CN1322762A - Humanized antibody with specificity to HBV surface antigen PRE-S1 and its producing process - Google Patents
Humanized antibody with specificity to HBV surface antigen PRE-S1 and its producing process Download PDFInfo
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Abstract
The present invention relates to humanized antibody with specificity to HBV surface antigen pre-S1, and it exhibits affinity similar to mouse monoclonal antibody and obviously lower immunogenicity owing to less amino acid residue from mouse. Therefore, the humanized antibody of the present invention may be used in preventing HBV infection and treating hepatitis B.
Description
The present invention relates to that HBV surface antigen pre-S1 is had specific humanized antibody.
Specifically, the present invention relates to that HBV surface antigen pre-S1 is had specific humanized antibody, this antibody contains humanized heavy chain and light chain; The gene that relates to humanized heavy chain of coding or light chain; Relate to the intestinal bacteria transformant that contains said expression carrier and contain said expression vector; And relating to the pharmaceutical composition that contains said humanized antibody, it can be used for preventing that HBV from infecting or the treatment chronic hepatitis B.
HBV (hepatitis B virus) is the cause of disease of human chronic or acute hepatitis, can cause liver cirrhosis and liver cancer when serious.Estimate that about in the world 300,000,000 people suffer from hepatitis (Tiollais and Buendia, Sci.Am.264:48,1991).
Have three kinds of HBV surface proteins, they contain different types of surface antigen.Specifically, these surface antigen protein matter comprise and contain the antigenic main protein of S, contain the antigenic medium protein of S and pre-S2 (middle protein), with contain S, the antigenic large-scale protein of pre-S2 and pre-S1 (large protein) (Neurath and Kent, the virus research progress, 34:65-142,1988).During all surface antigen protein matter can be induced and the antibody of HBV, particularly, the rehabilitation of infecting at the removing and the HBV of the antigenic antibody of HBV pre-S and virus, overcome the relevant (Iwarson etc. of the antigenic non-responsiveness of S, the medical virology magazine, 16:89-96,1985; Itoh etc., PNAS, 85:9174-9178,1986; Budkowska etc., medical virology magazine, 20:111-125,1986; Milich etc., PNAS, 82:8168-8172,1985; Milich etc., Journal of Immunology, 137:315-322,1986).
Be different from pre-S2 and S antigen, pre-S1 antigen exists only in (Heerman etc., Journal of Virology, 52:396-402,1984) in the virion of infection and participates in infection to human hepatocytes.Thereby, existing report be specific to the antigenic monoclonal antibody of pre-S1 can be effectively in and HBV (Neurath etc., cell (Cell), 46:429,1986; Pontisso etc., virusology (Virology, 173:533,1989; Neurath etc., vaccine (Vaccine), 7:234,1989), and this monoclonal antibody is considered to can be used for preventing that HBV from infecting and the treatment chronic hepatitis B.
Up to the present, hepatitis B immune globulin has been used to prevent that HBV from infecting, and it can be protected, for example, the positive mother's of HBV newborn infant is exposed to the medical personnel of HBV and the liver transplantation patient (Beasley etc. of the hepatopathy relevant with Chronic HBV, Lancet, 2:1099,1983; Todo etc., Hepatology, 13:619,1991).But hepatitis B immune globulin has some shortcomings, for example, is difficult to obtain, and low ratio is lived and may infected former pollution.Moreover another shortcoming of hepatitis B immune globulin is to continue to provide blood plasma.
As a kind of alternative of hepatitis B immune globulin, developed a kind of mouse monoclonal antibody at the HBV surface antigen.Though but mouse monoclonal antibody demonstrates high antigen affinity and mass preparation, they induce human antimouse antibody response (Shawler etc., Journal of Immunology, 135:1530,1985) in patient's body.The trial of existing preparation human monoclonal antibodies, but the seldom part in these antibody demonstrates high affinity.
Yet people have carried out the exploitation of humanized antibody.Humanized antibody has the high affinity and the specificity of the murine antibody of being similar to, and its immunogenicity is greatly diminished.Humanized antibody is a kind of hybrid antibody, and wherein, the CDR of murine antibody (complementarity-determining region territory) is transplanted in people's antibody by gene engineering.It can prepare on a large scale, and seldom causes immune response in the people, because most of dna sequence dna of coding humanized antibody is from human DNA sequence (Riechman etc., nature (Nature), 332:323,1988); Nakatani etc., protein engineering (Protein Engineering), 7:435,1994).
In order to overcome the above-mentioned of mouse or people HBV immunoglobulin (Ig) and other shortcoming, the present inventor once attempted preparing and can be used for preventing that HBV from infecting and the humanized antibody of treatment chronic hepatitis B.Before the present invention, we have prepared the mouse monoclonal antibody KR127 at HBV surface antigen pre-S1.In addition, we have separated the gene of the heavy chain of encoded K R127 antibody and variable region of light chain and have measured the sequence (korean patent application 1997-30696) of these genes.The present invention is by the human immunoglobulin gene of screening with the sequence homology of KR127 light chain of antibody and variable region of heavy chain; Make up the gene of humanized antibody; These genes are inserted expression vector; Carrier is imported host cell; From the culture of cell transformed, obtain humanized antibody; Confirm that this humanized antibody has high affinity to HV pre-S1 antigen, is similar to mouse monoclonal antibody KR127.
An object of the present invention is to provide humanized antibody, it has specificity to the CDR of mouse HBV surface antigen pre-S1, in the people antigen is had high affinity and low immunogenicity.
The present invention has reached above-mentioned purpose.
The invention provides HBV surface antigen pre-S1 is had specific humanized antibody, comprise humanized heavy chain and light chain.
The present invention also provides the gene of the variable region of encode said humanized heavy chain or light chain.
