WO2004013278A2

WO2004013278A2 - Expression vectors

Info

Publication number: WO2004013278A2
Application number: PCT/KR2003/001555
Authority: WO
Inventors: Jong-Wook Lee; In-Young Ko; Moo-Young Song; Chang-Seok Kim; Jae-Sun Lee; Tae-Hyoung Yoo; Ji-Min Kim; Sang-Koo Park
Original assignee: Yuhan Corporation
Priority date: 2002-08-02
Filing date: 2003-08-01
Publication date: 2004-02-12
Also published as: AU2003253447A1; AU2003253447A8; WO2004013278A3

Abstract

Provided is an expression vector for a heavy chain variable region, a lambda (λ ) light chain variable region, or a kappa (κ ) light chain variable region of an antibody. The expression vector includes a restriction site which commonly exists in joining regions of genes encoding antibody variable regions; and a gene encoding an antibody constant region.

Description

EXPRESSION VECTORS

Technical Field

The present invention relates to an expression vector for a heavy^' chain variable region, a lambda (λ ) light chain variable region, or a kappa (K ) light chain variable region of an antibody. More particularly, the present invention relates to an expression vector comprising: a restriction site which commonly exists in joining regions of genes encoding antibody variable regions; and a gene encoding an antibody constant region. A gene encoding a variable region can be directly cloned in a cassette form into the expression vector, and thus, the expression vector can be efficiently used in the development of therapeutic antibodies.

Background Art Developments of various types of therapeutic antibodies against many different diseases have been actively performed [Reichmann, L, Clark, M., et al. Nature 322, 323-327 (1988); Paul Carter and H. Michael Shepard, Proc. Natl. Acad. Sci. U.S.A. 89, 4285-4289 (1992); John Hakimi, and William P. Schneider, J. Immunology 151 , 1075-1085 (1993)]. Examples of therapeutic antibodies include humanized antibodies and chimeric antibodies.

For example, in the development of chimeric antibodies, genes encoding antibody variable regions for subjects such as mice are genetically recombinated with genes encoding human constant regions to produce chimeric antibody genes, which are then inserted into suitable expression vectors. In order to evaluate the presence of gene expression, host cells such as animal cells are transfected with the expression vectors thus constructed.

The pCDNA vector system (Invitrogen, America) and the pMG vector system (InvivoGen, America) have widely been used as expression vectors. vector system (InvivoGen, America) have widely been used as expression vectors. The pMG vector system is known as a vector system suitable for expression of proteins consisting of two or more subunits, such as antibody, since two multi-cloning sites (MCSs) are contained in a single vector. However, in the case of screening a gene with desired characteristics among a number of variable region coding genes using such a conventional pCDNA or pMG vector system, there arises a problem in that the same or similar cloning steps for each candidate gene must be repeatedly performed, which unfavorably requires a prolonged time. Further, an expression vector may comprise a dihydrofofate reductase (dhfή gene to increase antibody productivity, i.e., to amplify an inserted gene. In this regard, however, use of such a conventional vector system is involved in such a problem that the dhfr gene must be separately inserted into the vector system for each amplification step.

Meanwhile, pKCdhfr, an expression vector for a light chain variable region comprising the dhfr gene required for amplification of an inserted gene is disclosed [J. Medical Virology 52: 226-233, 1997]. However, as for the pKCdhfr expression vector, the SV40 promoter, an origin of replication (ORl), and a poly A (polyadenine, polyadenylation site) sequence are contained several times in a single expression vector, and thus, there is a disadvantage in that self-recombination may occur in the expression vector.

Therefore, an expression vector capable of efficiently expressing a candidate group for variable region genes directly inserted thereinto is required to develop various types of therapeutic antibodies.

Disclosure of the Invention

The present invention provides an expression vector comprising: a restriction site which commonly exists in joining regions of genes encoding antibody variable regions; and a gene encoding an antibody constant region.

According to an aspect of the present invention, there is provided an expression vector for an antibody heavy chain variable region, which comprises: (i) a gene (gene A) comprising a Sacl recognition sequence; and a gene encoding an antibody heavy chain constant region, which is linked to the gene A; (ii) a gene encoding an antibody heavy chain constant region which comprises an Apa\ recognition sequence; or (iii) the gene A and the gene of (ii), which are linked to each other. According to another aspect of the present invention, there is provided a host cell transformed with the above expression vector for an antibody heavy chain variable region.

