CN109762068A

CN109762068A - A kind of single-gene bispecific antibody targeting CTLA4 and PD-1 and its application

Info

Publication number: CN109762068A
Application number: CN201811619419.XA
Authority: CN
Inventors: 吴稚伟; 吴喜林; 李彦磊; 黄碧莲; 马晓花
Original assignee: Source Daolong (suzhou) Medical Technology Co Ltd
Current assignee: Source Daolong (suzhou) Medical Technology Co Ltd
Priority date: 2018-08-09
Filing date: 2018-12-28
Publication date: 2019-05-17

Abstract

The present invention relates to the single-gene bispecific antibodies that one kind can target CTLA4 and PD-1, it successively include the first targeted integration area, the second targeted integration area and the area Fc from N-terminal to C-terminal, first targeted integration area is connect with the second targeted integration area by junction fragment, first targeted integration area targets one in CTLA4 and PD-1, and the second targeted integration area targets another；Single-gene bispecific antibody is further related in preparation for the application in anti-tumor drug；Further relate to a kind of AAV bispecific antibody expression vector.Single-gene bispecific antibody of the invention can be effectively targeted to PD1 and CTLA4, and the antibody sequence is short, and structure is simple, suitable for antibody expression vector of the building based on AAV virus, obtained antibody expression vector can be used for direct injection, and improve antibody holding time in vivo.

Description

A kind of single-gene bispecific antibody targeting CTLA4 and PD-1 and its application

Technical field

The present invention relates to tumor therapeutic agent fields, more specifically it relates to a kind of single base that can target CTLA4 and PD-1 Because of bispecific antibody and its application, and the AAV antibody of the DNA encoding sequence comprising above-mentioned single-gene bispecific antibody Expression vector.

Priority

This application claims the applicant submitted on August 9th, 2018 application No. is the invention of 201810903651X is special The priority of benefit application, entire contents are herein incorporated by reference.

Background technique

Bispecific antibody appear in earliest in the 1990s, had at that time scholar begin one's study have two it is anti- The antibody of former binding site can be classified as IgG class hypotype and non-IgG class hypotype two according to the structure of bispecific antibody Kind, all there are many different forms under of all categories.Wherein again with three function antibodies (Triomab) of IgG class hypotype and non- Bispecific T cell adapter (Bispecific T cell Engager, BiTE) technology of IgG class hypotype is the most mature. BiTE mainly passes through CD3 antibody as T cell adapter, is not suitable for exploitation for certain targets (such as PD1 and CTLA4 Deng) bispecific antibody.Also, current bispecific antibody exists, and structure is complicated, potency is not high, specificity is impacted The shortcomings that.

Cytotoxic T lymphocyte antigen-4 (cytotoxic T lymphocyte sociated antigen4, CTLA4) be costimulatory molecules B7 receptor, be mainly expressed in the T cell surface that is activated.B7 combination CTLA4 can inhibit T cell Activation, to inhibit function of immune system.Therefore, many tumor diseases, such as some melanomas, lung cancer, prostate cancer It is all related with the expression of CTLA4 with cervical carcinoma etc..Widely apply to treat melanoma for the monoclonal antibody medicine of CTLA4, Small Cell Lung Cancer, bladder cancer and prostate cancer etc., and show certain curative effect.

PD-1 can lower effector T cell response, via PD-1 inhibition signal transduction inducing T cell reactionless or Unresponsiveness state is unable to Clonal expansion so as to cause cell or generates effector cell's factor of optimum level.PD-1 may be used also Inhibit the ability of the survival-signal from costimulation by it to induce the Apoptosis of T cell.There is studies have shown that will target After the antibody of CTLA4 and the antibody of targeting PD1 are combined treatment, clinical efficacy promotes highest.

However, two kinds of antibody combined treatments lead to very high medical expense, but effect is not yet constructed in this field at present Good not only anti-CTLA 4 but also the bispecific antibody of anti-PD-1.Therefore, it is necessary to a kind of new double spies for CTLA4 and PD-1 Heterogenetic antibody.

