KR20000033009A - Preparation of humanized antibody on surface antigen pre-s1 of hepatitis b virus - Google Patents

Abstract

PURPOSE: Humanized antibody on surface antigen pre-S1 of hepatitis B virus (HBV) is provided which is derived from mouse monoclonal antibody (KR 359) The humanized antibody maintains the affinity on the antigen but decreases the antigenecity of mouse antibody on the human body by changing amino acid residues specific for mouse into human specific one. CONSTITUTION: Humanized variable region of heavy chain is derived from variable region of heavy chain of human antibody DP67 substituted into mouse framework residues and complementarity-determining region (CDR)1, and 3 and part of 2. Complete humanized heavy chain is constructed by joining leader sequence for secretion, constant region of heavy chain from pRC/CMV-HC-HuS, and humanized variable region. Humanized variable region of light chain is derived from variable region of light chain of human antibody DPK9 substituted into mouse framework residues and complementarity-determining region (CDR)1, 2, and 3. Complete humanized light chain is constructed by joining leader sequence for secretion, constant region of heavy chain from pKC-hr-HuS, and humanized variable region. Each complete chain is cloned into expression vector, pRc/CMV, and transfected into COS7 cell for the production of antibody.

Description

Humanized antibody against surface antigen free-S1 of hepatitis V virus and preparation method thereof

The present invention relates to humanized antibodies against surface antigen free-S1 of HBV.

More specifically, the human antibody genes most similar to the variable regions of the mouse monoclonal antibody KR359 against the surface antigen pre-S1 of HBV are selected and subjected to the heavy and light chain genes of humanized antibodies. It relates to an expression vector comprising the same, a transformant transformed with the expression vector, and a method of producing a humanized antibody against the surface antigen pre-S1 of HBV by culturing the transformant.

HBV is a pathogen that invades the human body and causes chronic and acute hepatitis, and when it worsens, causes cirrhosis and liver cancer. It is estimated that 300 million patients worldwide (Tiollais and Buendia, Sci. Am., 264). , 48, 1991). The coating of HBV consists of three proteins, specifically, a major protein containing S antigen, a middle protein including S antigen and pre-S2 antigen, and a S antigen, pre-S2 antigen and pre- It consists of a large protein containing the S1 antigen. (Neurath, A.R. and Kent, S.B.H., Adv. Virus 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 S antigen ( Non-responsiveness) can be overcome (Iwarson et al., J. Med. Virol. 16: 89-96, 1985; Itoh, et al., Proc. Natl. Acad. Sci. USA 84: 9174-9178, 1986; Neurath Vaccine 7: 234-236, 1989; Budkowska et al., J. Med. Virol. 20: 111-125, 1986; Milich et al., Proc. Natl. Acad. Sci. USA 82: 8168-8172, 1985; Milich et al. , J. Immunol. 137. 315-322, 1986).

Among them, the pre-S1 antigen is mainly present in infectious virus particles unlike the pre-S2 antigen or the S antigen (Heermann et al., J. Virol. 52: 396-402, 1984). Because of their involvement in infection, monoclonal antibodies specific for pre-S1 are highly effective at neutralizing viruses (Neurath et al., Cell, 46, 429, 1986; Pontisso, et al., Virol., 173. 533 1989; Neurath et al., Vaccine, 7, 234, 1989), which may be useful for the prevention of HBV infection and for the treatment of chronic hepatitis B.

HBV infection is currently used to prevent HBV infections, such as newborns born from HBV-positive mothers, health care workers exposed to HBV, or liver transplant patients with HBV-related liver disease. HBIG "(Beasley et al., Lancet, 2, 1099, 1983; Todo et al., Hepatol., 13, 619, 1991). However, since HBIG currently used is extracted from plasma, there is a problem of low specificity to antigen, exposure to pollutants during the manufacturing process, and continuous supply of human blood.

In order to solve the above problem, a method of using a monoclonal antibody against HBV surface antigen of mouse has been developed. Mouse monoclonal antibodies, however, generally have high antigen affinity and mass production, but have the disadvantage of inducing an immune response in humans when injected into humans (Shawler et al., J. Immunol., 135, 1530). , 1985).

In order to solve the above problems, a technique using a human monoclonal antibody has been reported, but a technology for producing a high affinity human monoclonal antibody has not been put to practical use.

