CN100435849C - Antitumor agent containing interleukin 23 gene - Google Patents

Antitumor agent containing interleukin 23 gene Download PDF

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CN100435849C
CN100435849C CNB2004100081062A CN200410008106A CN100435849C CN 100435849 C CN100435849 C CN 100435849C CN B2004100081062 A CNB2004100081062 A CN B2004100081062A CN 200410008106 A CN200410008106 A CN 200410008106A CN 100435849 C CN100435849 C CN 100435849C
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CN1701816A (en
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田川雅敏
王彦青
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Fudan University
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Abstract

The present invention relates to an antitumor agent containing an IL-23 gene or an IL-23 gene-introduced cell as an active ingredient, which belongs to the field of biotechnology. The antitumor agent specifically comprises the following substances as active ingredients: a virus or non-viral vector expressing IL-23 gene, a tumor cell into which IL-23 gene is introduced and which has lost its ability to divide, and an immune cell into which IL-23 gene is introduced. The antitumor agent of the present invention can be used alone or in combination with treatment means such as surgery, radiotherapy, chemotherapy, etc., for improving cellular immune response of the body, thereby achieving the purposes of reducing tumor volume, preventing metastasis and preventing tumor recurrence.

Description

A kind of antitumor agent that contains the interleukin 23 gene
Technical field
The invention belongs to biological technical field, relate to the IL-23 gene or import the antitumor agent of the cell of IL-23 gene as effective ingredient.Specifically comprise with the following substances being the antitumor agent of effective ingredient: express virus or non-virus carrier, the importing IL-23 gene of IL-23 gene and lose the tumor cell of splitting ability, the immunocyte of importing IL-23 gene.Antitumor agent of the present invention can use separately, also can combine with treatment meanss such as operation, X-ray therapy, chemotherapy, is used to improve the cellullar immunologic response of body, dwindles gross tumor volume, prevents to shift and the purpose of prophylaxis of tumours recurrence thereby reach.
Background technology
Existing tumor therapeuticing method (as X-ray therapy, chemotherapy, excision etc.) because have great toxic and side effects, the immune function that easily causes body descends, and therefore is unfavorable for patient's prognosis.Chemotherapy or X-ray therapy infringement bone marrow function, the body defense reaction is low, causes secondary infection on the one hand easily, and immune system is also low to the immunne response of tumor cell on the one hand in addition.Operation itself causes the wound to body, also causes immunosuppressant, therefore, the expansion of micro metastasis often takes place after the operation.In addition, for various reasons, the immune system of tumor patient itself lacks reaction to the attack of tumor, the immunologic tolerance phenomenon promptly occurs.Therefore, breaking immunologic tolerance, improve the immunologic hypofunction that various treatments cause, strengthen the immunne response of body to tumor, is the key subjects in the oncotherapy.
For addressing the above problem, utilize the various trial well afoots that act on each stages of cell factor regulation and control body's immunity of immunne response.A kind of method is directly to give recombinant cytokine, and another method is that the gene that gives cytokine carries out gene therapy.(Rosenberg?SA，J?ClinOncol，10；180-199，1992。Tepper?RI，Mule?JJ，Hum?Gene?Ther，5；153-164，1994)。Because body clearance rate height, cause the half-life of general cytokine very short, cytokine is difficult to keep certain concentration in the blood, and excessively gives cytokine toxic effect, therefore, directly gives recombinant cytokine and has certain limitation.And along with the technology of utilizing physics maneuvers such as liposome, virus or ultrasonic, electric shock with the allogenic gene transfered cell reaches its maturity, to tumor cell transfered cell factor gene, making its secrete cytokines, producing antitumor action is simple and effective method, and this is just belonging to the category of therapy of tumor.
In this Therapeutic Method, directly cause activation from the cytokine of tumor by local continuous release to the accumulative immunocyte group of tumor, therefore infer to have better antitumous effect and safety than directly giving recombinant cytokine to individuality.Because the key factor of performance Graft Versus Tumor is that tumor is had the cell killing T cell (CTL) of specific killing effect and has the natural killer cell (N of non-specific lethal effect Atural Killer cell, the NK cell), the cytokine of using acts on above-mentioned two class cells mostly at present, promotes its activation and/or propagation.
Some tumor antigen polypeptide is found in recent years, further provides practical theoretical foundation for utilizing immunne response treatment tumor.Through after the suitable processing, tumor antigen is a cell killing T cell by antigen presenting cell arborescent cell submission on-effect cell, thereby it is activated and brings into play the effect of specific killing tumor cell (Nature 392 for Banchereau J, Steiman RM; 245-252,1998).In the cytokine of always finding, has the important function of inducing I type helper T lymphocyte (Th1), inducing the generation Graft Versus Tumor by the secreted interleukin 12 of activatory arborescent cell (IL-12).Found again recently by the excretory a kind of new cytokine of arborescent cell, be named as interleukin 23 (Interlukine-23, IL-23) (Oppmann B et al, Immunity 13; 715-725,2000).The dimer that IL-23 is made up of two subunits (p19 molecule+p40 molecule), wherein the p40 molecule also is a subunit of IL-12 (p35 molecule+p40 molecule).IL-23 and IL-12 the two except the p40 subunit is all arranged, the amino acid sequence homology of the p19 subunit of IL-23 and the p35 subunit of IL-12 is also very high.
Summary of the invention
The cell that the objective of the invention is to utilize the IL-23 gene or import the IL-23 gene prepares antitumor agent for effective ingredient.
Though had not major general's recombinant cytokine IL-2, IFN-α, TNF-α etc. to be used for the treatment of the practice of tumor, curative effect does not also have clear and definite final conclusion.In addition, directly the clinical experimental study that carries out gene therapy to the tumor quiding gene is in the initial development stage, and therefore above-mentioned cytokine and gene thereof are applied to clinical treatment and do not obtain remarkable break-throughs.
The used cytokine of the present invention and therapy of tumor in the past is different, use newfound, the cytokine IL-23 that produces by activatory arborescent cell.And the activation of arborescent cell has pivotal role at the immunne response initial stage, is to induce the necessary process of cell killing t cell activation with specificity antineoplastic effect.The present invention observes these cells and causes stronger Graft Versus Tumor in the IL-23 gene transfered cell.Specifically be that external, IL-23 has facilitation to propagation, activation and the T emiocytosis IFN-γ of memory T cell; In vivo, IL-23 can cause that tumour-specific obtains immunity; Transplant people's tumor cell to the immunodeficiency nude mice that lacks the T cell, also observe the antitumor action of IL-23.
