KR100473713B1 - ANTI-CARCINOEMBRYONIC ANTIGEN MONOCLONAL ANTIBODY AND SINGLE CHAIN Fv PREPARED THEREFROM - Google Patents

Abstract

The present invention relates to anti-carcinoembryonic agtigen monoclonal antibodies and single-chain Fv, more particularly anti-carcinoma produced by hybridoma cells (Accession No. KCLRF-BP-00053). Comprising an antigenic monoclonal antibody and a VH, glycine or cystine having an amino acid sequence of SEQ ID NO: 2 of the Sequence Listing and a linker peptide having 13 to 17 amino acids and a VL having an amino acid sequence of SEQ ID NO: 4 of the Sequence Listing Anti-carcinoma fetal antigen single chain Fv. According to the present invention, an anti-cancer fetal antigen single chain Fv can be obtained that is soluble and can be expressed in a host cell and secreted out of the cell with affinity comparable to the parent antibody.

Description

ANTI-CARCINOEMBRYONIC ANTIGEN MONOCLONAL ANTIBODY AND SINGLE CHAIN Fv PREPARED THEREFROM}

The present invention includes an anti-carcinoembryonic agtigen monoclonal antibody, a heavy chain variable region polypeptide thereof and a DNA encoding the same, a light chain variable region and a DNA encoding the same, and the heavy chain variable region polypeptide and a light chain variable region polypeptide. The present invention relates to a single-chain Fv constituted by

Carcinoma fetal antigens were first described by Gold and Freedman ( J. Exp. Med. , 121, 439-462, 1965). Carcinoma fetal antigen is a glycoprotein with a molecular weight of about 200 kDa and the sugar content is known to be about 50 to 60% by weight. Carcinoma fetal antigens appear during normal fetal development, but at very low levels in the visceral tract of normal adults. Carcinoma fetal antigens are found in almost all colon cancers and epithelial cancers such as breast, lung and uterine cancers. Anti-tumor fetal antigens have been widely used for the diagnosis of these cancers, and recently, researches for developing cancer therapeutic agents using antibody engineering techniques have been actively conducted (Juweid M et al., Cancer , 78, 157-168). , 1996; Hu S et al., Cancer Res , 56, 3055-3061, 1996; Mayer A et al. , J. Immunol.Methods, 231, 261-273, 1999). Thus, anti-tumor fetal antigen monoclonal antibodies can be very useful in targeting various types of cancer known to express carcinoma fetal antigen (CEA).

Single-chain Fv refers to the binding of the heavy chain variable region (VH) and the light chain variable region (VL) of the antibody to a flexible peptide linker that facilitates the functional folding of the antigen binding site. It is maintained as it is (Bird RE et al . , Trends Biotechnol. , 9, 132-137, 1991). Here, Fv (25kDa) is a heterodimer of two chains of antibody variable region domains (VH and VL) and is the minimum structural component of the antibody required for antigen binding activity. However, Fv has a problem of dissociation at low protein concentration and physiological conditions (Glockshuber, R. et al., Biochemistry , 29, 1362, 1990). In order to solve this problem of stability, a single polypeptide Fv, a single polypeptide, has been studied by introducing a linker peptide such that separate VH and VL domains are linked (Bird, RE et al., Science , 1988, 242, 423). Since single-chain Fv is expressed as a single protein, it can be expressed in several hosts, such as E. coli or mammalian cell lines. It has also been studied not only as a function of tumor-position, but also as a target for fusion protein formation with therapeutic agents (Chester KA et al . , Cancer Chemother. Pharmacol. , 46, 8-12, 2000).

However, when expressed in a host such as E. coli , such single-chain Fv is aggregated with each other and produced in the form of insoluble aggregate or multimer, which must be denatured and refolded for purification. There was a problem. Such multimeric single-chain Fvs are reported to be produced by intermolecular VL / VH pairing between two or more single-chain Fvs (Kott, A. et al. Eur. J. Biochem. , 221, 151, 1994; Holliger, P. et al . , Proc. Natl Acad. Sci. USA , 90, 6444, 1993; Whitlow, M. et al . , Protein Eng. , 7, 1017, 1994; Desplancq, D. et al . , Protein Eng. , 7 , 1017, 1994). It is known that the formation of the multilayer depends on the length of the linker and each variable region domain sequence.

In order to solve the above problems, the present invention provides an anti-cancer fetal antigen monoclonal antibody with high affinity and high affinity when expressed in a host cell and expressed in a soluble form, and the parent antibody. It is an object of the present invention to provide an anti-carcinoma fetal antigen single chain Fv that can be expressed in a host cell and secreted out of the cell with an equivalent affinity.