Summary of the invention

In order to solve the above problem, the single-gene bispecific that can target CTLA4 and PD-1 the present invention provides one kind resists Body successively includes the first targeted integration area, the second targeted integration area and the area Fc, first targeted integration area from N-terminal to C-terminal It is connect with second targeted integration area by junction fragment, first targeted integration area targets one in CTLA4 and PD-1 A, second targeted integration area targets another.

In a preferred embodiment, the sequence in first or second targeted integration area is as shown in SEQ ID NO:1, The sequence in the described second or first targeted integration area is as shown in SEQ ID NO:2.

In a preferred embodiment, the sequence in the area Fc is as shown in SEQ ID NO:3.

In a specific embodiment, the junction fragment is made of one or more unit sequences, the unit sequence Column are as shown in SEQ ID NO:4.Preferably, the junction fragment sequence is as shown in SEQ ID NO:12.

In a preferred embodiment, the sequence of the single-gene bispecific antibody such as institute of SEQ ID NO:5 or 6 Show.

The present invention also provides above-mentioned single-gene bispecific antibody in preparation for the application in anti-tumor drug.

In a specific embodiment, the anti-tumor drug is for treating colon cancer, melanoma, lung cancer, forefront One of gland cancer, cervical carcinoma, bladder cancer are a variety of.

The present invention also provides a kind of AAV bispecific antibody expression vectors comprising above-mentioned single-gene bispecific is anti- The DNA encoding sequence expression cassette of body.

Preferably, the DNA encoding sequence in first or second targeted integration area is as shown in SEQ ID NO:9, and described Two or first targeted integration area DNA encoding sequence as shown in SEQ ID NO:10.The DNA encoding sequence in the area Fc is preferred As shown in SEQ ID NO:13.

In being preferably implemented at one, the DNA encoding sequence of the single-gene bispecific antibody such as SEQ ID NO:7 or 8 It is shown.

In a preferred embodiment, 5 ' ends of the DNA encoding sequence of single-gene bispecific antibody are also connected with letter Number peptide-coding sequence.Preferably, the DNA encoding sequence of the signal peptide is as shown in SEQ ID NO:11.

Single-gene bispecific antibody of the invention can be effectively targeted to PD1 and CTLA4, and the antibody sequence is short, knot Structure is simple, and suitable for antibody expression vector of the building based on AAV virus, obtained antibody expression vector can be used for directly Injection, and improve antibody holding time in vivo.

Detailed description of the invention

Fig. 1 is the primary structure schematic diagram of single-gene bispecific antibody of the invention；

Fig. 2 is that single-gene bispecific antibody of the invention carries out the structural schematic diagram after homodimerization；

Fig. 3 is that ELISA detects to obtain the statistical chart of the binding force of QV1-6 and PD1 and CTLA4 albumen；

The Western-blot that Fig. 4 is QV5 and QV6 detects photo；

Fig. 5 is the combination force curve of QV5 and QV6 to PD-1 of various concentration；

Fig. 6 is the combination force curve of QV5 and QV6 to CTLA4 of various concentration；

Fig. 7 is to use the processed T cell of QV5 and QV6 to the specific lysis statistical chart of target cell respectively；

Fig. 8 is bent using the time of AAV-QV6 tumor size during humanization mouse interior therapeutic HT29 colon cancer Line；

Fig. 9 is the survival rate time using AAV-QV6 mouse during humanization mouse interior therapeutic HT29 colon cancer Curve.

Specific embodiment

The principle and features of the present invention will be described below with reference to the accompanying drawings, and the given examples are served only to explain the present invention, and It is non-to be used to limit the scope of the invention.

1. the building and expression of single-gene tetravalence bispecific antibody

We obtain three strain antibodies in the course of the research, wherein 1 plant of targeting PD-1, other two plants of targeting CTLA4.It is logical Structure and the sequence analysis to this three strain antibody are crossed, we devise the protein domain AC (amino of a targeting CTLA4 Acid sequence SEQ ID NO:1, nucleotide sequence SEQ ID NO:9) and two targeting PD-1 protein structure domains name respectively For AP1 (sequence does not provide) and AP2 (SEQ ID NO:2, nucleotide sequence SEQ ID NO:10).

