KR100250831B1 - Variable region of murine antibody against pre-s1 surface antigen of hepatitis -b virus , its c-dna and dna sequence thereof - Google Patents

Abstract

PURPOSE: Provided is cDNA gene coding rat's monoclonal antibody KR359 which binds with 21-28 amino acids of antigen pre-S1 epitope of hepatitis B virus in order to use the gene to bind with human antibody genes and consequently develop human antibodies with fewer side effects. CONSTITUTION: cDNA gene coding rat's monoclonal antibody KR359 is obtained by the following steps of: i) extracting RNA from hybridoma cell line (KCTC 0284 BP) and synthesizing cDNA for light and heavy chains; ii) implementing PCR with cDNA as a template and a primer of sequence ID. No. 1 of 5'-GCAGTCGACT GAGGCACCTC CAGATGTTAA C-3' for a light chain and a primer of sequence ID. No. 2 of 5'-CGGTCGACAG GGATCCAGAG TTCCAGGTCA C-3' for a heavy chain; iii) and obtaining 420bp cDNA of sequence ID. No. 8 described as in the description as a light chain and 500bp cDNA of sequence ID. No. 9 described as in the description as a heavy chain. Wherein, cDNA is also used in constructing plasmid vectors pKR359H and pKR359K.

Description

Variable regions of mouse monoclonal antibodies recognizing surface antigen pre-ES1 epitopes of hepatitis virus, genes encoding them and their base sequences

The present invention relates to variable regions of mouse monoclonal antibodies, cDNA genes thereof and their nucleotide sequences that bind to the surface antigen pre-S1 epitope of hepatitis B virus (hereinafter abbreviated as "HBV").

More specifically, the present invention provides a variable region of a mouse monoclonal antibody that specifically binds to an epitope comprising amino acids 21-28 of surface antigen pre-S1 of HBV, heavy and light chain cDNA genes encoding them, and their bases. Regarding a sequence and plasmid vectors comprising the same, the gene can be used to develop humanized antibodies and the like for preventing and treating HBV infection.

Hepatitis B virus (HBV) is a pathogen that invades humans, causes chronic and acute hepatitis, and when it worsens, causes cirrhosis and liver cancer. It is estimated that as many as 300 million people worldwide are infected (Tiollais & Buendia, Sci. Am., 264, 48, 1991).

In the case of HBV infection, HB immunoglobulin (HBIG) may be used to prevent HBV infection, especially in newborns born to HBV-positive mothers, health care workers exposed to HBV, or patients undergoing liver transplantation due to HBV-related liver disease. (Beasley, et al., Lancet, 2: 1099, 1983; Todo, et al., Hepatol., 13: 619, 1991). However, currently used HBIG is extracted from plasma, which has a low specificity and exposure to contaminants, which requires a continuous supply of human blood for mass production. This disadvantage can be overcome by developing and using monoclonal antibodies against the surface antigens of HBV.

The coating of HBV consists of three proteins, specifically, a major protein containing S antigen, a middle protein containing S antigen and a pre-S2 antigen, and a S antigen, pre-S2 antigen and a pre- It consists of a large protein containing the S1 antigen (Neurath, AR and Kent, SBH, Adv. Vir. Res. 34: 65-142, 1988). All of these surface antigen proteins induce antibodies that neutralize and neutralize HBV, in particular antibodies induced by the pre-S site are associated with the removal of the virus and recovery from acute HBV infection and the immune response to the S antigen. Non-responsiveness) (Iwarson, S. et al., J. Med. Virol. 16: 89-96, 1985; Itoh, Y. et al., Proc. Natl. Acad. Sci. USA 84 : 9174-9178, 1986; Neurath, AR et al., Vaccine, 7: 234-236, 1989; Budkowska, A. et al., J. Med. Virol., 20: 111-125, 1986; Milich DR et al., Proc. Natl. Acad. Sci. USA, 82: 8168-8172, 1985; Neurath, AR et al., J. Med. Virol. 17: 119-125, 1985; Milich, DR et al., J Immunol., 137: 315-322, 1986). In particular, amino acid positions 21-47 of the pre-S1 protein bind to the HBV receptor of human hepatocytes and play a decisive role in infecting the hepatocytes, so monoclonal antibodies specific for these sites may play an important role in virus neutralization. Neurath, et al., Cell, 46: 429, 1986; Pontisso, et al., Virol., 173: 533, 1989; Neurath, et al., Vaccine, 7: 234, 1989).

