Humanized Antibody And Process For Preparing Same

Abstract

A humanized antibody is produced by process comprising the steps of: (a) selecting a specificity determining residue (SDR) of the complementarity determining region (CDR) of murine monoclonal antibody heavy chain and light chain variable regions; and (b) grafting said SDR to at least one of the corresponding amino acid sequences in human antibody variable regions.

Description

FIELD OF THE INVENTION

[0001] The present invention relates to a process for preparing a humanized antibody by grafting SDRs (specificity determining residues) in CDRs (complementary determining residues) of murine monoclonal antibody to human antibody and the humanized antibody prepared according to said process.

BACKGROUND OF THE INVENTION

[0002] For preventing infectious diseases such as hepatitis B, there has generally been used a method of administering immunoglobulins formed in blood plasma against a target antigen. However, the method has the problems that the immunoglobulins generally have low specificity and may contain contaminants.

[0003] Murine monoclonal antibody derived from mouse has been reported to have high affinity to antigen and is suitable for mass-production. However, repeated injection of murine monoclonal antibody induces an immune response because the murine antibody is regarded as a foreign antigen in humans (Shawler D. L. et al., J. Immunol., 135, 1530-1535 (1985)).

[0004] Accordingly, numerous efforts have been made to generate "humanized antibody" by: grafting the CDR (complementarity determining region) of murine monoclonal antibody variable region which directly binds to antigens, to a human antibody framwork (CDR-grafting method); and replacing the amino acid residues of the human antibody framework region (FR) that influence the CDR conformation with the amino acid residues of murine monoclonal antibody. The humanized antibody thus obtained maintains the affinity and specificity of original murine monoclonal antibody, and minimizes HAMA(human anti-mouse antibody) response in humans (Riechmann et al., Nature, 332, 323-327 (1988); Queen C. et al., Proc. Natl. Acad. Sci. USA, 86, 10029-10033 (1989); Nakatani et al., Protein Engineering, 7, 435-443 (1994)). However, this humanized antibody still causes problems when injected repeatedly into humans (Stephens et al., Immunology, 85, 668-674 (1995); Sharkey et al., Cancer Research, 55, 5935s-5945s(1995)).

[0005] Approximately 300 millions of world population carry hepatitis B virus ("HBV") which may cause chronic infection, leading to cirrhosis and hepatocellular carcinoma (Tiollais P. and Buendia M. A., Sci. Am., 264, 48 (1991)). The HBV envelope consists of three proteins, major protein containing S antigen, middle protein containing S and pre-S2 antigens, and large protein containing S, pre-S2 and pre-S 1 antigens (Neurath A. R. and Kent S. B., Adv. Vir Res., 34, 65-142 (1988)). These surface antigens have been known to play important roles in the process of forming antibodies against HBV in hepatitis patient. The pre-S1 region, in particular, is found on infectious viral particles (Heermann et al., J. Viral., 52, 396-402 (1984)) and plays a role in attachment to cell surface infection (Neurath et al., Cell, 46, 429 (1986); Pontisso et al., Viral., 173, 533, (1989); Neurath et al., Vaccine, 7, 234 (1989)). Thus a monoclonal antibody against the pre-S1 would be effective against viral infection.

[0006] The present inventors have previously reported a murine monoclonal antibody (KR127) against HBV pre-S1 (Korean Patent No. 246128), a murine monoclonal antibody KR127 gene encoding same (Korean Patent No. 250832) and a humanized antibody (HZKP 1271) of KR127 prepared by CDR-grafting method (Korean Patent No. 246128).

[0007] The present inventors have further endeavored to develop a humanized antibody having minimized adverse immune response (HAMA response) as well as enhanced affinity to antigen, and found that HAMA response can be reduced when the amino acid residues of CDR of mouse antibody are replaced with those of human antibody.

SUMMARY OF THE INVENTION

[0008] Accordingly, it is an object of the present invention to provide a process for preparing a humanized antibody which is expected to show lower HAMA response and has higher affinity than humanized antibody of the prior art.

[0009] It is another object of the present invention to provide a humanized antibody prepared according to said process.

[0010] It is a further another object of the present invention to provide a DNA encoding the heavy chain or light chain of said antibody and a vector 35 comprising said DNA.

[0011] It is a still further object of the present invention to provide a microorganism transformed with said vector.

[0012] In accordance with one aspect of the present invention, there is provided a process for preparing a humanized antibody comprising the steps of (a) selecting a specificity determining residue (SDR) of the complementarity determining region (CDR) of murine monoclonal antibody heavy chain and light chain variable regions; and (b) grafting the amino acid residues of said SDR to at least one of the corresponding amino acid sequences in human antibody variable regions.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The above and other objects and features of the present invention will become apparent from the following description of the invention taken in conjunction with the following accompanying drawings; which respectively show:

[0014] FIG. 1: the procedure for constructing an expression vector of a chimeric heavy chain;

[0015] FIGS. 2A, 2B and 2C: collectively teach the nucleotide and amino acid sequence of the humanized 20 heavy chain variable region;

[0016] FIG. 3: the procedure for constructing an expression vector of a chimeric light chain;

[0017] FIGS. 4A and 4B: the nucleotide and amino acid sequence of the humanized light chain variable region;

[0018] FIG. 5: the affinity to antigen of a humanized antibody having a heavy chain CDR mutant;

[0019] FIG. 6: the procedure for constructing an expression vector of the humanized antibody; and

[0020] FIG. 7: is a plot showing the results of analysis for MHC class II-binding peptide sequences in heavy chain variable regions of HuKR127 and light chain variable regions of HuKR127, respectively.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The humanized antibody of the present invention may be prepared by grafting the amino acid residues of SDR of murine monoclonal antibody to the corresponding amino acid sequences in human antibody variable regions.

[0022] SDRs of the murine monoclonal antibody used in the present invention may be determined by independently replacing each amino acid residue of CDR of the murine monoclonal antibody with alanine, selecting transformants which have lower affinity (k.sub.D) to antigen than the original murine antibody and determining the replaced CDR amino acid residues of said transformants as SDRs.

[0023] Further, in order to enhance the affinity to antigen, the CDR residues of a mouse antibody that increase the affinity and the framework residues that influence the conformation of CDR loops may also be grafted to the corresponding sites of human antibody.

[0024] For example, the present invention describes a process for preparing a humanized antibody for hepatitis B virus (HBV) pre-S1 by using murine monoclonal antibody KR127 (Korean Patent No. 250832) as follows:

[0025] After selecting SDR amino acid residues, which play important roles in binding with antigen, from CDR of the murine monoclonal antibody KR127 heavy and light chains, chimeric heavy chain and chimeric light chain genes may be prepared by combining either the variable region of ICR127 antibody heavy chain with the constant region (C.sub.1 1) of human antibody or the variable region of KR127 antibody light chain with the constant region (CO of human antibody.

[0026] SDRs of the murine monoclonal antibody for HBV pre-S 1 are determined by replacing each amino acid residue of CDR HCDRI (aa 31-35), HCDR2 (aa 50-65) and HCDR3 (aa 95-102) of the heavy chain (SEQ ID NO: 2) and CDR LCDR1 (aa 24-34), LCDR2(aa 50-56) and LCDR3(aa 89-97) of the light chain (SEQ ID NO: 4) of the murine monoclonal antibody KR127 with alanine according to the alanine scanning mutagenesis method and selecting the amino acid residues (SDRs) whose replacement with alanine bring about more than 3 times reduction in the affinity to antigen(KD) as compared with the original murine antibody. Throughout this description, amino acid residue number is assigned according to Kabat numbering scheme (Kabat, E. A. et al, Sequences of Proteins of Immunological Interest. National Institute of Health, Bethesda, Md., 1991).

[0027] Examples of preferred SDR include tryptophan at position 33 (it is represented as "Trp33"), Met34, and Asn35 of HCDR1; Arg50, Tyr52, and Pro52a of HCDR2; Glu95, Tyr96, and Glu98 of HCDR3 of the murine monoclonal antibody KR127 heavy chain; Leu27b, Tyr27d, Ser27e; Asn28, Lys30, Tyr32, and Asn34 of LCDR1; Leu50 and Asp55 of LCDR2; and Va189, Gln90, Gly91, Thr92, His93, Phe94, Pro95, and Gln96 of LCDR3 of the murine monoclonal antibody KR127 light chain.

