CN100444895C - Immunomodulator for treating malignant tumor - Google Patents

Abstract

The present invention discloses one kind of immunomodulator preparation for treating malignant tumor, and belongs to the field of biological preparation technology. The immunomodulator preparation is water solution containing interleukin-12 in 5-30 mcg/mg and bacillus pyocyaneus in 1-3 billion/ml. The immunomodulator preparation has obvious effects of raising the IFN-gamma generating level of peripheral blood mononuclear cell and inhibiting tumor, and is used clinically in auxiliary treatment of malignant tumor.

Description

The immunomodulator that is used for treating malignant tumor

Technical field

The present invention relates to a kind of biological preparation, be meant a kind of immunomodulator that is used for treating malignant tumor especially.

Technical background

Malignant tumor is commonly encountered diseases, frequently-occurring disease, is one of principal disease of serious threat human health.The early discovery of malignant tumor, the early treatment is the key factor that obtains good therapeutic effect, but because the concealment of tumor invasion process lacks corresponding sings and symptoms in early days sometimes.Many in addition crowds lack and have regular physical checkups, thus non-early stage when considerable patient finds, so just be to treat to bring many difficulties.The main treatment means of tumor remains operative therapy, chemotherapy and X-ray therapy at present.But centering, the above-mentioned therapy of patients with terminal often do not reach the purpose of radical cure tumor, because remain residual focus in the body in tumor, and these focuses are exactly the root of tumor local recur and metastasis, therefore before and after operative therapy, chemotherapy or X-ray therapy, implement immune modulating treatment to the radical cure of tumor or the residual tumor focus that exists is thoroughly removed is to improve cure rate to prevent the important means that recurs and shift.Transfer body's immunological function this moment, and self the anti-tumor ability that improves body is crucial.Therefore the immunomodulating therapy of tumor more and more is subject to people's attention.Though the cause of disease of tumor is not definite fully as yet, its occurrence cause is multifactorial.Wherein the immunity of organism function for monitoring descends or imbalance, and the decline of immunne response ability exists considerable patient.Clinical examination shows that every immune indexes of cancer patient is how relatively low or is in abnormal state, therefore use appropriate and effective immunomodulator, improve the body fluid and the cellular immunization of body, the ability of activation killing tumor cells target cell then helps cancer patient's prognosis.So the effect of biological immune therapy in the clinical treatment of tumor that grows up based on immunization therapy extensively paid attention to gradually.The biological immune therapy mainly is specificity and the nonspecific immunity by adjusting and enhancing body, the propagation of the anti-cancer ability anticancer of enhancing body and then kill and wound cancerous cell it is withered away, this has become and has improved cancer patient's cure rate or prolong one of time-to-live important means of tumor patient.

But effectively immunomodulator is very rare really on the clinical tumor at present, extremely needs so develop and develop new tumour immunity adjusting preparation.Tumour immunity is regulated preparation and is killed tumor cell residual in the body as a kind of auxiliary treatment means of tumor on the one hand, by the specificity of raising body and the recurrence and the transfer of nonspecific immunity function prophylaxis of tumours significant values is arranged also on the other hand.

RhIL-12 is the important cytokine of occupying core position in immunological network.The main product survivor of IL-12 is the Monocytes to the microorganism stimulation responses, antigen presenting cell (APC), B cell, neutrophil cell and horn cell.IL-12 is the light chain (P35 by 35kd, IL-12 α) and the heterodimer of two chains of the different genes coding formed of the heavy chain of 40kd (P40, IL-12 β), connect by a plurality of disulfide bond, relative molecular weight is 70～75kd, and isoelectric point, IP is between pH3.5～pH5.5.Because two subunits are by different gene codes, the gene transfection result of the test shows to have only the IL-12 that the transfection simultaneously of two subunits could be obtained biologically active, abiology activity during two subunit individualisms.

The biologic activity of IL-12 has specificity, must be mediated by the receptor of high-affinity.IL-12R is made of β 1 and β 2 chains, the affinity of IL-12 and independent β 1 or β 2 chains a little less than, have only β 1 and β 2 coexpressions could form the receptor complex that has high-affinity with IL-12.