Instead, "humanized antibodies" have been developed as a way of minimizing immune induction in humans while maintaining the high affinity and specificity of mouse-derived monoclonal antibodies. Humanized antibodies are implanted with mouse antigen-binding sites (CDRs) into human antibodies. They can be mass-produced genetically and humanized most of genes, which greatly reduces the immune response in humans. Riechmann et al., Nature, 332, 323, 1988; Nakatani et al., Protein Engineering, 7,435, 1994).

The present inventors have developed a mouse monoclonal antibody KR359 against the surface antigen free-S1 of HBV (Korean Patent Application No. 97-30695), and isolated the gene encoding the variable region of the KR359 antibody and sequenced It was decided (Korean Patent Application No. 97-30694).

In the present invention, to prepare a humanized antibody from the mouse monoclonal antibody KR359 to use for the treatment of HBV infection and chronic hepatitis B, the human genes most similar to the variable region sequences of the light and heavy chains of the KR359 antibody are searched and humanized. The present invention was completed by preparing light and heavy chain genes of antibodies and cloning them into expression vectors to transform host cells, and then culturing them to obtain humanized antibodies to maintain antigen binding ability similar to that of mouse monoclonal antibody KR359. .

An object of the present invention is to HBV surface antigen-free which reduces the induction of immune response in human body by humanizing most of amino acid residues derived from mouse while maintaining antigen-binding ability against mouse monoclonal antibody KR359 against HBV surface antigen free-S1. To provide a humanized antibody against -S1.

1 shows a comparison of the amino acid sequence and the nucleotide sequence of the mouse monoclonal antibody KR359 and humanized antibody heavy chain against HBV surface antigen pre-S1,

2 schematically shows a process for obtaining the heavy chain gene HKR359HC of the humanized antibody against the mouse monoclonal antibody KR359 against HBV surface antigen pre-S1,

3 shows a comparison of amino acid sequences and nucleotide sequences of mouse monoclonal antibody KR359 and humanized antibody light chains against HBV surface antigen pre-S1,

Figure 4 schematically shows the process of obtaining the light chain gene HKR359KC (I) of the humanized antibody against the mouse monoclonal antibody KR359 against the surface antigen free-S1 of HBV,

5 schematically shows a process for obtaining the light chain gene HKR359KC (II) of the humanized antibody against the mouse monoclonal antibody KR359 against the surface antigen free-S1 of HBV,

Figure 6a shows the heavy chain expression vector pCMV-HKR359HC of the humanized antibody against the surface antigen free-S1 of HBV of the present invention,

Figure 6b shows the light chain expression vector pKC-dhfr-HKR359 of the humanized antibody against the surface antigen free-S1 of HBV of the present invention,

Figure 7 shows the antigen binding affinity of the humanized antibody HZKR359 of the present invention in comparison with the mouse monoclonal antibody KR359.

◆: KR359; ▲: HZKR359 (II);

■: HZKR359 (Ⅰ)

In order to achieve the above object, the present invention selects the human antibody gene most similar to the variable region of the mouse monoclonal antibody KR359 against the surface antigen pre-S1 of HBV and the amino acid of the humanized antibody and light or heavy chain thereof Provide the sequence.

The present invention also provides humanized antibody light and heavy chain genes encoding the light and heavy chains of the humanized antibodies against surface antigen free-S1 of HBV.

The present invention provides a method for producing a humanized antibody against HBV surface antigen free-S1 by culturing a transformant transformed with an expression vector comprising the light and heavy chain genes of the humanized antibody against surface antigen pre-S1 of HBV. To provide.

Hereinafter, the present invention will be described in detail.

In the present invention, the surface antigen free of humanized antibody H-V of the humanized heavy chain comprising the variable region of the amino acid sequence of SEQ ID NO: 31 and the surface antigen free of HBV of the humanized light chain comprising the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33 Provide a humanized antibody against -S1.

In addition, the present invention comprises a humanized heavy chain gene HKR359HC for the surface antigen pre-S1 of HBV encoding a humanized heavy chain comprising the variable region of the amino acid sequence of SEQ ID NO: 31 and a variable region of the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 33, respectively. Humanized light chain genes HKR359KC (I) or HKR359KC (II) for surface antigen free-S1 of HBV encoding humanized light chains are provided.

The present invention also provides an expression vector pC MV-HKR359HC (Accession No .: KCTC 0530BP) comprising the gene HKR359HC and an expression vector pKC-dhfr-HKR359 (Accession No .: KCTC 0532BP) comprising the gene HKR359KC (II). do.