By these three structural domains be combined with linker be connected and then with IgG Fc4 constant region (SEQ ID NO:3) It connects (as shown in Figure 1), obtains six plants of new antibodies, as shown in table 1.

The composition of 1 six plants of new antibodies of table

Antibody

QV1

QV2

QV3

QV4

QV5

QV6

Composition

AC+AP1

AP1+AC

AP2+AP1

AP1+AP2

AP2+AC

AC+AP2

Note: in composition described above, write on+left side is located at N-terminal, between two structural domains with linker (SEQ ID NO:12) Connection.

According to the sequence of designed antibody, we, which design, has synthesized corresponding gene coded sequence, and adds in front end Sequence to be expressed, is then inserted on pVAX1 carrier and forms expression plasmid by signal peptide (SEQ ID NO:11).It will be upper The correct plasmid amplification of building is stated, 293F cell is transfected by transfection reagent PEI, cell conditioned medium is collected after 6 days and is carried out Protein G purifies to obtain corresponding antibody, can pass through the cysteine shape on IgG Fc4 between every two antibody protein chain Together at disulfide-bonded, the homodimer by Y word configuration is formed, to become the tetravalent antibody (figure of bispecific 2)。

2. the screening of functional antibody

Enzyme-linked immunosorbent assay (enzyme-linked is carried out to the antibody QV1-6 that above purifying obtains Immunoassay, ELISA) detection antibody for PD-1 and CTLA4 affinity, with commercially available Mono-specific antibodies Yer and Opd is as control.Detection plate is with 0.5 μ gml^-1Concentration coating, every hole 100ul, 4 DEG C be incubated overnight after, sealed with 4%BSA It closes, every hole 200 μ l, 37 DEG C of incubation 1h, washs 4 times, the 100 μ l of antibody or cell conditioned medium of gradient dilution is added in every hole, is incubated for 1h is washed 4 times, and secondary antibody 100ul, the 37 DEG C of incubation 1h that the diluted HRP label of 1:1000 is added in every hole add bottom after washing 4 times Object, the HCL stopped reaction of 37 DEG C of incubations 15min, 50ul 2M measure the OD value (OD 450nm) of sample.

As a result as shown in figure 3, QV5 (amino acid sequence SEQ ID NO:5, nucleotide coding sequence SEQ ID NO:7) and QV6 (amino acid sequence SEQ ID NO:6, nucleotide coding sequence SEQ ID NO:8) is shown to PD-1's and CTLA4 High-affinity, QV1 and QV2 show the weak affinity to PD-1 and CTLA4, and QV3 and QV4 are hardly shown to target site Affinity.

3. the identification of bispecific antibody

According to previous step experiment as a result, selection QV5 and QV6 is further identified.Pass through Western Blotting Detect the size of QV5 and QV6.Method is as follows: protein sample adds a certain amount of 5xSDS sample loading buffer and heats in 100 DEG C After 5min, routine SDS-PAGE electrophoresis, resolving gel concentration 7.5% are carried out.After electrophoresis, electricity is gone on pvdf membrane, and 5% Skimmed milk power close 1 hour, primary antibody be incubated at room temperature 1 hour, wash film, diluted two with fluorescent marker of 1:10000 1 hour of anti-incubation at room temperature, wash film, ODyssey fluorescence detection.As a result as shown in figure 4, QV5 and QV6 are 80kDa or so Band, meet expection.

In order to further identify the combination activity of QV5 and QV6 and PD1 and CTLA4 albumen, the antibody of gradient concentration is used ELISA detection is carried out, as the result is shown QV5 and QV6 energy gradient combination PD1 and CTLA4 albumen, and the combination readings of QV6 is high In QV5 (Figures 5 and 6).

The detection of 4.T cell killing

In order to further verify the antibody enhancing T cell killing function of the tetravalence bispecific antibody QV5 and QV6 that newly construct Can, we have carried out cell killing experiment in vitro.