IL-12 occupies the very core cytokine of critical role in immunological network, can make the activated T cell proliferation and increase its cytotoxic activity, induces the secretion of IFN-γ, regulates the Th1/Th2 cell development, impels it to the Th1 cell differentiation.IL-12 is that the optimum cell factor that induces the CTL immunne response starts cell-mediated immunoreation.Not only can activate the innate immunity cell, promote the APC function, promote its maturation, improve angtigen presentation efficient.Also can be used as the 3rd signal direct activation T cells, make it change T effector cell into.Proved that responsiveness and Memorability CD8+T cell are lacking under the isogeneic stimulation, IL-12, IL-18 can impel it to have the ability of secretion of gamma-IFN.IL-12 still keeps a kind of candidate's factor of Memorability CD4+T cell.Prompting IL-12 works in promoting Memorability cell-stimulating process.Be worth especially pointing out that the not only collaborative IL-18 of IL-12 promotes the propagation activation NK cell of NK cell to produce cytotoxicity, induce the NK cell and produce IFN-γ, and IL-12 is most important to the activation of NKT and the secretion of IFN-γ subsequently, the IL-12 receptor complex that the NKT cellular expression is abundant becomes the primary goal of IL-12 effect.NKT is amplified by IL-12 the faint reaction of self part, thereby starts immunne response, and visible IL-12 replys the important function of removing in the malignant cell at the startup inherent immunity.

The biological effect of IL-12 is its antitumor, the basis that suppresses tumor growth and transfer.Its main biological effect is replied for regulating Th1/Th2, inducing early stage Th0 cell differentiation is the Th1 cell, induce its growth and propagation, the killing ability, the IL-12 that strengthen the T cell can carry out proliferation and differentiation as the 3rd signal at initial CD8+T cell, but when antigen levels was low, the effect of the 3rd signal was very important, and whether IL-12 exists as the 3rd signal, having determined T cells further to break up in varying degrees, whether produce immunne response.IL-12 stimulates the secretion of CD4+ Memorability cell and keeps and plays an important role.Schluns etc. think that IL-12 may be a kind of candidate's factor of keeping Memorability CD4+ cell.The collaborative IL-18 of IL-12 promotes the memory t cell activation, promotes the propagation of NK cell, and activation NK cell produces cytotoxicity, induces the NK cell and produces IFN-γ, strengthens the NK/LAK cell lysis activity.

A large amount of zooperies show that rhIL-12 can not only suppress growth of tumor; prevent sending out and shifting of tumor, cause the prolongation of tumor animal life span, can also induce established tumor tuberosity to disappear; and, has protective immunological reaction to the invasion and attack once more of tumor of the same race.IL-12 not only can suppress newborn growth of tumor, and can make already present tumor regression.Discovery whole bodies such as Brunda are used the growth that IL-12 can significantly reduce the number of mouse tumor lung transfer and suppress subcutaneous solid tumor, local injection IL-12 better effects if, even Subcutaneous tumor is disappeared.In addition, after the Mus intravenous injection B16F10 melanoma, with the IL-12 intraperitoneal injection lung is shifted obviously and reduces, though the injection oncocyte after 7 days begin treatment also obtain similar effect.The number of the renal cell carcinoma hepatic metastases of same IL-12 treatment minimizing the experimental M5076 reticulosarcoma and Renca.Prolonged the life-span of animal.

The clinical trial of using rhIL-12 treatment malignant tumor is existing abroad more than 10, is recruiting the volunteer or is carrying out clinical I phase, II phase clinical observation.The main malignant tumor of selecting comprises that intractable solid tumor of progressive stage, the hepatic metastases that is secondary to colorectal carcinoma, breast carcinoma involve liver, injection rhIL-12 treatment malignant melanoma, recurrent ovarian carcinoma in the carcinoma of prostate after the radiotherapy, cancer.Metastatic evil is black, the rectal cancer of recurrence in late period, advanced cervical cancer and malignant lymphoma etc.Treatment is broadly divided into independent use rhIL-12 or rhIL-12 and other cytokines and antitumor monoclonal antibody use in conjunction.The approach of administration comprises intravenously administrable, intraperitoneal administration, locally injected into tumor and subcutaneous injection.Dosage 100～1000ng/kg, patient's maximum tolerated dose (MTD) is 1000～1500ng/kg.The dosage and the course of treatment reach in different tumors, different way of administration, and inconsistent.Produce the dosage and the course of treatment of the rhIL-12 of best clinical effectiveness, not definite fully as yet.In therapeutic process, answer emphasis monitoring differential blood count and platelet count and bone marrow biopsy or marrow aspiration inspection.Comprise the detection of peripheral blood lymphocyte and cell subsets, serum anti-angiogenesis, VEGF (VEGF) and the basic fibroblast factor (bFGF), serum I FN-γ, IL-15, IL-18 level in addition.The oncotherapy reaction was generally assessed after treatment in the 8th week, and assessment in per 2～March after this once.

