CN1312182C - Monoclone antibody for anti HLA-G and hybrid tumour cell secreting same, cancer dignosis method, diagnosis reagent box and its application - Google Patents
Monoclone antibody for anti HLA-G and hybrid tumour cell secreting same, cancer dignosis method, diagnosis reagent box and its application Download PDFInfo
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Abstract
The present invention discloses a monoclonal antibody which relates to a monoclonal antibody for resisting a human leukocyte antigen G(HLA-G), and a hybridoma cell strain for secreting the antibody. The present invention has the purpose that a novel monoclonal antibody is provided, the name of the monoclonal antibody is HGY, the monoclonal antibody is a G monoclonal antibody for resisting the human leukocyte antigen, and belongs to an IgG3 subtype, and the monoclonal antibody can generate antigen-antibody reaction with 7 kinds of HLA-G isomers. The present invention also provides a hybridoma cell strain for secreting the monoclonal antibody. Because the monoclonal antibody of the present invention has high specificity for the HLA-G, and all HLA-G isomers are combined with each other, the present invention has high sensitivity in cancer pathology detection. Simultaneously, the present invention also discloses a method for using the antibody to diagnose malignant tumuors, and an immunohistochemical reagent kit prepared from the antibody and the application thereof.
Description
Technical field
The present invention relates to a kind of monoclonal antibody, specifically, relate to the monoclonal antibody of a kind of anti-Leucocyte Antigen G (HLA-G) and secrete the hybridoma cell strain of this antibody.
The invention still further relates to the method that the HLA-G unconventionality expression in the common cancer pathology comes diagnosing cancer that detects.
The invention still further relates to the immunohistochemical methods test kit of the diagnosing malignant tumor that utilizes this Antibody Preparation, and this test kit is estimated the application in cancer metastasis and prognosis and the treatment guidance at cancer diagnosis.
Background technology
In order to prevent that body is subjected to the invasion and attack such as external cause of diseases such as virus and bacteriums, Mammals has formed the complex system that is called main histocompatibility complex (MHC) in its evolutionary process, can will distinguish mutually with allosome from body.Human mhc gene group is also referred to as human leucocyte antigen (HLA) system, is positioned on No. 6 chromosomal galianconism, is separated into three different families: I, II and III family.Classical H LAI family antigen (HLA-A ,-B and-C) be the cell surface glycoprotein of polymorphism highly, molecular weight is 40～50kD.Histocyte in the overwhelming majority all has expression.Classical H LA I family antigen with in cell, handle after polypeptide combine and bring it into cell surface then and be and pass the toxic T lymphocyte of CD8 positive cells (CTLs).The latter's (CTLs) function is to check that these cells have or not foreign protein synthetic, and will synthesize the cell destruction of foreign protein.Therefore, classical H LA I family antigen is important for eliminating following cell polar through immunity system: the cell of virus infection (infection), the cell (cancer) of experience canceration, or allotransplantation cell (organ transplantation).(about the summary of the biological procedures relevant, referring to the article of Sherman etc., AmRev.Immunol.11:385,1993 with the HLA system.)
Leucocyte Antigen G (all is called: HLA-G) be non-classical I class antigen in the present invention.In normal circumstances, its expression is confined to the chorionic trophoblast cell of placenta.And at the chorionic trophoblast cell, classical H IA I family and II family antigen is expression deletion then.(see McMaster etc., J.Immunol.154:3771,1995.)
The gene order of HLA-G and structure and classical H LA I gene height homology are made up of 8 exons, 7 introns and 13 ' terminal non-translational region.The proteic amino-acid sequence of HLA-G the B2M zone of α 1, the α 2 of heavy chain and α 3 zones and light chain and HLA-A ,-B and-homology of C albumen order surpasses 86%.The molecule that it is 37～39 KD that HLA-G albumen has been accredited as one group of molecular weight (is seen Geraghty etc., Proc.Natl.Acad.Sci.U.S.A.84:945,1987; Parham etc., Proc.Natl.Acad.Sci.U.S.A.85:4005,1988; Shimizu etc., Proc.Natl.Acad.Sci.U.S.A.85:227,1988.) opposite with classical H LA I family gene, the polymorphism of HLA-G gene is lower, only finds 4 allelotrope so far.(see Ellis etc., Am J.Reprod.Immunol.23:84,1990; Kovats etc., Science 248:220,1990.) but, HLA-G gene transcription product can be sheared and become isomer.Four kinds of isomer of expressing the HLA-G mRNA of membranin have now been observed: HLA-G1, HLA-G2, HLA-G3 and HLA-G4 and three kinds of isomer of expressing the HLA-G mRNA of soluble proteins: HLA-G5, HLA-G6 and HLA-G7.(see Ishitani etc., Proc.Natl.Acad.Sci.U.S.A.85:3947,1992; Kirszenbaum etc., Proc.Natl.Acad.Sci.U.S.A.91:4029,1994; Fujii etc., J.Immunol.153:5516.) these mRNA encoded protein have total length or lack the zone one or two, and then cause the molecular weight of protein product and function that very big variation is all arranged.
Half gene of fetus comes to father, thereby is a kind of half allogeneic thing to mother.Mother's immunity system may produce fetus to be repelled.Big quantity research proves that HLA-G can suppress the maternal immunity reaction at the chorionic trophoblast cell expressing of placenta.In other words, the effect of HLA-G is to keep the immunotolerance of double allogeneic fetus of parent by suppressing the maternal immunity reaction.(about the summary of HLA-G function in gestation, can referring to Rouas-Freiss etc., J.Biol.Regul.Homeost.Agents.14:93,2000.)
