CN101378783A - Methods and compositions for expanding T regulatory cells - Google Patents

Abstract

The present invention provides methods and compositions for expanding Treg cells ex vivo or in vivo using one or more conjugates comprising a costimulatory moiety that stimulates at least one of three signals involved in Treg cell development and/or using dendritic cells pulsed with antigens and modified to display TGF-beta, or hematopoetic stem cells or bone marrow cells modified to display TGF-beta. The methods and compositions are useful, for example, in the treatment and prevention of autoimmune disease, including Type 1 diabetes and in preventing foreign graft rejection, as well as to establish mixed chimerism, induce tolerance to autoantigens, alloantigens or xenoantigens, beta cell regeneration, prevention of foreign graft rejection, and treatment of a genetically inherited hematopoietic disorder.

Description

The method and composition that is used for expanding T regulatory cells

NIH authorizes fund

This working portion obtains NIH (R21 DK61333, R01 AI47864, R21 AI057903, R21 HL080108), juvenile-onset diabetes WARF (1-2001-328), what ADA (1-05-JF-56) and the Commonwealth of Kentucky Research ChallengeTrust Fund provided supports.

The cross reference of related application

The application according to 35U.S.C. § 199 (e) require following U.S. provisional application in please day rights and interests: 60/748,177 (December was applied on the 8th in 2005); 60/758,391 (application on January 12nd, 2006); 60/799,642 (December in 2006 application on the 12nd); With 60/799,643 (application on May 12nd, 2006).At this aforesaid each piece application is incorporated herein by reference with its integral body.

Invention field

The present invention generally relates to the field of immunization therapy.Especially, the invention provides the method and composition that is used for expanding T regulatory cells.This method and composition can be used for for example preventing and treats disease based on immunity, comprises diabetes and prevention external source transplant rejection.

Background of invention

T regulates the CD4 of 5-10% in (Treg) cellularity people and the rodent ⁺T cell, and the expression of composing type ground CD25, CD28, CTLA-4, GITR, CD62L and 4-1BB, and transcription factor FoxP3, it relates to their generation and function.As if IL-2 play an important role in generation of Treg cell and homoiostasis, produce T cell hyperproliferation and autoimmune disease in default of the animal of IL-2 or its receptor component, these diseases can obtain medical treatment by the acquired transfer from the Treg cell of natural animal.Similarly, lack quantity by the interactional signal of CD28/CD80 and the Treg cell of reduction and functional relevant, show that this receptor/Fas lignand system plays an important role in the generation of Treg cell and function.

The CD4 of natural appearance ⁺CD25 ⁺FoxP3 ⁺The Treg cell is the dendritic cell population of cell, and these cells are positive that select and demonstrated in the foundation of the antigenic immunologic tolerance of oneself with in keeping and play an important role on the high-affinity part in thymus.Violent autoimmune in the animal model of the generation of these cells and/or the shortage of function and people and various congenital or inductive autoimmune is relevant.

The Treg cell is by cell and the intercellular toleration effect that contacts directly or present them by soluble factor indirectly.Although these cell inhibiting mechanism are illustrated as yet fully, the blocking-up that IL-2 in the effector T cell (Teff) expresses, the physics of Teff cell eliminates, the toleration dendritic cell (DC) by the CTLA-4/B7 axle induce and the Teff cell inhibiting by TGF-β and IL-10 is some mechanism that related to up to now.Also shown by CTLA-4/CD80 the reverse Teff of the being passed to cell of signal has been played an important role in by the Treg cell inhibiting.Similarly, CTLA-4 on the Treg cell and the interaction between the CD80 on the DC can cause reverse signal and raise indole amine dioxygenase, and this enzyme relates to by regulating the toleration of tryptophan mechanism.

Except in the natural action of setting up and keep self-antigen tolerance, also demonstrated the Treg cell and in peripheral tolerance, played effect by the inductive exogenous antigen of various immunomodulating methods.For example, demonstrate the common ground that the Treg cell relates to the mechanism of transplantation antigen peripheral tolerance, the immunomodulating method of acquired tolerance is irrelevant with being used for.The Treg cell also relates to the immune evasion mechanism of tumor and various pathogen.

The importance of Treg cell in the toleration of setting up and keep self-antigenic toleration and inductive exogenous antigen produced keen interest to the method for (ex vivo) amplification Treg cell that exsomatizes for therapeutic purposes.Referring to, for example, Tang etc., 2004, J.Exp.Med.199:1455-65; Battaglia etc., 2005, Blood 105:4743-48; Earle etc., 2005, Clin.Immunol.115:3-9; Godgrey etc., 2004, Blood 104:453-61; Hoffmann etc., 2004, Blood 104:895-903.Take place by the signal by TXi Baoshouti (TCR), CD28 and IL-2 because the Treg cell produces, the method for the Treg cell that therefore increases focuses on this three kinds of signals is provided.Referring to, for example, Tang etc., above; Godfrey etc., above; Hoffimann etc., above.For example, Tang etc., above, having reported can be from nonobese diabetes (NOD) animal amplification Treg cell, by stimulating with the pearl of puting together anti-CD3 and CD28 antibody in the presence of high dose IL-2 (2000IU/ml).The acquired transfer of the specific TCR transgenic of self antigen Treg cell of amplification has prevented the diabetes in the acquired metastasis model and has reversed diabetes in the new diabetes NOD mice.Produced other amplification experimental programs recently, in amplification rodent and people's Treg cell, obtained some successes based on the limited quantity of this experimental program.Referring to, for example, Earle etc., above; Godfrey etc., above.For example, Godfrey etc. have reported that using Fc γ RII (CD32) express cell as the pearl alternative is the people Treg cell that increases, and this cell line is fixed on the cell surface by the antibody that the Fc receptor will resist CD3 and CD28.The stripped amplification experimental program of nearly all report is based on similar scheme, and needs to use the IL-2 of high dose, makes it effective.

Although still there is the method and composition that need be used for stripped amplification Treg cell in these progress.Use the method for solid support to exist special needs to not needing.To need not existing special needs for the method for the IL-2 of the high dose of effect yet.The method and composition that is used for amplification Treg cell in the body is also existed special needs.

Type i diabetes remains the main cause that the whole world surpasses the medium-term and long-term M ﹠ M of one of percentage crowd.Although insulinize and islet transplantation are the most effective therapeutic schemes at present, these two kinds of methods all exist serious defective.Therefore, still exist and to induce the specific method of islets of langerhans, be used for effectively and lastingly treating of type i diabetes from body, allos and external source toleration.

As mentioned above, the Treg cell plays an important role in the foundation of control that self-reactivity is replied and exogenous antigen toleration with in keeping.Therefore, the Treg cell has presented important therapeutic goal, is used for prevention and treats various autoimmune diseases, comprises type 1 diabetes, and the repulsion of solid organ, tissue, stem cell, medullary cell, hematopoietic stem cell and transplanting are to host disease (GVHD).

Still there is amplification method in the body that needs the controlled of Treg cell and have a mind to, is used for the treatment of these diseases.

Summary of the invention

The present invention always provides the method and composition that is used for expanding T regulatory cells.

According to an aspect, the invention provides a kind of combination, it comprises that (A) is one or more and is selected from following conjugate: (a) first conjugate, and it comprises that (i) comprises the first conjugate member of 4-1BBL polypeptide and (ii) comprise the second conjugate member in conjunction with first right member; (b) second conjugate, it comprises that (i) comprises the first conjugate member of CD80 polypeptide and (ii) comprise the second conjugate member in conjunction with first right member; (c) the 3rd conjugate, it comprises that (i) comprises the first conjugate member of TGF-beta polypeptides and (ii) comprise the described second conjugate member in conjunction with first right member; (B) one or morely be selected from following conjugate: (a ') the 4th conjugate, it comprises that (i) comprises the first conjugate member of anti-CD 3 antibodies and (ii) comprise the second conjugate member of second combination to the member; (b ') the 5th conjugate, it comprises that (i) comprises the first conjugate member of cytokine and (ii) comprise the second conjugate member of second combination to the member; (c ') the 6th conjugate, it comprises that (i) comprises the antigenic first conjugate member and (ii) comprise the second conjugate member of second combination to the member; (d ') the 7th conjugate, the first conjugate member of it comprises that (i) comprises anti--CD28 antibody and (ii) comprise the second conjugate member of second combination to the member.

In a specific embodiment, comprise avidin or Succ-PEG-DSPE (as the core Succ-PEG-DSPE) in conjunction with first right member, and comprise biotin in conjunction with second right member.In another specific embodiment, at least one in first, second or the 3rd conjugate comprises fused polypeptide, and this fused polypeptide comprises first and the second conjugate member.In further embodiment, at least one in first, second or the 3rd conjugate combines in the combination between the member and the 4th, the 5th, the 6th or the 7th conjugate at least one by first and second combination.

In a specific embodiment, cytokine is selected from IL-2 and IL-4.In another specific embodiment, antigen is self antigen.In another specific embodiment, antigen is selected from insulin, collagen, myelin basic protein and MHC/ antigenic compound.In further specific embodiment, antigen is selected from glutamate decarboxylase (GAD), islet cells self antigen (ICA) and self antigen NRP-A7.

According to another aspect, the invention provides the method for amplification Treg cell, comprise and be selected from following conjugate with Treg cell colony contact (A) is one or more: (a) first conjugate, it comprises that (i) comprises the first conjugate member of 4-1BBL polypeptide and (ii) comprise the second conjugate member in conjunction with first right member; (b) second conjugate, it comprises that (i) comprises the first conjugate member of CD80 polypeptide and (ii) comprise the second conjugate member in conjunction with first right member; (c) the 3rd conjugate, it comprises that (i) comprises the first conjugate member of TGF-beta polypeptides and (ii) comprise the described second conjugate member in conjunction with first right member; (B) one or morely be selected from following conjugate: (a ') the 4th conjugate, it comprises that (i) comprises the first conjugate member of anti-CD 3 antibodies and (ii) comprise the second conjugate member of second combination to the member; (b ') the 5th conjugate, it comprises that (i) comprises the first conjugate member of cytokine and (ii) comprise the second conjugate member of second combination to the member; (c ') the 6th conjugate, it comprises that (i) comprises the antigenic first conjugate member and (ii) comprise the second conjugate member of second combination to the member; (d ') the 7th conjugate, the first conjugate member of it comprises that (i) comprises anti--CD28 antibody and (ii) comprise the second conjugate member of second combination to the member.

In a specific embodiment, the Treg cell comprises in first, second or the 3rd conjugate receptor of at least one, and at least one in first, second or the 3rd conjugate puted together by combination between the first conjugate member and the receptor and Treg cell, and in the 4th, the 5th, the 6th and the 7th conjugate at least one puted together combination between the member and Treg cell by first and second combination.In further embodiment, the Treg cell colony comprises the Treg cell that is selected from CD4+ cell, CD25+ cell and FoxP3+ cell.In further embodiment, the Treg cell colony comprises the CD4+CD25+FoxP3+ cell.

In one embodiment, this method further comprises the free IL-2 of Treg cells contacting.In another embodiment, this method further comprises the free anti-CD antibody of Treg cells contacting.

In one embodiment, this contact is exsomatized and is realized.In further embodiment, this method comprises that further the Treg cell that will increase gives the patient.

In another embodiment, realize contact in vivo by conjugate being given the patient.In further embodiment, the patient suffers from autoimmune disease or is in the risk of autoimmune disease autoimmune disease such as type i diabetes.In another embodiment, the patient is the external source transplant patient.

In one embodiment, this method further comprises and gives the patient with rapamycin.In another embodiment, this method comprises that the compositions that will comprise the foreign cell of showing TGF-β gives the patient.In a specific embodiment, foreign cell is selected from splenocyte, islet tissue and medullary cell.In another specific embodiment, obtain foreign cell by the following method, this method comprises that (a) comprises that with foreign cell contact forming the foreign cell of modification and (b) in conjunction with first right member and bifunctional molecule in conjunction with the molecule of described cell surface contacts the foreign cell of modifying and comprise that TGF-β and second combination form the foreign cell of displaying TGF-β to member's conjugate.

According to another aspect, the invention provides the method for the dendritic cell of the displaying TGF-β that obtains pulse, this method comprises that (a) obtains the dendritic cell of pulse with the immature dendritic cell of antigen pulse; (b) contact of the dendritic cell of pulse is comprised in conjunction with first right member and form the pulse dendritic cell of modification in conjunction with the bifunctional molecule of the molecule of cell surface; (c) the pulse dendritic cell contact of modifying is comprised TGF-β and second combination form the dendritic cell of the displaying TGF-β of pulse to member's conjugate.In one embodiment, this method further comprises the dendritic cell maturation that orders about pulse.

In one embodiment, antigen is diabetogenic self antigen, as glutamate decarboxylase (GAD), islet cells self antigen (ICA) or self antigen NRP-A7.In another embodiment, antigen is collagen.In another embodiment, antigen is myelin basic protein.

According to another aspect, the invention provides the dendritic cell population of the displaying TGF-β of antigen pulse, those as making by said method.

According to another aspect, the invention provides in the patient method of amplification Treg cell, comprise the compositions of dendritic cell population of the displaying TGF-β of antigen pulse, as those cell colonys that make by said method.In one embodiment, this method further comprises and gives the patient with rapamycin.

According to another aspect, the invention provides the hematopoietic stem cell that to show TGF-β or the method for medullary cell, comprise hematopoietic stem cell or medullary cell contact comprised in conjunction with first right member with in conjunction with the bifunctional molecule of the molecule of cell surface forming the cell of modification and (b) cells contacting of modifying being comprised TGF-β and second combination form the cell of showing TGF-β to member's conjugate.

According to another aspect, the invention provides in the patient method of amplification Treg cell, comprise the compositions of the medullary cell that contains the hematopoietic stem cell of showing TGF-β or TGF-β, as those cells that make by said method.In one embodiment, this method further comprises and gives the patient with rapamycin.In specific embodiment, needs of patients is to the induction of tolerance of self antigen, heterologous antigen or exogenous antigen; β cell regeneration; Prevent the external source transplant rejection; Or the treatment of heritability hematopoietic disease.

According to another aspect, the invention provides the hematopoietic stem cell populations or the medullary cell colony that show TGF-β, those as making by said method.

The accompanying drawing summary

Figure 1A and 1B have listed the nucleotide sequence (SEQ ID NO:1) and the aminoacid sequence (SEQID NO:2) of the fusion rotein that comprises core Succ-PEG-DSPE and Mus LIGHT albumen ectodomain.Core Succ-PEG-DSPE sequence is a underscore.

Fig. 2 A and 2B have listed the nucleotide sequence (SEQ ID NO:3) and the amino acid sequence coded (SEQID NO:4) of the fusion rotein that comprises people CD80 ectodomain and core Succ-PEG-DSPE.Core Succ-PEG-DSPE sequence is a underscore.

Fig. 3 A and 3B have listed the nucleotide sequence (SEQ ID NO:5) and the amino acid sequence coded (SEQ ID NO:6) of the fusion rotein that comprises Mus 4-1BBL ectodomain and core Succ-PEG-DSPE.Core Succ-PEG-DSPE sequence is a underscore.

Fig. 4 has listed the aminoacid sequence (SEQ ID NO:7) of the fusion rotein that comprises core Succ-PEG-DSPE and people 4-1BBL ectodomain.Core Succ-PEG-DSPE sequence is a underscore.

Fig. 5 A and 5B have listed the nucleotide sequence (SEQ ID NO:8) and the amino acid sequence coded (SEQID NO:9) of the fusion rotein that comprises core Succ-PEG-DSPE and people B7.2 ectodomain.

Fig. 6 A and 6B have listed the nucleotide sequence (SEQ ID NO:10) and the amino acid sequence coded (SEQID NO:11) of the fusion rotein of the active fragment that comprises IL-2 and core Succ-PEG-DSPE.In Fig. 6 B, the sequence of IL-2 is an italic, and core Succ-PEG-DSPE sequence is a underscore.

Fig. 7 A and Fig. 7 B have listed the nucleotide sequence (SEQ ID NO:12) and the amino acid sequence coded (SEQ IDNO:13) of the fusion rotein that comprises core Succ-PEG-DSPE and mature T GF-β.In Fig. 7 B, TGF-β sequence is an italic, and core Succ-PEG-DSPE sequence is a underscore.

Fig. 8 A-D has illustrated the structure and the sign of chimeric CSA-4-1BBL fusion rotein.

(A) ectodomain of mice 4-1BBL is held the core Succ-PEG-DSPE (SA) that is cloned in the PMT/BiP/V5-HisA carrier at C.

(B) western blot analysis of the chimeric 4-1BBL albumen (CSA-4-1BBL) of purification under degeneration (swimming lane 2) and natural (swimming lane 3) condition.4-1BBL is shown as the monomer of 37kDa under the degeneration condition, under natural endowment, be shown as tetramer and the high stage structure of 150kDa.

(C) chimeric 4-1BBL (CSA-4-1BBL) and 4-1BB receptor combines.With CSA-4-1BBL (200ng/l * 10 ⁶Cell) or the contrast CSA albumen of equimolar amounts (Lycoperdon polymorphum Vitt is filled) hatch BALB/c tranquillization or the activated splenocyte of ConA, and use anti-4-1BBL Ab to detect CD4 by flow cytometry ⁺And CD8 ⁺4-1BBL on the T cell is in conjunction with (black line).Hatch some activated cells with anti-CD137 and block receptor.

(D) stimulate the T cell with CSA-4-1BBL.Shown in the presence of the CSA of the solubility CSA-4-1BBL of concentration (ng/ml) or equimolar amounts, use anti-CD3Ab (0.5 μ g/ml) and the splenocyte that shone stimulates the CD4 of sorting ⁺The T cell.The anti-CD3 Ab of 5 μ g/ml is used as positive control.Relatively and with contrast CSA albumen compare mutually, ^*P＜0.05.The data of C and D are (average ± SD) 3 experiments that independently have analog result of expression.

Fig. 9. use the stripped amplification of the Treg cell of 4-1BBL.Fig. 9 has illustrated the long-term amplification in vitro according to Treg cell of the present invention, uses CSA-4-BBL in the presence of APC, anti-CD 3 antibodies and IL-2.Spleen and lymph node sorting CD4 from natural B ALB/c mice ⁺CD25 ⁺The Treg cell, and in the flat board of 6-hole at the anti-CD3 Ab of 0.5 μ g/ml solubility, 1 * 10 ⁶The homology splenocyte of individual irradiation and the existence of 25U/mlIL-2 are cultivated down, use or do not use 1 μ g/ml solubility 4-1BBL.Every 3-4 days, with the fresh culture that replenishes IL-2 and with 1 * 10 ⁶The concentration of cell/ml is coated with somatoblast.(A) before the sorting and with or after 4-1BBL useless increases, CD4+CD25+ group's flow cytometry.(B) for using () or (■) useless 4-1BBL cultured cells, the amplification at double that natural Treg cell exsomatizes.Described result from last 3 independent amplifications.Arrow represents to have with the conduct of anti-CD3, IL-2,4-1BBL and APC (the second and the 3rd activator) cell-stimulating CSA-4-1BBL useless the contrast of minimum amplification.

Figure 10 has illustrated according to the present invention 4-1BB receptor expression on the stripped Treg cell that increases.To be maintained in the culture 18-24 days Teff and Treg cell with the antibody staining of 4-1BB and pass through flow cytometry.The group that black is filled is the homotype contrast.

Figure 11 has proved and has prevented the heteroplastic transplantation repulsion according to the present invention by the Treg cell of ev vivo amplification.Implanting allos C57BL/6 islets of langerhans the previous day, give the natural B ALB/c mice acquired transferase 45-8 * 10 that causes diabetes by the single injection streptozotocin ⁶The Treg cell (O) of individual amplification.Control animal is not accepted the Treg cell, but has implanted allos islets of langerhans (●).Confirmed repulsion by the twice continuous blood sugar reading that surpasses 300mg/dL.Use Kaplan-Meier logarithm level test (p＜0.05) relatively to survive.