In addition, the invention provides intestinal bacteria (E.Coli) transformant that contains said expression carrier and contain said expression vector.
The present invention also provides the pharmaceutical composition that contains said humanized antibody, and it can be used for preventing that HBV from infecting or the treatment chronic hepatitis B.
Other characteristics of the present invention will be described hereinafter.
Brief Description Of Drawings:
Fig. 1 a and Fig. 1 b describe V in mouse monoclonal antibody KR127 and two humanized antibodies of the present invention comparatively
HThe amino acid and the nucleotide sequence in zone (Variable Area of heavy chain);
Fig. 2 a describes the process of HKR127HC (I) gene of preparation coding humanized antibody heavy chain of the present invention;
Fig. 2 b describes the process of HKR127HC (III) gene of preparation coding humanized antibody heavy chain of the present invention;
Fig. 3 a and Fig. 3 b describe V in mouse monoclonal antibody KR127 and the humanized antibody of the present invention comparatively
LThe amino acid and the nucleotide sequence in zone (Variable Area of light chain);
Fig. 4 describes the process of HKR127KC (I) gene of preparation coding humanized antibody of the present invention;
Fig. 5 a describes the expression carrier pCMV-HKR127HC of the heavy chain that contains humanized antibody;
Fig. 5 b describes the expression carrier pKC-dhfr-HKR127 of the light chain that contains humanized antibody;
Fig. 5 c describes the expression carrier pCMV-HKR127HC (III) of the heavy chain that contains humanized antibody;
Fig. 6 a shows the binding affinity of humanized antibody (HZKR127 I) and mouse monoclonal antibody (KR127) comparatively; With
Fig. 6 b shows the binding affinity of humanized antibody (HZKR127 III) and humanized antibody (HZKR127 I) comparatively.
Hereinafter will describe the present invention in detail.
The invention provides HBsAg pre-S1 is had specific humanized antibody, Comprise humanized heavy chain and light chain.
The present invention also provides the gene of the variable region of encode said humanized heavy chain or light chain.
Said humanized heavy chain contains a kind of Variable Area, and it is from the V of mouse KR127 antibodyHThe zone. The V of mouse KR127 antibodyHRegion description is in SEQ ID NO:19, the V of humanized antibody of the present inventionHThe zone can be passed through mouse KR127 VHThe CDR in zone is transplanted to the human immunoglobulin(HIg) V of homologyHThe zone prepares.
And said humanization light chain contains a kind of Variable Area, and it is from the V of mouse KR127 antibodyLThe zone. The V of mouse KR127 antibodyLRegion description is in SEQ ID NO:22, the V of humanized antibody of the present inventionLThe zone can be passed through mouse KR127 VLThe CDR in zone is transplanted to the human immunoglobulin(HIg) V of homologyLThe zone prepares.
In preferred embodiments, our examination with the heavy chain of mouse monoclonal antibody KR127 Or the amino acid sequence of light chain demonstrates the human immunoglobulin(HIg) of the highest similitude. As a result, from Examination has gone out human immunoglobulin(HIg) bacterium strain (germline) gene DP7 in the GenBank database And DPK12. DP7 demonstrates the V with mouse-anti body KR127HThe zone has the highest homology, and the V of DPK12 and KR127LThe zone is the most similar.
Humanized antibody of the present invention can be from the humanized V that encodesHZone or VLThe restructuring base in zone Because of preparation. These genes are by CDR employment DP7 or DPK12 antibody with mouse KR127 CDR replace making up. In making up these genes, big corresponding to humanized CDR Partial amino-acid series is from the CDR of mouse-anti body KR127. But some are from the CDR of mouse Residue is replaced by the homologue among the people, because their corresponding amino acid residue is not considered to participate in Antigen is in conjunction with (seeing Fig. 1). In the same way, the non-CDR frame area (FR) of variable region In some amino acid residues that derive from the people replaced by the homologue of mouse because it is believed that these FR Residue can affect the configuration of CDR.
Specifically, coding humanization VHThe HKR127HCv (HZ II) in zone is by with mouse The part CDR1 of KR127 heavy chain, 2,3 and FR residues (site 72) are transplanted to people DP7 (the seeing Fig. 1) for preparing on the gene.
But, corresponding antigen is not shown significant binding ability from the antibody of HKR127HCv (HZ II) genetic expression.In order to improve HKR127HCv (HZ II) gene, we have also prepared HKR127HCv (HZ I) gene and HKR127HCv (HZ III) gene, and they contain more codon (see figure 1) from mouse than HKR127HCv (HZ II) gene.
HKR127HCv (HZ I) contains CDR1, portion C DR2, and CDR3 and mouse KR127V
H11 FR residues, and HKR127HCv (HZ III) contains identical mouse CDR codon and 2 mouse FR residue (see figure 1)s.
In order to make up HKR127HC (I) gene of total length heavy chain of coding humanized antibody of the present invention, carry out the PCR reaction, wherein, the template that is used to increase is HKR127HCv (HZ II) gene or pRC/CMV-HC-HuS (KCTC0229BP), and it contains heavy chain homing sequence and the constant region sequence of human immunoglobulin heavy chain γ 1.
Six couples of oligonucleotide (SEQ ID NO:1 and 2; 3 and 4; 5 and 6; 7 and 8; 9 and 10; 11 and 12) be used as the PCR primer and (see Fig. 2 a).
Five initial PCR products are carried out annealing reaction.Then, the dna fragmentation that contains these five PCR products is used as the template of recombinant PCR, wherein uses two primers that are described in SEQ IDNO:1 and 10.Carry out another recombinant PCR, be connected with the dna fragmentation that obtains by the PCR that uses two primers (SEQ ID NO:11 and 12) with the dna fragmentation of 431 base pairs that will increase.This recombinant PCR uses two primers that are described in SEQ ID NO:1 and 12.To the encode PCR product of 1431 final base pairs of humanized antibody (HZKR127 I) heavy chain, HKR127HC (I) imports pBluescript SK (+) carrier (Clontech), with the plasmid vector called after pHKR127HC (I) that produces.