In oncobiology, the downward modulation of HLAI family genetic expression is a common phenomena.(relevant summary is seen Garrido etc., Immunology Today 18:89,1997.) because HLA I family molecule is brought into play central role in the lymphocytic activation of Cytotoxic T, the downward modulation of HLA I family genetic expression just becomes one of tumor escape mechanism.But and since the NK cell can hydrolysis HLA I family developed by molecule reduce or the cell of disappearance, and make the cancer cells of HLA I family down regulation of gene expression may be subjected to the attack of NK cell.(see Lanier LL and Philips JH, Immunol.Today 17:86-9,1996.) but because HLA-G has the function that suppresses the NK cell, if, just may be that it provide another kind of mechanism of escaping the host immune supervision because of the unconventionality expression of HLA-G appears in tumour cell.(see Dietl etc., Gynecol.Obstet.Invest.33:197,1992; Chumbley etc., Cell Immunol.155:312,1993.) therefore, whether understanding HLA-G has participated in tumour immunity escape mechanism just has great importance.Since the nineties, various countries are to the big quantity research of HLA-G expression having carried out of various cancers type and various JEG-3.Most research groups confirm that being expressed on the transcriptional level of HLA-G is positive.Yet the result of study of on protein level HLA-G being expressed is not quite identical.In these researchs, measure the proteic method of HLA-G and comprise qualitative and quantitative multiple technologies, as flow cytometry, immunity seal stain, immunocytochemistry and enzyme linked immunological (ELISA) etc.Part adopts the research of anti-HLA-G monoclonal antibody (87G, 01G, 223G and MEMG/9) to draw negative findings.(see Amiot etc., Human Immunol.59:524,1998; Real etc., Int.J.Cancer 81:512,1999; Frumento etc., Tissue Antigens 56:30,2000; Hurks etc., Invest.Ophthalmol.Vis.Sci.42:3081,2001.) mainly being to adopt in other research of the anti-HLA-G monoclonal antibody of 4H84, have 21%～51% result then positive.(see Mizuno etc., Br.J.Haematol.111:280,2000; Urosevic etc., Am.J.Pathol.159:817,2001; Urosevic etc., Blood 99:609,2002; Lefebvre etc., J.Pathol.196:266,2002; Yang etc., Chinese J.Histochem.Cytochem.10:70,2001.) according to document and investigation, up to now, the anti-HLA-G monoclonal antibody that all HLA-G protein determinations to cancerous tissue or JEG-3 all adopt synthetic polypeptide or bacterium synthetic reorganization HLA-G protein immunization mouse to produce is carried out.All antibody all combines with heavy chain, main α 1 district or α 2 districts at the HLA-G molecule.
As mentioned above, in most cancerous tissues, all detect the expression of the mRNA of HLA-G.Also further prove the isomer of transcribing that to express different HLA-G in dissimilar malignant tumours recently.(see Ritau etc., Transplant Proc.33:2360,2001.) and the HLA-G protein translation after the isomer of various glycoprotein (see McMaster etc., J.Immunol.160:5922,1998.)。These find prompting, all occur the proteic expression of HLA-G in the cancer of most types, just occur with different isomer in the front and back of translation.So for the HLA-G protein expression that detects cancer, detection reagent is as antibody, the result that must while just can obtain with multiple isomerization reaction.And these by synthetic polypeptide and or the antibody that produces of bacterium synthetic reorganization HLA-G protein immunization mouse be unsafty, bacterium synthetic reorganization HLA-G albumen can not glycosylation because synthetic polypeptide has only the amino acid planar sequence, the antibody that produces can not be simultaneously and the isomer generation immune response of multiple natural form (near the form under the physiological condition), thereby cause in certain cancers detects and be positive the result and the result that is negative in other cancer detection, so accuracy rate is not high in the tumour pathology detection.Therefore, be necessary to improve the monoclonal antibody of anti-HLA-G, make it that all isomer are all had higher detection specificity and sensitivity.The most important thing is that HLA-G does not express, and expresses in nogestational normal adult tissue, have the specificity of the malignant tumour of height in multiple malignant tumor tissue, suitable to cancer diagnosis, the new tumor markers that prognosis and treatment are instructed.Studies show that the expression of HLA-G in cancer is relevant with the disease progression stage with cancer cell tissue, its application prospect is very optimistic.(see Ugurel etc., Cancer 92:369,2001, Urosevic etc., Am.J.Patho1.159:817,2001; Urosevic etc., Blood99:609,2002.)
Summary of the invention
At the defective of existing anti-HLA-G monoclonal antibody, the purpose of this invention is to provide a kind of new monoclonal antibody, called after HGY as the cancer detection mark.This monoclonal antibody is anti-Leucocyte Antigen G monoclonal antibody, and promptly anti-HLA-G monoclonal antibody belongs to the IgG3 hypotype.Further, this monoclonal antibody can with 7 kinds of HLA-G isomer generation antigen antibody reactions.
Monoclonal antibody of the present invention can produce by hybridoma cell strain of the present invention, therefore can from the nutrient solution of cultivating hybridoma cell strain of the present invention, obtain, or with the hybridoma cell strain cell seeding to laboratory animal abdominal cavity generation ascites and obtain.Yet, produce monoclonal antibody method of the present invention and be not particularly limited, as what obtain by gene engineering method can antigen antibody reaction take place with 7 kinds of HLA-G, belong to the antibody of IgG3 hypotype, also belong within the scope of the present invention.
Another object of the present invention provides a kind of hybridoma cell strain, and it can produce the anti-HLA-G monoclonal antibody that belongs to the IgG3 hypotype.Further, it can produce the monoclonal antibody with 7 kinds of HLA-G isomer generation antigen antibody reactions.
Hybridoma cell strain of the present invention can produce with cell-fusion techniques well-known to those skilled in the art, therefore, with (natural HLA-G) under the approximate physiological condition as animals such as antigen immune mouse, splenocyte or lymphocyte and myeloma cell's fusion with this animal produce hybridoma cell strain.And therefrom filter out and produce the IgG3 hypotype, can with the hybridoma cell strain of the monoclonal antibody of 7 kinds of HLA-G isomer generation antigen antibody reactions, thereby obtain hybridoma cell strain of the present invention.
Described hybridoma cell strain, preferably the deposit number by China's typical culture collection center preservation is the hybridoma cell strain clone of CCTCC NO.C200416.