Figure 12 A-B.The Treg cell of amplification suppresses the polyclone or the antigenic specificity propagation of stripped Teff cell.(A) in the existence of 1 μ g/ml4-1BBL or not, use the Treg cell (Exp-DP) of amplification, suppress test according to the polyclone (anti-CD3 Ab) that carries out described in Fig. 9.(B) isoantigen suppresses test.Will from the spleen of natural B ALB/c mice and periphery lymph-node cell (replying agent) with from the splenocyte (stimulant) of the irradiation of natural C57BL/6 mice and the Treg cell (Exp-DP) that increases with shown in ratio cultivated altogether 5 days.Mutually relatively also compared with the control, ^*P＜0.05.Data (average ± SD) for A, be the expression of 4 independent experiments, for B, be the expression of 2 independent experiments, have similar result.

Figure 13 A-C.TCR, 4-1BB and IL-2R signal are to the synergism of Treg cell amplification.Figure 13 A ﹠amp; 13B has illustrated that according to the present invention stimulus signal 1,2 and 3 is by TCR, 4-1BB and the IL-2R synergism to the Treg cell amplification.

(A) in the presence of 0.5 μ g/ml CD3 Ab, with the natural DP Treg cell of sorting and the homology splenocyte co-cultivation of shining 3 days.As directed, replenish 25U/mlIL-2 and/or 1 μ g/ml 4-1BBL to culture.(B) cultivate the natural DP Treg cell of sorting, use or do not use the splenocyte that shine, use or do not use solubility CD3 Ab, 4-1BBL and IL-2, as directed.Mutually relatively also compared with the control, ^*P＜0.05.Data (average ± SD) 2 independently experiments of expression, have similar result.(C) be untreated or in the presence of IL-2 and/or APC, the DP of sorting and SP cell culture 2 days.Some cells and APC and IL-2 were cultivated 2 days, and IL-2 is removed in washing, and cultivates 2 days again, some cells is untreated cultivated 2 days, and add IL-2, and cultivated 2 days again.With homotype contrast (Lycoperdon polymorphum Vitt is filled), use the expression (black line) of flow cytometry 4-1BB.

Figure 14 shown TGF-β-CSA fusion rotein in vitro inhibition of the same race replying.With CFSE labelling ACI splenocyte and with the WF cell culture of the irradiation of equivalent, use (B) or do not use (A) TGF-β.At the 5th day, collecting cell also passed through flow cytometry, was used for CFSE dilution test.

Figure 15 A-B.Signal suppressing by the 4-1BB receptor Treg cell inhibiting function and drive the propagation of two kinds of cell colonys.(A) from spleen and the periphery lymph node sorting CD4 of natural B ALB/c mice ⁺CD25 ^-(SP) Teff cell and CD4 ⁺CD25 ⁺(DP) Treg cell was cultivated 3 days separately or with the 1:1 ratio.To culture replenish splenocyte shine, anti-CD3Ab (0.5 μ g/ml) and shown in concentration (μ g/ml) 4-1BBL or etc. mole contrast SA albumen.(B) CFSE that measures SP and DP cell proliferation tests.With CFSE labelling SP or DP cell, and be used for aforesaid inhibition test.Each rectangular histogram has shown the percentage ratio of somatoblast.Mutually relatively, ^*P＜0.05.Data (average ± SD) 3 independently experiments of expression, have similar result.

Figure 16 A-C.The phenotype of the Treg cell of amplification characterizes.(A) with or 4-1BBL expanded cells useless on analyze expression for the important cell surface marker of Treg cell function.Insert vertical line arbitrarily as use or 4-1BBL sample useless between reference relatively.(B) RT-PCR has shown labelling (M, the labelling of the FoxP3 of the Treg cell by amplification; H=HPRT, F=FoxP3; SP=CD4 ⁺CD25 ^-DP=CD4 ⁺CD25 ⁺The Treg cell of Exp-DP=amplification).(C) dyeing in the born of the same parents, demonstration are passed through with FoxP3 expression in the born of the same parents of the Treg cell of (dotted line) or (solid line) useless 4-1BBL amplification.The homotype contrast that is used for FoxP3 is with comparing (interstitial wire).3 independently experiments of data representation have similar result.

Detailed Description Of The Invention

The invention provides the method and composition by stimulating three kinds to relate at least a amplification Treg cell in the signal that the Treg cell produces. Signal 1 relates to TCR, and can stimulate with antibody such as anti-CD 3 antibodies, or uses the antigen that sends signal by TCR to stimulate. Can mediate signal 2 by several different molecules, comprise co-stimulators, such as CD80 and 4-1BBL. Signal 3 is transduceed by cell factor, such as IL-2 or TGF-β. The invention provides the method that can exsomatize or realize in vivo amplification Treg cell, the composition that is used for carrying out the method also is provided. In one embodiment, the method and composition stimulate in these signals two kinds. In another embodiment, the method and composition stimulate in these signals three kinds.

In one aspect of the method, the invention provides the method and composition of amplification Treg cell, with the DC that shows TGF-β with antigen pulse and modification, and with modifying candidate stem cell or the bone marrow cell of showing TGF-β.

Many autoimmune diseases in people or other animals are relevant with a small amount of Treg cell and/or shortage regulatory function. Therefore, suffer from the preferentially amplification that Treg cell among autoimmune disease (such as the type 1 diabetes) patient surpasses the Teff cell and guaranteed basic treatment benefit. Therefore, method and composition of the present invention can be used for for example preventing and treats disease based on immunity, comprises type 1 diabetes, and can be used for preventing heteroplastic transplantation to repel.

For purposes of this application, following term has these definition:

As used in this, " one " or " a kind of " meaning is one or more, unless specially point out only to represent one.

" give " as used in this suitable method that comprises that all provide material to the patient. That approach commonly used comprises is oral, the hypogloeeis, through mucous membrane, in skin, rectum, vagina, subcutaneous, intramuscular, intravenous, artery, in the sheath, by conduit, by implanting etc.

" in conjunction with to " refers to by interactional two molecules of any molecular force, and molecular force comprises, for example, ion, covalency, hydrophobic, Van der Waals and hydrogen bond are so that this combination is to having the characteristic of mutual specific binding. The meaning of specific binding is less than under the condition in conjunction with another molecule, mutually combines in conjunction with the member is presented. Biotin-avidin, hormone-acceptor, receptor-ligand, enzyme-substrate, IgG-albumin A, antigen-antibody etc. in conjunction with right example.

" patient " comprises any vertebrate as used in this, comprises horse, sheep, he-goat, ox, pig, birds, dog, cat and primate species. In one embodiment, the patient is the people. Those skilled in the art will recognize that specific co-stimulators, signaling molecule, cell marking, cell type, infectious agent etc., when discussing about species, in different species, have corresponding analog, and the present invention includes such analog and the purposes in corresponding and relative species thereof.

According to an aspect, the invention provides the conjugate that comprises that at least one stimulates part altogether, this common stimulation unit is divided at least one in stimulus signal 1, signal 2 or the signal 3. In a specific embodiment, conjugate comprises that common stimulation part and combination are to the member. The part of one of any stimulus signal can be used according to the present invention, and any combination is like this equally to the member. Below will discuss example in more detail stimulates part and combination to the member altogether.

Unless be appointed as " total length " at this, divide segment or the part that comprises total length part (for example, full-length polypeptide) and present common stimulatory function at this about common stimulation unit, include but not limited to, below those segments and the part specially pointed out. Therefore, for example, about 4-1BBL mean comprise total length 4-1BBL present common stimulatory function segment or the part polypeptide, such as ectodomain or the total length 4-1BBL albumen of 4-1BBL.

Signal 1

The example that is used for stimulus signal 1 altogether stimulation unit is divided and is comprised the antibody that resists CD3 or CD3 and TCR compound, antigen/MHC compound and send the medicament of signal by TCR, such as the antibody of any composition of ionomycin and phorbol myristate acetate (PMA).

The anti-CD 3 antibodies that is used for immunotherapy method is known in the art. Referring to, for example, Eeale etc., 2005, Clin.Immunol.115:3-9. The suitable anti-CD 3 antibodies of example comprises antibody, single-chain antibody and the CD3-binding antibody fragment that people or mouse-anti body, humanized antibody, restructuring produce. Can obtain such anti-CD 3 antibodies by methods known in the art.

As mentioned above, can also use the antibody of any composition of antagonism TCR compound, such as the antibody of antagonism TCR α or TCR β chain, and the antibody of antagonism CD3 composition. Referring to, for example, Niederberger etc., 2005, J.Leukoc.Biol.77:830-41; Hamano etc., 2000, J.Immunol.164:6113-19. Moreover, can use the antibody (people, mouse, restructuring, strand etc.) of any type.

The common antigen of part that stimulates as stimulus signal 1 comprises the antigen relevant with target disease or illness. For example, self-antigen and insulin (particularly being suitable for treating type 1 diabetes), collagen (being particularly suitable for treating rheumatoid arthritis), MBP (being particularly suitable for treating multiple sclerosis) and MHC (be used for the treatment of and prevent external source graft rejection). Can be used as and comprise that combination comes administration antigen to the part of member's conjugate. Optional, provide antigen as the part of MHC/ antigenic compound. In this embodiment, MHC and antigen can be external source or homology individually. For example, can use donor MHC and allos or isogeneic.

According to an aspect of the present invention, antigen is self-antigen. For example, antigen is glutamate decarboxylase (GAD), islet cells self-antigen (ICA) or self-antigen NRP-A7 (be derived from the relevant self-antigen of pancreas islet specificity G-6-Pase catalytic subunit, and show recently in diabetes it is important). These antigens have represented quite a few among the whole members of pancreas islet specificity self-antigen, therefore, may be effectively in the tolerance that other potential self-antigen are provided, and distribute or the dominant immune regulation mechanism of Treg by epitope. In a specific embodiment, self-antigen is GAD65, ICA512 or NRP-A7.

According to an embodiment, the invention provides and comprise as mentioned above as the anti-CD 3 antibodies that stimulates altogether part or antigen/MHC compound with as in conjunction with the conjugate to member's biotin. Can make such conjugate by biotinylation anti-CD 3 antibodies or antigen/MHC compound by methods known in the art, and in following embodiment illustrated. Perhaps, can be with the link of antibody or antigen in conjunction with being the fusion that contains in conjunction with to member such as core streptavidin to member such as core streptavidin or with antibody or antigen presentation, with form can be used according to the invention other conjugate.

Signal 2

The example that is used for stimulus signal 2 stimulates part to comprise the member of B7 and TNF family altogether, includes but not limited to following those that list.

B7 and CD28 family member

Part	Receptor
		B7.1(CD80)	CD28，CTA-4(CD152)
B7.2(CD86)	CD28，CTA-4
		ICOSL(B7h，B7-H2，B7RP-1，GL50，LICOS)	ICOS(AILIM)
PD-L1(B7-H1)	PD-1
		PD-L2(B7-DC)	PD-1
B7-H3	Unknown
		B7-H4(B7x；B7S1)	Unknown (BTLA?)
Unknown (HVEM *)	BTLA

^*Be TNF member

The TNF family member

Part	Receptor
		OX40L	OX40(CD134)
4-1BBL	4-1BB(CD137)
		CD40L(CD154)	CD40
CD27L(CD70)	CD27
		CD30L	CD30
LIGHT	HVEM、LTβR、DcR3
		GITRL	GITR
BAFF(BLyS) **	BAFF-R、TACI、BCMA
		APRIL **	TACI、BCMA

^*These are that the B cell is relevant

Find the nucleotide and/or the aminoacid sequence of these antibody in the prior art, as follows:

Part (people)	List of references
		B7.1	Freeman G.J., Freeman A.S., Segil J.M., Lee G., Whitman J.F., Nadler L.M.B7, a new member of theIg superfamily with unique expression on activated andneoplastic B cell (B7, the newcomer of Ig superfamily has unique expression on activated and tumor B cell).J.Immunol.143:2714-2722(1989)。
B7.2	Freeman G.J., Gribben J.G., Boussiotis V.A., Ng J.W., Restivo V.A., Jr., Lombard L.A., Gray G.S., the Nadler L.M.Clong of B7-2:a proliferation (clone of B7-2: propagation).Science?262:909-911(1993)。
		ICOSL	Wang S., Zhu G., Chapoval A.I., Dong H., TamadaK., Ni J., Chen L.Costimulation of T cells by B7-H2, a B7-like molecule that binds ICOS (by the T cell co-stimulatory of B7-H2, B7-H2 is the B7-sample molecule in conjunction with ICOS).Blood?96:2808-2813(2000)。Yoshinaga S.K., Zhang M., Pistillo J., Horan T., KhareS.D., Miner K., Sonnenberg M., Boone T., BrankowD., Dai T., Delaney J., Han H., Hui A., Kohno T., Manoukian R, Whoriskey J.S., Coccia M.A.Characterization of a new human B7-related protein:B7RP-1 is the ligand to the Co-stimulatory proteinICOS (sign of new people B7-associated protein: B7RP-1 is the part of common stimulatory protein(SP) ICOS).Int.Immunol.12:1439-1447(2000)。

PD-L1	Dong H., Zhu G., Tamada K., Chen L.B7-H1, a thirdmember of the B7 family, co-stimulates T-cellproliferation and interleukin-10 secretion (B7-H1, the 3rd member of B7 family stimulate T-cell proliferation and IL-1 0 secretion altogether).Nat.Med.5:1365-1369(1999)。Freeman G.J., Long A.J., Iwai Y., Bourque K., Chernova T., Nishimura H., Fitz L.J., Malenkovich N., Okazaki T., Byrne M.C., Horton H.F., Fouser L., Carter L., Ling V., Bowman M.R., Carreno B.M., Collins M., Wood C.R., Honjo T.Engagement of thePD-1 immunoinhibitory receptor by a novel B7-familymember leads to negative regulation of lymphocyteactivation (negative regulation that causes lymphocyte activator by new B7-family member in conjunction with PD-1 immunosuppressant receptor).J.Exp.Med.192:1027-1034(2000)。
		PD-L2	Tseng S.-Y., Otsuji M., Gorski K., Huang X., SlanskyJ.E., Pai S.I., Shalabi A., Shin T., Pardoll D.M., Tsuchiya H.B7-DC, a new dendritic cell molecularwith potent costimulatory properties for T cell (B7-DC, new dendritic cell molecule has effective T cell co-stimulatory characteristic).J.Exp.Med.193:839-846(2001)。Latchman Y., Wood C.R., Chernova T., Chaudhary D., Borde M., Chernova I., Iwai Y., Long A.J., Brown J.A., Nunes R., Greenfied E.A., Bourque K., BoussiotisV.A., Carter L.L., Carreno B.M., Malenkovich N., Nishimura H., Okazaki T., Honjo T., Sharpe A.H., Freeman G.J.PD-L2 is a second ligand for PD-1 andinhibits T cell activation (PD-L2 is second part of PD-1 and suppresses the T-cell-stimulating).Nat.Immunol.2:261-268(2001)。

B7-H3	Steinberger P, Maidic O., Derdak S.V., Pfistershammer K., Kirchberger S., Klauser C., Zlabinger G., Pickl W.F., Stoeckl J., Knapp W.Molecular characterization of human 4-Ig-B7-H3, amember of the B7 family with fourimmunoglobulin-like domains (characterization of molecules of people Ig-B7-H3 has the member of the B7 family of four immunoglobulin like domain).(in JIUYUE, 2003) is committed to the EMBL/GenBank/DDBJ data base.Mingyi Sun, Sabrina Richards, Durbaka V.R.Prasad, Xoi Muoi Mai, Alexnader Rudensky and Chen Dong.Characterization of Mouse and Human B7-H3 Genes (sign of Mus and human B 7-H 3 gene).J.Immunol?168:6294-6297(2002)。
		B7-H4 (B7x; B7S1)	Zang X., Loke P., Kim J., Murphy K., Waitz R., Allison J.P.B7x:a widely expressed B7 familymember that inhibits T cell activation (B7x: the activated B7 family member of the suppressor T cell of wide expression).Natl.Acad，.Sci.U.S.A.100:10388-10392(2003)。Sica G.L., Choi I.-H., Zhu G., Tamada K., WangS.-D., Tamura H., Chapoval A.I., Flies D.B., BajorathJ., Chen L. (in April, 2003) is committed to the EMBL/GenBank/DDBJ data base.

Part	List of references
		OX40L	Baum P.R., Gayle R.B.III, Ramsdell F., Srinivasan S., Sorensen R.A., Watson M.L., Seldin M.F., Clifford K.N., Grabstein K., Alserson M.R.Identification of OX40ligand and preliminary characterization of its activatieson OX40 receptor (evaluation of OX40 part and the active preliminary sign on the OX40 receptor thereof).Circ.Shock?44:30-34(1994)。Mirua S., Ohtani K., Numata N., Niki M., Ohbo K., Ina Y., Gojobori T., Tanaka Y., Tozawa H., NakamuraM., Sugamura K.Molecular cloning andcharacterization of a novel glycoprotein, gp34, that isspecifically induced by the human T-cell leukemia virustype I transactivator p40tax (molecular cloning of neoglycoprotein gp34 and sign, this albumen are inductive by the sub-p40tax specificity of the anti-activation of I type adult T-cell leukosis virus).Mol.Cell.Biol.11:1313-1325(1991)。Godfrey W.R., Fagnoni F.F., Harara M.A., Buck D., Engleman E.G.Identification of a human OX-40ligand, a costimulator of CD4+T cell with homology totumor necrosis factor (evaluation of people OX-40 part has the common stimulant of the CD4+T cell of tumor necrosis factor homology).J.Exp.Med.180:757-762(1994)。
4-1BBL	Alderson M.R., Smith C.A., Tough T.W., Davis-SmithT., Armitage R.J., Falk B., Roux E., Baker E., Sutherland G.R., Din W.S., Goodwin R.G.Molecularand biological characterization of human 4-1BB and itsligand (molecule of people 4-1BB and part thereof and biological the sign).Eur.J.Immunol.24:2219-2227(1994)。

CD40L	Graf D., Korthaeuer U., Mages H.W., Senger G., Kroczek R.A.Cloning of TRAP, a ligand for CD40 onhuman T cells (clone of TRAP, the part of the last CD40 of human T-cell).Eur.J.Immunol.22:3191-3194(1992)。11:4313-4321(1992)。Holllenbaugh D., Grosmaire L.S., Kullas C.D., Chalupny J.N., Braesch-Andersen S., Noelle R.J., Stamenkovic I., Ledbetter J.A., Aruffo A., The humanT cell antigen gp39, a member of the TNF gene family, is a ligand for the CD40 receptor:expression of asoluble form of gp39 with B cell co-stimulatory activity (human T-cell's antigen gp39, the member of tnf gene family, be the part of CD40 receptor: the expression of the gp39 of soluble form has B cell co-stimulatory activity).EMBO?J.11:4313-4321(1992)。
		CD27L (CD70)	Goodwin R.G., Alderson M.R., Smith C.A., ArmitageR.J., Vandenbos T., Jerzy R., Tough T.W., SchoenbornM.A., David-Smith T., Hennen K., Farrah T., Giri J.G., Beckman M.P.Molecular and biologicalcharacterization of a ligand for CD27 defines a newfamily of cytokine with homology to tumor necrosisfactor (characterize the molecule of CD27 part and biology and define the new cytokine family that has homology with tumor necrosis factor).Cell?73:447-456(1993)。
CD30L	Smith C.A., Gruess H.-J., Davis T., Anderdon D., Farrah T., Baker E., Sutherland G.R., Brannan C.I., Copeland N.G., Jenkins N.A., Grabstein K.H., GliniakB., McAlister I.B., Franslow W., Alderson M., Falk B., Gimpsel S., Gillis S., Din W.S., Goodwin R.G., Armitage R.J., CD30 antigen, a marker for Hodgkin ' slymphoma, is a receptor whose ligand defines anemerging family of cytokines with homology to TNF (CD30 antigen, the labelling of Hodgkin lymphoma, be a kind of receptor, its part defines the emerging cytokine family with TNF homology).Cell?73:1349-1360(1993)。

LIGHT	Mauri D.N., Ebner R., Montgomery R.I., Kochel K.D., Cheung T.C., Yu G.-L., Ruben S., Murphy M., Eisenberg R.J., Cohen G.H., Spear P.G., Ware C.F.LIGHT, a new member of the TNF superfamily, andlymphotoxin alpha are ligands for herpesvirus entrymediator (LIGHT, the newcomer of TNF superfamily, and lymphotoxin α is the part that herpesvirus enters medium) Immunity 8:21-30 (1998).
		GITRL	Gurney A.L., Marsters S.A., Huang R.M., Pitti R.M., Mark D.T., Baldwin D.T., Gray A.M., Dowd A.D., Brush A.D., Heldens A.D., Schow A.D., Goddard A.D., Wood W.I., Baker K.P, Godowski P.J., AshkenaziA.Identification of a new member of the tumor necrosisfactor family and its receptor, a human ortholog ofmouse GITR (newcomer's of tnf family cytokines and receptor thereof evaluation, the people ortholog of Mus GITR).Curr.Biol.9:215-218(1999)。
BLyS	Moore P.A., Belvedere O., Orr A., Pieri K., LaFleurD.W., Feng P., Soppet D., Charters M., Gentz R., Parmelee D., Li Y., Galperina O., Giri J., Roschke V., Nardelli B., Carrell J., Sosnovtseva S., Greenfield W., Ruben S.M., Hilbert D.M., Lys:member of the tumornecrosis factor family and B lymphocyte stimulator (Lys: the member of tnf family cytokines and bone-marrow-derived lymphocyte stimulant).Science?285:260-263(1999)。
		APRIL	Hahne M., Kataoka T., Schroeter M., Hofmann K., Irmler M., Bodmer J-L., Schneider P., Bornand T., Holler N., French L E., Sordat B., Rimoldi D., TschoppJ., APRIL, a new ligand of the tumor necrosis factorfamily, stimulates tumor cell growth (APRIL, the new part of tnf family cytokines stimulate growth of tumour cell).J.Exp.Med.188:1185-1190(1998)。

The suitable common particular instance of part that stimulates comprises 4-1BBL, CD80, OX40L and CD86, below will describe in more detail.Yet, should be appreciated that above-mentioned any being total to stimulates part to use according to the present invention.