RhIL-12 is as single medicine, and from the clinical observation of the kinds of tumors of having carried out, the result shows and shows different curative effects at different tumors.The I phase clinical observation of rhIL-12 treatment renal cell carcinoma and malignant melanoma demonstrates infusive effect.Have good tolerability and the anti-tumor activity stronger after the application than other cytokines, show that rhIL-12 can rebuild the cellular immune function of patients with advanced cancer, the potentiality that late tumor shown treatment, confirm the really secretion of the energy inducing cell immunologic function promotion cells involved factor of rhIL-12, cause disappearing of tumor, Motzer etc. observe rhIL-12 treatment renal cell carcinoma 51 examples in late period, and treatment finishes back 1 routine patient alleviates 34 routine stable disease fully.The rhIL-12 treatment malignant melanoma in late period that Mortarini etc. carry out, 2 routine metastasis all disappear after 2 courses of treatment, and 1 example part disappears.But II phase clinical observation rhIL-12 treatment renal cell carcinoma and malignant melanoma effect can not be satisfactory.Anas Younes etc. use rhIL-12 treatment recurrent and intractable non-Hodgkin lymphomas and hodgkin's lymphomas, recruit 42 patients that treated altogether, wherein 32 are called the non-Hodgkin lymphomas, 10 hodgkin's tumors by name.The patient accepts rhIL-12 intravenous injection (n=11) or subcutaneous injection (n=31), and intravenous dosage is 250ng/kg, and continuous 5 days, per 3 weeks, once hypodermic dosage was 500ng/kg, and 2 times weekly, the treatment back was assessed curative effect in the 8th week.39 patients (93%) can carry out curative effect assessment, and 6 (21%) in 29 non-Hodgkin lymphomas have reaction partially or completely, and therapeutic response does not all appear in 10 lymphogranulomatosises.The plateau that further observations indicate that the patient has continued 54 months.

About the toxic reaction of rhIL-12 treatment, most of patient can tolerate preferably, shows as toxic reaction 1 or 2 grades, and 19% patient reaches 3 grades.Heating appears in 95% patient, myalgia, the arthralgia of general appears in 81% patient, feeling sick appears in 58% patient, 21% patient low-grade infection occurs and comprises sinusitis, influenza, conjunctivitis and pharyngitis, the hepatotoxicity reaction appears in individual patient, need to reduce the consumption of rhIL-12, do not see the antibacterial and the fungal infection of general.Most of in a word patient accepts intravenous injection or subcutaneous injection rhIL-12 can well tolerate, and the toxicity that part patient occurs is resolution of symptoms after reusing rhIL-12.

Carried out exploring widely about the antitumor mechanism of rhIL-12, but do not illustrated fully as yet so far, it suppresses growth of tumor and transfer, prolongs the main cause of patient's time-to-live, and each author's understanding is not the same.1. majority think that the antitumor action of rhIL-12 mainly relies on its immunoregulation effect, because IL-12 is the core cytokine in the immunological network, particularly by inducing Th1 cell development and propagation, strengthen the killing ability of T cell, activation NK cell impels it to produce cytotoxicity, strengthens the NK/LAK cell lysis activity.Chen etc. think that the antitumor action of IL-12 mainly passes through the secretion increase of the antigenic specificity IFN-γ of CD4+T cell generation, realization kills and wounds tumor cell, Van Herpen etc. thinks the Graft Versus Tumor of IL-12 mainly with to induce the NK cell to soak into relevant in primary tumo(u)r, Xu etc. think that the antitumor action of IL-12 needs the fellowship of NK cell and CD8+T cell.And Kobayashi thinks that the Graft Versus Tumor of IL-12 mainly relies on the T cell, the comprehensive function of NK cell and natural killer T cells (NKT).2. the other author thinks that the antitumor action of IL-12 mainly relies on its antineoplastic vascular nucleus formation, discoveries such as Sunamura, under no immune system participation situation, the angiogenesis inhibitor effect that IL-12 is simple enough stops the growth of human pancreas cancer in the SCID mice (PK.1).Reports such as Dian, in athymism Mus tumor experiment, IL-12 can make angiogenesis reduce, melanoma, colon cancer, Burkitt lymphoma, ovarian cancer, breast carcinoma etc. all there is therapeutical effect, and think that IL-12 suppresses the participation that angiogenesis needs the NK cell, the NK cell may be the mechanism that suppresses angiogenesis to the toxic action of endotheliocyte.3. comprehensive the above views thinks that the antitumor action of IL-12 is the coefficient result of multiple factor, has both comprised immunoregulation effect, comprises blood vessel formation against function again.Should be understood that IL-12 has complicated network regulation in vivo, very complex interactions is arranged between the various cytokines, exist the heterogeneity of antitumor mechanism probably, promptly under different situations, different tumors is existed different action principles.Therefore rhIL-12 and other chemotherapeutics, cytokine, antitumor monoclonal antibody can be united use, and the Comprehensive Treatment of branch period, sequential will be a developing direction from now on.IL-12 antineoplastic effect will well be embodied in the control of human malignancies surely.