The monoclonal antibody that hybridoma cell strain of the present invention produces has constituted one aspect of the present invention.
The immunologic detection method of a kind of malignant tumour of the present invention has constituted another aspect of the present invention, and this method uses at least a above-mentioned monoclonal antibody to detect.
Further, this method uses any one above-mentioned monoclonal antibody to detect.This method can be used for diagnosing malignant tumor; Also can be used to estimate malignant progression, transfer and prognosis, can also instruct treatment for cancer.
Immunologic detection method preferably of the present invention is to use immune imprinting to carry out, and also can carry out with elisa technique.
Another aspect of the present invention is, contains the test kit of at least a above-mentioned monoclonal antibody, and preferred test kit is to contain any one above-mentioned monoclonal antibody.Test kit of the present invention can be used for diagnosing malignant tumor, also can be used for malignant progression, transfer and prognosis, and treatment is instructed.Test kit of the present invention preferably adopts immune imprinting or elisa technique to detect.
Because there is not cross reaction in the HLA I family antigen of monoclonal antibody of the present invention and other types, therefore monoclonal antibody of the present invention has the specificity of height for HLA-G.And monoclonal antibody of the present invention combines with all HLA-G isomer, though dissimilar cancers may give expression to different HLA-G isomer, but to the various expression products of dissimilar cancers, monoclonal antibody of the present invention can both be detected, and therefore has very high susceptibility.Simultaneously, monoclonal antibody of the present invention is stable, can be by method known in those skilled in the art, Monoclonal Antibody of the present invention is become various forms of test kits, with convenient clinical extensive use, be the diagnosis of cancer, a kind of reliable valuable approach that provides is provided for evaluation that development is shifted and treatment.
Description of drawings
Fig. 1. proteic SDS-PAGE of purifying natural HLA-G and immunity seal stain.
With the HLA-G albumen (A) of antibody affinity chromatography technical point in first phase gestation placenta tissue lysate.The albumen SDS-PAGE of purifying, coomassie brilliant blue staining analysis (B) also is transferred to pvdf membrane, prints stain reaction (C) with anti--HLA-G monoclonal antibody 4H84.
Fig. 2. the comparison of antibody HGY of the present invention and anti--HLA-G monoclonal antibody 4H84.
Spend the night bag by elisa plate with 4 ℃ in the HLA-G albumen of purifying natural.Washing plate uses anti--HLA-G monoclonal antibody HGY of various dose and 4H84 room temperature to be incubated 1 hour with the sealing back.Wash plate 4 times, be incubated 1 hour with sheep anti-mouse antibody-HRP mixture room temperature.With TMB is the substrate colour developing, the reaction of 1M HCl color development stopping.On microplate reader, read the optical density(OD) of 450/630nm wavelength.When antibody concentration was identical, HGY and the proteic bonding force of purifying HLA-G were higher than 4H84.
Fig. 3. antibody competition ELISA.With purifying natural HLA-G bag by elisa plate, biotin labeled anti--HLA-G monoclonal antibody 4H84 with the unmarked 4H84 of various dose and of the present invention anti--HLA-G monoclonal antibody HGY room temperature insulation 1 hour again.Be incubated 1 hour with antibiotin-HRP binding substances room temperature after washing plate.Color reaction is carried out with tmb substrate liquid, 1M HCl termination reaction.The competition per-cent of unmarked antibody 4H84 and HGY is calculated with comparing (100% combination) in the hole that does not add antibody.HGY does not compete with biotin labeled 4H84, shows that the binding site of HGY is different from 4H84.
Fig. 4. immunity seal stain: the character of anti--HLA-G monoclonal antibody HGY and 4H84 is relatively.With the villioma cell strain JEG-3 cell of SDS-PAGE separation and Culture and the placenta tissue lysate (PL) of miscarriage in pregnant three months, be transferred to pvdf membrane, print the stain reaction with HGY and 4H84 respectively.For the JEG-3 cell lysate, the seal of two kinds of antibody stain be corresponding to the HLA-G1 isomer, molecular weight is single band of 38KD.From the placenta lysate, 4H84 can combine and the solubility isomerization reaction with the film of HLA-G, and HGY can discern all HLA-G isomer proteins: HLA-G1 (37～39KD), G2 (～30KD), G3 (～20KD), G4 (～29KD) and G5 (～34KD).
Fig. 5. the immune blot analysis of anti--HLA-G monoclonal antibody HGY pair cell strain.With breast carcinoma cell strain (MB231 and MB468), adenocarcinoma ovaries cell strain (SK-OV-3), the prostate cancer cell strain of shifting (LnCap) and the culturing cell cracking of villioma cell strain (JEG-3), SDS-PAGE separates the back and carries out immune blot analysis with anti--HLA-G monoclonal antibody.The band that a 38KD is arranged in JEG-3 and LnCap cell strain corresponding to HLA-G1.At MB231, in MB468 and the SK-OV-3 cell strain, seal stain band is greater than 38KD, and is corresponding with the HLA-G molecule of high glycosylation.In MB468, a band corresponding to the soluble form of HLA-G is arranged, and, have other isomer to occur in SK-OV-3 and the LnCap cell strain at MB231.
Fig. 6. the cell strain immune blot analysis.Earlier carry out immunoprecipitation, carry out immune blot analysis with same antibody then with the culturing cell lysate of the affine sepharose 4B of HGY antibody after to homogenate.1=MB231,2=people's epithelium of cervix uteri JEG-3 (CaSki), 3=SK-OV-3,4=LnCap, 5=people's embryonal carcinoma strain (CaTes-1B), 6=JEG-3,7=LCL221 (lymphoma cell strain).In JEG-3, identify discovery expression in the negative LCL221 cell strain of HLA-G expression corresponding to the list band of HLA-G1.The isomer that HLA-G is different in other cell strain is expressed.
