CN1844146A - Monoclonal antibody against human B7-1 molecule and its use - Google Patents
Monoclonal antibody against human B7-1 molecule and its use Download PDFInfo
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Abstract
The invention relates to the conjugated proteins or polypeptides of the immune globulin super family members, more specifically, the invention relates to anti-human B7-1 monoclonal antibody 4E5, its preparing process and the use of the monoclonal antibody in inhibiting accretion and breeding of malignant lymphoma cells and treating kidney diseases of folic acid.
Description
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-people B7-1 molecule monoclonal antibody 4E5, its preparation method and this strain monoclonal antibody are in growth and breeding that suppresses the malignant lymphatic oncocyte and the application in the folic acid ephrosis.
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 an immunoglobulin superfamily, as (Korthauer U et al., Nature such as B7-CD28/CTLA4, LAF1-ICAM-1/ICAM-2/ICAM-3, ICOS-GL50, CD2/LFA-3,361:539,1993).Another kind of 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.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.
The B7-1 molecule is the B cell activation antigen of the about 50KD of certified molecular weight of nineteen eighty-two, is contactin member's transmembrane glycoprotein, and the assignment of genes gene mapping is in 3q21.The B7-1 molecule mainly is expressed in the surface of most of antigen presenting cell with the form of oligomer, on activatory B cell, T cell, dendritic cell and monocyte.B7-1 molecule and the signal that the CD28 interaction of molecules that is expressed on the T cell produces are acknowledged as the most basic costimulatory signal of T cell activation.The B7-CD28 signal relates generally to the initial period of APC and T cell interaction, and this signal is modified and replenished the TCR signal, and with behind the multiple signal integration, starts immunne response.In the progress of immunne response, further regulate and keep the survival of an amount of T cell by CD28, continue to produce specific immune response.Simultaneously, by the balance of cytokine network and the expression of adjusting CTLA-4, keep the validity and the appropriateness of immunne response.Strengthen this signal, help the repulsion of tumour and killing and wounding of tumour cell; And the second signal of blocking-up B7-1 mediation, but the generation of inducing T cell specific immunologic tolerance, the prevention and the treatment that help autoimmune disorder or allergy and allogeneic to repel.
Studies show that in recent years, the B7:CD28 signal path can play a significant role in the process of mediation anti-tumor immune response, graft-rejection and autoimmune disorder.Change B7-1 over to kinds of tumor cells, as seen all these tumours all are ostracised behind transfection B7-1 molecule.Experiment confirm, B7-1/B7-2 is two to be struck dna murine and serious diabetes and pancreas islet inflammation can occur, shows the B7-1 of antigen presenting cell surface low expression level and the formation that the B7-2 molecule helps autoantigen inductive immunological tolerance thus.If the generation with the antagonist CTLA-Ig of B7-1 and B7-2 can block autoantibody improves the brightic symptom that causes in glomerular basement membrane owing to immune complex deposit.As seen, B7-1 may be the target molecule of an ideal immunologic intervention.Anti-B7-1 monoclonal antibody then will promise to be the ideal diagnosis and the therapeutical agent of clinical intervention autoimmune disorder, graft-rejection and tumour.
Although relevant B7/CD28 receptors ligand all is known in conjunction with preparation and their other immunologic functions of right T cytositimulation function, CD28 or B7-1 antibody, do not appear in the newspapers as new anti-people B7-1 monoclonal antibody of the present invention and relatively simple preparation method thereof.And anti-people B7-1 monoclonal antibody 4E5 of the present invention does not also appear in the newspapers to the potential therapeutic action of folic acid ephrosis.
Goal of the invention
An object of the present invention is to provide anti-people B7-1 monoclonal antibody 4E5.
According to the preferred embodiments of the invention, the anti-people B7-1 monoclonal antibody 4E5 light chain that is defined as above has the aminoacid sequence of inferring shown in SEQ ID NO:3, and heavy chain has the aminoacid sequence of inferring shown in SEQ ID NO:4.
According to the preferred embodiments of the invention, the anti-people B7-1 monoclonal antibody 4E5 that is defined as above is the monoclonal antibody of anti-soluble human B7-1.
Another object of the present invention provides the anti-people B7-1 monoclonal antibody method that preparation is defined as above, and this method comprises:
(1) the commentaries on classics people B7-1 gene cell strain XG7-B7 of the high expression level people B7-1 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, wherein said anti-people B7-1 monoclonal antibody 4E5 is that deposit number is the hybridoma generation justacrine of CGMCC No.1166.
