CN108728464A - A kind of minicircle dna carrier and its preparation method and application of expression AntiHIV1 RT activity bispecific antibody - Google Patents

A kind of minicircle dna carrier and its preparation method and application of expression AntiHIV1 RT activity bispecific antibody Download PDF

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Publication number
CN108728464A
CN108728464A CN201710245144.7A CN201710245144A CN108728464A CN 108728464 A CN108728464 A CN 108728464A CN 201710245144 A CN201710245144 A CN 201710245144A CN 108728464 A CN108728464 A CN 108728464A
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cell
variable region
carrier
chain variable
bispecific antibody
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谢亦武
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Shenzhen New Connaught Micro Ring Biological Technology Co Ltd
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Shenzhen New Connaught Micro Ring Biological Technology Co Ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
    • C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
    • C07K16/1036—Retroviridae, e.g. leukemia viruses
    • C07K16/1045—Lentiviridae, e.g. HIV, FIV, SIV
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2809—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against the T-cell receptor (TcR)-CD3 complex
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C07K16/468—Immunoglobulins having two or more different antigen binding sites, e.g. multifunctional antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/73—Inducing cell death, e.g. apoptosis, necrosis or inhibition of cell proliferation

Abstract

This application involves a kind of minicircle dna carriers and its preparation method and application of expression AntiHIV1 RT activity bispecific antibody.More particularly to a kind of minicircle dna carrier for expressing AntiHIV1 RT activity bispecific antibody in vivo.The AntiHIV1 RT activity bispecific antibody combination HIV infection cell, moreover it is possible to neutralize free inhibition of HIV.The minicircle dna carrier, bispecific antibody are for preventing or treating the illnesss such as AIDS or HIV infection.

Description

A kind of minicircle dna carrier and preparation method thereof of expression AntiHIV1 RT activity bispecific antibody and Using
Technical field
The invention belongs to biomedicine fields, and in particular to a kind of recombination carrier, it is more specifically a kind of in body The minicircle dna carrier (MC.anti-HIV.BsAb) of interior expression AntiHIV1 RT activity bispecific antibody.
Background technology
Human immunodeficiency virus (HIV) infection seriously threatens human health.After inhibition of HIV infection cell, viral genome It can be integrated into cellular genome, form provirus (Protovirus), the template replicated as viral persistence.Existing antiviral therapy Means energy suppressing virus replication controls virus load, however can not but utterly destroy the cell of carrier virus, to remove provirus Library.Infected cell is only removed, could realize the purpose for removing provirus library.Studies have shown that part HIV infection person's body is exempted from Epidemic disease system can generate the HIV neutralizing antibodies of wide spectrum, can remove free virion, and it is infected also to act on removing by ADCC Cell (such as：CD4+Cell etc.).But the killing functions of immunocytes that ADCC is mediated is limited, can not remove all infection cells.
Evidence suggests by connecting target cell and effector cell (such as simultaneously：CD3+T cell or CD16+NK cells), it is double Specific antibody can mediate effector cell to act on the Efficient killing effect of target cell, more thousands of by force than the fragmentation effect that ADCC is mediated Times, particularly suitable for anti-hiv therapy.In addition, when HIV is in dormant, extremely low expression is not expressed or be in viral antigen, Immunity of organism and drug can not work.And CD3 antibody has activation to T cell, the T cell transcriptional activity being active Increase, target cellular protein (including being integrated in the viral antigen of cellular genome) can be promoted to express so that bispecific antibody can The infection cell that targeting is hidden in these.It is to express HIV neutralizing antibodies using AAV carriers with the immediate technology of the invention AAV-anti-HIV (WO2012115980), or expression and purification HIV bispecific antibodies albumen (WO2016054053) in vitro.