Fig. 7 A primary breast cancer immune blot analysis.Earlier carry out immunoprecipitation, carry out immune blot analysis with same antibody then with the sample of the affine sepharose 4B of HGY antibody after to homogenate.Sample 1-4 and 7 is a mammary cancer, and 5 is mammary gland fibroma, and 6 is normal galactophore tissue.Mammary cancer 1-3 has the 34KD band corresponding to the solvable isomer of HLA-G, a little less than the expression.Mammary cancer 4 has 37KD and two strong bands of 39KD, and is corresponding with HLA-G1, and mammary cancer 7 has the higher district's band of a part amount, corresponding with the isomer of the HLA-G1 that contains different glycosylation.Mammary gland fibroma and healthy tissues do not see Table and reach.
The immune blot analysis of Fig. 7 B primary breast cancer.The homogenate sample is directly used in immune blot analysis.Anti--the HLA-G monoclonal antibody is HGY.Sample 2 is the primary breast cancer of strong expression HLA-G1 and other isomer.Sample 5 is for only expressing the mammary cancer of HLA-G1.Mammary gland fibroma (3 and 4 row) and normal galactophore tissue's (1 row) do not have and express.6 is the placenta tissue lysate of miscarriage in pregnant three months, and HLA-G1 and HLA-G5 isomer all have expression.
Fig. 8. the histochemical stain section of primary breast cancer
Fig. 9. transitional cell carcinoma of bladder and normal bladder tissue HLA-G histochemical stain section contrast
Figure 10. large bowel cancer and normal bowel gland tissue HLA-G histochemical stain section contrast
Figure 11. lung squamous cancer and the HLA-G of normal lung tissue histochemical stain section contrast
Figure 12. breast ductal cancer and the HLA-G of normal breast tracheal tissue histochemical stain section contrast
Figure 13. carcinoma of endometrium and normal uterine tissue HLA-G histochemical stain section contrast
The embodiment of invention
Below the invention will be further described by specific embodiment, and the special applications example of cited antibody of the present invention in cancer detection and diagnosis helps to understand characteristics of the present invention better, is not limitation of the present invention.
[embodiment 1]
MONOCLONAL ANTIBODIES SPECIFIC FOR of the present invention
Basis of the present invention is that the natural HLA-G protein of purifying wherein contains whole HLA-G and transcribes isomer and glycoprotein isomer the placenta tissue of employing miscarriage in pregnant three months, prepares the monoclonal antibody of anti-HLA-G.(see Ishitani etc., Proc.Natl.Acad.Sci.U.S.A.85:3947,1992; Kirszenbaun etc., Acad.Sci.U.S.A.91:4209,1994; Fujii etc., J.Immunol.153:5516,1994; McMaster etc., J.Immunol.160:5922,1998; Blastchitze etc., Early Pregnancy 5:67,2001.)
1, the proteic purifying of HLA-G
● collect the postoperative embryonic tissue of miscarriage patient in three months immediately ,-80 ℃ are frozen standby.
● during purifying HLA-G albumen, frozen tissue is thawed on ice and be cut into small pieces.With 50mM Tris-HC1 damping fluid (pH 7.4) suspended tissue, carry out tissue homogenate with Brinkmaan Polytron homogenizer in 4 ℃.
● homogenate is through 39,000g, 4 ℃ centrifugal 10 minutes, obtain precipitation, with above-mentioned centrifugal and suspended pattern washing precipitation 3 to 4 times.
● with the lysis buffer of the 10 times of throw out volumes precipitation that suspends again, put into the jolting device and slowly mix 4 ℃ and spend the night.
● 39,000g, 4 ℃ centrifugal 30 minutes and collect supernatant.
● adopt two successive antibody affinity columns that the HLA-G albumen in the supernatant liquor is carried out purifying: with the 4H84 of purifying (anti--HLA-G) and BMM1 (resisting-β2Wei Qiudanbai) monoclonal antibody directly be coupled at respectively on the ball of activatory carrier emblem and be prepared into antibody affinity chromatography, with the supernatant liquor upper prop, wash and wash-out then.The purity of the HLA-G of purifying is checked by electrophoresis and Western blot.10%PAGE and ECL Western blot the results are shown in accompanying drawing 1.The result shows that through double antibody affinity chromatography, the proteic purity of HLA-G that comprises the solubility isomer is more than 90%, and the HLA-G albumen of inactivation also can be used as immunogen and uses.
2, the natural HLA-G MONOCLONAL ANTIBODIES SPECIFIC FOR of antivenom purification
● carry out peritonaeum under immune to 3 week female BALB/c mouse in age (Charles River) with the HLA-G albumen of above-mentioned purifying by standard method.(see Harlow and Lane, Antibodies, aLaboratory Manual, Cold Spring Harbor, NY, pp139,1988.) promptly, the HLA-G albumen of purifying is diluted to 500ug/ml with physiological saline, first, with complete Freund's adjuvant with 1: 1 mixed, injecting immune under the peritonaeum then.Afterwards, 1: 1 proportioning concentration immune animal of the HLA-G albumen of too many or too much for use full freund's adjuvant and purifying, 3～4 weeks of interval, continuous three times.Stop immunity January, 1: 1 proportioning concentration of the HLA-G albumen of too many or too much for use at last full freund's adjuvant and purifying carries out enhancing immunity under 1 peritonaeum.After the enhancing immunity 10 days, with antibody absorption ELISA method detect in the mice serum with purifying after the titre of natural HLA-G protein-specific binding antibody.After the anti-HLA-G antibody titers of mice serum reach a certain height, select titre the higher person, proceed by the following method;
● put to death mouse, its splenocyte and SP2 myeloma cell strain are merged hybridization with techniques well known.With limiting dilution assay and antibody capture ELISA method the hybridoma cell strain cell is carried out screening and cloning then.Filtered out a hybridoma cell strain.