Perhaps, can use these to stimulate the antibody of any receptor of part altogether.Such antibody is known in the art, and can commercially obtain and obtain by methods known in the art.In one embodiment of the invention, use anti--CD28 antibody.Be used for immunotherapy method anti--CD28 antibody is known in the art.Referring to, for example, Earle etc., above.Suitable anti--CD28 the antibody of example comprises people or murine antibody, humanized antibody, antibody, single-chain antibody and CD28 binding antibody fragment that reorganization produces.Can obtain so anti--CD28 antibody by methods known in the art.

4-1BBL (being also referred to as 4-BB-L, 4-BB part, TNFSF9, ILA part) is the member of TNF receptor family, and after activation 2-3 days, at activated antigen presenting cell (APC), comprises that activated B cell, macrophage and DC go up to express.4-1BB (being also referred to as CD137), it is the receptor of 4-1BBL, at activated CD4 ⁺And CD8 ⁺On the T cell surface, on natural killer cell, mononuclear cell and tranquillization DC, obtain expressing.Also verified recently Treg cell constitutive expression 4-1BB receptor.Referring to, for example, Choi etc., 2004, J.Leukoc.Biol.75:785-91; McHugh etc., 2002, Immunity 16:311-23.

4-1BBL contains 254 aminoacid (26624Da).Referring to, Alderson etc., Eur JImmunol.1994 JIUYUE; 24 (9): 2219-27.Can in the Swiss-Prot data base, find whole aminoacid sequences of people 4-1BBL according to accession number no.P41273.4-1BBL is an II type glycoprotein, forms potential Cytoplasm domain by residue 1-28, and residue 29-49 forms the membrane spaning domain of single prediction, and residue 50-254 forms potential ectodomain, and residue 35-41 represents to gather-and the Leu chain.Can in GenBank accession number no.NM_003811, find the nucleotide sequence of coding people 4-1BBL.

Can connect in conjunction with to the member or be expressed as the fusion rotein that contains in conjunction with to the member in conjunction with the residue 50-254 of the 4-1BBL of its homoreceptor 4-1BB or its fragment, use according to the present invention.For example, Fig. 3 A and 3B have listed nucleotide and the aminoacid sequence (SEQ ID NO:5 and 6) of CSA-Mus 4-1BBL.Fig. 4 has shown the aminoacid sequence (SEQ ID NO:7) of the chimeric protein that comprises people 4-1BBL ectodomain and core Succ-PEG-DSPE.Perhaps, the 4-1BBL biotinylation can be formed and comprise as the 4-1BBL that stimulates altogether part with as the conjugate of combination to member's biotin.

(be also referred to as B7.2, CD28LG2 is that example stimulates polypeptide altogether LAB72), and the both is in conjunction with the CD28/CTLA4 co-receptor by the T cellular expression for CD80 (being also referred to as B7.1, CD28LG or LAB7) and CD86.CD80 contains 288 aminoacid (33048Da).Referring to Freeman etc., J.Immunol.143 (8), 2714-2722 (1989).Can in the Swiss-Prot data base, find whole aminoacid sequences of people CD80 according to accession number no.P33681.

B7.1 is an I type glycoprotein, and 1-34 forms secretion signal by residue, and residue 35-242 forms potential ectodomain, and residue 243-263 forms potential membrane spaning domain, and residue 264-288 forms potential Cytoplasm domain.Therefore, there is not the ripe B7.1 of secretory signal sequence to divide subrepresentation aminoacid 35-288.Can in GenBank accession number no.NM_005191, find the nucleotide sequence of coding people B7.1.

Can connect in conjunction with to the member or be expressed as the fusion rotein that contains in conjunction with to the member in conjunction with the residue 35-242 of the B7.1 of its homoreceptor CD28 or its fragment, use according to the present invention.For example, Fig. 2 A and 2B have listed the nucleotide (SEQ ID NO:3) and the aminoacid sequence (SEQ ID NO:4) of the chimeric protein that comprises people B7.1 (CD80) ectodomain and core Succ-PEG-DSPE.Perhaps, the CD80 biotinylation can be formed and comprise as stimulating partial C D80 altogether and as in conjunction with conjugate to member's biotin.

B7.2 contains 329 aminoacid (37696Da).Referring to Freeman etc., Science 262 (5135), 909-911 (1993).Can in the Swiss-Prot data base, find whole aminoacid sequences of people B7.2 according to accession number no.P42081.B7.2 is an I type glycoprotein, and 1-23 forms secretion signal by residue, and residue 24-247 forms potential ectodomain, and residue 248-268 forms potential membrane spaning domain, and residue 269-329 forms potential Cytoplasm domain.Therefore, there is not the ripe B7.2 of secretory signal sequence to divide subrepresentation aminoacid 24-329.Can in GenBank accession number no.NM_175862, find the nucleotide sequence of coding people CD86.

Can connect in conjunction with to the member or be expressed as the fusion rotein that contains in conjunction with to the member in conjunction with the residue 24-247 of the B7.2 of its homoreceptor CD28 or its fragment, use according to the present invention.For example, Fig. 5 A and 5B have listed the nucleotide (SEQ ID NO:8) and the aminoacid sequence (SEQ ID NO:9) of the chimeric protein that comprises people B7.2 (CD86) ectodomain and core Succ-PEG-DSPE.Perhaps, the CD86 biotinylation can be formed and comprise as stimulating partial C D86 altogether and as in conjunction with conjugate to member's biotin.

B7.2 does not express on tranquillization B cell usually, but goes up with low expression level in peripheral blood lymphocytes (PBC) and DC; Yet after activation, the expression on B cell and other APC such as macrophage and the DC obtains raising.On the contrary, CD86 obtains constitutive expression on PBC and DC, and is raised faster on the B cell.The interaction of the MHC/ peptide complexes on TXi Baoshouti (TCR) and the APC strengthens CD80/86 and CD28 on the T cell simultaneously, this causes the tyrosine phosphorylation of lipid kinase phosphatidyl-inositol 3-kinase, and it suppresses the inducing of a series of IL-2 of causing gene expressions, cell proliferation subsequently and is divided into incident in the complicated born of the same parents of effector function.Signal 2 can further increase generation property immunne response by regulating anti-apoptotic genes expression such as Bal-xL to prevent cell death.

OX40L is expressed by dendritic cell and other APC, and in conjunction with the OC40 that is present on the activated T cells.OX40L contains 183 aminoacid (21950Da).Referring to Miura etc., Mol.Cell.Biol.11:1313-1325 (1991).Can in the Swiss-Prot data base, find whole aminoacid sequences of OX40L according to accession number no.P23510.OX40L is an II type glycoprotein, and residue 1-23 is the Cytoplasm domain, and residue 24-50 is a membrane spaning domain, and residue 51-183 is an ectodomain.The nucleotides sequence of OX40L is classified 3510bp as, and coded sequence is 157-708 (referring to GenBank accession number no.NM_003326.2).Can connect in conjunction with to the member or be expressed as the C-end fusant that contains in conjunction with to the member in conjunction with the residue 51-183 of the OX40L of its homoreceptor OX40 or its fragment, use according to the present invention.

LIGHT polypeptide (being also referred to as TNFS14, HVEM-L, LTg, TR2) is and the homologous TNF superfamily member of lymphotoxin.Referring to Mauri etc., Immunity 8 (1), 21-30 (1998).Can in the Swiss-Prot data base, find whole aminoacid sequences of people LIGHT according to accession number no.O43557.LIGHT contains 240 aminoacid (26351Da) and is II type glycoprotein, forms potential Cytoplasm domain by residue 1-37, and residue 38-58 forms the membrane spaning domain of single prediction, and residue 59-240 forms potential ectodomain.The division site relates to residue 82-83.Can in GenBank accession number no.NM_172014, find the nucleotide sequence of coding people LIGHT.

Can connect in conjunction with to the member or be expressed as the fusant that contains in conjunction with to the member in conjunction with the residue 59-240 of the LIGHT of its homoreceptor HVEM, LT β R or TR6 or its fragment, use according to the present invention.For example, Figure 1A and lB have listed the nucleotide (SEQ ID NO:1) and the aminoacid sequence (SEQ ID NO:2) of the chimeric protein that comprises core strepto-antibiotic and Mus LIGHT ectodomain.Perhaps, the LIGHT biotinylation can be formed and comprise as the LIGHT that stimulates altogether part with as the conjugate of combination to member's biotin.

LIGHT mainly expresses on activated T cells, NK cell and immature dendritic cell, and is used for regulating the various aspects of immunne response.Synthesize LIGHT as embrane-associated protein, but its cell surface expression is subjected to the adjusting of several translations back mechanism.In a few minutes of expressing, separate LIGHT and accumulate (obform body 1 from cell surface as shla molecule by matrix metalloproteinase; General expression residue 83-240; Swiss-Prot O43557-1).Cell surface Cytoplasm fragment is represented obform body 2 (Swiss-Prot O43557-2).In addition, various cell types are stored LIGHT and are secreted out by various biostimulations when activating in vesicle.Although the effect of the soluble form of LIGHT is not characterized fully, can be used as negative feedback loop, by competing the function that suppresses film combining form with HVEM and LT β R.

LIGHT and three different acceptor interactions: the herpesvirus on (1) T cell enters medium (HVEM), (2) LT β R, its mainly express on epithelial cell and the stromal cell and (3) various cells on solubility inveigle receptor 3.LIGHT is given in these interactions different functions.Take place relevant with the interaction of LT β R on the stromal cell and generation, lymph node (LN) organ of various cytokine/chemotactic factors with the recovery of secondary lymph structure.On the other hand, the interaction of the HVEM receptor on LIGHT and the lymphocyte causes the activation and the generation of cytokine, is mainly IFN-γ and GM-CSF.As if about this point, the LIGHT/HVEM axle transmits with the Th1 type and replys the relevant costimulatory signal of activation, this plays pivotal role in oncolysis.

Signal 3

The example that is used for stimulus signal 3 stimulates cytokine and the somatomedin that partly comprises stimulus signal 3 altogether, as IL-2, IL-4 and TGF-β (comprising TGF-β 1, TGF-β 2 and TGF-β 3).The IL-2 and the IL-4 that are used for the treatment of in the method partly are known in the art.Referring to, for example, Earle etc., 2005, above; Thorton etc., 2004, J.Immunol.172:6519-23; Thorton etc., 2004, Eur.J.Immunol.34:366-76.According to an embodiment, use the maturing part of cytokine.

For example, IL-2 or IL-4, or its active fragment can link in conjunction with to the member or be expressed as and contain in conjunction with to member's fusion rotein, use according to the present invention.For example, common unsettled U.S. patent application 10/312,245 discloses the maturing part that comprises IL-2 or IL-4 and the chimeric protein of core Succ-PEG-DSPE, is useful according to the present invention.Can also be referring to Fig. 6 A and 6B, it has listed the nucleotide (SEQ ID NO:10) and aminoacid (the SEQ ID NO:11) sequence of IL-2-CSA chimeric protein.Perhaps, can pass through methods known in the art, IL-1 or IL-4 biotinylation be provided comprise as the IL-2 that stimulates part altogether or IL-4 with as the conjugate of combination to member's biotin.Referring to, for example, Jordan etc., 2003, Clin.Diag.Lab.Immunol.10:339-44; DeJong etc., 1995, J.Immunol.Methods 184:101-12.

TGF-β 1 (being also referred to as TGF-β, TGF1, CED, DPD1, HGNC:2997, the key sexual abnormality 1 of progressivity, transforminggrowthfactor-) is a multifunctional polypeptide, controls propagation, differentiation and other functions in many cell types.Many cells synthesize (justacrine) TGF-β 1, and all have the special receptor of this peptide basically.TGF-β 1 regulates the effect of many other peptide growth factors, and measures the forward or the negative sense of these effects.It plays an important role in bone remodeling, and is potential skeletonization bone formulation stimulant, causes the osteoblastic chemotaxis, propagation and the differentiation that relate to.

TGF-β 1 molecule comprises 390 aminoacid (44,341 dalton).This is the precursor substance that splits into the mature T GF-β 1 and related peptides (LAP) that hide.There is not active form to constitute by non-covalent TGF-β 1 homotype dimer in conjunction with LAP homotype dimer.There do not have active complex can contain potential TGF-β to be conjugated protein.Activity form is the homotype dimer with 12 amino acid whose ripe β of monomeric form, and this is that disulphide connects.The aminoacid sequence that finds according to accession number P01137 among the SwissProt data base comprises 29 amino acid whose signal peptides at residue 1-29 place, 249 amino acid whose latency-associated peptides at residue 30-278 place, the nucleotide sequence of 112 amino acid whose active TGF-β 1 genes at residue 279-390 place, wherein coded sequence is at base 842-2017.The initial TGF-β 1 of nucleotide sequence, 3 amino acid whose cell connection site at residue 244-246 place.There are many variant sequences, the present invention includes these.Find expression nucleotide 1-2745 to be disclosed in Dernyk etc. at GenBank according to accession number no.NM-000660,1987, Nucl.Acids.Res.

TGF-β 1 exists as the film binding growth factor of solubility, and relates generally to organ generation and embryo's early stage pattern.TGF-β plays an important role in immune system.For example, the mouse life of shortage TGF-β 1 is lacked and death relates to the TGF-β 1 in the peripheral tolerance owing to the extensive infiltration of inflammatory lymph cell in the critical organ and macrophage.

A series of nearest researchs have shown that TGF-β can mediate in various autoimmune and the transplanting situation the toleration from body and isoantibody.For example, in rat, the allograft of use donor specific transfusion is relevant with the high-level TGF-β in the graft, by the toleration of infiltration lymphocyte and the abolishment of blocking-up anti-TGF-beta.Josien etc., 1998, J.Clin.Invest.102:1920-26.In addition, the TGF-β unconventionality expression in the cardiac transplantation makes their long-term survivings in the allogeneic receiver.Ditto.In addition, in NOD, find the self tolerance dependent T GF-β that uses anti-CD 3 antibodies to set up.Belghith etc., 2003, Nat.Med.9:12-02-08.The transient expression that has also shown TGF-β in the islet cells can effectively prevent autoimmune diabetes among the NOD by the Treg cell.Peng etc., 2004, Proc.Nat ' lAcad.Sci.USA 101:4572-77.Similarly, the whole body gene therapy of use TGF-β 1 causes the toleration among the obvious diabetes NOD, the cell tolerance of β, and treatment of diabetes.Luo etc., 2005, Transplantation 79:1091-96.

TGF-β has also demonstrated in generation, homoiostasis and the amplification of Treg cell and has played an important role.For example, owing to pass through the adjusting of the FoxP3 of TGF-β, the mice of defective TGF-β 1 has the Treg cell that reduces quantity in periphery.Peng etc., above.The Treg cell is transferred to the wild type animal and causes their persistent existence and functions from TGF-β deficient mice is acquired, may be because the paracrine effect of TGF-β 1.Also demonstrated TGF-β in NOD homoiostasis and the function of Treg cell in play an important role.Referring to, for example, Pop etc., J.Exp.Med.201:1333-46.Compare with disease-resistant strain, the NOD mice has the Treg cell of significantly reduced absolute quantity.This reduction is reduced by TGF-β cell surface expression and causes, this causes the function of FoxP3 expression that reduces and the Treg cell that changes subsequently, and this outbreak with disease is consistent.Referring to, for example, Gregg etc., 2004, J.Immunol.173:7308-16; You etc., 2005, Diabetes 54:1415-22; Pop etc., above.

TGF-β is also by inducing FoxP3 to express natural CD4 ⁺CD25 ^-The T cell transformation becomes in the Treg cell to play an important role.The Treg cell that TGF-β transforms can suppressor T cell propagation and prevent antigen stimulation after the clonal expansion of T cell.Although considerably less research focuses on the mechanism that the beta induced FoxP3 of TGF-expresses, as if by suppressing the following mediation of transferring of Smad7, this downward modulation is caused by FoxP3, therefore makes the TGF-signal beta by positive modulators Smad3 and 4 in this effect.

TGF-β, or its active fragment can connect in conjunction with to the member or be expressed as and contain in conjunction with to member's fusion rotein, use according to the present invention.For example, common unsettled U.S. patent application 10/312,245 discloses the active structure domain that comprises people TGF-β and the chimeric protein of core Succ-PEG-DSPE, uses according to the present invention.See also Fig. 7 A and 7B, it has listed the nucleotide (SEQ ID NO:12) and aminoacid (the SEQ ID NO:13) sequence of this chimeric protein separately.Perhaps, can comprise as the TGF-β that stimulates part altogether with as the conjugate of combination to provide by means commonly known in the art with TGF-β or its active fragment biotinylation member's biotin.Can also commercially obtain biotinylated IL-2 (R﹠amp; D Systems).