According to another aspect of the present invention, there is provided an expression vector for an antibody lambda (λ ) light chain variable region, which comprises: a gene (gene B) comprising an Avή\ recognition sequence; and a gene encoding an antibody λ light chain constant region, which is linked to the gene B.

According to another aspect of the present invention, there is provided a host cell transformed with the above expression vector for an antibody λ light chain variable region.

According to another aspect of the present invention, there is provided an expression vector for an antibody kappa (K ) light chain variable region, which comprises: a gene (gene C) comprising a BsiWl recognition sequence; and a gene encoding an antibody K light chain constant region, which is linked to the gene C.

According to yet another aspect of the present invention, there is provided a host cell transformed with the above expression vector for an antibody K light chain variable region.

Brief Description of the Drawings The above and other features and advantages of the present invention will become more apparent by describing in detail exemplary embodiments thereof with reference to the attached drawings in which:

FIG. 1 is a genetic map of an expression vector for a heavy chain variable region according to the present invention; FIG. 2 is a schematic view illustrating a preparation process of the expression vector for a heavy chain variable region shown in FIG. 1 ;

FIG. 3 is an electrophoretic photograph of an entire heavy chain gene inserted in an expression vector according to the present invention;

FIG. 4 is a genetic map of an expression vector for a lambda (λ ) light chain variable region according to the present invention;

FIG. 5 is a schematic view illustrating a preparation process of the expression vector for a λ light chain variable region shown in FIG. 4;

FIG. 6 is an electrophoretic photograph of an entire λ light chain gene inserted in an expression vector according to the present invention; FIG. 7 is a genetic map of an expression vector for a kappa (K ) light chain variable region according to the present invention;

FIG. 8 is a schematic view illustrating a preparation process of the expression vector for a light chain variable region shown in FIG. 7; and

FIG. 9 is an electrophoretic photograph of an entire light chain gene inserted in an expression vector according to the present invention.

Best mode for carrying out the Invention

The present inventors performed sequencing of a variety of genes encoding the human antibody variable regions and genes encoding human antibody constant regions. According to the sequencing results, as for antibody heavy chains, the recognition sequence (GAGCTC) of a Sacl restriction enzyme is present in all of joining regions (JRs) at the ends of genes encoding variable regions and the recognition sequence (GGGCCC) of an Apal restriction enzyme is present in all of starting positions of constant regions. As for antibody lambda (λ ) light chains, the recognition sequence (CCTAGG) of an

restriction enzyme is present in all of joining regions at the ends of genes encoding variable regions. As for antibody kappa (K ) light chains, the recognition sequence (CGTACG) of a BsiW\ restriction enzyme is present in all of joining regions at the ends of genes encoding variable regions.

Therefore, according to an aspect of the present invention, there is provided an expression vector for an antibody heavy chain variable region, which comprises: (i) a gene (gene A) comprising a Sacl recognition sequence; and a gene encoding an antibody heavy chain constant region, which is linked to the gene A; (ii) a gene encoding an antibody heavy chain constant region which comprises an Apa\ recognition sequence; or (iii) the gene A and the gene of (ii), which are linked to each other. Preferably, there is provided an expression vector for an antibody heavy chain variable region, having the nucleotide sequence set forth in SEQ ID NO: 1. A gene encoding an antibody heavy chain variable region can be efficiently inserted into the Sacl or Apal restriction site of the expression vector for an antibody heavy chain variable region of the present invention.

The Sacl recognition sequence-containing gene (gene A) is about 15 to 50 bp long, preferably about 20 to 35 bp long, and more preferably about 27 bp long. On the other hand, the gene encoding an antibody heavy chain constant region which comprises the Apa\ recognition sequence may be a full-length gene, or if necessary, a gene having a deletion at the 3'-end thereof. One of skilled persons in the art could use gene fragments with different lengths within the scope of the present invention.

The expression vector for an antibody heavy chain variable region of the present invention can be prepared by deletion of an >Apal recognition site from pCDNA (Invitrogen, America) and then insertion of the above gene of (i), (ii), or (iii) thereinto. The expression vector contains a CMV promoter, which is present in the upstream of the inserted gene, and BGH poly A (BGH polyadenine, BGH polyadenylation site), which is present in the downstream of the inserted gene.

