CN101104639A - Preparation for anti human B7-H3 monoclonal antibody and application thereof - Google Patents

Abstract

The invention relates to binding proteins or polypeptides of a member of the immunoglobulin superfamily, specifically relates to an anti-human B7-H3 monoclonal antibodies 4H7 and 21D4, a preparation method of the monoclonal antibodies, and the applications of the monoclonal antibodies in tumor (gastric cancer) prognostic evaluation and in being used as a marked molecule for identification of human dendritic cells (DC).

Description

The preparation of anti human B 7-H 3 monoclonal antibody and application thereof

FIELD OF THE INVENTION

The present invention relates to immunoglobulin superfamily member's conjugated protein or polypeptide, more particularly, the present invention relates to anti human B 7-H 3 monoclonal antibody 4H7 and 21D4, its preparation method and these monoclonal antibodies are in tumour (cancer of the stomach) prognostic evaluation and the application of molecule in identifier's dendritic cell (DC) that serve as a mark.

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 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.

To be calendar year 2001 stride film sphaeroprotein (comprise signal peptide, a pair of VC immunoglobulin (Ig) extracellular fragment, stride film district and cytoplasmic domain) by people such as Chapoval clone's the I type of being made up of 316 amino acid to human B 7-H 3 (CD276).On space structure, the ligand-receptor structural domain of this molecule and other members' of B7 family homology is respectively 27% (B7-H1), 25% (B7-H2), 21% (B7-1) and 20% (B7-2).The Northern engram analysis shows that B7-H3 mRNA has very high expression at a lot of tissues of the mankind as heart, liver, prostate gland, placenta, testis, pancreas, small intestine and large intestine.Initial discovers, B7-H3 can work in coordination with stimulates CD4 ⁺, CD8 ⁺The propagation of T cell increases the active and raising IFN-γ excretory expression of CTL.And B7-H3 is to IL-8, and TNF-α produces also the rise effect.Therefore, think B7-H3 be a kind of positivity regulatory molecule (Chapoval AI et al.Nat.Immunol., 2001,2:269-274.).But find that in research subsequently 2IgB7-H3 and 4IgB7-H3 fusion rotein all can suppress CD4 ⁺The lymphocytic in-vitro multiplication of T, downward modulation T cell to the secretion of cytokines such as IFN-γ, I1-10, IL-2, IL-6 and IL-8 (Ling V et alGenomics, 2003,82:365-377.).People such as Suh are by setting up the B7-H3 gene knock-out mice model, in experimental autoimmune encephalomyelitis (EAE) experimental study, find, helper cell is difficult to be divided into I type helper cell in the B7-H3 knock out mice body, and can be to II type helper cell normal differentiation; CD4 in the knock out mice body ⁺, CD8 ⁺The T cell quantity all is lower than wild-type mice.Therefore think, and the immunne response of B7-H3 by the immune response downward modulation body of downward modulation I type helper cell mediation (Suh WK et al.Nat.Immunol., 2003,4:899-906.).

The research of the biological action that is risen in tumour immunity about the B7-H3 molecule is also at the early-stage.For example, the expression vector that people such as Sun at first will be contained mouse B7-H3 injects the EL-4 lymphoma tissue, then the tumor tissues after handling is transplanted in the mouse body, the result shows, B7-H3 can effectively suppress diameter less than 0.3 centimetre growth of tumor even make its disappear fully (＞50%), to diameter greater than then can obviously the slow down speed of its propagation of 0.3 centimetre tumour.Studies show that further B7-H3 is by regulation and control CD8 ⁺CTL and the NK cell realize its anti-tumor in vivo immunne response (Sun X et al.Gene Ther., 2003,10:1728-1734.).People such as Luo find that the B7-H3 molecular energy obviously suppresses growth of tumor speed.Analyze the intravital lymphocytic variation of inoculation mouse, find that B7-H3 mainly is by acting on CD8 ⁺The T lymphocyte bring into play its antineoplastic biological action (Luo L et al.J.Immunol., 2004,173:5445-5450.).

But also there is the scholar to find, B7-H3 can reduce the biological function of NK cell by certain unknown signal path, thereby make the neuroblastoma cell of expressing this molecule avoid by NK cell killing (Roberta C et al.Proc.Natl.Acad.Sci., 2004,101:12640-12645.).

