JPH01131122A - Purified tfs-4 antigen derived from human brain and method for purification - Google Patents

Abstract

PURPOSE:To provide TFS-4 antigen separated from human brain and useful for the efficient production of an antibody useful for the investigation of the nature and origin of pulmonary parvicellular cancer and the diagnosis and remedy of the cancer. CONSTITUTION:The objective TFS-4 antigen having a partial structure of formula from an amino terminal and having a molecular weight of about 124 K-dalton can be produced by (1) precipitating a solubilized human brain with sulfuric acid, (2) treating the precipitated substance having antigen activity with a DEAE CL-6B sepharose column, (3) treating the column with a solution containing 150-300m-mol of sodium chloride in a tris-buffer solution to elute a substance having antigen activity, (4) concentrating the eluted fraction, (5) bonding to immuno-affinity column, (6) eluting with a glycine-HCl buffer solution and (7) subjecting to gel-filtration high-performance liquid chromatography.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a method for isolating and purifying TFS-4 antigen, which is a small cell lung cancer antigen derived from human brain, and TFS-4.
Concerning antigens. Regarding the production of TFS-4 hybridoma, see Okabe et al.
al antlbodies to 5urf'ace
an-tlgens of'sguall cell
l carclnoia o ('the lung)
'[Cancer Research (Cancer Res)
, Vol. 44, pp. 5273-5278, 1984].

That is, the antibody is produced as a culture supernatant or ascites fluid of Ba1b/c mice. 10% TFS-4 hybridoma cells
RPM11640 medium supplemented with beef tallow serum [Flow Labo
(Ra-Torles Inc., Rockville, MD) at a concentration of 1 x 105 cells/ml for 3 days. The prepared medium is centrifuged at 10009 for 10 minutes and the supernatant is frozen until use. I x 10'
TFS-4 hybridoma cells are injected intraperitoneally into Ba1b/c mice pretreated with 0.5 mAb of Bristane.

After 10 days, ascites fluid is collected and subjected to 10009 centrifugation for 20 minutes to remove cell debris. Place the supernatant on ice for 50 min.
Clarify by precipitation with 20.000% ammonium sulfate and centrifugation for 20 minutes. Precipitate, 4
It was dissolved in 20 mmol Tris-HCl containing 0 mmol sodium chloride (buffer A) and dialyzed against 20 mmol Tris-hydrochloric acid containing 20 mmol sodium chloride (buffer B).

The dialyzed sample was applied to a DB52 column equilibrated with Buffer B and eluted with a gradient of 20-200 mmol sodium chloride. Both parts were immunoblotted (io
+unoblotting) and the peak fractions are concentrated by Amicon ultrafiltration using YMIO membranes to produce the TFS-4 monoclonal antibody.

The biochemical characteristics of small cell lung cancer have been extensively studied.
Earrings are APUD (Aa+ine)
Precursor Uptake and Decar
revealed a wide range of peripheral lineages of cells with boxylator properties [Pierce, N.G.
rse, A, G, E,) The Cytochemistry and Ultrastructural Polypeptides Hormone Broducing Cells of Genie Pnee Day CiJ-s and the Embryologic Physiologic and Pathologic Implications of the Concept, (T
he Cyto-chemistry and utt
rastructure of polypcpttd
chormon-producing cells o
f the APUD 5eries and the
embryologic physiologic a
nd patholo-gic implications
ns of' the concept)'Journal of Histochemistry and Cytochemistry (J, Hlstochem, Cytochem,
), Vol. 17, pp. 303-313, 1989].

It has been shown that small cell lung cancer exhibits APUD cell characteristics (Hattori et al., “Oat cell carcinoma of the lung”, Cancer, vol. 30, 101).
4-1024-, 1972).

Although the biochemical characteristics of small cell lung cancer are highly studied,
There is very little knowledge about surface antigens specifically expressed on small cell lung cancer cells.

The TFS-4 monoclonal antibody recognizes a unique antigen with a molecular weight of approximately 124 kilodaltons that is selectively expressed on small cell lung carcinoma and neural tissue ta (brain-associated cancer antigen: BACA). Although small cell lung cancer cells exhibit many APUD cell characteristics, it is not known whether small cell lung cancer cells arise from neural crest or hepatic lobar structures like other bronchial cancers.

Brain-associated cancer antigens are expressed on oligotinoid tumors, neuroblastoma, and retinoblastoma [Watanabe et al.
Small Cell Lang Cancer from Non-
Small Cell Lang Cancer (Monocl.