Bacillus pyocyaneus (PA-MSHA) is at the nature ubiquity, basic research and clinical observation show that PA-MSHA vaccine preparation is not only effective to nosocomial infection, and tumor treatment also there is better action, this dead thalline by the discovery bacillus pyocyaneus of Japanese scholar had immunoregulation effect in 1975, humoral immunization and the cellular immunization of energy activation experiment animal, induce IFN-γ to generate, and the growth of cancer cells of opposing inoculation, but can't use because of toxicity is big and clinical, domestic scholars etc. provide a kind of MSHA the construction method of positive bacillus pyocyaneus pilus strain, it is characterized in that many very thin and upright and outspoken pili are arranged around thalline, MSHA (Mannose Sensitive Hemagglu tination) is positive, and flat-plate bacterial colony adheres to erythrocyte test strong positive.Its biological behavior performance at first is the virulence that has reduced bacillus pyocyaneus, and virulence only is 1/80 of original flagellum strain.Next is a whole body property MSHA pili, makes it have the immunogenicity that the broad-spectrum high efficacy valency is crossed over Pseudomonas.Use the dead bacterium of this bacterial strain to make hypotoxic broad-spectrum immunomodulator, but activating immune system is resisted the infection of multiple pathogenic microorganism, also adjustable whole body's immunological function activates body fluid and cellular immunization so that produce antineoplastic action.

The report of domestic and international application PA-MSHA vaccine preparation for treating animal experiment tumor is much.The injection flagellin can reduce the speed of tumor growth when Sfondrini in 2006 etc. reported to experiment mice immunoprophylaxis originality tumor.If inoculate the treatment that flagellin is united the Deoxydization nucleotide that contains CPG in early days, the growth of experimental tumor is suppressed fully, and showing between flagellin and the CPG has synergism.Report PA-MSHA vaccines such as domestic Zhang Mingce---a kind of anti-tumor activity of the vaccine by the low toxicity MSHA bacillus pyocyaneus preparation of carrying common I type flagellum, the result shows that the immunoloregulation function of vaccine can significantly strengthen the growth and the transfer of planting the local metastatic tumor of tumor mouse anti, immunization therapy can improve survival rate, prolongs average survival period.Preimmunization inoculation PA-MSHA vaccine can improve the percentage rate of transplanting mice spleen CD3 cell and cd4 cell in addition.Regulate CD3/CD4 ratio in normal condition, stimulate the NKT activity that strengthens the NK cell, induce to produce multiple antibody response reaction, the anti-tumor activity of prompting PA-MSHA vaccine is realized by influencing a plurality of approach such as T lymphocyte, NK cell function and humoral Immunological State.

