CN1844145A - Preparation and use of monoclonal antibody against human OX40L - Google Patents
Preparation and use of monoclonal antibody against human OX40L Download PDFInfo
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Abstract
The invention relates to the conjugated proteins or polypeptides of the tumor necrosis factor super family members, more specifically, the invention relates to anti-human OX40L monoclonal antibody 9H10, 4C12 and 1E7, their preparing process and the use of these monoclonal antibodies in inhibiting hybrid lymphocyte reaction, stimulating T cell proliferation, inducing dendritic cell differentiation and maturity, stimulating T cell dependent B cell differentiation and antibody secretion.
Description
FIELD OF THE INVENTION
The present invention relates to tumor necrosis factor superfamily member's conjugated protein or polypeptide, more particularly, the present invention relates to monoclonal antibody against human OX 40 L 9H10,4C12 and 1E7, its preparation method and these monoclonal antibodies are suppressing mixed lymphocyte reacion, are stimulating T cell proliferation, are inducing the differentiation of dendritic cells maturation, are stimulating the application in T cell dependency B cytodifferentiation and the antibody-secreting.
The background of invention
Known have many receptor-ligand binding all to participate in inducing, setting up and regulate antigen specific immune reaction.For activated T cell reaction effectively, need two signals usually at least.Wherein the MHC-antigenic compound on T cell antigen receptor (TCR) identification antigen presenting cell (APC) is the antigen-specific signal so that first signal to be provided, also necessary non-antigen-specific, the restrictive second signal of non-MHC that obtains the costimulatory molecules interaction back generation of T cell and APC expression.Second signal is costimulatory signal or costimulatory signal.Lack costimulatory signal if the antigen-specific signal is only arranged, the T cell will show as reactionless or immune tolerance state, even cause apoptosis.As seen, costimulatory signal is that the amplification of T cell clone, differentiation and performance biological effect institute are requisite.Therefore can think, first signal deciding specificity of T cell activation, can second signal then determine the T cell-mediated immune responses effectively carry out (Noelle RJ, et al., Proc.Natl.Acad.Sci.USA.89:6550,1992; Allen RC et al., Science.259:990,1993).
In recent years, Protocols in Molecular Biology is widely used in immunology research, and costimulatory molecules is constantly found.According to its structure, these costimulatory moleculeses can be divided into two classes: a class is tumour necrosis factor/Tumor Necrosis Factor Receptors (TNF/TNFR) superfamily, comprises CD40/CD154, CD27/CD27L, CD30/CD30L, 4-1BB/4-1BBL, OX40/OX40L, RANK/RANKL and Fas/FasL etc.Another kind of is immunoglobulin superfamily, as (Korthauer U et al., Nature, 361:539,1993) such as B7-CD28/CTLA4, LAF1-ICAM-1/ICAM-2/ICAM-3, ICOS-GL50, CD2/LFA-3.These costimulatory moleculeses are with the mode conducted signal of acceptor and ligand interaction.Acceptor generally is expressed in different cell surfaces with part, and common one is the persistence expression, and one is the inducible expression.In the different steps of immunne response, these molecules participate in the signal conduction in mode unique and that be associated separately, regulate and control the immunne response of body jointly.
People OX40 part (OX40L or CD134L) be 1991 by people such as Miura clone, its belongs to TNF superfamily, is the II type transmembrane glycoprotein of the about 34-40KD of molecular weight.Human OX 40 L and is expressed in the histoorgans such as the heart, skeletal muscle, testis and lung mainly on cell surfaces such as sophisticated dendritic cell (DC), activatory B cell, vascular endothelial cell (ECV), umbilical cord vein blood vessel endotheliocyte and scavenger cell.The OX40/OX40L approach mainly plays a role in the late period of t cell responses, has the activatory of promotion CD4
+T cell clone hyperplasia, the functions such as quantity that strengthen cytological effect, prolong the cell survival phase and increase memory T cell.In addition, OX40/OX40L can also transmit reverse signal and promote the B cell amplification to be divided into plasmocyte, secretes various antibody.Under the effect of OX40/OX40L signal, make M
0Phase dendritic cell (M
0-DC cell) up-regulated of phenotype CD80, CD86, CD54, CD40 promotes secretor type CD40L (sCD40L) activatory IL-4-M
0-DC emiocytosis IL-12, TNF-α, cytokines such as IL-1 β, IL-6 show that OX40L can strengthen the ability of sCD40L activation prematurity IL-4-Mo-DC, promotes the DC maturation.
Recent study shows that the OX40/OX40L signal path can play a significant role in the process of mediation anti-tumor immune response, resisting transplant rejection reaction and autoimmune disorder.At the inflammation part of various autoimmune disease, can both be separated to OX40
+CD4
+T cell, and the OX40 of these inflammation parts
+The T cell is exactly the autoantigen specific T-cells.These T cells with antigenic specificity finally can cause autoimmune response, clinical symptom occurs.In mouse experiment cerebrospinal meningitis (EAE) model, blocking-up OX40/OX40L path can be alleviated the symptom of animal significantly.Equally, blocking-up OX40/OX40L path also can prolong the survival time of dermatoplasty and heart transplantation mouse significantly.In addition, the tumour-specific OX40 of tumor tissues corresponding site
+The T cell also has vital role in body is antitumor.Experimentation on animals confirms, strengthens the OX40 signal and can stop mouse tumor to form, and improves the survival rate of tumor-bearing mice simultaneously.OX40 is specificity overexpression on the T cells with antigenic specificity at inflammation and tumor-infiltrated position only, makes it become the target molecule of an ideal immunologic intervention.Therefore, the anti-OX 40 l monoclonal antibody will promise to be the ideal diagnosis and the therapeutical agent of clinical intervention autoimmune disorder, graft-rejection and tumour.
Although relevant OX40/OX40L receptors ligand all is known in conjunction with preparation and their other immunologic functions of right T cytositimulation function, OX40 or OX40L antibody, do not appear in the newspapers as new monoclonal antibody against human OX 40 L of the present invention and relatively simple preparation method thereof.And the monoclonal antibody against human OX 40 L of bibliographical information is to be used for the blocking-up type antibody of suppressor T cell propagation, blocking-up B emiocytosis mostly, and monoclonal antibody against human OX 40 L of the present invention then has different biological functions.
Goal of the invention
An object of the present invention is to provide one group of monoclonal antibody against human OX 40 L that is selected from 9H10,4C12 and 1E7.
According to the preferred embodiments of the invention, the heavy chain of monoclonal antibody against human OX 40 L 9H10 and 4C12 and light chain have aminoacid sequence shown in SEQ ID NO:6 to SEQ ID NO:9 in the sequence table respectively, and the heavy chain of monoclonal antibody against human OX 40 L 1E7 has the aminoacid sequence shown in the SEQ ID NO:10.