However, there are disadvantages, including with high costs, safety risks for existing AAV expression antibody technique.It specifically includes： (a) packaging, purification process of AAV viral vectors are complicated, need a large amount of cell culture, take time and effort；(b) preservation of AAV viruses, The processes such as transport need low temperature environment, cause cost increase and inconvenient for use；(c) AAV virus coats are easy to evoke strong exempt from Epidemic disease is reacted, and the DNA sequence dna radom insertion carried in carrier is easy to cause gene mutation or even carcinogenic risk.
Likewise, by cell culture in vitro expression and purification HIV antibody the shortcomings that it is mainly with high costs, it is inconvenient for use. It specifically includes：(a) antibody in vitro expression and purification complex process, it is costly；(b) albumen preserves, transport usually requires low temperature environment, Ask harsher；(c) antibody use need to need to be used for a long time by vein or hypodermic injection, limited half-life, increase patient burden.
It is anti-the present invention is directed to express AntiHIV1 RT activity bispecific in vivo using recombination carrier, especially minicircle dna carrier Body (MC.anti-HIV.BsAb) removes HIV infection for preventing or treating AIDS.
Invention content
The present invention relates to a kind of recombination carrier for expression AntiHIV1 RT activity bispecific antibody in vivo is (especially micro- Circular DNA carrier MC.anti-HIV.BsAb), play the role of removing HIV infection cell.Due to the portion of the antibody AntiHIV1 RT activity of the present invention Divide and derive from extensive neutralizing antibody, therefore the AntiHIV1 RT activity bispecific antibody expressed can also play the role of neutralizing the inhibition of HIV that dissociates.
The present invention provides a kind of recombination carrier of expression AntiHIV1 RT activity bispecific antibody, the bispecific antibodies Include the first antigen-binding domains combined with HIV infection cell-specific, and second with effector cell's specific binding Antigen-binding domains；Preferably, the bispecific antibody can also neutralize free inhibition of HIV.
In one embodiment, it is preferred to, the recombination carrier is selected from non-viral gene vector；It is furthermore preferred that The recombination carrier is selected from minicircle dna carrier.
In one embodiment, the effector cell includes T cell or NK cells.Preferably, the second antigen knot It closes structural domain and specifically binds action target spot selected from the following：CD3,CD28,4-1BB,OX40,TCR,CD16,CD56,NKG2D, NCR and other.
In one embodiment, the recombination carrier is recombinant expression carrier, selected from prokaryotic expression carrier or very Nuclear expression carrier；It is preferred that carrier for expression of eukaryon；It is more preferably used for the recombinant expression carrier of mammalian cell eukaryotic expression.
In one embodiment, the first antigen-binding domains of the bispecific antibody include：Such as SEQ ID NO:The heavy chain variable region of amino acid sequence shown in 1, and such as SEQ ID NO:The light chain variable region of amino acid sequence shown in 2；With And the second antigen-binding domains of the bispecific antibody include：Such as SEQ ID NO:The heavy chain of amino acid sequence shown in 3 Variable region, and such as SEQ ID NO:The light chain variable region of amino acid sequence shown in 4.
In one embodiment, the heavy chain variable region that first antigen-binding domains include has and SEQ ID NO:Sequence shown in 1 at least with 90%, 95%, 98% or 99% homology amino acid sequence, including light chain variable region tool Have and SEQ ID NO:At least amino acid sequence with 90%, 95%, 98% or 99% homology of sequence shown in 2.
In one embodiment, the heavy chain variable region that second antigen-binding domains include has and SEQ ID NO:Sequence shown in 3 at least with 90%, 95%, 98% or 99% homology amino acid sequence, including light chain variable region tool Have and SEQ ID NO:At least amino acid sequence with 90%, 95%, 98% or 99% homology of sequence shown in 4.
The present invention provides a kind of recombination carrier of expression AntiHIV1 RT activity bispecific antibody, the recombination carriers Include the encoding gene of the bispecific antibody.
In one embodiment, the nucleotide sequence of encoding gene such as SEQ ID described in the recombination carrier NO:Shown in 5.
