KR100666238B1 - Extended Type 1 Chain Sphingoglycolipids As Tumor-associated Antigens - Google Patents

Abstract

Various compounds are provided that are useful as antigens and tumor markers. The present invention discloses a method associated with the detection of cancer. Extension of the lacto-series type 1 chain appears to be present in various cancer tissues. The invention also provides cell lines and monoclonal antibodies made from them. Such antibodies have a variety of uses, including diagnostic or therapeutic methods.

Description

Extended type 1 chain glycosphingolipids as tumor-associated antigens

The present invention relates to novel human tumor-associated antigens. The present invention more particularly relates to extended type 1 chain sphingolipids and their use, for example as antigens and tumor markers.

Despite huge investments in financial and human resources, cancer is still one of the leading causes of death. Current cancer therapies treat only about 50% of patients with malignant tumors. In most human malignancy, metastasis is the leading cause of death.

Metastasis is the formation of a secondary tumor colony at a distant site. In most human malignancies, distant metastasis is often too small to detect in the treatment of the primary tumor. Moreover, extensive initiation of metastatic colonies generally occurs before the clinical symptoms of metastatic disease become evident. Variation in the size and lifespan of metastases, their distributed anatomical location and their heterogeneous composition all interfere with surgical removal and limit the concentration of anticancer agents that can be delivered to metastatic colonies It's all about that. Thus, identifying malignancies prior to transmission of tumor cells from the primary site is necessary to enhance the effectiveness of current cancer therapies.

Aberrant glycosylation has been observed to be a common feature of most cancer forms. Most of the carbohydrate antigens used for the diagnosis of human cancer have a polylactosamine structure, including, for example, Galβ1 → 3 / 4GlcNAc. Polylactosamines are usually classified into two categories according to their polylactosamine unit structure. Polylactosamines having a Galβ1 → 2GlcNAc structure are called type 1 chains and those having a Galβ1 → 4GlcNAc structure are called type 2 chains. The most common tumor-associated antigens found in the majority of human cancers usually have lacto-series 2 type chain structures that are sialylated and / or fucosylated. Type 1 chain antigens are present in normal cells and tissues and are also associated with cancer. For example, the 2 → 3 sialylated Le ^a antigen [CA 19-9 antigen defined by N 19-9 antibody] is a cancer-associated type 1 chain-antigen. However, cancer diagnostic methods by detection of these known antigens have been hindered by high false positives and / or high false negative incidences.

Due to the difficulties in current approaches to cancer diagnosis, improved compositions and methods are needed. The present invention fulfills this need and further provides other related advantages.

Summary of the Invention

In brief, the present invention provides an isolated compound and a method for screening cancer by detecting such a compound. In one aspect, the present invention provides free compounds having the following formulas, with or without fucosyl and / or sialyl residues:

1 →4 결합(linkage)을 통해 GlcNAc 잔기 및/또는

1 →2 결합을 통해 터미날 Gal 잔기에 결합되고, 상기 하나 이상의 시알릴 잔기들은

2 →3 결합을 통해 터미날 Gal 잔기 및/또는

2 →6 결합을 통해 하나 이상의 서브터미널 GlcNAc 잔기에 결합된다.Wherein n is an integer equal to or greater than 0 or 1, and there are at least two fucosyl and / or one or more sialyl residues, Gal is galactose, Glc is glucose, and GlcNAc is N-acetylglucosamine Where Cer represents ceramide, wherein the at least two fucosyl residues

GlcNAc residues and / or via 1 → 4 linkage

1 → 2 bond to a terminal Gal residue, wherein the one or more sialyl residues

Terminal Gal residues and / or via 2 → 3 bonds

Binding to one or more subterminal GlcNAc residues via 2 → 6 binding.

In another aspect, the present invention provides the above-mentioned free compound having the following formula:

Here, Fuc represents fucose and NeuAc represents N-acetylneuraminic acid.

In another aspect, the present invention provides the first described compound having the following formula:

Here, Fuc represents fucose and NeuAc represents N-acetylneuraminic acid.

In another aspect, the invention provides a first described compound having the formula:

Here, Fuc represents fucose and NeuAc represents N-acetylneuraminic acid.

In yet another embodiment, the present invention provides a free compound having the following formula.

In another aspect, the present invention provides a free compound comprising an epitope having the formula:

In another aspect, the compounds of the present invention can be used as antigens for making polyclonal or monoclonal antibodies.

In another aspect of the invention, a method of screening for cancer is provided. The method comprises the steps of (a) separating a biological sample from a warm blooded animal; (b) testing this sample to know the presence or amount of a compound.

1 is sugar I (Glc), II (Gal), III (GlcNAc). 4.20 ppm to 5.60, encompassing IV (Gal), V (GlcNAc) and VI [also in addition to fucose bound to III GlcNAc represented by F _III and fucose bound to V GlcNAc represented by F _IV ] ¹ H-NMR spectrum of extended sialyl Le ^a with chemical shift at ppm. In this spectrum, F _V and F _III All anomeric proton spectrum (anomeric proton spectrum) of _V- 1 is indicated by F and F _III- 1. On the other hand, fucose spectral C5 protons are represented by multiple bonds labeled F _III- 5 and F _V- 5. The spectrum labeled Cis is a double bond of one Cis spingosine, and R-5 and R-4 represent the spectra of sphingosine.

Detailed description of the invention

The present invention relates generally to compounds and methods related to the detection of cancer. More specifically, the description of the present invention shows that lacto-series type 1 chains occur in extended form in cancerous tissues.

As mentioned above, type 1 chain lactosamine (Galβ1 → 3GlcNAc) is known to be abundant in normal cells and tissues. Although polylactosamine antigens with one extended type 2 chain (eg Galβ1 → 4GlcNAc core structure repeated) were detected, those with extended type 1 chains were not detected. Thus, it has conventionally been thought that lacto-series type 1 chains do not occur in the extended form.

