CN100406061C - Cancer treatment through suction effect T-cell and loss regulating T-cell - Google Patents

Abstract

The present invention relates to a purpose of loss agents of regulating T-cells in preparing medicine, wherein medicine treats or prevents tumors (especially cancer, such as solid cancer) by losing regulating T-cells of patients (which comprise animals, mammals and especially human beings). The examples of loss agents of the regulating T-cells are CD4<+> antibodies such as anti-CD4<+>CD25<+> antibodies and IL2-toxin. The present invention also relates to a medicinal composition which comprises the loss agents of the regulating T-cells and a method for treating the cancer by the loss agents of the regulating T-cells or the medicinal composition.

Description

The purposes of regulatory T cells consume agent in the preparation medicine

Technical field

The application relates generally to field of cancer, relates more specifically to be used for the method and composition of cancer immunotherapy.

Background technology

The radiotherapy of tumor and chemotherapy can not be removed tumor cell fully, have also damaged immune system simultaneously.Remaining tumor cell (comprising metastatic tumour) has often finally been captured patient's life.Immunization therapy provides the optimal method of removing remaining tumor fully, but tumor is often escaped immune attack because of immune activation is not enough.The not enough reason of immune activation is to immerse the tumor cell deficiency of immuning tissue, or lacks enough strong to soaking the activation signals of Run tumor lymphatic cell.Increase T lymphocyte usually brings good prognosis to the infiltration of tumor.The immunity that how to increase tumor becomes the removing tumor and don't damages immune key.

CD4 ⁺CD25 ⁺The T cell subsets is a kind of effective instrumentality (Sakaguchi, 2004 that are proved to be t cell response in organ specificity autoimmune and chronic infection; Shevach, 2002; Maloy and Powrie, 2001; People such as Casares, 2003; People such as Golgher, 2002).Such viewpoint has been supported in recent research, and the effectiveness of promptly vaccine-induced anti-tumor immune response is subjected to CD4 ⁺CD25 ⁺The restriction of T cell (people such as Machiels, 2001; People such as Sutmuller, 2001; People such as Steitz, 2001; People such as Yu, 2003; People such as Houghton, 2001; People such as Toes, 1999).

Therefore, exist for the needs of understanding the immunne response component that relates in the cancer and mechanism better with for needs based on other cancer therapies of this understanding.

Summary of the invention

The inventor has observed CD4 ⁺CD25 ⁺The selective aggregation of T cell in people and murine tumor.These cells have constituted the major part of tumor infiltrating lymphocyte at the later stage of tumor development.At this, the inventor demonstrates, and has found in tumor and CD8 ⁺The T cell is the CD4 of contact closely ⁺CD25 ⁺The T cell, these T cells have suppressed CD8 in the inherent partial tumor locus of body ⁺The propagation of T cell and interferon-produce.The effect of blocking-up IL-10 and TGF-β can partly reverse by CD4 ⁺The inhibition that cell causes.In addition, the CD4 in the tumor ⁺The partially spent of cell (depletion) causes eradicating the high aggressivity tumor that is completed into and causes forming secular antitumor memory.Therefore, CD4 ⁺CD25 ⁺The T cell has kept such environment in tumor, promptly this environment has been hidden the immunogenicity of tumor cell.This research for example understands, the inhibition of being regulated the antineoplastic immune that cell causes by T mainly appears at tumor locus, and the part of inhibition is reversed even formed late stage in tumor also can be effective treatment for the cancer that is completed into.

One aspect of the present invention relates to such method, and it comprises by use at least a consume regulatory T cells (CD4 particularly to the experimenter ⁺The T cell) first chemical compound consumes regulatory T cells among the experimenter who suffers from cancer (CD4 particularly ⁺The T cell).In most embodiment, this consume cause the experimenter anticancer disease immunity activation and cause the guiding anticancer disease immunne response.In some embodiments of the present invention, the experimenter can have tumor, such as but not limited to solid tumor.The CD4 of consume ⁺The T cell can be positioned at the position of cancer and/or tumor.In some embodiments, the CD4 of consume ⁺The T cell is in tumor.In some embodiments, the CD4 of consume ⁺The T cell is in the outside of tumor.In other embodiments, find the CD4 of consume ⁺The T cell is positioned at the inside and outside of tumor.Consume can only be confined near tumor and/or the CD4 in tumor ⁺The T cell, perhaps consume can be a general." consume (depletion) " is illustrated in regulatory T cells (CD4 particularly ⁺The T cell) any minimizing in localization or the general colony." consume " do not require does not have CD4 ⁺The T cell is retained in some localized areas among the experimenter or in the experimenter.In addition, " consume " do not require that elimination is inner or near all of cancer location or all basically CD4 in experimenter's cancer location ⁺The T cell.In most of embodiments of the present invention, " consume " requires CD4 ⁺The minimizing of the sufficient amount of T cell colony is to cause therapeutic effect.

In certain embodiments, the CD8 among the experimenter ⁺The T cell is with respect to CD4 ⁺The T cell is not consumed.In certain embodiments, CD8 ⁺The T cell has participated in the immunne response that leads anticancer disease.Be noted that " the CD8 among the experimenter ⁺The T cell is with respect to CD4 ⁺The T cell is not consumed " expression, with respect to CD4 ⁺The T cell colony, CD8 ⁺The size of T cell colony is with respect to the CD8 among the experimenter ⁺The T cell does not have the situation of consume not reduce.Embodiment preferred more of the present invention will relate to the CD8 among the experimenter ⁺The T cell colony is with respect to the CD4 among the experimenter ⁺The enrichment of T cell colony.

First chemical compound can be any at present known or measure after a while and be used to consume CD4 ⁺The chemical compound of T cell.In some embodiments, first chemical compound is an anti-CD 4 antibodies.Antibody can be the antibody of anti-people CD4.Antibody can be monoclonal antibody.Antibody can be humanized antibody.In other embodiments, first chemical compound is anti-CD 25 antibody or CD4 ⁺CD25 ⁺The IL-2 toxin.The IL-2 toxin can preferentially consume CD4 ⁺CD25 ⁺Cell.

In certain embodiments, this method is further defined as to the experimenter and uses at least a second chemical compound.Second compositions can be CD8 ⁺The activator of T cell.CD8 ⁺The activator of T cell can be an antibody, including, but not limited to monoclonal antibody and/or humanized antibody.CD8 ⁺The activator of T cell can be that coding activates CD8 ⁺The peptide of T cell or proteinic nucleic acid.For example, activator can be the nucleic acid of coding LIGHT.In certain embodiments, second chemical compound is a chemotherapeutics.Chemotherapeutics can be to CD8 ⁺The chemotherapeutics that the T cell is nontoxic basically.Chemotherapeutics can be for CD8 ⁺The T cell is compared to CD4 ⁺The T cell has littler toxic chemotherapeutics.Second chemical compound can be an anti-cancer vaccine.Anti-cancer vaccine can be gene vaccine or peptide vaccine.Second chemical compound can be the nucleic acid of coding therapeutic peptide.In certain embodiments, first chemical compound and second chemical compound are sent simultaneously.In certain embodiments, first chemical compound is sent in different with second chemical compound.First chemical compound is used according to the dosage that the size of tumor can 60 μ g-600 μ g anti-CD 4 antibodies.Can not produce significantly influence like this for the cd4 cell of whole body.

The experimenter can be a mammal, including, but not limited to the people.Cancer can be any type of cancer.For example, in some cases, cancer is the solid cancer, such as, but be not limited to breast carcinoma, ovarian cancer, pulmonary carcinoma, head and neck cancer, carcinoma of prostate, thyroid carcinoma, bladder cancer, gastric cancer, the brain cancer, melanoma, nasopharyngeal carcinoma, skin carcinoma or renal carcinoma.Cancer can be the cancer that shifts, for example breast carcinoma of Zhuan Yiing.Cancer can be hemapoietic cancer, for example lymphoma or leukemia.Certainly, those of skill in the art will appreciate that the cancer that can treat any number according to the present invention.

Another aspect of the present invention relates to the diagnosis or the method for prognosis of cancer, and it comprises the CD4 that analyzes among the experimenter ⁺The T cell colony.CD4 ⁺The T cell can be arranged in experimenter's tumor.In certain embodiments, CD4 in the tumor ⁺The level of the rising of T adjusting cell shows the poor prognosis for the experimenter.In certain embodiments, this method further comprises the CD8 that analyzes among the experimenter who suffers from cancer ⁺The T cell colony.With respect to normal CD8 ⁺The T cellular level, CD8 among the experimenter ⁺The rising of T cell or normal level can be the positive prognostic indicator and/or the diagnosis of cancer.

Another aspect of the present invention relates to the purposes of regulatory T cells consume agent in the preparation medicine, wherein said medicine (comprises animal by the consume patient, mammal, particularly human) regulatory T cells and treat or prophylaxis of tumours (particularly cancer, for example solid carcinoma).

The present invention relates to pharmaceutical composition on the other hand, it comprises regulatory T cells consume agent as active component, described pharmaceutical composition (comprises animal by the consume patient, mammal, particularly human) regulatory T cells and treat or prophylaxis of tumours (particularly cancer, for example solid carcinoma).

Regulatory T cells consume agent is chemical compound, compositions, mixture or the combination formulations of any or multiple consume regulatory T cells." consume " speech is above having explanation.Regulatory T cells consume agent includes, but not limited to anti-regulatory T cells antibody, as humanized anti-CD 4 antibodies; Perhaps disclosed herein, prior art is known or will found consume regulatory T cells (CD4 particularly ⁺The T cell) any other chemical compound or one or more this combination of compounds.

In these areas, described regulatory T cells CD4 preferably ⁺T cell, particularly CD4 ⁺CD25 ⁺The T cell.Described regulatory T cells can be at the tumor locus place, and/or in tumor, and/or outside tumor.Described consume can be a tumor by local, and/or is general.

In one embodiment, described regulatory T cells consume agent is the antibody of regulatory T cells, particularly anti-CD4 ⁺Antibody is as anti-CD4 ⁺CD25 ⁺Antibody; Perhaps, described regulatory T cells consume agent is the IL2-toxin.The preferably anti-people CD4 of described antibody ⁺Antibody.Described antibody can be monoclonal antibody or polyclonal antibody.Preferably, antibody is humanized antibody.

In one embodiment, described patient's CD8+T cell is not consumed.Preferably, the CD8+T cell participates in the immunne response to tumor.

