JPH10165184A - Antibody, gene and production of chimera antibody - Google Patents

Abstract

PROBLEM TO BE SOLVED: To obtain a new gene coding H chain V region or L chain V region of an antibody capable of recognizing a structure containing sialic acid and fucose, and capable of recognizing a cancer-concerning antigen, useful for production, etc., of an antibody useful for a serodiagnosis of the cancer, etc. SOLUTION: This gene is a new gene coding H chain V region or L chain V region of an antibody capable of recognizing a structure containing sialic acid and fucose, capable of recognizing a cancer concerning antigen and useful for production, etc., of an antibody recognizing a structure containing slalic acid and fucose, an application, etc., of which to a serodiagnosis of the cancer are expected. The gene is obtained by producing hybridoma 2D3 by using an acidic glycolipid extracted from meconium as an immunogen, extracting mRNA from the cells by a conventional method, converting the mRNA to a cDNA, performing a polymerase chain reaction(PCR) by using the obtained cDNA as a template and further using a primer, and amplifying the DNA fragment coding H chain V region or L chain V region of the antibody.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a gene for an antibody recognizing a structure containing sialic acid and fucose.

[0002]

2. Description of the Related Art Antibodies that recognize specific sugar chains produced by cancer cells have been used for serodiagnosis of cancer (Yakahira et al.,
Latest inspection volume 2, p61, 1984). Antibodies that recognize cancer-specific sugar chains are used in cancer cells (Koprowski et al.,
Somatic Cell Genet, Volume 5, p95
7, 1979), a tissue-extracted glycoconjugate (Japanese Unexamined Patent Publication No.
4294) or a synthetic sugar chain (JP-A-3-280894)
) Is used as an antigen to immunize mice and the like, to prepare hybridomas, and to make them monoclonal.
The monoclonal antibody thus obtained is characterized on the basis of its antigen binding and affinity.

[0003]

The sugar chains expressed in gastrointestinal epithelial cells during the fetal period and excreted in the meconium are similar in structure to the sugar chains expressed on the cancer cell membrane, and include sialic acid and fucose. It is said that it is a structure including. Therefore,
Antibodies that recognize structures containing sialic acid and fucose
It is expected to recognize cancer-related antigens. In addition, since antigens having sugar chains containing sialic acid are difficult to be eliminated from the blood,
Antibodies that recognize it are expected to be applied to serodiagnosis.

Accordingly, an object of the present invention is to provide a gene encoding an antibody recognizing a structure containing sialic acid and fucose, and the like.

[0005]

Means for Solving the Problems In view of the above problems, the present inventors have made intensive studies on the gene encoding the V region of an antibody recognizing a structure containing sialic acid and fucose. Reached.

[0006] That is, the present invention is a gene characterized by encoding the H chain V region or the L chain V region of an antibody that recognizes a structure containing sialic acid and fucose. The present invention also relates to an antibody H chain V region or L chain V region represented by the amino acid sequence of SEQ ID NO: 1 or 2.
Furthermore, the present invention is a method for producing a chimeric antibody, comprising inserting the above-described gene into a chimeric antibody expression plasmid, culturing a host transformed with the plasmid, and collecting the produced antibody. Hereinafter, the present invention will be described in more detail.

[0007] The gene according to the present invention encodes an H chain V region or an L chain V region of an antibody that recognizes a structure containing sialic acid and fucose. Examples of sugar chain antigens containing sialic acid and fucose include, but are not limited to, sialyl Lewis a antigen and sialyl Lewis x antigen. These are said to be similar in structure to sugar chains expressed on cancer cell membranes, and can be extracted from meconium. The method for extracting acidic glycolipid from meconium is not particularly limited, and extraction with various solvents, purification by column chromatography, and the like may be appropriately employed.

[0008] Such an antibody recognizing a structure containing sialic acid and fucose is not particularly limited, and can be prepared by a conventional method. For example, Japanese Patent Application Laid-Open No. 2-10
The antibody produced by the hybridoma 2D3 described in Japanese Patent No. 4294 can be exemplified.