-nal Autibody That Dlstin
guishcs Small-CellLung Ca
ncer From Non-3ffall Ce1
Lung Cancer), Cancer Research, scheduled for publication in February 1987].

In normal tissues, brain-associated cancer antigens were detected in neural tissue, cardiac muscle, and endocrine cells. This observation suggests that small cell lung cancer specifically expresses antigens common to brain and endocrine tissue (Cancer Research, supra). In normal tissues, TFS-4 monoclonal antibody reacted with central tissues. It has common features in antigenic properties in human brain and lung small cell carcinomas: trypsin sensitivity, thermostability, and neuraminidase resistance. It suggests that both are the same peptide.

The study of antigens that are selectively expressed in small cell lung cancer is of great importance to understanding cancer.

TFS-4 antigen was successfully solubilized from human brain cell membrane fraction with 0.5% Nonidet P-40. A comparison of the anti-TFS-4 activity in the lysate and the anti-TFS-4 activity harvested daily in ammonium sulfate precipitation shows that about 16% of the antigen was solubilized with Nonidet P-40. Since the solubilizing agent interferes with the adsorption of proteins to the nitrocellulose membrane, the antigen concentration in the Nonidet P-40 solubilized fraction can be determined by immunoplotting.
(oblotting).

The 50% ammonium sulfate precipitate is believed to contain most of the antigenic activity since only trace activity is detected in the supernatant. In this step, the lipids contained in the brain homogeneous material are separated by forming a layer floating in the ammonium sulfate solution after centrifugation. 60% antigen applied to a DEAE Sepharose CL-8B column was eluted in fractions containing 150-300 mmol sodium chloride in Tris buffer (
Figure 1). TFS-4 antigen was bound to an immunoaffinity column and eluted with glycine-HCl buffer (al12.5). The active fraction was concentrated by freezing and transferred to TSK G40.
00SW column. Gel permeation high performance liquid chromatography (IIPLc) revealed three main peaks (Figure 2). Only the first peak fraction showed antigenic activity. Purified protein from gel permeation HPLC showed a single protein band on 5DS-PAGE by silver staining technique. The molecular weight is estimated to be approximately 124 kilodaltons (Figure 3)
. In the total purification step, 8.6% of the antigen was recovered, and the specific activity was increased by 1000 times.

As described above, TFS-4 antigen from human brain tissue was purified by a series of ion exchange chromatography, FE, immunoaffinity chromatography and gel permeation HPLC.

Purified TFS-4 antigen showed a single protein band with a molecular weight of 124 kilodaltons in 5O8-polyacrylamide gel electrophoresis under reduced conditions.

A previous study demonstrated that the antigen in small cell lung cancer has a molecular weight of approximately 124 kilodaltons in SOS polyacrylamide gel electrophoresis in reduced conditions (Cancer Research, supra). This observation supports the idea that the antigen in small cell lung cancer and the antigen in the brain represent structurally related peptides. Antigens isolated from human brain are of interest in the study of the nature and origin of small cell lung cancer. Furthermore, the purified TFS-4 antigen of the present invention is useful for efficiently producing TFS-4 antibodies that are effectively used in the diagnosis or treatment of small cell lung cancer.

Determination of the antigen titer of TFS-4 antigen is shown below.

Two microliters of serially diluted samples were adsorbed onto a nitrocellulose membrane (Blo-Rad, Richmond, Calif.) and air dried. Membranes were pretreated with 1% gelatin in phosphate-buffered saline for 20 minutes to prevent nonspecific binding of antibodies. The membrane was then reacted with TFS-4 antibody for 1 hour at room temperature. After washing three times with phosphate-buffered saline, the membrane was treated with biotinylated mouse immunoglobulins (blotinylated and
ti-mouse IgG) [Vec
tor), Burllngame,
California] (at a ratio of 1:2000 in phosphate-buffered saline containing 1% gelatin) for 30 minutes, washed three times, then avidin D peroxidase [Vector] (1% % gelatin in phosphate buffered saline at a ratio of 1:2000).

Finally, the membrane was reacted with HRP reagent (Bio-Rad) according to the manufacturer's instructions. Refers to the dilution of the sample that gives the same antigen titer and reaction intensity as the standard sample. 1 unit means undiluted Nonldet P-
This defines the antigenic activity of the 40 solubilized fractions.

The measurement of protein concentration is shown below.

