CN104592390A - Bispecific recombinant anti-HBsAg antibody, and preparation method and application thereof - Google Patents

Abstract

The invention belongs to the technical field of biology, and discloses a bispecific antibody specifically bond to different epitopes of hepatitis b surface antigen (HBsAg). The invention provides the amino acid sequence of the antibody, a DNA sequence encoding the antibody, an expression vector containing the DNA sequence, and a host cell containing the expression vector. The invention also discloses a preparation method of the antibody, and application of the antibody to prevent hepatitis B virus infection.

Description

One class two special restructuring AntiHBsAg antibody, Preparation Method And The Use

Technical field

The invention belongs to biological technical field, more specifically, the invention discloses the two special restructuring AntiHBsAg antibody of a class, its preparation method and prevention, treat hepatitis b virus infected in application.

Background technology

Hepatitis B virus (Hepatitis B virus, HBV) is double-stranded DNA virus, causes chronic hepatitis B, and causes the generation of liver failure, liver cirrhosis and liver cancer.The nearly 300,015,000 HBV infection persons in the whole world, and this also becomes world public health problem.Nearest decades, succeeding in developing of Hepatitis B virus vaccine, brings very large hope to the prevention of hepatitis B.But truly have a minority's HB vaccination that immunity system but can not be excited to produce anti-hepatitis b antibody, this part population, once touch the high risk factor of hepatitis B, can only be injected Human Hepatitis B Immune Globulin to prevent.But human normal immunoglobulin composition relative complex, the Hazard Factor of use are relatively uncertain.Therefore Human Hepatitis B Immune Globulin is substituted in the urgent need to developing that a kind of people originates, that Hazard Factor are relatively controlled biological products.

Along with the development of biotechnology, the development of genetic engineering antibody is maked rapid progress.Human monoclonal antibody produces and comprises EBV immortalization bone-marrow-derived lymphocyte, human B lymphocyte hybridoma technology, the humanization of other kind antibody, based on the human antibody technology that phage library is shown, based on human antibody technology and the Single cell RT-PCR monoclonal antibody technology of humanization mouse.Due to EBV immortalization bone-marrow-derived lymphocyte low conversion rate; seriously limit popularization [the Guan Y of this technology; Sajadi MM; Kamin-Lewis R, et al. Discordant memory B cell and circulating anti-Env antibody responses in HIV-1 infection. Proc Natl Acad Sci U S A 2009; 106:3952-7].Human B lymphocyte and myeloma cell fusion efficiency extremely low, cause human B lymphocyte hybridoma technology to be seldom used to prepare human monoclonal antibody.Phage display library human antibody technology is the random pair based on human normal immunoglobulin light chain and heavy chain gene, do not experience somatic mutation and immune pair of Sexual behavior mode, low (the KD > 10 of the general avidity of antibody causing its screening to obtain ^-7); dissociation rate fast (V < 60min); antibody Tm value low (< 65 DEG C); poor heat stability; potential allergy and autoimmunization risk [Mohapatra S, Juan HS. Designer monoclonal antibodies as drugs:the state of the art. Expert Rev Clin Immunol 2008; 4:305-7].Because the patent of the human antibody technology-humanization mouse of most potential quality only rests in minority international pharmacy giant hand, and cost of development is high, seriously constrains the popularization of this technology.

Single cell RT-PCR monoclonal antibody technology is directly separated the B cell containing object antibody from human peripheral, uses single-cell RT-PCR technology Direct Cloning light chain of antibody and heavy chain, carrys out Dispersal risk through vitro recombination.Prepared antibody sources is natural, and Hazard Factor are controlled.Because monoclonal antibody is all clone from the bone-marrow-derived lymphocyte of human peripheral, experienced by two Sexual behavior mode of human immune system, long-term body fluid circulatory and somatic mutation, therefore, demonstrate its fabulous avidity (KD < 10 ^-9); dissociation rate slow (V > 6hours); Heat stability is good; potential allergy and low [the Smith K of autoimmunization risk; Garman L; Wrammert J, et al. Rapid generation of fully human monoclonal antibodies specific to a vaccinating antigen. Nat Protoc 2009; 4:372-84].The technological development of Single cell RT-PCR monoclonal antibody is with low cost, especially more can show its superiority for exotic antigen.

