CN104558194A - Anti-CD20-Flex bi-functional fusion protein as well as preparation method and application thereof - Google Patents
Anti-CD20-Flex bi-functional fusion protein as well as preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses anti-CD20-Flex bi-functional fusion protein as well as a preparation method of the protein and an application of the protein in preparing antineoplastic drug. Specifically, the invention discloses bi-functional fusion protein CD20-Flex with structure and function similar to those of a complete antibody, an amino acid sequence and a preparation method of the protein and an application of the protein in preparing antineoplastic drug. The bi-functional fusion protein can be combined with CD20 and further has the function of Flt3 ligand, thereby being capable of exciting tumor specific immunity of an organism effectively while having the killing efficiency.
Description
Technical field
The invention belongs to biological technical field, more specifically, the invention discloses the anti-CD20 bifunctional fusion proteins of a class, its preparation method and the application in Dispersal risk tumour medicine thereof.Anti-CD20 bifunctional fusion proteins disclosed by the invention, has the CD20-Flex bifunctional fusion proteins of similar whole antibody structure and fuction, both can be combined with CD20, have again the function of Flt3 part.
Background technology
Tumour especially malignant tumour is the disease of world today's serious harm human health, in various disease, occupy second in caused death.And in recent years, its sickness rate is obvious ascendant trend.Therapeutic effect of malignant tumour is poor, and late period, the rate of transform was high, and prognosis is how not good.Although conventional treatments adopted clinically alleviates slight illness to a great extent as radiotherapy, chemotherapy and operative treatment at present, extending survival time, all there is significant limitation in these methods, and its curative effect is difficult to further raising.
Antibody target tropism medicine has the advantages such as specificity is good, side effect is little, the cycle of partly declining is long, has been widely used in the clinical treatment of tumour now.Antibody drug has achieved challenging result for the treatment of in clinical treatment.But, due to heterogeneity and the complicacy of tumorigenesis process, the antibody target medicine of single target spot only specific target target protein play kill and wound, the effect such as blocking-up, be difficult to more effective killing tumor cell, excitating organism immunity, overcome tumor recurrence.Therefore, current antibody research field mainly concentrate on improve antibody avidity, find that new antibody target is marked with and prepares the aspects such as many targeting antibodies.Adopt the method for computer aided design (CAD) and library technology raising affinity of antibody very ripe, adopt proteomic techniques to find new drug targets with the use of mass spectrum detection, be also achieved.Method now can prepare the antibody that avidity reaches 1nM fast, new drug targets also constantly carry out clinical before and in clinical trial.But antibody variable region is mainly transformed into single-chain antibody by the method preparing multivalent antibody at present, the general affinity of antibody of multivalent antibody that this method obtains is not high, and curative effect does not also get a desired effect.The DVD antibody of nearest appearance be by the variable region of heavy chain of two kinds of antibody and variable region of light chain respectively correspondence merge, form the structure of HV1-linker-HV2 and LV1-linker-LV2.Although this DVD antibody overcomes the low shortcoming of single-chain antibody avidity, because molecular weight is excessive, antibody structure changes greatly, its expression amount can be caused not high; And, when protein target molecule amount is comparatively large or when forming larger mixture on cytolemma, can affect the combination of DVD antibody to two target spot.
Rituximab(Rituximab) commodity are called Mabthera, it is the chimeric mAb of a kind of anti-CD20, within 1997, ratified to be used for the treatment of B cell non-Hodgkin lymphoma by U.S. FDA, follicular lymphoma, can be used for treating other B cell lymphomas such as diffuse large B cell lymphoma with CHOP combined utilization.
Although the not so further investigation of antagonist treatment tumour mechanism, think at present: antibody can the growth of killing tumor cell, Tumor suppression significantly by CDC, ADCC and inducing cell death, but more and more evidence shows that the body specific immunity of antibody induction is killed and wounded and plays an important role overcoming in tumor recurrence process.
Flt3 ligand extracellular region (Flex) can not only make marrow increase the quantity of producing DC, and the functional trend of DC can be made ripe, the latter is made to have stronger antigen presentation and antitumor action, the more important thing is, Flex can also make ripe DC discharge from marrow and enter peripheral tissues, after experiment in vivo confirms application Flex, in mouse spleen, marrow, lymphoglandula, liver, DC is that time-dependent manner increases.Therefore, preparing a class can the antibody of effective excitating organism tumor-specific immunity having lethal while, is a focus of current antibody research.