Primer is described in the SEQ ID NO:1 to 12 in the sequence table, and specifically, the primer of being described by SEQ IDNO:1 contains EcoR I sequence at 5 ' end, and contains Sal I sequence by the primer that SEQ ID NO:12 describes at 3 ' end.
Variable Area in HKR127HC (I) gene contains 11 FR residues from mouse, and its site is 12,28,30,48,67,68,70,72,74,79 and 95 (see figure 1)s.Variable region of heavy chain has 87 FR residues, and the residue that does not have to change is 76.Thereby the aminoacid sequence of the variable region of heavy chain FR of HKR127HC (I) gene has 87% homology with the aminoacid sequence of the variable region of heavy chain FR of the mankind's DP7 gene.
In order further to make HZKR127 (I) humanization, made up HZKR127 (III) gene, it contains HKR127HCv (HZ III) gene, its 72 and 74 the FR residue (see figure 1)s with 2 from mouse.
In order to make up HKR127HC (III) gene of total length heavy chain of coding humanized antibody of the present invention, carry out the PCR reaction, wherein, the template that is used to increase is HKR127HCv (HZ II) gene or pRC/CMV-HC-HuS (KCTC0229BP), and it contains heavy chain homing sequence and the constant region sequence (seeing Fig. 2 b) of human immunoglobulin heavy chain γ 1.
Four couples of oligonucleotide (SEQID NO:1 and 24; 25 and 26; 27 and 28; 11 and 12) be used as PCR primer (seeing Fig. 2 b).Three initial PCR products are carried out annealing reaction.Then, the dna fragmentation that contains these three PCR products is used as the template of recombinant PCR, wherein uses two primers that are described in SEQ ID NO:1 and 28.Carry out another recombinant PCR, be connected with the dna fragmentation that obtains by the PCR that uses two primers (SEQ ID NO:11 and 12) with the dna fragmentation of 431 base pairs that will increase.This recombinant PCR uses two primers that are described in SEQID NO:1 and 12.
To the encode PCR product of 1431 final base pairs of humanized antibody (HZKR127 III) heavy chain, HKR127HC (III) imports pBluescript SK (+) carrier (Clontech), with the plasmid vector called after pHKR127HC (III) that produces.
In another embodiment, coding humanization V
LThe HKR127KCv (HZ II) in zone is by the portion C DR1 with mouse KR127 light chain, and CDR3 and portion C DR2 are transplanted to the (see figure 3) for preparing on the people DPK12 gene.
But, corresponding antigen is not shown significant binding ability from the antibody of HKR127KCv (HZ II) genetic expression.In order to improve the binding ability of HKR127KCv (HZ II), we have also prepared HKR127KCv (HZ I) gene, and it contains more codon (see figure 3) from mouse than HKR127KCv (HZ II) gene.
In order to make up HKR127KC (I) gene of full-length light chains of coding humanized antibody of the present invention, carry out the PCR reaction, wherein, the template that is used to increase is HKR127KCv (HZ II) gene or pKC-dfhr-HuS (KCTC0230BP), and it contains light chain homing sequence and the constant region sequence of human immunoglobulin light chain κ.
Three couples of oligonucleotide (SEQ ID NO:13 and 14; 15 and 16; 17 and 18) be used as PCR primer (see figure 4).
Two initial PCR products are carried out annealing reaction.Then, the dna fragmentation that contains these two PCR products is used as the template of recombinant PCR, wherein uses two primers that are described in SEQ IDNO:13 and 16.Carry out another recombinant PCR, be connected with the dna fragmentation that obtains by the PCR that uses two primers (SEQ ID NO:17 and 18) with the dna fragmentation of 360 base pairs that will increase.This recombinant PCR uses two primers that are described in SEQ ID NO:13 and 18.To the encode PCR product of 739 final base pairs of humanized antibody (HZKR127 I) light chain, HKR127KC (I) imports pBluescript SK (+) carrier (Clontech), with the plasmid vector called after pHKR127KC (I) that produces.
Primer is described in the SEQ ID NO:13 to 18 in the sequence table, and specifically, the primer of being described by SEQIDNO:13 contains Hind III sequence at 5 ' end, and contains Sal I sequence by the primer that SEQ ID NO:18 describes at 3 ' end.
The Variable Area FR of HKR127KC gene contains 5 codon (see figure 3)s from mouse KR127.This light chain has 83 FR residues, and the FR residue that does not have to change is 78.Thereby the aminoacid sequence of the variable region of light chain FR of HKR127KC gene has 94% homology with the aminoacid sequence of the variable region of light chain FR of the mankind's DP7 gene.
In addition, the invention provides contain the coding humanized heavy chain (V
H) or light chain (V
L) the expression carrier of variable region and intestinal bacteria (E.Coli) transformant that contains said expression vector.
In other embodiment preferred, prepared expression vector, it contains the encode heavy chain of humanized antibody or the gene of light chain (seeing Fig. 5 a, 5b or 5c).
Specifically, from plasmid pHKR127HC (I) and pHKR127HC (III) by obtain two kinds of dna fragmentations of corresponding and humanized heavy chain respectively with restriction enzyme treatment, be inserted into pRc/CMV (Invitrogen) then, obtain expression vector pCMV-HKR127HC respectively and (see that Fig. 5 a) and pCMV-HKR127 (III) HC (seeing Fig. 5 c).
In addition, the dna fragmentation from pHKR127KC carrier separation coding humanization light chain imports pCMV-dfhr (KCTC8671P) construction of expression vector pKC-dhfr-HKR127 (seeing Fig. 5 b) with it then.