● preparation antibody, available
A, hybridoma cell strain culture medium culturing cell strain
Optimize substratum 410ml (Gibco), TANGGO 5ml (Sigma), ITS (Sigma) 0.5ml and FBS (Sigma) 75ml.Cultivate 7～10 days until all hybridoma cell strain necrocytosiss.400xg removed cell debris in centrifugal 10 minutes, and supernatant merges.Adopt Protein G affinity column (Sigma) to carry out purifying antibody by catalogue.It is frozen standby that antibody purified is through the molecular weight that dams that 4 ℃ of 3000rpm of filter membrane (Millipore) of 10,000 were concentrated-80 ℃ of backs in centrifugal 30 minutes.
B or produce ascites to the BALB/c mouse abdominal cavity with the hybridoma cell strain cell seeding
Then is to be expelled to the BALB/c mouse abdominal cavity behind the HGY hybridoma cell strain cell washing of cultivation with the hybridoma cell strain cell seeding to the ascitogenous method in BALB/c mouse abdominal cavity.Collect ascites after 7-10 days ,-80 ℃ frozen standby.
[embodiment 2]
The characteristic of monoclonal antibody of the present invention
Monoclonal antibody with above preparation is carried out following experiment, proves:
1, mouse antibodies somatotype reagent kit (Amersh) is measured, and the monoclonal antibody hypotype that cell strain produces is IgG3.
2, it has the specificity of height to HLA-G
Antibody capture ELISA measures and shows that there is not cross reaction in the HLA I family antigen of antibody of the present invention and other types, (sees Gosling and Basso, Immunoassay, Butterworth-Hainemann, London) confirmed that antibody of the present invention has the specificity of height for HLA-G.(seeing Table 1)
The cross reaction of table 1HGY antibody
| Compound | Cross reaction % |
| HLA-G HLA-A2 HLA-B4 HLA-C mixing |
100 0.01 0.01 0.001 0.005 |
3, itself and existing anti-HLA-G monoclonal antibody relatively have higher antigen affinity
With antibody capture ELISA method HLA-G albumen affinity has been compared to two kinds of antibody of antibody of the present invention and 4H84.(see Gosling and Basso, Immunoassay, Butterworth-Hainemann, London) result shows that the affinity of antibody of the present invention is higher than 4H84 (Fig. 2).
4, the antigen-binding site of monoclonal antibody of the present invention closes on glycosylated zone in α 1 district
In order to determine the antigen-binding site of monoclonal antibody of the present invention, designed the competitive immunometric assay with 4H84, (referring to Harlow and Lane, Antibodies, aLaboratory Manual, Cold Spring Harbor, NY, pp567,1988.) monoclonal antibody 4H84 is by preparing with one section synthetic polypeptide immune mouse corresponding to the 61st～83 amino acids in α 1 district on the HLA-G molecule.Fig. 3 shows that two kinds of antibody combine with the different antigenic determinants of HLA-G molecule.Because antibody capable of the present invention is discerned the isomer protein of all HLA-G, comprise the isomer (seeing Fig. 4 and Fig. 5) of the glycoprotein after the translation.And the isomer protein of all HLA-G all has α 1 district, and the glycosylation that HLA-G albumen also generates the glycoprotein after the translation also (is seen Bouteiller PL﹠amp on 86 aspartic acids in α 1 district; MalletV (1997) JReprodFertil:2:7-13.McMaster et al (1998) J.Immunol:160:5922.), so the antigen-binding site of antibody of the present invention should close on glycosylated zone in α 1 district.
5, monoclonal antibody of the present invention can combine with all the HLA-G isomer in the same tissue preparation thing
Monoclonal antibody of the present invention and 4H84 steep a single district band going up 38kD with JEG-3 cell hydrolyzate Western seal and combine (Fig. 4), confirm that further antibody of the present invention is the same with 4Hg4, has specificity to HLA-G.Only for the placenta hydrolysate, 4H84 only discerns soluble form and the film combining site (HLA-G1 and HLA-G5) of HLA-G, and monoclonal antibody of the present invention then combines with all HLA-G isomer in the same tissue preparation thing.
6, use monoclonal antibody of the present invention can detect dissimilar cancers
Fig. 5 demonstrates immunoblotting (Westem blot) collection of illustrative plates of antibody of the present invention and part JEG-3.Villioma cell strain JEG-3 and prostate cancer cell strain LnCap have the immunoblotting district band of a 38kD.And with the reaction of the extract of breast carcinoma cell strain MDA-MB231, MDA-MB468 and ovarian cancer cell strain SK-OV-3, antibody of the present invention produces district's band of many HLA-G isomer.These data presentation, dissimilar cancers may give expression to different HLA-G isomer, and to the various expression products of dissimilar cancers, antibody of the present invention can both be detected.
[embodiment 3]
The immunity of HLA-G seal stain detects in cell strain and the mammary cancer sample
Introduction
The downward modulation that classical human leucocyte antigen (HLA) (HLA) I family expresses is the common phenomenon in the oncobiology.The change that this classical H LAI family expresses may make tumour cell escape the lymphocytic immunity monitoring of host toxicity.Because the cell that NK cell energy hydrolysis classical H LA I family lacks, above-mentioned change can make the tumour cell of the downward modulation of human leucocyte antigen (HLA) (HLA) I family expression be subjected to the attack of NK cell.Garrido etc., Immunology Today 18:89,1997.Have been found that the antigen as a kind of non-classical I family, one of immunologic function of HLA-G is exactly to suppress the NK cell activity.If therefore tumour cell is expressed HLA-G, just can make it exempt from the elimination of NK.In order to verify this hypothesis, we adopt at the immunity seal stain method of the specific monoclonal antibody (HGY) of HLA-G 5 kinds of human cancer cell strains and primary breast cancerous tissue are investigated.
Material and method
Human breast carcinoma and normal galactophore tissue are from surgical patient.Put immediately after the tissue sample collection-80C deposits until mensuration.Human breast cancer cell strain (MDA-MB-231), people's epithelium of cervix uteri JEG-3 (CASK1), metastatic human prostate cancer strain (LNCAP, FGC), people's adenocarcinoma ovaries strain (SK-OV-3) and people's embryonal carcinoma strain (CATES-1B) are available from ATCC (Roekville, Maryland, USA).According to the ATCC specification sheets with different substratum culturing cell in culture dish.