Summary of the invention:
In order to solve the problem, the present inventor studies for a long period of time, through lot of experiments, utilize genetic engineering technique to construct CD20-Flex bifunctional fusion proteins that a class has similar whole antibody structure and fuction, this bifunctional fusion proteins both can be combined with CD20, had again the function of Flt3 part.
The invention discloses:
1. there is a CD20-Flex bifunctional fusion proteins for similar whole antibody structure and fuction, it is characterized in that, both can be combined with CD20 antigen, there is again the function of Flt3 part.
2. above-mentioned bifunctional fusion proteins, be made up of three peptide chains, be respectively Flex-CL-Hinge-CH2-CH3(SEQ ID NO: 18) rituximab heavy chain knob mutant (SEQ ID NO: 16) rituximab light chain (SEQ ID NO: 10).
3. the nucleic acid molecule be separated, three peptide chains of encoding above-mentioned, have nucleotide sequence described in SEQ ID NO: 17, SEQ ID NO:15, SEQ ID NO:9.
4. a carrier, containing the expression regulation sequence that upper described nucleic acid molecule is connected with the sequence being operational of described nucleic acid molecule, 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 host cell, containing above-mentioned carrier, is eukaryotic cell.
7. above-mentioned host cell is mammalian cell.
8. above-mentioned host cell is Chinese hamster ovary celI.
9. prepare an above-mentioned CD20-Flex bifunctional fusion proteins, the method comprises:
A) CD20 antibody rituximab variable region and Flt3 ligand membrane outskirt is cloned respectively;
B) knob mutant is built respectively in antibody Fc region: T366W, S354C; Hole mutant: T366S, L368A, Y407V and Y394C;
C) Flt3 ligand membrane outskirt and antibody light chain constant region are constructed Flex-CL and merge segment;
D) merged in rituximab variable region of heavy chain and knob mutant respectively, Flex-CL and hole mutant merges, and loads expression vector;
E) build two expression vectors and the common transfection of expression vector that rituximab light chain gene is housed are expressed, separation and purification.
10. a composition, containing above-mentioned CD20-Flex bifunctional fusion proteins, and pharmaceutically acceptable carrier.
The purposes of 11. above-mentioned CD20-Flex bifunctional fusion proteins in Dispersal risk tumour medicine.
12. above-mentioned compositions are preparing the purposes in antitumor drug.
13. above-mentioned arbitrary purposes, also comprise the antitumor drug conbined usage with other.
The object of this invention is to provide the CD20-Flex bifunctional fusion proteins that a class has similar whole antibody structure and fuction, both can be combined with CD20, there is again the function of Flt3 part.This antibody-like had both remained structure and the size of similar antibody, there is the avidity similar with parental antibody and transformation period, also the ability of CD20 antibody killing tumor cells similar CDC, ADCC and inducing cell death is remained, combine again Flt3 part can induce DC and NK cell propagation and maturation at tumor vicinity, excitating organism tumor-specific immunity, overcomes the recurrence of tumour.
Antagonist structure of the present invention has carried out deep understanding, and on this basis, establish a kind of not only reserved category like the structure and fuction of antibody and have a fusion rotein of Flt3 ligand function, its structure as shown in Figure 1.In order to prove that the method has curative effect more better than rituximab antibody and Flex-Ig drug combination, the present invention carries out the experiment of following protein antitumor action.In principle, method of the present invention can be widely used in various antibody and Flex builds bifunctional fusion proteins, accelerates the fusion rotein that research and development have biology and medical significance.Meanwhile, antibody variable region is combined with Flex and builds the method for bifunctional fusion proteins, for the design carrying out bifunctional fusion proteins provides a kind of new thinking later.
In the present invention, any suitable carrier can use, and can be pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), one of pDHFR, expression vector comprises and is connected with the suitable fusion dna sequence with translational regulation sequence of transcribing.
The expression of Mammals or insect host cell culture systems bifunctional fusion proteins used in the present invention, the cells such as COS, CHO, NS0, sf9 and sf21 are all applicable to the present invention.
Available host cell is the prokaryotic cell prokaryocyte containing above-mentioned carrier, can be DH5a, BL21 (DE3), one of TG1.
