JPH01120289A - Nonspecific cross reaction antigen - Google Patents

Abstract

PURPOSE:To make it possible to obtain a carcinoembryonic antigen specific antibody, by expressing a human nonspecific crossreacting antigen, etc., in a cell using a genetic manipulation, preparing the antigen in large quantities and absorbing an anticancer embryonal antibody using the antigen. CONSTITUTION:mRNA extracted from a cell strain HLC-1 derived from human lung cancer is fractionated by sugar density gradient centrifugation so as to subject a human NCA (nonspecific crossreacting antigen) gene to cloning and cDNA library is prepared using the 2.5kb-4.0kb mRNA. The NCA gene is liberated and identified therefrom and base sequence thereof is analyzed and determined. Total structure of human NCA is revealed based on the solved base sequence.

Description

[Detailed description of the invention] (Industrial application field) The present invention relates to human carcinoembryonic antigen (carctn).

embryonic antigen: CE
Non-specific cross-antigen (n
onspecific cross reacting
Antigen (hereinafter abbreviated as NCA) protein and genes encoding the protein.

BACKGROUND OF THE INVENTION Carcinoembryonic antigen (CEA) was identified in 1965 by Gotd and Freedman as human colon cancer.
It was discovered as an antigen commonly present in the gastrointestinal tract of a 6-month-old fetus (Gold, P. and Freedman, S.O.
: J, Exp, Med, Kamimata, 439, 1965;
Gold, P., and Freedman, S. O.: J.
, Exp, Med.

, 122, 467. 1965): Currently, it is one of the most widely used tumor markers in clinical testing.

However, a serious problem in using CEA as a tumor marker is the presence of CEA-related antigens that are also present in normal tissues. CEA-related antigens are antigens that are protein-chemically very similar to CEA and immunologically thought to have common antigenic determinants that cannot be distinguished from CEA using ordinary anti-CEA antibodies. A typical CEA-related antigen is known as non-specific cross-antigen (NCA), a glycoprotein with a molecular weight of approximately 90,000 found in the lungs of normal people (v .

n Kleist, S., et al., Proc, Nat.
l.

Acad, Scf, USA, 69.2492.1972
). In addition, NCA-2 (B
Ur t in, P, et al., J., Immuno.
Hl, 111, 1926.1973), biliary glycoprotein-1 (Hl
ycoprotein-1 (BGP-1)) (Sven
berg, T., Int., J.

Cancer, supra 7.588 596.1976) and NFA (normal
facial antigen) (Matsuoka,
Y. et al., Gann, 6th Sat., 203.1973). NFA consists of three molecular species, each of which
-1, NFA-2 and NFcA (normal f
ecal cr.

ss-reacting antigen), but their actual structure and physicochemical properties have not been clarified (Kurok
t, M. et al., Cancer Res.

, supra, 713.1981).

By the way, most or most of the antibodies used in the currently commercially available radioimmunoassay (RIA) or enzyme immunoassay (EIA) kits for measuring CEA recognize the common antigen part of CEA and CEA-related antigens. Therefore, these CEA-related antigens and C
There is a drawback that EA cannot be measured separately. Furthermore, when the same CEA sample is measured using conventional kits, the problem has been raised that the measured values differ between the kits (Kuroki, M., et al., J., Immunol, M.
ethos, 60, 221.1983).

(Problems to be solved by the invention) In order to solve the above problems and develop a method to specifically measure only CEA, it is necessary to elucidate the structure of CEA and CEA-related antigens and develop highly specific antibodies. It is requested that it be manufactured. The primary structure of CEA has already been revealed by Oikawa et al. (Oikawa, S. et al., Biochem.

Biophy3 Res、Commun、、Upper soil、
511.1987 and Japanese Unexamined Patent Publication No. 62-6851)
.

Therefore, the next question is the structure of CEA-related antigens. Among the CEA-related antigens mentioned above, NCA is abundantly contained in the lungs and lungs (von Kad, Sci, US
A, 69, 2492. 1972), and are produced in the white fast cells (mainly granulocytes and monocytes) (Bordes
, M. et al., Eur, J. Cancer, Sat±, 783
．． (1975), and the blood concentration is also quite high, so CEA
This is the antigen that requires the most attention when measuring the blood concentration of CEA or when detecting CEA immunohistochemically.

The amino acid composition of NCA is similar to that of CEA, but NCA contains relatively more methionine, whereas CEA has less. Also, according to a comparison of the N-terminal amino acid sequence, it is identical to CEA up to the 24th position, except that the 21st valine is replaced with alanine (Engva
l l, E, et al., Proc, Natl, A.
CAD, Sci, USA.

