CN104610453A - Anti-HER2 dual-targeting antibodies, and preparation method and application thereof - Google Patents
Anti-HER2 dual-targeting antibodies, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses anti-HER2 dual-targeting antibodies, a preparation method thereof and application of the anti-HER2 dual-targeting antibodies in the preparation of antibody tumor drugs. More specifically, the invention discloses the dual-targeting antibodies Tras-Per CrossMab and Tras-Permut CrossMab with structures and functions similar to the structures and functions of complete antibodies, and amino acid sequences and the preparation method thereof, and application of the dual-targeting antibodies in the preparation of antitumor drugs. According to the invention, the functions similar to the functions of the complete antibodies are retained; HER2 target protein is dually targeted; and antitumor activity of the anti-HER2 dual-targeting antibodies is better than the antitumor activity of single-targeting anti-HER2 antibodies-Trastuzumab and Pertuzumab.
Description
Technical field
The invention belongs to biological technical field, more specifically, the invention discloses the two targeting antibodies of the anti-HER2 of a class, its preparation method and the application in Dispersal risk tumour medicine thereof.The two targeting antibodies of anti-HER2 disclosed by the invention, there is the two targeting antibodies of HER2 of similar whole antibody structure and fuction, both the function that whole antibody is similar had been remained, again can two target HER2 target protein, there is anti-tumor activity more better than the HER2 antibody trastuzumab of single target, Pertuzumab.
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 successfully applied to the clinical treatment of tumour now.Antibody drug has achieved challenging result for the treatment of in clinical treatment.Within 1998, U.S. FDA have approved anti-HER 2 humanized antibody Herceptin (Trastuzumab, Trastuzumab, herceptin) be used for the treatment of the metastatic breast cancer of the HER2 positive, become first by the Humanized monoclonal antibodies of the treatment mammary cancer of FDA approval listing.It is by gene engineering method, is combined in non-specific human IgG stable region and special mouse-anti HER2 antigenic determinant and designs humanization monoclonal antibody, can with HER2 ectodomain IV specific binding.Trastuzumab, since within 1998, obtaining U.S. FDA approval listing, has got permission the treatment for HER2 positive breast cancer in new auxiliary, auxiliary and late period at present.Trastuzumab and chemotherapy drugs in combination apply the result for the treatment of that can further improve HER2 breast cancer patients with positive; Trastuzumab and target therapeutic agent, as: the combined utilization such as lapatinibditosylate, rhuMAb-VEGF, handkerchief trastuzumab, T-DM1, HKI-272 also show good effect.Further research and exploitation Trastuzumab is combined with other targeted drug, for HER2 breast cancer patients with positive provides more more options to be also clinical required.But in treatment clinical course, the reactivity of the mono-medicine of Trastuzumab is not high, about 12%-34%, its median survival time is average of 9 months.Therefore, most of HER2 process LAN tumour tolerates Trastuzumab single therapy.In fact, in the metastatic breast cancer patient of process LAN HER2 albumen, to the primary drug resistance of Trastuzumab single therapy up to 66% to 88%.The further clinical III phase tests confirmation, and Trastuzumab and taxol or docetaxel drug combination can increase reactivity, delays disease progression and extends survival time.Even if but applying with chemotherapy drugs in combination, patient also can produce resistance to this methods for the treatment of and tolerance within 1 year.Therefore, design has the novel HER2 antibody of more powerful antitumor activity, is a focus of present breast cancer treatment research.
Pertuzumab is recently by FDA approval another HER2 Humanized monoclonal antibodies for clinical treatment, it can be combined in HER2 ectodomain II region, by suppressing the formation of HER2/HER3 heterodimer, lower the activation levels of PI3K/Akt signal path.And Trastuzumab is in conjunction with HER2 ectodomain IV, when there is part, only showing the effect of more weak suppression HER2/HER3 dimerization, thinking that the mechanism of action of Pertuzumab mechanism of action and Trastuzumab is complementary at present.Pertuzumab shows good anti-tumor activity to metastatic breast cancer in clinical trial, now carries out clinical trial as assisting therapy.In addition, show with external preclinical test data in body, the breast cancer cell line of drug combination to HER2 process LAN of Trastuzumab and Pertuzumab shows the antitumous effect of cooperative compensating.It should be noted that in the mouse model of human breast carcinoma, the mouse that after treating Trastuzumab, tumour is still in progress accepts Trastuzumab and Pertuzumab treatment can work in coordination with the inhibit activities increased tumour.Recent report, one adopts the data of the II clinical trial phase of Trastuzumab and Pertuzumab drug combination consistent with above-mentioned preclinical test result for HER2 process LAN metastatic breast cancer.Their result shows, combined utilization better tolerance in clinical treatment of two antibody, its clinical Benefit rate is 50%.On this basis, devise another clinical trial and obtain role in challenging clinical efficacy like this for evaluating Trastuzumab and Pertuzumab drug combination further.Their result shows, in the HER2 positive metastatic patient with breast cancer be in progress after Trastuzumab treatment in early days, adopt Pertuzumab single therapy curative effect not good enough, its clinical Benefit rate is only 10.3%.And adopting Trastuzumab and Pertuzumab drug combination to show the anti-tumor activity stronger than Pertuzumab single therapy, its clinical Benefit rate is 41.2%.In the recent period, the clinical data adopting Trastuzumab, Pertuzumab and docetaxel drug combination to be used for the treatment of HER2 positive metastatic mammary cancer as a line medication shows, compare with docetaxel drug combination with placebo, Trastuzumab, triple combination's medication does not increase cardiac toxic, and can Progression free survival phase of significant prolongation patient.After treating resistance with clinical testing data prompting Trastuzumab before these are clinical, HER2 albumen still can have the target spot of therapeutic value as one, and HER2 ectodomain IV and II of target Trastuzumab and Pertuzumab effect simultaneously can overcome Trastuzumab and treat tolerance.