Primary breast cancerous tissue and normal galactophore tissue after the homogenate, and JEG-3 centrifugation 1ml lysate cracking.Lysate contains: 0.5%NP40,0.15M NaC1,5mM EDTA, 50mM Tris-HC1, pH7.2 and 1.0mMPMSF.Lysate is mixed with the affine sepharose 4B of HGY antibody, and 4 ℃ are spent the night and carry out immunoprecipitation and handle.Use immunoprecipitation lavation buffer solution (0.1M PBS, 0.05%NP-40 and 0.02%NaN3) washing precipitation 5 times (seeing Sigma :) afterwards with the specification sheets of antibody coupling activatory sepharose 4B, 1966.With the sample sex change, 12%SDS-PAGE separates then, and room temperature was transferred on the PVDA film in 3 hours.With 5% milk closing membrane,, be incubated 1 hour with sheep anti-mouse igg-HRP mixture (1: 2000) again with insulation under the HGY antibody room temperature 1 hour.With the fluorescence immunoassay imprinting analyze (AmershemArlington, Heights, IL).The hydrolyzate of the primary breast cancerous tissue after the part homogenate is directly analyzed with the fluorescence immunoassay imprinting without the heavy ingot of immunity.
The result
JEG-3 is the villioma cell strain, expresses HLA-G and classical I family antigen (HLA-A, B and C) molecule simultaneously.LCL-211 is a lymphoma cell strain, only expresses Ia family antigen and does not express HLA-G.In this research, JEG-3 cell hydrolyzate is used as positive control, and the LCL-221 hydrolyzate is then as negative control.(Kovalts etc., Science 248:220,1990.) in JEG-3, detecting an albumen list band that is equivalent to HLA-G1 (38kDa), 211 of LCL do not detect.All detect the protein of molecular weight corresponding to HLA-G1 and other isomer in other all cells strain hydrolyzate, its kind is complied with different cell strains and different (Fig. 6).For breast cancer tissue's hydrolyzate, a part has been carried out immunoprecipitation processing (Fig. 7 A) with the affine pearl of HGY antibody coupling earlier, and another part then directly separates (Fig. 7 B) with SDS-PAGE.Normal galactophore tissue is as negative control, and the placenta tissue of gestation miscarriage in three months is as positive control.Directly bursting to test with indirect immunity seal does not all detect HLA-G albumen in normal galactophore tissue and mammary gland fibroma, but then identifies the isomer protein of various HLA-G in different mammary gland cancer patients.
Discuss
Adopt the above data of anti-HLA-G monoclonal antibody HGY of the present invention to show that JEG-3 and primary breast cancer are all expressed HLA-G albumen.The result shows that except JEG-3 villioma cell strain and fetal tissues tissue, the cancer of some type also will be expressed HLA-G.HLA-G especially makes it escape the hydrolysis of NK cell in the escape mechanism that cancer cells unconventionality expression prompting HLA-G may participate in tumour cell.Illustrate this hypothesis fully and remain more deep research.
[embodiment 4]
The immunohistochemical analysis of mammary cancer and malignant galactophore disease HLA-G
Adopt immunity seal stain method to we have found that the expression of HLA-G is arranged in the primary breast cancerous tissue.The purpose of this research is exactly to adopt the HLA-G protein expression in the histochemical method check breast tumor and understand the relation that may exist between expression and the histology result.
Material and method
Human breast carcinoma and normal galactophore tissue are from surgical patient.Tissue sample is fixed with 4% Paraformaldehyde 96, paraffin embedding.22 parts of breast tumor that cancerate are studied.By differentiation degree tumour is divided into 3 groups: I level (n=1), II level (n=8) and III level (n=13).Other has normal galactophore tissue (n=3) and mammary gland fibroma tissue (n=2).
During immunohistochemical analysis,, carry out the direct immunization peroxidase stain by standard operation to the wax embedding block section.(Bullock GR，Petrusz P：Techniques in Immunocytochemistry，Academic Press，New Tork，1982。) section with the HGY monoclonal antibody in 4 ℃ of incubated overnight.After this, endogenous peroxidase activity and non-differential protein binding site sealed 30 minutes with the PBS solution of 1% hydrogen peroxide treatment and 10% normal goats serum respectively.Rinse with PBS then and wash section, with goat anti-mouse igg-HRP (Sigma) room temperature insulation of dilution in 1: 2,000 1 hour.PBS develops a film, and makes binding antibody colour developing 5 minutes with diaminobenzidine (Sigma).In addition, use HLA-G albumen and 4 ℃ of incubated overnight of HGY antibody of the purifying of 100ug/ml, purpose is to allow HLA-G albumen adsorb anti-HLA-G monoclonal antibody in advance, and contrasts as the specificity Quality Control.Specificity Quality Control contrast also comprises with PBS or normal mouse IgG (Sigma company product) replaces antibody.
The result
In I level cancerous tissue a little less than the immune response of HLA-G, on the contrary in II level tumour the HLA-G immunoreactivity between medium and strong reaction.Have 6 examples to show medium HLA-G immunoreactivity in the 13 routine III level samples to intensity, the reactivity of all the other 7 examples for do not have or a little less than.Organize in normal galactophore tissue and mammary gland fibroma and then not detect HLA-G albumen.The positive dyeing example of Fig. 8.
Discuss
Above result shows that 68% mammary cancer has the proteic expression of HLA-G in various degree.Between HLA-G expression and the cancer cells differentiation positively related trend is arranged.
[embodiment 5]
The HLA-G immunohistochemical methods of cancer common type is surveyed the chamber
Before find that the expression of HLA-G is arranged in part primary breast cancer patient, and have relevant trend between between expression and histological type.The purpose of this research is whether the cancer of investigating other type also has the unconventionality expression of HLA-G and pathological diagnosis and the prognosis whether this expression can be used for cancer.