Method is as follows: tumour cell and 0.5 μM of CFSE being carried out room temperature and dye 25min, terminates CFSE with ice-cold FBS Dyeing, PBS wash cell；5x10 is inoculated in 6 orifice plates⁶A tumour target cell for being infected with CFSE, according to tumour cell: PBMC is thin The ratio of born of the same parents is that 1:10 adds fresh PBMC cell, while adding the YER of 10nM, OPD, QV5, QV6 and control antibodies, is mixed Cell is closed in 37 DEG C of 5%CO₂7-AAD dyeing is carried out after being incubated for 48 hours altogether；Positive thin of the flow cytometer detection CFSE positive and 7-AAD Born of the same parents are specific killer cells.

The results show that the experimental group Cell killing efficacy of addition QV6 antibody is significantly higher than Yer and the Opd processing of female generation group Group, also conspicuousness is higher than QV5 processing group (Fig. 7).Experimental result prompt QV6 tetravalence bispecific antibody significantly improves T cell Cytotoxicity in vitro effect.

5.AAV viral vectors loads QV6

AAV Helper-Free Viral Packaging System is purchased from Cell Biolabs, San Diego USA.Above-mentioned building QV6 is building up to pAAV-MCS plasmid by molecule clone technology；It is proved after constructing successfully by sequencing, the plasmid that will be built PAAV-Ab and pHelper and pAAV-DJ plasmid use PEI transfection reagent cotransfection AAV- in the way of mass ratio 1:1:1 293T cell.Respectively at 48,72,96 and 120 hours collection supernatants after transfection, and it is concentrated with 5xPEG8000 (sigma), Finally purified with 1.37g/ml cesium chloride.The AAV of purifying is dissolved in PBS, is identified and is stored in -80 after being dispensed ℃。

6. carrying out experiment in vivo using the QV6 that AAV viral vectors loads

The half-life period of existing antibody and the like generally at 3-7 days, by taking Yer Antybody therapy as an example, therapeutic dose For 3mg/kg, treatment in every three weeks is primary, and four times altogether, treatment cycle is 12 weeks, therefore one antibody of the patient of a 60kg is controlled It treats the course for the treatment of to get off, takes around 720mg antibody, greatly increase treatment cost in this way.UniQure ' s based on AAV1 carrier Drug Glybera has been approved in European Union in clinical use in 2012 as first carrier medicine.It is carried by AAV carrier Antibody can maintain the time for a long time in vivo.

Intramuscular injection is carried out after whether can carrying by AAV to further verify QV6 tetravalence bispecific antibody to reach To therapeutic effect, we express QV6 using AAV viral vectors submission, and in the humanization mouse of inoculation colon cancer HT29 cell Interior curative effect assessment is carried out in vivo.

ImmunodeficientNOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NCG) mouse is purchased from Nanjing University Model animal institute, the mouse have lacked IL2 acceptor gene on the basis of SCID mice, lead to do not have mouse T cell in vivo, B is thin Born of the same parents and minimal amount of NK cell.1.0x10⁷PBMC is injected intraperitoneally into 4-6 weeks NCG Mice Body, and inoculates simultaneously 3.6x10⁶Personal colon cancer tumours cell HT29；It takes a blood sample after three weeks, by the CD45 for dyeing people⁺,CD3⁺,CD4⁺And CD8⁺Detection Human T-cell.The ratio of the CD45 positive cell of people reaches 30% or more, is determined as mouse humanized success.It measures simultaneously swollen Tumor size, the subcutaneous volume of tumour are greater than 80m³Judge that humanization HT29 mouse tumor model is successful.

The successful humanization HT29 mice with tumor of above-mentioned judgement is grouped at random, receives AAV-QV6 (1x10¹¹ gc/100μ L) intramuscular injection, using AAV-GFP as control group, the physiological and biochemical index and tumour of subsequent different time points detection mouse are big It is small.

The results show that AAV-QV6, after tumor inoculation 55 days, tumour growth is slowly (Fig. 8).In addition receive AAV-QV6's Survival rate of the mouse after tumor inoculation 69 days is still 100%, and AAV-GFP control group receives the mouse of AAV-GFP in tumour The 55th day of inoculation, it is all dead.It can be seen that AAV-QV6 can significantly improve the survival rate (Fig. 9) of inoculated tumour mouse.