As disclosed herein, extended forms of lacto-series type 1 chains (eg Galβ1 → 2GlcNAcβ1 → (3Galβ1 → 2GlcNAcβ →) _n 3Galβ1 → R) with or without sialyl and / or fucosyl residues are present in cancer tissues. exist. Two representative extensions of lacto-series type 1 chains were separated by preparative column and thin layer chromatography (glycolipid fraction) extracted from tumor cells.

-sLe^a 단일클론성 항체 (MAb) NKH-1를 사용하는 TLC 면역스테이닝(immunostainning)에 의해 강하게 스테이닝되었다(stained). 마일드 산(mild acid) 가수분해(1% 아세트산, 1시간동안 100℃)는, 이량체-Le^a 표준 GSL과 같이 이동되는 고속이동성분(faster migration component)을 수득하였고,

-이량체 Le^a MAb ST-421에 의해 진하게 스테이닝되었다. 그 구조는 ¹H-NMR 분광기에 의해 시알릴-이량체 Le^a(하기 구조 참조)로 확인되었다.
Slow migrating sialyl-Lewis ^a (sLe _a ) active glycosphinglipids (GSLs) are homogenized from the monosialyl ganglioside fraction of the colonic adenocarcinoma cell line Colo205. Purified. This compound is purified by HPLC and preparative HPTLC of two different solvent systems,

-SLe ^a monoclonal antibody (MAb) was strongly stained by TLC immunostaining using NKH-1. Mild acid hydrolysis (1% acetic acid, 100 ° C. for 1 hour) yielded ^a fast migration component that migrates with dimer-Le ^a standard GSL,

Deeply stained with dimer Le ^a MAb ST-421. The structure was identified as sialyl-dimer Le ^a (see structure below) by ¹ H-NMR spectroscopy.

In addition to the specific glycolipids shown above, Le ^a -Le ^a and Le ^b -Le ^a epitopes can exist as extended type 1 chains with additional [3Galβ1 → 3GlcNAcβ1 →] n units. Moreover, glycoproteins, such as high molecular weight mucine-like sera glycoproteins, may have Le ^a -Le ^a and Le ^b -Le ^a epitopes.

According to the teachings of this specification, to those of ordinary skill in the art, other extended forms of lacto-series type 1 chain compounds may be freed from biological starting materials such as cancerous tissues or chemically after structural identification (structural identification). And / or enzymatically). In simple terms, the structure of carbohydrates bound to either lipids or proteins is characterized by degradation, electron-impact direct-probe (EI) and fast atom bombardment (FAB). Mass spectrometry and methylation analysis [see, eg, Nudelman et al . , J. Biol. Chem. 261: 5487-5495, 1986]. Degradation assays can be performed chemically and / or enzymatically, for example by glycosidases. The carbohydrate sequences proposed by the decomposition analysis are described by chemical ionization mass spectrometry of hypermethylated sugars (eg, Stellner et al., Arch. Biochem. Biophys. 155: 464-472, 1974; Revere et al., Meth. Enzymol . 138 : 13-25, 1987], followed by methylation analysis (eg Hakomori, J. Biochem . 55 : 205-208, 1964]. Alternatively, or in connection with this technique, E1 mass spectroscopy may be performed on hypermethylated glycans or after appropriate degradation of intact glycans [eg, Kannagi et al., J. Biol. Chem . 259 : 8444-8451, 1984; Nudelman et al., J. Biol. Chem . 263 : 13942-13951, 1988]. Homogeneity of carbohydrate sequences can be demonstrated based on various chemical and physical criteria, including atomic NMR spectroscopy and FAB mass spectrometry of intact or methylated glycans. Once the carbohydrate structure is established, carbohydrates or derivatives thereof or non-carbohydrate functional equivalents thereof can be synthesized using techniques well known in the art.

The compounds of the present invention can be used as antigens for making polyclonal and monoclonal antibodies (MAbs). Polyclonal antibodies can be made by standard methodologies. For example, in brief, polyclonal antibodies can be made by immunization of an animal with a compound of the present invention followed by collection of its serum. In general, early immunization (method) using one or more boosters prior to serum collection is preferred. These monoclonal antibodies (MAbs) are generally described by Kohler and Milstein methods [ Nature 256 : 495-497, 1975; Eur. J. Immunol . 6 : 511-519, 1976]. In brief, lymph nodes and / or spleens of animals immunized with the compounds of the present invention are fused with myeloma cells to form a hybrid cell line (hybridomas or clones). Each hybridoma secretes a single type of immunoglobulin and, like myeloma cells, has the potential for indefinite cell division. Instead of making MAbs through hybridomas, bacteriophages and bacteria are used to create MAb expression libraries (eg, Sastry et al., Proc. Natl. Acad. USA 86 : 5728, 1989; Huuse et al., Science 246 : 1275, 1989]. The selection of antibodies that exhibit the desired specificity can be performed in a variety of ways well known to those of ordinary skill in the art.

In order to increase their immunogenicity, it may be desirable to combine the compounds of the present invention with one carrier. Suitable carriers include inert bacteria, keyhole limpet hemocyanin, thyroglobulin, bovine serum albumin and derivatives thereof. For example, all or part of the carbohydrate residues of GSLs Le ^a -Le ^a or Le ^b -Le ^a may be associated with one carrier. The compounds of the present invention may be associated with one carrier by a variety of methods, including adsorption and covalent attachment.

A representative example of the use of a compound of the present invention as an antigen is immunization of mice (rats) with Le ^b / Le ^a antigen. In short, the Le ^b / Le ^a separation from Colo205 cells, were injected into a BALB / c mouse through the tail vein after the suspension and the mixture of acid-treated Salmonella laminate sotae (acid-treated Salmonella minnesotae), at 10 day intervals Injections were repeated three times. After the last injection, antibody-producing cells (splenocytes) of immunized mice were collected and fused with myeloma cells. Hybridomas that exhibit preferential reactivity with antigen, IMH2, have been established and have been established in the American Type Bank (American Type Culture Collection, United States, Virginia 20110, Manassas, University Boulevard 10801). Deposited in an arc. The hybridomas produce MAb 1MH2 with an IgG3 isotype.

