CN104558194B

CN104558194B - A kind of anti-CD20-Flex bifunctional fusion proteins, preparation method and the usage

Info

Publication number: CN104558194B
Application number: CN201310486424.9A
Authority: CN
Inventors: 钱卫珠
Original assignee: Taizhou Mai Bartec Pharmaceutical Co Ltd
Current assignee: Taizhou Mai BARTEC Pharmaceutical Co. Ltd.
Priority date: 2013-10-17
Filing date: 2013-10-17
Publication date: 2018-04-27
Anticipated expiration: 2033-10-17
Also published as: AU2013403112B2; CN104558194A; WO2015054958A1; AU2013403112A1

Abstract

The invention discloses a kind of anti-CD20 Flex bifunctional fusion proteins, its preparation method and its application in antitumor drug is prepared.More specifically, the invention discloses have the function of similar whole antibody structure and bifunctional fusion proteins CD20 Flex, its amino acid sequence, preparation method and its application in antitumor drug is prepared, it can both be combined with CD20, there is Flt3 ligands again, with anti-personnel while can effective excitating organism tumor-specific immunity.

Description

A kind of anti-CD20-Flex bifunctional fusion proteins, preparation method and the usage

Technical field

The invention belongs to biological technical field, more specifically, the invention discloses a kind of anti-CD20 bifunctional fusion proteins, Its preparation method and its application in antibody tumor medicine is prepared.Anti- CD20 bifunctional fusion proteins disclosed by the invention, tool There are the CD20-Flex bifunctional fusion proteins of similar whole antibody structure and function, can not only be combined with CD20, but also match somebody with somebody with Flt3 The function of body.

Background technology

Tumour especially malignant tumour is the disease that the world today seriously endangers human health, and institute is lethal in various diseases Second is occupied in dying.And in recent years, its incidence is in obvious ascendant trend.Therapeutic effect of malignant tumour is poor, the late period rate of transform How bad height, prognosis be.Although current clinically used conventional treatments such as radiotherapy, chemotherapy and operative treatment are in very great Cheng Slight illness is alleviated on degree, extends life span, but these methods, there are significant limitation, its curative effect is difficult to further carry It is high.

Antibody target tropism medicine has the advantages that good specificity, Small side effects, cycle of partly declining are long, widely should now Clinical treatment for tumour.Antibody drug has been achieved for encouraging therapeutic effect in clinical treatment.But due to The heterogeneity and complexity of tumorigenesis process, the antibody target medicine only specific targeting one of single target spot Target protein plays the effects such as killing, blocking, it is difficult to significantly more efficient killing tumor cell, excitating organism be immunized, overcome it is swollen Knurl recurs.Therefore, antibody research field is concentrated mainly on the affinity of raising antibody, finds that new antibody target is marked with and makes at present Standby more targeting antibodies etc..The method of affinity of antibody has been improved very using CAD and library technology Maturation, finds new drug targets with the use of mass spectrum detection using proteomic techniques, has also been achieved.Now Method have been able to quickly to prepare affinity and reach the antibody of 1nM, new drug targets also constantly carry out it is preclinical and In clinical test.However, antibody variable region is mainly transformed into single-chain antibody, this side by the method for preparing multivalent antibody at present The universal affinity of antibody of multivalent antibody that method obtains is not high, and curative effect is also not reaching to expected effect.The DVD antibody occurred recently That correspondence is merged respectively by the heavy chain variable region of two kinds of antibody and light chain variable region, formed HV1-linker-HV2 and The structure of LV1-linker-LV2.Although this DVD antibody overcomes the shortcomings that single-chain antibody affinity is low, but due to molecule Measure excessive, antibody structure to change greatly, its expression quantity can be caused not high；Moreover, when protein target molecule amount is larger or in cell When being formed on film compared with large complex, combination of the DVD antibody to double target spots can be influenced.

Rituximab（Rituximab）Trade name Mabthera, is a kind of chimeric mAb of anti-CD20, It is used to treat B cell non-Hodgkin lymphoma by U.S. FDA approval within 1997, follicular lymphoma can with CHOP use in conjunction For treating other B cell lymphomas such as diffusivity large B cell lymphoid tumor.

Although the not so further investigation to antibodies for antitumor therapy mechanism, it is now recognized that：Antibody is by CDC, ADCC and lures Guided cell death can significantly killing tumor cell, suppress the growth of tumour, but more and more evidence shows that antibody lures The body specific immunity killing led plays an important role during tumor recurrence is overcome.

Flt3 ligand extracellular regions（Flex）It can not only make the quantity of marrow increase production DC, and can tend to the function of DC Maturation, makes the latter have stronger antigen submission and antitumor action, it is often more important that, Flex can also make ripe DC from bone Marrow is discharged into peripheral tissues, and experiment in vivo confirms after applying Flex that DC is in mouse spleen, marrow, lymph node, liver Time dependence increase.Therefore, prepare a kind of with lethal while effectively excitating organism tumor-specific immunity can resist Body, is a hot spot of current antibody research.

The content of the invention：

To solve the above-mentioned problems, the present inventor is studied for a long period of time, through a large number of experiments, utilizes genetic engineering Technique construction is a kind of have the function of similar whole antibody structure and CD20-Flex bifunctional fusion proteins, the bifunctional fusion Albumen can not only be combined with CD20, but also have the function of Flt3 ligands.

The invention discloses：

1. it is a kind of have the function of similar whole antibody structure and CD20-Flex bifunctional fusion proteins, it is characterized in that, both may be used With with CD20 antigen bindings, and there is Flt3 ligands.