The present inventors have attempted to obtain monoclonal antibodies directed against amino acid sites 21-47 of the surface antigen pre-S1 protein of HBV, and have developed various monoclonal antibodies that specifically recognize these epitopes. In particular, the monoclonal antibody KR359, which recognizes epitopes at amino acid positions 21-28, has been widely applied to various types of HBV such as adr, adw, and ayw, and thus may be useful for preventing and treating HBV infection.

However, the monoclonal antibodies produced in the mice show side effects that cause an immune response when injected into humans. Thus, attempts have been made to reduce this problem by using humanized antibodies that humanized mouse antibodies. Humanized antibodies can be prepared by implanting a region of complementarity determining regions (CDRs) in the variable regions of a mouse monoclonal antibody into human antibodies. To this end, the gene of the mouse antibody variable region should be obtained and expressed in animal cells by fusion with the gene of human antibody by genetic recombination (Verhoeyen, et al., Science, 239: 1534-1536, 1988).

Therefore, the inventors of the present invention provide a hybridoma cell line producing a monoclonal antibody KR359 that binds to an epitope at amino acid positions 21-28 of the HBV surface antigen pre-S1 to prepare a humanized monoclonal antibody that effectively prevents and treats HBV infection. The present invention was completed by obtaining the heavy and light chain cDNAs of the antibody variable regions using DNA sequences and confirming their DNA base sequences.

The present invention relates to epitopes comprising amino acids 21-28 of surface antigen free-S1 of HBV.

The object is to provide a variable region of the mouse monoclonal antibody KR359 that specifically binds to a human antibody, heavy and light chain cDNA genes encoding the same, and their nucleotide sequences.

1 is a cDNA base sequence of the light chain gene encoding the variable region of the mouse monoclonal antibody KR359 of the present invention and an amino acid sequence deduced therefrom,

2 is a cDNA base sequence of the heavy chain gene encoding the variable region of the mouse monoclonal antibody KR359 of the present invention and an amino acid sequence deduced therefrom.

The present invention provides a heavy chain cDNA gene encoding the variable region of mouse monoclonal antibody KR359 and the nucleotide sequence of SEQ ID NO: 9. The present invention also provides a light chain cDNA gene encoding the variable region of the mouse monoclonal antibody KR359 and the nucleotide sequence of SEQ ID NO: 8.

In addition, the present invention provides the plasmid vector pKR359H comprising the heavy chain gene of the monoclonal antibody and its E. coli transformant (Accession No .: KCTC 0355 BP) and the plasmid vector pKR359K comprising the light chain gene and its E. coli transformant (Accession No. KCTC 0336 BP).

The present invention also provides a variable region of the mouse monoclonal antibody KR359 consisting of the heavy chain of SEQ ID NO: 11 and the light chain of SEQ ID NO: 10.

Hereinafter, the present invention will be described in detail.

The present invention provides a monoclonal variable region of a mouse monoclonal antibody that binds to an epitope comprising amino acids 21-28 of amino acid sites 21-47 that play an important role in the antigenicity of surface antigen pre-S1 of HBV. A hybridoma cell line (Accession Number: KCTC 0284 BP) that produces the antibody KR359 is used.

Specifically, RNA was extracted from the hybridoma cell line to synthesize cDNAs of light and heavy chain genes, and the polymerase chain reaction was performed using the primer of SEQ ID NO: 1 for the light chain and the primer of SEQ ID NO: 2 for the heavy chain. (polymerase chain reaction, PCR) was performed. As a result, a light chain cDNA gene of about 420 bp including the nucleotide sequence of SEQ ID NO: 8 of the present invention and a heavy chain cDNA gene of about 500 bp comprising the nucleotide sequence of SEQ ID NO: 9 were isolated.

The cDNA genes were cloned into plasmid vectors pBluescript KS +, respectively, to prepare plasmid vectors pKR359H and pKR359K of the present invention.

The transformant obtained by transforming the plasmid vectors into Escherichia coli was deposited on May 29, 1997 to the Gene Bank of the Institute of Biotechnology, Korea Institute of Science and Technology (Accession No .: KCTC 0335 BP, KCTC 0336 BP).

The present invention also provides a variable region of a mouse monoclonal antibody consisting of a heavy chain variable region having an amino acid sequence of SEQ ID NO: 11 and a light chain variable region having an amino acid sequence of SEQ ID NO: 10.

