CN1572801A - Double functional fusion protein possessing antineoplastic functions, method for making same and use thereof - Google Patents

Double functional fusion protein possessing antineoplastic functions, method for making same and use thereof Download PDF

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CN1572801A
CN1572801A CN 200310119930 CN200310119930A CN1572801A CN 1572801 A CN1572801 A CN 1572801A CN 200310119930 CN200310119930 CN 200310119930 CN 200310119930 A CN200310119930 A CN 200310119930A CN 1572801 A CN1572801 A CN 1572801A
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fusion rotein
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CN1297567C (en
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马菁
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Shanghai Maitai Junao Biotechnology Co ltd
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Priority to EP04776301A priority patent/EP1633398A4/en
Priority to CA002528595A priority patent/CA2528595A1/en
Priority to PCT/US2004/017765 priority patent/WO2005001048A2/en
Priority to US11/004,639 priority patent/US20050232931A1/en
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Abstract

The present invention discloses a double functional fusion protein possessing antineoplastic functions which includes a first part and a second part, wherein the first part is molecule possessing induce tumour cell apoptosis function, the second part is Flt ligand. The invention also discloses a encode nucleic acid for the double functional fusion protein and a preparation method. The invention further discloses the application of the double functional fusion protein in preparing antineoplastic medicine.

Description

Has bifunctional fusion proteins of anti-tumor function and its production and application
Technical field
The present invention relates to the tumor immunology field, be specifically related to a kind of bifunctional fusion proteins and coding nucleic acid, preparation method and application in the preparation antitumor drug with anti-tumor function.
Background technology
The immunotherapy of tumour is meant main work by tumor host defense mechanism or biotechnological formulation in order to regulating the biologically of body self, thereby suppresses or kill tumour cell.It has become the 4th kind of treatment pattern after operation, radiotherapy, chemotherapy, is described as " improving therapy " of conventional means, for tumor treatment provides new hope.In China, liver cancer, the lymphocytomatous sickness rate of mammary cancer is very high, is one of modal malignant tumour.Liver cancer patient has 2/3 to lose operation opportunity approximately when going to a doctor, promptly enable row excision person, because can't excise the invisible tumor tissues of naked eyes, the postoperative recurrence rate is higher, and main is that operation can not be removed tumorigenic basic reason.Chemicotherapy all has serious restraining effect to hematopoiesis and immunity system, even causes immunity of organisms further to descend, and causes tumour extensively to shift.Thereby, seek effective immunotherapy of tumors novel method and be extremely important for the thorough healing of liver cancer.
Studies show that in recent years: at the cell surface molecule of tumor death or cell proliferation (as FAS, EGFR, HER2 etc.) monoclonal antibody is the apoptosis of inducing tumor cell (Shimizu M etc., Biochem Biophys ResCommun.1996,228 (2): 375-9) directly.The traditional idea of antigen antibody reaction in the oncotherapy has been abandoned in this discovery, and turn to the signal transduction pathway that utilizes mAb target regulating cell apoptosis and propagation, the monoclonal antibody Herceptin of anti-people HER2 becomes first by the monoclonal antibody of the treatment mammary cancer of FDA approval listing, its curative effect has obtained fully certainly, for a new field has been opened up in the immunotherapy of tumour.Though the target gene of the tumor-targeting of research treatment at present is more, mainly concentrates among the two bars conduction paths.The first activates Tumor Necrosis Factor Receptors (TNFR) family, and the apoptosis pathway that mediates by Caspase plays a role; It two is Ras approach of blocking-up EGF-R ELISA (EGFR) family mediation.
TNF-R family mainly comprises Fas, TNF acceptor and TRAIL acceptor.High expression level is in some large bowel cancer, mammary cancer, prostate cancer, neuroblastoma histocyte.They do not have activity under free state, but at respective ligand (as FAS-part, TNF or TRAIL), or under the effect of monoclonal antibody (mAb), can finally activate Caspase3.Caspase3 can cut the apoptosis substrate and make its activation, causes programmed cell death.The FAS part also can cause the apoptosis of part liver cancer and melanoma cell.But, because FAS also is expressed in thymus gland, liver, the heart and kidney simultaneously.In the experiment, anti-FAS mAb or FAS part can produce injury (especially liver) to above-mentioned normal tissue cell, thereby have limited their application in antineoplaston greatly in vivo.
The EGFR subfamily is made up of 4 closely-related acceptors: EGFR, HER2, HER3 and HER4.Be the transmembrane protein with tyrosine kinase activity, high expression level is in some prostate cancer, large bowel cancer, mammary cancer, carcinoma of the pancreas, kidney, ovarian cancer, nonsmall-cell lung cancer histocyte.They are bringing into play crucial effect in the generation of tumour and evolution.Similar with FAS, under free state, there is not activity, in case under the part effect, take place can activate the signal transduction pathway in downstream by strong tyrosine kinase activity, the generation of induced tumor in conjunction with forming dimer.The blocking-up of special monoclonal antibody (mAb) the target ground of EGFR or HER2 they with the combining of its part, thereby the signal transduction pathway of blocking-up tumor cell proliferation, the inhibition tumor growth also brings out apoptosis.
No matter be FAS or EGFR or HER-2, though in some tumour cell high expression level, also be expressed in (as liver, kidney, lymphoglandula etc.) in some important healthy tissues.Interference to these healthy tissues signal transduction pathway might bring bigger side effect.P230 is the protein molecular of specifically expressing in people's liver cancer, mammary cancer and melanoma tissue cell.Since it in Western bloting can with monoclonal antibody SM5-1 (US number of patent application 09/915,746) combination, and manifest specific band so gain the name at the 230KD place.In the nutrient solution of external no complement effect, SM5-1 can induce hepatoma cell strain (XJC) and melanoma cell strain (U10) that apoptosis takes place by p230.The experiment of nude mice lotus knurl confirms that also p230 can suppress tumor growth under the inducing of SM5-1.Flow cytometry shows that SM5-1 can not mark derives from melanoma cells B16 and the liver cancer cell hepal-6 of C57BL/6 mouse, with human antigen's gene p230 transfection B16 and hepal-6 cell, confirmed that with methods such as flow cytometries the p230 antigen protein can be expressed in the surface of mouse tumor cell, used SM5-1 and can induce transfection that the B16 (B16p230) and hepal-6 (hepap230) the cell generation apoptosis of p230 gene are arranged.B16p230 and hepap230 cell are inoculated the C57BL/6 mouse respectively, find that they all can become knurl in the mouse body, give SM5-1 and can cause tumour cell downright bad in a large number, suppress growth of tumor and cause tumor regression.
Above result of study shows that p230 is a newfound target gene that can be used for liver cancer treatment, anti-people p230 monoclonal antibody can by with the effect Mediated Human hepatoma cell apoptosis of p230, be a new hepatoma-targeting treatment monoclonal antibody.
Along with therapeutic monoclonal antibody continuous advancement in technology, there is increasing monoclonal antibody medicine to be used to the clinical treatment of tumour.But, a large amount of experimental results show, give Antybody therapy merely and can not solve the problem that there is high relapse rate in present malignant tumour, this also is that other therapeutic monoclonal antibody comprises the common difficulty that the anti-HER-2 monoclonal antibody Herceptin that gone on the market and monoclonal antibody Rituximab of anti-CD20 etc. are run into.Cause the major obstacle of this problem to be, with these antibody drugs execute control after, always have a small amount of tumour cell not to be killed.In case these remaining cell conditions suitable (finishing as Antybody therapy) then can form tumour again.At this problem, if can be in the good antitumor curative effect of performance monoclonal antibody, fully excitating organism is replied the specificity active immunity of tumour, passive immunization and the response to active immunization of body to tumour organically combined, just can bring into play antitumor action and generation " immunizing power " more strongly, solve the practical problems of the tumor recurrence that exists in Antybody therapy or the other treatment method tumour of the same race.
As the passive immunotherapy agent, past 10 years monoclonal antibody medicine has obtained immense success, has been used to major diseases such as clinical treatment tumour.The preparation in mouse source, people source, technology such as mouse source antibody humanization transformation, recombinant cell strain structure, recombinant antibodies purifying are increasingly mature, and the screening method of the structure of design of primers, rite-directed mutagenesis, expression vector, the transfection of recipient cell and reconstitution cell, cell culture technology have become current molecular biological mature technology.
Tumor vaccine is as a kind of mode of tumour-specific active immunity treatment, is to show the vaccine that the body input has tumour antigen, stimulates body immune system to produce the antineoplastic immune effect, is used for the treatment of tumour and immune body the recurrence of tumour is prevented.What study morely at present is that (Dendritic cells DC) is the fused cell tumor vaccine on basis with dendritic cell.DC is sole duty antigen presenting cell (the Antigen-presenting cells that function is the strongest in the body immune system, APC), its maximum characteristics are to activate primary tape T cell (CD4+Th cell and CD8+CTL cell), generate helper T cell and killer T cell, DC can discern, process, offers antigen, expresses high-level MHC quasi-molecule, costimulatory molecules, adhesion molecule, simultaneously the IL-12 of secreting high levels, activate corresponding immunocyte, in antineoplastic immune, play an important role.The intravital DC of tumour patient may the factor amount or function is former thereby lack effectively the offering of tumour specific antigen, and makes tumour escape the immunosurveillance of body.If the DC of body is extracted, activation through external various immunomodulators, modify DC with the various forms tumour antigen, feed back in the body then, by DC efficiently offering to act on and then can break the immunological tolerance of body, the effectively specificity antineoplastic immunity of excitating organism reaction to tumour antigen to tumour.At present, represented outstanding curative effect based on the tumor vaccine of DC in anti-tumor immunotherapy, clinical application launches rapidly.But the DC vaccine applies to clinical many problem demanding prompt solutions that also have: 1. because the strict restriction of MHC, must separate from patient's peripheral blood, increase is fed back into blood after preparing a large amount of DC again, and this technical difficulty is higher.2. behind the amplification in vitro, whether the character of DC, angtigen presentation ability change and thus the influence of result of study are still waiting checking.3. at different oncotherapy schemes, need to determine the immunization route, dosage, number of times, pitch time of the required antigen/RNA dosage of sensitization DC, DC etc.At above practical problems, consider if amplification, activation and the antigen of DC cell are modified in the liver cancer patient body to be finished, will simplify the preparation procedure of DC vaccine greatly.But the realization of this imagination is based on the solution of two problems, the one, how to finish amplification and the activation of DC in vivo, and the 2nd, how for providing enough tumour antigens, to modify the DC that function is arranged in vivo.For second above problem, the hepatoma targeting character monoclonal antibody SM5-1 cell lysate that liver cancer apoptosis reducing produced effectively of preparation and apoptotic body etc. can be used as tumour antigen and modify DC.Therefore, how to finish the amplification of DC and activation and gather in the tumor by local selectivity and just to become distinct issues in vivo.
Therefore, this area presses for the medicine of the new effective treatment tumour of exploitation.
The summary of the invention summary
Purpose of the present invention is exactly a kind of material of new effective treatment tumour, it be contain molecule that (a) have an inducing apoptosis of tumour cell function as first part and (b) the Flt3 part as the bifunctional fusion proteins of second section.
In a first aspect of the present invention, a kind of bifunctional fusion proteins with anti-tumor function is provided, described fusion rotein comprises first part and second section, and first part is the molecule with inducing apoptosis of tumour cell function, and second section is the Flt3 part.
In a preference, the molecule of described inducing apoptosis of tumour cell is selected from: the apoptotic protein molecular of the variable region of monoclonal antibody, monoclonal antibody or induced tumor or its varient.
In another preference, the molecule of described inducing apoptosis of tumour cell is selected from:
(a) SM5-1 antibody, CD20 antibody, Her2 antibody, Her3 antibody, Her4 antibody, EGFR antibody or its variable region;
(b) Fas, TNF acceptor, Trail acceptor or its extracellular region.
In another preference, also containing length between first part and second section is 10-25 amino acid whose joint sequence or isoleucine zipper sequence.
In another preference, described fusion rotein contains SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQID NO:32, SEQ ID NO:34, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, the aminoacid sequence shown in SEQ ID NO:66 or the SEQ ID NO:68.
In a second aspect of the present invention, provide a kind of isolated nucleic acid molecule, its bifunctional fusion proteins of the present invention of encoding.Preferably, described nucleic acid molecule has SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, the nucleotide sequence shown in SEQ ID NO:65 or the SEQ IDNO:67.
In a third aspect of the present invention, a kind of carrier is provided, it contains said nucleic acid molecule of the present invention, and the expression regulation sequence that links to each other with the series of operations of described nucleic acid molecule.
In a fourth aspect of the present invention, a kind of host cell is provided, it contains the above-mentioned carrier of the present invention.
In a fifth aspect of the present invention, a kind of method for preparing bifunctional fusion proteins of the present invention is provided, this method comprises:
A) under expression condition, cultivate above-mentioned host cell, thereby express bifunctional fusion proteins,
B) the described bifunctional fusion proteins of isolated or purified.
In a sixth aspect of the present invention, the purposes of bifunctional fusion proteins of the present invention is provided, it is used to prepare antitumor drug.
In a seventh aspect of the present invention, a kind of pharmaceutical composition is provided, it contains bifunctional fusion proteins of the present invention and pharmaceutically acceptable carrier.
Description of drawings
Fig. 1 has shown two special tetravalent antibody molecules (A) and bifunctional protein Flex/Fc/Fv (B) antigen-4 fusion protein gene structure.
Fig. 2 has shown Nucleotide (SEQ ID NO:1) and amino acid (the SEQ ID NO:2) sequence of FLex.SP is a signal peptide sequence.
Fig. 3 has shown Nucleotide (SEQ ID NO:3) and amino acid (the SEQ ID NO:4) sequence of FLex-Ig.SP is a signal peptide sequence; FLex, people FL film outskirt gene.
Fig. 4 has shown joint (Gly4Ser) 3Nucleotide (SEQ ID NO:5) and amino acid (SEQ ID NO:6) sequence.SP is a signal peptide sequence.
Fig. 5 has shown 1% agarose gel analysis of anti-people p230 monoclonal antibody (SM5-1) variable region gene PCR product.Swimming lane 1:SM5-1 variable region of heavy chain wherein; Swimming lane 2:SM5-1 variable region of light chain; Swimming lane 3:DL2000 DNA standard substance.
Fig. 6 has shown the variable region of heavy chain Nucleotide (SEQ ID NO:7) and amino acid (the SEQ ID NO:8) sequence of SM5-1 monoclonal antibody.SP is a signal peptide sequence.
Fig. 7 has shown the variable region of light chain Nucleotide (SEQ ID NO:9) and amino acid (the SEQ ID NO:10) sequence of SM5-1 monoclonal antibody.SP is a signal peptide sequence.
Fig. 8 has shown the Nucleotide (SEQ ID NO:11) and amino acid (SEQ IDNO:12) sequence of SM5-1 chimeric antibody (chSM) heavy chain.SP is a signal peptide sequence, and intron sequences is represented in the shadow zone; Stop, translation stop codon;
Fig. 9 has shown the Nucleotide (SEQ ID NO:13) and amino acid (SEQ IDNO:14) sequence of SM5-1 chimeric antibody (chSM) light chain.SP is a signal peptide sequence; Stop, translation stop codon;
Figure 10 has shown SM5-1 chimeric heavy chain expression vector synoptic diagram; Wherein HCMV pro represents human cytomegalovirus M ajor Immediate early promoter; VH represents the heavy chain variable region gene of humanization SM5-1; CH represents people γ 1 chain constant region encoding gene; BGH pA represents Bovine growth hormone polyadenous glycosidation signal; SV40 ori represents the early stage replication orgin of SV40 virus; DHFR represents dihydrofolate reductase gene; PUC origin is the plasmid replication starting point; Amp is an ampicillin resistance gene.
Figure 11 has shown SM5-1 light chain expression vector synoptic diagram, and wherein VL represents the chain variable region gene of humanization SM5-1; CL represents people's chain constant region gene, the same Figure 10 of all the other elements.
Figure 12 has shown SM5-1 humanized antibody (huSM) variable region of heavy chain Nucleotide (SEQ ID NO:15) and amino acid (SEQ ID NO:16) sequence.SP is a signal peptide sequence.
Figure 13 has shown SM5-1 humanized antibody (huSM) variable region of light chain Nucleotide (SEQ ID NO:17) and amino acid (SEQ ID NO:18) sequence.SP is a signal peptide sequence.
Figure 14 has shown the Nucleotide (SEQ ID NO:19) and amino acid (SEQID NO:20) sequence of SM5-1 humanized antibody (huSM) heavy chain.SP is a signal peptide sequence, and intron sequences is represented in the shadow zone; Stop, translation stop codon;
Figure 15 has shown the Nucleotide (SEQ ID NO:21) and amino acid (SEQID NO:22) sequence of SM5-1 humanized antibody (huSM) light chain.SP is a signal peptide sequence; Stop, translation stop codon;
Figure 16 has shown the Nucleotide (SEQ ID NO:23) and amino acid (SEQ IDNO:24) sequence of HuSMVH/Fc/FL fusion rotein.SP is a signal peptide sequence, and intron sequences is represented in the shadow zone; Stop, translation stop codon;
Figure 17 has shown the Nucleotide (SEQ ID NO:25) and amino acid (SEQID NO:26) sequence of huSMVH/Fc/Link/FL fusion rotein.SP is a signal peptide sequence, and intron sequences is represented in the shadow zone; Stop, translation stop codon;
Figure 18 has shown the Nucleotide (SEQ ID NO:27) and amino acid (SEQ IDNO:28) sequence of FL/Fc/huSMFv fusion rotein.SP is a signal peptide sequence, Stop, translation stop codon;
Figure 19 has shown the hFLex/Fc/Fv antigen-4 fusion protein gene structure after SM5-1 and Flex merge.
Figure 20 has shown the Nucleotide (SEQ ID NO:29) and amino acid (SEQ IDNO:30) sequence of chSMVH/Fc/FL fusion rotein.SP is a signal peptide sequence, and intron sequences is represented in the shadow zone; Stop, translation stop codon;
Figure 21 has shown the Nucleotide (SEQ ID NO:31) and amino acid (SEQID NO:32) sequence of chSMVH/Fc/Link/FL fusion rotein.SP is a signal peptide sequence, and intron sequences is represented in the shadow zone; Stop, translation stop codon;
Figure 22 has shown the Nucleotide (SEQ ID NO:33) and amino acid (SEQ IDNO:34) sequence of FL/Fc/chSMFv fusion rotein.SP is a signal peptide sequence, Stop, translation stop codon;
Figure 23 has shown the variable region of heavy chain Nucleotide (SEQ ID NO:35) and amino acid (the SEQ ID NO:36) sequence of 2B8 monoclonal antibody.SP is a signal peptide sequence.
Figure 24 has shown the variable region of light chain Nucleotide (SEQ ID NO:37) and amino acid (the SEQ ID NO:38) sequence of 2B8 monoclonal antibody.SP is a signal peptide sequence.
Figure 25 has shown the Nucleotide (SEQ ID NO:39) and amino acid (the SEQ ID NO:40) sequence of CD20 chimeric antibody heavy chain.SP is a signal peptide sequence, and intron sequences is represented in the shadow zone; Stop, translation stop codon;
Figure 26 has shown the Nucleotide (SEQ ID NO:41) and amino acid (the SEQ ID NO:42) sequence of CD20 chimeric antibody light chain.SP is a signal peptide sequence; Stop, translation stop codon;
Figure 27 has shown the Nucleotide (SEQ ID NO:43) and amino acid (SEQ IDNO:44) sequence of CD20VH/Fc/FL fusion rotein.SP is a signal peptide sequence, and intron sequences is represented in the shadow zone; Stop, translation stop codon;
Figure 28 has shown the Nucleotide (SEQ ID NO:45) and amino acid (SEQID NO:46) sequence of CD20VH/Fc/Link/FL fusion rotein.SP is a signal peptide sequence, and intron sequences is represented in the shadow zone; Stop, translation stop codon;
Figure 29 has shown the Nucleotide (SEQ ID NO:47) and amino acid (SEQ IDNO:48) sequence of FL/Fc/CD20Fv fusion rotein.SP is a signal peptide sequence, Stop, translation stop codon;
Figure 30 has shown the hFLex/Fc/scFv antigen-4 fusion protein gene structure after CD20 and Flex merge
Figure 31 has shown Her2 humanized antibody variable region of heavy chain Nucleotide (SEQ ID NO:49) and amino acid (SEQ IDNO:50) sequence.SP is a signal peptide sequence.
Figure 32 has shown Her2 humanized antibody variable region of light chain Nucleotide (SEQ ID NO:51) and amino acid (SEQ IDNO:52) sequence.SP is a signal peptide sequence.