According to another aspect of the present invention, there is provided a host cell transformed with the above expression vector for an antibody heavy chain variable region. Preferably, the host cell is E.coli XL1-Blue/pHAB-HC (accession number: KCTC 10229BP).

According to another aspect of the present invention, there is provided an expression vector for an antibody λ light chain variable region, which comprises: a gene (gene B) comprising an Awil recognition sequence; and a gene encoding an antibody λ light chain constant region, which is linked to the gene B. Preferably, there is provided an expression vector for an antibody λ light chain variable region, having the nucleotide sequence set forth in SEQ ID NO: 3. A gene encoding an antibody λ light chain variable region can be efficiently inserted into the Avή\ restriction site of the expression vector for an antibody λ light chain variable region of the present invention.

The

recognition sequence-containing gene (gene B) is about 15 to 55 bp long, preferably about 30 to 45 bp long, and more preferably about 39 bp long. The gene encoding an antibody λ light chain constant region may be a full-length gene, or if necessary, a gene having a deletion at the 3'-end thereof. One of skilled persons in the art could use gene fragments with different lengths within the scope of the present invention. The expression vector for an antibody λ light chain variable region of the present invention can be prepared by deletion of an Awl I recognition site from pCDNA (Invitrogen, America) and then insertion of the Avή\ recognition sequence-containing gene and the λ light chain constant region gene thereinto. The expression vector contains a CMV promoter, which is present in the upstream of the inserted genes, and BGH poly A, which is present in the downstream of the inserted genes. In addition, a dehydrofolate reductase (dhfr) gene can be inserted into the expression vector to ensure gene amplification. According to another aspect of the present invention, there is provided a host cell transformed with the above expression vector for an antibody λ light chain variable region. Preferably, the host cell is E.coli XL1-Blue/pHAB-LC (accession number: KCTC 10231BP).

According to another aspect of the present invention, there is provided an expression vector for an antibody light chain variable region, which comprises: a gene (gene C) comprising a BsiW\ recognition sequence; and a gene encoding an antibody K light chain constant region, which is linked to the gene C. Preferably, there is provided an expression vector for an antibody light chain variable region, having the nucleotide sequence set forth in SEQ ID NO: 5. A gene encoding an antibody K. light chain variable region can ,be efficiently inserted into the BsiW\ restriction site of the expression vector for an antibody light chain variable region of the present invention.

The BsiWl recognition sequence-containing gene (gene C) is about 15 to 50 bp long, preferably about 20 to 30 bp long, and more preferably about 25 bp long. The gene encoding an antibody light chain constant region may be a full-length gene, or if necessary, a gene having a deletion at the 3'-end thereof. One of skilled persons in the art could use gene fragments with different lengths within the scope of the present invention.

The expression vector for an antibody light chain variable region of the present invention can be prepared by deletion of a BsiW\ recognition site from pCDNA (Invitrogen, America) and then insertion of the BsiWl recognition sequence-containing gene and the K light chain constant region gene thereinto. The expression vector contains a CMV promoter, which is present in the upstream of the inserted genes, and BGH poly A, which is present in the downstream of the inserted genes. In addition, a dhfr gene can be inserted into the expression vector to ensure gene amplification.

According to yet another aspect of the present invention, there is provided a host cell transformed with the above expression vector for an antibody K light chain variable region. Preferably, the host cell is E.coli XL1-Blue/pHAB-KC (accession number: KCTC 10230BP). The expression vectors of the present invention can be efficiently used as expression cassettes capable of expressing corresponding variable region genes inserted into the corresponding restriction sites.

Hereinafter, the present invention will be described more specifically by examples. However, the following examples are provided only for illustrations and thus the present invention is not limited to or by them.

Example 1 : Sequencing of human antibody heavy chains The amino acid sequences and nucleotide sequences of the human antibody heavy chains were determined [NIH 91 , 3242, 1991]. According to the sequencing results, all of joining regions (JRs) at ends of genes encoding variable regions contained a Sacl recognition sequence (GAGCTC) and all of starting positions of constant regions contained an >Apal recognition sequence (GGGCCC), as shown in Table 1 below. Table 1 : Amino acid and nucleotide sequences in joining regions at ends of genes encoding heavy chain variable regions and starting positions of genes encoding heavy chain constant regions

Example 2: Synthesis of primers for heavy chains In order to use the recognition sequences of the restriction enzymes observed in Example 1 , the primers set forth in SEQ ID NOs: 7 and 8 were synthesized using an ABI DNA/RNA synthesizer.