It is also an object of the present invention to provide an expression vector and transformant for expression of the anti-carcinoma fetal antigen single chain Fv.

The anti-carcinomatous antigen monoclonal antibody of the present invention comprises a heavy chain variable region polypeptide and a light chain variable region polypeptide having the amino acid sequence of SEQ ID NO: 4 of SEQ ID NO: 2 of the sequence listing It is characterized in that the configuration. Preferably, it is an anti-tumor fetal antigen monoclonal antibody produced by said hybridoma cells (Accession No. KCLRF-BP-00053).

In addition, the DNA of the present invention is characterized by encoding the heavy chain variable region polypeptide of the anti-carcinoma fetal antigen monoclonal antibody. Preferably, the nucleotide sequence of SEQ ID NO: 1 in the sequence listing.

In addition, the DNA of the present invention is characterized by encoding the light chain variable region polypeptide of the anti-carcinoma fetal antigen monoclonal antibody. Preferably, the nucleotide sequence of SEQ ID NO: 3 in the sequence listing.

In addition, the anti-carcinomatous antigen single chain Fv of the present invention is composed of VH, glycine or cystine having the amino acid sequence of SEQ ID NO: 2 of the Sequence Listing, and a linker peptide having 13 to 17 amino acids and of SEQ ID NO: 4 of the Sequence Listing. It is characterized by comprising a VL having an amino acid sequence. Said anti-carcinomatous antigen single chain Fv preferably has the amino acid sequence of SEQ ID NO: 6 in Sequence Listing.

The present invention also relates to a DNA having a nucleotide sequence encoding the amino acid sequence of SEQ ID NO: 6 in the sequence listing. The DNA preferably has the nucleotide sequence of SEQ ID NO: 5 in Sequence Listing.

The present invention also relates to an expression vector comprising the DNA. Preferably the expression vector is a pCANTAB 5E vector.

The present invention also relates to a transformant transformed into a host cell using the expression vector. The host cell may be any of bacteria, plant cells, insect cells and animal cells, preferably Gram negative bacteria, more preferably E. coli .

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are described for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example

Example 1 Construction of Hybridomas Producing Anti-tumor Fetal Antigen Monoclonal Antibodies

Human carcinoembryonic antigen (CEA) (Calbiochem, Darmstadt, Germany) was immunized to Balb / c mice, and spleens of immunized mice were extracted to separate B cells. The isolated B cells were fused to SP2 / O myeloma cells using PEG to prepare hybridomas ( Kor J Biochem , Oh YS et al ., 1988, 20, 43-48). Clones secreting anti-carcinoma fetal antigens were selected from the hybridomas.

Screening of the clones was carried out by measuring the antibody binding to fetal cancer antigens using ELISA method as follows. First, carcinoma fetal antigen (Calbiochem, Darmstadt, Germany) was diluted with 2 μg / ml phosphate buffered saline (PBS), and 100 μl of this diluted carcinoma antigen was plated in a 96-well plate (Nunc Immuno Module, Maxisorp, Roskilde, Denmark). Aliquots and incubated overnight at 4 ° C. Plates were washed once with PBST (PBS with 0.05% Tween 20, pH 7.4), then 200 μl of 1% bovine serum albumin (BSA) PBS was added and incubated for 1 hour at room temperature. After washing, 100 μl of hybridoma culture solution was added thereto, and the cells were incubated at room temperature for 2 hours. After a second wash with PBST, diluted goat anti-mouse IgG (Fab specific) -peroxidase conjugate (Sigma, St Louis, MO, USA) in 1% BSA-PBS was added and kept at room temperature for 1 hour. Incubated. Wash once more with PBST, then add 100 μl of 2,2′-azino-bis (3-ethylbenzothiazoline) -6-sulfonic acid (ABTS) solution (KPL, Gaithersburg, MD, USA) and light density at 405 nm (OD) was measured. In this way, eight clones were selected. These clones were named C1 to C8, respectively.

DMEM (Dulbecco's modified Eagle's minimal essential medium: Life Technologies, Gaithersburg, D.), containing 10% bovine serum (FBS) from Life Technologies, Gaithersburg, MD, USA MD, USA) The culture was carried out in a medium, and the isotype of the antibody obtained therefrom was determined using a mouse monoclonal antibody isotype determination kit (Roche Diagnostics Corp., Indianapolis, IN, USA). Isotype determination results for two clones of the eight antibodies are shown in Table 1 below.