But the drug combination of several monoclonal antibody considerably increases family and social health care cost and economical load.More importantly, because the therapeutic antibodies quantity of having gone on the market at present is few, the selection of drug combination is restricted.The more important thing is, due to tumorigenic complicacy, only rely on the monoclonal antibody of target Single locus to be difficult to more effective killing tumor cells, overcome oncotherapy recurrence.Therefore, single for better solving monoclonal antibody target, strengthen the problems such as the anti-tumor activity of antibody, design one and novel both there is two targeting antibodies advantage, there is again similar whole antibody structure and fuction advantage, having two target spot Anti-HER 2s of strong anti-tumor activity, is current problem demanding prompt solution.
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 a class and both there is similar whole antibody structure and fuction, again can the novel HER2 antibody of two target target protein, this antibody had both remained the advantage of similar whole antibody killing tumor cells, again can two target HER2 albumen, show than single medicine parental antibody, even better than Trastuzumab and Pertuzumab drug combination anti-tumor activity.
The invention discloses:
1. there is a two targeting antibodies Tras-Per CrossMab and Tras-Permut CrossMab of novel HER2 of similar whole antibody structure and fuction, it is characterized in that both there is the structure and fuction similar with HER2 whole antibody, again can two target HER2 albumen.
2. above-mentioned two targeting antibodies Tras-Per CrossMab, are made up of four peptide chains, are respectively Trastuzumab heavy chain knob mutant, Trastuzumab light chain, Pertuzumab heavy chain hole mutant, Pertuzumab light chain mutant PerLV-CH1.
3. above-mentioned two targeting antibodies Tras-Permut CrossMab, be made up of four peptide chains, be respectively Trastuzumab heavy chain knob mutant, Trastuzumab light chain, Pertuzumab heavy chain hole mutant, Pertuzumab light chain mutant PerLV (L56TY)-CH1.
4. the nucleic acid molecule be separated, four peptide chains of the above-mentioned Tras-Per CrossMab of coding composition, be respectively Trastuzumab heavy chain knob mutant, nucleotide sequence described in Pertuzumab heavy chain hole mutant and Trastuzumab light chain and Pertuzumab light chain mutant PerLV-CH1.
5. the nucleic acid molecule be separated, four peptide chains of the above-mentioned Tras-Permut CrossMab of coding composition, be respectively Trastuzumab heavy chain knob mutant, Pertuzumab heavy chain hole mutant and Trastuzumab light chain and Pertuzumab light chain mutant PerLV (L56TY)-CH1.
6. a carrier, containing the expression regulation sequence that upper described Tras-Per CrossMab or Tras-Permut CrossMab nucleic acid molecule are connected with the sequence being operational of described nucleic acid molecule, wherein carrier can be pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), one of pDHFR.
7. above-mentioned carrier is pDR1, pcDNA3.1(+), pcDNA3.1/ZEO(+), one of pDHFR.
8. a host cell, containing above-mentioned carrier, is eukaryotic cell.
9. above-mentioned host cell is mammalian cell.
10. above-mentioned host cell is Chinese hamster ovary celI.
Prepare the two targeting antibodies of above-mentioned Tras-Per for 11. 1 kinds, the method comprises:
A) clone that Her2 antibody trastuzumab variable region of heavy chain and Pertuzumab are heavy, variable region of light chain respectively;
B) knob mutant is built respectively in antibody Fc region: T366W, S354C; Hole mutant: T366S, L368A, Y407V and Y394C;
C) by Pertuzumab variable region of light chain and heavy chain constant region 1(CH1) construct PerLV-CH1 and merge segment, Pertuzumab variable region of light chain mutant L56TY and heavy chain constant region CH1 is constructed PerLV (L56TY)-CH1 and merges segment;
D) merged in Trastuzumab variable region of heavy chain and knob mutant respectively, Pertuzumab variable region of heavy chain and antibody light chain constant region (CL) and hole mutant merge PerHV-CL-Hinge-CH2-CH3, load expression vector;
E) Trastuzumab heavy chain knob mutant (SEQ ID NO:17) is respectively charged into by what build, the common transfection of expression vector of Pertuzumab heavy chain hole mutant (SEQ ID NO:19) and Trastuzumab light chain (SEQ ID NO:25) and Pertuzumab light chain mutant (SEQ ID NO:21) is expressed, separation and purification.
F) Trastuzumab heavy chain knob mutant (SEQ ID NO:17) is respectively charged into by what build, the common transfection of expression vector of Pertuzumab heavy chain hole mutant (SEQ ID NO:19) and Trastuzumab light chain (SEQ ID NO:25) and Pertuzumab light chain mutant PerLV (L56TY)-CH1 (SEQ ID NO:23) is expressed, separation and purification.