In another embodiment, described medicine also comprises other active component, and described medicine is formulated into the form that is suitable for described regulatory T cells consume agent and administration simultaneously of described other active component or separate administration.Described other active component can be the activator of CD8+T cell.The example of described activator is an antibody, monoclonal antibody especially, humanized antibody etc.In a specific embodiments, described activator is the nucleic acid that coding activates the polypeptide of CD8+T cell.Especially, described activator is nucleic acid, the especially nucleic acid of encoding mutant LIGHT of coding LIGHT.

In one embodiment, described other active component is to comprise and express the tumor cell of nucleic acid of encoding mutant LIGHT or tumor (preferably, this tumor cell or tumor also comprise and expressing tumor antigen) or comprise Pharmaceutical composition, particularly therapeutic vaccine or the preventative vaccine of tumor cell or tumor.In another embodiment, described other active component is recombinant virus (preferred recombinant adenovirus or fowlpox virus) that comprises and express the nucleic acid of encoding mutant LIGHT or the Pharmaceutical composition that comprises this recombinant virus.Preferably, described sudden change LIGHT comprises and stops the sudden change of protease to its degraded, for example, described sudden change LIGHT is the LIGHT that does not contain protease cracking site, does not particularly contain the sudden change LIGHT of the proteolysis site EKLI (EQLI among the mankind) of LIGHT albumen 79-82 position.Perhaps, described sudden change LIGHT preferably has extracellular domain amino acid sequence and sequence label (for example being convenient to the label of purification), as has the aminoacid sequence and the flag sequence of LIGHT 85-239 position.

In one embodiment, described other active component is a chemotherapeutic agent, especially the CD8+T cell is not had substantive toxic chemotherapeutics, perhaps the toxicity of CD8+T cell is compared the little chemotherapeutics of toxicity of CD4+T cell.For example, described other active component is the radiotherapy medicine.

In one embodiment, described other active component is an anti-cancer vaccine, for example gene vaccine or peptide vaccine.

In one embodiment, described other active component is the nucleic acid of therapeutical peptide or coding therapeutical peptide.

In one embodiment, described tumor is a solid carcinoma, for example breast carcinoma, ovarian cancer, pulmonary carcinoma, head and neck cancer, carcinoma of prostate, thyroid carcinoma, bladder cancer, gastric cancer, the brain cancer, melanoma, esophageal carcinoma, skin carcinoma or renal carcinoma, particularly breast carcinoma, metastatic breast cancer; Perhaps described tumor is the blood cancer, for example lymphoma or leukemia.

The present invention also relates on the other hand and can be used for analyzing regulatory T cells in patient's body (particularly CD4+T cell, particularly CD4 ⁺CD25 ⁺The T cell) reagent of colony is used for the purposes of the medicament or the kit of cancer diagnosis or prognosis in preparation.Preferably, described regulatory T cells is in patient tumors.At this moment, especially, the regulatory T cells level raises and shows that patient's prognosis is not good in the tumor.

In one embodiment, described medicament or kit also comprise the reagent that is used to analyze CD8+T cell colony in cancer patient's body.In this embodiment, especially, with respect to the normal level of CD8+T cell, rising of CD8+T cellular level or normal level are the positive prognostic indicator and/or the diagnosis of cancer in described patient's body.

The invention still further relates to the recombinant virus that comprises and express the nucleic acid cDNA of encoding mutant LIGHT on the other hand, preferably, this recombinant virus is recombinant adenovirus or fowlpox virus.Preferably, described sudden change LIGHT comprises and stops the sudden change of protease to its degraded, for example, described sudden change LIGHT is the LIGHT that does not contain protease cracking site, does not particularly contain the sudden change LIGHT of the proteolysis site EKLI (EQLI among the mankind) of LIGHT albumen 79-82 position.In another embodiment, described sudden change LIGHT has extracellular domain amino acid sequence and sequence label (for example being convenient to the label of purification), as has the aminoacid sequence and the flag sequence of LIGHT 85-239 position.

The invention still further relates to the Pharmaceutical composition that comprises recombinant virus of the present invention, particularly therapeutic vaccine or preventative vaccine on the other hand.

The invention still further relates to virus of the present invention or compositions on the other hand and be used for the treatment of purposes in the medicine of cancer in preparation.

On the other hand, the invention still further relates to the treatment cancer or destroy the method for tumor, comprising: comprise and expresses the recombinant virus (preferably recombinant adenovirus or fowlpox virus) of the nucleic acid of encoding mutant LIGHT or comprise the Pharmaceutical composition of this recombinant virus to the cancer patient; Perhaps comprise and express the tumor cell of nucleic acid of encoding mutant LIGHT or tumor (preferably to the cancer patient, this tumor cell or tumor also comprise and expressing tumor antigen) or comprise Pharmaceutical composition, particularly therapeutic vaccine or the preventative vaccine of this tumor cell or tumor.In a preferred embodiment, described is to give in tumor.

On the other hand, the invention still further relates to the tumor cell or the tumor that comprise and express the nucleic acid of encoding mutant LIGHT, preferably, this tumor cell or tumor also comprise and expressing tumor antigen; Also relate to the Pharmaceutical composition that comprises tumor cell of the present invention or tumor, particularly therapeutic vaccine or preventative vaccine.

On the other hand, the invention still further relates to the method that is used to screen or test cancer therapeutic agent, comprise by in the body or experiment in vitro test candidate substances whether can consume regulatory T cells.

When in claim and/or description, " containing " when being used in combination with term, make word " a " or " an " can represent " one ", but it also is that the implication with " one or more ", " at least one " and " or more than " is consistent." another " of Shi Yonging can be represented at least the second or more a plurality of herein.

Can consider that any embodiment of being discussed in this description can be implemented with regard to any method of the present invention or compositions, vice versa.In addition, compositions of the present invention can be used to obtain method of the present invention.

In whole this application, term " approximately " is used to show that value comprises the equipment that is used to measure this value, the constant error of method changes, and perhaps is included in the variation that exists among the research experimenter.

Use in the claims term " or " be used for expression " and/or ", unless show clearly and only relate to a kind of selection, perhaps two kinds of selections are to repel mutually.

When using in this description and claim, speech " contains ", " having ", " comprising " or " comprising " be wide in range or unconfined, and they do not discharge composition or method step other, that do not describe.

Other targets of the present invention, feature and advantage will be well understood to from following detailed description.; pointed out particular embodiment of the present invention though should be appreciated that detailed description and special embodiment, just explanation for example; because from this describes in detail, various variations within the spirit and scope of the present invention and modification all can be conspicuous to those skilled in the art.

Description of drawings

Following accompanying drawing has formed the part of this description, comprises that these accompanying drawings are in order further to prove some aspect of the present invention.The present invention may be better understood by the detailed description with reference to one or more in these accompanying drawings and the particular embodiment that provides of associating herein.

Fig. 1 .T cellular infiltration breast cancer tissue.Inspection is from 20 patients' breast carcinoma sample, and demonstrates representative picture.Original position (a and b) or the h and E (H﹠amp of (c and d) breast cancer tissue that invades; E) (a and c) or anti-CD4 (brown) and anti-CD8 (blueness) (b and d) dyeing.Arrow in plate b and d indicates the zone that is positioned at the lower right corner that shows with higher enlargement ratio.In the high power field (* 40) of random choose, in 10 original positions and breast cancer tissues 10 intrusions, count CD8 ⁺Cell and CD4 ⁺The quantity of cell, and for each cancerous tissue calculating CD4 ⁺Cell and CD8 ⁺The ratio of cell.

Fig. 2 .CD4 ⁺CD25 ⁺Thereby the T cell is assembled the rejection that suppresses tumor in inside tumor.A, strong antigen L ^dDo not stop tumor growth.With 5 * 10 ⁵Ag104 and Ag104L ^dThe tumor cell subcutaneous vaccination is to the C3B6F1 mice.Detect growth of tumor, and following volume calculated: volume=length * wide * height/2.(b-c), with 10 ⁶Ag104L ^dTo the C3B6F1 mice, and groin LN, spleen and tumor tissues are isolated in after tumor inoculation 7 or 16 days to tumor cell from mice in the base portion subcutaneous vaccination of tail.Measure CD4 by FACS ⁺CD25 ⁺The T cell in lymphocyte percent (b) and CD45RB at CD4 ⁺CD25 ⁺Or CD4 ⁺CD25 ^-Expression on the T cell (c).(d-e), spleen and tumor tissues are collected in after tumor inoculation 16 days from these have the mice of tumor.By the negative selectivity globule method of using magnetic systems from spleen enrichment CD4 ⁺The T cell.Further will be with magnetic systems from the CD4 of spleen ⁺CD25 ^-T cell and CD4 ⁺CD25 ⁺The T cell separation is opened.Tumor-infiltrated T cell is with biotinylated anti-Thy1.2 antibody and carry out enrichment with the antibiotin magnetic bead subsequently.Soak into the CD4 of tumor ⁺The T cell further separates with FACS, and this is to classify after the anti-CD 4 antibodies that uses PE to put together dyes.D is from the CD4 of these purification ⁺Separate obtaining total RNA in the T cell colony, and the cDNA that is derived from RNA is implemented PCR in real time about foxp-3.E is with every hole 5 * 10 in the 96 hole flat boards ⁴Individual purified CD4 from not tried mice originally ⁺CD25 ^-The T cell stimulated 72 hours with the anti-cd 3 antibodies through the bag quilt of optimal dose (2 μ g/ml).Perhaps add 5 * 10 ⁴Individual from the tumor tissues of the mice with tumor or the CD4 of spleen ⁺The T cell, so as with the CD4 of purification ⁺CD25 ^-T co-culture of cells and the addings in last 24 hours of cultivating ³H, and measure mixing of its.(f), after tumor inoculation, give the mice anti-CD 25 antibody in 14 days, collect spleen and tumor tissues after 2 days, will be at the CD25 in CD4 or the cd8 cell ⁺Or CD25 ^-The percent of cell and untreated comparing.When consuming CD4 with antibody in vivo ⁺(g) or CD25 ⁺(h) during cell, the monitoring growth of tumor.