The preparation method disclosed in the present invention with the bifunctional fusion proteins of antibody structure and functionality advantage, under expression condition, cultivates above-mentioned host cell, thus expresses bifunctional fusion proteins, the bifunctional fusion proteins described in isolated or purified.
The method of affinity chromatography can be utilized to carry out separation and purification to bifunctional fusion proteins disclosed by the invention, according to the characteristic of utilized affinity column, the conventional method such as bifunctional fusion proteins of method elution of bound on affinity column such as high-salt buffer, change PH can be used.
Utilize aforesaid method, can be substantially homogeneous material by bifunctional fusion proteins purifying, such as, be single band on SDS-PAGE electrophoresis.
According to a preferred embodiment of the present invention, a class bifunctional fusion proteins comprises antigen binding domain and the Flt3 ligand extracellular region of rituximab.
One class bifunctional fusion proteins has antibody structure and functionality advantage, and its preparation method comprises the following steps:
1) rituximab antibody heavy chain variable region and Flt3 ligand extracellular region gene is cloned respectively;
2) gene of Flt3 ligand extracellular region and antibody light chain constant region are merged, build Flex-CL and merge fragment;
3) antagonist Fc region builds knob mutant respectively: T366W, S354C; Hole mutant: T366S, L368A, Y407V and Y394C;
4) merged in rituximab variable region of heavy chain and knob mutant respectively, Flex-CL and hole mutant merges, and loads expression vector;
5) above-mentioned expression vector and rituximab light chain of antibody are carried out corotation expression, obtain bifunctional fusion proteins by separation and purification.
The complete weight built above-mentioned, light chain gene are respectively charged into eukaryotic expression vector pcDNA3.1 (+) (Invitrogen Products).Above-mentioned plasmid one reinstates liposome transfection CHO-K1 cell (ATCC), and screens the cell clone of stably express bifunctional fusion proteins with the Selective agar medium containing 600 μ g/ml G418.Utilize Protein A chromatography column, obtain bifunctional fusion proteins by affinity chromatography purifying from the supernatant of cell culture.
Above-mentioned bifunctional fusion proteins or preparation are applied in the medicine that preparation is anticancer, also comprise the antitumor drug conbined usage with other.
The present invention discloses above-mentioned bifunctional fusion proteins; can pharmaceutical preparations composition be formed thus more play consistently curative effect together with pharmaceutically acceptable auxiliary material; these preparations can ensure the conformation integrity of bifunctional fusion proteins amino acid core sequence disclosed by the invention, also want the polyfunctional group of protected protein matter to prevent its degraded (including but not limited to cohesion, deamination or oxidation) simultaneously.Under normal circumstances, for liquid preparation, usually can preserve under 2 ° of C-8 ° of C conditions and at least stablize 1 year, for freeze-dried preparation, within least six months, keep stable at 30 ° of C.Here preparation can be the conventional preparation such as suspendible, liquid drugs injection, freeze-drying of pharmacy field, preferred liquid drugs injection or freeze-dried preparation, for liquid drugs injection or the freeze-dried preparation of above-mentioned bifunctional fusion proteins disclosed by the invention, pharmaceutically acceptable auxiliary material comprises tensio-active agent, solution stabilizer, isotonic regulator and damping fluid one or a combination set of, and wherein tensio-active agent comprises nonionic surface active agent as Polyoxyethylene Sorbitol Fatty Acid Esters (polysorbas20 or 80), poloxamer(is as poloxamer 188), Triton, sodium lauryl sulphate (SDS), Sulfuric acid,monododecyl ester, sodium salt, tetradecyl, sub-oil base or octadecyl sarkosine, Pluronics, MONAQUAT
tMdeng, its add-on should make the granulating trend of bifunctional fusion proteins minimum, solution stabilizer can be carbohydrate, comprise reducing sugar and nonreducing sugar, amino acids comprises msg powder type or Histidine, alcohols comprises trivalent alcohol, senior sugar alcohol, propylene glycol, polyoxyethylene glycol one or a combination set of, steady state is kept in the time that the add-on of solution stabilizer should make last preparation those skilled in the art formed think to reach stable, isotonic regulator can be sodium-chlor, one of N.F,USP MANNITOL, damping fluid can be TRIS, histidine buffering liquid, one of phosphate buffered saline buffer.