Table 1. Subtypes of Anti-tumor Fetal Antigen Monoclonal Antibodies

Clone number Isotype C2 IgG1, κ C7 IgG1, κ

As shown in Table 1, both clones consisted of heavy chain IgG1 and light chain κ. Among the eight clones established, the C7 clone showed the highest affinity (Hybridoma, Kim SH et al., 2001, 20, 265-272), and the human colon cancer cell line LS 174T (obtained by Seoul National University Cancer Institute Cell Line Bank) In the western blot used, C7 showed the best response (FIG. 1). In Figure 1, lane A represents clone C2, lane B represents clone C7, lane C represents clone C1, and the amount of antibody used was 45 ng / ml.

In the above, hybridomas producing antibody C7 showing the highest affinity were deposited on the Korea Cell Line Research Foundation (Accession No. KCLRF-BP-00053) on December 22, 2001.

Example 2 Cloning of Anti-tumor Fetal Antigen Monoclonal Antibody Gene

Hybridomas were incubated in DMEM medium at 1 × ^{10 8 and} RNA was isolated using Trizol (Life Technologies) according to the instructions. For cloning and expression of antibody genes such as cDNA synthesis, amplification of antibody VH and VL DNA, binding of two VH and VL DNAs, and binding to pCANTAB 5E vectors, the Mouse ScFv and Expression Module (Amersham Pharmacia) It was performed according to the method presented. The detailed process is as follows.

1. Isolation of RNA

1 ml of trizol per 5-10 × 10 ⁶ hybridoma cells were added, the cells were freed by pipette, and then waited at room temperature (15 ° C.-30 ° C.) for 5 minutes. Next, 200ul of chloroform per 1ml of Trizol was mixed well, the lid was closed and shaken vigorously for 15 seconds by hand, and then waited at room temperature for 3 minutes. Thereafter, the mixture was centrifuged at 15,000 rpm for 15 minutes at 2 to 8 ° C, and only the supernatant was transferred to a new tube. 500 ml of isopropyl alcohol was added per 1 ml of Trizol, mixed well, and then waited at room temperature for 10 minutes. Thereafter, the mixture was centrifuged at 15,000 rpm for 10 minutes at 2 to 8 ° C, and the supernatant was removed. 1 ml of 75% ethanol was added per 1 ml of Trizol, and centrifuged at 15,000 rpm for 5 minutes at 2 ° C to 8 ° C. After 150ul of distilled water was dissolved in RNA, the absorbance was measured and measured at 260nm. It was then stored at -20 ° C. The amount of RNA isolated was 1.0 to 2.5 ug / ul.

2. Synthesis and Amplification of cDNA

CDNA was synthesized from the RNA obtained above. First, mRNA was reacted at 65 ° C. for 10 minutes and immediately cooled on ice. 10ul of mRNA, 10ul of distilled water, 11ul of first-strand mix, and 1ul of DTT were added and reacted at 37 ° C for 1 hour. Herein, the first strand mixture is included in the Mouse ScFv and Expression Module (Amersham Pharmacia), a mouse reverse transcriptase, random hexadeoxyribonucleotides (pd (N) ₆ ) , BSA, dATP, dCTP, dGTP, dTTP without RNase / DNase.

Next, PCR was performed using the obtained cDNA. Two PCR tubes were prepared, and one tube was filled with a PCR kit, 33 µl of cDNA, 2 µl of a light primer mix and 15 µl of distilled water, and the other tube contained a PCR kit, 33 µl of a cDNA and a heavy chain primer. 2 μl of the mixture (heavy primer mix) and 15 μl of distilled water were added thereto, and the reaction was performed by a PCR machine. The PCR reaction was carried out by reacting for 5 minutes at 95 ° C., then 40 times with 1 minute at 95 ° C., 2 minutes at 56 ° C., 2 minutes at 72 ° C., and finally at 72 ° C. for 15 minutes. The light chain primer mixture refers to a mixture of primers corresponding to ten variable regions of the light chain, and includes a 5 'primer and a 3' primer. The heavy chain primer mixture is a mixture of primers corresponding to the variable region of the heavy chain, each present as a 5 'primer and a 3' primer, and was mixed and used together during the reaction. The mixtures themselves are provided to the Mouse ScFv and Expression Module (Amersham Pharmacia).

The PCR product DNA thus obtained was confirmed by electrophoresis on 1.2% agarose gel (see FIG. 3), and purified using a QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). The purified DNA was electrophoresed with a mass control DNA (mass ladder) on a 2% agarose gel to quantify the amount of light and heavy chains. As shown in FIG. 3, the bands indicating C2 VH and VL were identified in lanes 1 and 2, and the bands indicating C7 VH and VL were identified in lanes 3 and 4.