It is respectively Flex-CL-Hinge-CH2-CH3 2. above-mentioned bifunctional fusion proteins, are made of three peptide chains（SEQ ID NO ：18 ）Rituximab heavy chain knob mutant（SEQ ID NO ：16 ）Rituximab light chains（SEQ ID NO ： 10 ）.

3. a kind of separated nucleic acid molecule, encodes three above-mentioned peptide chains, there is SEQ ID NO：17、SEQ ID NO：15 、SEQ ID NO：9 nucleotide sequences.

4. a kind of carrier, the expression tune being connected containing the upper nucleic acid molecules with the series of operations of the nucleic acid molecules Sequence is controlled, wherein carrier can be pDR1, pcDNA3.1（+）, pcDNA3.1/ZEO（+）One of, pDHFR.

5. above-mentioned carrier, is pcDNA3.1（+）Or pcDNA3.1/ZEO（+）.

6. a kind of host cell, is eukaryotic containing above-mentioned carrier.

7. above-mentioned host cell, is mammalian cell.

8. above-mentioned host cell, is Chinese hamster ovary celI.

9. one kind prepares above-mentioned CD20-Flex bifunctional fusion proteins, this method includes：

A) CD20 antibody rituximab variable regions and Flt3 ligand membrane outskirts are cloned respectively；

B) knob mutant is built respectively in antibody Fc region：T366W, S354C；Hole mutant：T366S, L368A, Y407V and Y394C；

C) Flt3 ligand membranes outskirt is constructed into Flex-CL with antibody light chain constant region and merges segment；

D) rituximab heavy chain variable regions being merged with knob mutant respectively, Flex-CL is merged with hole mutant, Load expression vector；

E) build two expression vectors and the expression vector equipped with rituximab light chain genes are transfected into progress jointly Expression, isolates and purifies.

10. a kind of composition, contains above-mentioned CD20-Flex bifunctional fusion proteins, and pharmaceutically acceptable carrier.

11. purposes of the above-mentioned CD20-Flex bifunctional fusion proteins in antibody tumor medicine is prepared.

12. purposes of the above-mentioned composition in antitumor drug is prepared.

13. any of the above-described purposes, further includes and other antitumor drugs are used in combination.

The object of the present invention is to provide one kind have the function of similar whole antibody structure and CD20-Flex bifunctional fusion eggs In vain, it can not only be combined with CD20, but also there is Flt3 ligands.The antibody-like had both remained the structure of similar antibody and big It is small, there is the affinity similar with parental antibody and half-life period, also remain CD20 antibody killing tumour similar CDC, ADCC With the ability of inducing cell death, and combine Flt3 ligands can tumor vicinity induce DC and NK cells propagation and into Ripe, excitating organism tumor-specific immunity, overcomes the recurrence of tumour.

The present invention has carried out deep understanding to antibody structure, on this basis, establishes a kind of both retained similar to antibody Structure has the fusion protein of Flt3 ligand functions with function again, its structure is as shown in Figure 1.In order to prove this method have than The effect of rituximab antibody and more preferable Flex-Ig drug combinations, the present invention carries out the experiment of following protein antitumor action. In principle, method of the invention can be widely used in various antibody and Flex structure bifunctional fusion proteins, accelerate research and development Fusion protein with biology and medical significance.Meanwhile antibody variable region is combined structure bifunctional fusion egg with Flex White method, a kind of new thinking is provided to carry out the design of bifunctional fusion proteins later.

In the present invention, any suitable carrier can use, and can be pDR1, pcDNA3.1（+）, pcDNA3.1/ZEO （+）One of, pDHFR, expression vector includes being connected with the fusion dna sequence of suitable transcription and translation regulatory sequence.

Mammal or the expression of insect host cell culture systems bifunctional fusion proteins for use in the present invention, COS , CHO, NS0, the cell such as sf9 and sf21 may be applicable to the present invention.

Available host cell is the prokaryotic containing above-mentioned carrier, can be DH5a, BL21 (DE3), one of TG1.

The preparation method of the bifunctional fusion proteins with antibody structure and functionality advantage disclosed in the present invention is in table Under the conditions of reaching, above-mentioned host cell is cultivated, so that bifunctional fusion proteins are expressed, the bifunctional fusion described in isolated or purified Albumen.

Bifunctional fusion proteins disclosed by the invention can be isolated and purified using the method for affinity chromatography, according to institute The characteristic of the affinity column utilized, can use conventional method such as high-salt buffer, change the methods of PH elution of bound in parent With the bifunctional fusion proteins on column.

Using the above method, it is substantially uniform material that can purify bifunctional fusion proteins, such as in SDS-PAGE It is single band on electrophoresis.

According to a preferred embodiment of the present invention, a kind of bifunctional fusion proteins include the antigen binding of rituximab Area and Flt3 ligand extracellular regions.

A kind of bifunctional fusion proteins have antibody structure and functionality advantage, and its preparation method comprises the following steps：

1）Rituximab heavy chain of antibody variable region and Flt3 ligand extracellular regions gene are cloned respectively；

2）The gene of Flt3 ligand extracellular regions is merged with antibody light chain constant region, structure Flex-CL fusion fragments；

3）Knob mutant is built to antibody Fc region respectively：T366W, S354C；Hole mutant：T366S, L368A, Y407V and Y394C；

4）Rituximab heavy chain variable regions are merged with knob mutant respectively, Flex-CL is merged with hole mutant, Load expression vector；

5）Above-mentioned expression vector and rituximab antibody light chains are subjected to corotation expression, by isolating and purifying to obtain double work( Can fusion protein.