Above-mentioned preparation is the composition comprising bifunctional fusion proteins, and after to the animals administer comprising people, antitumous effect is obvious.Specifically, preventing and/or treating effectively tumour, can use as antitumor drug.
In the present invention, bifunctional fusion proteins and composition thereof to the animals administer comprising people are being, dosage because of age of patient and body weight, disease traits and seriousness, and route of administration and different, can with reference to zooperal result and all situations, total dosage can not exceed certain limit.Specifically intravenous dosage is 0.1 ~ 3000mg/ days.
Antitumor drug alleged by the present invention, refer to that there is the medicine suppressing and/or treat tumour, the reduction of delay and/or these severity of symptom developed with tumor growth related symptoms can be comprised, it further comprises alleviating of already present tumor growth simultaneous phenomenon and prevents the appearance of other symptoms, still reduces or prevents transfer.
Bifunctional fusion proteins disclosed by the invention and composition thereof can also and other antitumour drug Combined Preparation, for the treatment of tumour, the medicine that these antitumour drugs comprise 1, cytotoxic drug (1) acts on DNA chemical structure: alkylating agent is as nitrogen mustards, nitrous urine class, methanesulfonate ester class; Platinum compound is as cis-platinum, carboplatin and RP-54780 etc.; Mitomycin (MMC); (2) medicine of nucleic acid synthesis is affected: dihydrofolate reductase inhibitor is as Rheumatrex (MTX) and Alimta etc.; Thymus nucleoside synthetase inhibitors is as fluorouracil (5FU, FT-207, capecitabine) etc.; Purine nucleoside synthetase inhibitors is as Ismipur (6-MP) and 6-TG etc.; Ribonucleotide reductase inhibitor is as hydroxyurea (HU) etc.; DNA polymerase inhibitor is as cytosine arabinoside (Ara-C) and gemzar (Gemz) etc.; (3) act on the medicine of transcribed nucleic acid: selectively acting, in DNA profiling, suppresses DNA dependenc RNA polysaccharase, thus suppress the medicine of RNA synthesis as dactinomycin, daunorubicin, Zorubicin, pidorubicin, aclacinomycin, Plicamycin etc.; (4) medicine of tubulin synthesis is mainly acted on: taxol, taxotere, vincaleucoblastine, vinorelbine, Podophyllum emodi var chinense alkali class, homoharringtonine; (5) other Cytotoxic drugs: the synthesis of the main arrestin matter of Asparaginase; 2, hormones estrogen antagonist: tamoxifen, droloxifene, Exemestane etc.; Arimedex: aminoglutethimide, Lan Telong, letrozole, Rui Ningde etc.; Androgen antagonist: its ammonia RH-LH agonist/antagonist of fluorine: Zoladex, enatone etc.; 3, biological response modifier: mainly through body's immunity Tumor suppression Interferon, rabbit; Interleukin II; Thymic; 4, monoclonal antibody: Mabthera (MabThera); Cetuximab (C225); Trastuzumab (Trastuzumab) Bevacizumab (Avastin); 5, other draw together some machine-processed medicines failed to understand and need research further at present; Cell differentiation inducer is as retinoids; Cell death inducer.Bifunctional fusion proteins disclosed by the invention and composition thereof can with above-mentioned antitumor drug one or a combination set of drug combination.
Accompanying drawing explanation
Fig. 1. CD20-Flex BiFP bifunctional fusion proteins structural representation.
Fig. 2 .Rituximab heavy chain knob mutant structure figure.
Fig. 3. Flex-CL-Hinge-CH2-CH3 structure iron.
Fig. 4. CD20-Flex BiFP is to the avidity of CD20.
Fig. 5. the CDC killing activity of CD20-Flex BiFP.
Fig. 6. the ADCC killing activity of CD20-Flex BiFP.
Fig. 7. the apoptotic activity of CD20-Flex BiFP.
Fig. 8. the mouse interior tumor prolection of CD20-Flex BiFP.
