The present invention relates to humanized antibodies specific for HBV surface antigen pre-S1, which show binding affinity similar to mouse monoclonal antibody and which show remarkably reduced immunogenicity since they have less mouse-derived amino acid residues. Thus, the humanized antibodies of the present invention may be useful for the prevention of HBV infection and for the treatment of hepatitis B.

SUMMARY OF THE INVENTION

It is an object of this invention to provide humanized antibodies specific for CDRs of mouse HBV surface antigen pre-S1, having high affinity to the antigen and reduced immunogenicity in human.

In accordance with the present invention, the foregoing objects and advantages are readily obtained.

The present invention provides humanized antibodies specific for HBV surface antigen pre-S1, comprising humanized heavy and light chains.

This invention also provides genes encoding the variable regions of said humanized heavy or light chain.

In addition, this invention provides expression vectors containing said genes and E. coli transformants containing said expression vectors.

This invention further provides pharmaceutical compositions comprising said humanized antibody, which may be administered in order to prevent HBV infection or to treat chronic hepatitis B.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Hereinafter, the present invention is described in detail.

This invention provides humanized antibodies specific for HBV surface antigen pre-S1, comprising humanized heavy and humanized light chains.

This invention also provides genes encoding the variable regions of said humanized heavy or light chain.

Said humanized heavy chain contains variable region which is derived from the V.sub.H region of mouse KR127 antibody. The V.sub.H region of mouse KR127 antibody is described by SEQ ID NO: 19, and the V.sub.H region of the humanized antibody of this invention can be prepared by grafting the CDRs of mouse KR127 V.sub.H region to homologous human immunoglobulin V.sub.H region.

And said humanized light chain contains variable region which is derived from the V.sub.L region of mouse KR127 antibody. The V.sub.L region of mouse KR127 antibody is described by SEQ ID NO: 22, and the V.sub.L region of the humanized antibody of this invention can be prepared by grafting the CDRs of mouse KR127 V.sub.L region to homologous human immunoglobulin V.sub.L region.

In preferred embodiments, we screened human immunoglobulin that show the highest similarities of amino acid sequence to the heavy or light chain of the mouse monoclonal antibody KR127. In result, human immunoglobulin germ line genes DP7 and DPK12 were screened from GenBank database. DP7 shows the highest homology to the V.sub.H region of mouse antibody KR127, while DPK12 is most similar to the V.sub.L region of KR127.

The humanized antibodies of this invention can be produced from recombinant genes encoding humanized V.sub.H region or V.sub.L region. These genes are constructed by substituting CDRs of mouse KR127 for those of the human DP7 or DPK12 antibody. In constructing these genes, most of the amino acid residues corresponding to the humanized CDRs are derived from the CDRs of mouse antibody KR127. However, some mouse-derived CDRs residues are replaced by human counterparts, since their corresponding amino acid residues are expected not to be involved in the antigen binding (see FIG. 1). In the same way, some human-derived amino acid residues for the non-CDR framework regions (FR) of variable region are replaced with mouse counterparts, since it is expected that these FR residues may affect the conformation of CDRs.

Particularly, HKR127HCv(HZII) gene encoding a humanized V.sub.H region was prepared by grafting the partial CDR1, 2, 3 and a FR residue (at position 72) of mouse KR127 heavy chain to the human DP7 gene (see FIG. 1).

However, antibody expressed from HKR127HCv(HZII) gene did not show any significant level of binding capacity to corresponding antigen. To improve the HKR127HCv(HZII) gene, we also prepared HKR127HCv(HZI) gene and HKR127HCv(HZIII) gene which contain more mouse-derived codons than HKR127HCv(HZII) gene (see FIG. 1).

HKR127HCv(HZI) contains CDR1, partial CDR2, and CDR3, and 11 FR residues of mouse KR127 V.sub.H, while HKR127HCv(HzIII) contains the same mouse CDR codons and 2 mouse FR residues (see FIG. 1).

To construct HKR127HC(I) gene encoding a full-length heavy chain of the humanized antibody of this invention, PCRs were conducted, in which template for amplification is either the HKR127HCv(HZII) gene or pRC/CMV-HC-HuS (KCTC 0229BP) containing the heavy chain leader sequence and the constant region sequence of human immunoglobulin heavy chain .gamma.l.

Six pairs of oligonucleotides (SEQ ID NO: 1 and 2; 3 and 4; 5 and 6; 7 and 8; 9 and 10; and 11 and 12) were used as PCR primers (see FIG. 2a).