In one embodiment, encoding gene described in the recombination carrier has and SEQ ID NO:Shown in 5 The sequence at least nucleotide sequence with 90%, 95%, 98% or 99% homology.It is well known to those skilled in the art, do not changing In the case of becoming encoded amino acid, the justice such as one or more of described coding gene sequence codon can carry out are replaced It changes, such as one or several codons, such as 1,2,3,4,5,6,8,9,10,15,20,30,40,50 codon.
The present invention provides the bispecifics expressed by a kind of recombination carrier by before described in embodiment to resist Body.Preferably, the recombination carrier is selected from non-viral gene vector；It is furthermore preferred that the recombination carrier is selected from Minicircle dna carrier.
The present invention provides a kind of preparation methods of recombination carrier described in embodiment before, include the following steps：
(1) HIV neutralizing antibodies and its amino acid sequence of heavy chain variable region and light chain variable region are obtained；
(2) antibody of recognition effect cell and its amino acid sequence of heavy chain variable region and light chain variable region are obtained；
(3) the recombination carrier of structure expression AntiHIV1 RT activity bispecific antibody；
Optional, (4) identify the expression of the bispecific antibody in vivo and in vitro, and it is thin to HIV infection to detect it The scavenging effect of born of the same parents.
In one embodiment, in the step (1) of the preparation method, (a) obtains HIV from the prior art The heavy chain variable region of neutralizing antibody and the amino acid sequence of light chain variable region, or (b) detach in HIV antibody positive patient sera HIV specificity neutralizing monoclonal antibodies, then to the variable region of the antibody be sequenced；In the step (2), from existing skill The amino acid sequence of the heavy chain variable region and light chain variable region of the antibody of recognition effect cell is obtained in art.
In one embodiment, it is preferred to, the effector cell includes T cell or NK cells；Preferably, the identification The antibody specificity of effector cell combines action target spot selected from the following：CD3,CD28,4-1BB,OX40,TCR,CD16,CD56, NKG2D, NCR and other.
The present invention provides a kind of host cells, including the recombination carrier described in embodiment before, or by it The obtained recombination carrier of preparation method described in preceding embodiment.
In one embodiment, the host cell includes bacterial cell, yeast cell, mammalian cell or elder brother Worm cell.
The present invention provides a kind of preparation method of bispecific antibody described in embodiment before, specific steps include：
(1) the recombination carrier is built；
(2) by the recombination vector introduction host cell；
(3) under conditions suitable for the expression, host cell is cultivated, expression isolates and purifies, obtains the bispecific antibody.
The present invention provides a kind of pharmaceutical compositions, including before recombination carrier described in embodiment or before Bispecific antibody described in embodiment and pharmaceutically acceptable carrier.
In one embodiment, pharmaceutical preparation can be made in described pharmaceutical composition according to conventional methods.In production process, It is preferred that recombination carrier or bispecific antibody are mixed with pharmaceutically acceptable carrier or diluted with carrier.When carrier is made For diluent when, can be solid, semisolid or liquid.Preparation be selected from tablet, pill, pulvis, capsule, elixir, suspension, The forms such as emulsion, solution, aerosol, capsule, injection solution.Suitable carrier, excipient or diluent include water, breast Sugar, glucose, sucrose, sorbierite, mannitol, calcium silicates, cellulose, polyvinylpyrrolidone, methyl hydroxybenzoate, hydroxyl Propyl benzoate, talcum powder, magnesium stearate and mineral oil etc..Preparation can also include filler, anticoagulant, lubricant, profit Humectant, flavoring agent, emulsifier, preservative etc..
The present invention provides recombination carrier described in embodiment before, the bispecific antibody is described Host cell or the pharmaceutical composition are preparing the application in preventing or treating the drug of AIDS or HIV infection.
