JPH0234601A - Novel transferrin - Google Patents

Abstract

NEW MATERIAL:A transferrin represented by formula I [wherein R1 and R2 are each a saccharide of formulae I-IV (except both R1 and R2 are saccharides of formula IV when fucose does not bind to GIcNAc adjacent to Asn); and Tb is a transferrin protein moiety]. USE:The title compound is used as an antigen for the preparation of an antihepatoma variant transferrin monoclonal antibody used for a diagnostic agent for liver cancer, a positive control which is used in the step of preparing the antibody and a standard of a diagnosis kit for liver cancer. PREPARATION:The aimed compound is produced by isolating by means of the specific linkage with AAL lectin or DSA lectin.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] This invention relates to a novel transferrin having a sugar chain that has undergone liver cancerous changes. The novel transferrin of this invention can be used as an antigen for producing an anti-liver cancer mutant transferrin monoclonal antibody that has uses as a liver cancer diagnostic agent, as a positive control used during the production process, and as a standard for a liver cancer diagnostic kit. has.

[Prior art] Traditionally, as an in vitro clinical testing method for liver cancer, γ-
BACKGROUND ART A method of detecting so-called tumor markers such as glutamyl transpeptidase and α-fetoprotein (AFP) using polyclonal antibodies or monoclonal antibodies that specifically bind to these markers has been put into practical use. However, tumor markers are generally substances biosynthesized by tumor cells that are closely related to tumors (cancerases).
mediated 5 ubstance), is produced only at tumor sites, and is not present in normal tissues. Tumor-specific substances or tumor-specific antigens have not yet been discovered, and therefore the reliability of the liver cancer detection method using the JII marker cannot be fully satisfied.

On the other hand, cancerous changes in the sugar chains of many glycoproteins have been elucidated. Examples of such glycoproteins include AFP (Yoshima et al., Canc.
er Res, 404276, '80. Yama
Shita et al.

Cancer Res, 434691. '83)
, the above γ-glutamyl transpeptidase, human chorionic gonadotropin (Yo Kobata, Oncologia
14, Fall Issue, 77-88, 1985), α-antitrypsin, and des-γ-carboxyprothrombin.

Therefore, monoclonal antibodies having the sugar chains of these glycoproteins as epitopes can be used as cancer diagnostic agents. Examples of such monoclonal antibodies include the conventionally known CA19-9, CA12S and C3LEX-
I can name one. If a glycoprotein with a cancerous sugar chain, which is rarely or not present in a normal human body, exists in a liver cancer patient, monoclonal antibodies can be produced using that glycoprotein as an antigen to treat liver cancer. Monoclonal antibodies that have use as diagnostic agents can be obtained.

[Problems to be Solved by the Invention] Therefore, an object of the present invention is to provide a novel glycoprotein having a sugar chain that has undergone liver cancerous changes.

[Means for Solving the Problems] As a result of intensive research, the present inventors have discovered that the sugar chains of transferrin in the serum of liver cancer patients have undergone cancerous changes, and have found that the sugar chains of transferrin in the serum of patients with liver cancer have undergone cancerous changes. The present invention was completed by successfully isolating transferrin having the sugar chain and identifying the sugar chain.

That is, this invention provides a sugar chain represented by any one of the following general formulas [I] [+1] to [IVI], provided that fucose is not bound to GlcNAc adjacent to Asn, and R1 and A novel transferrin, in which R2 and R2 are simultaneously represented by the formula (excluding sugar chains represented by IVI), and Tf represents a transferrin protein moiety.

(However, R, and R2 are each independently represented by the following general formula +
1 +1 +1 +1 +1 Size +1 +1 [Effects of the Invention] According to this invention, although it is hardly present in the serum of normal people (approximately 3 to 4%), it is present in large amounts in the serum of patients with liver cancer (approximately 35 to 65%). , a novel transferrin having a sugar chain that has undergone cancerous changes was provided. The transferrin of the present invention is present in large amounts in the serum of patients with liver cancer, but hardly present in the serum of normal people, and therefore the monoclonal antibody of the present invention, which has a sugar chain epitope of transferrin, is useful as a diagnostic agent for liver cancer. Therefore, the transferrin of the present invention is useful as an antigen for producing an anti-hepatoma mutant transferrin monoclonal antibody that has use as a liver cancer diagnostic agent, as a positive control used during the production process, and as a standard for a diagnostic kit. be.