Embodiment
the clone of embodiment 1. rituximab antibody heavy chain variable region gene
With rituximab heavy chain of antibody for template clones rituximab heavy chain variable region gene by PCR, reaction conditions is: 95 DEG C 15 minutes; 94 DEG C 50 seconds, 58 DEG C 50 seconds, 72 DEG C 50 seconds, 30 circulations; 72 DEG C 10 minutes, obtain PCR primer rituximab HV.Antibody signal peptide amino acid sequence MGWSCIILFLVATATGVHS.Correct cloned sequence is obtained for subsequent use through order-checking.SEQ ID NO:2 shows the aminoacid sequence of rituximab variable region of heavy chain, and its nucleotides sequence is classified as SEQ ID NO:1.
the clone of embodiment 2. Flt3 part variable region gene
With lymphocyte separation medium (ancient cooking vessel state biotech development Products) separating health human lymphocyte; total serum IgE is extracted with Trizol reagent (Invitrogen Products); 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; 197-207.) and document (The nucleotide sequence of a human immunoglobulin C gamma1 gene. Ellison JW 22 (1 Pt 1):; Berson BJ, Hood LE. Nucleic Acids Res. 1982 Jul 10; 10 (13): 4071-9.) RT-PCR is adopted to react amplification Flt3 part extracellular fragment gene.PCR primer reclaims through agarose gel electrophoresis purifying and is cloned in pGEM-T carrier, confirms to obtain correct clone after sequence verification.SEQ ID NO:4 shows Flex aminoacid sequence, and its nucleotides sequence is classified as SEQ ID NO:3.
the clone in embodiment 3. antibody light chain constant region, Fc district
With lymphocyte separation medium (ancient cooking vessel state biotech development Products) separating health human lymphocyte; total serum IgE is extracted with Trizol reagent (Invitrogen Products); 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; 197-207.) and document (The nucleotide sequence of a human immunoglobulin C gamma1 gene. Ellison JW 22 (1 Pt 1):; Berson BJ, Hood LE. Nucleic Acids Res. 1982 Jul 10; 10 (13): 4071-9.) RT-PCR is adopted to react amplification antibody light chain constant region and Fc district gene.PCR primer reclaims through agarose gel electrophoresis purifying and is cloned in pGEM-T carrier, confirms to obtain correct clone after sequence verification.SEQ ID NO:6 shows CL aminoacid sequence, and its nucleotides sequence is classified as SEQ ID NO:5; SEQ ID NO:8 shows Fc aminoacid sequence, and its nucleotides sequence is classified as SEQ ID NO:7.
the structure of embodiment 4. antibody Fc district knob mutant
By the antibody Fc district obtained in embodiment 3, the method of overlap PCR is adopted to introduce catastrophe point: T366W, S354C (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 primer reclaims through agarose gel electrophoresis purifying and is cloned in pGEM-T carrier, confirms to obtain correct clone after sequence verification.SEQ ID NO:12 shows Fc-knob aminoacid sequence, and its nucleotides sequence is classified as SEQ ID NO:11.
the structure of embodiment 5. antibody Fc district hole mutant
By the antibody Fc district obtained in embodiment 3, the method of overlap PCR is adopted to introduce catastrophe point: 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 primer reclaims through agarose gel electrophoresis purifying and is cloned in pGEM-T carrier, confirms to obtain correct clone after sequence verification.SEQ ID NO:14 shows Fc-hole aminoacid sequence, and its nucleotides sequence is classified as SEQ ID NO:13.
the structure of embodiment 6. rituximab heavy chain mutant
The Fc district knob mutant that the rituximab variable region of heavy chain obtained with embodiment 1 and embodiment 4 obtain is for template, the method of overlap PCR is adopted to be merged in rituximab variable region of heavy chain and Fc district knob mutant, then expression vector is loaded, introduce two sudden change S354C and T366W in the Fc constant region of Rituximab heavy chain, build Rituximab heavy chain knob mutant (Fig. 2).SEQ ID NO:16 shows rituximab heavy chain knob variant amino acid sequence, and its nucleotides sequence is classified as SEQ ID NO:15.
the structure of embodiment 7. Flex mutant
With the Flex that embodiment 2 obtains, the CL that embodiment 3 obtains and the Fc district hole mutant that embodiment 5 obtains are template, adopt the method for overlap PCR that Flex and CL and Fc district hole is carried out segment composition, then load expression vector, build Flex-CL-Hinge-CH2-CH3(Fig. 3).SEQ ID NO:18 shows Flex-CL-Hinge-CH2-CH3 aminoacid sequence, and its nucleotides sequence is classified as SEQ ID NO:17.
