CN1460022A - Extended type 1 chain glycosphingolipids as tumer-associated antigens - Google Patents

Extended type 1 chain glycosphingolipids as tumer-associated antigens Download PDF

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CN1460022A
CN1460022A CN00819788A CN00819788A CN1460022A CN 1460022 A CN1460022 A CN 1460022A CN 00819788 A CN00819788 A CN 00819788A CN 00819788 A CN00819788 A CN 00819788A CN 1460022 A CN1460022 A CN 1460022A
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CN1291723C (en
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S·莱弗里
S－I·哈克莫利
M·斯特劳德
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Abstract

A variety of compounds are provided which are useful as immunogens and as tumor markers. The present invention discloses methods relating to the detection of cancer. Extended forms of the lacto-series type 1 chain are shown to the present in various cancer tissues. The present invention also provides a cell line and the monoclonal antibody produced therefrom. Such as antibody has a number of uses, in including in diagnostic or therapeutic methods.

Description

Stretching, extension 1 type chain glycosyl sphingolipid as tumor associated antigen
Technical field
The present invention relates generally to the Novel Human tumor associated antigen.The invention particularly relates to and stretch 1 type chain glycosyl sphingolipid and uses thereof, for example, as immunogen and tumor marker.
Background of invention
Although dropped into huge financial resources and human resources, tumor remains one of major causes of death.Current oncotherapy can only be cured the patient who develops into malignant tumor about 50%.In most of malignant tumor, transfer is the main cause of death.
Transfer is the formation of secondary tumor colony in distant site.In most of human malignant lesions, when the treatment primary tumor, be difficult to be found to such an extent as to remote transfer is too little usually.And the spreading property of transitivity colony is made a start, and takes place before the metastatic disease clinical symptoms is obvious usually.The anatomical location of the size of secondary tumor, life-span, its distribution and heterogeneous composition the thereof all are to hinder excision and restriction cancer therapy drug to be delivered to the factor of the concentration at transitivity colony place.Therefore, improve the effectiveness of current oncotherapy, need before tumor cell is propagated from former position, detect malignant tumor.
The universals that unusual glycosylation is most of tumor types have been observed.The most sugars antigen that is used for people's diagnosing tumor has poly lactose amine (polylactosamine) structure, and promptly it comprises Gal β 1 → 3/4GlcNAc.Poly lactose amine is divided into two classes according to its poly lactose amine unit structure usually.Poly lactose amine with Gal β 1 → 3GlcNAc structure is called 1 type chain, and the poly lactose amine with Gal β 1 → 4GlcNAc structure is 2 type chains.The most common tumor related antigen of finding in most of cancers has breast system (lacto-series) 2 type chain structures, usually by sialylated and/or fucosylation.Be rich in 1 type chain antigen in normal cell and the tissue, it also is a cancer associated antigen.For example: 2 → 3 sialylated Le aAntigen (the CA19-9 antigen that N19-9 antibody is determined) is exactly 1 relevant type chain antigen of a kind of cancer.Yet, be subjected to false positive rate height and/or the high obstruction of false negative rate based on the cancerous diagnose method that detects these known antigens.
In view of the difficulty that current cancerous diagnose method exists, this area need be improved compositions and method.The present invention has satisfied this needs, and other related advantages further is provided.
Summary of the invention
In brief, the invention provides isolated compound reaches by detecting the method that these chemical compounds screen tumor.On the one hand, the invention provides a kind of isolated compound, be with or without fucosido and/or sialic acid residues, have general formula:
Galβ1→3GlcNAcβ1→3Galβ1→3GlcNAcβ1→(3Galβ1→3GlcNAcβ1→) n3Galβ1→4Glcβ1→1Cer
Wherein n is 0 or 1 or above integer, have two fucosidos and/or one or more sialic acid residues at least, Gal represents galactose, Glc represents glucose, GlcNAc represents N-acetyl-glucosamine, Cer represents ceramide, wherein said at least two fucosyl residues are connected with the GlcNAc residue with α 1 → 4 key, and/or be connected with terminal Gal residue with α 1 → 2 key, described one or more sialic acid residues is connected with the Gal residue of α 2 → 3 keys with end, and/or is connected with α 2 → 6 keys and one or more inferior terminal GlcNAc residues.
On the other hand, the invention provides above-claimed cpd with following general formula:
Wherein Fuc represents fucose, and NeuAc represents the N-n acetylneuraminic acid n.
On the other hand, the invention provides the chemical compound of describing for the first time with following general formula:
Wherein Fuc represents fucose, and NeuAc represents the N-n acetylneuraminic acid n.
On the other hand, the invention provides the chemical compound of describing for the first time with following general formula:
Wherein Fuc represents fucose, and NeuAc represents the N-n acetylneuraminic acid n.
In another embodiment, the present invention also provides the isolated compound with following general formula:
On the one hand, the present invention also provides and comprises the epi-position isolated compound with following general formula again:
Figure A0081978800063
In others, any chemical compound all can be used as the immunogen of preparation polyclone or monoclonal antibody among the present invention.
The present invention also provides the tumor method for screening in addition.These methods comprise that (a) is from homoiothermic animal separating bio specimen; (b) existence of chemical compound or amount in the working sample.