It is existing historical for many years that PA-MSHA vaccine preparation is applied to the clinical treatment malignant tumor.Sun Wen equality has been observed 12 immune indexes in the therapeutic process of PA-MSHA bacterination 301 routine cancers (acute leukemia, malignant lymphoma and pulmonary carcinoma) and has been changed and compared with clinical efficacy.The result shows the immunization therapy total effective rate: acute leukemia is 87.1%, and malignant lymphoma is 77.78%, and pulmonary carcinoma is 84.09%.Experimental group after the treatment in 3 groups and every immune indexes (as IgG, M, A, C3, C4, CD4/CD8, NK cytoactive, IL-2 level etc.) of matched group are compared, and the P value is all less than 0.05.Think that the PA-MSHA vaccine can improve comprehensively and adjust cancer patient's humoral immunization and cellular immune function, strengthen the effect of killing and wounding with anticancer.Cheng Ran in 2002 uses PA-MSHA bacterination pulmonary carcinoma to compare complete remission rate with the chemotherapy matched group and rises to 66.09% by 6.67%.Lab testing shows treatment back NK cell activity, and the remarkable rising of the level of CD4/CD8 and IL-2 all reaches normal level.In addition the big and Zhang Chenghui of Yao Zhi etc. respectively with green Mu An promptly " Pseudomonos aeruginosa MSHA fimbria strain vaccine " cooperate radiotherapy in the treatment middle and advanced stage cervical cancer and cooperation CHOP scheme to treat intractable malignant lymphoma respectively, obtained good effect.

The toxic and side effects of PA-MSHA vaccine is very unobvious, and disposable heating appears in indivedual cases, and redness, pain, scleroma in various degree, transference cure after anti symptom treatment appear in the some cases injection site.

Mechanism about PA-MSHA bacterination tumor has proved that the flagellin of bacillus pyocyaneus is as a kind of ectogenic part, can specificly be discerned by Toll sample receptor-5 (TLR5), the body various kinds of cell can be expressed TLRs, as dendritic cell, macrophage etc., identification by the pattern recognition receptor, start very complicated signal transduction pathway, trigger innate immunity and acquired immunity, thereby produce corresponding immunoregulation effect.Laboratory work shows that bacillus pyocyaneus flagellin antineoplastic dominant mechanism is by transferring, strengthen host's autoimmune function, suppressing to kill cancerous cell.The patient IL-2 level of application PA-MSHA bacterination, the ratio of NK cytoactive CD4+/CD8+ are all apparently higher than matched group, the flagellin of prompting bacillus pyocyaneus, impel the T cell activation to produce a series of cytokines such as IL-2, stimulate NK cell and macrophage proliferation, increase its cytotoxicity, improve the function that it kills and wounds cancerous cell, activation CD4+ cell is strengthened the synthetic and secretion of IFN-γ, and IFN-γ plays an important role in antagonism cancerous cell and immunomodulating.

In sum, rhIL-12 treatment late period, intractable solid tumor had certain curative effect, the patient's that well-known late malignant tumour is particularly performed the operation, radiation and chemotherapy recurs later on or occur shifting treatment is very thorny, and the effect of rhIL-12 therapeutic advance phase, intractable tumor I phase clinical observation is challenging.But should point out that the therapeutic effect of rhIL-12 and toxicity all exist dose-effect relationship to a certain extent.Be that the using dosage of rhIL-12 and curative effect and toxic and side effects increase along with the increase of dosage, therefore limited bigger its effect of performance of rhIL-12 to a certain extent.RhIL-12 can be used as the initial property of the 3rd signal direct activation T cell theoretically in addition, if but increase an antigenic stimulation again, this antigen just can increase the biological effect of rhIL-12 greatly as the 1st signal and rhIL-12 synergism.On the other hand, use the PA-MSHA vaccine separately, the immunogenicity of its flagellar antigen is lower, and the immune response that brings out particularly cellular immunization is strong not enough, and rhIL-12 then can obviously strengthen the effect of PA-MSHA vaccine as the 1st signal.

Summary of the invention

The present invention is for solving the technical problem that present oncotherapy immunomodulator kind is few, therapeutic effect is poor, side effect is big, providing a kind of therapeutic effect the good immunomodulator that is used for treating malignant tumor.

The present inventor thinks, rhIL-12 treatment malignant tumor preferably and other TPAs or other have the antigen combined application of active cell immunity, so both can reduce the therapeutic dose of rhIL-12, reduce toxic and side effects and can strengthen other immunogenicity of antigens effects again, thereby obviously improve working in coordination with and complementary action of the two.