Nearest discovers, the B7-H3 molecular energy that is expressed on DCs and the T cell promotes the lymphocytic external activation and proliferation of T.Using under the precursor of immunosuppressor, blocking-up B7-H3 signal can significantly improve homology heteroplastic transplantation plant survival time, weaken the acute and chronic transplant rejection (Wang L et al.J.Immunol., 2005,35:428-438.).

B7-H3 is as a costimulatory molecules of recently sending out new, and the research of its biological function is at the early-stage and have a bigger dispute.In view of other members in this family (as B7-1, B7-2, B7-H1, B7-H2, B7-H4 etc.) have play a part importantly, therefore infer that B7-H3 also has vital role in tumor development in the developing of tumour.The inventor utilizes the two strain mouse-anti human B 7-H 3 monoclonal antibodies of developing voluntarily, and (4H7 21D4), has carried out large-scale clinical tumor sample screening, found that the expression and the cancer of the stomach prognosis relation of being proportionate of B7-H3 molecule.Therefore, the expression level of B7-H3 on tumor tissues might be as an important evaluation index of estimating the existence of tumour patient postoperative.DC is the APC most important in the entity, that function is the strongest, and its maximum characteristics are to excite unsensitized T lymphocyte activation.Therefore, DC has unique status in immunne response.Studies show that in recent years, body might be regulated immunne response by the function of regulating DC, thereby to tumour, plays a significant role in the treatment of transplant rejection and autoimmune disease and the prevention.Yet,, limited further investigation greatly to the DC function because DC quantity in the middle of tissue is few and cell surface lacks the characteristic mark.Although continue after set up the amplification in vitro method of DC, but still characteristic surface mark for want of, and isolating purifying of DC and phenotypic evaluation are met difficulty.

We find that also the B7-H3 molecule is sustainable stably at CD14 ⁺/ CD34 ⁺High expression level on the dendritic cell in source, and at peripheral blood T, B, NK, then do not express on the monocyte or expression level very low.We think in view of the above, and the B7-H3 molecule may be a kind of comparatively special DC surface markers molecule.In view of this, the B7-H3 monoclonal antibody is expected performance important use value on DC phenotypic evaluation, separation and purifying.

Goal of the invention

An object of the present invention is to provide one group of anti human B 7-H 3 monoclonal antibody that is selected from 4H7 and 21D4.

According to the preferred embodiments of the invention, anti human B 7-H 3 monoclonal antibody 4H7 that is defined as above and 21D4 are the monoclonal antibodies of anti-soluble human B7-H3.

Another object of the present invention provides the method for preparing the anti human B 7-H 3 monoclonal antibody that is defined as above, and this method comprises:

(1) transgenic cell of the high expression level human B 7-H 3 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 anti human B 7-H 3 monoclonal antibody 4H7 and 21D4 is respectively CGMCC No.1638 and CGMCC No.1639.

A further object of the present invention provides anti human B 7-H 3 monoclonal antibody 4H7 and the application of 21D4 in the cancer of the stomach prognostic evaluation that is defined as above.

A further object of the present invention provides the anti human B 7-H 3 monoclonal antibody 21D4 that is defined as above and the 4H7 application of molecule in DC phenotypic evaluation and screening that serve as a mark.

Brief Description Of Drawings

Fig. 1 shows the identification to B7-H3 molecule on the transgenic cell with flow cytometry anti human B 7-H 3 monoclonal antibody 4H7 and 21D4.The wherein negative contrast in grey peak, first antibody (being called for short resists) is a rabbit igg, second antibody (being called for short two resists) is the goat anti-rabbit igg of fluorescein PE mark.Transparent peak shows transgenic cell and rabbit anti human B 7-H 3 resist the result of reaction more, and wherein first antibody is respectively a rabbit anti human B 7-H 3 polyclone anti-antibody, and second antibody is the goat anti-rabbit igg of fluorescein PE mark.

Fig. 2 shows that Flow Cytometry analyzes the specificity of the anti-B7-H3 monoclonal antibody of two strain specificitys and identify.(the L929 cell strain comes from American type culture collection (ATCC, CCL1)), with dividing in the streaming pipe (5 * 10 after the PBS washing that contains 2% calf serum with well-grown L929/B7-H3 cell or L929/mock cell ⁵Individual/pipe).The wherein negative contrast in grey peak, first antibody (following be called for short sometimes anti-) is mouse IgG, second antibody is the sheep anti-mouse igg of fluorescein PE mark.Transparent peak shows the result of transgenic cell and mouse-anti human B 7-H 3 monoclonal antibody (4H7 or 21D4) reaction, wherein first antibody is respectively rabbit anti human B 7-H 3 monoclonal antibody (the following monoclonal antibody that is called for short sometimes) (4H7 or 21D4), and second antibody (following be called for short sometimes two anti-) is the sheep anti-mouse igg of fluorescein PE mark.