Due to the multifactor property of disease, single target treatment can not meet clinical demand.The report of existing conjunctive use two kinds of monoclonal antibody preclinical laboratory in clinical trial; but this combined utilization monoclonal antibody has much restricted [Kou G; Shi J; Chen L, et al. A bispecific antibody effectively inhibits tumor growth and metastasis by simultaneous blocking vascular endothelial growth factor A and osteopontin. Cancer Lett 2010; 299:130-6].Such as; combined utilization two kinds of monoclonal antibodies security and effect need to go respectively to evaluate; consequent long period; high development cost limit its development process; bi-specific antibody solves this bottleneck [Wu C just; Ying H, Grinnell C, et al. Simultaneous targeting of multiple disease mediators by a dual-variable-domain immunoglobulin. Nat Biotechnol 2007; 25:1290-7].In addition; bispecific antibody can also obtain the characteristic of some novelties not available for its parental monoclonal antibody sometimes: as often having the dissociate speed lower than former parental antibody; [the Lu D such as stronger sealing process; Kotanides H; Jimenez X, et al. Acquired antagonistic activity of a bispecific diabody directed against two different epitopes on vascular endothelial growth factor receptor 2. J Immunol Methods 1999; 230:159-71].

Summary of the invention

For above problem, the present invention's design based on Single Cell RT-PCR monoclonal antibody technological development one strain have fabulous in and the Anti-HBsAg bi-specific antibody of HBV virus.The present invention adopts ultra-high speed streaming separation system from healthy volunteer's peripheral blood of HB vaccination, be separated the special memory B cells (HBsAg of HBsAg ⁺igG ⁺cD19 ⁺), and be prepared into unicellular sample.Employing Single Cell RT-PCR technology clones the gene that the antibody in each unicellular sample is correlated with, and is cloned in Mammals efficient expression vector, the corresponding antibody of in-vitro recombination expression.

The invention discloses:

1. a class bi-specific antibody, it contains two different variable region of heavy chain, variable region of light chain, can respectively in conjunction with the functional epitope that HBsAg is different.

2. the class bi-specific antibody described in, it is characterized in that variable region of heavy chain has the aminoacid sequence shown in SEQ NO:2, variable region of light chain has the aminoacid sequence shown in SEQ NO:4.

3. the nucleic acid molecule be separated, encode such amino acid sequences, has nucleotide sequence shown in nucleotide sequence shown in SEQ NO:1 and SEQ NO:3.

4. a carrier, containing described nucleic acid molecule, with the expression regulation sequence that the sequence being operational of described nucleic acid molecule is connected, wherein carrier can be pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), one of pDHFR.

5. the carrier described in is pcDNA3.1(+).

6. a host cell, containing described carrier, is eukaryotic cell.

7. the host cell described in is mammalian cell.

8. the host cell described in is Freestyle 293F cell.

9. prepare a method for described bi-specific antibody, the method comprises:

A) adopt the memory B cells that in volunteer's peripheral blood of ultra high rate airflow classification systematic position HB vaccination, HBsAg is special, and be prepared into unicellular sample;

B) Single Cell RT-PCR technology is adopted to clone antibody genes involved in each sample, in-vitro recombination expression corresponding antibodies;

C) measure through functional epitope, avidity measure and in and HBV determination of activity, filter out monoclonal antibody C4G4 and the D2H2 of strong, active high two anti-HBsAgs in conjunction with difference in functionality epi-position of avidity;

D) utilize biotechnology to be connected respectively through heavy chain linker, light chain linker in the heavy chain of these two monoclonal antibodies of C4G4, D2H2, variable region of light chain, be prepared into and there is bifunctional antibody;

E) under expression condition, cultivate arbitrary described host cell, express bi-specific antibody C4D2-BsAb, separation and purification.

10. a composition, containing described bi-specific antibody, and pharmaceutically acceptable carrier.

Bi-specific antibody described in 11. is preparing the application in anti HBV infecting.

Composition described in 12. is preparing the application in anti HBV infecting.

13. arbitrary described purposes, also comprise and the Drug combination in other anti HBV infecting.

In the present invention, the Anti-HBsAg monoclonal antibody that successful clone 42 strains are brand-new, adopts the method for ELISA to identify its functional epitope, avidity and in and HBV active.Through a series of test experience, from wherein have selected 2 strains and the active best Anti-HBsAg monoclonal antibody C4G4 and D2H2 of HBV, utilize biotechnology that 2 strain antibodies are prepared into bi-specific antibody C4D2-BsAb.