IL-23 is excretory by activatory arborescent cell, by the p40 subunit of IL-12 and the cytokine be made up of the p19 of high homology with the p35 subunit of IL-12.The used IL-23 gene of the present invention shown in sequence table, listed aminoacid sequence in the sequence table of base sequence code book shown in the sequence table 1 (being people p19 molecule); Listed aminoacid sequence in the sequence table of base sequence code book shown in the sequence table 2 (being people p40 molecule);
Coding also can be used for genetic treatment of tumor with the gene that IL-23 has the IL-23 mutant of same pharmacological action.Such as, on sequence table 1 listed aminoacid sequence (p19) basis, disappearance one is to several aminoacid, displacement or add several aminoacid, and generation has the mutant of p19 pharmacological action; Again such as, on aminoacid sequence shown in the sequence table 2 (p40) basis, disappearance one is to several aminoacid, displacement or add several aminoacid, generation has the mutant of p40 pharmacological action; Also comprise in addition, by under stringent condition, obtain with the gene recombination that contains sequence 1 listed base sequence, coded molecule and p19 (or p40) molecule have the gene of mutant of the p19 (or p40) of same pharmacological action.Here the stringent condition of indication is such as hybridizing under following condition: containing 6 * SSPE, in the hybridization solution of 2 * Denharts solution, 0.5%SDS, 0.1mg/ml milt DNA, under 65 ℃, carrying out Southern hybridization in 12 hours.
Said gene can obtain with methods such as well-known PCR according to the let others have a look at sequence of p19 molecule and p40 molecule of sequence table.These methods, convenient and easy under the guidance of " molecular cloning experiment guide " books such as (second editions) of publishing at the cold spring port.The making of said mutation body utilizes the method for PCR or hybridization also simple.
For reaching the purpose of treatment tumor, the present invention need utilize virus or non-virus carrier to express IL-23 albumen.Representative viral vector has adenovirus, retrovirus retrovirus etc., retrovirus retrovirus, adenovirus, adeno-associated virus, herpesvirus, vaccinia virus, poxvirus, sindbis alphavirus, poliovirus, poliovirus, Sendai virus, SV40, HIV (human immunodeficiency virus) DNA viruses such as (HIV) and some RNA viruses of poisoning as nothing.In above-mentioned viral vector,, thereby use more because adenovirus vector has higher efficiency of infection.
Non-virus carrier refers to can be at some carriers of mammal expression in vivo genes of interest, pcDNA3.1 for example, pZeoSV, pBK-CMV (Invitrogene company, Strategene company), pCAGGS (Gene 108,193-200,1991) etc.
For the included antitumor agent of preparation the present invention, must in above-mentioned all kinds of carriers, insert said gene or mutant in effable mode.The preparation method that is used for these viral vector or non-virus carrier is that medication all is to utilize disclosed technology, has a lot of documents or the guide book of experiment can reference, as " experimental medicine fascicle ", the basic fundamental of gene therapy, sheep Tu She, Japan, 1996; " experimental medicine fascicle ", the experimental technique that gene imports and expresses, Yang Tushe, Japan, 1997; " gene therapy developmental research handbook, 1999 etc. are compiled by gene therapy association.
These are inserted into the IL-23 gene and can express virus or the both directly administrations of non-virus carrier of IL-23, also can add excipient in medical permissible range, make dosage forms such as solution, suspension, colloid.Administering mode can carry out topical to the position that the tumor focus or the corresponding easy generation of different tumor of tumor patient are shifted, or be administered systemically by vein or tremulous pulse.The dosage of administration is according to the difference of patient's symptom, age, sex, route of administration, dosage form and difference, with the Mass Calculation of IL-23 gene, can give 10 micrograms to 500 every day of generally being grown up and milligram not wait.
The present invention also is included in and externally imports said gene to patient's cell and make it express IL-23, then these cells is imported in patient's body again.This cell mainly comprises kinds of tumor cells and immunocyte.Tumor cell such as colorectal cancer cells, lung carcinoma cell, kidney cancer cell, melanoma cell, breast cancer cell, squamous epithelium cancerous cell, transitional epithelium cancerous cell, sarcoma cell, neuroglial cytoma etc.What particularly point out is that the tumor cell that is used for the treatment of purposes preferably derives from same tumor; In addition, homologous tumor cell of the same race is also proper.Immunocyte then comprises the activated killer cell of lymphokine (LAK cell), cell killing T cell (CTL), tumor infiltrating lymphocyte (TIL), natural killer cell (NK cell), NKT cell, B cell or peripheral blood lymphocytes.Immunocyte of the same race more is applicable to treatment, promptly treats the immunocyte that human tumor is answered the end user.
Can utilize the above-mentioned virus or the non-virus carrier that contain the IL-23 gene to import the IL-23 gene to tumor cell.As (BioTechniques 7 for Miller AD, Rosman GJ to LXSN; 980-990,1989), the multiple clone site of MCF, α-SCG, PLJ, pEm (6-503968 patent announcement in 1994) isoinversion record viral vector, but insert purpose IL-23 gene with expression way, the retrovirus vector that will make up then imports incasing cells, cultivation is imported into the incasing cells of IL-23 gene, get its supernatant infected tumor cell, thereby import the IL-23 gene to tumor cell.The tumor cell that imports the IL-23 gene passes through the irradiation of lonizing radiation usually, imports in the body after making it lose splitting ability.
In addition, can utilize the above-mentioned virus or the non-virus carrier that contain the IL-23 gene, import the IL-23 gene to immunocyte.For example, to Adexl (Saito, I.et al. from human 5 type adenoviruss source, J Viol., 54,711-719,1985) but etc. cloning site insert the IL-23 gene with expression way, the adenovirus vector that will make up is with in the conventional method importing immunocyte then.
Tumor cell or immunocyte that these import the IL-23 gene can add excipient in medical permissible range, make dosage forms such as solution, suspension, colloid.Administering mode can carry out topical to the position that the tumor focus or the corresponding easy generation of different tumor of tumor patient are shifted, or be administered systemically by vein or tremulous pulse.The dosage of administration is according to the difference of patient's symptom, age, sex, route of administration, dosage form and difference is calculated with the tumor cell or the immunocyte number that give, and can give from 10 the every day of generally being grown up 5Individual to 10 9Individually do not wait.
