KR100528772B1 - A lambda light chain expression vector - Google Patents

Abstract

The present invention provides a light chain lambda expression vector pHAB-LC having a human light chain lambda constant gene linked to an Avr II cognitive sequence (CCTAGG). According to the expression vector pHAB-LC of the present invention, the light chain lambda variable genes of various antibodies can be inserted directly into the vector in the form of a cassette to express the entire light chain lambda gene.

Description

Antibody Light Chain Lambda Expression Vector {A LAMBDA LIGHT CHAIN EXPRESSION VECTOR}

The present invention relates to light chain lambda expression vectors of various antibodies.

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, 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, a polya (polyadenine, polyadenylation, poly A) used for the origin of replication (ORI) for termination of transcription and the SV40 promoter and vector replication in the same vector. The gene of the same SV40 virus should be used to insert the sequence, which has a disadvantage that recombination may occur in the 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, the development of a general expression vector capable of expressing a whole gene linked with a variable region gene and a constant region gene in one step is required by directly inserting a variable region gene candidate group into the vector. Do.

Accordingly, the present inventors have repeatedly studied to solve the above problems, and as a result, a restriction enzyme site which is commonly present in the joining region of the light chain lambda variable region genes (hereinafter, “variable genes”) of various antibodies is present. ), A general expression vector including a light chain lambda constant region gene (hereinafter referred to as "constant gene") linked to the restriction enzyme recognition sequence was developed. It is possible to express various light chain lambda genes by inserting the light chain lambda variable genes of various antibodies directly into the expression vector without having to undergo several steps of cloning using the expression vectors as described above.

An object of the present invention is to provide a general light chain lambda expression vector capable of expressing the light chain lambda whole gene by inserting the light chain lambda variable genes of various antibodies directly into the expression vector. According to one aspect of the present invention, In the antibody expression vector, there is provided an expression vector pHAB-LC having a human light chain lambda constant gene linked to an Avr II cognitive sequence (CCTAGG) under the promoter.

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The expression vector pHAB-LC of the present invention comprises a human light chain lambda constant gene downstream of the promoter.

We have analyzed the variable and constant genes of various human antibodies. As a result, in the case of light chain lambda (λ), it was found that the junction region of the variable gene terminal contains the restriction enzyme Avr II recognition sequence (CCTAGG) in common, and this is a restriction enzyme recognition position for linkage of the variable gene and the constant gene. Used as. Thus, the initiation site of the light chain lambda constant gene, which is a site directly linked to the light chain lambda variable gene, is linked to the Avr II cognitive sequence (CCTAGG). As described above, the light chain lambda constant gene linked to the Avr II cognitive sequence (CCTAGG) is included in the lower part of the promoter, and from this configuration, the variable chain light lambda variable genes of various antibodies are simply inserted into the expression vector pHAB-LC in a cassette form. It is possible to express whole genes in which genes and constant genes are linked. The human light chain lambda constant gene associated with Avr II cognitive sequence (CCTAGG) may have an amino acid sequence of SEQ ID NO: 4. In addition, the human light chain lambda constant gene includes the nucleotide sequence of SEQ ID NO: 3. In the expression vector pHAB-LC of the present invention, a promoter may be used as a CMV (CMV) promoter, and in order to increase the gene termination efficiency, it is subordinated to BG polyadenyse ((BGH polyadenine, BGH polyadenylation, BGH poly A)). In addition, the dhfr (dehydrofolate reductase) gene may be inserted to enable amplification of the gene. Preferably, the expression vector pHAB-LC of the present invention may have the structure of FIG. 1. The expression vector pHAB-LC according to the present invention was prepared by removing the restriction enzyme Avr II site from pCDNA (Invitrogen, USA) and introducing a human light chain lambda constant gene linked to Avr II cognitive sequence (CCTAGG). The method for expressing the light chain lambda whole gene using the expression vector pHAB-LC of the present invention prepared as described above comprises the steps of cleaving the expression vector pHAB-LC with restriction enzyme Avr II and the cleavage site. Directly inserting the light chain lambda variable genes of various antibodies According to another aspect of the present invention, host cells transformed with the expression vector pHAB-LC are provided, preferably E. coli XL1-Blue / A host cell which is pHAB-LC (Accession No .: KCTC 10231BP) is provided. The expression vector pHAB-LC according to the present invention includes a light chain lambda constant gene linked to an Avr II cognitive sequence (CCTAGG) in the lower part of the promoter and thus various antibodies. The light chain lambda variable gene of can be inserted directly into the Avr II restriction enzyme site in the form of a cassette, it is possible to express the light chain lambda whole genes of various antibodies at once without having to go through several cloning steps.