In conjunction with to the member

The example combination is to being biotin and Succ-PEG-DSPE (SA) or avidin.Can also use maintenance to biotin substantially in conjunction with active SA or avidin fragment, as separately at least 50% or how natural SA or avidin in conjunction with active.Such fragment comprises " core Succ-PEG-DSPE " (" CSA "), the clipped form of total length Succ-PEG-DSPE polypeptide, and it comprises Succ-PEG-DSPE residue 13-138,14-138,13-139 or 14-139.Referring to, for example, Pahler etc., J.Biol.Chem.1987.262:13933-37.Can also use and keep strong in conjunction with other Succ-PEG-DSPE of biotin and the clipped form of avidin.Referring to, for example, Sano etc., J Biol Chem.1995 November 24,270 (47): 28204-09 (having described core Succ-PEG-DSPE variant 16-133 and 14-138) (U.S. patent no.6.022,951).Can also use keep basic biotin in conjunction with the active or biotin that improves in conjunction with active Succ-PEG-DSPE mutant and Succ-PEG-DSPE core form.Referring to, Chilcoti etc., Proc NatlAcad Sci U S is Feb28 A.1995; 92 (5): 1754-8; Reznik etc., Nat Biotechnol.1996 August; 14 (8): 1007-11.For example, can use to have the immunogenic mutant of reduction, as remove the mutant of potential T cellular antigens decision position or lymphocyte antigen decision position sudden change by direct mutagenesis.Referring to Meyer etc., Protein Sci.2001 10:491-503.Equally, also can use keep basic biotin in conjunction with the active or biotin that improves in conjunction with the mutant of active avidin and the core form of avidin.Referring to, Hiller etc., J.Biochem. (1991) 278:573-85; Livnah etc., Proc Natl Acad Sci USA 90:5076-80 (1993).For convenience, in the description at the moment, term " avidin " and " Succ-PEG-DSPE " comprise that these are in conjunction with biotin binding fragment, mutant and core form to the member as used in this.Avidin and Succ-PEG-DSPE can obtain from commercial supplier.In addition, can find nucleotide sequence and the Succ-PEG-DSPE and the avidin aminoacid sequence of coding Succ-PEG-DSPE and avidin, for example, at GenBank accession number No.X65082; X03591; NM_205320; X05343; Z21611 and Z21554.

As used in this " biotin " comprise can mating surface such as cell surface (comprising tumor cell surface) contain biotin moiety, as NHS-biotin and EZ-Link ^TMSulfo group-NHS-LC-biotin (Pierce).The albumino reaction form of such biotin can be buied.

In scope of the present invention, several advantages have been given in the interaction between biotin and binding partners avidin or the Succ-PEG-DSPE.For example, biotin is for Succ-PEG-DSPE (10 ¹³M ^-1) and avidin (10 ¹⁵M ^-1) have a very high affinity.In addition, Succ-PEG-DSPE and avidin are separately in conjunction with four polymerization polypeptide of four biotin molecules.Therefore, the immunity that comprises Succ-PEG-DSPE or avidin stimulates part to have the trend that forms tetramer and higher structure altogether.Therefore, they can crosslinked its corresponding immunocyte receptor, is used for the more efficient signal transduction, as the accumulation by receptor.

Those skilled in the art will recognize that and (for example to use other mechanism, other conjugation methods, for example use other coupling part or chemistry or the gene crosslinked) the high stage structure of immune costimulatory molecules is provided, as comprising the conjugate of dimer, trimer, tetramer and the high-order polymer of immune costimulatory molecules, it presents favourable characteristic equally.Such conjugate comprises within the scope of the invention.

Conjugate

As mentioned above, one aspect of the present invention relates to and comprises that at least one stimulates part and combination puting together the member altogether.Can make such conjugate by means commonly known in the art.For example, stimulate altogether part and in conjunction with can be directly to the member or by the mutual covalent bond of junctional complex or put together mutually.

According to one embodiment of the invention, conjugate is to comprise common stimulation polypeptide and in conjunction with to the fusion rotein of member such as CSA.Can make fusion rotein by in the multiple distinct methods known in the art any.For example, can chemosynthesis maybe can use known recombinant nucleic acid technology to produce the composition polypeptide of one or more fusion rotein.(as used in this, " nucleic acid " refers to RNA or DNA).For example can using, polymerase chain reaction (PCR) obtains nucleotide sequence useful among the present invention.Various PCR method are described in, for example, PCRPrimer:A Laboratory Manual, Dieffenbach 7 Dveksler edit, Cold SpringHarbor Laboratoey Press, 1995.

According to an embodiment, stimulate polypeptide to pass through its C-end and the N-end combination that combines the member altogether.For example, the present invention includes the CD80-CSA fusion rotein, TGF-β-CSA fusion rotein, IL-2-CSA fusion rotein and IL-4-CSA fusion rotein, wherein CD80, TGF-β, IL-2 or IL-4 partly combine with the N-end of CSA by its C-end.According to another embodiment, stimulate polypeptide to pass through its N-end and the C-end combination that combines the member altogether.For example, the present invention includes the CSA-4-1BBL fusion rotein, wherein CSA partly stimulates the N-end combination of part together by its C-end.Stimulate altogether polypeptide can with in conjunction with to the member directly in conjunction with maybe can such as one or morely being connected the polypeptide combination by one or more coupling parts.

Comprise common stimulation polypeptide fragment and/or, need only the activity of fragment maintenance with reference to full-length polypeptide in conjunction with segmental nucleic acid of member and polypeptide be can be used among the present invention.Therefore, stimulate part should keep its common stimulating activity (for example, keeping its ability) altogether in conjunction with its receptor or part, and in conjunction with keeping its ability in conjunction with its binding members to fragment.Conventional method by this area is screened fragment for the activity that keeps, and is included in those of following examples illustrated.Below listed exemplary common stimulation polypeptide fragment.

Conjugate can comprise junctional complex such as peptide junctional complex between to member and stimulation part altogether.Can select junctional complex length and form the activity that improves the arbitrary function end of part.Junctional complex is generally about 3 to about 15 amino acid longs, and more preferably from about 5 to about 10 amino acid longs, yet, shorter or longer junctional complex can be used, or junctional complex can be all distributed.In this, can use and be used for connecting the heavy chain of single-chain antibody and elasticity junctional complex (for example, (Gly of light chain ₄Ser) ₃).Referring to, Huston etc. (1988) Proc.Nat.Acad.Sci.USA, 85:5879-5883; U.S. patent No.5,091,513,5,132,405,4,956,778; 5,258,498 and 5,482,858.Other junctional complex is FENDAQAPKS or LQNDAQAPKS.The segmental one or more domains of immunoglobulin Fc (for example, CH1, CH2 and/or CH3) also can be used as junctional complex.Can also use chemical linkers.

Nucleic acid and polypeptide that modify, that change or sudden change also are useful in the present invention, as long as they keep the activity with reference to nucleic acid or polypeptide.For example, be applicable to nucleic acid of the present invention and peptide sequence and, that is, stimulate polypeptide altogether with the known immunity of coding or combine nucleotide sequence to have sequence homogeneity (comprising at least 80% sequence homogeneity) at least about 80% to the member with reference to nucleic acid or polypeptide.In some embodiments, nucleotide sequence or polypeptide and with reference to nucleic acid or polypeptide have at least about 85%, at least about 90%, at least about 95% or at least about 99% sequence homogeneity.

The present invention includes the nucleic acid of sequence change " silence ", identical aminoacid (promptly because their are encoded.The degenerate core acid sequence).The present invention comprises that also coding has the nucleic acid of the polypeptide of conservative amino acid replacement, and such polypeptide.Conservative amino acid replacement (for example, substituting a hydrophobic residue with different hydrophobic residue) is well known in the art and can for example waits by point mutation and realize.Can use receptors bind known in the art and/or biological screening method to confirm the suitability of given modification sequence, variant or mutant, as above about described those methods of fragment.

As used in this, the quantity of mated position in nucleic acid by measuring comparison or the peptide sequence, with the quantity of mated position divided by comparison nucleotide or aminoacid separately sum and multiply by 100 and calculate " % sequence homogeneity ".Identical nucleotide or amino acid whose position appear in the same position that paired position refers in aligned sequences.Comparison nucleotide or amino acid whose sum refer to nucleotide or the amino acid whose minimum number that second sequence of comparison needs, the comparison that does not comprise non-homogeneous sequence (for example, the comparison that forces), as those sequences (that is, coding immunity the stimulation altogether polypeptide or combination are to member's sequence) that can hold at the N-of target sequence or the C-end merges.Comparison nucleotide or amino acid whose sum can be corresponding to complete coded sequences, or can be corresponding to the fragment of full length sequence, as defined in this.

Can use Altschul etc. (1997, Nucleic Acids Res., 25:3389-3402) algorithm of Miao Shuing comes aligned sequences, because introduced in BLAST (the basic local comparison research tool) program, can obtain at the ncbi.nlm.hih.gov of World Wide Web.Can carry out blast search or compare the percentage ratio sequence homogeneity of measuring between nucleic acid molecules (" inquiry sequence ") and any other sequence or its fragment, use the Altschul algorithm.Can use BLASTN to compare and the homogeneity between the nucleotide sequence relatively, and use BLASTP to compare and the comparing amino acid sequence between homogeneity.When the nucleotide sequence of use blast program calculation code therapeutical peptide and the percentage ratio homogeneity between another sequence, can use the default parameter of program separately, comprise the default of breach punishment.

Can be by detecting nucleic acid of the present invention as DNA or rna blot analysis (that is hybridization), PCR or the such method of in situ hybridization analysis.Usually by the immunocytochemistry in the transfectional cell series or by sodium lauryl sulphate (SDS)-polyacrylamide gel electrophoresis follows Coomassie blue stain or western blot analysis detects polypeptide, use the antibody (monoclonal or polyclone) of specificity binding affinity with specific polypeptide.Many Sambrook etc. (1989 that are described in greater detail in these methods, Molecular Cloning:A Laboratory Manual (molecular cloning: laboratory manual), the 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

Coding immunity the stimulation altogether polypeptide and combination can operationally interconnect in construct member's nucleotide sequence, use conventional Protocols in Molecular Biology.Referring to, for example, MolecularCloning:A Laboratory Manual (molecular cloning: (Sambrook etc. laboratory manual), 2001, the 2nd edition, Cold Spring Harbor Laboratory Press) or Short Protocolsin Molecular Biology (molecular biological succinct experimental program) (Ausubel etc., 2002, the 5th edition, Current Protocols).Be applicable to construct in these methods be can buy and be that this area is used in everyday.Construct can comprise the sequence of expression needs, and as promoter sequence, regulating and controlling sequence as enhancer sequence and response sequence and/or derivable sequence, is regulated the expression of nucleotide sequence.As used in this, " be operably connected " and refer to (i) and place promoter and/or other regulating and controlling sequence with respect to nucleotide sequence in the mode that instructs or regulate expression of nucleic acid; And/or (ii) place the nucleic acid of coding immunity the stimulation altogether polypeptide and coding in conjunction with nucleic acid to the member, and make coded sequence " in frame ", that is, make the construct coding comprise that immunity stimulates polypeptide altogether and in conjunction with fusion rotein to the member.

Can be by methods known in the art with construct breeding or expression, in host cell, to produce polypeptide.As used in this, term " host " or " host cell " meaning are not only to comprise prokaryote, as escherichia coli, and comprise eukaryote, as yeast, insecticide, plant and animal cell.Zooblast comprises, for example, and COS cell and HeLa cell.Can use dna molecular (for example, construct) to transform or transfection host cell, use and well known to a person skilled in the art any technology, as calcium phosphate or lithium acetate precipitation, electroporation, fat transfection and microparticle bombardment.The host cell that contains carrier of the present invention can be used for as the breeding carrier, produces nucleic acid (for example, DNA or RNA), express the purpose that immunity stimulates polypeptide or its fragment or expressed fusion protein altogether, as mentioned above.

Tu1A ﹠amp; 1B, 2A ﹠amp; 2B, 3A ﹠amp; 3B, 4A ﹠amp; 4B, 5A ﹠amp; 5B and 7A ﹠amp; 7B has shown representational nucleotide sequence (SEQ ID NO.1,3,5,8,10 ﹠amp; 12), these sequential codings comprise the core Succ-PEG-DSPE and stimulate the conjugate of polypeptide altogether, and the aminoacid sequence of corresponding encoded (SEQ ID NO.2,4,6,7,9,11; 13).

The present invention also provides and has comprised and stimulate part and as in conjunction with the conjugate to member's biotin altogether.Can will stimulate the part biological elementization altogether by methods known in the art, and in following embodiment illustrated.

For example, from Avidity, (Denver, biotin AviTag technology CO) can be used for producing biotinylated albumen to Inc..Biotin AviTag is made of 15 unique amino acid peptides, and this peptide is by biotin ligase BirA identification, and biotin enzyme connects the lysine residue in biotin and the peptide sequence.Schatz，1993，Biotechnology，11:1138-43。Biotin AviTag can merge in heredity to any target protein, makes and can use the biotin molecule labelled protein.

A latent defect of biotin AviTag technology is the probability of low biotinylation degree, because the single unique lysine residue biotinylated protein of system in the label fragment.In order to overcome any such problem, can use the albumen that obtains labelling of the biotin ligase of purification at external modification purification.Because carry out biotinylation by enzyme, reaction condition is gentle, and labelling is a high degree of specificity, and reaction is more effective than the albumen chemical modification of using biotin derivative.Perhaps, above described method such as Jordan can be used for producing the protein of through engineering approaches and bioid.

The conjugate combination

According to an embodiment, the invention provides the conjugate combination of the Treg cell method that is used for increasing.In a specific embodiment, combination comprise (a) at least one comprise in (i) stimulus signal 1, signal 2 or the signal 3 part of at least one and (ii) in conjunction with first right member conjugate and (b) at least one comprise in (i) stimulus signal 1, signal 2 or the signal 3 part of at least one and (ii) second combination to member's conjugate.In one embodiment, combination comprises in stimulus signal 1, signal 2 or the signal 3 part of at least one.In a specific embodiment, this part stimulus signal 3.For example, this part can comprise IL-2 or IL-4.In another embodiment, compositions comprises in stimulus signal 1,2 and 3 at least two part.In a specific embodiment, this part stimulus signal 2 and 3.In another specific embodiment, this part stimulus signal 1 and 3.In another embodiment, compositions comprises the part that stimulus signal 1,2 and 3 is whole.

In one embodiment, combination comprises:

(A) one or morely be selected from following conjugate: (a) first conjugate, it comprises that (i) comprises the first conjugate member of 4-1BBL polypeptide and (ii) comprise the second conjugate member in conjunction with first right member; (b) second conjugate, it comprises that (i) comprises the first conjugate member of CD80 polypeptide and (ii) comprise in conjunction with first right member second conjugate member and (c) the 3rd conjugate, it comprise (i) comprise the first conjugate member of TGF-beta polypeptides and (ii) comprise described in conjunction with first right member the second conjugate member and

(B) one or morely be selected from following conjugate: (a ') the 4th conjugate, it comprises that (i) comprises the first conjugate member of anti-CD 3 antibodies and (ii) comprise the second conjugate member of described combination to second member of member; (b ') the 5th conjugate, it comprises that (i) comprises the first conjugate member of cytokine and (ii) comprise the described second conjugate member in conjunction with second right member; (c ') the 6th conjugate, it comprises that (i) comprises the antigenic first conjugate member and (ii) comprise the second conjugate member of second combination to the member; (d ') the 7th conjugate, the first conjugate member of it comprises that (i) comprises anti--CD28 antibody and (ii) comprise the described second conjugate member in conjunction with second right member.

As mentioned above, any one or more conjugate can comprise fused polypeptide, and this fused polypeptide comprises first conjugate (for example, stimulating part altogether) and the second conjugate member (for example, in conjunction with to the member).In one embodiment, comprise avidin or Succ-PEG-DSPE (comprising the core Succ-PEG-DSPE), and comprise biotin in conjunction with second right member in conjunction with first right member.

Can be in the compositions of separating, or in single compositions, provide conjugate.Each compositions may further include acceptable carrier on the materia medica, excipient or diluent, as known in the art.Acceptable carrier is can be as the material of compositions medium on the materia medica, because in the administration scope, this material is inert or medically is being acceptable in addition, and with activating agent be compatible.Carrier can be accepted on the materia medica and conventional medicine additive well known in the art can be contained.

When combination is provided in single compositions, and at least one in first, second or the 3rd conjugate can combine in the combination between the member and the 4th, the 5th, the 6th or the 7th conjugate at least one by first and second combination.

Example combinations comprises following one or more:

The conjugate or the fusion rotein that comprise 4-1BBL and core Succ-PEG-DSPE;

The conjugate or the fusion rotein that comprise CD80 and core Succ-PEG-DSPE;

The conjugate or the fusion rotein that comprise TGF-β and core Succ-PEG-DSPE;

The conjugate that comprises antigen (or antigen/MHC complex) and biotin;

The conjugate that comprises IL-2 and biotin;

The conjugate that comprises IL-4 and biotin;

Comprise anti-CD 3 antibodies and biotin conjugate and

The conjugate that comprises anti--CD28 antibody and biotin.

Method

The present invention also provides the method for amplification Treg cell.In one embodiment, this method relate to Treg cells contacting (a) at least one comprise in (i) stimulus signal 1, signal 2 or the signal 3 part of at least one and (ii) in conjunction with first right member conjugate and (b) at least one comprise in (i) stimulus signal 1, signal 2 or the signal 3 part of at least one and (ii) second combination to member's conjugate.In one embodiment, this method comprises at least one part in Treg cells contacting stimulus signal 1, signal 2 or the signal 3.In a specific embodiment, this part stimulus signal 3.For example, this part can comprise IL-2 or IL-4.In another embodiment, this method comprises at least two part in Treg cells contacting stimulus signal 1,2 and 3.In a specific embodiment, this part stimulus signal 2 and 3.In another specific embodiment, this method stimulus signal 1 and 3.In another embodiment, this method comprises the part with whole signals in Treg cells contacting stimulus signal 1,2 and 3.

According to a specific embodiment, this method comprises the Treg cell colony is contacted:

(A) one or morely be selected from following conjugate: (a) first conjugate, it comprises that (i) comprises the first conjugate member of 4-1BBL polypeptide and (ii) comprise the second conjugate member in conjunction with first right member; (b) second conjugate, it comprises that (i) comprises the first conjugate member of CD80 polypeptide and (ii) comprise in conjunction with first right member second conjugate member and (c) the 3rd conjugate, it comprise (i) comprise the first conjugate member of TGF-beta polypeptides and (ii) comprise described in conjunction with first right member the second conjugate member and

(B) one or morely be selected from following conjugate: (a ') the 4th conjugate, it comprises that (i) comprises the first conjugate member of anti-CD 3 antibodies and (ii) comprise the second conjugate member of described combination to second member of member; (b ') the 5th conjugate, it comprises that (i) comprises the first conjugate member of cytokine and (ii) comprise the described second conjugate member in conjunction with second right member; (c ') the 6th conjugate, it comprises that (i) comprises the antigenic first conjugate member and (ii) comprise the second conjugate member of second combination to the member; (d ') the 7th conjugate, the first conjugate member of it comprises that (i) comprises anti--CD28 antibody and (ii) comprise the described second conjugate member in conjunction with second right member.

As mentioned above, have been found that the receptor of Treg cellular expression 4-1BBL, CD80 and TGF-β.Therefore, according to an embodiment, the Treg cell comprises in first, second or the 3rd conjugate receptor of at least one, and at least one in first, second or the 3rd conjugate puted together by combination between the first conjugate member and the receptor and Treg cell thus.According to this embodiment, conjugate will be used for Treg activation and amplification in conjunction with its receptor and crosslinked with receptor.In aspect this embodiment is further, at least one in the 4th, the 5th, the 6th and the 7th conjugate puted together by at least one and Treg cell in first or second conjugate, by first and second combination to the combination between the member.