Table 2: Synthetic primers

Example 3: Cloning of genes encoding heavy chain constant regions flanked by restriction sites

Step 1 : Extraction of entire mRNA

Bloods from normal humans were centrifuged to recover lymphocytes. mRNA was obtained from the recovered lymphocytes using the FastTrack 2.0 mRNA isolation kit (Invitrogen, America). mRNA was quantified by measuring absorbance at 260 nm using spectrophotometer.

Step 2: Synthesis of entire cDNA Synthesis of entire cDNA was performed using a Thermotranscript kit (GibcoBRL, America). First, 0.5 μg of mRNA was mixed with oligo-dTs (to reach 10 μJt of a total volume), followed by denaturation at

65 ^°C for about 5 minutes and primer annealing. The resultant mixture was incubated at 50 ^°C for one hour under the following conditions: reverse transcriptase (1μd), buffered solution (5 μl) (50 mM Tris-CI, pH 7.6, 50 mM KCI, 10 mM MgCI₂), DTT (0.1 M, 2.5 μl), dNTPs (2.5 β ), RNase inhibitor (1 μl), and distilled deionized water (to total volume of 25 μl), and then inactivated at 95 ^°C for 5 minutes.

Step 3: PCR amplification of genes encoding heavy chain constant regions flanked by restriction sites

Genes encoding heavy chain constant regions flanked by restriction sites were amplified by PCR (polymerase chain reaction) using the cDNA (3 μl) obtained in Step 2 and the primers set forth in SEQ ID NOs: 7 and 8. PCR was performed under the following conditions: first step - 95 ^°C for 5 minutes for 1 cycle; second step - 94 ^°C for 2 minutes, 55 ^°C for 2 minutes, and 72 ^°C for 3 minutes, for 30 cycles; and third step - 94 ^°C for 2 minutes, 55 ^°C for 2 minutes, and 72 ^°C for 10 minutes, for 1 cycle. After the PCR amplification, gene fragments with about 1 ,000 bp were identified by agarose gel electrophoresis and ethidium bromide (EtBr) staining.

Step 4: Cloning of genes encoding heavy chain constant regions flanked by restriction sites into PCR vectors.

After electrophoresis on a 1.5% agarose gel, the gene fragments encoding heavy chain constant regions flanked by restriction sites were recovered using a QIAgen extraction kit (Qiagen, America). The recovered gene fragments were subjected to impurity removal using phenol (200 μl) and chloroform (200 μl) and then purified using ethanol (2.5 ml). The purified gene fragments were subcloned into pCRII vectors (Invitrogen, America) and then transformed into E.co//^' XL1-Blue [Cohen, S. N. et al., Proc. Natl. Acad. Sci. 69, 2110, 1972] to thereby obtain transformants. The obtained transformants were incubated in LB (Luria-Bertani) media containing 100 μglvnl of ampicillin for overnight and then plasmids were recovered. The recovered plasmids were digested with a restriction enzyme, EcoR\ (Biolab, America), which revealed the presence of the above gene fragments. Furthermore, the sequencing results showed that the gene fragments contain the nucleotide sequence set forth in SEQ ID NO: 1 and the amino acid sequence set forth in SEQ ID NO: 2. The recovered plasmids were designated as pCR-HC.

Example 4: Cloning of heavy chain expression vectors Step 1 : Construction of pCDNA3.1A vectors with deletion of >4pal pCDNA3.1A vectors (Invitrogen, America) were digested with an Apa\ restriction enzyme at 37 ^°C for 2 hours and then incubated with 1 unit of Klenow enzyme and dNTPs at 30 ^°C for 1 hour to thereby fill overhanging single-stranded nucleotides into double-stranded nucleotides. Then, gene fragments were recovered in the same manner as in Step 4 of Example 3. Then, 1 unit of T4-DNA ligase was added to the recovered gene fragments and let stand at 16^°C for overnight to induce self-ligation. The resultant gene fragments were transformed into E.coli XL1-Blue in the same manner as in Step 4 of Example 3 to obtain transformants. The obtained transformants were incubated in LB media containing 100 μg/ of ampicillin for overnight and then plasmids were recovered. When 4pal restriction enzymes (Biolab, America) were added to the recovered plasmids, the recovered plasmids were not digested, which was revealed by electrophoresis. The recovered plasmids were designated as pHAB.