3. Preparation of Single Chain Fv.

PCR was performed to prepare single-chain Fv comprising C2 VH, VL and linker obtained above and single-chain Fv DNA comprising C7 VH, VL and linker. First, the amount of the heavy and light chain DNA obtained in the PCR kit is similar to the above, add 4ul of linker-primer mix (filler-primer mix) and filled with distilled water so that the total amount is 50ul, then 1 minute at 94 ℃, 63 ℃ The reaction was carried out 7 times in 4 minutes at. The linker-primer mixture refers to a mixture of 3 ′ heavy chain linker primers and 5 ′ light chain linker primers in the same molar ratio, which is itself provided to the Mouse ScFv and Expression Module (Amersham Pharmacia). By such a linker-primer mixture, single-chain Fv having linker DNA introduced into each heavy and light chain DNA was prepared.

4 μl of RS primer mix was added to the single-chain Fv DNA generated as described above, followed by PCR. The PCR reaction was performed by reacting at 95 ° C. for 5 minutes, then 40 times with 1 minute at 95 ° C., 2 minutes at 56 ° C., 2 minutes at 72 ° C., and finally at 72 ° C. for 15 minutes. The RS primer mixture is a mixture of 5 ′ heavy chain primer having SfiI restriction enzyme site and 3 ′ light chain primer having NotI restriction enzyme site, which is itself provided to Mouse ScFv and Expression Module (Amersham Pharmacia). By using such a RS primer mixture, restriction enzyme sites were introduced into the sock end of the single-chain Fv.

In summary, the manufacturing process of the single-chain Fv as described above is shown in FIG. As shown in FIG. 4, VH and VL added with a linker are produced in the first PCR using HB mix and HF mix, which are 3 ′ heavy chain linker primers, and LF mix and LB mix, which are 5 ′ light chain linker primers. Thereafter, single chain Fv DNA of the VH-linker-VL structure is combined by secondary PCR using an RS mix primer.

Electrophoresis of the PCR product is shown in FIG. As shown in FIG. 5, it was possible to identify C2 single-chain Fv DNA in lane 1 and C7 single-chain Fv DNA in lane 2.

4. Preparation of Recombinant Vectors.

Single-chain Fv DNA of C2 and C7 obtained above was inserted into an expression vector using a restriction enzyme or the like to prepare a recombinant vector.

First, the C2 and C7 single-chain Fv DNA obtained above were put in Sfi I and Not I, reacted at 37 ° C. for 3 to 4 hours to cleave the restriction enzyme site, and the DNA cut by the restriction enzyme was then transferred onto an agarose gel. After performing the phorescence, the band was cut (Fig. 6).

Single chain Fv DNA digested with restriction enzymes were isolated and purified from the cut gel using a QIAquick PCR purification kit (Qiagen, Valencia, CA, USA). It was inserted using pCANTAB 5E vector (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and ligase treated with the same enzyme, to prepare a recombinant vector (Fig. 7). Figure 7 shows a single chain Fv recombined with a pCANTAB 5E vector.

Example 3 Preparation of Transformant

The recombinant pCANTAB 5E prepared above was mixed well with E. coli HB2151, and then transformed by reacting at 42 ° C. for 1 minute.

The transformed cells were incubated at 37 ° C. in LB-agarose plate-carbenicillin (50 ug / ml), and each cultured colony was incubated in 2 ml of LB medium for 20 hours. . Bacteria pellets were prepared from the culture medium, and plasmids were isolated using a Qiagen plasmid mini kit (Qiagen, Valencia, CA, USA). The separated plasmids were digested with Sfi I and Not I, and electrophoresed to confirm that the DNA was inserted.

Example 4 Expression of Escherichia Coli in Single Chain Fv (scFv)

From the electrophoresis results, E. coli HB2151 cells containing pCANTAB 5E vector with the correct size of the antibody DNA insert were selected, spread on LB-carbenicillin plate and incubated at 37 ° C. for 16 hours.

Each colony on the resulting plate was incubated at 37 ° C. in 2 ml of LB-carbenicillin medium, grown until OD ₆₀₀ = 1, then added IPTG to 1 mM, and then transferred to 30 ° C. for culture.

The bacterial culture was centrifuged to remove the pellet, and the culture was examined for expression of single-chain Fv antibody using ELISA (FIG. 8). As shown in FIG. 8, it was found that both C2 and C7 express single chain Fv in the culture solution.