Fig. 3 .CD4 ⁺Cell suppresses tumor-infiltrated CD8 ⁺The propagation of T cell and IFN-γ produce.With 10 ⁶Ag104L ^dThe tumor cell subcutaneous vaccination is to the C3B6F1 mice, and after tumor inoculation 14 days intraperitoneal (i.p.) injection anti-CD 4 antibodies (GK1.5).After the CD4 consume, 1 week from mice, isolated spleen and tumor tissues.Measure CD8 in the tumor tissues by FACS ⁺The percent of T cell (a).With anti-Thy1.2 magnetic bead system's tumor-infiltrated T cell of enrichment (TIL).The TI L of splenocyte and purification stimulated 4 hours in the presence of brefeldin A at external use PMA and ionomycin.Measure total CD8 in the tumor by FACS ⁺The CD8 of generation IFN-γ among the T cell ⁺The T percentage of cells.With 10 ⁶The MC57-SIY tumor cell at a plurality of sites subcutaneous injection to being in Rag-1 ^-/-The 2C TCR transgenic mice of background is to activate the 2CT cell.Separated 2C T cell in 96 hours after activating, it has CD62L ^LoCD44 ^HighPhenotype.The activated 2C T cell of CFSE labelling is transferred to these in the mode of entrusting one's child to the care of sb. has Ag104L ^dIn the mice of the process of tumor or not process CD4 consume.After transferase 12 C T cell, from mice, collected tumor tissues in 2 days.Monitor the propagation of the 2C T cell of CFSE labelling by FACS.

Fig. 4 .CD4+ cell keeps the antiinflammatory environment of inside tumor.(a-b), between tumor inoculation 1 day, consume CD4 by handling mice with anti-CD 4 antibodies intraperitoneal (i.p.) ⁺Cell.With 10 ⁶Ag104L ^dThe tumor cell subcutaneous vaccination is collected spleen (a) and tumor tissues (b) and homogenate 2 weeks to the C3B6F1 mice from mice after tumor inoculation.With fragment rotation precipitation, collect supernatant and its dosed cells is measured globule array test kit (CBA).C is with 10 ⁵Ag104L ^dThe tumor cell subcutaneous vaccination is to the C3B6F1 mice, and 1 day and 7 days afterwards inject the 100 anti-IL-10 receptor of μ g barrier (IL-10R) antibody to mice before tumor inoculation.Unite anti-IL-10R and handle, after tumor inoculation 7 days, give mice in intraperitoneal (i.p.) mode 100 μ gLPS or 100 μ g antagonism anti-CD 40 antibodies.D is with 10 ⁵Ag104L ^dThe tumor cell subcutaneous vaccination is to the C3B6F1 mice, and 1 day and 7 days afterwards inject anti-CD 4 antibodies or 250 μ g anti-TGF-beta antibodies to mice before tumor inoculation.The monitoring growth of tumor.

Fig. 5 .CD4 ⁺Consume causes the rejection of the tumor that forms in the tumor of cell.With 10 ⁵Ag104L ^dThe tumor cell subcutaneous vaccination is to the C3B6F1 mice, and injects 12.5 μ g anti-CD 4 antibodies (GK1.5) in the mode in the tumor in 14 days after tumor inoculation.After CD4 consume 2 days, from mice, isolate spleen and tumor tissues.By the CD4 among FACS mensuration spleen or the tumor tissues medium-sized lymphocyte ⁺The percent of T cell (a).Monitoring growth of tumor (b).

The specific embodiment

The present invention provides in certain embodiments and has comprised consume CD4 ⁺The T cell interior for treatment for cancer.In some embodiment, anti-CD 4 antibodies such as humanization anti-CD 4 antibodies can be used at the position of tumor consume CD4 ⁺Therefore the T cell makes CD8 ⁺Tumor can more effectively be discerned and destroy to the T cell.

Humanized antibody

Certain embodiments of the present invention are intended to the use of antibody, comprise anti-CD 4 antibodies.Relate to preparation monoclonal and polyclonal antibody, comprise that the method for preparing humanized antibody is well-known (people such as Smith, 2004 in the art; People such as Hinoda, 2004).The method that is used to prepare humanized antibody comprises and relates to immunity produces the animal of humanized antibody owing to gene alteration (for example transgenic is integrated into the gene that at least one causes producing humanized antibody) method.

" humanization " of Shi Yonging is defined as such chemical compound herein, in the time of promptly in being injected into human body, can not produce significant immunne response in the treatment that resists this chemical compound of leading.For example, in the time of in being injected into human body, the humanization anti-CD 4 antibodies can produce anti-CD4 ⁺The significant immunne response of T cell; , in human body, there is not to produce significant immunne response in the treatment that resists this humanization anti-CD 4 antibodies.

The method that is used to produce polyclonal antibody is well-known in the art.Polyclonal antibody can wish to produce the target of antibody and adjuvant and form in animal for it by the injection of repeatedly subcutaneous (sc) or intraperitoneal (ip).In some cases; with having in the fragment of target or target amino acid sequence and the species for the treatment of immunity at this that immunogenic protein (for example, using keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin or the soybean trypsin inhibitor of difunctionality or derivatization reagent such as dimaleoyl imino benzoyl sulfosuccinimide ester, N-hydroxy-succinamide, glytaraldehyde or succinic anhydrides) puts together is useful.Then, the antibody of the anti-target of guiding that animal can be produced carries out purification subsequently, and is used for the treatment of.

Being used to produce monoclonal antibody method also is well-known in the art.Obtain monoclonal antibody from the colony of the antibody of homogeneous basically, the antibody that promptly comprises this colony is same, except the sudden change of the possible natural appearance that can lesser amt exists.Therefore, modifier " monoclonal " has shown that the feature of antibody is not the mixture of unrelated antibody.Be used to prepare monoclonal antibody method and generally include the use hybridoma.

The antibody of other types also can be used for the present invention.For example, can use bi-specific antibody in certain embodiments of the invention, it has binding specificity at least two kinds of different antigens, and/or mixes and put together antibody, and it is made up of two covalently bound antibody.

Pharmaceutical composition

Pharmaceutical composition of the present invention contains one or more consumes CD4 of effective dose ⁺The chemical compound of T cell (for example, humanized anti-CD 4 antibodies; Perhaps disclosed herein, prior art is known or will found consume CD4 ⁺Any other chemical compound of cell) or be dissolved or dispersed in additional material in the medicine acceptable carrier.Term " medicine or pharmacology are acceptable " is meant in the time being applied to animal (if suitable, for example be the mankind), does not produce the molecular entity and the compositions of disadvantageous, hypersensitive or other undesired reaction.According to disclosure of the present invention and Remington ' s Pharmaceutical Sciences, 18th Ed.MackPrinting Company, the explanation of 1990 (herein quoting as a reference), those of ordinary skills know how to prepare contains at least a consume CD4 ⁺The pharmaceutical composition of the chemical compound of T cell or extra active component.And, use for animal (for example human), should understand the desired aseptic of biologic criteria, pyrogen, overall security and purity rubric that said preparation should satisfy FDA.

As used herein, " medicine acceptable carrier " is meant any and all solvents known to those of ordinary skills, disperse medium, coating materials, surfactant, antioxidant, antiseptic (antibacterial agent for example, antifungal), isotonic agent, absorption delays reagent, salt, antiseptic, medicine, the medicine stabilizing agent, gel, bonding agent, excipient, disintegrating agent, lubricant, sweeting agent, flavoring agent, dyestuff, similar substance and combination thereof (referring to, Remington ' sPharmaceutical Sciences for example, 18th Ed.Mack Printing Company, 1990, pp.1289-1329 quotes as a reference herein).Unless conventional carriers and active component are incompatible, otherwise they may be used in the described pharmaceutical composition.

Consume CD4 ⁺The chemical compound of T cell can contain dissimilar carriers, and this depends on that it is to use and whether it needs to sterilize to be applicable to the route of administration of injection and so on the form of solid, liquid or aerosol.The present composition can be as known to persons of ordinary skill in the art in intravenous, Intradermal, transdermal, sheath, intra-arterial, intraperitoneal, intranasal, internal rectum, epidermis (topical), intramuscular, subcutaneous, mucosa ground, oral, epidermis ground, partly, the modes such as (for example aerosol suction), injection, infusion, continuous infusion that suck use, or directly, through conduit, through douche and regional perfusion is spread in target cell, or use with cream, fluid composition (for example liposome) or alternate manner or above-mentioned any combination.Referring to, Remington ' s Pharmaceutical Sciences for example, 18th Ed.Mack PrintingCompany, 1990, quote as a reference) herein.

The combination treatment that is used for cancer

In order to strengthen consume CD4 ⁺The usefulness of the chemical compound of T cell (for example humanized anti-CD 4 antibodies) it is desirable to these compositionss of the present invention and method and effectively material (for example antitumor and anticancer agent) coupling of treatment hyperplasia disease." anticancer " reagent can produce negatively influencing to the cancer among the experimenter, for example, by kill one or more cancerous cell, in one or more cancerous cell cell death inducing, reduce one or more cancerous cell growth rate, reduce metastasis rate or number, minimizing tumor size, suppress tumor growth, reduce blood supply, improve immunne response, prevent or suppress the development of tumor or increase life-span of cancer patient to one or more cancerous cell or tumor to tumor or one or more cancerous cell.Antitumor and anticancer agent comprises, for example, chemotherapy agents (chemotherapy), radiotherapy reagent (X-ray therapy), operation method (operation), immunization therapy reagent (immunotherapy), genetic therapy reagent (gene therapy), hormonotherapy, other biological reagent (biotherapy) and/or substituting therapy.

More generally, such reagent will with consume CD4 ⁺The chemical compound of T cell provides with effective kill cancer cell or the combined amount that suppresses its propagation together.Such method can comprise: with cell and reagent and consume CD4 ⁺The chemical compound of T cell contacts simultaneously, and perhaps contact in a period of time wherein will be consumed CD4 ⁺The chemical compound of T cell and reagent separate administration have produced required therapeutic effect for cell, tissue or biology.This also can realize by following manner: with cell, tissue or biologically consume CD4 with containing simultaneously ⁺The single compositions or the pharmaceutical preparation of the chemical compound of T cell and one or more reagent contact, perhaps that cell is different with two or more compositionss or preparation contact, and wherein a kind of compositions comprises consume CD4 ⁺The chemical compound of T cell, and another kind of compositions comprises one or more reagent.