With coli strain DH5 α expression vector pCMV-HKR127HC, pCMV-HKR127 (III) HC or pKC-dhfr-HKR127 transform.The intestinal bacteria transformant that contains pCMV-HKR127HC and pKC-dhfr-HKR127 that obtains is deposited in KCTC (Korea S typical case's culture collection center) (preserving number is respectively: KCTC0531BP and KCTC0529BP), and preservation day is on October 12nd, 1998.The intestinal bacteria transformant that contains pCMV-HKR127 (III) HC is deposited in KCTC (preserving number: KCTC0691BP) on November 15th, 1999.
In another preferred embodiment, HBV surface antigen pre-S1 is had specific humanized antibody in zooblast, express, and from the substratum of this cell, obtain.The COS7 cell with expression vector pCMV-HKR127HC and pKC-dhfr-HKR127 transient cotransfection, and is cultivated the cells transfected that obtains and culture supernatants is used to identify humanized antibody HZKR127 I of the present invention.Also use expression vector pCMV-HKR127 (III) HC and pKC-dhfr-HKR127 cotransfection COS7 cell, and the culture supernatant of cells transfected is used to identify humanized antibody HZKR127 III.
The present invention also provides the pharmaceutical composition that contains said humanized antibody.
According to other embodiment preferred, verified, HZKR127 I of the present invention and HZKR127 III humanized antibody be when with mouse monoclonal antibody KR127 relatively the time, demonstrates much at one antigen binding affinity and (see Table 1,2 and Fig. 6 a, 6b).
Said composition comprises the humanized antibody at HBV antigen pre-S1 for the treatment of significant quantity, contains/do not contain pharmaceutically acceptable delivery carrier.And said composition can comprise other Antihepatitis medicament, for example anti--S monoclonal antibody or lamivudin.
Humanized antibody at HBV antigen pre-S1 can be prepared with pharmaceutical carrier or thinner, is used for intravenously, and subcutaneous, intramuscular is used.This pharmaceutical composition can use solid or the liquid support that is suitable for required form of medication in a conventional manner, thinner and additive preparation.
The dosage range of humanized antibody administration of the present invention is about 1-10mg/kg, preferred 3-5 mg/kg, and can be administered once weekly.
Embodiment
The preferred embodiments of the invention are described in the following embodiments.
But those of ordinary skill in the art it will be appreciated that, does not depart from the scope of the present invention according to openly can the carrying out a lot of changes and improvements of this paper and spirit.Embodiment 1: the preparation of the gene of coding humanization heavy chain
In order to make up the humanization heavy chain variable region gene, at first, we select the human immunoglobulin heavy chain's gene that has the highest amino acid identity with the variable region of heavy chain of mouse monoclonal antibody KR127.As a result, from the GenBank database, selected (germline) gene DP7 of human normal immunoglobulin bacterium system.Then, we have made up humanized V by the DNA recombinant technology
HRegional gene HKR127HCv (HZ II), it is based on mouse KR127 V
HZone and people DP7 V
HThe comparison in zone.Because humanized heavy chain does not show significant antigen-binding activity, we have prepared another V of coding
HHKR127HCv (HZ I) gene in zone is to improve HKR127HCv (HZ II) gene (see figure 1).
Specifically, HKR127HCv (HZ II) gene is by the V with people DP7 gene
HZone and mouse KR127 V
HThe portion C DR1 in zone, 2,3 and FR residues (site 72) transplant preparation.It is believed that people CDR and FR amino-acid residue influence the antigen binding affinity of antibody.
Thereby, make up HKR127HCv (HZ I) gene by using HKR127HCv (HZ II) gene as the PCR of template.
On the other hand, carrier pRc/CMV-HC-Hus (preserving number: KCTC0229BP) be used to composite coding people C
HThe dna sequence dna of zone and heavy chain homing sequence, it is essential to the suitable secretion of heavy chain.
At last, by with the heavy chain homing sequence, HKR127HCv (HZ I) gene and people C
HGene carries out the annealed recombinant PCR and makes up the HKR127HC (I) of coding humanization heavy chain (Fig. 2 a).
Primer in these PCR is the synthetic oligonucleotide, is described in SEQ ID NO:1 to 12.The Taq archaeal dna polymerase is used in PCR reaction, and its thermal cycling repeats 30 times, by 94 ℃ 1 minute, 55 ℃ of 1 minute and 72 ℃ of 1 minute compositions.Five pairs of oligonucleotide (SEQ ID NO:1 and 2,3 and 4,5 and 6,7 and 8,9 and 10) are used as the PCR primer, and five PCR products (being respectively 113 base pairs, 96 base pairs, 120 base pairs, 78 base pairs and 87 base pairs) are annealed.Then, the dna fragmentation that contains five PCR products is used as the template of recombinant PCR, wherein, has used the primer that is described in SEQ ID NO:1 and 10.Carry out another recombinant PCR, be connected with the dna fragmentation of 1015 base pairs that obtain by the PCR that uses two primers (SEQ ID NO:11 and 12) with the dna fragmentation of 431 base pairs that will increase.This recombinant PCR uses two primers that are described in SEQ ID NO:1 and 12.
The final PCR product (HKR127HC (I) of reorganization heavy chain with the coding humanized antibody, about 1431 base pairs) the EcoR I-Sal I site of importing pBluescript SK (+) carrier (Clontech) is with the plasmid vector called after pHKR127HC (I) that produces.The dna sequence dna of the gene that inserts is measured by the dideoxy nucleotide method.
In order further to make HKR127HC (I) humanization, made up another humanized heavy chain gene, HKR127 (III), it contains the mouse FR residue of lesser amt.