In HepaRG cell model, C4D2-BsAb demonstrate stronger than C4G4 and D2H2 coupling in and the activity of HBV.In addition, research display FcRn mediates the release suppressing HBsAg and virion in C4D2-BsAb endocytosis to liver cell.

Accompanying drawing explanation

Fig. 1, streaming CD19 ⁺igG ⁺hBsAg ⁺b cell analyzes.

Fig. 2, pcr amplification antibody variable gene figure.

Under Fig. 3, reduced state, SDS-PAGE analyzes recombinant human monoclonal antibody electrophorogram.

Fig. 4, bi-specific antibody structural representation.

Under Fig. 5, reductive condition, SDS-PAGE analyzes the bi-specific antibody electrophorogram of purifying.

The feature of Fig. 6, anti-HBsAg monoclonal antibody, the position of synthetic peptide on HBsAg.

Fig. 7, HepaRG cell is assessed in monoclonal antibody and the activity of HBV.

Fig. 8, monoclonal antibody C4G4 and D2H2 coordinate repression.

Fig. 9, bi-specific antibody C4D2-BsAb and D2C4-BsAb are in conjunction with improvement on synthesis P2, P3 activation analysis.

Figure 10, bi-specific antibody C4D2-BsAb and D2C4-BsAb are in conjunction with HBsAg activation analysis.

In Figure 11, bi-specific antibody C4D2-BsAb and HBV activation analysis.

Embodiment

Below in conjunction with embodiment, experimental example, the present invention will be described further, and these embodiments, experimental example should not be construed as limitation of the present invention.

The preparation of the single B cell of embodiment 1:HBsAg specificity

By being separated the lymphocyte injecting the peripheral blood of Hepatitis B virus vaccine volunteer, be separated HBsAg with CD19-PE/Cy5, IgG-PE, HBsAg-biotin, Streptavidin-FITC fluorescence antibody ⁺cD19 ⁺igG ⁺b cell.As shown in Figure 1, the cell (accounting for whole B cell group 0.01-0.02%) of the visible a small set of HBsAg positive.Each HBsAg of MoFlo XDP hypervelocity fluidic cell separation system sorting ⁺cD19 ⁺igG ⁺b cell injects 96-well Single cell PCR plates, guarantees a cell in each PCR hole.Then add lysate, after liquid nitrogen flash freezer, be stored in-80 DEG C of ice for subsequent use.

Embodiment 2:RT-PCR and Chao Shi PCR clonal antibody variable region gene and genetic expression

Get and freeze 96-well Single cell PCR plates in-80 DEG C of refrigerators, make on ice slowly to melt, add reversed transcriptive enzyme synthesis cDNA.Because the cDNA of individual cells synthesis is rare especially, the method for Chao Shi PCR must be adopted to increase its antibody gene contained.Visible significantly PCR band amplification (size is about 400bp) after two-wheeled PCR.Order-checking after the PCR band recovery that weight chain is paired.As shown in Figure 2, analyze specific PCR amplification antibody variable region band after its sequence, and introduce restriction enzyme site.Expression vector pcDNA3.1 (+) is inserted after amplification antibody variable region PCR primer is connected with antibody constant region.

Embodiment 3: monoclonal antibody gene is expressed

The Freestyle 293F cell that paired light chain of antibody and heavy chain plasmid transient transfection serum free medium are cultivated, transfection is collecting cell supernatant after 7 days, IgG antibody in Protein A purifying cells supernatant.Obtain anti-hbs monoclonal antibodies 42 strain altogether.As shown in Figure 3, the IgG antibody of purifying demonstrates two bands at 10% SDS-PAGE to SDS-PAGE electroresis appraisal, and wherein one is about 50KD (heavy chain), and other one is about 25KD (light chain).

Embodiment 4: anti-hbs monoclonal antibodies HBsAg avidity measures

Get certain density monoclonal antibody to mix with the antigen HBsAg of serial doubling dilution, 4 DEG C are spent the night, thus ensure that reaction reaches balance.Immune complex is transferred to bag by HBsAg enzyme plate, hatches 1 hour for 37 DEG C.The sheep anti-human kappa chain antibody adding HRP mark after washing hatches 1 hour.After washing, TMB colour developing 450nm wavelength measures the specific absorption in each hole, and the avidity of calculating antibody, result is as shown in table 1.