The accompanying drawing summary
The sialic acid Le of Fig. 1 for stretching a 1H-NMR (nuclear magnetic resonance, NMR) spectrum, chemical shift comprise that (galactose and mark are made F for sugared I (Glc), II (Gal), III (GlcNAc), IV (Gal), V (GlcNAc) and VI from 4.20ppm to 5.60ppm IIIThe fucose and the mark that are connected with III GlcNAc make F IVThe fucose that is connected with V GlcNAc).In this spectrum, all Fv and F IIIHeterogeneous sub-spectrum Fy-1 and FIII-1 labelling.In addition, the C5 proton spectrum F of fucose III-5 and many couplings labelling of Fv-5 labelling.The spectrum that is labeled as Cis is meant the two keys of Cis of sphingol, represents sphingol spectrum with R-5 and R-4.
Detailed Description Of The Invention
The present invention relates generally to detect relevant compound and method with cancer. More specifically, originally Summary of the invention shows that the breast that has the stretching, extension form in the cancerous tissue is 1 type chain.
As mentioned above, known normal cell and the tissue in be rich in lactose amine I type chain (Gal β 1 → 3GlcNAc). Although detected and had that to stretch 2 type chains (be Gal β 1 → 4GlcNAc core The repetition of structure) poly lactose amine antigen, stretch the poly-of 1 type chain but also detect to have Lactose amine antigen. Therefore it is generally acknowledged that breast is that 1 type chain does not exist the stretching, extension form.
As disclosed in the present invention, the breast of stretching, extension form is that I type chain (is Gal β 1 → 3GlcNAc β 1 → [3Gal β 1 → 3GlcNAc β →]n3Gal β 1 → R, be with or without sialic acid and / or fucosyl residues) be present in the tumor tissues. By with the glycolipid component (from tumour cell The middle extraction) cross preparative column and thin-layer chromatography, being separated to two representational stretching, extension form breasts is 1 The type chain.
From being GM1 (monosialyl ganglioside) component of Colo205, colon adenocarcinoma cell is purified into the homogeneous sialylated-Lewis (sLe that moves slowlya) active glycosyl sphingolipid (GSL). This compound is by HPLC (high performance liquid chroma-tography) Be able to purifying with the preparation HPTLC (High Performance Thin Layer Chromatography) in two kinds of different solvents, use A-sLeaMonoclonal antibody (MAb) NKH-1 dyes by force through the TLC immunostaining. The weak acid hydrolysis (1% acetic acid, 100 ℃ 1 hour) produces and dimerization LeaStandard GSL moves altogether, and α-dimerization LeaMAb ST-421 can make the fast migration of its strong dyeing component. By1Its structure of H-NMR spectra is sialic acid-dimerization Lea(seeing following structure).
Figure A0081978800071
Except above-mentioned special glycolipid, Lea-Le aAnd Leb-Le aEpi-position with may be additional to contain The stretching, extension 1 type chain of [3Gal β 1 → 3GlcNAc β 1 →] n unit exist. In addition, Lea-Le aAnd Leb-Le aEpi-position may be entrained by glycoprotein such as the clear glycoprotein of high-molecular weight mucins sample of blood.
Consider instruction provided herein, other breast that stretches form be 1 type chain compound can from Separate in biological raw material such as the tumor tissues, or behind Structural Identification chemical (and/or enzymatic) Synthetic, this is apparent to those of ordinary skills. Briefly, with lipid or egg The structure of the sugar of white matter combination can based on degraded, mass spectral analysis, comprise the direct probe of electron collision (electron-impact direct-probe) (EI) and fast atom bombardment (FAB), methylation analysis (method as described below and, such as people such as Nudelman, J. Biol.Chem.261:5487-5495,1986) measure. Degradation analysis can be by changing And/or enzymatic are finished, as degrading by glycosidase. The glycosylation sequence that degradation analysis shows can By methylation analysis (Hakomori for example, J.Biochem. 55:205-208, 1964), again with the sugared chemical ionization mass spectrum of Hypermethylation (permethylated) (such as people such as Stellner, Arch.Biochem.Biophys.155:464-472, 1974; The people such as Levery, Meth.Enzymol.138:13-25,1987) determine. Perhaps, unite these methods of use, to the complete glycan after Hypermethylation glycan or the suitable degraded Carry out EI mass spectral analysis (for example, the people such as kannagi, J.Biol.Chem.259:8444-8451,1984; The people such as Nudelman, J.Biol.Chem.263:13942-13951, 1988). The homogeney of glycosylation sequence can be illustrated according to number of chemical and physical criterion, has comprised Proton N MR and the FAB mass spectral analysis of the whole or glycan that methylates. In case determined sugared structure, Can be non-by the synthetic sugared or derivatives thereof of the known technology of those of ordinary skill in the art or its The carbohydrate function equivalent.
Compound of the present invention can be used as the immunity of preparation polyclone and monoclonal antibody (MAb) Former. Polyclonal antibody can prepare by standard method. For example: in brief, polyclonal antibody can lead to Cross with the compound immune animal among the present invention, gather its serum then and prepare. Usually preferred Before gathering serum, after first immunisation, carry out again the booster immunization of one or many. MAb can adopt the method for Kohler and Milstein usually(Nature 256：495-497， 1975； Eur.J.Immunol.6:511-519,1976) preparation. In brief, take from through The lymph node of the animal of compound immunity and/or spleen among the present invention merge product with the myeloma cell Give birth to hybrid cell line (" hybridoma " or " clone's "). The single immune ball of each hybridoma secretion Albumen, and as the myeloma cell, have the potential of unlimited division. Replace hybridoma system The other method of standby monoclonal antibody is to set up the expression library (example of MAb with bacteriophage and bacterium Such as people such as Sastry, Proc.Natl.Acad.Sci.USA 86:5728,1989; The people such as Huse, Science 246:1275,1989). Can use the ordinary skill people The known several different methods of member is screened has the specific antibody of purpose.