According to the present invention, give the IL-23 gene or be imported into tumor cell, the immunocyte of IL-23 gene, by activating anti-tumor immune response performance antitumor action, reach the treatment tumor, suppressing the effect of neoplasm metastasis.The tumor cell that imports the IL-23 gene can cause that specificity antineoplastic immunity replys, and the patient who is particularly advantageous at immunologic hypofunction brings into play antitumor action.
Description of drawings
Fig. 1 .CD40Ligand (CD40L) activates arborescent cell (DCs) and causes the secretion of P40 subunit.After expressing the mice lung cancer A11 and tree-shaped (DC) cell co-cultivation of Fas L or CD40L, measure the amount of the p40 molecule of tree-shaped (DC) emiocytosis.
Fig. 2 .CD40Ligand (CD40L) activates arborescent cell (DCs) and causes cell p19 and the excretory situation of p40 subunit.Detect the situation of tree-shaped (DC) cellular expression cytokine with the RT-PCR method.Mig is the content that the inductive monokine.GAPDH of IFN γ is used to demarcate used cDNA.
Fig. 3. the expression of p19 and p35 Subunit mRNA in the activation arborescent cell.Detect p19 and p35 expression of gene situation in the activated DC cell of CD40L with the RT-PCR method.The p19mRNA expression is significantly higher than p35mRNA.
Fig. 4. the expression of p19 and p35 Subunit mRNA in the Colon26 cell of importing IL-23 gene.With retrovirus vector after Colon 26 imports IL-23 genes (p19-IRES-p40), Colon26/IL-23#1 and Colon 26/IL-23#9) express the situation of IL-23 gene.
Fig. 5. import the antitumous effect (1) of the Colon26 cell of IL-23 gene.(Colon 26/IL-23#1 and #9 are respectively 1 * 10 to change Colon 26 cells of IL-23 gene over to 6Individual) antitumous effect that produces.Matched group is Colon 26 cells (1 * 10 that change the hole carrier DNA over to 6Individual).Cell is inoculated into subcutaneous.Standard error is marked among the figure.
Fig. 6. import the antitumous effect (2) of the Colon26 cell of IL-23 gene.(Colon 26/IL-23#4 and #12 are respectively 5 * 10 to change Colon 26 cells of IL-23 gene over to 5Individual) antitumous effect that produces.Matched group parental plant Colon 26 cells (5 * 10 6Individual).Cell is inoculated into subcutaneous.Standard error is marked among the figure.
Fig. 7. import the antitumous effect (3) of the Colon26 cell of IL-23 gene.Mix parental plant Colon 26 (5 * 10 5) with change cell (Colon 26/IL-23#9,1 * 10 of IL-23 gene over to 5Individual, 10%) antitumous effect that produces.Matched group parental plant Colon 26 cells (1 * 10 6Individual, 100%).Cell is inoculated into subcutaneous.Standard error is marked among the figure.
Fig. 8. import the antitumous effect (4) of the Colon26 cell of IL-23 gene.The parental plant Colon that opposite side is inoculated simultaneously to BALB/C mice subcutaneous abdomen one side joint kind Colon 26/IL-23#9
The influence of 26 tumor growth situations.
Fig. 9. the amount of tumor regression mouse boosting cell secretion of gamma-IFN behind the inoculation Colon26/IL-23 cell.The situation of mouse spleen emiocytosis IFN-γ after the eliminating Colon 26/IL-23 tumor.Spleen cell passes through anti-CD4 in advance, and anti-CD8 and/or anti-ansialo GM1 antibody are handled, then Colon 26 or the RL-male-1 cell co-cultivation of crossing with treatment with irradiation.
The antitumous effect of the Colon26 cell inoculation of Figure 10 .IL-23 gene behind the nude mice.Change Colon 26 cells (Colon 26/IL-23#9,1 * 10 of IL-23 gene over to 6Individual) antitumous effect that produces.It is subcutaneous that cell is inoculated into the BALB/c nude mice.Standard error is marked among the figure.
Figure 11. the expression of p19 and p35 subunit in the ASPC-1 cell of importing IL-23 gene.Utilize retrovirus vector to change IL-23 gene (p19-internal over to human pancreatic cancer cell AsPc-1
Ribosomal rentry site-p40) after, p19, p35, the isogenic expression of p40.
Figure 12. the antitumous effect of the ASPC-1 cell inoculation that imports the IL-23 gene behind the nude mice.Utilize retrovirus vector to change the IL-23 gene over to, the Graft Versus Tumor of AsPc-1/IL-23 cell to human pancreatic cancer cell AsPc-1.It is subcutaneous that cell is inoculated into the BALB/c nude mice.Standard error is marked among the figure.
Figure 13. the antitumous effect of the ASPC-1 cell inoculation that imports the IL-23 gene behind the SCID mice.With the AsPc-1/IL-23 cell inoculation to the serious immunodeficiency of BALB/c (SCID) mice subcutaneous after, the growth of tumor situation.Standard error is marked among the figure.
Figure 14. the cell killing activity of nude mice spleen cells.With 51The Cr method for releasing is measured the cell killing activity of splenocyte.Spleen cell source is for contrast nude mice (not inoculated tumour cell) or inoculated parental plant AsPc-1 or changed the nude mice of the AsPc-1 cell (AsPc-1/IL-23) of IL-23 gene over to.With these spleen cells with 51The AsPc-1 cell co-cultivation of Cr labelling.
The specific embodiment
Following about implementation example of the present invention, just, the present invention is not constituted any qualification for illustrating in greater detail the concrete condition of invention.
Embodiment 1
The comparison of activation arborescent cell secretion IL-23 and IL-12 amount
Behind antibody method removal mouse bone marrow cells T cell, B cell and macrophage, in the training liquid that adds GM-CSF and IL-4 (20ng/ml, PeproTech company), cultivated non-adherent cell 10 days, thereby obtain arborescent cell.Utilize Cell Sorter to analyze the surface marker of gained cell, cell activation signs such as II type main tissue intersolubility antigen and CD86 show the weak positive, thereby are defined as immature arborescent cell.
On 24 well culture plates with these cells (6 * 10 5Individual/hole) with the lung carcinoma cell A11 cell (2 * 10 that has imported the CD40 gene 5Individual/hole) after the co-cultivation, cell activation signs such as CD86 increase, and determine that cell activates.From culture supernatant detect p40 with ELISA method (BioSourse company test kit) this moment, and in contrast, with arborescent cell and after importing the A11 co-culture of cells of FasLigand gene, supernatant does not detect p40.(Fig. 1).Detect cytokine expression with the RT-PCR method, find that the RNA of IL-23 subunit p19 and IL-12 subunit p35 has expression.(Fig. 2).The expression height of the expression ratio IL-12p35 of IL-23 subunit p19RNA.(Fig. 3).