The present invention provides a kind of recombination carriers for expressing bispecific antibody in vivo, especially micro-loop DNA vector has the advantage that：
(a) the case where not expressed for inhibition of HIV antigen or being in extremely low expression, the T cell being active Transcriptional activity increases, and target cellular protein (including being integrated in the viral antigen of cellular genome) can be promoted to express so that double special Property antibody can act on these concealment infection cell.
(b) compared with prior art, especially AAV carriers express neutralizing antibody, cell culture technology vivoexpression antibody Compare, safety of the invention is more preferable, and cost is cheaper, carrier and antibody it is more convenient to use.
Description of the drawings
Fig. 1：The structure chart of minicircle dna carrier Anti-HIV.BsAb.
Specific implementation mode
Following experimental methods are conventional method unless otherwise instructed, used experiment material unless otherwise instructed, It can easily be obtained from commercial company.
The structure of embodiment 1, AntiHIV1 RT activity bispecific antibody (Anti-HIV.BsAb)
(a) Anti-HIV.BsAb is built, includes the first antigen-binding domains combined with HIV infection cell-specific With the second antigen-binding domains specifically bound with effector cell (action target spot CD3).
(b) the first antigen-binding domains include：Heavy chain variable region, amino acid sequence such as SEQ ID NO:Shown in 1, and Light chain variable region, amino acid sequence such as SEQ ID NO:Shown in 2.
(c) the second antigen-binding domains include：Heavy chain variable region, amino acid sequence such as SEQ ID NO:Shown in 3, and Light chain variable region, amino acid sequence such as SEQ ID NO:Shown in 4.
(d) the minicircle dna carrier of structure expression Anti-HIV.BsAb, includes the coding of first antigen-binding domains The encoding gene of gene and second antigen-binding domains；And also comprising suitable Expression element in minicircle dna carrier.
(e) include the expression cassette (shown in Figure 1) of Anti-HIV.BsAb, including formula in minicircle dna carrier：
Kozak-SP-CD3.VL-Linker-HIV.VH-ASTKGP-K4-Furin-GSG-2A-SP-HIV.VL- Linker-CD3.VH-ASTKGP-E4-His6
Wherein, Kozak is Kozak sequences, and SP is signal peptide (Signal Peptide)；Linker is catenation sequence, Furin is furin protease cleavages, 2A is 2A self cleavages site.
SP can be Secrecon, and amino acid sequence is：MWWRLWWLLLLLLLLWPMVWA or other；Furin is sheared The amino acid sequence in site is R-X- [R/K]-R (such as RRKR), and X refers to any amino acid；The amino acid sequence of GSG-2A For GSGEGRGSLLTCGDVEENPGP；The amino acid sequence of K coli (K4) is KVSALKEKVSALKEKVSALKEKVSALKE； The amino acid sequence of E coli (E4) is EVSALEKEVSALEKEVSALEKEVSALEK；2A includes E2A, F2A, P2A and T2A Deng, wherein the amino acid sequence of T2A be EGRGSLLTCGDVEENPGP.
The nucleotide sequence of above-mentioned expression cassette such as SEQ ID NO:Shown in 5.
(f) expression cassette of the BsAb it is translated, shearing after formed two chains：
First chain：CD3.VL-Linker-HIV.VH-ASTKGP-K4；With
Second chain：HIV.VL-Linker-CD3.VH-ASTKGP-E4-His6；
First chain and the second chain can form stable heterodimer by the E/K-coli of pairing.
The preparation of embodiment 2, the structure of minicircle dna carrier and micro-loop (MC)
(a) encoding gene of Anti-HIV.BsAb is synthesized.
(b) suitable site double digestion minicircle dna empty carrier pMC.BESPX is selected.
(c) use seamless clones or conventional cloning methods (digestion-connection) by bispecific antibody (BsAb) In empty carrier pMC.BESPX after encoding gene insertion double digestion, it is built into BsAb minicircle dnas carrier (pMC.BsAb).
