive gene fragments, can anneal subsequently and amplification again, produce chimeric gene order (for example referring to John Wiley ﹠amp such as " molecular biology updated plan " eds.Ausubel; Sons:1992).And a lot of expression vectors all are commercial available, and the fusion part (for example gst polypeptide) of having encoded.The nucleic acid molecules of coding B7 can be cloned in such expression vector, so that this fusion partly with in B7 protein carries out framework is connected.For example, can in peptide, add six-histidine, being used for fixing metal ion affinity chromatography purification (Hochuli, E. etc. (1988) " biotechnology " 6:1321-1325).In addition, in order to promote not contain the antigenic separation of bone-marrow-derived lymphocyte of irrelevant sequence, can between the sequence that merges part and peptide, introduce specificity endoproteinase cracking site.The dissolubility that increases peptide may be necessary, for example adds functional group in peptide, perhaps leaves out the hydrophobic region of peptide.

In one embodiment, the DNA of coding B7 molecule or its part is carried out being connected in the framework with the required antigenic DNA of immunne response that encodes, this antigen is virus antigen or tumor-cell antigen for example.

In one embodiment, the DNA that coding is equivalent to the aminoacid sequence in B7 cell antigen external structure territory is connected (for example referring to United States Patent (USP) 5 with the DNA that coding is equivalent to the aminoacid sequence of immunoglobulin molecules constant region, 580,756 or WO 97/28267).Second kind of peptide can comprise constant region for immunoglobulin, for example (for example hinge region, CH2 and the CH3 district of people IgC γ 1 or people IgC γ 4 are for example referring to US such as Capon 5,116 for people C γ 1 domain or C γ 4 domains or C μ or its part, 964, quote at this as a reference).Gained B7Ig fusion rotein can have altered dissolubility, in conjunction with affinity, stability and/or quantivalence (the available binding site number of each molecule just), and can increase the efficient of protein purification.

In preferred embodiment, the cysteine residues in the constant region for immunoglobulin is guarded, and helps the bonding and the proteic generation of solubility dimerization B7Ig of disulphide.In one embodiment, the constant region of the part of B7 molecule and IgM antibody or its part merges, and helps the proteic generation of B7Ig of solubility poly form.

Particularly preferred B7Ig fusion rotein comprises extracellular domain part or the spline structure territory, variable region with link coupled people B7-1 of constant region for immunoglobulin or B7-2.The constant region for immunoglobulin that is used in the solubility B7 molecule can contain genetic modification, and these modifications reduce or eliminate the inherent effector activity of immunoglobulin structure (for example referring to WO 97/28267).For example, the DNA in the constant region spline structure territory of the spline structure territory, variable region of the DNA of the extracellular part of coding B7-1 or B7-2 and coding B7-1 or B7-2 or B7-1 or B7-2 can be connected through the people IgC γ 1 of site-directed mutation modification and/or hinge region, CH2 and the DNA in CH3 district of IgC γ 4 with coding.

If use the non-human immunoglobulin constant region, preferably this constant region is by humanization.The technology that is used to prepare chimeric or humanized antibody is well known in the art (for example referring to " institute of NAS newspaper " 81:6851 (1985) such as Morrison; Takeda etc. " nature " 314:452 (1985); U.S. Patent No.s such as Cabilly 4,816,567; U.S. Patent No.s such as Boss 4,816,397; European patent communique EP 171496 such as Tanaguchi; European patent communique 0173494; British patent GB 2177096B; Teng etc. " institute of NAS newspaper " 80:7308-7312 (1983); " immunology today " 4:7279 such as Kozbor (1983); Olsson etc. " Enzymology method " 92:3-16 (1982); PCT communique WO 92/06193 and EP 0239400).

Be used to assemble and express technology for example synthetic, the PCR, transformant, carrier construction, expression system etc. of oligonucleotide of DNA that coding is equivalent to the aminoacid sequence of B7 antigen and solubility B7Ig molecule, all obtained the abundant affirmation of this area.

By the B7 fusion rotein of recombinant technique production and polypeptide can be from the mixture of the cell that contains protein or peptide and culture medium secretion and separating.Select as an alternative, protein or peptide can be retained in the Cytoplasm, harvesting, dissolving, isolated protein.Cell culture generally includes host cell, culture medium and other compositions.The culture medium that is suitable for cell culture is well known in the art.Protein can separate from cell culture medium, host cell with polypeptide, perhaps utilizes the technology that is used for protein purification and polypeptide known in the art.The technology that is used for transfection host cell and protein purification and peptide describes in further detail in this article.

The screening active ingredients of the solubility co stimulatory molecule of III, structurally associated

Utilize one or more some different algoscopys can finish the B7 molecular screening of structurally associated, as described herein they keep distinctive bone-marrow-derived lymphocyte antigen active.For example, the screening of peptide as can be seen their keep to naturally occurring B7 molecule bonded anti--idiosyncrasy of B7 monoclonal antibody.Specifically, the DNA transfection that suitable cell, for example COS cell can be supplied the examination polypeptide with coding.In immunoprecipitation assay, can utilize anti--B7 monoclonal antibody or CTLA4Ig or CD28 fusion rotein to estimate the generation of secreted form B7.By moving, can also test cell and CTLA4 or the CD28 bonded ability on flat board of expressing relevant peptide.Select as an alternative, can also test for examination peptide and the competition of naturally occurring B7 molecule and CD28 or the bonded ability of CTLA4.

The antigenic functional character of other preferred algoscopy test B7.As described above, the ability of the T cell synthetic cell factor depends on that not only TXi Baoshouti occupies or crosslinked (" the elementary activation signals " that is provided by anti--CD3 or Buddhist ripple ester for example antigenic, produce " activated T cell "), and depend on inducing of common stimulus signal, be by inductive in this case with the antigenic interaction of bone-marrow-derived lymphocyte, for example CD28 on B7-1 or B7-2 and the T cell and/or the interaction of CTLA4 molecule.On B7 molecule and the T cell via the combining to have and transmit the effect that signal is given the T cell of its native ligand of TXi Baoshouti received signal, induce the level that produces cytokine, particularly interleukin-2 to increase, the latter and then stimulate the lymphocytic propagation of T.Other are about the algoscopy of B7 function thereby relate to and measure the synthetic of cytokine, for example interleukin-2, interleukin-4 or other cytokines, and/or measure the CD28 that has received elementary activation signals ⁺The T cel l proliferation of T cell.

Externally can be like this provide first or elementary activation signals to the T cell, make they and anti--T3 monoclonal antibody (for example anti--CD3) or Buddhist ripple ester contact, perhaps more preferably, provide by the antigen relevant with I class or II class MHC molecule.The T cell that has received elementary activation signals is referred to herein as the activated T cell.The B7 function is to measure like this, in the T cell culture, add B7 source (for example expressing the cell of secreted form) and elementary activation signals (for example with I class or the relevant antigen of II class MHC) with the active peptide of B7 or B7, measurement function result for example measures interleukin-2, gamma interferon or other known or unknown cytokines in the culture supernatants.For example, can adopt any one of interleukin-2 algoscopy of some routines, " institute of NAS newspaper " described algoscopy of 86:1333 (1989) for example, relative section is quoted at this as a reference.The test kit that is used for interferon generation mensuration can be from Genzyme Corporation (Cambridge, MA) acquisition.Can also the described measurement of following embodiment T cell proliferation.Compare with the negative control that lacks common stimulus signal, the cytokine that the peptide of reservation B7 antigen property as described herein can cause each T cell to be produced increases, and for example IL-2 can also cause the T cel l proliferation to strengthen.

The medication of IV, solubility co stimulatory molecule

Compositions as herein described and/or medicine promote curee's administration of immunne response to needs.In one embodiment, solubility B7 molecule and antigen prevention administration are for example before by pathogenic infection or to not cancered curee's administration.In another embodiment, solubility B7 molecule is the therapeutic administration, for example to will be from strengthening ill curee's administration that common stimulation benefits, for example suffers from cancer or by the curee of pathogenic infection.

In one embodiment, co stimulatory molecule and antigen preparation co-administered.Antigen can be protein, polysaccharide, lipopolysaccharide, lipopeptid, and perhaps it can be these combination arbitrarily.For example, antigen can comprise native protein or protein fragments, synthetic proteins or protein fragments or peptide; It can comprise glycoprotein, glycopeptide, lipoprotein, lipopeptid, nucleoprotein, nuclear peptide; It can comprise peptide-peptide conjugate; Perhaps it can comprise the expression of nucleic acid product of reorganization.

In one embodiment, antigen preparation comprises antigenic mixture, and for example antigen is with the form administration of radiation treatment cell (for example cell of tumor cell or viral infection), virion or thick homogenate.In another embodiment, with the purification preparation of the recombinant forms of antigenic peptides or antigenic peptides to curee's administration, for example viral peptide or tumor associated antigen.In one embodiment, antigen preparation comprises I class MHC restriction peptide.In another embodiment, antigen preparation comprises II class MHC restriction peptide.In another embodiment, antigen preparation comprises the combination of I class restriction peptide and II class restriction peptide, is used for the administration to the curee.

In one embodiment, antigen is by " heredoimmunity " administration.In this embodiment, the DNA expression vector injection host animal with the relevant peptide of coding for example injects curee's skin or muscle.Gene outcome is by synthetic and glycosylation correctly, and is folding, expressed by the curee.Utilize this method, can will be difficult to the antigen administration of capacity acquisition or purification.In one embodiment, by particle bombardment device " particle gun " DNA is injected muscle or is discharged into skin, DNA is coated on the golden microgranule.Shown that heredoimmunity inducing specific humoral response and cellullar immunologic response are (for example referring to 1995 " IMMUNOLOGY KEY WORDS INDEX 155:2039 such as Mor; Xu and Liew.1995 " immunology " 84:173; Davis etc. 1994 " vaccine " 12:1503).

The optimum administration process of solubility B7 molecule can be different because of the curee who is treated.For example, solubility B7 molecule can with antigen administration and/or can be individually dosed before the antigen administration, perhaps can be after the antigen administration some days individually dosed.In one embodiment, solubility B7 molecule can " heredity " administration, the nucleic acid molecules administration of solubility B7 molecule or its part of just will encoding.In another embodiment, solubility B7 molecule and antigen are with the form administration of conjugate.

The dosage scheme of solubility B7 molecule can provide optimum therapeutic response for every curee through adjusting, and need not to carry out over-drastic experiment.For example, can measure to antigenic antibody titer or to antigenic cellullar immunologic response (for example DTH replys (Puccetti etc. 1994 " European IMMUNOLOGY KEY WORDS INDEX 24:1446)), to determine that whether the curee is forming immunne response to antigen or showing the immunne response that has strengthened, and correspondingly can adjust dosage.

Active medicine or compositions can also be passed through parenteral or the administration of intraperitoneal mode.Medicine for example can pass through intranasal, oral, intravenous, intramuscular, subcutaneous or mucosa mode administration.Can also in glycerol, liquid macrogol and composition thereof and oil, prepare disperse system.Under common storage and service condition, these preparations can contain antiseptic, to prevent microbial growth.

The pharmaceutical composition that is fit to inject comprises aseptic aqueous solution (if water miscible) or disperse system and is used for preparing the sterilized powder of aseptic injectable solution or disperse system temporarily.Pharmaceutical composition of the present invention can be suitable for specific route of administration through preparation.For example, in various embodiments, pharmaceutical composition of the present invention can be suitable for injection, suck or be blown into (through port or nose), perhaps is suitable for intranasal, mucosa, oral, cheek, parenteral, rectum, intramuscular, intravenous, intraperitoneal and subcutaneous release.

Medicine or compositions will be aseptic.In addition, they should be stable under preparation and storage requirement, should prevent the contamination of microorganism, for example antibacterial and fungus.Carrier can be solvent or disperse medium, the mixture that for example contains water, ethanol, polyhydric alcohol (for example glycerol, propylene glycol and liquid macrogol etc.) and be fit to.Suitable flowability can keep like this, for example utilizes coating, for example lecithin, keeps required particle diameter and utilize surfactant under the situation of disperse system.Various antibacteriums and antifungal can prevent action of microorganisms, for example p-Hydroxybenzoate, methaform, phenol, ascorbic acid, thimerosal etc.Under many circumstances, preferably in compositions, comprise isotonic agent, for example sugared, polyhydric alcohol, for example mannitol, Sorbitol, sodium chloride.In compositions, comprise the absorption that the reagent that postpone to absorb can prolong Injectable composition, for example monostearate aluminum and gelatin.