12. 1 kinds of compositions, containing the two targeting antibodies of above-mentioned Tras-Per, and pharmaceutically acceptable carrier.
The two purposes of targeting antibodies in Dispersal risk tumour medicine of 13. above-mentioned Tras-Per.
14. above-mentioned compositions are preparing the purposes in antitumor drug.
15. above-mentioned arbitrary purposes, also comprise the antitumor drug conbined usage with other.
The object of this invention is to provide the two targeting antibodies of Tras-Per that a class has similar whole antibody structure and fuction.This antibody-like had both remained structure and the size of similar antibody, there is the avidity similar with parental antibody, again can two target HER2 target protein, show than single medicine parental antibody, even better than Trastuzumab and Pertuzumab drug combination anti-tumor activity.
Antagonist structure of the present invention has carried out deep understanding, and on this basis, establish the two targeting antibodies of Tras-Per that a class has similar whole antibody structure and fuction, its structure as shown in Figure 1.In order to prove that the method has and has better curative effect than HER2 antibody trastuzumab and the mono-medicinal medicine of Pertuzumab or Trastuzumab and Pertuzumab 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 the structure of various pairs of targeting antibodies, accelerates the antibody drug that research and development have biology and medical significance.Meanwhile, the method for the two targeting antibodies of this structure, 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 two targeting antibodies of antibody structure and functionality advantage, under expression condition, cultivates above-mentioned host cell, thus expresses two targeting antibodies, the two targeting antibodies described in isolated or purified.
The method of affinity chromatography can be utilized to carry out separation and purification to disclosed by the invention pair of targeting antibodies, according to the characteristic of utilized affinity column, the conventional method such as two targeting antibodies 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 two targeting antibodies purifying, such as, be single band on SDS-PAGE electrophoresis.
According to a preferred embodiment of the present invention, the two targeting antibodies of a class comprises the antigen binding domain of Trastuzumab and the antigen binding domain of Pertuzumab.
The two targeting antibodies of one class has antibody structure and functionality advantage, and its preparation method comprises the following steps:
1) HER2 antibody trastuzumab and Pertuzumab weight, chain variable region gene is cloned respectively;
2) Pertuzumab variable region of heavy chain and antibody light chain constant region are constructed PerHV-CL and merge fragment;
3) Pertuzumab variable region of light chain and heavy chain of antibody CH1 district are constructed PerLV-CH1 and merge fragment;
4) Pertuzumab light chain mutant variable region and heavy chain of antibody CH1 district are constructed PerLV (L56TY)-CH1 and merge fragment;
5) antagonist Fc region builds knob mutant respectively: T366W, S354C; Hole mutant: T366S, L368A, Y407V and Y394C;
6) merged in Trastuzumab variable region of heavy chain and knob mutant respectively, PerHV-CL and hole mutant merges, PerLV-CH1 and Trastuzumab light chain, loads expression vector;
7) merged in Trastuzumab variable region of heavy chain and knob mutant respectively, PerHV-CL and hole mutant merges, PerLV (L56TY)-CH1 and Trastuzumab light chain, loads expression vector;
5) kind of the expression vector of four in above-mentioned 6 or 7 is carried out corotation expression, obtain two targeting antibodies Tras-Per CrossMab or Tras-Permut CrossMab 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 two targeting antibodies by affinity chromatography purifying from the supernatant of cell culture.
Above-mentioned pair of targeting antibodies 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 pair of targeting antibodies; 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 disclosed by the invention pair of targeting antibodies amino acid core sequence, 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 pair of targeting antibodies 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, MONAQUATTM etc., 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 two targeting antibodies, 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, two targeting antibodies 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.
Disclosed by the invention pair of targeting antibodies 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.Disclosed by the invention pair of targeting antibodies and composition thereof can with above-mentioned antitumor drug one or a combination set of drug combination.
Accompanying drawing explanation
Fig. 1. the two targeting antibodies structural representation of Tras-Per CrossMab.
Fig. 2. Trastuzumab heavy chain knob mutant structure figure.
Fig. 3. Pertuzumab heavy chain hole mutant PerHV-CL-Hinge-CH2-CH3 structure iron.
Fig. 4. the antiproliferative activity of the two targeting antibodies of Tras-Per CrossMab.
Fig. 5. the ADCC killing activity of the two targeting antibodies of Tras-Per CrossMab.
Fig. 6. the two targeting antibodies inducing cell death of Tras-Per CrossMab is active.
Fig. 7. anti-tumor activity in the Mice Body of the two targeting antibodies of Tras-Per CrossMab.
Embodiment
Embodiment
1. Trastuzumab and Pertuzumabantibody heavy chain variable region,
pertuzumabthe clone of chain variable region gene
With Trastuzumab or Pertuzumab heavy chain of antibody for template clones Trastuzumab and Pertuzumab heavy chain variable region gene respectively by PCR, and Pertuzumab chain variable region gene, 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 Trastuzumab HV, Pertuzumab HV and Pertuzumab LV.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 Trastuzumab variable region of heavy chain, and its nucleotides sequence is classified as SEQ ID NO:1; SEQ ID NO:3 is the nucleotide sequence of Pertuzumab variable region of heavy chain, and its aminoacid sequence is SEQ ID NO:4; SEQ ID NO:6 shows the aminoacid sequence of Pertuzumab variable region of light chain, and its nucleotides sequence is classified as SEQ ID NO:5; SEQ ID NO:26 is the aminoacid sequence of Trastuzumab variable region of light chain, and its nucleotides sequence is classified as SEQ ID NO:25.