In addition, the expression of the single-chain Fv antibody was confirmed by SDS-PAGE and Western blotting. Cell lysates and culture supernatants were electrophoresed on 10% polyacrylamide gels. The cell lysate was taken in 1 ml of cultured E. coli cells, placed in a microcentrifuge tube, centrifuged to discard the upper solution, and the remaining cells were washed once with 1 ml of PBS, and the remaining cells were suspended in 0.8 ml of PBS. To this, 0.2 ml of sample buffer for SDS-PAGE was added and reacted with boiling water for 5 minutes to use for analysis. As such, proteins separated on SDS-PAGE gels were transferred to nitrocellulose membranes. The membrane was blocked for 1 hour in 5% skim milk / PBS at room temperature. After washing several times with PBST buffer, the HRP / E-tag conjugate was reacted at 4 ° C. for 16 hours. The HRP / E-tag conjugate is a mouse anti-E-tag monoclonal antibody conjugated with horseradish peroxidase and binds to a peptide tag (E-tag) located at the C terminus of a single-chain Fv. Antibody-enzyme complex. Thereafter, after washing with PBST buffer several times, the membrane was identified using an ECL Western blotting detection reagent (Amersham Pharmcia) (Fig. 9). Figure 9 shows SDS-PAGE (Figure 9A) and Western blotting results (Figure 9B) for C7 single chain Fv. As can be seen in Figure 9, the SDS-PAGE and Western blotting confirmed that the single chain Fv was expressed at a molecular weight of about 29,000.

E. coli HB2151 cells transformed with the pCANTAB 5E vector containing the C7 single chain Fv were deposited on December 17, 2001 in the Korea Institute of Biotechnology Gene Resource Center Gene Bank (Accession No. KCTC 10141BP).

Example 5: Determination of the affinity of the single-chain Fv of the present invention and the nucleotide sequence of the antibody gene

1. Determination of the Affinity of Monoclonal Antibodies and Single-chain Fv According to the Invention

The affinity of the anti-carcinoma fetal antigen monoclonal antibody and its single chain Fv derived from hybridomas was measured by the competition ELISA method. The detailed process is as follows.

(1) Determination of Proper Antibody Concentration

A. Preparation of Plates

Carcinoma fetal antigen (Calbiochem, Darmstadt, Germany) was diluted to 2ug / ml with PBS buffer and 100ul aliquot, and incubated overnight at 4 ℃. Plates were washed once with PBST, then 200ul of 1% BSA-PBS solution was dispensed and stored at room temperature for 1 hour.

B. First Reaction

The E. coli culture containing the expressed antibody was diluted with PBS in several steps and placed in 100 ul in each well of the plate, reacted for 2 hours at room temperature, and washed 5 times with PBST.

C. Secondary Reaction

HRP / anti-E tag conjugate (manufactured by Amersham Pharmacia Biotech) was diluted 5,000-fold with 1% BSA-PBS solution and added to 100 ul in each well, followed by 1 hour of reaction at room temperature, followed by washing 5 times with PBST.

D. Substrate Reaction

100 ul of ABTS (KPL, MD, USA) solution was added to each well and O.D. was measured at 405 nm. A half concentration of the antibody concentration showing the maximum binding value was determined as an appropriate antibody concentration.

(2) competitive ELISA

A. Preparation of Plates

Carcinoma fetal antigens (Calbiochem, Darmstadt, Germany) were diluted to 2 ug / ml with PBS buffer, then 100 ul were dispensed into each well and incubated overnight at 4 ° C. The plate was washed once with PBST, then 200ul of 1% BSA-PBS solution was dispensed into each well and stored at room temperature for 1 hour.

B. First Reaction

Carcinoma fetal antigen was diluted to 2.8x10 ^-6 M to 1.8x10 ^-10 M and placed in ¹⁰ ul prepared plates. Then, the antibody was diluted to the appropriate antibody concentration determined in (1), 90 ul each, and reacted at room temperature for 2 hours, followed by PBST. Washed 5 times.

C. Secondary Reaction

HRP / anti-E tag conjugate (manufactured by Amersham Pharmacia Biotech) was diluted 5,000-fold with 1% BSA-PBS solution and added to each well by 100ul, and reacted at room temperature for 1 hour, followed by washing 5 times with PBST.

D. Substrate Reaction

100 ul of ABTS (KPL, MD, USA) solution was added to each well and O.D. was measured at 405 nm. Carcinogenic antigen concentrations that inhibit 50% of maximal binding (ELISA O.D values without competing carcinoma antigens) were calculated as affinity (Kd). The affinity of anti-tumor fetal antigen monoclonal antibodies derived from hybridomas was determined only by different conjugates used for secondary reactions.

The results of the affinity measurement of the hybridoma monoclonal antibody and its single chain Fv are shown in Figure 10 and Table 2.

Table 2. Results of affinity measurements of anti-carcinomatous antigen monoclonal antibodies and their single chain Fv.