Embodiment 5: antibody epitope is identified

The hepatitis B surface antigen small peptide of synthesizing biotinylated mark measures the calmodulin binding domain CaM of anti-HBsAg monoclonal antibody on HBsAg.As shown in Figure 6, four biotin labeling hepatitis B surface antigen small peptide P1(aa:104-120 of synthesis), P2(aa:121-137), P3(aa:139-148), P4(aa:149-163), and on HBsAg mode chart, mark antigen small peptide P1, the structure in the region corresponding to P2, P3, P4; Wherein antigen small peptide P1 and P4 is linear structure, P2 and P3 is ring texture.[Jin A, Ozawa T, Tajiri K, et al. A rapid and efficient single-cell manipulation method for screening antigen-specific antibody-secreting cells from human peripheral blood. Nat Med 2009;15:1088-92]。

	Amino acid sequence number	Aminoacid sequence	Structure
				P1	104-120	LPVCPLLPGTSTTSTGP	Linear peptides
P2	123-137	T CTIPAQGTSMFPSC	Cyclic peptide
				P3	135-144	CTKPSDGNCT	Cyclic peptide
P4	149-163	CIPIPSSWAFARFLW	Linear peptides

Adopt the epitope of the combination of the methods analyst of ELISA 42 strain Anti-HBsAg monoclonal antibodies.As shown in table 1, what result display was combined in P1 accounts for 7.1% (3/42), what be combined in P2 accounts for 38.1% (16/42), be combined in P3 account for 31.0% (13/42) and be combined in P4 account for 9.5% (4/42), but have the monoclonal antibody of 14.3% (6/42) not to be combined with four synthetic peptides.

Embodiment 6: with the qualification of HBV activity during anti-hbs monoclonal antibodies is external

HepaRG clone is current uniquely by the HBV Infection in Vitro model extensively approved.Therefore, we adopt the ability with HBV infection HepaRG cell in HepaRG raji cell assay Raji 42 strain Anti-HBsAg monoclonal antibody.With Anti-HBsAg monoclonal antibody (1 μ g) and HBV(1 × 10 ⁶copies) preincubate, then be added to DMSO and the differentiation-inducing HepaRG cell of hydrocortisone, measures in it and the ability of HBV activity.Infect the 7th day we determine HBV-DNA copy number in the content of HBsAg in cells and supernatant and cell.As shown in Figure 8 A, infect the 7th day, in most cells cells and supernatant there is obviously declining (* in HBsAg p< 0.05).Equally as shown in Figure 7, infect the 7th day, in most cells cell there is obviously declining (* in HBV-DNA copy number p< 0.05).As shown in table 1, comprehensively multifactorly to carry out in 42 strain Anti-HBsAg monoclonal antibodies and the analysis of HBV infection HepaRG cell ability, good two mono-clonal C4G4 and D2H2 of final Selection effect.

Embodiment 7: with HBV determination of activity during anti-hbs monoclonal antibodies is collaborative

Select to detect its synergistic effect with the best monoclonal antibody (C4G4 and D2H2) of HBV activity in two strains.

HBV virion (1 × 10 ⁶copies) respectively with 1 μ g monoclonal antibody C4G4, D2H2 and Control human IgG, preincubate 1 hour, join (containing 4%PEG8000 in substratum) incubated overnight in differentiation-inducing good HepaRG clone, wash PBS next day tri-times, renew fresh substratum.HBsAg content in the HBV-DNA copy number detected for the 6th day in cell and culture supernatant.HBV-DNA detects and uses HBV-DNA PCR kit for fluorescence quantitative (China of Shanghai section is biological).HBsAg measures and collects culture supernatant, detects with hepatitis B surface antigen quantification kit (Electrochemiluminescince) (Beijing section is U.S. biological).As shown in Figure 8, C4G4 with D2H2 coupling is compared with C4G4 with D2H2 is alone, significantly can reduce (the * of HBV-DNA copy number in the expression of HBsAg in cell conditioned medium and cell p< 0.05).