In the present invention, the transformant HZIKR359 (I) and the expression vector pCMV-HKR359HC and the expression vector pKC-dhfr-HKR359 obtained by transforming COS7 cells with the expression vector pCMV-HKR359HC and the expression vector pKC-dhfr-HKR359 (I). (II) provides transformant HZIKR359 (II) obtained by transforming COS7 cells.

The present invention also provides a method for producing a humanized antibody HZKR359 (I) or HZKR359 (II) against HBV surface antigen free-S1 by culturing the transformant HZIKR359 (I) or HZIKR359 (II).

In the present invention, in order to prepare a humanized antibody against the surface antigen pre-S1 of HBV, a human antibody is first searched for the variable region sequence of the mouse monoclonal antibody KR359 against the surface antigen pre-S1 of HBV.

Human immunoglobulin germline genes were selected from the GenBank data base, and DP67 was the most similar human antibody gene to the variable region gene of the mouse antibody KR359 heavy chain, and DPK9 was the mouse antibody KR359 light chain. It was confirmed that the human antibody gene most similar to the variable region gene.

Therefore, the CDRs of the mouse antibody KR359 were transplanted into the human antibody genes DP67 and DPK9 to prepare light and heavy chain variable region genes of the humanized antibody of the present invention.

In the present invention, in the production of light and heavy chain variable region genes of humanized antibodies, most of the CDRs of the heavy and light chains of the mouse antibody corresponding to the antigen-binding site were used as they are of the mouse antibody KR359, but are not expected to participate in antigen binding. The resulting amino acid was substituted with the amino acid of the human antibody, even if it was a CDR residue. In addition, several amino acid residues of the framework region (FR) which affect the conformation of CDRs among the amino acid of the human antibody variable region were changed to those of the variable region amino acid of the mouse antibody KR359.

Specifically, in order to prepare a heavy chain gene of the humanized antibody, a portion of CDR1, 2 and some FR residues of the heavy chain variable region of the mouse monoclonal antibody KR359 were transplanted into the human antibody heavy chain variable region DP67 to humanized heavy chain variable region gene HKR359HC _V (HZII) was prepared (see FIG. 1).

However, humanized antibodies prepared using HKR359HC _V (HZII) showed no antigen binding capacity. Therefore, in the present invention, the humanized heavy chain variable region gene HKR359HC _V (HZI), in which more amino acid residues of the mouse antibody KR359 were transplanted in order to maintain antigen binding ability, was designed (see FIG. 1).

To prepare full-length HKR359HC (I), pRC / CMV-HC with the new humanized heavy chain variable region gene HKR359HC _V (HZII), the leader sequence of the heavy chain, and the constant region sequence of the human antibody heavy chain γ1 PCR is performed using the primers of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and SEQ ID NO: 7 and SEQ ID NO: 8, respectively, using -HuS (KCTC 0229BP) as a template (see FIG. 2).

Annealing the PCR products according to SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, and SEQ ID NO: 8 to form a template, and recombination using the primers of SEQ ID NO: 1 and SEQ ID NO: 8 By PCR, a fragment of about 383 bp is obtained. PCR was carried out using PCR products using the primers of SEQ ID NO: 9 and SEQ ID NO: 10, respectively, to obtain 1453 bp of humanized heavy chain gene HKR359HC, which was cloned into pBluescript SK (+), and referred to as pHKR359HC. Named it.

The base sequences of the primers of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 used in the process of synthesizing the humanized heavy chain gene are shown in the sequence list. Specifically, the primer of SEQ ID NO: 1 has a restriction enzyme EcoRI sequence at its end and the primer of SEQ ID NO: 10 has a restriction enzyme SalI sequence at its terminal.

The variable region of the humanized heavy chain gene HKR359HC prepared as described above encodes the substitution of 18 variable region amino acid residues of the KR359 mouse antibody with that of the human antibody DP67 (see FIG. 1).

In addition, in order to prepare the light chain gene of the humanized antibody, CDR1, 2, 3 whole of the light chain variable region of the mouse monoclonal antibody KR359 was transplanted to the human antibody light chain DPK9 to prepare a humanized light chain variable region gene HKR359KC _V (HZI). 3).