The functional activity of activation arborescent cell is the key factor that excites the Graft Versus Tumor cytoactive.The amount of the excretory IL-23 of activation arborescent cell is significantly more than IL-12, and results suggest IL-23 may be more important than IL-12.
Embodiment 2
The foundation of IL-23 gene transfered cell
From the cell of BALB/c mouse subcutaneous transplantation tumor, extract total RNA, use the RT-PCR method, obtain p19 (attached sequence 3) and p40 (sequence 4) full length sequence cDNA, and be inserted into retrovirus vector LXSN (Miller AD, Rosman GJ, BioTechniques 7; 980-990,1989) multiple clone site (EcoRI/Bam HI), (intemal ribosome entry sites IRES) connects (Duke GM etc., J Virol 66:1602-1609,1992) by internal ribosome entry site between two kinds of cDNA.Therefore, retrovirus retrovirus can be transcribed p19 (being positioned at IRES 5 ' end) and p40 (being positioned at IRES 3 ' end) under the effect of the promoter that is positioned at long terminal repeat (LTR), and transcription product can be respectively by independent translation.
Utilize Lipofectin reagent (Invitrogen company) with above-mentioned retrovirus vector transfection in incasing cells ψ 2 cells, use G418 (400 μ g/l, Invitrogen company) to add in the culture fluid then and screen.Contain and add Polybrene (8mg/ml, Aldrich company) in the retrovirus retrovirus culture supernatant, be used to infect PA317 cell (ATCC), add G418 (400 μ g/ml, Invitrogen company) and select.Have in the PA317 cells and supernatant of G418 resistance and contain required retrovirus retrovirus.
With above-mentioned retroviral infection colorectal cancer cells (Colon 26), the cloning of passing through going forward side by side in IL-23 gene transfered cell screening.Set up cell strain Colon 26/IL-23#1, #4, #9, #12.Use Northern
Blot confirms that these cell strains that imported exogenous gene express p19, p35, and the situation of p40 (representative clone sees figure four), (secretory volume is 0.8-1.1ng/ml/1 * 10 to have confirmed to secrete in the culture supernatant situation of p40 with the ELISA method in addition 6/ 48 hours).The expression of the major histocompatibility antigen H-2K/H-2D that the tumor antigen submission is played an important role and the tumor cell of wild strain do not have significant difference.The in-vitro multiplication situation of parental plant and the strain of gene transfered cell is also identical.
Embodiment 3
The antitumous effect of IL-23 gene transfered cell
To homology BALB/c mouse inoculating two kinds import the IL-23 gene Colon 26 cells (Colon26/IL-23#1, #9), the tumor growth of generation is postponed, after after a while, tumor disappears fully.And in contrast, inoculation imports empty carrier, and not behind the parental plant cell of quiding gene, tumor growth is rapid,, finally cause dead mouse.Other clones also have similar antitumous effect.Inoculation Colon 26 cells (1 * 10 in the homology mouse peritoneal 6Individual) in back 20 days, mice is all dead.And behind the Colon 26/IL-23#9 or #12 of the same amount of inoculation, most of mice survivals surpass 40 days (x 2=3.5, df=1, P=0.037; Log-rank test, P=0.046).
Colon 26/IL-23#1 closes that (total cell number was 1 * 10 with 1: 9 mixed with Colon 26/IL-23#9 6Individual) after be inoculated in the homology mice, tumor form and the speed of growth than simple inoculation Colon 26 parental plants obviously slow down (P＜0.05).Show that the cell of secreting IL-23 can cause antitumor action at the tumor inoculation position.
[embodiment 4]
The therapeutic effect of IL-23 gene transfered cell
To homology mouse web portion one side subcutaneous vaccination Colon 26 cells (1 * 10 6Individual), abdominal part opposite side inoculation Colon 26/IL-23#9 (1 * 10 6Individual).Colon 26/IL-23#9 tumor finally disappears, and offside Colon 26 growth of tumor obviously slow down than the mouse tumor of only inoculating Colon 26.Therefore, behind the inoculation IL-23 gene transfered cell, can cause the immunne response of the parental plant tumor cell that exists at the distally, thereby reach therapeutic effect.
[embodiment 5]
Tumour-specific obtains the foundation of immunologic function behind the inoculation IL-23 gene transfered cell
IL-23 gene transfered cell inoculation back forms tumor and is refused by mice, and behind the initial inoculated tumour cell 60 days, inoculate Colon 26 cells of lethal dose once more, tumor no longer forms, and survival was above 120 days.And the mice of refusing Colon 26/IL-23 tumor, subcutaneous vaccination lethal dose xenograft tumor RL
Behind the male-1, tumor forms and grows rapidly most of dead mouse.Above result shows after tumor cell imports the IL-23 gene, causes the specific immune response with the T mediation.Table 1 is the situation of setting up that tumour-specific obtains immunity.
Table 1.
Figure C20041000810600121
1Be subcutaneous vaccination
2The total number of mice of number of mice/inoculate for survival
Embodiment 6
Induce IFN-γ to produce behind the inoculation IL-23 gene transfered cell
The spleen cell (1 * 10 of tumor regression mice behind the inoculation Colon 26/IL-23#9 7/ ml) with Colon 26 cells of handling through the lonizing radiation of 50Gy (1 * 10 6/ ml) co-cultivation is secreted in a large number with the closely-related cytokine IFN-γ of antitumous effect in the culture supernatant.And after cultivating altogether with RLmale-1 that lonizing radiation were handled, have only a small amount of IFN-γ secretion in the supernatant.Never inoculated after the spleen cell in mice source of tumor and the above-mentioned co-culture of cells in the supernatant substantially not secretion of gamma-IFN.The result shows, after inoculation can produce the tumor cell of IL-23, can induce generation at tumour-specific cell killing T cell.Table 2 is situations of BALB/c mouse spleen cell secretion of gamma-IFN.