the expression and purification of embodiment 8. CD20-Flex bifunctional fusion proteins
3 × 10 are inoculated in 3.5cm tissue culture dishes
5cHO-K1 cell (ATCC CRL-9618); cell cultures carries out transfection when 90%-95% merges: get plasmid 10 μ g rituximab heavy chain knob mutant; Flex-CL-Hinge-CH2-CH3 and 4 μ g rituximab light chain (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.) and 20 μ l Lipofectamine2000 Reagent(Invitrogen Products) be dissolved in 500 μ l plasma-free DMEM medium respectively, room temperature leaves standstill 5 minutes, by above 2 kinds of liquid mixing, incubated at room is formed to make DNA-liposome complex for 20 minutes, therebetween with the DMEM substratum of 3ml serum-free replace in culture dish containing blood serum medium, then the DNA-liposome complex of formation is joined in plate, CO
2incubator adds the DMEM perfect medium of 2ml containing 10% serum after cultivating 4 hours, be placed in CO
2continue in incubator to cultivate.After 24h is carried out in transfection, cell changes containing 600 μ g/ml G418 Selective agar medium screening resistance clones.Get cells and supernatant ELISA and detect screening high-expression clone: goat anti-human igg (Fc) is coated in elisa plate, 4 ° of C spend the night, 2h is closed in 37 ° of C with 2%BSA-PBS, add resistance clone culture supernatant to be measured or standard substance (Human myeloma IgG1, κ), 37 ° of C incubation 2h, add HRP-goat anti-human igg (κ) and carry out association reaction, 37 ° of C incubation 1h, add TMB in 37 ° of C effect 5min, finally use H
2sO
4termination reaction, surveys A
450value.By screening the high-expression clone serum free medium enlarged culturing obtained, with Protein A affinity column (GE Products) separation and purification bifunctional fusion proteins.Antibody purification PBS is dialysed, finally with the concentration of uv-absorbing standard measure determination purified antibodies.
experimental example 1. fusion rotein avidity detects
The avidity of all fusion roteins is undertaken measuring [Cragg MS by radioimmunoassays, 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, the fusion rotein after purifying, is marked by iodine pearl method (iodobead method).By the mark fusion rotein of 125 iodine and Daudi (ATCC CCL-213) and 8266 cell incubation 2 hours, 37 ° of C.Pass through centrifugation with the antibody of Cell binding and free iodine labeling, detect the radioactivity (see figure 4) of the antibody of 125 iodine labelings be combined on Daudi cell.Fusion rotein affinity constant is tried to achieve by Hill equation curve matching titration binding curve.CD20-Flex bifunctional fusion proteins is to the avidity of CD20 and Flt3 [Title:Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Author:Reff, M. E. similar with the avidity of Flt3 part to the parent rituximab of bibliographical information; 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, be 4.82 ± 0.25 nM to the avidity numerical value of CD20 albumen; Be 230 ± 40 pmol/L to the avidity of Flt3.
the CDC Function detection of experimental example 2. CD20-Flex fusion rotein
Raji and Daudi cell is without phenol red medium and fusion rotein 37 degree of cultivations 1 hour, and the normal human serum 37 degree then adding 10% in the medium hatches 4 hours, then adopts the LDH test kit (Promega) of standard to carry out detection CDC active.As shown in Figure 5, fusion rotein CD20-Flex BiFP has the CDC killing activity similar to rituximab.
the ADCC Function detection of experimental example 3. CD20-Flex fusion rotein
Daudi cell (ATCC CCL-213), under different fusion rotein concentration, carries out cultivating (37 degree, 4 hours) than the ratio of 25:1 according to E:T, then adopts the LDH test kit (Promega) of standard to carry out detection ADCC activity.As shown in Figure 6, fusion rotein CD20-Flex BiFP has the ADCC killing activity similar to rituximab.
the apoptosis function of experimental example 4. CD20-Flex fusion rotein detects
Ramos cell and the CD20-Flex fusion rotein of 10 μ g/ml and the caspase inhibitor of different concns are hatched altogether, adds the goat-anti people F (ab ') of 20 μ g/ml simultaneously
2fragment (Southern Biotechnology).After 16 hours, the cell of apoptosis adopts Annexin V-Fluos test kit (BD company) to carry out streaming list dye.As shown in Figure 7, CD20-Flex fusion rotein two anti-crosslinked after, can induce the necrocytosis of similarity degree being cross-linked with rituximab after, its necrocytosis of inducing shows the dose-dependently of caspase inhibitor.