In order to make up HKR127HC (III), make up HKR127HCv (HZ III) as the PCR of template by using HKR127HCv (HZ II) gene.On the other hand, with carrier pRc/CMV-HC-HuS (preserving number: KCTC0229BP) be used for composite coding people C
HThe dna sequence dna of zone and weight homing sequence, it is essential to the suitable secretion of heavy chain.
At last, by with the heavy chain homing sequence, HKR127HCv (HZ III) gene and people C
HGene carries out the HKR127HC (III) (Fig. 2 b) that the annealed recombinant PCR makes up coding humanization heavy chain.
Primer in these PCR is the synthetic oligonucleotide, is described in SEQ ID NO:24 to 28.The Taq archaeal dna polymerase is used in PCR reaction, and its thermal cycling repeats 30 times, by 94 ℃ 1 minute, 55 ℃ of 1 minute and 72 ℃ of 1 minute compositions.Three pairs of oligonucleotide (SEQ ID NO:1 and 24,25 and 26,27 and 28) are used as the PCR primer, and three PCR products (being respectively 179 base pairs, 141 base pairs and 87 base pairs) are annealed.Then, the dna fragmentation that contains three PCR products is used as the template of recombinant PCR, wherein, has used the primer that is described in SEQ ID NO:1 and 28.Carry out another recombinant PCR, be connected with the dna fragmentation of 1015 base pairs that obtain by the PCR that uses two primers (SEQ ID NO:11 and 12) with the dna fragmentation of 431 base pairs that will increase.This recombinant PCR uses two primers that are described in SEQ ID NO:1 and 12.
The final PCR product (HKR127HC (III) of reorganization heavy chain with the coding humanized antibody, about 1431 base pairs) the EcoR I-Sal I site of importing pBluescript SK (+) carrier (Clontech) is with the plasmid vector called after pHKR127HC (III) that produces.The dna sequence dna of the gene that inserts is measured by the dideoxy nucleotide method.Embodiment 2: the preparation of the gene of coding humanization light chain
In order to make up the humanization light chain that contains the variable region, we have designed the gene of coding light chain.At first, we select the people κ immunoglobulin gene that has the highest amino acid sequence homology with the light chain of mouse monoclonal antibody KR127.As a result, from the GenBank database, selected people κ immunoglobulin gene DPK12.Then, we pass through mouse KR127 V
LThe CDR1 in zone, portion C DR2 and CDR3 and one in the site 41 FR residue be transplanted to people DPK12V
LThe zone makes up coding humanization V
LHKR127KCv (HZ II) gene in zone.The humanization VL that obtains does not have the antigen combined function.In order to improve HKR127KCv (HZ II) gene, we have made up another V of coding
LHKR127KCv (HZ I) the gene (see figure 3) in zone.
By V with people DPK12 antibody
LZone and mouse KR127 V
LSeveral FR residues and CDR1, CDR2 and CDR3 transplant and make up HKR127KCv (HZ I) gene.
On the other hand, carrier pKC-dhfr-Hus (preserving number: KCTC0230BP) be used to composite coding people C
LThe dna sequence dna of zone and light chain homing sequence, it is essential to the suitable secretion of light chain.
At last, by with the PCR product, light chain homing sequence, HKR127KCv (HZ I) gene and people C
LThe HKR127KC (I) that gene carries out annealed recombinant PCR preparation coding humanization light chain (Fig. 4).
Primer in these PCR is the synthetic oligonucleotide, is described in SEQ ID NO:13 to 18.The Taq archaeal dna polymerase is used in PCR reaction, and its thermal cycling repeats 30 times, by 94 ℃ 1 minute, 55 ℃ of 1 minute and 72 ℃ of 1 minute compositions.Two pairs of oligonucleotide (SEQ ID NO:13 and 14 and 15 and 16) are used as the PCR primer, and two PCR products (being respectively 101 base pairs and 159 base pairs) are annealed.Then, the dna fragmentation that contains these two PCR products is used as the template of recombinant PCR, wherein, has used the primer that is described in SEQ ID NO:13 and 16.Carry out another recombinant PCR, be connected with the dna fragmentation of 515 base pairs that obtain by the PCR that uses two primers (SEQIDNO:17 and 18) with the dna fragmentation of 248 base pairs that will increase.This recombinant PCR uses two primers that are described in SEQID NO:13 and 18.
The final PCR product (HKR127KC (I) of reorganization light chain with the coding humanized antibody, 736 base pairs) the Hind III-Sal I site of importing pBluescript SK (+) carrier (Clontech) is with the plasmid vector called after pHKR127KC (I) that produces.The dna sequence dna of the gene that inserts is measured by the dideoxy nucleotide method.Embodiment 3: the structure that contains the expression vector of humanization heavy chain gene
With pHKR127HC (I) or the Sal I enzymic digestion of pHKR127HC (III) plasmid of embodiment 1, use Klenow enzyme is handled two terminal flush endizations with carrier.With this dna fragmentation with further digestion obtain the encoding gene of humanization heavy chain of Not I enzyme.
On the other hand, pRc/CMV (Invitrogen) is cut with Xba I enzyme, and the end of carrier undertaken flush endization by handling with the Klenow enzyme, then with the digestion of Not I.
The humanization heavy chain gene is connected with linearizing carrier obtains expression vector pCMV-HKR127HC or pCMV-HKR127 (III) HC.The intestinal bacteria transformant that will contain pCMV-HKR127HC or pCMV-HKR127 (III) HC is deposited in KCTC (Korea S typical case's culture collection center) (preserving number is respectively: KCTC0531BP and KCTC0691BP), and expression vector pCMV-HKR127HC and pCMV-HKR127 (III) HC is shown in Fig. 5 a and 5c respectively.Embodiment 4: the structure that contains the expression vector of humanization light chain gene
With pHKR127KC carrier Hind III and the enzymic digestion of Apa I of embodiment 2, and the fragment that obtains is inserted pCMV-dhfr, and (preserving number: Hind III KCTC8671P)-Apa I site obtains expression vector pKC-dhfr-HKR127.The intestinal bacteria transformant that will contain pKC-dhfr-HKR127 is deposited in KCTC (Korea S typical case's culture collection center), and (preserving number is: KCTC0529BP), expression vector pKC-dhfr-HKR127 is shown in Fig. 5 b.Embodiment 5: the expression of humanized antibody in the COS7 cell
The COS7 cell is being maintained among the DMEM (Gibco) that has added 10% calf serum at 37 ℃ under 5% carbon dioxide conditions.These cells are seeded in the culture dish of 100mm, bathe 37 ℃ of temperature of spending the night then.