According to the preferred embodiments of the invention, monoclonal antibody against human OX 40 L 9H10,4C12 that is defined as above and 1E7 are the monoclonal antibodies of anti-soluble human OX40L.
Another object of the present invention provides the method for preparing the monoclonal antibody against human OX 40 L that is defined as above, and this method comprises:
(1) transgenic cell of the high expression level human OX 40 L molecule of usefulness pre-preparation is as the immunogen immune animal;
(2) separate the splenic lymphocyte of immunized animal and merge with suitable myeloma cell being suitable for producing under the condition of hybridoma;
(3) screen and cultivate the hybridoma that as above obtains;
(4) from the ascites fluid of animal of cell culture fluid or inoculation hybridoma, separate and the required monoclonal antibody of purifying.
According to the preferred embodiments of the invention, the deposit number that wherein produces the hybridoma cell strain of monoclonal antibody against human OX 40 L 9H10,4C12 and 1E7 is respectively CGMCC No.1167, CGMCCNo.1168 and CGMCC No.1169.
A further object of the present invention provides monoclonal antibody against human OX 40 L 9H10 and the application of 4C12 in suppressing unidirectional and two-way mixed lymphocyte reacion that is defined as above.
A further object of the present invention provides the application of monoclonal antibody against human OX 40 L 9H10 in inducing differentiation of dendritic cells and maturation that is defined as above.
A further object of the present invention provides monoclonal antibody against human OX 40 L 9H10 and the application of 4C12 in stimulating T cell dependency B emiocytosis antibody that is defined as above.
A further object of the present invention provides the application of monoclonal antibody against human OX 40 L 1E7 in external activated T cell that is defined as above.
Brief Description Of Drawings
Fig. 1 shows the structure synoptic diagram of L929/OX40L transgenic cell.
Fig. 2 shows the identification to OX40L molecule on the transgenic cell with flow cytometry monoclonal antibody against human OX 40 L 9H10,4C12 and 1E7.The wherein negative contrast in grey peak, one anti-is mouse IgG, two anti-ly are the sheep anti-mouse igg of fluorescein PE mark.Transparent peak shows the result of transgenic cell and the reaction of anti-human OX 40 L monoclonal antibody, wherein first antibody is respectively the anti-human OX 40 L monoclonal antibody of 3 strains 9H10,4C12 and the 1E7 of the anti-human OX 40 L monoclonal antibody of commercialization TAG-34 (positive control) and we oneself preparation, and second antibody is the sheep anti-mouse igg of fluorescein PE mark.
Fig. 3 shows 9H10,4C12 and the chromosomal karyotyping of 1E7 hybridoma cell strain (* 1000 times).
Fig. 4 shows with flow cytometry 9H10,4C12 and 1E7 and discerns OX40L molecule on ripe DC and the activatory B cell.A: 3 days B cell of dyers' grapes (PWM) activation.The wherein negative contrast in grey peak, B cell are at first reacted with mouse IgG, add the sheep anti-mouse igg of two anti-fluorescein FITC marks then, and the mouse-anti people CD19 that adds fluorescein PE mark at last directly marks monoclonal antibody; Transparent peak is the sheep anti-mouse igg effect that adds fluorescein FITC mark after the B cell reacts with the anti-human OX 40 L monoclonal antibody of 3 strains 9H10,4C12 and 1E7 respectively again, and the mouse-anti people CD19 that adds fluorescein PE mark at last directly marks monoclonal antibody.B: anti-people CD40 monoclonal antibody excites 2 days DC.The wherein negative contrast in grey peak, one anti-is biotin labeled mouse IgG, two anti-ly are the streptavidin (Streptavidin-PE) of PE mark; Transparent peak is that DC adds the Streptavidin-PE effect respectively with after the anti-human OX 40 L monoclonal antibody of biotin labeled 3 strains 9H10,4C12 and the 1E7 reaction again.
Fig. 5 shows the competitive inhibition with the antigen site of flow cytometry monoclonal antibody 9H10,4C12 and 1E7 identification.1: the negative contrast in grey peak, wherein one anti-is biotin labeled mouse IgG, two anti-ly are Streptavidin-PE.The positive contrast in the transparent peak of dotted line, wherein one anti-is biotin labeled 4C12, two anti-ly are Streptavidin-PE; The transparent peak of solid line shows the competitive inhibition of 9H10 and 4C12 loci.9H10 is added after the L929/OX40L reaction and biotin labeled 4C12 effect again, add Streptavidin-PE at last.2: the negative contrast in grey peak, wherein one anti-is biotin labeled mouse IgG, two anti-ly are Streptavidin-PE; The positive contrast in the transparent peak of dotted line, wherein one anti-is biotin labeled 1E7, two anti-ly are Streptavidin-PE; The transparent peak of solid line shows the competitive inhibition of 9H10 and 1E7 loci, with 9H10 add L929/OX40L react after again with biotin labeled 1E7 effect, add Streptavidin-PE at last.3: the negative contrast in grey peak, wherein one anti-is biotin labeled mouse IgG, two anti-ly are Streptavidin-PE; The positive contrast in the transparent peak of dotted line, wherein one anti-is biotin labeled 1E7, two anti-ly are Streptavidin-PE; The transparent peak of solid line shows the competitive inhibition of 4C12 and 1E7 loci, with 4C12 add L929/OX40L react after again with biotin labeled 1E7 effect, add Streptavidin-PE at last again.
Fig. 6 shows Western blot immunoblotting assay.M is molecular weight standard (KD); 2,4,6 is the tropina of abduction delivering GST-OX40L fusion rotein engineering bacteria, and wherein the antibody 2 of Jia Ruing is 9H10, and 4 is 4C12, and 6 is 1E7; 1,3,5 is that wherein the antibody 1 of Jia Ruing is 9H10 so that the inductive tropina is not as negative control, and 3 is 4C12, and 5 is 1E7.
Fig. 7 show with
3The H-TdR method of mixing analyzes monoclonal antibody 9H10 and 4C12 suppresses mixed lymphocyte reacion.Wherein ordinate zou is the cpm value, and X-coordinate is the differential responses group.A: be unidirectional mixed lymphocyte reacion, promptly anti-human OX 40 L monoclonal antibody 9H10 and 4C12 and unite anti-people B7-1 monoclonal antibody and suppress the short proliferation function of mature dendritic cell (CD) to the T cell.B: be two-way mixed lymphocyte reacion.Wherein 1: human peripheral blood mononuclear karyolymph cell (PBMC1) sample; 2: another part human peripheral blood mononuclear karyolymph cell (PBMC2) sample; 3:PBMC1+PBMC2; 4:PBMC1+PBMC2+IgG; 5:PBMC1+PBMC2+9H10; 6:PBMC1+PBMC2+4C12.