A further object of the present invention provides the application of anti-people B7-1 monoclonal antibody 4E5 in the folic acid treatment of kidney disease that is defined as above.
Brief Description Of Drawings
Fig. 1 shows with the identification of the anti-people B7-1 of flow cytometry monoclonal antibody 4E5 to B7-1 molecule on the transgenic cell.Wherein one anti-is mouse IgG1 and monoclonal antibody 4E5 of the present invention, and two anti-ly are the sheep anti-mouse igg of fluorescein PE mark.
Fig. 2 shows the chromosomal karyotyping of monoclonal antibody 4E5 hybridoma cell strain (* 1000 times).
Fig. 3 shows with the identification of flow cytometry monoclonal antibody 4E5 to the B7-1 molecule of PBLs, Raji and Daudi cell surface.With people PBLs separately and respectively with divide in small test tube (5 * 10 after Raji and Daudi cell (1: 1) mix
5/ pipe), adding 4E5 monoclonal antibody and sheep anti mouse fluorescence two are anti-, use flow cytometry analysis.The result shows, the B7-1 molecule on the recognizing cells surface that monoclonal antibody of the present invention can be good.The positive combination rate of 4E5 and people PBLs, Raji and Daudi is respectively 10.2%, 92.7%, 89.2%.Raji and Daudi cell respectively with people PBL with 1: 1 mixed after, the positive combination rate of corresponding antigens is respectively 45.1% and 42.9% on 4E5 and the target cell.
Fig. 4 shows the competitive inhibition of monoclonal antibody 4E5 to antigen binding site.With XG7-B7 cell (5 * 10
5/ pipe), add 4 ℃ of effects of 4E5 antibody (10 μ g/50 μ l/ pipe) 45 minutes with PBS washing back, the washing back adds standard CD 80-PE and directly marks monoclonal antibody (2 μ g/50 μ l/ pipe), uses flow cytometry analysis after the effect.The positive and negative contrast is set simultaneously.Results suggest, 4E5 can block standard anti-people B7-1 monoclonal antibody and antigenic reaction fully.Show that this monoclonal antibody is the specific antibody of B7-1, and discern same or analogous antigen site with standard antibody.
Fig. 5 shows the growth-inhibiting effect of monoclonal antibody 4E5 to malignant lymphatic oncocyte Raji and Daudi: with Raji (1 * 10
5/ ml) and Daudi (2 * 10
5/ ml) be incubated in 24 orifice plates, add the 4E5 monoclonal antibody, make its final concentration be respectively 1 μ g/ml, 2.5 μ g/ml, 5.0 μ g/ml, 10.0 μ g/ml and 20.0 μ g/ml.Cultivate 24,48,72,96h time point, counting cells concentration respectively.The result shows, when the concentration of 4E5 is 2.5 μ g/ml, the obvious growth restraining effect promptly occurs.
Fig. 6 show monoclonal antibody 4E5 in and activation analysis: XG1-B7 cell (1 * 10 that will be after mitomycin is handled
5/ ml) with human peripheral lymphocyte (PBL) (1 * 10
6/ ml equal-volume (each 100 microlitre) is mixed in 96 orifice plates, cultivates 72 hours, and every hole adds 20 microlitre 5%MTT solution, continues to cultivate 4-6 hour, measures OD
570, the XG1 cell of handling with mitomycin compares simultaneously.Results suggest, 4E5 can block the conduction of the numerator mediated costimulatory signal of B7-1, thereby suppresses the PBL propagation that XG1-B7 stimulates.
Fig. 7 shows the influence that monoclonal antibody 4E5 changes mouse folic acid nephropathy model animal nephridial tissue.Injection the 21st day behind the folic acid is contracted kidney, rarely seen normal region of about 7.45 ± 11.77%, and the local pyknosis of the rarely seen kidney of combined utilization 4E5, above-mentioned pathology is visible significantly to be improved, expansion and the tubule of atrophy, the zone of tubule interstitial fibrosis obviously dwindle, and the kidney normal region reaches 66.51 ± 25.81%.