75.1670.1987). Furthermore, Oikawa et al.
awa, S. et al., Biochem, Biophys
, Res, Commun,, 146-.

464.1987) and Thompson et al.
mpson, J.A., et al., Proc., Natl., A.
cad, Sci, USA, 84, 2965.1987)
has cloned the 5” terminal @ region of the NCA chromosomal gene, and when this gene was compared with CEA cDNA, the amino acid sequence at position 10,707 from the N-terminus was found to be CE
A and NCA showed high homology of about 89%. N.C.
The molecular weight of A differs depending on the texture, and there are reports that the molecular weight of A is 1/50 for small ones and 130 for large ones.For example, Buchegger et al.
There are 95 NCAs in humans, but 55 in epithelial cells.
(Buch
Egger, F. et al.

Int. J. Cancer, 33,643.1984)
In addition, Grunert et al. isolated 75 NCAs from colon cancer in addition to the above two types of NCAs (Grunert et al.
rt, F., et al., Int., J.

Cancer, 36, 357.1985)* This difference in molecular weight may be due to the slow sugar chain structure, considering that NCA is a glycoprotein like CEA and contains 20 to 50% sugar by weight. First of all, we cannot deny the possibility that multiple NCAs exist as proteins.There are many aspects of the overall structure of NCAs that have not been clarified at present.Therefore, the present inventors By cloning the NCA gene and determining its DNA base sequence, we conducted extensive research in order to clarify the entire amino acid sequence of human NCA.

(Means for Solving the Problems) In order to clone the human NCA gene, the present inventors investigated mR extracted from the human lung cancer-derived cell line HLC-1.
NA was fractionated by sucrose density gradient centrifugation, and 2.5 kb ~
4. A CDNA library was created using Okb mRNA. From there, the NCA gene was isolated and identified, and its nucleotide sequence was further analyzed and determined. Based on the elucidated base sequence, the entire structure of human NCA was clarified, leading to the completion of the present invention.

The gene encoding the human NCA protein of the present invention can be obtained as follows.

First, we used the human lung cancer cell line HLC-1 (Ic
hiki, S. et al., Jpn, J.

Cancer Res, (Gann), 7th, 462
．． 1986) was fractionated by sucrose density gradient centrifugation, and the resulting 2.5 kb to 4. A CDNA library is prepared using Okb mRNA. The cDNA library may be prepared by any known appropriate method, but for example, the method of Young et al. (Young, R.A., et al., Proc. Na.
L 1. 'Acad, Sci, USA, 80,119
4.1983). The outline is shown in Figure 1. Screening for the target clone (clone expressing human CEA protein) from the CDNA library (E. coli) prepared in this way was carried out using anti-NC
This can be done using the A antibody.

That is, it is sufficient to examine the presence or absence of a reaction between the protein expressed in the prepared CDNA library and the anti-NCA antibody.For the expression of the human-NCA protein in E. coli, the NCA protein itself may be expressed. The protein may be expressed as a fusion protein between the NCA protein and another protein, preferably E. coli β-galactosidase. In this case, the activity of β-galactosidase can be used as an indicator.

Next, phage DNA was isolated from the positive clone (E. coli) screened as described above by a conventional method, and the target cDNA (human NC) was isolated by restriction enzyme analysis.
It is confirmed that the cDNA of A) has been inserted into the phage DNA, and the base sequence of the inserted cDNA is determined by a conventional method.

Next, the base sequence of the cDNA obtained as described above was determined by the method of Sanger et al., and the translatable region (open reading area),
By determining the base sequence, the structure (amino acid sequence) of the human NCA gene and human NCA protein can be determined. Human NCA whose nucleotide sequence was thus determined
The structural gene can also be obtained by chemical synthesis.

Furthermore, using the human NCA gene obtained as described above, it is possible to produce human NCA protein in microorganisms such as Escherichia coli and yeast, or in animal cells. That is,
After adding an appropriate promoter region to the 5'' side of the human NCA structural gene obtained above and inserting this into an appropriate plasmid, it is introduced into microorganisms such as E. coli or yeast or animal cells and cultured. Such hill cultivation can be carried out using known techniques, and by producing specific antigenic peptides using microorganisms or animal cells, excluding unnecessary peptide regions, such as peptides common to CEA. Using these peptides as antigens, polyclonal or monoclonal antibodies that specifically react only to human NCA can be produced by conventional methods.Also, by comparing the base sequence with cDNA encoding CEA, Therefore, it is possible to prepare a specific probe that can distinguish between CEA and NCA, and by using such a probe, normal i, 11
It is also possible to accurately determine the mRNA expression levels of CEA and NCA contained in tissue and cancer tissue.