When being used for cell, tissue or biochron, term " contact " and " exposure " are meant a kind of like this process herein, by means of this process, and consume CD4 ⁺The treatment construction of the chemical compound of T cell and/or other reagent (for example chemotherapy agents or radiotherapy reagent) is sent in target cell, or is directly placed described target cell, tissue or biology side by side.For cell killing or make the cell tranquillization, consume CD4 ⁺The chemical compound of T cell and/or additional agents are effectively to kill described cell or to prevent that their splitted combined amount are delivered to one or more cells.

Consume CD4 ⁺The chemical compound of T cell can be before other reagent, with its simultaneously and/or after it, use, interval therebetween be several minutes to a few weeks longer.In some embodiments, consume CD4 ⁺The chemical compound of T cell and other reagent separate administration are in cell, tissue or biology, and in such embodiments, the interval between should guaranteeing usually at every turn to send should be not oversize, thereby make consume CD4 ⁺The chemical compound of T cell and reagent still can apply favourable combined effect to described cell, tissue or biology.For example, under these circumstances, imagination can be with two, three, four or more features (modality) and consume CD4 ⁺Almost (promptly being less than about 1 minute) and described cell, tissue or biology contact the chemical compound of T cell simultaneously.In others, one or more reagent can with the consume CD4 ⁺The chemical compound of T cell is almost used simultaneously, or is using consume CD4 ⁺Before the chemical compound of T cell and/or about afterwards 1 minute, about 5 minutes, about 10 minutes, about 20 minutes, about 30 minutes, about 45 minutes, about 60 minutes, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, about 20 hours, about 21 hours, about 22 hours, about 22 hours, about 23 hours, about 24 hours, about 25 hours, about 26 hours, about 27 hours, about 28 hours, about 29 hours, about 30 hours, about 31 hours, about 32 hours, about 33 hours, about 34 hours, about 35 hours, about 36 hours, about 37 hours, about 38 hours, about 39 hours, about 40 hours, about 41 hours, about 42 hours, about 43 hours, about 44 hours, about 45 hours, about 46 hours, about 47 hours, about 48 hours, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 7 days, about 8 days, about 9 days, about 10 days, about 11 days, about 12 days, about 13 days, about 14 days, about 15 days, about 16 days, about 17 days, about 18 days, about 19 days, about 20 days, about 21 days, about 1 week, about 2 weeks, about 3 weeks, about 4 weeks, about 5 weeks, about 6 weeks, use within about 8 weeks of about 7 all or or more weeks, or in any time scope that derives by these time, use.

Can use consume CD4 ⁺The various combination scheme of the chemical compound of T cell and one or more reagent.The non-limitative example of this kind combination is as follows, wherein contains consume CD4 ⁺The compound compositions of T cell is " A ", and reagent is " B ":

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/B

B/B/B/A B/B/A/B A/A/B/B A/B/A/B A/B/B/A B/B/A/A

B/A/B/A B/A/A/B A/A/A/B B/A/A/A A/B/A/A A/A/B/A

Contain consume CD4 ⁺The compound compositions of T cell can be followed the general approach of using chemotherapeutics to cell, tissue or biological using and carries out, and considers toxicity (if virose words) simultaneously.Expection, if necessary, should the repetitive therapy cycle.In specific embodiment, imagine multiple additional agents can with any applied in any combination of the present invention.LIGHT treatment and LIGHT treatment are consumed the associating that agent is treated with regulatory T cells

LIGHT is one and newfoundly acts on immunoregulatory gene.Thereby the sudden change that we carried out can improve it at the stability of cell surface expression immune response stimulating better.Four amino acid whose sudden changes to LIGHT have stoped this proteinic degraded.We are converted into thought and new mutant gene that adenoid invention has comprised a new genetic immunization treatment about adopting LIGHT with tumor tissues.

The immunotherapy method that we are newly clear and definite a kind of: make lymphocyte can directly contact tumor cell by in tumor tissues, bringing out lymphoid tissue, thereby induce suitable immunoreation to eradicate tumor in inside tumor to tumor.LIGHT is the ligand as the HVEM of the lymph poison receptor of stromal cell expression and T cellular expression.Among LIGHT introducing tumor tissues, will induce high-caliber chemokines and adhesion molecule, and follow the Jin Run of a large amount of not activated T lymphocytes sudden change.The mutant of LIGHT has stoped the degraded of protease to it, makes that LIGH can be in the surface of tumor cells expressed intact.LIGHT will have great help to the selective activation of TS T lymphocyte reaction to immunoreactive synergistic activation activity, remove the existing high progressive primary tumo(u)r and the tumor at other positions of deriving thereby be of value to.The tumor cell of expressing LIGHT can be used as clinical relevant therapeutic vaccine, can be used for eradicating the primary tumo(u)r of having deposited.The tumor cell of expressing LIGHT will attract and activate lymphoblast as clinical relevant therapeutic vaccine in inside tumor, be used to remove tumor thereby produce more antitumor lymphocyte.Our research has proved that tumor necrosis factor superfamily (TNFSF14) and LIGHT can form receptor signal and attract the T cell of tumour-specific at the stromal cell of inside tumor and lymphocytic cell surface.We find that the tumor cell that LIGHT transforms can strengthen by the new effect of collaborative its two receptoroid depositing the immune clearance reaction of primary tumo(u)r.Our data shows that the LIGHT of expression of tumor tissue has the stronger scavenging action that excites former tumor progression, be better than excitation (Nature Immunology, Vol.5, the No.2 of other cytokines and collaborative stimulation molecule, in February, 2004, p.141-149).Up to the present, resemble LIGHT like this, have so dual calling up not see the report that any other is arranged as yet with the molecule of the usefulness of activated T cell.Such characteristic makes us can develop a kind of brand-new immunotherapy method to tumor.

In a preferred embodiment, described LIGHT is human LIGHT.In a further preferred embodiment, LIGHT is sudden change LIGHT.Particularly preferably be, described sudden change LIGHT comprises and stops the sudden change of protease to its degraded, for example, described sudden change LIGHT is the LIGHT that does not contain protease cracking site, does not particularly contain the sudden change LIGHT of the proteolysis site EQLI (for the people) of LIGHT albumen 79-82 position.In another embodiment, described sudden change LIGHT has extracellular domain amino acid sequence and sequence label (for example being convenient to the label of purification), as has the aminoacid sequence and the flag sequence of LIGHT 85-239 position.LIGHT aminoacid sequence and amino acid residue are numbered referring to Nature Immunology, Vol.5, and No.2, in February, 2004, p.141-149.

The administration of LIGHT or mutant LIGHT is preferably by various viral delivery systems or express that the tumor cell of LIGHT carries out.Particularly, comprising: comprise and express the recombinant virus (preferred recombinant adenovirus or fowlpox virus) of the nucleic acid of encoding mutant LIGHT or comprise the Pharmaceutical composition of this recombinant virus to the cancer patient; Perhaps comprise and express the tumor cell of nucleic acid of encoding mutant LIGHT or tumor (preferably to the cancer patient, this tumor cell or tumor also comprise and expressing tumor antigen) or comprise Pharmaceutical composition, particularly therapeutic vaccine or the preventative vaccine of this tumor cell or tumor.In a preferred embodiment, described is to give in tumor.Particularly, above-mentioned recombinant virus or tumor cell injection are arrived former position of tumor

Adenovirus that preferred viral delivery system includes, but not limited to recombinate and fowlpox virus (fowlpox virus) wait other virus.In particularly preferred embodiments, mutant LIGHT comes administration by the adenovirus and the fowlpox virus of reorganization.If the primary tumo(u)r cell fails in the month to be eliminated at 1-2, tumor will be by excision or radiating irradiation.Our previous experiments is found to handle through the treatment of LIGHT, and animal body has been set up intensive tumour immunity memory, can remove the tumor at tip position.Thereby we estimate that same scavenging action be arranged the tumor at diffusion transfer position.

Use the adenovirus and the fowlpox virus of reorganization to come administration to have special advantage, for example, efficient is higher and safer.Owing to be easy to produce and the output height, recombinant adenovirus has been widely used in the humans and animals experiment, but Most patients may have anti-adenovirus antibody, the adenovirus that they may neutralize rapidly and inject.Whether the injection adenovirus can avoid this quick removing in the not clear tumor.Seldom there is the patient that anti-fowlpox virus antibody is arranged in serum.Therefore, this recombinant virus to continue be a potential advantage.But it is difficult producing high titre fowlpox virus.Might initially use recombinant adenovirus, treat the patient with fowlpox virus then, to reduce the host removing the reaction of virus.Our PRELIMINARY RESULTS shows, uses to have 5 * 10 of reorganization LIGHT ¹⁰Virion is expelled in tumor in the Ag104Ld tumor of having set up, causes local and the repulsion (5/5) of tumor at a distance, and not obviously protection (being ostracised for 0 in 5 mices) of contrast virus (not having LIGHT).

CD4 ⁺CD25 ⁺The T cell is a regulatory T cells, and they can depression effect T cell kill the ability of tumor cell.The consume regulatory T cells is CD4 particularly ⁺ CD2 5+ cell can strengthen anti-tumor activity.To give LIGHT and the consume regulatory T cells combines in tumor, be a kind of effective Therapeutic Method of new immunization therapy.

The CD4 that raises ⁺Control T cellular level is the indicator of diagnosis and prognosis

The present invention can be used as the diagnosis and/or the prognostic indicator of cancer, and the indicator of cancer prognosis.For example, the CD4 that raises in the tumor ⁺The T cell may show some cancer patient's relatively poor prognosis.In addition, the present invention also can be used for determining the effect of potential novel treatment to cancer, and the present invention also can be used for drug development.For example, cause CD4 in the cancerous tumours ⁺The hypocellular chemical compound of T may show that this chemical compound has the remarkable potentiality as cancer therapeutic agent.In other cases, use the testing in vitro method of cell line can be used for determining that chemical compound is to CD4 ⁺The effect of T cell.In certain embodiments, test compounds is to CD4 in animal or human's class tumor ⁺The minimizing of T cell may show the effect of improvement.In addition, the present invention also provides and has been used for determining whether the experimenter should accept to cause CD4 ⁺The method of the medicine of the hypocellular treatment effective dose of T.For example, CD4 ⁺The patient that the T cell quantity increases especially can be from causing experimenter's tumor sites CD4 ⁺Obtain an advantage in the therapy that T reduces.