Extended detection methods of type 1 chain antigens, such as Le ^a -Le ^a and / or Le ^b -Le ^a antigens, can be used to screen cancer. For example, settling by MAb 1MH2 and MAb NCC-ST-421 [Watanabe et al., Jpn. GSL Le ^b -Le ^a and GSL Le of neutral glycolipid fractions made from various tumor samples, respectively, detected by TLC immunostainning using J. Cancer Res (Gann) 76 : 43-52, 1985]. ^{There is a} -Le ^a . Such tumor samples include tissues of colon cancer, breast cancer, Hodgkin's disease, gallbladder cancer and embryonal rhabdomyosarcoma. For example, GSL Le ^a -Le ^a was not detected in the glycolipid fraction of normal tissues of the spleen, liver, kidney, placenta and lung. In accordance with the teachings of this specification, various methods for detecting tumor-associated prolonged type 1 antigens are known to those of ordinary skill in the art [tumor-related prolonged such as GSL Le ^a -Le ^a and GSL Le ^b -Le ^a. Including the use of binding partners specific for type 1 antigens] may be used in the method of the present invention. For example, antibodies specific for Le ^a -Le ^a or Le ^b -Le ^a epitopes can be made as described above, and the presence of immunocomplexes allows the formation of the immunocomplexes. Under sufficient time and conditions, such antibodies can be tested after contact (eg, culture) with a biological sample.

The detection of the presence of an immunocomplex formed between the antigen described above and an antibody specific for the antigen is performed by various known techniques, such as radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA). Can be. Suitable immunoassays include, for example, David et al., Double monoclonal antibody sandwich immunoassay technique (US Pat. No. 4,376,110); Monoclonal-polyclonal antibody sandwich assays [Wide et al., Kirkhan and Hunter, eds., Radioimmunoassay Methods E. and S. Livingstone ( Livingstone, Edinburgh, 1970]; Western blot method such as Gordon et al. (US Pat. No. 4,452,901); Immunoprecipitation of labeled ligands (Brown et al., J. Biol. Chem . 255 : 4980-4983, 1980; Enzyme-linked immunosorbent assay as described for example by Raines and Ross [ J. Biol. Chem . 257 : 5154-5160, 1982; Immunocytochemical techniques involving the use of fluorochromes (Brooks et al., Clin. Ecp. Immunol . 39: 477, 1980; And neutralization of activity [Bowen-Pope et al., Proc. Natl. Acad. Sci. USA 81 : 2396-2400, 1984]. In addition to the immunological assays described above, US Pat. No. 3,817,827; 3,850,752; 3,850,752; 3,901,654; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 3,996,345; Various other immunological assays are available, including those described in 4,034,074 and 4,098,876.

For detection purposes, the antibody may or may not be labeled. If not labeled, antibodies are used in agglutination assays. Unlabeled antibodies, on the other hand, can be used in combination with a labeled molecule that is reactive with an immunocomplex or with a directed against antibody (second antibody), such as an antibody specific for immunoglobulins. . Alternatively, antibodies can be labeled directly. If they are labeled, the reporter group may comprise radioisotopes, fluorophores, enzymes, luminescers, or dye particles. These and other labels are well known in the art, see for example US Pat. No. 3,766,162; 3,791,932; 3,817,837; 3,817,837; 3,996,345; 3,996,345; And 4,233,402.

In one preferred embodiment for detecting immunocomplexes, one informative group is bound to the antibody. Detecting the immunocomplex comprises substantially removing the unbound antibody and then detecting the presence of the informative group. Unbound antibody is an antibody that is not bound to an antigen.

In another preferred embodiment, one informative group is bound to a second antibody capable of binding to an antibody specific for the antigen. Detecting the immunocomplex comprises: (a) substantially removing unbound antibody (eg, an antibody not bound to an antigen), (b) adding a second antibody, and (c) Substantially removing the unbound second antibody, and (d) detecting the presence of the informative group. For example, when an antibody specific for an antigen is derived from a mouse, the second antibody is one anti-murine antibody.

In a third preferred embodiment for detecting immunoglobulins, one informative group is bound to one molecule capable of binding to an immunocomplex. The detecting step includes (a) adding a molecule, (b) substantially removing the unbound molecule, and then (c) detecting the presence of an informative group. An example of a molecule capable of binding to an immunocomplex is protein A.

Other uses of labeled antibodies, labeled second antibodies or labeled molecules that are generally reactive with the immunocomplex are immunological measurements using the labeled antigen. In such assays ("indirect" or "competitive"), the antigen present in the sample will compete with the labeled antigen for the antibody.

It will be apparent to those skilled in the art that various methods for detecting immunocomplexes can be used within the scope of the present invention. Suitable informative groups for use in the method include radioisotopes, fluoropores, enzymes, cold bodies and dye particles. On the other hand, a binding partner (non-antibody) specific for tumor-associated extended type 1 antigens of the present invention can be used for testing for such antigens, and the complex formed between such binding partner and antigen can be used for such immunity. It can be detected by the same techniques as those described above for the complex.

The following examples are provided for illustration and not limitation.

Example

Example 1

Prepared from tumors and normal tissue

Using neutral glycolipid MAB NCC-ST-421

HPTLC immunostaining and immunological measurements

A. Monoclonal Antibodies and Immunological Measurements

MAb ST-421 is described above (Watanabe et al., Jpn. J. Cancer Res (Gann) 76 : 43-52, 1985]. MAb MNH-1, which defines the type 1 chain N-acetyllactosamine [Galβ1 → 3GlcNAcβ1 → R], was prepared in our laboratory; MAb 1B2, which defines the type 2 chain N-acetyllactosamine [Galβ1 → 4GlcNAcβ1 → R], is described above by Young et al., J. Biol. Chem . 256 : 10967-10972, 1981]. Was purchased from anti ^{-Le a MAb (anti-Le a} MAb) is el-Chem Bio Med tidi. (Chembiomed Ltd .; Edmonton, Alberta, Canada). Anti-Le ^y MAb AH6 is described as described above (Abe et al., J. Biol. Chem. 258 : 11793-11797, 1983] and did not exhibit cross-reactivity with Le ^b . Anti-Le ^b MAb was purchased from Chembiomed Eltidi. (Edmonton, Alberta, Canada) and exhibited cross-reactivity with type 1 chain H. Another anti-Le ^b MAb was purchased from Monocarb (Lund, Sweden) and showed reactivity with Le ^b , type 1 chain H and Le ^y . HPTLC immunostaining is the method described first by Magnini et al. [Magnani et al., Anal. Biochem . 109 : 399-402, 1980] (Kannagi et al., J. Biol. Chem . 257 : 4438-4442, 1982; Kannagi et al . , J. Biol. Chem . 257 : 14865-14874, 1982] using Whatman HPTLC plates (HP-KF).