Embodiment
2.antibody light chain constant region, CH1,
fcthe clone in 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, CH CH1 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:8 shows CL aminoacid sequence, and its nucleotides sequence is classified as SEQ ID NO:7; SEQ ID NO:10 shows Fc aminoacid sequence, and its nucleotides sequence is classified as SEQ ID NO:9; SEQ ID NO:12 shows CH1 aminoacid sequence, and its nucleotides sequence is classified as SEQ ID NO:11.
Embodiment
3.antibody
fcdistrict
knobthe structure of mutant
By the antibody Fc district obtained in embodiment 2, 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:14 shows Fc-knob aminoacid sequence, and its nucleotides sequence is classified as SEQ ID NO:13.
Embodiment
4.antibody
fcdistrict
holethe structure of 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:16 shows Fc-hole aminoacid sequence, and its nucleotides sequence is classified as SEQ ID NO:15.
Embodiment
5. Trastuzumabthe structure of heavy chain mutant
The Fc district knob mutant that the Trastuzumab variable region of heavy chain obtained with embodiment 1 and embodiment 3 obtain is for template, the method of overlap PCR is adopted to be merged in Trastuzumab 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 Trastuzumab heavy chain, build Trastuzumab heavy chain knob mutant (Fig. 2).SEQ ID NO:18 shows Trastuzumab heavy chain knob variant amino acid sequence, and its nucleotides sequence is classified as SEQ ID NO:17.
Embodiment
6. Pertuzumab heavy chainthe structure of mutant
With the Pertuzumab variable region of heavy chain that embodiment 1 obtains, the CL that embodiment 2 obtains and the Fc district hole mutant that embodiment 4 obtains are template, adopt the method for overlap PCR that segment composition is carried out in Pertuzumab variable region of heavy chain and CL and Fc district hole, then load expression vector, build PerHV-CL-Hinge-CH2-CH3(Fig. 3).SEQ ID NO:20 shows Flex-CL-Hinge-CH2-CH3 aminoacid sequence, and its nucleotides sequence is classified as SEQ ID NO:19.
Embodiment
7. Pertuzumabthe structure of light chain mutant
With the Pertuzumab variable region of light chain that embodiment 1 obtains, the CH1 that embodiment 2 obtains is template, adopts the method for overlap PCR that segment composition is carried out in Pertuzumab variable region of light chain and CH1, then loads expression vector, build PerLV-CH1.Adopt the method for overlap PCR, introduce mutation T yr(L56TY Pertuzumab chain variable region amino acid the 56th).SEQ ID NO:22 shows the aminoacid sequence of PerLV-CH1, and its nucleotides sequence is classified as SEQ ID NO:21; SEQ ID NO:24 shows the aminoacid sequence of PerLV (L56TY)-CH1, and its nucleotides sequence is classified as SEQ ID NO:23.
Embodiment
8. Tras-Per CrossMabexpression and purification
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 Trastuzumab heavy chain knob mutant (SEQ ID NO:18), Pertuzumab heavy chain hole mutant (SEQ ID NO:20) and 4 μ g Trastuzumab light chains (SEQ ID NO:26) and Pertuzumab light chain mutant (SEQ ID NO:22) (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.The high-expression clone serum free medium enlarged culturing obtained will be screened, with the two targeting antibodies of Protein A affinity column (GE Products) separation and purification.Antibody purification PBS is dialysed, finally with the concentration of uv-absorbing standard measure determination purified antibodies.
Embodiment
9. Tras-Permut CrossMabexpression and purification
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 Trastuzumab heavy chain knob mutant (SEQ ID NO:18), Pertuzumab heavy chain hole mutant (SEQ ID NO:20) and 4 μ g Trastuzumab light chains (SEQ ID NO:26) and Pertuzumab light chain mutant PerLV (L56TY)-CH1 (SEQ ID NO:24) (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.The high-expression clone serum free medium enlarged culturing obtained will be screened, with the two targeting antibodies of Protein A affinity column (GE Products) separation and purification.Antibody purification PBS is dialysed, finally with the concentration of uv-absorbing standard measure determination purified antibodies.
Experimental example
1.two targeting antibodies avidity detects
The avidity of all fusion roteins is undertaken measuring [Li B by ELISA method; Zhao L; Wang C; Guo H; Wu L, Zhang X, Qian W; Wang H, Guo Y. The protein-protein interface evolution acts in a similar way to antibody affinity maturation. J Biol Chem 2010; 285:3865 – 71.].Briefly, the antibody after purifying with to be coated on elisa plate and the HER2-ECD albumen that concentration increases gradually hatches 1 hour.Then ELISA method is adopted to detect the concentration of free HER2 antibody and then calculate the avidity of antibody.Wherein, the avidity numerical value of Tras-Per CrossMab to HER2 albumen is the avidity numerical value of 2.57 ± 0.68 nM, Tras-Permut CrossMab to HER2 albumen is 0.53 ± 0.08 nM.