Table 2
Figure C20041000810600131
1Lonizing radiation are handled
2Do not detect
For further determining The above results, to mouse hypodermic inoculation Colon 26/IL-23#9 cell, after treating that tumor disappears fully, getting spleen cell cultivates, with anti-GM1 antibody, anti-CD 4 antibodies, anti-CD8 antibody treatment, remove NKT (NK) cell, CD4 positive T cell, CD8 positive T cell respectively, detect the secretion situation (Fig. 9) of IFN-γ.After the spleen cell of the anti-CD8 antibody treatment of process and Colon 26 co-cultivation that radiation exposure is crossed, the amount of its secretion of gamma-IFN significantly descends.Show that the inductive Graft Versus Tumor of IL-23 is mainly mediated by CD8 positive cell killer T cell.
Embodiment 7
Under the immune deficiency state, the Graft Versus Tumor of IL-23 gene transfered cell
The BALB/c nude mice subcutaneous vaccination Colon 26/IL-23#9 cell (1 * 10 that lacks the T cell 6Individual), different with the normal BALB/c mouse of immunologic function, tumor forms the back and continues growth.Show that α/functional activity of β T cell and the antitumous effect of IL-23 are closely related.In addition, Colon 26/IL-23#9 growth of tumor speed still than parental plant Colon 26 tumors slow down (P＜0.001, Figure 10).This result shows, under the immune deficiency state that lacks α/β T cell, imports the IL-23 gene to mice and also can cause certain antitumous effect.
Utilize method shown in the embodiment 2, change IL-23 over to human pancreatic adenocarcinoma (AsPC-1) cell, obtain two stably express IL-23 cell clone (AsPC-1/IL-23#6, #10).These two clones' cellular expression p19 and p40 gene 11), the amount of secretion p40 is 2ng//ml/5 * 10 in the culture supernatant 5/ 48 hours.By measuring these cells 35S OOO metaboilic level also carries out co-immunoprecipitation with anti-p40 antibody and tests, and has proved conclusively that to have the p19+p40 complex in the culture supernatant be IL-23.(expression C) and parental plant AsPC-1 cell do not have significant difference for HLA-A, B for body outer cell proliferation and Class I major histocompatibility antigen.
With AsPC-1/IL-23#6 and #10 (1 * 10 6Individual) to be inoculated in the BALB/c nude mouse respectively subcutaneous, and growth of tumor is than parental plant AsPC-1 (1 * 10 6Individual) obviously slow down.And AsPC-1/IL-23#10 is inoculated in serious acquired immunodeficiency syndrome (SCID) mice that lacks T, B and NK cell, do not observe antitumous effect (Figure 13).
The above results prompting gamma/delta T, B and NK cell all participate in the antitumor action of IL-23.The splenocyte (2 * 10 of the mice of AsPC-1/IL-23#10 will have been inoculated 7Individual) with the parental plant AsPC-1 cell of crossing with the 50Gy radiation exposure (2 * 10 6) co-cultivation 5 days, the cell killing activity ratio was inoculated parental plant AsPC-1 mouse spleen cell obviously raise (Figure 14).In addition, cultivated altogether under the similarity condition 24 hours, detect IFN-γ in the supernatant, and have only a small amount of IFN-γ secretion in the supernatant of cultivating altogether with human lung carcinoma cell QG-56 that the 50Gy radiation exposure is crossed.In a word, the tumor cell of personnel selection experimentizes, and the cell that confirm to import the IL-23 gene also can the inducing cell lethal effect in the nude mice that lacks gamma/delta T cell, plays antitumor action.Table 3 is situations of BALB/c nude mouse splenocyte secretion of gamma-IFN.
Table 3
Figure C20041000810600141
1 lonizing radiation are handled (50Gy)
The present invention imports the special immunoreation of tumor cell energy induced tumor of IL-23 gene, thereby reaches significant antitumous effect.Introduced among the present invention IL-23 leading-in technique and IL-23 gene transfered cell in the huge applications potentiality aspect the treatment tumor.The present invention can share separately or with other therapies, reaches the purpose of safe and effective treatment cancer, has important practical value.