Clone Affinity (Kd: mole / liter) Monoclonal Antibodies from Hybridomas Single chain Fv C2 6.1x10 ^-9 8.9 x 10 ^-9 C7 2.4 x 10 ^-9 3.6 x 10 ^-9

As can be seen from Table 2, the single-chain Fv C2 single-chain Fv and C7 single-chain Fv according to the present invention has a very strong affinity without significant difference compared to the affinity of the hybridoma-derived mono monoclonal antibody It is showing.

2. Determination of the DNA base sequence of the single-chain Fv according to the present invention

As a result of Example 4 E. coli cells expressing single-chain Fv were incubated overnight in 10 ml of LB medium containing 50 ug / ml carbenicillin, followed by Qiagen plasmid mini kit (Qiagen, Valencia, CA, USA). Recombinant plasmids were isolated using. The recombinant plasmid DNA thus obtained was used for sequencing.

Primers used for sequence determination were pCANTAB5-S1 (SEQ ID NO. 7: 5′-CAACGTGAAAAAATTATTATTCGC-3 ′) and pCANTAB5-R2 (SEQ ID NO. 8: 5′-) provided in the Mouse ScFv and Expression Module (manufactured by Amersham Pharmacia Biotech). CGATCTAAAGTTTTGTCGTCTTTCC-3 ′) was used, and the sequencing was analyzed by Macrogen (Seoul, Korea) according to a known method.

As a result of sequencing, the base sequence of the VH of C7 and the amino acid sequence estimated therefrom are shown in SEQ ID NOs: 1 (GenBank Accession No. AF393386) and 2 of the Sequence Listing, and the base sequence of the VL of C7 and the amino acid sequence estimated therefrom are Shown in SEQ ID NO: 3 (GenBank Accession No. AF393387) and 4 of the list. The full base sequence of the single chain Fv of C7 and the amino acid sequence estimated therefrom are shown in SEQ ID NOs: 5 and 6 of the Sequence Listing, respectively.

The amino acid sequences identified above were compared to the amino acid sequences of the mouse immunoglobulins described in Kabat et al., Department of Health and Human Services (NIH (1991)), and the VH of the C2 clone was X. Found to belong to the IIIA subgroup of the family, the VL belongs to the VI subgroup of the MISCELLANEOUS family, the VH of the C7 clone belongs to the IIIA subgroup of the X family, and the VL to the VI of the MISCELLANEOUS family. Found to belong to a subgroup. In addition, the C7 single-chain Fv had a VH-linker-VL structure, and the linker sequence had 15 amino acid sequences in the form of three repeated GS linkers [(Glys ₄ S) ₃ ].

According to the C7 hybridoma according to the present invention, it is possible to provide a C7 monoclonal antibody having excellent affinity for anti-carcinoma fetal antigen.

According to the single chain Fv according to the present invention, it can be expressed in soluble form in E. coli .

Further, according to the single chain Fv according to the present invention, single chain Fv can be expressed with high affinity.

1 is a human colon cancer cell line LS 174T (prepared by the Bank of Cancer Research Center of Seoul National University) and subjected to SDS-PAGE of cell debris, which is transferred to a nitrocellulose membrane and reacted with a purified anti-carcinoma antigenic antibody to obtain Western blots. As a result showing the results of the lot, lane A represents clone C2, lane B represents clone C7, lane C represents clone C1,

Figure 2 is a diagram showing a pCANTAB 5E vector map, ampicilin refers to the ampicillin resistance gene, M13 ori is the origin of replication of bacteriophage M13, ORI is the origin of replication, PLACZ is a promoter, Signal is a signal peptide of gene 3 of phage M13 E-tag is an E tag sequence for detection and purification, amber is an amber stop codon,

Figure 3 shows the results of electrophoresis of DNA amplified from cDNA, lane 1 is C2 VH DNA, lane 2 is C2 VL DNA, lane 3 is C7 VH DNA, lane 4 is C7 VL DNA and lane M is molecular weight marker. To represent

4 is a view showing a process for producing a single-chain Fv of the present invention,

5 is a diagram showing the results of electrophoresis of the prepared single-chain Fv DNA, M is the molecular weight marker, lane 1 is C2 single-chain Fv DNA, lane 2 is C7 single-chain Fv DNA,

Figure 6 is a diagram showing the result of treatment of the prepared single-chain Fv DNA with Not I and Sfi I, electrophoresis to cut the site, lane 1 is C2 single-chain Fv, lane 2 is C7 single-chain Fv,

FIG. 7 is a recombinant pCANTAB 5E vector map showing the recombination of single chain Fv and pCANTAB 5E treated with restriction enzymes, LacZ is a promoter, g3 is a signal peptide of gene 3 of phage M13, and E is detection and purification Is an E tag for A, A is an amber stop codon, Gene 3 is gene 3 of phage M13,

8 is a diagram showing the results of an ELISA in which a culture of E. coli HB2151 transformed with recombinant pCANTAB 5E containing C2 single-chain Fv and C7 single-chain Fv was used to confirm expression of single-chain Fv.