Hereinafter, the present invention will be explained in more detail with reference to Examples.

(Example) From the cell line HLC-1 (2X109 cells) derived from human lung cancer, the method of Schaerblein et al.
M. et al., Biochemistry, 8,529
4.1979) and extracted 5.53 μm of RNA using a 4M guanidium thiocyanate solution. Next, from 4.6I1g of total RNA, 0.5M LiCl, 1
0mM EDTA.

Poly(A)” RNA (mRNA) 8B bound to oligo-dT cellulose using 10 mM 1-Lis-HCl (pH 7°2) containing 0.5% SDS as the binding buffer.
3ttg was isolated (Nakazato, 1.

et al., Meth, Enzym, 29, 431.1974
), 500 pg of the obtained BoIJ(A) + RNA was added to 1
It was subjected to 5% to 30% sucrose density gradient centrifugation and size fractionated. Next, the fractionated 2.5kb to 4゜Okb Po! J
(A) + RNA 10 pg and 0.25 g (7
), f-Rico(dT) +2-18 was used as a primer to insert the cDNA into the λgt, 11 vector to create a cDNA library (Young
,R,A,et al.,Proc,Natl,Acad,
Set, USA, 1st, 1194.1983),
Approximately 60,000 recombinant phage plaques (cDNA library) were obtained per 1 μg of poly(A) + RNA. Incidentally, the outline of the cDNA library preparation method using the above-mentioned λgtll vector is shown in FIG.

Antibody screening was performed using the following method.

A nitrocellulose filter impregnated with 10IIM IPTG and air-dried was placed on the plate containing approximately 50,000 phage plaques obtained in Example (1) above, and the gene product from the inserted cDNA (i.e., β-galactosidase and fusion protein) was induced. After about 2 hours, remove the filter from the plate and add TBS solution (2(1+M
I-Lis hydrochloric acid, pl (7,5°500wM NaCl
) for 5 minutes.The filter was then gently shaken for 20-30 minutes in a blocking solution (TBS solution containing 3% gelatin). Next 2 times T-TB
S solution (TBS solution containing 0.05% Tween-20)
The filters were washed by shaking gently for 5 minutes in a water bottle.

This filter was then soaked in the primary antibody solution and
Shake gently for an hour. In addition, -next antibody solution is 11500 volumes of anti-CEA antibody (next antibody), 1% Escherichia coli extract (Bio-Rad, Catalog No. 170-320).
5) in T-TB containing 1/200 volume and 1% gelatin (Bioland, Catalog No. 170-6537)
As the secondary antibody for the S solution, a rabbit anti-CEA antibody (DAKOPATTS, Denmark, code A115) was used. In addition, this anti-CEA antibody is N
It is already known that it also reacts with CA.

Next, the filter was gently shaken twice in the T-TBS solution for 5 minutes, then immersed in the secondary antibody solution, and gently shaken for 2 hours. The secondary antibody solution was peroxidase-labeled goat anti-heron Ig antibody (Bioland, Catalog No.
, 170-6513) in T-TBS solution containing 1/3000 volume and 1% gelatin.

Next, the filter was gently shaken twice in the T-TBS solution for 5 minutes, then immersed in the coloring solution and gently shaken for 100 minutes. , Catalog No., 170-320
16.5ml of 1)! A liquid dissolved in cold methanol,
83.5 containing 30% H2o2 (hydrogen peroxide) 50 pN
ml of TBS solution was mixed immediately before use.

Three positive clones with purple color were obtained by the above cultivation, and they were λNCAIL λNCA14 and λNCA14, respectively.
It was named 15. These positive clones were plated again and a second screening was performed using the same method as described above, except that - anti-CEA antibody and I
NCA-specific anti-NCA antibody (I
chiki, S., et al., Jpn, J., Cnacer
Res, (Gann), 77.462.1986) was used. This antibody is obtained by absorbing an anti-NCA antibody with a sufficient amount of CEA antigen, and removing the anti-NCA antibody that reacts with CEA. As a result of the second screening, λNCAl
4 reacts only with anti-CEA antibody, and λNCAII and λNC
Since A15 reacted with both antibodies, λNCAl4
contains CEA cDNA, and also λNCAII and λNCA1.
No. 5 was determined to contain NCA cDNA.