Step 2: Construction of pHAB-HC expression vectors The pHAB vectors obtained in Step 1 were digested with restriction enzymes, HindUUNot (Biolab, America). On the other hand, the pCR-HC vectors obtained in Example 3 were digested with Hind\\\INot\ to thereby obtain target gene fragments and then the obtained gene fragments were inserted into the recognition sequences of the Hind\\\/Not\ restriction enzymes of the pHAB vectors. The resultant pHAB vectors were transformed into E. coli XL1-Blue in the same manner as in Step 4 of Example 3 to thereby obtain transformants. Plasmids were recovered from the obtained transformants and digested with Hind\\\lNot\, followed by electrophoresis. The electrophoresis results showed that the recovered plasmids contain genes encoding heavy chain constant regions flanked by restriction sites set forth in SEQ ID NO: 1. The above transformants were deposited at the Gene Bank in Korea Research Institute of Bioscience & Biotechnology (KRIBB) on April 18, 2002 (accession number: KCTC10229BP) under the accession name of E. coli XL1 -Blue/pHAB-HC.

Example 5: Cloning of heavy chain variable region The recombinant vectors, pCRA9Hv, which contains genes encoding heavy chain variable regions (WO01/092529), and the expression vectors obtained in Example 4, the E. coli XL1-Blue/pHAB-HC (accession number: KCTC10229BP) were respectively digested with the H/>7θτll//4pal (Biolab, America). The obtained two types of gene fragments were incubated with T4 DNA ligase at 16°C for overnight and transformed into E. coli XL1-Blue to obtain transformants. The obtained transformants were incubated in LB media containing 100 μgM of ampicillin for overnight and plasmids were recovered. The recovered plasmids were digested with the HindUUNotl, followed by electrophoresis. The results of electrophoresis revealed the presence of entire antibody heavy chain genes of about 1.4 kb (see FIG. 3). Example 6: Seouencing of human antibody λ light chains

The amino acid sequences and nucleotide sequences of the human antibody λ light chains were determined [NIH 91 , 3242, 1991]. According to the sequencing results, all of joining regions (JRs) at ends of genes encoding variable regions contained the recognition sequence

(CCTAGG) of an Avή\ restriction enzyme, as shown in Table 3 below.

Table 3: Amino acid and nucleotide sequences in joining regions (JL) at ends of genes encoding λ light chain variable regions

In the λ light chain JL4 sequence, ATT and TTA coding for isoleucine and leucine, respectively, can be changed into ATC and CTA, respectively, without change of amino acids. Therefore, the recognition sequence of the >Avrll restriction enzyme can be applied to the λ light chain JL4 sequence. Similarly, CTC in the JL6 and JL7 sequences can also be changed into CTA without change of amino acid.

Example 7: Synthesis of primers for λ light chains In order to use the recognition sequence of the restriction enzyme observed in Example 6, the primers set forth in SEQ ID NOs: 9 and 10 were synthesized using an ABI DNA/RNA synthesizer. Table 4: Synthetic primers

Example 8: Cloning of genes encoding λ light chain constant regions

Step 1 : Extraction of entire mRNA

Step 2: Synthesis of entire cDNA

Synthesis of entire cDNA was performed using a Thermotranscript kit (GibcoBRL, America). First, 0.5 μg of mRNA was mixed with oligo-dTs (to reach 10 μl of a total volume), followed by denaturation at 65 °C for about 5 minutes and primer annealing. The resultant mixture was incubated at 50 °C for one hour under the following conditions: reverse transcriptase C\μl), buffered solution (5 μl) (50 mM Tris-CI, pH 7.6, 50 mM KCI, 10 mM MgCI₂), DTT (0.1 M, 2.5 μl), dNTPs (2.5 μl), RNase inhibitor (1 μl), and distilled deionized water (to total volume of 25 μl), and then inactivated at 95 °C for 5 minutes.