The first five PCR products were brought to annealing reaction. Then, the DNA fragment containing the five PCR products was employed as a template of recombinant PCR wherein two primers described by SEQ ID NO: 1 and 10 were used. Another recombinant PCR was conducted to link the amplified 431-bp DNA fragment to DNA fragment which was obtained by PCR using two primers (described by SEQ ID NO: 11 and 12). The recombinant PCR employed two primers described by SEQ ID NO: 1 and 12. The final 1431-bp PCR product, HKR127HC(I), encoding the heavy chain of a humanized antibody (HZKR127I) was introduced into pBluescript SK(+) vector (Clontech), and the resulting vector was designated pHKR127HC(I).

The primers are described in SEQ ID NO: 1 to 12 in SEQUENCE LISTING, and particularly, primer described by SEQ ID NO: 1 contains EcoRI sequence at the 5' end, while primer described by SEQ ID NO: 12 does SalI sequence at the 3' end.

The variable region in the HKR127HC(I) gene contains 11 mouse-derived FR residues at positions 12, 28, 30, 48, 67, 68, 70, 72, 74, 79 and 95 (see FIG. 1). The heavy chain variable region has 87 FR residues, and the unmodified FR residues is 76. Thus, the amino acid sequence of the heavy chain variable FR of the HKR127HC(I) gene is 87% homologous to that of human DP7 gene.

To more humanize the HZKR127(I), HZKR127(III) gene was constructed, which contains HKR127HCv(HZIII) gene with 2 mouse-derived FR residues at position 72 and 74 (see FIG. 1).

To construct HKR127HC(III) gene encoding a full-length heavy chain of the humanized antibody of this invention, PCRs were conducted, in which template for amplification is either the HKR127HCv(HZII) gene or pRC/CMV-HC-HuS (KCTC 0229BP) containing the heavy chain leader sequence and the constant region sequence of human immunoglobulin heavy chain .gamma.1 (see FIG. 2b).

Four pairs of oligonucleotides (SEQ ID NO: 1 and 24; 25 and 26; 27 and 28; and 11 and 12) were used as PCR primers (see FIG. 2b). The first three PCR products were brought to annealing reaction. Then, the DNA fragment containing the three PCR products was employed as a template of recombinant PCR wherein two primers described by SEQ ID NO: 1 and 28 were used. Another recombinant PCR was conducted to link the amplified 431-bp DNA fragment to DNA fragment which was obtained by PCR using two primers (described by SEQ ID NO: 11 and 12). The recombinant PCR employed two primers described by SEQ ID NO: 1 and 12.

The final 1431-bp PCR product, HKR127HC(III), encoding the heavy chain of a humanized antibody (HZKR127III) was introduced into pBluescript SK(+) vector (Clontech), and the resulting vector was designated pHKR127HC(III).

In a further embodiment, HKR127KCv(HZII) gene encoding a humanized V.sub.L region was prepared by grafting the CDR1, CDR3 and partial CDR2 of mouse KR127 light chain to the human DPK12 gene (see FIG. 3).

However, antibody expressed by using the HKR127KCv(HZII) gene did not show any significant level of binding capacity to corresponding antigen. To improve the binding capacity of HKR127KCv(HZII), we also prepared HKR127KCv(HZI) gene which contains more mouse-derived amino acid residues (see FIG. 3) than HKR127HKCv(HZII) (see FIG. 3).

To construct HKR127KC(I) gene encoding a full-length light chain of the humanized antibody of this invention, PCRs were conducted, in which template for amplification is either the HKR127KCv(HZII) gene or pKC-dfhr-HuS (KCTC 0230BP) containing the light chain leader sequence and the constant region sequence of human immunoglobulin light chain .

Three pairs of oligonucleotides (SEQ ID NO: 13 and 14; 15 and 16; and 17 and 18) were used as PCR primers (see FIG. 4).

The first two PCR products were brought to annealing reaction. Then, the DNA fragment containing the two PCR products was employed as a template of recombinant PCR wherein two primers described by SEQ ID NO: 13 and 16 were used. Another recombinant PCR was conducted to link the amplified 360-bp DNA fragment to DNA fragment which was obtained by PCR using two primers (described by SEQ ID NO: 17 and 18). The recombinant PCR employed two primers which are described by SEQ ID NO: 13 and 18. The final 739-bp PCR product, HKR127KC(I), encoding the light chain of a humanized antibody (HZKR127I) was introduced into pBluescript SK(+) vector (Clontech), and the resulting vector was designated pHKR127KC(I).