Fig. 8 shows the promoter action of flow cytometry analysis 9H10 to the DC cell maturation.The grey peak shows negative control, wherein uses the mouse IgG of fluorescein PE mark directly to mark monoclonal antibody; Transparent peak shows the reaction of DC and the different straight mark monoclonal antibody (CD80-PE, CD86-PE, CD83-PE and CXCR4) of PE mark.
Fig. 9 shows that the DC that euzymelinked immunosorbent assay (ELISA) analysis 9H10 excites promotes T emiocytosis cytokine.X-coordinate is the cell response fate, and ordinate zou is the content of cytokine.
Figure 10 shows that euzymelinked immunosorbent assay (ELISA) is analyzed monoclonal antibody 9H10 and 4C12 promotes T cell dependency B emiocytosis IgG.Ordinate zou is a human IgG content, and X-coordinate is the differential responses group.
Figure 11 show with
3The H-TdR method of mixing is analyzed the short proliferation function of monoclonal antibody 1E7 to the T cell.Ordinate zou is the cpm value, and X-coordinate is the differential responses group.
The particular content of invention
The present invention relates to tumor necrosis factor superfamily member's conjugated protein or polypeptide, more particularly, The present invention relates to monoclonal antibody against human OX 40 L 9H10,4C12 and 1E7, its preparation method and this A little monoclonal antibodies in external inhibition mixed lymphocyte reaction (MLP), stimulate T cell proliferation, induce the dendron shape thin Application in born of the same parents' differentiation and maturation, stimulation T cell dependence B Cell Differentiation and the antibody-secreting.
The invention further relates to and use the anti-OX 40 l monoclonal antibody to produce for improving people or mammal Immunoreactive pharmaceutical composition. After the T cell is excited by the specific antigen that it contacted, come into operation like this Pharmaceutical composition can effectively increase the generation of the host inner cell factor and the number of memory T cell, The recognition capability of antigen is improved immune system (for example tumour is thin for specific antigen by strengthening the T cell Born of the same parents or other pathogenic factors) immune response.
Said in this specification " OX40 acceptor " refers to the CD4 of antigen activation+Show on the T cell surface The protein that reaches; The OX40 part is can tying with the OX40 receptor-specific of expressing on some mammalian cell The protein that closes.
Term " OX40L " comprises complete OX40 part, solubility OX40 part and comprises OX40 The fusion of the functional activity part of part. Term " anti-OX 40 l antibodies " can be that OX40 is special Property monoclonal or polyclonal antibody or their immunologic competence part. Among the present invention, monoclonal preferably Antibody, particularly mouse anti human OX40L antibody.
Can prepare monoclonal antibody against human OX 40 L of the present invention according to conventional method known in the art 9H10,4C12 and 1E7 are (referring to Kohler and Milrtein, Nature 256:495-96,1975; Harlow And Lane, Aatibodies, A Laboratory, Cold Spring Harbor Laboritory, 1988). Must When wanting, also can be according to United States Patent (USP) 5,585, the method described in 089 prepares corresponding humanization form Anti-OX40 monoclonal antibody.
According to the preferred embodiments of the invention, preferably use the expression vector that is carried the human OX 40 L gene What transform also can efficiently express the recombinant cell of people OX40 polypeptide as preparation under suitable condition of culture The immunogene of monoclonal antibody of the present invention.
Therefore, in order to prepare monoclonal antibody against human OX 40 L of the present invention, at first make up the high expressed people The transgenic cell of OX40L (L929/OX40L). The transgenic cell of high expressed human OX 40 L molecule L929/OX40L has stronger immunogenicity, and the steric configuration of expressed antigen molecule can be with oneself Right state is exposed to surface of cell membrane, thus the more effectively immune response of excitating organism. In addition, because The L929 cell is the fibroblast of mouse, and other molecules of cell surface have relatively weak antigenicity, Therefore will reduce the generation of non-specific antibody with this cell as immunogene, make the screening of monoclonal antibody More convenient and greatly improve positive rate.
In order to prepare the L929/OX40L cell, can be according to DNA operation well known to those skilled in the art Technology (for example referring to Sambrook et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbour, 1989) carry out gene separation, nucleotide fragments cutting be connected, clone and table Reach identification, transformation and the cultivation of Vector construction and amplification, nucleotide sequence. For example, At first, from the BMDC of maturation, amplify the human OX 40 L gene by the RT-PCR technology. Will The OX40L gene Retroviral Vector pEGZ-Term that packs into obtains recombinant retroviral vector PEGZ-Term/OX40L. Then, use recombinant retroviral vector pEGZ-Term/OX40L and auxiliary Help together cotransfection 293T cell of viral pHIT456 and pHIT60. After 48 hours, collection contains viral The 293T cell culture supernatant of grain infects L929 cell, consecutive infection 3 days with it. At last, with containing The selection Screening of Media of Zeocin, it is enough big to treat that the resistance monoclonal grows to, and picking monoclonal colony carries out Enlarge and cultivate. The OX40L expression rate of the transgenic cell that obtains as stated above is up to more than 97%.
According to the regulation of the 25th of budapest treaty and patent law of china detailed rules for the implementation, in 2004 6 The hybridoma cell strain that will produce monoclonal antibody against human OX 40 L 9H10,4C12 and 1E7 the moon on the 2nd is protected Ensconce BeiJing, China China common micro-organisms preservation administrative center (CGMCC), deposit number is respectively CGMCC No.1167, CGMCC No.1168 and CGMCC No.1169.
Can be according to conventional method known in the art (Kohler and Milstein, Nature 265:495-497,1975) preparation monoclonal antibody against human OX 40 L 9H10 of the present invention, 4C12 and 1E7. Simply Ground is said, with transgenic cell L929/OX40L immune balb/c mice. When the serum of immunized animal anti-When the body level reached peak value, the splenocyte of separating animal's also prepared single cell suspension. In case of necessity, can use and exempt from Epidemic disease adsorption method screening splenocyte. For example, splenocyte suspension can be added to OX40L antigen protein bag In the flat board or aperture of quilt, the B cell of expressing OX40L polypeptid specificity immunoglobulin (Ig) namely is attached to flat On the plate, and can not be washed off by remaining suspension. Can collect then that resulting B cell or all dissociate Splenocyte, and for example merge to form hybridization with myeloma under the inducing of polyethylene glycol at suitable fusion agent Knurl, and for example cultivate the hybridoma that merges with screening in the HAT culture medium at selective medium. So After, identify required positive resistance with methods such as flow cytometry, Western blotting, immuno-precipitations Cell line. In external (for example organizing in blake bottle or the porous fibre reactor) or body (as mouse Ascites) hybridoma of the anti-human OX 40 L antibody of the selected secretion of cultivation, and from cell culture fluid or mouse Collect and the required antibody of purifying in the ascites fluid.