PKC-dhfr with pKR359K (Korean Patent Application No. 97-30694), the leader sequence of the light chain, and the constant region sequence of the human antibody light chain κ for the production of a new full-length humanized light chain gene HKR359KC (I) PCR is performed using primers of SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 15, 16, and SEQ ID NO: 17 and SEQ ID NO: 18, respectively, using -HuS (KCTC 0230BP) as a template (see FIG. 4).

Specifically, PCR was performed using the primers of SEQ ID NO: 11 and SEQ ID NO: 16 using the PCR products using primers of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and SEQ ID NO: 18 together, respectively. Synthesize a fragment of 380bp. PCR was carried out using the PCR products using the primers of SEQ ID NO: 17 and SEQ ID NO: 18, followed by PCR using the primers of SEQ ID NO: 11 and SEQ ID NO: 18 to obtain 724bp of humanized light chain gene HKR359KC (I), which was cloned into pBluescript SK (+). And pHKR359KC (I).

The base sequences of the primers of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, 18 used in the process of synthesizing the humanized light chain gene are shown in the sequence list. Specifically, the primer of SEQ ID NO: 11 has a restriction enzyme HindIII sequence at its terminal and the primer of SEQ ID NO: 18 has a restriction enzyme SalI sequence at its terminal.

The variable region of the humanized light chain gene HKR359KC (I) prepared as described above encodes the substitution of 12 amino acid residues of the mouse monoclonal antibody KR359 light chain with those of the human antibody DPK9 (see FIG. 4).

In addition, a new humanized light chain gene, HKR359KC _V (II), was designed to reduce the human immunogenic ability by changing the amino acid residues of mouse antibodies to amino acid residues of human antibodies.

To prepare a new full-length humanized light chain gene HKR359KC (II), pKC-dhfr-HuS (KCTC 0230BP) containing pKR359K and the light chain leader sequence and the constant region sequence of human antibody light chain κ was used as a template. PCR is performed using the primers of SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, and SEQ ID NO: 29 and SEQ ID NO: 30, respectively (see Figure 5). .

Specifically, the primers of SEQ ID NO: 11 and SEQ ID NO: 20 were prepared by using PCR products using SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, and SEQ ID NO: 27, SEQ ID NO: 28, respectively. PCR is carried out using a 345bp fragment is synthesized. Using the PCR product using the primers of SEQ ID NO: 29 and SEQ ID NO: 30 as a template, PCR was performed using the primers of SEQ ID NO: 19 and SEQ ID NO: 30 to obtain 724 bp of humanized light chain gene HKR359KC (II), which was cloned into pBluescript SK (+). And pHKR359KC (II).

The base sequences of the primers of SEQ ID NO: 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 used in the synthesis of the humanized light chain gene are shown in the sequence list. Specifically, the primer of SEQ ID NO: 19 has a restriction enzyme HindIII sequence at its terminal and the primer of SEQ ID NO: 30 has a restriction enzyme SalI sequence at its terminal.

The variable region of the humanized light chain gene HKR359KC (II) prepared as described above encodes the substitution of 23 amino acid residues of the mouse monoclonal antibody KR359 light chain with that of the human antibody DPK9 (see FIG. 3).

The present invention is a method for producing a humanized antibody against the surface antigen pre-S1 of HBV by culturing a transformant transformed with an expression vector comprising the light and heavy chain genes of the humanized antibody against the surface antigen pre-S1 of HBV. To provide.

In order to prepare a humanized antibody against the surface antigen free-S1 of HBV, first of all, in the present invention, an expression vector including light and heavy chain genes of the humanized antibody is prepared.

Specifically, the humanized heavy chain of the present invention was isolated from the pHKR359HC containing a humanized heavy chain using a restriction enzyme and then inserted into pRc / CMV (Invitrogen) to prepare a humanized heavy chain expression plasmid pCMV-HKR359HC of the present invention. In addition, the humanized light chain of the present invention is isolated from the above-mentioned pHKR359KC (I) or pHKR359KC (II) containing a humanized light chain using a restriction enzyme and then inserted into pCMV-dhfr (KCTC 8671P), which has been developed by the present inventors. Humanized light chain expression plasmids pKC-dhfr-HKR359 (I) or pKC-dhfr-HKR359 (II) were prepared.

E. coli DH5α strains were transformed with the expression vectors pCMV-HKR359HC or pKC-dhfr-HKR359 (II), respectively, to prepare E. coli transformants, which were established in 1998 by the Korea Institute of Science and Technology. Deposited October 17 (Accession No .: KCTC 0530BP, KCTC 0532BP).