Material and method
1007 specimens paraffin embedding slices that comprise various common cancers.First antibody is anti--HLA-G monoclonal antibody HGY, and second antibody is Envision
TM(available from DAKO).As positive control, various corresponding healthy tissuess are as negative control with the paraffin section of the fetal tissues tissue of taking from abortion operation.
The result
Painted section is divided into 4 classes: the nothing expression (, the cancer cells of no positive staining), the low expression (+, positive staining cancer cells＜25%), the moderate expression (++, the positive staining cancer cells is between 25% to 50%), strongly expressed (+++, positive staining cancer cells＞50%).Be listed in the table 2 according to this sorting result.Observed all cancers of this research all have the proteic expression of HLA-G.Positive staining per-cent is between 42～100%.Then do not detect the proteic expression of HLA-G (Fig. 9) in corresponding healthy tissues.Simultaneously, the result also proves HLA-G positive expression and mammary cancer, lung cancer, and esophagus cancer, cancer of the stomach, straight colorectal carcinoma, uterus carcinoma, the cell transfer of cancers such as bladder cancer has significant correlation with invading to moisten.Analytical results sees Table 3.Several cancers of other of this research fail to carry out statistical analysis because of sample is too for a short time.
Table 2:HLA-G is in the expression of the malignant tumour of various common types
| HLA-G expresses | ||||||
| The example number | Negative | + | ++ | +++ | Positive % | |
| Colon-rectum cancer | 200 | 70 | 66 | 39 | 25 | 64.7 |
| Cancer of the stomach | 101 | 37 | 32 | 19 | 13 | 62.7 |
| Esophagus cancer | 61 | 10 | 19 | 16 | 16 | 83.6 |
| The liver and gall cancer | 6 | 2 | 2 | 1 | 1 | 66.7 |
| Lung cancer | 42 | 14 | 9 | 10 | 9 | 66.6 |
| Mammary cancer | 178 | 59 | 55 | 35 | 29 | 66.8 |
| Uterus carcinoma | 70 | 23 | 18 | 24 | 5 | 67.1 |
| Cervical cancer | 41 | 24 | 9 | 5 | 3 | 41.5 |
| |
30 | 16 | 7 | 5 | 2 | 46.7 |
| Prostate cancer | 31 | 14 | 8 | 5 | 4 | 54.8 |
| Carcinoma of testis | 6 | 3 | 2 | 1 | 0 | 50.0 |
| |
10 | 5 | 1 | 1 | 3 | 50.0 |
| |
36 | 13 | 11 | 8 | 4 | 63.9 |
| |
5 | 1 | 2 | 1 | 1 | 80.0 |
| Laryngocarcinoma | 18 | 1 | 9 | 3 | 5 | 94.4 |
| Innocent tumour | 172 | 172 | 0 | 0 | 0 | 0 |
The clinical detection result's of table 3.HLA-G immunohistochemical methods clinical meaning
| HLA-G expresses positive | Clinical meaning | |
| Mammary cancer | The high recall rate of metastasis of cancer 54.4% cancer (78.2% is higher by 25.8% than medical image method recall rate) | Prognosis mala accuracy rate of diagnosis height |
| Esophagus cancer | Metastasis of cancer/high the recall rate of |
Prognosis mala accuracy rate of diagnosis height |
| Cancer of the stomach | Metastasis of cancer 71.9% | Prognosis mala |
| Rectum-colorectal carcinoma | Metastasis of cancer/cell alienation/high the recall rate of infiltration 67% cancer (64.7% is higher by 18% than medical image method recall rate) | Prognosis mala accuracy rate of diagnosis height |
| Lung cancer | Metastasis of cancer/cell alienation 78.5% high pathological staging | Prognosis mala |
| Uterus carcinoma | Metastasis of cancer/cell alienation 75% high pathological staging | Prognosis mala |
| Bladder cancer | High pathological staging | Prognosis mala |
| Laryngocarcinoma | The high recall rate of cancer (94.4% is higher by 55% than medical image method recall rate) | The accuracy rate of diagnosis height |
Discuss
This research is used of the present invention resisting-HLA-G monoclonal antibody HGY and is found first, and the unconventionality expression of HLA-G is a phenomenon that extensively exists in the oncobiology.The also strong prompting of the result of this research is measured the new tumor markers that HLA-G can become common all kinds of cancer diagnosis and prognosis with the immunohistochemical methods method.Participated in Immune escape of tumor because of HLA-G in the expression of cancer again, and the expression of HLA-G is subjected to the adjusting of various immune molecules; Can strengthen the expression of HLA-G as Interferon, rabbit.Cancer patient to the HLA-G high expression level, may run counter to desire (referring to Wagner SN with interferon alpha-treatment, Rebmann V, Wi llers CP, Grosse-Wilde H, Goos M._Expression analysisof classic and non-classic HLA molecules before interferon alfa-2b treatment ofmelanoma.Lancet.2000 Jul 15; 356 (9225): 220-1. and Ugurel S, Rebmann V, FerroneS, Tilgen W, Grosse-Wilde H, Reinhold U.Soluble human leukocyte antigen--G serumlevel is elevated in melanoma patients and is further increased byinterferon-alpha immunotherapy.Cancer.2001 Jul 15; 92 (2): 369-76.), therefore, measuring HLA-G might become the index that instructs the treatment of tumour personalized biological.
Invention is summed up
The present invention is with to produce anti--HLA-G monoclonal antibody with the natural HLA-G protein immunization mouse of purifying relevant.By the inventive method, HLA-G antibody compares and antigen HLA-A2 with the selective binding of HLA-G, and it is strong more than 1000 times that HLA-B4, HLA-C or blended white corpuscle prepare the combination of liquid.No matter how (placenta or tumour) antibody of the present invention can detect proteic all isomer of HLA-G in the source of HLA-G molecule.The combining site of antibody of the present invention and HLA-G is different from other antibody commonly used, as 4H84.Adopt antibody of the present invention, in most of cancers, detected the unconventionality expression of HLA-G.To the cancer of common type, adopt immunohistochemical methods of the present invention and other method of immunity, its measurement result has the using value of clinical cancer diagnosis and prognosis.