Above-mentioned complete weight, the light chain gene built is respectively charged into eukaryotic expression vector pcDNA3.1（+） (Invitrogen Products).Above-mentioned plasmid one reinstates liposome transfection CHO-K1 cells（ATCC）, and with containing 600 μ g/ The cell clone of expression bifunctional fusion proteins is stablized in the Selective agar medium screening of ml G418.Using Protein A chromatographic columns, Purified by affinity chromatography from the supernatant of cell culture and obtain bifunctional fusion proteins.

Above-mentioned bifunctional fusion proteins or preparation are applied in the medicine for preparing anticancer, are further included and other antineoplastics Thing is used in combination.

The present invention discloses above-mentioned bifunctional fusion proteins, can form medicine system together with pharmaceutically acceptable auxiliary material For agent composition so as to more stably play curative effect, these preparations can ensure bifunctional fusion proteins amino acid disclosed by the invention The conformation integrality of core sequence, while also want the polyfunctional group of protected protein matter to prevent its degraded（Including but not limited to agglomerate, Deamination or oxidation）.Under normal conditions, for liquid preparation, it can usually preserve and at least stablize 1 year under the conditions of 2 °C -8 °C, For lyophilized formulations, keep within least six months stablizing at 30 °C.Herein preparation can be the common suspension of pharmaceutical field, liquid drugs injection, It is lyophilized to wait preparation, preferably liquid drugs injection or lyophilized formulations, the liquid drugs injection or lyophilized for above-mentioned bifunctional fusion proteins disclosed by the invention Preparation, pharmaceutically acceptable auxiliary material include one of surfactant, solution stabilizer, isotonic regulator and buffer solution or its Combination, wherein surfactant includes nonionic surface active agent such as Polyoxyethylene Sorbitol Fatty Acid Esters（Polysorbas20 or 80）；poloxamer（Such as poloxamer 188）；Triton；Lauryl sodium sulfate（SDS）；Sodium Laurylsulfate；The tetradecane Base, sub- oil base or octadecyl methyl amimoacetic acid；Pluronics；MONAQUAT^TMDeng its addition should make bifunctional fusion proteins Granulating trend is minimum, and solution stabilizer can be carbohydrate, including reducing sugar and nonreducing sugar, amino acids include paddy ammonia Sour list sodium or histidine, alcohols include one or a combination set of trihydroxylic alcohol, advanced sugar alcohol, propane diols, polyethylene glycol, solution stabilizer The addition preparation those skilled in the art that should make to eventually form think to reach and keep stablizing shape in the stable time State, isotonic regulator can be one of sodium chloride, mannitol, and buffer solution can be TRIS, histidine buffering liquid, phosphate-buffered One of liquid.

Above-mentioned preparation is the composition comprising bifunctional fusion proteins, after to animal administration including people, is resisted swollen Knurl positive effect.Specifically, the prevention to tumour and/or treatment are effective, can be used as antitumor drug.

Bifunctional fusion proteins and combinations thereof are being to animal administration including people in the present invention, dosage because The age of patient and weight, disease traits and seriousness, and method of administration and it is different, may be referred to the result and kind of zoopery Kind situation, total dosage is no more than a certain range.The dosage being specifically injected intravenously is 0.1~3000mg/ days.

Antitumor drug alleged by the present invention, refers to the medicine for suppressing and/or treating tumour, can include with tumour The delay of related symptoms development and/or the reduction of these severity of symptom are grown, it further comprises already present tumour Grow the mitigation of simultaneous phenomenon and prevent the appearance of other symptoms, still reduce or prevent to shift.

Bifunctional fusion proteins disclosed by the invention and combinations thereof can also be used with other antineoplastic administering drug combinations In the treatment of tumour, these antineoplastics include 1, cytotoxic drug（1）Act on the medicine of DNA chemical constitutions：Alkanisation Agent such as nitrogen mustards, nitrous urine class, pyrovinic acid esters；Platinum-like compounds such as cis-platinum, carboplatin and oxalic acid platinum etc.；Mitomycin （MMC）；（2）Influence the medicine of nucleic acid synthesis：Dihydrofolate reductase inhibitor such as methopterin（MTX）With Alimta etc.； Thymus nucleoside synthetase inhibitors such as fluorouracil（5FU, FT-207, capecitabine）Deng；Purine nucleosides synthetase inhibitors Such as Ismipur（6-MP）With 6-TG etc.；Ribonucleotide reductase inhibitor such as hydroxycarbamide（HU）Deng；DNA polymerases suppress Agent such as cytarabine（Ara-C）And gemzar（Gemz）Deng；（3）Act on the medicine of transcribed nucleic acid：Selectively acting is in DNA moulds Plate, suppresses DNA dependenc RNA polymerases, so as to suppress the medicine of RNA synthesis such as：Actinomycin D, daunorubicin, adriamycin, table Adriamycin, aclacinomycin, mithramycin etc.；（4）Mainly act on the medicine of tubulin synthesis：Taxol, taxotere, length Spring flower alkali, vinorelbine, Podophyllum emodi var chinense alkali class, homoharringtonine；（5）Other Cytotoxic drugs：L-Asparaginasum mainly suppresses albumen The synthesis of matter；2nd, steroids antiestrogenic：Tamoxifen, Droloxifene, Exemestane etc.；Arimedex：Ammonia Shandong Meter Te, Lactel are grand, Letrozole, auspicious Ningde etc.；Antiandrogen：Its ammonia RH-LH agonist/antagonist of fluorine：Zoladex, enatone Deng；3rd, biological response modifiers：Tumour interferon is mainly suppressed by body's immunity；Interleukin 2；Thymus gland Peptides；4th, monoclonal antibody：Mabthera (MabThera)； Cetuximab (C225) ；Trastuzumab (Trastuzumab) Bevacizumab (Avastin) ；5th, it is unknown and need the medicine further studied to include some current mechanism for other；Cell Differentiating inducer such as retinoids；Cell death inducer.Bifunctional fusion proteins disclosed by the invention and combinations thereof can be with One or a combination set of above-mentioned antitumor drug drug combination.