SEQUENCE?LISTING
＜110〉Fudan University's Chiba,Japan county government
＜120〉a kind of antitumor agent that contains the interleukin 23 gene
<130>
<160>4
<210>1
<211>570
<212>DNA
<213>Homo?sapience
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Thr?Asn?Asp?Val?Pro?His?Ile?Gln?Cys?Gly?Asp?Gly?Cys?Asp?Pro?Gln
65 70 75 80
gga?ctc?agg?gac?aac?agt?cag?ttc?tgc?ttg?caa?agg?atc?cac?cag?ggt 288
Gly?Leu?Arg?Asp?Asn?Ser?Gln?Phe?Cys?Leu?Gln?Arg?Ile?His?Gln?Gly
85 90 95
ctg?att?ttt?tat?gag?aag?ctg?cta?gga?tcg?gat?att?ttc?aca?ggg?gag 336
Leu?Ile?Phe?Tyr?Glu?Lys?Leu?Leu?Gly?Ser?Asp?Ile?Phe?Thr?Gly?Glu
100 105 110
cct?tct?ctg?ctc?cct?gat?agc?cct?gtg?ggc?cag?ctt?cat?gcc?tcc?cta 384
Pro?Ser?Leu?Leu?Pro?Asp?Ser?Pro?Val?Gly?Gln?Leu?His?Ala?Ser?Leu
115 120 125
ctg?ggc?ctc?agc?caa?ctc?ctg?cag?cct?gag?ggt?cac?cac?tgg?gag?act 432
Leu?Gly?Leu?Ser?Gln?Leu?Leu?Gln?Pro?Glu?Gly?His?His?Trp?Glu?Thr
130 135 140
cag?cag?att?cca?agc?ctc?agt?ccc?agc?cag?cca?tgg?cag?cgt?ctc?ctt 480
Gln?Gln?Ile?Pro?Ser?Leu?Ser?Pro?Ser?Gln?Pro?Trp?Gln?Arg?Leu?Leu
145 150 155 160
ctc?cgc?ttc?aaa?atc?ctt?cgc?agc?ctc?cag?gcc?ttt?gtg?gct?gta?gcc 528
Leu?Arg?Phe?Lys?Ile?Leu?Arg?Ser?Leu?Gln?Ala?Phe?Val?Ala?Val?Ala
165 170 175
gcc?cgg?gtc?ttt?gcc?cat?gga?gca?gca?acc?ctg?agt?ccc?taa 570
Ala?Arg?Val?Phe?Ala?His?Gly?Ala?Ala?Thr?Leu?Ser?Pro?Sto
180 185 190
<210>2
<211>987
<212>DNA
<213>Homo?sapience
<400>2
atg?tgt?cac?cag?cag?ttg?gtc?atc?tct?tgg?ttt?tcc?ctg?gtt?ttt?ctg 48
Met?Cys?His?Gln?Gln?Leu?Val?Ile?Ser?Trp?Phe?Ser?Leu?Val?Phe?Leu
5 10 15
gca?tct?ccc?ctc?gtg?gcc?ata?tgg?gaa?ctg?aag?aaa?gat?gtt?tat?gtc 96
Ala?Ser?Pro?Leu?Val?Ala?Ile?Trp?Glu?Leu?Lys?Lys?Asp?Val?Tyr?Val
20 25 30
gta?gaa?ttg?gat?tgg?tat?ccg?gat?gcc?cct?gga?gaa?atg?gtg?gtc?ctc 144
Val?Glu?Leu?Asp?Trp?Tyr?Pro?Asp?Ala?Pro?Gly?Glu?Met?Val?Val?Leu
35 40 45
acc?tgt?gac?acc?cct?gaa?gaa?gat?ggt?atc?acc?tgg?acc?ttg?gac?cag 192
Thr?Cys?Asp?Thr?Pro?Glu?Glu?Asp?Gly?Ile?Thr?Trp?Thr?Leu?Asp?Gln
50 55 60
agc?agt?gag?gtc?tta?ggc?tct?ggc?aaa?acc?ctg?acc?atc?caa?gtc?aaa 240
Ser?Ser?Glu?Val?Leu?Gly?Ser?Gly?Lys?Thr?Leu?Thr?Ile?Gln?Val?Lys
65 70 75 80
gag?ttt?gga?gat?gct?ggc?cag?tac?acc?tgt?cac?aaa?gga?ggc?gag?gtt 288
Glu?Phe?Gly?Asp?Ala?Gly?Gln?Tyr?Thr?Cys?His?Lys?Gly?Gly?Glu?Val
85 90 95
cta?agc?cat?tcg?ctc?ctg?ctg?ctt?cac?aaa?aag?gaa?gat?gga?att?tgg 336
Leu?Ser?His?Ser?Leu?Leu?Leu?Leu?His?Lys?Lys?Glu?Asp?Gly?Ile?Trp
100 105 110
tcc?act?gat?att?tta?aag?gac?cag?aaa?gaa?ccc?aaa?aat?aag?acc?ttt 384
Ser?Thr?Asp?Ile?Leu?Lys?Asp?Gln?Lys?Glu?Pro?Lys?Asn?Lys?Thr?Phe
115 120 125
cta?aga?tgc?gag?gcc?aag?aat?tat?tct?gga?cgt?ttc?acc?tgc?tgg?tgg 432
Leu?Arg?Cys?Glu?Ala?Lys?Asn?Tyr?Ser?Gly?Arg?Phe?Thr?Cys?Trp?Trp
130 135 140
ctg?acg?aca?atc?agt?act?gat?ttg?aca?ttc?agt?gtc?aaa?agc?agc?aga 480
Leu?Thr?Thr?Ile?Ser?Thr?Asp?Leu?Thr?Phe?Ser?Val?Lys?Ser?Ser?Arg
145 150 155 160
ggc?tct?tct?gac?ccc?caa?ggg?gtg?acg?tgc?gga?gct?gct?aca?ctc?tct 528
Gly?Ser?Ser?Asp?Pro?Gln?Gly?Val?Thr?Cys?Gly?Ala?Ala?Thr?Leu?Ser
165 170 175
gca?gag?aga?gtc?aga?ggg?gac?aac?aag?gag?tat?gag?tac?tca?gtg?gag 570
Ala?Glu?Arg?Val?Arg?Gly?Asp?Asn?Lys?Glu?Tyr?Glu?Tyr?Ser?Val?Glu
180 185 190
tgc?cag?gag?gac?agt?gcc?tgc?cca?gct?gct?gag?gag?agt?ctg?ccc?att 624
Cys?Gln?Glu?Asp?Ser?Ala?Cys?Pro?Ala?Ala?Glu?Glu?Ser?Leu?Pro?Ile
195 200 205
gag?gtc?atg?gtg?gat?gcc?gtt?cac?aag?ctc?aag?tat?gaa?aac?tac?acc 672
Glu?Val?Met?Val?Asp?Ala?Val?His?Lys?Leu?Lys?Tyr?Glu?Asn?Tyr?Thr
210 215 220
agc?agc?ttc?ttc?atc?agg?gac?atc?atc?aaa?cct?gac?cca?ccc?aag?aac 720
Ser?Ser?Phe?Phe?Ile?Arg?Asp?Ile?Ile?Lys?Pro?Asp?Pro?Pro?Lys?Asn
225 230 235 240
ttg?cag?ctg?aag?cca?tta?aag?aat?tct?cgg?cag?gtg?gag?gtc?agc?tgg 768
Leu?Gln?Leu?Lys?Pro?Leu?Lys?Asn?Ser?Arg?Gln?Val?Glu?Val?Ser?Trp
245 250 255
gag?tac?cct?gac?acc?tgg?agt?act?cca?cat?tcc?tac?ttc?tcc?ctg?aca 816
Glu?Tyr?Pro?Asp?Thr?Trp?Ser?Thr?Pro?His?Ser?Tyr?Phe?Ser?Leu?Thr
260 265 270
ttc?tgc?gtt?cag?gtc?cag?ggc?aag?agc?aag?aga?gaa?aag?aaa?gat?aga 864