The variable regions of the mouse antibodies of the invention and their genes can be used to humanize mouse antibodies. Humanized antibodies prepared by implanting CDRs regions directly binding to antigens among the variable regions of mouse monoclonal antibodies can produce monoclonal antibodies that can minimize immune responses when injected into humans, particularly monoclonal antibodies. Humanized antibodies using the antibody KR359 can be widely applied to the prevention of several types of HBV, such as adr, adw, ayw.

Hereinafter, the present invention will be described in detail with reference to Examples.

However, the embodiments are only illustrative of the present invention and the present invention is not limited by the embodiments.

<Example 1> Isolation and Total cDNA Synthesis of Total RNA from Mouse Hybridoma KR359 Cell Line

Single step method; Chomczynski and Sacchi, Anal. Biochem., 162: 10 ⁷ hybridoma KR359 cells producing mouse monoclonal antibodies that recognize amino acids 21-28 of the HBV surface antigen pre-S1 156-159, 1987) using a total of 10 μg of RNA extracted as a template, a light chain synthetic DNA oligomer (31-mer) having the nucleotide sequence of SEQ ID NO: 1, a heavy chain having a nucleotide sequence of SEQ ID NO: 2 50 pmol of each of the DNA oligomers (31-mers) was annealed at 70 ° C. for 10 minutes using a primer complementary to the antibody constant region. The primary cDNA was synthesized by adding a Superscript II (Gibcos) enzyme to perform reverse transcription.

The gene of the monoclonal antibody was amplified by PCR using a fifth fraction of the synthesized primary cDNA as a template and using Vent DNA polymerase (New England Biolabs). In this case, the primer used in the light chain, the 3 'side primer is a synthetic DNA oligomer having a nucleotide sequence of SEQ ID NO: 1 complementary to the immunogenic kappa chain constant region, the 5' primer is considered in consideration of the various N-terminal end of the variable region As the mixed primers designed, two types of primers were used: a synthetic DNA oligomer having a nucleotide sequence of SEQ ID NO: 3 (33-mer) and a synthetic DNA oligomer having a nucleotide sequence of SEQ ID NO: 4 (33-mer). In the case of the heavy chain, the synthetic DNA oligomer (31-mer) having the base sequence of SEQ ID NO: 2 complementary to the immunogenic Cγ1 region for the 3 'side primer, and the various N-terminus of the variable region for the 5' side primer are considered. A mixed primer designed to synthesize a synthetic DNA oligomer (46-mer) having a nucleotide sequence of SEQ ID NO: 5, a synthetic DNA oligomer (32-mer) having a nucleotide sequence of SEQ ID NO: 6, and a synthetic DNA oligomer having a nucleotide sequence of SEQ ID NO: 7 (32-mer), three kinds of primers were used.

For efficient cloning of PCR products, the light chain was assigned Sal I restriction enzyme sites at the 3'- primer end, and the heavy chain was assigned Sal I restriction enzyme sites at both the 5'- primer and 3'- primer. It was. PCR was repeated 30 times under reaction conditions of 1 minute at 94 ° C, 1 minute at 55 ° C, and 1 minute at 72 ° C. As a result, an amplified DNA having a length of about 420 bp and a heavy chain of about 500 bp in size estimated from the primer position was obtained.

<Example 2> Cloning of the mouse monoclonal antibody KR359 gene

In order to clone the light chain gene amplified in Example 1, the PCR product was first phosphorylated with T ₄ polynucleotide kinase (polynucleotide kinase, Boehringer Mannheim), treated with Sal I, and then developed on 1% agarose gel. About 420 bp of DNA was isolated using GeneClean II (GeneClean II, Bio101). To clone it, the plasmid vector pBluescript SK + was treated with Sma I and again with Sal I and then isolated using a kit of GeneClean II (Bio101). The two DNAs were linked using a T ₄ DNA ligase (New England Biolabs), transformed into E. coli NM522 by CaCl ₂ method, and then cloned K2, K3, K4, K5 clones with DNA inserts of about 420 bp. Selected.

In addition, in order to clone the heavy chain gene, the heavy chain gene amplified in Example 1 was treated with Sal I, developed on a 1% agarose gel, and the DNA corresponding to about 500 bp was cloned into GeneClean II (GeneClean II, Bio101). Separated using. The plasmid vector pBluescript KS + to be used as a vector to be cloned was treated with Sal I and subjected to dephosphorylation with calf intestinal phosphatase (New England Biolabs), followed by GeneClean II (GeneClean II, Bio101). Separated. The two DNAs were linked using a T ₄ DNA ligase (New England Biolabs) and transformed into Escherichia coli NM522 by CaCl ₂ method, and H2, H8 and H10 clones with 500 bp DNA insert were selected.