The primers are described in SEQ ID NO: 13 to 18 in SEQUENCE LISTING, and particularly, primer described by SEQ ID NO: 13 contains HindIII sequence at the 5' end, while primer described by SEQ ID NO: 18 does SalI sequence at the 3' end.

The variable region FR of the HKR127KC gene contains 5 mouse KR127-derived codons (see FIG. 3). The light chain has 83 FR residues, and the unmodified FR residues is 78. Thus, the amino acid sequence of the light chain variable FR of the HKR127KC gene is 94% identical to that of human DP7 gene.

In addition, this invention provides expression vectors containing genes encoding the humanized V.sub.H or V.sub.L region and provides E. coli transformants containing said expression vector.

In other preferred embodiments, expression vectors are prepared, which contain the gene encoding the heavy or light chain of humanized antibody (see FIGS. 5a, 5b or 5c).

Particularly, two kinds of DNA fragment corresponding to humanized heavy chain was respectively obtained from the plasmids pHKR127HC(I) and pHKR127HC(III) by treatment of restriction enzymes, and then inserted into pRc/CMV (Invitrogen) to give expression vector pCMV-HKR127HC (see FIG. 5a) and pCMV-HKR127(III)HC (see FIG. 5c), respectively.

In addition, DNA fragment encoding the humanized light chain was isolated from the pHKR127KC vector, and then introduced into pCMV-dfhr (KCTC 8671P) to construct expression vector pKC-dhfr-HKR127 (see FIG. 5b).

E. coli strain DH5.alpha. was transformed with the expression vector pCMV-HKR127HC, pCMV-HKR127(III)HC or pKC-dhfr-HKR127. The resulting E. coli transformants containing pCMV-HKR127HC or pKC-dhfr-HKR127 were deposited in KCTC (Korean Collection for Type Culture) (Accession Number: KCTC 0531BP and KCTC 0529BP, respectively) on Oct. 12, 1998. The E. coli transformant containing pCMV-HKR127(III)HC was deposited in KCTC (Accession Number: KCTC 0691BP, respectively) on Nov. 15, 1999.

In another preferred embodiment, humanized antibodies specific for HBV surface antigen pre-S1 were expressed in animal cells and obtained from culture media of the cells. COS7 cells were transiently cotransfected with the expression vectors pCMV-HKR127HC and pKC-dhfr-HKR127, and the resulting transfected cells was cultured and the culture supernatant was used to characterize a humanized antibody HZKR127I of the present invention. COS7 cells were also cotransfected with the expression vectors pCMV-HKR127(III)HC and pKC-dhfr-HKR127, and the culture supernatant of transfected cells was used to characterize a humanized antibody HZKR127III.

This invention further provides pharmaceutical compositions containing said humanized antibody.

According to still other preferred embodiments, it was verified that HZKR127I and HZKR127III humanized antibodies of the present invention, showed almost same antigen-binding affinity when compared with mouse monoclonal antibody KR127 (see Table 1, 2 and FIGS. 6a, 6b).

The composition includes a therapeutically effective amounts of the humanized antibody against HBV antigen pre-S1, with/without a pharmaceutically acceptable delivery vehicle. Moreover, the compositions may include other anti-hepatitis drug(s), such as anti-S monoclonal antibody or lamivudin.

The humanized antibody against HBV antigen pre-S1 may be formulated with a pharmaceutical vehicle or diluent for intravenous, subcutaneous, intramuscular administration. The pharmaceutical composition can be formulated in a classical manner using solid or liquid vehicles, diluents and additives appropriate to the desired mode of administration.

The humanized antibody of this invention may be administered in a dosage range of about 1.about.10 mg/kg, preferably 3.about.5 mg/kg, and may be administered once a week.
 

Claim 1 of 8 Claims

1. A gene encoding a humanized heavy chain which comprises a humanized heavy chain variable region having an amino acid sequence selected from the group consisting of: the amino acid sequence of SEQ ID NO: 21; and the amino acid sequence of SEQ ID NO: 21 which is modified by at least one amino acid substitution selected from the group comprising: Lys.sup.12.fwdarw.Val.sup.12, Thr.sup.28.fwdarw.Ala.sup.28, Thr.sup.30.fwdarw.Ser.sup.30, Met.sup.48.fwdarw.Ile.sup.48, Arg.sup.67.fwdarw.Lys.sup.67, Val.sup.68.fwdarw.Ala.sup.68, Met.sup.70.fwdarw.Leu.sup.70, Val.sup.79.fwdarw.Ala.sup.79, and Tyr.sup.95.fwdarw.Phe.sup.95.

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