(d) pMC.BsAb converts Escherichia coli E coli.ZYCY10P3S2T, and micro-loop is obtained by standard micro-loop preparation method (MC.BsAb)。
Wherein, minicircle dna empty carrier pMC.BESPX, engineering bacteria E coli.ZYCY10P3S2T and micro-loop preparation method See document Nat Biotechnol.2010,28 (12):1287-9.
The expression and purifying of embodiment 3, AntiHIV1 RT activity bispecific antibody (Anti-HIV.BsAb)
One, expression of the Anti-HIV.BsAb in 293T cells
Above-mentioned minicircle dna is transfected by 293T cells using superfect plasmid transfections kit (Invitrogen companies), 293T cell supernatants are collected respectively after being cultivated three days in serum free medium, and bispecific is detected using Western Blot The expression of antibody (BsAb).
Two, the purifying of Anti-HIV.BsAb
293 cell culture supernatants are first subjected to low temperature ultracentrifugation, take supernatant, then pure using His-Tag affine resins Change (cOmplete His-Tag Purification Resin, Roche), albumen after purification is fixed using Western Blot Property detection, and use ELISA method quantitative detection of protein concentration.Purified product is placed in -20 DEG C of long-term preservations.
The Binding experiment of embodiment 4, AntiHIV1 RT activity bispecific antibody (Anti-HIV.BsAb)
Using Flow cytometry Anti-HIV.BsAb and target cell (Targets, T), effector cell (Effector, E combination activity), includes the following steps：
(a) cell culture：Target cell (HIV positive cells)；Effector cell's (T cell or NK cells).
(b) cell is collected：Attached cell is digested using pancreatin, and after adding serum-containing media to neutralize, centrifugation is abandoned supernatant and obtained carefully Born of the same parents；Suspension cell is directly collected by centrifugation.
(c) two kinds of cells wash tears 2 times with precooling PBS, and 200g centrifuges 4min, collect cell, count respectively.
(d) each experimental group distributes 1 × 10 respectively5A target cell and effector cell, are grouped as follows：Blank group (is not added with ) and BsAb groups BsAb
(e) 100 μ L/ groups of BsAb solution are added and are incubated 30min on ice.
(f) 1mL precooling PBS washings are added, 200g centrifuges 4min, collects cell, adds precooling and mixed anti-flag in advance The 100 μ L of PBS of antibody, are incubated 30min on ice.
(g) 1mL precooling PBS washings are added, 200g centrifuges 4min, collects cell, and 100 μ L PBS are added and are resuspended.Up flow type Observation combines situation.
Embodiment 5, AntiHIV1 RT activity bispecific antibody (Anti-HIV.BsAb) mediate effector cell to kill target cell
(a) shift to an earlier date 1 day target cell is inoculated on 96 orifice plates, cell inoculation amount is 2 × 104/ hole；Grouping is set simultaneously (as shown in table 1；Every group of 3 multiple holes)；
Table 1BsAb mediates the different grouping of effector cell killing target cell
(b) fresh opti-MEM culture mediums, 100 holes μ L/ are replaced within second day.
(c) effector cell (E) counts and is resuspended in opti-MEM culture mediums, according to T：E=1：8 ratio, according in (a) The effector cell (1.6 × 10 of respective numbers is added in grouping5/ hole, 100 holes μ L/).
(d) according to grouping, be added per hole into corresponding hole 10 μ L purifying BsAb (concentration is respectively 0.02,0.04, 0.06 μ g/ μ L), mixing.
(e) 37 DEG C of cell incubators are incubated 6 hours.
(f) cck8 (Dojindo, Japan) reagent (20 holes μ L/) is added, after 37 DEG C of cell incubators are incubated 2 hours, Absorbance is measured at 450nm.
(g) it according to cck8 kits (Dojindo, Japan) specification, counts number of viable cells and calculates killing-efficiency：
Killing rate=[((T+E)-T&E)/T] × 100%
Wherein, T represents target cell numbers living；E represents effector cell's quantity living；Then T+E be equal to target cell living and The sum of effector cell；T&E represents living cells quantity of the target cell after effector cell kills.