[0028] The humanized antibody of the present invention can be prepared by grafting one. or more SDRs determined as above onto the human antibody heavy chain and/or light chain. The human antibody heavy chain which may be used in the present invention is human heavy chain DP7-JH4 consisting of human immunoglobulin germline VH gene segment DP7 (Tomlinson et al., J. Mol. Biol., 227, 776-798, 1992) and JH4 segment (Ravetch et al., Cell, 27, 583-591, 1981). The human antibody light chain which may be used in the present invention is human light chain DPK12-JH4 consisting of human immunoglobulin germline VK gene segment DPK12 (Cox et al., Eur. J. Irnmunol., 24, 827-836 (1994)) and JH4 segment (Hieter et al., J. Biol. Chem., 257, 1516-1522 (1982)).

[0029] The humanized heavy chain of the present invention may be prepared by grafting at least one of Trp33, Met34, and Asn35 of HCDR1; Arg50, Tyr52, and Pro52a of HCDR2; Glu95, Tyr96, and Glu98 of HCDR3 of the murine monoclonal antibody KR127 heavy chain to the corresponding amino acid sequences in human antibody heavy chain. The inventive humanized light chain may be prepared by grafting at least one of Leu27b, Tyr27d, Ser27e; Asn28, Lys30, Tyr32, and Asn34 of LCDR1; Leu50 and Asp55 of LCDR2; and Va189, Gln90, Gly91, Thr92, His93, Phe94, Pro95, and Gln96 of LCDR3 of the murine monoclonal antibody KR127 light chain to the corresponding amino acid sequences in human antibody light chain DPH1241(4.

[0030] Moreover, the affinity to antigen of the humanized antibody can be enhanced by the follow substitutions:

[0031] (a) the amino acid residue at position 32 in HCDR1 of the modified human heavy chain DP7-JH4 by Ala;

[0032] (b) the amino acid residue at position 97 in HCDR3 of the modified human heavy chain DP7-JH4 by Arg or Ala;

[0033] (c) the amino acid residue at position 98 in HCDR3 of the modified human heavy chain DP7-JH4 by Val; and

[0034] (d) the amino acid residue at position 102 in HCDR3 of the modified human heavy chain DP7-3H4 by Arg or Ala.

[0035] In addition, Ala71 and Lys73 in framework region 3 in the heavy chain variable region of KR127, which affects the conformation of the CDR loop, may further be grafted to human heavy chain DP7-JH4. Also, Leu36 and Arg46 in framework region 2 in the light chain variable region of KR127, which affects conformation of CDR loop, may be further grafted to human light chain DPH12-3K4.

[0036] The heavy chain variable region of humanized antibody of the present invention has the amino acid sequence of SEQ ID NO: 2, preferably encoded by the nucleotide sequence of SEQ ID NO: 1 and the inventive light chain variable region of humanized antibody has the amino acid sequence of SEQ ID NO: 4, preferably encoded by the nucleotide sequence of SEQ ID NO: 3.

[0037] The humanized antibody heavy chain and light chain of the present invention may be encoded by a gene comprising a nucleotide sequence deduced from the humanized antibody heavy chain and light chain according to the genetic code. It is known that several different codons encoding a specific amino acid may exist due to the codon degeneracy, and, therefore, the present invention includes in its scope all nucleotide sequences deduced from the humanized antibody heavy chain and light chain amino acid sequence. Preferably, the humanized antibody heavy chain and light chain gene sequences include one or more preferred codons of host cell.

[0038] The humanized antibody consisted of the humanized heavy chain HuKR127HC of the present invention and humanized light chain HZKR127I prepared by CDR-grafting has an affinity to antigen of about over 50 times higher than that of the humanized antibody HZKR1271.

[0039] The humanized antibody consisting of the humanized heavy chain HuKR127KC of the present invention and humanized light chain HZKR127I prepared by CDR-grafting has an affinity to antigen equal to that of the humanized antibody HZICR127I.

[0040] The genes of humanized antibody heavy chain and light chain thus prepared may be inserted to pdCMV-dhfrC-HAV6 vector (KCTC 10028BP) to obtain an expression vector pdCMV-dhfrC-HuKR127 which can express both humanized antibody heavy chain HuKR127HC and light chain HZKR127I. The expression vector of the present invention may be introduced into microorganism, e.g., E. coli DH5a according to a conventional transformation method to obtain transformants E. coli DH5a/pdCMV-dhfrC-HuKR127. The transformants E. coli DH5a pdCMVdhfrC-HuKR127 was deposited on Mar. 13, 2002 with the Korean Collection for Type Cultures(KCTC)(Address: Korea Research Institute of Bioscience and Biotechnology(KRIBB), #52, Oun-dong, Yusong-ku, Taejon, 305-333, Republic of Korea) under the accession number, KCTC 10198BP, in accordance with the terms of Budapest Treaty on the International . . . Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure.

[0041] Meanwhile, CHO/HuKR127, CHO (Chinese hamster ovary) cell line transfected with vector pdCMV-dhfrC-HuKR127, was deposited on Mar. 13, 2002 with the Korean Collection for Type Cultures(KCTC) under the accession number, KCTC 10199BP, in accordance with the terms of Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure.

[0042] The humanized antibody HuKR127 of the present invention produced by culturing the CHO/HuKR127 cell line has a higher affinity to antigen and is expected to reduce HAMA (human anti-mouse antibody) response to a greater extent than the conventional antibody prepared according to the CDR-grafting method.

[0043] Accordingly, the humanized antibody of the present invention can be used in preventing hepatitis B virus infection and treating chronic Hepatitis B.

[0044] Thus, for preventing hepatitis B virus infection and treating chronic Hepatitis B, a pharmaceutical formulation of the inventive humanized antibody may be prepared in accordance with any of the conventional procedures.

[0045] The pharmaceutical composition of the present invention can be administered via various routes including intravenous and intramuscular introduction. It should be understood that the amount of the active ingredient actually administered ought to be determined in light of various relevant factors including the condition to be treated, the chosen route of administration, the age, sex and body weight of the individual patient, and the severity of the patient's symptom; and, therefore, the above dose should not be intended to limit the scope of the invention in any way.

[0046] The following Examples are intended to further illustrate the present invention without limiting its scope.

Example 1

Preparation of Mouse/Human Chimeric Heavy Chain Gene

[0047] The gene encoding leader sequence and the yl constant region of the human antibody heavy chain were separately prepared by carrying out PCR using pCMV-HKR127HC (Korean Patent No. 246128, KCTC 0531BP) as a template and a primer set of Ryu94 (SEQ ID NO: 5) and HUR43-1 (SEQ ID NO: 6) or HUR46-1 (SEQ ID NO: 9) and HUR31 (SEQ ID NO: 10). The gene encoding heavy chain variable region of the murine monoclonal antibody KR127 was prepared by carrying out PCR using pKR127H (Korean. Patent No. 250832, KCTC 0333BP) as a template and primers HUR44-1 (SEQ ID NO: 7) and HUR45-1 (SEQ ID NO: 8).

TABLE-US-00001 Ryu94: 5'-GAG AAT TCA CAT TCA CGA TGT ACT TG-3' HUR43-1: CTG CAG CTG GAC CTG ACT CTG GAC ACC ATT-3' HUR44-1: 5'-CAG GTC CAG CTG CAG CAG TCT GGA CCT GAA CTG-3' HUR45-1: 5'-TGG GCC CTT GGT GGA GGC TGC AGA GAC AGTGAC-3' HUR46-1: 5'-GCC TCC ACC AAG GGC CCA TCG GTC TTC CCC CTG-3' HUR31: 5'-CAG CGG CCG CTC ATT TAC CCG GGG ACA G-3'

[0048] Each PCR reaction was carried out using 10 ng of template, 1 ie of each primer (50 ppm), 0.5 p.t of .sub.Pfu DNA polymerase (Promega), 4 a of 2.5 mM dNTPmix and 5, ull of 10.times.Pfu reaction buffer solution. After pre-denaturation at 95 t for 5 minutes, a PCR cycle was repeated 25 times, the cycle being composed of 95 t for 30 sec., 50.degree. C. for 30 sec. and 72.degree. C. for 45 sec. After annealing the DNA fragment obtained by using primers Ryu94 and HUR43-1, the DNA fragment obtained by using primers HUR44-1 and HUR45-1, and the DNA fragment obtained by using primers HUR46-1 and HUR31 were recombined by conducting recombinant PCR using primers Ryu94 and HUR31. The recombinant PCR reaction was carried out using the same reaction buffer solution as used above. After pre-denaturation at 95 t for 5 minutes, a PCR cycle was repeated 30 times, the cycle being composed of: 95 t for 30 sec., 50t for 30 sec. and 72 t for 60 sec., and finally, the extension reaction was carried out at 72 t for 5 min.