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 Light Chain Sequencing

As a result of analyzing human antibody light chain lambda amino acid and nucleotide sequence ( NIH 91, 3242, 1991), the restriction enzyme recognition sequence (CCTAGG) was identified at the joining region (JR) of the end of the gene encoding the variable region. Avr II was found to be common.

Junction region (JL) at the end of the gene encoding the light chain lambda variable region Amino acid sequence Base sequence JL1 --VLG --- GTC CTA GGC JL2 --VLG --- GTC CTA GGC JL3 --VLG --- GTC CTA GGC JL4 --ILG --- ATT TTA GGC JL5 --VLG --- GTC CTA GGC JL6 --VLG --- GTC CTC GGC JL7 --ALG --- GCC CTC GGC

In the case of the light chain lambda JL4 sequence, since the ATT TTA encoding Ile / Leu can be changed to ATC CTA without changing the amino acid, the Avr II restriction enzyme recognition sequence can be used. Similarly, in the case of JL6 and JL7, it is possible to change CTC to CTA without changing amino acids.

Example 2. Synthesis of Light 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 FL320 Light chain lambda start GT C CTA GGC CAG CCC AAG GCT GCC CCT TCG GTC ACT CTG 2 LDCT Light chain lambda ending CTA TGA ACA TTC TGT AGG GGC CAC TGT C

Example 3. Cloning of Light Chain Lambda 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 (LDCT) (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 Light Chain Lambda Constants

In order to amplify the light chain lambda constant gene using the cDNA obtained in step 2, PCR (polymerase chain reaction) was performed using 3 μl cDNA and the primers of Table 2, and in the first step, 1 hour at 95 ° C. for 5 minutes. In the second 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, 2 minutes at 94 ° C, 2 minutes at 55 ° C, and 10 minutes at 72 ° C. It was confirmed by agarose electrophoresis and ethidium bromide (EtBr) that the light chain lambda constant gene fragment of about 320bp was amplified by one time under the condition of.

Step 4. Cloning of Light Chain Lambda Constants into PCR Vectors

After performing 1.5% agarose gel electrophoresis, phenol (200 μl) and chloroform (200 μl) were added to 320bp gene fragments recovered using a QIAgen extraction kit (Qiagen, USA) to remove impurities. Then, ethanol (2.5 ml) was added and purified. 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 transformants were incubated overnight in a Luria-bertani (LB) medium containing 100 μg / ml of ampicillin to recover the plasmid. The recovered plasmid was digested with the restriction enzyme EcoR I (Biorepsa, USA) to confirm the presence of the gene fragment. 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-LC.

Example 4 Cloning of Light Chain Lambda Expression Vectors

Step 1. Development of pCDNA3.1A Vector Without Neo

The pCDNA3.1A vector (Invitrogen, USA) was reacted with the restriction enzyme Pvu II for 2 hours at 37 ° C for the neomycin resistance gene expression system (SV40 promoter, ori, Neomycin, SV40 poly A) gene (hereinafter , Called 'Neo') was cleaved from the entire vector gene and confirmed by 1.0% electrophoresis, only about 3.5Kb vector gene from which Neo was removed was isolated. In order to self-ligation the vector fragment from which Neo was removed, 1 unit of T4-DNA ligase was added and left overnight at 16 ° C. to connect the truncated vector ends. E. coli XL1-Blue was transformed in the same manner as in Step 4 of Example 3 (Cohen, SN et al., Proc. Nat. Acad. Sci. 69, 2110, 1972) 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. The recovered plasmid was digested with restriction enzyme Pvu II (Biorep, USA) and confirmed that the Neo gene was removed by agarose electrophoresis.

Step 2. Insertion of dhfr gene into pCDNA3.1A vector without Neo

The vector obtained in step 1 was digested with restriction enzyme HindIII / PvuI (Bioreps, USA) to recover a gene fragment of about 2 Kb from which a portion of the CMV promoter and ampicillin gene was removed. In addition, pKCdhfr animal cell expression vector ( J. Medical Virology 52: 226-233, 1997) were digested with the restriction enzyme HindIII / PvuI to recover a gene fragment of about 3 Kb containing a CMV promoter, a dhfr amplification gene, and a portion of the ampicillin resistance gene. Each of the recovered about 2Kb gene fragment and about 3Kb gene fragment were recovered using the T4-DNA ligase, and then E. coli XL1-Blue was transfected in the same manner as in Step 4 of Example 3. Conversion was made to transformants. The resulting transformants were cultured overnight in a medium containing ampicillin to recover the plasmid. The recovered plasmid was digested with restriction enzyme HindIII / PvuI, and agarose electrophoresis confirmed that gene fragments of about 2 kb and about 3 kb were inserted. The recovered plasmid was named pCDNA-dhfr vector.