The nucleotide sequence analysis result shows, three strain monoclonal antibody against human OX 40 L of purifying of the present invention The nucleotide coding sequence of 9H10 and 4C12 heavy chain and light chain respectively such as SEQ ID NO:1 in the sequence table extremely Shown in the SEQ ID NO:4, and monoclonal antibody against human OX 40 L 1E7 light chain has SEQ ID NO: Nucleotide coding sequence shown in 5; Nucleotide coding sequence according to monoclonal antibody 9H10 and 4C12 pushes away The heavy chain of surveying and light-chain amino acid sequence are respectively such as SEQ ID NO:6 to SEQ ID NO:9 in the sequence table Shown in, and monoclonal antibody against human OX 40 L 1E7 light chain has the amino shown in the SEQ ID NO:10 Acid sequence. Wherein all amino acid sequences are all with the expression of amino acid whose standard single-letter abbreviation symbol.
Flow cytometry analysis result demonstration, monoclonal antibody against human OX 40 L 9H10 provided by the invention, 4C12 and 1E7 all can identify the OX40L that expresses on ripe DC and the activating B cell surface to some extent Molecule. Competition inhibition test result further shows monoclonal antibody against human OX 40 L 9H10 of the present invention But the antigen site identical with the 4C12 identification division, 1E7 then identifies diverse antigen site. Cause This might utilize 9H10 and 1E7 or 4C12 and 1E7 for the preparation of detecting soluble human OX40L Kit.
In order to identify the biological function of monoclonal antibody against human OX 40 L of the present invention, the present invention at first advances Gone mixed lymphocyte reaction (MLP). Mixed lymphocyte reaction (MLP) be a kind of for judge transplantation whether be fit to into The important tests of the degree of rejection after row and monitoring are transplanted. Our unidirectional and two-way mixed lymphocytes Reaction result shows that monoclonal antibody against human OX 40 L 9H10 of the present invention and 4C12 all can block The OX40/OX40L combination suppresses the T Proliferation of lymphocytes in dosage dependence mode, and can with anti-people B7-1 blocking-up type monoclonal antibody performance synergy. Graft-rejection and autoimmune disease all show as T Therefore the overactivity of cell, can utilize the anti-human OX 40 L monoclonal of this two strains blocking-up type of the present invention anti-The activation and proliferation of body suppressor T cell, and then suppressor T cell immune response are with prevention and treatment graft rejection Reaction and autoimmune disease.
Another strain monoclonal antibody against human OX 40 L 1E7 of the present invention then has opposite biologic activity.Experiment shows that this antibody can be worked in coordination with the propagation that promotes the T cell with excitated type CD3 monoclonal antibody.As everyone knows, tumour is to escape the result that malignant proliferation takes place in immunosurveillance because of cell.One of immune evasion mechanism is because body antigen presenting cell dendritic cell unusual on quantity and function particularly causes the specific for tumour antigen T cell of body to be in immune tolerance state.Therefore, might utilize exciting type anti-human OX40L monoclonal antibody 1E7 of the present invention to change T cellular immunization tolerance status, and the reaction of excitating organism specificity antineoplastic immunity, to remove and the control tumour cell.
Dendritic cell (DC) is in vivo specialized antigen presenting cell, is former and the immunoreactive opener of secondary.DC is unique antigen presenting cell that can excite naivety T cell, and its antigen presentation ability is big more than 100 times than other cell antigens (comprising scavenger cell and B cell), therefore plays an important role in inducing the body specific cellular immunity.On the basis of using low dosage CD40 monoclonal antibody to excite, monoclonal antibody against human OX 40 L 9H10 of the present invention can further raise the expression of costimulatory molecules CD80, CD86, CD83 and chemotactic cytokine receptor CXCR 4 molecule on the DC.That is to say that 9H10 can promote the differentiation and maturation of DC.And the DC that excites with independent use CD40 monoclonal antibody compares, and unites the DC that anti-human OX 40 L monoclonal antibody (9H10) excites and can promote more IL-2 of T emiocytosis and IFN-γ, and the variation of IL-4 and IL-10 is then little.These result of study explanations, anti-human OX 40 L monoclonal antibody can further promote the T cell to break up to the Th1 direction.
Use that DC is external to be excited, or the DC of load tumour antigen or goal gene transfection simultaneously is as vaccine, the specificity antineoplastic response to active immunization of excitating organism effectively after the immunization produces the specific killing effect to tumour cell.Carrying out one of technology crucial in the oncotherapy at application DC is the DC that manages to be used for the treatment of in a large number in external acquisition purpose.The method that the amplification in vitro dendritic cell is adopted is external cultivation monocyte in the presence of rHuGM-CSF (GM-CSF) and people's recombinant interleukin 4 (IL-4) normally, stimulates to add exciting type anti-human CD 40 monoclonal antibody again after 6 days.We discover, if add anti-human OX 40 L monoclonal antibody 9H10 simultaneously, can more effectively promote the maturation of DC.These DC not only can strengthen the excitation to the T cell, but also can promote the T cell to break up to Th1, thereby strengthen the anti tumor immune response of T cell.
The B cell plays an important role in humoral immunization.In the B cell culture system that the dyers' grapes (PWM) that the T cell relies on excite, the culture supernatant IgG content of monoclonal antibody against human OX 40 L 9H10 of the present invention or 4C12 obviously increases, and shows that this two strains monoclonal antibody helps the differentiation of B cell also can promote the antibody-secreting of T cell dependency B cell.
Below describe the present invention for example in detail by non-limiting example.Those skilled in the art can not deviate under essence spirit of the present invention and the principle prerequisite, change or change some technology contents or the sport technique segment of describing in this specification sheets.But one will understand that change that these are parallel or change all will be included within the claim scope that awaits the reply of the present invention.
Embodiment 1: the preparation of monoclonal antibody against human OX 40 L
Present embodiment is described the preparation of monoclonal antibody against human OX 40 L 9H10 of the present invention, 4C12 and 1E7.
(1) foundation of transgenic cell L929/OX40L and L929/mock
(a) clone of human OX 40 L gene: use TRIZOL reagent (Gibco, BRL) total RNA of the sophisticated DC of extracting antigen induction.Method according to MBI reverse transcription test kit working instructions are recommended becomes cDNA with the mRNA reverse transcription.Getting 5 μ l cDNA is template, use upstream primer 5 '-AACTG CAGATG GAA AGG GTC CAA CCC-3 ' (SEQ ID NO:11), carry out pcr amplification (94 ℃ of sex change 30 seconds with downstream primer 5 '-CG GGATTC TCA AAG GAC ACA GAA TTC-3 ' (SEQ ID NO:12), annealed 30 seconds for 55 ℃, 72 ℃ were extended 1 minute, and 30 circulations were extended 5 minutes for back 72 ℃).Behind the purifying, at T
4Directly the PCR product is connected with the pMD18-T carrier under the dna ligase effect and spends the night.Connect product transformed competence colibacillus bacterium TOP10 with gained, and the positive colony of screening is carried out PCR and enzyme cut and identify and dna sequencing.The result shows that the human OX 40 L cDNA sequence of registering among cDNA sequence that is obtained and the GenBank is in full accord.