Phe?Cys?Val?Gln?Val?Gln?Gly?Lys?Ser?Lys?Arg?Glu?Lys?Lys?Asp?Arg
275 280 285
gtc?ttc?acg?gac?aag?acc?tca?gcc?acg?gtc?atc?tgc?cgc?aaa?aat?gcc 912
Val?Phe?Thr?Asp?Lys?Thr?Ser?Ala?Thr?Val?Ile?Cys?Arg?Lys?Asn?Ala
290 295 300
agc?att?agc?gtg?cgg?gcc?cag?gac?cgc?tac?tat?agc?tca?tct?tgg?agc 960
Ser?Ile?Ser?Val?Arg?Ala?Gln?Asp?Arg?Tyr?Tyr?Ser?Ser?Ser?Trp?Ser
305 310 315 320
gaa?tgg?gca?tct?gtg?ccc?tgc?agt?tag 987
Glu?Trp?Ala?Ser?Val?Pro?Cys?Ser?Sto
325
<210>3
<211>591
<212>DNA
<213>Mouse
<400>3
atg?ctg?gat?tgc?aga?gca?gta?ata?atg?cta?tgg?ctg?ttg?ccc?tgg?gtc 48
Met?Leu?Asp?Cys?Arg?Ala?Val?Ile?Met?Leu?Trp?Leu?Leu?Pro?Trp?Val
5 10 15
act?cag?ggc?ctg?gct?gtg?cct?agg?agt?agc?agt?cct?gac?tgg?gct?cag 96
Thr?Gln?Gly?Leu?Ala?Val?Pro?Arg?Ser?Ser?Ser?Pro?Asp?Trp?Ala?Gln
20 25 30
tgc?cag?cag?ctc?tct?cgg?aat?ctc?tgc?atg?cta?gcc?tgg?aac?gca?cat 144
Cys?Gln?Gln?Leu?Ser?Arg?Asn?Leu?Cys?Met?Leu?Ala?Trp?Asn?Ala?His
35 40 45
gca?cca?gcg?gga?cat?atg?aat?cta?cta?aga?gaa?gaa?gag?gat?gaa?gag 192
Ala?Pro?Ala?Gly?His?Met?Asn?Leu?Leu?Arg?Glu?Glu?Glu?Asp?Glu?Glu
50 55 60
act?aaa?aat?aat?gtg?ccc?cgt?atc?cag?tgt?gaa?gat?ggt?tgt?gac?cca 240
Thr?Lys?Asn?Asn?Val?Pro?Arg?Ile?Gln?Cys?Glu?Asp?Gly?Cys?Asp?Pro
65 70 75 80
caa?gga?ctc?aag?gac?aac?agc?cag?ttc?tgc?ttg?caa?agg?atc?cgc?caa 288
Gln?Gly?Leu?Lys?Asp?Asn?Ser?Gln?Phe?Cys?Leu?Gln?Arg?Ile?Arg?Gln
85 90 95
ggt?ctg?gct?ttt?tat?aag?cac?ctg?ctt?gac?tct?gac?atc?ttc?aaa?ggg 336
Gly?Leu?Ala?Phe?Tyr?Lys?His?Leu?Leu?Asp?Ser?Asp?Ile?Phe?Lys?Gly
100 105 110
gag?cct?gct?cta?ctc?cct?gat?agc?ccc?atg?gag?caa?ctt?cac?acc?tcc 384
Glu?Pro?Ala?Leu?Leu?Pro?Asp?Ser?Pro?Met?Glu?Gln?Leu?His?Thr?Ser
115 120 125
cta?cta?gga?ctc?agc?caa?ctc?ctc?cag?cca?gag?gat?cac?ccc?cgg?gag 432
Leu?Leu?Gly?Leu?Ser?Gln?Leu?Leu?Gln?Pro?Glu?Asp?His?Pro?Arg?Glu
130 135 140
acc?caa?cag?atg?ccc?agc?ctg?agt?tct?agt?cag?cag?tgg?cag?cgc?ccc 480
Thr?Gln?Gln?Met?Pro?Ser?Leu?Ser?Ser?Ser?Gln?Gln?Trp?Gln?Arg?Pro
145 150 155 160
ctt?ctc?cgt?tcc?aag?atc?ctt?cga?agc?ctc?cag?gcc?ttt?ttg?gcc?ata 528
Leu?Leu?Arg?Ser?Lys?Ile?Leu?Arg?Ser?Leu?Gln?Ala?Phe?Leu?Ala?Ile
165 170 175
gct?gcc?cgg?gtc?ttt?gcc?cac?gga?gca?gca?act?ctg?act?gag?ccc?tta 576
Ala?Ala?Arg?Val?Phe?Ala?His?Gly?Ala?Ala?Thr?Leu?Thr?Glu?Pro?Leu
180 185 190
gtg?cca?aca?gct?taa 591
Val?Pro?Thr?Ala?Sto
195
<210>4
<211>1008
<212>DNA
<213>Mouse
<400>4
atg?tgt?cct?cag?aag?cta?acc?atc?tcc?tgg?ttt?gcc?atc?gtt?ttg?ctg 48
Met?Cys?Pro?Gln?Lys?Leu?Thr?Ile?Ser?Trp?Phe?Ala?Ile?Val?Leu?Leu
5 10 15
gtg?tct?cca?ctc?atg?gcc?atg?tgg?gag?ctg?gag?aaa?gac?gtt?tat?gtt 96
Val?Ser?Pro?Leu?Met?Ala?Met?Trp?Glu?Leu?Glu?Lys?Asp?Val?Tyr?Val
20 25 30
gta?gag?gtg?gac?tgg?act?ccc?gat?gcc?cct?gga?gaa?aca?gtg?aac?ctc 144
Val?Glu?Val?Asp?Trp?Thr?Pro?Asp?Ala?Pro?Gly?Glu?Thr?Val?Asn?Leu
35 40 45
acc?tgt?gac?acg?cct?gaa?gaa?gat?gac?atc?acc?tgg?acc?tca?gac?cag 192
Thr?Cys?Asp?Thr?Pro?Glu?Glu?Asp?Asp?Ile?Thr?Trp?Thr?Ser?Asp?Gln
50 55 60
aga?cat?gga?gtc?ata?ggc?tct?gga?aag?acc?ctg?acc?atc?act?gtc?aaa 240
Arg?His?Gly?Val?Ile?Gly?Ser?Gly?Lys?Thr?Leu?Thr?Ile?Thr?Val?Lys
65 70 75 80
gag?ttt?cta?gat?gct?ggc?cag?tac?acc?tgc?cac?aaa?gga?ggc?gag?act 288
Glu?Phe?Leu?Asp?Ala?Gly?Gln?Tyr?Thr?Cys?His?Lys?Gly?Gly?Glu?Thr
85 90 95
ctg?agc?cac?tca?cat?ctg?ctg?ctc?cac?aag?aag?gaa?aat?gga?att?tgg 336
Leu?Ser?His?Ser?His?Leu?Leu?Leu?His?Lys?Lys?Glu?Asn?Gly?Ile?Trp
100 105 110
tcc?act?gaa?att?tta?aaa?aat?ttc?aaa?aac?aag?act?ttc?ctg?aag?tgt 384
Ser?Thr?Glu?Ile?Leu?Lys?Asn?Phe?Lys?Asn?Lys?Thr?Phe?Leu?Lys?Cys
115 120 125
gaa?gca?cca?aat?tac?tcc?gga?cgg?ttc?acg?tgc?tca?tgg?ctg?gtg?caa 432
Glu?Ala?Pro?Asn?Tyr?Ser?Gly?Arg?Phe?Thr?Cys?Ser?Trp?Leu?Val?Gln
130 135 140
aga?aac?atg?gac?ttg?aag?ttc?aac?atc?aag?agc?agt?agc?agt?tcc?cct 480
Arg?Asn?Met?Asp?Leu?Lys?Phe?Asn?Ile?Lys?Ser?Ser?Ser?Ser?Ser?Pro
145 150 155 160
gac?tct?cgg?gca?gtg?aca?tgt?gga?atg?gcg?tct?ctg?tct?gca?gag?aag 528
Asp?Ser?Arg?Ala?Val?Thr?Cys?Gly?Met?Ala?Ser?Leu?Ser?Ala?Glu?Lys
165 170 175
gtc?aca?ctg?gac?caa?agg?gac?tat?gag?aag?tat?tca?gtg?tcc?tgc?cag 576
Val?Thr?Leu?Asp?Gln?Arg?Asp?Tyr?Glu?Lys?Tyr?Ser?Val?Ser?Cys?Gln