Fig. 3 shows the specificity of analyzing the anti-B7-H3 monoclonal antibody of two strains with Western blotting (Western blot).(each is 5 * 10 years old to collect well-grown 293T, L929/B7-H3 and L929/mock cell ⁷Individual), with the PBS washed cell of precooling and after hatching, add cytolemma lysate (RIPA) 0.5ml and proteinase inhibitor (PMSF) 100 μ l successively, respectively the membranin of 3 kinds of cells of extracting.Three kinds of protein examples are carried out SDS-PAGE electrophoresis and electrotransfer to nitrocellulose filter.After the sealing of 5% skim-milk, 4 ℃ are spent the night.Next day, antibody 4H7 (dilution in 1: 1000) or 21D4 (dilution in 1: 1000) with PBS-T washing and adding dilution sealed 1 hour.Then, with the PBS-T thorough washing and to add the rabbit anti-mouse igg two of alkaline phosphatase (AP) mark anti-, incubated at room 2 hours.Wash cell once more and use the colour developing of NBT/BCIP Color Appearance System.

Fig. 4 shows the competitive inhibition with the antigen site of flow cytometry monoclonal antibody 4H7 and 21D4 identification.L929/B7-H3 cell (5 * 10 ⁵/ pipe) with after the PBS washing, adds monoclonal antibody (every pipe 2g), biotin labeled monoclonal antibody, streptavidin peroxidase solution successively, and use flow cytometry analysis behind washing and the incubation, establish the positive and negative control group simultaneously.1: the B7-H3 site that antibody 21D4 competition binding antibody 4H7 is discerned.The negative contrast in transparent peak, wherein one anti-is biotin labeled mouse IgG, two anti-ly are Streptavidin-PE.Grey peak and black peak show the competitiveness of 4H7 and 21D4 loci.After 4H7 added L929/B7-H3 reaction, again with biotin labeled 4H7 (grey peak) or 21D4 (black peak) effect, the streptavidin (Streptavidin-PE) that adds the phycoerythrin mark at last, flow cytometer detects 2: with method 1, observe the B7-H3 site that antibody 4H7 competition binding antibody 21D4 is discerned.

Fig. 5 shows the identification to the B7-H3 molecule on the peripheral blood lymphocyte with flow cytometry 4H7 and 21d4.The conventional healthy human peripheral blood mononuclearcell (PBMCs) that separates, reacted 30 minutes for 4 ℃ with the PBMCs of B7-H3 monoclonal antibody (21D4) or homotype contrast mouse IgG1 and fresh separated or through the PBMCs that handles after activating, the sheep anti mouse two that adds the FITC mark behind the centrifugal clean antibody that is not attached on the cytolemma is anti-, and 4 ℃ of lucifuges were reacted 30 minutes.Then cell is divided into the number pipe, reacted 30 minutes with anti-CD3, CD14 and 4 ℃ of lucifuges of CD19 monoclonal antibody of PE mark respectively.Behind the centrifuge washing, detect the expression of B7-H3 on peripheral blood lymphocyte with Flow Cytometry.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 transgenic cell and mouse-anti human B 7-H 3 monoclonal antibody (4H7 or 21D4) reaction result, and wherein first antibody is respectively rabbit anti human B 7-H 3 monoclonal antibody 21D4, and second antibody is the sheep anti-mouse igg of fluorescein PE mark.