λNCAII and λNCA1 containing NCA cDNA
The phage DNA of No. 5 was isolated according to a conventional method. insert DNA
Restriction enzyme Ec attached to both ends of.

There are two types of cDNA fragments obtained by cutting at the R1 site.2. 0.9kbp for tNcA11. 4kbp
In 1λNCA15, they were 1.1 kbp and 1.4 kbp. This indicates that Ec in the NCA cDNA.

This shows that the R1 break point exists. CEA and N
As a result of Southern hybridization using an oligonucleotide probe corresponding to the 26th to 31st amino acids of CA, 0.9 kbp was found for λNCAl1.
(7), and in λNCAl5, a 1.1 kbp fragment hybridized with the probe. Therefore, there is a region encoding the N-terminus of NCA in these fragments, and λ
NCA15 has a longer cDNA. Therefore, two EcoR1 fragments obtained from λNCA15 were subcloned into pUc19, and the entire nucleotide sequence of the cDNA inserted in the resulting plasmid was determined.

The nucleotide sequence was determined using the method of Sanger et al. (Proc, Na
t1. Acad, Sc t, USA, U4,5
463.1977).

Of the two EcoR1 fragments from λNCA15, the plasmid into which the fragment containing the entire structural gene of human NCA was cloned was named pNcA15-3, and Escherichia coli J transformed with this plasmid
M109 strain (JMI09/pNcA15-3) is S
Named 8M294, FERM P-96 was sent to the Microtech Institute.
It has been deposited under the number 87.

The inserted cDNA of λNCA15 consists of 2533 bases,
The DNA sequence contained a long translatable sequence (open reading frame) that could code for 344 amino acids (base sequence number 1 to 1032 in Figure 2).
number).

In Figure 2, starting from Met (-34), Al
The 34 amino acid sequence ending with a(-1) is considered to correspond to a signal peptide based on its structure. Therefore,
h) The structural gene of NCA starts from Lys(1). 1~ from fact Lys to 35th Lys
The 35 amino acid sequence is based on the previously reported N-terminal amino acid sequence of NCA (Paxton, R.J., et al., Pr.
oc.

Natl, Acad, Sci, USA, 8th Sat, 920.
1987). The same authors also reported the amino acid sequences of tryptic or chymotryptic peptides of NCA, but some of them are completely identical, while others have some sequence differences.

These results indicate that human NCA is biosynthesized as a precursor consisting of at least 344 amino acids, and when it passes through the endoplasmic reticulum membrane, the signal peptide consisting of 34 amino acids is removed, resulting in a mature protein consisting of 310 amino acids. It is thought that it is generated as . Furthermore, the 24 amino acids at the C-terminus are highly hydrophobic, and it is thought that this region is buried in the cell membrane, while the 286 amino acids at the N-terminus are exposed to the outside of the cell.

(Effects of the Invention) (11) It has become possible to produce tA in large quantities by expressing human NCA or various NCA fragments in cells (E. coli, yeast, animal cells, etc.) using the gene of the present invention.

CEA-specific antibodies can be obtained by absorbing anti-CEA antibodies using the resulting NCA or NCA fragments.

(2) Structural differences between CEA and NCA have been clarified by the present invention. For example, the amino acid sequence from position 212 to position 214 of NCA is different from the corresponding amino acid sequence of any domain of CEA. Therefore, if a peptide fragment containing this portion is produced using genetic engineering or synthetic methods and used as an antigen, CEA
Alternatively, it is possible to obtain NCA-specific antibodies.

(3) As mentioned above, the present inventors are able to produce human CEA- and NCA-specific antibodies, which can be used for cancer diagnosis, such as cancer screening, definitive diagnosis, determining the progression of cancer, monitoring treatment, and labeled antibodies. It is now possible to localize cancer using the CEA antibody, and it is also expected to be used in treatments such as missile therapy.

(4) According to the present invention, CEA and NCA-specific
A probe can be prepared. If such a probe is used, tissues such as tumors that produce human I-CEA can be detected more specifically by in 5itU hybridization instead of conventional immunohistochemical means using antibodies. It is also possible to do so.

As described above, the fields of application of the present invention are wide-ranging, and its utility value is great.