[0049] The chimeric heavy chain gene thus prepared was cleaved with EcoRI (GAATTC) and Ndel (GCGGCCGC) and inserted at the EcoRTINdel section of vector pcDdA (plasmid which is removed Apal site in the multiple cloning site of pcDNA received from Invitrogen), to obtain, vector pcDdAchKR127HC (FIG. 1). The base sequence of the chimeric heavy chain variable region gene (KR127VH) was confirmed by DNA sequence analysis (FIG. 2).

Example 2

Preparation of Mouse/Human Chimeric Light Chain Gene

[0050] The gene encoding reader sequence and the constant region of the human antibody light chain were each prepared by carrying out PCR using pKC-dhfr-FIKR127 (Korean Patent No. 2000-33008, KCTC 0529BP) as a template and a primer set of Ryu86 (SEQ ID NO: 11) and HUR48 (SEQ ID NO: 12) or HUR51 (SEQ ID NO: 15) and CK1D (SEQ ID NO: 16).

[0051] The gene encoding light chain variable region of the murine monoclonal antibody KR127 was prepared by carrying out PCR using pKR127K (Korean Patent No. 250832, KCTC 0334BP) as a template and primers HUR49 (SEQ ID NO: 13) and HUR50 (SEQ ID NO: 14).

TABLE-US-00002 Ryu86: 5'-CAA AGC TTG GAA GCA AGA TGG ATT CA-3' HUR48: 5'-CAA GAT ATC CCC ACA GGT ACC AGA TAC-3' HUR49: 5'-TGT GGG GAT ATC TTG ATG ACC CAA ACT-3' HUR50: 5'-CAC AGA TCT TTT GAT TTC CAG CTT GGT-3' HUR51: 5'-ATC AAA AGA TCT GTG GCT GCA CCA TCT-3' CK1D: 5'-GCG CCG TCT AGA ATT AAC ACT CIC CCC TGT TGA AGC TCT TTG TGA CGG GCG AACTCAG-3'

[0052] Each PCR reaction was carried out according to the method described in Example 1 except that primers Ryu86 and CK1D were used to ligate the annealed DNA fragments obtained by PCR reactions.

The chimeric light chain gene thus prepared was cleaved with HindlII (AAGCTT) and Xbal (TCTAGA) and inserted at the HindMIXbal section of vector pBluescript KS, to obtain a recombinant plasmid. Subsequently, the recombinant plasmind was cleaved with Hindilll and Apal and inserted at the HindlrII Apal section. of vector pCMV-dhfr (KCTC 8671P), to obtain plasmid pKC-dhfr-chKR127 (FIG. 3). The base sequence of the chimeric light chain varible region gene (KR127VK) was confirmed by DNA sequence analysis (FIG. 4).

Example 3

Mutation of CDR. of Chimeric KR127 Antibody Heavy Chain by Alanine Injection

[0053] To examine whether each amino acid residue of KR127 heavy chain HCDR1 (aa 31-35), HCDR2(aa 50-65) and HCDR3 (aa 95-102) binds to antigen, PCR reaction was carried out using vector pcDdA-chKR127HC as a template to prepare a modified gene, wherein an amino acid residue of CDR was replaced with alanine (the replaced amino acid residue No. was indicated as Kabat number) (see FIG. 2).

[0054] A forward primer YMOO1N of SEQ ID NO: 17 was designed to provide the sequence corresponding to the reader sequence at the 5'-end of the chimeric heavy chain gene and EcoRI restrition site, and a reverse primer YM003 of SEQ ID NO: 18 was designed to have the sequence corresponding to the N-terminal downstream of CH1 domain of human heavy chain gene and Apal restriction site.

TABLE-US-00003 YMOO1N: 5'-CCG GAA TTC ACA TTC ACG ATG TAC TTG-3' YM003: 5'-TGC CCC CAG AGG TGC T-3'

[0055] The 5'-end primer ym257 of SEQ ID NO: 19 (corresponding to nucleotide Nos. 80 to 112 of SEQ ID NO: 1) was designed to replace Ser31 of HCDR1 with alanine (S31A) and the 3'-end primer YM258 of SEQ ID NO: 20 (corresponding to nucleotide Nos. 101 to 71 of SEQ ID NO: 1), to replace AGT (coding for Ser) of nucleotide Nos. 91 to 93 of HCDRI gene with GCT (coding for alanine).

[0056] Each PCR reaction was carried out according to the method described in Example 1 except that primer sets, YMOO1N and YM258; and ym258 and YM003, were used and also that primers YMOO1N and YM003 were used to recombine the annealed DNA fragments obtained by PCR.

[0057] The chimeric light chain gene thus prepared was cleaved with EcoRI and Apal and inserted at the EcoRTIApal section of vector pcDdA-chICR127HC prepared in Example 1, to obtain peDdA-chICR127HC-S31A. The base sequence of the humanized antibody heavy chain variable region gene was confirmed by DNA sequence analysis. Vectors containing mutants thus prepared are shown in Table 1.

[0058] In Table 1, primer and mutation positions are numbered based on the base sequence of SEQ ID NO: 1.

TABLE-US-00004 TABLE 1 primer mutation CDR primer position position mutant vector F ym257 80-112 91-93 S er CACTI-?+0 R YM258 101-71 Ala(GCT) pcDdA-chICR127HC-S31A F yrn259 83-112 Sre CTCT1-?+0 R YM260 106-73 94-96 Ala(GCT) pcDdA-chKR127HC-S32A F ym261 86-117 Trp(TGG)- pcDdA-chKR127HC-1Y33A HCDR1 R YM262 108-76 97-99 Ala(GCG) F ym263 90-118 Met(ATCF)- R YM264 111-79 100-102 Al a(GCG) pcDdA-chKR127HC-M33A F ym265 94-120 Asn(AAC)- R ym266 112-81 103-105 Ala(GCC) pcDdA-chKR127HC-N35A F YIV1221 139-174 Arg(CGG)-' pcalA-chICR.127HC-R50A R YM222 158-128 148-150 Ala(GCC) F YM225 143-178 151-153 I I e(A 1 1)- pcDdA-chKR127HC-I 51A R YM226 162-131 Ala(GCT) F YM227 145-180 Tvr CT'AT)- R YM228 165-135 154-156 Ala(GCT) pcDdA-chKR127HC-Y52A F ym229 148_181 Pri-dn'T 1-?+0 R YM230 167-136 157-159 AI a(GCT) pcDdA-chICR127HC-P52aA F ym231 150-186 G lv(GGA)- R YM232 173-145 160-162 A 1 a(GCA) pcDdA-chICR127HC-G53A HCDR2 F vm233 152-188 Asn(C1AT4- R YM234 176-144 163-165 Ala(GCT) pcDdA-chKR127HC-D54A F ym235 155-193 Glv(GGA)-?+0 R YM236 178-146 166-168 A Ia(GCA) peDdA-chKR127HC-G55A F ym2 3 7 158-194 A cn(GAT'-?+0 ym238 184-149 169-171 A I a(GCT) pcDdA-chKR127HC-D56A F ym239 160-195 Thr(M. 1 '1- R ym240 185-150 172-174 A 1 a(GCT) pcDdA-chICR127HC-T57A F yrn241 164-196 Acn(A AC1- R ym242 187-150 175-177 Ala(GCC) pcDdA-ch1CR127HC-N58A F YM207 286-317 Glu(GAG1- R YM208 305-274 295-297 A 1 a(GCG) pcDdA-chICR127HC-E95A F YN1209 289-316 Tvr(TAC'.)-. R YNI210 307-276 298-300 A 1 a(GCC) pcDdA-chKR127HC-Y96A F YM211 292-318 Asn(GAC)-?+0 HCDR1 R YN1217 111-279 Al a CGCG) F YM213 296-321 Gln(CIAG)-. R YM214 315-285 304-306 A I a(GCG) pcDdA-chKR127HC-E98A F YM255 303-327 Tvr(TAO-. R YM256 319-289 310-312 Ala(GCC) peDdA-chKR127HC-Y102A

Test Example 1

Expression of Chimeric Antibody Having a Modified Heavy Chain and its Affinity to Antigen

(Step 1) Expression of Chimeric Antibody

[0059] COS7 cells (ATCC CRL-1651) were seeded to DMEM media (GIBCO) containing 10% bovine serum and subcultured in an incubator at 37.degree. C. under an atmosphere of 5% CO.sub.2. 1.times.10.sup.6 cells thus obtained were seeded to the same media and incubated at 37.degree. C. overnight. Thus, 5 lig of plasmid pICC-dhfr-chKR.127 (expressing chimeric light chain) obtained in Example 2, 5 jig of plasmid obtained in Example 3 were diluted with OPTI-1VLEMAGLBCO) to 800 ge. 50 of Lipofectamine (GIBCO) were diluted with the same solution to 800 .sub.Ia. The resulting solutions were added to a 15 rae tube, mixed and then, kept at room temperature for more than 15 minutes. Meanwhile, COS7 cells incubated as above were washed three times with OPTI-MEM I. Then, 6.4 me of OPTI-MEM I was added to the DNA-Lipofectamine mixture and the resulting solution was evenly distributed on the COS7 cells, which were cultured for 48 hours in a 5% CO.sub.2 incubator to obtain a supernatant. The resulting solution was subjected to sandwich ELISA analysis using anti-human IgG (Sigma) as a capture antibody and anti-human antigen (Fc-specific)-horseradish peroxidase (PIERCE) as a secondary antibody to confirm the expression of the chimeric antibody.