To analyze the DNA sequencing of the immune genes, each clone was grown to OD 0.5 in 1 ml 2 x YT medium containing 150 μg / ml ampicillin and then 10 times more helper phage M13K07 (helper) than E. coli. phage M13K07) was incubated for 6 hours in 10 ml of 2 x YT medium containing 7 μg / ml kanamycin, and the supernatant was concentrated with 5% polyethylene glycol (PEG) in a supernatant and phenol / chloroform. Extracted several times and separated. Using the T ₃ or T ₇ primer as a template, the nucleotide sequences of the individual clones were analyzed using a Sequenase version II kit (Amersham). As a result, all four clones of the light chain had the same nucleotide sequence, and the plasmid vector isolated from the clone was named pKR359K. The base sequences of the three heavy chain clones were identical, and the plasmid vector isolated from the clone was named pKR359H. The base sequence results confirmed above are shown in FIG. 1.

The transformant obtained by transforming the plasmid vector into Escherichia coli was deposited on May 29, 1997, at the Genetic Bank of Korea Institute of Science and Technology (accession number: KCTC 0335 BP, KCTC 0336 BP).

<Example 3> Sequence analysis of cDNA

Analysis of the DNA sequences encoding the variable regions of the mouse monoclonal antibody KR359 revealed that these immunogenes have complete residues and arrangements characteristic of the antibody structure, specifically the heavy chains of mouse subgroup I among several groups of immunoglobulins. Belongs to (A) and the light chain belongs to mouse subgroup V. CDR residues that recognize the antigen are heavy chain, CDR1 for 30-36, CDR2 for 51-66, CDR3 for 99-108, for light chain CDR1 for 24-34, CDR2 for 50-56, CDR3 for 89- It corresponded to 97. The structurally essential disulfide bonds involved cysteines 22 and 96 in the heavy chain and cysteines 23 and 88 in the light chain. The J (Joining) minigene, the terminal portion of the variable region, was located at 109-119 for the heavy chain and the light chain at 98-108. From the analysis results of this gene, it was confirmed that the light and heavy chain genes cloned into the plasmid vectors pKR359K and pKR359H of the present invention are functional.

As described above, the cDNA genes encoding the heavy and light chains of the variable region of the novel monoclonal antibody KR359, which bind to amino acid positions 21-28 of the surface antigen pre-S1 of HBV, are functional. In other words, humanized antibodies having low side effects in various types of HBV neutralization such as adr, adw, and ayw can be developed, which can be usefully used for the prevention and treatment of HBV infection.

(Sequence list)

SEQ ID NO: 1

Sequence length: 31

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 2

Sequence length: 31

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 3

Sequence length: 33

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 4

Sequence length: 33

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 5

Sequence length: 46

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 6

Sequence length: 32

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 7

Sequence length: 32

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 8

Length of sequence: 324

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 9

Sequence length: 357

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

(Sequence list)

SEQ ID NO: 10

Sequence length: 108

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: protein

(Sequence list)

SEQ ID NO: 11

Sequence length: 119

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: protein

Claims (8)

A cDNA gene comprising the nucleotide sequence of SEQ ID NO: 9 encoding the variable region of the heavy chain of a mouse monoclonal antibody that specifically binds an epitope comprising amino acids 21-28 of surface antigen pre-S1 of hepatitis B virus. A cDNA gene comprising the nucleotide sequence of SEQ ID NO: 8 encoding the variable region of the light chain of a mouse monoclonal antibody that specifically binds an epitope comprising amino acids 21-28 of surface antigen pre-S1 of hepatitis B virus. A plasmid vector pKR359H comprising the cDNA gene of claim 1. E. coli transformant DH5α / pKR359H transformed with the plasmid vector of claim 3 (Accession No .: KCTC 0335 BP). A plasmid vector pKR359K comprising the cDNA gene of claim 2. Escherichia coli transformant transformed with the plasmid vector of claim 5 DH5α / pKR359K (Accession Number: KCTC 0336 BP). A variable region of a mouse monoclonal antibody comprising a light chain of SEQ ID NO: 10 and a heavy chain of SEQ ID NO: 11 that specifically bind to an epitope comprising amino acids 21-28 of surface antigen pre-S1 of hepatitis B virus. Use of the variable region genes of the mouse monoclonal antibodies of claims 1 and 2 in combination with human antibody genes to produce humanized antibodies that recognize HBV surface antigen pre-S1.

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