Protein concentrations were calculated using Bovine Serum Albumin as a standard unless otherwise stated.
Lovry, O.H. et al., Protein Determination with Folln Phenol Reagent, Journal of Biological Chemistry, J.Bol. Chem, 193
Vol. 265-275, 1951)].

Table 1 below shows the effect of each purification step on TFS-4 antigen from human brain.

Table 1 Purification process of TFS-4 antigen from human brain
Quantity Precipitation concentration Precipitation value Efficacy Specification Efficacy value Recovery rate Homogeneous materialization 140ml 7.7wg/
ml 1080mg 3211 4.2
4411011 -Note 1 NP-401307,71000 (1 unit) -m=DE
AE 56 1.0 56 8 8
．． 0 450 81 anti-TFS-440,0530,2
1264120026035HPLCl O,01B
0.016 64 4000 64
8.8 Note 1 Nonidet P-40 interfered with the adsorption of the antigen to the nitrocellulose paper, so its efficacy in both parts was the least. The reaction of both undiluted samples is defined as 1 unit. Specific and total activities were not shown due to obvious underestimation.

Note 2 Protein concentration was determined by absorbance at 280 nm.

The amino acid sequence of the TFS-4 antigen purified by the method of the present invention was analyzed using an ABI Gas Face 470A sequencer, and its identification was performed using an ABI 120A HPLC.
u-G 1 n-Va 1-As p-Ile-Va
IP ro-8e r-G 1 n-G 1y-G 1
It was found to have a partial structure of u-Ile-8cr-Val-Gly-Glu.

FIG. 1 depicts DEAE chromatography of TFS-4 antigen.

Ammonium sulfate to precipitate proteins was added to 2 mmol phenylmethylsulfonyl fluoride, 0.01% EDTA.
, 0.005% Nonidet P-40 and 0.02%
The sample was applied to a DEAE Sepharose CL-6B column (4×8 cm) equilibrated with 100 mmol of Tris buffer (pH 7.4) containing 50% sodium azide. Elution is carried out with a linear gradient of 50-500 mmol sodium chloride in Tris buffer. Antigenic activity is detected by immunoblotting using 1'FS-4 as the initial antibody.

Figure 2 represents gel permeation HPLC of T)''S-4 antigen.

Frozen material from the immunoaffinity column was resolved into 10.0 microliters of distilled water and loaded onto a TSK G400OSW column (0.7 microliters) equilibrated with phosphate-buffered saline.
II PLC at 5XlGcm). Elution was performed with phosphate buffered saline at a flow rate of 0.5 ml/min, and 0.5 IIli of each fraction was collected.

Figure 3 shows purified TFS from human brain in SDS polyacrylamide gel electrophoresis detected by silver staining.
-4 antigen analysis.

0.16 micrograms of purified TFS-4 antigen under reducing conditions (with 2.5% mercaptoethanol) 5DS-P
Subjected to AGE (T-7, 5%). The gel was stained by silver staining technique. The molecular weight marker is myosin,
It contained 01.2 micrograms each of β-galactosidase, phosphorylase B1, bovine serum albumin, and ovalbumin.

Examples of the isolation and purification of TFS-4 antigen from human brain according to the present invention are shown below.

Example 1) Method for Solubilizing TFS-4 Antigen from Human Brain Human brains are obtained at autopsy and frozen at -70°C within 2 hours after death. 5H brain in 0.25 mol sucrose, 2 mmol phenylmethylsulfonyl fluoride, 0.01% ED.
Contains TA and 0,02% sodium azide 15
Solubilization was carried out using a Polytron mixer in the presence of 0 milliliters of phosphate buffered saline. The solubilized material was centrifuged at 10,000 g for 20 minutes, and the membrane fraction was collected as a supernatant.

Nonidet (No.
nidet) P-40 is added to the supernatant to 0.5% over 30 minutes with stirring at 4°C.

The mixture was centrifuged at 100.0009 for 60 min and drained.

Ammonium sulfate precipitated as described below [Khosravl, M. et al.
Cultyard Human Melanoma Cells (Pur
ification of melanoma-ass
induced oncof'ctal antige
n gP87 from spent medium a
nd cell homogenate of' cu
Ltured hul! lanmelanoma ce
lls), European Journal on Cancer Clinical Oncology (Eur, J. Cancer
r C11n, 0neo1. ), vol. 20, 1163-1
p. 175, 1984].