[Detailed Description of the Invention] Transferrin is a glycoprotein with a molecular weight of approximately 80,000, also called diterophyllin or iron-binding globulin. As mentioned above, the inventors of the present invention found that the sugar chains of transferrin in the blood of liver cancer patients have cancerous changes. The transferrin of the present invention that has undergone cancerous changes due to liver cancer has an asparagine-linked sugar chain represented by the above general formula [1]. As specific examples of those represented by general formula [I], the following formulas [V] to [XV
]CQc! ll ζ1 qyuca cQ (ri -koro aC cQ q ri cQ a d Sasa size size Sasa TT ↑ ↑ TT above formula [V]
Among the transferrins represented by formulas [V] to [XV], those represented by formulas [V] to [X] are particularly preferred as antigens for inducing monoclonal antibodies as diagnostic agents for liver cancer.

Among the transferrins of this invention, those having fucose branches are AAL lectin (Aleuriaauran).
Lia 1ect, in, N, Kochib
e and K, Furukawa.

Biochemistry, vol. 19, pp. 2
841-2848) and those without DSA lectin (Reference: Crowly et al. 1Metho
ds Enzymol, 8:t a ), but normal transferrin is not bound to these lectins; therefore, one transferrin of the present invention is bound to AA
For example, transferrin recovered from the serum of a liver cancer patient can be isolated using AAL lectin or DSA lectin.
The transferrin of the present invention can be isolated by passing it through a column containing lectin A so that only the transferrin of the present invention is adsorbed onto the column, and then eluting with an eluent. The column can be filled with AAL lectin or DSA lectin bound to a suitable carrier and immobilized. Methods for immobilizing lectins are well known, such as by Yasashita.

Kochibc, O1+kura, Lleda an
d KobaLa, “JBiological Ch.
emistry, vol, 260. pp, 4
688-4693 (+985). Preferred carriers include Sepharose beads, and the concentration of lectin to be immobilized is usually 20 g/sl to 1 mg/l, preferably about 3 B/ml.

Moreover, f (including the carrier) of the immobilized lectin packed in the column is not particularly limited, or approximately 1-1 is sufficient. Note that the ms method for measuring transferrin in serum is well known.
For example, Volume 8 of "Sequential Biochemistry Experiment Course" edited by the Japanese Biochemical Society (Blood) (
Above) 462-464. As the eluent, in the case of AAL lectin, a buffer containing, for example, 10 μl of fucose may be mentioned, and in the case of DSA lectin, a buffer containing, for example, 1% N-acetyl-glucosamine oligomer may be mentioned. I can do it.

The transferrin of this invention can be used as an antigen in a suitable test such as a mouse! A hybridoma that produces a monoclonal antibody specific to the transferrin of the present invention can be produced by immunization with the transferrin of the present invention by the conventional method of Köhler and Milstein. Monoclonal antibodies produced by such hybridomas can be used as diagnostic agents for liver cancer. The transferrin of the present invention also has use as a positive control used when producing such hybridomas. That is, it can be used to examine whether a monoclonal antibody produced by the prepared hybridoma specifically binds to the transferrin of the present invention.

In addition, in order to be used as a liver cancer diagnostic agent, the monoclonal antibody must have an epitope that is a sugar chain that has undergone cancerous changes. This can be determined by examining the connectivity with

Further, the transferrin of the present invention has the same usefulness even when it is in the form of a mixture containing a plurality of transferrins as when it is used alone. Furthermore, even only sugar chains separated from transferrin have utility. That is,
Separated sugar chains bound to a suitable carrier such as a protein such as albumin also have the above-mentioned usefulness.

[Example] Reference Example 1 Protein levels in serum of 4 liver cancer patients used for determination of carbohydrate chain structure
ma) The findings of liver cancer tissues, serum proteins, and especially cancer marker values of the four patients were as shown in Table 1. The value of AFP, which is widely used for diagnosis as a marker for liver cancer, is approximately 0.10,000.30,000.200.
000 ng/ml, which could be used as a model. The transferrin concentration was approximately 200 to 300 mg/old in all cases.

The total protein amount was determined by colorimetric method, transferrin 1 was determined by 8g immunoassay, GOT and GOP were determined by enzyme activity measurement method, and AF
P and CEA were measured by enzyme immunoassay. Example 1 Analysis (1) Preparation of transferrin Using affinity chromatography using immobilized anti-transferrin antibody (manufactured by E-Y Laboratory) from serum 51 of patients and healthy individuals. Transferrin was separated to a high degree of purity.The separated transferrin was dialyzed against distilled water and then lyophilized.