Experimental example
2. Tras-Per CrossMab and Tras-Permut CrossMabthe antiproliferative activity of two targeting antibodies
BT-474 cell (ATCC HTB-20) cell is without phenol red medium and antibody 37 degree of cultivations 2 hours, then add in the medium final concentration be respectively 5 and the people of 1nmol/L to recombinate EGF and HRG, continue cultivation 4 days, then employing CellTiter 96 AQueous One Solution Cell Proliferation Assay(Promega) carry out the antiproliferative activity detecting antibody.As shown in Figure 4, two targeting antibodies Tras-Per CrossMab and Tras-Permut CrossMab shows than Trastuzumab, Pertuzumab and the better tumour antiproliferative activity of Trastuzumab and Pertuzumab drug combination.Further research shows, when there is HER2 part, Tras-Permut CrossMab shows the antiproliferative activity stronger than Tras-Per CrossMab.
Experimental example
3. Tras-Per CrossMab and Tras-Permut CrossMabtwo targeting antibodies
aDCCkilling activity
BT-474 cell (ATCC HTB-20), 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 5, two targeting antibodies Tras-Per CrossMab shows and has the ADCC killing activity similar to parental antibody Trastuzumab and Pertuzumab.But Tras-Permut CrossMab shows the ADCC killing activity stronger than Tras-Per CrossMab.
Experimental example
4. Tras-Per CrossMab and Tras-Permut CrossMabtwo targeting antibodies inducing cell death is active
By two to HER2 positive breast cancer cells and Tras-Per CrossMab targeting antibodies and Trastuzumab and Pertuzumab according to 1:1 drug combination co-cultivation 5 days.After adopting SYTOX Red (Life technologies) to dye 15 minutes in washed cell, carry out flow cytometer showed.As shown in Figure 6, the two targeting antibodies of Tras-Per CrossMab and Tras-Permut CrossMab shows the ability than single medicine parental antibody Trastuzumab, Pertuzumab and the stronger inducing death of neoplastic cells of Trastuzumab and Pertuzumab drug combination.
Experimental example
5. Tras-Per CrossMab and Tras-Permut CrossMabanti-tumor activity in the Mice Body of two targeting antibodies
Choose the 17b-estradiol pellets (Innovative Research of America) that female BAl BIc/c nude mice implants 0.72mg 60 days slowly-releasings, after 6 days, adopt PBS:Matrigel according to the resuspended BT-474 cell (1 × 10 of 1:1 ratio in the inoculation of mouse mammary pad
7individual).When gross tumor volume reaches 100mm
3after adopt randomized blocks to divide into groups tumor-bearing mice after, according to twice inoculation HER2 antibody weekly, vaccinization surrounding.By anti-tumor activity in the Mice Body of the two targeting antibodies of measurement gross tumor volume size variation evaluation.As shown in Figure 7, the two targeting antibodies of Tras-Per CrossMab and Tras-Permut CrossMab shows the ability than killing tumor cells in single medicine parental antibody Trastuzumab, Pertuzumab and the stronger Mice Body of Trastuzumab and Pertuzumab drug combination.Further research shows, Tras-Permut CrossMab shows the mouse interior tumor kill capability stronger than Tras-Per CrossMab.
SEQUENCE LISTING
<110> Chinese People's Liberation Army General Hospital
, sail
Zhao, of heap of stone
Poplar, pretty blue
The anti-HER2 of <120> mono-class two targeting antibodies, Preparation Method And The Use
<130> Reference Number
<160> 26
<170> PatentIn version 3.5
<210> 1
<211> 360
<212> DNA
<213> synthetic
<400> 1
gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
tcctgtgcag cttctggctt caacattaaa gacacctata tacactgggt gcgtcaggcc 120
ccgggtaagg gcctggaatg ggttgcaagg atttatccta cgaatggtta tactagatat 180
gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240
ctgcagatga acagcctgcg tgctgaggac actgccgtct attattgttc tagatgggga 300
ggggacggct tctatgctat ggactactgg ggtcaaggaa ccctggtcac cgtctcctcg 360
<210> 2
<211> 120
<212> PRT
<213> synthetic
<400> 2
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 3
<211> 357
<212> DNA
<213> synthetic
<400> 3
gaagttcaat tagtcgagtc tggtggcgga ttggtacagc ctgggggttc ccttcgtctc 60
tcatgtgctg cctcgggctt tactttcacc gattatacaa tggactgggt gcgccaagca 120
cccggaaaag ggctagaatg ggttgcggat gtcaatccaa acagtggtgg cagcatttac 180
aatcagcgat ttaagggacg gttcaccctg tctgtagaca gatccaaaaa cactttatat 240
ttgcaaatga actcacttag ggctgaggat accgccgtgt actattgcgc acgtaacctc 300
gggccgtcgt tttacttcga ctattggggt cagggcacac tagttaccgt cagtagc 357
<210> 4
<211> 119
<212> PRT
<213> synthetic
<400> 4
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser
115
<210> 5
<211> 324
<212> DNA
<213> synthetic
<400> 5
gatattcaaa tgactcagtc tccttcctca ttatcggcta gtgttggtga ccgtgtcacc 60
atcacatgta aagccagcca agatgtatct ataggcgtgg catggtatca gcaaaagccc 120
ggaaaagcgc caaagttgct tatttactcc gcttcatatc gctacacggg ggttccgtcg 180
cgatttagtg gtagcggctc tggaactgac ttcaccctca caatctcctc actacagcct 240
gaagattttg ccacctatta ctgccaacag tattacatat atccctacac tttcgggcaa 300
ggtaccaaag tcgagattaa gcgg 324
<210> 6
<211> 108
<212> PRT
<213> synthetic
<400> 6
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
<210> 7
<211> 318
<212> DNA
<213> synthetic
<400> 7
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 agtgttag 318
<210> 8
<211> 105
<212> PRT
<213> synthetic
<400> 8
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 Cys
100 105
<210> 9
<211> 996
<212> DNA
<213> synthetic
<400> 9
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc gcgggatgag 720
ctgaccaaga accaggtcag cctgacgtgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggtaaa tgatga 996