The Treg cell colony can comprise CD4+ cell, CD25+ cell and FoxP3+ cell.In a specific embodiment, the Treg cell colony comprises the CD4+CD25+FoxP3+ cell.

According to an embodiment, can exsomatize and realize this method, by exsomatizing with Treg cells contacting conjugate.In aspect of this embodiment, basically simultaneously will at least two conjugate contact Treg cells.For example, in the single compositions of contact Treg cell colony, provide at least two conjugates.In a specific embodiment, at least one in first, second or the 3rd conjugate combines in the combination between the member and the 4th, the 5th, the 6th and the 7th conjugate at least one by first and second combination.

According to another stripped embodiment of the inventive method, in order with at least two conjugates and Treg cells contacting.For example, can in contacting the compositions of separating of Treg cell colony in order, provide at least two conjugates.According to this back embodiment, by 4-1BBL and/or CD80 and/or TGF-β and the combination between the receptor on the Treg cell thereof, first, second and/or the 3rd conjugate at first contact and make it in conjunction with the Treg cell.Then, the 4th, the 5th, the 6th and/or the 7th conjugate can contact and make it in conjunction with the Treg cell, by first and second combination to the combination between the member.

For example, can obtain the Treg cell and purification is realized the stripped amplification of Treg cell from the patient by using standard technique, as use the antibody of anti-CD25 and CD4.Then comprise the common conjugate that stimulates part (as CSA-4-1BBL), comprise anti-CD 3 antibodies (as biotinylated anti-CD 3 antibodies) and IL-2 (comprising biotinylated IL-2) conjugate in the presence of the Treg cell of culture purified.Stimulate after 3 days, replenish IL-2 (containing or do not contain other 4-1BBL), continue 7 days to culture.In one embodiment, from beginning also to have used the conjugate (as CSA-TGF-β or biotinylated TGF-β) that comprises TGF-β, continue several weeks, or run through whole incubation.Repeat this circulation once more, as long as cell is remained in the culture.

According to another embodiment, by conjugate being delivered medicine to patient's realize in vivo increasing method of Treg cell.According to this embodiment, can be in order or administration conjugate simultaneously basically.For example, can be in order or come administration composition as the compositions of separating simultaneously, or can be used as single compositions basically to come administration composition.

In the embodiment according to administration composition in order, by 4-1BBL and/or CD80 and/or TGF-β and the combination between the receptor on the Treg cell thereof, the at first administration and make it be positioned the Treg cell of first, second and/or the 3rd conjugate.Then, can administration the the 4th, the 5th, the 6th and/or the 7th conjugate, and make it be positioned the Treg cell to the combination between the member by first and second combination.

In the embodiment of while administration composition, first, second and/or the 3rd conjugate can combine with the 4th, the 5th, the 6th and/or conjugate the combination between the member by first and second combination in single compositions.According to this embodiment, by 4-1BBL and/or CD80 and/or TGF-β and the combination between the receptor on the Treg cell thereof, conjugate can be positioned the Treg cell.

Can whole body or topical conjugate, as by intravenous, peritoneum or subcutaneous injection.In one embodiment, can pass through the different one or more conjugates of administration.For example, can one or more conjugates of topical, and can the one or more conjugates of whole body administration.

Using with conjugate during the present invention also is included in and puts together free stimulates part altogether, and promptly this part is not the embodiment of above-mentioned conjugate composition.Therefore, for example, the Treg cell can contact one or more conjugates, as mentioned above, and contacts and one or morely free stimulates part altogether, as external source IL-2, IL-4 or anti-CD 3 antibodies.Can with contact one or more conjugates simultaneously, before or after, with the one or more free parts that stimulate altogether of Treg cells contacting.As mentioned above, can exsomatize or realize this contact in vivo.

According to this embodiment, the amount of used external source IL-2 is more much lower than a large amount that art methods needs.For example, use in the situation of 2000IU/mL in art methods, we demonstrate lower amount, comprise 25IU/mL IL-2, are effective.Therefore, the present invention includes and be lower than about 25IU/mL at least about 1000IU/mL or the method for the IL-2 of a large amount more.For example, the present invention includes to use and be lower than about 25IU/mL, about 25IU/mL, about 50IU/mL, about 75IU/mL, about 100IU/mL, about 150IU/mL, about 200IU/mL, about 250IU/mL, about 300IU/mL, about 350IU/mL, about 400IU/mL, about 450IU/mL, about 500IU/mL, about 600IU/mL, about 700IU/mL, about 800IU/mL, about 900IU/mL, about 1000IU/mL or the method for the IL-2 of a large amount more.

Amplification is important to Treg in the stimulation of the tables of data clear signal of discussing in following examples 3.In scope of the present invention, can use the conjugate of the part that comprises stimulus signal 3 to come stimulus signal 3, such part comprises IL-2, IL-4 or another cytokine.Perhaps, can use the free part that stimulates altogether of stimulus signal 3 to come stimulus signal 3 as external source IL-2 or IL-4.In another interchangeable embodiment, can pass through any other mode, other modes are as known in the art come stimulus signal 3.

The present invention also provides the method that the Treg cell is delivered medicine to the patient, wherein according to the stripped amplification of above-mentioned method Treg cell.According to this embodiment, by above-mentioned any approach such as intravenous come administration Treg cell.

Suitable patient comprises people or other animals that needs the Treg cell amplification.For example, according to the present invention, suffer from autoimmune disease such as type i diabetes or be in patient in the autoimmune disease risk, or accept patient that external source transplants implant (promptly, allograft patient and xenotransplantation), be the target patient of Treg amplification, accept the tumor patient (preventing GVHD) of bone marrow transplantation and accept external source hemopoietic or the patient of other stem cell (as treat produce enough mixochimaeras of being good at treat hemopoietic genetic defect or autoimmune disease) also is like this.The present invention can also be used for the treatment of that the amplification of suffering from by the Teff cell that causes a disease causes or any disease relevant with the amplification of pathogenic Teff cell or be in patient in such disease risks.

In one embodiment, this method further comprises rapamycin is delivered medicine to the patient.Rapamycin has effective immunosuppressive activity, and be widely used for preventing testing with clinical setting in transplant rejection.Rapamycin does not disturb the activation of T cell, but is used for preventing the signal of IL-2 mediation, and cell cycle stops at the G1-S border, therefore causes T cell anergy and/or apoptosis, and induces the operation toleration.Rapamycin works by suppressing mTOR, and mTOR relates to the synthetic serine/threonine kinase that starts and survive the signal transmission of albumen.

In the scope of the invention importantly with regard to produce, keep with function with regard to, rapamycin is to the not same-action of Treg cell and Teff cell.Different with tacrolimus with calcineurin inhibitor such as ciclosporin, rapamycin does not disturb activation and the high expression level of FoxP3 in the Treg cell.Referring to, for example, Baan etc., 2005, Transplantation 80:110-17.Use rapamycin to demonstrate in vivo and can induce CD4 ⁺CD8 ⁺The apoptosis of thymocyte cell also causes periphery to regulate CD4 ⁺CD25 ⁺The amplification of T cell.Tian etc., 2004, Transplantation 77:183-89.In isolated culture, demonstrate the function that rapamycin does not disturb people Treg cell, but suppressed the propagation and the cytokine secretion of Teff cell, and opposite with the Teff cell, the Treg cell can be resisted the inductive apoptosis of rapamycin.Game etc., 2005, Am.J.Transplant.5:454-64.Consistent with these researchs, also demonstrate, in the presence of rapamycin, the Teff cell is by the antigenic external cell death (AICD) that activates back experience activation-inducing, and the Treg cell is preferentially increased and be transferred to the repulsion that can block the allos islets of langerhans when suppressing among the receiver acquired.Battaglia etc., 2005, Blood 105:4743-48.Rapamycin also demonstrate can change DC immunostimulation function towards the phenotype that causes tolerance more, this can be used as positive feedback loop subsequently, is used for the generation and the amplification of Treg cell.Chiang etc., 2004, J.Immunol.172:1355-63.These receptors are consistent with popular idea, and this idea is: different with the calcineurin inhibitor, rapamycin not interference tolerance is induced.Therefore, rapamycin is applicable in the method for the present invention that wherein the Treg amplification is required.

In another embodiment, this method further comprises the compositions that contains the foreign cell (for example, allogeneic or xenogenesis) of showing TGF-β.For example, can modify foreign cell such as splenocyte, dendritic cell, medullary cell, hematopoietic stem cell, solid organ and islet cells and show TGF-β with the antigen pulse.In a particular aspects of this embodiment, also rapamycin is delivered medicine to the patient.

In one embodiment, obtain the foreign cell of modification by following method, this method comprises that (a) comprises that with foreign cell contact forming the foreign cell of modification and (b) in conjunction with first right member and bifunctional molecule in conjunction with the molecule of cell surface contacts the foreign cell of modifying and comprise TGF-β and second combination conjugate to the member, forms the foreign cell of displaying TGF-β.TGF-β can be total length TGF-β or its any active fragment, as mentioned above.The design bifunctional molecule, make cell surface bound fraction that bifunctional molecule passes through bifunctional molecule in conjunction with cell surface after, keep it for the affinity of second combination basically in conjunction with first right member to the member.When bifunctional molecule comprises biotin, can be located in cell surface by following illustrational method or by additive method (those described in WO02/02751).In a specific embodiment, bifunctional molecule is the proteinic biotin form that can put together in vivo on the cell surface, as the NHS-biotin, comprises sulfo group-NHS-LC-biotin.Comprise in the embodiment of biotin at bifunctional molecule, comprise avidin or Succ-PEG-DSPE (or its above-mentioned any variant, comprise the core Succ-PEG-DSPE) in conjunction with second right member.

The present invention also provides the method for amplification Treg cell, and using, ev vivo modifies the DC of the antigen pulse of showing TGF-β.According to this aspect of the invention, come pulsed D C, exsomatize then to modify and show TGF-β, as mentioned above with one or more antigens such as one or more self antigen.In one embodiment, cause the diabetes self antigen such as GAD, islet cells self antigen (ICA) or self antigen NRP-A7 come pulsed D C with one or more.In a specific embodiment, come pulsed D C with among GAD65, ICA512 and the NRP-A7 each.Perhaps, with the immature DC of islets of langerhans lysate pulse.In another embodiment, come pulsed D C (for example, being used for the treatment of arthritis) with collagen.In another embodiment, come pulsed D C (for example, being used for the treatment of multiple sclerosis) with myelin basic protein.

An embodiment of the method for the pulsed D C of acquisition displaying TGF-β comprises that (a) uses the immature dendritic cell of antigen pulse, obtains the dendritic cell of pulse; (b) contact of the dendritic cell of pulse is comprised in conjunction with first right member and form the pulse dendritic cell of modification in conjunction with the bifunctional molecule of the molecule of cell surface; (c) the pulse dendritic cell contact of modifying is comprised TGF-β and second combination form the pulse dendritic cell of showing TGF-β to member's conjugate.TGF-β can be total length TGF-β or its active fragment arbitrarily, as mentioned above.In a specific embodiment, bifunctional molecule comprises sulfo group-NHS-LC-biotin.Comprise in the embodiment of biotin that at bifunctional molecule second combination comprises avidin or Succ-PEG-DSPE (or its above-mentioned any variant, comprise the core Succ-PEG-DSPE) to the member.

In one embodiment, order about the DC maturation of pulse by methods known in the art.For example, can be by hatch the DC maturation of ordering about pulse with 4-1BBL.

According to this aspect of the invention, DC pulse, that show TGF-β can be needed the patient of Treg amplification, as those above-mentioned target patient.Can give DC by above-mentioned any approach, as pass through intravenous administration.Can measure the optimal dose of DC by experiment, described in hereinafter.Use in the NOD mice before that pulsed D C's studies show that 5 * 10 ⁵Cell/animal and 2 * 10 ⁵Cell/animal is effective in inducing the Treg amplification.

The present invention also comprises DC pulse, displaying TGF-β in conjunction with one or more above-mentioned methods that stimulates conjugate altogether and/or come administration in conjunction with rapamycin.Therefore, for example, can be with DC pulse, that show TGF-β in conjunction with comprising that 4-1BBL, CD80 and/or IL-2 or above-mentioned any other stimulate part to deliver medicine to the patient altogether.Additionally or replacedly, rapamycin can be delivered medicine to the patient.According to these embodiments, can with DC basically simultaneously, or before or after DC, one or more conjugates of administration and/or rapamycin.

In one embodiment of the invention, use the conjugate and the optional TGF-β that comprise 4-1BBL and/or CD80, IL-2 to obtain nonspecific Treg amplification.In another embodiment, use comprises the conjugate of 4-1BBL and/or CD80 and IL-2, in conjunction with external source islets of langerhans or organ or the dendritic cell also modified with the related antigen pulse, temporarily using under the covering up of rapamycin the Treg amplification of acquisition antigenic specificity with TGF-β.

The present invention also provides the method for amplification Treg cell, and with above-mentioned same way as for pulsed D C, the hematopoietic stem cell or the medullary cell (BMC) that use TGF-β to modify comprise external source (for example, allogeneic or xenogenesis) BMC.This method can be used for setting up blended chimera, for example, is used to induce toleration and treatment autoimmune power and hemopoietic genetic defect to self antigen, isoantigen and heteroantigen.Can use this method separately, or in conjunction with above-mentioned common stimulation conjugate and/or in conjunction with rapamycin, as mentioned above.Use hematopoietic stem cell or the BMC Treg cell that not only increases, and the blended chimera of will doing the best, this chimera will control autoimmune power and allow pancreatic beta cell regeneration, the feasible diabetes that prevent and/or treat.Use hematopoietic stem cell that TGF-β modifies or the external source BMC Treg cell that will increase in conjunction with conjugate, its will make subsequently prevention stem cell or BMC repulsion and set up the mixochimaera of controlling autoreactivity and homogeneous reactivity.

According to this embodiment of the present invention, hematopoietic stem cell or BMC biotinylation are also modified with TGF-β-CSA, as mentioned above, and intravenous injection under rapamycin is covered up.One or more that can as above make in conjunction with administration stimulate conjugate (comprising CD80/IL-2 and/or 4-1BBL/IL-2 conjugate) to realize this processing altogether, so that the effect of amplification tolerogenesis.With the processing of hematopoietic stem cell unmodified or TGF-β-modification or BMC and the rapamycin Treg cell that will increase, make prevent diabetes.

As mentioned above, TGF-β and rapamycin can synergism be blocked the activation and the amplification of self antigen specificity T eff cell, promote the activation and the amplification of Treg cell simultaneously or it is from CD4 ⁺CD25 ^-The T transformation.One or more stimulate the optional use of conjugate this effect of can further increasing altogether.

Following examples are understood the present invention in more detail, and not in office where face limits the scope of the invention.

Embodiment

Conventional method

Animal: (Bar Harbor Maine) and according to NIH and Guidelines raises non-diabetic mice (NOB) available from Jackson Laboraories.(Bar Harbor ME), raises under the SPF condition and instructs and nurse according to association and NIH in University of Louisville available from Jackson Laboratory for BALB/c and C57BL/6 mice.

Use insecticide DES expression system to express and purification IL-2 and 4-1BBL, CD80 and TGF-β chimeric protein: can use fruit bat DES expression system (Invitrogen; Carlsbad CA) sets up stable transfection of expressing these molecules, as Singh etc., and 2003, described in the Cancer Res.63:4067-73.In being set at the cultivation agitator of 25 ℃ and 105rpm, at the fruit bat serum-free medium (Gibco that replenishes 1mM copper sulfate; Carlsbad CA) was hatched transfectant 72 hours, was used for expression of recombinant proteins.By centrifugal collection culture supernatants and accept large scale purification, use the crosslinked fixed metal affinity of the agarose resin (BD-Talon of cobalt (II)-carboxymethyl asparagic acid salt, BD Biosciences) or Ni-NTA metal affinity resin (Qiagen), utilize be engineered to 6 in the protein *-the His-label.In brief, produce 10% alcoholic acid final concentration, contain the culture medium of recombiant protein with precipitation by dropwise adding 95% ethanol.After 4 ℃ of overnight incubation, sedimentary albumen is dissolved in again (50mM sodium phosphate pH7.0 in the binding buffer liquid of 1/10 initial volume; 500mM sodium chloride; 0.5% tween 20; 1% glycerol; The 5mM 2 mercapto ethanol).Use the binding buffer liquid of 5 * gel bed volume to come balance metal affinity resin, add in the dissolved again protein solution, and hatched 45 minutes with putting upside down rotation in room temperature.With 50-100ml lavation buffer solution (50mM sodium phosphate pH7.0; 500mM sodium chloride) with the metal affinity resin of conjugated protein washing 2 *.Use elution buffer (the 50mM sodium phosphate pH7.0 of 2 * gel bed volume; 500mM sodium chloride; The 150mM imidazoles) from the bonded protein of metal affinity resin elution.

Merge the albumen eluent of purification and be loaded into Amicon UltraTM (Millipore; Bedford Mass) in the centrifugal filter device, uses 30kD molecular weight mwco membrane.(2000 * g) 4 ℃ centrifugal 15 minutes at 3000rpm with centrifugal filter device.Add aseptic PBS in the trapped substance and with filter at 3000rpm (2000 * g) recentrifuge.Sucking-off contains the proteic trapped substance of concentrated/desalination from centrifugal filter device, places aseptic low temperature bottle, and is stored in the liquid nitrogen.Measure the concentration of protein isolate by the SDS-polyacrylamide gel electrophoresis.Use BCA albumen to test according to the explanation (Pierce) of manufacturer and measure protein concentration.

The expression of biotinylation GAD and purification: collect the islets of langerhans of NOD and homogenizing in the 4M guanidine thiocyanate immediately.According to the total RNA of described separation before, referring to, for example, Shiwan etc., 1993, J.Immunol.150:2295-304.Purified RNA is dissolved in the water of diethyl coke hydrochlorate processing, is distributed into little equal portions, and is stored in-70 ℃ before use.With the part of this RNA template, use the specific primer of Mus GAD coded sequence as RT-PCR.Referring to, for example, Lee etc., 1993, Biochim.Biophys.Acta 1216:157-60.With PCR product cloning (Invitrogen) to the TA cloning vehicle.Identify functional clone and be used for sub-clone to the carrier pAC (Avidity) that is used to express.Antibacterial transforms and after selecting on the ampicillin medium, several clones is accepted minimum plasmid preparation and digest with suitable restriction endonuclease to identify positive colony.To have the segmental clone of insertion and be used for big plasmid preparation.Plasmid is used to transform the AVB100 escherichia coli, and this bacterial strain has the birA ligase gene of stable integration to the chromosome.Express with the L-arabinose induced protein, be used to have the high level expression of the GAD of biotin labeling thing.Use Western blotting and measure concentration, level of endotoxin and the biotinylation of purification GAD as the Succ-PEG-DSPE of the alkali phosphatase conjugate of probe.If desired, use is removed endotoxin from the Detoxi-gel endotoxin removal test kit of Pierce.With biotinylation GAD five equilibrium and be frozen in-70 ℃ until use.

Immunomodulating: will comprise that by mixed in molar ratio in PBS the common conjugate of part and core biological chain mycin that stimulates is in conjunction with biotinylated IL-2 and/or biotinylated GAD albumen with 1:1.With all conjugates and the DC among the dosage intravenous injection PBS shown below.

The blended lymphocyte reaction of allosome.In test in 5 days, cultivate from natural B ALB/c mice (1 * 10 as replying agent ⁵/ hole) spleen and lymph-node cell, (2000cGy) that use was shone is from the splenocyte (1 * 10 of natural C57BL/6 mice ⁵/ hole) as stimulant.Add in the culture with the Treg cell of the different ratios of replying agent and Treg amplification.In last 16 hours that cultivate, use H ³-thymidine comes the pulse cell.