In order to strengthen their immunogenicity, may want the compound among the present invention and carrier The associating use. Suitable carrier comprises bacterium, keyhole limpet hemocyanin, the thyroid gland ball egg of deactivation In vain, bovine serum albumin(BSA) and their derivative. For example: the GSL Le of all or parta-Le aOr Leb-Le aSaccharide residue can unite with carrier. Compound among the present invention can pass through several different methods With the carrier associating, comprise absorption and covalently bound.
Compound is to use Le as immunogenic representative instance among use the present inventionb/Le aAntigen immune Mouse. In brief, the Le that from the Colo205 cell, separatesb/Le aWith acid-treated bright Buddhist nun's soda The associating of salmonella (Salmonella minnesotae) suspension is given through tail vein injection BALB/c mouse, interval duplicate injection in 10 days three times. After the last injection, gather immune little The splenocyte of mouse merges with the myeloma cell. Setting up hybridoma IMH2 (shows immunogenic And be deposited in ATCC (American Type Culture selective reaction), Collection, 10801 University Boulevard, Manassas, Virginia 20110 USA), be ATCC NO.HB 11026. This hybridoma produces has the IgG3 isotype MAb 1MH2.
1 type chain antigen such as Le for detection of the form of stretching, extensiona-Le aAnd/or Leb-Le aThe method of antigen Can be used to screen tumour. GSL Le for exampleb-Le aWith GSL Lea-Le a, can use respectively Mab 1MH2 from the neutral glycolipid component of kinds of tumors sample preparation and Mab ST-421 (according to people such as Watanabe,Jpn.J.Cancer Res(Gann)76:43-52,1985 build Vertical) carry out the TLC immunostaining and detect. These samples comprise from colon cancer, breast cancer, The group of Hodgkin's disease (Hodgkin ' s disease), carcinoma of gallbladder and embryonal rhabdomyosarcoma Knit. For example, GSL Lea-Le aAt the glycolipid from spleen, liver, kidney, placenta and lung normal structure Can't detect in the component. Consider instruction provided herein, can adopt many in the inventive method The stretching, extension 1 type antigen that kind method detects Tumor-assaciated (comprises utilizing and specially stretches for Tumor-assaciated Open up the binding partners of 1 type antigen; GSL Le for examplea-Le aAnd Leb-Le a), this is for this The field those of ordinary skill is apparent. For example, can prepare as stated above Lea- Le aOr Leb-Le aThe specific antibody of epi-position makes antibody and biological specimen connect under certain condition Touch (for example incubation), and continue for some time, be enough to make immune complex to form after, measure The existence of immune complex.
Can detect between above-mentioned antigen and this antigen-specific antibodies by multiple known method and form The existence of immune complex, for example radiommunoassay (RIA) and Enzyme-linked Immunosorbent Assay are surveyed Fixed (ELISA). Suitable immunoassays comprise people's (United States Patent (USP)s such as David 4,376,110) double-monoclonal antibody sandwich method of immunity; Monoclonal-polyclonal antibody Sandwich test (people such as Wide, in Kirkham and Hunter, eds., Radioimmunoassay Methods E.and S.Livingstone, Edinburgh, 1970); The people's such as Gordon (United States Patent (USP) 4,452,901) " western trace " Method; The immuno-precipitation of mark aglucon (people such as Brown, J.Biol.Chem. 255:4980-4983,1980); Such as Raines and Ross (J.Biol.Chem. 257:5154-5160,1982) enzyme linked immunosorbent assay (ELISA) of describing; ImmunohistochemistryMethods Methods, bag Draw together use fluorescent dye (people such as Brooks, Clin.Exp.Immunol.39:477, 1980); And active the inhibition (people such as Bowen-Pope, Proc.Natl.Acad.Sci. USA 81:2396-2400,1984). Except above-mentioned immunoassays, also can adopt many other Method of immunity comprises United States Patent (USP): 3,817,827; 3,850,752; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; With 4,098,876 is described.
According to testing goal, antibody can mark also mark not. During mark, antibody can not Be used for agglutination test. In addition, unmarked antibody also can with can with the mark of immune complex reaction Molecule is united use, perhaps with can with directly for the labelled antibody of the antibody response of compound (SA) (such as immunoglobulin (Ig) specific antibody) united use. Perhaps, can be straight Connect labelled antibody. Reporter group during mark can comprise radio isotope, fluorogen, enzyme, Shiner (luminescers) or dye granule. These and other mark all is the many institutes in this area Known, and be described in for example at following United States Patent (USP): 3,766,162; 3,791,932; 3,817,837; 3,996,345; With 4,233, in 402.
In the preferred embodiment that immune complex detects, reporter group and antibody knot Close. The step that detects immune complex comprises removes any unconjugated antibody basically, then The existence of examining report group. Binding antibody does not refer to the antibody that those are not combined with antigen.
In a further preferred embodiment, reporter group is with the second antibody combination of energy conjugated antigen specific antibody.The detection step of immune complex comprises that (a) removes any unconjugated antibody (promptly not with the bonded antibody of antigen) basically, (b) adds second antibody, (C) removes any unconjugated second antibody basically, then the existence of (d) examining report group.For example, when antigen-specific antibodies during from mice, second antibody is anti--murine antibody.