In the present invention, in order to express humanized antibodies against the surface antigen pre-S1 of HBV, expression vectors pCMV-HKR359HC and pKC-dhfr-HKR359 (I) or pCMV-HKR359HC comprising the humanized heavy and humanized light chain genes of the present invention. COS7 cells were transiently transformed with pKC-dhfr-HKR359 (II) to obtain transformant HZKR359 (I) or HZKR359 (II) and then cultured to isolate the humanized antibody of the present invention.

As a result of comparing the antigen-binding affinity of the humanized antibody HZKR359 (I) and HZKR359 (II) and the mouse monoclonal antibody KR359 obtained by the above procedure, the mouse monoclonal antibody KR359 to HBV surface antigen free-S1 of the present invention The humanized antibodies HZKR359 (I) and HZKR359 (II) were not significantly different from the mouse monoclonal antibody KR359.

Hereinafter, the present invention will be described in detail by examples.

However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

<Example 1> Preparation of humanized heavy chain gene

To prepare a humanized heavy chain variable region, first, a human antibody DP67 having the amino acid sequence most similar to the heavy chain variable region gene of the mouse monoclonal antibody KR359 was selected from the GenBank database, and the heavy chain variable region of the KR359 and heavy chain variable region of the DP67 antibody were selected. of the amino acid sequence and the nucleotide sequence compared to the first humanized heavy chain variable region gene as basis and then devising HKR359HC _V (HZⅡ), a second humanized heavy chain variable region gene by improving the first humanized heavy chain designed HKR359HC _V (HZI) Devised.

Specifically, the humanized heavy chain variable region gene HKR359HC _V (HZI) is used to transplant several mouse framework amino acid residues and portions of CDR1,3 and CDR2 into human antibody heavy chain variable region DP67, which are believed to affect the structure of the antibody in humanized antibodies. HKR359HC _V (HZII) was prepared, and then prepared by the following PCR.

The heavy chain leader sequence and human antibody heavy chain constant region gene required for secretion of the heavy chain were synthesized from pRC / CMV-HC-HuS (KCTC 0229BP).

The leader sequence, humanized heavy chain variable region gene HKR359HC _V (HZII), and humanized heavy chain constant region gene were linked by recombinant PCR to finally produce humanized heavy chain gene HKR359HC (I) (see FIG. 2).

The primers used for PCR are described in SEQ ID NOs: 1-10.

PCR reaction conditions were performed 30 times using Taq DNA polymerase (1 minute at 94 ℃, 1 minute at 55 ℃, 1 minute at 72 ℃). First, among the DNA fragments obtained using the primers of SEQ ID NO: 1 and SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and SEQ ID NO: 7 and SEQ ID NO: 8, SEQ ID NO: 1 and SEQ ID NO: 2 (173 bp), SEQ ID NO: 3 and SEQ ID NO: 4 75 bp), DNA fragments obtained using the primers of SEQ ID NO: 5 and SEQ ID NO: 6 (90 bp), SEQ ID NO: 7 and SEQ ID NO: 8 (90 bp) were annealed and linked by recombinant PCR with the primers of SEQ ID NO: 1 and SEQ ID NO: 8, and the synthesized fragments (383 bp) was linked to the DNA fragment (1085 bp) synthesized using the primers of SEQ ID NO: 9 and SEQ ID NO: 10 using the primers of SEQ ID NO: 1 and SEQ ID NO: 10.

Finally, the synthesized humanized antibody heavy chain gene (about 1453bp) was cut at both ends by restriction enzyme EcoRI and restriction enzyme SalI, cloned into pBluescript SK (+), and named as pHKR359HC. The base sequence of the prepared humanized antibody heavy chain gene was confirmed by DNA sequencing.

<Example 2> Preparation of humanized light chain gene

In order to prepare a humanized light chain variable region, first, a human antibody DPK9 having the most similar amino acid sequence to the variable region gene of KR359 was selected from the GenBank database, and the amino acid sequence and base of the light chain variable region of KR359 and the light chain variable region of DPK9 antibody. By comparing the sequences, the first humanized light chain variable region genes HKR359KC _V (HZI) and HKR359KC _V (HZII) were designed.

The HKR359KC _V (HZI) is a whole of CDR1, 2, 3 of the mouse antibody KR359 is transplanted into the human antibody light chain DPK9 variable region (see FIG. 3).

