KR100604038B1 - A heavy chain expression vector - Google Patents

Abstract

The present invention relates to a heavy chain expression vector of an antibody, and more particularly to encoding a restriction site and a constant region of an antibody which are commonly present in a joining region of a gene encoding a variable region of an antibody. It relates to an expression vector containing the gene to be. The expression vector is capable of expressing the entire heavy chain gene by inserting genes encoding various variable regions in a direct cassette format, which is useful for developing various therapeutic antibodies.

Therapeutic antibody, expression vector, variable region, constant region, restriction enzyme, heavy chain

Description

Antibody heavy chain expression vector {A HEAVY CHAIN EXPRESSION VECTOR}

Figure 1 shows a genetic map of the expression vector of the present invention.

Figure 2 schematically shows the manufacturing process of the expression vector of the present invention.

The present invention relates to a heavy chain expression vector of an antibody, and more particularly to encoding a restriction site and a constant region of an antibody which are commonly present in a joining region of a gene encoding a variable region of an antibody. It relates to an expression vector containing the gene to be.

The development of therapeutic antibodies has been made in many fields around the world, and antibodies have been developed for the treatment of numerous diseases (Reichmann, L., Clark, M., et al. Nature 322, 323-327 (1988). Paul Carter and H. Michael Shepard. Proc. Natl. Acad. Sci. USA 89, 4285-4289 (1992); John Hakimi, and William P. Schneider. J. Immunology 151, 1075-1085 (1993).

To develop humanized antibodies and chimeric antibodies, cloned the variable region genes of mouse antibodies into human constant genes by genetic recombination to make humanized antibodies, and then cloned them into animal cell expression vectors to transform them into animal cells and then express them. I'm using the method of analysis.

As such, in the case of the antibody expression vector, a specific vector system is not present, but the antibody gene is expressed by using an animal cell expression vector that has been used previously, and a system that is widely used as an animal cell expression vector is pCDNA (Invitrogen). Inc., USA) As a vector system, various protein genes are cloned and expressed in animal cells. However, the above-described pCDNA vector system (Invitrogen, USA) has a problem of inserting the dhfr gene into the light chain or heavy chain expression vector after several steps of cloning and amplifying the antibody gene.

In addition, in order to express antibody genes using animal cell expression vectors, two kinds of expression vectors for expressing light and heavy chain genes are required.In the case of the expression vector pKCdhfr developed by the Biotechnology Research Institute, amplification of antibody genes is required. The required dhfr gene is inserted into an expression vector containing a light chain, and a heavy chain-inserted expression vector is constructed except for the light and dhfr genes ( J. Medical Virology 52: 226-233, 1997). It was.

However, in the case of the pKCdhfr light chain expression vector described above, an origin of replication (ORI) for the SV40 promoter and vector replication in the same vector, and a polya (polyadenine, polyadenylation, poly A) used for termination of transcription ) The same SV40 virus gene should be used to insert the sequence, which has a disadvantage in that recombination may occur in a vector.

The recently developed animal cell expression vector pMG (Invivogen, USA) contains two multicloning sequences (MCS) in the same vector, which is a protein composed of two or more subunits such as antibodies. It is a vector system suitable for expression. However, cloning the antibody gene using such a vector system requires a long research period for cloning work in developing a new therapeutic antibody, and in order to amplify the antibody gene, the dhfr gene must be inserted from an external vector. It is implicated.

Therefore, in order to develop various therapeutic antibodies, it is necessary to develop a new expression vector system capable of efficiently expressing all genes linked with variable region-constant regions by inserting various variable region gene candidate groups directly into a vector.

Accordingly, the present inventors have conducted studies to solve the above problems, and as a result, the inventors have identified genes encoding restriction chains and heavy chain constant regions that are commonly present in the joining region of the gene encoding the heavy chain variable region. The expression vector of the present invention was developed, and since the expression vector of the present invention can produce the entire heavy chain gene by directly cloning the gene encoding the variable region in a cassette format, it can be usefully used for developing various therapeutic antibodies.
Accordingly, it is an object of the present invention to provide an expression vector having the nucleotide sequence of SEQ ID NO: 3 under the promoter.
It is also an object of the present invention to provide a host cell transformed with the expression vector.