Figure 33 has shown the Nucleotide (SEQ ID NO:53) and amino acid (the SEQ ID NO:54) sequence of Her2 humanized antibody heavy chain.SP is a signal peptide sequence, and intron sequences is represented in the shadow zone; Stop, translation stop codon;
Figure 34 has shown the Nucleotide (SEQ ID NO:55) and amino acid (the SEQ ID NO:56) sequence of Her2 humanized antibody light chain.SP is a signal peptide sequence; Stop, translation stop codon;
Figure 35 has shown the Nucleotide (SEQ ID NO:57) and amino acid (SEQ IDNO:58) sequence of her2VH/Fc/FL fusion rotein.SP is a signal peptide sequence, and intron sequences is represented in the shadow zone; Stop, translation stop codon;
Figure 36 has shown the Nucleotide (SEQ ID NO:59) and amino acid (SEQID NO:60) sequence of her2VH/Fc/Link/FL fusion rotein.SP is a signal peptide sequence, and intron sequences is represented in the shadow zone; Stop, translation stop codon;
Figure 37 has shown the Nucleotide (SEQ ID NO:61) and amino acid (SEQ IDNO:62) sequence of FL/Fc/her2Fv fusion rotein.SP is a signal peptide sequence, Stop, translation stop codon;
Figure 38 has shown the hFLex/Fc/Fv antigen-4 fusion protein gene structure after Her2 and FL merge
Figure 39 has shown the Nucleotide (SEQ ID NO:63) and amino acid (SEQ IDNO:64) sequence of hFLex/Trailex fusion rotein.SP is a signal peptide sequence, Stop, translation stop codon;
Figure 40 has shown hFLex/TRAILex antigen-4 fusion protein gene structure
Figure 41 has shown the Nucleotide (SEQ ID NO:65) and amino acid (SEQ IDNO:66) sequence of hFLex/IZ/Trailex fusion rotein.SP is a signal peptide sequence, Stop, translation stop codon;
Figure 42 has shown the Nucleotide (SEQ ID NO:67) and amino acid (SEQ IDNO:68) sequence of hFLex/Fc/Trailex fusion rotein.SP is a signal peptide sequence, Stop, translation stop codon;
Figure 43 has shown hFLex/Fc/Trailex antigen-4 fusion protein gene structure
Figure 44 has shown that SM/FL is to people's bleeding of the umbilicus CD34+ cells in vitro amplification effect.
Figure 45 has shown that SM/FL is to amplification effect in the body of NK and DC cell.
Figure 46 has shown that anti-people SM5-1 mosaic monoclonal antibody/FL fusion rotein (A), anti-SM5-1 humanization monoclonal antibody/FL fusion rotein (B) suppress growth curve to various cells in vitro
Figure 47 has shown that the FL fusion rotein is to B16 (A), Hepal-6 (B), B16p230 (C) and the isocellular vitro inhibition growth curve of Hepal-6p230 (D)
Figure 48 has shown Her2/FL fusion rotein (A) and heceptin (B) the vitro inhibition growth curve to various tumour cells
Figure 49 has shown Her2/FL fusion rotein (A) and heceptin (B) the external killing activity to various tumour cells
Figure 50 has shown the external killing activity of CD20/FL fusion rotein to various tumour cells
Figure 51 has shown Trail/FL fusion rotein (A) and Trail (B) the vitro inhibition growth curve to various tumour cells
Figure 52 has shown Trail/FL fusion rotein (A) and Trail (B) the external killing activity to various tumour cells
Figure 53 has shown anti-HER-2/FL fusion rotein anti-tumor in vivo activity.
Figure 54 has shown CD20/FL fusion rotein anti-tumor in vivo activity.
Figure 55 has shown TRAIL/FL anti-tumor in vivo activity.
Figure 56 has shown in the body of chSM/FL, huSM/FL fusion rotein and has distributed.
Figure 57 has shown the interior experiment that distributes of the body of three kinds of fusion roteins that anti-human Her 2 monoclonal antibody, anti-humen CD 20 monoclonal antibody and TRAIL and FL form.
Detailed Description Of The Invention
The detailed content of invention will be divided following a few part introduction.
A, definition:
Unless detailed description is arranged in addition, technology used herein and scientific terminology are with conventional term.
B, contain molecule that (a) have an inducing apoptosis of tumour cell function as first part and (b) the Flt3 part as the bifunctional fusion proteins of second section.
On the one hand, the present invention is a kind of bifunctional fusion proteins, and this albumen comprises Flt3 part or bioactive fragment wherein and a kind of protein or peptide molecule, and this molecular energy inducing tumor cell is transferred and died.Be more preferably, this fusion rotein is an independently albumen, does not have with other albumen, polypeptide or molecule and gets in touch, and is the purified product cultivated of recombinant host cell or as a kind of extract of purifying.
Any suitable Flt3 part all relates to composition provided herein and method.Term used herein " Flt3 part " is meant a peptide species, can be in conjunction with Flt3 acceptor and inducement signal conduction.Flt3 part or bioactive fragment wherein comprise a part of conserved amino acid alternative sequence, and this sequence does not influence its activity.Suitably replacing amino acid is the known technology of this area, and this work can be easy to carry out and guarantee not change the gained molecular biological activity.These skills are recognized people, in general, can not change biological activity basically at the inessential area change single amino acids of a peptide species.See Molecular Biology of The Gene such as Watson, the 4th edition, 1987, TheBenjamin/Cummings Pub.Co.P224.Table 1 is some examples of this replacement.
Table 1
Original acid residue preservative replacement
Ala(A)???????????????????Gly；Ser
Arg(R)???????????????????Lys
Asn(N)???????????????????Gln；His
Cys(C)???????????????????Ser
Gln(Q)???????????????????Asn
Glu(E)???????????????????Asp
Gly(G)???????????????????Ala；Pro
His(H)???????????????????Asn；Gln
Ile(I)???????????????????Leu；Val
Leu(L)???????????????????Ile；Val
Original acid residue preservative replacement
Lys(K)???????????????????Arg；Gln；Glu
Met(M)???????????????????Leu；Tyr；Ile
Phe(F)???????????????????Met；Leu；Tyr
Ser(S)???????????????????Thr
Thr(T)???????????????????Ser
Trp(W)???????????????????Tyr
Tyr(Y)???????????????????Trp；Phe
Val(V)???????????????????Ile；Leu
The similar replaceability that may also have other, this replaceability often can rule of thumb be determined or be determined according to known conserved sequence.
The Flt3 part is an I type transmembrane protein, is discharged into the extracellular with the form of solubility homodimer.See Lyman et al., Flt3 ligand in THE CYTOKINE HANDBOOK (Thomson et al.ed., 4th ed (2003)).A kind of particular case, Flt3 part or its bioactive fragment are soluble.At amino acid levels people Flt3 part and mouse Flt3 part 72% homology is arranged, have a lot of characteristics of mouse source protein, as glycosylation site, crucial cysteine residues and shearing tie point.The albumen of suitable Flt3 part has all been pointed out in a lot of researchs.As Cell 75: 1157-67 (1993), Hannum et al., Nature, 368: 364-67 (1996); U.S.Patent No.5,843,423; U.S.Patent Application Serial Nos:200030113341and 20030148516; And Genebank AccessionNos.NM 001459, U2 9874, U03858, and U04806. etc.
The acceptor of Flt3 part, promptly Flt3 is the member of three receptor tyrosine kinase receptor families.At normal cell, Flt3 is expressed in immature hematopoietic cell, as the CD34+ cell, and placenta, sexual gland and cerebral tissue.See e.g., Rosnet, et al., Blood 82:1110-19 (1993); Small et al., Proc.Natl.Acad.Sci.U.S.A.91:459-63 (1994); And Rosnet et al., Leukemia 10:238-48 (1996) .Flt3 high expression level is at pernicious blood tissues, as acute BM form leukemia, acute pre B cell Lymphocytic leukemia, acute T cellularity Lymphocytic leukemia and chronic lymphocytic leukemia.Signal transduction pathway after Flt3 part and its receptors bind in the active cells, as STAT5, PI 3 '-kinases, PLC γ, MAPK, SHC, SHP2, and SHIP.See e.g., Gilliand et al., Curr.Opin.Hematol.9:274-81 (2002). membranous type FL and solubility FL can in conjunction with and activate the Flt3 acceptor.
Particularly, the fragment of Flt3 part (FL) or biologically active can both the hemopoietic stem cell or the propagation of progenitor cell.Distinguishingly, the fragment of Flt3 part or biologically active can optionally stimulate bone marrow precursor or monocyte, scavenger cell, B cell, dendritic cell and natural killer cell propagation.FL is expressed in hematopoietic cell and other cells of bone marrow microenvironment at first, comprises the B cell, T cell and bone marrow precursor.FL is the growth-stimulating factor of CD34+ progenitor cell, promotes the propagation and the differentiation of dendritic cell and natural killer cell.Studies show that such as us Flt3 passes through the NK cell activity and mediates antitumor signal.Péron?et?al.，J.Immunol.161：6164-70(1998).
The Flt3 part also promotes the maturation of dendritic cell, makes it more effectively to bring into play at tumour cell the effect of antigen presenting cell.See e.g., Fong et al., Gene Ther. 9 (17): 1127-38 (2002). the more important thing is through the Flt3 part and induce that sophisticated dendritic cell is discharged into peripheral blood from marrow, the dendritic cell quantity that immune stimulatory is replied increases.But the existence of this other hemopoieticgrowth factors of effective derived need and cytokine.
The bioactive fragment of arbitrary FL can be applied in the present research method.Here, the segmental bioactive implication of FL is meant the stimulating factor as Flt3, and it still can keep function.Generally, the Flt3 part combines at cell surface with Flt3, stimulates one or more signal transduction pathway or approach and causes cell response, for example propagation.Under the normal circumstances, active fragments keeps the activity of 50% stimulation Flt3 at least.Under condition preferably, active fragments can keep 60%, 70%, 80%, 90%, 95%, 99% and 100% stimulating activity.The activity of Flt3 depends on a kind of suitable method that adopts, and comprises but is not limited only to the activity of signaling molecule, and these signaling molecules comprise STAT5, PLC γ, perhaps the in-vitro multiplication activity of the cells in vitro that Flt3 is relied on.For example, the BAF/BO3 cell strain lacks the Flt3 acceptor, is IL-3 dependent form cell strain.But,, it is produced Flt3 part inductive propagation reply with Flt3 transfection BAF/BO3 cell strain.See Hatakeyama, et al., Cell 59: 837-45 (1989).
Specifically, Flt3 part or bioactive fragment have SEQ ID NO:2 aminoacid sequence in fusion rotein.The Flt3 part can specificity with and the antibody that combines of SEQ ID NO:2 aminoacid sequence combine, and the Flt3 part can continue to keep its biologic activity, any suitable Flt3 ligand specificity antibody can both be used.In addition, the Flt3 part comprises one section sequence, and 28 to 128 of this sequence and SEQ ID NO:2 have 80% homology.The Flt3 part also comprises one section aminoacid sequence, and this sequence is to choose from 28 to 160 of SEQ ID NO:2 and 28 to 182 s' amino-acid residue.Under special circumstances, the Flt3 part also comprises 28 to 128 the amino-acid residue of one section SEQ ID NO:2.The Flt3 part comprises at least 100 amino-acid residues, and Flt3 part and SEQ ID NO:2 have at least 40% homology, this homology depends on the size of the same acid sequence among the SEQ ID NO:2, and the Flt3 part remains its biologic activity.
In the middle of the tumour method that any bioactive fragment all may be used on here mentioning.Be applied to this, " tumour medicine " refers to the medicine that can cause death of neoplastic cells.This medicine normally protein or the polypeptide class, this necrocytosis can be apoptosis, necrosis or analogue.Generally speaking, necrocytosis causes by apoptosis, this apoptosis or directly by the apoptotic signal approach that can induce the Fas part or indirectly inactivation by somatomedin produce.Here, apoptosis refers to the programmed death of tumour cell and finally causes the cracking of chromatinic aggegation and DNA.Any suitable method may be used to apoptotic assessment, comprises but is not limited only to flow cytometer showed, and for example TUNEL analyzes, agarose gel analysis and caspase 3 activation analysiss.In addition, anti-tumor activity takes place in the tumour medicine of fusion rotein under natural situation.This medicine comprises the part of the acceptor of energy inducing tumor cell cessation of growth cessation or death, comprises occurring the part equimolecular under the nature situation, can induce to comprise TNF-α, Fas (CD95) part, TRAIL, lymphotoxin (LT), TWEAK, and the apoptosis of other tnf ligand superfamily members.Specifically, later tumour medicine is from the FAS part, and TNF is select in the extracellular motif of TRAIL or biologically active.In addition, the bioactive fragment of tumour medicine has at least 50% apoptosis activity, and under suitable situation, derived fragment has 60%, 70%, 80%, 90%, 95%, 99% and 100% apoptosis activity.
In addition, tumour medicine is a kind ofly can suppress tumor proliferation, in some cases, and also albumen that can be apoptosis-induced.The targeted molecular of these antibody comprises that for example epithelical cell growth factor acceptor subfamily (comprises EGFR, HER2, HER3 and HER4) these all are the membrane-spanning proteins with tyrosine kinase activity, these albumen (comprise prostate cancer in most of malignant tumours, colorectal carcinoma, mammary cancer, kidney, ovarian cancer, lung cancer, carcinoma of the pancreas) express in, thereby anti-specifically EGFR or Anti-HER 2 can be blocked the signal transduction path of blocking tumor cell proliferation that combines of these albumen and EGFR and HER2, directly or indirectly suppress tumor growth, the apoptosis of inducing tumor cell.See e.g., Clin.CancerRes. 8: 1720-30 (2002); Brodowicz et al.Br.J.Cancer 85: 1764-70 (2001); Crombet-Ramos et al., Int.J.Cancer 101: 567-75 (2002); Herbst et al., Expert Opin.Biol.Ther. 1: 719-32 (2001).
In addition, tumour medicine be a kind of can with tomour specific or the apoptosis-induced albumen of related antigen bonded.For example, p230 is a kind of in people's liver cancer, mammary cancer, and the albumen of specifically expressing in the lymphoma cell, its name comes from the band of the tangible special 230KD of of using that the SM5-1 monoclonal antibody obtains by Western blotting detection method.See U.S.PatentApplication Serial No.:09/915,746.P230 is because its specificity points out it to can be used as the target target gene of immunotherapy of tumors, and P230 can cell death inducing with combining of part or antibody.Some anti-SM5-1 antibody can obtain describing in example 3.At some in particular cases, use Serial No. (Attomey Docket No.54906-2000100) albumen and can seal SM5-1.
In addition, tumour medicine is a kind of antibody or the fragment with biologic activity, and here, antibody is meant complete antibody, Fab fragment, Fab ' fragment, F (ab ') 2Fragment, the Fv fragment, disome, the multi-specificity antibody that single-chain antibody and antibody fragment are formed, its molecule still can keep biologic activity.The antibody that is applied in now in the whole bag of tricks can pass through cell cultures, produces in microbiotic and the animal, and these animals include but are not limited to ox, rabbit, goat, mouse, rat, hamster, cavy, sheep, dog, cat, monkey, orangutan, ape etc.Therefore, the antibody of using at present all is the antibody in Mammals source.
Phage technology can be used for separating initial antibodies or producing different specificitys or avidity.This technology is ripe.Specifically, can obtain antibody by well-known recombinant technology.For example, can obtain recombinant antibodies by the carrier transfection host cell that contains the encoding antibody dna sequence dna.One or more carriers can be used at least one V of transfection expression LWith a V HDistinguish to host cell.Comprise Delves, ANTIBODY PRODUCTION:ESSENTIAL TECHNIQUES (Wiley, 1997) for the description that obtains and produce the recombinant technology standardization of antibody; Shephard, et al., MONOCLONAL ANTIBODIES (Oxford University Press, 2000); And Goding, MONOCLONAL ANTIBODIES:PRINCIPLES AND PRACTICE (Academic Press, 1993).
Can modify to improve the validity of antibody required function by recombination method in the use of antibody at present.Can expect simultaneously that antibody can be modified by the reorganization replacement.Typical this replacing with guarded replacement.For example, the amino acid of at least one constant region replaces with other residues.See U.S.Patent No.5 for details, 624,821, U.S.Patent No.6,194,551, Application No.WO 9958572; And Angal et al., Mol.Immunol.30:105-08 (1993).Amino acid whose modification comprises disappearance, adds and replaces.In some cases, this change can reduce unwanted activity, as complement dependent cytotoxicity.
Antibody can be humanized antibody.Said here " humanized antibody " refers to that the amino acid of non-antigen binding domain is changed in the antibody, and antibody is similar to people's antibody more like this, and still keeps its primary antigen-specific.Being typically the variable region is a kind of species such as mouse, and constant region is behaved.Antibody also can be chimeric antibody.Said here " chimeric antibody " refers to that aminoacid sequence is changed in the antibody, and the sequence that antibody contains is from more than one Mammals, and still keeps its primary antigen-specific.Said here " single-chain antibody (ScFv) " refers to genetic engineering antibody, contains the variable region of heavy chain and the variable region of light chain of immunoglobulin (Ig), connects by flexible polypeptide.
Better, the antibody in the method and composition described here is monoclonal antibody.Here said " monoclonal antibody " refers to the monospecific antibody that obtained by a B cell.
Antibody can be people's antibody.Said here " people's antibody " refers to that the complete light chain and the heavy chain district of antibody comprise that complementarity-determining region is all from the people basically.For example, by people's hybridoma technology, human B cell technology can obtain human monoclonal antibodies (see Kozbor for details, et al., Immunol.Today 4; 72 (1983), EBV transformation technology (seeing Cole et al.MONOCLONAL ANTIBODIES AND CANCER THERAPY 77-96 (1985) for details), perhaps use phage display techniques (seeing Marks et al. for details, J.Mol.Biol.222:581 (1991)).A special example has obtained people's antibody by transgenic mice.The existing document of this people's antibody is partially or completely delivered, and any this technology can be used.According to an example that particularly points out, in transgenic mice, obtained whole person's antibody sequence of expressing human light chain of antibody and heavy chain gene.The transgenic mice of people's antibody of normative description produce to(for) preparation sees Application No.WO 02/43478.B cell from the transgenic mice of producing required antibody can merge with hybridoma again, like this can continuous production antibody.See U.S.Patent Nos.5 for details, 569,825; 5,625,126; 5,633,425; 5,661,016; And 5,545, and 806; And Jakobovits, Adv.Drug Del.Rev.31:33-42 (1998); Green, et al., J.Exp.Med.188:483-495 (1998).
A concrete example, used here antibody can suppress the propagation of target tumor cell.If cell proliferation suppresses when certain antibody exists, and not this antibody or cell proliferation when being non-binding antibody thinks that then this antibody suppresses for cell proliferation.Propagation can be by any suitable method quantitative assay.Typical method be by the radiolabeled Nucleotide of external test (as 3The H-thymidine) breeds with combining quantitatively of DNA.Specifically, can be luminous by ATP, as CellT iter-Glo TMLuminescent Cell Viability Assay (Promega) measures propagation.Like this, that antibody can be special or at the molecule of any adjusting cells survival or growth.
A concrete example contains anti-p230, anti-CD20, anti-Her-2, anti-Her-3, anti-Her-4, anti-egfr antibodies or its bioactive fragment from one group and to screen antibody.Comprise that for these antibody the example normative description sees U.S.Patent Nos.5,677,171; 6,399,061; 6,458,356; 6,455,043; And 5,705, and 157.
Containing the fusion rotein of Flt3 part or the chimeric antibody of its bioactive fragment and killing cancer cell reagent can connect with suitable connexon.For example, the Flt3 part can be connected by the polypeptide connexon with killing cancer cell reagent.A special example, connecting polypeptide is (Gly 4Ser) 3
The composition of the fusion rotein of saying here and method can contain the Flt3 part and killing cancer cell reagent is linked in sequence with any.For example, the Flt3 part is positioned at the N end of fusion rotein.Again for example, the Flt3 part also can be positioned at the C end of fusion rotein.
Fusion rotein can further comprise: the polypeptide label that contains a polypeptide fragment at its C end.Any suitable label can use.For example, label can be FLAG, HA, HA1, c-Myc, 6-His, AU1, EE, T7,4A6, ε, B, gE and Ty1 (seeing Table 2).These labels can be used for the purifying of fusion rotein.