Aseptic injectable solution can prepare like this, adds the active compound or the medicine of aequum in appropriate solvent, and as required a kind of composition of as above enumerating or its combination, aseptic filtration then.Usually, disperse system is preparation like this, adds reactive compound (for example co stimulatory molecule and/or antigen and other medicine arbitrarily) in the sterile carrier of required other compositions that contain basic disperse medium and as above enumerate.Under the situation of the sterilized powder that is used for the aseptic injectable solution preparation, preferred manufacturing procedure is vacuum drying and lyophilization, obtains the powder that active component (for example medicine or compositions) adds other arbitrarily required composition from the solution of its aseptic filtration in advance.Medicine or compositions can be the form that is applicable to the Needleless injection medical instrument (such medical instrument is known in the art, for example referring to 5,383,851,5,581,198,5,846,233) when administration, and for example " molecular medicine " 1998.4:109 is described.

When active medicine or compositions during by due care, as mentioned above, protein for example can be with inert diluent or assimilable edible carrier oral administration." pharmaceutically acceptable carrier " used herein comprises all solvent, disperse medium, coating materials, antibacterium and antifungal, isotonic agent and absorption delay agent etc. arbitrarily.This class medium and reagent are well known in the art about the purposes of pharmaceutically active substances.Except with inconsistent those conventional media of reactive compound or reagent, its purposes in therapeutic combination is expected.Also can in compositions, add the reactive compound that replenishes.

Especially advantageously prepare the dosage unit form of parenteral composition, help the even of administration and dosage.Dosage unit form used herein refers to physically discontinuous unit, is suitable as the dosage unit of mammalian subject; Each unit contains the reactive compound of scheduled volume and required pharmaceutical carrier, and this scheduled volume produces required therapeutic effect through calculating.The standard of dosage unit form of the present invention is taken orders from and is directly depended on the specific characteristic of (a) reactive compound and the therapeutic effect that will reach and (b) compound a kind of like this active medicine or compositions are used for the individual treatment inherent restriction in field.

" pharmaceutically acceptable carrier " used herein for example comprises solvent, disperse medium, coating materials, antibacterium and antifungal, isotonic agent and absorption delay agent etc.This class medium and reagent are well known in the art about the purposes of pharmaceutically active substances.Also can add additional reagent.

Medicine of the present invention, is replied with enhance immunity curee's administration with the biologically compatible form that is suitable for the drug disposition administration." the biologically compatible form that is suitable for vivo medicine-feeding " expression protein form to be administered, the therapeutic effect of its Chinese medicine overweights any toxic action.Term curee plan comprises the live organism that wherein can cause immunne response, for example mammal.Curee's example comprises people, non-human primates, Canis familiaris L., cat, mice, rat and genetically modified organism thereof.The administration of the peptide of the B7 of having molecular activity as described herein can be any pharmacology's form, comprising independent solubility B7 peptide, solubility B7 peptide and the antigenic combination of treatment effective dose and the combination of solubility B7 peptide and pharmaceutically acceptable carrier.

The administration of the present composition of treatment or prevention effective dose is defined in necessary dosage of required result and the effective down amount of time phase of reaching.Preferably, the administration of solubility B7 molecule (containing or do not contain antigen) causes the immunne response of antigen (for example virus or tumor-cell antigen) is strengthened.

The curee can utilize art-recognized technology to be measured to antigenic immunne response (for example cell and/or humoral immunoresponse(HI)).Antigenic immunne response is measured can be in vivo or external carrying out.For example, by carrying out biopsy and seeking cellular infiltration, can measure the reactivity of immunocyte to tumor cell.Near infection site or tumor locus or in draining lymph node, can carry out the dyeing of original position cytokine.Can carry out cells in vitro and cultivate, with the test cell to antigenic reactivity (for example propagation in the presence of antigen and/or cytokine generation, and/or to antigenic cytotoxic activity).For example, treatment or prevention effective dose with the active polypeptide of B7 can be different because of various factors, and for example Ge Ti morbid state, age, sex and body weight and peptide cause individual required ability of replying.Dosage can provide best treatment or prevention to reply through adjusting.For example, can divide the several times administration every day or can suitably reduce dosage, this urgency level on the treatment situation is decided.

Reactive compound can be by suitable mode administration, for example injection (subcutaneous, intravenous etc.), oral administration, suction, transdermal medication or rectally.According to route of administration, reactive compound can be a coating, is not subjected to the effect of enzyme, acid and other natural conditions to protect chemical compound, and these may make chemical compound lose activity.

The invention further relates to the reactive compound of medicament forms, be used in the therapy as herein described.Reactive compound can also be used for the treatment of the preparation of medicine.

The administration of V, other drug

Treatment can obtain replenishing of other drug administration, with further increase immunne response.For example, can be with adjuvant and/or cytokine to curee's administration.

In one embodiment, can be with the cytokine administration, for example: granulocyte-macrophage colony stimutaing factor, M-CSF, granulocyte colony-stimulating factor, interleukin-11, interleukin-22, interleukin-13, interleukin 4, interleukin-15, interleukin 6, interleukin 7, interleukin-11 0 and/or interleukin 12.Also can be with the interferon administration, for example α, β and/or IFN-.Preferably, will be such as cytokine administrations such as IL-12, they help the auxiliary formation of replying of Th1-type T and the formation of cellular immunization.In another embodiment, the medicine that other can be modulated common stimulus signal for example resists-CD28 antibody curee's administration.

In another embodiment, can the drug administration of adjuvant will be known as.Nowadays unique people's of being widely used in adjuvant is Alumen (aluminum phosphate or an aluminium hydroxide).Other adjuvants for example Saponin and purified components Quil A thereof, Freund Freund's complete adjuvant and other are used for studying the adjuvant of using with the veterinary and have potential purposes at vaccine for man.But; also can use new chemically defined preparation, for example muramyldipeptide, a phosphoryl lipid A, phospholipid conjugates (for example Goodman-Snitkoff etc. " IMMUNOLOGY KEY WORDS INDEX 147:410-415 (1991) described those), resorcinol, non-ionic surface active agent (for example polyoxyethylene oleoyl ether and n-hexadecyl polyvinylether), enzyme inhibitor (comprising trypsin inhibitor), DFP (DEP) and aprotinin.In embodiment with the antigen administration, antigen for example can be encapsulated in the proteoliposome, as described in " The Journal of Experimental Medicine " 176:1739-1744 (1992) such as Miller, quote as a reference, perhaps be encapsulated in the lipid capsule, for example Novasome TM lipid capsule (Micro Vescular Systems at this, Inc., Nashua N.H.), replys with further enhance immunity.

Unless indication is arranged in addition, enforcement of the present invention will be adopted cytobiology, cell culture, molecular biology, microbiology, recombinant DNA and the immunological technique of this area routine.Such technology has detailed explanation in the literature.For example referring to " hereditism: molecular cloning laboratory manual " the 2nd edition Sambrook, J. etc. compile (Cold Spring Harbor Laboratory Press (1989)); " the simple and clear scheme of molecular biology " the 3rd edition Ausubel, F. etc. compile (Wiley, NY (1995)); " dna clone " I and II volume (D.N.Glover compiles, 1985); " oligonucleotide is synthetic " (M.J.Gait compiles (1984)); US Patent No such as Mullis: 4,683,195; " nucleic acid hybridization " (B.D.Hames ﹠amp; S.J.Higgins compiles (1984)); Monograph " Enzymology method " (Academic Press, Inc., N.Y.); " immuno-chemical method in cell and the molecular biology " (Mayer and Walker compile, Academic Press, London (1987)); " experiment immunization is learned handbook I-IV volume (D.M.Weir and C.C.Blackwell compile (1986)); Miller, J. " molecular genetics experiment " (Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1972)).

The content of the patent of all citation list of references, pending application application and announcements is in this application specially quoted as reference in view of the above.Every piece of list of references disclosed herein all quotes in full.All patent applications as the application's basis for priority also all quote in full at this as a reference.

The following example is further set forth the present invention, and they should not be interpreted as further restriction.

Embodiment

Embodiment 1: solubility co stimulatory molecule enhanced CT L replys

Best T cell activation effect had both required to signal by TXi Baoshouti, also required common stimulation.B7-1 and B7-2 are the lip-deep two kinds of strong co stimulatory molecules of APC.On inspection in the soluble form body of B7-2 to the effect of t cell response.Shla molecule is a kind of chimeric protein, contains the extracellular domain of B7-2, merges with the Fc district of mice IgG2a.With the administration of B7-2Ig fusion rotein, significantly enhancement antigen-specific T-cells is bred and cytokine response in the immunity of usefulness II class restriction peptide.The CTL that the B7-2Ig administration also strengthens the immunity of I class restriction peptide replys.In the presence of the t helper cell of II class restriction peptide was replied, B7-2Ig significantly increased the potentiation that CTL replys.These discoveries have proved the immunostimulatory activity of the soluble protein form of co stimulatory molecule, for example B7-2 to many T cellullar immunologic response parameter, and proof, and these molecules have clinical practice in infectious disease and vaccine indication.

Embodiment 1 material therefor and method:

Mice

This research use 7 to 10 ages in week female BALB/cJ mice (Jackson Labs, BarHarbor, ME).

The preparation of peptide based immunogens

Used whole peptide all is the immunodominant epitope of H-2d-restriction, from the nucleoprotein (NP) of influenza virus A/PR18/34.By the HOBT/DCC amino acid activation, utilize the Fmoc/NMP chemistry of standard, synthetic I class restriction peptide aa 147-155 TYQRTRALV (Taylor on PE Biosystems 430A peptide synthesizer, P.M. wait 1987 " immunogenetics " 26:267) and II class restriction peptide aa 55-77 RLIQNSLTIERMVLSAFDERRNK-OH and aa 206-229 FWRGENGRKTRIAYERMCNILKGK (Brett, S.J. etc. 1991 " IMMUNOLOGY KEY WORDS INDEX 147:1647).On Beckman HPLC, analyze and purification, utilize Bruker MALDI mass spectrograph to confirm quality.

The preparation of AIPR18/34 virus original seed

In order to prepare and titration AIPR18/34 influenza virus original seed, will to plant virus and be expelled to 10 days by 104 thinner ratios and contain in the embryo ovum gallinaceum.Cultivate after 42 hours, gather in the crops the allantoic fluid of infected ovum one by one, aseptic affirmation in addition on the blood agar plate.Compile aseptic allantoic fluid, the preparation aliquot, quick-freezing is stored under-70 ℃.Plaque titration (Bucher, D.J. etc. 1991 " clinical microbiology magazine " 29:2484) is carried out in quick thawing and dilution (in allantoic fluid) in virus afterwards.

The B7-2Ig fusion rotein

As expression as described in the forefathers and purification B7-2Ig fusion rotein (Fields, P.E. etc. 1998 " IMMUNOLOGY KEY WORDS INDEX 161:5268).In brief, expression plasmid pED makes up like this, connects signal and the cDNA of extracellular domain and the genomic DNA in encoding murine IgG2a hinge region, CH2 and CH3 district of encoding murine B7-2.The expression vector transfection in Chinese hamster ovary celI system, is used the described technology amplifications of forefathers (Kaufman, R.J. etc. 1988 " journal of biological chemistry " 263:6352).Make the cell culture supernatant liquid after concentrating pass through the mobile fast post (PharmaciaBiotech) of protein A agarose.B7-2Ig with 20mM citrate pH3.0 eluting, with 1M Tris (Sigma) pH8.0 neutralization, is formulated among the PBS pH7.2 by the buffering exchange.Utilize the 280nm absorbance to calculate protein concentration, extinction coefficient are 1.33cm/mg/ml.Level of endotoxin is less than 0.25EU/mg, and this measures (Cape Cod Associates) with gel grumeleuse algoscopy.The percentage rate of polymer formal protein is less than 1%, this with TSK 3000 SWXL posts measure (TosoHaas USA, Mongomeryville, PA).The external common stimulating activity of B7-2Ig be confirmed (Fields, P.E. etc. 1998 " IMMUNOLOGY KEY WORDS INDEX 161:5268).Here the experiment of enumerating is carried out three times at least, and the result is similar.Every experiment repeats once at least, uses purification mouse monoclonal IgG2a (Genetics Institute, CambridgeMA) contrast of handling as B7-2Ig of anti-irrelevant people's Protein G DF-9.