Fig. 8 shows that monoclonal antibody 4E5 handles effect of immunologic function.Folic acid nephropathy model immune function of mice is obviously hyperfunction in acute phase, then shows as tangible immunocompromised in the later stage.Utilization 4E5 antibody therapeutic intervention was found that the expression level of mouse CD3, CD4 is higher than the FA group, and with the FA group marked difference is arranged after 3 days.Behind the therapeutic intervention of 4E5 antibody the 14th day, the CD3 of the FA group mouse of immunologic hypofunction, the expression of CD4 obviously increased, and with the FA group marked difference are arranged, and have recovered the expression of CD3, CD4 to a certain extent.Therefore the result shows, 4E5 antibody can make the immunologic function of animal recover normal substantially after to folic acid ephrosis mouse therapeutic intervention.
The particular content of invention
The present invention relates to conjugated protein or the polypeptide of immunoglobulin superfamily superfamily member, more particularly, the present invention relates to anti-human B7-1 molecule monoclonal antibody 4E5, its preparation method and this strain monoclonal antibody are in the growth and breeding that suppresses the malignant lymphatic oncocyte and the application in the folic acid ephrosis.
Said in this specification " B7-1 part " refers to the protein of expressing on the cell surface; The B7-1 acceptor refer to express on some mammalian cell can with the protein of B7-1 part specific bond.
Term " B7-1 " comprises complete B7-1, solubility B7-1 and comprises the fusion of the functional activity part of B7-1. Term " anti-B7-1 antibody " can be B7-1 specific monoclonal or polyclonal antibody or their immunologic competence part. Among the present invention, preferably monoclonal antibody, particularly mouse anti human B7-1 antibody.
Can prepare anti-human B7-1 monoclonal antibody 4E5 of the present invention (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-B7-1 monoclonal antibody of corresponding humanization form.
The present invention further provides the method for producing the anti-human B7-1 monoclonal antibody that is defined as above, the method comprises:
(1) with the transgenic cell XG7-B7 of pre-prepared high expressed people B7-1 molecule as the immunogen immune animal;
(2) separate the SPL 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, preferably use the expression vector that is carried people XG7-B7 gene recombinant cell that transform and that can under suitable condition of culture, efficiently express people XG7-B7 polypeptide as the immunogene of preparation monoclonal antibody of the present invention.
Therefore, in order to prepare anti-people XG7-B7 monoclonal antibody of the present invention, at first make up the transgenic cell XG7-B7 of high expression level people B7-1.The transgenic cell XG7-B7 of high expression level people B7-1 molecule has stronger immunogenicity, and the sterie configuration of expressed antigen molecule can be exposed to surface of cell membrane with state of nature, thus the more effectively immune response of excitating organism.
Can be according to ordinary method known in the art (Kohler and Milstein, Nature 265:495-497,1975) preparation anti-people B7-1 monoclonal antibody 4E5 of the present invention.Briefly, with transgenic cell X67-B7 immune balb/c mice.When the serum antibody level of immunized animal reached peak value, the splenocyte of separating animal's also prepared single cell suspension.In case of necessity, can use immunosorption method screening splenocyte.For example, splenocyte suspension can be added in the flat board or aperture of B7-1 antigen protein bag quilt, the B cell of expressing B7-1 polypeptid specificity immunoglobulin (Ig) promptly is attached on the flat board, and can not be washed off by remaining suspension.Can collect resulting B cell or all dissociated splenocytes then, and for example merge forming hybridoma with myelomatosis under the inducing of polyoxyethylene glycol, and for example cultivate the hybridoma that merges with screening in the HAT substratum at selective medium at suitable fusogen.Then, identify required positive resistant cell strain with methods such as flow cytometry, Western blotting, immuno-precipitations.(as mouse ascites) cultivates the hybridoma of the anti-people B7-1 of selected secretion antibody in external (for example in tissue culture flasks or multiporous fiber reactor) or body, and collects and the required antibody of purifying from cell culture fluid or mouse ascites liquid.
According to the regulation of the 25th of budapest treaty and patent law of china detailed rules for the implementation, the hybridoma cell strain that will produce anti-people B7-1 monoclonal antibody 4E5 on June 2nd, 2004 is deposited in BeiJing, China China common micro-organisms preservation administrative center (CGMCC), and deposit number is CGMCC No.1166.