The spendable tag system of table 2
Epi-position Peptide Antibody Document
FLAG ????DYKDDDK ???4E11 Prickett 1
HA ????YPYDVPDYA ???12Ca5 Xie 2
HA1 ????CQDLPGNDNST ???mouse?MAb Nagelkerken 3
c-Myc ????EQKLISEEDL ???9E10 Xie 2
6-His ????HHHHHH ???BAbCO *
AU1 ????DTYRYI ???BAbCO
Epi-position Peptide Antibody Document
EE ????EYMPME ??anti-EE ??Tolbert 4
T7 ????ASMTGGQQMGR ??Invitrogen ??Chen 5??Tseng 6
4A6 ????SFPQFKPQEI ??4A6 ??Rudiger 7
????KGFSYFGEDLMP ??anti-PKC ??Olah 8
B ????QYPALT ??D11，F10 ??Wang 9
gE ????QRQYGDVFKGD ??3B3 ??Grose 10
Ty1 ????EVHTNQDPLD ??BB2，TYG5 ??Bastin11
1.Prickett，et?al.，BioTechniques， 7(6)：580-584(1989)
2.Xie，et?al.，Endocrinology， 139(11)：4563-4567(1998)
3.Nagelkerke，et?al.，Electrophoresis， 18：2694-2698(1997)
4.Tolbert?and?Lameh，J.Neurochem.， 70：113-119(1998)
5.Chen?and?Katz，BioTechniques， 25(1)：22-24(1998)
6.Tseng?and?Verma，Gene， 169：287-288(1996)
7.Rudiger，et?al.，BioTechniques， 23(1)：96-97(1997)
8.Olah，et?al.，Biochem.， 221：94-102(1994)
9.Wang，et?al.，Gene， 169(1)：53-58(1996)
10.Grose，U.S.Patent?No.5,710,248
11.Bastin，et?al.，Mol.Biochem.Parasitology， 77：235-239(1996)Invitrogen，Sigma，Santa?Cruz?Biotech
On the other hand, the invention provides the isolating nucleic acid of a kind of fusion rotein of coding, also can be its complementary strand; This fusion rotein comprises the protein and the polypeptide of Flt3 part, its activated fragment or energy killing tumor cells.In an example, the nucleic acid of separated coding fusion rotein have SEQ ID NO:1,9,13,17,21 or, the nucleotide sequence shown in 25.The present invention also provides the carrier of the nucleic acid molecule that comprises the separated coding fusion rotein.Carrier also may comprise the continuous expression regulation sequence of series of operations of described nucleic acid molecule.
Any coding Flt3 part or its activated segmental suitable dna structure all are applicable to the present invention.Such structure includes but not limited to the nucleotide sequence of Genbank coding U03858 and ATCC coding ATCC 69382.Can be applicable to the United States Patent (USP) 5,843 that also comprises of the present invention, 423 and the U.S. use dna sequence dna and corresponding protein thereof in the patent 200030113341 and 20030148516.
The protein of any coding energy killing tumor cells and the dna structure of polypeptide also are applicable to present method.Hereinafter the sequence of mentioning in the example all is applicable to present method.
Any suitable carriers can be used.Be used for the clone of bacterium, fungi, yeast and mammalian cell and expression vector has all been chatted and, as Pouwels et al., CLONING VECTORS: laboratory manual (Elsevier latest edition).
Expression vector comprises and is connected with the suitable fusion rotein dna sequence dna of regulating sequence of transcribing and translate, as Mammals, microorganism, virus or insect genes.Regulate sequence and comprise that transcripting promoter, operon, enhanser, ribosome bind site or control transcribes and translation initiation and terminated proper sequence.When the fusion rotein sequence need be regulated functional nucleotide sequence, then connect proper regulation sequence.Like this, promoter sequence is connected encoding fusion protein dna sequence dna front end.The ability of duplicating in host cell is usually by the replication initiation point control.The screening-gene that is used for transformant identification also can add expression vector
In addition, the encoding sequence of the signal peptide of the protein of non-natural Flt3 part or energy killing tumor cells and polypeptide can be introduced expression vector.For example: signal peptide (secretion guidance) sequence can with merge with the fusion rotein encoding sequence, thereby the fusion rotein of translation is had.Signal peptide can strengthen host cell to the exocytosis chimeric polyeptides.Signal peptide can be cut from the process that the cell internal secretion is come out at chimeric polyeptides.
Mammals or insect host cell culture systems also can be used for the expression of recombination fusion protein.It is that cell also can be used that Luckow andSummers has summarized the building of situation (Bio/Technology6:47 (1988) .) Mammals source that the poxvirus system is used for producing at insect cell foreign protein.Mammal cell line comprises monkey-kidney cells COS-7 (ATCC CRL 1651), L cell, C127 cell, 3T3 cell (ATCC CCL 163), Chinesehamster ovary (CHO) cell, HeLa cell, and BHK (ATCC CRL 10) cell, derives from CV-1/EBNA-1 clone (the EMBO J. of African green monkey kidney cell line CVI (ATCC CCL 70) 10: 2821,1991) and NSO clone Galfre et al., Methods Enzymol. 73: 3-46 (1981))
Be used for transcribing and translate and regulating sequence and can obtain of mammalian cell expression vector from viral genome.Promotor and operon sequence commonly used derive from polyomavirus, adenovirus 2, SV40 and human cytomegalic inclusion disease virus.Derive from the virus genomic sequence of SV40,, can be that the expression of other genes sequence provides the gene element in mammalian cell as SV40 replication origin, early stage and late promoter, enhanser, connexon, POLYA site.Virus is early stage and late promoter is especially suitable, because the two obtains from viral genome easily, and comprises the replication origin of a virus.Reference, Fiers et al., Nature 273: 113 (1978).Big SV40 fragment and little SV40 fragment also can be used, and it can provide the sequence of the about 250bp from Hind III site to Bgl I site, has comprised the replication origin of SV40.
The available mammalian cell expression vector can as Okayama and Berg (as described in structure Mol.Cell.Biol. 3: 280 (1983).It is a kind of that the system of high expression level can be as structure (Mol.Immunol.23:935,1986) as described in the Cosman et al in C127 mouse mammary epithelial cell.A kind of high-expression vector PMLSV N1/N4 has been stored in ATCC, coding 39890, (Cosman et al., Nature 312:768,1984).Another useful Mammals is expressed in and sees EP-A-0367566 and U.S.Pat.application Ser.No.07/701, and 415, also be suitable at this.Retroviral vector also is suitable for.Position at the natural signals peptide sequence, can add allogenic signal peptide sequence, as U.S.Pat.No.4,965, the signal peptide sequence of the IL-2 acceptor that the signal peptide sequence of 195 IL-7 that describe, Cosman etc. describe (signal peptide sequence, the U.S.Pat.No.4 of the IL-4 that Nature 312:768 (1984), EP 367,566 describe, 968, the signal peptide sequence of the III type IL-1 acceptor that the signal peptide sequence of the 607 I type IL-1 acceptors of describing and EP 460,846 describe.
The method of producing fusion rotein also comprises.This method comprises cultivates the reconstitution cell that contains the fusion rotein coding nucleic acid.This fusion rotein comprises the protein and the polypeptide of Flt3 part, its activated fragment or energy killing tumor cells.The fusion rotein of cell expressing coding, the renaturation of the fusion rotein of expression.In an example, method also comprises the separation and/or the purifying of the fusion rotein of renaturation.The product of this method is also protected.Can be the character of basic homogeneous with the fusion rotein purifying, for example on the SDS-PAGE electrophoresis, be single band.For example, when recombinant protein is secreting, expressing, can adopt commercial ultra-filtration membrane, for example company's products such as Millipore, Amicon, Pellicon at first will be expressed supernatant and be concentrated.The method that concentrated solution can adopt gel chromatography is purifying in addition further, or adopts the method purifying of ion exchange chromatography.For example anion-exchange chromatography (DEAE etc.) or cation-exchange chromatography.Gel matrix can be the matrix that agarose, dextran, polymeric amide etc. are usually used in protein purification.The SP group is an ideal ion-exchange group comparatively.At last, also available RPLC methods such as (RP-HPLC) is to the further refining purifying of above-mentioned purified product.Above-mentioned all purification steps can utilize different combinations, finally make purity of protein reach basic homogeneous.
Can utilize the affinity column that contains the flt3 aglucon that the amalgamation polypeptide of expressing is carried out purifying.According to the characteristic of employed affinity column, can utilize conventional method, as the amalgamation polypeptide of method elution of bound on affinity column such as high-salt buffer, change pH.Another kind of alternate method is to utilize this amalgamation polypeptide of antibody affinity column purifying that contains in conjunction with Flt3-L.
For simplifying purification step, can utilize transformed yeast cells as the secretory host cell expressed fusion protein.The method of purification of Recombinant polypeptide can be with reference to (J.Chromatog.296:171,1984) treatises such as Urdal from yeast cell fermentation supernatant.
In the reconstitution cell expressed fusion protein that utilization contains specific nucleic acid sequence is also included within.One of example, this cell can be eukaryotic cell.In one of example that refers in particular to, this cell can be Chinese hamster ovary celI, COS cell, or the NSO cell.
The nucleic acid of recombinant protein and this recombinant protein of encoding can utilize the appropriate means preparation, for example, chemosynthesis, recombinant expressed, or it merges application.Referring to John Wiley ﹠amp; Sons, " the CurrentProtocols in Molecular Biology " that Inc published in 2000, and " the Molecular Coning:A Laboratory Manual " of Cold Spring Harbor Laboratory Press1989 publication.In an example, the nucleotide sequence of encoding fusion protein utilizes the round pcr preparation, and method is referring to (Protein Eng.5 (8): 827-29 such as Prodromou; 1992) treatise.
The pharmaceutical cpd of submitting to protect contains a kind of fusion rotein, and this fusion rotein promptly contains Flt3-L fusion rotein or its activeconstituents, contains albumen or polypeptide composition with anti-tumor activity again.This pharmaceutical cpd also can contain the vehicle composition.The described medicinal ingredients of this patent can utilize one or more conventional methods to make medicinal formula, is beneficial to active constituents of medicine and brings into play its pharmacological action.This pharmaceutical formulation technology can be used preparation process such as conventional suspendible, aqueous injection, particle, tablet, sugar-pill, capsule, ointment, spraying drying, freeze-drying.How to select suitable formulation to depend on the route of administration of employing.When adopting liquid preparation, optional with the aqueous solution, oil (containing animal, plant, mineral oil) solution, as auxiliary material.Can contain physiological salts solution, glycerine, PEG etc. in the solution.When using liquid preparation, wherein the mentioned protein content from 0.5% to 90% (w/w) of the present invention refers in particular to from 1-50%.
Be a kind of composition on the other hand, said composition is by forming with the lower section: a) the Flt3 part of effective dose and the fusion rotein be made up of the peptide or the albumen of tumor-killing effect; B) effective antitumour preparation.The anti-tumor agent of our indication is the formation that can suppress melanoma, mammary cancer or liver cancer.They play a role by the growth-inhibiting or the apoptosis of inducing tumor cell.Also have some anti-tumor agents to comprise cytokine, part, antibody, radionuclide and chemotherapy composition.Such as IL-2, TNF etc.; 131I, 90Y etc.; Zorubicin, daunorubicin etc.; Also have bacterium, plant and other toxin; Tyrosine kinase inhibitor; Ly207702; The liposome that contains antineoplastic component; The part of other antibody or antibody (Fab).
The test kit of working method is provided.Such test kit is made up of the fusion rotein of one or more effective doses and the Action Specification of these fusion roteins of use.Fusion rotein is formed by the Flt3 part with by the peptide or the albumen of tumor-killing effect.This test kit also will comprise above said effective antitumour preparation specifically.Best medicinal forms is to be dissolved in stroke-physiological saline solution, glucose, and other balance liquid, or other can be brought into play in the sterile liquid of drug effect.In addition can also freeze-drying or drying.This test kit also should illustrate preferably aseptic physiological saline and the Glucose Liquid of solvent that does not influence drug effect in this case.Also have some test kits preferably to also have sterile packed syringe needle and syringe.Can also contain and instruct doctor or patient's operation instructions.
As used herein, so-called treatment effective dose or effective composition are meant independent use fusion rotein or can effectively prevent or tumor remission growth or tumour relative disease with the other treatment combined utilization.The effective dose of treatment is meant the dosage of the effective relief of symptoms of compound physical efficiency.Such as treatment, to cure, prevention, or the medical condition of alleviating are cured in prevention, or the related medical conditioned disjunction of alleviating raising treatment.When only using activated composition at individuality, the effective dose of treatment only refers to effective constituent.When using mixture, the effective dose of treatment is meant the combined amount of activeconstituents, no matter is the associating use, and continuous passing through used or application simultaneously.
C, contain the fusion rotein of Flt3 part and kill the usage of tumour preparation
The present invention also comprises to needs treatments or the animal that meets the requirements and takes the fusion rotein that the Flt3 part of effective dose and albumen or the antitumor thing of polypeptide class are formed.The cell that is suitable for comprises mammalian cell.It in particular cases is the mammals tumour cell.Also can be the Mammals cells in vivo specifically.
The Caspase activated channel plays an important role in the apoptosis of cell, (budihardjo etc., ann.Reev。cell?dev。Biol。15:269-90), caspase family has a very high specific L-Cysteine HCL Anhydrous family, and its cracking must be discerned 4 Tianmen radon propylhomoserins near cracked N-end (Grutter, Curr.Op.Struct.Biol.10:649-55 (2000) .).Caspase3 is called CPP32 again, YAMA and apopain, its specific cracking site WEHD.It is downstream or executive caspase, the multiple substrate of cleavable is as lamins, PARP, DFF etc.Caspase3 is present in the born of the same parents with unactivated proenzyme, and postactivated by the caspase9 of upstream and Apaf-1 cracking, the activation of caspase9 and Apaf-1 then depends on the stimulation of extracellular apoptotic signal, as Fas-L, and TNF and TRAIL.The Caspase activated channel can confirm by the kinds of experiments method, concrete certain methods see APOPTOSIS:APRACTICAL APPROACH (Studzinski, ed.1999).
Caspase3 is one of L-Cysteine HCL Anhydrous family member, and it plays an important role in apoptosis or apoptosis.(Gr ü tter, Curr.Opin.Structural Biol.10:649-55 (2000)), it is present in the cell with the form of proenzyme, and its typical active mode is by startup person caspase such as caspase8, and 9,10 are activated by proteolysis.Caspase3 after the activation follows other substrate protein of cracking, comprises participating in the albumen that DNA repairs, and cytoskeleton and nuclear skeleton albumen, these albumen all contain its recognition sequence DEVD behind the radon propylhomoserin of Tianmen.The detection of caspase3 active state can be with reference to following article: United States Patent (USP): 6,342,611; 6,391,575; 6,335,429; With U.S. Patent application No.20030186214.Therefore, any detection caspase 3 active appropriate method all can be used for the present invention.
Here we provide a kind of method with embedding and protein for treatment tumour.Promptly with containing FLt3-L, or the tumor-killing molecular composition fusion rotein of its active fragments and albumen or polypeptide, being used for the treatment of mammal tumor, this method has also been set forth its necessity and effective dose.See the second section of SUPRA, its concrete application is that tumour is a melanoma, when mammary cancer and hepatocellular carcinoma.
On the other hand, this method can produce the lymphocyte of tumour-specific.Promptly the above-mentioned fusion rotein by giving effective dose (with containing FLt3-L, or the tumor-killing molecular composition fusion rotein of its active fragments and albumen or polypeptide) is the lymphocyte that Mammals produces tumour-specific.
Treat the Mammals that needs this processing by the above-mentioned fusion rotein and the antibumor molecules that give effective dose.
Any Mammals all is suitable for this methods of treatment, especially people.As long as this object suffers from tumour, need accept this kind treatment.
This method can be used for suffering from gland cancer, the object of leukemia, lymphoma, melanoma, sarcoma.The source of tumor tissues can be: suprarenal gland, gall-bladder, bone, marrow, brain, mammary gland, bile duct, gi tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid gland, penis, prostate gland, skin, sialisterium, spleen, testis, thymus gland, Tiroidina and uterus.Except above-mentioned tumour, the tumour that also can be used for central nervous system as: spongiocyte diversity knurl, astrocyte slip, few dendron shape glioma, ependyma with choroid plexus tumour, pineal gland tumour, neurone tumour, become neurocele tumour, Schwann cell knurl, meningioma, meninges sarcoma; The tumour of eye such as rodent cancer, squamous cell carcinoma, melanoma in addition, rhabdosarcoma, retinoblastoma; Also have incretory gland tumour such as pituitary tumor, Tiroidina pomegranate, adrenal cortical tumor, neuroendocrine system tumour, gi tract pancreatic endocrine system tumor, genital system tumor; Head and tumor colli: neck tumour, mouth neoplasm, pharyngeal tumour, laryngeal neoplasm, odontogenic tumor; Breast tumor: maxicell lung cancer, small cell lung cancer, lung cancer in non-cellule type, thoracic cavity tumour, malignant mesothe, thymus gland pomegranate, the original embryonal cell lipoma of chest; The tumour at digestive tube position: esophagus knurl, tumor stomach, liver neoplasm, tumor of biliary tract, pancreatic neoplasm, knot rectal adenocarcinoma, anus neoplasm; Urogenital tract tumour: renal cell carcinoma, kidney tumor of ureter, tumor of bladder, tumor of urethra, tumor of prostate, tumor of penis, tumor of testis; Female genital organ tumor: vaginal tumor, tumor of cervix, body of uterus adenoma, ovarian cancer, gynaecology's sarcoma, breast tumor; Dermatoma: base cell carcinoma, squamous cell carcinoma, one-tenth fibrosarcoma, MERKEL glucagonoma, malignant melanoma; Bone and soft tissue neoplasm: osteosarcoma, malignant fibrous histiocytoma, chrondrosarcoma, ewing sarcoma, neuroectodermal tumors, angiosarcoma; Hematopoietic system cancer: myelodysplasia syndromes, acute myeloid leukemia, chronic myelogenous leukemia, acute lymphoblastic are leukemia, HTLV-1, T chronic myeloid leukemia/lymphoma, slow LL, hairy cell leukemia, Hokdkin disease, non_hodgkin lymphoma, giant cells leukemia; Pediatric tumor: acute lymphoblastic plasmocyte sample leukemia, acute myelocytic leukemia, neuroblastoma, bone tumor, rhabdosarcoma, lymphoma, kidney and liver neoplasm.
Here, " suppressing or treatment " effect that we say comprises that the delay of following the development of tumor growth related symptoms is or/and the reduction of these severity of symptom, it also further comprises alleviating of already present tumor growth simultaneous phenomenon, and prevent the appearance of other symptoms, also can reduce or prevent to shift.Therefore, suppress or treatment " effect refer to those are had pernicious or have the favourable effect of Mammals that develops into these diseases or symptom.
When implementing this methods of treatment, this proteic effective dose determines according to the personal considerations.As method provided by the invention, this fusion rotein can be used or unite other method of use separately and for example be used other immune molecule (cytokine), chemotherapeutic and antitumour drug etc.When in the other medicines coupling, this albumen can be injected simultaneously with other medicines or injection subsequently.If injection subsequently determines their order successively by implementing the doctor.The validity of this therapeutic component and toxicity are to be decided by basic pharmacological kinetics or laboratory animal, for example their medium lethal dose and median effective dose, and this can represent with the ratio of LD50 and ED50.The curative effect that fusion rotein does very well is needed.Reference when cell cultures and the resulting data of zooscopy can be used as the people.The change of dosage decides according to the approach and the type of service of injection.The doctor selects injecting pathway, medicament forms and accurate dose according to patient's situation.Active drug level in blood plasma according to the individual determines the dosage and the pitch time of medicine, thereby reaches ideal curative effect or minimum effective concentration.Every kind of composition can change according to effective concentration, also can change according to external data.
Adopt suitable injecting pathway.Dosage form comprises tablet, tablet, powder, capsule etc., for example sees Lei Mingdun pharmacology.
According to each patient's age, body weight and reaction, doctor's routine will determine administered antibodies dosage here.
The method and technology that forms albumen and injection rapidly can be referring to REMINGTON ' S PHAR-MACEUTICALScience.The form of the fusion rotein of here mentioning is similar to antibody with injecting method.Suitable way has oral, subcutaneous and directly intra-arterial injection, intravenous injection or intraocular injection etc.Observing the pharmacology of this proteinoid and its implements this method and can study according to traditional method.
In addition, the people can be according to circumstances at this albumen of local injection rather than systemic injection, such as antibody is directly injected tumour.And the people can be with targeted drug delivery system injection albumen, for example with liposome tissue specificity antibody target input tumour.