(2) Release of asparagine-linked sugar chain from transferrin For sample 3B, add 0.3 ml of anhydrous hydrazine to lyophilized transferrin in a sealed test tube.
The reaction was carried out at 00°C for 8 hours. Unreacted hydrazine in the reaction product was distilled off and finally removed by absorption in sulfuric acid under reduced pressure to obtain a partially N-acetylated oligosaccharide.

(3) N-acetylation of asparagine-linked sugar chains The oligosaccharide obtained in this way was mixed with a saturated aqueous solution of sodium bicarbonate at 31°C.
and 300 IL+ of acetic anhydride were added to N-acetylate the amino group of the sugar chain.

Furthermore, paper chromatography was used to remove amino acids by disrupting the protein portion of the glycoprotein.

(4) Reducing end labeling of the oligosaccharide group Add 0.08 N sodium hydroxide at 100° C. to the solidified oligosaccharide group at 0°C to adjust the pH to NaB'1.
14 (25sCi dissolved in dimethylsulfoxide 31)
Add 201L of 201L of 201L, stir, and react at 30°C for 4 hours.After the reaction, add a few drops of IN acetic acid to decompose the excess )laB and 0.5mL of Dowex 5OW-
Desalting was performed using a H◆ (manufactured by Bio-Rad) column. Boric acid was removed by adding methyl alcohol, and 1H-labeled oligosaccharide was obtained by vapor chromatography according to a conventional method.

(5) High-pressure filter paper electrophoresis of tritium-labeled oligosaccharides The tritium [3H]-labeled oligosaccharides were analyzed using Whatman No.

: Spotted on a 1M 1 liter filter paper (manufactured by Whatman) and placed in a high-pressure filter aerophoresis device (manufactured by Shanton Southern, model L-) with pyridine-vinegar #buffer (pH 5,4).
Electrophoresis was performed using 24) under the conditions of 73V/c layer and 1 hour.

The radioactivity pattern was detected with a radiochromatography scanner (Paracard, model 7201). Extract the neutral sugar chain fraction and acidic sugar chain fraction for each peak with distilled water,
Recovered.

(6) Gel filtration chromatography of asparagine-linked sugar chains The terminal sialic acid of acidic oligosaccharides was removed by treatment with sialidase (manufactured by Daikin Oil Co., Ltd.) (50 millimeters, 18 hours).

Bio Ge
t P-4 (hydrophilic gel formed into beads of a copolymer of acrylamide and N,N-methylenebisacrylamide, manufactured by Bio-Rad), diameter 2. Oc■X height 100c
(2) to elute. Gel filtration chromatography is 5
It was carried out at a constant temperature of 5℃ and a flow rate of 181/hour, and the eluate was 1.8■
It was fractionated in liter portions. The elution pattern was measured using a scintillation counter (Beckman LS 7) using tritium as a landmark.
000, manufactured by Beckman).

(7) Separation and fractionation of oligosaccharides using a lectin column The obtained oligosaccharides were first applied to a Con A column (manufactured by Pharmacia),
It was divided into an adsorbing fraction and a non-adsorbing fraction. Elution of the adsorbed fraction was performed with 5mM α-methyl D-mannoside.

Next, each of the obtained fractions was applied to an AAL column (Bioorganism Laboratory, Institute of Medical Science, University of Tokyo) to separate the non-adsorbed fraction, weakly adsorbed fraction,
Divided into adsorption fractions, fraction I was separated from the ConA adsorption fraction.
(AAL non-adsorption), fraction IT (AAL adsorption)
I got it.

Next, the ConA non-adsorbed fraction was divided into three fractions using AAL, and each of the fractions was applied to a DSA column (Bioorganism Laboratory, Institute of Medical Science, University of Tokyo) to obtain fractions III, rv, and v. moreover,
The non-adsorbed fraction and delayed fraction of the DSA column were applied to an E-PHA column (manufactured by Hounen), and fractions Vl, ■,
■, Obtained IX. Fractions IV and ■ were further applied to a DSA column in the presence of almond alpha fucosidase ■.

These procedures and elution patterns are shown in Figure 1.

The eluents used for elution from the AAL column, DSA column, and E-PIIA column were 5 mM fucose, 1% N-acetylglucosamine oligomer, and T.
ris-buffer. In addition, 1 liter of each fraction was collected.

(8) Sequential decomposition of tritiated oligosaccharides by exoglycosidase In order to determine the sequence order and anomeric structure of sugar residues in sugar chains, the 0 reaction in which sequential decomposition by β-galactosidase was performed was performed using β- Galactosidase (
Bioorganic Research Laboratory, Institute of Medical Science, The University of Tokyo) was added to the dried sugar chain in 0.15 M citric acid-phosphate buffer solution pH 6.0.
One drop of toluene was added to the 0 reaction solution, which was carried out by adding in 30 µl of F and reacting overnight, to suppress bacterial growth.