B. Preparing Lipids

-푸코시다제(

-fucosidase) 처리에 의해 만들어졌다[예를 들어 100㎍의 당지질이 0.05 유니트(units)의 소의 신장

-L-푸코시다제[시그마 케미컬 씨오.(Sigma Chemical Co.), 미주리주 세이트 루이스(St. Louis, Mo.)]를 포함하는 0.2M 구연산염 완충액(pH 4.5)으로 37℃에서 2시간동안 배양되었다]. IV²III⁴Fuc₂Lc ₄, V³III³Fuc₂nLc₆ 및 VI²V³Fuc₂nLc ₆ 는, Colo205로부터의

1 →3/4 푸코실전이효소(fucosyltransferase)를 사용하는 기질로서 IV²FucLc₄, nLc₆와 VI²FucnLc₆ (각각)의

1 →3 푸코실화(fucosylation)에 의해 생합성적으로 만들어졌다.

1 →3/4 푸코실전이효소는, 4℃의 포터-엘브젬 균질기(Potter-Elvehjem homogenizer)에서 50mM 헤페스 완충액(Hepes buffer)[pH 7.0], 0.5 M 수크로즈, 1 mM의 EDTA, 그리고 1% 트리톤(Triton) CF-54의 두 볼륨(volumes)으로의 균질화(homogenization)에 의해 Colo205 세포로부터 용해되었다(solubilized). 균질액이 100,000 X g 에서 1시간동안 원심분리되었고, 상층액이 여막분석(dialysis)에 의해 세포의 원래 부피로 농축되었다. 효소 조제물(enzyme preparation)은 필요할 때까지 -80℃로 저장되었다.All glycolipid samples used were enzymatically isolated or synthesized. VI ³ NeuAcnLc ₆ , IV ³ NeuAcIII ⁴ FucLc ₄ , VI ² FucnLc ₆ , and IV ² FucLc ₄ , Folch partition, DEAE-Sephadex chromatography, and Iatrobeads 6RS HPTLC on a -8010 column [Magnani et al., J. Biol. Chem . 257 : 14365-14369, 1982; Watanabe et al., J. Biol. Chem . 254 : 8223-8229, 1979; Hakomori et al., J. Immunol . 98 : 31-38, 1967; Human placenta, hepatic adenocarcinoma, human type O erythrocytes, and swine intestine after extraction with IHW (55:25:20), followed by Stellenner et al., Biochemistry 12 : 656-661, 1973]. From each. nLc ₆ and III ⁴ FucLc ₄ were made by desalylylation of VI ³ NeuAcnLc ₆ and IV ³ NeuAcIII ⁴ FucLc ₄ , respectively, by heating the samples in 1% acetic acid at 100 ° C. for 1 hour. . IV ³ GlcNAcnLc ₄ , IV ³ Galβ1 → 3-GlcNAcnLc ₄ , IV ³ Galβ1 → 3 [Fuc1 → 4] GlcNAcnLc ₄ and IV ³ Galβ1 → 3 [Fuc1 → 4] GlcNAcIII ³ FucnLc ₄ (Le ^a -Le ^x ) are enzymes It was made by ever synthesis. IV ³ Galβ1 → 3GlcNAcIII ³ FucnLc ₄ is a combination of IV ³ Galβ1 → 3 [Fuc1 → 4] GlcNAcIII ³ FucnLc ₄ .

Fucosidase (

-fucosidase treatment (e.g. 100 μg of glycolipids of 0.05 units of bovine kidney)

Incubate for 2 hours at 37 ° C. with 0.2M citrate buffer (pH 4.5) containing -L-fucosidase (Sigma Chemical Co., St. Louis, Mo.) Became]. IV ² III ⁴ Fuc ₂ Lc ₄ , V ³ III ³ Fuc ₂ nLc ₆ and VI ² V ³ Fuc ₂ nLc ₆ from Colo205

1 → 3/4 substrate of IV ² FucLc ₄ , nLc ₆ and VI ² FucnLc ₆ (each) as fucosyltransferase

Biosynthesized by 1 → 3 fucosylation.

1 → 3/4 fucosyltransferases were obtained in 50 mM Hepes buffer [pH 7.0], 0.5 M sucrose, 1 mM EDTA, at 4 ° C. Potter-Elvehjem homogenizer. And solubilized from Colo205 cells by homogenization with two volumes of 1% Triton CF-54. The homogenate was centrifuged at 100,000 X g for 1 hour and the supernatant was concentrated to the original volume of the cells by dialysis. Enzyme preparations were stored at -80 ° C until needed.