When by vein, during the albumen of intracutaneous or subcutaneous injection significant quantity, it is the acceptable aqueous solution of apyrogenic parenteral.According to its PH, preparation such as stability corresponding proteins.Vein, the albumen that intracutaneous and hypodermic pharmaceutical cpd are introduced except us also have for example sodium-chlor etc. of isotonic solution.For intramuscular injection, how permeate agent infiltrates is an obstacle.
For inhalation, the fusion rotein routine is to suck with aerosol form with suitable propulsive force.With regard to the aerosol of compression, the dosage of suction is to be decided by the valve that the amount of sending into is provided.Injection form can be single dosage or a plurality of dosage, also can be solution or suppository.
Parenteral medicament forms includes the active aqueous solution.In addition, the suspended substance in the activeconstituents can prepare with the finish suspended substance.Lipotropy solution comprises fat such as sesame oil, or the synthetic fatty acid ester is such as liposome.The injection suspension composition of water comprises the material that can increase visibility.Optionally, suspension agent can be the material that can increase the liquid water-soluble and the stability of high density.Can alternate be that activeconstituents can be a powder type, for example aseptic apyrogenic water.
The used treatment condition of the condition of the usage quantity dependent patient of chimeric antibody and patient decide in the composition of medicine.At last, implementing the doctor determines with the different different patients of protein content treatment.Originally, the doctor observes the reaction situation with the albumen of low dosage, and dosage increases gradually up to reaching with only dosage and obtains only result of treatment then.Dosage no longer increases then.Different pharmaceutical cpds should use between 0.01ug and 1mg/kg.These compositions are apyrogenic when using, the physical form acceptable.Medicable material also has other composition except fusion rotein, they can be optionally or other and albumen simultaneously or the injection of system.The cartridge bag of acting on behalf of exemplary contains antitumour drug.
Fusion rotein as herein described can use separately, also can unite use with other methods of treatment.For example, carry out radiotherapy at fusion rotein institute bonded tumor by local.Radiocurable dosage should be enough to kill the tumour cell of propagation phase.Ideal situation is that the effect of combination therapy must be higher than the single therapy effect of radiotherapy or fusion rotein.Typical radiation therapy method comprises beta-ray, X-ray, alpha-particle etc.Radioactive source to the tumour irradiation can be external radiation exposure, also can be to utilize radionuclide to produce internal radiation.The method that the method for implementing internal radiation can be in the industry to be familiar with.The method of tumour radiotherapy can be referring to " Cancer:Principles ﹠amp that compiles such as V.T DaVita Jr.; Practice of Oncology 248 " S.Hellman in the 4th edition showed " Principles of RadiationTherapy " chapters and sections 1993 years.Radiocurable typical doses is 1 to 500cGy (for example, by 1 to 500rads).
Can will contain Flt3-L or its effective constituent, and the fusion rotein of anti-tumor protein/polypeptide, use as vaccine with immunostimulant.
On the other hand, this paper has listed to utilize and has contained Flt3-L or its effective constituent, and the fusion rotein of anti-tumor protein/polypeptide, use as vaccine with immunostimulant, thus the using method of effective antitumour immunity in the activation mammalian body.
As used herein " immunostimulant " speech mean can strengthen or extension pin to certain target antigen, as the immunoreactive material of tumour antigen.This immuno-potentiation can be synergetic or collaborative.The connotation of " immune response " speech comprises the immune response that immune response that the cell-mediated immune response of B, T are cell-mediated and T, B cell all participate in as used herein.The example of immune response toughener such as cytokine, as IL-12, IL-2, IFN-γ, immunological adjuvant, immunostimulation polypeptide, or the like.In composition of the present invention and the method, immunostimulant and fusion rotein can use simultaneously or successively use, and can use identical route of administration or different route of administration.
Can prepare vaccine with traditional method.For example, with suitable liquid such as salt solution, fully or Freund dilute immunogen.In addition, the immunogen molecule also can with or be not connected with carrier molecule the preparation proteantigen.Such carrier molecule is not limited to bSA, KLH, tetanus toxin or the like.Immunogen also can be injected with lipoprotein or liposome, adjuvant.Approach immune animals such as approach that immunogen can produce with any suitable antibody such as vein, subcutaneous, intracutaneous, intramuscular injection.Immunogen can be once or rule intermittently administration up to having significant antitumor t cell responses or antitumor cell antibody to produce.The evaluation that the antitumor cell reaction occurs can realize by the appearance ratio that detects the precursor cell of tumor-resistant antigen cytotoxic T lymphocyte before and after immunity.Detection of antibodies can record from serum as the method for bibliographical information with immunodetection.
Current traditional vaccinate is the purpose in order to prevent and to treat.When being purpose with the prevention, fusion rotein gives before any symptom of tumour or the appearance of any sign.Giving the particularly human preventative injection fusion rotein of Mammals is in order to prevent or to alleviate pernicious symptom.When being purpose with the treatment, fusion rotein begin in disease (or just having begun) or any symptom occur before administration, its objective is in order to palliate a disease.
The method of bibliographical information can be used in the position that topical to pathology is got involved, but is not limited thereto, as, local injection is implanted porous infiltration device, and the inside comprises fusion molecule, fusion rotein or the immunostimulant of cell expressing.
The evaluation of vaccine can at first be carried out in animal model, from rodent, arrives inhuman spirit palm class animal then, arrives the people at last.The security of immune programme for children is to decide by the general health situation (variation of body weight, body temperature, gastrointestinal function or the like) and the pathological change of postmortem of observing immune animal.After the experimentation on animals, can detect on one's body at tumour patient.Traditional method can decide the usefulness of vaccine by the immune response of evaluate patient.For example, in body, be separated to T lymphocyte, peripheral blood lymphocyte, lymphoglandula or neoplasm invasiveness lymphocyte.Such lymphocyte can be separated to and do vitro culture with the method for bibliographical information on one's body from the patient or the volunteer of treatment.Its activity detects with trypan blue dyeing.Evaluating is used for determining producing the situation of immunocyte and the development of tumour in the lymphocytic usefulness of T, the treatment back animal body.Can assess these parameters with traditional method, comprise cytotoxicity experiment, mixed lymphocyte reacion, production of cytokines experiment.
Any suitable tumor model how is used as the test model of fusion rotein.Muroid can be accepted tumour and become suitable strain.Tumour can be of the same race or allotypic or allosome.The receptor can be that one or more immune correlation functions have activity or disappearance, includes, but are not limited to nu/nu, scid, and beige mouse.For example the receptor is Balb/c or C57BL/6 mouse.Any suitable tumour all can be used for animal model experiment, comprises building the cell that is the cell of getting in the tumour biological tissue, short-term polyclone tumour cell.Tumor cell line comprises Renca, B16 melanoma cell, Hepal, BT-474, Raji, QYC, D2F2,4T1, A20.The dosage of fusion rotein is 1 μ g/ mouse～1mg/ mouse, is administered once at least.Antibody can pass through any suitable method afford.A concrete example gives antibody 100 μ g/mouse/ mouse biweekly.A special example, subcutaneous plantation tumour is measured tumor size with vernier callipers in the time of the 0th day in the specific time.Any suitable reference protein all can use.For example, reference protein is sublimed IgG 1The homotype control antibodies.
Following implementation example is for the use range of better this invention of explanation is extensive, and the also use of unrestricted this invention.
Embodiment 1
FL film outskirt fragment coding DNA's is synthetic
FL is an I type transmembrane protein, its N end is the film outskirt, such protein molecular is modified its N end and is had a strong impact on biologic activity sometimes, Morrison (Coloma MJ, Morrison S.Design and production of noveltetravalent bispecific antibodies.Nat Biotech, 1997,15:159-163) a single-chain antibody molecule is placed the C of another complete antibody molecule CH hold, the dual specific tetravalent antibody molecule (shown in Figure 1A) that success makes up is given provides new thinking.In order to guarantee to obtain highly active bifunctional protein, consider to make up FL film outskirt (Flex) at the N end and antibody molecule at the fusion rotein of C end, with 3 of Flex gene '-end and people's antibody constant region (5 of the gene of hinge area+CH2+CH3) '-link to each other, be built into the Flex/Fc fusion gene, and then the SM5-1 single-chain antibody is connected on the C end of Flex/Fc, be built into bifunctional fusion proteins (Flex/Fc/Fv sees also Figure 1B).
People FLT3 ligand gene cDNA sequence is known (referring to the GenBank database Http:// www.ncbi.nkn.nih.gov/entrez/The sequence of middle accession number U03858), wherein 84-161 is a signal peptide coding region, and 162-629 is film outskirt fragment coding district, and both additions are totally 546 bases.
According to Prodromou C, Pearl LH. (Recursive PCR:a novel technique for total genesynthesis.Protein Eng.1992 Dec; 5 (8): it is synthetic that method 827-829.) is carried out full gene.Entrust Shanghai to give birth to worker bio-engineering corporation and carry out the complete synthesis and PAGE purifying of gene.
For the DNA that reclaims, be connected to the multiple clone site of pGEM-T carrier (Promega company product), confirm to have obtained correct clone (Fig. 2, SEQ ID NOS:1 and 2) after the sequence verification.Correct clone's note in this example is made pGEM-T/hFlex.
Embodiment 2
The clone and the evaluation of people's IgG antibody 1 constant region (Fc) encoding sequence
The acquisition and the clone of total length human IgG1 cDNA sequence (5 ' → 3 '): natural total length IgG1 contains 1416bp in the coding region, 471 amino acid and the terminator codon of encoding (referring to Genbank accession number BC024289).
Obtain IgG1 cDNA sequence by reverse transcription, and be cloned on the carrier pGEM-T.Specific operation process is: 100 milliliters of human peripherals, separate the acquisition white corpuscle with lymphocyte separation medium (Sigma product), mRNA extracting and purifying test kit extracting mRNA with Qiagen, RT-PCR test kit with GIBCO company after identifying its integrity on 1.2% agarose gel electrophoresis carries out reverse transcription and pcr amplification human IgG1 Fc fragment, obtained required cDNA, electrophoresis is identified rear clone to the pGEM-T carrier, and note is made pGEM-T/IgFc, and the errorless back of sequence verification is preserved standby.
Embodiment 3
The structure of chimeric and humanization SM5-1 antibody
1.SM5-1 the variable region gene of monoclonal antibody and sequencing
Extract the total RNA of hybridoma SM5-1 with Trizol reagent [Gibco company product], then by 5 '-RACE test kit [Gibco company product] specification sheets increase respectively heavy chain of antibody and chain variable region gene.Warm start, reaction conditions: 94 C 5 minutes are all adopted in first run PCR and nest-type PRC reaction; 94 C 45 seconds, 60 C45 seconds, 72 C 1 minute and 10 seconds, 30 circulations; 72 C 7 minutes.More than 2 PCR products respectively through 1% agarose gel electrophoresis analysis, first run pcr amplification product does not all have tangible specific band, the nest-type PRC amplification shows the special band that a treaty 590bp is arranged in the heavy chain product, and the special band (referring to Fig. 5) of a treaty 530bp is arranged in the light chain product.The PCR product cloning that obtains is arrived pGEM-T[Promega company product] carrier, screening positive clone checks order, and has determined to obtain correct SM5-1 variable region of heavy chain (Fig. 6 SEQ ID NO:7) and chain variable region gene (Fig. 7 SEQ ID NO:9).And infer its protein sequence (referring to Fig. 6 SEQ ID NO:8 and Fig. 7 SEQ ID NO:10) according to above-mentioned gene order.In order to clone conveniently, adopt PCR at 5 ' of antibody heavy chain variable region gene-end design EcoRV and XbaI, 3 '-end adds the NheI site, in chain variable region gene 5 '-end design HindIII site.Above variable region gene is cloned into the pGEM-T carrier respectively, errorless through sequence verification.
2.SM5-1 the structure of chimeric antibody expression vector
Through order-checking identify the back with EcoR V and Nhe I with the heavy chain variable region gene VH of SM5-1 from the pGEM-T-VH cutting-out and be cloned into the pAH4604[Coloma MJ that cuts through same enzyme, Hastings A, Wims LA, Morrison SL.Novelvectors for the expression of antibody molecules using variable regionsgenerated by polymerase chain reaction.J Immunol Methods, 1992,152:89] in the carrier, constituted the expressed intact framework pAH4604-VH of chimeric antibody heavy chain gene, Fig. 8 has shown the Nucleotide (SEQ ID NO:11) and amino acid (the SEQ ID NO:12) sequence of SM5-1 chimeric antibody (chSM) heavy chain. with XbaI and BamHI enzymolysis pAH4604-VH, separation and purification wherein about 3200bp chimeric heavy chain band (VHCH) and be cloned among the pDR, obtain chimeric antibody heavy chain expression carrier pDR-VHCH (Figure 10).
Because light chain carrier pAG4622[Coloma MJ, Hastings A, Wims LA, Morrison SL.Novelvectors for the expression of antibody molecules using variable regionsgenerated by polymerase chain reaction.J Immunol Methods, 1992,152:89] on the κ chain gene in big section intron arranged, be not easy to later vector construction, adopt Overlapping PCR that humanization variable region of light chain and the human antibody light chain constant region cDNA direct splicing of SM5-1 are got up, Overlapping PCR reaction conditions is: 94 C 5 minutes; 94 C 55 seconds, 55 C 55 seconds, 72 C 1 minute and 30 seconds, 30 circulations; 72 C 7 minutes obtain product VLCL at last, and it 5 ' contains restriction enzyme sites HindIII, and 3 ' end contains restriction enzyme sites EcoRI.Reclaim and be cloned in the pGEM-T carrier through the agarose gel electrophoresis purifying, get pGEM-T-VLCL, the screening positive clone order-checking.Fig. 9 has shown the Nucleotide (SEQ ID NO:13) and amino acid (the SEQ ID NO:14) sequence of SM5-1 chimeric antibody (chSM) light chain. use the two enzymic digestions of Hind III and EcoR I at last, VLCL is downcut from the pGEM-T-VLCL carrier, be cloned among the pDR of same enzymolysis, get pDR-VLCL (Figure 11), cut the expression vector of determining structure through enzyme.
The expression of 3 chimeric antibodies and purifying
In 3.5cm tissue culture ware, inoculate 3.5 * 10 5The CHO-dhfr in/hole -Cell, cell is containing xanthoglobulin (H), be cultured in the DMEM perfect medium of Thymine deoxyriboside (T) and glycine (G) when 90-95% merges and carry out transfection: get plasmid 10 μ g (chimeric heavy chain expression plasmid 4 μ g, chimeric light chain expression plasmid 6 μ g) and 20 μ lLipofectamine2000 Reagent[Gibco company products] be dissolved in 500 μ l serum-free DMEM substratum respectively, room temperature left standstill 5 minutes, with above 2 kinds of liquid mixing, incubated at room 20 minutes is so that the formation of DNA-liposome complex, therebetween with the blood serum medium that contains in the DMEM substratum replacement culture dish of 3ml serum-free, then the DNA-liposome complex that forms is joined in the plate CO 2Incubator is cultivated after 4 hours and is added the DMEM perfect medium that 3ml contains 10% serum, places CO 2Continue in the incubator to cultivate.Transfection is carried out after 24 hours cell by 0.8 * 10 5/ ware passes in the 10cm culture dish, changes the selection substratum that does not contain H, T, G, treats that the clone forms the back with dihydrofolate reductase inhibitor methotrexate (MTX) screening of pressurizeing, and concentration is from 2 * 10 -8Mol/L to 5 * 10 -7The pressurization of mol/L gradient is carried out subclone with limiting dilution assay.Get cells and supernatant and detect the screening high-expression clone with ELISA.With the high-expression clone serum free medium enlarged culturing that screening obtains, use Protein A affinity column separation and purification chimeric antibody.
4 synthetic SM5-1 humanized antibody variable region genes
At first according to the principle of the heavy chain of above-mentioned mouse source SM5-1 antibody and light chain variable region amino acid sequence and humanization design, with KOL and REI (referring to Morea V et al., Immunotechnology 1997 Mar; 3 (1): 71-81) make the template that humanization designs, the aminoacid sequence of design humanization SM5-1 antibody.In order to clone conveniently, at 5 ' of humanized antibody heavy chain variable region gene-end design EcoRV and XbaI, 3 '-end adds the NheI site, in chain variable region gene 5 '-end design HindIII site.At first heavy chain and the light chain CDR district with SM5-1 is grafted directly to respectively on people's From Template KOL and the REI, constitute humanized variable region heavy chain and light chain gene, with with make up the same method of chimeric antibody expression vector, obtained the heavy chain and the light chain expression vector of SM5-1 humanized antibody.Because not only participating in the space in CDR district, antibody FR district constitutes, what have participates in mutually combining of antigen-antibody directly, so simple CDR transplants and tends to destroy antibody and antigenic effect, the avidity of antibody is descended greatly even disappear, at this moment have only to become mouse source residue could recover the activity of antibody some important residue reverse mutation in the FR district, people source.The humanization that obtains is heavy, light chain expression vector Lipofectamine 2000[Gibco company product] cotransfection COS cell, the antigen-binding activity with flow cytometry mensuration antibody found that the activity of direct CDR grafted antibody is almost completely lost.In order to obtain the humanized antibody of high-affinity, we analyze the antigen-binding activity of measuring them with reverse mutation with flow cytometry to the important FR residue that may influence antibody binding activity.Obtain and the active suitable humanized antibody huSM5-1 of mouse source SM5-1 monoclonal antibody through screening is final, its heavy chain (huSM5VH) variable region sequences is seen Figure 12 (SEQ ID NOS:15 and 16), and light chain (huSM5VL) variable region sequences sees that its humanization heavy chain full length sequence of Figure 13 (SEQ ID NOS:17 and 18) sees Figure 14 (SEQ ID NOS:19 and 20).Its humanization light chain full-length gene order and aminoacid sequence are seen Figure 15 (SEQ IDNOS:21 and 22).
4. the expression of humanized antibody and purifying
In 3.5cm tissue culture ware, inoculate 3.5 * 10 5The CHO-dhfr-cell in/hole, cell is containing xanthoglobulin (H), be cultured in the DMEM perfect medium of Thymine deoxyriboside (T) and glycine (G) when 90-95% merges and carry out transfection: get plasmid 10 μ g (heavy chain expression plasmid 4 μ g, light chain expression plasmid 6 μ g) and 20 μ l Lipofectamine2000Reagent[Gibco company products] be dissolved in 500 μ l serum-free DMEM substratum respectively, room temperature left standstill 5 minutes, with above 2 kinds of liquid mixing, incubated at room 20 minutes is so that the formation of DNA-liposome complex, therebetween with the blood serum medium that contains in the DMEM substratum replacement culture dish of 3ml serum-free, then the DNA-liposome complex that forms is joined in the plate CO 2Incubator is cultivated after 4 hours and is added the DMEM perfect medium that 3ml contains 10% serum, places CO 2Continue in the incubator to cultivate.Transfection is carried out after 24 hours cell by 0.8 * 10 5/ ware passes in the 10cm culture dish, changes the selection substratum that does not contain H, T, G, treats that the clone forms the back with dihydrofolate reductase inhibitor methotrexate (MTX) screening of pressurizeing, and concentration is from 2 * 10 -8Mol/L to 5 * 10 -7The pressurization of mol/L gradient is carried out subclone with limiting dilution assay.Get cells and supernatant and detect the screening high-expression clone with ELISA.With the high-expression clone serum free medium enlarged culturing that screening obtains, use ProteinA affinity column separation and purification humanized antibody.
5. the avidity of humanization and inosculating antibody people SM5-1 antibody is relatively:
BIAcore with Pharmacia company, with reference to (Karlsson such as Karlsson, R., Michaelsson, A., and Mattsson, L. (1991) Kinetic analysis of monoclonal antibody-antigeninteractions with a new biosensor based analytical system.J.Immunol.Methods145, the method that 229-240) provides is measured both affinity constant Kd (Kon/Koff), the avidity of finding humanized antibody and chimeric antibody is approaching, is respectively 9.31 * 10 -9M and 3.78 * 10 -9M.
The The above results explanation, the humanization design improvement of this antagonism SM5-1 is successful, does not reduce avidity substantially, has reached the requirement of treatment with the avidity of monoclonal antibody.
Embodiment 4
The structure of FL/SM bifunctional fusion proteins gene (huSM/FL)
The antigen-4 fusion protein gene that makes up 3 kinds of structures is used for later biological function research.