In the 3rd preferred embodiment that immune complex detects, reporter group is with combining with the bonded molecule of immune complex.Detect step and comprise that (a) adds this molecule, (b) remove any not binding molecule basically, then the existence of (C) examining report group.Can be a-protein with bonded minute sub-instance of immune complex.
The immunoassay of employing labelled antigen can replace the second antibody of common traget antibody, labelling or the use of molecule labelling and the immune complex reaction.This type of mensuration (" indirectly " or " in the competitiveness "), the antigen in the sample will with this antibody of antigenic competition of labelling.
Those of ordinary skills obviously can adopt multiple detection immune complex method in the present invention.The reporter group that is applicable to arbitrary method comprises, radiosiotope, fluorogen, enzyme, shiner and dye granule.In addition, should recognize and to use the antigenic specific binding ligand of stretching, extension 1 type (rather than antibody) that tumor is relevant among the present invention to detect this antigen, and can detect the complex that forms between this binding partner and antigen by the detection method of similar above-mentioned immune complex.
It is never to be construed as limiting for explanation that following examples are provided.
Embodiment
Embodiment 1 usefulness is carried out HPTLC immunostaining and immunoassay from the monoclonal antibody NCC-ST-421 of the neutral glycolipid of tumor and normal structure preparation
A. monoclonal antibody and immunoassay
Mab ST-421 sets up (people such as watanabe, Jpn.J.CancerRes. (Gann) 76:43-52,1985) as previously mentioned.(the MAb MNH-1 of Gal β 1 → 3GlcNAc β 1 → R) is in inventor's prepared in laboratory to discern 1 type chain N-acetyl lactosamine; (the MAb 1B2 of Gal β 1 → 4GlcNAc β 1 → R) is by former described foundation people such as (, J.Biol.Chem.256:10967-10972,1981) Young to discern 2 type chain N-acetyl lactosamines.Anti--Le aMAb available from Chembiomed Ltd (Edmonton, Alberta, Canada).Anti--Le yMAb AH6 set up by describing in the past (people such as Abe, J.Biol.Chem.258: 11793-11797,1983), do not show and Le bAny cross reactivity.Anti--Le b(Edmonton, Alberta Canada), show the cross reactivity with 1 type H chain to MAb available from Chembiomed Ltd..Another anti--Le b(Lund Sweden), shows and Le MAb available from Monocarb b, 1 type H chain and Le yCross reactivity.By at first by improve one's methods (people such as Kannagi, J.Biol.Chem., 257:4438-4442,1982 of people such as Magnani (people such as Magnani, Anal.Biochem.109:399-402,1980) institute's describing method; People such as Kannagi, J.Biol.Chem.257:14865-14874,1982), adopt Whatman HPTLC plate (HP-KF) to carry out the HPTLC immunostaining.
B. glycolipid preparation
Used glycolipid sample is isolating or synthetic by enzyme catalysis.VI 3NeuAcnLc 6, IV 3NeuAcIII 4FucLc 4, VI 2FucnLc 6And IV 2FucLc 4From people's Placenta Hominis, liver adenocarcinoma, people O type erythrocyte and Intestinum Sus domestica, separate respectively, after IHW (55: 25: 20) extracting, carry out again that Folch distributes, the HPTLC of DEAE-Sephadex chromatography and Iatrobeads 6RS 8010 posts (people such as Magnani, J.Biol.Chem.257:14365-14369,1982; People such as Watanabe, J.Biol.Chem.254:8223-8229,1979; People such as Hakomori, J.Immunol.98:31-38,1967; People such as Stellner, Biochemistry 12:656-661,1973).NLc6 and III 4FucLc 4By 100 ℃ of heated sample in 1% acetic acid 1 hour and make VI respectively 3NeuAcnLc 6And IV 3NeuAcIII 4FucLc 4The asialoglycoprotein preparation.IV 3GlcNAcnLc 4, IV 3Gal β 1 → 3-GlcNAcnLc 4, IV 3Gal β 1 → 3[Fucl → 4] GlcNAcnLc 4And IV 3Gal β 1 → 3[Fucl → 4] GlcNAcIII 3FucnLc 4(Le a-Le x) all by the synthetic preparation of enzymatic.IV 3Gal β 1 → 3GlcNAcIII 3FucnLc 4By handling IV with fucosidase 3Gal β 1 → 3[Fucl → 4] GlcNAcIII 3FucnLc 4, promptly (0.2M citrate buffer Mo) (pH 4.5) prepared in 37 ℃ of incubations 100 μ g glycolipids in 2 hours for Sigma Chemical Co., St.Louis with the Ren Bovis seu Bubali a-L-fucosidase that contains 0.05 unit.IV 2III 4Fuc 2Lc 4, V 3III 3Fuc 2NLc 6And VI 2V 3Fuc 2NLc 6The preparation of employing biosynthesis, method is with IV 2FucLc 4, nLc 6And VI 2FucnLc 6Be substrate, use a1 → 3/4 fucosyltransferase (difference) to make its a1 → 3 fucosylated from Colo205.By long-pending 50 mMHepes buffer (pH 7.0), 0.5M sucrose, 1mM EDTA and the 1%Triton CF-54 of diploid in Potter Elvehjem homogenizer in 4 ℃ of homogenate, dissolving obtains a1 → 3/4 fucosyltransferase from the Colo2o5 cell.Homogenate is with 100, centrifugal 1 hour of 000xg, and supernatant is concentrated into the cell initial volume after dialysis.Enzyme preparation is standby in-80 ℃ of preservations.