knurl experiment is become in Mice Body in experimental example 5. CD20-Flex fusion rotein body
By 5 × 10
6daudi or A20-CD20 cell tail vein injection BALB/c mouse, the 0th, the treatment of 4 and 8 days fusion roteins to mouse tail vein injection 200 μ g.42nd day, fusion rotein treated non-tumor-bearing mice in its opposite side subcutaneous vaccination 1 × 10
4a20-CD20 or A20 cell.Observe the size of tumor-bearing mice tumour, evaluate its result for the treatment of.As shown in Figure 8, CD20-Flex fusion rotein not only can have the lethal effect similar to rituximab to the tumour of primary vaccination, and it is active still to show long-term tumor protection to the tumour again inoculated.
SEQUENCE LISTING
Shanghai Hai Sitaike pharmaceutcal corporation, Ltd of <110> Shanghai Zhangjiang Biological Technology Co
The anti-CD20-Flex bifunctional fusion proteins of <120> mono-class, Preparation Method And The Use
<130> 2013
<160> 18
<170> PatentIn version 3.5
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cctggtcggg gcctggaatg gattggagct atttatccag gaaatggtga tacttcctac 180
aatcagaagt tcaagggcaa ggccacactg actgcagaca aatcctccag cacagcctac 240
atgcagctca gcagcctgac atctgaagac tctgcggtct attactgtgc aagatcgact 300
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Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala
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Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe
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Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr
65 70 75 80
Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly
100 105 110
Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser
115 120
<210> 3
<211> 627
<212> DNA
<213> synthetic
<400> 3
acccaggact gctccttcca acacagcccc atctcctccg acttcgctgt caaaatccgt 60
gagctgtctg actacctgct tcaagattac ccagtcaccg tggcctccaa cctgcaggac 120
gaggagctct gcgggggcct ctggcggctg gtcctggcac agcgctggat ggagcggctc 180
aagactgtcg ctgggtccaa gatgcaaggc ttgctggagc gcgtgaacac ggagatacac 240
tttgtcacca aatgtgcctt tcagcccccc cccagctgtc ttcgcttcgt ccagaccaac 300
atctcccgcc tcctgcagga gacctccgag cagctggtgg cgctgaagcc ctggatcact 360
cgccagaact tctcccggtg cctggagctg cagtgtcagc ccgactcctc aaccctgcca 420
cccccatgga gtccccggcc cctggaggcc acagccccga cagccccgca gccccctctg 480
ctcctcctac tgctgctgcc cgtgggcctc ctgctgctgg ccgctgcctg gtgcctgcac 540
tggcagagga cgcggcggag gacaccccgc cctggggagc aggtgccccc cgtccccagt 600
ccccaggacc tgctgcttgt ggagcac 627
<210> 4
<211> 209
<212> PRT
<213> synthetic
<400> 4
Thr Gln Asp Cys Ser Phe Gln His Ser Pro Ile Ser Ser Asp Phe Ala
1 5 10 15
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> synthetic
<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> synthetic
<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> synthetic
<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> synthetic
<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> synthetic
<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> synthetic
<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> synthetic
<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> synthetic
<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> synthetic
<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> synthetic
<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> synthetic
<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> synthetic
<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> synthetic
<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> synthetic
<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 (13)
1. there is a CD20-Flex bifunctional fusion proteins for similar whole antibody structure and fuction, it is characterized in that, both can be combined with CD20 antigen, there is again the function of Flt3 part.
2. bifunctional fusion proteins according to claim 1, be made up of three peptide chains, be respectively Flex-CL-Hinge-CH2-CH3(SEQ ID NO: 18) rituximab heavy chain knob mutant (SEQ ID NO: 16) rituximab light chain (SEQ ID NO: 10).
3. the nucleic acid molecule be separated, three peptide chains according to claim 2 of encoding, have nucleotide sequence described in SEQ ID NO: 17, SEQ ID NO:15, SEQ ID NO:9.