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The present invention relates to an expression vector comprising a restriction region and a gene encoding a heavy chain constant region, which are commonly present in a joining region of a gene encoding a heavy chain variable region.

We analyzed genes encoding variable regions and genes encoding constant regions of various human antibodies. As a result, in the case of the heavy chain, the joining region (JR) at the end of the gene encoding the variable region contains the restriction enzyme Sac I recognition sequence (GAGCTC) in common, and the starting position of the constant region is the restriction enzyme recognition sequence ( GGGCCC) was found to include Apa I in common.

Thus, according to one aspect of the invention, there is provided an expression vector comprising the nucleotide sequence of SEQ ID NO: 3 below the promoter, preferably an expression vector having the genetic map of FIG. In the expression vector of the present invention, a gene encoding a heavy chain variable region of the antibody can be effectively introduced into a Sac I or Apa I restriction site.
The expression vector according to the present invention can be prepared by removing the restriction enzyme Apa I site from pCDNA (Invitrogen, USA), and introducing the nucleotide sequence of SEQ ID NO. The expression vector includes a CMV promoter upstream as a promoter and a BZ polyadenyse (BGH polyadenine, BGH polyadenylation, BGH poly A) downstream.

According to another aspect of the present invention, a host cell transformed with the expression vector is provided, and preferably, a host cell which is E. coli XL1-Blue / pHAB-HC (Accession Number: KCTC 10229BP) is provided.
The expression vector according to the present invention can produce the entire heavy chain gene in which the variable region gene and the constant region gene are linked by inserting (cloning) the gene encoding the heavy chain variable region into the restriction enzyme region directly.

Variable region genes that can be inserted into the expression vector according to the present invention includes a human, mouse or humanized variable region gene.
Hereinafter, the present invention will be described in more detail with reference to Examples. However, this does not limit the present invention.

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Example

Example 1 Human Antibody Heavy Chain Sequencing

As a result of analyzing human antibody heavy chain amino acid and nucleotide sequence ( NIH 91, 3242, 1991), restriction enzyme recognition sequence Sac I (GAGCTC) which is common in all joining regions (JR) of all variable gene ends as shown in Table 1 ) And the restriction enzyme Apa I (GGGCCC) at the start of the constant region were commonly found.

The junction region (JH) at the end of the gene encoding the heavy chain variable region and the starting position (CH) encoding the constant region Amino acid sequence Base sequence JH1 / CH1 --VSSASTKGP --- GTG AGC TCT GCC TCC ACC AAG GGC CCA JH2 / CH1 --VSSASTKGP --- GTG AGC TCT GCC TCC ACC AAG GGC CCA JH3 / CH1 --VSSASTKGP --- GTG AGC TCT GCC TCC ACC AAG GGC CCA JH4 / CH1 --VSSASTKGP --- GTG AGC TCT GCC TCC ACC AAG GGC CCA JH5 / CH1 --VSSASTKGP --- GTG AGC TCT GCC TCC ACC AAG GGC CCA JH6 / CH1 --VSSASTKGP --- GTG AGC TCT GCC TCC ACC AAG GGC CCA

Example 2. Synthesis of Heavy Chain Linked Primer

In order to use the restriction enzyme recognition sequence found in Example 1, primers as shown in Table 2 were synthesized. Synthesis of the primer was synthesized using an ABI DNA / RNA synthesizer.

Synthetic primer SEQ ID NO: Primer Name location Sequence One FH365 Heavy chain start GT G AGC TC T GCC TCC ACC AA G GGC CC A TCG GTC TTC C 2 HCT Heavy chain termination C TCA TTT ACC CGG GGA CAG GGA GAG GCT C

Example 3 Cloning of Heavy Chain Constants

Step 1. Full mRNA extraction

Lymphocytes were recovered by centrifugation of normal human blood and mRNA was obtained from recovered lymphocytes using a FastTrack 2.0 mRNA isolation kit (Invitrogen, USA). Quantitative mRNA was measured using a spectrophotometer, and mRNA was quantified by measuring absorbance at 260 nm.