Enzymatic a1 → 3/4 fucosylation cumulative volume be 1ml contain 1mg glycosyl sphingolipid (GSL) substrate, 1mg deoxidation taurocholic acid, 10 μ mol MnCl 2, carry out in 25 μ mol Hepes buffer (pH7.0), 5 μ molCDP-choline, 6 μ mol GDP-fucosees and the 500 μ l enzyme preparation reaction mixture.This reactant mixture is in 37 ℃ of incubations 16 hours, and lyophilizing then is with the ultrasonic extracting of isopropyl alcohol-hexane-water (IHW) (55: 25: 20), centrifugal.Supernatant is through Iatrobeads 6RS-8010 post HPLC, uses from 55: 40: 5 to 55: 25: 20 IHW gradient elution 200 minutes.Collect component by 2 milliliters,, merge the collecting pipe that contains end-product according to the migration of the HPTLC in chloroform-methanol-water of 50: 40: 10.With orcin spray colour developing GSL band.
Various GSL with definite structure be characterised in that can with the reactivity of specificity MAb, i.e. Le b/ Le aAntigen and anti--Le bMAb reaction and not with anti--Le yMAb AH6 reaction; Le y/ Le xWith AH6 reaction and not with anti--Le bOr anti--Le xThe MAb reaction; Le a/ Le aAnd Le a/ Le xWith anti--Le aMAb and MAb ST-421 reaction.
The C.TLC immunostaining
Show from the TLC immunostaining of the neutral glycolipid component of kinds of tumors sample preparation and to have ratio-Le aThe migration of-active ceramide pentasaccharides slow and with anti--Le aThe These positive bands of MAb cross reaction.This band can be dyeed by force by MAb NCC-ST-421, and sees in most of tumors of checking so far.Example is from colon cancer, breast carcinoma, Hokdkin disease, carcinoma of gallbladder and embryonal rhabdomyosarcoma.
Embodiment 2, dimerization Le aAntigen and Le b-Le aAntigenic separation
A. the preparation of tumor tissues
Colo205 cell (ATCC) (people such as semple, Cancer Res.38:1345-1355,1978) is cultivated in RPMI 1640 culture medium that contain 10% hyclone.Collecting cell went down to posterity once in per approximately 7 days.The cell trypsinization of collecting, centrifugal, wash twice with phosphate buffered saline (PBS) (pH 7.4), count with cell counter.Give 6 no breasts (naked) mice every injection 4 * 10 through subcutaneous 6Cell.2 week back tumor resections (each about 2ml) and be stored in-80 ℃ standby.
B. from the Colo205 tumor, separate and move Le slowly a -active component (dimerization Le a )
About 200g tumor is used isopropyl alcohol-hexane-water (IHW) (55: 25: 20) extracting, carries out Folch distribution, DEAE-sephadex chromatography again and carry out HPLC on Iatrobeads 6RS-8010 post.With from 55: 40: 5 to 55: 25: 20 IHW gradient elution upper strata phase neutral component 200 minutes.Collect components by 2 milliliters, move according to the HPTLC in chloroform-methanol-water (50: 40: 10) and merge component.Slowly move Le a-active component (showing) by the TLC immunostaining further by Merck HPTLC plate (Silica Gel 60, Merck, Darmstadt, Germany) the preparation TLC on comes purification, and the character that is used for structure is identified.
C.Le b -Le a Antigenic separation
Migration is just in time at dimerization Le aThe method purification that These positive bands under the antigen (according to embodiment 1 MAbNCC-ST-421 immunostaining) is partly described with top B.
Embodiment 3 dimerization Le aAntigen and Le b-Le aAntigenic character is identified
A. enzymatic degradation
(Mo) the dimerization Le of 1mg is carried out in hydrolysis for SigmaChemical Co., St.Louis to use a-fucosidase (Ren Bovis seu Bubali), 0.5 unit beta galactosidase (Semen Canavaliae) and the β of the 0.5 unit-N-acetylglucosamine enzyme (cattle epididymis) of 0.5 unit successively aEnzymatic degradation.Institute responds and all carries out in 0.2M sodium citrate (pH 4.5), 37 ℃ of shaking water baths 4 hours.With every kind of catabolite of preparation property HPTLC purification.
The vitro cytotoxicity of B.IMH2
1. cell line
Colo205 (ATCC) obtains from U.S. typical case culture center (American Type CultnreCollection) at first, cultivates in the RPMI-1640 culture medium that is supplemented with 10% hyclone, mM L-glutaminate, 100IU/ml penicillin and 10 μ g/ml streptomycins.(San Diego CA) gives people's epidermoid carcinoma A431 cell line (people such as Macleod, J.Cell.Physiol.127:175-182,1986) for GildredCancer Facility, UCSD School of Medicine by Dr.Carol MacLeod at first.Le on this expression of cell lines EGF-R ELISA a, Le b, Le x, Le yAnd ALe b(people such as Gooi., Biosci.Reports 5:83-94,1985).The A431 cell is being supplemented with Dulbecco ' s modified Eagle ' smedium (the Irvine Scientific of 5% hyclone, 1mM glutamine, 110mg/l Sodium Pyruvate, 100IU/ml penicillin and l0 μ g/ml streptomycin, Santa Ana, CA) the middle cultivation.Inoculating cell (about 5 * 10 5/ ml), cover with the back and collect, to handle with EDTA earlier, reuse contains Ca 2+And Mg 2+PBS washing.The target cell that these cells are measured as vitro cytotoxicity, or be used for subcutaneous vaccination 5 * 106 cells and measure oncogenicity in nude mice.Human erythroleukemia K562 cell (people such as Lozzio; Blood 45:321-334,1975) as the active contrast of mensuration system medium-sized lymphocyte NKT (NK).