Islet transplantation.By single intravenous injection 200mg/kg streptozotocin (Biomol, Plymouth Meeting PA) make male BALB/c mouse (22-26g, 6-8 week big) suffer from diabetes, and by twice〉300mg/dl continuous blood sugar reading confirms diabetes.In islet transplantation the previous day, by intravenous injection with 5-8 * 10 ⁶The Treg cell transfer of individual stripped amplification is to every animal.The release enzymatic solution (Roche) of use 0.2mg/ml is inculcated from the C57BL/6 mice of complete mispairing by the original position of pancreas and is collected donor islet.37 ℃ of digestion after 17 minutes, use Ficoll gradient purification islets of langerhans and in complete medium 37 ℃ keep spend the night (replenishing 10% FBS, 2mM L-glutaminate, the RPMI of 100U/ml penicillin/streptomycin and 50mM 2 mercapto ethanol).Collected islets of langerhans in second day, transplant under receiver's the left side scrotum to each BALB/c with the PBS washing and with 400-600 islet transplantation.Control animal is transplanted the allogeneic islets of langerhans, but does not accept the Treg cell.Animal that accept to transplant of monitoring is three times weekly, and by twice〉the continuous blood sugar reading of 300mg/dl confirms to repel.

Islets of langerhans infiltration lymphocyte: collect the infiltration lymphocyte by at first discharging enzyme (Roche) pancreas digested from the islets of langerhans of NOD animal with 0.2mg/ml, for example, and as Yolcu etc., 2002, described in the Immunity 17:795-808.In case obtain single islets of langerhans, according to described with they digestion before, for example, and Green etc., 2002, described in the Immunity 16:183-91, obtain islets of langerhans infiltration lymphocyte.Latter's dyeing is used for flow cytometry.

Flow cytometry: at first anti-and two anti-by titration target one, in flow cytometry, use optium concentration to carry out flow cytometry then, use standardization program.Referring to, for example, Mhoyan etc., 1997, Transplantation 64:1665-70.Homotype pairing antibody is as negative control.With sample at FACS Calibur or Vantage (Becton Dickinson; Mountain View CA) goes up operation and use FlowJo (TreeSoft) or CellQuest (BD Biosciences) software and analyzes.

Use the intracellular cytokine analysis of flow cytometry: in flow cytometry, use the specific monoclonal antibodies of these cytokines (PharMingen) to carry out the analysis of IL-2, IL-4 in the born of the same parents, IL-10 and IFN-γ.Referring to, for example, Elson etc., 1995, J.Immunol.154:4294-301.37 ℃ with 4% paraformaldehyde with cell fixation 5 minutes, and with PBS/1% BSA washed twice.(PBS in the PBS-S buffer that replenishes 0.1%BSA and 10%FCS, 0.01M HEPES, 0.1% saponarin) after overnight incubation is blocked non-specific binding on ice, hatched 30 minutes with the cytokine specific antibody with twice of cell washing and on ice with the PBS-S buffer.After PBS-S buffer washed twice, the anti-mice IgG rat antibody incubation that they are puted together with FITC-on ice 30 minutes, and analyze by flow cytometry.The paired uncorrelated antibody of homotype is as negative control.Experimental program (eBioscience) according to manufacturer carries out FoxP3 analysis in the born of the same parents.

T cell sorting and mensuration phenotype by flow cytometry.Collect spleen and lymph-node cell from natural B ALB/c mice, be processed into single-cell suspension liquid, and use ACK solution to come the cracking erythrocyte.For cell sorting, with anti-CD4-FITC, anti-CD25-PE and anti-CD8-APC with cell dyeing.(BD Bioscience, San Jose CA) come sorting CD4 to use the FACSVantage cell sorter ⁺CD25 ^-Single positive (SP) and CD4 ⁺CD25 ⁺Two positives (DP) T cell.The cell of sorting is〉95% pure.In flow cytometry, use the Abs of the avidin of CD4-APC, CD4-FITC, CD8-PE, CD8-PerCP, 4-1BBL-PE, CD25-PE, CD95-FITC, biotin-CD137, biotin-CD28, biotin-GITR, biotin-TGF-β and FITC labelling to measure originally phenotype with the Treg cell of amplification.The homotype Abs that will have the fluorescent dye that matches is with comparing.(eBiosciences, San Diego CA) carry out FoxP3 dyeing in the born of the same parents according to the experimental program of manufacturer.

For expression of receptor test, with the CD4+CD25+ of sorting or CD4+CD25-T cell in the flat board of 96-hole separately, or at IL-2 (25U/ml), the splenocyte (1 * 10 that shone ⁵/ hole) or under both existence cultivated 2 days.After cultivating 2 days, collect a part of cell, use the PBS washed twice, and be divided into two cultures that separate.Replenish IL-2 for simultaneously a culture, and another maintenance does not have IL-2.Cultivate after 2 days, with cell dyeing, and use flow cytometry to analyze with the antibody of anti-4-1BB or CD28.

After 2 days, collecting one group of cell with IL-2 and spleen cell cultures, washed twice, and be untreated and cultivate 2 days again.In another group cell, added IL-2 at the 2nd day and also cultivated again 2 days.With cell dyeing, and use flow cytometry to analyze with the antibody of anti-4-1BB or CD28.

The RT-PCR of FoxP3.Use TRI reagent from the originally spleen of BALB/c mouse and the CD4 of the fresh sorting of lymph node ⁺CD25 ^-And CD4 ⁺CD25 ⁺The total RNA of Treg cell separation of T cell or amplification.Use two μ gRNA to produce the first chain cDNA, FoxP3 and HPRT Auele Specific Primer are used in the performing PCR of going forward side by side amplification, carry out 33 and 27 circulations separately.Primer sequence is:

FoxP3 forward 5 '-CAG CTG CCT ACA GTG CCC CTA G-3 '

FoxP3 negative sense 5 '-CAT TTG CCA GCA GTG GGT AG-3 '

HPRT forward 5 '-GAA GTG TTG GAT ACA GCC CAG AC-3 '

HPRT negative sense 5 '-GAG GGT AGG CTG GCA TCT AGG CT-3 '

Histopathology: estimate the autoimmune histology's signal of islets of langerhans and the ability of excreting insulin thereof.Different time points after processing is taken out islets of langerhans from what handled with contrast NOD animal.With each islets of langerhans sheet of 10% buffered formalin fixed, be embedded in the paraffin, and with 4 μ M section, and with hematoxylin and eosin (H﹠amp; E) general physiological change is measured in dyeing.According to described immunohistochemistry of carrying out cytokine, T cell and insulin before.Referring to, for example, Green etc., 2002, Immunity 16:183-91.

The preparation of DC and with causing the pulse of diabetes antigen: use GM-CSF and IL-4 in culture, to produce immature cell 4-5 days from bone marrow (BM) cell of NOD.(30 μ g/ peptides/ml) order about to break up with the cell pulse and with 100ng/ml mice CSA-4-1BBL conjugate or CD40L and spend the night with the mixture of GAD65, ICA512 and NRP-A7 peptide.Buy peptide from GenScript Corporation.With thoroughly washing and of cell with TGF-β modification (or core Succ-PEG-DSPE albumen) in contrast, according to method above-mentioned and described in detail below.In some embodiments, the NP40 lysate of the thorough dialysis of NOD APC and various concentration is cultivated altogether, prepared this lysate from NOD islets of langerhans as the self antigen source.After the PBS washing, in flow cytometry, analyze a part of cell, be used for before immunomodulating, on cell surface, show TGF-β, mediation CD80/CD86 costimulatory molecules on the II class.

The displaying of TGF-β on cell surface: containing 5 μ M EZ-Link with ice-cold PBS washing DC and with 2.5 * 106 cells/ml ^TMIncubated at room is 20 minutes among the PBS of the pH8.0 of sulfo group-NHS-LC-biotin (Pierce).With the thorough washing of cell and with 1 * 10 ⁶Cell/ml was hatched 15 minutes at 4 ℃, used the TGF-β-core Succ-PEG-DSPE conjugate (or the core Succ-PEG-DSPE in contrast) of 50-100ng purification.After the thorough washing of PBS, the cell processing is used for flow cytometry and is used for further research, as described below.

Statistics: use the effect of Kaplan-Meier curve estimation treatment to the type 1 diabetes prevention.Use the survival difference of logarithm level testing (generalized Savage/Mantel Cox) between measuring not on the same group.Relate to comparison and at first have the F of use check (two groups) or the detected equal difference of Levent ' s check (many groups) from the program of single animal groups data.When difference is unequal, carries out log and transform.When comparing the sample mean of normal distribution, use Student ' s check (two groups) or Newman-Keuls check (organize) more.When data do not have normal distribution, use Mann-Whitney U check (two groups) or Kruskal-Wallis check (many groups).Significance,statistical is defined as p＜0.05.

The clone and the expression of embodiment 1:CSA-4-1BBL conjugate

(Molecular Research Center, Cincinnati OH), separate total RNA from use 2 days mouse boosting cell of LPS (5 μ g/ml) stimulation to use TRI reagent.This RNA of two micrograms is used to produce the first chain cDNA, used as the increase ectodomain (aa 104-309) of 4-1BBL of the template of PCR, use justice (5 '-ATC GAA TTC CGC ACC GAGCCT CGG CCA GCG-3 ') and antisense (5 '-GGA CTC GAG CAT AGC AGCTTG AGG ACT TAG C-3 ') primer.With the primer through engineering approaches comprise EcoRI and XhoI site promote directed and frame in be cloned in the DES expression vector (Invitrogen, San Diego, CA).The PCR product cloning is accepted dna sequencing to the PCR2.1TOPO carrier and with several positive colonies.Contain the single clone of correct 4-1BBL sequence and sub-clone to the pMT/BiP/V5-His expression vector that contains 6 * His label and core Succ-PEG-DSPE (CSA) with EcoRI and XhoI digestion, make the ectodomain of mice 4-1BBL form domain at the biotin-combination and the tetramer that the C-end is cloned into CSA.Fig. 8 A, but in the metal inducible expression carrier, the secretion signal that uses fruit bat is with sub-clone in the mosaic gene frame, and use fruit bat DES expression system at S2 expressed in insect cells CSA-4-1BBL conjugate, use the agarose column purification, and use limulus amebocyte lysate test kit (Charles River) test endotoxin.

The CSA-4-BBL conjugate exists as the tetramer and Geng Gao stage structure, measures by PAGE under at natural endowment.Only under the degeneration condition, four poly-/oligomerization structures are dissociated into～the 37kDa monomer, and by the SDS-PAGE classification.Fig. 8 B has shown the western blot analysis of purification CSA-4-1BBL under degeneration (swimming lane 2) and natural (swimming lane 3) condition.Under the degeneration condition, CSA-4-1BBL occurs as the monomer of 37kDa, and under natural endowment, protein as the tetramer and the high stage structure of 150kDa occurs.

Followingly measured combining of CSA-4-1BBL and 4-1BB receptor.At total blended lymphocyte reaction (MLR) culture medium (replenishing the DMEM of 5%FBS, 2mM L-glutaminate, 100U/ml penicillin/streptomycin, 10mM HEPES and 100mM MEM-Sodium Pyruvate) (Invitrogen, Carlsbad, CA) and 50mM 2 mercapto ethanol (SIGMA, St.Louis.MO) middle with 5 μ g/ml ConA (Sigma-Aldrich, St.Louis MO) stimulates from the splenocyte of BALB/c or C57BL/6 mice 48 hours.Use CSA-4-BBL conjugate (200ng/l * 10 then ⁶Cell) or CSA reference protein (76ng/l * 10 of equimolar amounts ⁶Cell) on gyrate shaker, activated and/or resting cell were hatched 30 minutes for 4 ℃.After hatching, with PBS several times,, and pass through flow cytometry with the antibody staining of anti-CD4 (APC), CD8 (PerCP), 4-1BBL (PE) and SA (FITC) with cell washing.The cell that tranquillization and CSA-are hatched is as negative control.

For the blocking-up test, also use excessive (50 μ g/l * 10 ⁶Cell) (3H3 is provided by the R.Mittler friendship of Emery university anti-4-1BB antibody, CA) 1,000,000 activated cells is hatched 30 minutes.Then with cell washing several times, use the chimeric 4-1BBL of 200ng (CSA-4-1BBL) to hatch again then 30 minutes with PBS.Then with the PBS washed cell and with the antibody staining of anti-CD4-APC, CD8-PerCP, 4-1BBL-PE and CSA-FITC, and pass through flow cytometry.The cell that tranquillization and CSA-are hatched is as negative control.

With 4-1BBL (200ng/l * 10 ⁶Cell) or contrast CSA albumen (76ng/l * 10 ⁶Cell) will hatch 30 minutes at 4 ℃ from tranquillization or the activated splenocyte of ConA of BALS/c mice.Use the antibody test CD4 of anti-mice 4-1BBL ⁺And CD8 ⁺4-1BBL on the T cell is in conjunction with (black line).The cell usefulness of hatching with CSA albumen compares (Lycoperdon polymorphum Vitt is filled).As shown in Fig. 8 C, CSA-4-BBL is in conjunction with the activation CD8 that expresses the 4-1BB receptor ⁺And CD4 ⁺The T cell.This combination is specific for 4-1BB, because natural T cell lacks the receptor sorting feminine gender, in addition, at first causes having lost in conjunction with (Fig. 8 C, right figure) with anti-4-1BB antibody blocking receptor.

Whether in order to test 4-1BBL is functional, in the presence of the solubility CSA-4-1BBL conjugate of various concentration (showing with ng/ml among Fig. 8 D) or contrast CSA are proteic, will be with the anti-CD 3 antibodies of time good concentration from the spleen of natural B ALB/c and total CD4 of lymph node purification ⁺T cytositimulation 4 days.Fig. 8 D.With the common stimulation of CSA-4-BBL conjugate at CD4 ⁺Produced strong propagation in the T cell and replied, this be concentration dependent and be significant (p＜0.05) statistically.To reply be that 4-1BBL is dependent to propagation, because do not measuring replying that the anti-CD 3 antibodies that increases by inferior good dosage obtains at the CSA albumen that waits mol level to use.Generally speaking, these results the CSA-4-1BBL conjugate have the architectural feature of core Succ-PEG-DSPE, because it has formed the tetramer and oligomer, in conjunction with the 4-1BB on the activated T cells, and under inferior good anti-CD 3 antibodies stimulates as effective activator of T cell.

Embodiment 2: use the Treg amplification of CSA-4-BBL conjugate

Use the cell sorting (FACS) of fluorescence-activation from the spleen of BALB/c mouse, to sub-elect CD4 ⁺CD25 ⁺Treg cell (Fig. 9 A), and in the presence of homology APC, activate with anti-CD 3 antibodies (0.5 μ g/ml), CSA-4-BBL (1 μ g/ml) and IL-2 (25IU/ml).Then with cell with～1 * 10 ⁶Cell/ml keeps, and uses the culture medium of replenishing IL-2, and continues 10-12 days in per 3 days.Then culture is accepted the activation that another is taken turns, then kept with IL-2.As shown in Figure 9, this scheme makes Treg cell 55 to 110 times of Treg cell amplifications in 18-24 days, 110 times (Fig. 9 B) of amplification in 25 days.Under the same conditions but the Treg cell that does not have the CSA-4-1BBL conjugate to keep has been had to minimum amplification.The Treg cell of amplification has formed by CD4 ⁺CD25 ^BrightSame population (Fig. 9 A of cellularity, bottom-right graph), the high-caliber FoxP3 albumen of this cellular expression (by RT-PCR or anti-FoxP3 TPPA) and Fas, CD62L, GITR, CD25, CD28 and cell surface TGF-β (data not shown), and suppress allos and reply and use the polyclone of the T cell that anti-CD3 stimulates and activate (data not shown).On the contrary, the culture of 4-1BBL of no use only demonstrates 2.5 times of amplifications of DP cell quantity, and demonstrates by CD4 ⁺CD25 ^SecretlyCell and CD4 ⁺CD25 ^BrightThe xenogenesis group of cellularity (Fig. 9 A, top right plot).CD4 with amplification ⁺SP cell difference, the high-caliber 4-1BB of Treg cellular expression.Figure 10 (the not homotype contrast of group that black is filled).

Embodiment 3: the survival of the Treg cell elongation islets of langerhans allograft of amplification

In order to test the therapeutic effect of the Treg cell that polyclone increases, with (using streptozotocin) diabetes BALS/c mice acquired transferase 45-10 * 10 of chemical induction ⁶The individual aforesaid Treg cell that increases in culture continues 20-25 days, after 24 hours, gives the transplanting of the C57BL/6 allogeneic islets of langerhans of complete mispairing then.The blood sugar level of monitor animal, inferior on every Wendesdays.The animal (o) that all Treg handle has the survival (MST=68.7 ± 10 day) of prolongation, the stage of observation～85 days in, surpass 1/3 (66%) and do not repel graft.Figure 11.Obviously opposite, the control animal (●) of all Treg cell therapies of no use repels graft (MST=16.6 ± 2.7 day) strongly.

Embodiment 4: the CD4+CD25+Treg cell of amplification is an inhibition

In traditional inhibition test, use CD3 to stimulate the function of the Treg cell that characterizes amplification.Similar to natural Treg cell, in replying the CD3 stimulation, keep the Treg cell of anergic amplification can suppress CD4 ⁺The polyclone propagation of Teff cell, and this inhibit feature should be subjected to the inhibition (Figure 12 A) of 4-1BBL.

Use mixed lymphocyte reaction that more evidences of the aforesaid Treg cell inhibiting function that increases according to the present invention are provided.With the spleen of natural B ALS/c mice and periphery lymph-node cell with the agent that responses, and will be from the splenocyte of the irradiation of natural C57BL/6 mice as stimulant.Effective inhibition of the Teff propagation that the alloantigen that exists significant (p＜0.05) to be caused by the Treg cell that increases orders about is even reply the ratio (Figure 12 B) of agent than Treg at 10:1.This has shown that the Treg cell of amplification given traditional inhibit feature of the Treg cell that belongs to natural generation.Generally speaking, these digital proofs be inhibition and Treg cell way of act and natural generation similar with the Treg cell of CSA-4-1BBL conjugate amplification.

Embodiment 5

A.4-1BBL with the synergism of IL-2 for the Treg cell

In the presence of 0.5 μ g/ml anti-CD 3 antibodies, the natural DP cell and the homology splenocyte that shone of sorting were cultivated 3 days altogether.Replenish 25U/ml IL-2,1 μ g/mlCSA-4-1BBL or both mixture to culture.Be enough to induce Treg cell (DP) propagation for Treg culture adding IL-2 or 4-1BBL; Yet, with replenish 1 μ g/ml 4-1BBL those compare, in the culture that replenishes 25U/ml IL-2, has 4 times propagation raising.Figure 13 A.Being used in combination 4-1BBL and IL-2 has produced maximum propagation and replys and (surpass independent IL-2 twice; Figure 13 A), show that these two albumen synergism promote the propagation of Treg cell.