The H chain V region or L chain V of such an antibody
The gene encoding the region is not particularly limited. For example, a hybridoma producing such an antibody may have a mR
After extracting NA and converting it to cDNA by a conventional method, PC
It can be prepared by amplifying the DNA with R or the like. For example, the nucleotide sequence represented by SEQ ID NO: 1 or 2 can be exemplified as the gene sequence of the H chain V region or L chain V region of the antibody produced by hybridoma 2D3. In addition, the amino acid sequence of the H chain V region or L chain V region of the antibody can be deduced from this nucleotide sequence.
As the chain V region or L chain V region, the amino acid sequence represented by SEQ ID NO: 1 or 2 can be exemplified. Further, a nucleic acid sequence encoding the amino acid sequence represented by SEQ ID NO: 1 or 2 is also included in the present invention.

[0010] Using such a gene of the present invention, a chimeric antibody can be produced. For example, the gene of the present invention may be inserted into a plasmid capable of expressing a chimeric antibody, a host may be transformed with the plasmid, the host may be cultured, and the produced chimeric antibody may be recovered. The specific conditions are not particularly limited, and can be performed by a conventional method.

[0011]

EXAMPLES The present invention will be described below in more detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Isolation of 2D3 Cell Antibody Gene According to JP-A-2-104294, a hybridoma 2D3 was prepared using an acidic glycolipid extracted from meconium as an immunogen. From 1 × 10 ⁶ hybridoma 2D3 cells cultured in RPMI 1640 (manufactured by Gibco), mRN
MRNA of 2D3 cells was prepared using an A extraction kit (manufactured by Pharmacia) and converted to cDNA according to a conventional method.

Next, using this cDNA as a template, polymerase chain reaction (PCR) was performed using the nucleotide sequences of oligonucleotide primers VH1BACK; SEQ ID NO: 3 and VHFOR2SAL1; SEQ ID NO: 4 to obtain the V region of the H chain of the antibody. The plasmid pUC19 was amplified by amplifying a DNA fragment encoding
Was inserted. On the other hand, using the same cDNA as a template, PCR was performed using the nucleotide sequences of the oligonucleotide primers Vκ1BACK; SEQ ID NO: 5, and Vκ1FOR; Plasmid pUC digested with restriction enzyme SmaI
19 was inserted.

Example 2 Determination of Nucleotide Sequence of 2D3 Cell Antibody Gene The nucleotide sequence encoding the V region of the H chain and the V region of the L chain cloned into the plasmid pUC19 prepared in Example 1 was obtained by using a fluorescent DNA sequencer Model 373A (Applied Biosystem) and Taq dye terminator cycle sequencing kit (Applied Biosystem). The nucleotide sequence of each V region of the H and L chains and the amino acid sequence deduced from the nucleotide sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

Example 3 Preparation of Mouse / Human Chimeric Antibody p cloned V region of H chain prepared in Example 1
UC19 was digested with restriction enzymes PstI and SalI, and the digested DNA fragment was digested with restriction enzymes Sse8387I and Sse8387I.
allI digested chimeric antibody H chain expression plasmid pEd
The plasmid was inserted into HCG1 (see JP-A-8-51995) to construct pEdHCG1-2D3. The pUC19 cloned with the V region of the L chain prepared in Example 1 was digested with restriction enzymes PvuII and BglII, and
The NA fragment was inserted into a chimeric antibody light chain expression plasmid pEdHCκ (see JP-A-8-51995) digested with the restriction enzymes PvuII and BclI to obtain pEdHCκ.
-2D3 was constructed. The plasmid used here is
A mouse / human chimeric antibody is produced in a culture supernatant.

[0016] Transient expression in COS cells was confirmed by the following procedure. pEdHCG1-2D3 and pEdHCκ-
2D3 was co-transfected into COS cells by the calcium phosphate method, and the culture supernatant was collected three days later.
The same operation was performed without DNA, and the collected culture supernatant was used as a control. The chimeric antibody titer in the collected culture supernatant was measured.