The peptide immunity

100 μ g specified polypeptides or peptide mixer by volume 1: 1 are mixed emulsifying with IFA (Sigma).To the subcutaneous 100 μ l antigen/IFA Emulsions of giving of root of the tail portion, cause mouse immune.At the position at contiguous peptide immunity position with 100 μ l B7-2Ig subcutaneous administrations.In the experiment of results lymph-node cell, mice is all accepted peptide and B7-2Ig administration in root of the tail portion and nape, as mentioned above.Each treatment group is made up of 5 mices.Institute's administration be B7-2Ig 0.1 aluminium hydroxide solution (Rehydragel, Rehies, Dublin, Ireland).

Live virus immunity and attack

(Mallinckrodt Veterinary, Mundelein IL) by the administration of nose cone body, reply until mice forfeiture reflection with Metafane.With the live virus intranasal administration that is diluted among the PBS, ultimate density is 40 μ l.Virus fatal dose (4, the 000PFU/ mice) causes not immune mouse 100% death.The immunizing dose of 40PFU/ mice causes there is not death, protects mice not to be subjected to cause death fully and attacks.

Proliferation assay

Collect lymph node (except the mesenteric node) and spleen at appointed day, the preparation single cell suspension is in 200 μ l RPMI 1640 (Gibco/BRL), by 5 * 10 ⁵Cells/well is cultivated in flat 96 hole flat boards, and culture media supplemented has 5 * 10 ^-5M 2ME, 2mM L-glutaminate, 100U/ml penicillin, 100 μ g/ml streptomycin (Gibco/BRL) and 10%FCS.When cultivating beginning, add II class restriction peptide by prescribed concentration.Utilize 1450 Microbeta plate readers (Wallac), measure proliferation function by the 3H thymidine combination after 18 hours burst process (1 μ C/ hole).The propagation of carrying out lymph-node cell on the the 3rd, 5,7 and 9 day behind the initial immunity is replied comparison.Immune once more back three days results spleens, assessment is replied once more.

Cytokine assay

As above about the described cultured cell of proliferation assay.At the 3rd day results supernatant, utilize commercial available test kit, measure level (IFN-γ Genzyme, Cambridge MA mensuration, IL-5 Endogen, Woburn MA mensuration, the IL-13 R﹠amp of IFN-γ, IL-5 and IL-13 by the ELISA method; D Systems, Minneapolis, MN measures).

The CTL of indivedual unsegregated mice blood cell samples measures

Two to three weeks behind the initial immunity are from Aerrane (Ohmeda Caribe, Inc, Guayama, PR) blood-letting behind Ma Zui the mouse orbit, collection blood sample (every part 150 μ l to 200 μ l).Sample is contained the PBS dilution of 50U heparin with 100 μ l.Get 40 μ l samples and carry out numeration of leukocyte.The differential count of sample is not customary method.Different time is got 100 increments and is originally carried out differential count during studying, and the percentage rate that lymphocyte accounts for the leukocyte part is 69% ± 8%.Washed cell is to remove heparin.To amount to 8 * 105WBC/ mice and be suspended in 10ml again as above about in additional RPMI 1640 culture medium of the described process of proliferation assay, in addition, replying and do following replenish for producing external first CTL: 4 μ g/ml are anti--CD28 (PV1.17) and 10U/ml reorganization Mus IL-12 (rmIL-12, Genetics Institute, Cambridge, MA), 10U/ml IL-2 and 200U/mlIL-6 (Genzyme, Cambridge, MA), (TYQRTRALV is aa147-155) with 1 * 10 for 0.1pM I class restriction peptide ⁶/ ml radiation treatment (2,000rads) syngeneic spleen cell, splenocyte buffered 0.17M NH through 0.017M Tris ₄Cl handled, with dissolving RBC (Gajewski, T.F.1996 " IMMUNOLOGY KEY WORDS INDEX 156:465).

With volume be the cell plating of 100 μ l in Costar flat board at the bottom of 96 hole circles, every hole amounts to 8 * 10 ³Individual leukocyte.Every part of blood sample is 80 holes of plating altogether.There are 16 holes only to accept to contain the culture medium of radiation treatment splenocyte on each flat board.At the 7th day that cultivates, be inverted flat board with the supernatant that comes down in torrents, stir dull and stereotyped with suspension cell granule.The P815 cell is spent the night preparation antigen-positive (the Ag-positive) target with 10 μ g/mlI classes restriction peptide burst process.In the 100 μ l culture medium in 50% hole, add 1 * 10 ³The Ag-positive, ⁵¹The P815 target of Cr labelling is to all the other 50% addings 1 * 10 ³The Ag-feminine gender, ⁵¹The contrast P815 target of Cr labelling.Each flat board does not contain in 16 holes of effector lymphocyte, and 8 are used for spontaneous release, and 8 are used for maximum release and measure.Add 20 μ l 10%Triton X100 and reach all release.Cultivate after 4 hours, 50 μ l supernatant/holes are gathered in the crops in the Wallac 96 hole flat boards (1450-401), (Turku Finland), counts dull and stereotyped in Wallac Microbeta 1450 for OptiPhase SuperMix, Wallac to add 125 μ l scintillators.

The calculating that the average molten born of the same parents of specificity lead

The molten born of the same parents that calculate every hole according to normalized form lead:

Wherein all release is the average cpm in all holes of accepting target and triton-X100, and it is the average cpm+SD in all holes of only accepting target and culture medium that culture medium discharges.The molten born of the same parents of average specificity that calculate every hole according to following formula lead:

Data represent that with the molten born of the same parents ± SD of average specificity this is averaged by five the every group every hole of the mice gained molten born of the same parents' values of average specificity again and calculates.Utilize two tail student t checks to measure significance,statistical.

The result:

The B7-2Ig enhancing is replied the first CD4+T cell proliferation of II class restriction peptide immunity

Whether can strengthen or suppress first t cell response in order to measure B7-2Ig to the peptide immunity, be with or without the B7-2Ig administration in the presence of mice is used the peptide immunity.Will be from II class restriction peptide aa 55-77 and the mixture subcutaneous administration of aa 206-229 in IFA of reassortant influenza virus N P.These peptides have shown it is immundominance CD4+Th cell epitope (Brett, S.J. etc. 1991 " IMMUNOLOGY KEY WORDS INDEX 147:1647, Brett, S.J. and J.P.Tite.1996).With and the gene that is not connected with H-2 all influence the nucleoprotein influenza epi-position by CD4+T cell recognition (" immunology " 87:42).Be close to the position with B7-2Ig (0.1% alum solution) administration.In preliminary study, after immunity, measured the peptide specific propagation of lymph-node cell on the the 3rd, 5,7 and 9 day and reply.The kinetics of replying is not subjected to the influence of B7-2Ig administration.After immunity the 7th and 9 day, the mice of B7-2Ig and control treatment all observed best propagation and replys.As shown in Figure 1, reply remarkable increase (p=0.014, external use 50 μ g/ml peptides stimulate again) handling the antigenic specificity propagation lead immunity lymph-node cell of results after 9 days with B7-2Ig in the peptide immunity.Among Fig. 1, in the IFA Emulsion immunity of two subcutaneous location, the IFA Emulsion of per injection respectively contains two kinds of II classes restriction peptides of 100 μ g or PBS with every group of five mices.At the position at contiguous immune position with 0.1% alum solution of 100 μ g B7-2IgG2a or 0.1% independent Alumen administration.The mouse lymph nodal cell with independent peptide immunity (zero-), with independent B7-2Ig handles (△-) or with the peptide immunity and with B7-2Ig handles (■-), in immunity results after 9 days, is externally respectively stimulated with immune used two kinds of peptides of prescribed concentration again.Do not accept the peptide immune mouse of B7-2Ig and accept Alumen in contrast.By ³H-thymidine combination is measured proliferation function.Data are represented with average cpm/ hole ± SD of every group of 5 mices.When using IgG2a mAb mice in contrast, obtain analog result.Data are from one of three representative experiments.There not being the B7-2Ig administration under the immunization not cause propagation to be replied, proved the antigen dependency after B7-2Ig acts on initial immunity.These results prove that the vivo medicine-feeding of B7-2Ig strengthens the CD4 to the peptide immunity ⁺The Th cell response.

B7-2Ig strengthens anamnestic response

Whether strengthen anamnestic response in order to test the B7-2Ig dependency potentiation that first antigenic specificity propagation replys, in the presence of being with or without that B7-2Ig handles, mice use the peptide immunity, reinforcement after 46 days, and do not have further B7-2Ig administration.Collected splenocyte in immune once more back 3 days, measure peptide specific propagation and reply.As shown in Figure 2, having accepted mice that single B7-2Ig handles in initial immunity has bigger propagation in immunity back once more than the mice of never accepting B7-2Ig and replys (p=0.0006, external use 100pg/ml peptide stimulates again).With every group of five mices with independent peptide immunity (zero-), with independent B7-2Ig handles (△-) or with the peptide immunity and with B7-2Ig processing (■-).Behind the initial immunity the 46th day, do not have the B7-2Ig co-administered in the presence of with two groups of peptide immune mouses all with peptide immunity again.Non-immune mouse is only accepted IFA.Again immune back three days, external each the immune used two kinds of peptide with prescribed concentration of splenocyte are stimulated again.By ³H-thymidine combination is measured proliferation function.Data are represented with average cpm/ hole ± SD of every group of 5 mices.Data are from one of twice repeated experiments.These data show that B7-2Ig strengthens anamnestic response when being used as the adjuvant of first t cell response.

B7-2Ig strengthens Th1 and the first cytokine response of Th2 dependency

For whether the B7-2Ig that measures as immunological adjuvant differentially promotes the formation that Th1 or Th2 reply, in the peptide specific cytokine response of the 3rd, 5,7 and 9 day analysis immune mouse lymph-node cell behind initial immunity after the immunity.As propagation is replied, observed best cytokine response at the 9th day.As shown in Table I, cause with Th1 related cytokine IFN-γ (p=0.017), significantly increase in the B7-2Ig administration in the peptide immunity with the generation level of Th2 related cytokine IL-5 (p=0.011) and IL-13 (p=0.002).In the culture of the not immune mouse that the B7-2Ig that uses by oneself handles, do not observe antigenic specificity cytokine generation effect.These results show that B7-2Ig strengthens the first t cell response of Th1 and Th2 phenotype.

The CTL that B7-2Ig strengthens I class restriction peptide replys

Film in conjunction with the interaction of B7 and CD28 shown can not have the CD4+ cell in the presence of outside the inductor CTL reply (Harding, F.A. etc. 1993 " The Journal of Experimental Medicine " 177:1791).In order to test the effect that B7-2Ig replys first CTL,, follow or do not follow the B7-2Ig administration with the IFA Emulsion immunity of mice with immundominance I class restriction peptide.Utilize a small amount of blood sample to measure the peptide specific CTL that does not separate peripheral blood cells and reply, CTL measures as described in material and the method.This mensuration might be implemented in the single experiment statistical significant difference analyzed between a large amount of mices, evaluation group, repeat indivedual mices during immunne response CTL measures and research CTL replys and body in mutual relation between the result.

Data are with single numerical value (the average molten born of the same parents of the specificity lead) expression (Fig. 3) of every mice.IFA Emulsion administration (100 μ g/ mice) with peptide.With the subcutaneous co-administered of B7-2Ig (0.1% alum solution of 100 μ g/mice).Three weeks of immunity back are collected unsegregated blood, measure CTL.Data with the molten born of the same parents of average specificity lead ± SD represents.The numerical value of live virus immune group is from the single thing that compiles of 2 mices, and 9 18 mices of test in the experiment independently, it is 52 ± 12 that the molten born of the same parents of average specificity that obtain this group lead.In every other group, every group has five mices.Shown in data from one of three repeated experiments.The not obvious background that is higher than of replying to I class restriction peptide IFA Emulsion." discovery of IMMUNOLOGY KEY WORDS INDEX 147:406 is consistent to this discovery and Fayolle etc. 1991, and they point out under the help that does not have the T cell, the CTL of I class restriction peptide IFA Emulsion is replied approach background level.When in the immunity of I class restriction peptide IFA Emulsion, during with the B7-2Ig administration, observing and replying small and the significantly increase (Fig. 3) of (p=0.013) of statistics.But, compare with replying with the peptide specific of live virus mice immunized, little with the peptide immunity and the amplitude of replying with the peptide specific CTL of the mice of B7-2Ig co-administered.Therefore, the co-administered of 100 μ g B7-2Ig does not raise to the maximum horizontal that is reached to effective live virus immunization of replying of I class restriction peptide immunization.