Fig. 6 shows the recognition capability that Mo-DCs is gone up the B7-H3 molecule with flow cytometry monoclonal antibody 21D4.Reference literature separates healthy human peripheral blood mononuclearcell (PBMC) (Jin Baiquan, press of The Fourth Military Medical University, 2002), wash cell after, with cell (3 * 10 ⁶/ ml) add in 6 well culture plates (2ml/ hole), cultivated 2 hours for 37 ℃.Removing cell after the cultivation adds in culture plate and contains GM-CSF (100ng/ml), IL-4 (50ng/ml), 10%FCS, 0.02mmol/LL-glutamine, 3-mercaptoethanol (2-ME, 5 * 10 ^-5Mol/L) RPMI-1640 continues to cultivate (changing liquid once in per 3 days).Cultivate after 7 days, add 5C11 (5 μ g/ml) or TNF-α (10ng/ml) and induced 3 days.Then, collect the DCs that respectively induces group to be in different developmental phases, by the expression of flow cytometry analysis cell surface B7-H3.(a) monoclonal antibody 21D4 is to the recognition capability of prematurity Mo-DCs surface molecular B7-H3.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 mouse-anti human B 7-H 3 monoclonal antibody (4H7 or 21D4) reaction, and wherein first antibody is respectively rabbit anti human B 7-H 3 monoclonal antibody 21D4, and second antibody is the sheep anti-mouse igg of fluorescein PE mark.Monoclonal antibody 4H7 also has similar recognition capability (figure slightly).(b) monoclonal antibody 21D4 is to the recognition capability of ripe Mo-DCs surface molecular B7-H3.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 mouse-anti human B 7-H 3 monoclonal antibody (4H7 or 21D4) reaction, and wherein first antibody is respectively rabbit anti human B 7-H 3 monoclonal antibody 21D4, and second antibody is the sheep anti-mouse igg of fluorescein PE mark.

Fig. 7 shows with Western blot immunoblotting and analyzes monoclonal antibody 21D4 and the 4H7 identification to Mo-DCs.Collect the good Mo-DCs (about 5 * 10 of growth conditions ⁷Individual), after educating with twice of the PBS of precooling washing and ice, add successively the cytolemma lysate (RIPA, 0.5ml) and proteinase inhibitor (PMSF, 100 μ l) back difference extracting epicyte protein.Behind the electrophoresis with required isolate electrotransfer to nitrocellulose filter.5% skim-milk sealing nitrocellulose membrane, 4 ℃ of concussion incubated overnight.Next day, PBS-T washing back adds antibody 4H7 (dilution in 1: 1000) or the 21D4 (dilution in 1: 1000) or the homotype contrast mouse IgG of dilution, and behind sealing and the thorough washing, the rabbit anti-mouse igg two that adds alkaline phosphatase (AP) mark is anti-, incubated at room 2 hours is with the colour developing of NBT/BCIP Color Appearance System.

Fig. 8 shows the identification of monoclonal antibody 21D4 to B7-H3 molecule on the tumour cell.Adopt indirect immunofluorescence to detect the expression of B7-H3 molecule on B lymphoma cell line, multiple myeloma cells system, epithelium tumor clone and tonsilla and peripheral blood lymphocyte.The result shows that the B7-H3 molecule has the expression of higher level on the tumor cell surface of epithelial origin.And be U266,8266 and XG series cell at multiple myeloma cells; Leukemia cell HL60, K562; T cell line cell Jurkat; Express hardly on B cell line cell Raji, the Daudi.Monoclonal antibody 4H7 also has similar recognition capability (figure slightly).

Fig. 9 monoclonal antibody 21D4 is to the identification of B7-H3 molecule on the flesh tissue.This tissue of taking a sample, conventional preparation paraffin section (4 μ m).Section with distilled water wash and add 0.3% hydrogen peroxide-methyl alcohol to block endogenous peroxidase, was hatched under room temperature 30 minutes after dewaxing and repairing antigen.Once more after the washing, every section is added dropwise to 10% normal sheep serum, specific murine anti human B 7-H 3 antibody 21D4 (1: 50, anti-as) successively, goes into biotin labeled rabbit anti-mouse igg (second antibody), streptavidin peroxidase solution.Add as all washing cell before the reagent and hatching 40-60 minute in 37 ℃ with PBS at every turn.After the reflection, add 0.04%DAB+0.03%H ₂O ₂Develop the color, and dye with the Hematorylin lining.Mounting after washing, dehydration and the transparent processing.Microscopic examination as seen, all visible B7-H3 molecule positive expression in stomach cancer cell, adenoma of stomach cytolemma and the cytoplasm.A, 10 routine normal gastric mucosas are not seen the expression of B7-H3 molecule; B, B7-H3 developed by molecule positive rate 100.0% in the 10 routine adenoma of stomach cells; B7-H3 developed by molecule positive rate is 58.8% among the C, 102 routine stomach cancer cells (cytolemma and cytoplasm).