The nucleotide sequence analysis result shows that the nucleotide coding sequence of the anti-people B7-1 monoclonal antibody 4E5 light chain of purifying of the present invention is shown in SEQ ID NO:1 in the sequence table, and the nucleotide coding sequence of heavy chain is shown in SEQ ID NO:2 in the sequence table; The aminoacid sequence of inferring according to their nucleotide coding sequence is respectively shown in SEQ ID NO:3 in the sequence table and SEQ ID NO:4.Wherein all aminoacid sequences are all with the expression of amino acid whose standard single-letter abbreviation symbol.
The flow cytometry analysis result shows that anti-people B7-1 monoclonal antibody 4E5 provided by the invention can discern the B7-1 molecule of expressing on PBLs, Raji and the Daudi cell surface to some extent.Competition inhibition test result shows that further anti-people B7-1 monoclonal antibody 4E5 of the present invention can discern same or similar antigen site.
In order to identify the biological function of anti-people B7-1 monoclonal antibody of the present invention, the present invention has at first carried out the growth-inhibiting experiment of 4E5 to malignant lymphatic oncocyte Raji and Daudi.The result shows that when the concentration of 4E5 was 2.5 μ g/ml, the obvious growth that target cell promptly occurs suppressed.With the activation analysis results suggest, monoclonal antibody 4E5 of the present invention can block the conduction of the numerator mediated costimulatory signal of B7-1 further, thereby suppresses the effect (referring to embodiment 1) of the PBL propagation of X61-B7 stimulation.
Because graft-rejection and autoimmune disorder all show as the overactivity of T cell, therefore, can utilize of the present invention in and the activation and proliferation of the anti-people B7-1 of type monoclonal antibody suppressor T cell, and then the suppressor T cell immunne response, with prevention and treatment graft-rejection and autoimmune disorder.
Anti-people B7-1 monoclonal antibody of the present invention (B7-1MAb) has vital role in the folic acid ephrosis.Use in the experiment that the folic acid nephrosis animal model carries out at us, give B7-1MAb (B7-1MAb group) simultaneously for experimental therapy treated animal abdominal injection folic acid, (after 3 days duplicate injection once, dosage is the same); Negative control group injection mouse IgG+ folic acid (FA group); Normal control group (Control group) injection NaHCO
3As seen the result adds with behind the B7-1MAb the 21st day, and blood urea nitrogen of animal (Bun) and inosine (Cr) level are starkly lower than the control group that does not add with B7-1MAb.Microscopic examination showed is used B7-1MAb and is caused the local pyknosis of animal kidney, gives B7-1MAb in the time of the 21st day, and pathology is significantly improved, and the expansion and the tubule of atrophy and the zone of tubule interstitial fibrosis obviously dwindle, and the kidney normal region reaches 66.51 ± 25.81%.On the contrary, do not add with the rarely seen normal region that has about 7.45 ± 11.77% of the control group of B7-1MAb.Medication the 21st day, the mortality ratio of experimental therapy treated animal is 11.01%, and does not add mortality ratio with the control animals of B7-1MAb up to 47.83% (P＜0.01).As seen the weight of animals changes also analogue.In addition, the expression of experimental therapy treated animal CD3, CD4 is higher than the FA group, and difference of them is remarkable.At the 14th day, the FA of immunologic hypofunction group mouse was behind the therapeutic intervention of two strain antibodies, and the expression of CD3 and CD4 obviously increases, and has recovered the expression level (referring to embodiment 2) of CD3, CD4 basically.
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: anti-people B7-1 MONOCLONAL ANTIBODIES SPECIFIC FOR
Present embodiment is described the preparation of anti-people B7-1 monoclonal antibody 4E5 of the present invention.
(1) anti-people B7-1 MONOCLONAL ANTIBODIES SPECIFIC FOR
With the transgenic cell (XG7-B7) of high expression level B7-1 molecule 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 XG7-B7 of high expression level B7-1 molecule and with the negative contrast of the XG7 that does not express the B7-1 molecule, hybridoma culture supernatant is carried out preliminary screening, Identification of Fusion Protein and Ig subgroup identification with indirect immunofluorescence.Positive colony is behind 3 limiting dilutions, and clone's positive rate reaches about 100%.Through the flow cytometry repeated screening, the result shows, the monoclonal anti physical efficiency that we prepare is well discerned the XG7-B7 molecule (referring to Fig. 1) of transgenic cell XG7-B7 cell surface, behind subclone repeatedly, obtain stably to secrete the hybridoma cell strain of specificity mouse-anti people B7-1, be named as 4E5 (CGMCC No.1166).These hybridomas after external continue to go down to posterity (40 generation), secreting specificity antibody stably still.Chromosome analysis to hybridoma cell strain 4E5 shows that chromosome number is 80-100 (referring to Fig. 2).