(step 2) Affinity to Antigen

[0060] 150 ng of HBV recombinant antigen GST-pre-S1(1-56) (H. S. Kim and H. 3. Hong, Biotechnology Letters, 17, 871-876 (1995)) was coated to each well of a microplate and 5 ng of the supernatant obtained in Step 1 was added to each well. The resulting solution was subjected to indirect ELISA using the same secondary antibody as used in step 1, followed by measuring the absorbance at 450 nm. Further, the affinity to antigen (K.sub.D) of each modified heavy chain was determined by competitive ELISA method (Ryu et al., J. Med. Viral., 52, 226 (1997)) and compared with that of pCK-dhfr-chKR.127 containing wildtype chimeric heavy chain. The result is shown in Table 2.

TABLE-US-00005 TABLE 2 KD CDR Mutant (nM) WT 11.0 .+-. 1.664 ii1 S31A 14.67 .+-. 2.386 S32A 8.455 .+-. 0.840 W33A >10000 M34A >10000 N35A >10000 H2 R50A .sup. >10000 .cndot. I51A 12.8 .+-. 1.05 Y52A 276.8 .+-. 23.60 P52aA 170.3 .+-. 5.318 G53A 7.697 .+-. 0.980 D54A 1.663 .+-. 0.477 G55A 5.766 .+-. 0.211 D56A 6.59 .+-. 1.09 T57A 13.68 .+-. 4.016 N58A 1.568 .+-. 0.085 H3 E95A .cndot. >10000 .sup. Y96A >10000 D97A 0.57 .+-. 0.03 E98A 64.2 .+-. 7.78 Y102A 3.581 .+-. 0.457

[0061] As shown in Table 2, the affinities to antigen of the mutants obtained by replacing Trp33, Met34, or Asn35 of HCDR1; Arg50, Tyr52, or Pro52a of HCDR2; Glu95, Tyr96, or Glu98 of HCDR3 with alanine were more than 3 times lower than that of wild type. However, a mutant having alanine substituting for Asp97 or Tyr102 residue of HCDR3 exhibited an enhanced affinity to antigen.

Example 4

Preparation of HCDR3 Mutants and Their Affinities to Antigen

[0062] (step 1) D97R and E98V Mutants

[0063] Each mutant was prepared by replacing Asp97 or Glu98 of HCDR3 with arginine as a positively charged amino acid (it is represented as "D97R") or valine as a neutral amino acid (it is represented as "E98V") according to the site-directed mutagenesis as used in Example 3. Vectors containing mutants prepared as above are shown in Table 3.

TABLE-US-00006 TABLE 3 mutation CDR primer position position mutant vector HCDR3 R P1 312-279 301-303 Asp(GAC).fwdarw. pcDdA-chKR127HC-D97R F P2 295-326 Arg(CGG) R PC 312-279 301-303 Asp(GAC).fwdarw. pcDdA-chKR127HC-D97V F P4 295-326 Val(GTT) R P5 312-279 304-306 Glu(GAG).fwdarw. pcDdA-chKR127HC-E98R F P6 295-326 Arg(CGG R P7 312-297 304-306 Glu(GAG).fwdarw. pcDdA-chKR127hc-E98V F P8 295-326 Val(GTT)

[0064] Then, each mutant thus obtained was measured for its affinity to antigen in according to the method described in Test Example 1 and compared with that of the wild type.

[0065] As shown in FIG. 5, the affinity to antigen of D97R was more than 3 times higher than that of the wild type, which the affinity to antigen of E98V, more than 4 times higher than that of the wild type. However, mutant E98R showed a low affinity to antigen.

(Step 2) D97R/E98V Mutant

[0066] To prepare D97R/E98V mutant containing both D97R and E98V, which were found to be mutants having high affinity to antigen, PCR reaction was carried out using pcDdA-chICR127HC-D97R which contains D97R gene as a template and primers P7 and P8.

[0067] Then, the D97R/E98V mutant thus obtained was measured for its affinity to antigen in according to the method described in Test Example 1.

[0068] As shown in FIG. 5, the affinity to antigen of D97R/E98V was more than 15 times higher than that of the wild type.

(Step 3) D97R/E98V/Y102A Mutant

[0069] To prepare D97R/E98V/Y102A mutant containing D97R, E98V and Y102A, PCR reaction was carried out using pcDdA-chICR127HC-RV containing D97R/E98V as a template and primers YM255 and YM256.

[0070] Then, the D97R/E98V/Y102A mutant (hereinafter "RVAA") thus obtained was measured for its affinity to antigen in according to the method described in Test Example 1.

[0071] As shown in FIG. 5, the affinity to antigen of D97R/E98V/Y102A was similar to that of D97R/E98V.

(Step 4) D97R/E98V/Y102E and D97R/E98V/Y102R mutants

[0072] To prepare D97R/E98V/Y102E mutant and D97R/E98V/Y102R mutant, PCR reaction was carried out using pcDdA-chKR127HC-RV containing D97R/E98V as a template, and primer sets P17/P18 and P19/P20, respectively.

[0073] Vctor containing mutants prepared above are shown in Table 4.

TABLE-US-00007 TABLE 4 primer mutation primer position postion mutant vector HCDR3 R P17 312-279 307-309 TYr(TAC)- pcDdA-chKR12711C-RVAE F P18 295-326 Glu(GAG) R P19 312-279 307-309 TYr(TAC)- pcDdA-chKR127HC-RVAR F P20 295-326 Arg(CGT)

[0074] Then, D97R/E98V/Y102E mutant (hereinafter "RVAE") and D97R/E98V/Y102R mutant (hereinafter "RVAR") thus obtained were measured for respective affinities to antigen in according to the method described in Test Example 1.

[0075] As shown in FIG. 5, the affinity to antigen of RVAE was similar to that of RVAA, while the affinity to antigen of RVAR was higher than that of RVAA.

Test Example 2

Measurement of Affinity to Antigen of RVAR

[0076] The RVAR mutant prepared in step 4 of Example 4 was subjected to competitive ELISA to measure its affinity to antigen as follows:

[0077] COS7 cells were transfected with the plasmid prepared in step 4 of Example 4 and the plasmid expressing chimeric light chain (pKC-dhfr-chKR127) prepared in Example 2 to produce an antibody. 5 ng of the antibody thus obtained was reacted with pre-S1 antigen (1.times.10.sup.-7 to 1.times.10-12 M) at 37.degree. C. for 2 hours. The resulting solution was added to each well of a 96-well microplate coated with pre-S1 antigen and reacted at 37.degree. C. for 30 minutes, and then the resulting solution was subjected to ELISA analysis according to the method described in Example 4. Used as a control is chimeric antibody (chICR127) obtained from COST cells transfected with pcDdA-chKR127HC and pKC-dhfr-chKR127.

[0078] The affinity to antigen of RVAR was about 1.8.times.10.sup.-10 M, which is 45 times higher than that of chKR127, about 8.2.times.10-9M

Example 5

Mutation of CDR of Chimeric KR127 Antibody Light Chain by Alanine Injection

[0079] To examine the affinity of each amino acid residue of KR127 light chain LCDR1 (aa 24-34), LCDR2(aa 50-60) and LCDR3 (aa 89-97) to antigen, PCR reaction was carried out using vector pKC-dhfr-chKR127 as a template to prepare a modified gene having each amino acid residue of CDR replaced with alanine (the replaced amino acid residue Number was indicated as Kabat number) (see FIG. 2).

[0080] Forward primer YM004 of SEQ ID NO: 21 was designed to provide the sequence corresponding to the reader sequence at the 5'-end of the chimeric light chain gene and the HindIII restrition site, and a reverse primer YM009 of SEQ ID NO: 22 was designed to have the sequence corresponding to the N-terminal region of human light chain gene and the BsiWI(CGTACG) restriction site. These primers were used in preparation of mutants of light chain CDR residue.