(b) construction of recombinant defective retroviral vector: respectively with endonuclease PstI and correct pMD18-T/OX40L plasmid and retroviral vector pEGZ-Term (the modern immunology of BamHI digestion order-checking, 2004,24 (2): 99-103) (37 ℃, 4 hours).Recovery contains the fragment of goal gene, and with connecting product transformed competence colibacillus bacterium.After identify confirming with aforesaid method, the recombinant retroviral vector that is obtained named be pEGZ-Term/OX40L.
(c) structure of the L929 transgenic cell of stably express human OX 40 L: according to flow process shown in Figure 1, the liposome method of recommending with the test kit operational manual at first, with recombinant retroviral vector pEGZ-Term/OX40L and helper virus pHIT456 and pHIT60 (modern immunology, 2004,24 (2): 99-103) 60-70% confluent growth 293T cell in blocks in (volume ratio 2: 1: 1) cotransfection 6 orifice plates.Collect the 293T culture supernatant that contains virion after 48 hours, infect the L929 cell with it.Add polybrene to final concentration be 8ng/ml, continues 37 ℃ of infection 6 hours.Add 1640 substratum (liquid is changed in the 2ml/ hole every other day) that contain 10% foetal calf serum (FCS) again.Collecting cell after the superinfection 3 times places 2 weeks of screening and culturing in the selection substratum that contains Zeocin (Invitrogen, 500 μ g/ml) with the cell culture of dilution in 1: 6.It is enough big to treat that the resistance mono-clonal grows to, and picking mono-clonal bacterium colony carries out enlarged culturing.Preparation is as the transgenic cell L929/mock of negative control simultaneously.Flow cytometer detects and shows that the proteic expression rate of human OX 40 L is about 97% on the L929/OX40L cytolemma.
(2) preparation of monoclonal antibody against human OX 40 L
The transgenic cell (L929/OX40L) that uses the high expression level OX40L molecule as above obtain is as immunogen, three immunization Balb/c mouse (10
7/ 500 μ l/ are only) (3 weeks at interval).After the last immunity the 4th day, get mouse spleen cell and SP2/0 myeloma cell strain and carry out cytogamy (totally 20 96 orifice plates).With the positive contrast of L929/OX40L of high expression level OX40L molecule and the negative contrast of L929/mock of expression OX40L molecule, hybridoma culture supernatant is carried out preliminary screening, Identification of Fusion Protein and Ig subgroup identification (referring to Fig. 2) with indirect immunofluorescence.Positive colony is behind 3 limiting dilutions, and clone's positive rate reaches about 95%.Behind multiple sieve and subclone, obtain stably to secrete the hybridoma cell strain of specificity mouse-anti human OX 40 L, and be named as 9H10 (CGMCC No.1167), 4C12 (CGMCCNo.1168) and 1E7 (CGMCC No.1169) respectively.These hybridomas after external continue to go down to posterity (40 generation), secreting specificity antibody stably still.Chromosome analysis to hybridoma cell strain 9H10,4C12 and 1E7 shows that the chromosome number of this three strain of hybridoma is 80-110 (referring to Fig. 3).
(3) production of monoclonal antibody against human OX 40 L and CHARACTERISTICS IDENTIFICATION
(a) induce method manufacture order clonal antibody in the ascites body that adopts this chamber to set up.Get the 6-8 female Balb/c mouse in age in week, intraperitoneal injects Pristane (0.5ml/ only).Inoculation hybridoma (1 * 10 in one all pneumoretroperitoneums
7/ only), the equal-volume mixture (0.2ml/) of intraperitoneal injection Pristane and freund 's incomplete adjuvant once more simultaneously.Gather in the crops ascites after 5-10 days, and the centrifuging and taking supernatant is in-80 ℃ of preservations.
(b) purifying of ascitic type monoclonal antibody and quantitative.Ascites fluid is after the removal scleroproein and the processing of saltouing, with Protein G affinity column chromatography method purifying.Collect the protein peak effluent liquid, it is 0.8～10mg/ml that antibody protein concentration is measured with 751 ultraviolet spectrophotometers in phosphate buffered saline buffer (PBS) dialysis back.Indirect immunofluorescence is analyzed, and tiring of monoclonal antibody is more than 1: 1000 behind the purifying.
(c) Ig subgroup identification.Adopt test paper rapid determination (Argen company) method, identify the Ig subclass, the result shows that 9H10 and 1E7 are mouse IgG1 type, and 4C12 is the IgG2b type.
(d) indirect immunofluorescence is analyzed the expression of OX40L on activating B cell and ripe DC.Take out 3 days lymphocytes in tonsil of dyers' grapes (PWM) activation, add monoclonal antibody 9H10,4C12 or 1E7 (every pipe 2 μ g), hatched 30 minutes for 4 ℃.Cell is handled also with the direct traget antibody of CD19 of PE mark with the sheep anti-mouse igg of FITC mark in the washing back, hatches after 30 minutes for 4 ℃ and carries out flow cytometry.The result as seen, three strain monoclonal antibodies all can be discerned the OX40L molecule (Fig. 3) of expressing on the activating B cell.Equally, the result who detects with similarity method confirms that three strain monoclonal antibodies of the present invention also all can be discerned ripe DC and go up the OX40L molecule (referring to Fig. 4) of expressing.
(e) competitive inhibition of antibody recognition antigen site test.At L929/OX40L cell (5 * 10
5/ pipe) suspension adds monoclonal antibody (every pipe 2 μ g) respectively, hatches 45 minutes for 4 ℃.Add biotin labeled monoclonal antibody successively after washing cell, and streptavidine-PE, and respectively 4 ℃ hatched 30 minutes.Flow cytometry analysis is used in the washing back once more, establishes the positive and negative control simultaneously.As shown in Figure 5, monoclonal antibody 9H10 partly blocks combining of OX40L molecule on monoclonal antibody 4C12 and the L929/OX40L cell, and monoclonal antibody 9H10 and 4C12 all can not block combining of OX40L molecule on monoclonal antibody 1E7 and the L929/OX40L cell.Show that thus the OX40L molecular antigen site of 9H10 and 4C12 identification is incomplete same, 1E7 then has the antigen recognition site that is different from 9H10 and 4C12.
(f) the Western trace of monoclonal antibody is identified.0.1 μ g expresses the tropina of GST-OX40L engineering bacteria through the 12%SDS-PAGE electrophoresis, and electrotransfer (0.65mA/cm
2) to nitrocellulose membrane, spend the night with 4 ℃ of sealings of 5% skim-milk.Added the antibody purified incubated at room next day 1 hour.With the unconjugated antibody of TBST solution flush away, add two of AP mark and resist 30 minutes.Hatch the back and add the substrate colour developing.As shown in Figure 6,9H10,4C12 and 1E7 all can combine with the OX40L protein-specific, form positive band.Show that this three strains monoclonal antibody all has the good specificity of identification OX40L molecule.