[embodiment 6]
The preparation of cancer diagnosing kit
By above result of study, we have developed HLA-G immunohistochemical methods test kit, because anti-HLA-G monoclonal antibody of the present invention has high degree of specificity and susceptibility, and physico-chemical property is stable, and can prepare easily according to standard method becomes cancer diagnosing kit.
Below be concrete composition with a kind of diagnostic kit of the anti-HLA-G Monoclonal Antibody of the present invention:
The anti-HLA-G antibody of HGY 18ul
The anti-HLA-G antibody diluent of HGY 2.5ml
Rabbit anti-mouse antibody-horseradish peroxidase complex 2.5ml
3% hydrogen peroxide 10ml
20X phosphoric acid buffer 50ml
5X citrate buffer solution 50ml
Chromogen substrate solution (DAB) 0.1ml
Chromogen substrate buffer solution 2.5ml
1 of positive control sheet
[calibrating principle]
This test kit is to use the very high anti-HLA-G monoclonal antibody (the anti-HLA-G antibody of HGY) of a specific specificity to be combined in the HLA-G antigen in the tumor tissue section, adopts rabbit anti-mouse antibody-horseradish peroxidase complex in conjunction with anti-HLA-G antibody then.Horseradish peroxidase catalysis chromogen substrate (DAB) colour developing detects the expression that has or not HLA-G in the histopathologic slide.
Claims (10)
1, a kind of hybridoma cell strain is characterized in that: this hybridoma is that the deposit number by China's typical culture collection center preservation is the hybridoma cell line of CCTCC NO.C200416.
2, the monoclonal antibody of the described hybridoma cell strain generation of a kind of claim 1.
3, monoclonal antibody according to claim 2 is characterized in that: it belongs to the IgG3 hypotype.
4, a kind of test kit is characterized in that: contain at least a as claim 2,3 described monoclonal antibodies.
5, test kit according to claim 4 is characterized in that: contain just like claim 2 or 3 described any one monoclonal antibodies.
6, according to claim 4 or 5 described test kits, it is characterized in that: be used for diagnosing malignant tumor.
7, according to claim 4 or 5 described test kits, it is characterized in that: be used to estimate malignant progression, transfer and prognosis.
8, according to claim 4 or 5 described test kits, it is characterized in that: be used to instruct treatment for cancer.
9, according to any described test kit of claim 4-8, it is characterized in that: described test kit adopts elisa technique.
10, according to any described test kit of claim 4-8, it is characterized in that: described test kit adopts immunity seal stain technology.
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| CN101358964B (en) * | 2007-07-31 | 2012-06-20 | 叶尚勉 | Cancer diagnosing kit containing HLA-G monoclonal antibodies and use thereof |
| US20120177671A1 (en) * | 2009-06-18 | 2012-07-12 | Hla-G Technologies | Hla-g alpha 1 multimers and pharmaceutical uses thereof |
| CN103185803A (en) | 2011-12-31 | 2013-07-03 | 深圳迈瑞生物医疗电子股份有限公司 | Method and kit for identifying sensitivity of antibody and clone cell strain |
| EP2730588A1 (en) | 2012-11-12 | 2014-05-14 | Intelectys | Antibodies and fragments thereof raised against the alpha-3 domain of HLA-G protein, methods and means for their preparation, and uses thereof |
| CN103756967B (en) * | 2013-12-31 | 2018-09-21 | 卢英 | Application of the monoclonal antibody coupling immunomagnetic beads of anti-HLA-G in tumour cell sorting |
| CN107533051B (en) * | 2015-03-27 | 2020-11-13 | 南加利福尼亚大学 | HLA-G as a novel target for CAR T cell immunotherapy |
| CA3025681A1 (en) * | 2016-06-03 | 2017-12-07 | Invectys | Anti hla-g specific antibodies |
| TW201829463A (en) | 2016-11-18 | 2018-08-16 | 瑞士商赫孚孟拉羅股份公司 | Anti-hla-g antibodies and use thereof |
| KR20210098956A (en) | 2018-09-27 | 2021-08-11 | 티조나 테라퓨틱스 | Anti-HLA-G Antibodies, Compositions Containing Anti-HLA-G Antibodies, and Methods of Using Anti-HLA-G Antibodies |
| CN113045656B (en) * | 2020-07-27 | 2022-03-08 | 台州恩泽医疗中心(集团) | Monoclonal antibodies against HLA-G isoform molecules HLA-G5 and HLA-G6 and uses thereof |
| CN112794908A (en) * | 2020-12-31 | 2021-05-14 | 杭州冰湖生物科技有限公司 | Preparation and analysis method of anti-HLA-G antibody |
| CN114605543B (en) * | 2021-12-17 | 2023-11-07 | 台州恩泽医疗中心(集团) | Monoclonal antibody of HLA-G isomer molecule HLA-G5 and HLA-G6 and application thereof |
| CN115819583A (en) * | 2022-03-31 | 2023-03-21 | 台州恩泽医疗中心(集团) | Monoclonal antibody for resisting HLA-G molecule and application thereof |
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Non-Patent Citations (3)
| Title |
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| HLA-G class I gene expression in normal and malignanthematopoietic cells Amiot.Laurence et al,Human.Immunology,Vol.59 No.8 1998 * |
| HLA-G class I gene expression in normal and malignanthematopoietic cells Amiot.Laurence et al,Human.Immunology,Vol.59 No.8 1998;Specific activation of the non-classical class Ihistocompatibility HLA-G antigen and expression of the ILT2inhibitory receptor in human breast cancer Lefebvre.Sophie et al,Journal.of.Pathology,Vol.196 No.3 2002 * |
| Specific activation of the non-classical class Ihistocompatibility HLA-G antigen and expression of the ILT2inhibitory receptor in human breast cancer Lefebvre.Sophie et al,Journal.of.Pathology,Vol.196 No.3 2002 * |
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