A. make up bifunctional fusion proteins gene huSMFv/Fc/FL
Construction process according to above-mentioned fusion gene, by OverlapPCR with the Flex gene directly with 3 of huSM5-1 heavy chain of antibody gene (SMH) '-the end fusion, obtain PCR product huSMFv/Fc/FL, its 5 '-contain restriction enzyme sites HindIII, 3 ' end contains restriction enzyme sites EcoRI.Reclaim the PCR product and be cloned in the pGEM-T carrier (Promega company product) the screening positive clone order-checking through the agarose gel electrophoresis purifying.The heavy chain fusion gene that will check order correct then downcuts from the pGEMT carrier with HindIII and the two enzymic digestions of EcoRI, is cloned in the pDR carrier, is built into pDR (huSMFv/Fc/FL) expression vector.The aminoacid sequence of huSMFv/Fc/FL and gene order are listed in SEQ ID NOS:23 and 24 (Figure 16) respectively.
In 3.5cm tissue culture ware, inoculate 3.5 * 10 5The CHO-dhfr in/hole -Cell, cell is containing xanthoglobulin (H), be cultured in the DMEM perfect medium of Thymine deoxyriboside (T) and glycine (G) when 90-95% merges and carry out transfection: get plasmid 10 μ g (fusion gene expression plasmid pDR (huSMFv/Fc/FL) 4 μ g, humanized antibody light chain huSM5 expression plasmid 6 μ g) and 20 μ l Lipofectamine2000 Reagent[Gibco company products] be dissolved in 500 μ, 1 serum-free DMEM substratum respectively, room temperature left standstill 5 minutes, with above 2 kinds of liquid mixing, incubated at room 20 minutes is so that the formation of DNA-liposome complex, therebetween with the blood serum medium that contains in the DMEM substratum replacement culture dish of 3ml serum-free, then the DNA-liposome complex that forms is joined in the plate CO 2Incubator is cultivated after 4 hours and is added the DMEM perfect medium that 3ml contains 10% serum, places CO 2Continue in the incubator to cultivate.Transfection is carried out after 24 hours cell by 0.8 * 10 5/ ware passes in the 10cm culture dish, changes the selection substratum that does not contain H, T, G, treats that the clone forms the back with dihydrofolate reductase inhibitor methotrexate (MTX) screening of pressurizeing, and concentration is from 2 * 10 -8Mol/L to 5 * 10 -7The pressurization of mol/L gradient is carried out subclone with limiting dilution assay.Get cells and supernatant and detect the screening high-expression clone with ELISA: goat anti-human igg (Fc) bag quilt is in elisa plate, 4 ℃ are spent the night, sealed 2 hours in 37 ℃ with 2%BSA-PBS, (Human myeloma IgG1 κ), was hatched 2 hours for 37 ℃ to add culture supernatant to be measured and standard substance, add HRP-goat anti-human igg (κ) and carry out association reaction, hatched 1 hour for 37 ℃, add TMB, use H at last in 37 ℃ of effects 5 minutes 2SO 4Termination reaction is surveyed OD 450Value.。With the high-expression clone serum free medium enlarged culturing that screening obtains, use Protein A affinity column separation and purification fusion rotein.
B. make up the bifunctional fusion proteins gene (huSMFv/Fc/Link/FL) of belt lacing sequence
According to the construction process of aforementioned fusion gene, with Overlap PCR with 3 of huSM5-1 heavy chain gene '-end is by (Gly 4Ser 1) 3Joint (Fig4, SEQ ID NO:5 and 6) obtains PCR product huSMFv/Fc/Link/FL with 5 of FLex '-fusion, its 5 '-contain restriction enzyme sites HindIII, 3 ' end contains restriction enzyme sites EcoRI.Reclaim the PCR product and be cloned in the pGEM-T carrier (Promega company product) the screening positive clone order-checking through the agarose gel electrophoresis purifying.The heavy chain fusion gene that will check order correct then downcuts from the pGEMT carrier with HindIII and the two enzymic digestions of EcoRI, is cloned in the pDR carrier, is built into pDR (huSMFv/Fc/Link/FL) expression vector.It is identical with huSMFv/Fc/FL fusion rotein method therefor with 26 (Figure 17) this Expression of Fusion Protein and purification process that the aminoacid sequence of huSMFv/Fc/Link/FL and gene order are listed in SEQ ID NOS:25 respectively.
C. make up bifunctional fusion proteins gene (FL/Fc/huSMFv)
Be that template obtains the hFLex gene with overlapping PCR and people Fc gene (contains hinge area with pGEM-T/hFLex carrier and the carrier pGEM-T/IgFc that contains human IgG1's constant region cDNA respectively, CH2, CH3), again by overlapping PCR, with 3 of hFLex gene '-end and 5 of people Fc gene '-end merges, obtained the FL/Fc fusion gene, reclaim the PCR product and be cloned in the pGEM-T carrier through the agarose gel electrophoresis purifying, note is made pGEM-T (FL/Fc), the screening positive clone order-checking, Fig. 3 has shown Nucleotide (SEQ ID NO:3) and amino acid (the SEQ ID NO:4) sequence of FL/Fc..
Adopt aforementioned antigen-4 fusion protein gene construction process, with 3 of the variable region of heavy chain of huSM5-1 '-end is by (Gly 4Ser) 35 of joint and its variable region of light chain '-the end fusion, constitute huSM5-1 single-chain antibody gene (Fv).
Adopt aforementioned antigen-4 fusion protein gene construction process that 3 of hFLex/Fc '-end and 5 of SM5-1 single-chain antibody Fv '-end are merged, make up bifunctional fusion proteins gene FL/Fc/huSMFv (Figure 18, SEQ ID NOS:27 and 28.), the synoptic diagram of this fusion gene is seen Figure 19.
Fusion gene cloning to the pDR carrier, is built into pDR (FL/Fc/huSMFv) expression vector.With liposome method with the expression vector transfection CHO cell, with the MTX screening of pressurizeing, get cells and supernatant and detect the screening high-expression clone with ELISA: goat anti-human igg (Fc) bag quilt is in elisa plate, and 4 ℃ are spent the night, and uses 2%BSA-PBS in 37 ℃ of sealings 2 hours, add culture supernatant to be measured, hatched 2 hours for 37 ℃, add HRP-goat-anti FL and carry out association reaction, hatched 1 hour for 37 ℃, add TMB in 37 ℃ of effects 5 minutes, use H at last 2SO 4Termination reaction is surveyed the OD450 value.,, use Protein A affinity column separation and purification fusion rotein with the high-expression clone serum free medium enlarged culturing that screening obtains.
Adopt-made up above-mentioned three kinds of fusion roteins of SM5-1 chimeric antibody and FL with the same construction process of huSM/FL.ChSMV wherein HThe sequence of/Fc/FL is seen Figure 20 (SEQ ID NOS:29 and 30), chSMV HThe sequence of/Fc/Link/FL is seen Figure 21 (SEQ ID NOS:31 and 32), and the sequence of FL/Fc/chSMFv is seen Figure 22 (SEQ ID NOS:33 and34)
Embodiment 5
The structure of anti-humen CD 20 monoclonal antibody and FL bifunctional fusion proteins gene
1. the construction and expression of anti-humen CD 20 chimeric antibody
With reference to United States Patent (USP) 6,399,061 anti-humen CD 20 monoclonal antibody data and the sequence that provides, the method for using embodiment 1 to describe is synthesized the variable region of heavy chain (V of anti-humen CD 20 H) and variable region of light chain (V L) cDNA.Figure 23 has shown the variable region of heavy chain Nucleotide (SEQ ID NO:35) and amino acid (the SEQ ID NO:36) sequence of 238 monoclonal antibodies. Figure 24 has shown the variable region of light chain Nucleotide (SEQ ID NO:37) and amino acid (the SEQ ID NO:38) sequence of 2B8 monoclonal antibody. at 5 ' end design EcoRV of antibody heavy chain variable region gene, 3 ' end adds the NheI site, in chain variable region gene 5 ' end design HindIII site.Through order-checking identify the back with EcoR V and Nhe I with the heavy chain variable region gene VH of CD20 monoclonal antibody from the pGEM-T-VH cutting-out and be cloned into through the pAH4604 carrier that same enzyme is cut, constituted the expressed intact framework pAH4604-VH of chimeric antibody heavy chain gene, with XbaI and BamHI enzymolysis pAH4604-VH, separation and purification wherein about 3200bp chimeric heavy chain band (VHCH) and be cloned among the pDR, obtain chimeric antibody heavy chain expression carrier pDR-VHCH.Figure 25 has shown the Nucleotide (SEQ ID NO:39) and amino acid (the SEQ ID NO:40) sequence of CD20 chimeric antibody heavy chain.
Adopt Overlapping PCR that CD20 monoclonal antibody variable region of light chain and human antibody light chain constant region cDNA direct splicing are got up, Overlapping PCR reaction conditions is: 94 C 5 minutes; 94 C 55 seconds, 55 C 55 seconds, 72 C 1 minute and 30 seconds, 30 circulations; 72 C 7 minutes obtain product VLCL at last, and it 5 ' contains restriction enzyme sites HindIII, and 3 ' end contains restriction enzyme sites EcoRI.Reclaim and be cloned in the pGEM-T carrier through the agarose gel electrophoresis purifying, get pGEM-T-VLCL, the screening positive clone order-checking.Use the two enzymic digestions of Hind III and EcoR I at last, VLCL is downcut from the pGEM-T-VLCL carrier, be cloned among the pDR of same enzymolysis, get pDR-VLCL, cut the expression vector of determining structure through enzyme.Figure 26 has shown the Nucleotide (SEQ ID NO:41) and amino acid (the SEQ ID NO:42) sequence of CD20 chimeric antibody light chain.
In 3.5cm tissue culture ware, inoculate 3.5 * 10 5The CHO-dhfr in/hole -Cell, cell is containing xanthoglobulin (H), be cultured in the DMEM perfect medium of Thymine deoxyriboside (T) and glycine (G) when 90-95% merges and carry out transfection: get plasmid 10 μ g (chimeric heavy chain expression plasmid 4 μ g, chimeric light chain expression plasmid 6 μ g) and 20 μ lLipofectamine2000 Reagent[Gibco company products] be dissolved in 500 μ l serum-free DMEM substratum respectively, room temperature left standstill 5 minutes, with above 2 kinds of liquid mixing, incubated at room 20 minutes is so that the formation of DNA-liposome complex, therebetween with the blood serum medium that contains in the DMEM substratum replacement culture dish of 3ml serum-free, then the DNA-liposome complex that forms is joined in the plate CO 2Incubator is cultivated after 4 hours and is added the DMEM perfect medium that 3ml contains 10% serum, places CO 2Continue in the incubator to cultivate.Transfection is carried out after 24 hours cell by 0.8 * 10 5/ ware passes in the 10cm culture dish, changes the selection substratum that does not contain H, T, G, treats that the clone forms the back with dihydrofolate reductase inhibitor methotrexate (MTX) screening of pressurizeing, and concentration is from 2 * 10 -8Mol/L to 5 * 10 -7The pressurization of mol/L gradient is carried out subclone with limiting dilution assay.Get cells and supernatant and detect the screening high-expression clone with ELISA.With the high-expression clone serum free medium enlarged culturing that screening obtains, use Protein A affinity column separation and purification chimeric antibody.
2. the antigen-4 fusion protein gene that makes up 3 kinds of structures is used for later biological function research.
A. make up bifunctional fusion proteins gene C D20Fv/Fc/FL
Construction process according to above-mentioned fusion gene, by Overlap PCR with the Flex gene directly with 3 of CD20 heavy chain of antibody gene '-the end fusion, obtain PCR product huSMFv/Fc/FL, its 5 '-contain restriction enzyme sites HindIII, 3 ' end contains restriction enzyme sites EcoRI.Reclaim the PCR product and be cloned in the pGEM-T carrier (Promega company product) the screening positive clone order-checking through the agarose gel electrophoresis purifying.The heavy chain fusion gene that will check order correct then downcuts from the pGEMT carrier with HindIII and the two enzymic digestions of EcoRI, is cloned in the pDR carrier, is built into pDR (huSMFv/Fc/FL) expression vector.Figure 27 has shown the Nucleotide (SEQ ID NO:43) and amino acid (the SEQ ID NO:44) sequence of CD20VH/Fc/FL fusion rotein.
B. make up the bifunctional fusion proteins gene (CD20Fv/Fc/Link/FL) of belt lacing sequence
According to the construction process of aforementioned fusion gene, with Overlap PCR with 3 of CD20 heavy chain gene '-end is by (Gly 4Ser 1) 3Joint obtains PCR product C D20Fv/Fc/Link/FL with 5 of FLex '-fusion, its 5 '-contain restriction enzyme sites HindIII, 3 ' end contains restriction enzyme sites EcoRI.Reclaim the PCR product and be cloned in the pGEM-T carrier (Promega company product) the screening positive clone order-checking through the agarose gel electrophoresis purifying.The heavy chain fusion gene that will check order correct then downcuts from the pGEMT carrier with HindIII and the two enzymic digestions of EcoRI, is cloned in the pDR carrier, is built into pDR (CD20Fv/Fc/Link/FL) expression vector.Figure 28 has shown the Nucleotide (SEQ ID NO:45) and amino acid (the SEQ ID NO:46) sequence of CD20VH/Fc/Link/FL fusion rotein.
C. make up bifunctional fusion proteins gene (FL/Fc/CD20Fv)
Adopt aforementioned antigen-4 fusion protein gene construction process, with 3 of the variable region of heavy chain of CD20 monoclonal antibody '-end is by (Gly 4Ser) 35 of joint and its variable region of light chain '-the end fusion, constitute CD20 single-chain antibody gene (Fv).
Adopt aforementioned antigen-4 fusion protein gene construction process with 3 of FL/Fc gene '-end merges with 5 of CD20 single-chain antibody Fv '-end, make up bifunctional fusion proteins gene FL/Fc/CD20Fv, Figure 29 has shown the Nucleotide (SEQ ID NO:47) and amino acid (the SEQ ID NO:48) sequence of FL/Fc/CD20Fv fusion rotein. the synoptic diagram of this fusion gene is seen Figure 30.
More than three kinds of Expression of Fusion Protein identical with purification process with embodiment 4.
Embodiment 6
The structure of anti-Her2 humanized antibody and FL bifunctional fusion proteins gene
1. the construction and expression of anti-human Her 2 humanized antibody
With reference to the complete synthesis anti-her2 humanized antibody of the method for embodiment 1 (hcrceptin) [referring to Carter P, Presta L, Gorman CM, Ridgway JBB, Henner D, Wong WLT, Rwoland AM, Kotts C, Carver ME andShepard HM.Humanization of an anti-p185HER2 antibody for human cancer therapy.ProcNatl Acad Sci USA, 1992,89:4285] variable region of heavy chain (VH) and variable region of light chain (VL) gene.Figure 31 has shown Her2 humanized antibody variable region of heavy chain Nucleotide (SEQ ID NO:49) and amino acid (SEQ ID NO:50) sequence.Figure 32 has shown Her2 humanized antibody variable region of light chain Nucleotide (SEQ ID NO:51) and amino acid (SEQ ID NO:52) sequence.5 of humanized antibody heavy chain variable region gene '-end design EcoRV, the XbaI site, 3 '-end adds the NheI site, chain variable region gene 5 '-end design HindHI site.Agarose gel electrophoresis separates amplified production, reclaims the purpose band and is cloned in the pGEM-T carrier screening positive clone order-checking.PCR adopts the high-fidelity amplification system of Roche company, and reaction conditions is: 95 ℃ 5 minutes; 94 ℃ 45 seconds, 55 ℃ 45 seconds, 72 ℃ 55 seconds, 30 circulations; 72 ℃ 7 minutes.After order-checking is identified, humanization VH is downcut and is cloned into through the pAH4604 carrier that same enzyme is cut from pGEM-T/VH, constituted the expressed intact framework pAH4604-VH of humanized antibody heavy chain gene with EcoRV and NheI.
With XbaI and BamHI enzymolysis pAH4604-VH, separation and purification wherein about 3200bp chimeric heavy chain band (VHCH) and be cloned among the pDR, obtain humanized antibody heavy chain expression carrier pDR-VHCH.Figure 33 has shown the Nucleotide (SEQ ID NO:53) and amino acid (the SEQ ID NO:54) sequence of Her2 humanized antibody heavy chain.
Adopt Overlapping PCR that her2 humanized antibody variable region of light chain and human antibody light chain constant region cDNA direct splicing are got up, Overlapping PCR reaction conditions is: 94C 5 minutes; 94C 55 seconds, 55C 55 seconds, 72C 1 minute and 30 seconds, 30 circulations; 72C 7 minutes obtains product VLCL at last, and it 5 ' contains restriction enzyme sites HindIII, and 3 ' end contains restriction enzyme sites EcoRI.Reclaim and be cloned in the pGEM-T carrier through the agarose gel electrophoresis purifying, get pGEM-T-VLCL, the screening positive clone order-checking.Use the two enzymic digestions of Hind III and EcoR I at last, VLCL is downcut from the pGEM-T-VLCL carrier, be cloned among the pDR of same enzymolysis, get pDR-VLCL, cut the expression vector of determining structure through enzyme.Figure 34 has shown the Nucleotide (SEQ ID NO:55) and amino acid (SEQ IDNO:56) sequence of Her2 humanized antibody light chain.
In 3.5cm tissue culture ware, inoculate 3.5 * 10 5The CHO-dhfr in/hole -Cell, cell is containing xanthoglobulin (H), be cultured in the DMEM perfect medium of Thymine deoxyriboside (T) and glycine (G) when 90-95% merges and carry out transfection: get plasmid 10 μ g (her2 humanized antibody heavy chain expression plasmid 4 μ g, her2 humanized antibody light chain expression plasmid 6 μ g) and 20 μ l Lipofectamine2000 Reagent[Gibco company products] be dissolved in 500 μ l serum-free DMEM substratum respectively, room temperature left standstill 5 minutes, with above 2 kinds of liquid mixing, incubated at room 20 minutes is so that the formation of DNA-liposome complex, therebetween with the blood serum medium that contains in the DMEM substratum replacement culture dish of 3ml serum-free, then the DNA-liposome complex that forms is joined in the plate CO 2Incubator is cultivated after 4 hours and is added the DMEM perfect medium that 3ml contains 10% serum, places CO 2Continue in the incubator to cultivate.Transfection is carried out after 24 hours cell by 0.8 * 10 5/ ware passes in the 10cm culture dish, changes the selection substratum that does not contain H, T, G, treats that the clone forms the back with dihydrofolate reductase inhibitor methotrexate (MTX) screening of pressurizeing, and concentration is from 2 * 10 -8Mol/L to 5 * 10 -7The pressurization of mol/L gradient is carried out subclone with limiting dilution assay.Get cells and supernatant and detect the screening high-expression clone with ELISA.With the high-expression clone serum free medium enlarged culturing that screening obtains, use Protein A affinity column separation and purification humanized antibody.
2. the antigen-4 fusion protein gene that makes up 3 kinds of structures is used for later biological function research.
A. make up bifunctional fusion proteins gene her2Fv/Fc/FL
Construction process according to above-mentioned fusion gene, by OverlapPCR with the Flex gene directly with 3 of her2 heavy chain of antibody gene '-the end fusion, obtain PCR product her2/Fc/FL, its 5 '-contain restriction enzyme sites HindIII, 3 ' end contains restriction enzyme sites EcoRI.Reclaim the PCR product and be cloned in the pGEM-T carrier (Promega company product) the screening positive clone order-checking through the agarose gel electrophoresis purifying.The heavy chain fusion gene that will check order correct then downcuts from the pGEMT carrier with HindIII and the two enzymic digestions of EcoRI, is cloned in the pDR carrier, is built into pDR (her2/Fc/FL) expression vector.Figure 35 has shown the Nucleotide (SEQ ID NO:57) and amino acid (the SEQ ID NO:58) sequence of her2VH/Fc/FL fusion rotein.
B. make up the bifunctional fusion proteins gene (her2Fv/Fc/Link/FL) of belt lacing sequence
According to the construction process of aforementioned fusion gene, with Overlap PCR with 3 of her2 heavy chain gene '-end is by (Gly 4Ser 1) 3Joint obtains PCR product C D20Fv/Fc/Link/FL with 5 of FLex '-fusion, its 5 '-contain restriction enzyme sites HindIII, 3 ' end contains restriction enzyme sites EcoRI.Reclaim the PCR product and be cloned in the pGEM-T carrier (Promega company product) the screening positive clone order-checking through the agarose gel electrophoresis purifying.The heavy chain fusion gene that will check order correct then downcuts from the pGEMT carrier with HindIII and the two enzymic digestions of EcoRI, is cloned in the pDR carrier, is built into pDR (her2Fv/Fc/Link/FL) expression vector.Figure 36 has shown the Nucleotide (SEQ ID NO:59) and amino acid (the SEQ ID NO:60) sequence of her2VH/Fc/Link/FL fusion rotein.