Sequence table

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Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Ser Ser Asn Trp Pro Arg

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

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

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

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

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

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Tyr Cys Ala Arg Thr Gly Trp Leu Gly Pro Phe Asp Tyr Trp Gly Gln

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Gly Thr Leu Val Thr Val Ser Ser Glu Ser Lys Tyr Gly Pro Pro Cys

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

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Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

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Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

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Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

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Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu

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

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Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro

290 295 300

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

305 310 315 320

Gly Ile Pro Ala Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr

325 330 335

Leu Thr Ile Ser Ser Leu Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys

340 345 350

Gln Gln Ser Ser Asn Trp Pro Arg Thr Phe Gly Gln Gly Thr Lys Val

355 360 365

Glu Ile Lys Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly

370 375 380

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

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Pro Gly Arg Ser Leu Arg Leu Asp Cys Lys Ala Ser Gly Ile Thr Phe

405 410 415

Ser Asn Ser Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu

420 425 430

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

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

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

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Tyr Tyr Cys Ala Thr Asn Asp Asp Tyr Trp Gly Gln Gly Thr Leu Val

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Thr Val Ser Ser Ala Ser Thr Lys Glu Ser Lys Tyr Gly Pro Pro Cys

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

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Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu

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Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln

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Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys

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Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu

580 585 590

Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys

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Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys

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Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser

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Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys

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Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln

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Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly

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Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln

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Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn

705 710 715 720

His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys

725 730

<210> 7

<211> 2202

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 7

gagatcgtgc tgacccagtc ccccgctaca ctgtccctgt cccctggaga gagggccacc 60

ctgtcctgca gggctagcca gagcgtgtcc tcctacctgg cctggtatca gcagaagccc 120

ggccaagctc ccaggctgct gatctacgac gcctccaaca gggccacagg catccctgcc 180

aggttctccg gttctggttc tggcaccgac ttcaccctga ccatctcctc cctggagccc 240

gaggacttcg ccgtgtacta ctgccagcag tcctccaact ggcccaggac cttcggccag 300

ggcaccaagg tggagatcaa gaggggagga ggaggatccg gaggaggagg atctggcggc 360

ggcggcagcc aggtgcagct ggtggagtcc ggaggaggcg tggtgcaacc tggcaggtcc 420

ctgaggctgg actgcaaggc ctccggcatc accttctcca acagcggcat gcactgggtg 480

aggcaggctc ctggaaaggg cctggagtgg gtggccgtga tctggtacga cggctccaag 540

aggtactacg ccgactccgt gaagggcagg ttcaccatct ccagggacaa ctccaagaac 600

accctgttcc tgcagatgaa ctccctgagg gccgaggaca ccgccgtgta ctactgcgcc 660

accaacgacg actactgggg acagggcacc ctggtgaccg tgtcctccgc ttccaccaag 720

ggcggaggcg ggtcaggagg aggagggagc ggaggaggag ggagcggagg cgggggaagt 780

ggatccgaaa tcgtgctgac acagtctcca ggaacactgt ctctgtctcc aggagaaaga 840

gctaccctgt cttgtagagc ttctcagtca gtgggatcct cttacctggc ttggtaccag 900

cagaaaccag gacaggctcc tagactgctg atttatggcg ctttttctag agctaccggc 960

atccccgata gattttccgg ctccggatcc ggaacagatt tcacactgac catctccaga 1020

ctggagccag aagactttgc cgtgtactat tgccagcagt acggctcttc tccttggaca 1080

tttggacagg gaacaaaggt ggagatcaag agaggaggag gaggttccgg aggaggagga 1140

tctggcggcg gcggcagcga ggtgcagctg ctggagtccg gcggcggcgt ggtgcagcca 1200

ggcaggtctc tgaggctgag ctgtgcagca tccggcttca ccttttctag ctacacaatg 1260

cactgggtgc gccaggcacc tggcaaggga ctggagtggg tgaccttcat ctcttatgac 1320

ggcaacaata agtactatgc cgatagcgtg aagggcaggt ttaccatctc tcgcgataac 1380

agcaagaata cactgtacct gcagatgaac agcctgcggg ccgaggacac cgccatctac 1440

tattgcgcaa gaacaggatg gctgggaccc ttcgattact ggggccaggg caccctggtg 1500

acagtgtcct ctgagagcaa gtatggacca ccttgcccac catgtcctgc accagagttt 1560

ctgggcggcc catccgtgtt cctgtttcct ccaaagccca aggacaccct gatgatctcc 1620

cggaccccag aggtgacatg cgtggtggtg gacgtgtctc aggaggatcc cgaggtgcag 1680

ttcaactggt acgtggatgg cgtggaggtg cacaatgcca agacaaagcc aagggaggag 1740

cagtttaatt ccacctaccg cgtggtgtct gtgctgacag tgctgcacca ggactggctg 1800

aacggcaagg agtataagtg caaggtgagc aataagggcc tgcccagctc catcgagaag 1860

accatctcca aggcaaaggg acagcccagg gagcctcagg tgtacacact gccccctagc 1920

caggaggaga tgaccaagaa ccaggtgtcc ctgacatgtc tggtgaaggg cttctatccc 1980

tccgacatcg ccgtggagtg ggagtctaat ggccagcctg agaacaatta caagaccaca 2040

ccacccgtgc tggactccga tggctctttc tttctgtata gccggctgac cgtggataag 2100

tccagatggc aggagggcaa cgtgttttct tgcagcgtga tgcacgaagc actgcacaat 2160

cactacactc agaagtccct gtccctgtcc ctgggcaaat ga 2202

<210> 8

<211> 2202

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 8

gaaatcgtgc tgacacagtc tccaggaaca ctgtctctgt ctccaggaga aagagctacc 60

ctgtcttgta gagcttctca gtcagtggga tcctcttacc tggcttggta ccagcagaaa 120

ccaggacagg ctcctagact gctgatttat ggcgcttttt ctagagctac cggcatcccc 180

gatagatttt ccggctccgg atccggaaca gatttcacac tgaccatctc cagactggag 240

ccagaagact ttgccgtgta ctattgccag cagtacggct cttctccttg gacatttgga 300

cagggaacaa aggtggagat caagagagga ggaggaggat ccggaggagg aggatctggc 360

ggcggcggca gcgaggtgca gctgctggag tccggcggcg gcgtggtgca gccaggcagg 420

tctctgaggc tgagctgtgc agcatccggc ttcacctttt ctagctacac aatgcactgg 480

gtgcgccagg cacctggcaa gggactggag tgggtgacct tcatctctta tgacggcaac 540

aataagtact atgccgatag cgtgaagggc aggtttacca tctctcgcga taacagcaag 600

aatacactgt acctgcagat gaacagcctg cgggccgagg acaccgccat ctactattgc 660