In order to express humanized antibody HZKR127 I, the pCMV-HKR127HC or the pKC-dhfr-HKR127 of 5 micrograms are diluted with the OPTI MEM I (Gibco) of 800 microlitres, and the Lipofectamin (Gibco) of 50 microlitres is also diluted with the OPTI MEM I of 800 microlitres.With these mixtures in 15 milliliters of test tubes at room temperature temperature bathe 15 minutes or longer time.Simultaneously, the COS7 cell is used OPTI MEM I washed twice.
OPTI MEM I (6.4 milliliters) is added in the DNA-Lipofectamin mixture, and thorough mixing is poured on the COS7 cell.Cell is cultivated 72 hours in CO2gas incubator after, substratum is centrifugal, and supernatant liquor concentrated by ultra-filtration equipment.Use anti-human IgG and anti-human IgG-HRP (horseradish peroxidase) association to measure the concentration of antibody by SandwichELISA.
In order to express and to obtain humanized antibody HZKR127 III, repeat identical method, only be to use pCMV-HKR127 (III) HC, rather than use pCMV-HKR127HC.Embodiment 6: humanized antibody is active to the combination of HBV surface antigen pre-S1
We have prepared HBV surface antigen pre-S1 (amino-acid residue 1-56; Kim and Hong, Biotechnology letters, 17:871-876,1995), and the pre-S1 of the purifying of 1 microgram is coated in each hole of droplet plate.Adding 0,0.25,0.5,1,2,3,4,5,7.5,10,20 of embodiment 5 preparations, or behind the humanized antibody of 40ng, carrying out indirect ELISA, wherein second antibody is the specific anti-human IgG of Fc--HRP association.The combination activity of antibody is to measure by the OD value of measuring 492nm.
With comparing, and use the anti-mouse IgG-HRP of Fc-specificity association to carry out the ELISA of KR127 antibody as second antibody the mouse KR127 antibody of purifying.The results are shown in table 1 and 2.Table 1KR127 and HZKR127 I are to the combination active (in the OD of 492nm value) of HBV surface antigen pre-S1
Continuous table 1
Table 2HZKR127 I and HZKR127 III are to the combination active (in the OD of 492nm value) of HBV surface antigen pre-S1
Continuous table 2
Embodiment 7: humanized antibody is to the antigen binding affinity of the anti-pre-S1 in HBV surface
Antigen binding affinity to the anti-pre-S1 in HBV surface is measured (Ryu etc., J.Med.Virol., 52:226,1997) by the competitive ELISA method.
Pre-S1 antigen (1 * 10
-7-1 * 10
-12M) and between the humanized antibody of embodiment 5 (5ng), perhaps antigen (1 * 10
-7-1 * 10
-12M) and the binding reactive between the control antibodies KR127 (5ng) be to carry out 2 hours at 37 ℃.Then reaction mixture is added on the 96 hole droplet plates of 96 holes with the antigen pre-S1 bag quilt of 250ng.
Fig. 6 a shows the affinity of two kinds of antibody.Verified, the binding affinity of humanized antibody HZKR127 I almost identical (7 * 10 with the binding affinity of murine antibody KR127
7M
-1).
The comparison of the affinity of Fig. 6 b demonstration HZKR127 III and the affinity of HZKR127 I.The affinity (5 * 10 of HZKR127 III
7M
-1) with the difference little (7 * 10 of the affinity of HZKR127 I
7M
-1).Industrial applicibility
As indicated above, the invention provides humanized antibody at HBV surface antigen pre-S1, compare it with mouse monoclonal antibody and demonstrate similar binding affinity, and the immunogenicity of humanized antibody reduces greatly.Thereby humanized antibody of the present invention can be used for preventing that HBV from infecting and the treatment hepatitis B.
Those of ordinary skill in the art it will be appreciated that notion disclosed herein and specific embodiment can be used as further improvement or design the basis of other embodiment, to reach the purpose identical with the present invention.Those of ordinary skill in the art it will be appreciated that these equivalent embodiments do not break away from the spirit and scope of the present invention described in the accompanying Claim.