Brief description of the drawings

Fig. 1 CD20-Flex BiFP bifunctional fusion proteins structure diagrams.

Fig. 2 .Rituximab heavy chain knob mutant structure figures.

Fig. 3 Flex-CL-Hinge-CH2-CH3 structure charts.

Affinity of Fig. 4 CD20-Flex BiFP to CD20.

The CDC killing activities of Fig. 5 CD20-Flex BiFP.

The ADCC killing activities of Fig. 6 CD20-Flex BiFP.

The apoptotic activity of Fig. 7 CD20-Flex BiFP.

The mouse interior tumor protection activity of Fig. 8 CD20-Flex BiFP.

Embodiment

The clone of 1. rituximab antibody heavy chain variable region genes of embodiment

Rituximab heavy chain variable region genes, reaction condition are cloned by PCR using rituximab heavy chain of antibody as template For：95 DEG C 15 minutes；94 DEG C 50 seconds, 58 DEG C 50 seconds, 72 DEG C 50 seconds, 30 circulation；72 DEG C 10 minutes, obtain PCR product rituximab HV.Antibody signal peptide amino acid sequence MGWSCIILFLVATATGVHS.Correct cloned sequence is obtained through sequencing It is spare.SEQ ID NO：The amino acid sequence of 2 display rituximab heavy chain variable regions, its nucleotides sequence are classified as SEQ ID NO： 1。

The clone of 2. Flt3 ligand variable region genes of embodiment

Use lymphocyte separation medium（Ancient cooking vessel state biotech development Products）Separating health human lymphocyte, uses Trizol Reagent (Invitrogen Products) extracts total serum IgE, according to document（Cloned human and mouse kappa immunoglobulin constant and J region genes conserve homology in functional segments. Hieter PA, Max EE, Seidman JG, Maizel JV Jr, Leder P. Cell. 1980 Nov;22(1 Pt 1):197-207.）And document（The nucleotide sequence of a human immunoglobulin C gamma1 gene. Ellison JW, Berson BJ, Hood LE. Nucleic Acids Res. 1982 Jul 10;10(13):4071-9.）Amplification Flt3 ligand extracellular fragment genes are reacted using RT-PCR.PCR product Purified through agarose gel electrophoresis and recycle and be cloned into pGEM-T carriers, confirm to obtain correct clone after sequence verification. SEQ ID NO：4 display Flex amino acid sequences, its nucleotides sequence are classified as SEQ ID NO：3.

3. antibody light chain constant region of embodiment, the clone in Fc areas

Use lymphocyte separation medium（Ancient cooking vessel state biotech development Products）Separating health human lymphocyte, uses Trizol Reagent (Invitrogen Products) extracts total serum IgE, according to document（Cloned human and mouse kappa immunoglobulin constant and J region genes conserve homology in functional segments. Hieter PA, Max EE, Seidman JG, Maizel JV Jr, Leder P. Cell. 1980 Nov;22(1 Pt 1):197-207.）And document（The nucleotide sequence of a human immunoglobulin C gamma1 gene. Ellison JW, Berson BJ, Hood LE. Nucleic Acids Res. 1982 Jul 10;10(13):4071-9.）Amplification antibody light chain constant region and Fc areas gene are reacted using RT-PCR. PCR product purifies through agarose gel electrophoresis and recycles and be cloned into pGEM-T carriers, confirms to obtain after sequence verification correct Clone.SEQ ID NO：6 display CL amino acid sequences, its nucleotides sequence are classified as SEQ ID NO：5；SEQ ID NO：8 displays Fc amino acid sequences, its nucleotides sequence are classified as SEQ ID NO：7.

The structure of 4. antibody Fc district knob mutant of embodiment

The antibody Fc district that will be obtained in embodiment 3, catastrophe point is introduced using the method for overlap PCR： T366W, S354C(Schaefer WW, Regula JTJ, Bähner MM, et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies. Proc Natl Acad Sci U S A.2011;108(27):11187–11192.).PCR product is through fine jade Sepharose Purified in electrophoresis is recycled and is cloned into pGEM-T carriers, confirms to obtain correct clone after sequence verification.SEQ ID NO：12 display Fc-knob amino acid sequences, its nucleotides sequence are classified as SEQ ID NO：11.

The structure of 5. antibody Fc district hole mutant of embodiment

The antibody Fc district that will be obtained in embodiment 3, catastrophe point is introduced using the method for overlap PCR：T366S, L368A, Y407V and Y394C (Schaefer WW, Regula JTJ, B hner MM, et al. Immunoglobulin domain crossover as a generic approach for the production of bispecific IgG antibodies. Proc Natl Acad Sci U S A.2011;108(27):11187–11192.).PCR product is through fine jade Sepharose Purified in electrophoresis is recycled and is cloned into pGEM-T carriers, confirms to obtain correct clone after sequence verification.SEQ ID NO：14 display Fc-hole amino acid sequences, its nucleotides sequence are classified as SEQ ID NO：13.