180 185 190
gag?gat?gtc?acc?tgc?cca?act?gcc?gag?gag?acc?ctg?ccc?att?gaa?ctg 624
Glu?Asp?Val?Thr?Cys?Pro?Thr?Ala?Glu?Glu?Thr?Leu?Pro?Ile?Glu?Leu
195 200 205
gcg?ttg?gaa?gca?cgg?cag?cag?aat?aaa?tat?gag?aac?tac?agc?acc?agc 672
Ala?Leu?Glu?Ala?Arg?Gln?Gln?Asn?Lys?Tyr?Glu?Asn?Tyr?Ser?Thr?Ser
210 215 220 225
ttc?ttc?atc?agg?gac?atc?atc?aaa?cca?gac?ccg?ccc?aag?aac?ttg?cag 720
Phe?Phe?Ile?Arg?Asp?Ile?Ile?Lys?Pro?Asp?Pro?Pro?Lys?Asn?Leu?Gln
230 235 240
atg?aag?cct?ttg?aag?aac?tca?cag?gtg?gag?gtc?agc?tgg?gag?tac?cct 768
Met?Lys?Pro?Leu?Lys?Asn?Ser?Gln?Val?Glu?Val?Ser?Trp?Glu?Tyr?Pro
245 250 255
gac?tcc?tgg?agc?act?ccc?cat?tcc?tac?ttc?tcc?ctc?aag?ttc?ttt?gtt 816
Asp?Ser?Trp?Ser?Thr?Pro?His?Ser?Tyr?Phe?Ser?Leu?Lys?Phe?Phe?Val
260 265 270
cga?atc?cag?cgc?aag?aaa?gaa?aag?atg?aag?gag?aca?gag?gag?ggg?tgt 864
Arg?Ile?Gln?Arg?Lys?Lys?Glu?Lys?Met?Lys?Glu?Thr?Glu?Glu?Gly?Cys
275 280 285
aac?cag?aaa?ggt?gcg?ttc?ctc?gta?gag?aag?aca?tct?acc?gaa?gtc?caa 912
Asn?Gln?Lys?Gly?Ala?Phe?Leu?Val?Glu?Lys?Thr?Ser?Thr?Glu?Val?Gln
290 295 300 305
tgc?aaa?ggc?ggg?aat?gtc?tgc?gtg?caa?gct?cag?gat?cgc?tat?tac?aat 960
Cys?Lys?Gly?Gly?Asn?Val?Cys?Val?Gln?Ala?Gln?Asp?Arg?Tyr?Tyr?Asn
310 315 320
tcc?tca?tgc?agc?aag?tgg?gca?tgt?gtt?ccc?tgc?agg?gtc?cga?tcc?tag?1008
Ser?Ser?Cys?Ser?Lys?Trp?Ala?Cys?Val?Pro?Cys?Arg?Val?Arg?Ser?Sto
325 330 335

Claims (6)

1, a kind of antitumor agent that contains the interleukin 23 gene, it is characterized in that the cell to express the interleukin 23 gene is an effective ingredient, described interleukin gene is made up of the structure of sequence 1 and sequence 2, and described interleukin 23 gene is expressed after importing described cell by virus or non-virus carrier.
2, antitumor agent according to claim 1 is characterized in that, described antitumor agent is the dosage form that can directly inject in tumor.
3, antitumor agent according to claim 1 and 2 is characterized in that described antitumor agent is made up of cell of expressing the interleukin 23 gene and the excipient in the medical permissible range.
4, antitumor agent according to claim 1, the cell that it is characterized in that described expression interleukin 23 gene are the tumor cells of forfeiture splitting ability, and described tumor cell is to belong to tumor cell of the same race with the target tumor.
5, antitumor agent according to claim 1 is characterized in that the cell of described expression interleukin 23 gene is an immunocyte.
6, the purposes of the described antitumor agent of claim 1 in preparation activation anti-tumor immune response preparation.
CNB2004100081062A 2003-02-28 2004-02-28 Antitumor agent containing interleukin 23 gene Expired - Fee Related CN100435849C (en)

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AU2006265898A1 (en) * 2005-06-30 2007-01-11 Archemix Corp. Polynucleotides and polypeptides of the IL-12 family of cytokines
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Non-Patent Citations (6)

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Title
NOVEL P19protein engages il-12O40to form a cytokin,i-L-23,with biological activites similar as well as destinct from IL-12. Brigit Oppmann et al.Immunity,Vol.13 . 2000
NOVEL P19protein engages il-12O40to form a cytokin,i-L-23,with biological activites similar as well as destinct from IL-12. Brigit Oppmann et al.Immunity,Vol.13 . 2000 *
Regulation of Virus-Induced IL-12 and IL-23 Expression inHuman Macrophages. Jaana Pirhonen et al.The Journal of Immunology,Vol.169 . 2002
Regulation of Virus-Induced IL-12 and IL-23 Expression inHuman Macrophages. Jaana Pirhonen et al.The Journal of Immunology,Vol.169 . 2002 *
Regulation of virus-i-nducedIL-12 and IL-23Expression inhuman macrophages. Jaana Prihnen et al.The Jouranl of Immunology,Vol.169 . 2002
Regulation of virus-i-nducedIL-12 and IL-23Expression inhuman macrophages. Jaana Prihnen et al.The Jouranl of Immunology,Vol.169 . 2002 *

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