gcaagaacag gatggctggg acccttcgat tactggggcc agggcaccct ggtgacagtg 720

tcctctggcg gaggcgggtc aggaggagga gggagcggag gaggagggag cggaggcggg 780

ggaagtggat ccgagatcgt gctgacccag tcccccgcta cactgtccct gtcccctgga 840

gagagggcca ccctgtcctg cagggctagc cagagcgtgt cctcctacct ggcctggtat 900

cagcagaagc ccggccaagc tcccaggctg ctgatctacg acgcctccaa cagggccaca 960

ggcatccctg ccaggttctc cggttctggt tctggcaccg acttcaccct gaccatctcc 1020

tccctggagc ccgaggactt cgccgtgtac tactgccagc agtcctccaa ctggcccagg 1080

accttcggcc agggcaccaa ggtggagatc aagaggggcg gcggcggctc tggaggagga 1140

ggaagcggag gaggaggatc ccaggtgcag ctggtggagt ccggaggagg cgtggtgcaa 1200

cctggcaggt ccctgaggct ggactgcaag gcctccggca tcaccttctc caacagcggc 1260

atgcactggg tgaggcaggc tcctggaaag ggcctggagt gggtggccgt gatctggtac 1320

gacggctcca agaggtacta cgccgactcc gtgaagggca ggttcaccat ctccagggac 1380

aactccaaga acaccctgtt cctgcagatg aactccctga gggccgagga caccgccgtg 1440

tactactgcg ccaccaacga cgactactgg ggacagggca ccctggtgac cgtgtcctcc 1500

gcttccacca aggagtccaa gtatggacca ccttgcccac catgtcctgc accagagttt 1560

ctgggcggcc ctagcgtgtt cctgtttcct ccaaagccaa aggatacact gatgatcagc 1620

cgcacacctg aggtgacctg cgtggtggtg gacgtgagcc aggaggaccc agaggtgcag 1680

ttcaactggt acgtggacgg cgtggaggtg cacaatgcca agaccaagcc acgggaggag 1740

cagtttaact ctacatacag agtggtgagc gtgctgaccg tgctgcacca ggactggctg 1800

aacggcaagg agtataagtg caaggtgtct aataagggcc tgccttcctc tatcgagaag 1860

acaatcagca aggccaaggg ccagcccaga gagcctcagg tgtacaccct gcccccttct 1920

caggaggaga tgacaaagaa ccaggtgagc ctgacctgtc tggtgaaggg cttctatcca 1980

agcgatatcg ccgtggagtg ggagtccaat ggccagcccg agaacaatta caagacaacc 2040

ccacccgtgc tggactccga tggctctttc tttctgtatt ccaggctgac cgtggacaag 2100

tctcgctggc aggagggcaa cgtgttcagc tgcagcgtga tgcacgaagc actgcacaat 2160

cactacactc agaaatcact gtcactgtca ctggggaagt ga 2202

<210> 9

<211> 720

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 9

gagatcgtgc tgacccagtc ccccgctaca ctgtccctgt cccctggaga gagggccacc 60

ctgtcctgca gggctagcca gagcgtgtcc tcctacctgg cctggtatca gcagaagccc 120

ggccaagctc ccaggctgct gatctacgac gcctccaaca gggccacagg catccctgcc 180

aggttctccg gttctggttc tggcaccgac ttcaccctga ccatctcctc cctggagccc 240

gaggacttcg ccgtgtacta ctgccagcag tcctccaact ggcccaggac cttcggccag 300

ggcaccaagg tggagatcaa gaggggcggc ggcggctctg gaggaggagg aagcggagga 360

ggaggatccc aggtgcagct ggtggagtcc ggaggaggcg tggtgcaacc tggcaggtcc 420

ctgaggctgg actgcaaggc ctccggcatc accttctcca acagcggcat gcactgggtg 480

aggcaggctc ctggaaaggg cctggagtgg gtggccgtga tctggtacga cggctccaag 540

aggtactacg ccgactccgt gaagggcagg ttcaccatct ccagggacaa ctccaagaac 600

accctgttcc tgcagatgaa ctccctgagg gccgaggaca ccgccgtgta ctactgcgcc 660

accaacgacg actactgggg acagggcacc ctggtgaccg tgtcctccgc ttccaccaag 720

<210> 10

<211> 726

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 10

gaaatcgtgc tgacacagtc tccaggaaca ctgtctctgt ctccaggaga aagagctacc 60

ctgtcttgta gagcttctca gtcagtggga tcctcttacc tggcttggta ccagcagaaa 120

ccaggacagg ctcctagact gctgatttat ggcgcttttt ctagagctac cggcatcccc 180

gatagatttt ccggctccgg atccggaaca gatttcacac tgaccatctc cagactggag 240

ccagaagact ttgccgtgta ctattgccag cagtacggct cttctccttg gacatttgga 300

cagggaacaa aggtggagat caagagagga ggaggaggat ccggaggagg aggatctggc 360

ggcggcggca gcgaggtgca gctgctggag tccggcggcg gcgtggtgca gccaggcagg 420

tctctgaggc tgagctgtgc agcatccggc ttcacctttt ctagctacac aatgcactgg 480

gtgcgccagg cacctggcaa gggactggag tgggtgacct tcatctctta tgacggcaac 540

aataagtact atgccgatag cgtgaagggc aggtttacca tctctcgcga taacagcaag 600

aatacactgt acctgcagat gaacagcctg cgggccgagg acaccgccat ctactattgc 660