Embodiment 2: monoclonal antibody against human OX 40 L 9H10 and 4C12 suppress unidirectional and two-way mixed lymphocyte reacion
Present embodiment describe with
3The H-TdR method of mixing detects monoclonal antibody 9H10 and the 4C12 restraining effect to mixed lymphocyte reacion.
(1) preparation of peripheral blood mononuclear cell (PBMC), T cell and dendritic cell (DC): get normal volunteer's peripheral blood 100ml (deriving from Red Cross blood station, Suzhou), separate (Ficoll) and obtain mononuclearcell.After washing cell, be diluted to 3 * 10 with the RPMI1640 substratum that contains 10% calf serum
6/ ml.Place 6 well culture plates (2ml/ hole) to cultivate 2 hours (5%CO
2, after 37 ℃) and sucking-off suspension cell gently, promptly obtain PBMC.In culture plate, add the RPMI1640 substratum continuation cultivation that contains GM-CSF (100ng/ml), IL-4 (50ng/ml) and contain 10%FCS, changed liquid once in 3 days.Adding exciting type anti-human CD 40 monoclonal antibody (this chamber development, 5 μ g/ml) on the 6th day continues to cultivate.Collected sophisticated DC with ordinary method on the 8th day.The T cell is taken from normal volunteer's peripheral blood equally and is separated acquisition (purity＞90%) according to conventional sheep red blood cell (SRBC) combined techniques.
(2) unidirectional mixed lymphocyte reacion: with T cell (1 * 10
5/ hole) and external evoked sophisticated DC add in 96 orifice plates with 20: 1 ratios.The monoclonal antibody 9H10 or the 4C12 that add different concns establish 3 multiple holes, establish the contrast of mouse IgG homotype simultaneously for every group.Add in cultivating the 5th day
3H-TdR (0.5 μ Ci/ hole) continues to cultivate 16-18 hour.After the cultivation with cell harvesting in 49
#On the glass fiber paper, the per minute scintillometer numerical value (cpm) of oven dry and mensuration radionuclide.The result as seen, two strain monoclonal antibodies all can according to dosage rely on mode and suppress the short proliferation function (referring to Fig. 7 A) of ripe DC to the T cell to some extent.
(3) two-way mixed lymphocyte reacion: peripheral blood mononuclear cell (PBMC) equal proportion that will be obtained from two blood donors adds in 96 orifice plates (1 * 10
5/ hole), in each hole, adds monoclonal antibody 9H10 or 4C12 (10 μ g/ml) then, establish 3 multiple holes for every group.Add again in cultivating the 7th day
3H-TdR (0.5 μ Ci/ hole) also continues to cultivate 16-18 hour.After the cultivation with cell harvesting to 49
#On the glass fiber paper, the per minute scintillometer numerical value (cpm) of oven dry and mensuration radionuclide.The result as seen, two strain monoclonal antibodies all can suppress two-way mixed lymphocyte reacion (referring to Fig. 7 B) to some extent.
Embodiment 3: monoclonal antibody against human OX 40 L 9H10 promotes ripe and and then the activated t cell of dendritic cell (DC)
Present embodiment is described respectively and is detected the short Differentiation of monoclonal antibody 9H10 to DC with flow cytometry and euzymelinked immunosorbent assay (ELISA).
(1) monoclonal antibody against human OX 40 L 9H10 promotes the DC differentiation and maturation: get normal volunteer's peripheral blood 200m1 (deriving from Red Cross blood station, Suzhou), separate obtaining mononuclearcell.After washing cell, with containing the RPMI1640 substratum of 10% calf serum (FCS) with cell dilution to 3 * 10
6/ ml, and place 6 well culture plates (2ml/ hole) to cultivate 2 hours (5%CO
2, 37 ℃).Sucking-off suspension cell gently adds the RPMI1640 substratum that contains GM-CSF (100ng/ml), IL-4 (50ng/ml) and 10%FCS and continues cultivation in culture plate then, changes liquid once in 3 days.Adding exciting type anti-human CD 40 monoclonal antibody (for this chamber development, 2 μ g/ml) and anti-human OX 40 L monoclonal antibody 9H10 (5 μ g/ml) on the 6th day continues to cultivate.Take out DC two days later, detect the expression of CD80, CD86, CD83 and CXCR4 with flow cytometer.The result as seen, on the basis that anti-people CD40 monoclonal antibody excites, monoclonal antibody 9H10 can raise the expression of some molecule on the DC surface such as CD80, CD86, CD83 and CXCR4,, show that 9H10 can further promote DC maturation (referring to Fig. 8).
(2) in the DC cell culture, add the T cell with 1: 20 ratio, get the culture supernatant respectively at the 1st, 3,5,7 day, detect the wherein existence of cytokine with euzymelinked immunosorbent assay (ELISA).As shown in Figure 9, compare, unite the DC that use 9H10 and CD40 monoclonal antibody excite and to promote T emiocytosis IL-2 and IFN-γ, the secretion level of IL-4 and IL-10 is then changed not quite with single DC that excites with anti-people CD40 monoclonal antibody or OX40L monoclonal antibody.This result shows that the DC that anti-human OX 40 L monoclonal antibody excites can promote the T cell to break up to the Th1 direction.
Embodiment 4: monoclonal antibody against human OX 40 L 9H10 and 4C12 promote the antibody-secreting of T cell dependency B cell
Present embodiment is described with euzymelinked immunosorbent assay (ELISA) and is detected monoclonal antibody 9H10 and the 4C12 influence to the B cell IgG secretion level that is stimulated.
(1) producing of lymphocytes in tonsil: peel off the amygdaline coating of excision, conventional preparation single cell suspension, and therefrom separate (Ficoll) lymphocytes in tonsil (the B cell purity is about 50%).
(2) euzymelinked immunosorbent assay (ELISA) detects the human IgG level: resulting B cell is added in 24 orifice plates (5 * 10
5/ hole), add PWM (4 μ g/ml) simultaneously with activating B cell.In each hole, add monoclonal antibody against human OX 40 L 9H10 or 4C12, cultivate and collect the culture supernatant after 10 days, and with euzymelinked immunosorbent assay (ELISA) detection human IgG level wherein.The result shows that anti-human OX 40 L monoclonal antibody 9H10 and 4C12 all can promote the IgG secretion (referring to Figure 10) of T cell dependency B cell to some extent
Embodiment 5: monoclonal antibody against human OX 40 L 1E7 is used for amplification in vitro T cell
Present embodiment describe with
3The H-TdR method of mixing detects the short proliferation function of monoclonal antibody 1E7 to the T cell.