2) Ammonium Sulfate Precipitation Preliminary tests showed that 50% ammonium sulfate precipitated most antigenic activity. Ammonium sulfate powder was added smoothly with constant stirring until a 50% concentration was reached. After stirring for 30 minutes at 4°C, the mixture was
It was left for 0 minutes. The precipitated protein is 20.0009 and 2
Centrifuge for 0 min to collect 2 mmol of phenylmethylsulfonyl fluoride, 0.01% EDTA, 0.005%
100 mmol of Tris-HCl (pH 7.4) containing Nonidet P-40 and 0.02% thorium azide.
) 23a+I (Tris buffer).

This suspension was then dialyzed against four changes of the Tris buffer.

3) DHAE ion-exchange chromatography Both ammonium sulfate precipitated fractions were pre-equilibrated with Tris buffer and applied to a DEAE CL-6I3 Sepharose column (4×3 cm). The column was washed with 300 ml of Tris buffer containing 50 mmol sodium chloride and the bound components were washed in 8 ml increments at a flow rate of 80 ml/hr using a linear gradient of 150-300 mmol sodium chloride in the buffer. It eluted in the image. The fractions were evaluated for antigenic activity against TFS-4 by immunoblotting, and the fractions with efficacy were identified as YM100.
It was concentrated by Amicon ultrafiltration using a membrane.

4) Immunoaffinity chromatography BALB/
The TFS-4 monoclonal antibody produced as C mouse ascites was purified by ammonium sulfate precipitation and DE-52 ion exchange chromatography as described above.

Five milligrams of purified immunoglobulin was combined with 1+al of cyanogen bromide-activated Sepharose 4B according to the manufacturer's instructions. The enriched fraction containing TFS-4 antigen activity was
Sepharose 4B conjugated with -4 antibody (IX3(7))
was punished. After extensive washing with Tris buffer, the antigen was
Extracted with millimolar glycine-hydrochloric acid (pH 2,5).

The active fractions were pooled, immediately dialyzed against 100 mmol phosphate buffer, and lyophilized.

5) Gel permeation HPLC The frozen material from immunoaffinity chromatography was resolved into 100 microliters of distilled water and TSKc4ooosw column (0,75X 60cm) equilibrated with phosphate buffered saline (Toyo Soda, Tokyo, Japan). He was given IIP and LC.

Elution was performed with the same buffer at a flow rate of 0.5 ml/min, and 0.51 fractions were collected. Each fraction was evaluated for antigenic activity by immunoblotting.

6) 5O3-PAGE analysis of purified antigen The active fraction from gel-permeable HPLC was analyzed under reducing conditions (25
% mercaptoethanol), analyzed by 5DS-polyacrylamide gel electrophoresis (T-7, 5%) [Laemll, Cleavage of Structural Proteins Doering the Assembly]
of the head of bacterophage Ta (
Cleavage ol' 5structural p
roteinsduring the asseLIl
bly of' the head ol' bact
ero-phago T4), Nature (Nat
ure) Vol. 227, pp. 680-885, 1970]. Proteins were detected by silver staining (Gin Staining Daiichi Kagaku, Tokyo, Japan). Molecular weight markers were Bio-Rad (Bio-
Rad).

[Brief explanation of the drawing]

Figure 1 is the DEAE chromatogram of the TFS-4 antigen, Figure 2 is the gel permeation high performance liquid chromatogram of the TFS-4 antigen, and Figure 3 is the SDS polyacrylamide gel electrophoresis detected by silver staining. Figure 2 is an analysis photograph of purified TFS-4 antigen from human brain. 7 engravings (no change in content) Fig. 1 - Go, force iii book (7': no change in content) Retention time Fig. 2 Fig. 3 hand fi'll: i'+1] iTE DD 1986 3
month t3

Claims (2)

[Claims] (1) Leu-Gln-Val-Asp from the amino terminal
-Ile-Val-Pro-Ser-Gln-Gly-
A TFS-4 antigen having a partial structure of Glu-Ile-Ser-Val-Gly-Glu and a molecular weight of about 124 kilodaltons. (2) After solubilizing human brain, antigen active substances are precipitated with ammonium sulfate, and the precipitated substances are precipitated with DEAECL-6.
The antigen active substance was eluted from the column using a buffer containing sodium chloride, and the eluted fraction was concentrated and bound to an immunoaffinity column. After elution with a glycine-hydrochloric acid buffer, the column was subjected to gel permeation high performance liquid chromatography (HPLC). TFS from human brain by subjecting to
-4 Method of purifying antigen.