C. make up bifunctional fusion proteins gene (FL/Fc/her2Fv)
Adopt aforementioned antigen-4 fusion protein gene construction process, with 3 of the variable region of heavy chain of her2 monoclonal antibody '-end is by (Gly 4Ser) 35 of joint and its variable region of light chain '-the end fusion, constitute her2 single-chain antibody gene (Fv).
Adopt aforementioned antigen-4 fusion protein gene construction process with 3 of FL/Fc gene '-end merges with 5 of her2 single-chain antibody Fv '-end, make up bifunctional fusion proteins gene FL/Fc/her2Fv, Figure 37 has shown the Nucleotide (SEQ ID NO:61) and amino acid (the SEQ ID NO:62) sequence of FL/Fc/her2Fv fusion rotein. Figure 38 has shown the hFLex/Fc/Fv antigen-4 fusion protein gene structure after Her2 and FL merge
More than three kinds of Expression of Fusion Protein identical with purification process with embodiment 4.
Embodiment 7
The structure of TRAIL and Flt3L bifunctional fusion proteins gene
1. making up hFLex/TRAILex merges
Synthesizing of trail dna: the cDNA sequence of people TRAIL is referring to GenBan accession number HSU37518, and wherein 88-933 is the coding region of TRAIL.281 amino acid whose protein of this genes encoding, wherein the 95th amino acids rise to 281 for the cytolemma outskirt.
According to the DNA sequences encoding of above-mentioned film outskirt, according to synthetic this gene of embodiment 1 described method, the plasmid after the sequence verification (pGEM-T/hTRAIL) is preserved standby.
Synthesizing of fusion gene: according to the method for embodiment 4 construction of fusion protein genes, to contain the segmental pGEM-T carrier of people TRAIL and Flt3L film outskirt (pGEM-T/hTRAIL and pGEM-T/hFLex) is template, by Overlap PCR 3 ' of FLt3L film outskirt (hFLex)-end is passed through (Gly 4Ser 1) 3Joint is with 5 ' of TRAIL film outskirt gene (TRAILex)-fusion.Merge fragment purifying purpose segment after agarose gel electrophoresis separates, glue reclaims purifying and reclaims rear clone in the pGEM-T carrier, get pGEM-T (hFLex/TRAILex), the screening positive clone order-checking, Figure 39 has shown the Nucleotide (SEQ ID NO:63) and amino acid (the SEQ ID NO:64) sequence of hFLex/Trai lex fusion rotein, and Figure 40 has shown hFLex/TRAILex antigen-4 fusion protein gene structure
The structure of expression vector: with HindIII and the two enzymic digestions of EcoRI, hFLex/TRAILex is downcut from pGEM-T (hFLex/TRAILex) carrier, be cloned in the pDR carrier, get pDR (hFLex/TRAILex), cut through enzyme and identify the expression vector that makes up.
In 3.5cm tissue culture ware, inoculate 3.5 * 10 5The CHO-dhfr in/hole -Cell, cell is containing xanthoglobulin (H), be cultured in the DMEM perfect medium of Thymine deoxyriboside (T) and glycine (G) when 90-95% merges and carry out transfection: get expression vector plasmid 10 μ g and 20 μ l Lipofectamine2000 Reagent[Gibco company products] be dissolved in 500 μ l serum-free DMEM substratum respectively, room temperature left standstill 5 minutes, with above 2 kinds of liquid mixing, incubated at room 20 minutes is so that the formation of DNA-liposome complex, therebetween with the blood serum medium that contains in the DMEM substratum replacement culture dish of 3ml serum-free, then the DNA-liposome complex that forms is joined in the plate CO 2Incubator is cultivated after 4 hours and is added the DMEM perfect medium that 3ml contains 10% serum, places CO 2Continue in the incubator to cultivate.Transfection is carried out after 24 hours cell by 0.8 * 10 5/ ware passes in the 10cm culture dish, changes the selection substratum that does not contain H, T, G, treats that the clone forms the back with dihydrofolate reductase inhibitor methotrexate (MTX) screening of pressurizeing, and concentration is from 2 * 10 -8Mol/L to 5 * 10 -7The pressurization of mol/L gradient is carried out subclone with limiting dilution assay.Get cells and supernatant and detect the screening high-expression clone with ELISA:: goat-anti people Trail antibody sandwich is in elisa plate, 4 ℃ are spent the night, sealed 2 hours in 37 ℃ with 2%BSA-PBS, add resistance clone culture supernatant to be measured, hatched 2 hours for 37 ℃, add HRP-goat-anti people FL and carry out association reaction, hatched 1 hour for 37 ℃, add TMB in 37 ℃ of effects 5 minutes, use H at last 2SO 4Termination reaction is surveyed OD 450Value.The high-expression clone serum free medium enlarged culturing that screening is obtained is with the affinity column separation and purification fusion rotein of anti-people Trail.
2. make up the hFLex/IZ/TRAILex antigen-4 fusion protein gene
Its activity was the strongest when TRAIL was tripolymer, we add an isoleucine zipper (IZ at design between hFLex and TRAILex, isoleucine zipper) [Harbury PB, Zhang T, Kim PS, et al.A sitch between two, three, and four-stranded coiled coils in GCN4 leucine zipper mutants.Science, 1993,262:1401-1407], can make this fusion rotein form tripolymer.Gene constructed and protein expression purifying are with reference to the method described in 1, just with (Gly 4Ser 1) 3Replace to the sequence of IZ.Figure 41 has shown the Nucleotide (SEQ ID NO:65) and amino acid (the SEQ ID NO:66) sequence of hFLex/IZ/Trailex fusion rotein.
3. make up the hFLex/Fc/TRAILex antigen-4 fusion protein gene
With the plasmid pGEM-T/hTRAIL that contains people Trail film outskirt gene is template, pcr amplification obtains the Trailex gene segment, by overlapping PCR 5 of Trailex gene ' end is merged mutually with 3 of FL/Fc gene ' end, obtain the hFLex/Fc/TRAILex fusion gene, its 5 ' contain restriction enzyme sites HindIII, 3 ' end contains restriction enzyme sites EcoRI.Reclaim the fusion gene segment and be cloned among the pGEM-T vector (Promega) through the agarose gel electrophoresis purifying, pGEMT-(hFLex/Fc/TRAILex), the screening positive clone order-checking.Figure 42 has shown the Nucleotide (SEQ ID NO:67) and amino acid (the SEQ ID NO:68) sequence of hFLex/Fc/Trailex fusion rotein.Figure 43 has shown hFLex/Fc/Trailex antigen-4 fusion protein gene structure.
From following embodiment 8-16, chSM/FL, huSM/FL, CD20/FL, her2/FL, Trail/FL represent fusion roteins such as FL/Fc/chSMFv, FL/Fc/huSMFv, FL/Fc/CD20Fv, FL/Fc/HER2Fv and hFLex/IZ/Trailex respectively.
Embodiment 8
The biological function research of bifunctional molecule SM/FL
One .SM/FL is to people's bleeding of the umbilicus CD34+ cells in vitro amplification effect
In order to measure the external short proliferation activity of SM/FL to marrow CD34+ progenitor cell, use cytokine FL, SCF, G-CSF, GM-CSF, IL-3, CSF-1 and FLT3 part (below be abbreviated as FL) or chSM/FL, huSM/FL tests.The cell culture medium principal constituent of using in the experiment is IMDM, contains 20% foetal calf serum (GIBCO), 1% methylcellulose gum, 0.05mmol beta-mercaptoethanol, 300mg/ml glutamine, 66mg/L penicillin, 100mg/L Streptomycin sulphate and cytokine to be measured.
CD34 in immunomagnetic beads (available from Pharmacia company) the method separation of human bleeding of the umbilicus +Cell, the operation carry out with reference to shop instruction, its purity of flow cytometry, with above-mentioned isolating CD34+ cell inoculation to the 96 porocyte culture plates that contain serum, methylcellulose gum and cytokine to be measured, every hole 2 * 10 3Cell.；
Above-mentioned cell plate place 37 ℃, 5%CO 2Incubator is cultivated, and the counting colony forms number after 2 weeks.
Detected result is seen Figure 44, and presentation of results, SM/FL have the effect of obvious stimulation marrow CD34+ progenitor cell proliferation, and the SM/FL fusion rotein has the hormesis identical with FL.
Two .SM/FL measure NK, DC activity in vivo
This tests used C57BL/6 mouse available from the Shanghai City Experimental Animal Center, and FITC-anti CD3, PE-anti NK1.1 and FITC-anti CD11c monoclonal antibody are available from U.S. R﹠amp; D and SIGMA company.Process of the test is as follows:
The C57BL/6 mouse gives SM/FL, every animal 10ug every day.Animal subject is put to death mouse after accepting SM/FL certain hour (0 day, 3 days, 6 days, 8 days, 10 days, 12 days, 15 days, 18 days), gets its marrow, spleen and liver organization, separates the mononuclearcell in these tissues.FITC-anti CD3 and PE-antiNK1.1 monoclonal antibody are dyed above-mentioned mononuclearcell is two, perhaps singly dye the back and carry out analyzing and testing with flow cytometer with the FITC-antiCD11c monoclonal antibody, according to the mononuclearcell number of each organ and be labeled as the number of each group of positive cells percentage calculation cell, detected result (* 10 6/ ml) see Figure 45.
The above results shows that the SM/FL bifunctional molecule is the same with FL, has the effect of inducing spleen, liver and marrow NK cell and DC cell proliferation in the mouse body, peaks at 10-13 days.This inducing action helps to improve the resistivity of body to tumour.
Three .SM/FL bifunctional fusion proteins are external to the tumor growth restraining effect
Clone is selected SMMU for use, Raji, Hepal-6/230, B16p230 (institute of oncology provides by the Shanghai international co-operation).
Main agents is selected for use: anti-people SM5-1 chimeric antibody/FL bifunctional fusion proteins (chSM5/FL), anti-people SM5-1 humanization monoclonal antibody/FL bifunctional fusion proteins (huSM/FL), foetal calf serum (GIBCO company product), DMEM and RPMI-1640 (GIBCO company product), CellTiter96AQueous cell proliferation detecting kit (Promega company product), 0.05% trypsinase+0.02% disodium ethylene diamine tetraacetate (solvent is 1 * PBS, filtration sterilization).
The above-mentioned various cells of logarithmic phase with 0.05% trypsinase+0.02%EDTA digestion, are counted, and adjusting cell density is 6 * 10 4/ ml is resuspended in the 10%FCS 1640/DMEM nutrient solution standby.
Dilute anti-people SM5-1 chimeric antibody/FL fusion rotein and anti-people SM5-1 humanization monoclonal antibody/FL fusion rotein as follows:
The mother liquid concentration of anti-people SM5-1 mosaic monoclonal antibody/FL fusion rotein is the 20mg/ml aqueous solution.With 10 times of stepwise dilutions of 10%FCS1640/DMEM, be 16ug/ml until concentration, the 200ul/ hole adds 96 orifice plates.Make 2 doubling dilutions with 100ul10%FCS 1640/DMEM nutrient solution then, totally ten four concentration.
Anti-people SM5-1 humanized antibody/FL fusion rotein mother liquid concentration is the 20mg/ml aqueous solution.Dilution process is done 2 doubling dilutions, totally ten four concentration.10%FCS 1640/DMEM is as blank.
Above-mentioned various cells are added in 96 orifice plates that contain two kinds of antibody the 100ul/ hole respectively.One 96 orifice plate of every kind of cell.
For preventing the fringing effect of porous plate, do not use the hole at 96 porocyte culture plate edges, but must add the PBS in 200ul/ hole.At 37 ℃, 7%CO 2Cell culture incubator is cultivated the reading that develops the color after 7 days.
96 orifice plates are taken out from cell culture incubator.Add PMS: MTS=1: 20 developer, 20ul/ hole.Putting into cell culture incubator after 96 orifice plates lid is opened continues to cultivate 3 hours.
Microplate reader detects 96 orifice plate OD490nm, according to the mapping of absorbance value data, the results are shown in Figure 46 with Excel software.These two kinds of fusion roteins are described to B16p230, hepal-6p230, three kinds three kinds increments of expressing the proteic tumor cell line of p230f such as SMMU all can produce effective restraining effect.And this restraining effect is specific, to the not effect of irrelevant cell.
Four. inosculating antibody people SM5-1/FL and humanization SM5-1/FL extracorporeal anti-tumor function
Survey the viable cell strain and use SKBR-3, QYC clone, and Hepal-6, B16 clone (available from ATCC).Above-mentioned cell all goes down to posterity and is incubated at 25cm 2In the culturing bottle, use RPMI-1640/DMEM (V:V=1) substratum that contains 10% foetal calf serum.And change these two kinds of clones over to the P230 gene respectively.Make its high expression level people p230 gene.
Main agents is selected for use: anti-people CD3 mosaic monoclonal antibody/FL fusion rotein (chCD3/FL), anti-people CD3 humanization monoclonal antibody/FL fusion rotein (huCD3/FL), anti-people SM5-1 mosaic monoclonal antibody/FL fusion rotein (chSM/FL), anti-people SM5-1 humanization monoclonal antibody/FL (huSM/FL) fusion rotein, foetal calf serum (GIBCO company product), DMEM and RPMI-1640
(GIBCO company product), CellTiter96AQueous cell proliferation detecting kit (Promega company product), 0.05% trypsinase+0.02% disodium ethylene diamine tetraacetate (solvent is 1 * PBS, filtration sterilization).
With Hepal-6, B16 and the hepal-6p230 of logarithmic phase, B16p230 clone, with 0.05% trypsinase+0.02%EDTA digestion, counting, adjusting cell density is 6 * 10 4/ ml is resuspended in the 10%FCS 1640/DMEM nutrient solution standby.
Dilute the full humanization monoclonal antibody of anti-people CD3 mosaic monoclonal antibody/FL and anti-people CD3/FL fusion rotein and anti-people SM5-1 mosaic monoclonal antibody/FL and anti-people SM5-1 humanization monoclonal antibody/FL fusion rotein.
The mother liquid concentration of anti-people CD3 mosaic monoclonal antibody/FL and anti-people SM5-1 mosaic monoclonal antibody/FL is respectively the 20mg/ml aqueous solution.With 10 times of stepwise dilutions of 10%FCS 1640/DMEM, be 16ug/ml until concentration, the 200ul/ hole adds 96 orifice plates.Make 2 doubling dilutions with 10%FCS 1640/DMEM nutrient solution then, totally ten four concentration.
Anti-people CD3 humanized antibody/FL and anti-people SM5-1 humanization monoclonal antibody/FL mother liquid concentration are respectively the 20mg/ml aqueous solution.Dilution process is the same.The 200ul/ hole adds 96 orifice plates, makes 2 doubling dilutions with 100ul 10%FCS 1640/DMEM nutrient solution then, totally ten four concentration.
Above-mentioned various cells are added in 96 orifice plates that contain two kinds of antibody the 100ul/ hole respectively.Two 96 orifice plates of every kind of cell.Anti-people CD3 chimeric antibody/FL and anti-people CD3 humanized antibody/one 96 orifice plate of every kind of cell of FL fusion rotein.Anti-people SM5-1 chimeric antibody/FL and anti-people SM5-1 humanized antibody/a kind of 96 orifice plates of every kind of cell of FL fusion rotein.
For preventing the fringing effect of porous plate, do not use the hole at 96 porocyte culture plate edges, but must add the PBS in 200ul/ hole.37 ℃, the 7%CO2 cell culture incubator is cultivated the reading that develops the color after 7 days.
96 orifice plates are taken out from cell culture incubator.Add PMS: MTS=1: 20 developer, 20ul/ hole.Putting into cell culture incubator after 96 orifice plates lid is opened continues to cultivate 3 hours.
Microplate reader detects 96 orifice plate OD490nm, with the mapping of absorbance data, the results are shown in Figure 47 with Excel.Presentation of results, under isolated condition, chimeric SM/FL fusion rotein and humanization hSM/FL fusion rotein have the Hepal-6 of obvious suppression people P230 transfection and the effect of B16 cell, and the Hepal-6 and the B16 cell of not expressing the p230 molecule do not had restraining effect.The irrelevant anti-CD3/FL of antibody fusion protein does not all have effect to above 4 kinds of cells.
Embodiment 9
Anti-people HER-2 monoclonal antibody/FL, anti-humen CD 20/FL and the external killing activity of TRAIL/FL fusion rotein
For further research FL with other can inducing apoptosis of tumour cell antibody or the effect that also has above-mentioned external killing tumor cell of the fusion rotein of other molecules formation, carried out following experiment.These result of experiment prove, as the fusion rotein that SM5-1 monoclonal antibody and FL constitute, have the fusion protein molecule that apoptotic anti-humen CD 20 monoclonal antibody, anti-human Her 2 monoclonal antibody and TRAIL and FL formation take place induced tumor and also have the activity of external killing tumor cell.
Anti-Her2 monoclonal antibody/FL fusion rotein is to the growth-inhibiting of target cell and the in vitro study of killing activity
1.Her2/FL bifunctional molecule body outer suppressioning experiment
Survey the viable cell strain and use SK-BR-3, BT-474, D2F2,4T1 cell (above-mentioned cell is all from ATCC).BT-474 wherein, SK-BR-3 expresses the HER2 antigen molecule at its surface of cell membrane, and D2F2,4T1, Jarket cell are not expressed the HER2 antigen molecule.
Above-mentioned cell all goes down to posterity and is incubated at 25cm 2In the culturing bottle, use RPMI-1640/DMEM (V:V=1) substratum that contains 10% foetal calf serum.Adopt QIAGEN Corporation's Super Fect Transfection Reagent (Qiagen CAT.301307) liposome reagent with D2F2 and two kinds of clones difference of 4T1 transfection people her2 gene, and make it obtain high expression level at cell surface.The clone D2F2/e2 and the 4T1/her2 that change the her2 gene over to are used for this experiment.
Main agents is selected for use: anti-human Her 2/FL bifunctional molecule, Herceptin (available from Genetech company), foetal calf serum (GIBCO company product), DMEM and RPMI-1640 (GIBCO company product), CellTiter96AQueous cell proliferation detecting kit (Promega company product), 0.05% trypsinase+0.02% disodium ethylene diamine tetraacetate (solvent is 1XPBS, filtration sterilization).
The above-mentioned various cells of logarithmic phase with 0.05% trypsinase+0.02%EDTA digestion, are counted, and adjusting cell density is 6 * 10 4/ ml is resuspended in the 10%FCS 1640/DMEM nutrient solution standby.
Anti-human Her 2/FL bifunctional molecule and positive control Herceptin dilute as follows:
The mother liquid concentration of anti-human Her 2/FL bifunctional molecule is the 20mg/ml aqueous solution.With 10 times of stepwise dilutions of 10%FCS 1640/DMEM, be 16ug/ml until concentration, the 200ul/ hole adds 96 orifice plates.Carry out 2 doubling dilutions with 10%FCS 1640/DMEM nutrient solution then, totally ten four concentration.
Herceptin standard substance mother liquid concentration is the 20mg/ml aqueous solution.Dilution process is the same, makes 2 doubling dilutions successively, totally ten four concentration.
Above-mentioned various cells are added in 96 orifice plates that contain fusion rotein and anti-human Her 2 antibody (Herceptin) the 100ul/ hole respectively.One 96 orifice plate of every kind of cell.
Experimental procedure is carried out according to CellTiter96AQueous cell proliferation detecting kit shop instruction.
Experimental result such as Figure 48.
From The above results as can be seen, anti-people HER2 monoclonal antibody/FL fusion rotein is variously tried target cell the obvious growth restraining effect is all arranged what expression had a people Her2.This fusion molecule does not have restraining effect to irrelevant clone, illustrates that this restraining effect is specific.
2. the external killing activity of anti-people HER-2/FL fusion rotein
Survey the viable cell strain and use SK-BR-3, BT-474, D2F2,4T1 cell (above-mentioned cell is all from ATCC) change the clone D2F2/e2 and the 4T1/her2 of her2 gene over to.BT-474 wherein, SK-BR-3, D2F2/e2 and 4T1/her2 express the HER2 antigen molecule at its surface of cell membrane, and D2F2,4T1, Jarket cell are not expressed the HER2 antigen molecule,
Test kit: use CytoTox 96 heterotope cytotoxicity experiment test kits (Promega company product) to detect cell viability.Step is carried out according to producer's explanation.
Get the anti-Her2 monoclonal antibody/FL of detected sample, progressively being diluted to concentration with nutrient solution is 4 μ g/mL.Then be initial concentration, with nutrient solution two doubling dilutions to 15.6ng/ml.