In case the patient is suffered from cancer by diagnosis, we will check the control CD4 in the vivisection of tumor sample ⁺The T cell.If CD4 occupies dominant position, the patient will use the anti-CD 4 antibodies topical therapeutic.If be very difficult to the local injection tumor, then can carry out the general injection.If the permeability lymphocyte is limited, 50ul to 100ul ad-LIGHT (1-5 * 10 then ¹⁰Vp) will be injected into tumor tissues, and after 3 days, use anti-CD 4 antibodies, to allow in two weeks thereafter, to produce strong immune response (carrying out or do not carry out combined therapy).Perhaps, anti--CD4 unites use with anti--CD137 or other CD8 stimulus object.

Consume CD4 ⁺The chemical compound of T cell and/or treatment cancer and the external confirmation of combination

Those of ordinary skills obtain and a large amount of chemical compounds of testing in vitro or chemical compound combination, and they can consume CD4 ⁺T cell, the immunne response that stimulates the antagonism cancer and/or useful in the present invention any other effect of generation.This class testing can with as described in the following Examples and/or those methods of knowing of those of ordinary skills carry out.

In brief, by obtaining and test the external consume of candidate compound CD4 with human CD4 peptide or protein immune mouse ⁺The ability of T cell and/or treatment cancer.Selectively, we have obtained to contain the little murine hybridoma in anti-human CD4 antibody ground from the non-commercial American Tissue Culture Center of mechanism.As described in the early time, can obtain humanized CD4 by replace the CD4 gene with human immunoglobulin gene.The another kind of selection is that humanized anti-CD 4 antibodies can obtain from some private companys.

In case determined candidate compound or be combined in external demonstration to can be used for the present invention, then tested them with method in the following body usually.

Confirm in the body of the chemical compound of consume CD4+T cell

Can obtain and the body build-in test has the chemical compound of following feature or chemical compound combination in a large number: consume CD4+T cell; Stimulate the immunne response of antagonism cancer; And/or cause any other effect of the present invention that can be used for.Usually, only showing that with aforesaid in vitro method a certain particular candidate thing or combination have when hope, just carry out this type of body build-in test.Can be described among the following embodiment and/or known those methods of those of ordinary skills carry out this class testing.

In brief, will obtain candidate substances, and test the ability of consuming CD4+T cell and/or treatment cancer in its body.

Can be used for the present invention in case candidate compound or combination show in vivo, it is usually as the theme of following clinical trial.

Therefore, the invention still further relates to the method that is used to screen or test cancer therapeutic agent, comprise by in the body or experiment in vitro test candidate substances whether can consume regulatory T cells.

Clinical trial

Use method in the external and body, for example those are described herein and/or methods known in the art, identify promising chemical compound or combination after, this compounds and combination can be called the theme of the clinical trial of following institute general description.

In case the patient is suffered from cancer and potential transfer by diagnosis, we will check the control CD4 in the vivisection of tumor sample ⁺The T cell.If CD4 occupies dominant position, the patient will use the anti-CD 4 antibodies topical therapeutic.If be very difficult to the local injection tumor, even be difficult to intervene (guidance), then can carry out the general injection with radiation.If the permeability lymphocyte is limited, 50ul to 100ul ad-LIGHT (1-5 * 10 then ¹⁰Vp) will be injected into tumor tissues, and after 3 days, use anti-CD 4 antibodies, to allow in two weeks thereafter, to produce strong immune response (carrying out or do not carry out combined therapy).Perhaps, anti--CD4 unites use with anti--CD137 or other CD8 stimulus object.

The beneficial effect of the invention

In inventor's experiment, cause the immunity of tumor is improved 500 times with sudden change LIGHT treatment.The consume regulatory T cells provides other reinforced effects, and this treatment has shown very effectively (increases 100-500 doubly) to establishing tumor.To in tumor, give LIGHT and consume regulatory T cells to combine, better effect will be provided.

Embodiment

Embodiment proves the preferred embodiments of the invention below introducing.Persons of ordinary skill in the art will recognize that the fine enforcement technology of the present invention that is used for that on behalf of the inventor, the disclosed technology of following embodiment find, can think that therefore they have constituted enforcement optimal way of the present invention.But after reading the disclosure of invention, those of ordinary skills also it should be understood that and can carry out many changes and still can obtain similar result disclosed specific embodiments, but do not deviate from the spirit and scope of the present invention.

Embodiment 1

Material and method

Mice and cell line.The female C3HxB6F1 (C3B6F1) of 5-8 week size and C3H mouse are available from the Frederick CancerResearch Facility of US National Cancer Institute, and quilt is raised in the particular device that does not contain pathogen of Chicago University.Regulation according to institute and National Institutes of Health (NIH) is taken care of and is studied animal, and uses the approval of committee through the animal of Chicago University.Expression Mus H-2L has been described ^d(AG104-L ^d) Ag104 cell line (Wang, 2001) and Ag104 fibrosarcoma (people such as Wick, 1997).

Histology and immunology.The mankind tumor tissue that is used for histological examination is fixing in 10% buffered neutral formalin, paraffin embedding, and use standard method hematoxylin and eosin (H﹠amp; E) dyeing, or according to the explanation of manufacturer be specific to CD4 (NovocastraLaboratories, UK) and CD8 (DAKO, monoclonal antibody CA) dyes.(Sigma-Aldrich, (Vector, APAAP technology CA) discloses the combination of anti--CD4 or anti--CD8 antibody for peroxide zymotechnic MO) or use Vector Blue by using DAB respectively.For the breast carcinoma sample, 40 a times high of picked at random by the visual field in to CD4 ⁺And CD8 ⁺Cell carries out manual counting.

Measure the cytokine in spleen and the tumor.According to describing people such as (, 2003) Janssen, the inventor has prepared the homogenate of tumor and spleen.In brief, collect a considerable amount of tumors or spleen tissue, weigh, and in containing the PBS of protease inhibitor, carry out homogenate, by centrifugal collection supernatant.According to the explanation of manufacturer, in the FACSCaliber hematimeter of being furnished with cell QuestPro and CBA software (BD Pharmingen), use the cytokine quantity in the next quantitative supernatant of cytometer beads array test kit (CBA).

Flow cytometry.Use Fluorescein isothiocyanate (FITC) is puted together, that phycoerythrin (PE) is puted together, that Cy-chromium is puted together or the biological antibody of puting together at mice CD45RB, CD4, CD8, CD25 and IFN (all from BD Pharmingen) carries out facs analysis.Strepto-affinity element-Cy-chromium conjugate is from BD Pharmingen; At clonotype antibody (1B2) cell dyeing of the biotin-conjugated of 2C TCR according to described carrying out (Sun and Bevan, 2003).Dye for the cell within a cell factor, in the presence of Brefeldin A (Sigma-Aldrich), with PMA (50ng/ml, Sigma-Aldrich) and ionomycin (500ng/ml Sigma-Aldrich) stimulated 4 hours from the T of tumor tissues cell purification again.For the analysis of the 2C T cell of CFSE-labelling, with biotinylated 1B2 antibody isolating tumor or spleen single-cell suspension liquid are dyeed, washing, and with PE link coupled anti--mixture of CD8 and the link coupled strepto-affinity of CyC element dyes.Analyze the FACS data with FlowJo software (BD Pharmingen).

Cell separation and vitro detection.For separation of C D4 from tumor tissues ⁺The T cell makes the mice blood-letting to reduce the pollution of blood to tumor tissues earlier.Collect tumor tissues, wash among the PBS, cut into slices, and be resuspended in Dulbecco ' s improvement Eagle ' s culture medium 25 minutes in 37 ℃ of shaking tables, described culture medium has been added 2% hyclone and 1.5mg/ml collagenase D (collagenase D solution).Collecting cell suspension after 25 minutes, the pair cell piece digests and resolved into single-cell suspension liquid up to all tumor tissues in 25 minutes in collagenase D solution.(Miltenyi Biotech CA), then uses antibiotin magnetic bead tumor-infiltrated type T cell of enrichment from described single-cell suspension liquid with the Thy-1.2 antibody of biotin-conjugated to use the AutoMACS system.Anti--the CD4 that puts together with PE dyes to the tumor-infiltrated type T cell of enrichment, and elects CD4 as with the FACS branch on MoFlo ⁺Colony.By AutoMACS system (Miltenyi Biotech), use CD4 ⁺T cell or CD4 ⁺CD25 ⁺Control T cell separation magnetic bead system (Miltenyi Biotech) is purification CD4 from spleen single-cell suspension liquid respectively ⁺Or CD4 ⁺CD25 ^-And CD4 ⁺CD25 ^-Cell.For all cell separation, can obtain surpassing the required colony of 90% purity usually.Detect for the T cell co-stimulatory, the inventor is spent the night by 96 orifice plates with the anti-CD 3 antibodies bag of 50 μ l2 μ g/ml.Then wash described plate, with 6 * 10 with PBS ⁴The CD4 of individual purification ⁺CD25 ^-The T cell culture wherein adds or does not add 6 * 10 of the spleen that separates the mice that carries tumor or tumor tissues in every hole ⁴Individual CD ⁴+ T cell.In all detect, in last 24 hours of 3 days culture periods to every hole add 1 μ Ci [ ³H] thymidine, thus estimation T cell proliferation.TopCount micro plate scintillation counter (Packard, measure in MA) [ ³H] integration of thymidine.

The adaptability of 2C T cell shifts.The inventor at first passes through 10 ⁶MC57-SIY tumor cell subcutaneous vaccination is in B6/Rag-1 ^-/-2C T cell (Sun and Bevan, 2003) is activated in a plurality of sites of the 2C mice of background (2C mice).The inventor then separates lymph-node cell and splenocyte from the 2C mice that these are attacked, and uses CD8 behind tumor inoculation in 5 days ⁺, T cell enrichment test kit (Miltenyi Biotec) is to CD8 ⁺The T cell is born selection.When analyzing, most of 2C cellular expression CD44 ^HighCD62L ^LowPhenotype.According to described (Sun and Bevan, 2003), then these 2C T cells are carried out labelling with CFSE.The inventor is with 3 * 10 ⁶The T cell of individual CFSE-labelling (volume 0.2-ml) intravenous injection vascular plexus (retro-orbital plexus) behind the eye socket of the mice that has tumor.Shift back 48 hours isolated cells from spleen and tumor, and carry out facs analysis.