To produce a full-length new humanized light chain gene HKR359KC (I), the humanized light chain variable region gene HKR359KC _V (HZI) was prepared by recombinant PCR from pKR359K (see FIG. 4), and the leader sequence of the light chain required for light chain secretion. (leader sequence) and human antibody light chain constant region gene were synthesized from pKC-dhfr-HuS (KCTC 0230BP). SEQ ID NO: 11 and SEQ ID NO: 12 using the humanized light chain variable region gene HKR359KC _V (HZI), a pKC-dhfr-HuS (KCTC 0230BP) having a constant sequence of the light chain leader sequence and the human antibody light chain κ PCR is performed using the primers of SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, and SEQ ID NO: 17 and SEQ ID NO: 18, respectively.

PCR reaction conditions were performed 30 times in 1 minute at 94 ℃, 1 minute at 55 ℃, 1 minute at 72 ℃.

Specifically, the PCR products using the primers of SEQ ID NO: 11 and SEQ ID NO: 12 (110 bp), SEQ ID NO: 13 and SEQ ID NO: 14 (220 bp), and SEQ ID NO: 15 and SEQ ID NO: 16 (99 bp), respectively, were used as templates. PCR was performed to synthesize 380 bp fragments. Using the PCR product using the primers of SEQ ID NO: 17 and SEQ ID NO: 18 as a template, PCR was performed using the primers of SEQ ID NO: 11 and SEQ ID NO: 18 to obtain 724 bp of humanized light chain gene HKR359KC (I). The synthesized humanized antibody light chain gene HKR359KC (I) was cut at both ends by restriction enzymes HindIII and SalI, cloned into Bluescript SK (+), and named as pHKR359KC (I).

The base sequences of the primers of SEQ ID NOs: 11, 12, 13, 14, 15, 16, 17, and 18 used in the process of synthesizing the humanized light chain gene are shown in the sequence list. Specifically, the primer of SEQ ID NO: 11 has a restriction enzyme HindIII sequence at its terminal and the primer of SEQ ID NO: 18 has a restriction enzyme SalI sequence at its terminal.

The variable region of the humanized light chain gene HKR359KC (I) prepared as described above encodes the substitution of 12 amino acid residues of the mouse monoclonal antibody KR359 light chain with that of the human antibody DPK9 (see FIG. 4).

In addition, a new humanized light chain gene, HKR359KC _V (HZII), was designed to reduce the immunogenic ability in humans by changing amino acid residues of mouse antibodies to amino acid residues of human antibodies. The HKR359KC _V (HZII) is a part of CDR1, 2 and all of CDR3 and several FR residues of the mouse antibody KR359, which are transplanted into a human antibody light chain DPK9 variable region.

To prepare a full-length new humanized light chain gene HKR359KC (II), the humanized light chain variable region gene HKR359KC _V (HZII) was prepared by recombinant PCR from pKR359K (see FIG. 5), and the leader sequence of the light chain required for light chain secretion. (leader sequence) and human antibody light chain constant region gene were synthesized from pKC-dhfr-HuS (KCTC 0230BP). The leader sequence, humanized light chain variable region gene, and humanized light chain constant region gene were linked by recombinant PCR to prepare humanized light chain gene HKR359KC (II).

The primers used for PCR are described in SEQ ID NOs: 19-30.

PCR reaction conditions were performed 30 times using Taq DNA polymerase (1 minute at 94 ℃, 1 minute at 55 ℃, 1 minute at 72 ℃). First, among the fragments obtained using the primers of SEQ ID NO: 19 and SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27 and SEQ ID NO: 28, and SEQ ID NO: 29 and SEQ ID NO: 30, respectively, DNA fragments obtained using the primers of SEQ ID NO: 20 (110 bp), SEQ ID NO: 21 and SEQ ID NO: 22 (75 bp), SEQ ID NO: 23 and SEQ ID NO: 24 (90 bp), SEQ ID NO: 25 and SEQ ID NO: 26 (87 bp), and SEQ ID NO: 27 and SEQ ID NO: 28 (99 bp), respectively. The fragment (345bp) synthesized by recombination PCR by annealing the primers of SEQ ID NO: 19 and SEQ ID NO: 28 was then used again using the DNA fragment (379 bp) synthesized with the primer of SEQ ID NO: 29 and SEQ ID NO: 30, and the primer of SEQ ID NO: 19 and SEQ ID NO: 30. PCR was performed to synthesize 724bp of humanized light chain gene HKR359KC (II).