9 is a diagram showing the results of SDS-PAGE and Western blotting of the culture medium and cell lysates cultured with E. coli HB2151 transformed with recombinant pCANTAB 5E containing C2 single-chain Fv and C7 single-chain Fv. 1 is a culture media, lane 2 is E.coli lysate 5㎕, E.coli lysate 10㎕, E.coli lysate 15㎕, lane 5 is the non-induced E.coli lysate, M is a pre-stained molecular weight markers (Life Technologies G)

Fig. 10 shows the competition ELISA results of C2 single chain Fv and C7 single chain Fv, where? Is the result for C7 single chain Fv, and, is the result for C2 single chain Fv.

<110> Korea Green Cross Company <120> ANTI-CARCINOEMBRYONIC ANTIGEN SINGLE CHAIN Fv <160> 8 <170> KopatentIn 1.71 <210> 1 <211> 354 <212> DNA <213> mouse <220> <221> CDS (222) (1) .. (354) <223> C7 ANTI-CEA scFv VH SEQUENCE <400> 1 cag gtg aag ctg cag gag tct gga cct gag ctg aag aag cct gga gag 48 Gln Val Lys Leu Gln Glu Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 aca gtc aag atc tcc tgc aag gct tct ggg tat acc ttc aca gac tat 96 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gga atg aac tgg gtg aag ctg gct cca gga aag ggt tta aag tgg atg 144 Gly Met Asn Trp Val Lys Leu Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 ggc tgg ata aac acc tac act gga gag cca aca tat gct gat gac ttc 192 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 aag gga cgg ttt gcc ttc tct ttg gag acc tct gcc agc act gcc tat 240 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 ttg cag atc aac aac ctc aaa aat gag gac acg gct aca tat ttc tgt 288 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 gca aga aaa gac cta cta cgc tac ttt gac tac tgg ggc caa ggg acc 336 Ala Arg Lys Asp Leu Leu Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 acg gtc acc gtc tcc tca 354 Thr Val Thr Val Ser Ser 115 <210> 2 <211> 118 <212> PRT <213> mouse <400> 2 Gln Val Lys Leu Gln Glu Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Gly Met Asn Trp Val Lys Leu Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Lys Asp Leu Leu Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 3 <211> 321 <212> DNA <213> mouse <220> <221> CDS (222) (1) .. (321) <223> C7 ANTI-CEA scFv VL SEQUENCE <400> 3 gac atc gag ctc act cag tct cca tcc tca ctg tct gca tct ctg gga 48 Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 ggc aaa gtc acc atc act tgc aag gca agc caa gac att aac aag tat 96 Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 ata gct tgg tac caa cac aag cct ggt aaa ggt cct agg ctt ctc ata 144 Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile 35 40 45 cgt tac aca tct aca tta cag cca ggc atc cca tca agg ttc agt gga 192 Arg Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 agt ggg tct ggg aga gat tat tcc ttc agc atc agc aac ctg gag cct 240 Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro 65 70 75 80 gaa gat att gca act tat tat tgt cta cat tat aat aat ctt cac acg 288 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu His Tyr Asn Asn Leu His Thr 85 90 95 ttc gga ggg ggg acc aag ctg gag ctg aaa cgg 321 Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg 100 105 <210> 4 <211> 107 <212> PRT <213> mouse <400> 4 Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Lys Tyr 20 25 30 Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly Pro Arg Leu Leu Ile 35 40 45 Arg Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile Ser Asn Leu Glu Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Leu His Tyr Asn Asn Leu His Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg 100 105 <210> 5 <211> 720 <212> DNA <213> Artificial Sequence <220> C7 scFv SEQUENCE: VH-LINKER-VL SEQUENCE TYPE <220> <221> CDS (222) (1) .. (720) <400> 5 cag gtg aag ctg cag gag tct gga cct gag ctg aag aag cct gga gag 48 Gln Val Lys Leu Gln Glu Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 aca gtc aag atc tcc tgc aag gct tct ggg tat acc ttc aca gac tat 96 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 gga atg aac tgg gtg aag ctg gct cca gga aag ggt tta aag tgg atg 144 Gly Met Asn Trp Val Lys Leu Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 ggc tgg ata aac acc tac act gga gag cca aca tat gct gat gac ttc 192 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 aag gga cgg ttt gcc ttc tct ttg gag acc tct gcc agc act gcc tat 240 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 ttg cag atc aac aac ctc aaa aat gag gac acg gct aca tat ttc tgt 288 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 gca aga aaa gac cta cta cgc tac ttt gac tac tgg ggc caa ggg acc 336 Ala Arg Lys Asp Leu Leu Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 acg gtc acc gtc tcc tca ggt gga ggc ggt tca ggc gga ggt ggc tct 384 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 ggc ggt ggc gga tcg gac atc gag ctc act cag tct cca tcc tca ctg 432 Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu 130 135 140 tct gca tct ctg gga ggc aaa gtc acc atc act tgc aag gca agc caa 480 Ser Ala Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln 145 150 155 160 gac att aac aag tat ata gct tgg tac caa cac aag cct ggt aaa ggt 528 Asp Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly 165 170 175 cct agg ctt ctc ata cgt tac aca tct aca tta cag cca ggc atc cca 576 Pro Arg Leu Leu Ile Arg Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro 180 185 190 tca agg ttc agt gga agt ggg tct ggg aga gat tat tcc ttc agc atc 624 Ser Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile 195 200 205 agc aac ctg gag cct gaa gat att gca act tat tat tgt cta cat tat 672 Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu His Tyr 210 215 220 aat aat ctt cac acg ttc gga ggg ggg acc aag ctg gag ctg aaa cgg 720 Asn Asn Leu His Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg 225 230 235 240 <210> 6 <211> 240 <212> PRT <213> Artificial Sequence <400> 6 Gln Val Lys Leu Gln Glu Ser Gly Pro Glu Leu Lys Lys Pro Gly Glu 1 5 10 15 Thr Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Gly Met Asn Trp Val Lys Leu Ala Pro Gly Lys Gly Leu Lys Trp Met 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Asp Asp Phe 50 55 60 Lys Gly Arg Phe Ala Phe Ser Leu Glu Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Leu Gln Ile Asn Asn Leu Lys Asn Glu Asp Thr Ala Thr Tyr Phe Cys 85 90 95 Ala Arg Lys Asp Leu Leu Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Asp Ile Glu Leu Thr Gln Ser Pro Ser Ser Leu 130 135 140 Ser Ala Ser Leu Gly Gly Lys Val Thr Ile Thr Cys Lys Ala Ser Gln 145 150 155 160 Asp Ile Asn Lys Tyr Ile Ala Trp Tyr Gln His Lys Pro Gly Lys Gly 165 170 175 Pro Arg Leu Leu Ile Arg Tyr Thr Ser Thr Leu Gln Pro Gly Ile Pro 180 185 190 Ser Arg Phe Ser Gly Ser Gly Ser Gly Arg Asp Tyr Ser Phe Ser Ile 195 200 205 Ser Asn Leu Glu Pro Glu Asp Ile Ala Thr Tyr Tyr Cys Leu His Tyr 210 215 220 Asn Asn Leu His Thr Phe Gly Gly Gly Thr Lys Leu Glu Leu Lys Arg 225 230 235 240 <210> 7 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SEQUENCING PRIMER pCANTAB5-S1 <400> 7 caacgtgaaa aaattattat tcgc 24 <210> 8 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SEQUENCING PRIMER pCANTAB5-R2 <400> 8 cgatctaaag ttttgtcgtc tttcc 25