The best potentiation that B7-2Ig replys CTL needs the help of T cell

Because best CTL replys and depends on Th cell (Fayolle, 1991 " IMMUNOLOGY KEY WORDS INDEX 147:4069 such as C.; Keene, J.A. etc. 1982 " The Journal of Experimental Medicine " 155:768; VonHerrath, M.G. wait 1996 " Journal of Virology " 70:1072 and Ossendorp, F. wait 1998 " The Journal of Experimental Medicine " 187:693), therefore under with the condition of mice, test the effect that B7-2Ig replys CTL with the common immunity of peptide of the known Th of causing cell response.With mice with containing the IFA Emulsion immunity of independent I class restriction peptide or I class restriction peptide with the mixture of two kinds of II class restriction peptides, as Fig. 1 and 2 with following Table I as described in.The CTL that measures unsegregated peripheral blood cells replys (Fig. 4).Carry out the peptide immunization with single IFA Emulsion form.Press designation method with mouse immune and processing.After three weeks, the CTL that measures peripheral blood replys.Data with the molten born of the same parents of average specificity lead ± SD represents.The numerical value of live virus immune group is from the single thing that compiles of 2 mices, and 9 18 mices of test in the experiment independently, it is 52 ± 12 that the molten born of the same parents of average specificity that obtain this group lead.In every other group, every group has five mices.The molten born of the same parents of average specificity that accept the group of II class restriction peptide IFA Emulsion and contrast Ig alum solution lead the molten born of the same parents of average specificity that deduct the group of accepting contrast Ig and lead.The molten born of the same parents of average specificity that accept the group of II class restriction peptide IFA Emulsion and B7-2Ig alum solution lead the molten born of the same parents of average specificity that deduct the group of accepting B7-2Ig and lead.Shown in data from one of four repeated experiments.

The molten born of the same parents of average specificity that limit the mixture mice immunized cell of peptide with I class and II class lead the I class restriction peptide mice immunized cell (experiment shows p=0.046, and repeated experiments is .004) that is significantly higher than with independent.Cause the molten born of the same parents of average specificity to lead significantly with the mixture immunity of I class and II class restriction peptide and with 100 μ g B7-2Ig processing greater than the peptide immune mouse of not accepting B7-2Ig (p=0.003, p=0.0001, p=0.045 and p=0.0012 in four repeated experiments).When the dosage of B7-2Ig factors two to 200 μ g/ mices, do not increase this replying, illustrate that 100 μ g dosage induce maximum B7-2Ig dependency potentiation.

Peptide mixer immunity and combining of B7-2Ig processing cause peptide specific CTL to reply being similar to that the live virus immunization is caused replys on amplitude.After 18 live virus immune mouse institute values are averaged in 9 independent experiments, obtaining the molten born of the same parents' value of average specificity is 52 ± 12, has proved that the reproducibility of a small amount of peripheral blood sample CTL algoscopy and caused the replying of immunization of the peptide in the presence of the help of T cell and B7-2Ig are equivalent to the caused effectiveness of replying this conclusion of effective live virus immunization.The CTL that detects from peripheral blood cells replys in 2 to 3 weeks and reaches peak value, in the desalination of the 5th week, is not always the case with the live virus mice immunized with the peptide immunity and with the mice of B7-2Ig processing, and the similarity of replying is described once more.But just as discussed below, the attack of the protected virus of avoiding causing death of the mice of handling with the peptide mixer immunity and with B7-2Ig.

Fig. 3 and 4 data are produced by the B7-2Ig that is formulated in 0.1% Alumen, and the former itself is the immunological adjuvant (Aprile, M.A. etc. 1966 " Canadian public health magazine " 57:343) of the clinical approval of a kind of process.The alum solution administration of independent Alumen or contrast Ig does not influence anti-peptide and replys, and the Alumen preparation is neither B7-2Ig dependent immunity potentiation required.But, the Alumen preparation is strengthened the effect of B7-2Ig really.If the directly relatively effect of the PBS solution of the effect of the alum solution of B7-2Ig and B7-2Ig, observing bigger CTL with the alum solution administration of B7-2Ig the time replys potentiation (average molten born of the same parents leads that to contain Alumen be 72 ± 13, not containing Alumen is 38 ± 12, p=0.007).

The best antigen-specific, activated effect of T cell and regulating action need common stimulus signal to be discharged into CD28 and CTLA-4 (Boussiotis, V.A. etc. 1996 " immunology comment " 153:5 on the T cell surface from the lip-deep B7 molecule of APC; Lenschow, D.J. etc. 1996 " immunity is commented academic year " 14:233; Green, J.M. etc. 1994 " immunity " 1:501; Tivol, E.A. etc. 1995 " immunity " 3:541 and Waterhouse, P.1995 " science " 270:985).Multiple mouse model studies show that, can enhance immunity reply by the cell surface expression that increases B7.In these researchs, the increase that B7 expresses is following inductive: tumor cell transduction (Baskar, S. etc. 1993 " institute of NAS newspaper " 90:5687; Cavallo, F. etc. 1995 " European IMMUNOLOGY KEY WORDS INDEX 25:1154; Coughlin, C.M. etc. 1995 " cancer research " 55:4980; Chen, L. etc. 1992 " cell " 71:1093; Chen, L. etc. 1994 " The Journal of Experimental Medicine " 179:523; Gajewski, 1996 " IMMUNOLOGY KEY WORDS INDEX 8:2909 such as T.F.; Yang, G. etc. 1995 " 1996 " blood " 87:2938 such as IMMUNOLOGY KEY WORDS INDEX 154:279 and Dunussi-Joannopoulos K.), cDNA injects (Kim, J.J. etc. 1997 " natural biological engineering " 15:641; Kim, J.J. etc. 1998 " novel vaccine " 16:1828 and Horspool, J.H. etc. 1998 " IMMUNOLOGY KEY WORDS INDEX 160:2706) or with viral vector infection (Chamberlain, R.S. etc. 1996 " cancer research " 56:2832; Emtage, 1998 " IMMUNOLOGY KEY WORDS INDEX 160:2531 such as P.C.; Hodge, J.W. etc. 1994 " cancer research " 54:5552 and Putzer, B.M. etc. 1997 " institute of NAS newspaper " 94:10889).Utilize to increase the Different Strategies of B7 level in the body, developed the soluble protein form B7-2Ig of B7-2, forms by the Fc of mice IgG2a, the extracellular part fusion of this Fc and B7-2, this part is connected with every Ig chain.Vivo medicine-feeding enhancement antigen specificity T h cell and the CTL of B7-2Ig reply.Thereby the vivo medicine-feeding of B7-2Ig is transduceing or use virus or the dna vector optimization B7-2 mediation simple alternative of stimulation jointly from external B7 of tumor cell.

Because the consequence of infectious disease and autoimmune disease is subjected to the influence that responsiveness Th cell produces the pattern of cytokine greatly, determine whether the B7 approach can be used for replying deflection (Kuchroo, V.K. etc. 1995 " cell " 80:10 to Th1 or Th2 cytokines so people are interesting; Lenschow, D.J. etc. 1995 " The Journal of Experimental Medicine " 181:1145; Corry, 1994 " IMMUNOLOGY KEY WORDS INDEX 153:4142 such as D.B.; Freeman, G.J. etc. 1995 " immunity " 2:523; Schweitzer, 1997 " IMMUNOLOGY KEY WORDS INDEX 158:713 such as A.N.; Rulifson, I.C. etc. 1997 " IMMUNOLOGY KEY WORDS INDEX 158:658 and McAdam, A.J. etc. 1998 " immunology comment " 165:231).Big quantity experiment system shows that the Th2 differentiation more depends on common stimulation (Lenschow, D.J. etc. 1995 " The Journal of Experimental Medicine " 181:1145 of B7 than the Th1 differentiation; Corry, 1994 " IMMUNOLOGY KEY WORDS INDEX 153:4142 such as D.B.; Freeman, G.J. etc. 1995 " immunity " 2:523 and Rulifson, I.C. etc. 1997 " IMMUNOLOGY KEY WORDS INDEX 158:658).Anti--B7 mAb has been successfully used to handle in the body T cell differentiation.B7-1 be it is reported by the mAb blocking-up and helps the Th2 differentiation, and the blocking-up of B7-2 helps Th1 differentiation (Kuchroo, V.K. etc. 1995 " cell " 80:Mar 10 (5); Lenschow, D.J. etc. 1995 " The Journal of Experimental Medicine " 181:1145; Corry, 1994 " IMMUNOLOGY KEY WORDS INDEX 153:4142 such as D.B.; Freeman, G.J. etc. 1995 " immunity " 2:523 and Rulifson, I.C. etc. 1997 " IMMUNOLOGY KEY WORDS INDEX 158:658).On the contrary, Schweitzer etc. utilize B7 inefficacy mice APC to find, the external as if all differentiation of non preference adjusting Th1 or Th2 of B7-1 or B7-2 (Schweitzer, A.N. etc. 1997 " IMMUNOLOGY KEY WORDS INDEX 158:713).This example data show, the vivo medicine-feeding of B7-2Ig strengthens the antigenic specificity cytokine response of Th1 and Th2 type.Therefore, treatment potentiality B7-2Ig probably to raise but not deflection to reply be to be best under the situation of target.

In order to test the effect that B7-2Ig replys CTL, the caused peptide specific CTL of peptide of the NP of the susceptible malicious A/PR/8/34 that relatively is used for flowing automatically replys and caused the replying of using from the live virus immune mouse of identical peptide.B7-2Ig handles and to induce reply little of I class restriction peptide IFA Emulsion and significant potentiation.These data are consistent, prove outside not, help in the presence of, be enough to produce external CTL with the tumor cell of B7 transfection and reply (Harding, F.A. etc. 1993 " The Journal of Experimental Medicine " 177:1791).

But, replied by the enhanced CTL that significantly is lower than the identical peptide of live virus immune mouse that replys of B7-2Ig.These data show, mice can be replied than replying the much bigger anti-peptide of generation by restriction peptide immunity of I class and the caused mice of B7-2Ig co-administered.

In the effort of replying that further strengthens I class restriction peptide, adding Th cell, the antigenic effect of II class restriction peptide in containing the antigenic IFA Emulsion of I class restriction ctl peptide have been studied.Forefathers report that best CTL replys depends on Th cell (Fayolle, 1991 " IMMUNOLOGY KEY WORDS INDEX 147:4069 such as C.; Keene, J.A. etc. 1982 " The Journal of Experimental Medicine " 155:768; Von Herrath, M.G. etc. 1996 " Journal of Virology " 70:1072 and Ossendorp, F. etc. 1998 " The Journal of Experimental Medicine " 187:693).In with I class and II class restriction peptide mice immunized, CTL replys remarkable rising.These data show that the common immunity of Th cellular antigens can provide the help that antigenic CTL replys of antagonism peptide.Covalently bound between CTL and the Th peptide epitopes is not needed, points out any immunogenic protein can be used as common immunogen, so that the help that CTL is replied to be provided.This conclusion has further been supported in our discovery, just adds KLH, the remarkable potentiation that causes peptide specific CTL to reply in the IFA Emulsion that contains I class restriction peptide.

Although be enhanced, the peptide specific CTL that limits the peptide mice immunized with I class and II class replys also less than using the live virus mice immunized.The CTL that adding B7-2Ig in scheme increases with the peptide mixer mice immunized replys to the level with the live virus mice immunized.This two groups replys is suitable, but the attack of the former the not protected virus of avoiding causing death.The result of this result and Lawson etc. is consistent (Lawson, C.M. etc. 1994 " Journal of Virology " 68:3505), and they cause that by the recombinant vaccine virus administration with expression of peptides the violent CTL to being used in the identical peptide in the research replys.At present, the violent CTL of independent this peptide is replied be not enough to give BALB/c mouse with protective immunity.