In 96 orifice plates, add the good testing sample of dilution, every pore volume 100 μ l, each concentration is established 2 multiple holes.The negative contrast of nutrient solution.
The cell of taking the logarithm vegetative period is adjusted cell density to 6 * 10 4/ ml adds in above-mentioned 96 well culture plates 0.1ml/ hole.
Detected result as shown in figure 49.The result shows that Her-2 antibody/FL fusion rotein is to BT-474, SK-BR-3, and D2F2/e2 and 4T1/her2 cell have the specific killing effect.
Two. anti-humen CD 20 monoclonal antibody/FL fusion rotein is to the external killing activity of target cell
Principle: human B cell lymphoma cell line Raji cell surface high expression level CD20 antigen molecule, IDEC-C2B8 can combine with it.Then can make the target cell dissolving that combines antibody, the concentration of antibody and cytolytic degree are amount-result relation within the specific limits.Cytolytic degree can detect by detecting the serum lactic dehydrogenase that discharges after the cytolysis.
Main agents is selected for use: anti-humen CD 20/FL bifunctional molecule, IDEC-C2B8 rituximab.
Cell strain: Raji cell (ATCC), do not have mycoplasma contamination after testing.Go down to posterity with the RPMI-1640/DMEM that contains 10% newborn calf serum (NBS) (1: 1) nutrient solution and to be incubated at 25-75cm 2In the square vase.
Nutrient solution: A is for containing RPMI-1640/DMEM (1: the 1) nutrient solution of 10% newborn calf serum (NBS).B is not for containing the no phenol red RPMI-1640 nutrient solution of serum.C is the nutrient solution B that contains 5% normal human serum.
Detection kit: CytoTox 96 heterotope cytotoxicity experiment test kits (Promega company product)
The Raji cell of taking the logarithm vegetative period adds nutrient solution B and adjusts cell density to 2 * 10 5/ ml adds in 96 well culture plates 0.1ml/ hole.
Get testing sample and progressively be diluted to 4ug/mL with nutrient solution C, and be that initial concentration two doubling dilutions are to 7.8ng/ml.
With the negative contrast of nutrient solution C, in the culture plate of inoculating the Raji cell, the nutrient solution of step gradient dilution liquid 0.1mL/ hole and negative control in the adding.Each extent of dilution is established 2 multiple holes.Place 37 ℃ of 5% CO2gas incubator to cultivate 3-4 hour.
Detected result is seen Figure 50, the result as can be seen, under isolated culture condition, anti-humen CD 20 monoclonal antibody/FL fusion rotein and IDEC-C2B8 are similar, to being tried target cell tangible lethal effect are arranged.
Three .TRAIL/FL are to the in vitro study of target cell growth-inhibiting and lethal effect
The external survey of TRAIL/FL is lived and is tested
Survey viable cell select good strains in the field for seed usefulness: L929, MDA-MB-231 (available from ATCC), Renca (available from the Korea S institute of oncology).The negative control cells of hepG2.
Above-mentioned cell all goes down to posterity and is incubated at 25cm 2In the culturing bottle, use RPMI-1640/DMEM (V:V=1) substratum that contains 10% foetal calf serum.
Test kit uses CellTiter 96 Aqueous heterotope cell proliferation experiment test kits, and (Promega company product Catalog#G5430) detects cell viability.By two kinds of solution compositions of MTS/PMS, but multigelation is stored in below-70 ℃.Before using the two 20: 1 are by volume mixed.Mixed reagent can be stored in-20 ℃ temporarily.
The L929 that takes the logarithm vegetative period, MDA-MB-231, Renca, the hepG2 cell, tryptic digestion, counting, each survey live (1 96 orifice plate) gets about 5 * 10 6Individual cell, the centrifugal supernatant of abandoning adds nutrient solution B and adjusts cell density to 5 * 10 5/ ml adds in 96 well culture plates, 0.1ml/ hole, overnight incubation (18-24h), one 96 orifice plate of every kind of cell.
Get sample to be determined, be diluted to 10ug/ml with nutrient solution, and be that initial concentration two doubling dilutions are to 19.5ng/ml.
With the negative contrast of nutrient solution, in the culture plate of inoculating cell, the nutrient solution of step gradient dilution liquid 0.1mL/ hole and negative control in the adding.Each extent of dilution is established 2 multiple holes.
Place 37 ℃ of 5% carbonic acid gas incubator to cultivate 12h.
Endpoint determination: 96 orifice plates add freshly prepared 20: 1 blended MTS/PMS solution 20 μ L/ holes, continue to hatch 2-4 hour (96 orifice plates are lid not) in incubator, measure its A with microplate reader 490-A 630Value.
As can be seen from Figure 51, the TRAIL/FL fusion rotein has the obvious growth restraining effect to the various target cells of isolated culture, but to the not effect of irrelevant cell.
2.TRAIL/FL external killing activity
Cell strain: L929 cell, hepG2 cell go down to posterity with RPMI-1640/D ' MEM (1: the 1) nutrient solution that contains 10% newborn calf serum (NBS) and are incubated at 25-75cm 2In the square vase;
Test kit: use CytoTox 96 heterotope cytotoxicity experiment test kits (Promega company product)
The L929 cell of taking the logarithm vegetative period, tryptic digestion, counting, each survey live (1 96 orifice plate) gets about 2 * 10 6Individual cell, the centrifugal supernatant of abandoning adds nutrient solution B and adjusts cell density to 1.5 * 10 5/ ml adds in 96 well culture plates, 0.1ml/ hole, overnight incubation (18-24h).Inferior daily nutrient solution dilution dactinomycin to final concentration is 20 μ g/mL, gets 0.5mL as negative control.
Get sample to be determined, be diluted to 1600ng/ml with the nutrient solution that contains dactinomycin, and be that initial concentration two doubling dilutions are to 3.1ng/ml.
With the negative contrast of nutrient solution C, in the culture plate of inoculating the L929 cell, the nutrient solution of step gradient dilution liquid 0.1mL/ hole and negative control in the adding.Each extent of dilution is established 2 multiple holes.Place 37 ℃ of 5% carbonic acid gas incubator to cultivate 14-16 hour.
Experimental procedure is according to using CytoTox 96 heterotope cytotoxicity experiment test kit shop instructions to carry out.
Experimental result is seen Figure 52, shows that TRAIL/FL has the killing activity similar to Trail to the L929 cell.
Embodiment 10
SM/FL, hSM/FL fusion rotein are to the therapeutic action of B16/p230 and Hepal-6/p230 tumor-bearing mice
Main agents is used: anti-people SM5-1 mosaic monoclonal antibody, the full humanization monoclonal antibody of anti-people SM5-1, anti-people CD3 mosaic monoclonal antibody/FL fusion rotein, the full humanization monoclonal antibody of anti-people CD3/FL fusion rotein, anti-people SM5-1 mosaic monoclonal antibody/FL and anti-people SM5-1 humanization monoclonal antibody/FL fusion rotein.
Male C57BL/6 mouse is treated diameter of tumor average out to 0.5cm after inoculating, grouping experimentizes.Be divided into 7 big groups at random.One group is PBS group, totally 8 mouse.One group is the irrelevant anti-people CD3 of antibody mosaic monoclonal antibody/FL fusion rotein.One group is anti-people CD3 humanization monoclonal antibody/FL fusion rotein.One group is anti-people SM5-1 mosaic monoclonal antibody.One group is anti-people SM5-1 humanization monoclonal antibody.One group is anti-people SM5-1 chimeric antibody/FL fusion rotein, and one group is anti-people SM5-1 humanization monoclonal antibody/FL fusion rotein, with the 4mg/kg tail vein injection, and every group of ten mouse.
Various monoclonal antibodies all are diluted to final concentration 0.4mg/ml respectively with PBS, four kinds of different concns.Tail vein injection dosage is pressed 4mg/kg/ week, control group injection equal-volume PBS.According to the mouse body weight, average every mouse tail vein injection volume is about 250ul.
Observed for 6 weeks continuously, carry out statistical study.The results are shown in Table 3.
Tumor regression situation after table 3 oncotherapy
The cell title Contrast anti-people CD3 monoclonal antibody/FL fusion rotein Anti-people SM5-1 monoclonal antibody Anti-people SM5-1/FL fusion rotein ????PBS
Chimeric The people source Chimeric The people source Chimeric The people source
??Hepal-6 ??0/10 ????0/10 ????0/10 ????0/10 ????0/10 ????0/10 ????0/10
??Hepal-6/p230 ??0/10 ????0/10 ????7/10 ????8/10 ????10/10 ????10/10 ????0/10
??B16 ??0/10 ????0/10 ????0/10 ????0/10 ????0/10 ????0/10 ????0/10
??B16/p230 ??0/10 ????0/10 ????8/10 ????7/10 ????10/10 ????10/10 ????0/10
From above-mentioned experimental result as can be seen, when the p230 antigen presentation is arranged, no matter be chimeric or humanized SM5-1 monoclonal antibody, no matter also whether merge, all have the ability that significantly makes tumor regression with FL.And irrelevant antibody does not have this ability.But, merge the anti-tumor capacity that can largely improve in vivo with FL.
Embodiment 11
SM/FL and hSM/FL fusion rotein induce B16/P230 and Hepal-6/P230 tumor-bearing mice to produce specific immune response
Main agents is selected for use: anti-people SM5-1 mosaic monoclonal antibody, the full humanization monoclonal antibody of anti-people SM5-1, anti-people CD3 mosaic monoclonal antibody/FL fusion rotein, the full humanization monoclonal antibody of anti-people CD3/FL fusion rotein, anti-people SM5-1 mosaic monoclonal antibody/FL and anti-people SM5-1 humanization monoclonal antibody/FL fusion rotein.
With female C57BL/6 mouse difference hepal-6/P230 tumour cell, after inoculation, treat diameter average out to 0.5cm.Be divided into 7 big groups at random.One group is PBS group, totally 8 mouse.One group is the irrelevant anti-people SM5-1 of antibody mosaic monoclonal antibody.One group is anti-people SM5-1 humanization monoclonal antibody.One group is that anti-people SM5-1 mosaic monoclonal antibody and FL merge use.One group is that anti-people SM5-1 humanization monoclonal antibody and FL merge use.One group is anti-people SM5-1 chimeric antibody/FL fusion rotein, and one group is anti-people SM5-1 humanization monoclonal antibody/FL fusion rotein, with the 2mg/kg tail vein injection, and every group of 10 mouse.
Various monoclonal antibodies all are diluted to final concentration 0.4mg/ml respectively with PBS, four kinds of different concns.Tail vein injection dosage is pressed 4mg/kg/ week, control group injection equal-volume PBS.According to the mouse body weight, average every mouse tail vein injection volume is about 250ul.
Observe continuously to tumour and disappear, the mouse of tumor regression is carried out the parental tumor cell inoculation, observe mouse and become knurl situation, result such as table 4.
The antitumor action of table 4 fusion rotein
The therapeutical agent kind The tumour lapse count Inoculate the no knurl existence in back number once more
????chSM ????5/10，5/10，6/10 ??0/10
????huSM ????4/10，5/10，4/10 ??0/10
The chSM+FL coupling ????6/10，6/10，8/10 ??6/10
The huSM+FL coupling ????7/10，5/10，6/10 ??6/10
ChSM/FL merges ????10/10，8/10，10/10 ??28/30
HuSM/FL merges ????10/10，10/10，10/10 ??30/30
Above-mentioned data declaration, SM or hSM and FL are united when using, and the anti-tumor in vivo ability is improved to some extent.But the amplitude that fusion rotein SM/FL and hSM/FL improve is bigger, and mouse tumor is all disappeared, and inoculation back disease free survival is up to 100% once more.This explanation, fusion rotein SM/FL and hSM/FL make mouse obtain the active immunity ability of opposing tumour.
Anti-people SM5-1 chimeric antibody/FL and the anti-immune response that people SM5-1 humanized antibody/the FL fusion rotein is produced are specific.Get anti-people SM5-1 chimeric antibody/FL and anti-people SM5-1 humanized antibody/FL fusion rotein treatment B16p230 tumor-bearing mice and Hepal-6p230 tumor-bearing mice, after treating that tumour disappears, inoculate B16, Hepal-6, B16p230 or Hepal-6p230 tumour cell respectively once more, experimental result sees Table 5.The recurrence that the B16p230 mouse of anti-people SM5-1 chimeric antibody/FL and anti-people SM5-1 humanized antibody/FL fusion rotein treatment only can be resisted the B16 tumour cell, and to the effect of Hepal-6 passivity, vice versa.
Table 5 fusion rotein induces B16/P230 and Hepal-6/P230 tumor-bearing mice to produce specific immune response
The tumour cell title Treatment antibody Inoculation back tumour forms for the second time
????B16 ????Hepal-6
?B16/p230 Anti-people SM5-1 chimeric antibody/FL ????0/6 ????5/6
?B16/p230 Anti-people SM5-1 humanized antibody/FL ????0/6 ????5/5
?Hepal-6/p230 Anti-people SM5-1 chimeric antibody/FL ????5/5 ????0/5
?Hepal-6/p230 Anti-people SM5-1 humanized antibody/FL ????5/5 ????1/5
The above results shows, the B16p230 mouse of the fusion rotein treatment that constitutes with chSM and huSM and FL has obtained the B16 tumour cell that tangible opposing inoculates and formed the ability of tumour, but irrelevant Hepal-6 tumour cell is not obviously had this effect.Vice versa.This further specifies, and the immune response that two kinds of fusion roteins bring out is specific.
Embodiment 12
Anti-people HER-2/FL, the anti-tumor in vivo effect of anti-humen CD 20/FL and TRAIL/FL fusion rotein
For further research FL with other can inducing apoptosis of tumour cell antibody or the effect that also has killing tumor cell in the body of the fusion rotein of other molecules formation, carried out following experiment.These result of experiment prove, as the fusion rotein that SM or hSM and FL constitute, have induced tumor and apoptotic anti-humen CD 20 monoclonal antibody, anti-human Her 2 monoclonal antibody and TRAIL take place also have the activity of the interior killing tumor cell of body with the fusion protein molecule that FL constitutes.
One. killing activity in the anti-HER-2/FL fusion rotein body
Material: MCF-7 BT-474, male BALB/c nude mice (available from the Shanghai City Experimental Animal Center).
The BT-474 cell is with 2.5 * 10 7/ ml is suspended among the PBS, and it is subcutaneous that 200ul/ only inoculates armpit, and promptly 5 * 10 6/ only; Nude mice during seven all diameter of tumor average out to 0.5cm, is divided into experimental group and control group, respectively anti-HER-2 fusion rotein and isopyknic PBS in intravenous injection 10mg/kg/ week in inoculation back at random.Observed for 6 weeks continuously, investigation gained data are carried out statistical study.
Detected result is seen Figure 53.Two groups of data are carried out significance test of difference, t=2.64944, the result shows that at 0.05 horizontal p=0.03806, difference reaches conspicuous level, illustrates that anti-people HER2 monoclonal antibody/FL fusion rotein has the ability of tangible cells in vivo.
Two. killing activity in anti-humen CD 20/FL fusion rotein body
Clone behaviour lymph slide clone Raji; Female BALB/c nude mice is available from the Shanghai City Experimental Animal Center.
The Balb/c nude mice is prepared: the BALB/c nude mice 602GY is weekly in the Co irradiation, continuous 3 weeks.It is 4 * 10 that the Raji cell is adjusted density 7/ ml, 0.5ml is subcutaneous in oxter or the back of nude mice in inoculation, and promptly inoculating cell adds up to 2 * 10 7Tumor growth is to 0.5cm when size, and the aseptic tumor tissue of getting cuts 8mm 3Tissue block, multiple cropping 60The nude mice of Co irradiation.Nude mice during one all diameter average out to 0.5cm, is divided into experimental group and control group in inoculation back at random, the anti-CD20/FL fusion rotein and the PBS in intravenous injection 10mg/kg/ week respectively, 4 weeks of successive administration.6 weeks of observations are also carried out statistical study.
Detected result is seen Figure 54.Two groups of data are carried out significance of difference test, t=2.82287, at 0.05 horizontal p=0.03024, difference reaches conspicuous level.This explanation, anti-humen CD 20 monoclonal antibody/FL fusion rotein has significantly in vivo tumor-inhibiting action.
Killing activity in the three .TRAIL/FL bodies
Experiment material is selected Bel7402 QYC for use; The BALB/c nude mice.
The QYC cell is with 5 * 10 7/ ml is suspended among the PBS, and it is subcutaneous that 200ul/ only inoculates armpit, and promptly 1 * 10 7/ only; Nude mice is in back 10 days diameter average out to 0.5cm of inoculation.Be divided into experimental group and control group at random, respectively abdominal injection 0.5mg//day TRAIL and isopyknic PBS.Observed for 6 weeks continuously, carry out statistical study.
The result is shown in Figure 55, and test of significance shows, the t=2.63092 of two groups of data, and at the p=0.03901 of 0.05 level, difference reaches conspicuous level.This explanation, in vivo, the TRAIL/FL fusion rotein has tangible tumor-inhibiting action.
Embodiment 13
HER-2/FL, CD20/FL and the reaction of TRAIL/FL fusion rotein inductive specificity antineoplastic immunity
In order further to study anti-people HER-2/FL, whether anti-humen CD 20/FL and TRAIL/FL bifunctional fusion proteins have specific killing effect to tumour in vivo as the fusion rotein of chimeric and humanized SM monoclonal antibody and FL, and have the antitumor response to active immunization of inductor internal specific, with above-mentioned three kinds of fusion roteins tumor-bearing mice has been carried out the interior therapeutic experiment.
One. the anti-tumor in vivo experiment of anti-human Her 2 monoclonal antibody/FL fusion rotein
Experiment material: female BALB/C mice; D2F2, D2F2/e2,4T1,4T1/her2 tumor cell line.
With 2 * 10 6Wild-type D2F2,4T1 and D2F2/e2, the 4T1/her2 cell is inoculated BALB/C mice respectively, the tumor growth situation is measured in subcutaneous injection weekly, when tumor growth is treated during to 0.5 centimetre of diameter, dosage is 4mg/kg, carried out for 3 weeks continuously after, observe the growth of tumor situation.The mouse of getting the tumour disappearance inoculates B16, Hepal-6, B16p230 or Hepal-6p230 tumour cell respectively once more, after 6 weeks tumor growth, disappearance situation is investigated.Experimental result sees Table 6.
The antitumor action of table 6Her2/FL fusion rotein
Grouping Band knurl number of animals The no tumour number of animals in treatment back Curative ratio (%) Be with the knurl number of animals once more Shared per-cent
?PBS ????8 ??8 ????0 ??8 ??100
?FL ????16 ??4 ????25 ??14 ??87.5
?herceptin ????16 ??13 ????81 ??16 ??100
?herceptin+FL ????16 ??14 ????87 ??12 ??75
The Her2/FL fusion rotein ????24 ??21 ????87 ??2 ??8
From The above results as can be seen, Her2 antibody is the same, and anti-human Her 2 antibody/FL fusion rotein suppresses D2F2/e2 and the growth of 4T1/her2 cell in vivo, resists established tumour, induces the generation antineoplastic immune.Tumour to inoculation for the second time has powerful lethal effect, makes it can not form tumour.And give Her2 antibody merely, only can treat established tumour, but can not effectively resist tumor inoculation once more.
Two. the anti-tumor in vivo experiment of anti-humen CD 20 monoclonal antibody/FL fusion rotein
Test materials adopts A20 cell (ATCC) and BALB/C mice.The A20 cell changes people CD20 gene over to the transfection reagent box of Qiagen, and makes people CD20 antigen obtain high expression level at cell surface, is prepared into the A20/CD20 cell and is used for later experiment.
With 2 * 10 6A20/CD20 cell inoculation BALB/C mice, subcutaneous injection, measure the tumor growth situation weekly, when tumor growth during to 0.5 centimetre of diameter, treat with FL, anti-humen CD 20 monoclonal antibody, FL+ anti-humen CD 20 monoclonal antibody (rituximab) coupling, CD20/FL fusion rotein, dosage is 4mg/kg, carried out for 3 weeks continuously after, observe the growth of tumor situation.The mouse of getting the tumour disappearance inoculates the A20/CD20 tumour cell respectively once more, and tumor growth, disappearance situation are investigated.Experimental result sees Table 6.
After 6 weeks tumor growth, disappearance situation are investigated.
Experimental result sees Table 7.