The structure of 6. rituximab heavy chain mutants of embodiment

The Fc areas knob mutant that the rituximab heavy chain variable regions and embodiment 4 obtained using embodiment 1 obtains is mould Plate, rituximab heavy chain variable regions are merged, then filled using the method for overlap PCR with Fc areas knob mutant Enter expression vector, introduce double mutation S354C and T366W in the Fc constant regions of Rituximab heavy chains, build Rituximab heavy chains Knob mutant（Fig. 2）.SEQ ID NO：16 display rituximab heavy chain knob variant amino acid sequences, its nucleotides sequence It is classified as SEQ ID NO：15.

The structure of 7. Flex mutant of embodiment

The Flex obtained with embodiment 2, the Fc areas hole mutant that the CL and embodiment 5 that embodiment 3 obtains are obtained are Template, carries out segment composition by Flex and CL and Fc areas hole using the method for overlap PCR, is then charged into expression vector, Build Flex-CL-Hinge-CH2-CH3（Fig. 3）.SEQ ID NO：18 display Flex-CL-Hinge-CH2-CH3 amino acid sequences Row, its nucleotides sequence are classified as SEQ ID NO：17.

The expression and purification of 8. CD20-Flex bifunctional fusion proteins of embodiment

3 × 10 are inoculated with 3.5cm tissue culture dishes⁵CHO-K1 cells（ATCC CRL-9618）, cell culture Transfected when being merged to 90%-95%：Take 10 μ g rituximab heavy chain knob mutant of plasmid, Flex-CL-Hinge- CH2-CH3 and 4 μ g rituximab light chains（Li B, Zhao L, Guo H, et al. Characterization of a rituximab variant with potent antitumor activity against rituximab- resistant B-cell lymphoma. Blood. 2009;114(24):5007–5015.）With 20 μ l Lipofectamine2000 Reagent（Invitrogen Products）500 μ l plasma-free DMEM mediums are dissolved in respectively, Be stored at room temperature 5 minutes, will more than 2 kinds of liquid mix, incubation at room temperature 20 minutes is used so that DNA- liposome complexes are formed therebetween The DMEM culture mediums of 3ml serum-frees replace the serum-containing media in culture dish, then by the DNA- liposome complexes of formation It is added in plate, CO₂The DMEM complete mediums that 2ml contains 10% serum are added after when incubator culture 4 is small, are placed in CO₂Incubator relays Continuous culture.Cell is changed containing 600 μ g/ml G418 Selective agar mediums screening resistance clone after transfection carries out 24h.Take cell culture Supernatant detects screening high-expression clone with ELISA：Goat anti-human igg (Fc) is coated in elisa plate, and 4 °C overnight, are used 2%BSA- PBS closes 2h in 37 °C, adds resistance clone culture supernatant to be measured or standard items (Human myeloma IgG1, κ), 37 °C 2h is incubated, HRP- goat anti-human iggs (κ) is added and is combined reaction, 37 °C of incubation 1h, add TMB and act on 5min in 37 °C, most After use H₂SO₄Reaction is terminated, surveys A₄₅₀Value.Obtained high-expression clone serum free medium expansion culture will be screened, used Protein A affinity columns（GE Products）Isolate and purify bifunctional fusion proteins.Antibody purification is dialysed with PBS, most The concentration of purified antibodies is quantitatively determined with ultraviolet absorption method afterwards.

1. fusion protein affinity of experimental example detects

The affinity of all fusion proteins is measured by radioimmunoassays【Cragg MS, Morgan SM, Chan HT, Morgan BP, Filatov AV, Johnson PW, French RR, Glennie MJ (2003) Complement-mediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts. Blood 101(3): 1045-1052】.Briefly, fusion protein after purification, passes through iodine pearl method （iodobead method）It is marked.The fusion protein and Daudi (ATCC CCL-213) and 8266 of 125 iodine will be marked When cell incubation 2 is small, 37 °C.By centrifuging, detection is incorporated in antibody with cell combination and free iodine labeling The radioactivity of the antibody of 125 iodine labelings on Daudi cells is (see Fig. 4）.Fusion protein affinity constant passes through Hill equations Curve matching titration binding curve is tried to achieve.Affinity and document report of the CD20-Flex bifunctional fusion proteins to CD20 and Flt3 The parent rituximab in road is similar with the affinity of Flt3 ligands【Title: Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Author: Reff, M. E.; Carner, K.; Chambers, K. S. (...) Source: Blood, 1994, 83(2): 435-445；Turner AM, Lin NL, Issarachai S, Lyman SD, Broudy VC. FLT3 receptor expression on the surface of normal and malignant human hematopoietic cells. Blood. 1996;88 (9):3383-3390. ］.Wherein, the affinity numerical value to CD20 albumen is 4.82 ± 0.25 nM；To the affinity of Flt3 For 230 ± 40 pmol/L.

The CDC Function detections of 2. CD20-Flex fusion proteins of experimental example

Then Raji and Daudi cells add in the medium in no phenol red medium and small 37 degree of cultures 1 of fusion protein Enter 10% 37 degree of normal human serum be incubated 4 it is small when, then using the LDH kits of standard（Promega）It is detected CDC work Property.As shown in figure 5, fusion protein CD20-Flex BiFP have the CDC killing activity similar to rituximab.

The ADCC Function detections of 3. CD20-Flex fusion proteins of experimental example

Daudi cells（ATCC CCL-213）Under different fusion protein concentration, according to E:T is than 25：1 ratio is trained Support（37 degree, 4 it is small when）, then using the LDH kits of standard（Promega）It is detected ADCC activity.As shown in fig. 6, melt Hop protein CD20-Flex BiFP have the ADCC killing activity similar to rituximab.