Although the immunological enhancement that is not B7-2Ig is needed, the Alumen preparation still causes the potentiation bigger than PBS preparation.The alum solution administration of contrast Ig does not influence and replys, and illustrates that independent Alumen does not strengthen to reply.

Infecting with live virus is the natural and valid approach (Zinkernagel, R.M. etc. 1979 " immunology progress " 27:51) that activation is replied in the restriction of I class.Therefore, list here, show from obtaining the effectiveness that data that suitable anti-peptide replys have been established latter's immunization protocol with the live virus mice immunized with the mixed emulsion immunity of I class and II class restriction peptide and with the mice that B7-2Ig handles.At present, cause that it is very interesting that CTL to I class restriction peptide replys.Can discern the proof prompting of the I class relevant restriction peptide about people CTL as mice CTL in the last few years, reply at the CTL of these peptides and can confirm people's tumor effectively (van derBruggen, P. etc. 1991 " science " 254:1643 with tumor; Wolfel, T. etc. 1993 " international journal of cancer " 55:237; Maeurer, M.J.1996 " melanoma research " 6:11; Cormier, J.N. etc. 1997 " Scientific Beauty state human cancer magazine " 3:37; Apostolopoulos, 1997 " IMMUNOLOGY KEY WORDS INDEX 159:5211 such as V.; Rosenberg, S.A.1998 " natural medicaments " 4:321 and Nestle, F.O. etc. 1998 " natural medicaments " 4:328).This probability has stimulated the bigger enthusiasm of people, and design optimization is to the immunization protocol of replying of I class restriction peptide vaccine.It is reported, multiple different strategy has all been obtained success, comprise modification, the cDNA of the oligomerization that in recombinant virus and antibacterial, inserts mini-gene, peptide, lipid immunity, use toxin and cytokine as adjuvant and antigen impulse stimulation arborescent cell (Allsopp, C.E. etc. 1996 " European IMMUNOLOGY KEY WORDS INDEX 26:1951; Rotzschke, O. etc. 1997 " institute of NAS newspaper " 94:14642; Alving, C.A. etc. 1995 " immunology comment " 145:1; Ciernik, 1996 " IMMUNOLOGY KEY WORDS INDEX 156:2369 such as I.F.; Porgador, 1997 " IMMUNOLOGY KEY WORDS INDEX 158:834 such as A.; Noguchi, Y. etc. 1995 " institute of NAS newspaper " 92:2219 and Takahashi, H. etc. 1992 " international immunology " 5:849).Here the advantage of institute's reported method is, it can be applied to any peptide consistently, need not further modification, replys all effective to I class and the restriction of II class.

B7-2Ig replys to being equivalent to have pointed out the proteic soluble form of B7-2 also can strengthen immunogenic replying a little less than other by the caused level of live virus immunity with the relative weak I class restriction of the co-administered rising peptide of peptide immunity.These find to show that the soluble protein form of B7-2 can be replied enhancement antigen specific immune response in invalid cancer or the infectious disease therapy in immune system.

Table I: the B7-2Ig dependency potentiation of Th cytokine response ^a

Cytokine is handled IFN γ IL-5 IL-13 in external post-stimulatory again expression body

(((the immune b.d of peptide of pg/ml ± SD) of pg/ml ± SD) of pg/ml ± SD) ^b54 ± 44 136 ± 125 peptide immunity add B7-2Ig 1720 ± 986 2479 ± 1,104 5365 ± 1795IFA and add B7-2Ig b.d ^bB.d ^bB.d ^b

^aIn the IFA Emulsion immunity of two subcutaneous location, the Emulsion of per injection contains two kinds of II classes restriction peptides of 100 μ g or PBS with every group of five mices.At the position at contiguous immune position with 0.1% alum solution of 100 μ gB7-2IgG2a or 0.1% independent Alumen administration.Behind the Ninth Heaven, the results lymph-node cell stimulated three days again with the various peptides of 5 μ g/ml in culture medium.Be averaged from triplicate aperture gained result, obtain numerical value about every mice.Data are represented with the mean concentration ± SD of the mouse cell factor in organizing.The result is similar to and uses IgG2a mAb control mice gained result in contrast.Shown in data are one of three repeated experiments. ^bBe lower than the detection limit of algoscopy.

Embodiment 2:B7-IgG fusion rotein strengthens anti-tumor immune response, induces and sets up disappearing of tumor

Estimated the cell outskirt of B7-1 or B7-2 and in four kinds of different Mus tumor models, strengthened the ability that antitumor is replied about them, comprised weak immunogenicity melanin tumour b16/F10 with fusion rotein between the IgG2a.When mixing with B7-1 or B7-2 IgG, carry out single vaccination with the radiation treatment tumor cell, the protection mice avoids the attack of tumor alive.More significantly, use carry out vaccination with the blended radiation treatment tumor cell of B7-IgG after, the tumor regression of having grown 7 days.Even the therapeutic administration of independent B7-IgG has also reduced the burden of tumor similarly.Having repelled the animal counterweight new attack of having set up tumor is toleration, has pointed out the antitumor action of B7-IgG to be mediated by the tumour-specific immune mechanism strongly.In addition, use with B7-1 or the blended radiation treatment tumor cell of B7-2 IgG and carry out single vaccination, produce the anti-antigenic CD8 relevant and reply with tumor.Thereby, B7-IgG and exogenous release and the adjuvanticity existing power of endogenous antigen associative list.These discoveries have pointed out B7-Ig to strengthen the clinical value of antitumor and antiviral response.

Embodiment 2 material therefors and method

Mus B7-1-IgGIgG and B7-2-IgG2a Expression of Fusion Protein plasmid are to make up like this, connect the signal of coding Mus B7-1 or B7-2 and the DNA and the DNA of coding from the deutero-hinge region of Mus IgG2a antibody-CH2-CH3 domain of extracellular domain.It is conservative that cysteine residues in the antibody twisting district keeps, and the mB7-1-IgGIgG2a or the mB7-2-IgG2a that produce with toilet are dimer and bivalence.In the fusion rotein that is generated, in order to prevent by high-affinity Fc receptors bind and to prevent to replenish activation, sudden change IgG2a district (being called B7-IgG2mut).Amino acid residue in the following CH2 domain is replaced by alanine: #235 position leucine, #318 position glutamic acid, #320 position lysine and #322 position lysine.About the proteic generation of B7-IgG, expression plasmid in Chinese hamster ovary celI system, isolated protein.

P815 is from the deutero-tumor cell line of mastocytoma, is injecting 5 * 10 to DBA/2 mice (JacksonLaboratories) intradermal (i.d.) ⁴Be grown to serve as solid tumor behind the cell.The i.d. that is cloned in that is used in these experiments injects the spontaneous transfer in back, causes dead mouse after 25-35 days.MethA is from the deutero-tumor cell line of Balb/C murine sarcoma, in i.d. injection 5 * 10 ⁵Be grown to serve as solid tumor (from the Balb/C mice of Taconic) behind the cell.B16/F10 is from the deutero-non-immunogenic tumor of C57BL/6 mice (Taconic) melanoma system, in i.d. injection 1 * 10 ⁵Be grown to serve as solid tumor behind the cell, cause death because of after 25-35 days, shifting.Bladder cancer MB49 is also from the C57BL/6 mouse-derived, and i.d. injects 1 * 10 ⁵Cell, 100% mice grew solid tumor after 5-7 days.

External common stimulation algoscopy

With 2 * 10 ⁵Children's mice spleen cell triplicate plating on 96 hole flat boards, flat board scribble low concentration anti--CD3 mAb (0-500ng-ml-2C11, Pharmingen).In order to provide common stimulation necessary signal, flat board scribbles simultaneously anti--CD28 mAb (0-1 μ g/ml) perhaps scribbles B7-1-IgG or B7-2-IgG (0-9 μ g/m1) as positive control.Also add B7-IgG soluble form or with secondary anti--the crosslinked form of mice IgG2a Ab.Splenocyte was stimulated 72 hours.Get the supernatant sample and be used for IFN-γ analysis, with cell H ³-thymidine burst process 8 hours.In standard ELISA, carry out IFN-γ and analyze, with catching Ab R46A4 and biotinyl XGM1.2 as detecting Ab.

The vaccination scheme

In preventative tumor vaccine model,, when being attacked by tumor cell, mice carries out vaccination at the 0th day.Mice is used radiation treatment tumor cell (1 * 10 ⁷Cell) PBS solution immunity wherein is mixed with 75-100 μ g mB7-1-IgG, B7-2-IgG, Mus IgG or does not have whatever.Two hind legs are given (i.fp.) injection in the palmula.Repeated the injection of a B7-IgG at the 3rd or 5 day.At the 7th day, attack animal with the tumor cell alive of limiting dose, side of body i.d. injects 50 μ l on the right side.Monitor the growth of the primary tumor in 40-60 days and the survival rate of animal.

In therapeutic tumor vaccine model, primary tumor is to set up by the tumor cell of i.d. inoculation qualification quantity.5-7 days (this moment tumor obviously stereognosis), with 1 * 10 ⁷Radiation treatment tumor cell i.fp. inoculates mice, wherein is mixed with 100 μ g B7-1-IgG, B7-2-IgG or does not have whatever.After three days once more i.fp. inject 100 μ g B7-IgG.This vaccination scheme repeated for two or three weeks.Monitor the growth of the primary tumo(u)r in the 7th day to 40-70 days and the survival rate of mice.

In the treatment model, the mice B7-IgG that carries tumor is handled with independent fusion rotein.I.fp. inject independent B7-1-IgG of 50-100 μ g or three weeks of B7-2-IgG, biweekly.Monitor tumor growth and survival rate in 40 days.

The detection that the P1A specific CTL is replied

With being mixed with or not mixing behind the radiation treatment P815 cell single immunization of B7-1-IgG or B7-2-IgG 10-14 days, from the DBA/2 mice, collect spleen.After the erythrocytolysis, in the T25 flask with 20 * 10 ⁶Splenocyte stimulated 6 days again with 0.1ng/ml P1A peptide and 5U/ml IL-2 (Pharmingen).Then, carry out the 5h Cr of standard ⁵¹-discharge and measure,, carry out the peptide burst process or do not carry out burst process as target with the A20 cell with 10 μ g/ml P1A.The molten born of the same parents' percentage rate of P1A-peptide specific with the molten born of the same parents' percentage rate of the target of peptide-burst process and not the difference between the molten born of the same parents' percentage rate of target of burst process represent.

The result:

Immobilization or the crosslinked external mice spleen cell that stimulates for suboptimum of B7-IgG provide common stimulus signal

FACS  or Biocore  assay determination B7-1-IgG and B7-2-IgG fusion rotein combine with Mus CD28 and CTLA-4.In order to measure enhancing of B7-IgG fusion rotein or suppressor T cell activation, cultivate and dull and stereotyped bonded resisting-CD3 mAb and the B7-IgG that is fixed on the flat board, stimulated in vitro mice spleen cell by uniting.With flat board bonded anti--the common stimulation of CD28 mAb serves as positive control.B7-1-IgG and B7-2-IgG similarly induce propagation and the excretory dose dependent of IFN-γ to increase (Fig. 5).With 2 * 10 ⁵Children's splenocyte in triplicate with 50ng/ml anti--CD3 monoclonal antibody and the immobilization of quantity of increasing progressively be anti--CD28 antibody or B7-1 or B7-2 IgG stimulated 72 hours.Add the 3H-thymidine after the pulse in 6 hours, measure propagation and reply.B and C hurdle show the quantity of IFN γ or IL2 respectively, and they discharge after the immobilization B7-2-IgG with 50ng/ml anti-CD 3 antibodies and specified quantity stimulates 72 hours.Measure cytokine with standard ELISA.In the presence of not having that anti--CD3 stimulates, B7-IgG does not induce proliferation function.The B7-IgG molecule and secondary anti--immobilization of Mus IgG mAb or crosslinked be effectively common stimulate necessary because solubility B7-IgG albumen does not strengthen the generation of proliferation function or IFN-γ.B7-IgG albumen (B7-1-IgG and B7-2-IgG2mut) effectively common splenocyte that stimulates when being fixed on dull and stereotyped going up in its Fc land sudden change.