After the reaction, the enzyme reaction was stopped by heating with boiling water for 30 seconds.

The results of this sequential decomposition analysis using β-galactosidase are shown in Figure 2.In Figure 2, the solid curve shows the number of galactose liberated by β-galactosidase decomposition, and the dashed curve shows the number of galactose released by the β-galactosidase decomposition. You can learn the structure of the product. These results are summarized in Table 2.

Table 2 Table 2 (continued) R,: 4GlcNAc B l →4G1cNAcR
t: 4GlcNAc B 1-14 (fuca
l-e6) G1cNAc The structure of transferrin determined as above, adsorption to each lectin column, and abundance ratio (mol%) in total transferrin are summarized in Table 3. In Table 3, F (T, NA, etc. indicate the initials of the patient's or normal person's name)6 As shown in Table 3, normal transferrin, shown at the top of Table 3, accounts for 96% or 97% in normal people, In patients with liver cancer, the amount decreases to 37% to 63%, and abnormal transferrin (transferrin of the present invention) shown in numbers 11 and below in Table 3 appears.Therefore, it is possible to diagnose liver cancer by quantifying the transferrin of the present invention. Table of results (continued) al -14G1cNAc al β -14G1cNAc al β -14GICNAC al β -14GICNAC (9) Methylation analysis of oligosaccharide groups In order to confirm that there are no errors in the identification of the above sugar chain structure, various lectin columns were used. Methylation analysis was performed on the oligosaccharide group before fractionation according to a conventional method.The results are shown in Table 4.

As shown in Table 4, no results inconsistent with the β-galactosidase sequential degradation analysis results were obtained, confirming that the sugar chain structure identified as described above is accurate.

Separation of cancerous transferrin 1 µg of transferrin separated from the blood of a liver cancer patient was dissolved in distilled water containing 0.1% bovine serum albumin, and then added to an immobilized AAL-Sepharose column (1 ml, inner diameter: 5 ml).
n+m) or immobilized DSA-Sepharose column (1
ml, internal diameter 7.5++ m). 3 to 2℃
After a 0 min hold, the sample in the AAL lectin-Sepharose column was eluted with 5 ml of phosphate buffered saline containing 0.1% bovine serum albumin (pH 7.43) and then with the same buffer containing 50 mM fucose at room temperature. The results of this affinity chromatography are shown in Figure 3 A and B. In Figure 3 A and B, the first peak is normal transferrin, and the second peak is the transferrin of this invention having a fucose sugar chain. On the other hand, the sample in the DSA lectin-Sepharose column was mixed with 10+IJ Tr containing 0.1% bovine serum albumin.
It was eluted with is-HCIFJ buffer, pH 7.4 at 2°C, then with a buffer containing 1% N-acetylglucosamine oligomer mixture at room temperature, and then with 0.1N of acetic acid. The results are shown in FIGS. 3C and D. C and D in Figure 3
The first peak is normal transferrin, the second peak is transferrin of this invention having a C-2,4-branched sugar chain, and the third peak is transferrin of this invention having a C-2,6-branched structure. In addition, the transferrin content is determined by the conventional EL method of 1 ml of each portion.
Measured by ISA. Furthermore, A and C in FIG. 3 show the results for transferrin from liver cancer patient HT, and Bi5 and D in FIG. 3 show the results for transferrin from liver cancer patient HH.

[Brief explanation of the drawing]

Figure 1 is a diagram showing the separation of WM chains of transferrin using various lectin columns and the elution pattern of lectin column chromatography, and Figure 2 is a diagram showing the results of β-galactosidase sequential degradation analysis of separated sugar chains. . FIG. 3 is a diagram showing the results of affinity chromatography for separating transferrin of the present invention from transferrin in the blood of a liver cancer patient.

Claims (1)

[Claims] (1) The following general formula [I] ▲There are mathematical formulas, chemical formulas, tables, etc.▼ (However, R_1 and R_2 each independently represent a sugar chain represented by one of the following general formulas [II] to [IV]. (However, cases in which fucose is not bound to GlcNAc adjacent to Asn and R_1 and R_2 simultaneously represent a sugar chain represented by formula [IV] are excluded), and Tf represents a transferrin protein moiety) A novel transferrin indicated by . ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [II] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [III] ▲ There are mathematical formulas, chemical formulas, tables, etc. ▼ [IV]