Step 3: PCR amplification of genes encoding λ light chain constant regions flanked by restriction sites Genes encoding λ light chain constant regions flanked by restriction sites were amplified by PCR using the cDNA (3 μl) obtained in Step 2 and the primers set forth in SEQ ID NOs: 9 and 10. PCR was performed under the following conditions: first step - 95 ^°C for 5 minutes for 1 cycle; second step - 94 °C for 2 minutes, 55 ^°C for 2 minutes, and 72 ^°C for 3 minutes, for 30 cycles; and third step - 94 ^°C for 2 minutes, 55 ^°C for 2 minutes, and 72 ^°C for 10 minutes, for 1 cycle. After the PCR amplification, gene fragments with about 320 bp were identified by agarose gel electrophoresis and ethidium bromide (EtBr) staining.

Step 4: Cloning of genes encoding λ light chain constant regions flanked by restriction sites into PCR vectors.

After electrophoresis on a 1.5% agarose gel, the gene fragments encoding λ light chain constant regions flanked by restriction sites were recovered using a QIAgen extraction kit (Qiagen, America). The recovered gene fragments were subjected to impurity removal using phenol (200 μl) and chloroform (200 μl) and then purified using ethanol (2.5 ml). The purified gene fragments were subcloned into pCRII vectors (Invitrogen, America) and then transformed into E.co// XL1-Blue [Cohen, S. N. et al., Proc. Natl. Acad. Sci. 69, 2110, 1972] to thereby obtain transformants. The obtained transformants were incubated in LB media containing 100 μgf l of ampicillin for overnight and then plasmids were recovered. The recovered plasmids were digested with an EcoRl restriction enzyme (Biolab, America), which revealed the presence of the above gene fragments. Furthermore, the sequencing results showed that the gene fragments contain the nucleotide sequence set forth in SEQ ID NO: 3 and the amino acid sequence set forth in SEQ ID NO: 4. The recovered plasmids were designated as pCR-LC. Example 9: Cloning of λ light chain expression vectors

Step 1 : Construction of pCDNA3.1A vectors with deletion of Neo pCDNA3.1A vectors (Invitrogen, America) were incubated with

PvuW restriction enzymes at 37^°C for 2 hours to digest the gene (hereinafter, referred to as "Neo") for neomycin-resistance gene expression system (SV40 promoter, ori, Neomycin, SV40 poly A). After the deletion of the Neo was identified by electrophoresis, only the genes of about 3.5 kb of the vectors except for the Neo were isolated. Then, 1 unit of T4-DNA ligase was added to the isolated gene fragments and let stand at 16^°C for overnight to induce self-ligation. The resultant gene fragments were transformed into E.coli XL1-Blue in the same manner as in Step 4 of Example 8 to obtain transformants [Cohen, S. N. et al., Proc.

Nat. Acad. Sci. 69, 21 10, 1972]. The obtained transformants were incubated in LB media containing 100 μg/m of ampicillin for overnight and then plasmids were recovered. The recovered plasmids were treated with PvuW restriction enzymes (Biolab, America), followed by agarose gel electrophoresis, to reveal the deletion of the Neo gene.

Step 2: Insertion of dhfr gene into the Λ/eo-deleted pCDNA3.1A The plasmids obtained in Step 1 were digested with HindWUPvu restriction enzymes (Biolab, America) to obtain gene fragments of about

2 kb with deletion of CMV promoter and a part of ampicillin gene. On the other hand, pKCdhfr expression vectors [J. Medical Virology

52:226-233, 1997] were digested with HindWUPvul restriction enzymes to obtain gene fragments of about 3 kb with CMV promoter, dhfr amplification gene, and a part of ampicillin resistance gene. The obtained two types of fragments were joined by T4-DNA ligase and then transformed into E. coli XL1-Blue in the same manner as in Step 4 of

Example 8 to obtain transformants. The obtained transformants were incubated in ampicillin-containing media for overnight to recover plasmids. The recovered plasmids were digested with HindWUPvul restriction enzymes, followed by agarose gel electrophoresis, which revealed the presence of the gene fragments of about 2 kb and 3 kb. The recovered plasmids were designated as pCDNA-dhfr.