TABLE-US-00008 YM004: 5'-CCA AAG CTT GGA AAG ATG GAT TCA CAG-3' YM009: 5'-GCA GCC ACC GTA CGT TTG ATT TCC ACC TTG GT-3'

[0081] Forward primer YM135 was designed to replace Ser26 of LCDR1 with alanine (S26A) and a reverse primer YM136, to replace AGT coding for Ser at the nucleotide Nos. 76 to 78 of LCDRI gene with GCT coding for alanine.

[0082] PCR reactions were carried out according to the method described in Example 1 except that primer sets, YM004/YM135, and YM136/YM009, were used and that primers YM004 and YM009 were used to recombine the annealed DNA fragments obtained by PCR.

[0083] The variable region gene of the mutant thus prepared was cleaved with Hind111 and BsiWI and inserted at the HinaTillBsiWI section of vector pKC-dhfr-chKR127, to obtain pKC-dhfr-chKR12713S-S26A. The base sequence of the modified chimeric light chain variable region gene was 5 confirmed by DNA sequence analysis. The vectors containing mutants prepared above are shown in Table 5.

[0084] In Table 5, the primer and mutation positions are numbered based on the base sequence of SEQ ID NO: 3.

TABLE-US-00009 TABLE 5 primer mutation Primer position position mutant vector LCDR1 F YM135 67-102 76-78 Ser(AGT)- pKC-dhfr-chKR127BS- R YM136 86-54 Ala(GCT) S26A F YM137 69-107 79-81 Gln(CAG)- pKC-dhfr-chKR127BS- R YM138 91-56 Ala(GCG Q27A F YM139 70-111 82-84 Ser(AGC)- pKC-dhfr-chKR127BS- R YM140 94-58 Ala(GCC) S27aA F YM141 73-114 85-87 Leu(CTC)- pKC-dhfr-chKR127BS- R YM142 98-64 Ala(GCC) L27bA F YM143 73-116 Leu(TTA)- pKC-dhfr-chKR127BS- R YM144 102-68 88-91 Ala(GCA) L276A F YM145 79-118 91-93 Tyr(TAT)- pKC-dhfr-chKR127BS- R YM146 103-69 Ala(GCA) Y27dA F YM147 83-119 94-96 Ser(AGT)- pKC-dhfr-chKR127BS- R YM148 107-69 Ala(GCT) S27eA F YM149 84-120 97-99 Asn(AAT)- pice-dhfr-chKR127BS- R YM150 110-70 Ala(GCT) N28A F YM151 88-127 100-102 Gly(GGA)- pKC-dhfr-chKR127BS- R YM152 114-74 Ala(GCA) G29A F YM153 91-130 103-105 Lys(AAA)- pKC-dhfr-chKR127BS- R YM154 116-77 Ala(GCA) K30A F YM155 93-132 106-108 Thr(ACC)- pKC-dhfr-chKR127BS- R YM156 118-80 Ala(GCC) T31A F YM103 99-133 109-111 Tyr(TAT)- pKC-dhfr-chKR127BS- R YM104 120-83 Ala(GCT) Y32A F N34A-F 106-132 115-118 Asn(AAT)- pKC-dhfr-chKR127BS- R N34A-R 126-100 Ala(GCT) Y34A LCDR2 F YM129 151-188 163-165 Leu(CTG)- pKC-dhfr-chKR127BS- R YM130 175-140 Ala(GCG) L50A F YM131 153-191 166-168 Val(GTG)- pKC-dhfr-chKR127BS- R YM132 179-145 Ala(GCG) V51A F YM133 157-192 169-171 Ser(TCT)- pKC-dhfr-chKR127BS- R YM134 181-147 Ala(GCT) S52A F K53A-F 163-187 172-174 Lys(AAA)- pKC-dhfr-chKR127BS- R K53A-R 178-154 Ala(GCA) K53A F L54A-F 163-189 175-177 Leu(CTG)- pKC-dhfr-chKR127BS- R L54A-R 180-159 Ala(GCG) LS4A F D55A-F 170-195 178-180 Asp(GAC)- pKC-dhfr-chKR127BS- R D55A-R 184-163 Ala(GCC) D55A F K56A-F 175-198 181-183 Ser(TCT)- pKC-dhfr-chKR127BS- R K56A-R 190-168 Ala(GCT) S56A LCDR3 F YM113 270-304 280-282 Val(GTG)- pKC-dhfr-chKR127BS- R YM114 292-258 Ala(GCG) V89A F YM115 274-307 283-285 Gln(CAA)- pKC-dhfr-chKR127BS- R YM116 294-259 Ala(GCA) Q90A F YM117 277-310 286-288 Gly(GGT)- pKC-dhfr-chKR127BS- R YM118 296-265 Ala(GCT) G91A F YM119 281-310 289-291 Thr(ACA)- pKC-dhfr-chKR127BS- R YMI20 302-266 Ala(CCA) T92A F YM121 282-313 292-294 His(CAT)- pKC-dhfr-chKR127BS- R YM122 304-271 Ala(GCT) H93A F YM111 286-314 295-297 Phe(TTT)- pKC-dhfr-chKR127BS- R YM112 307-274 Ala(GCT) F94A F YM123 286-317 298-300 Pro(CCT)- pKC-dhfr-chKR127BS- R YM124 308-278 Ala(GCT) P95A F 114125 292-319 301-303 Gln(CAG)- pKC-dhfr-chKR127BS- R YM126 311-279 Ala(GCG) Q96A F YM127 294-320 304-306 Thr(ACG)- pKC-dhfr-chKR127BS- R YM128 313-282 Ala(GCG) T97A

Test Example 3

Measurement of Affinity to Antigen of Light Chain Mutant

[0085] COST cell was transfected with each of the light chain mutants prepared in Example 5 and the plasmid expressing chimeric heavy chain (pcDdA-chKR127HC) to produce an antibody. The antibody obtained was measured for its affinity to antigen in accordance with the method described in Test Example 1.

[0086] Table 6 shows the results obtained for the mutants and pdDA-chKR127HC containing wildtype chimeric KR127 heavy chain.

TABLE-US-00010 TABLE 6 CDR mutant Kloow) L1 S26A 6.49 .+-. 0.244 027A 14.2 + 2.29 S27aA 37.9 * 6.66 L27bA >10000 L27cA 36.8 * 11.01 Y27dA 1032.7 + 56.1 S27eA >10000 N28A >10000 G29A 23.94 * 2.62 K30A >10000 T31A 13.19 * 1.98 Y32A >10000 N34A >10000 L2 LSOA 159.4 + 21.37 V51A 37.00 + 10.33 S52A 14.08 * 0.509 K53A 7.928 * 0.976 L54A 12.57 + 2.453 D55A 225.2 * 2.970 S56A 12.95 .+-. 0.367 L3 V89A 121.2 + 4.62 490A >10000 G91A >10000 T92A 74.2 + 2.90 H93A 54.5 + 4.48 F94A >10000 P95A >10000 O96A 293.6 * 7.13 T97A 17.3 .+-. 2.56

[0087] As shown in Table 6, the affinities to antigen of the mutants obtained by replacing the Leu27b, Tyr27d, Ser27e, Asn28, Lys30, Tyr32, and Asn34 of LCDR1; Leu50 and Asp55 of LCDR2; and Va189, Gln90, Gly91, Thr92, His 93, Phe94, Pro95, and Gln96 of LCDR3 with alanine, respectively, were more than 3 times lower than that of the wild type. Therefore, these residues was determined as SDR.

Example 6

Preparation of humanized heavy chain by SDR-grafting method

[0088] A humanized heavy chain was prepared using DP7-JH4, a human heavy chain constructed by combining human immunoglobulin germline VH gene segment DP7 (Tomlinson et al., J. Mol. Biol., 227, 776-798, 1992) having an amino acid sequence similar to KR127 heavy chain variable regions and human immunoglobulin germline JH4 segment (Ravetch et al., Cell, 27, 583-591 (1981)).

[0089] The Trp33 and Asn35 in HCDR1 of the KR127 were grafted into the DP7-JH4. The Met34 in HCDR1 of the KR127 is identical to that of DP7-JIM. Further, to inhibit lowering the affinity to antigen, Tyr32 in HCDR1 of the KR127 was replaced with alanine of HCDR1 of a human antibody (Gen Bank data base 75023 (SAVVMN)).

[0090] The Arg50 and Tyr52 in HCDR2 of the KR127 were grafted onto the DP7-1H4. The Pro52a in HCDR2 of the KR127 is identical to that of DP715 JH4.