<210> 10
<211> 330
<212> PRT
<213> synthetic
<400> 10
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 11
<211> 312
<212> DNA
<213> synthetic
<400> 11
gcctccacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtt ga 312
<210> 12
<211> 330
<212> PRT
<213> synthetic
<400> 12
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 13
<211> 996
<212> DNA
<213> synthetic
<400> 13
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 60
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 120
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 180
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 240
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 300
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 360
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 420
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 480
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 540
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 600
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 660
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatg ccgggatgag 720
ctgaccaaga accaggtcag cctgtggtgc ctggtcaaag gcttctatcc cagcgacatc 780
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 840
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 900
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 960
cagaagagcc tctccctgtc tccgggtaaa tgatga 996
<210> 14
<211> 330
<212> PRT
<213> synthetic
<400> 14
Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys
1 5 10 15
Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr
20 25 30
Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser
35 40 45
Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60
Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr
65 70 75 80
Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95
Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys
100 105 110
Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125
Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
130 135 140
Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp
145 150 155 160
Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu
165 170 175
Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
180 185 190
His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205
Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220
Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg Asp Glu
225 230 235 240
Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr
245 250 255
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
260 265 270
Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe
275 280 285
Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn
290 295 300
Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr
305 310 315 320
Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
325 330
<210> 15
<211> 687
<212> DNA
<213> synthetic
<400> 15
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> 16
<211> 227
<212> PRT
<213> synthetic
<400> 16
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> 17
<211> 1356
<212> DNA
<213> synthetic
<400> 17
gaggttcagc tggtggagtc tggcggtggc ctggtgcagc cagggggctc actccgtttg 60
tcctgtgcag cttctggctt caacattaaa gacacctata tacactgggt gcgtcaggcc 120
ccgggtaagg gcctggaatg ggttgcaagg atttatccta cgaatggtta tactagatat 180
gccgatagcg tcaagggccg tttcactata agcgcagaca catccaaaaa cacagcctac 240
ctgcagatga acagcctgcg tgctgaggac actgccgtct attattgttc tagatgggga 300
ggggacggct tctatgctat ggactactgg ggtcaaggaa ccctggtcac cgtctcctcg 360
gcgtcgacca agggcccatc ggtcttcccc ctggcaccct cctccaagag cacctctggg 420
ggcacagcgg ccctgggctg cctggtcaag gactacttcc ccgaaccggt gacggtgtcg 480
tggaactcag gcgccctgac cagcggcgtg cacaccttcc cggctgtcct acagtcctca 540
ggactctact ccctcagcag cgtggtgacc gtgccctcca gcagcttggg cacccagacc 600
tacatctgca acgtgaatca caagcccagc aacaccaagg tggacaagaa agttgagccc 660
aaatcttgtg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 720
ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 780
gaggtcacat gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 840
tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 900
agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 960
gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 1020
aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatg ccgggatgag 1080
ctgaccaaga accaggtcag cctgtggtgc ctggtcaaag gcttctatcc cagcgacatc 1140
gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 1200
ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1260
cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1320
cagaagagcc tctccctgtc tccgggtaaa tgatga 1356
<210> 18
<211> 450
<212> PRT
<213> synthetic
<400> 18
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala
130 135 140
Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser
145 150 155 160
Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val
165 170 175
Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro
180 185 190
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 19
<211> 1359
<212> DNA
<213> synthetic
<400> 19
gaagttcaat tagtcgagtc tggtggcgga ttggtacagc ctgggggttc ccttcgtctc 60
tcatgtgctg cctcgggctt tactttcacc gattatacaa tggactgggt gcgccaagca 120
cccggaaaag ggctagaatg ggttgcggat gtcaatccaa acagtggtgg cagcatttac 180
aatcagcgat ttaagggacg gttcaccctg tctgtagaca gatccaaaaa cactttatat 240
ttgcaaatga actcacttag ggctgaggat accgccgtgt actattgcgc acgtaacctc 300
gggccgtcgt tttacttcga ctattggggt cagggcacac tagttaccgt cagtagcgtg 360
gctgcaccat ctgtcttcat cttcccgcca tctgatgagc agttgaaatc tggaactgcc 420