The measurement of the titer of the chimeric antibody in the culture supernatant was performed as follows. 96 with immobilized anti-human IgG1 antibody
The well immunoplate was blocked with phosphate buffered saline (PBS) containing 0.5% BSA. After washing the plate with PBS, 100 μl of the culture supernatant was added and left at room temperature for 2 hours. After washing the plate with PBS, horseradish peroxidase (HRP) -labeled anti-human Igκ antibody was added and left at room temperature for 2 hours. After washing the plate with PBS,
A commercially available coloring reagent (ABTS, manufactured by Dainippon Pharmaceutical Co., Ltd.) was added, and the degree of color development was measured using a commercially available measuring device (microtiter plate reader MPR4i, manufactured by Tosoh Corporation). As a result, it was found that about 40 ng / ml of the chimeric antibody was produced in the culture supernatant.

Example 4 Reactivity to sialyl Lewis a antigen Cotransfected COS recovered in Example 3
The following procedure confirmed that the cell culture supernatant recognized a structure containing sialic acid and fucose. As a control sample,
The culture supernatant of COS cells into which DNA was not used and the COS cell culture supernatant containing about 60 ng / ml of the anti-T3 chimeric antibody used in Example 3 were used.

A 96-well immunoplate on which sialyl Lewis a antigen was immobilized was blocked with PBS containing 0.5% BSA. After washing the plate with PBS, culture supernatant 1
After adding 00 μl, the mixture was allowed to stand at room temperature for 2 hours. Pre-loaded with PBS
After washing, the sample was added with an HRP-labeled anti-human Igκ antibody, and allowed to stand at room temperature for 2 hours. After the plate was washed with PBS, ABTS was added, and the degree of color development was measured using a microtiter plate-reader.
It was measured by MPR4i. The results are shown in FIG. The vertical axis is 41
The absorbance at 5 nm is shown, and the horizontal axis shows the logarithmic value of the concentration of sialyl Lewis a antigen (ng / well), respectively. In the figure, black circles indicate the chimeric antibody, and white circles indicate the control. As a result, the chimeric antibody bound to sialyl Lewis a antigen containing sialic acid and fucos.

[0020]

According to the present invention, the genes encoding the V region of the H chain and the V region of the L chain of an antibody that recognizes a structure containing sialic acid and fucose are inserted into a chimeric antibody expression plasmid. By culturing a host transformed with the plasmid, it is possible to produce a large amount of an antibody that recognizes a structure containing sialic acid and fucose.

[0021]

[Sequence list]

 SEQ ID NO: 1 Sequence length: 345 Sequence type: Nucleic acid Number of strands: Double strand Topology: Linear Sequence type: cDNA to mRNA Origin Cell line: Hybridoma 2D3 Sequence GTC CAG CTG CAG GAG TCT GGA GGA GGC TTG GTG CAA CCT GGA GGA 45 Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 TCC ATG AAA CTC TCT TGT GCT GCC TCT GGA TTC ACT TTT AGT GAC 90 Ser Met Lys Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp 20 25 30 GCC TGG ATG GAC TGG GTC CGC CAG TCT CCA GAG AAG GGG CTT GAG 135 Ala Trp Met Asp Trp Val Arg Gln Ser Pro Glu Lys Gly Leu Glu 35 40 45 TGG GTT GCT GAA ATT TAT AAC AAA GCT GGT AAT CAT GCA ACA TTC 180 Trp Val Ala Glu Ile Tyr Asn Lys Ala Gly Asn His Ala Thr Phe 50 55 60 TAT GCT GAG TCT GGG AAA GGG AGG TTC ACC ATC TCA AGA GAT GAT 225 Tyr Ala Glu Ser Gly Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp 65 70 75 TCC AAA AGT AGT GTC TAC CTG CAA ATG AAC AGC TTG AGA GCT GAA 270 Ser Lys Ser Ser Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu 80 85 90 GAC GCT GGC ATT TAT TAC TGT ACC TCC CGG TTT GCT TAC TGG GGC 315 Asp Ala Gly Ile Tyr Tyr Cys Thr Ser Arg Phe Ala Tyr Trp Gly 95 100 105 CAA GGG ACC ACG GTC ACC GTC TCC TCA GCG 345 Gln Gly Thr Thr Val Thr Thr Ser Ser Ala 110 115.