1 →3/4 푸코실화는 1mg의 스핑고당지질(glycospingolipids; GSL) 기질, 1mg의 데옥시타우로담즙산염 (deoxytaurocholate), 10 μmol의 MnCl₂, 25 μmol의 헤퍼스 완충액(Hepes buffer)[pH 7.0], 5 μmol의 CDP-콜린(CDP-choline), 6 μmol의 GDP-푸코스, 그리고 500 ㎕의 효소 조제물(enzyme preparation)을 전체 부피 1㎖로 함유하는 반응 혼합물에서 수행되었다. 반응 혼합물은 37℃에서 16시간동안 배양된 다음에 동결탈수되고, 초음파 분해(sonication)에 의해 이소프로판올-헥산-물 (isopropanol-hexane-water; IHW)(55:25:20)로 추출된 다음 원심분리되었다. 상층액은 200분 이상 55:40:4 부터 55:25:20 까지의 IHW의 기울기 용리(gradient elution)을 사용하는 아이애트로비즈(Iatrobeads) 6RS-8010 컬럼에서 HPLC 되었다. 2㎖ 분획이 수집되었고 최종 생성물을 포함하는 튜브가 클로로포름-메탄올-물 50:40:10에서의 HPTLC 이동에 따라 채워졌다. GSL 밴드는 오르시놀 스프레이 시약(orcinol spray reagent)에 의해 시각화되었다(visualized).Enzymatic

1 → 3/4 fucosylation results in 1 mg of glycospingolipids (GSL) substrate, 1 mg of deoxytaurocholate, 10 μmol of MnCl ₂ , 25 μmol of Hepes buffer [pH 7.0], 5 μmol of CDP-choline, 6 μmol of GDP-fucose, and 500 μl of enzyme preparation in a reaction volume containing 1 ml of total volume. The reaction mixture was incubated for 16 hours at 37 ° C., followed by freeze dehydration, extraction with isopropanol-hexane-water (IHW) (55:25:20) by sonication and centrifugation. Separated. Supernatants were HPLC on an Iatrobeads 6RS-8010 column using a gradient elution of IHW from 55: 40: 4 to 55:25:20 for at least 200 minutes. A 2 ml fraction was collected and the tube containing the final product was filled following the HPTLC shift in chloroform-methanol-water 50:40:10. GSL bands were visualized by orcinol spray reagent.

Each feature has a built GSL (defined) structure is specific MAb (s), for example, wherein (anti) -Le ^y MAb wherein non-AH6 -Le ^b MAbs reactive with the Le ^b / Le ^a antigen; Le ^y / Le ^x reacted with AH6 but neither anti-Le ^b nor anti-Le ^x MAbs; Characterized by reactivity with Le ^a / Le ^a and Le ^a / Le ^x reacted with anti-Le ^a MAb with MAb ST-421.

C. TLC Immunostaining

TLC immune stacking of neutral glycolipid fraction prepared from a variety of tumor samples inning is Le ^a - active ceramide penta saccharide (Le ^a -active ceramide pentasaccharide), wherein a slower moving than the (migrating) -Le ^a MAb with the cross-reacting (cross -reacting) showed the presence of a positive band. This band was strongly stained by NAb NCC-ST-421 and appeared in most tumors tested. Examples are colon cancer, breast cancer, Hodgkin's disease, gallbladder cancer and embryogenic rhabdomyosarcoma.

Example 2

Isolation of Dimeric Le ^a and Le ^b -Le ^a Antigens

A. Preparation of Tumor Tissue

Colo205 cells (ATCC) [Semple et al., Cancer Res . 38 : 1345-1355, 1978] was grown in RPMI 1640 medium containing 10% fetal calf serum. Cells were harvested and passed almost every seven days. Harvested cells were trypsinized, centrifuged, washed twice with phosphate-buffered saline (pH 7.4) and then counted using a hemocytometer. 4 × 10 ⁶ cells were injected subcutaneously into each of six athymic (nude) mice. Tumors (approximately 2 ml each) were excised after 2 weeks and frozen at −80 ° C. until needed.

B. Slow-Move, Le ^a -Active Component [Dimer Le ^a ] from Colo205 Tumors

Approximately 200 g of tumor was extracted with isopropanol-hexane-water (IHW) (55:25:20), followed by Folch partition, DEAE-Sephadex chromatography, and iAtrobiz ( Iatrobeads) HPTLC treated on 6RS-8010 column. Gradient elution of the upper-phase neutral fraction was carried out for at least 200 minutes in IHW from 55: 40: 5 to 55:25:20. A 2-ml fraction was collected and filled according to the HPTLC shift in chloroform-methanol-water (50:40:10). The slow-moving Le ^a -active fraction (represented by TLC immunostaining) is further purified by preliminary TLC on a Merck HPTLC plate [Germany, Damstatt, Merck, Silica gel 60], resulting in structural characterization Was used for).

Isolation of C. Le ^b -Le ^a Antigen

Positive bands (by immunostaining with MAb NCC-ST-421 according to Example 1) that migrate directly below the dimer Le ^a antigen were purified using the method described in section B above.

Example 3

Identification of Dimer Le ^a and Le ^b -Le ^a Antigens

A. Enzymatic Degradation

-푸코시다제(소의 신장), 0.5 유니트의 β-갈락토시다제[잭빈(jackbean)], 그리고 0.5 유니트의 β-N-아세틸글루코사미니다제(소의 부고환)[시그마 캐미칼 씨오.(Sigma Chemical Co.),미주리 세인트 루이스]를 사용한 연속적인 가수분해에 의해 수행되었다. 모든 반응이 37℃의 수조에서 4시간동안 0.2M 구연산나트륨(pH 4.5)에서 저어주면서 수행되었다. 각 분해 생성물의 정제가 예비 HPTLC에 의해 수행되었다.
Enzymatic digestion of 1 mg of dimer Le ^a

Fucosidase (bovine kidney), 0.5 unit of β-galactosidase (jackbean), and 0.5 unit of β-N-acetylglucosaminase (bovine epididymis) [Sigma Chemical C. (Sigma) Chemical Co.), St. Louis, Missouri]. All reactions were performed with stirring in 0.2 M sodium citrate (pH 4.5) for 4 h in a 37 ° C. water bath. Purification of each degradation product was performed by preparative HPTLC.