The apoptosis function detection of 4. CD20-Flex fusion proteins of experimental example

Ramos cells and the CD20-Flex fusion proteins of 10 μ g/ml and the caspase inhibitor of various concentrations are incubated altogether Educate, while add the goat-anti people F (ab ') of 20 μ g/ml₂Fragment（Southern Biotechnology）.16 hours with Afterwards, the cell of apoptosis uses Annexin V-Fluos kits（BD companies）Carry out streaming list dye.As shown in fig. 7, CD20- After the crosslinking of Flex fusion proteins secondary antibody, the cell death of similarity degree after being crosslinked with rituximab can be induced, it is induced Cell death show the dose dependent of caspase inhibitor.

Tested in experimental example 5.CD20-Flex fusion protein bodies in Mice Body into knurl

By 5 × 10⁶Daudi or A20-CD20 cell tail vein injection BALB/c mouses, the 0th, 4 and 8 day to mouse The fusion protein treatment of 200 μ g of tail vein injection.42nd day, fusion protein treated non-tumor-bearing mice in its opposite side subcutaneous vaccination 1×10⁴A20-CD20 or A20 cells.The size of tumor-bearing mice tumour is observed, evaluates its therapeutic effect.As shown in figure 8, CD20-Flex fusion proteins can not only have the tumour of primary vaccination the lethal effect similar to rituximab, and Long-term tumor protection activity is still shown to the tumour being inoculated with again.

SEQUENCE LISTING

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

<120>A kind of anti-CD20-Flex bifunctional fusion proteins, preparation method and the usage

<130> 2013

<160> 18

<170> PatentIn version 3.5

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Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp Tyr Pro Val

20 25 30

Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly Gly Leu Trp

35 40 45

Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys Thr Val Ala

50 55 60

Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr Glu Ile His

65 70 75 80

Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys Leu Arg Phe

85 90 95

Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser Glu Gln Leu

100 105 110

Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser Arg Cys Leu

115 120 125

Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro Pro Trp Ser

130 135 140

Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln Pro Pro Leu

145 150 155 160

Leu Leu Leu Leu Leu Leu Pro Val Gly Leu Leu Leu Leu Ala Ala Ala

165 170 175

Trp Cys Leu His Trp Gln Arg Thr Arg Arg Arg Thr Pro Arg Pro Gly

180 185 190

Glu Gln Val Pro Pro Val Pro Ser Pro Gln Asp Leu Leu Leu Val Glu

195 200 205

His

<210> 5

<211> 318

<212> DNA

<213>It is artificial synthesized

<400> 5

gtggctgcac catctgtctt catcttcccg ccatctgatg agcagttgaa atctggaact 60

gcctctgttg tgtgcctgct gaataacttc tatcccagag aggccaaagt acagtggaag 120

gtggataacg ccctccaatc gggtaactcc caggagagtg tcacagagca ggacagcaag 180

gacagcacct acagcctcag cagcaccctg acgctgagca aagcagacta cgagaaacac 240

aaagtctacg cctgcgaagt cacccatcag ggcctgagct cgcccgtcac aaagagcttc 300

aacaggggag agtgttga 318

<210> 6

<211> 105

<212> PRT

<213>It is artificial synthesized

<400> 6

Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu

1 5 10 15

Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

Thr Lys Ser Phe Asn Arg Gly Glu Ser

100 105

<210> 7

<211> 684

<212> DNA

<213>It is artificial synthesized

<400> 7

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat ctcgggagga gatgaccaag 420

aaccaggtca gcctgacctg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta tagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ccccgggtaa atga 684

<210> 8

<211> 227

<212> PRT

<213>It is artificial synthesized

<400> 8

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Gly Lys

225

<210> 9

<211> 705

<212> DNA

<213>It is artificial synthesized

<400> 9

atggattttc aggtgcagat tttcagcttc ctgctaatca gtgcttcagt cataatgtcc 60

agaggacaaa ttgttctctc ccagtctcca gcaatcctgt ctgcatctcc aggggagaag 120

gtcacaatga cttgcagggc cagctcaagt gtaagttaca tccactggtt ccagcagaag 180

ccaggatcct cccccaaacc ctggatttat gccacatcca acctggcttc tggagtccct 240

gttcgcttca gtggcagtgg gtctgggacc tcttactctc tcacaatcag tagagtggag 300

gctgaagatg ctgccactta ttactgccag cagtggacta gtaacccacc cacgttcggt 360

ggtgggacca agctggagat caaacgaact gtggctgcac catctgtctt catcttcccg 420

ccatctgatg agcagttgaa atctggaact gcctctgttg tgtgcctgct gaataacttc 480

tatcccagag aggccaaagt acagtggaag gtggataacg ccctccaatc gggtaactcc 540

caggagagtg tcacagagca ggacagcaag gacagcacct acagcctcag cagcaccctg 600

acgctgagca aagcagacta cgagaaacac aaagtctacg cctgcgaagt cacccatcag 660

ggcctgagct cgcccgtcac aaagagcttc aacaggggag agtgt 705

<210> 10

<211> 213

<212> PRT

<213>It is artificial synthesized

<400> 10

Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly

1 5 10 15

Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr Ile

20 25 30

His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr

35 40 45

Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser

50 55 60

Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu

65 70 75 80

Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro Thr

85 90 95

Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

Asn Arg Gly Glu Cys

210

<210> 11

<211> 687

<212> DNA

<213>It is artificial synthesized

<400> 11

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtacacc ctgcccccat gccgggatga gctgaccaag 420

aaccaggtca gcctgtggtg cctggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcta cagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa atgatga 687