[Brief explanation of the drawing]

Figure 1 shows cDNA using the λgtll phage vector.
FIG. 2 is a diagram showing an outline of the library construction method. FIG. 2 is a series of sequence diagrams showing the cDNA base sequence and the amino acid sequence of the translatable region of H)NCA. 1 "-Ichirei Tsumugi 2 items (ρu......GG AGCTCA AGCTCCTCT
ACA AAG AGG TGG ACA-32T
CA ATCCCA ACG TTT TAC
ATA AAA TAA GAGGCA GC
A TCT TTA ACA CACCCG
TGT GTT CAATCT 8GA CT
CACCCTGT TCT CACTCCCTG
TTTATA GAA TTG CTCCCT ACC
AGCTGA ACA GGGTTG AACATG
GCT AAA TACAAT GGG TAT CG
CGCT GCT TGG TTA AAA TGG
CTA CACTCA TCTTGT ATCTTG
CCT AAG GTG CGT AGT CCA A
CTGTA GTCATA CTCCCT GGT G
TA GTG TAT TCTGCCATCAAA
TACTGA ATG GTCTCT CTT
TGGATCAGG AGA ACA TCA
TAA CCCATG AAG GAT. GCA CTA 8TG CTT TAA
GAT TTG GTCACA CTCGCT
AAA TGCTAC8TA CTCCAAC
TG AAA TGT “ATT AAA AC
CAAT TTA AM AAA AAA AAG A
ACrTAA TACAGA AGT CCCC
TCTACTTT AACTTT 'TTA AC
CAAT GTA TTT ATT TCT GTG
GTT CTG"GTT CTT GTA TTG T
AT TGCCCA GGG GGA GCTCAAT
TCA TAA ATCACA AAT AAA A
GCCAA TTA (ATCCTT TAG T
GCACCCAG TCA-CTG 8CATTA
1542ATG TACAGT GGT
CCT TTT CAG AGT TGG A
CT 1602TAA TTCAAACCCA
GCCATG CAA AATA to TGCC
1662AGG AGT CTG TGCAGT TT
CTGA CACTTG TTG 1722TGA
GACTAA GTT GTA GAA AT
T AACAAA TGT 1782GACT
CA TTCTTT ATT CTA TTT TAG
TTG GTT 1842CTT GGT ATT
ACCCTCCTA ATA GTCATACTA
1902CTA AAA GCT TTA AAT
GTCTGCATG CAG CCA 1962C
TG GAA TTA CAA AACTCA GAG
AAA TGT GTC2022AAA AGCC
CCAAA TGG TGG TAA CTG
ATA ATA 2082TCA CCT
AGG TGA GCG GAT TGA
GCCAGT GGT 2142TAA
GGA AGA AGA TAG ATCCA
A TTA to AAAA 2202 to C8 G
GA GAT TCCAGT CTA CTT
GAG TTA GCA 2262rAC
AAA AAA GTA ACCTGA AC
T AAT CTG ATG 2322nd T
T CCT TGT TCCAAT TTG
8CA 8M CCCACT 2382 (TC
ACT GTA CTT GTA GAG
TGG TGCTGCTT 2442;CT
CTA TAA CT(A)n,,,,,3'
2533 procedure
Amendment February 2, 1986

Claims (8)

[Claims] (1) Human carcinoembryonic antigen (carcinoembryonic antigen) selected from cDNA prepared from messenger RNA produced by human lung cancer-derived cell line HLC-1.
nonspecific cross react, which is one of the related antigens
A gene encoding an amino acid sequence comprising at least a specific antigenic portion of a protein. (2) The DNA sequence of the above gene is a DNA sequence represented by the following formula I: [There is a gene sequence] or the DNA sequence encodes the same amino acid sequence as the protein encoded by the DNA sequence, as set forth in claim 1. gene. (3) At least a specific antigen of a non-specific cross-antigen protein, which is selected from cDNA prepared from messenger RNA produced by the human lung cancer-derived cell line HLC-1 and is one of the human carcinoembryonic antigen-related antigens. A phage or plasmid that contains a gene encoding an amino acid sequence that contains a sexual moiety. (4) A claim in which the gene contains a DNA sequence represented by the following formula I: [There is a gene sequence] or a DNA fragment containing a DNA sequence that encodes the same amino acid sequence as the protein encoded by the DNA sequence. The plasmid described in Section 3. (5) Claim 3 represented by pNCA15-3
The plasmid described in Section 4 or Section 4. (6) A non-specific amino acid whose amino acid sequence is represented by the following formula II: [There is a gene sequence] (In the formula, X represents a hydrogen atom, a formylmethionine residue, an acetylmethionine residue, or a methionine residue.) Cross-antigen protein. (7) Cultivating microorganisms or animal cells transformed with the plasmids set forth in claims 3 to 5;
A method for producing a peptide or protein with non-specific cross-antigenic activity. (8) The transformed microorganism is JM109/pNCA1
5-3 or SBM294 (FERMP deposit number
-9687).