Without departing from the spirit of the invention, those skilled in the art combine known technology, can be done to the present invention Go out many modifications, such modification also falls into the scope of the present invention.
Sequence table
SEQUENCE LISTING
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<120>A kind of minicircle dna carrier and its preparation method and application of expression AntiHIV1 RT activity bispecific antibody
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Gln Val Gln Leu Leu Gln Ser Gly Ala Ala Val Thr Lys Pro Gly Ala
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atgtggtggc gcctgtggtg gctgctgctg ctgctgctgc tgctgtggcc catggtgtgg 60
gccgccgccg acattgtact gacccagtct ccagcaactc tgtctctgtc tccaggggag 120
cgtgccaccc tgagctgcag agccagtcaa agtgtaagtt acatgaactg gtaccagcag 180
aagccgggca aggcacccaa aagatggatt tatgacacat ccaaagtggc ttctggagtc 240
cctgctcgct tcagtggcag tgggtctggg accgactact ctctcacaat caacagcttg 300
gaggctgaag atgctgccac ttattactgc caacagtgga gtagtaaccc gctcacgttc 360
ggtggcggga ccaaggtgga gatcaaaggt ggtggtggtt ctggcggcgg cggctccggt 420
ggtggtggtt ctcaggtgca gctcctccag tcaggagcag cagtgaccaa gccaggagcc 480
agcgtgagag tgtcttgcga agccagcggc tacaacatca gggactactt catccattgg 540
tggcggcagg ctccaggaca gggactccag tgggtcggtt ggattaaccc caagaccggc 600
cagcctaaca atcccaggca gttccagggc agggtgtctc tgacaaggca cgcctcttgg 660
gacttcgaca cctacagctt ctacatggac ctgaaggccc tgaggagcga cgataccgcc 720
gtgtactttt gcgccaggca gcggagcgac tattgggact tcgacgtctg gggaagcgga 780
acacaggtca cagtgtctag cgccagcaca aagggaccta aggtgtcagc tctcaaggag 840
aaggtctctg ctcttaaaga aaaagtctca gcactgaaag agaaggtttc tgcattgaag 900
gagcggagaa agagaggcag cggcgaggga agaggatctc tgctgacatg cggcgacgtg 960
gaagagaatc caggacctat gtggtggcgc ctgtggtggc tgctgctgct gctgctgctg 1020
ctgtggccca tggtgtgggc cgccgccgat atccagatga cccagagccc tagctctctg 1080
agcgctagcg tgggcgatac agtgaccatc acttgccagg ccaacggcta cctgaattgg 1140
taccagcaga ggcggggcaa ggctcctaag ctgctgatct acgacggctc caagctggag 1200
aggggcgtgc ccagcagatt cagcggaaga cgctggggcc aggagtacaa tctgaccatc 1260
aacaacctgc agcccgagga catcgccacc tacttttgcc aggtgtacga gttcgtggtg 1320
ccaggaacca ggctggatct gaagagaggt ggtggtggtt ctggcggcgg cggctccggt 1380
ggtggtggtt ctgacgtcca actggtgcag tcaggggctg aagtgaaaaa acctggggcc 1440
tcagtgaagg tgtcctgcaa ggcttctggc tacaccttta ctaggtacac gatgcactgg 1500
gtaaggcagg cacctggaca gggtctggaa tggattggat acattaatcc tagccgtggt 1560
tatactaatt acgcagacag cgtcaagggc cgcttcacaa tcactacaga caaatccacc 1620
agcacagcct acatggaact gagcagcctg cgttctgagg acactgcaac ctattactgt 1680
gcaagatatt atgatgatca ttactgcctt gactactggg gccaaggcac cacggtcacc 1740
gtctcctcag ccagcacaaa gggacctgag gttagtgcat tggagaaaga agtaagcgca 1800
ttggaaaaag aagtgtctgc attggagaaa gaggtctccg cgctcgaaaa g 1851

Claims (10)

1. a kind of recombination carrier of expression AntiHIV1 RT activity bispecific antibody, which is characterized in that the bispecific antibody includes The first antigen-binding domains combined with HIV infection cell-specific, and the second antigen with effector cell's specific binding Binding structural domain；Preferably, the recombination carrier is selected from non-viral gene vector；It is furthermore preferred that the recombination carries Body is selected from minicircle dna carrier；Further preferably, the bispecific antibody can also neutralize free inhibition of HIV；It is furthermore preferred that institute It includes T cell or NK cells to state effector cell；Most preferably, the second antigen-binding domains specific binding is selected from following Action target spot：CD3, CD28,4-1BB, OX40, TCR, CD16, CD56, NKG2D, NCR and other.