<210> 12

<211> 227

<212> PRT

<213>It is artificial synthesized

<400> 12

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Gly Lys

225

<210> 13

<211> 687

<212> DNA

<213>It is artificial synthesized

<400> 13

gacaaaactc acacatgccc accgtgccca gcacctgaac tcctgggggg accgtcagtc 60

ttcctcttcc ccccaaaacc caaggacacc ctcatgatct cccggacccc tgaggtcaca 120

tgcgtggtgg tggacgtgag ccacgaagac cctgaggtca agttcaactg gtacgtggac 180

ggcgtggagg tgcataatgc caagacaaag ccgcgggagg agcagtacaa cagcacgtac 240

cgtgtggtca gcgtcctcac cgtcctgcac caggactggc tgaatggcaa ggagtacaag 300

tgcaaggtct ccaacaaagc cctcccagcc cccatcgaga aaaccatctc caaagccaaa 360

gggcagcccc gagaaccaca ggtgtgcacc ctgcccccat cccgggatga gctgaccaag 420

aaccaggtca gcctgtcctg cgcggtcaaa ggcttctatc ccagcgacat cgccgtggag 480

tgggagagca atgggcagcc ggagaacaac tacaagacca cgcctcccgt gctggactcc 540

gacggctcct tcttcctcgt gagcaagctc accgtggaca agagcaggtg gcagcagggg 600

aacgtcttct catgctccgt gatgcatgag gctctgcaca accactacac gcagaagagc 660

ctctccctgt ctccgggtaa atgatga 687

<210> 14

<211> 227

<212> PRT

<213>It is artificial synthesized

<400> 14

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

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

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

115 120 125

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

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

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

195 200 205

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

210 215 220

Pro Gly Lys

225

<210> 15

<211> 1416

<212> DNA

<213>It is artificial synthesized

<400> 15

atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattcccag 60

gtacaactac agcagcctgg ggctgagctg gtgaagcctg gggcctcagt gaagatgtcc 120

tgcaaggctt ctggctacac atttaccagt tacaatatgc actgggtaaa gcagacacct 180

ggtcggggcc tggaatggat tggagctatt tatccaggaa atggtgatac ttcctacaat 240

cagaagttca agggcaaggc cacactgact gcagacaaat cctccagcac agcctacatg 300

cagctcagca gcctgacatc tgaagactct gcggtctatt actgtgcaag atcgacttac 360

tacggcggtg actggtactt caatgtctgg ggcgcaggga ccacggtcac cgtctctgca 420

gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 480

ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 540

tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 600

ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 660

tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 720

aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 780

ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 840

gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 900

tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 960

agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 1020

gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1080

aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatg ccgggatgag 1140

ctgaccaaga accaggtcag cctgtggtgc ctggtcaaag gcttctatcc cagcgacatc 1200

gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1260

ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1320

cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1380

cagaagagcc tctccctgtc tccgggtaaa tgatga 1416

<210> 16

<211> 470

<212> PRT

<213>It is artificial synthesized

<400> 16

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 Gln Gln Pro Gly Ala Glu Leu Val Lys

20 25 30

Pro Gly Ala Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe

35 40 45

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

50 55 60

Glu Trp Ile Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn

65 70 75 80

Gln Lys Phe Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser

85 90 95

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

100 105 110

Tyr Tyr Cys Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn

115 120 125

Val Trp Gly Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys

130 135 140

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

145 150 155 160

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

165 170 175

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

180 185 190

Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val

195 200 205

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

210 215 220

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

225 230 235 240

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

245 250 255

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

260 265 270

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

275 280 285

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

290 295 300

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

305 310 315 320

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

325 330 335

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

340 345 350

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

355 360 365

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

370 375 380

Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile

385 390 395 400

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

405 410 415

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

420 425 430

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

435 440 445

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

450 455 460

Ser Leu Ser Pro Gly Lys

465 470

<210> 17

<211> 1686

<212> DNA

<213>It is artificial synthesized

<400> 17

atgggatggt catgtatcat cctttttcta gtagcaactg caaccggtgt acattccacc 60

caggactgct ccttccaaca cagccccatc tcctccgact tcgctgtcaa aatccgtgag 120

ctgtctgact acctgcttca agattaccca gtcaccgtgg cctccaacct gcaggacgag 180

gagctctgcg ggggcctctg gcggctggtc ctggcacagc gctggatgga gcggctcaag 240

actgtcgctg ggtccaagat gcaaggcttg ctggagcgcg tgaacacgga gatacacttt 300

gtcaccaaat gtgcctttca gccccccccc agctgtcttc gcttcgtcca gaccaacatc 360

tcccgcctcc tgcaggagac ctccgagcag ctggtggcgc tgaagccctg gatcactcgc 420

cagaacttct cccggtgcct ggagctgcag tgtcagcccg actcctcaac cctgccaccc 480

ccatggagtc cccggcccct ggaggccaca gccccgacag ccccgcagcc ccctctgctc 540

ctcctactgc tgctgcccgt gggcctcctg ctgctggccg ctgcctggtg cctgcactgg 600

cagaggacgc ggcggaggac accccgccct ggggagcagg tgccccccgt ccccagtccc 660

caggacctgc tgcttgtgga gcacgtggct gcaccatctg tcttcatctt cccgccatct 720

gatgagcagt tgaaatctgg aactgcctct gttgtgtgcc tgctgaataa cttctacccc 780

agagaagcca aagtgcagtg gaaggtggac aacgccctgc agagcggaaa cagccaggaa 840

agcgtgacag agcaggattc caaggattcc acatacagcc tgagcagcac actgacactg 900

tccaaggccg actacgagaa gcacaaggtg tacgcctgcg aagtgacaca ccagggactg 960

tcctcccctg tgacaaagag cttcaacaga ggagaatgcg acaaaactca cacatgccca 1020

ccgtgcccag cacctgaact cctgggggga ccgtcagtct tcctcttccc cccaaaaccc 1080

aaggacaccc tcatgatctc ccggacccct gaggtcacat gcgtggtggt ggacgtgagc 1140

cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg gcgtggaggt gcataatgcc 1200