Claims (15)

delete delete delete A light chain variable region polypeptide of a mouse anti-carcinoma fetal antigen antibody, having the amino acid sequence of SEQ ID NO: 4 in Sequence Listing. DNA encoding the polypeptide of claim 4. The DNA of claim 4, wherein the DNA has a nucleotide sequence of SEQ ID NO: 3 in Sequence Listing. A hybridoma cell characterized by producing a mouse anti-carcinoma fetal antigen antibody comprising the heavy chain variable region polypeptide having the amino acid sequence of SEQ ID NO: 2 of the Sequence Listing and the polypeptide of claim 4 (Accession No. KCLRF-BP-00053) . Mouse anti-carcinoma embryos comprising VH, glycine or cystine having the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing and a linker peptide having 13 to 17 amino acids and VL having the amino acid sequence of SEQ ID NO: 4 in the Sequence Listing Antigen single chain Fv. The anti-carcinomatous antigen single chain Fv of claim 8, having the amino acid sequence of SEQ ID NO: 6 in Sequence Listing. DNA having a nucleotide sequence encoding the amino acid sequence of claim 9. The DNA of claim 10, having a nucleotide sequence of SEQ ID NO: 5 in Sequence Listing. An expression vector comprising the DNA of claim 10 or claim 11. The expression vector of claim 12, wherein the expression vector is a pCANTAB 5E vector. The transformant is prepared by transforming the host cell with a recombinant pCANTAB 5E expression vector comprising the DNA of claim 10 or claim 11. The transformant of claim 14, which is E. coli HB2151 (Accession No. KCTC 10141BP).