Figure 10 shows the positive correlation of expression and the patients with gastric cancer prognosis of B7-H3.Univariate analysis prompting, tumor invasive depth, lifetime, types of organization are relevant with the expression of B7-H3.Multivariate analysis shows that the expression of B7-H3 is that 2.803,95% credibility intervals are 1.051-7.477 to the relative risk of cancer of the stomach lifetime, and the P value is 0.040.And it is closely related with tumor invasive depth, types of organization.Therefore can think that the expression of B7-H3 is to influence the cancer of the stomach independent factor of lifetime.Show that with the relationship analysis of patient's postoperative survival time be significantly higher than lifetime less than the patients with gastric cancer (43.1%) in 2 year greater than the The positive expression rate (74.5%) of the patients with gastric cancer B7-H3 in 5 years lifetime.As seen, the expression of B7-H3 is relevant with the patients with gastric cancer prognosis.

Summary of the invention

The present invention relates to immunoglobulin superfamily member's conjugated protein or polypeptide, more particularly, the present invention relates to anti human B 7-H 3 monoclonal antibody 4H7 and 21D4, its preparation method and these monoclonal antibodies are as the application of the index of DCs surface markers Molecular Identification and sorting DCs and the evaluation of conduct evaluation tumor patient (cancer of the stomach) Postoperative determination.

Said in this specification " acceptor " refers to the protein of expressing on the T cell surface of antigen activation; Part is the protein that can be combined with receptor-specific of expressing on some mammalian cell.

Term " B7-H3 " comprises complete part, solvable part and comprises the fusion of the functional activity part of part. Term " anti-B7-H3 antibody " can be specific monoclonal or polyclonal antibody or their immunologic competence part. Among the present invention, preferably monoclonal antibody, particularly mouse anti human B7-H3 antibody.

Can prepare anti human B 7-H 3 monoclonal antibody 4H7 of the present invention and 21D4 (referring to Kohler and Milrtein, Nature 256:495-96,1975 according to conventional method known in the art; Harlow and Lane, Aatibodies, A Laboratory, Cold Spring Harbor Laboritory, 1988). In case of necessity, also can be according to United States Patent (USP) 5,585, the method described in 089 prepares the anti-monoclonal antibody of corresponding humanization form.

According to the preferred embodiments of the invention, preferably use the expression vector that is carried human B 7-H 3 gene recombinant cell that transform and that can under suitable condition of culture, efficiently express human polypeptides as the immunogene of preparation monoclonal antibody of the present invention.

Therefore, in order to prepare anti human B 7-H 3 monoclonal antibody of the present invention, at first make up the transgenic cell (L929/B7-H3) of high expressed human B 7-H 3. The transgenic cell L929/B7-H3 of high expressed human B 7-H 3 molecule has stronger immunogenicity, and the steric configuration of expressed antigen molecule can be exposed to surface of cell membrane with nature, thus the more effectively immune response of excitating organism. In addition, because the L929 cell is the fibroblast of mouse, 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/B7-H3 cell, can be according to DNA operating technology well known to those skilled in the art (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, identification, transformation and the cultivation of the structure of clone and expression vector and amplification, nucleotide sequence

Can be according to conventional method known in the art (Kohler and Milstein, Nature 265:495-497,1975) preparation anti human B 7-H 3 monoclonal antibody 4H7 of the present invention and 21D4. Briefly, with transgenic cell L929/B7-H3 immune balb/c mice. When the antibody level of serum of immunized animal reached peak value, the splenocyte of separating animal's also prepared single cell suspension. For example, splenocyte suspension can be added in B7-H3 the antigen protein coated flat board or aperture, the B cell of expressing B7-H3 polypeptid specificity immunoglobulin (Ig) namely is attached on the flat board, and can not be washed off by remaining suspension. Then can collect resulting B cell or splenocyte that all dissociate, and for example merge to form hybridoma with myeloma under the inducing of polyethylene glycol at suitable fusion agent, and for example cultivate the hybridoma that merges with screening in the HAT culture medium at selective medium. Then, identify required positive resisting cell strain with methods such as flow cytometry, Western blotting, immuno-precipitations. (as mouse ascites) cultivates the hybridoma of selected secretion anti human B 7-H 3 antibody in external (for example in tissue culture flasks or porous fibre reactor) or body, and collects and the required antibody of purifying from cell culture fluid or mouse ascites liquid.