B. In vitro Cytotoxicity of IMH2

  1. Cell line

Colo205 was originally purchased from the American Type Culture Collection (ATCC) and supplemented with RPMI-1640 medium supplemented with 10% fetal bovine serum, mM L-glutamine, 100 IU / ml penicillin and 10 μg / ml streptomycin. Incubated at. Human epidermoid carcinoma A431 cell line [MacLeod et al., J. Cell. Physiol . 127 : 175-182, 1986, was initially donated by Dr. Carol MacLeod (Canada, San Diego, UCSD Medical Department, Gildred Cancer Facility). This cell line represents Le ^a , Le ^b , Le ^x , Le ^y , and ALe ^b of EGF receptors (Gooi et al., Biosci. Reports 5 : 83-94, 1985]. A431 cells were filled with 5% fetal bovine serum, 1 mM glutamine, 110 mg / l sodium pyruvate, 100 IU / ml penicillin and 10 μg / ml streptomycin (Dulbecco's modified Eagle's medium) (Santa Ana, Canada). , Irvine Scientific. Cells (approximately 5 × 10 ⁵ / ml) were sparged and obtained at confluency by EDTA treatment and then washed with PBS containing Ca ²⁺ and Mg ²⁺ . These were used as target cells in in vitro cytotoxicity (cytotoxicity) measurements or to test tumorigenicity in nude mice by subcutaneous inoculation of 5 × 10 ⁶ cells. Human erythroleukemia K562 cells (Lozzio et al., Blood 45 : 321-334, 1975) used natural killer (NK) killing of lymphocytes used in the assay system. It was used as a control for activity.

2. Antibody-Dependent Cellular Cytotoxicity (ADCC) and Complement-Dependent Cytotoxicity (CDC)

For ADCC measurements, human peripheral blood leukocytes (HPBL) (used as effector cells) were obtained from the buffy coat fraction of blood of healthy volunteer donors. Briefly, monoclonal cells were separated by centrifugation through Ficoll Hypaque gradient elution at 2000 rpm for 20 minutes [Michell et al., Mishell, BB and Shiigi, SM (plates). ), Selected Methods in Cellular Immunology , pp. 3-27, WH Freeman & Co., San Francisco, Canada, 1980]. Mouse antibody-producing cells and mouse peritoneal macrophages (effector cells) were prepared, somewhat modified as follows, as described above by micelles. Target cells (5 × 10 ⁶ ) were labeled by incubation with 100 μl of ⁵¹ Cr at 37 ° C. for 90 minutes. After washing (3 ×) and incubation (1 h at 37 ° C.), cells (1 × 10 ⁶ / ml) were suspended in RPMI-1640 supplemented with 25 mM HEPES buffer and 3% bovine serum albumin. 20 μl of labeled cells, 100 μl of IMH2 or ST-421, and 100 μl of effector cell suspension were applied to Microtiter U-bottom plates (Corning, NY). Mixed with. Non-specific mouse Ig (Sigma, Missouri, St. Louis) was used as a negative control. After 4 hours of incubation, the plates were centrifuged with a hanging plate holder assembled to a centrifuge (500 X g, 2 minutes) and the radioactivity of 100 μl supernatant in each well was gamma counter. (gamma counter). Each test group was tested three times. The percent specific lysis is expressed by the formula “([AB] × 100) / C, A = cpm in lysed test cells; B = cpm in unlyzed target cells; C = cpm ″ in total target cells. Spontaneous release never exceeded 15% of the maximum release label radioactivity.

For CDC, a Cr-release assay was performed using the same method as that of ADCC, except that 100 μl of dilute human serum was added as a complement source instead of effector cells. Serum was inactivated at 56 ° C. for 30 minutes and used as a control. Percent specific dissolution was calculated as described above.

Cell disruption of IMH2 against Colo205 because Colo205 cells were characterized to express extended type 1 chains Le ^a / Le ^a and Le ^b / Le ^a antigens, which react strongly with MAbs ST-421 and IMH2, respectively. The effect was evaluated and compared to that of ST-421. Both MAbs showed striking ADCC killing of Colo205 cells. This killing was correlated with the effector: target cell (E: T) ratio and MAb concentration. The cytotoxic effect was maximal at an E: T ratio of 100: 1-200: 1 and at a MAb concentration of 35-70 μg / ml. Control mouse IgG and other non-specific MAbs did not show cytotoxic effects regardless of the E: T ratio or MAb concentration. When the same cell disruption test was performed on mouse antibody producing cells, the corresponding values were only 7% and 17% lysis (E: T ratio 200: 1, MAb concentration 30 μg / ml). MAb showed a weak cytotoxic effect on A431 cells (Table). A comparison of the maximum IMH2-dependent lysis of Colo205, A431 and K562 cells is shown in the table. High lysis values (eg, Colo205 cells 65% and 94% lysis with IMH2 and ST421, respectively) were only pronounced in the presence of HPBL; As previously observed with ST-421 [Watanabe et al., Cancer Res . 51 : 2199-2204, 1991], the values were smaller than those with mouse antibody producing cells. CDCs mediated by IMH2 and ST-421 correlated similarly to the complement concentration and MAb concentration.

.

(Table)

MAb-dependent Cytotoxic Activity in Colo205, A431, and K562 Cells by MAbs ST-421 and IMH2

  Target cell Percent dissolution ^a Antibody / reactive ^b Effector Cells + MAb + Effector Cells + MAb- Effector Cells-MAb + Colo205 A431 K562 ^c ST-421 + IMH2 +                                          ST-421 ± IMH2 ±                                          ST-421- IMH2- 94.5 65.0                                          14.4 7.6                                          48.2 44.8 2.7 2.7                                          10.9 9.2                                          36.2 36.2 0.8 0.7                                          1.1 0.6                                          0.5 0.3

a. Percent lysis at an E: T (effector: target cell) ratio of 100: 1 using IMH2 (35 μg / ml) and ST-421 [X100 diluted ascites].

b. Determined by flow cytometry. +, Positive; ± weakly positive; -, voice.

c. The high cytotoxic effects of K562 cells are also observed without MAb and are believed to reflect natural killer cell activity.