B7-1-IgG or B7-2-IgG be the protection effect of enhanced rad processing tumour-cell vaccine

In preventative tumor vaccine model, estimated B7-IgG fusion rotein as adjuvant.Inoculation 5 * 10 ⁴P815 tumor cell alive, 100% young DBA/2 mice generated solid tumor after 5-7 days.With every group of 10 DBA/2 mices with 1 * 10 ⁷Radiation treatment P815 tumor cell i.fp. immunity once.Cell vaccine is individually dosed, perhaps with B7-1-IgG, the B7-2-IgG of 100 μ g or their mutain mixing administration.At the 5th day with the B7-IgG rechallenge.In contrast, B7-1-IgG or the B7-2-IgG i.fp. administration that 100 μ g are independent at the 0th and 5 day.At the 7th day with 5 * 10 ⁴The P815 tumor cell of living is attacked mice.Do not have palpable tumor after 24 days, determine that attack is had protective effect.Fig. 6 shows the result of one of five representative experiments.Live and tumor challenge the last week mice not to be caused protective effect to tumor growth with the immunity of P815 tumor cell.But, with the radiation treatment tumor cell single immunization that is mixed with B7-1-IgG or B7-2-IgG, induce 60-70% protective effect (Fig. 6, table 2).On the contrary, do not provide protective effect (Fig. 6) with independent B7-1-IgG or B7-2-IgG immunity.There are not solid tumor and/or survival all to confirm protective effect at least in 60 days after 14-24 days.There is not metastatic disease in latter's explanation.

In order to estimate the role of IgG domain in the fusion rotein function, mice is used the radiation treatment P815 tumor cell immunity that is mixed with sudden change fusion rotein B7-IgGmut, the fusion rotein of this sudden change is not in conjunction with Fc receptor, not complement activation.The molecule of sudden change is not so good as wild type effectively (Fig. 6, table 2), the role in the Fc that has pointed out at the B7-IgG molecule combination.

Also compared with the blended radiation treatment tumor cell of B7-IgG and by the effect of the tumor cell of B7-1 or B7-2 transfection.Carry out vaccination with B7-transfection body, induce significantly lower antineoplastic immune, only protected 30% mice, and with having protected 65% (table 2) behind the radiation treatment wild type P815 cellular immunization that is mixed with B7-IgG.These find explanation, and B7-IgG may be the adjuvant that effectively generates the antitumor protective effect, can be more effective than the tumor cell of B7-transfection.

Carry out vaccination with the radiation treatment P815 tumor cell that is mixed with B7-1-IgG or B7-2-IgG, that cures mice sets up the P815 tumor

In order to test the adjuvanticity of B7-IgG in therapeutic tumor vaccine model, with P815 tumor cell i.d. injection DBA/2 mice, dosage is to generate palpable solid tumor after five to seven days.Injected 5 * 10 at the 0th day by i.d. ⁴The P815 cell is set up solid P815 tumor.After forming palpable tumor, beginning vaccination in the 7th day.Fig. 7 shows below the result of i.fp. injection mice: PBS contrasts (A, E), independent radiation treatment P815 tumor cell (B), the blended radiation treatment P815 tumor cell of nothing to do with mice IgG2a Ab (F) is with B7-1-IgG (C) or the blended radiation treatment P815 tumor cell of B7-2-IgG (G).Repeated immunity at the 7th, 14,21 day.After three days respectively with PBS, irrelevant Ab or B7-IgG rechallenge.Monitor the tumor growth in 40 days.After 25-30 days, control animals begins to die from spontaneous metastatic tumor.D and H hurdle show the time-to-live of different treatment groups.Data are representatives of four independent experiments.The kinetics of tumor growth and the survival after the vaccination are the representatives of four independent experiments as shown in Figure 7.From immunity back about is all for the first time, observes the tumor cell immune group mouse tumor growth that is mixed with B7-IgG and reduce, just tumor regression.Independent radiation treatment tumor cell or the radiation treatment tumor cell processed group tumor growth that is mixed with irrelevant mice IgG2a Ab do not reduce.Be mixed with after three cycles of radiation treatment P815 tumor cell immunity of B7-IgG, the rat primary tumor of 60-80% disappears.The long-term surviving correlate with mice of disappearing of primary tumor.The radiation treatment P815 cellular immunization that is mixed with B7-1-IgG or B7-2-IgG increase long-term surviving about 80% (Fig. 7 D, H).Most of mice of not treatment group or radiation treatment tumor vaccine matched group is generally dead between 25 and 35 days, obviously is caused by the spontaneous metastatic tumor that forms in liver, spleen and the lymph node.

In different tumor models as the B7-IgG of therapeutic tumor vaccine adjuvant

Whether consistent in order to determine B7-IgG result with P815 tumor or the strain of DBA/2 mice, utilize two kinds of different mice strains to test B7-IgG as the effect of adjuvant in three kinds of other tumor models.In all four kinds of models, all obtained similar positive findings.

To carry the Balb/c mice of setting up the MethA sarcoma on the 7th with PBS, independent radiation treatment MethA cell or be mixed with B7-1-IgG or the radiation treatment MethA cellular immunization of B7-2-IgG.In Balb/c or C57BL/6 mice, set up solid MethA or B16/F10 tumor respectively.Fig. 8 show with every group of ten mices use respectively independent radiation treatment tumor cell (B, E) or be mixed with 25 μ g (C, D) or 100 μ g (F, G) the radiation treatment tumor cell i.fp. immunity of B7-1-IgG or B7-2-IgG.Give PBS, B7-1-IgG or B7-2-IgG injection after 3-4 days again.Handle (A does not show about B16/F10) with independent PBS for one group.Monitor 35 days tumor growths.In case tumor reaches 400mm ², make MethA mice with tumor euthanasia, perhaps animal dies from spontaneous metastatic tumor.The percentage rate of surviving animals is shown in the H hurdle.Experiment repeats three times at least.With twice healing of radiation treatment MethA cellular immunization, 100% mice that is mixed with B7-IgG, and matched group 10-15% mice is spontaneously fully recovered (Fig. 8).In carrying the C57BL/6 mice of bladder cancer MB49, observe similar result.In the C57BL/6 mice, also studied replying of high transitivity and weak immunogenicity melanin tumour b16/F10.With the radiation treatment B16 tumor cell immunity that is mixed with one of two kinds of B7-IgG albumen three times, relative comparison minimizing tumor growth improves long-term surviving rate (Fig. 8).Control animals is all dead in 35-40 days, and has at least the 40-80% survival to surpass 60 days (Fig. 8) with the mice that the B7-IgG vaccine was handled.The observed result in the P815 tumor model has been supported in these discoveries, has proved the strong activity of B7-IgG as therapeutic tumor vaccine adjuvant.

The therapeutic administration inducing antitumor of independent B7-IgG is replied

As mentioned above, do not have the radiation treatment tumor cell in the presence of young Mus is carried out preventative immunity with B7-IgG, do not protect mice to avoid tumor challenge.But, in all four kinds of therapeutic tumor models, the mice of carrying tumor is handled with independent B7-1-IgG or B7-2-IgG, reduce tumor growth, increase survival rate (Fig. 9).At the 0th day with living P815 (A), MethA (B), MB49 (C) or C57BL/6 (D) tumor cell inoculation mice.Began immunity as mentioned above at 6-8 days.Mice is handled with PBS (), independent radiation treatment tumor cell (◇), the radiation treatment tumor cell that is mixed with B7-1-IgG (△) or B7-2-IgG (zero) or independent B7-1-IgG (*) or B7-2-IgG (+).The meansigma methods of every group of 7-10 mouse tumor size is drawn.In case tumor reaches 400mm ²Size makes mice euthanasia, if perhaps they are died from metastatic disease and just give this value.To some extent the test model in, with independent B7-IgG the mice of carrying tumor is carried out therapeutic treatment, delay tumor growth, induced tumor disappears, the increase survival rate.But, the B16/F10 tumor model Notes of Key Data adds the antitumor curative effect that B7-IgG carries out vaccination with the radiation treatment tumor cell and is better than independent B7-IgG, at least concerning weak immunogenic cancer so.

The result of these solubilities B7-IgG shockingly provides and utilizes the co stimulatory molecule gained result (solid phase stimulates jointly) who presents on cell surface.The tumor cell of having estimated in whole three kinds of these models with the B7-transfection carries out the therapeutic vaccine inoculation, shows tumor growth and survival rate are not acted on to having the appropriateness effect.Here utilize effect shown in the radiation treatment tumour-cell vaccine that is mixed with solubility B7-Ig shockingly much bigger by force.Mix 100 μ g B7-IgG and vaccine and can provide about 10 ¹⁶The B7 molecule, and 10 ⁷About total 10 is only arranged on the transfection tumor cell surface ¹⁰-10 ¹¹The B7 molecule.What a kind of so quantitative difference can be interpreted as and be significantly less than B7-transfection body alive with the efficient that radiation treatment B7-transfection body carries out vaccination, and wherein living cells can increase and increase available B7 molecular amounts.Other explanations may relate to the B7-IgG molecular proportion and be expressed in B7 molecule on the radiation treatment tumor cell surface and prolonged the time that exists.And shla molecule can differently distribute in vivo, arrives more suitable immunostimulation position.

The tumor recovery from illness of B7-IgG mediation is the dependent and IFN-γ independence of CD8 T cell

In order further to describe the feature that adaptive immune response participates in the immunostimulation of B7-IgG mediation, estimated the B7-IgG in the SCID mice that lacks mature T and B cell.In the SCID mice, set up solid tumor, then they are added B7-IgG with independent B7-IgG radiation treatment cell vaccine alive and handle.Do not have a kind of treatment measures that tumor growth is worked, proved the dependency (Figure 10) of the tumor response of B7-IgG mediation T or B cell.After making CD8 or cd4 t cell depletion, handle the mice of carrying tumor in addition.Begin the previous day in the B7-IgG therapy, injection of antibodies makes CD8 or CD4 T cell failure.The depleted success of facs analysis checking by PBL in the 28th day.In the mice of CD4 depletion, the B7-IgG induced tumor disappears and fully recovers, and with normal mouse as broad as long (Figure 11), and the anti-tumor activity of B7-IgG mediation has been abolished in CD8 depletion.Tumor growth is slower than the not treatment mice of CD8 depletion, does not have tumor recovery from illness (Figure 11) but observe.

Although IFN-γ monitors and antitumor is played an important role in replying at antineoplastic immune, can determine also that B7-IgG cures to have set up tumor and have nothing to do with IFN-γ.In IFN-γ inefficacy mice, set up solid tumor, handle with B7-IgG or the radiation treatment tumour-cell vaccine that is mixed with B7-IgG.Two kinds of equal induced tumors of processing disappear and recovery from illness about the 28th day, are equivalent to wild-type mice, have proved the IFN-γ independence (Figure 12) of B7-IgG tumor therapy.And, because of its IFN-γ receptor mutation tumor that is not responsiveness to IFN-γ handle with B7-IgG after still recovery from illness.

Also measured B7-IgG in antitumor therapy or stronger than blocking-up CTLA4 antibody as vaccine adjuvant.In three kinds of different tumor models, B7-IgG handles and cures tumor or be protected from tumor challenge, wherein not effect of anti-CTLA 4 antibody, although it has much higher affinity to CTLA4, forefathers have also reported its blocking-up activity.These digital proofs, the mechanism that the B7-IgG enhance immunity is replied are not limited only to and depend on the negative signal of blocking-up by the CTLA4 mediation.

Table 2: (under fire animal is tested number to provide protective immunity immunization protection percentage rate number of animals with the blended B7-IgG2a of radiation treatment tumor cell

The B7-2IgG 20% 2,/10 1 radiation treatment P815+ of B7-1IgG 28% (4) the 5/18 2 radiation treatment P815+ sudden change of (average+/-SD) protection number/sum) young mouse 8% (11) 3,/42 5 radiation treatment P815 2% (4) 1,/43 5 radiation treatment P815-B7-1 transfection body 23% (4) 3,/13 2 radiation treatment P815+B7-1IgG 65% (18) 29,/45 4 radiation treatment P815+B7-2IgG 60% (24) 23,/37 4 radiation treatment P815+ sudden change is anti--and CD28 0% 0,/10 1 radiation treatment P815+ is anti--CTLA-4 40% 4,/10 1 radiation treatment L1210-P1A 0% (0) 0,/19 2 radiation treatment L1210-P1A+ 10% (14) 2/20 2B7-1-IgGIgG

Equivalents

Those skilled in the art only need utilize conventional experimental technique will be familiar with or can determine the equivalents of the specific embodiment of a lot of inventions described herein.Such equivalent method plans to be contained by following claim.