Step 2. Synthesis of Whole cDNA

Synthesis of whole cDNA was performed using a Thermotranscript Kit (GibcoBRL, USA). First, 0.5 μg mRNA was mixed with oligo dT or Gene specific primer, SEQ ID NO: 2 (HCT) (the total volume is 10 μl), and denatured at 65 ° C. for about 5 minutes. (denaturation) and primer annealing (primer annealing). cDNA synthesis was performed using reverse transcriptase (1 μl) in buffer (5 μl), Diti (DTT, 0.1M, 2.5 μl), DNTP (2.5 μl), RNA degrading enzyme inhibitor (RNase inhibitor, 1 μl), and sterile secondary distilled water (wherein, the total volume is 25 μl) was reacted at 50 ° C. for 1 hour, and then inactivated at 95 ° C. for 5 minutes.

Step 3. PCR amplification of heavy chain constant genes

In order to amplify the antibody constant gene using cDNA, PCR (polymerase chain reaction) was performed using 3 μl cDNA and the primers of Table 2, and the first step was performed once at 95 ° C. for 5 minutes. In the step, 30 times were carried out under reaction conditions of 2 minutes at 55 ° C, 2 minutes at 55 ° C, and 3 minutes at 72 ° C. In the third step, once at the conditions of 2 minutes at 55 ° C, 2 minutes at 55 ° C and 10 minutes at 72 ° C. The amplification of the heavy chain constant gene fragment of about 1,000bp was confirmed by agarose electrophoresis and ethidium bromide (EtBr) staining.

Step 4. Cloning of Heavy Chain Constants into PCR Vectors

After performing 1.5% agarose gel electrophoresis, phenol (200 μl) and chloroform (200 μl) were added to the 1,000 bp heavy chain gene fragment recovered using a QIAgen extraction kit (Qiagen, USA). Was removed and purified by the addition of ethanol (2.5 ml). The purified gene fragment was subcloned into a pCRII vector (Invitrogen, USA) and then transformed into E. coli XL1-Blue (Cohen, SN et al, Proc. Natl. Acad. Sci. 69, 2110, 1972). ) Transformants were obtained. The resulting transformant was incubated overnight in a Luria-bertani (LB) medium containing 100 μg / ml of ampicillin, and then the plasmid was recovered. Then, the gene fragment was cut by restriction enzyme EcoR I (Biorepsa, USA). It was confirmed that this exists. In addition, it was confirmed that the sequence analysis had the nucleotide sequence of SEQ ID NO: 3 and the amino acid sequence of SEQ ID NO: 4. The plasmid obtained above was named pCR-HC.

Example 4 Cloning of Heavy Chain Expression Vectors

Step 1. Development of pCDNA3.1A Vector Without Apa I

The pCDNA3.1A vector (Invitrogen, USA) was digested with restriction enzyme Apa I for 2 hours at 37 ° C, and 1 unit of Klenow enzyme and dNTP were added for 1 hour at 30 ° C. The single nucleotide exposed by cleavage with Apa I restriction enzyme was filled with double nucleotides. After the reaction, the gene fragment was recovered in the same manner as in Step 4 of Example 3. In order to self-ligation of the recovered gene fragment, 1 unit of T4-DNA ligase is added to the recovered gene fragment, which is left overnight at 16 ° C., and the cut vector ends are connected. E. coli XL1-Blue was transformed in the same manner as in Step 4 of Example 3 to obtain a transformant. The resulting transformants were incubated overnight in a Luria-bertani (LB) medium containing 100 μg / ml of ampicillin to recover the plasmid. As a result of reacting the recovered plasmid with restriction enzyme Apa I (Biorep, USA), it was confirmed by electrophoresis that the recovered plasmid was not cleaved. The plasmid recovered above was named pHAB vector.