The synthesized humanized antibody light chain gene HKR359KC (II) was cut at both ends by restriction enzymes HindIII and SalI, cloned into Bluescript SK (+), and named as pHKR359KC (II).

The base sequence of the prepared humanized antibody light chain gene was confirmed by DNA sequencing.

<Example 3> Preparation of humanized heavy chain expression plasmid

The plasmid pHKR359HC was cut with restriction enzyme SalI and blunted with Klenow enzyme, and then cut again with restriction enzyme NotI to isolate the humanized heavy chain gene.

Meanwhile, pRc / CMV (Invitrogen) was cut with restriction enzyme XbaI, blunted with Klenau enzyme, and cut again with restriction enzyme NotI to separate the vector.

Humanized heavy chain expression vector pCMV-HKR359HC was produced by linking the humanized heavy chain gene to the isolated vector. The constructed humanized heavy chain expression vector pCMV-HKR359HC was deposited in KCTC (Accession No.:KCTC 0530BP), and the completed vector is shown in FIG. 6A.

<Example 4> Preparation of humanized light chain expression plasmid

The plasmids pHKR359KC (I) and pHKR359KC (II) were cut with restriction enzymes HindIII and ApaI to separate the humanized light chain genes, and inserted into the HindIII and ApaI positions of pCMV-dhfr (KCTC8671P) to express the humanized light chain expression vector pKC-dhfr-HKR359 (I). ) And pKC-dhfr-HKR359 (II).

The prepared humanized light chain expression vector pKC-dhfr-HKR359 (II) was deposited in KCTC (Accession No .: KCTC 0532BP), and the completed vector is shown in FIG. 6B.

Example 5 Expression in COS7 Cells of Humanized Antibodies

COS7 cells were passaged while maintaining 37 ° C. and 5% carbon dioxide in DMEM culture medium (GIBCO Co., Ltd.) to which 10% of calf serum was added. The cells were seeded 1 × 10 ⁶ in a 100 mm dish and incubated at 37 ° C. overnight.

On the other hand, 5 μg of the expression vectors pCMV-HKR359HC and pKC-dhfr-HKR359 (I) or pCMV-HKR359HC and pKC-dhfr-HKR359 (II) obtained in Examples 3 and 4 were respectively 800 μl of OPTI MEM I. 50 μl of lipofectamine (Lipofectamine, manufactured by GIBCO) was also diluted with 800 μl of OPTI MEM I. The mixtures were mixed in a 15 ml tube and then left at room temperature for at least 15 minutes. COS7 cells inoculated the day before incubating the mixture were washed twice with OPTI MEM I (manufactured by GIBCO).

6.4 ml of OPTI MEM I was added to the DNA-lipopeptamine mixture, mixed and poured uniformly on the washed COS7 cells. After incubation for 72 hours in a carbon dioxide gas incubator, the supernatant of the culture was collected and concentrated using an ultrafiltration kit, followed by an anti-human antibody and an anti-human antibody-HRP conjugate. The concentration of the antibody was measured by Sandwich ELIS1A using a conjugate).

<Example 6> Binding Capacity of Humanized Antibody to HBV Surface Antigen Free-S1

1 μg of surface antigen free-S1 (amino acids 1-56) of HBV prepared in our laboratory was coated in each well of a microplate, and antibodies 0, 0.25, 0.5, based on the concentration of the antibody quantified in Example 5 After adding 1, 2, 3, 4, 5, 7.5, 10, 20, 50 ng each, the antibody conjugated with horseradish peroxidase to the anti-human antibody (Fc specific) Indirect ELISA binding to the primary antibody was performed to measure absorbance at 492 nm.

As a control, purified KR359 was used, and the results are shown in Table 1. When the antigen-binding ability of KR359 was analyzed, an antibody conjugated with horseradish peroxidase to an anti-mouse antibody (Fc-specific) was used as a secondary antibody.