Sequence table＜110〉(the Genetics Institute of Genetics Institute, INC.; Inc)＜120〉purposes of soluble cosmetimulatory molecules to enhance immune responses＜130〉GNN-003＜140〉09/565; 316＜141〉2000-05-05＜150〉60/132,944＜151〉1999-05-06＜160〉7＜170〉PatentIn Ver.2.0＜210〉1＜211〉1491＜212〉DNA＜213〉 ( Homo sapiens )＜220〉＜221〉CDS＜222〉 ( 318 ) .. ( 1181 )＜220〉＜221〉＜222〉 ( 420 )＜220〉＜223〉3181181bp＜220〉＜223〉14741479bp＜400〉1ccaaagaaaa agtgatttgt cattgcttta tagactgtaa gaagagaaca tctcagaagt 60ggagtcttac cctgaaatca aaggatttaa agaaaaagtg gaatttttct tcagcaagct 120gtgaaactaa atccacaacc tttggagacc caggaacacc ctccaatctc tgtgtgtttt 180gtaaacatca ctggagggtc ttctacgtga gcaattggat tgtcatcagc cctgcctgtt 240ttgcacctgg gaagtgccct ggtcttactt gggtccaaat tgttggcttt cacttttgac 300cctaagcatc tgaagcc atg ggc cac aca cgg agg cag gga aca tca cca 350

Met?Gly?His?Thr?Arg?Arg?Gln?Gly?Thr?Ser?Pro

-30 -25tcc?aag?tgt?cca?tac?ctg?aat?ttc?ttt?cag?ctc?ttg?gtg?ctg?gct?ggt 398Ser?Lys?Cys?Pro?Tyr?Leu?Asn?Phe?Phe?Gln?Leu?Leu?Val?Leu?Ala?Gly

-20 -15 -10ctt?tct?cac?ttc?tgt?tca?ggt?gtt?atc?cac?gtg?acc?aag?gaa?gtg?aaa 446Leu?Ser?His?Phe?Cys?Ser?Gly?Val?Ile?His?Val?Thr?Lys?Glu?Val?Lys

-5 -1 1 5gaa?gtg?gca?acg?ctg?tcc?tgt?ggt?cac?aat?gtt?tct?gtt?gaa?gag?ctg 494Glu?Val?Ala?Thr?Leu?Ser?Cys?Gly?His?Asn?Val?Ser?Val?Glu?Glu?Leu?10 15 20 25gca?caa?act?cgc?atc?tac?tgg?caa?aag?gag?aag?aaa?atg?gtg?ctg?act 542Ala?Gln?Thr?Arg?Ile?Tyr?Trp?Gln?Lys?Glu?Lys?Lys?Met?Val?Leu?Thr

30 35 40atg?atg?tct?ggg?gac?atg?aat?ata?tgg?ccc?gag?tac?aag?aac?cgg?acc 590Met?Met?Ser?Gly?Asp?Met?Asn?Ile?Trp?Pro?Glu?Tyr?Lys?Asn?Arg?Thr

45 50 55atc?ttt?gat?atc?act?aat?aac?ctc?tcc?att?gtg?atc?ctg?gct?ctg?cgc 638Ile?Phe?Asp?Ile?Thr?Asn?Asn?Leu?Ser?Ile?Val?Ile?Leu?Ala?Leu?Arg

60 65 70cca?tct?gac?gag?ggc?aca?tac?gag?tgt?gtt?gtt?ctg?aag?tat?gaa?aaa 686Pro?Ser?Asp?Glu?Gly?Thr?Tyr?Glu?Cys?Val?Val?Leu?Lys?Tyr?Glu?Lys

75 80 85gac?gct?ttc?aag?cgg?gaa?cac?ctg?gct?gaa?gtg?acg?tta?tca?gtc?aaa 734Asp?Ala?Phe?Lys?Arg?Glu?His?Leu?Ala?Glu?Val?Thr?Leu?Ser?Val?Lys?90 95 100 105gct?gac?ttc?cct?aca?cct?agt?ata?tct?gac?ttt?gaa?att?cca?act?tct 782Ala?Asp?Phe?Pro?Thr?Pro?Ser?Ile?Ser?Asp?Phe?Glu?Ile?Pro?Thr?Ser

110 115 120aat?att?aga?agg?ata?att?tgc?tca?acc?tct?gga?ggt?ttt?cca?gag?cct 830Asn?Ile?Arg?Arg?Ile?Ile?Cys?Ser?Thr?Ser?Gly?Gly?Phe?Pro?Glu?Pro

125 130 135cac?ctc?tcc?tgg?ttg?gaa?aat?gga?gaa?gaa?tta?aat?gcc?atc?aac?aca 878His?Leu?Ser?Trp?Leu?Glu?Asn?Gly?Glu?Glu?Leu?Asn?Ala?Ile?Asn?Thr

140 145 150aca?gtt?tcc?caa?gat?cct?gaa?act?gag?ctc?tat?gct?gtt?agc?agc?aaa 926Thr?Val?Ser?Gln?Asp?Pro?Glu?Thr?Glu?Leu?Tyr?Ala?Val?Ser?Ser?Lys

155 160 165ctg?gat?ttc?aat?atg?aca?acc?aac?cac?agc?ttc?atg?tgt?ctc?atc?aag 974Leu?Asp?Phe?Asn?Met?Thr?Thr?Asn?His?Ser?Phe?Met?Cys?Leu?Ile?Lys170 175 180 185tat?gga?cat?tta?aga?gtg?aat?cag?acc?ttc?aac?tgg?aat?aca?acc?aag 1022Tyr?Gly?His?Leu?Arg?Val?Asn?Gln?Thr?Phe?Asn?Trp?Asn?Thr?Thr?Lys

190 195 200caa?gag?cat?ttt?cct?gat?aac?ctg?ctc?cca?tcc?tgg?gcc?att?acc?tta 1070Gln?Glu?His?Phe?Pro?Asp?Asn?Leu?Leu?Pro?Ser?Trp?Ala?Ile?Thr?Leu

205 210 215atc?tca?gta?aat?gga?att?ttt?gtg?ata?tgc?tgc?ctg?acc?tac?tgc?ttt 1118Ile?Ser?Val?Asn?Gly?Ile?Phe?Val?Ile?Cys?Cys?Leu?Thr?Tyr?Cys?Phe

220 225 230gcc?cca?aga?tgc?aga?gag?aga?agg?agg?aat?gag?aga?ttg?aga?agg?gaa 1166Ala?Pro?Arg?Cys?Arg?Glu?Arg?Arg?Arg?Asn?Glu?Arg?Leu?Arg?Arg?Glu

235 240 245agt gta cgc cct gta taacagtgtc cgcagaagca aggggctgaa aagatctgaa 1221Ser Val Arg Pro Val250ggtagcctcc gtcatctctt ctgggataca tggatcgtgg ggatcatgag gcattcttcc 1281cttaacaaat ttaagctgtt ttacccacta cctcaccttc ttaaaaacct ctttcagatt 1341aagctgaaca gttacaagat ggctggcatc cctctccttt ctccccatat gcaatttgct 1401taatgtaacc tcttcttttg ccatgtttcc attctgccat cttgaattgt cttgtcagcc 1461aattcattat ctattaaaca ctaatttgag, 1491＜210〉2＜211〉288＜212〉PRT＜213〉people (Homo sapiens)＜220〉＜223 〉-34 to-12 burst: the amino terminal order-checking＜220 of soluble protein〉＜223〉1 to 208 extracellular domain;

Similarity＜220 with known array〉＜223〉209 to 235 membrane spaning domain;

Similarity＜220 with known array〉＜223〉236 to 254 cell intracellular domain;

Similarity＜220 with known array〉＜223〉19-21,55-57,64-66,152-154,173-175,177-179,192-194,

The glycosylation that the N of 198-200 position connects is with similarity＜220 of known array〉＜223〉1 to 104 Ig V-set domain;

Similarity＜220 with known array〉＜223〉105 to 202 Ig C-set domain;

Similarity＜400 with known array〉2Met Gly His Thr Arg Arg Gln Gly Thr Ser Pro Ser Lys Cys Pro Tyr

-30 -25 -20Leu?Asn?Phe?Phe?Gln?Leu?Leu?Val?Leu?Ala?Gly?Leu?Ser?His?Phe?Cys

-15 -10 -5Ser?Gly?Val?Ile?His?Val?Thr?Lys?Glu?Val?Lys?Glu?Val?Ala?Thr?Leu

-1 1 5 10Ser?Cys?Gly?His?Asn?Val?Ser?Val?Glu?Glu?Leu?Ala?Gln?Thr?Arg?Ile?15 20 25 30Tyr?Trp?Gln?Lys?Glu?Lys?Lys?Met?Val?Leu?Thr?Met?Met?Ser?Gly?Asp

35 40 45Met?Asn?Ile?Trp?Pro?Glu?Tyr?Lys?Asn?Arg?Thr?Ile?Phe?Asp?Ile?Thr

50 55 60Asn?Asn?Leu?Ser?Ile?Val?Ile?Leu?Ala?Leu?Arg?Pro?Ser?Asp?Glu?Gly

65 70 75Thr?Tyr?Glu?Cys?Val?Val?Leu?Lys?Tyr?Glu?Lys?Asp?Ala?Phe?Lys?Arg

80 85 90Glu?His?Leu?Ala?Glu?Val?Thr?Leu?Ser?Val?Lys?Ala?Asp?Phe?Pro?Thr?95 100 105 110Pro?Ser?Ile?Ser?Asp?Phe?Glu?Ile?Pro?Thr?Ser?Asn?Ile?Arg?Arg?Ile

115 120 125Ile?Cys?Ser?Thr?Ser?Gly?Gly?Phe?Pro?Glu?Pro?His?Leu?Ser?Trp?Leu

130 135 140Glu?Asn?Gly?Glu?Glu?Leu?Asn?Ala?Ile?Asn?Thr?Thr?Val?Ser?Gln?Asp

145 150 155Pro?Glu?Thr?Glu?Leu?Tyr?Ala?Val?Ser?Ser?Lys?Leu?Asp?Phe?Asn?Met

160 165 170Thr?Thr?Asn?His?Ser?Phe?Met?Cys?Leu?Ile?Lys?Tyr?Gly?His?Leu?Arg175 180 185 190Val?Asn?Gln?Thr?Phe?Asn?Trp?Asn?Thr?Thr?Lys?Gln?Glu?His?Phe?Pro

195 200 205Asp?Asn?Leu?Leu?Pro?Ser?Trp?Ala?Ile?Thr?Leu?Ile?Ser?Val?Asn?Gly

210 215 220Ile?Phe?Val?Ile?Cys?Cys?Leu?Thr?Tyr?Cys?Phe?Ala?Pro?Arg?Cys?Arg

225 230 235Glu?Arg?Arg?Arg?Asn?Glu?Arg?Leu?Arg?Arg?Glu?Ser?Val?Arg?Pro?Val

240 245 250＜210〉3＜211〉1120＜212〉DNA＜213〉people (Homo sapiens)＜220〉＜221〉CDS＜222〉(107) .. (1093)＜400〉3cacagggtga aagctttgct tctctgctgc tgtaacaggg actagcacag acacacggat 60gagtggggtc atttccagat attaggtcac agcagaagca gccaaa atg gat ccc 115

Met?Asp?Pro

1cag?tgc?act?atg?gga?ctg?agt?aac?att?ctc?ttt?gtg?atg?gcc?ttc?ctg 163Gln?Cys?Thr?Met?Gly?Leu?Ser?Asn?Ile?Leu?Phe?Val?Met?Ala?Phe?Leu

5 10 15ctc?tct?ggt?gct?gct?cct?ctg?aag?att?caa?gct?tat?ttc?aat?gag?act 211Leu?Ser?Gly?Ala?Ala?Pro?Leu?Lys?Ile?Gln?Ala?Tyr?Phe?Asn?Glu?Thr?20 25 30 35gca?gac?ctg?cca?tgc?caa?ttt?gca?aac?tct?caa?aac?caa?agc?ctg?agt 259Ala?Asp?Leu?Pro?Cys?Gln?Phe?Ala?Asn?Ser?Gln?Asn?Gln?Ser?Leu?Ser