4. a carrier, containing the expression regulation sequence that nucleic acid molecule according to claim 3 is connected with the sequence being operational of described nucleic acid molecule, wherein carrier can be pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), one of pDHFR.
5. carrier according to claim 4 is pcDNA3.1(+) or pcDNA3.1/ZEO(+).
6. a host cell, containing carrier according to claim 5, is eukaryotic cell.
7. host cell described in claim 6 is mammalian cell.
8. host cell described in claim 7 is Chinese hamster ovary celI.
9. prepare a CD20-Flex bifunctional fusion proteins according to claim 1, the method comprises:
Clone CD20 antibody rituximab variable region and Flt3 ligand membrane outskirt respectively;
Knob mutant is built respectively: T366W, S354C in antibody Fc region; Hole mutant: T366S, L368A, Y407V and Y394C;
Flt3 ligand membrane outskirt and antibody light chain constant region are constructed Flex-CL and merges segment;
Merged in rituximab variable region of heavy chain and knob mutant respectively, Flex-CL and hole mutant merges, and loads expression vector;
Build two expression vectors and the common transfection of expression vector that rituximab light chain gene is housed are expressed, separation and purification.
10. a composition, containing CD20-Flex bifunctional fusion proteins according to claim 1, and pharmaceutically acceptable carrier.
11. purposes of CD20-Flex bifunctional fusion proteins in Dispersal risk tumour medicine according to claim 1.
12. compositions according to claim 10 are preparing the purposes in antitumor drug.
The arbitrary described purposes of 13. claim 11,12, also comprises the antitumor drug conbined usage with other.
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| CN201310486424.9A CN104558194B (en) | 2013-10-17 | 2013-10-17 | A kind of anti-CD20-Flex bifunctional fusion proteins, preparation method and the usage |
| PCT/CN2013/090277 WO2015054958A1 (en) | 2013-10-17 | 2013-12-24 | Anti-cd20-flex bifunctional fusion protein, and preparation method and use thereof |
| AU2013403112A AU2013403112B2 (en) | 2013-10-17 | 2013-12-24 | Anti-CD20-Flex bifunctional fusion protein, and preparation method and use thereof |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468956A (en) * | 2002-07-15 | 2004-01-21 | 杨 琴 | Recombinant virus of antibody for high-efficiency expression to treat tumor and its use |
| CN1572801A (en) * | 2003-06-13 | 2005-02-02 | 马菁 | Double functional fusion protein possessing antineoplastic functions, method for making same and use thereof |
| CN102448985A (en) * | 2009-05-27 | 2012-05-09 | 霍夫曼-拉罗奇有限公司 | Tri- or tetraspecific antibodies |
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| AU2004252465A1 (en) * | 2003-06-13 | 2005-01-06 | Oncomax Acquisition Corp. | Preparation and application of anti-tumor bifunctional fusion proteins |
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- 2013-10-17 CN CN201310486424.9A patent/CN104558194B/en active Active
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1468956A (en) * | 2002-07-15 | 2004-01-21 | 杨 琴 | Recombinant virus of antibody for high-efficiency expression to treat tumor and its use |
| CN1572801A (en) * | 2003-06-13 | 2005-02-02 | 马菁 | Double functional fusion protein possessing antineoplastic functions, method for making same and use thereof |
| CN102448985A (en) * | 2009-05-27 | 2012-05-09 | 霍夫曼-拉罗奇有限公司 | Tri- or tetraspecific antibodies |
Non-Patent Citations (3)
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| GONG Q, OU Q, YE S, ET AL.: "Importance of cellular microenvironment and circulatory dynamics in B cell immunotherapy..", 《J IMMUNOL》 * |
| ZHAO L, TONG Q, QIAN W, ET AL.: "Eradication of non-Hodgkin lymphoma through the induction of tumor-specific T-cell immunity by CD20-Flex BiFP.", 《BLOOD》 * |
| 谈珉: "抗CD20抗体/Flt3L双功能融合蛋白的抗肿瘤作用机理研究", 《中国博士学位论文医药卫生科技辑》 * |
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| Publication number | Publication date |
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| WO2015054958A1 (en) | 2015-04-23 |
| CN104558194B (en) | 2018-04-27 |
| AU2013403112B2 (en) | 2017-05-04 |
| AU2013403112A1 (en) | 2016-05-12 |
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