(3) production and the CHARACTERISTICS IDENTIFICATION of anti-people B7-1 monoclonal antibody
(a) the method manufacture order clonal antibody that adopts this chamber to set up to induce ascites in the body.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 4E5 is a mouse IgG1 subclass.
(d) 4E5 is to the identification of the B7-1 molecule of PBLs, Raji and Daudi cell surface.With people PBL separately and respectively with divide in small test tube (5 * 10 after Raji and Daudi cell mix at 1: 1
5/ pipe), adding 4E5 monoclonal antibody and sheep anti mouse fluorescence two are anti-, use flow cytometry analysis.The result shows that the positive combination rate of 4E5 and people PBLs, Raji and Daudi cell is respectively 10.2%, 92.7%, 89.2%.When Raji and Daudi respectively with people PBLs with 1: 1 mixed after, the positive combination rate of corresponding antigens is respectively 45.1% and 42.9% on 4E5 and the target cell.These results show, the B7-1 molecule (referring to Fig. 3) on the recognizing cells surface that monoclonal anti physical efficiency of the present invention is good.
(e) competitive inhibition of the antigen site of monoclonal antibody 4E5 identification.With XG7-B7 cell (5 * 10
5/ pipe), add 4E5 antibody (10 μ g/50 μ l/ pipe) with PBS washing back, 4 ℃ act on 45 minutes, and the washing back adds standard CD 80-PE and directly marks monoclonal antibody (2 μ g/50 μ l/ pipe), uses flow cytometry analysis after the effect.The positive and negative contrast is set simultaneously.Results suggest, 4E5 can block standard anti-people B7-1 monoclonal antibody and antigenic reaction fully.
Show
This monoclonal antibody is the specific antibody of B7-1, and discerns same or analogous antigen site (Fig. 4) with standard antibody.
(f) 4E5 is to the growth-inhibiting effect of malignant lymphatic oncocyte Raji and Daudi: with Raji (1 * 10
5/ ml) and Daudi cell (2 * 10
5/ ml) be incubated in 24 orifice plates, add the 4E5 monoclonal antibody and make its final concentration be respectively 1 μ g/ml, 2.5 μ g/ml, 5.0 μ g/ml, 10.0 μ g/ml and 20.0 μ g/ml.Cultivate 24,48,72,96h time point, counting cells concentration respectively.The result shows, when the concentration of 4E5 is 2.5 μ g/ml, obvious growth restraining effect (referring to Fig. 5) promptly occurs.
(e) monoclonal antibody 4E5 in and activation analysis: KG1-B7 cell (1 * 10 that will be after mitomycin is handled
5/ ml) with people PBLs (1 * 10
6/ ml equal-volume (each 100 microlitre) is mixed in 96 orifice plates, cultivates 72 hours every holes and adds 20 microlitre 5%MTT solution, continues to cultivate 4-6 hour, measures OD
570, the XG1 cell of handling with mitomycin compares simultaneously.Results suggest, monoclonal antibody 4E5 can block the conduction of the numerator mediated costimulatory signal of B7-1, thereby suppresses the effect (referring to Fig. 6) that XG1-B7 stimulates PBLs propagation.
Embodiment 2: monoclonal antibody 4E5 of the present invention is to the therapeutic action of folic acid ephrosis mouse model
(1) preparation of experimental animal model and experimental design.(diluent is NaHCO to CD1 mouse disposable celiac injection in 8 ages in week folic acid (FA) 240mg/Kg of body weight 18-22 gram
3), control group (3 of each time points) gives equivalent NaHCO
3The disposable celiac injection.1st, dead 3 animals in 3,7,14,21 natural gift other places are got its kidney and carry out immunohistochemical staining, and the detection of kidney function parameter is carried out in blood sampling, and collect spleen cell and experimentize and learn experiment.