tctgttgtgt gcctgctgaa taacttctat cccagagagg ccaaagtaca gtggaaggtg 480
gataacgccc tccaatcggg taactcccag gagagtgtca cagagcagga cagcaaggac 540
agcacctaca gcctcagcag caccctgacg ctgagcaaag cagactacga gaaacacaaa 600
gtctacgcct gcgaagtcac ccatcagggc ctgagctcgc ccgtcacaaa gagcttcaac 660
aggggagagt gtgacaaaac tcacacatgc ccaccgtgcc cagcacctga actcctgggg 720
ggaccgtcag tcttcctctt ccccccaaaa cccaaggaca ccctcatgat ctcccggacc 780
cctgaggtca catgcgtggt ggtggacgtg agccacgaag accctgaggt caagttcaac 840
tggtacgtgg acggcgtgga ggtgcataat gccaagacaa agccgcggga ggagcagtac 900
aacagcacgt accgtgtggt cagcgtcctc accgtcctgc accaggactg gctgaatggc 960
aaggagtaca agtgcaaggt ctccaacaaa gccctcccag cccccatcga gaaaaccatc 1020
tccaaagcca aagggcagcc ccgagaacca caggtgtgca ccctgccccc atcccgggat 1080
gagctgacca agaaccaggt cagcctgtcc tgcgcggtca aaggcttcta tcccagcgac 1140
atcgccgtgg agtgggagag caatgggcag ccggagaaca actacaagac cacgcctccc 1200
gtgctggact ccgacggctc cttcttcctc gtgagcaagc tcaccgtgga caagagcagg 1260
tggcagcagg ggaacgtctt ctcatgctcc gtgatgcatg aggctctgca caaccactac 1320
acgcagaaga gcctctccct gtctccgggt aaatgatga 1359
<210> 20
<211> 451
<212> PRT
<213> synthetic
<400> 20
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr
20 25 30
Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe
50 55 60
Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110
Thr Leu Val Thr Val Ser Ser Val Ala Ala Pro Ser Val Phe Ile Phe
115 120 125
Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys
130 135 140
Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val
145 150 155 160
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
165 170 175
Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
180 185 190
Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His
195 200 205
Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Cys Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365
Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Lys
450
<210> 21
<211> 636
<212> DNA
<213> synthetic
<400> 21
gatattcaaa tgactcagtc tccttcctca ttatcggcta gtgttggtga ccgtgtcacc 60
atcacatgta aagccagcca agatgtatct ataggcgtgg catggtatca gcaaaagccc 120
ggaaaagcgc caaagttgct tatttactcc gcttcatatc gctacacggg ggttccgtcg 180
cgatttagtg gtagcggctc tggaactgac ttcaccctca caatctcctc actacagcct 240
gaagattttg ccacctatta ctgccaacag tattacatat atccctacac tttcgggcaa 300
ggtaccaaag tcgagattaa gcgggcctcc accaagggcc catcggtctt ccccctggca 360
ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt caaggactac 420
ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc 480
ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc 540
tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc cagcaacacc 600
aaggtggaca agaaagttga gcccaaatct tgttga 636
<210> 22
<211> 211
<212> PRT
<213> synthetic
<400> 22
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ala Ser Thr Lys
100 105 110
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
115 120 125
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
130 135 140
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
145 150 155 160
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
165 170 175
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
180 185 190
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
195 200 205
Lys Ser Cys
210
<210> 23
<211> 636
<212> DNA
<213> synthetic
<400> 23
gatattcaaa tgactcagtc tccttcctca ttatcggcta gtgttggtga ccgtgtcacc 60
atcacatgta aagccagcca agatgtatct ataggcgtgg catggtatca gcaaaagccc 120
ggaaaagcgc caaagttgct tatttactcc gcttcatatc gctactacgg ggttccgtcg 180
cgatttagtg gtagcggctc tggaactgac ttcaccctca caatctcctc actacagcct 240
gaagattttg ccacctatta ctgccaacag tattacatat atccctacac tttcgggcaa 300
ggtaccaaag tcgagattaa gcgggcctcc accaagggcc catcggtctt ccccctggca 360
ccctcctcca agagcacctc tgggggcaca gcggccctgg gctgcctggt caaggactac 420
ttccccgaac cggtgacggt gtcgtggaac tcaggcgccc tgaccagcgg cgtgcacacc 480
ttcccggctg tcctacagtc ctcaggactc tactccctca gcagcgtggt gaccgtgccc 540
tccagcagct tgggcaccca gacctacatc tgcaacgtga atcacaagcc cagcaacacc 600
aaggtggaca agaaagttga gcccaaatct tgttga 636
<210> 24
<211> 211
<212> PRT
<213> synthetic
<400> 24
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Tyr Arg Tyr Tyr Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ala Ser Thr Lys
100 105 110
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
115 120 125
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro
130 135 140
Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr
145 150 155 160
Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val
165 170 175
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn
180 185 190
Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro
195 200 205
Lys Ser Cys
210
<210> 25
<211> 324
<212> DNA
<213> synthetic
<400> 25
gacatccaga tgacccagtc cccgagctcc ctgtccgcct ctgtgggcga tagggttacc 60
atcacctgcc gtgccagtca ggatgtgaat actgctgtag cctggtatca acagaaacca 120
ggaaaagctc cgaaactact gatttactcg gcatccttcc tctactctgg agtcccttct 180
cgcttctctg gctccagatc tgggacggat ttcactctga ccatcagcag tctgcagccg 240
gaagacttcg caacttatta ctgtcagcaa cattatacta ctcctcccac gttcggacag 300
ggtaccaagg tggagatcaa acgt 324
<210> 26
<211> 108
<212> PRT
<213> synthetic
<400> 26
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
100 105
Claims (19)
1. there is Tras-Per CrossMab and the two targeting antibodies of Tras-Permut CrossMab of similar whole antibody structure and fuction, it is characterized in that, both can combine with Trastuzumab binding site, can combine with Pertuzumab binding site again.