2. recombination carrier as described in claim 1, which is characterized in that first antigen-binding domains include：Such as SEQ ID NO:The heavy chain variable region of amino acid sequence shown in 1, and such as SEQ ID NO:The light chain variable of amino acid sequence shown in 2 Area；And second antigen-binding domains include：Such as SEQ ID NO:The heavy chain variable region of amino acid sequence shown in 3, and Such as SEQ ID NO:The light chain variable region of amino acid sequence shown in 4.
3. recombination carrier as claimed in claim 1 or 2, which is characterized in that the recombination carrier includes described double The encoding gene of specific antibody.
4. recombination carrier as claimed in claim 3, which is characterized in that the nucleotide sequence of the encoding gene such as SEQ ID NO:Shown in 5.
5. the bispecific antibody expressed by recombination carrier as described in any one of claim 1-4.
6. such as the preparation method of 1-4 any one of them recombination carriers in claim, which is characterized in that including walking as follows Suddenly：
(1) HIV neutralizing antibodies and its amino acid sequence of heavy chain variable region and light chain variable region are obtained；
(2) antibody of recognition effect cell and its amino acid sequence of heavy chain variable region and light chain variable region are obtained；
(3) the recombination carrier of structure expression AntiHIV1 RT activity bispecific antibody；
Optional, (4) identify the expression of the bispecific antibody in vivo and in vitro, and detect it to HIV infection cell Scavenging effect.
7. preparation method as claimed in claim 6, which is characterized in that in the step (1), (a) is obtained from the prior art The heavy chain variable region of HIV neutralizing antibodies and the amino acid sequence of light chain variable region are taken, or (b) detaches HIV antibody positive patient HIV specificity neutralizing monoclonal antibodies in serum are then sequenced the variable region of the antibody；In the step (2), from The amino acid sequence of the heavy chain variable region and light chain variable region of the antibody of recognition effect cell is obtained in the prior art；Preferably, The effector cell includes T cell or NK cells；It is furthermore preferred that the antibody specificity of the recognition effect cell combine selected from Under action target spot：CD3, CD28,4-1BB, OX40, TCR, CD16, CD56, NKG2D, NCR and other.
8. a kind of host cell, which is characterized in that comprising the recombination carrier as described in any one of claim 1-4, or The obtained recombination carrier of preparation method as claimed in claims 6 or 7.
9. a kind of pharmaceutical composition, which is characterized in that comprising as described in any one of 1-4 recombination carrier or as right is wanted Seek the bispecific antibody and pharmaceutically acceptable carrier described in 5.
10. the recombination carrier as described in any one of claim 1-4, bispecific antibody as claimed in claim 5, Host cell as claimed in claim 8 or pharmaceutical composition as claimed in claim 9 are preparing prevention or treatment AIDS Or the application in the drug of HIV infection.
CN201710245144.7A 2017-04-14 2017-04-14 A kind of minicircle dna carrier and its preparation method and application of expression AntiHIV1 RT activity bispecific antibody Pending CN108728464A (en)

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