The present inventor finds rhIL-12 and PA-MSHA vaccine preparation are combined into a kind of new immunomodulator, make the rhIL-12 that contains 5～30 μ g in every ml of formulation, the PA-MSHA vaccine preparation of 1,030 hundred million units and the aqueous solution of surplus, the level that significantly improves PERIPHERAL BLOOD MONONUCLEAR CELL generation IFN-γ that this combination preparation can be fairly obvious.

The preparation method of immunomodulator of the present invention is also very simple, makes compositions and gets final product rhIL-12 and PA-MSHA vaccine are directly mixed, and preparation is a water preparation.

Immunomodulator of the present invention shows to have tangible tumor-inhibiting action through the observation of laboratory animal tumor model.

The specific embodiment

The invention will be further described below in conjunction with embodiment, but do not constitute any limitation of the invention

Embodiment

The PA-MSHA vaccine is from Hainan microgram west Bioceuticals Inc. in the embodiment of the present invention.

RhIL-12 is developed voluntarily by the inventor, is positioned on the different chromosomes by the encode gene of P35 and P40 subunit of many heterologous protein dimerizations that disulfide bond is formed by two subunits of P40 and P35 because IL-12 is one.The P35 subunit can not be secreted into the extracellular separately, and the P40 subunit can be secreted into the extracellular separately.Therefore expressing two subunits in same cell inner equilibrium is keys of expressing the IL-12 of biologically active, and therefore the Chinese hamster ovary celI strain that makes up must make two subunit balances of IL-12 express.We insert two carriers that expression efficiency is different respectively with P40cDNA and P35cDNA for this reason, and cotransfection Chinese hamster ovary celI then can make the expression of two different subunits of IL-12 in a basic balance like this.Further improve the expression of P35 subunit in addition by the amplification system on the carrier.So just make the balance of P40 and P35 express further reinforcement.The CHO-SFM-III that successfully constructs the Chinese hamster ovary celI strain application Gibco that expresses IL-12 carries out static cultivation, use Sartorius BiosSTAT plus cell culture jar again and criticize cultivation and continuous culture, cell strain has reached generation more than 30, cell growth state is good, and protein expression is stable, adopt the ELISA method to detect content and the activity of rhIL-12, static cultivation mean concentration is 4 μ g/ml, cell culture jar continuous culture mean concentration is 10 μ g/ml, and the IL-12 determination of activity shows that having good IFN-γ induces ability.

The purification basic skills of rhIL-12 is that the supernatant with culture fluid carries out centrifugal treating, remove cell and fragment thereof, carry out ultrafiltration and concentration again through cation chromatography, hydrophobic chromatography, anion chromatography, obtain the rhIL-12 of purification at last by sieve chromatography, high performance liquid chromatography (HPLC) detects and shows that purity is near 98%.

It below is the preliminary pharmacodynamic experiment of novel immunomodulator.

At first observe different stimulated source and rhIL-12 and united the malignant tumor patient peripheral blood PBMCs secretion of gamma-IFN level that induces, the result shows that rhIL-12 associating PA-MSHA vaccine induces IFN-γ and obviously surpasses other stimulus or use rhIL-12 separately and the PA-MSHA vaccine, and experimental technique and result are as follows.

1. method

Malignant tumor patient 10 people, healthy people 10 people get the 5ml peripheral blood respectively, after the anticoagulant heparin, separate PBMCs with the FicoHHypaque density gradient centrifugation method.Collecting cell is also used the PRMI-1640 washed twice.The RPMI-1640 that contains 10% hyclone with 1ml is with cell suspension then, and counting is 2.0 * 10 with above-mentioned culture fluid adjustment cell concentration ⁶Individual/ml, getting 100 μ l adds in each hole of 96 well culture plates, the various stimulus object 100 μ l that add the RPMI-1640 preparation of 10% hyclone respectively, as follows, anti-cd 3 antibodies (0.2 μ g/ml), PA-MSHA vaccine (dilution in 1: 100), PA-MSHA vaccine (dilution in 1: 100)+rhIL-12 (0.1ng/ml), PA-MSHA vaccine (dilution in 1: 100)+rhIL-12 (1ng/ml), PA-MSHA vaccine (dilution in 1: 100)+rhIL-12 (2ng/ml) and barren RPMI-1640 are in contrast, each stimulus object is all done 3 multiple holes, at 5%CO ₂Cultivate 48h under the wet condition, get afterwards its supernatant put in-20 ℃ of refrigerators preserve to be checked.Detect PBMCs culture supernatant IFN-γ level with the ELISA method.