Step 3: Construction of pHAB-LC expression vectors The pCDNA-dhfr vectors obtained in Step 2 were digested with Hind ll/Notl restriction enzymes (Biolab, America). On the other hand, the pCR-LC vectors obtained in Step 4 of Example 8 were digested with Hindlll/Notl to obtain target gene fragments and then the obtained gene fragments were inserted into the recognition sequences of the Hindlll/Notl restriction enzymes of the pCDNA-dhfr vectors. The resultant vectors were transformed into E. coli XL1-Blue in the same manner as in Step 4 of Example 8 to thereby obtain transformants. Plasmids were recovered from the obtained transformants and digested with Hindlll/Notl, followed by electrophoresis. As a result, it was demonstrated that the recovered plasmids contain genes encoding λ light chain constant regions flanked by restriction sites set forth in SEQ ID NO: 3. The above transformants were deposited at the Gene Bank in KRIBB on April 18, 2002 (accession number: KCTC10231BP) under the accession name of E. coli XL1-Blue/pHAB-LC.

Example 10: Cloning of λ light chain variable region

The recombinant vectors, pCRA9Lv, which contains genes encoding λ light chain variable regions (WO01/092529), and the expression vectors obtained in Example 9, E. coli XL1-Blue/pHAB-LC

(accession number: KCTC10231BP) were respectively digested with the

Hindlll/Avήl (Biolab, America). The obtained two types of gene fragments were incubated with T4 DNA ligase at 16^°C for overnight and transformed into E. coli XL1-Blue to obtain transformants. The obtained transformants were incubated in LB media containing 100 μgM of ampicillin for overnight and plasmids were recovered. The recovered plasmids were digested with Hindlll/Notl, followed by electrophoresis, which revealed the presence of entire λ light chain genes of about 0.7 kb (see FIG. 6).

Example 11 : Sequencing of human antibody K light chains The amino acid sequences and nucleotide sequences of the human antibody K light chains were determined [NIH 91 , 3242, 1991]. According to the sequencing results, all of joining regions (JRs) at ends of genes encoding variable regions contained the recognition sequence (CGTACG) of a BsiWl restriction enzyme, as shown in Table 5 below.

Table 5: Amino acid and nucleotide sequences in joining regions (JK) at ends of genes encoding K light chain variable regions and starting positions (CK) of genes encoding light chain constant regions

Example 12: Synthesis of primers for K light chains

In order to use the recognition sequence of the restriction enzyme observed in Example 11 , the primers set forth in SEQ ID NOs: 11 and 12 were synthesized using an ABI DNA/RNA synthesizer.

Table 6: Synthetic primers

Example 13: Cloning of genes encoding K light chain constant regions

Step 1 : Extraction of entire mRNA

Step 2: Synthesis of entire cDNA

Synthesis of entire cDNA was performed using a Thermotranscript kit (GibcoBRL, America). First, 0.5 μg of mRNA was mixed with oligo-dTs (to reach 10 μl of a total volume), followed by denaturation at 65 ^°C for about 5 minutes and primer annealing. The resultant mixture was incubated at 50 ^°C for one hour under the following conditions: reverse transcriptase ("\≠), buffered solution (5 μl) (50 mM Tris-CI, pH 7.6, 50 mM KCI, 10 mM MgCI₂), DTT (0.1 M, 2.5 μl), dNTPs (2.5 ≠), RNase inhibitor (1 μl), and distilled deionized water (to total volume of 25 μl), and then inactivated at 95 ^°C for 5 minutes.

Step 3: PCR amplification of genes encoding light chain constant regions flanked by restriction sites

Genes encoding K light chain constant regions flanked by restriction sites were amplified by PCR using the cDNA (3 μl) obtained in Step 2 and the primers set forth in SEQ ID NOs: 11 and 12 (PCR conditions: 94 ^°C for 1 minute 30 seconds, 55^°C for 2 minutes, and 72 °C for 3 minutes, for 30 cycles). After the PCR amplification, gene fragments with about 350 bp were identified by agarose gel electrophoresis and ethidium bromide (EtBr) staining.