(1) T cell preparation: use conventional sheep red blood cell (SRBC) combined techniques from normal volunteer's peripheral blood lymphocyte, to separate T cell (purity＞90%).
(2) monoclonal antibody 1E7 is to the short proliferation function of T cell: exciting type anti-human CD3 monoclonal antibody (0.5 μ g/ml) is added wrapper sheet in 96 orifice plates, 4 ℃ of overnight incubation.After washing cell (twice) with PBS, to wherein adding the T cell, and add the anti-human OX 40 L monoclonal antibody 1E7 (2.5,5 and 10 μ g/ml) of different concns.Add again in cultivating the 3rd day
3H-TdR (0.5 μ Ci/ hole) also continues to cultivate 16-18 hour.Then with cell harvesting to 49
#On the glass fiber paper, the per minute scintillometer numerical value (cpm) of oven dry and mensuration radionuclide.The result shows that anti-human OX 40 L monoclonal antibody 1E7 of the present invention can rely on the propagation that mode promotes the T cell significantly with dosage, (referring to Figure 11).
The hybridoma sample preservation
Particularly produce the accidental opportunity of the hybridoma screening of monoclonal antibody of the present invention with high yield in view of positive hybridoma, also be as replenishing to this specification sheets text description part, the applicant is when preservation is used as immunogenic transgenic cell, also in same microbial preservation mechanism (Chinese common micro-organisms preservation administrative center, CGMCC) preservation produce three hybridoma cell strains of monoclonal antibody 9H10 of the present invention, 4C12 and 1E7 respectively, their deposit number is respectively CGMCC No.1167, CGMCCNo.1168 and CGMCC No.1169.
Sequence table
＜110〉University Of Suzhou's biotechnology research institute
＜120〉anti-OX 40 l Monoclonal Antibody and application
<140>
<141>
<160>12
<210>1
<211>468
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide coding sequence of synthetic monoclonal antibody against human OX 40 L 9H10 heavy chain.
<400>1
1?GTCAAGCTGC?AGCAGTCTGG?GCCTGAGCTG?GTAAAGCCTG?GGGCTTCAGT
51?GAAGATGTCC?TGTAAGGCTT?CTGGATACAG?ATTCACTAGC?TATGTTATGC
101?ACTGGGTGAA?GCAGAAGCCT?GGGCAGGGCC?TTGAGTGGAT?TGGATATATT
151?AATCCTTACA?ATGATGGAAC?TAAGTACAAT?GAGAAGTTCA?AAGGCAAGGC
201?CACACTGACT?TCAGACAAAT?CCTCCAGCAC?AGCCTACATG?GAGCTCAGCA
251?GCCTGACCTC?TGAGGACTCT?GCGTTCTATT?ACTGTGCAAG?AGAGCCGGGA
301?ATTTACTTTG?ATTACGGCGG?GGTGGGCCGG?TTTCCTTACT?GGGGCCAAGG
351?GACTCTGGTC?ACTGTCTCTG?CAGCCAAAAC?GACACCCCCA?TCTGTCTATC
401?CACTGGCCCC?TGGATCTGCT?GCCCAAACTA?ACTCCATGGT?GACCCTGGGA
451?TGCCTGGTCA AGGGCTAT
<210>2
<211>345
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide coding sequence of synthetic monoclonal antibody against human OX 40 L 9H10 light chain.
<400>2
1?ATTGACATTC?TGATGACCCA?GTCTCCAGCA?ATCATGCCTG?CATCTCCAGG
51?GGAGAAGGTC?ACCATGACCT?GCAGTGCCAG?CTCAAGTGTA?AGTTATATGC
101?ACTGGTACCA?GCAGAAGTCA?GGCACCTCCC?CCACAAGATG?GATTTATGAC
151?ACATCCAAAG?TGGCTTCTGG?AGTCCCTGCT?CGCTTCAGTG?GCAGTGGGTC
201?TGGGACCTCT?TACTCTCTCA?CAATCAGCAG?CATGGAGGCT?GAAGATGCTG
251?CCACCTATTA?CTGCCAGCAG?TGGAGTAGTA?ACCCACCGAC?GTTCGGTGGA
301?GGGACCAAGC?TGGAGATCAA?AAGTGCTGAT?GCTGCACCAA?CTGTA
<210>3
<211>456
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide coding sequence of synthetic monoclonal antibody against human OX 40 L 4C12 heavy chain.
<400>3
1?GTCCAACTGC?AGGAGTCAGG?AGGAGGCTTG?GTACAGCCTG?GGGGTTCTCT
51?GAGACTCTCC?TGTGCAACTT?CTGGGTTCAC?CTTAAGTGAT?TTTTACATGG
101?ACTGGGTCCG?CCAGCCTCCA?GGGAAGAGAC?TGGAGTGGAT?TGCTGCAAGT
151?AGAAATAAAG?CTAATAATTA?TACAACAGAG?TACAGTGCAT?CTGTGAAGGG
201?TCGGTTCATC?GTCTCCAGAG?ACACTTCCCA?CAGCATCCTC?TACCTTCAGA
251?TGAAAGCCCT?GAGACCTGAG?GACACTGCCG?TTTATTACTG?TACAAGAGTC
301?TATTATAAGT?CCTGGTACTT?CGATGTCTGG?GGCGCAGGGA?CCACGGTCAC
351?CGTCTCCTCA?GCCAAAACAA?CACCCCCATC?AGTCTATCCA?CTGGCCCCTG
401?GGTGTGGAGA?TACAACTGGT?TCCTCCGTGA?CTCTGGGATG?CCTGGTCAAG
451?GGCTAT
<210>4
<211>348
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide coding sequence of synthetic monoclonal antibody against human OX 40 L 4C12 light chain.
<400>4
1?CATTGTGATG?ACCCAGTCTC?CAGATTCATG?TCCACATCAA?TAGGAGACAG
51?GGTCAGTATC?ACCTGCACGG?CCAGTCAGGA?TGTGGGTAGG?ACTGTAGCCT
101?GGTATCAACA?GAAACCAGGG?CAATCTCCTA?AACTACTGAT?TTACTGGGCT
151?TCCACCCGGC?ACACTGGAGT?CCCTGATCGC?TTCACAGGCA?GTGGATCTGG
201?GACAGATTTC?ACTCTCACCA?TTAGCAATGT?GCAGTCTGAA?GACTTGGCAG
251?ATTATTTCTG?TCAGCAATAC?ACCAGCTATC?CGTATATATA?CACGTTCGGA
301?GGGGGGACCA?AGCTGGAGAT?CAAAAGTGCT?GATGCTGCAC?CAACTGTA
<210>5
<211>342
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide coding sequence of synthetic monoclonal antibody against human OX 40 L 1E7 light chain.