(2) Pathologic specimen is made and immunohistochemical analysis.Get kidney, through 10% formalin fixed dehydration after 72 hours, wax embedding, section (5mm); With section preparation dewaxing, gradient alcohol dehydration; Proteinase K carries out antigen retrieval to section preparation, hydrogen peroxide sealing endogenous peroxydase, protein blocking agent sealing nonspecific binding site.Add rat anti mouse 4-1BB monoclonal antibody (1: 100), rat anti-mouse CD3 monoclonal antibody (1: 1000) and rat anti-mouse CD11a monoclonal antibody (1: 1000) then in 4 ℃ of overnight incubation, added the rabbit mouse IgG of Chinese People's Anti-Japanese Military and Political College antibody incubated at room 30 minutes, add anti-rabbit igg antibody (the anti-rabbit IgG-HRP of horseradish peroxidase-labeled, Envision Kit, 1: 100), incubated at room 30 minutes.DAB colour developing back section is redyed through Hematorylin, and the resinene mounting is observed down and the Taking Pictures recording observations in opticmicroscope.
(3) variation of the renal function of folic acid nephropathy model model mice, pathomorphism and mortality ratio.Promptly showed as blood urea nitrogen (Bun) in first day behind the injected in mice folic acid, creatinine (Cr) obviously increases; descend to some extent after the 3rd day; and show as chronic renal insufficiency (injection folic acid after 14 days) in the later stage; chronic phase,, the pathology nephridial tissue presented obvious kidney pyknosis; color is pale, surface particlesization, the visible significantly sclerosis of tangent plane; the 3rd day mouse death rate reaches 48.57%, the 14 day general mortality rate and reaches 60% (table 1).。The visible sclerosis of conventional H E dyeing and fibrosis zone (Fig. 7).And the local pyknosis of the rarely seen kidney of combined utilization 4E5 monoclonal antibody of the present invention treated animal, above-mentioned pathology is visible significantly to be improved, and expansion and the tubule of atrophy, the zone of tubule interstitial fibrosis obviously dwindle, and the kidney normal region reaches 66.51 ± 25.81% (Fig. 7).These results show that monoclonal antibody of the present invention has obvious therapeutic action to the folic acid ephrosis.
The comparison of table 1 animal pattern treatment back the 3rd and 14 day blood urea nitrogen, Cr and mortality ratio
| Index/Group | control | D3 | D14 |
| Cr(μmol/L) Bun(mmol/L) Mortality | 36.6±6.3 11.03±1.08 0 | 148.17±34.22 83.79±1.2 48.57％(17/35) | 73.79±3.93 20.02±2.25 60％(21/35) |
Annotate: D3 represents folic acid injection back the 3rd day, and D14 represents folic acid injection back the 14th day
(4) monoclonal antibody 4E5 is to the influence of mouse spleen medium size lymphocyte phenotype.Immunofluorescence label and flow cytometry result show: (folic acid ephrosis acute phase) in early days, in the folic acid ephrosis mouse spleen the lymphocytic expression of CD3, CD4 apparently higher than control group, CD3
+Lymphocyte expresses 88.6 ± 2.9%, CD4
+Lymphocyte expresses 71.03 ± 4.65%; And control group CD3
+Lymphocyte expresses 49.8 ± 2.3%, CD4
+Lymphocyte expresses 36.3 ± 2.5%; But testing the 14th day (folic acid ephrosis chronic phase), CD3
+Lymphocyte expresses 14.1 ± 3.7%, CD4
+Lymphocyte expresses 9.5 ± 3.1%, significantly is lower than normal group.And in the whole lysis, CD8
+Lymphocyte percentage does not but have significance and changes.Results suggest, treatment are intervened the back immune function of mice and are recovered normal (referring to Fig. 8) substantially.
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 monoclonal antibody 4E5 hybridoma cell strain of the present invention respectively, its deposit number is CGMCC No.1166.
Sequence table
＜110〉University Of Suzhou's biotechnology research institute
＜120〉anti-people B7-1 molecule monoclonal antibody 4E5 and application thereof
<140>
<141>
<160>1
<210>1
<211>306
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide coding sequence of the anti-people B7-1 of synthetic molecule monoclonal antibody 4E5 light chain.