Also detected and do not had the independent contribution that signal 1,2 and 3 is bred Treg separately in the culture of the APC system.In this test, provide signal 1 by the solubility anti-CD 3 antibodies, provide signal 2 by 4-1BBL, provide signal 3 by IL-2.Use the natural DP cell of sorting separately, do not use the splenocyte that shone, and replenish anti-CD 3 antibodies, CSA-4-1BBL and/or IL-2 to culture.As shown in Figure 13 B with table 1 in institute generalized, stimulating independent signal 1 or 2 is invalid in inducing the Treg cell proliferation, and stimulus signal 3 causes appropriate amplification.Stimulus signal 1 and 2 both also have minimum Treg expanding effect.Yet stimulus signal 1 or 2 has significant cultivation effect in conjunction with signal 3 stimulations by IL-2, when all three signals obtain stimulating, observes the most violent effect (2-3 reaches 110-and doubly increases in week) to Treg propagation.The Treg cell of amplification all is CD25 ^BrightAnd express high-caliber CD28,4-1BB, GITR, Fas, CD62L, film in conjunction with TGF-β and FoxP3, compare with the DP cell of 4-1BBL amplification of no use.

Table 1

Signal	1	2	3	1+2	1+3	2+3	1+2+3
								Effect	-	-	+	+	++	+++	++++++

These results show in the effect of 1,2 and 3 pairs of Treg cell proliferation of stimulus signal and have hierarchy.Separately in fact stimulus signal 1 or 2 does not have effect, and separately stimulus signal 3 (passing through IL-2) or except signal 1 and/or 2 stimulus signal 3 have significant effect.Stimulating all three signals to have the most significant effect, then is the stimulation of signal 2 and 3, is 1 and 3 then.

B.IL-2 raises CD4 ⁺CD25 ⁺4-1BB receptor expression on the Treg cell

In the existence of IL-2 and/or the APC that shone or not, with the CD4 of sorting ⁺CD25 ⁺(DP) Treg and CD4 ⁺CD25 ^-(SP) the Treg cell culture is 2 days.Collecting cell then, and in flow cytometry, analyze the expression of 4-1BB.Have only 22% fresh sorting the Treg cellular expression 4-1BB, and the Teff cell of neither one sorting is male to this receptor.When cell single culture in the time of 2 days, the down-regulated expression of 4-1BB is to background level (2%).Cultivating the Treg cell has minimum to the constitutive expression of keeping the 4-1BB on the Treg cell effect (8%) in the presence of the APC that shone.Obviously opposite is, IL-2 is added in the culture of Treg cell and not only cause keeping of 4-1BB receptor, and (29% vs 22% of new fresh cell) is raised in mediation.The adding of the APC that shone has further improved the effect (53%) of IL-2 to the 4-1BB up-regulated.

Keeping/is raising effect among the 4-1BB on the Treg cell in order further to survey IL-2, APC is being added IL-2 exist to descend cultured cells thoroughly to wash, and cultivating 2 days (Figure 13 C, last column among the last figure) again in the presence of not at IL-2.All Treg cells with the 4-1BB down-regulated expression to background level.The adjusting that 4-1BB expresses by IL-2 is the Treg cell-specific, because in the change of the 4-1BB expression pattern of similar processing on the Teff cell be invalid (Figure 13 C, figure below).These results just are to have proved that for the first time the 4-1BB that IL-2 keeps/raises on the Treg cell expresses for ours, and the synergistic mechanism based to Treg cell vitro proliferation is provided between observation IL-2 and the 4-1BBL.

Embodiment 6: the structure of functional TGF-β-CSA conjugate

Produced the conjugate that comprises CSA and TGF-'beta ' activity form according to above-mentioned conventional method.When being used for MLR test (Figure 14) or activating (data not shown) with anti-CD3 polyclone, TGF-β-CSA conjugate demonstrates effective inhibition activity to the propagation of T cell.These digital proofs TGF-β-CSA be activated, and can be used for blocking antigenic specificity propagation, and be useful in vitro and in vivo the Treg cell amplification therefore.

Embodiment 7: with 4-1BBL, CD80 ﹠amp; The non-selective Treg amplification of IL-2

As mentioned above, the generation of understanding diabetes in the NOD mice relates to the destruction of the function of the Treg cell of controlling morbific Teff cell.Compare with the insulin resistance strain, the NOD mice has the Treg cell of lesser amt in periphery, and along with the age demonstrates the decline of Treg cell quantity and function, this outbreak with diabetes is relevant.Really cutter reason is still unknown although cause this situation, and APC regulates the generation of Treg cell and the shortage of the ability kept may play effect.Corresponding to this argument is following observed result: (i) DC among the NOD demonstrates the expression of the costimulatory molecules such as the CD80 of reduction; these costimulatory moleculeses are that important and (ii) various sickness rate to diabetes in the NOD mice has the biological substance of protective effect such as anthelmintic and virus and may induce the Treg cell by the adjusting of DC for the generation of Treg cell and function.

This embodiment has illustrated and used the conjugate that comprises CD80 and/or 4-1BBL and the IL-2 Treg cell that optionally increases in the NOD mice, and shown the effect of this method for the type 1 diabetes among the protection NOD.Conjugate will preferentially increase them in conjunction with the Treg cell and with therapeutic modality.

Diabetes phase coarse among NOD ground can be divided into (1-3 week), insulitis (4-8 week) before the insulitis, prediabetes (8-24 week) and 28 weeks of diabetes (〉).These stages are according to the structure of captive animal and difference.Select prediabetic female NOD mice,, prevent the diabetes in these animals will have clinical effectiveness because these animals will have the Treg cell and the APC deficiency of full-blown autoimmune power and expection.

For the Treg cell that increases in vivo,, comprise the conjugate of CD80 and IL-2 or 4-1BBL and IL-2 for the animal intravenous injection with various combinations, frequency and dosage.Core Succ-PEG-DSPE (CSA)/IL-2 conjugate will be with comparing.By before use CSA-CD80 or CSA-4-1BBL conjugate and biotinylated IL-2 being prepared conjugate with the mixed in molar ratio of 1:1.

Use super excitement (superagonistic) antibody of CD28, find that producing peak value Treg amplification in back 3 days in injection replys.Therefore, measured the level of Treg amplification to animal injection conjugate and just blood-letting before conjugate injection next time in per 3 days.In flow cytometry, use the antibody of anti-CD4 and FoxP3 to finalize the design.In case measured time of peak value Treg cell amplification from this hemanalysis, animal is sacrificed, be used to collect the periphery lymph node, comprise pancreas lymph node, spleen and pancreas.Spleen and lymph node and the GIL that collects from the islets of langerhans of every animal are processed into single-cell suspension liquid, and use various cell surface CD4, CD8,4-1BB, CD62L, TGF-β, CD25 and born of the same parents interior FoxP3, IL-10 and IFN-γ labelling to finalize the design by flow cytometry, to have the comprehensive observing of T cell state.

The processing of prediabetic NOD mice as described herein surpasses rapid amplifying Treg cell in the Teff cell.Expection stimulate altogether by CD80,4-1BBL and IL-2 and the signal of surviving the rapid amplifying that causes the Treg cell is provided.This effect can be further strengthened in activation by DC, and this can give the credit to the amplification and/or the recovery of Treg cell function.Resulting amplification can be a whole body, or the Treg cell can preferably get back to pancreas lymph node and pancreas, is used for protective response.The Treg cell of amplification will be expressed all typical Treg cell markings, as IL-10 in cell surface TGF-β, CD25,4-1BB and the born of the same parents.

Embodiment 8: by non-selective Treg amplification prevention type 1 diabetes

The following ability that has proved that Treg increases and prevents or postpone the type 1 diabetes outbreak in the prediabetic NOD animal.Handle prediabetic NOD (12 weeks are big) animal with the conjugate that comprises CD80 and IL-2 or 4-1BBL and IL-2, as mentioned above, and under the condition that allows the strong amplification of Treg cell, handle, measure as above institute.The diabetes development of monitor animal continued for 25 weeks (till this time, surpassing 85% the unsteered female diabetes that produced in our colony).Think twice continuous every day measurement of glucose levels to surpass 250mg/dl be the confirmation of diabetes.Handle the animal of failure and those sacrifices that do not produce diabetes during with 28 weeks, and collect various tissues, be used for Treg phenotype test and immunohistochemistry, with the state (vs. insulitis before the no vs. insulitis) of measuring disease.Staying the animal of being untreated or handling with the CSA-IL-2 conjugate will be as the contrast of onset diabetes rate.

According to the present invention, the Treg cell amplification in the prediabetes animal will prevent the generation of diabetes.The preventive effect that continues may need to use the periodicity treatment of conjugate, to keep a high proportion of Treg than autoimmune Teff cell.Long-term non-diabetic animal can not have disease maybe may have does not fully have the clinical preceding insulitis that presents.Expect that ill animal has the Treg cell that reduces quantity, express the film TGF-β and the excretory IL-10 of low amount.These animals may also contain the CD4 of a large amount of secretion of gamma-IFN ⁺And CD8 ⁺The Teff cell.

Embodiment 9: use 4-1BBL ﹠amp; The selectivity Treg amplification of self antigen

Although the Treg cell can suppress immunne response in the antigen non-specific mode, the activation of the Treg cell that antigen orders about and amplification can give a lot of advantages with regard to the effect of specificity and raising.This embodiment has illustrated the selective amplification of self antigen specificity T reg cell, uses the conjugate that comprises 4-1BBL and self antigen GAD, in conjunction with the conjugate that comprises CD80 and IL-2 and TGF-β and IL-2, is used for self antigen is sent to DC effectively.Also used rapamycin to improve effect.

As mentioned above, 4-1BB is expressed on DC composing type ground, and will cause the activation of DC by the 4-1BBL/GAD conjugate is bonded by the signal of this receptor, the rise of costimulatory molecules and the effectively synthetic and secretion of the various cytokines that need of t cell response.In addition, CD80 and the IL-2 Treg cell that will preferentially increase, CD28 and CD25 are expressed in its composing type ground.Simultaneously, use TGF-β and IL-2 will block the function and the amplification of Teff cell, and improve the function and the amplification of Treg cell.Rapamycin will be by blocking-up Teff cell propagation and promote its apoptosis to increase the effect of TGF-β, and the amplification of Treg cell is not had main influence.Therefore, in the presence of TGF-β and rapamycin, the activation and the function of Teff cell will be optionally blocked in the identification of self antigen, promote the generation and the amplification of antigen specific T reg cell simultaneously, and 4-1BBL will activate DC, and the Treg that is used to continue replys.

With various combinations, frequency and dosage, comprise 4-1BBL and GAD, CD80 and IL-2 for the prediabetes animals administer (by intravenous injection) in 12 ages in week, and the conjugate of TGF-β and IL-2.By the CSA-4-1BBL conjugate being mixed (as above-mentioned prepared) with biotinylated GAD or preparing conjugate by CSA-CD80 or TGF-β-CSA conjugate are mixed with biotinylated IL-2 as mentioned above.With conjugate treatment whole the duration, give the rapamycin of 1.5mg/kg dosage every day by intraperitoneal.

The peak value of Treg cell amplification in the antibody monitor animal blood of use CD4 and FoxP3 in flow cytometry.In case be measured to the time that peak value Treg replys, make animal euthanasia, be used to collect the periphery lymph node, comprise the pancreas lymph node, spleen and pancreas, and, various cell surfaces and born of the same parents' internal labeling are carried out the multiparameter typing according to described in the above embodiment 7.Stay and be untreated or will be with comparing with the NOD receiver of various conjugate combined treatment., measured before, they have been used to handle another treated animal, be used for prevent diabetes for the most effective condition of Treg cell amplification.

Expect with whole four conjugate (4-1BBL/GAD; 4-1BBL/IL-2; CD80/IL-2; TGF-β/IL-2) therapeutic alliance with rapamycin is the effective scheme that is used for the diabetes of the Treg cell amplification of antigenic specificity and prevention prediabetes animal.The 4-1BBL/GAD conjugate is sent to DC with the GAD self antigen, causes protein processing, the activation of DC and with the GAD submission to Treg cell and morbific Teff cell.TGF-β and rapamycin are blocked the activation and the amplification of self antigen specificity T eff cell with synergism, promote activation and amplification by the Treg cell of CD80/IL-2 and 4-1BBL/IL-2 and/or 4-1BBL/GAD conjugate simultaneously.TGF-β and rapamycin also will promote CD4 by inducing FoxP3 to express ⁺CD25 ^-T cell transformation originally becomes the Treg cell.In with the group of handling one of at least in TGF-β or the rapamycin, will obtain the specific amplification of self antigen specificity T reg cell,, and the amplification of Treg cell significantly not influenced because these two kinds of materials are preferentially blocked the Teff cell proliferation.

The amplification of Treg cell is relevant with the prevention of diabetes.It is useful that the periodicity of use conjugate repeats to treat for keeping the Treg set.Use that other self antigen (conjugate that for example, comprises the self antigen that 4-1BBL is different with other) can other Treg cell of width variety improves effect by increasing more.

Embodiment 10: use DC selective amplification Treg cell that modify, pulse

This embodiment has illustrated the use DC Treg cell that increases, and this DC is with three kinds of mixed pulses mistakes that cause diabetes self antigen (GAD, ICA152 and NRP-A7), and with TGF-β modified.Can use this method separately, or use in conjunction with above-mentioned common stimulation conjugate and/or in conjunction with above-mentioned rapamycin.Use the Treg cell with inducing different types with the DC of three kinds of self antigen pulses, and the direct displaying of the last TGF-β of DC has not only limited anyly may use the relevant toxicity of soluble protein with whole body, but also will increase and/or recover the function of Treg cell effectively, the feasible diabetes that prevent among the NOD.

Use GM-CSF and IL-4 to produce immature DC, as mentioned above from the bone marrow of NOD.With the mixture that causes diabetes antigen: GAD65, islet cells self antigen (ICA512) peptide and NRP-A7 peptide with the cell pulse.By ordering about the immature DC maturation, and characterize, use antibody and the various ripe labelling of anti-CD11c, as high-caliber II class MHC, CD80 and CD86 molecule by flow cytometry with the 4-1BBL night incubation.

(5 μ M EZ-Link Sulfo-NHS-LC-Btioin are Pierce) also with TGF-β-CSA (100ng/10 with the DC biotinylation ⁶Cell) modify, as mentioned above, and under the covering up of rapamycin, intravenous injection is to the prediabetes animal.With different dosage intravenous injection DC, beginning is 5 * 10 ⁵Cell/animal.(DC of two GAD peptides of the usefulness of this dosage and the pulse of a hsp60 peptide has demonstrated the onset diabetes rate that can effectively reduce in the prediabetes NOD mice).

In relevant experiment, also given the CD80/IL-2 and the 4-1BBL/IL-2 conjugate (in conjunction with DC pulse, that modify) that as above make, increase the tolerogenesis effect.

The cell of modifying with the cell of unmodified with CSA is with comparing.As mentioned above, the Treg cell amplification of analyzing animal and the prevention of diabetes.

With the processing of DC unmodified or the pulse that TGF-β modifies and the rapamycin Treg cell that will increase, make prevent diabetes.As mentioned above, TGF-β and rapamycin can collaborative work be blocked the activation and the amplification of self antigen specificity T eff cell, promote the activation and the amplification of Treg cell simultaneously, or it are from CD4 ⁺CD25 ^-The T transformation.Optional CD80/IL-2 and the 4-1BBL/IL-2 conjugate this effect that can further increase of using.

Embodiment 11: use the BMC selective amplification Treg cell of modifying

This embodiment has illustrated external source (allogeneic or the xenogenesis) medullary cell (BMC) that uses with the TGF-β modification Treg cell that increases.Can use this method separately, or in conjunction with above-mentioned common stimulation conjugate and/or in conjunction with rapamycin, as mentioned above.Use the BMC Treg cell that not only increases, but also set up blended chimera, this will control autoimmune power and allow beta Cell of islet regeneration, the feasible diabetes that prevent and/or treat.Use the external source BMC that modifies with TGF-β in conjunction with the conjugate Treg cell that will increase, it will make prevention BMC repulsion and set up mixochimaera subsequently, and this will control from body and two kinds of reactivities of xenogenesis.

(5 μ M EZ-Link Sulfo-NHS-LC-Btioin are Pierce) also with TGF-β-CSA (100ng/10 with the BMC biotinylation as mentioned above ⁶Cell) modify, and under the covering up of rapamycin, intravenous injection is to prediabetic animal.With different dosage intravenous injection BMC, beginning is 5 * 10 ⁵Cell/animal.

In relevant experiment, also given the CD80/IL-2 and the 4-1BBL/IL-2 conjugate (in conjunction with the BMC that modifies) that as above make, increase the tolerogenesis effect.

The cell of modifying with the cell of unmodified with CSA is with comparing.As mentioned above, the Treg cell amplification of analyzing animal and the prevention of diabetes.

With the processing of BMC unmodified or that TGF-β modifies and the rapamycin Treg cell that will increase, make prevent diabetes.As mentioned above, TGF-β and rapamycin can collaborative work be blocked the activation and the amplification of self antigen specificity T eff cell, promote the activation and the amplification of Treg cell simultaneously, or it are from CD4 ⁺CD25 ^-The T transformation.Optional CD80/IL-2 and the 4-1BBL/IL-2 conjugate this effect that can further increase of using.

Embodiment 12: chimeric 4-1BBL (CSA-4-BBL) suppresses Treg cell inhibiting sexual function, orders about Treg cell and Teff cell proliferation simultaneously

We use chimeric 4-1BBL albumen (CSA-4-BBL) to study the effect of 4-1BB signal in the Treg function.Based on [ ³H] in the co-culture experiments introduced of thymidine, from the CD4 of natural B ALB/c mice sorting ⁺CD25 ⁺Two positives (DP) T cell has significantly suppressed to stimulate inductive single positive (SP) CD4 by CD3 ⁺CD25 ^-The proliferative of Teff cell is replied (Figure 15 A).By replenishing the chimeric 4-1BBL of 1 μ g/ml (CSA-4-1BBL) effectively (p＜0.05) and suppressed this inhibition effect specifically to culture, but the CSA reference protein of molar concentration such as use not.

In order to test whether the viewed inhibiting inhibition that is caused by chimeric 4-1BBL is because CD4 ⁺The propagation of recovery that the Teff cell proliferation is replied or inductive Treg cell is caused, uses CFSE (CF 5(6)-Carboxyfluorescein succinimide ester) with the SP cell marking, and is used for co-culture experiments (Fig. 2 B, last figure).(56%) compared with the control stimulates with chimeric 4-1BBL to cause the CD4 that improves altogether ⁺Teff cell proliferation (75%).To significantly reduce CD4 in the Treg cell adding culture ⁺The propagation of Teff cell (30%), this has obtained partly recovering (62%) by 4-1BBL.Lack in the co-culture experiments and reply the CD4 that 4-1BBL stimulates altogether ⁺The full recovery of Teff cell proliferation may be owing to Treg cell and Teff cell competition chimeric protein and/or other factors, as IL-2.In parallel experiment, the DP cell of CFSE labelling is used for co-culture experiments, test the Treg cell and whether also demonstrate the propagation that 4-1BBL is stimulated and reply (Figure 15 B, figure below).When cultivating, there is the remarkable propagation of Treg cell response 4-1BBL when single culture (44%) or in conjunction with SP Teff cell (58%vs. contrast 28%) with 17% comparing of contrast.Generally speaking, these results have proved that 4-1BBL orders about the Treg cell proliferation, and suppress its inhibition function.

Embodiment 13: the CD4 of amplification ⁺CD25 ⁺The phenotype of Treg cell

Use flow cytometry that expanded cells among the embodiment 2 is further characterized traditional Treg cell marking.Treg cellular expression CD25,4-1BB, CD28, GITR, Fas, CD62L and the cell surface TGF-β of amplification.Importantly, compare with those of 4-1BBL useless amplification, all these are marked on the Treg cell of 4-1BBL amplification and are significantly raised (Fig. 9 A and 16A).The Treg cell of amplification has also been expressed signal transcription factor FoxP3, as by (Figure 16 B) that RT-PCR measured, and intracellular signal (Figure 16 C).Importantly, compare with the Treg cell that 4-1BB useless stimulates, the Treg cell of amplification has the FoxP3 albumen of improving the standard in the presence of 4-1BBL.Generally speaking, these digital proofs 4-1BBL stimulate all cells surface markers and the FoxP3 raised the generation/function that relates to natural generation Treg cell.