SEQ ID NO: 2 Sequence length: 318 Sequence type: number of nucleic acid strands: double-stranded Topology: linear Sequence type: cDNA to mRNA origin Cell line: hybridoma 2D3 sequence GAC ATT CAG CTG ACC CAG TCT CCA TCT TCC ATG TAT GCA TCT CTA 45 Asp Ile Gln Leu Thr Gln Ser Pro Ser Ser Met Tyr Ala Ser Leu 1 5 10 15 GGA GAG AGA GTC ACT ATC ACT TGC AAG GCG AGT CAG GAC ATT AAT 90 Gly Glu Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn 20 25 30 AGG TAT TTA AGC TGG TTC CAG CAT AAA CCA GGG AAA TCT CCT AAG 135 Ser Tyr Leu Ser Trp Phe Gln His Lys Pro Gly Lys Ser Pro Lys 35 40 45 ACC CTG ATC TAT CGT GCA AAC AGA TTG GTA GAT GGG GTC CCA TCA 180 Thr Leu Ile Tyr Arg Ala Asn Arg Leu Val Asp Gly Val Pro Ser 50 55 60 AGG TTC AGT GGC AGT GGA TCT GGG CAA GAT TAT TCT CTC ACC ATC 225 Arg Phe Ser Gly Ser Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile 65 70 75 AGC AGC CTG GAG TAT GAA GAT ATG GGA ATT TAT TAC TGT CTA CAG 270 Ser Ser Leu Glu Tyr Glu Asp Met Gly Ile Tyr Tyr Cys Leu Gln 80 85 90 TAT GAT GAG TCT CCT CGG ACG TTC GGT GGA GGG ACC AAG CTG GAG 315 Tyr Asp Glu Ser Pro Arg Thr Phe Gly Gly Gly Thr Lys Leu Glu 95 100 105 ATC 318 Ile 106.

SEQ ID NO: 3 Sequence length: 22 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence AGGTSMARCT GCAGSAGTCW GG22.

SEQ ID NO: 4 Sequence length: 46 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GGGGGTCGAC GCTGAGGAGA CGGTGACCGT GGTCCCTTGG CCCCAC 46.

SEQ ID NO: 5 Sequence length: 24 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GACATTCAGC TGACCCAGTC TCCA24.

SEQ ID NO: 6 Sequence length: 22 Sequence type: Number of nucleic acid strands: Single strand Topology: Linear Sequence type: Other nucleic acid Synthetic DNA sequence GTTAGATCTC CAGCTTGGTC CC22.

[Brief description of the drawings]

FIG. 1 is a diagram showing the reactivity of a chimeric antibody with a sialyl Lewis a antigen performed in Example 4.

Claims (7)

[Claims] 1. A gene encoding an H chain V region or an L chain V region of an antibody that recognizes a structure containing sialic acid and fucose. 2. The gene according to claim 1, wherein the structure containing sialic acid and fucose is present in acidic glycolipid. 3. The gene according to claim 2, wherein the acidic glycolipid is extracted from meconium. 4. The gene according to claim 1, which codes for the amino acid sequence of SEQ ID NO: 1 or 2. 5. The gene according to claim 1, which is represented by the nucleotide sequence of SEQ ID NO: 1 or 2. 6. An antibody H chain V region or L chain V region represented by the amino acid sequence of SEQ ID NO: 1 or 2. 7. The method according to claim 1, wherein the gene according to any one of claims 1 to 5 is inserted into a chimeric antibody expression plasmid, a host transformed with the plasmid is cultured, and the produced antibody is recovered. A method for producing a chimeric antibody.