The flow cytometry analysis result shows that anti human B 7-H 3 monoclonal antibody 4H7 provided by the invention and 21D4 all can identify the B7-H3 molecule of expressing on ripe DC and the activated monocyte surface. Competition inhibition test result shows that further anti human B 7-H 3 monoclonal antibody 4H7 of the present invention and 21D4 identify diverse antigen site. Therefore, might utilize 4H7 and 21D4 for the preparation of the kit that detects soluble human B7-H3.

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 anti human B 7-H 3 monoclonal antibody

Present embodiment is described the preparation of anti human B 7-H 3 monoclonal antibody 4H7 of the present invention and 21d4.

(1) foundation of transgenic cell L929/B7-H3 and L929/mock

(a) clone of human B 7-H 3 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 '-AA CTG CAGATG GAA AGG GTC CAA CCC-3 ', carry out pcr amplification (94 ℃ of sex change 30 seconds with downstream primer 5 '-CG GGA TTC TCA AAGGAC ACA GAA TTC-3 ', 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 B 7-H 3 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/B7-H3 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/B7-H3.

(c) structure of the L929 transgenic cell of stably express human B 7-H 3: the liposome method of recommending with the test kit operational manual at first, with recombinant retroviral vector pEGZ-Term/B7-H3 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 (ATCC, CRL-1573).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 B 7-H 3 is about 97% on the L929/B7-H3 cytolemma.

(2) preparation of anti human B 7-H 3 monoclonal antibody

The transgenic cell (L929/B7-H3) that uses the high expression level B7-H3 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/B7-H3 of high expression level B7-H3 molecule and the negative contrast of L929/mock of expression B7-H3 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 specific murine anti human B 7-H 3, and be named as 4H7 (CGMCC No.1638) and 21D4 (CGMCC No.1639) respectively.These hybridomas after external continue to go down to posterity (40 generation), secreting specificity antibody stably still.Chromosome analysis to hybridoma cell strain 4H7 and 21d4 shows that the chromosome number of this three strain of hybridoma is 80-110 (referring to Fig. 3).

(3) production of anti human B 7-H 3 monoclonal antibody 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.The indirect immunofluorescence analytical results shows, the tiring more than 1: 1000 of the monoclonal antibody of purifying.

(c) Ig subgroup identification.Adopt test paper rapid determination (Argen company) method to identify the Ig subclass, the result shows that 4H7 and 21D4 are mouse IgG1 type antibody.

(d) competitive inhibition of antibody recognition antigen site test.At L929/B7-H3 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 and streptavidin peroxidase solution (Streptavidine-PE) successively after washing cell, 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 4H7 partly blocks combining of B7-H3 molecule on monoclonal antibody and the L929/B7-H3 cell, monoclonal antibody 4H7 and all can not block combining of B7-H3 molecule on monoclonal antibody 21d4 and the L929/B7-H3 cell.Show that thus the B7-H3 molecular antigen site of 4H7 and identification is incomplete same, 21d4 then have be different from 4H7 and antigen recognition site.

(e) the Western trace of monoclonal antibody is identified.The 12%SDS-PAGE electrophoretic separation is expressed the reorganization tropina of GST-B7-H3, and electrotransfer also spends the night with 4 ℃ of sealings of 5% skim-milk to nitrocellulose filter.Second day, added the antibody purified incubated at room 1 hour.With the unconjugated antibody of TBST solution flush away, add two of AP mark and resist.Hatch the back and add the substrate colour developing.The result confirms that 4H7 and 21D4 all can combine with the B7-H3 protein-specific, form positive band.Show that this two strains monoclonal antibody all has the good specificity (referring to Fig. 5) of identification B7-H3 molecule.

Embodiment 2: monoclonal antibody 21D4 is to the recognition capability of the B7-H3 molecule that is expressed in the Mo-DCs surface

1) the conventional healthy human peripheral blood that separates obtains mononuclearcell (PBMC), after RPMI-1640 washs twice, adjusts cell density to 3 * 10 with the RPMI-1640 that contains 10%FCS ⁶/ ml adds in 6 well culture plates (2ml/ hole).5%CO then ₂, 37 ℃ cultivated 2 hours.Contain GM-CSF (100ng/ml), IL-4 (50ng/ml), 10%FCS, 0.02mmol/LL-glutamine, 5 * 10 with in culture plate, adding after the suspension cell sucking-off ^-5The RPMI-1640 of/mol/L2-ME continues to cultivate, and changes liquid once in per 3 days.Cultivate after 7 days, add 5C11 (5 μ g/ml) or TNF-α (10ng/ml) and induced 3 days.