Step 2. Development of pHAB-HC Expression Vector

The vector pHAB obtained in step 1 was digested with restriction enzyme HindIII / NotI (Bioreps, USA). In addition, the vector pCR-HC containing the heavy chain constant gene was digested with HindIII / NotI, and a gene fragment of about 1 Kb was inserted into the HindIII / NotI restriction enzyme recognition sequence of the vector pHAB. E. coli XL1-Blue was transformed in the same manner as in Step 4 of Example 3 to obtain a transformant. The plasmid was recovered from the obtained transformant, digested with restriction enzyme HindIII / NotI, and confirmed by electrophoresis that the gene encoding the heavy chain constant region containing the restriction enzyme site of SEQ ID NO: 3 was inserted. The sieve was deposited as E. coli XL1-Blue / pHAB-HC (Accession No .: KCTC10229BP) with the Korea Biotechnology Research Institute on April 18, 2002.

The expression vector according to the present invention can efficiently produce the entire heavy chain gene by inserting the heavy chain variable region genes of various antibodies in a direct cassette format. Therefore, the expression vector according to the present invention can be usefully used for the development of various therapeutic antibodies.

<110> YUHAN CORPORATION <120> THE HEAVY CHAIN EXPRESSION VECTOR FOR DEVELOPING THERAPEUTIC          ANTIBODY <130> YU200205 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gtgagctctg cctccaccaa gggcccatcg gtcttcc 37 <210> 2 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ctcatttacc cggggacagg gagaggctc 29 <210> 3 <211> 1002 <212> DNA <213> Homo sapiens <400> 3 gtgagctctg cctccaccaa gggcccatcg gtcttccccc tggcaccctc ctccaagagc 60 acctctgggg gcacagcggc cctgggctgc ctggtcaagg actacttccc cgaaccggtg 120 acggtgtcgt ggaactcagg cgccctgacc agcggcgtgc acaccttccc ggctgtccta 180 cagtcctcag gactctactc cctcagcagc gtggtgaccg tgccctccag cagcttgggc 240 acccagacct acatctgcaa cgtgaatcac aagcccagca acaccaaggt ggacaagaaa 300 gttgagccca aatcttgtga caaaactcac acatgcccac cgtgcccagc acctgaactc 360 ctggggggac cgtcagtctt cctcttcccc ccaaaaccca aggacaccct catgatctcc 420 cggacccctg aggtcacatg cgtggtggtg gacgtgagcc acgaagaccc tgaggtcaag 480 ttcaactggt acgtggacgg cgtggaggtg cataatgcca agacaaagcc gcgggaggag 540 cagtacaaca gcacgtaccg tgtggtcagc gtcctcaccg tcctgcacca ggactggctg 600 aatggcaagg agtacaagtg caaggtctcc aacaaagccc tcccagcccc catcgagaaa 660 accatctcca aagccaaagg gcagccccga gaaccacagg tgtacaccct gcccccatcc 720 cgggatgagc tgaccaagaa ccaggtcagc ctgacctgcc tggtcaaagg cttctatccc 780 agcgacatcg ccgtggagtg ggagagcaat gggcagccgg agaacaacta caagaccacg 840 cctcccgtgc tggactccga cggctccttc ttcctctaca gcaagctcac cgtggacaag 900 agcaggtggc agcaggggaa cgtcttctca tgctccgtga tgcatgaggc tctgcacaac 960 cactacacgc agaagagcct ctccctgtct ccgggtaaat ga 1002 <210> 4 <211> 333 <212> PRT <213> Homo sapiens <400> 4 Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro   1 5 10 15 Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val              20 25 30 Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala          35 40 45 Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly      50 55 60 Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly  65 70 75 80 Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys                  85 90 95 Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys             100 105 110 Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu         115 120 125 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu     130 135 140 Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys 145 150 155 160 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys                 165 170 175 Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu             180 185 190 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys         195 200 205 Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys     210 215 220 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 225 230 235 240 Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys                 245 250 255 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln             260 265 270 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly         275 280 285 Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln     290 295 300 Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 305 310 315 320 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys                 325 330

Claims (8)

The expression vector of an antibody expression vector which has the base sequence of SEQ ID NO: 3 under a promoter. The expression vector of claim 1 having the gene map of FIG. 1. A host cell transformed with the expression vector according to claim 1. The host cell according to claim 3, which is E. coli XL1-Blue / pHAB-HC (Accession Number: KCTC 10229BP). delete delete delete delete

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