C. Tumor Suppression in Vivo

Colo205 and A431 cells used for in vivo testing were grown in vitro, washed 2 × with medium and reconstituted in PBS at the desired cell concentration. Cells (5 X 10 ⁶ / 100㎕) are 5-7 weeks old (5- to 7-week-old ) were subcutaneously injected into thymus-free, etc. (backs) of BALB / c mice, the intraperitoneal injection of MAb It started immediately after. IMH2 (1.1 mg / ml) purified in ascites fluid with a concentration of IgG (1.1-1.2 ng / ml) at a dosage of 0.2 ml / animal ) Or ST-421 was injected intraperitoneally at 1 × / day for 2 weeks. Tumor width and length were measured three times a week (3X / week) by the same observer. Tumor weight was calculated as "(width ² x length) / 2". Control animals received multiple proteins produced by mouse myeloma cell line NSI in BALB / c mice. Seven mice per group were used for each test and the test was run twice. Mean values of tumor weight based on two tests were plotted.

Both MAb IMH2 and ST-421 showed significant inhibition of Colo205 tumor growth in nude mice. In contrast, both MAbs showed a minimal inhibitory effect on A431 tumor growth. Thus, high expression of defined antigens appears to be essential for susceptibility to antibody-dependent inhibition of tumor growth in vivo.

D. Reactivity of various tumors with normal tissues of IMH2

Various tumors and adjacent normal tissues were obtained from formalin-fixed and paraffin-embedded surgical specimens. Meanwhile, the necropsy of the victims of normal tissues and tumor tissues from the brain, thymus, lungs, liver, stomach, colon, kidneys, adrenal glands, spleen, colon, uterus (with endometrium) and skin Obtained by Surgical and autopsy specimens are all available from Washington, Seattle, Swedish Medical Center, Department of Pathology and The Biomembrane Institute. It is provided with permission from Ms. Debbie Bennet. Samples were incised (3 μM thick) to remove paraffins with zylene, dehydrated in ethanol, treated with primary MAb, then washed with biotinylated secondary MAb. Treated with peroxidase-conjugated avidin and stained with 3 ', 3'-diaminobenzidine. Endogenous peroxidase activity was blocked by 20 minute cleavage treatment with 0.3% H ₂ O ₂ of the sections. Some sections were incubated with mouse IgG as negative control. Biotinylated goat anti-mouse IgM, avidin, and biotin were products of Vectastain (Burlingame, Calif.).

MAb IMH2 responded strongly and with high incidence to tumors from colon, rectum, liver, pancreas and endometrium (Table 1). In contrast, MAb IMH2 did not respond to terminal colon and rectal normal mucosae, including crypt regions and goblet cells, which did not react with large cell or small cell carcinoma, Reacted. One of five cases of squamous cell carcinoma showed strong positive reactivity. MAb IMH2 did not react with various blood cells, including normal brain, lung, spleen, skin or granulocytes.

The locations observed to be strongly stained in normal tissue are: Hasel's bodies and thymus epithelial cells (thymus cells are negative); Secretory glands of mucous epithelial cells and gastric mucosa (lamina propria, serous, and muscle layers are negative); Both medulla and cortex of the adrenal glands. The location of normal tissues with positive staining from moderate to weak is: epithelial cells of the body and terminal convolution of the kidneys (negative portions are negative); Cells of Langerhans islet of the pancreas (other parts of the intestine are negative); Cecal mucosal layer; Urinary tract epithelium. Very weak staining was observed for hepatocytes (negative parts of liver, infralobular connective tissue, central veins, bile ducts and Kupffer's cells). These results are summarized in Table 1.

Immunohistological staining by MAb IMH2 in normal tissues and carcinomas group dyeing Localization / annotation Normal brain lung spleen rectal colon cecum skin granulocytes lymphocytes pancreas liver thymus stomach adrenal gland uterine / endometrial carcinoma colon / rectal liver (primary) pancreatic lung adenocarcinoma squamous cell versus small cell endometrium  -----+---+ ± ++ +++ + +++-/ + + / +++ ++ +++ ++ +---/ +++    Including broncheolar epithelium, including crypt area -11/12, +1/12 on Langerhans Island; Others-hepatocytes faintly + / ±; Others are-+ in Hastel's bodies, epithelial and reticular cells; In thymic cells-mucosal layer, glandular cells (see text) weakly in tubular epithelia (see text) in the endometrium-or weakly +; -9/15, ± 2/15, +4/15 (total positive case 4/15 = 27%) ++++ 4, ++ 6, +4, ± 1, -1 (total positive case 14/16 = 88%) 2/3 2/2 2/4 1/5 0/3 0/5 +++ 4, ++ 11, +6/24, ± -3 (total positive case 21/24 = 88%)

E. Reactivity of IMH2 with Normal and Malignant Colon and Bladder Tissues in Patients with Known Lewis and Secretor Stats

rntoft et al.), Lab. Invest. 58:576-583, 1988년; (

rntoft et al.), Blood 77:1389-1396, 1991년], Le^b 및 Le^y 결정자(determinants)의 발현은 개체의 분비자 상태(secretor status)와 상호 관련되어 있다[사카모토 등(Sakamoto et al.), Molec. Immun. 21:1093-1098, 1984년; (

rntoft et al.), J. Urol. 138:171-176, 1987년]. 따라서, 알려진 루이스(Lewis) 및 분비자 상태를 가진 환자의 정상 및 악성의 대장 및 방광 조직과 MAb IMH2의 반응성을 연구하였다. 그 결과들이 표 2 및 3에 요약되어 있다. IMH2는 정상 직장 조직이 아닌 직장 종양과 반응성이 있었고, 이 반응성은 분비자 상태와 관련이 없었다. 역으로, IMH2는 정상 맹장과 반응성이 있었으며, 연구된 단일 맹장 종양 샘플과는 반응성이 더 적었다. 이 결과들은 정상 및 악성 대장 조직에서의 IMH2 에피토프 발현의 경향이 ABH 항원의 잘-정착된(well-established) 발현 패턴과 유사함을 암시한다. 진성 루이스-음성(Le^a-b-) 개체들[(