2) collect the DCs that respectively induces group to be in different developmental phases, by the expression of flow cytometry analysis cell surface B7-H3.The result shows: B7-H3 presents very stable high level expression on Mo-DCs.Monocyte inducing culture 24 hours in containing 1640 substratum of GM-CSF, IL-4 can detect the expression of B7-H3 molecule, expresses to last till the Mo-DCs apoptosis always.In view of on periphery blood T cell, B cell and monocyte, not detecting the B7-H3 developed by molecule or expression level is very low,, be used for evaluation and the screening of Mo-Dcs so B7-H3 is expected to very important surface markers molecule as Mo-DCs.

The expression of embodiment 3:B7-H3 on tumour cell and the relation of tumor prognosis

1) case source

102 routine cancer of the stomach samples are taken from the cancer of the stomach patient with operation that attached the 3rd hospital of University Of Suzhou hospitalizes in the period of 1998-1999.The male sex's 75 examples wherein, women's 27 examples.Age 28-77 year, average 55 years old.Tumour is positioned at 10 examples under mucous membrane and the mucous membrane, is positioned at shallow flesh layer 20 example, and tumor invasive depth meets or exceeds dark flesh layer 72 example.Histological type is divided into: differentiated gland cancer (papillary carcinoma, high differentiation tubular adenocarcinoma, middle differentiation tubular adenocarcinoma) and low differentiated gland cancer (poorly differentiated adenocarcinoma, signet ring cell cancer, carcinoma muco-cellulare).Do not accept chemotherapy or radiotherapy before all patient's arts.

10 routine adenoma of stomach samples are taken from the adenoma of stomach patient with operation of hospitalizing in the period of attached the 3rd 1998-1999 of hospital of University Of Suzhou, the male sex's 7 examples wherein, women's 3 examples.Age 55-67 year, average 60 years old.

2) immunostaining and assessment

The conventional tumor tissue section for preparing after dewaxing and the aquation, washes three times and repairs accordingly with PBS (PH 7.4).Every section drips (50 μ l%) H ₂O ₂(blocking-up endogenous peroxydase), first antibody (50 μ l%), polymkeric substance toughener (reagent A, 50 μ l), enzyme are marked anti-mouse/rabbit polymkeric substance (reagent B, 50 μ l).Equal thorough washing and under 37 ℃ or room temperature, hatching behind the new reagent of every adding.At last, in section, drip freshly prepared DBA colour developing liquid (about 100 μ l) and examine under a microscope the result.The obvious tinter's note of tumour cell is made B7-H3 positive cell (it is negative that the B7-H3 positive cell is less than 20% note do expression in the stomach organization, makes the expression positive greater than 20% note).

3) statistical procedures

Adopt the SPSS10.0 statistical software to carry out data analysis, B7-H3 expresses the relatively employing X between positive and negative group ²Check, the relation of prognosis and factors adopt multivariate Logistic regression to analyze

4) expression of B7-H3 in cancer of the stomach and adenoma of stomach

All visible B7-H3 molecule positive expression in stomach cancer cell and adenoma of stomach cytolemma and the cytoplasm, B7-H3 developed by molecule positive rate 100.0% in the 10 routine adenoma of stomach cells, B7-H3 developed by molecule positive rate 58.8% in the stomach cancer cell.

5) relation of B7-H3 expression and patient age, sex and prognosis

The expression of B7-H3 and patient age, sex irrelevant (p＞0.05).Show that with the relationship analysis of patient's postoperative survival time the expression of B7-H3 is relevant with the patients with gastric cancer prognosis, be significantly higher than lifetime less than patients with gastric cancer (the 43.1%) (x in 2 year greater than the The positive expression rate (74.5%) of the patients with gastric cancer B7-H3 in 5 years lifetime ²=10.36, P＜0.01).