Embodiment 8: the structure of bi-specific antibody and qualification

Two strain antibody C4G4 and D2H2 best with HBV activity in selection, design bispecific antibody, antibody structure is as Fig. 4.The variable region of heavy chain of a monoclonal antibody and variable region of light chain connect peptide respectively by heavy chain connection peptides and light chain and are fused to the variable region of heavy chain of another monoclonal antibody and 5 ' end of variable region of light chain.It is ASTKGPSVFPLAP that heavy chain connects peptide amino acid sequence, and it is TVAAPSVFIFPP that light chain connects peptide amino acid sequence.Correct bispecific antibody heavy chain, chain variable region gene are cloned in IgG1-AbVec plasmid and Ig κ-AbVec plasmid respectively.Bispecific antibody C4D2-BsAb and D2C4-BsAb is expressed in Freestyle 293F clone.SDS-PAGE electroresis appraisal bi-specific antibody C4D2-BsAb and D2C4-BsAb, result is as Fig. 5.

Experimental example 1: the avidity of bi-specific antibody measures

Wrap by elisa plate with synthetic peptide P2 and P3 respectively, use ELISA method detects the avidity difference between the same parental monoclonal antibody of bi-specific antibody (C4G4 and D2H2) of two kinds of different structures.As Fig. 9 display: when C4G4 variable region is placed on before D2H2 variable region, the C4D2-BsAb of formation all maintains the avidity similar to parental monoclonal antibody (C4G4 with D2H2) to synthetic antigen small peptide P2 with P3; And when forming D2C4-BsAb before D2H2 variable region is put in C4G4, D2C4-BsAb and the binding activities of synthetic antigen small peptide P2 decline obviously compared with C4G4; The binding activities of D2C4-BsAb and synthetic antigen small peptide P3 and D2H2 are without significant difference.

The avidity of same mensuration two bi-specific antibody C4D2-BsAb and D2C4-BsAb and HBsAg.As shown in Figure 10, C4D2-BsAb and D2C4-BsAb can well be combined with HBsAg.

Experimental example 2: with HBV determination of activity in bi-specific antibody C4D2-BsAb

HBV virion (1 × 10 ⁶copies) respectively with C4G4, D2H2, C4G4+D2H2 and C4D2-BsAb preincubate 1 hour of different concns, join (containing 4%PEG8000 in substratum) incubated overnight in differentiation-inducing good HepaRG clone, wash PBS next day tri-times, renew fresh substratum.HBsAg content in the HBV-DNA copy number detected for the 6th day in cell and culture supernatant.HBV-DNA detects and uses HBV-DNA PCR kit for fluorescence quantitative (China of Shanghai section is biological).HBsAg measures and collects culture supernatant, detects with hepatitis B surface antigen quantification kit (Electrochemiluminescince) (Beijing section is U.S. biological).As Figure 11, result display bi-specific antibody C4D2-BsAb show fabulous in and the ability of HBV, exceeded the effect of C4G4 and D2H2 coupling.

Characterization of anti-HBsAg antibodies.