2. the cytotoxicity (CDC) of the cytotoxicity of antibody-dependence (ADCC) and complement-dependence
Being used for human peripheral leucocytes (HPBL) (as the effector lymphocyte) that ADCC measures obtains from leukocyte (buffy coat) component of health aspiration donor blood.In brief, use the Ficoll-Hypaque gradient solution, with 2000rpm centrifugal 20 minutes, separating monocytic cell (people such as Mishell, in Mishell, B.B and Shiigi, S.M. (eds.), Selected Methods in Cellular Immunology, pp.3-27, W.H.Freeman ﹠amp; Co., San Francisco, CA, 1980).Mouse boosting cell and mouse peritoneum macrophage (effector lymphocyte) are pressed people's description preparations in the past such as Mishell, but make the following modification.Target cell (5 * 10 6) by with 100 μ l's 51Cr carried out labelling in 90 minutes in the 37C incubation.Behind washing (3x) and the incubation (37 ℃, 1 hour), cell (1 * 10 6/ ml) be suspended among the RPMI-1640 that is supplemented with 25mM HEPES buffer and 3% bovine serum albumin.Microtitration plate (Corning, NY) middle mix of effector lymphocyte's suspension of the IMH2 of the labeled cell of 20 μ l, 100 μ l or ST-421 and 100 μ l at the bottom of the U type.(Sigma, St.Louis MO) make negative control with non-specific mouse immuning ball protein.Behind the incubation 4 hours,, get 100 μ l supernatant in every hole, measure radioactivity with gamma counter with centrifugal titer plate (500xg, 2 minutes) in the centrifuge that hangs dull and stereotyped holder (hanging plate-holder) is housed.Every experimental group is measured three times.Calculate specificity cracking percent according to formula ([A-B] * 100)/C, the cpm of A=cracking experimental cell wherein, B=is the cpm of cracking target cell not; The cpm of the whole target cells of C=.Spontaneous release is no more than 15% of maximum releasable labelling radioactivity.
For CDC, 51Cr-discharges to measure to use with the ADCC similar methods and carries out, except the human serum substitution effects cell that adds 100 μ l dilution is originated as complement.Serum is 56 ℃ of deactivations 30 minutes, with comparing.Specificity cracking percent is by method calculating as mentioned above.
Because being accredited as to express, the Colo205 cell stretches 1 type chain Le a/ Le aAnd Le b/ Le aAntigen, it reacts by force with MAb ST-421 and MAb IMH2 respectively, so estimated the cellulotoxic effect of IMH2 to Colo205, and with the comparing of ST-421.Two monoclonal antibodies all show significant ADCC to the Colo205 cell and kill and wound.This kill and wound with effector lymphocyte and target cell ratio (E: T) and the concentration of monoclonal antibody relevant.Cellulotoxic effect is at E: the T ratio is 100: 1-200: 1, and maximum when the monoclonal anti bulk concentration is 35-70 μ g/ml.Regardless of E: T ratio or monoclonal anti bulk concentration, control mice IgG and other non-specific monoclonal antibodies all do not show cellulotoxic effect.When implementing same cell toxicity test with mouse boosting cell, corresponding value only is 7% and 17% cracking (E: T ratio 200: 1, monoclonal anti bulk concentration 30 μ g/ml).Monoclonal antibody shows weak cellulotoxic effect (table) to the A431 cell.The maximum cracking of having compared the IMH2-dependence of Colo205, A431 and K562 cell in the table.High cracking value (for example, IMH2 and ST-421 are respectively 65% and 94% cracking to Colo205) is only remarkable when HPBL exists; When using mouse boosting cell, cracking value is much smaller, as former during with ST-421 viewed people such as (, Cancer Res.51:2199-2204,1991) Watanabe.Relevant approx with complement concentration by IMH2 with used monoclonal anti bulk concentration with the CDC of ST-421 mediation.
Table MAb ST-421 and IMH2 are to the cellulotoxic effect of the MAb-dependence of Colo205, A431 and K562 cell
Cracking percent target cell antibody/reactivity bEffector lymphocyte+MAb+ effector lymphocyte+MAb-effector lymphocyte-MAb+Colo205 ST-421+ 94.5 2.7 0.8
IMH2+ 65.0 2.7 0.7A431 ST-421± 14.4 10.9 1.1
IMH2± 7.6 9.2 0.6K562 c ST-421- 48.2 36.2 0.5
IMH2- 44.8 36.2 0.3
A. at E: (effector lymphocyte: target cell) ratio is the cracking percent of 100: 1 o'clock IMH2 (35 μ g/ml) and ST-421 (ascites of 100 times of dilutions) to T.
B. measure with fluidic cell wood ,+, the positive; ±, the weak positive;-feminine gender.
C. also can be observed the high cytotoxin effect to the K562 cell under the situation that does not have MAb to exist, this is considered to reflect natural killer cell activity.