Step 3. Development of pHAB-LC Expression Vector

The pCDNA-dhfr vector obtained in step 2 was digested with restriction enzyme HindIII / NotI (Biorepth, USA). In addition, a gene fragment obtained by cutting the pCR-LC vector in which the gene encoding the light chain lambda constant region including the restriction site was inserted with HindIII / NotI was inserted into the HindIII / NotI restriction enzyme recognition sequence of the pCDNA-dhfr vector. . 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 light chain lambda constant region containing the restriction enzyme site of SEQ ID NO: 3 was inserted. The transformants were deposited as E. coli XL1-Blue / pHAB-LC (Accession No .: KCTC10231BP) to the Korea Biotechnology Research Institute Gene Bank on April 18, 2002.

The expression vector pHAB-LC according to the present invention can insert the light chain lambda variable region genes of various antibodies directly into the vector in the form of a cassette so that the entire light chain lambda gene can be expressed at one time without going through several cloning steps and various treatments. It can be usefully used for antibody development.

Figure 1 shows a genetic map of the light chain lambda (λ) variable region expression vector of the present invention.

Figure 2 schematically shows the manufacturing process of the light chain lambda (λ) expression vector.

<110> YUHAN CORPORATION <120> THE LIGHT CHAIN EXPRESSION VECTOR FOR DEVELOPING THERAPEUTIC ANTIBODY <130> YU200206 <160> 4 <170> KopatentIn 1.71 <210> 1 <211> 39 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 1 gtcctaggcc agcccaaggc tgccccttcg gtcactctg 39 <210> 2 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> primer <400> 2 ctatgaacat tctgtagggg ccactgtc 28 <210> 3 <211> 327 <212> DNA <213> Homo sapiens <400> 3 gtcctaggcc agcccaaggc tgccccttcg gtcactctgt tcccaccctc ctctgaggag 60 cttcaagcca acaaggccac actggtgtgt ctcataagtg acttctaccc gggagccgtg 120 acagtggcct ggaacggaga tagcagcccc gtcaaggcgg gagtggagac caccacaccc 180 tccaaacaaa gcaacaacaa gtacgcggcc agcagctacc tgagcctgac gcctgagcag 240 tggaagtccc acaaaagcta cagctgccag gtcacgcatg aagggagcac cgtggagaag 300 acagtggccc ctacagaatg ttcatag 327 <210> 4 <211> 108 <212> PRT <213> Homo sapiens <400> 4 Val Leu Gly Gln Pro Lys Ala Ala Pro Ser Val Thr Leu Phe Pro Pro 1 5 10 15 Ser Ser Glu Glu Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile 20 25 30 Ser Asp Phe Tyr Pro Gly Ala Val Thr Val Ala Trp Asn Gly Asp Ser 35 40 45 Ser Pro Val Lys Ala Gly Val Glu Thr Thr Thr Pro Pro Lys Gln Ser 50 55 60 Asn Asn Lys Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln 65 70 75 80 Trp Lys Ser His Lys Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser 85 90 95 Thr Val Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100 105

Claims (8)

The expression vector pHAB-LC of the antibody expression vector, which has a human light chain lambda constant gene linked to the Avr II cognitive sequence (CCTAGG) under the promoter. The expression vector pHAB-LC according to claim 1, wherein the amino acid sequence of the constant gene is SEQ ID NO: 4. The expression vector pHAB-LC according to claim 2, wherein the nucleotide sequence of the constant gene is SEQ ID NO: 3. A host cell transformed with the expression vector according to any one of claims 1 to 3. The host cell according to claim 4, which is E. coli XL1-Blue / pHAB-LC (Accession Number: KCTC 10231BP). According to claim 1, Expression vector pHAB-LC having the structure of Figure 1. (a) cleaving the expression vector pHAB-LC with restriction enzyme Avr II; And (b) inserting a light chain lambda variable gene into the cleavage site Method for expressing the light chain lambda whole gene comprising a. delete