After electrophoresis on a 1.5% agarose gel, the gene fragments encoding K light chain constant regions flanked by restriction sites were recovered using a QIAgen extraction kit (Qiagen, America). The recovered gene fragments were subjected to impurity removal using phenol (200 μl) and chloroform (200 μl) and then purified using ethanol (2.5 ml). The purified gene fragments were subcloned into pCRII vectors (Invitrogen, America) and then transformed into E.coli XL1-Blue

[Cohen, S. N. et al., Proc. Natl. Acad. Sci. 69, 2110, 1972] to thereby obtain transformants. The obtained transformants were incubated in LB media containing 100 μgl l of ampicillin for overnight and then plasmids were recovered. The recovered plasmids were digested with an EcoRl restriction enzyme (Biolab, America), which revealed the presence of the above gene fragments. Furthermore, the sequencing results showed that the gene fragments have the nucleotide sequence set forth in SEQ ID NO: 5 and the amino acid sequence set forth in SEQ ID NO: 6. The recovered plasmids were designated as pCR-KC.

Example 14: Cloning of K light chain expression vectors

The pCDNA-dhfr vectors obtained in Step 2 of Example 9 were digested with HindUUNotl restriction enzymes (Biolab, America). On the other hand, the pCR-KC vectors obtained in Step 4 of Example 13 were digested with Hindlll/Notl to obtain target gene fragments and then the obtained gene fragments were inserted into the recognition sequences of the Hindlll/Notl restriction enzymes of the pCDNA-dhfr vectors. The resultant vectors were then transformed into E. co//XL1-Blue in the same manner as in Step 4 of Example 13 to thereby obtain transformants. Plasmids were recovered from the obtained transformants and digested with Hindlll/Notl, followed by electrophoresis. As a result, it was demonstrated that the recovered plasmids contained genes encoding light chain constant regions flanked by restriction sites set forth in SEQ ID NO: 5. The above transformants were deposited at the Gene Bank in KRIBB on April 18, 2002 (accession number: KCTC10230BP) under the accession name of E. coli XL1-Blue/pHAB-KC.

Example 15: Cloning of K light chain variable region

The recombinant vectors, pCRC6Lv which contains genes encoding ic light chain variable regions (WO02/092819), and the expression vectors obtained in Example 14, E. coli XL1-Blue/pHAB-KC (accession number: KCTC10230BP) were respectively digested with Hind U BsiWl. The obtained two types of gene fragments were incubated with T4 DNA ligase at 16^°C for overnight and transformed into E. coli XL1-Blue to obtain transformants. The obtained transformants were incubated in LB media containing 100 μg/ml of ampicillin for overnight and plasmids were recovered. The recovered plasmids were digested with Hindlll/Notl, followed by electrophoresis, which revealed the presence of entire light chain genes of about 0.7 kb (see FIG. 9).

Claims

What is claimed is:

1. An expression vector for an antibody heavy chain variable region, which comprises:

(i) a gene (gene A) comprising a Sacl recognition sequence; and a gene encoding an antibody heavy chain constant region, which is linked to the gene A;

(ii) a gene encoding an antibody heavy chain constant region which comprises an Apal recognition sequence; or

(iii) the gene A and the gene of (ii), which are linked to each other.

2. The expression vector according to claim 1 , which comprises the nucleotide sequence set forth in SEQ ID NO: 1.

3. A host cell transformed with the expression vector according to claims 1 or 2.

4. The host cell according to claim 3, which is E.coli XL1-Blue/pHAB-HC (accession number: KCTC10229BP).

5. An expression vector for an antibody lambda (λ ) light chain variable region, which comprises: a gene (gene B) comprising an Avήl recognition sequence; and a gene encoding an antibody λ light chain constant region, which is linked to the gene B.

6. The expression vector according to claim 5, further comprising a dehydrofolate reductase gene.

7. The expression vector according to claim 6, which comprises the nucleotide sequence set forth in SEQ ID NO: 3.

8. A host cell transformed with the expression vector according to any one of claims 5 to 7.

9. The host cell according to claim 8, which is E.coli XL1 -Blue/pHAB-LC (accession number: KCTC10231 BP).

10. An expression vector for an antibody kappa (K ) light chain variable region, which comprises: a gene (gene C) comprising a BsiWl recognition sequence; and a gene encoding an antibody K light chain constant region, which is linked to the gene C.

11. The expression vector according to claim 10, further comprising a dehydrofolate reductase gene.

12. The expression vector according to claim 11 , which comprises the nucleotide sequence set forth in SEQ ID NO: 5.

13. A host cell transformed with the expression vector according to any one of claims 10 to 12.

14. The host cell according to claim 13, which is E. coli XL1-Blue/pHAB-KC (accession number: KCTC10230BP).