2. not only possess the antitumor advantage of killing and wounding of Trastuzumab and Pertuzumab antibody, and show the tumor-killing effect stronger than Trastuzumab and Pertuzumab conbined usage.
3. according to claim 1 couple of targeting antibodies Tras-Per CrossMab, be made up of four peptide chains, be respectively Trastuzumab heavy chain knob mutant (SEQ ID NO:18), Pertuzumab heavy chain hole mutant (SEQ ID NO:20) and Trastuzumab light chain (SEQ ID NO:26) and Pertuzumab light chain mutant (SEQ ID NO:22).
4. the nucleic acid molecule be separated, four peptide chains according to claim 2 of encoding, have nucleotide sequence described in SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:21.
5. according to claim 1 couple of targeting antibodies Tras-Permut CrossMab, be made up of four peptide chains, be respectively Trastuzumab heavy chain knob mutant (SEQ ID NO:18), Pertuzumab heavy chain hole mutant (SEQ ID NO:20) and Trastuzumab light chain (SEQ ID NO:26) and Pertuzumab light chain mutant PerLV (L56TY)-CH1 (SEQ ID NO:24).
6. the nucleic acid molecule be separated, four peptide chains according to claim 4 of encoding, have nucleotide sequence described in SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:25, SEQ ID NO:23.
7. 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 (+) or pcDNA3.1/ZEO (+), one of pDHFR.
8. a carrier, containing the expression regulation sequence that nucleic acid molecule according to claim 5 is connected with the sequence being operational of described nucleic acid molecule, wherein carrier can be pDR1, pcDNA3.1 (+) or pcDNA3.1/ZEO (+), one of pDHFR.
9. carrier according to claim 6 is pcDNA3.1 (+) or pcDNA3.1/ZEO (+).
10. carrier according to claim 7 is pcDNA3.1 (+) or pcDNA3.1/ZEO (+).
11. a host cell, containing the carrier described in claim 6 or 7, it is eukaryotic cell.
12. host cells according to claim 10 are mammalian cell.
13. host cells according to claim 11 are Chinese hamster ovary celI.
14. prepare the two targeting antibodies of Tras-Per CrossMab according to claim 1 and Tras-Permut CrossMab, the method comprises:
A) clone that Her2 antibody trastuzumab variable region of heavy chain and Pertuzumab are heavy, variable region of light chain respectively;
B) knob mutant is built respectively in antibody Fc region: T366W, S354C; Hole mutant: T366S, L368A, Y407V and Y394C;
C) by Pertuzumab variable region of light chain and heavy chain constant region 1(CH1) construct PerLV-CH1 and merge segment, Pertuzumab variable region of light chain mutant L56TY and heavy chain constant region CH1 is constructed PerLV (L56TY)-CH1 and merges segment;
D) merged in Trastuzumab variable region of heavy chain and knob mutant respectively, Pertuzumab variable region of heavy chain and antibody light chain constant region (CL) and hole mutant merge PerHV-CL-Hinge-CH2-CH3, load expression vector;
E) Trastuzumab heavy chain knob mutant (SEQ ID NO:17) is respectively charged into by what build, the common transfection of expression vector of Pertuzumab heavy chain hole mutant (SEQ ID NO:19) and Trastuzumab light chain (SEQ ID NO:25) and Pertuzumab light chain mutant (SEQ ID NO:21) is expressed, separation and purification.
15.f) be respectively charged into Trastuzumab heavy chain knob mutant (SEQ ID NO:17) by what build, the common transfection of expression vector of Pertuzumab heavy chain hole mutant (SEQ ID NO:19) and Trastuzumab light chain (SEQ ID NO:25) and Pertuzumab light chain mutant PerLV (L56TY)-CH1 (SEQ ID NO:23) is expressed, separation and purification.
16. 1 kinds of compositions, containing the two targeting antibodies of Tras-Per CrossMab according to claim 1 and Tras-Permut CrossMab, and pharmaceutically acceptable carrier.
17. the two targeting antibodies of Tras-Per CrossMab according to claim 1 and Tras-Permut CrossMab is preparing the purposes in antitumor drug.
18. compositions according to claim 14 are preparing the purposes in antitumor drug.
The arbitrary described purposes of 19. claim 17,18, also comprises the antitumor drug conbined usage with other.
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| CN108948206A (en) * | 2017-05-23 | 2018-12-07 | 赵磊 | A kind of bis- targeting antibodies of anti-EGFR/PD-1, preparation method and the usage |
| CN109536476A (en) * | 2018-05-21 | 2019-03-29 | 沣潮医药科技（上海）有限公司 | Targent fused protein, the Preparation method and use for having hyaluronidase activity |
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| CN108948206A (en) * | 2017-05-23 | 2018-12-07 | 赵磊 | A kind of bis- targeting antibodies of anti-EGFR/PD-1, preparation method and the usage |
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| CN109536476A (en) * | 2018-05-21 | 2019-03-29 | 沣潮医药科技（上海）有限公司 | Targent fused protein, the Preparation method and use for having hyaluronidase activity |
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Application publication date: 20150513 |