6) B7-H3 expresses and the clinicopathologic relation of cancer of the stomach

B7-H3 expresses and tumour status of lymph node metastasis, tumor locus, size irrelevant (P＞0.05); And it is relevant with invasive depth, lifetime, histological type.In the stomach organization type is differentiated (x ²=4.55, P＜0.05), tumor-infiltratedly do not reach dark flesh layer (x ²=5.56, P＜0.05) the B7-H3 The positive expression rate is higher.

7) multivariate analysis of cancer of the stomach correlative factor lifetime

Include in the correlative factor variable analysis such as the expression of invasive depth, lifetime, types of organization, B7-H3 and show, the univariate analysis prompting, tumor invasive depth, lifetime and types of organization are relevant with the expression of B7-H3.Multivariate analysis shows that the expression of .B7-H3 is that 2.803,95% credibility intervals are 1.051-7.477 to the relative risk of cancer of the stomach lifetime, and the P value is 0.040.As seen, the expression of B7-H3 and tumor invasive depth, types of organization are relevant.Therefore can think that the expression of B7-H3 is to influence the cancer of the stomach independent factor of lifetime, has the important clinical meaning.

Relevant experimental data sees down tabulation 1 for details.

B7-H3 expresses and the clinicopathologic relation of cancer of the stomach (positive rate %)

Project	Total routine number	Positive routine number (positive rate %)	X 2	The p value
					Lifetime＜2, the sex men and women age＞60 year old＜60 years old year＞5 years low differentiated invasive depth of types of organization's differentiated is not invaded and dark flesh layer is invaded and Weishang, the negative positive gastric carcinoma position of dark flesh layer nodus lymphoideus transferring rate 1/3 stomach in 1/3 tumour size＜5cm＞5cm under 1/3 stomach	75 27 64 38 51 51 72 30 30 72 54 48 31 30 41 55 47	42(42.7) 18(44.4) 36(56.3) 24(63.2) 22(43.1) 38(74.5) 48(66.7) 12(40.0) 23(76.7) 37(51.3) 33(61.1) 27(56.3) 19(61.2) 17(56.7) 24(58.5) 35(63.6) 25(53.2)	0.993 0.470 10.362 6.217 5.586 0.248 0.137 1.141	0.334 0.493 0.001 0.013 0.018 0.619 0.934 0.285

By the result shown in the above table 1 as can be seen, the expression of B7-H3 and patient's age, sex irrelevant (p＞0.05), and with tumour tumour happening part and size, and status of lymph node metastasis irrelevant (P＞0.05).On the contrary, the expression level of B7-H3 is relevant with tumor invasive depth, lifetime and histological type.Particularly, the stomach organization type is differentiated (x ²=4.55, P＜0.05), tumor-infiltratedly do not reach dark flesh layer (x ²The B7-H3 The positive expression rate of cell=5.56, P＜0.05) obviously raises.

Therefore, can use Monoclonal Antibody of the present invention to be used for the cancer of the stomach prognostic evaluation, or the test kit of identifier's dendritic cell (DC).It is external that said test kit removes the monoclonal anti that comprises anti-cell of the present invention surface B7-H3 molecule, also can comprise blood sample collection device and related solvents, damping fluid and standard substance etc.

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 in the preservation of microbial preservation mechanism China common micro-organisms preservation administrative center (CGMCC) produce two hybridoma cell strains of monoclonal antibody 4H7 of the present invention and 21D4 respectively, their deposit number is respectively CGMCCNo.1638 and CGMCC No.1639.

Claims (6)

1. anti human B 7-H 3 monoclonal antibody 4H7 and 21D4.

2. according to the anti human B 7-H 3 monoclonal antibody of claim 1, be characterised in that these antibody are monoclonal antibodies of anti-soluble human B7-H3.

3. according to the monoclonal antibody of claim 1, wherein anti human B 7-H 3 monoclonal antibody 4H7 is to be the hybridoma cell line excretory of CGMCC No.1638 by deposit number.

4. according to the monoclonal antibody of claim 1, wherein anti human B 7-H 3 monoclonal antibody 21D4 is to be the hybridoma cell line excretory of CGMCC No.1639 by deposit number.

5. the application in tumor prognosis is estimated according to the anti human B 7-H 3 monoclonal antibody 4H7 of claim 1 and 21D4.

6. the application in identifying dendritic cell according to the anti human B 7-H 3 monoclonal antibody 4H7 of claim 1 and serving as a mark property of 21D4 molecule.

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