gcaagaacag gatggctggg acccttcgat tactggggcc agggcaccct ggtgacagtg 720

tcctct 726

<210> 11

<211> 114

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 11

atggacgcca tgctgcgcgg actgtgctgc gtgctgctac tgtgcggcgc cgtgttcgtg 60

agccccagcc aggagatcca cgcccgattc aggagaggag ccagaggagg atcc 114

<210> 12

<211> 22

<212> PRT

<213>artificial sequence (Artificial Sequence)

<400> 12

Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly

1 5 10 15

Gly Gly Gly Ser Gly Ser

20

<210> 13

<211> 690

<212> DNA

<213>artificial sequence (Artificial Sequence)

<400> 13

gagtccaagt atggaccacc ttgcccacca tgtcctgcac cagagtttct gggcggccct 60

agcgtgttcc tgtttcctcc aaagccaaag gatacactga tgatcagccg cacacctgag 120

gtgacctgcg tggtggtgga cgtgagccag gaggacccag aggtgcagtt caactggtac 180

gtggacggcg tggaggtgca caatgccaag accaagccac gggaggagca gtttaactct 240

acatacagag tggtgagcgt gctgaccgtg ctgcaccagg actggctgaa cggcaaggag 300

tataagtgca aggtgtctaa taagggcctg ccttcctcta tcgagaagac aatcagcaag 360

gccaagggcc agcccagaga gcctcaggtg tacaccctgc ccccttctca ggaggagatg 420

acaaagaacc aggtgagcct gacctgtctg gtgaagggct tctatccaag cgatatcgcc 480

gtggagtggg agtccaatgg ccagcccgag aacaattaca agacaacccc acccgtgctg 540

gactccgatg gctctttctt tctgtattcc aggctgaccg tggacaagtc tcgctggcag 600

gagggcaacg tgttcagctg cagcgtgatg cacgaagcac tgcacaatca ctacactcag 660

aaatcactgt cactgtcact ggggaagtga 690

Claims

1. the single-gene bispecific antibody that one kind can target CTLA4 and PD-1, which is characterized in that successively include from N-terminal to C-terminal First targeted integration area, the second targeted integration area and the area Fc, first targeted integration area and second targeted integration area are logical Junction fragment connection is crossed, first targeted integration area targets one in CTLA4 and PD-1, second targeted integration area target To another.

2. single-gene bispecific antibody according to claim 1, which is characterized in that the first or second targeted integration The sequence in area is as shown in SEQ ID NO:1, and the sequence in the described second or first targeted integration area is as shown in SEQ ID NO:2.

3. according to right want 1 described in single-gene bispecific antibody, which is characterized in that the sequence in the area Fc such as SEQ ID Shown in NO:3.

4. single-gene bispecific antibody according to any one of claim 1-3, which is characterized in that the junction fragment It is made of one or more unit sequences, the unit sequence is as shown in SEQ ID NO:4.

5. single-gene bispecific antibody according to claim 1, which is characterized in that the sequence such as institute of SEQ ID NO:5 or 6 Show.

6. single-gene bispecific antibody of any of claims 1-5 is in preparation for answering in anti-tumor drug With.

7. application according to claim 6, which is characterized in that the anti-tumor drug is for treating colon cancer, melanin One of tumor, lung cancer, prostate cancer, cervical carcinoma, bladder cancer are a variety of.

8. a kind of AAV bispecific antibody expression vector, which is characterized in that include list of any of claims 1-5 The DNA encoding sequence expression cassette of gene bispecific antibody.

9. AAV bispecific antibody expression vector according to claim 8, which is characterized in that the single-gene is double special The DNA encoding sequence of property antibody is as shown in SEQ ID NO:7 or 8.

10. AAV bispecific antibody expression vector according to claim 8 or claim 9, which is characterized in that single-gene is double special Property antibody DNA encoding sequence 5 ' end be also connected with signal coding sequence.