[0091] The Asp95, Tyr96, Arg97, Va198, and ArgI 02 of HCDR3 were grafted into DP7-JH4.

[0092] Further, Ala71 and Lys73 of FR 3 (framwork region 3) in the heavy chain variable region of KR127 antibody which affects the conformation of 20 CDR loops were grafted thereto.

[0093] Then, PCR reaction was carried out using primers Ryu 166 of SEQ ID NO: 23 and Hur37 of SEQ ID NO: 24 according to the method described in Example 3 to obtain a humanized heavy chain variable region gene, HuKR127VH-VII.

TABLE-US-00011 Ryu 166: 5'-GGA ITT GTC TGC AGT CAT TGT-GGC TCT GCC CTG GAA CTT-3' Hur 37: 5'-GAC AAA TCC ACG AGC ACA GTC TAC ATG-3'

[0094] The base sequence of the humanized heavy chain variable region gene was determined by DNA sequence analysis (FIG. 2). Then, the gene was cleaved with EcoRI and .Apal and inserted at the EcoRllApal section of vector pdDdA-chKR127HC to obtain pHuKR127HC.

[0095] A humanzied antibody was prepared by combining humanized heavy chain thus obtained and the humanized antibody HZKR127I light chain described in Korean. Patent No. 246128 and measured the affinity to antigen was numbered according to the method described in Test Example 2. Humanized antibody HZKR127I was used as a control.

[0096] The affinity to antigen of the humanized antibody of about 1.5.times.10-10 M was about 50 times higher than that of HZKR127I, about 8.2.times.le M.

Example 7

Preparation of Humanized Light Chain by SDR-Grafting Method

[0097] A humanized light chain was prepared using DP7-3134, a human light chian constructed by combining human immunoglobulin germline VK gene segment DPK12 (Cox et al., Eur. J. Immunol., 24, 827-836 (1994)) having an amino acid sequence similar to KR127 light chain variable regions and human immunoglobulin germline 0.1K4 segment (Hieter et al., J. Biol. Chem., 257, 1516-1522 (1982)).

[0098] The Tyr27d, Asn28 and Asn34 in LCDR1 of KR127 were grafted into the DPK12-3K4. The amino acid residues at position 27b, 27e, 30 and 15 32 of DP7 is identical to those of KR127 light chain.

[0099] The Leu50 and Asp55 in LCDR2 of KR.127 were grafted into the DPK12-3K4 gene.

[0100] The Va189, Gly91, Thr92, His93, Phe94, and Gln96 in LCDR3 of KR127 were grafted into the DPK12-JK4. The residues at positions 90 and 20 95 of DP7 is identical to those of KR127.

[0101] Further, Leu36 and Arg46 of FR 2 in the light chain variable region of KR127 antibody (which acts on interaction with heavy chain or CDR) were grafted thereto.

[0102] Then, PCR reaction was carried out using primers Ryul18 of SEQ ID NO: 25 and Ryu 119 of SEQ ID NO: 26 according to the method described in Example 3 to prepare a humanized light chain variable region gene, HuKR127VH-IV.

TABLE-US-00012 Ryu 118: 5'-CTG TGG AGG CTG GCC TGG CTT CTG TAA TAA CCA-3' Ryu 119: 5'-GGC CAG CCT CCA CAG CTC CTA ATC TAT CTG-3'

[0103] The base sequence of the humanized light chain variable region gene was determined by DNA sequence analysis (see HZIV of FIG. 4). Then, the gene was cleaved with HindJII and BsiWI and inserted at the HindlII/BsiWI section of vector pKC-dhfr-chKR127BS to obtain pHuK.R.127KC.

[0104] A humanzied antibody was prepared by combining humanized light chain thus obtained and the humanized antibody HZICR1271 heavy chain de(scribed in Korean Patent No. 246128 and its affinity to antigen was measured according to the method described in Test Example 2. Humanized antibody HZKR127I was used as a control.

[0105] The affinities to antigen of the humanized antibody of about 8.4.times.10''.sup.9 M was similar to that of HZICR1271, about 8.2.times.10''.sup.9 M.

Example 8

Preparation of Humanized Antibody and Measurement of the Affinity to Antigen

[0106] To prepare a plasmid containing humanized heavy chain plasmid pHuKR127HC and humanized light chain plasmid pHuKR127KC, the EcoRIMpal fragment containing humanized heavy chain variable region gene of pHuKR127HC and the HindMIBsiWI fragment containing humanized light chain variable region gene of pHuKR127KC were inserted at the EcoRIMpal and HindllBsiWI sections of vector pdCMV-dhfrC-HAVE (KCTC 10028BP), respectively, to obtain plasmid pdCIVW-dhfrC-HuKR127 (FIG. 6). E. coli DH5 a was transformed with the plasmid thus obtained and the transformed E. coli DH5a/pdCMC-dhfrC-HuKR127 was deposited on Mar. 13, 2002 with the Korean Collection for Type Cultures(KCTC) (Address: Korea Research Institute of Bioscience and Biotechnology(KRIBB), #52, Oun-dong, Yusong-ku, Taejon, 305-333, Republic of Korea) under the accession number, KCTC 10198BP, in accordance with the terms of Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure.

[0107] To prepare cell line expressing the humanized antibody, dhfr-defected CHO (chinese hamster ovary) cells were transformed with plasmid pdCMV-dhfrC-HuICR127 as follows:

[0108] CHO cells (ATCC CRL 9096) were seeded to DMEMJF12 media (GIBCO) containing 10% fetal bovine serum and subcultured in an incubator at 37.degree. C. under an atmosphere of 5% CO.sub.2. 5.times.10.sup.5 cells thus obtained were seeded to the same media and incubated at 37.degree. C. overnight, followed by washing 3 times with OPTI-MEMI solution (GIBCO).

[0109] Meanwhile, 5 gg of the plasmid pdCMV-dhfi-C-HuICR127 was diluted in 500 .alpha. of OPTI-MEIVII solution. 25 ihe of Lipofectamine was diluted in 500, u-e of the same solution. The resulting solutions were added to a 15 me tube, mixed, and then, kept at room temperature for more than 15 minutes. Then, 2 nit of OPTI-MEM I was added to by DNA-Lipofectamine mixture and the resulting solution was distributed evenly on the COST cells to be kept in a 5% CO.sub.2 incubator at 37.degree. C. for 6 hours. Added thereto was 3 in.c, of DMEM/F12 containing 20% fetal bovine serum and cultured for 48 hours.

[0110] Then, CHO cells were taken up with trypsin and cultured in-a-MEM media (GIBCO) of 10% dialyzed fetal bovine serum containing G418 (GIBCO BRL; 550. mit) for 2 weeks. After confirming of antibody-producing ability of the transformed clone, the clone was cultured in a-MEM media of 10% dialyzed fetal bovine serum containing 20 nM MTX to induce amplification of gene.

[0111] Cell line CHO/HuKR127 having the highest antibody-productivity was selected from the clones and deposited on Mar. 13, 2002 with the Korean Collection for Type Cultures(KCTC) under the accession number, KCTC 10199BP, in accordance with the terms of Budapest Treaty on the International Recognition of the Deposit of Microorganism for the Purpose of Patent Procedure.

[0112] To measure the affinity to antigen of the humanized antibody HuKR127, CHO cell line thus obtained was mass cultured in a serum-absence media (CHO-SFMII, GIBCO) and subjected to protein G-shepharose 413 column (Pharmacia). Then, the antibody absorbed on the column was eluted with 0.1 M glycine solution (pH 2.7) and neutralized with 1.0 M tris solution (pH 9.0), followed by dialyzing in PBS buffer (pH 7.0). Further, the affinity to antigen of the purified antibody was determined by the competitive ELISA method described in Test Example 2 and compared with that of a control, humanized HuKR1271. The result was shown in FIG. 7.

[0113] As shown in FIG. 7, the affinity to antigen of the humanized antibody of the present invention of 1.6.times.10.sup.I.sup.o M was about 50 times higher than 8.2.times.10.sup.-9 M3 of the control group.

Example 9

Confirmation of Immune-Response Induction of Humanized Antibody

[0114] To confirm whether the humanized antibody of the present invention (HuKR127) prevents HAMA response, an analysis was conducted according to the TEPITOPE method (Sturniolo et al., Nature Biotechnology, 17, 555-561, 1999) to examine whether a peptide sequences which can bind to MHC (major histocompatibility complex) class II exists in the heavy and light chain variable regions of the humanized antibody.

[0115] Tables 8a and 8b show the results of such analysis for MHC class II-5 binding peptide sequences in the heavy chain variable regions of HuKR127 and the light chain variable regions of HuKR127, respectively.