Cells in vivo is decreasing and the cytokine blocking-up.According to standardization program (Sun and Bevan, 2003), use at CD4 ⁺The mAb GK1.5 of cell, at CD8 ⁺The mAb 2.34 of cell and at CD25 ⁺Lymphocyte call subtype in the PC61 systematicness consume mice of cell.The CD4 that the inspection of splenocyte and lymph-node cell is shown consume with flow cytometry ⁺Or CD8 ⁺Subclass account for total lymphocyte less than 0.5%, the level of other subclass is normal.Carried out CD4 in the tumor with the 12.5-50 μ g GK1.5 that is dissolved in 50 μ l PBS ⁺The cell consume.In order to block IL-10 or TGF-β, behind preceding 1 day of tumor challenge and tumor challenge 7 days with 100 μ g anti--IL-10 receptor or 250 μ g are anti--TGF-β antibody is injected into mice through intraperitoneal (i.p.).Behind the tumor challenge 7 days, with 100 μ g lipopolysaccharide (LPS) or/and antagonism anti--CD4 0 antibody gives mice through intraperitoneal.

Real-time quantitative RT-PCR detects.According to describing (Shedlock and Shen, 2003), foxp-3 is carried out real-time quantitative sex reversal record PCR (RT-PCR) detect.In brief, separate total RNA from tumor, (Amersham Pharmacia NJ) becomes complementary with the total RNA reverse transcription of 5 μ g to use First Strand cDNA Synthesis Kit.(Cepheid carries out the real-time quantitative pcr analysis on CA) at the real-time thermal cycler of CepheidSmartCycler.According to the explanation of manufacturer (PE Applied Biosystems, MA), with the TaqMan Universal PCR master mixture that contains AmpliTaqGold DNA Polymerase increase foxp-3 and GAPDH in each cDNA sample.The inventor utilizes comparison type CT (the threshold cycle number at the intersection point place of amplification curve and threshold value) method to determine the concentration of target gene, and with numerical value to inner GAPDH contrast carrying out standardization.The primer sequence that is used for foxp-3 is 5 '-CCCAGGAAAGACAGCAACCTT-3 ' (forward primer) and 5 '-TTCTCACAACCAGGCCACTTG-3 ' (reverse primer), the probe that is used for FOXP-3 is 5 '-ATCCTACCCACTGCTGGCAAATGGAGTC-3 '.

Embodiment 2

The destruction that consume CD4+T cell causes tumor

CD4+T cellular infiltration aggressive breast carcinoma in people patient.Human cancer usually has antigenicity, but pondered-over be that most of human cancers can not be induced repulsion, even also be so (Rosenberg, 1999 when lymphocyte significantly soaks into tumor tissues; Beck-Engeser etc., 2001).In order to study the state and the character of soaking into the tumor lymphatic cell, the inventor has checked several human cancer tissues, comprises breast carcinoma, colon cancer, pulmonary carcinoma and melanoma, and observes the significant tumor lymphatic cell that soaks into of usually existence.In order further to analyze the T lymphocytic infiltration tumor tissues of which kind of type, the inventor uses anti--CD4 and anti--CD8 antibody staining breast carcinoma sample and discovery CD8+ and CD4+T cellular infiltration (Fig. 1 a-d) in various degree.Interesting is that in the breast carcinoma sample that the inventor checks, the inventor observes the early stage considerably less CD4+T cellular infiltration breast cancer tissue at tumour progression.Yet along with tumor development, the CD4+T cell colony of inside tumor sharply increases.In order to verify this observed result, the inventor has counted ten breast carcinoma of early stage tissues, the original position breast carcinoma quantity with respect to CD4+ that exists in the aggressive breast cancer tissue ten late periods and CD8+T cell, finds that the CD4+/CD8+T cell proportion sharply increases (Fig. 1 e). and the inventor also observes the CD4+T cell and usually closely contacts (Fig. 1 b and d) with the CD8+T cell.Consider the progressive character of these cancers, the inventor infers that the immunologic rejection lack cancer may not be to be caused but replied by abnormal immune by weak immunity identification to cause.

Strong antigen does not hinder tumor growth.Because for the limitation that exists aspect the basic role of human cancer, the inventor has developed a kind of mouse tumor model in research tumor-infiltrated property CD4+T cyton, this model is very similar to the human cancer with certain C D4+ and CD8+T cellular infiltration.Ag104 is a kind of low differentiation solid tumor of spontaneous formation in C3H mouse, and it has the immunogenicity of carrying out property highly, difference and immunization therapy is had resistance (Belkaid etc., 2002; Wang, 2001; Dudley etc., 2002).When inoculation was lacked to 104 cells, Ag104 is equal 100% growth in C3H and C3B6F1 mice.The reduced immunogenicity that causes this tumor of quick growth in the homogenic wild-type mice body of immunocompetence is due to may or not expressing by the weak expression of tumor antigen.In order to get rid of this explanation, the inventor uses coding L ^dThe gene transfection Ag104 tumor cell line of antigen (a kind of strong antigen).The high-caliber L of tumor cell line vivoexpression of transfection ^dUnexpectedly, express L ^dAg104 tumor (Ag104L ^d) 10 ⁴Growing in similar kinetics mode to parent's tumor during low dosage, (Fig. 2 a).It is reported that the variant that can select antigen to lose is avoided immunity identification (Pardoll, 2003).Yet the inventor finds, separates the Ag104L of the mice that carries tumor ^dCellular expression before tumor cell and the injection is the L of level quite ^d(data not shown).From this research, the inventor draws such conclusion: the antigenicity tumor can be made progress under the situation of not losing its tumor antigen.Therefore, by Ag104L ^dTumor can not be caused by low expression or antigenic the losing of I type MHC in the weak immunogenicity of this tumor of the intravital quick growth indication of the homogenic wild-type mice of immunocompetence.

Antigen L ^dA reason that does not hinder tumor growth may be that this antigen is ignored by immune system and never found by immune system.In order to detect this probability, the inventor is at inoculation Ag104L ^dRemoved this tumor by operation in 20 days after the tumor, 3 weeks were attacked mice once more with the tumor cell of high dose more after operation then.Surprisingly, all these mices all repel tumor challenge for the second time, and inmature mice dies from tumor load (table 1).This presentation of results, the T cell is by L ^dAntigen sensibilization, and after tumor was removed in operation, these T cells can develop into memory cell and give the anti-protective effect of tumor challenge for the second time.The inventor can prove that these memory T cells mainly are L ^dSpecific, because these mices can not repel parent's tumor Ag104 (table 1).These results suggest we, T cells with antigenic specificity exists really, but it can not suitably bring into play function in having the mice of tumor.This has proposed a kind of probability: the weak immunogenicity of Ag104Ld tumor may not be to be caused but when being existed by tumor by weak immunity identification, and especially the abnormal immune in the tumor microenvironment is replied and caused.

The CD4+CD25+T cell is built up in inside tumor.For study in addition under the situation that immunogenicity antigen exists the still impaired reason of tumor rejection, the inventor has at first checked infiltration tumor lymphatic cell (TIL) in animal model.Immunohistochemical staining discloses, Ag104L ^dTumor tissues is by CD4+ and CD8+ cellular infiltration, the similar manner co of these cells to see in mankind tumor tissue.Observe the DX5+NK or the NK T cell (data not shown) of considerably less infiltration tumor.Surprisingly, the lymphocyte that accumulates in the tumor is mainly formed (Fig. 2 b) by the CD4+CD25+T cell subsets.The inventor compared spleen, draining lymph node then on the the 0th, 7 or 16 day behind tumor challenge (draining lymph node, the CD4+CD25+T cell accounts for lymphocytic percent DLN) and in the tumor.The inventor finds that as time passes, the CD4+CD25+T percentage of cells of inside tumor sharply increases, and its maintenance constant (Fig. 2 b) in spleen and DLN.These data show, have the CD4 that increases percent at effective-site rather than in lymphatic organ ⁺CD25 ⁺The T cell.These CD4 ⁺CD25 ⁺The T cell is CD45RB ^Low(FIG.2c) of phenotype, this with to CD4 ⁺CD25 ⁺The observed result unanimity of regulatory T cells (Golgher etc., 2002).FOXP3 is to mice CD4 ⁺CD25 ⁺Forkhead/winged spiral transcription factor gene (Wang etc., 2002 of regulatory T cells differentiation and function key; Van Elsas etc., 2001; Dieckmann etc., 2001).Real-time polymerase chain reaction (PCR) proves, with the isolating CD4 of spleen from the mice that has tumor ⁺T cell or from the isolating CD4 of the spleen of inmature mice ⁺CD25 ^-The T cell is compared, from the isolating CD4 of this tumor ⁺The expression of FOXP3 mRNA much better than (FIG.2d) in the T cell.These observed results have proposed this probability, that is: in the tumor development process, accumulation constitutes effective-site 70%CD4 ⁺The CD4 of T cell ⁺CD25 ⁺Cell suppresses local immunity should.The inventor infers that cancer immunity originality difference may be because the CD4 that accumulates in tumor environment ⁺CD25 ⁺The immune regulation mechanism that the T cell produces causes.

CD4 ⁺CD25 ⁺The T cell suppresses antitumor immune.In order to check tumor-infiltrated CD4 ⁺CD25 ⁺Whether the T cell suppresses external t cell response, and the inventor is from setting up 20 days tumor tissues separation of C D4 the C3B6F1 mice ⁺The T cell, and under the situation that the dull and stereotyped bonded zest anti-cd 3 antibodies of optimal dose (2 μ g/ml) exists with them and the purification CD4 that obtains from inmature C3B6F1 mice ⁺CD25 ^-The T co-culture of cells.The inventor finds, these tumor-infiltrated CD4 ⁺The T cell significantly suppresses to mix by 3H the CD4 of measurement really ⁺CD25 ^-The propagation of T cell, and from the isolating CD4 of spleen of the mice that has tumor ⁺The T cell does not produce this inhibition (FIG.2e).Because CD4 ⁺And CD8 ⁺Cell can consume CD25 so produced in vivo at the early expression CD25 of T cell activation ⁺Whether cell can eliminate the CD8 that will be activated ⁺The query of T cell.But the inventor observes, 2 weeks behind tumor challenge, most of CD8 in spleen and tumor ⁺The T cell is CD25 ^-(FIG.2f).Consume CD25 ⁺Cell does not reduce CD8 ⁺Colony is because CD8 only seldom ⁺Cellular expression CD25 (FIG.2f).In tumor environment inside, the CD4 of preferred accumulation ⁺CD25 ⁺Cell and CD8 ⁺Cell is not all expressed CD25.In order directly to study CD4 ⁺The T cell is the basic effect in the tumour immunity in vivo, and the inventor has consumed CD4 ⁺Cell is observed tumor growth then.Be surprised to find, work as CD4 ⁺When cell is consumed, the Ag104L of height vascularization and reduced immunogenicity ^dTumor is repelled (FIG.2g andTable 2) rapidly.CD4 ⁺The tumor rejection that the cell consume produces is CD8 ⁺Cell is dependent, because tumor is not having CD4 ⁺Cell, there is not a CD8 yet ⁺(Table2) uncontrollably grows during cell.NK1.1 ⁺The consume of NK and NKT cell does not cause tumor rejection (Table 2).In order to confirm to express the CD4 of CD2 5 ⁺The T cell subsets has caused the inhibition of anti-tumor in vivo immunity, and the inventor handles two weeks of mice of band tumor with anti-CD 25 antibody.Behind tumor challenge 14 days with this antibody consume CD25 ⁺Cell suppresses growth of tumor (FIG.2h) significantly.These data show, in the later stage of anti-tumor immune response, mainly are CD25 ⁺The T cell plays inhibitory action.