aagacaaagc cgcgggagga gcagtacaac agcacgtacc gtgtggtcag cgtcctcacc 1260

gtcctgcacc aggactggct gaatggcaag gagtacaagt gcaaggtctc caacaaagcc 1320

ctcccagccc ccatcgagaa aaccatctcc aaagccaaag ggcagccccg agaaccacag 1380

gtgtgcaccc tgcccccatc ccgggatgag ctgaccaaga accaggtcag cctgtcctgc 1440

gcggtcaaag gcttctatcc cagcgacatc gccgtggagt gggagagcaa tgggcagccg 1500

gagaacaact acaagaccac gcctcccgtg ctggactccg acggctcctt cttcctcgtg 1560

agcaagctca ccgtggacaa gagcaggtgg cagcagggga acgtcttctc atgctccgtg 1620

atgcatgagg ctctgcacaa ccactacacg cagaagagcc tctccctgtc tccgggtaaa 1680

tgatga 1686

<210> 18

<211> 560

<212> PRT

<213>It is artificial synthesized

<400> 18

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

1 5 10 15

Val His Ser Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser

20 25 30

Asp Phe Ala Val Lys Ile Arg Glu Leu Ser Asp Tyr Leu Leu Gln Asp

35 40 45

Tyr Pro Val Thr Val Ala Ser Asn Leu Gln Asp Glu Glu Leu Cys Gly

50 55 60

Gly Leu Trp Arg Leu Val Leu Ala Gln Arg Trp Met Glu Arg Leu Lys

65 70 75 80

Thr Val Ala Gly Ser Lys Met Gln Gly Leu Leu Glu Arg Val Asn Thr

85 90 95

Glu Ile His Phe Val Thr Lys Cys Ala Phe Gln Pro Pro Pro Ser Cys

100 105 110

Leu Arg Phe Val Gln Thr Asn Ile Ser Arg Leu Leu Gln Glu Thr Ser

115 120 125

Glu Gln Leu Val Ala Leu Lys Pro Trp Ile Thr Arg Gln Asn Phe Ser

130 135 140

Arg Cys Leu Glu Leu Gln Cys Gln Pro Asp Ser Ser Thr Leu Pro Pro

145 150 155 160

Pro Trp Ser Pro Arg Pro Leu Glu Ala Thr Ala Pro Thr Ala Pro Gln

165 170 175

Pro Pro Leu Leu Leu Leu Leu Leu Leu Pro Val Gly Leu Leu Leu Leu

180 185 190

Ala Ala Ala Trp Cys Leu His Trp Gln Arg Thr Arg Arg Arg Thr Pro

195 200 205

Arg Pro Gly Glu Gln Val Pro Pro Val Pro Ser Pro Gln Asp Leu Leu

210 215 220

Leu Val Glu His Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser

225 230 235 240

Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn

245 250 255

Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala

260 265 270

Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys

275 280 285

Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp

290 295 300

Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu

305 310 315 320

Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Ser Asp Lys Thr

325 330 335

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

340 345 350

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

355 360 365

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

370 375 380

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

385 390 395 400

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

405 410 415

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

420 425 430

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

435 440 445

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

450 455 460

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

465 470 475 480

Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

485 490 495

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

500 505 510

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

515 520 525

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

530 535 540

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

545 550 555 560

Claims

1. it is a kind of have the function of similar whole antibody structure and CD20-Flex bifunctional fusion proteins, it is characterised in that by three Peptide chain is formed, and is respectively Flex-CL-Hinge-CH2-CH3, its gene order is SEQIDNO：18；Rituximab heavy chains knob Mutant, its gene order are SEQIDNO：16；Rituximab light chains, its gene order are SEQIDNO：10.

2. a kind of separated nucleic acid molecule, encodes three peptide chains described in claim 1, its nucleotides sequence is classified as SEQIDNO： 17, SEQIDNO：15, SEQIDNO：9.

3. a kind of carrier, the series of operations containing the nucleic acid molecule described in claim 2 with the nucleic acid molecule is connected Expression regulation sequence.

4. a kind of host cell, is eukaryotic containing the carrier described in claim 3.

5. host cell described in claim 4, is mammalian cell.

6. host cell described in claim 5, is Chinese hamster ovary celI.

7. it is a kind of prepare described in claim 1 have the function of similar whole antibody structure and CD20-Flex bifunctional fusion eggs White method, this method include：CD20 antibody rituximab variable regions and Flt3 ligand membrane outskirts are cloned respectively；In antibody Fc Region builds knob mutant respectively：T366W, S354C；Hole mutant：T366S, L368A, Y407V and Y394C；By Flt3 Ligand membrane outskirt constructs Flex-CL with antibody light chain constant region and merges fragment；Respectively by rituximab heavy chain variable regions with Knob mutant merges, and Flex-CL is merged with hole mutant, loads expression vector；By build two expression vectors with Expression vector equipped with rituximab light chain genes jointly expressed by transfection, is isolated and purified.

8. a kind of composition, containing described in claim 1 have the function of similar whole antibody structure and CD20-Flex it is difunctional Fusion protein, and pharmaceutically acceptable carrier.

9. described in claim 1 have the function of similar whole antibody structure and CD20-Flex bifunctional fusion proteins preparing Purposes in antibody tumor medicine.

10. purposes of the composition described in claim 8 in antitumor drug is prepared.

11. the purposes described in claim 9 or 10, further includes and other antitumor drugs are used in combination.