TABLE-US-00013 TABLE 7 HaKR1271 HuICR127 antiboby peptide MHC class II peptide MHC class II MHC class LVQSGAEVV DRB1_0306 LVQSGAEVK 0 II-binding DRB1_0307 DRB1_0308 DRB1_0311 DRB1_0421 DRB1_0701 DRB1_0703 VKPGASVKV DRB1_0102 lUtPGASVKV 0 FSSSWMNWV DRB1_0703 FTSAWMNWV 0 IfIGRIYPGD DRB1_0801 WMGRIYPSG 0 DRB1_0817 FQGRATLTA DRB1_0401 FQGRVIMTA ORB1_0305 DRB1_0402 DRB1 0408 DRB1_0405 DRB1_0401 DRB1_0421 DRI31_0402 DRB1_0426 DRB1_0408 DRB1_0801 DRB1_0426 DRI31_0802 DRB1_0801 DRB1_0B04 DRB1_0802 DRB1_0806 DRB1_0804 DRB1_0804 DRB1_0806 DR81_0813 DRB1_0813 DRB1_0806 DRB1_0617 DRBL0817 DRB1_1101 DRB1_1101 DRB1_1114 DRB1_1102 DRB1_1120 DRB1 1104 DRB1_1128 DRB1-1106 DRB1_1302 DRB1_1114 DRB1_1305 DRB1_1120 DRB1_1307 DRB1_1121 D/W1_1321 DRB1_1128 DRB1_1323 DRB1_1302 DRB1_1502 DRB1_1305 DRB1_1307 DRB1_1311 DRB1_1321 DRB1_1322 DRB1_1323 YWGQGILVT DRB1_0401 RWGQGTLVT 0 DRB1_0405 DRB1_0421 DRB1_0426 IGRIYPGDG DRI350101 MGRIYPSGG DRB1_0404 DRB5_0105 DRB1_0405 DRB1_0410 DRB1_0423 YAQKPQGRA DR1_0802 YAQKFQGRV 0 VYFCAREYD DRB1_1304 VYYCAREYR DRB1_0301 YWGQGTLVT DRB1_0401 RWGQGILVT 0 DRB1_0405 DRBU1421 DRB1_0426 total 50 26

TABLE-US-00014 TABLE 8a HzKR.127I HuKR127 antiboby Peptide MHC class II peptide MHC class II MHC class II- ILMTQTPLS DRBL0301 IVMTQTPLS 0 binding DRB1_0305 DRB1_0306 DRB1 0307 DRB1_0308 DRB1_0309 DRBL0311 DRBL0401 DRB1 0402 DRB1_0404 DRB1_0405 DRB1_0408 DRB1_0410 DRBL0421 DRB1 0423 DREli0426 DRB1_0804 ERBL1101 DRBL1102 DRB1_1104 DRBL1106 DRB1_1107 DRBL1114 DRB1_1121 DRB1_1128 DRB1_1301 DRB1_1304 DRB1_1305 DRB1_1307 DRB1_1311 DREIL1321 DRB1_1322 DRB1_1323 DRB1_1327 DRB1_1328 LMTQTPLSL DRBL0101 MCIQMISL 0 DRB1_0102 DRB1_0304 MUMS ORB1_0101 wILQXPGQP 0 DRB1_0305 DRBL0309 DRBL0401 DRB10408 ORM_0421 DRB10426 DRB1_0802 DRB1_1101 DRB1_1107 DR/11_1114 DRB1_1120 DRB1_1128 DRB1 1302 DRB1_1305 DRB1_1307 DR/a . . . 1321 DRB1_1323 DRB5_0101 DRB10105 YYCVQGTHF DRB1_0101 YYCVQGTHF DRBLOI01 DRB1_0701 DRBI_0701 DRB1_0703 DRBL0703 DRB1_0104 DRB5_0101 DRB1_0105 DRB5_0105 YCVQGTHFP DR&040/ YCVQGTHFP ORBL0401 DRB1_0421 DR/31_0421 ORB1_0426 DRBL0426

TABLE-US-00015 TABLE 8b HzKR127I HuKR127 antiboby Peptide MEC class II peptide MEC class II VGVYYCVQG DRB1 0806 VGVYYCVQG DRB1_0806 IYLVSKLDS DRB1 0301 DRB1_0402 D1631-0305 DR13_0404 DRB1-0306 DRB1_0405 DRB1-0307 DRB1-0308 DRB1 01301 DRB1-0309 DR131-0408 DRI31-0311 DRB1-0405 DRI31-0410 ORM 01302 DR131 0806 DRB -1)423 DB1-0813 DRBI-W04 DR131-0817 DB1-1102 19131-1101 DRB1-11(14 DRB1-1102 DRB -1106 DRB1-1104 DRB111114 DRB1-1106 IYLVSNRDS DRB -1121 DR13E1107 DRB1 1301 DR81 1114 DRB1-1307 121A3I-1120 DRB1-1311 DRB -1322 DR/31-r1 a B IliP DRB1-1301 DRB1-1302 X1 T3228 DRB1-1301- DR135 0101 DB1-1305 DRB5 0105 DRB1-1307 DRBE1311 DRB1 1321 DRB1-1322 DRB1-1323 DRB1-1327 DRB1-1328 DRB1-1501 DR13111506 DRB1 0401 DRB1-0404 DRB1-0405 DRB1 13046 _ D R B 1 0 DRDIum---nain LlYINSFID 8 0 1 0 DRB1 _ 1321 - DRB1 0421 8 D-0123 LIYLVSNRD DR131RB1-0126 DR1311304 YLVSNRDSG 0 YLVSNRDSG DRB1 0309 40 106 total

[0116] As can be seen from Tables 7 and 8, the number of the peptide Sequence in the humanized antibody HuKR127 which binds to MHC class II was fewer than of that the HzKR127I. These results suggest that 35 humanized antibody HuKR127 of the present invention is expected to reduce HAMA response to a greater extent than HzKR127I.

[0117] While the embodiments of the subject invention have been described and illustrated, it is obvious that various changes and modifications can be made therein without departing from the spirit of the present invention which should be limited only by the scope of the appended claims.