40 45 50gag?cta?gta?gta?ttt?tgg?cag?gac?cag?gaa?aac?ttg?gtt?ctg?aat?gag 307Glu?Leu?Val?Val?Phe?Trp?Gln?Asp?Gln?Glu?Asn?Leu?Val?Leu?Asn?Glu

55 60 65gta?tac?tta?ggc?aaa?gag?aaa?ttt?gac?agt?gtt?cat?tcc?aag?tat?atg 355Val?Tyr?Leu?Gly?Lys?Glu?Lys?Phe?Asp?Ser?Val?His?Ser?Lys?Tyr?Met

70 75 80ggc?cgc?aca?agt?ttt?gat?tcg?gac?agt?tgg?acc?ctg?aga?ctt?cac?aat 403Gly?Arg?Thr?Ser?Phe?Asp?Ser?Asp?Ser?Trp?Thr?Leu?Arg?Leu?His?Asn

85 90 95ctt?cag?atc?aag?gac?aag?ggc?ttg?tat?caa?tgt?atc?atc?cat?cac?aaa 451Leu?Gln?Ile?Lys?Asp?Lys?Gly?Leu?Tyr?Gln?Cys?Ile?Ile?His?His?Lys100 105 110 115aag?ccc?aca?gga?atg?att?cgc?atc?cac?cag?atg?aat?tct?gaa?ctg?tca 499Lys?Pro?Thr?Gly?Met?Ile?Arg?Ile?His?Gln?Met?Asn?Ser?Glu?Leu?Ser

120 125 130gtg?ctt?gct?aac?ttc?agt?caa?cct?gaa?ata?gta?cca?att?tct?aat?ata 547Val?Leu?Ala?Asn?Phe?Ser?Gln?Pro?Glu?Ile?Val?Pro?Ile?Ser?Asn?Ile

135 140 145aca?gaa?aat?gtg?tac?ata?aat?ttg?acc?tgc?tca?tct?ata?cac?ggt?tac 595Thr?Glu?Asn?Val?Tyr?Ile?Asn?Leu?Thr?Cys?Ser?Ser?Ile?His?Gly?Tyr

150 155 160cca?gaa?cct?aag?aag?atg?agt?gtt?ttg?cta?aga?acc?aag?aat?tca?act 643Pro?Glu?Pro?Lys?Lys?Met?Ser?Val?Leu?Leu?Arg?Thr?Lys?Asn?Ser?Thr

165 170 175atc?gag?tat?gat?ggt?att?atg?cag?aaa?tct?caa?gat?aat?gtc?aca?gaa 691Ile?Glu?Tyr?Asp?Gly?Ile?Met?Gln?Lys?Ser?Gln?Asp?Asn?Val?Thr?Glu180 185 190 195ctg?tac?gac?gtt?tcc?atc?agc?ttg?tct?gtt?tca?ttc?cct?gat?gtt?acg 739Leu?Tyr?Asp?Val?Ser?Ile?Ser?Leu?Ser?Val?Ser?Phe?Pro?Asp?Val?Thr

200 205 210agc?aat?atg?acc?atc?ttc?tgt?att?ctg?gaa?act?gac?aag?acg?cgg?ctt 787Ser?Asn?Met?Thr?Ile?Phe?Cys?Ile?Leu?Glu?Thr?Asp?Lys?Thr?Arg?Leu

215 220 225tta?tct?tca?cct?ttc?tct?ata?gag?ctt?gag?gac?cct?cag?cct?ccc?cca 835Leu?Ser?Ser?Pro?Phe?Ser?Ile?Glu?Leu?Glu?Asp?Pro?Gln?Pro?Pro?Pro

230 235 240gac?cac?att?cct?tgg?att?aca?gct?gta?ctt?cca?aca?gtt?att?ata?tgt 883Asp?His?Ile?Pro?Trp?Ile?Thr?Ala?Val?Leu?Pro?Thr?Val?Ile?Ile?Cys

245 250 255gtg?atg?gtt?ttc?tgt?cta?att?cta?tgg?aaa?tgg?aag?aag?aag?aag?cgg 931Val?Met?Val?Phe?Cys?Leu?Ile?Leu?Trp?Lys?Trp?Lys?Lys?Lys?Lys?Arg260 265 270 275cct?cgc?aac?tct?tat?aaa?tgt?gga?acc?aac?aca?atg?gag?agg?gaa?gag 979Pro?Arg?Asn?Ser?Tyr?Lys?Cys?Gly?Thr?Asn?Thr?Met?Glu?Arg?Glu?Glu

280 285 290agt?gaa?cag?acc?aag?aaa?aga?gaa?aaa?atc?cat?ata?cct?gaa?aga?tct 1027Ser?Glu?Gln?Thr?Lys?Lys?Arg?Glu?Lys?Ile?His?Ile?Pro?Glu?Arg?Ser

295 300 305gat?gaa?gcc?cag?cgt?gtt?ttt?aaa?agt?tcg?aag?aca?tct?tca?tgc?gac 1075Asp?Glu?Ala?Gln?Arg?Val?Phe?Lys?Ser?Ser?Lys?Thr?Ser?Ser?Cys?Asp

310 315 320aaa?agt?gat?aca?tgt?ttt?taattaaaga?gtaaagccca?aaaaaaa 1120Lys?Ser?Asp?Thr?Cys?Phe

325＜210〉4＜211〉329＜212〉PRT＜213〉people (Homo sapiens)＜400〉4Met Asp Pro Gln Cys Thr Met Gly Leu Ser Asn Ile Leu Phe Val Met, 15 10 15Ala Phe Leu Leu Ser Gly Ala Ala Pro Leu Lys Ile Gln Ala Tyr Phe

20 25 30Asn?Glu?Thr?Ala?Asp?Leu?Pro?Cys?Gln?Phe?Ala?Asn?Ser?Gln?Asn?Gln

35 40 45Ser?Leu?Ser?Glu?Leu?Val?Val?Phe?Trp?Gln?Asp?Gln?Glu?Asn?Leu?Val

50 55 60Leu?Asn?Glu?Val?Tyr?Leu?Gly?Lys?Glu?Lys?Phe?Asp?Ser?Val?His?Ser?65 70 75 80Lys?Tyr?Met?Gly?Arg?Thr?Ser?Phe?Asp?Ser?Asp?Ser?Trp?Thr?Leu?Arg

85 90 95Leu?His?Asn?Leu?Gln?Ile?Lys?Asp?Lys?Gly?Leu?Tyr?Gln?Cys?Ile?Ile

100 105 110His?His?Lys?Lys?Pro?Thr?Gly?Met?Ile?Arg?Ile?His?Gln?Met?Asn?Ser

115 120 125Glu?Leu?Ser?Val?Leu?Ala?Asn?Phe?Ser?Gln?Pro?Glu?Ile?Val?Pro?Ile

130 135 140Ser?Asn?Ile?Thr?Glu?Asn?Val?Tyr?Ile?Asn?Leu?Thr?Cys?Ser?Ser?Ile145 150 155 160His?Gly?Tyr?Pro?Glu?Pro?Lys?Lys?Met?Ser?Val?Leu?Leu?Arg?Thr?Lys

165 170 175Asn?Ser?Thr?Ile?Glu?Tyr?Asp?Gly?Ile?Met?Gln?Lys?Ser?Gln?Asp?Asn

180 185 190Val?Thr?Glu?Leu?Tyr?Asp?Val?Ser?Ile?Ser?Leu?Ser?Val?Ser?Phe?Pro

195 200 205Asp?Val?Thr?Ser?Asn?Met?Thr?Ile?Phe?Cys?Ile?Leu?Glu?Thr?Asp?Lys

210 215 220Thr?Arg?Leu?Leu?Ser?Ser?Pro?Phe?Ser?Ile?Glu?Leu?Glu?Asp?Pro?Gln225 230 235 240Pro?Pro?Pro?Asp?His?Ile?Pro?Trp?Ile?Thr?Ala?Val?Leu?Pro?Thr?Val

245 250 255Ile?Ile?Cys?Val?Met?Val?Phe?Cys?Leu?Ile?Leu?Trp?Lys?Trp?Lys?Lys

260 265 270Lys?Lys?Arg?Pro?Arg?Asn?Ser?Tyr?Lys?Cys?Gly?Thr?Asn?Thr?Met?Glu

275 280 285Arg?Glu?Glu?Ser?Glu?Gln?Thr?Lys?Lys?Arg?Glu?Lys?Ile?His?Ile?Pro

290 295 300Glu?Arg?Ser?Asp?Glu?Ala?Gln?Arg?Val?Phe?Lys?Ser?Ser?Lys?Thr?Ser305 310 315 320Ser?Cys?Asp?Lys?Ser?Asp?Thr?Cys?Phe

325＜210〉5＜211〉9＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence description: peptide＜400〉5Thr Tyr Gln Arg Thr Arg Ala Leu Val 15＜210〉6＜211〉23＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence description: peptide＜400〉6Arg Leu Ile Gln Asn Ser Leu Thr Ile Glu Arg Met Val Leu Ser Ala 15 10 15Phe Asp Glu Arg Arg Asn Lys

20＜210〉7＜211〉24＜212〉PRT＜213〉artificial sequence＜220〉＜223〉artificial sequence description: peptide＜400〉7Phe Trp Arg Gly Glu Asn Gly Arg Lys Thr Arg Ile Ala Tyr Glu Arg 15 10 15Met Cys Asn Ile Leu Lys Gly Lys

20

Claims (21)

1, preventative enhancing curee comprises the method for antigenic immunne response: will comprise the soluble composition administration of co stimulatory molecule extracellular domain, so that strengthen the curee to antigenic immunne response.

2, therapeutic strengthens the method for curee to antigenic immunne response, comprising: will comprise the soluble composition administration of co stimulatory molecule extracellular domain, so that strengthen the curee to antigenic immunne response.

3, claim 1 or 2 method, wherein this co stimulatory molecule is selected from the group of being made up of B7-1 and B7-2.

4, strengthen the method for curee to the CD8+T cell response of I class limited antigen, comprise: will comprise I class limited antigen or its segmental first kind of medicine and the soluble composition administration that comprises B7 molecular cell external structure territory, so that in case, promptly strengthen CD8+T cell response to I class limited antigen to curee's administration.

5, the method for claim 4 further comprises II class limited antigen curee's administration.

6, claim 1,2 or 4 each methods further comprise adjuvant curee's administration.

7, claim 1,2 or 4 each methods, wherein this B7 molecule is the B7-1 molecule.

8, claim 1,2 or 4 each methods, wherein this B7 molecule is the B7-2 molecule.

9, claim 1,2 or 4 each methods, wherein this co stimulatory molecule is a monospecific.

10, claim 1,2 or 4 each methods, wherein this co stimulatory molecule is dimer and bivalence.

11, claim 1,2 or 4 each methods, wherein this solubility co stimulatory molecule be monospecific with dimer and bivalence.

12, the method for claim 11, wherein the outer part of B7 molecular cell is to merge with second kind of protein that comprises an immunoglobulin molecules part or polypeptide.

13, the method for claim 12, wherein this part of this immunoglobulin molecules comprises cysteine residues.

14, the method for claim 12, wherein this part of this immunoglobulin molecules comprises hinge region, CH2 and the CH3 district of human normal immunoglobulin's molecule.

15, the method for claim 12, wherein this part of this immunoglobulin molecules comprises hinge region, CH1, CH2 and the CH3 district of human normal immunoglobulin's molecule.

16, the method for claim 12, wherein this immunoglobulin molecules through modifying, has reduced complement fixation and/or Fc receptors bind.

17, claim 1,2 or 4 each methods, wherein this antigen is tumor-cell antigen.

18, the method for claim 2, wherein this curee suffers from the cancer of the set type of being selected from down: colon cancer, breast carcinoma, carcinoma of prostate, renal cell carcinoma, leukemia, lymphoma, melanoma, mastocytoma, sarcoma and bladder cancer.

19, claim 1,2 or 4 each methods, wherein this antigen is selected from the group of being made up of bacterial antigens, virus antigen and parasite antigen.

20, claim 1,2 or 4 each methods, wherein this immunne response is a cellullar immunologic response.

21, claim 1,2 or 4 each methods, wherein this immunne response is a humoral immunoresponse(HI).

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