2. result

10 routine malignant tumor patients and 10 routine healthy people PA-MSHA vaccines (dilution in 1: 100) add that variable concentrations rhIL-12 induces the comparison of PBMC secretion of gamma-IFN level, the results are shown in subordinate list 1.Show that rhIL-12 associating PA-MSHA vaccine induces the PBMC secretion of gamma-IFN and obviously surpasses use rhIL-12, PA-MSHA vaccine separately.

Table 1 different stimulated source induces the result of IFN-γ to healthy people and malignant tumor patient PBMCs

Group	Healthy people	Malignant stages patient
			Blank	22.8±6.18	33.9±29.51
Anti--CD3 contrasts (0.2ug/ml)	29461.01±2739.10	23324.93±11755.52
			rhIL-12(0.1ng/ml)	556.60±145.21	440.90±314.88
rhIL-12(1ng/ml)	1229.63±725.17	870.13±384.43
			rhIL-12(2ng/ml)	1682.44±650.96	1194.38±912.90
PA-MSHA vaccine (1: 100)	212.27±88.52	131.05±124.09
			PA-MSHA vaccine (1: 100)+rhIL-12 (0.1ng/ml)	6363.34±4367.48	5597.38±4329.97
PA-MSHA vaccine (1: 100)+rhIL-12 (1ng/ml)	24369.65±8974.49	17150.02±16604.52
			PA-MSHA vaccine (1: 100)+rhIL-12 (2ng/ml)	26398.52±10854.95	18740.52±15932.15

RhIL-12 and the active observation of the anti-experimental tumor of PA-MSHA vaccine have been carried out in addition again.

1. method: laboratory animal is the SD rat of cleaning level, body weight 80～100g, and 40, male and female half and half, all animals are in right stomach wall inoculation 0.2ml Walker-256 tumor cell suspension (cell number 5.0 * 10 down ⁵/ ml).The rat of inoculated tumour cell is divided into 4 groups, 10 every group, the 1st group of rhIL-12 group, from the inoculated tumour cell, the next day lumbar injection rhIL-12 1.5 μ g/ time, to 21 days; The 2nd group of PA-MSHA vaccine group from inoculated tumour, injected PA-MSHA vaccine 0.1ml every day, to 21 days; The 3rd group of rhIL-12+PA-MSHA vaccine group, lumbar injection rhIL-121.5 μ is g/ time next day of from the inoculated tumour cell, injects PA-MSHA vaccine 0.1ml to the every day 21 days; The 4th group of matched group, every day, lumbar injection RPMI-1640 1ml to the was 21 days.

2. result: rhIL-12 associating PA-MSHA vaccine antitumous effect sees Table 2.The result shows that tumor incidence rate and the growth of tumor speed of rhIL-12 associating PA-MSHA vaccine after suppressing the Walk-256 tumor inoculation all obviously is better than using separately rhIL-12 or PA-MSHA vaccine.

Table 2rhIL-12, PA-MSHA vaccine lumbar injection are to inoculating the influence of Walker-256 tumor SD rat tumor incidence rate and tumor weight

Group	Number of animals	Tumor incidence rate (%)	Tumor weight (g)
				rhIL-12	20	80	6.1±2.4
The PA-MSHA vaccine	20	85	7.3±3.1
				The rhIL-12+PA-MSHA vaccine	20	45	3.2±1.8
Matched group	20	100	12.5±4.3

Show by above-mentioned experiment:, have very significant immunoregulation effect of having mutually promoted self by the novel immunomodulator of rhIL-12 and PA-MSHA vaccine preparation combination.The result who uses is that PA-MSHA vaccine preparation is obviously strengthened as the immunogenicity of first signal, and rhIL-12 is as the core cytokine of immunological network, and its 3rd signal effect has also obtained reinforcement especially.Its result causes the two function of bringing out cellular immunization significantly to be strengthened, and for suppressing growth of tumor, killing tumor cell has been created good internal condition.Therefore, this combination immunomodulator has very good prospects for application as the supplementary means of clinical treatment tumour.

Claims (1)

1. immunomodulator that is used for treating malignant tumor is characterized in that containing in every ml of formulation the Pseudomonas Vaccine preparation of recombinant human interleukin-11 2,10～3,000,000,000 units of 5～30 μ g and the aqueous solution of surplus.