<400>5
1?GACATTCAGA?TGACCCAGTC?TCCAGCAATC?ATGTCTGCAT?CTCCAGGGGA
51?GAAGGTCACC?ATGACCTGCA?GTGCCAGCTC?AAGTATAAGT?TACATGCACT
101?GGTACCAGCA?GAAGCCAGGC?ACCTCCCCCA?AAAAATGGAT?TTTTGACACA
151?TCCAAACTGG?CTTCTGGAGT?CCCTACTCGC?TTCAGTGGCA?GTGGGTCTGG
201?GACCTCTTAT?TCTCTCACAA?TCAGCAACAT?GGAGGCTGGA?GATGCTGCCA
251?CTTATTACTG?CCATCAGCAG?AATAATTACC?CGTACACGTT?CGGAGGGGGG
301?ACCAAGCTGG?AGATCAAAAG?TGCTGATGCT?GCACCAACTG?TA
<210>6
<211>156
＜212〉amino acid
＜213〉artificial sequence
<220>
＜223〉heavy chain amino acid sequence of inferring according to the heavy chain nucleotide coding sequence of synthetic monoclonal antibody against human OX 40 L 9H10.
<400>6
1?VKLQQSGPEL?VKPGASVKMS?CKASGYRFTS?YVMHWVKQKP?GQGLEWIGYI
51?NPYNDGTKYN?EKFKGKATLT?SDKSSSTAYM?ELSSLTSEDS?AFYYCAREPG
101?IYFDYGGVGR?FPYWGQGTLV?TVSAAKTTPP?SVYPLAPGSA?AQTNSMVTLG
151?CLVKGY
<210>7
<211>156
＜212〉amino acid
＜213〉artificial sequence
<220>
＜223〉light-chain amino acid sequence of inferring according to the light chain nucleotide coding sequence of synthetic monoclonal antibody against human OX 40 L 9H10.
<400>7
1?VKLQQSGPEL?VKPGASVKMS?CKASGYRFTS?YVMHWVKQKP?GQGLEWIGYI
51?NPYNDGTKYN?EKFKGKATLT?SDKSSSTAYM?ELSSLTSEDS?AFYYCAREPG
101?IYFDYGGVGR?FPYWGQGTLV?TVSAAKTTPP?SVYPLAPGSA?AQTNSMVTLG
151?CLVKGY
<210>8
<211>115
＜212〉amino acid
＜213〉artificial sequence
<220>
＜223〉heavy chain amino acid sequence of inferring according to the heavy chain nucleotide coding sequence of synthetic monoclonal antibody against human OX 40 L 4C12.
<400>8
1?IDILMTQSPA?IMPASPGEKV?TMTCSASSSV?SYMHWYQQKS?GTSPTRWIYD
51?TSKVASGVPA?RFSGSGSGTS?YSLTISSMEA?EDAATYYCQQ?WSSNPPTFGG
101?GTKLEIKSAD?AAPTV
<210>9
<211>115
＜212〉amino acid
＜213〉artificial sequence
<220>
＜223〉light-chain amino acid sequence of inferring according to the light chain nucleotide coding sequence of synthetic monoclonal antibody against human OX 40 L 4C12.
<400>9
1?IDILMTQSPA?IMPASPGEKV?TMTCSASSSV?SYMHWYQQKS?GTSPTRWIYD
51?TSKVASGVPA?RFSGSGSGTS?YSLTISSMEA?EDAATYYCQQ?WSSNPPTFGG
101?GTKLEIKSAD?AAPTV
<210>10
<211>
＜212〉amino acid
＜213〉artificial sequence
<220>
＜223〉light-chain amino acid sequence of inferring according to the light chain nucleotide coding sequence of synthetic monoclonal antibody against human OX 40 L 1E7.
<400>10
<210>11
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>7
AACTGCAGAT?GGAAAGGGTC?CAACCC
<210>12
<211>26
<212>DNA
＜213〉artificial sequence
<220>
＜223〉according to the specific nucleotide sequence design, with primer as pcr amplification.
<400>8
CGGGATTCTC?AAAGGACACA?GAATTC
Claims (9)
1, is selected from one group of monoclonal antibody against human OX 40 L that comprises 9H10,4C12 and 1E7.
2, according to the monoclonal antibody against human OX 40 L of claim 1, the heavy chain and the light chain that are characterised in that monoclonal antibody against human OX 40 L 9H10 and 4C12 have aminoacid sequence shown in SEQ ID NO:6 to SEQID NO:9 in the sequence table respectively, and the heavy chain of monoclonal antibody against human OX 40 L 1E7 has the aminoacid sequence shown in the SEQ IDNO:10.
3,, be characterised in that these antibody are monoclonal antibodies of anti-soluble human OX40L according to the monoclonal antibody against human OX 40 L of claim 1.
4, preparation claim 1 the method for monoclonal antibody against human OX 40 L, this method comprises:
(1) transgenic cell of the high expression level human OX 40 L molecule of usefulness pre-preparation is as the immunogen immune animal;
(2) separate the splenic lymphocyte of immunized animal and merge with suitable myeloma cell being suitable for producing under the condition of hybridoma;
(3) screen and cultivate the hybridoma that as above obtains;
(4) from the ascites fluid of animal of cell culture fluid or inoculation hybridoma, separate and the required monoclonal antibody of purifying.
5, according to the method for claim 6, the deposit number that wherein produces the hybridoma cell strain of monoclonal antibody against human OX 40 L 9H10,4C12 and 1E7 is respectively CGMCC No.1167, CGMCC No.1168 and CGMCC No.1169.
6, the application in suppressing unidirectional and two-way mixed lymphocyte reacion according to the monoclonal antibody against human OX 40 L 9H10 of claim 1 and 4C12.
7, the application in the antibody-secreting that stimulates T cell dependency B cell according to the monoclonal antibody against human OX 40 L 9H10 of claim 1 and 4C12.
8, according to the application of monoclonal antibody against human OX 40 L 9H10 in inducing differentiation of dendritic cells and maturation of claim 1.
9, according to the application of monoclonal antibody against human OX 40 L 1E7 in external activated T cell of claim 1.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533656A (en) * | 2010-12-29 | 2012-07-04 | 嘉和生物药业有限公司 | Method for screening cell line |
| CN108137686A (en) * | 2015-05-07 | 2018-06-08 | 阿吉纳斯公司 | Anti- OX40 antibody and its application method |
-
2005
- 2005-04-07 CN CNB2005100387299A patent/CN100509848C/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533656A (en) * | 2010-12-29 | 2012-07-04 | 嘉和生物药业有限公司 | Method for screening cell line |
| CN108137686A (en) * | 2015-05-07 | 2018-06-08 | 阿吉纳斯公司 | Anti- OX40 antibody and its application method |
| CN108137686B (en) * | 2015-05-07 | 2022-06-17 | 阿吉纳斯公司 | anti-OX 40 antibodies and methods of use thereof |
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