Binding Capacity of KR359 with HZKR359 (I) and HZKR359 (II) to HBV Surface Antigen Free-S1 (Absorbance, 492nm) Amount (ng) 0 0.25 0.5 One 2 3 4 5 7.5 10 20 40 KR359 0.11 0.22 0.28 0.43 0.62 0.84 0.99 1.10 1.40 1.76 2.53 3.03 HZKR359 (Ⅰ) 0.12 0.25 0.37 0.59 0.84 1.05 1.38 1.41 1.63 1.97 2.43 3.02 HZKR359 (Ⅱ) 0.15 0.32 0.43 0.65 0.96 1.23 1.34 1.45 1.67 1.95 2.90 3.15

<Example 7> Antigen-binding affinity of a humanized antibody

To determine the affinity of humanized antibodies for HBV free-S1 antigen, antigen binding affinity was measured using a competitive ELISA method (Competitive ELISA; Ryu et al., J. Med. Virol., 52, 226, 1997). It was.

First, HBV-free S1 at a concentration of 1 × 10 ^{−7 to} 1 × 10 ^-12 M, the antibody produced in Example 5, and 5 ng of KR359 as a control group were combined at 37 ° C. for 2 hours, and then the reaction was coated on 96 wells. In combination with the pre S1 antigen and indirect eliza as in Example 6, the results are shown in FIG. Comparing the antigen binding affinities of KR359 with HZKR359 (Ⅰ) and HZKR359 (II), KR359 was approximately 1.4 × 10 ⁸ M ^-1 , HZKR359 (Ⅰ) 3 × 10 ⁸ M ^-1 , and HZKR359 (Ⅱ) 6 × 10 ⁸ M ⁻¹ confirmed no significant difference in antigen binding affinity between the three antibodies (see FIG. 7).

As described above, the humanized antibody against the HBV surface antigen pre-S1 of the present invention exhibits similar antigen binding ability as that of the mouse monoclonal antibody, whereas the amino acid residues are more humanized than the mouse monoclonal antibody, resulting in increased immunogenic ability in the human body. Significantly reduced, it can be usefully used for the prevention and treatment of HBV infection.

[Sequence list]

SEQ ID NO: 1

Sequence length: 26

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: 24

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: 18

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: 42

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: 45

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: 42

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: 42

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

Sequence length: 39

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: 39

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: 26

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 11

Sequence length: 26

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 12

Sequence length: 42

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 13

Sequence length: 48

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 14

Sequence length: 36

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 15

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 16

Sequence length: 54

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 17

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 18

Sequence length: 45

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 19

Sequence length: 42

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 20

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 21

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 22

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 23

Sequence length: 26

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 24

Sequence length: 45

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 25

Sequence length: 48

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 26

Sequence length: 45

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 27

Sequence length: 42

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 28

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 29

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 30

Sequence length: 27

Type of sequence: nucleic acid

Number of chains: 1 chain

Form: Straight

Type of sequence: Other nucleic acid (oligonucleotide)

[Sequence list]

SEQ ID NO: 31

Sequence length: 119

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: protein

[Sequence list]

SEQ ID NO: 32

Sequence length: 108

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: protein

[Sequence list]

SEQ ID NO: 33

Sequence length: 108

Type of sequence: amino acid

Number of chains: 1 chain

Form: Straight

Type of sequence: protein

Claims (9)

Humanized antibody against the surface antigen pre-S1 of HBV in a humanized heavy chain comprising the variable region of the amino acid sequence of SEQ ID NO: 31. Humanized antibody against the surface antigen pre-S1 of HBV of a humanized light chain comprising the variable region of the amino acid sequence of SEQ ID NO: 32. Humanized antibody against the surface antigen pre-S1 of HBV of a humanized light chain comprising the variable region of the amino acid sequence of SEQ ID NO: 33. Humanized heavy chain gene for surface antigen pre-S1 of HBV encoding a humanized heavy chain comprising the variable region of the amino acid sequence of SEQ ID NO: 31 of claim 1. Humanized light chain gene for the surface antigen pre-S1 of HBV encoding the humanized light chain comprising the variable region of the amino acid sequence of SEQ ID NO: 32 of claim 2. Humanized light chain gene for the surface antigen pre-S1 of HBV encoding the humanized light chain comprising the variable region of the amino acid sequence of SEQ ID NO: 33 of claim 3. Heavy chain expression vector pCMV-HKR359HC of humanized antibody against surface antigen pre-S1 of HBV comprising the gene of claim 4 (Accession No .: KCTC 0530BP). Light chain expression vector pKC-dhfr-HKR359 of humanized antibody against surface antigen pre-S1 of HBV comprising the gene of claim 6 (Accession No .: KCTC 0532BP). Use of the humanized antibody against HBV surface antigen pre-S1 of claims 1, 2 or 3 for the prevention of HBV infection and the treatment of chronic hepatitis B.