Even there is not antigen L ^dSituation under, the consume CD4 ⁺Cell also allows 50% mice to repel parent Ag104 tumor (table 1) fully, and 50% remaining mice has postponed tumor growth kinetics (data not shown).These observed result promptings, in case remove the inhibition of CD4 mediation, other tumor antigen also may inducing antitumor immunity.Whether cause protective immunity in order to test repulsion, at first inoculate Ag104L at other tumor antigen ^dThe tumor cell consumed CD4 after 14 days ⁺Cell.Consume CD4 ⁺The tumor rejection that cell causes is not only at Ag104L ^dThe attack afterwards of tumor cell produces protective effect, and produces protective effect (table 1) at the attack afterwards of Ag104 tumor cell.These data show, inhibition CD4 ⁺The CD8 that the removal of cell makes expansion ⁺The immunity of cell colony participation protective.

Regulatory T cells suppresses the CD8+T cell in effector phase.In order to test CD4 ⁺Whether cell suppresses CD8 in sensitization phase or effector phase ⁺T cell response, the inventor is 20 days consume CD4 behind preceding 3 days of tumor challenge or tumor challenge ⁺Cell.The facs analysis of peripheral blood is confirmed each is handled all behind 20 days or tumor challenge and to eliminate CD4 in 20-40 days ⁺Cell.CD4 consume is limited in 20 days, perhaps in other words, in the sensitization phase of tumor growth, cause postponing but progressive tumor is grown (table 2).In effector phase, promptly behind the tumor challenge 20 days---the tumor of foundation reaches 500mm ³Carry out the CD4 consume during above mean size, cause tumor rejection (table 2) completely.Whole duration consume CD4 at tumour progression ⁺During cell, also observe repulsion (table 2).10 days injection anti-CD 4 antibodies behind preceding 3 days of tumor challenge and tumor challenge.The inventor must see height tumor progression ruined any other processing fully that may cause this type of large volume, fully vascularization.These Notes of Key Datas are in immunne response late period rather than lack CD4 in early days ⁺Cell is for strengthening CD8 ⁺The cell-mediated antineoplastic immune of T is more crucial.

As above-mentioned, the tumor rejection of CD4 consume mediation depends on CD8 ⁺The T cell, this points out at inside tumor CD4 ⁺Cell may suppress CD8 ⁺Cell.The present invention has still then checked CD4 ⁺How cell suppresses CD8 ⁺The function of T cell.At first, the inventor is at CD4 ⁺Detect CD8 in the tumor after the consume ⁺The T cell increases sharply that (Fig. 3 a) and detect CD8 ⁺The T cell produces the IFN-γ (Fig. 3 b) that increases.This prompting, CD4 ⁺Cell can suppress to reply the CD8 of tumor ⁺The amplification of cell.In order to detect this hypothesis, the inventor with the 2C T cell adoptive transfer of CFSE labelling to having Ag104L ^dThe C3B6F1 mice in, wherein said 2C T cell is at 2C/Rag-1 ^-/-Activate by MC57-SIY tumor cell (Sun and Bevasn, 2003) in the mice.2C T cell derives from TXi Baoshouti (TCR) transgenic 2C mice, and it can Direct Recognition Ag104L ^dOn L ^dAntigen.The high-caliber CD44 of effect 2C T cellular expression of these transfers and the expression (data not shown) of reducing CD62L.At CD4 ⁺Before the consume of T cell, only have the effect 2C cell of the CFSE labelling of minority to be present in the tumor, but after consume, the number and percentage of observing this effector lymphocyte sharply increase (Fig. 3 c).The more important thing is that these 2C T cells are mainly at CD4 ⁺Breed in effective-site T cell consume back, and this prompting inhibitory action mainly is present in local tumor position (Fig. 3 c).Therefore, these CD4 ⁺Cell has suppressed CD8 at tumor locus ⁺T cell proliferation, maturation and expansion.

IL-10 and TGF-β participate in intravital inhibition mechanism.In order to study CD4 ⁺Cell suppresses the mechanism of antineoplastic immune, and the inventor has compared CD4 ⁺Cytokine levels before and after the cell consume in spleen and the tumor.In spleen, there is CD4 ⁺During cell or do not have a CD4 ⁺Detect during cell, cytokine levels does not have significant difference, and (Fig. 4 a).The Th2 cytokine, there are or are lacking CD4 in IL-4 and IL-5 ⁺Do not show significant difference (Fig. 4 b) in tumor locus during the T cell.Then, lack CD4 ⁺During the T cell, the inflammatory cytokine in the tumor tissues (comprising IFN-γ, TNF, IL-6 and MCP-1) is compared to there being CD4 ⁺Level during cell sharply increases.At consume CD4 ⁺Behind the cell, many (Fig. 4 b) that the level of anti-inflammatory cytokines IL-10 is also low.These results suggest, CD4 ⁺Cell has been kept the anti-inflammatory environment that suppresses antineoplastic immune.

If the inventor infers the effect of blocking-up anti-inflammatory cytokines and may promote antineoplastic immune.In fact, after the inventor was with anti--IL-10 receptor (IL-10R) antibody treatment mice, tumor growth was sharply suppressed (Fig. 4 c).When handling mice with antibody combined inflammatory signal of anti-IL-10R (as LPS) or anti-CD 40 antibodies agonist, tumor growth is further received inhibition (Fig. 4 c).TGF-β is the another kind of cytokine that can suppress inflammatory reaction.For research TGF-β is in the effect aspect the mediation antineoplastic immune in model, the inventor is using Ag104L ^dWhen attacking, blocked tumor cell TGF-β.TGF-β blocking-up back tumor is repelled (Fig. 4 d) fully.Anti-inflammatory environment in these presentation of results, tumor is preventing that aspect the tumor rejection be crucial.CD4 ⁺Cell shows can keep this anti-inflammatory environment actively to suppress antineoplastic immune, promotes the antigenicity tumor growth thus.

At effective-site consume CD4 ⁺CD25 ⁺The T cell can be eradicated the tumor of having set up.Because CD4 ⁺CD25 ⁺The T cell is the main CD4 in the tumor during the tumor development ⁺The T cell colony is so the inventor proposes at tumor locus consume CD4 ⁺Cell will be enough to realize tumor rejection.In having the host of tumor, use a spot of anti-CD 4 antibodies optionally to consume CD4 at tumor section ⁺Cell can reverse CD8 effectively ⁺Cell is to the immunologic tolerance of tumor, and keeps having in the lymphoid tissue CD4 of normal quantity simultaneously ⁺Cell.In order to ensure setting up the back in tumor in effective-site consume CD4 ⁺SC can promote antineoplastic immune, and the inventor has used the anti-CD 4 antibodies (10-15 μ g) of low dosage to carry out in the tumor injection to guarantee in tumor rather than systemic the CD4 that consumes ⁺(Fig. 5 a) for the T cell.The inventor finds, consumes CD4 behind the inoculated tumour in 14 days in tumor ⁺Cell causes the complete repulsive interaction (Fig. 5 b) to the tumor of abundant foundation apace.This result confirms, CD4 in the tumor ⁺The consume of SC is enough to cause the repulsive interaction to the tumor of having set up, and prompting antitumor CD8 ⁺Effective inhibitory action of T cell mainly occurs in effective-site and can reverse.Importantly, Local treatment can become affordable effective immunotherapy of tumors processing method.

The undesirable immune response of weak tumor antigen usually is considered to the basis of carrying out property of tumor growth among the immunocompetence host, and is speculated as successfully the major obstacle of immunization therapy.In this research, the inventor is with strong L ^dIsoantigen is introduced in the mice Ag104 fibrosarcoma cell system, and this cell line is a kind ofly can kill host, the tumor of carrying out property, aggressive and vascularization highly in several weeks.Yet, exist strong antigen to fail to cause that anti-tumor immune response is to stop or to postpone tumor growth.In the humans and animals cancer, generally can observe lymphocytic infiltration (Gilboa, 1999; Clark etc., 1989; Clemente etc., 1996), still seldom observe immunologic rejection to cancer.Under the situation with obvious T cellular infiltration, it is a perplexing phenomenon that is always fully understood that the host can not eradicate tumor.Nearlyer observation to TIL in animal model discloses CD4 ⁺Subgroup CD4 ⁺CD25 ⁺Accumulate in the tumor T cell selective and comprised the great majority of all TIL.The inventor also confirms, in the tumour progression process, and CD4 ⁺CD25 ⁺The T cell mainly mediates the inhibition environment and abolishes CD8 in tumor ⁺The effector function of T cell.These regulatory T cells of consume make the immunogenicity of tumor be exposed and reverse the CTL toleration in the local tumor, cause the quick repulsive interaction to the tumor of fully having set up.The more important thing is that in this research, the inventor has illustrated, the T regulatory cell can be a kind of important local factors, and the immunne response that it can suppress at strong tumor antigen causes cancer carrying out property growth in the immunocompetence host.