Antibody

Affinity KD(M) a

Epitope b

Neutralization activity c

Antibody

Affinity KD(M) a

Epitope b

Neutralization activity c

A3E3

5.67×10 8

3

++

D12H12

2.04×10 7

2

++

A5E5

3.75×10 9

4

-/+

2A1E1

2.39×10 9

4

-/+

A6E6

3.24×10 8

ND

+

2A2E2

3.72×10 8

3

++

A7E7

6.64×10 9

1

-/+

2A9E9

4.35×10 8

ND

-/+

A10E10

2.12×10 8

2

+

2B4F4

1.71×10 9

2

++

A11E11

5.63×10 8

1

-/+

2B5F5

6.63×10 8

2

+

B1F1

2.95×10 7

2

++

2B6F6

5.11×10 8

3

++

B6F6

4.82×10 8

2

ND

2B10F10

8.35×10 9

4

++

B7F7

6.55×10 9

3

-/+

2B11F11

6.28×10 8

ND

-/+

B9F9

2.86×10 9

4

++

2C2G2

1.77×10 7

2

+

B12F12

1.33×10 8

3

+

2C3G3

3.66×10 8

ND

-/+

C1G1

6.74×10 7

3

ND

2C5G5

3.28×10 9

2

+

C2G2

1.85×10 9

2

++

2C6G6

1.11×10 8

3

++

C4G4

3.66×10 9

2

++

2C7G7

8.6×10 8

3

+

C8G8

3.03×10 7

2

-/+

2C8G8

8.28×10 7

3

++

C9G9

3.81×10 8

ND

+

2D1H1

5.04×10 8

1

++

D2H2

5.82×10 9

3

++

2D4H4

3.48×10 9

2

++

D3H3

4.14×10 8

2

ND

2D5H5

2.73×10 7

2

ND

D5H5

1.88×10 9

2

++

2D8H8

2.12×10 8

3

++

D6H6

4.86×10 7

3

++

2D9H9

5.29×10 8

3

+

D10H10

1.58×10 8

2

+

C4D2-BsAb

3.88×10 9

2,3

++

D11H11

8.92×10 9

ND

-/+

D2C4-BsAb

4.95×10 9

2,3

^aAffinities were analyzed with ELISA.

^bEpitopes were analyzed by ELISA using synthesized peptides (P1, amino acids 104–120; P2, amino acids 121–137; P3, amino acids 139–148; P4, amino acids 149–163) covering extracellular domain of HBsAg.

^cHBV-neutralization activity : ++, more than 60% inhibition; +, 40%–60% inhibition; +/–, 20-40% inhibition at day 7 post-infection from that with control IgG.

SEQUENCE LISTING

 

Shanghai Hai Sitaike pharmaceutcal corporation, Ltd of <110> Shanghai Zhangjiang Biological Technology Co

 

<120> mono-class two special restructuring AntiHBsAg antibody, Preparation Method And The Use

 

<130> 2013

 

<160> 4

 

<170> PatentIn version 3.5

 

<210> 1

<211> 1842

<212> DNA

<213> artificial sequence

 