rntoft et al.), Lab. Invest. 58:576-583, 1988년]은, 정상 및 악성 대장 조직에서 IMH2 에피토프를 발현했다(표 2 및 3).While expression of Lewis antigens in some tumors is not associated with host Lewis status [(

rntoft et al., Lab. Invest . 58 : 576-583, 1988; (

rntoft et al., Blood 77 : 1389-1396, 1991], the expression of Le ^b and Le ^y determinants is correlated with the secretor status of the individual [Sakamoto et al. .), Molec. Immun . 21 : 1093-1098, 1984; (

rntoft et al.), J. Urol . 138 : 171-176, 1987]. Therefore, the reactivity of MAb IMH2 with normal and malignant colon and bladder tissues of patients with known Lewis and secretory states was studied. The results are summarized in Tables 2 and 3. IMH2 was reactive with rectal tumors rather than normal rectal tissue, and this reactivity was not associated with secretory status. Conversely, IMH2 was reactive with normal cecum and less responsive to the single cecum tumor sample studied. These results suggest that the trend of IMH2 epitope expression in normal and malignant colon tissue is similar to the well-established expression pattern of the ABH antigen. Intrinsic Louis-speech (Le ^ab-) Objects [(

rntoft et al., Lab. Invest . 58 : 576-583, 1988] expressed IMH2 epitopes in normal and malignant colon tissues (Tables 2 and 3).

IMH2 epitopes were expressed in normal urinary tract epithelium, but their expression decreased to varying degrees in bladder tumors. There seems to be a correlation with the grade of atypia, for example IMH2 epitope expression is the lowest in highly invasive tumors. In other words, this trend is similar to that of ABH antigen expression in normal and malignant bladder tissues. However, compared to colon tissue, IMH2 epitope expression in bladder tissue from blood group A individuals is correlated with secretory state. Genuine Lewis-negative (Le ^ab- ) individuals expressed IMH2 epitopes in both normal and malignant bladder tissues.

Immunohistoscopic Staining by MAb IMH2 in Normal and Malignant Colon Tissues: Relationship to Host Lewis Status Normal ------------------ Rectal Caecum Malignant ------------------ rectal cecum A Le ^{a-b +} 0/5 1/1 3/4 1/1 A Le ^{a + b-} 0/4 ND 2/2 ND O Le ^{a-b +} 0/2 ND 2/3 ND O Le ^{a + b-} 0/2 ND 1/1 ND Genuine Le ^ab- 0/1 1/1 ^a 1/1 0/1 Non-genuine Le ^ab- 0/2 ND 1/1 0/1

1 →4 푸코실화전 이효소 활성과 항-Le^a 및 Le^b MAb와의 적혈구 반응성에 의해 확정되었다. 표현형(phenotypes)의 정의는 홈즈 등(Holmes et al.)[ Arch. Biochem. Biophys. 274:14-25, 1989년, 및 (

rntoft et al.), Lab. Invest. 58:576-583, 1988년]에서 찾을 수 있다.
The numbers represent positive samples divided by the entire sample tested. "ND" stands for "not determined". For Le ^ab- individuals (intrinsic and non-intrinsic), the phenotypic status is in saliva.

It was confirmed by 1 → 4 pre-fucosylated dienzyme activity and erythrocyte reactivity with anti-Le ^a and Le ^b MAb. The definition of phenotypes is described in Holmes et al. [ Arch. Biochem. Biophys . 274 : 14-25, 1989, and (

rntoft et al., Lab. Invest . 58 : 576-583, 1988].

^a non-secretor

Immunohistoscopic Staining by MAb IMH2 in Normal and Malignant Bladder Tissues: Relationship with Host Lewis Status   normal Bladder Carcinoma ------------------ Non-Invasive Invasive A Le ^{a-b +} 4/4 1/1 1/2 A Le ^{a + b-} 0/2 1/2 1/3 O Le ^{a-b +} 1/1 1/1 1/2 O Le ^{a + b-} 2/2 1/1 0/1 Intrinsic Le ^ab- 2/2 ND 0/1 Non-intrinsic Le ^ab- ND ND ND

Major footnotes are as in Table 2.

F. Extended sialyl -Le ^a separation of the (S or Le ^a -Le ^a)

Testing of monosialo-ganglioside fractions of Colo205 cells led to or separation of one major ganglioside by high performance thin layer chromatography techniques. Brought. Major bands were extracted and characterized. The structure was identified as follows.

This structure was validated by ¹ H-NMR.

SLe ^a - sialyl Le a -Le ^a continuation of ^a structure, a Le ^a check by the immune staining chromatography with the monoclonal antibodies ST-421 - Florida sialic to obtain the same compound with the Le ^a (sialidase By enzymatic degradation using The original sialyl Le ^a -Le ^a or extended Le ^a does not show reactivity with MAb ST-421. However, this compound showed reactivity with MAbs directed to sialyl-Le ^a such as N-19-9, NKH-1 and NKH-2.

Claims (8)

Has the following formula,

[Where n is an integer of 0 or 1 or greater and there are two or more fucosyl residues, Gal represents galactose, GlcNAc represents N-acetylglucosamine, Glc represents glucose, wherein fucos ( fucose)

GlcNAc residues via 1 → 4 binding or

1 → 2 bond to terminal Gal residues],

Terminal Gal residues via 2 → 3 bonds or

A free compound further comprising one or more N-acetylneuraminic acid (sialic acid) residues linked to one or more subterminal GlcNAc residues via a 2 → 6 bond. The free compound of claim 1, wherein there are two or more sialyl moieties. The glass compound of Claim 1 which has a following formula.

[Where Fuc stands for fucose and NeuAc stands for N-acetylneuraminic acid.] The glass compound of Claim 1 which has a following formula.

[Where Fuc stands for fucose and NeuAc stands for N-acetylneuraminic acid.] The glass compound of Claim 1 which has a following formula.

[Where Fuc stands for fucose and NeuAc stands for N-acetylneuraminic acid.] delete delete A glass compound having the following formula.

Where NeuAc represents N-acetylneuraminic acid and Fuc represents fucose.

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