C. intravital tumor suppression
Colo205 and A431 cells in vitro as experiment in the body are cultivated, and wash secondary with culture medium, meet the requirements of cell density again with PBS.Cell (5 * 10 6/ 100 μ l) subcutaneous injection is gone into 5-to the 7-athymism BALB/c mouse back in age in week, begins the monoclonal antibody intraperitoneal administration after the injection immediately.The IMH2 of purification (1.1mg/ml) or in ascites the ST-421 of corresponding IgG concentration (1.1-1.2ng/ml), with 0.2ml/ animal dosage lumbar injection once injected for 2 weeks every day.Measure the width and the length of tumor weekly for 3 times by same observer.Tumor weight is estimated as (width 2* length)/2.The ascites albumen that control animals received is produced in BALB/c mouse by mouse myeloma cell line NS1.Test every group with 7 mices at every turn, test and repeat twice.Meansigma methods with the tumor weight of twice experiment is drawn.
MAb IMH2 and ST-421 significantly suppress the growth of Colo205 tumor in nude mice.On the contrary, two kinds of MAb antibody all shows the minimum depression effect to the A431 tumor growth.Therefore, discerning antigenic high expressed, to seem the susceptibility that the tumor growth in vivo of antagonist-dependence suppresses be essential.
The reactivity of D.IMH2 and kinds of tumors and normal structure
Kinds of tumors and normal adjacent tissue are taken from formalin fixed and are used paraffin-embedded specimens from pri.In addition, cut open from disaster victim's fresh cadaver and get brain, thymus, lung, liver, stomach, colon, kidney, adrenal gland, spleen, pancreas, uterus (having endometrium) and skin normal structure and some tumor tissues.Operation and necropsy specimen are Department ofPathology, Swedish Medical Center, and Seattle, the Ms.Debbie Bennett favour of WA and Biomembrane Institute supplies.Sample section (3 μ M are thick), with the zylene dewaxing, ethanol dehydration is handled with a MAb, handle with the avidin of biotinylation the 2nd MAb and peroxidase conjugated then, with 3 ', 3 '-diaminobenzidine dyes.Use 0.3%H 2O 2Handle and cut into slices 20 minutes with the blocking-up endogenous peroxidase activity.Some are cut into slices with mice IgG incubation as negative control.Biotinylation sheep anti mouse IgM, avidin and biotin available from Vectastain (Burlingame, CA).
The reaction of MAb IMH2 and colon, rectum, liver, pancreas and endometrial tumors is strong and incidence rate is high (Table I).On the contrary, it does not show and the reactivity that comprises DC and the rectum normal mucosa of crypts district and goblet cell.Itself and adenocarcinoma of lung are reacted and are not reacted with big or small cell carcinoma.Example in the 5 routine scale cell cancers shows strong positive reaction.MAb IMH2 is not with normal brain activity, lung, spleen, skin histology or comprise the various hemocyte reactions of granulocyte.
Observed to have a strong painted normal structure position as follows: thymus corpuscle and thymus epithelial skein cell (thymocyte cell is negative); Mucous epithelium and gastric mucosa secreting gland (lamina propria, serous coat and Musclar layer are negative); Adrenal medulla and cortex.The normal structure position of by the time weak positive staining is in the intensity: the epithelial cell (other parts are negative) that kidney near-end and far-end curl; Islets of langerhans (Langerhan ' s islets) cell (the pancreas other parts are negative); Cecum mucosa; Urothelial.Observing very weak dyeing (connective tissue, central vein, bile duct and Kupffer cell under the other parts of liver, the lobule (Kupffer ' scell) in hepatocyte is negative).These results are summarized in Table I.
Table I. with the immunohistology dyeing of MAb IMH2 to normal structure and cancer.Tissue staining position/note normal brain activity-lung-comprise bronchus (broncheolar) epithelium spleen-rectum-comprise crypts district colon--11/12, ± 1/12 caecum+skin-granulocyte-lymphocyte-pancreas+islets of langerhans+, all the other-liver ± hepatocyte is weak+/ ±, all the other--thymus ++ thymus corpuscle, epithelium and skein cell ++, breast
Glandular cell-stomach +++mucosa, glandular cell (seeing text) kidney+tubulose epithelium be weak+(seeing text) adrenal gland +++uterus/Endometrio-/+Endometrio-or weak+;-9/15,
± 2/15 ,+4/15 (total positives rate 4/15=27%) cancer colon/rectum+/ +++++ ++ 4, ++ 6 ,+4, ± 1 ,-1 (total positives
Rate 14/16=88%) liver (former) ++ 2/3 pancreas +++2/2 adenocarcinoma of lung ++ 2/4 flaky+1/5 maxicell-0/3 minicell-0/5 Endometrio-/++++++4, ++ 11, + 6/24, ±/-3 (total sun
Property rate 21/24=88%)
E.IMH2 with from Lewis and the known patient's of secreting gland state normal and virulent colon Reactivity with bladder body
Le bAnd Le yThe expression of determinant relevant (people such as Sakamoto, Molec.Immun.21:1093-1098,1984 with individual secreting gland state;
Figure A0081978800191
Deng the people, J.Urol.138:171-176,1987), and Lewis antigen in some tumor expression and host Lewis state irrelevant ( Deng the people, Lab.Invest.58:576-583,1988;
Figure A0081978800193
Deng the people, Blood 77:1389-1396,1991).Therefore, studied MAb IMH2 with from Lewis and the known patient's of secreting gland state the normal and virulent colon and the reactivity of bladder body.The result is summarized in Table II and III.IMH2 and rectal neoplasm and not with the reaction of normal rectal tissue, this reactivity and secreting gland state have nothing to do.On the contrary, IMH2 and normal caecum reaction, and with the single cecal neoplasma example reaction of being studied a little less than.This result shows the expression trend of IMH2 epi-position in normal and pernicious colon and the ABH antigen presentation pattern similarity that everybody generally acknowledges.Real Lewis-feminine gender (Le A-b-) individual (
Figure A0081978800201
Deng the people, Lab.invest.58:576-583,1988), all express IMH2 epi-position (Table II and III) in normal and virulent colon.