<400> 1

atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt gcacagccag 60

gtgcagctgg tggaaagcgg cggaggagtg gtgagacccg gaagaagcct gagactgagc 120

tgcgccgcta gcggctttgc cttcagcgac tacagcatca actgggtgag gcaagctccc 180

ggcaagggac tggaatgggt ggccatcatc agctacgacg gcaggatcac ctactacagg 240

gacagcgtga agggcaggtt caccatctcc agggacgaca gcaagaacac cctgtacctg 300

cagatgaaca gcctgaggac cgaggacacc gccgtgtatt actgcgccag gcagtactac 360

gacttctgga gcggctcctc cgtgggcagg aactacgacg gaatggacgt gtggggcctg 420

ggcaccacag tcaccgtgag ctcagctagc accaagggac cttctgtgtt ccctctggcc 480

cctcaggtgc agctggtgga aagcggagga ggcgtggtgc aacccggagg aagcctgagg 540

ctgagctgcg ctcctagcgg cttcgtgttc aggagctacg gcatgcactg ggtgaggcag 600

acacccggca aaggcctgga gtgggtgagc ctgatctggc acgacggcag caacaggttc 660

tacgccgaca gcgtgaaggg caggttcacc atcagcaggg acaacagcaa gaacaccctg 720

tacctgcaga tgaacagcct gagggctgag gacaccgcca tgtacttctg cgccagggag 780

aggctgattg ccgctcctgc cgctttcgac ctgtggggac agggcaccct ggtgaccgtc 840

agctcggcgt cgaccaaggg cccatcggtc ttccccctgg caccctcctc caagagcacc 900

tctgggggca cagcggccct gggctgcctg gtcaaggact acttccccga accggtgacg 960

gtgtcgtgga actcaggcgc cctgaccagc ggcgtgcaca ccttcccggc tgtcctacag 1020

tcctcaggac tctactccct cagcagcgtg gtgaccgtgc cctccagcag cttgggcacc 1080

cagacctaca tctgcaacgt gaatcacaag cccagcaaca ccaaggtgga caagaaagtt 1140

gagcccaaat cttgtgacaa aactcacaca tgcccaccgt gcccagcacc tgaactcctg 1200

gggggaccgt cagtcttcct cttcccccca aaacccaagg acaccctcat gatctcccgg 1260

acccctgagg tcacatgcgt ggtggtggac gtgagccacg aagaccctga ggtcaagttc 1320

aactggtacg tggacggcgt ggaggtgcat aatgccaaga caaagccgcg ggaggagcag 1380

tacaacagca cgtaccgtgt ggtcagcgtc ctcaccgtcc tgcaccagga ctggctgaat 1440

ggcaaggagt acaagtgcaa ggtctccaac aaagccctcc cagcccccat cgagaaaacc 1500

atctccaaag ccaaagggca gccccgagaa ccacaggtgt acaccctgcc cccatcccgg 1560

gatgagctga ccaagaacca ggtcagcctg acctgcctgg tcaaaggctt ctatcccagc 1620

gacatcgccg tggagtggga gagcaatggg cagccggaga acaactacaa gaccacgcct 1680

cccgtgctgg actccgacgg ctccttcttc ctctacagca agctcaccgt ggacaagagc 1740

aggtggcagc aggggaacgt cttctcatgc tccgtgatgc atgaggctct gcacaaccac 1800

tacacgcaga agagcctctc cctgtctccg ggtaaatgat ga 1842

 

 

<210> 2

<211> 612

<212> PRT

<213> artificial sequence

 

<400> 2

 

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Ser Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Arg

20 25 30

Pro Gly Arg Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ala Phe

35 40 45

Ser Asp Tyr Ser Ile Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

50 55 60

Glu Trp Val Ala Ile Ile Ser Tyr Asp Gly Arg Ile Thr Tyr Tyr Arg

65 70 75 80

Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys Asn

85 90 95

Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Thr Glu Asp Thr Ala Val

100 105 110

Tyr Tyr Cys Ala Arg Gln Tyr Tyr Asp Phe Trp Ser Gly Ser Ser Val

115 120 125

Gly Arg Asn Tyr Asp Gly Met Asp Val Trp Gly Leu Gly Thr Thr Val

130 135 140

Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala

145 150 155 160

Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly

165 170 175

Gly Ser Leu Arg Leu Ser Cys Ala Pro Ser Gly Phe Val Phe Arg Ser

180 185 190

Tyr Gly Met His Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Trp

195 200 205

Val Ser Leu Ile Trp His Asp Gly Ser Asn Arg Phe Tyr Ala Asp Ser

210 215 220

Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu

225 230 235 240

Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Phe

245 250 255

Cys Ala Arg Glu Arg Leu Ile Ala Ala Pro Ala Ala Phe Asp Leu Trp

260 265 270

Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro

275 280 285

Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr

290 295 300

Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr

305 310 315 320

Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro

325 330 335

Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr

340 345 350

Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn

355 360 365

His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser

370 375 380

Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu

385 390 395 400

Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu

405 410 415

Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser

420 425 430

His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu

435 440 445

Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr

450 455 460

Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn

465 470 475 480

Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro

485 490 495

Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln

500 505 510

Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val

515 520 525

Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val

530 535 540

Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro

545 550 555 560

Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr

565 570 575

Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val

580 585 590

Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu

595 600 605

Ser Pro Gly Lys

610

 

 

<210> 3

<211> 1080

<212> DNA

<213> artificial sequence

 

<400> 3

atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt gcacggcgac 60

atcgtcatga cccagagccc tctcagcctg tccgtgaccc ctggagaacc cgccagcatc 120

tcctgcagga gcagccagag cctgctgcac aggagcggca acaactacct ggactggtac 180

ctgcagaagc ccggccatag cccccagctg ctgatctacg tgggcagcaa cagagcttcc 240

ggcgtgcccg acagattcag cggaagcgga tccggcaccg agtacaccct gaagatcagc 300

agagtggagg ccgaggacgt gggcgtgtac tactgcatgc aggccctgca gacccccagg 360

acattcggcc agggcaccaa gctggagatc aagcgtaccg tggctgcccc tagcgtgttc 420

atcttccctc ctgagctggt gatgacccag agcccttcca gcctgagcgc tagcgtggga 480

gacagggtga ccatcacctg cagggccagc cagggcatct acaacagcat cgcctggtac 540

cagcagaagc ccggcaaggc tcccaagctg ctgctgtaca gcaccagcac actgctgagc 600

ggcgtgccca gcagattcag cggaagcggc agcggcaccg attacaccct gaccatcacc 660

aacctgcagc ctgaggactt cgccacctac tactgccagc agtacttcgt gacccccgag 720

accttcggac agggcaccaa ggtggagatc aagcgtacgg tggctgcacc atctgtcttc 780

atcttcccgc catctgatga gcagttgaaa tctggaactg cctctgttgt gtgcctgctg 840

aataacttct accccagaga agccaaagtg cagtggaagg tggacaacgc cctgcagagc 900

ggaaacagcc aggaaagcgt gacagagcag gattccaagg attccacata cagcctgagc 960

agcacactga cactgtccaa ggccgactac gagaagcaca aggtgtacgc ctgcgaagtg 1020

acacaccagg gactgtcctc ccctgtgaca aagagcttca acagaggaga atgctgatga 1080

 