Sequence CWU 1

1

381345DNAArtificial SequenceVariable region of humanized heavey chain HZVII 1caggtccagc tggtgcagtc tggagctgaa gtgaagaagc ctggggcctc agtgaaggtt 60tcctgcaaag cttctggcta caccttcacc agtgcttgga tgaactgggt gcgacaggcc 120cctggacagg gtcttgagtg gatgggacgg atttatccta gtggtggaag cactagctac 180gcacagaagt tccagggcag agtcacaatg actgcagaca aatccacgag cacagtctac 240atggagctca gcagcctgag atctgaggac acggcggtgt attactgtgc aagagagtac 300cgggttgccc gttggggcca aggaactctg gtcactgtct cttca 3452115PRTArtificial SequenceVariable region of humanized heavey chain HZVII 2Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Ala Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ala 20 25 30 Trp Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Tyr Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Tyr Arg Val Ala Arg Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ala 115 3336DNAArtificial SequenceVariable region of humanized light chain HZIV 3gatatcgtga tgacccaaac tccactttct ttgtcggtta cccctggaca accagcctct 60atctcttgca agtcaagtca gagcctctta tatagtaatg gaaaaaccta tttgaattgg 120ttattacaga agccaggcca gcctccacag cgcctaatct atctggtgtc taatcgggac 180tctggagtcc ctgacaggtt cagtggcagt ggatcaggaa cagattttac actgaaaatc 240agcagagtgg aggctgagga tgttggagtt tattactgcg tgcaaggtac acattttcct 300cagacgttcg gtggaggcac caaggtggaa atcaaa 3364112PRTArtificial SequenceVariable region of humanized light chain HZIV 4Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Lys Pro Gly Gln Pro 35 40 45 Pro Gln Arg Leu Ile Tyr Leu Val Ser Asn Arg Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Gly 85 90 95 Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110 526DNAArtificial Sequenceoligomer Ryu94 5gagaattcac attcacgatg tacttg 26633DNAArtificial Sequenceoligomer HUR43-1 6ctgctgcagc tggacctgac tctggacacc att 33733DNAArtificial Sequenceoligomer HUR44-1 7caggtccagc tgcagcagtc tggacctgaa ctg 33833DNAArtificial Sequenceoligomer HUR45-1 8tgggcccttg gtggaggctg cagagacagt gac 33933DNAArtificial Sequenceoligomer HUR46-1 9gcctccacca agggcccatc ggtcttcccc ctg 331028DNAArtificial Sequenceoligomer HUR31 10cagcggccgc tcatttaccc ggggacag 281126DNAArtificial Sequenceoligomer Ryu86 11caaagcttgg aagcaagatg gattca 261227DNAArtificial Sequenceoligomer HUR48 12caagatatcc ccacaggtac cagatac 271327DNAArtificial Sequenceoligomer HUR49 13tgtggggata tcttgatgac ccaaact 271427DNAArtificial Sequenceoligomer HUR50 14cacagatctt ttgatttcca gcttggt 271527DNAArtificial Sequenceoligomer HUR51 15atcaaaagat ctgtggctgc accatct 271658DNAArtificial Sequenceoligomer CK1D 16gcgccgtcta gaattaacac tctcccctgt tgaagctctt tgtgacgggc gaactcag 581727DNAArtificial Sequenceoligomer YM001N 17ccggaattca cattcacgat gtacttg 271816DNAArtificial Sequenceoligomer YM003 18tgcccccaga ggtgct 161933DNAArtificial Sequenceoligomer ym257 19acgcattcag tgcttcttgg atgaactggg tga 332031DNAArtificial Sequenceoligomer YM258 20atccaagaag cactgaatgc gtagccagaa g 312138DNAArtificial Sequenceoligomer YM004 21ccaattcaaa gcggtttttc cattactata taagaggc 382232DNAArtificial Sequenceoligomer YM009 22gcagccaccg tacgtttgat ttccaccttg gt 322339DNAArtificial Sequenceoligomer Ryu 166 23ggatttgtct gcagtcattg tggctctgcc ctggaactt 392427DNAArtificial Sequenceoligomer Hur 37 24gacaaatcca cgagcacagt ctacatg 272533DNAArtificial Sequenceoligomer Ryu 118 25ctgtggaggc tggcctggct tctgtaataa cca 332630DNAArtificial Sequenceoligomer Ryu 119 26ggccagcctc cacagctcct aatctatctg 3027345DNAArtificial SequenceVariable region of humanized heavy chain KR127VH 27caggtccagc tgcagcagtc tggacctgaa ctggtgaagc ctggggcctc agtgaagatt 60tcctgcaaag cttctggcta cgcattcagt agttcttgga tgaactgggt gaagcagagg 120cctggacagg gtcttgagtg gattggacgg atttatcctg gagatggaga tactaactac 180aatgggaagt tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac 240atgcagctca gcagcctgac ctctgtggac tctgcggtct atttctgtgc aagagagtac 300gacgaggctt actggggcca agggactctg gtcactgtct ctgca 34528115PRTArtificial SequenceVariable region of humanized heavy chain KR127VH 28Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Asp Thr Asn Tyr Asn Gly Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Val Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Tyr Asp Glu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ala 115 29336DNAArtificial SequenceVariable region of humanized light chain KR127VK 29gatatcttga tgacccaaac tccacttatt ttgtcggtta ccattggaca accagcctct 60atctcttgca agtcaagtca gagcctctta tatagtaatg gaaaaaccta tttgaattgg 120ttattacaga ggccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180tctggagtcc ctgacaggtt cactggcagt ggatcaggaa cagattttac actgaaaatc 240atcagagtgg aggctgagga tttgggagtt tattactgcg tgcaaggtac acattttcct 300cagacgttcg gtggaggcac caagctggaa atcaaa 33630112PRTArtificial SequenceVariable region of humanized light chain KR127VK 30Asp Ile Leu Met Thr Gln Thr Pro Leu Ile Leu Ser Val Thr Ile Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Lys Thr Tyr Leu Asn Trp Leu Leu Gln Arg Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ile Arg Val Glu Ala Glu Asp Leu Gly Val Tyr Tyr Cys Val Gln Gly 85 90 95 Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 110 31294DNAArtificial SequenceVariable region of humanized heavy chain DP7 31caggtgcagc tggtgcagtc tggggctgag gtgaagaagc ctggggcctc agtgaaggtt 60tcctgcaagg catctggata caccttcacc agctactata tgcactgggt gcgacaggcc 120cctggacaag ggcttgagtg gatgggaata atcaacccta gtggtggtag cacaagctac 180gcacagaagt tccagggcag agtcaccatg accagggaca cgtccacgag cacagtctac 240atggagctga gcagcctgag atctgaggac acggccgtgt attactgtgc gaga 2943298PRTArtificial SequenceVariable region of humanized heavy chain DP7 32Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Tyr Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Asn Pro Ser Gly Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg 33302DNAArtificial SequenceVariable region of humanized light chain DPK12 33gatattgtga tgacccagac tccactctct ctgtccgtca cccctggaca gccggcctcc 60atctcctgca agtctagtca gagcctcctg catagtgatg gaaagaccta tttgtattgg 120tacctgcaga agccaggcca gcctccacag ctcctgatct atgaagtttc caaccggttc 180tctggagtgc cagataggtt cagtggcagc gggtcaggga cagatttcac actgaaaatc 240agccgggtgg aggctgagga tgttggggtt tattactgca tgcaaagtat acagcttcct 300cc 30234100PRTArtificial SequenceVariable region of humanized light chain DPK12 34Asp Ile Val Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu His Ser 20 25 30 Asp Gly Lys Thr Tyr Leu Tyr Trp Tyr Leu Gln Lys Pro Gly Gln Pro 35 40 45 Pro Gln Leu Leu Ile Tyr Glu Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln Ser 85 90 95 Ile Gln Leu Pro 100 35345DNAArtificial SequenceVariable region of humanized heavy chain HZI 35caggtccagc tggtgcagtc tggagctgaa gtggtgaagc ctggggcctc agtgaaggtt 60tcctgcaaag cttctggcta cgcattcagt agttcttgga tgaactgggt gcgacaggcc 120cctggacagg gtcttgagtg gattggacgg atttatcctg gagatggaga tactaactac 180gcacagaagt tccagggcaa ggccacactg actgcagaca aatccacgag cacagcctac 240atggagctca gcagcctgag atctgaggac acggcggtct atttctgtgc aagagagtac 300gacgaggctt actggggcca aggaactctg gtcactgtct cttca 34536115PRTArtificial SequenceVariable region of humanized heavy chain HZI 36Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Asp Gly Ser Thr Ser Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Glu Tyr Asp Glu Ala Tyr Trp Gly Gln Gly Thr Leu Val Thr 100 105 110 Val Ser Ser 115 37336DNAArtificial SequenceVariable region of humanized light chain HZI 37gatatcttga tgacccaaac tccactttct ttgtcggtta cccctggaca accagcctct 60atctcttgca agtcaagtca gagcctctta tatagtaatg gaaaaaccta tttgaattgg 120ttattacaga agccaggcca gtctccaaag cgcctaatct atctggtgtc taaactggac 180tctggagtcc ctgacaggtt cagtggcagt ggatcaggaa cagattttac actgaaaatc 240agcagagtgg aggctgagga tgttggagtt tattactgcg tgcaaggtac acattttcct 300cagacgttcg gtggaggcac caaggtggaa atcaaa 33638112PRTArtificial SequenceVariable region of humanized light chain HZI 38Asp Ile Leu Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Asn Gly Lys Thr Tyr Leu Tyr Trp Leu Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Lys Arg Leu Ile Tyr Leu Val Ser Lys Leu Asp Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Val Gln Gly 85 90 95 Thr His Phe Pro Gln Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 110

Claims

1. (canceled)

2. A process for preparing a humanized antibody consisting of the steps of: (a) first performing alanine scanning mutagenesis for replacing each amino acid residue in the entire complementarily determining region (CDR) of a murine monoclonal antibody heavy chain and light chain variable regions with alanine to produce a series of transformants, selecting a transformant that has a lower affinity to the human antigen (KD) than of the original murine antibody, and determining the replaced amino acid residue of said selected transformant as a specificity determining residue (SDR); and (b) subsequently grafting all of the said SDR to the corresponding amino acid residues into human antibody variable regions.

3. The process of claim 2, wherein the CDR is selected from the group consisting of HCDR1(aa 31-35), HCDR2(aa 50-65) and HCDR3(aa 95-102) of the heavy chain (SEQ ID NO: 2); and LCDR1(aa 24-34), LCDR2(aa 50-56) and LCDR3(aa 89-97) of the light chain (SEQ ID NO: 4) of the murine monoclonal antibody variable regions of that bind hepatitis B virus pre-S 1 antigen, selecting a transformant that has an affinity to antigen which is more than 3 times lower than the original murine antibody when replaced with alanine, determining the replaced amino acid residue of said transformant as an SDR, and grafting said SDR to the corresponding amino acid sequence in human antibody heavy chain and light chain.

4-24. (canceled)