Patent Application
20140121358
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.
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.
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)).
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)).
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.
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).
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.
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.
It is another object of the present invention to provide a humanized antibody prepared according to said process.
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.
It is a still further object of the present invention to provide a microorganism transformed with said vector.
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.
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:
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.
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 (kD) to antigen than the original murine antibody and determining the replaced CDR amino acid residues of said transformants as SDRs.
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.
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:
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 (C1 1) of human antibody or the variable region of KR127 antibody light chain with the constant region (CO of human antibody.
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).
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.
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)).
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.
Moreover, the affinity to antigen of the humanized antibody can be enhanced by the follow substitutions:
(a) the amino acid residue at position 32 in HCDR1 of the modified human heavy chain DP7-JH4 by Ala;
(b) the amino acid residue at position 97 in HCDR3 of the modified human heavy chain DP7-JH4 by Arg or Ala;
(c) the amino acid residue at position 98 in HCDR3 of the modified human heavy chain DP7-JH4 by Val; and
(d) the amino acid residue at position 102 in HCDR3 of the modified human heavy chain DP7-3H4 by Arg or Ala.
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.
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.
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.
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.
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.
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.
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.
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.
Accordingly, the humanized antibody of the present invention can be used in preventing hepatitis B virus infection and treating chronic Hepatitis B.
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.
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.
The following Examples are intended to further illustrate the present invention without limiting its scope.
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).
Each PCR reaction was carried out using 10 ng of template, 1 ie of each primer (50 ppm), 0.5 p.t of Pfu DNA polymerase (Promega), 4 a of 2.5 mM dNTPmix and 5, ull of 10×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° C. for 30 sec. and 72° 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.
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 (
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).
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).
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 (
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
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.
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).
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.
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.
In Table 1, primer and mutation positions are numbered based on the base sequence of SEQ ID NO: 1.
COS7 cells (ATCC CRL-1651) were seeded to DMEM media (GIBCO) containing 10% bovine serum and subcultured in an incubator at 37° C. under an atmosphere of 5% CO2. 1×106 cells thus obtained were seeded to the same media and incubated at 37° 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 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% CO2 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
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 (KD) 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.
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.
(step 1) D97R and E98V Mutants
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.
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.
As shown in
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.
Then, the D97R/E98V mutant thus obtained was measured for its affinity to antigen in according to the method described in Test Example 1.
As shown in
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.
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.
As shown in
(Step 4) D97R/E98V/Y102E and D97R/E98V/Y102R mutants
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.
Vctor containing mutants prepared above are shown in Table 4.
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.
As shown in
The RVAR mutant prepared in step 4 of Example 4 was subjected to competitive ELISA to measure its affinity to antigen as follows:
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 (1×10−7 to 1×10-12 M) at 37° 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° 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.
The affinity to antigen of RVAR was about 1.8×10−10 M, which is 45 times higher than that of chKR127, about 8.2×10-9M
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
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.
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.
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.
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.
In Table 5, the primer and mutation positions are numbered based on the base sequence of SEQ ID NO: 3.
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.
Table 6 shows the results obtained for the mutants and pdDA-chKR127HC containing wildtype chimeric KR127 heavy chain.
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.
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)).
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)).
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.
The Asp95, Tyr96, Arg97, Va198, and ArgI 02 of HCDR3 were grafted into DP7-JH4.
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.
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.
The base sequence of the humanized heavy chain variable region gene was determined by DNA sequence analysis (
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.
The affinity to antigen of the humanized antibody of about 1.5×10-10 M was about 50 times higher than that of HZKR127I, about 8.2×le M.
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)).
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.
The Leu50 and Asp55 in LCDR2 of KR.127 were grafted into the DPK12-3K4 gene.
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.
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.
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.
The base sequence of the humanized light chain variable region gene was determined by DNA sequence analysis (see HZIV of
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.
The affinities to antigen of the humanized antibody of about 8.4×10″9 M was similar to that of HZICR1271, about 8.2×10″9 M.
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 (
To prepare cell line expressing the humanized antibody, dhfr-defected CHO (chinese hamster ovary) cells were transformed with plasmid pdCMV-dhfrC-HuICR127 as follows:
CHO cells (ATCC CRL 9096) were seeded to DMEMJF12 media (GIBCO) containing 10% fetal bovine serum and subcultured in an incubator at 37° C. under an atmosphere of 5% CO2. 5×105 cells thus obtained were seeded to the same media and incubated at 37° C. overnight, followed by washing 3 times with OPTI-MEMI solution (GIBCO).
Meanwhile, 5 gg of the plasmid pdCMV-dhfi-C-HuICR127 was diluted in 500 α 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% CO2 incubator at 37° C. for 6 hours. Added thereto was 3 in.c, of DMEM/F12 containing 20% fetal bovine serum and cultured for 48 hours.
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.
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.
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
As shown in
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.
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.
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.
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.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 10508759 | Sep 2004 | US |
| Child | 13653481 | US |