To CD4 ⁺Broad research (Liyanage etc., 2002) has been carried out in the effect of T cell in tumour immunity.Studies show that CD4 ⁺The T cell is promoting CD8 ⁺T cell response (Woo etc., 2001; Woo etc., 2002; Wang etc., 2004) and development CD8 T cell memory (Yu etc., 2004; Ward etc., 1989; Shedlock and Shen, 2003) has important function in.In antineoplastic immune, CD4 ⁺The T cell is proved to be for keeping adoptive transfer CD8 ⁺The function of T cell is important (Sun and Bevan, 2003; Dudley etc., 2002; Wang etc., 2002).In mice, consume CD4 ⁺Cell significantly weakens granulocyte/huge to be had a liking for colony-stimulating factor (GM-CSF) vaccine/anti-CTLA 4 and handles and watch for animals not by the ability of follow-up tumor challenge (vanElsas etc., 2001).On the other hand.The such viewpoint of the strong support of nearest research, that is, and CD4 ⁺Colony in the cell, CD4 ⁺CD25 ⁺The T cell significantly limits vaccine-induced anti-tumor immune response, and suppresses or eliminated them significantly to strengthen immunotherapy of tumors (Machiels etc., 2001 before tumor challenge; Sutmuller etc., 2001; Steitz etc., 2001; Yu etc., 2003; Houghton etc., 2001).For example, in a research, when animal was used the anti-CD25 antibody treatment before tumor challenge, the tumor vaccine of GM-CSF transduction was more effectively eliminated the tumor of having set up with combining of anti-CTLA4.(Yu etc., 2003).Although these studies show that regulatory T cells can suppress CD8 ⁺The initial sensitization of T cell, but still common concern is to consume CD4 in having the host of tumor ⁺CD25 ⁺The T cell may have been eliminated CD4 unintentionally ⁺The miscellaneous function of T cell, CD4 ⁺The T cell also can be expressed CD25 after by tumor or immunity inoculation activation ⁺Sign.The inventor proposes, CD4 ⁺Cell mainly plays the effect of enhancing property accessory cell in the starting stage, in case but tumor becomes tumor lastingly long-term and that establish, CD4 ⁺T regulates the cell accumulation to be increased, and suppresses CD8 ⁺The function of cell is also covered the immunogenicity of tumor.Really, this research is verified, and regulatory T cells can participate in effector phase (effector phase) to a greater degree, mainly in the local tumor site, by the tumor-infiltrated property of direct inhibition CD8 ⁺Effector lymphocyte's (effector cell) realizes.Consume these regulatory T cells at tumor locus and reversed CTL toleranceization (tolerization) efficiently, cause establishing the quick repulsion of tumor.Therefore, this studies have shown that, CD4 ⁺CD25 ⁺The T cell (mainly is CD8 for antineoplastic immune ⁺T cell response) inhibition mainly takes place in effector phase at tumor locus, can not weaken possible t helper cell function at tumor development later stage consume T regulatory cell.In fact, consume T regulatory cell has disclosed the immunogenicity of tumor cell, and, in addition lack strong antigen L ^dThe attack once more of parent's tumor cell long-term protection is provided.This results suggest, inducing of immunity is not only at L ^d, on the contrary, the consume of regulatory T cells has disclosed there not being the immunity of immunogenic tumor antigen in the past, has expanded the CD8 with tumor response ⁺T cell aggregation (repertoire).Parent Ag104 (no L ^d) tumor system (highly progressive, as not have immunogenicity antigen) can not produce reaction to traditional immunization therapy, but show enhanced immunogenicity behind the consume regulatory T cells, causes the tumor development of tumor rejection or delay.Can expect, by consume CD4 ⁺The local inductive this powerful antitumor immunity of cell may cause the repulsion of tumor at a distance.

Tumor microenvironment may allow to activate later on or the amplification regulatory T cells at tumor development.The inventor infers, may be similar aspect some of protection of normal tissues immunity in chronic autoimmune inflammation or the long-lasting infection process at the chronic inflammation of tumor tissues inside, and the latter allows to accumulate CD4 ⁺CD25 ⁺The T cell subsets limits local inflammation and alleviates local organization and destroy (Sakaguchi, 2004 with the downward modulation immunne response; Shevach, 2002; Maloy andPowrie, 2001; Casares etc., 2003; Golgher etc., 2002).In addition, the inhibition factor of tumor mediation has the existence that is beneficial to regulatory T cells for effector T cell.As a result, design is used for preventing that the same regulatory mechanism of inflammatory response out of control from might suppress antineoplastic immune in normal structure.In various tumor tissues, find and contain CD4 ⁺CD25 ⁺The tumor-infiltrated T cell of colony is unrare (Dieckmann etc., 2001; Liyanage etc., 2002; Woo etc., 2001; Woo etc., 2002; Wang etc., 2004).Compose by surface marker and cytokine and to characterize better from the isolating CD4 of tumor tissues ⁺The character of T cell will be useful.

For the result who determines the inside tumor immunne response, the balance of understanding T effector lymphocyte and T regulatory cell may be more important.Nearest studies have shown that, raises inmature lymphocyte rapidly and expands CD8 in inside tumor ⁺The T cell may be that a kind of foundation mainly is the method (Janssen etc., 2003) of urging the inflammatory environment, causing repelling locality and distant place tumor.Now, the inventor is proof in this research further, and the consume regulatory T cells is the another kind of effective ways that inside tumor antiinflammatory environment are converted into short scorching environment.Topical therapeutic is compared with systemic treatment with the elimination regulatory T cells has three advantages: the first, and it needs the very antibody of low dosage, and therefore, this treatment expense clinically is affordable, even for developing country; The second, it has avoided whole body to consume all CD4 ⁺The inductive side effect of T cell, and whole body consumes all CD4 ⁺The T cell may be eliminated the protective immunity at pathogen of t helper cell mediation; The 3rd, it can not hinder auxiliary CD4 in lymphoid tissue ⁺The T cell is to CD8 ⁺Effective sensitization of T cell is because described consume is partial.Therefore, regulatory T cells is a kind of noticeable target in the tumor environment, and their consume may cause the improvement of current immunotherapy method.The effector lymphocyte of rapid expansion tumor locus, the therapeutic alliance of partially spent regulatory cell simultaneously may provide a kind of available strategy that strengthens antineoplastic immune and the cancer patient is produced favourable clinically result.

* * *

Based on content disclosed herein, all compositionss that this paper is open and claimed and method can both not need undo experimentation and prepare and implement.Although the compositions and methods of the invention are illustrated by embodiment preferred, but it will be apparent for a person skilled in the art that, the step of the present composition and method and this method and sequence of steps can be changed, and do not deviate from notion of the present invention, spirit and scope.More particularly, clearly, can substitute reagent disclosed herein, realize identical or similar result simultaneously with chemistry some reagent relevant with the physiology.All these those skilled in the art significantly substitute and revise and are believed to comprise within spirit of the present invention, scope and notion that claims limit.

Table 1 CD4 consume is induced at L ^dAntigenic immunne response in addition.

^aShown in tumor cell number be subcutaneously injected in the C3B6F1 mice.Exenterate or after anti-CD 4 antibodies handle to repel for the first time tumor 30 days, attack once more.

^b21 days exenterate tumors behind first time tumor challenge.

^cBehind first time tumor challenge, consumed CD4 with anti-CD 4 antibodies on the 14th day ⁺Cell.Consume is by checking that with FACS peripheral blood confirms.

Table 2 is by removing CD4 ⁺Cell increases tumor rejection

^aShown in tumor cell number be subcutaneously injected in the C3B6F1 mice.

^bThat obtain from independent experiment or from two results that independent experiment is comprehensive.

^c3 days and 7 days afterwards are with anti-CD 4 antibodies consume CD4+ cell before tumor challenge.

^d3 days and 7 days afterwards are with anti-CD 4 antibodies consume CD4+ cell before tumor challenge.Behind tumor challenge 14 days with anti-CD8 antibody consume CD8+ cell.

^ePreceding 3 days of tumor challenge with anti-asialo GM-1 antibody consume NK cell.

^f3 days or 7 days afterwards are with anti-NK1.1 antibody consume NK and NKT cell before tumor challenge.

The consume of various cell masses is by checking that with FACS peripheral blood confirms.

Table 3 CD4 ⁺The inhibition that cell causes is in effector phase (effector phase) rather than at sensitization phase (priming phase)

Tumor challenge a	Anti-CD4 +Handle [with respect to the natural law of tumor challenge (0)]	The incidence rate of tumor growth ° (%)
			Ag104L d1×10 5	-3 and 10-3 20	0/12 (0) 5/6 (83) 0/10 (0)
5×10 5	20 d	0/8 (0)

^aShown in tumor cell number be subcutaneously injected in the C3B6F1 mice.

^bSuppose that tumor challenge is at the 0th day.

CD4 ⁺Cell consumes with anti-CD 4 antibodies.The non-existent sky of CD4+ cell is by checking that with FACS peripheral blood confirms.

^cThe result sums up from a plurality of independent experiments.

^dThe average tumor size reaches 500mm ³More than.

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Claims (12)

1. comprise and express the recombinant virus of the nucleic acid cDNA of encoding mutant LIGHT.

2. the recombinant virus of claim 1, wherein this recombinant virus is recombinant adenovirus or fowlpox virus.

3. the recombinant virus of claim 1, wherein said LIGHT is human LIGHT.

4. the recombinant virus of claim 1, wherein said sudden change LIGHT comprise and stop the sudden change of protease to its degraded.

5. the recombinant virus of claim 4, wherein said sudden change LIGHT is the LIGHT that does not contain protease cracking site.

6. the recombinant virus of claim 5, wherein said sudden change LIGHT is the sudden change LIGHT that does not contain the proteolysis site EKLI of LIGHT albumen 79-82 position.

7. the recombinant virus of claim 5, wherein said sudden change LIGHT is the sudden change LIGHT that does not contain the proteic proteolysis of people LIGHT site EQLI.

8. the recombinant virus of claim 1, wherein said sudden change LIGHT has extracellular domain amino acid sequence and sequence label.

9. the recombinant virus of claim 8, wherein said sudden change LIGHT has the aminoacid sequence and the flag sequence of LIGHT 85-239 position.

10. the Pharmaceutical composition that comprises the recombinant virus of one of claim 1-9.

11. the Pharmaceutical composition of claim 10, wherein this Pharmaceutical composition is therapeutic vaccine or preventative vaccine.

12. the virus of one of claim 1-9 or the compositions of claim 10 are used for the treatment of purposes in the medicine of cancer in preparation.

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