*?*?*?*

Although enough describe in detail and for example understand the present invention, be used for those skilled in the art and prepare and use, various replacements, changes and improvements should be clearly, and do not break away from the spirit and scope of the present invention.In the expression that this embodiment that provides is a preferred embodiment, be not to plan to limit the scope of the invention.Those skilled in the art will produce change and other purposes wherein.These changes are included in the spirit of the present invention, and are limited by the scope of claim.

To the present invention disclosed herein form various substitute and change and do not depart from the scope of the present invention with spirit be that those skilled in the art are conspicuous.

All patents mentioned in the description and publication are the expressions of those skilled in the art's level.Be incorporated herein all patents and publication as a reference, to the same degree of specially and separately representing as every piece of independent publication to be incorporated herein by reference.

The present invention who is implemented in this illustrative description under the situation of this specially not disclosed any key element, restriction suitably can lacked.Therefore, for example, in this each situation, term " comprises ", " consisting essentially of " and in " comprising " any can be substituted by in other two terms any.Already used term and statement be unrestricted term as describing, do not use shown in such term and the statement eliminating and the intention of described any equivalent characteristics or its part, but think that various changes are possible in the desired scope of the invention.Therefore, although be to be understood that and disclose the present invention specifically by embodiment preferred and optional feature, the change of notion disclosed herein and change to rely on those skilled in the art, and think such change and changing in the scope of the present invention that claims limit.

Other embodiments are listed in following exemplary and the claim subsequently.

Exemplary:

1. combination comprises:

(A) one or morely be selected from following conjugate:

(a) first conjugate, it comprises that (i) comprises the first conjugate member of 4-1BBL polypeptide and (ii) comprise the second conjugate member in conjunction with first right member;

(b) second conjugate, it comprises that (i) comprises the first conjugate member of CD80 polypeptide and (ii) comprise the second conjugate member in conjunction with first right member;

(c) the 3rd conjugate, it comprises that (i) comprises the first conjugate member of TGF-beta polypeptides and (ii) comprise the described second conjugate member in conjunction with first right member;

With

(B) one or morely be selected from following conjugate:

(a ') the 4th conjugate, it comprises that (i) comprises the first conjugate member of anti-CD 3 antibodies and (ii) comprise the described second conjugate member in conjunction with second right member;

(b ') the 5th conjugate, it comprises that (i) comprises the first conjugate member of cytokine and (ii) comprise the described second conjugate member in conjunction with second right member;

(c ') the 6th conjugate, it comprises that (i) comprises the antigenic first conjugate member and (ii) comprise the described second conjugate member in conjunction with second right member; With

(d ') the 7th conjugate, the first conjugate member of it comprises that (i) comprises anti--CD28 antibody and (ii) comprise the described second conjugate member in conjunction with second right member.

In embodiment 1, the selection of cytokine is unrestricted.

2. the combination of embodiment 1 wherein saidly comprises avidin or Succ-PEG-DSPE in conjunction with right described first member, and describedly comprises biotin in conjunction with right described second member.

3. the combination of embodiment 1 wherein saidly comprises the core biotin in conjunction with right described first member.

4. the combination of embodiment 1, at least one in wherein said first, second or the 3rd conjugate comprises fused polypeptide, this fused polypeptide comprises described first conjugate member and the described second conjugate member.

5. the combination of embodiment 1, wherein said cytokine is selected from IL-2, IL-4 or IL-7.

6. the combination of embodiment 1, wherein said antigen is self antigen.

7. the combination of embodiment 1, wherein said antigen are selected from insulin, collagen, myelin basic protein and MHC/ antigenic compound.

8. the combination of embodiment 1, wherein said antigen are selected from glutamte dehydrogenase (GAD), islet cells self antigen (ICA) and self antigen card NRP-A7.

9. the combination of embodiment 1 wherein provides described conjugate in the compositions of separating.

10. the combination of embodiment 6, at least a acceptable carrier on the materia medica, excipient or the diluent of further comprising in the wherein said separate compositions.

11. the combination of embodiment 1 wherein provides described conjugate in single compositions.

12. the combination of embodiment 8, wherein said single compositions further comprises acceptable carrier on the materia medica, excipient or diluent.

13. the combination of embodiment 8, at least one in wherein said first, second or the 3rd conjugate combines with in described the 4th, the 5th, the 6th or the 7th conjugate at least one in conjunction with the combination between right described first and second member by described.

14. the method for amplification Treg cell, described method comprise the Treg cell colony is contacted:

(A) one or morely be selected from following conjugate:

(a) first conjugate, it comprises that (i) comprises the first conjugate member of 4-1BBL polypeptide and (ii) comprise the second conjugate member in conjunction with first right member;

(b) second conjugate, it comprises that (i) comprises the first conjugate member of CD80 polypeptide and (ii) comprise the second conjugate member in conjunction with first right member;

(c) the 3rd conjugate, it comprises that (i) comprises the first conjugate member of TGF-beta polypeptides and (ii) comprise the described second conjugate member in conjunction with first right member;

With

(B) one or morely be selected from following conjugate:

(a ') the 4th conjugate, it comprises that (i) comprises the first conjugate member of anti-CD 3 antibodies and (ii) comprise the described second conjugate member in conjunction with second right member;

(b ') the 5th conjugate, it comprises that (i) comprises the first conjugate member of cytokine and (ii) comprise the described second conjugate member in conjunction with second right member;

(c ') the 6th conjugate, it comprises that (i) comprises the antigenic first conjugate member and (ii) comprise the described second conjugate member in conjunction with second right member; With

(d ') the 7th conjugate, the first conjugate member of it comprises that (i) comprises anti--CD28 antibody and (ii) comprise the described second conjugate member in conjunction with second right member.15. the method for embodiment 14, wherein said Treg cell comprises in described first, second or the 3rd conjugate receptor of at least one, and at least one in wherein said first, second or the 3rd conjugate puted together by combination between described first conjugate member and the described receptor and described Treg cell, and in described the 4th, the 5th, the 6th and the 7th conjugate at least one puted together combination between the member and described Treg cell by described first and second combination.

16. the method for embodiment 14 wherein exsomatizes and realizes described contact.

17. the method for embodiment 16, at least two in the wherein said conjugate contact described Treg cell basically simultaneously.

18. the method for embodiment 16 wherein provides at least two in the described conjugate in single compositions.

19. the method for embodiment 18, at least one in wherein said first, second or the 3rd conjugate puted together in the combination between the member and described the 4th, the 5th, the 6th and the 7th conjugate at least one by described first and second combination.

20. the method for embodiment 16, at least two in the wherein said conjugate contact described Treg cell in order.

21. the method for claim 16 comprises that further the Treg cell with described amplification delivers medicine to the patient.

22. the method for claim 14 wherein realizes described contact in vivo by described conjugate is delivered medicine to the patient.

23. the method for embodiment 14, wherein said Treg cell colony comprises the Treg cell that is selected from CD4+ cell, CD25+ cell and FoxP3+ cell.

24. the method for embodiment 23, wherein said Treg cell colony comprises the CD4+CD25+FoxP3+ cell.

25. the method for embodiment 21 or 22, wherein said patient suffers from autoimmune disease or is in the risk of autoimmune disease.

26. the method for embodiment 25, wherein said patient suffers from type i diabetes or is in the risk of type i diabetes.

27. the method for embodiment 21 or 22, wherein said patient is the external source transplant patient.

28. the method for embodiment 21 or 22 further comprises rapamycin is delivered medicine to described patient.

29. the method for embodiment 21 or 22 comprises that further the compositions that will comprise the foreign cell of showing TGF-β delivers medicine to described patient.

30. the method for embodiment 29 further comprises rapamycin is delivered medicine to described patient.

31. the method for embodiment 29, wherein said foreign cell is selected from splenocyte, islet tissue and medullary cell.

32. the method for embodiment 29 wherein obtains described foreign cell by the following method:

(a) foreign cell contact is comprised in conjunction with first right member and form the foreign cell of modification in conjunction with the bifunctional molecule of the molecule of described cell surface; With

(b) contact of the foreign cell of described modification is comprised TGF-β and described conjugate in conjunction with second right member form the foreign cell of showing TGF-β.

33. the method for embodiment 14 wherein saidly comprises avidin or Succ-PEG-DSPE in conjunction with right described first member, and describedly comprises biotin in conjunction with right described second member.

34. the method for embodiment 14 wherein saidly comprises the core Succ-PEG-DSPE in conjunction with right described first member.

35. the method for embodiment 14, at least one in wherein said first, second or the 3rd conjugate comprises fused polypeptide, and this fused polypeptide comprises described first conjugate member and the described second conjugate member.

36. the method for embodiment 14, wherein said cytokine is selected from IL-2 and IL-4.

37. the method for embodiment 14, wherein said antigen is self antigen.

38. the method for embodiment 14, wherein said antigen are selected from insulin, collagen, myelin basic protein and MHC/ antigenic compound.

39. the method for embodiment 14, wherein said antigen are selected from glutamate decarboxylase (GAD), islet cells self antigen (ICA) and self antigen NRP-A7.

40. the method for embodiment 14 further comprises the free IL-2 of described Treg cells contacting.

41. the method for embodiment 14 further comprised free anti-CD 3 antibodies of described Treg cells contacting or free resisting-CD28 antibody.

42. obtain the method for the pulse dendritic cell of displaying TGF-β, comprising:

(a) with antigen pulse immaturity dendritic cell, to obtain the dendritic cell of pulse;

(b) contact of the dendritic cell of described pulse is comprised in conjunction with first right member and form the pulse dendritic cell of modification in conjunction with the bifunctional molecule of the molecule of described cell surface; With

(c) contact of the pulse dendritic cell of described modification is comprised TGF-β and described conjugate in conjunction with second right member form the pulse dendritic cell of showing TGF-β.

43. the method for embodiment 42, wherein said antigen are to cause the diabetes self antigen.

44. the method for embodiment 43, the wherein said diabetes self antigen that causes is selected from glutamate decarboxylase (GAD), islet cells self antigen (ICA) and self antigen NRP-A7.

45. the method for embodiment 44, the wherein said diabetes self antigen that causes is selected from GAD65 and ICA512.

46. the method for embodiment 45 comprises with the described immaturity dendritic cell of each pulse among GAD65, ICA512 and the NRP-A7.

47. the method for embodiment 42, wherein said antigen is collagen.

48. the method for embodiment 42, wherein said antigen is myelin basic protein.

49. the method for embodiment 42 further comprises the dendritic cell maturation that orders about described pulse.

50. the method for embodiment 49, wherein said ordering about comprises the dendritic cell of hatching described pulse with 4-1BBL.

51. show the dendritic cell population of the antigen pulse of TGF-β.

52. the dendritic cell population of the antigen pulse of the displaying TGF-β that the method by embodiment 42 makes.

53. the method for amplification Treg cell in the patient comprises that the compositions that will contain the dendritic cell of the antigen pulse of showing TGF-β delivers medicine to described patient.

54. the method for amplification Treg cell in the patient comprises that the compositions of the pulse dendritic cell of the displaying TGF-β that the method that will contain by embodiment 42 makes delivers medicine to described patient.

55. the method for embodiment 54 further comprises rapamycin is delivered medicine to described patient.

56. obtain to show the hematopoietic stem cell of TGF-β or the method for medullary cell, comprising:

(a) contact of hematopoietic stem cell or medullary cell is comprised in conjunction with first right member and form the cell of modification in conjunction with the bifunctional molecule of the molecule of described cell surface; With

(b) cells contacting of described modification is comprised TGF-β and described conjugate in conjunction with second right member form the cell of showing TGF-β.

57. the method for embodiment 56 wherein saidly comprises biotin and describedly comprises the core chain mycin in conjunction with right described second member in conjunction with right described first member.

58. the method for amplification Treg cell in the patient comprises delivering medicine to described patient with containing the hematopoietic stem cell of showing TGF-β or the compositions of showing the medullary cell of TGF-β.

59. the method for embodiment 58 wherein prepares the cell of showing TGF-β by the following method, this method comprises:

(a) contact of hematopoietic stem cell or medullary cell is comprised in conjunction with first right member and form the cell of modification in conjunction with the bifunctional molecule of the molecule of described cell surface; With

(b) cells contacting of described modification is comprised TGF-β and described conjugate in conjunction with second right member form the cell of showing TGF-β.

60. the method for embodiment 58 further comprises rapamycin is delivered medicine to described patient.

61. the method for embodiment 58, wherein said patient is need be to the induction of tolerance of self antigen, isoantigen or heteroantigen; β cell regeneration; The prevention of external source transplant rejection; Or the patient of intrinsic hemopoietic disease therapeutic is gone up in heredity.

62. show the medullary cell colony of TGF-β.

63. the medullary cell colony of the displaying TGF-β that makes by the following method, this method comprises:

(a) medullary cell contact is comprised in conjunction with first right member and form the cell of modification in conjunction with the bifunctional molecule of the molecule of described cell surface; With

(b) cells contacting of described modification is comprised TGF-β and described conjugate in conjunction with second right member form the cell of showing TGF-β.

64. show the hematopoietic stem cell populations of TGF-β.

65. the hematopoietic stem cell populations of the displaying TGF-β that makes by the following method, this method comprises:

(a) hematopoietic stem cell contact is comprised in conjunction with first right member and form the cell of modification in conjunction with the bifunctional molecule of the molecule of described cell surface; With

(b) cells contacting of described modification is comprised TGF-β and described conjugate in conjunction with second right member form the cell of showing TGF-β.

Claims (20)

1. combination, it comprises:

(A) one or morely be selected from following conjugate:

(a) first conjugate, it comprises that (i) comprises the first conjugate member of 4-1BBL polypeptide and (ii) comprise the second conjugate member in conjunction with first right member;

(b) second conjugate, it comprises that (i) comprises the first conjugate member of CD80 polypeptide and (ii) comprise the second conjugate member in conjunction with first right member; With

(c) the 3rd conjugate, it comprises that (i) comprises the first conjugate member of TGF-beta polypeptides and (ii) comprise the described second conjugate member in conjunction with first right member;

With

(B) one or morely be selected from following conjugate:

(a ') the 4th conjugate, it comprises that (i) comprises the first conjugate member of anti-CD 3 antibodies and (ii) comprise the described second conjugate member in conjunction with second right member;

(b ') the 5th conjugate, it comprises that (i) comprises the first conjugate member of cytokine and (ii) comprise the described second conjugate member in conjunction with second right member;

(c ') the 6th conjugate, it comprises that (i) comprises the antigenic first conjugate member and (ii) comprise the described second conjugate member in conjunction with second right member; With

(d ') the 7th conjugate, the first conjugate member of it comprises that (i) comprises anti--CD28 antibody and (ii) comprise the described second conjugate member in conjunction with second right member.

2. the combination of claim 1, at least one in wherein said first, second or the 3rd conjugate comprises fused polypeptide, this fused polypeptide comprises described first conjugate member and the described second conjugate member.

3. the combination of claim 1, wherein said cytokine is selected from IL-2, IL-4 or IL-7.

4. the combination of claim 1, wherein said antigen is self antigen.

5. the combination of claim 1, wherein said antigen are selected from insulin, collagen, myelin basic protein, MHC/ antigenic compound, glutamate decarboxylase (GAD), islet cells self antigen (ICA) and self antigen NRP-A7.

6. the combination of claim 1 wherein provides described conjugate in the compositions of separating.

7. the combination of claim 1 wherein provides described conjugate in single compositions.

8. the method for amplification Treg cell, described method comprise the Treg cell colony are contacted:

(A) one or morely be selected from following conjugate:

(a) first conjugate, it comprises that (i) comprises the first conjugate member of 4-1BBL polypeptide and (ii) comprise the second conjugate member in conjunction with first right member;

(b) second conjugate, it comprises that (i) comprises the first conjugate member of CD80 polypeptide and (ii) comprise the second conjugate member in conjunction with first right member;

(c) the 3rd conjugate, it comprises that (i) comprises the first conjugate member of TGF-beta polypeptides and (ii) comprise the described second conjugate member in conjunction with first right member;

With

(B) one or morely be selected from following conjugate:

(a ') the 4th conjugate, it comprises that (i) comprises the first conjugate member of anti-CD 3 antibodies and (ii) comprise the described second conjugate member in conjunction with second right member;

(b ') the 5th conjugate, it comprises that (i) comprises the first conjugate member of cytokine and (ii) comprise the described second conjugate member in conjunction with second right member;

(c ') the 6th conjugate, it comprises that (i) comprises the antigenic first conjugate member and (ii) comprise the described second conjugate member in conjunction with second right member; With

(d ') the 7th conjugate, the first conjugate member of it comprises that (i) comprises anti--CD28 antibody and (ii) comprise the described second conjugate member in conjunction with second right member.

9. the method for claim 8, wherein (a) exsomatizes and/or (b) realizes described contact in vivo.

10. the method for claim 8, wherein said Treg cell colony comprises the Treg cell that is selected from CD4+ cell, CD25+ cell and FoxP3+ cell.

11. the method for claim 8 comprises that further the compositions that will contain the foreign cell of showing TGF-β gives described patient, wherein said foreign cell is selected from splenocyte, islet tissue and medullary cell.

12. the method for claim 8, wherein said antigen are selected from insulin, collagen, myelin basic protein, MHC/ antigenic compound, glutamate decarboxylase (GAD), islet cells self antigen (ICA) and self antigen NRP-A7.

13. obtain the method for the pulse dendritic cell of displaying TGF-β, described method comprises:

(a), obtain the dendritic cell of pulse with antigen pulse immaturity dendritic cell;

(b) contact of described pulse dendritic cell is comprised in conjunction with first right member and form the pulse dendritic cell of modification in conjunction with the bifunctional molecule of the molecule of described cell surface; With

(c) contact of the pulse dendritic cell of described modification is comprised TGF-β and described conjugate in conjunction with second right member form the pulse dendritic cell of showing TGF-β.

14. the method for claim 13, wherein said antigen is selected from collagen; Myelin basic protein; With the diabetes self antigen, wherein said diabetes self antigen further is selected from glutamate decarboxylase (GAD), islet cells self antigen (ICA) and self antigen NRP-A7.

15. show the dendritic cell population of the antigen pulse of TGF-β.

16. the method for amplification Treg cell in the patient, described method comprises that the compositions that will contain the dendritic cell of the antigen pulse of showing TGF-β delivers medicine to described patient.

17. obtain to show the hematopoietic stem cell of TGF-β or the method for medullary cell, described method comprises:

(a) contact of hematopoietic stem cell or medullary cell is comprised in conjunction with first right member and form the cell of modification in conjunction with the bifunctional molecule of the molecule of described cell surface; With

(b) cells contacting of described modification is comprised TGF-β and described conjugate in conjunction with second right member form the cell of showing TGF-β.

18. comprising, the method for amplification Treg cell in the patient, described method deliver medicine to described patient with containing the hematopoietic stem cell of showing TGF-β or the compositions of showing the medullary cell of TGF-β.

19. show the cell colony of TGF-β, wherein said cell is selected from medullary cell and hematopoietic stem cell.

20. the cell colony of the displaying TGF-β of claim 19, described cell colony makes by the following method, and this method comprises:

(a) cells contacting is comprised in conjunction with first right member with in conjunction with the bifunctional molecule of the molecule of described cell surface and forms the cell of modification that wherein said cell is selected from medullary cell and hematopoietic stem cell; With

(b) cells contacting of described modification is comprised TGF-β and described conjugate in conjunction with second right member form the cell of showing TGF-β.

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