The IMH2 epi-position is being expressed in normal urothelial, but its expression weakens in various degree in bladder cancer.As if the dependency that the atypia degree is arranged, promptly the expression of IMH2 epi-position in the height invasive tumor is minimum.This trend is also similar in normal expression with virulent bladder body to ABH antigen.But opposite with colon, the expression of IMH2 epi-position in the individual bladder body of A type blood is relevant with the secreting gland state.Real Lewis-feminine gender (Le A-b-) individual in normal and virulent bladder body, all express the IMH2 epi-position.Table II. with MAb IMH2 normal and virulent colon immunohistology is dyeed: with the relation of host Lewis state
Normally pernicious
Rectum caecum rectum caecum A Le A-b+0/5 1/1 3/4 1/1 A Le A+b-0/4 ND, 2/2 ND, 0 Le A-b+0/2 ND, 2/3 ND, 0 Le A+b-The real Le of 0/2 ND, 1/1 ND A-b-0/1 1/1 a1/1 0/1 non-real Le A-b-0/2 ND 1/1 0/1 aNon-secreting gland numeral positive sample is divided by investigation specimen sum.ND=is undetermined.Le A-b-The phenotype state of individual (real and non-real), by saliva al → 4 fucosyltransferase activity, and erythrocyte and anti--Le aWith anti--LebMA bReactivity determine.The phenotype definition can be referring to people such as Holmes, Arch.Biochem.Biophys.274:14-25, and 1989, and
Figure A0081978800211
Deng the people, Lab.Invest.58:576-583,1988.Table III. with MAb IMH2 normal and virulent bladder body immunohistology is dyeed: with the relation of host Lewis state.
Bladder cancer
Normal Non-Invasive aggressive ALe A-b+4/4 1/1 1/2 ALe A+b-0/2 1/2 1/3 OLe A-b+1/1 1/1 1/2 OLe A+b-2/2 1/1 0/1 real Le A-b-2/2 ND, 0/1 non-real Le A-b-ND ND ND
The main same Table II of footnote
F. the sialylated Le of Shen Zhaning a (or SLe a -Le a ) separation
Cause or cause a kind of separation of main ganglioside with high performance thin layer chromatography technical checking Colo205 cell list sialyl-ganglioside component.This master tape of extracting also carries out character and identifies.Its structure is accredited as:
This structure warp 1H-NMR spectrum confirms.
SLe a-Le aOn stretching, extension sialic acid-Le aStructure produces and Le by the enzymatic degradation with sialidase a-Le aIdentical chemical compound (it is by thin-layer chromatographic analysis and monoclonal antibody ST-421 immunostaining conclusive evidence) is determined.Initial sialic acid Le a-Le aOr the Le that stretches aDo not show any reactivity with MAb ST-421.But this chemical compound show with at sialic acid-Le aMAb such as the reactivity of N-19-9, NKH-1 and NKH-2.

Claims (8)

1. an isolated compound is with or without fucosido and/or sialic acid residues, has general formula:
Gal β 1 → 3GlcNAc β 1 → 3Gal β 1 → 3GlcNAc β 1 → (3Gal β 1 → 3GlcNAc β 1 →) n3Gal β 1 → 4Glc β 1 → 1Cer wherein n is 0 or 1 or above integer, and when n=1, have two fucosidos and/or one or more sialic acid residues at least, Gal represents galactose, GlcNAc represents N-acetyl-glucosamine, Glc represents glucose, Cer represents ceramide, wherein said at least two fucosyl residues link to each other with the GlcNAc residue with a1 → 4 keys and/or are connected with terminal Gal residue with a1 → 2 keys, described one or more sialic acid residues links to each other with the Gal residue of end with a2 → 3 keys, and/or is connected with one or more inferior terminal GlcNAc residues with a2 → 6 keys.
2. isolated compound in the claim 1 wherein has two sialic acid residueses at least.
3. isolated compound in the claim 1 has general formula:
Wherein Fuc represents fucose, and NeuAc represents the N-n acetylneuraminic acid n.
4. isolated compound in the claim 1 has general formula:
Wherein Fuc represents fucose, and NeuAc represents the N-n acetylneuraminic acid n.
5. isolated compound in the claim 1 has general formula:
Wherein Fuc represents fucose, and NeuAc represents the N-n acetylneuraminic acid n.
6. isolated compound in the claim 1 has general formula:
Figure A0081978800032
Wherein Fuc represents fucose.
7. isolated compound in the claim 1 has general formula:
Wherein Fuc represents fucose.
8. isolated compound in the claim 1 has general formula:
Wherein NeuAc represents the N-n acetylneuraminic acid n, and Fuc represents fucose.
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