 

<210> 4

<211> 358

<212> PRT

<213> artificial sequence

 

<400> 4

 

Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly

1 5 10 15

Val His Gly Asp Ile Val Met Thr Gln Ser Pro Leu Ser Leu Ser Val

20 25 30

Thr Pro Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu

35 40 45

Leu His Arg Ser Gly Asn Asn Tyr Leu Asp Trp Tyr Leu Gln Lys Pro

50 55 60

Gly His Ser Pro Gln Leu Leu Ile Tyr Val Gly Ser Asn Arg Ala Ser

65 70 75 80

Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Tyr Thr

85 90 95

Leu Lys Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys

100 105 110

Met Gln Ala Leu Gln Thr Pro Arg Thr Phe Gly Gln Gly Thr Lys Leu

115 120 125

Glu Ile Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro

130 135 140

Glu Leu Val Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

145 150 155 160

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Tyr Asn Ser

165 170 175

Ile Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Leu

180 185 190

Tyr Ser Thr Ser Thr Leu Leu Ser Gly Val Pro Ser Arg Phe Ser Gly

195 200 205

Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Thr Asn Leu Gln Pro

210 215 220

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Phe Val Thr Pro Glu

225 230 235 240

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

245 250 255

Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly

260 265 270

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

275 280 285

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

290 295 300

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

305 310 315 320

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

325 330 335

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

340 345 350

Phe Asn Arg Gly Glu Cys

355

Claims (13)

1. a class bi-specific antibody, it contains two different variable region of heavy chain, variable region of light chain, can respectively in conjunction with the functional epitope that HBsAg is different.

2., described in claim 1, a class bi-specific antibody, it is characterized in that variable region of heavy chain has the aminoacid sequence shown in SEQ NO:2, variable region of light chain has the aminoacid sequence shown in SEQ NO:4.

3. the nucleic acid molecule be separated, aminoacid sequence described in coding claim 2, has nucleotide sequence shown in nucleotide sequence shown in SEQ NO:1 and SEQ NO:3.

4. a carrier, containing nucleic acid molecule according to claim 3, with the expression regulation sequence that the sequence being operational of described nucleic acid molecule is connected, wherein carrier can be pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), one of pDHFR.

5. carrier according to claim 4 is pcDNA3.1(+).

6. a host cell, containing carrier described in claim 5, is eukaryotic cell.

7. host cell described in claim 6 is mammalian cell.

8. host cell described in claim 7 is Freestyle 293F cell.

9. prepare a method for bi-specific antibody according to claim 1, the method comprises:

A), adopt the memory B cells that in volunteer's peripheral blood of ultra high rate airflow classification systematic position HB vaccination, HBsAg is special, and be prepared into unicellular sample;

B) Single Cell RT-PCR technology, is adopted to clone antibody genes involved in each sample, in-vitro recombination expression corresponding antibodies;

C), through functional epitope measure, avidity measure and in and HBV determination of activity, filter out monoclonal antibody C4G4 and the D2H2 of strong, active high two anti-HBsAgs in conjunction with difference in functionality epi-position of avidity;

D), utilize biotechnology to be connected respectively through heavy chain linker, light chain linker in the heavy chain of these two monoclonal antibodies of C4G4, D2H2, variable region of light chain, be prepared into and there is bifunctional antibody;

E), under expression condition, cultivate the arbitrary described host cell of claim 6-8, express bi-specific antibody C4D2-BsAb, separation and purification.

10. a composition, containing bi-specific antibody according to claim 1, and pharmaceutically acceptable carrier.

11. bi-specific antibodies according to claim 1 are preparing the application in anti HBV infecting.

12. compositions according to claim 9 are preparing the application in anti HBV infecting.

The arbitrary described purposes of 13. claim 10,11, also comprises and the Drug combination in other anti HBV infecting.