Sequence table＜110〉Korean Institute of Science and Technology
＜120〉 HBVPRE-S1＜130〉 9FPO-11-03＜150〉 KR1998-49663＜151〉 1998-11-19＜160〉 28＜170〉 KOPATIN1.5＜210〉 1＜211〉 26＜212〉 DNA＜213〉 ＜220〉＜223〉 1＜400〉 1gaattcac attcacgatg tacttg 26＜210〉 2＜211〉 27＜212〉 DNA＜213〉 ＜220〉＜223〉 2＜400〉 2ggccccaggc ttcaccactt cagctcc 27＜210〉 3＜211〉 18＜212〉 DNA＜213〉 ＜220〉＜223〉 3＜400〉 3gtgaagcctg gggcctca 18＜210〉 4＜211〉 27＜212〉 DNA＜213〉 ＜220〉＜223〉 4＜400〉 4agaactactg aatgcgtagc cagaagc 27＜210〉 5＜211〉 27＜212〉 DNA＜213〉 ＜220〉＜223〉 5＜400〉 5gcattcagta gttcttggat gaactgg 27＜210〉 6＜211〉 27＜212〉 DNA＜213〉 ＜220〉＜223〉 6＜400〉 6aatccgtcca accactcaa gaccctg 27＜210〉 7＜211〉 21＜212〉 DNA＜213〉 ＜220〉＜223〉 7＜400〉 7tggattggac ggatttatcc t 21＜210〉 8＜211〉 39＜212〉 DNA＜213〉 ＜220〉＜223〉 8＜400〉 8ggattgtct gcagtcagtg tggccttgcc ctggaactt 39＜210〉 9＜211〉 39＜212〉 DNA＜213〉 ＜220〉＜223〉 9＜400〉 9actgcagaca aatccacgag cacagcctac atggagctc 39＜210〉 10＜211〉 33＜212〉 DNA＜213〉 ＜220〉＜223〉 10＜400〉 10gtcgtactct cttgcacaga aatagaccgc cgt 33＜210〉 11＜211〉 33＜212〉 DNA＜213〉 ＜220〉＜223〉 11＜400〉 11gcaagagagt acgacgaggc ttactggggc caa 33＜210〉 12＜211〉 26＜212〉 DNA＜213〉 ＜220〉＜223〉 12＜400〉 12cggtcgactc attacccgg agacag 26＜210〉 13＜211〉 26＜212〉 DNA＜213〉 ＜220〉＜223〉 13＜400〉 13caaagcttgg aagcaagatg gattca 26＜210〉 14＜211〉 36＜212〉 DNA＜213〉 ＜220〉＜223〉 14＜400〉 14tggagtttgg gtcatcaaga tatccccaca ggtacc 36＜210〉 15＜211〉 48＜212〉 DNA＜213〉 ＜220〉＜223〉 15＜400〉 15atgacccaaa ctccactttc tttgtcggtt acccctggac aaccagcc 48＜210〉 16＜211〉 39＜212〉 DNA＜213〉 ＜220〉＜223〉 16＜400〉 16caccagatag attaggcgct ttggagactg gcctggctt 39＜210〉 17＜211〉 39＜212〉 DNA＜213〉 ＜220〉＜223〉 17＜400〉 17ctaatctatc tggtgtctaa actggactct ggagtccct 39＜210〉 18＜211〉 17＜212〉 DNA＜213〉 ＜220〉＜223〉 18＜400〉 18gaagtcgacc taacact 17＜210〉 19＜211〉 115＜212〉 PRT＜213〉 ＜220〉＜223〉 KR127＜400〉 19Gln Val Gln Leu Gln Gln Ser Gly Pro Glu Leu Val Lys Pro Gly Ala 1 5 10 15Ser Val Lys Ile Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser
20 25 30Trp?Met?Asn?Trp?Val?Lys?Gln?Arg?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Ile
35 40 45Gly?Arg?Ile?Tyr?Pro?Gly?Asp?Gly?Asp?Thr?Asn?Tyr?Asn?Gly?Lys?Phe
50 55 60Lys?Gly?Lys?Ala?Thr?Leu?Thr?Ala?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr?65 70 75 80Met?Gln?Leu?Ser?Ser?Leu?Thr?Ser?Val?Asp?Ser?Ala?Val?Tyr?Phe?Cys
85 90 95Ala?Arg?Glu?Tyr?Asp?Glu?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr
100 105 110Val?Ser?Ala
115＜210〉variable region of heavy chain HKR127HC (HZ I)＜400〉20Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Val Lys Pro Gly Ala, 15 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Ser Ser 20＜211〉115＜212〉PRT＜213〉artificial sequence＜220〉＜223〉humanization
20 25 30Trp?Met?Asn?Trp?Val?Arg?Gln?Ala?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Ile
35 40 45Gly?Arg?Ile?Tyr?Pro?Gly?Asp?Gly?Asp?Thr?Asn?Tyr?Ala?Gln?Lys?Phe
50 55 60Gln?Gly?Lys?Ala?Thr?Leu?Thr?Ala?Asp?Lys?Ser?Thr?Ser?Thr?Ala?Tyr?65 70 75 80Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Phe?Cys
85 90 95Ala?Arg?Glu?Tyr?Asp?Glu?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr
100 105 110Val?Ser?Ser
115＜210〉variable region of heavy chain HKRR127HC (HZ III)＜400〉21Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala, 15 10 15Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Ser 21＜211〉115＜212〉PRT＜213〉artificial sequence＜220〉＜223〉humanization
20 25 30Trp?Met?Asn?Trp?Val?Arg?Gln?Ala?Pro?Gly?Gln?Gly?Leu?Glu?Trp?Met
35 40 45Gly?Arg?Ile?Tyr?Pro?Gly?Asp?Gly?Asp?Thr?Asn?Tyr?Ala?Gln?Lys?Phe
50 55 60Gln?Gly?Arg?Val?Thr?Met?Thr?Ala?Asp?Lys?Ser?Thr?Ser?Thr?Val?Tyr?65 70 75 80Met?Glu?Leu?Ser?Ser?Leu?Arg?Ser?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?CysAla?Arg?Glu?Tyr?Asp?Glu?Ala?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr
100 105 110Val?Ser?Ser
115＜210〉variable region of light chain in the KR127 antibody＜400〉22Asp Ile Leu Met Thr Gln Thr Pro Leu Ile Leu Ser Val Thr Ile Gly, 15 10 15Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 22＜211〉113＜212〉PRT＜213〉artificial sequence＜220〉＜223〉mouse
20 25 30Asn?Gly?Lys?Thr?Tyr?Leu?Asn?Trp?Leu?Leu?Gln?Arg?Pro?Gly?Gln?Ser
35 40 45Pro?Lys?Arg?Leu?Ile?Tyr?Leu?Val?Ser?Lys?Leu?Asp?Ser?Gly?Val?Pro
50 55 60Asp?Arg?Phe?Thr?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?65 70 75 80Ile?Arg?Val?Glu?Ala?Glu?Asp?Leu?Gly?Val?Tyr?Tyr?Cys?Val?Gln?Gly
85 90 95Thr?His?Phe?Pro?Gln?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys
100 105 110Arg＜210〉23＜211〉113＜212〉PRT＜213〉artificial sequence＜220〉＜variable region of 223〉humanization light chains＜400〉23Asp Ile Leu Met Thr Gln Thr Pro Leu Ser Leu Ser Val Thr Pro Gly, 15 10 15Gln Pro Ala Ser Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser
20 25 30Asn?Gly?Lys?Thr?Tyr?Leu?Asn?Trp?Leu?Leu?Gln?Lys?Pro?Gly?Gln?Ser
35 40 45Pro?Lys?Arg?Leu?Ile?Tyr?Leu?Val?Ser?Lys?Leu?Asp?Ser?Gly?Val?Pro
50 55 60Asp?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Lys?Ile?65 70 75 80Ser?Arg?Val?Glu?Ala?Glu?Asp?Val?Gly?Val?Tyr?Tyr?Cys?Val?Gln?Gly
85 90 95Thr?His?Phe?Pro?Gln?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys
100 105 110Arg＜210〉 24＜211〉 27＜212〉 DNA＜213〉 ＜220〉＜223〉 24＜400〉 24gttcatccaa gaactggtga aggtgta 27＜210〉 25＜211〉 27＜212〉 DNA＜213〉 ＜220〉＜223〉 25＜400〉 25agttcttgga tgaactgggt gcgacga 27＜210〉 26＜211〉 27＜212〉 DNA＜213〉 ＜220〉＜223〉 26＜400〉 26gctcgtggat ttgtctgcag tcattgt 27＜210〉 27＜211〉 27＜212〉 DNA＜213〉 ＜220〉＜223〉 27＜400〉 27gacaaatcca cgagcacagt ctacatg 27＜210〉 28＜211〉 27＜212〉 DNA＜213〉 ＜220〉＜223〉 28＜400〉 28gtcgtactct ctcgcacagt aatacac 27
Claims (15)
1. one kind has specific humanized antibody to HBV surface antigen pre-S1, contains humanized heavy chain and light chain variable region.
2. according to the described humanized antibody of claim 1, wherein, the aminoacid sequence of being described by SEQ ID NO:20 is contained in this humanization weight chain variable zone.
3. according to the described humanized antibody of claim 1, wherein, the aminoacid sequence of being described by SEQ ID NO:21 is contained in this humanization weight chain variable zone.
4. according to the described humanized antibody of claim 1, wherein, a kind of aminoacid sequence is contained in this humanization weight chain variable zone, and it is to replace by at least one amino acid that is selected from down group from the amino-acid residue of SEQ ID NO:21 to change: Lys
12→ Val
12, Thr
28→ Ala
28, Thr
30→ Ser
30Met
48→ Ile
48, Arg
67→ Lys
67, Val
68→ Ala
68, Met
70→ Leu
70, Val
79→ Ala
79, and Tyr
95→ Phe
95
5. according to the described humanized antibody of claim 1, wherein, this humanization light chain variable region contains the aminoacid sequence of being described by SEQ ID NO:23.
6. gene of humanization heavy chain of encoding, this humanization heavy chain contains the humanization weight chain variable zone of claim 2,3 or 4.
7. according to the described gene of claim 6, wherein, the aminoacid sequence of being described by SEQ ID NO:20 is contained in this humanization weight chain variable zone.
8. according to the described gene of claim 6, wherein, the aminoacid sequence of being described by SEQ ID NO:21 is contained in this humanization weight chain variable zone.
9. a coding contains a kind of gene of humanization light chain of humanization light chain variable region, and this humanization light chain variable region contains the aminoacid sequence that is described in SEQ ID NO:23.
10. the expression carrier that contains claim 6.
11. according to the expression vector of claim 10, pCMV-HKR127HC, wherein, the gene of claim 7 is inserted into pRC/CMV (preserving number: KCTC0531BP).
12. according to the expression vector of claim 10, pCMV-HKR127 (III) HC, wherein, the gene of claim 8 is inserted into pRC/CMV (preserving number: KCTC0691BP).
13. expression carrier that contains claim 9.
14. according to the expression vector of claim 13, pKC-dhfr-HKR127, wherein, the gene of claim 9 is inserted into pCMV-dhfr (preserving number: KCTC0529BP).
15. contain the pharmaceutical composition of the humanized antibody of claim 1, it can be used for preventing that HBV from infecting or the treatment chronic hepatitis B.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1980956B (en) * | 2004-04-14 | 2011-09-28 | 株式会社柳韩洋行 | Humanized antibody against S-surface antigen of hepatitis b virus |
| CN107614525A (en) * | 2015-05-22 | 2018-01-19 | 华辉安健（北京）生物科技有限公司 | S1 HBV antibody before anti- |
-
2000
- 2000-05-17 CN CN 00107535 patent/CN1216914C/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1980956B (en) * | 2004-04-14 | 2011-09-28 | 株式会社柳韩洋行 | Humanized antibody against S-surface antigen of hepatitis b virus |
| CN107614525A (en) * | 2015-05-22 | 2018-01-19 | 华辉安健（北京）生物科技有限公司 | S1 HBV antibody before anti- |
| CN107614525B (en) * | 2015-05-22 | 2021-07-06 | 华辉安健（北京）生物科技有限公司 | Anti pre-S1 HBV antibodies |
| US11485774B2 (en) | 2015-05-22 | 2022-11-01 | Huahui Health Ltd. | Anti-pre-S1 HBV antibodies |
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| Publication number | Publication date |
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