<400>1
1 TTTCAGCTCC AGCTTGGTCC CAGCACCGAA CGTGAGCGGG TTACTACTCC
51 ACTGCTGGCA GTAATAAGTG GCAGCATCTT CAGCCTCCAT GCTGCTGATT
101 GTGAGAGAGT AAGAGGTCCC AGACCCACTG CCACTGAAGC GAGCAGGGAC
151 TCCAGAAGCC AGTTTGGATG TGTCATAAAT CCATCTTTTG GGGGAGGTGC
201 CTGACTTCTG CTGGTACCAG TGCATGTAAC TTACACTTGA GCTGGCACTG
251 CAGGTCATGG TGACCTTCTC CCCTGGAGAT GCAGACATGA TTGCTGGAGA
301 CTGGGT
<210>2
<211>281
<212>DNA
＜213〉artificial sequence
<220>
＜223〉nucleotide coding sequence of the anti-people B7-1 of synthetic molecule monoclonal antibody 4E5 heavy chain.
<400>2
1 AGGAGTCTGG ACCTGAGCTG GTAAAGCCTG GGGCTTCAGT GAAGATGTCC
51 TGCAAGGCTT CTGGATACAC ATTCACTAGC TATGTTATCC AATGGGTGAA
101 GCAGAAGCCT GGGCAGGGCC TTGAGTGGAT TGGAAGTATT AATCCTTACA
151 ATGATTATAC TAAGTACAAT GAGAAGTTCA AAGGCAAGGC CACACTGACT
201 TCAGACAAAT CCTCCATCAC AGCCTACATG GAGTTCAGCA GCCTGACCTC
251 TGAGGACTCT GCGCTCTATT ACTGTGCAAG A
<210>3
<211>102
＜212〉amino acid
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of inferring according to the nucleotide coding sequence of the anti-people B7-1 of synthetic molecule monoclonal antibody 4E5 light chain.
<400>3
1 TQSPAIMSAS PGEKVTMTCS ASSSVSYMHW YQQKSGTSPK RWIYDTSKLA
51 SGVPARFSGS GSGTSYSLTI SSMEAEDAAT YYCQQWSSNP LTFGAGTKLE
101 LK
<210>4
<211>93
＜212〉amino acid
＜213〉artificial sequence
<220>
＜223〉aminoacid sequence of inferring according to the nucleotide coding sequence of the anti-people B7-1 of synthetic molecule monoclonal antibody 4E5 heavy chain.
<400>4
1 ESGPELVKPG ASVKMSCKAS GYTFTSYVIQ WVKQKPGQGL EWIGSINPYN
51 DYTKYNEKFK GKATLTSDKS SITAYMEFSS LTSEDSALYY CAR
Claims (3)
1, has the light chain shown in SEQ ID NO:3 and SEQ ID NO:4 and the anti-people B7-1 monoclonal antibody 4E5 of heavy chain amino acid sequence respectively.
2, according to the antibody of claim 1, wherein said anti-people B7-1 monoclonal antibody 4E5 is that deposit number is the hybridoma generation justacrine of CGMCC No.1166.
3, according to the application in growth and breeding that suppresses the malignant lymphatic oncocyte and treatment folic acid ephrosis of the antibody of claim 1.
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| CNB2005100387301A CN100369931C (en) | 2005-04-07 | 2005-04-07 | Monoclonal antibody against human B7-1 molecule and its use |
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| CNB2005100387301A CN100369931C (en) | 2005-04-07 | 2005-04-07 | Monoclonal antibody against human B7-1 molecule and its use |
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| CN1844146A true CN1844146A (en) | 2006-10-11 |
| CN100369931C CN100369931C (en) | 2008-02-20 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102190729A (en) * | 2011-03-09 | 2011-09-21 | 苏州大学 | Anti-human CD80 bivalent dimer and application thereof |
Family Cites Families (3)
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|---|---|---|---|---|
| CN1180410A (en) * | 1995-02-10 | 1998-04-29 | 三菱化学株式会社 | Method of diagnosing immune diseases |
| US6113898A (en) * | 1995-06-07 | 2000-09-05 | Idec Pharmaceuticals Corporation | Human B7.1-specific primatized antibodies and transfectomas expressing said antibodies |
| BR9713498A (en) * | 1996-11-08 | 2000-02-29 | Idec Pharma Corp | Identification of unique binding interactions between some antibodies and human co-stimulatory antigens b7.1 and b7.2 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102190729A (en) * | 2011-03-09 | 2011-09-21 | 苏州大学 | Anti-human CD80 bivalent dimer and application thereof |
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| CN100369931C (en) | 2008-02-20 |
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