The antitumor action of table 7CD20/FL fusion rotein
Grouping Band knurl number of animals The no tumour number of animals in treatment back Curative ratio (%) Be with the knurl number of animals once more Shared per-cent
PBS ??8 ??8 ????100 ??8 ??100
FL ??16 ??4 ????25.0 ??14 ??87.5
The anti-humen CD 20 monoclonal antibody ??12 ??10 ????83.3 ??12 ??100
Anti-humen CD 20 monoclonal antibody+FL coupling ??14 ??12 ????85.7 ??10 ??71.4
Anti-humen CD 20 monoclonal antibody/FL fusion rotein ??20 ??18 ????90.0 ??2 ??10.0
From The above results as can be seen, the same with anti-humen CD 20 antibody, anti-humen CD 20 antibody/FL fusion rotein can be treated established tumour in vivo, induces the generation antineoplastic immune.And the tumour of inoculation for the second time had powerful lethal effect, make it can not form tumour.And give anti-humen CD 20 antibody merely, and only can treat established tumour, can not effectively resist tumor inoculation once more.
Three. people TRAIL/FL bifunctional molecule is to the anti-tumor in vivo experiment of tumor-bearing mice
Surveying viable cell selects good strains in the field for seed with Renca (available from Korea S tumor research institute)
Above-mentioned cell all goes down to posterity and is incubated at 25cm 2In the culturing bottle, use RPMI-1640/DMEM (V:V=1) substratum that contains 10% foetal calf serum.
Main agents:
People TRAIL/FL bifunctional molecule TRAIL
The people FL control group antibody control (anti-people CD3/FL) that has nothing to do
Get female BALB/c mouse respectively at 5 week of Renca inoculation back, two kinds of diameter of tumor average out to 0.5cm.Be divided into five big groups at random.One group is PBS group, totally 8 mouse.One group is the irrelevant anti-people CD3/FL of antibody control, and one group is people TRAIL/FL bifunctional molecule, and one group is the TRAIL positive controls, and one group is people FL control group, and respectively with every group of eight mouse, therapeutic dose is 2mg/kg.
Various samples all are diluted to final concentration 0.4mg/ml respectively with PBS, four kinds of different concns.Tail vein injection dosage is pressed 4mg/kg/ week, control group injection equal-volume PBS, carried out for 3 weeks continuously after, observe the growth of tumor situation.The mouse of getting the tumour disappearance inoculates the Renca tumour cell respectively once more, and tumor growth, disappearance situation are investigated.Experimental result sees Table 7.Experimental result is shown in Table 8.
The antitumor action of table 8TRAIL/FL fusion rotein
Therapeutical agent The tumour lapse count Inoculate the no knurl existence in back number once more
Inoculate the Renca cell once more Inoculate the D2F2 cell once more
PBS
0/10，0/10，0/10 0/10 0/10
Anti-people CD3/FL fusion rotein 0/10，0/10，0/10 0/10 0/10
TRAIL 6/10，5/10，6/10 0/10 0/10
FL 3/10，2/10，2/10 0/10 0/10
The TRAIL/FL fusion rotein 8/10，10/10，9/10 10/10 0/10
Last table presentation of results, the independent use of TRAIL and FL all has certain antitumor action, does not all show the effect of significantly inducing active immunity when still using separately.The TRAIL/FL fusion rotein has the obvious treatment effect to the Renca tumour that inoculation forms, and can induce the generation response to active immunization, makes it can resist tumor inoculation once more.Thisly induce the effect of active immunity that tangible specificity is arranged.
The explanation of above-mentioned experimental result, the bifunctional fusion proteins of forming for several and FL of examination not only has tangible interior therapeutic effect, and all has inducing mouse specific tumors is produced immunoreactive effect.Show that fusion rotein effectively induced DC and NK cell amplification in the body, not only improved the lethal effect of NK cell to tumour cell, and the DC cell improves the immunogenicity of some component of tumour cell or its release greatly, brings out body specific tumors has been produced response to active immunization.
Embodiment 14
The immunohistochemical analysis of tumor tissues
One .SM/FL and the hSM/FL fusion rotein immunohistochemical analysis after to the tumor tissues administration
In order further to illustrate the mechanism of action of SM monoclonal antibody/FL fusion rotein and hSM monoclonal antibody/FL fusion rotein, we have carried out immunohistochemical analysis to the mouse of treatment.Discovery has the massive tumor necrocytosis in administration animal tumor tissue, and with DC cell, NK cell and other lymphocytic infiltrations.This shows that SM monoclonal antibody/FL fusion rotein and hSM monoclonal antibody/FL fusion rotein have cells such as guiding DC, NK and concentrate and bring into play its effect to the tumor cytotoxicity function to tumor tissues.
1. inoculation
Recovery Hepal-6/P230 rat liver cancer clone and B16/P230 mouse melanin tumor cell system cultivate with 10%FCS16/DM.Treat cell amplification to certain number, with 0.05% trypsinase, 0.02%EDTA digestion, adjusting cell density is 2.7 * 10 7/ ml is respectively with 200ul/ subcutaneous vaccination C57BL/6 mouse.When treating that C57BL/6 tumor-bearing mice diameter of tumor reaches 0.5cm, with medicine tail vein injections such as SM5/FL fusion rotein, 4mg/kg.Observation of tumour size and immunohistochemical methods detection are carried out in treatment 3 weeks continuously.
2. group fractional analysis (HE staining)
Reagent uses 10% neutral formalin.
Take out tumor tissue down with eye scissors, tweezer aseptic condition.Half tumor tissue is fixed 24 hours with 10% neutral formalin.Tumor tissue after fixing is through a series of conventional dehydrations, transparent, waxdip, and last embedding becomes wax stone.Wax stone is cut into slices with slicing machine, and every wax stone is cut 10 10 μ M slabs, and 58 ℃ of roasting sheets after 18 hours, begin dewaxing.Dimethylbenzene I, II, III level each 10 minutes.Dehydrated alcohol, 95% ethanol, 85% ethanol, 75% ethanol respectively 2 minutes.Washed from the beginning 2-3 minute, and carried out HE dyeing.Section is examined under a microscope, count the quantity of various cells, statistical study.
Experimental result sees Table 9:
The experiment grouping The neoplasm necrosis situation Cell quantity (50X)
????NK ????DC ????T ????B
Anti-people CD3 monoclonal antibody/FL ????+ ????- ????+ ????+ ????+
FL ????+ ????++ ????++ ????++ ????++
Chimeric SM ????+++ ????- ????- ????- ????-
Humanization SM ????++++ ????+ ????- ????- ????-
Chimeric SM+FL associating ????+++ ????++ ????++ ????++ ????+
Humanization SM+FL associating ????+++ ????++ ????++ ????+++ ????+
Chimeric SM/FL fusion rotein ????++++ ????++++ ????++++ ????++++ ????++++
Humanization SM/FL fusion rotein ????++++ ????++++ ????++++ ????++++ ????++++
Wherein ,-be 0 cell/mm 2,+be 0-5 cell/mm 2, ++ be 5-10 cell/mm 2, +++be 10-15 cell/mm 2, ++ ++ be＞20 cells/mm 2
The The above results explanation, FL does not have the ability of tangible inducing death of neoplastic cells when using separately, but chimeric uniting when using with the independent use of humanized SM monoclonal antibody or with FL all can be induced tangible death of neoplastic cells.Simultaneously, observe and find, around tumor tissues neutralizes it, have a certain amount of lymphocytic infiltration, the cell type of infiltration to comprise NK, DC and other T cells and B cell.Chimeric or humanization SM/FL fusion rotein then has the more significantly ability of inducing death of neoplastic cells, and its tumor tissues and its have a large amount of lymphocytic infiltrations on every side.The irrelevant anti-CD3 monoclonal antibody/FL of fusion rotein does not have this ability.
The above results explanation, the fusion rotein that two kinds of antibody and FL form have significant guiding DC, NK and other lymphocytes to the concentrated ability of tumor tissues.
Two. the fusion rotein that anti-human Her 2, anti-humen CD 20 and TRAIL and FL form is to the immunohistochemical analysis of tumor tissues
In order further to illustrate the mechanism of action of other fusion roteins, adopt aforementioned same method the fusion rotein of anti-human Her 2, anti-humen CD 20 and TRAIL and FL composition to be carried out the immunohistochemical analysis of tumor tissues.The results are shown in Table 10:
Table 10
The experiment grouping The neoplasm necrosis situation Cell quantity (50X)
????NK ????DC ????T ????B
Anti-people CD3 monoclonal antibody/FL ????+ ????- ????+ ????+ ????+
????FL ????+ ????++ ????++ ????++ ????++
Anti-people HER2 monoclonal antibody ????+++ ????- ????- ????- ????-
Anti-humen CD 20 ????++++ ????+ ????- ????- ????-
????TRAIL ????++ ????++ ????+ ????++ ????+
Anti-human Her 2+FL coupling ????+++ ????++ ????++ ????++ ????+
Anti-humen CD 20+FL coupling ????+++ ????++ ????++ ????+++ ????+
The TRAIL+FL coupling ????++ ????+++ ????+++ ????+++ ????+++
Anti-human Her 2/FL fusion rotein ????++++ ????+++++ ????++++ ????++++ ????++++
Anti-humen CD 20/FL fusion rotein ????+++++ ????++++ ????++++ ????++++ ????++++
The TRAIL/FL fusion rotein ????++++ ????++++ ????++++ ????+++ ????++++
Wherein ,-be 0 cell/mm 2,+be 0-5 cell/mm 2, ++ be 5-10 cell/mm 2, +++be 10-15 cell/mm 2, ++ ++ be＞20 cells/mm 2
The test-results of The above results and aforementioned SM, hSM is in full accord, the fusion rotein that SM, hSM, IDEC-C2B8, anti-Her2 monoclonal antibody and TRAIL is described and forms with FL can both be by a large amount of killing tumor cells of approach of immune cell activated, after administration, it can bring into play very big activity, take NK and DC cell to tumor tissues, thereby reach the effect of treatment tumour.
Embodiment 15
Experiment distributes in the body of fusion rotein
In order to study chimeric whether can combining with tumour to specificity with the fusion rotein that FL constitutes with humanized SM5-1, we have studied fusion rotein distribution in vivo.
With chSM, chSM/FL, huSM and the huSM/FL fusion rotein difference tail vein injection tumor-bearing mice of labelled with radioisotope, get its various tissues after 48 hours, measure its radioactive intensity.Result such as Figure 56.
Result displayed explanation among the figure, the tissue distribution of the corresponding chimeric monoclonal antibody SM with hSM/FL fusion rotein and its of SM/FL, humanization monoclonal antibody is consistent, and they have all inherited the specificity of its corresponding monoclonal antibody, and all high concentration is distributed in tumor tissues.
This tissue distribution of above-mentioned monoclonal antibody and fusion rotein is that the specificity of this tissue distribution is significant on clinical treatment by the decision of the specificity of monoclonal antibody.On the one hand, this specific tissue distributes dosage is obviously reduced, and in addition, less medication and drug specificity are distributed in target tissue, have just reduced the damage to non-target tissue.
Employing is as above-mentioned method, distributes in the body of totally three kinds of fusion roteins that anti-human Her 2 monoclonal antibody, anti-humen CD 20 monoclonal antibody and TRAIL and FL are formed and studies.Experimental result is shown in Figure 57.
As the aforementioned result who sees, the fusion rotein that anti-people HER2, anti-humen CD 20 and three kinds of molecules of TRAIL and they and FL form all concentrates on tumor tissues in a large number, and the distribution in its hetero-organization is relatively low.This species specific distribution situation is identical with the situation of the fusion rotein that chSM/FL, huSM/FL form.
Embodiment 16
The lymphocyte infusion experiment
By the experimental program among the embodiment 1 B16/P230 and Hepal-6/P230 tumor-bearing mice are treated with chSM/FL or huSM/FL, treat that the tumor-bearing mice tumour disappears after, kill animals is taken out spleen, the ordinary method separating Morr. cell.Then, adjust cell concn and reach 1 * 10 9/ ml.The spleen cell for preparing is defeated by the mouse that did not inoculate tumour, every mouse infusion 5 * 10 7Cell, and then inoculation Hepap230 or B16p230 cell, the survival condition of 6 weeks back observation mouse.Experimental result sees Table 11.
The experiment of table 11 lymphocyte infusion
The splenocyte donor Acceptor mouse quantity Inoculate the death condition of the mouse of tumour cell behind the infusion
????Hepap230 ????B16p230
Anti-CD3 monoclonal antibody/FL ????15 ????15/15 ????15/15
????FL ????15 ????9/15 ????10/15
The SM monoclonal antibody ????15 ????12/15 ????14/15
The hSM monoclonal antibody ????15 ????13/15 ????14/15
SM monoclonal antibody+FL ????15 ????10/15 ????10/15
HSM monoclonal antibody+FL ????15 ????10/15 ????10/15
SM monoclonal antibody/FL fusion rotein ????15 ????0/15 ????1/15
HSM monoclonal antibody/FL fusion rotein ????15 ????1/15 ????0/15
The The above results explanation, passive infusion induces the splenocyte of the mouse of generation active immunity ability can make the attack that is tried mouse opposing institute inoculated tumour through chSM monoclonal antibody/FL fusion rotein and huSM monoclonal antibody/FL fusion rotein.The anti-CD3 monoclonal antibody/FL of fusion rotein of irrelevant antibody and use monoclonal antibody and FL all can not make the lymphocyte of infusion have antitumor action separately.This explanation, the lymphocyte of infusion has specific antitumor action really.
The above results also illustrates, the mechanism of SM monoclonal antibody/FL fusion rotein and hSM monoclonal antibody/FL fusion rotein antitumor action is that they are induced and has produced narrow spectrum antitumor response to active immunization.
Adopt above-mentioned identical method, by the experimental program among the embodiment 1 lotus is had 4T1/her2, A20/CD20; The mouse of Renca is treated with her2/FL, CD20/FL or Trail/FL respectively, treat that the tumor-bearing mice tumour disappears after, kill animals is taken out spleen, the ordinary method separating Morr. cell.Then, adjust cell concn and reach 1 * 10 9/ ml.The spleen cell for preparing is defeated by the mouse that did not inoculate tumour, every mouse infusion 5 * 10 7Cell, and then inoculate 4T1/her2, A20/CD20 respectively; The Renca cell, the survival condition of 6 weeks back observation mouse.Experimental result sees Table 12.
The experiment of table 12 lymphocyte infusion
The splenocyte donor Acceptor mouse quantity The dead mouse situation of inoculated tumour cell behind the infusion
Key cells is *
Anti-CD3 monoclonal antibody/FL ??15 ????15/15
????FL ??15 ????9/15
The anti-human Her 2 monoclonal antibody ??15 ????11/15
Anti-people Her monoclonal antibody+FL coupling ??15 ????9/15
The HER2/FL fusion rotein ??15 ????4/15
The anti-humen CD 20 monoclonal antibody ??15 ????13/15
Anti-humen CD 20 monoclonal antibody+FL coupling ??15 ????10/15
The CD20/FL fusion rotein ??15 ????2/15
????TRAIL ??15 ????8/15
The TRAIL+FL coupling ??15 ????10/15
The TRAIL/FL fusion rotein ??15 ????5/15
* testing used key cells is: Her related experiment 4T1/her2; CD20 related experiment A20/CD20; TRAIL related experiment Renca.
The test-results of The above results and aforesaid chSM/FL, huSM/FL is in full accord.Illustrate that fusion rotein that SM, huSM, IDEC-C2B8, anti-Her2 monoclonal antibody and TRAIL and FL form all is that approach by activated lymphocyte reaches the killing tumor cell effect, and this lethal effect has specificity, to the not effect of irrelevant clone.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Sequence table
＜110〉horse, cyanines
＜120〉has the bifunctional fusion proteins and its production and application of anti-tumor function
<130>037089
<150>CN?03129290.9
<151>2003-06-13
<160>68
<170>PatentIn?version?3.1
<210>1
<211>546
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<220>1
atgacagtgc?tggcgccagc?ctggagccca?acaacctatc?tcctcctgct?gctgctgctg?????60
agctcgggac?tcagtgggac?ccaggactgc?tccttccaac?acagccccat?ctcctccgac????120
ttcgctgtca?aaatccgtga?gctgtctgac?tacctgcttc?aagattaccc?agtcaccgtg????180
gcctccaacc?tgcaggacga?ggagctctgc?gggggcctct?ggcggctggt?cctggcacag????240
cgctggatgg?agcggctcaa?gactgtcgct?gggtccaaga?tgcaaggctt?gctggagcgc????300
gtgaacacgg?agatacactt?tgtcaccaaa?tgtgcctttc?agcccccccc?cagctgtctt????360
cgcttcgtcc?agaccaacat?ctcccgcctc?ctgcaggaga?cctccgagca?gctggtggcg????420
ctgaagccct?ggatcactcg?ccagaacttc?tcccggtgcc?tggagctgca?gtgtcagccc????480
gactcctcaa?ccctgccacc?cccatggagt?ccccggcccc?tggaggccac?agccccgaca????540
gccccg???????????????????????????????????????????????????????????????546
<210>2
<211>182
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<220>2
Met?Thr?Val?Leu?Ala?Pro?Ala?Trp?Ser?Pro?Thr?Thr?Tyr?Leu?Leu?Leu
1???????????????5???????????????????10??????????????????15
Leu?Leu?Leu?Leu?Ser?Ser?Gly?Leu?Ser?Gly?Thr?Gln?Asp?Cys?Ser?Phe
20??????????????????25??????????????????30
Gln?His?Ser?Pro?Ile?Ser?Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu
35??????????????????40??????????????????45
Ser?Asp?Tyr?Leu?Leu?Gln?Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu
50??????????????????55??????????????????60
Gln?Asp?Glu?Glu?Leu?Cys?Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln
65??????????????????70??????????????????75??????????????????80
Arg?Trp?Met?Glu?Arg?Leu?Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly
85??????????????????90??????????????????95
Leu?Leu?Glu?Arg?Val?Asn?Thr?Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala
100?????????????????105?????????????????110
Phe?Gln?Pro?Pro?Pro?Ser?Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser
115?????????????????120?????????????????125
Arg?Leu?Leu?Gln?Glu?Thr?Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp
130?????????????????135?????????????????140
Ile?Thr?Arg?Gln?Asn?Phe?Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro
145?????????????????150?????????????????155?????????????????160
Asp?Ser?Ser?Thr?Leu?Pro?Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala
165?????????????????170?????????????????175
Thr?Ala?Pro?Thr?Ala?Pro
180
<210>3
<211>1242
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(1242)
＜223〉anti-SM5-1 antibody nucleotide sequence
<220>3
atgacagtgc?tggcgccagc?ctggagccca?acaacctatc?tcctcctgct?gctgctgctg?????60
agctcgggac?tcagtgggac?ccaggactgc?tccttccaac?acagccccat?ctcctccgac????120
ttcgctgtca?aaatccgtga?gctgtctgac?tacctgcttc?aagattaccc?agtcaccgtg????180
gcctccaacc?tgcaggacga?ggagctctgc?gggggcctct?ggcggctggt?cctggcacag????240
cgctggatgg?agcggctcaa?gactgtcgct?gggtccaaga?tgcaaggctt?gctggagcgc????300
gtgaacacgg?agatacactt?tgtcaccaaa?tgtgcctttc?agcccccccc?cagctgtctt????360
cgcttcgtcc?agaccaacat?ctcccgcctc?ctgcaggaga?cctccgagca?gctggtggcg????420
ctgaagccct?ggatcactcg?ccagaacttc?tcccggtgcc?tggagctgca?gtgtcagccc????480
gactcctcaa?ccctgccacc?cccatggagt?ccccggcccc?tggaggccac?agccccgaca????540
gccccggagc?ccaaatcttg?tgacaaaact?cacacatgcc?caccgtgccc?agcacctgaa????600
ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac?ccaaggacac?cctcatgatc????660
tcccggaccc?ctgaggtcac?atgcgtggtg?gtggacgtga?gccacgaaga?ccctgaggtc????720
aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg?ccaagacaaa?gccgcgggag????780
gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca?ccgtcctgca?ccaggactgg?????840
ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag?ccctcccagc?ccccatcgag?????900
aaaaccatct?ccaaagccaa?agggcagccc?cgagaaccac?aggtgtacac?cctgccccca?????960
tcccgggatg?agctgaccaa?gaaccaggtc?agcctgacct?gcctggtcaa?aggcttctat????1020
cccagcgaca?tcgccgtgga?gtgggagagc?aatgggcagc?cggagaacaa?ctacaagacc????1080
acgcctcccg?tgctggactc?cgacggctcc?ttcttcctct?acagcaagct?caccgtggac????1140
aagagcaggt?ggcagcaggg?gaacgtcttc?tcatgctccg?tgatgcatga?ggctctgcac????1200
aaccactaca?cgcagaagag?cctctccctg?tctcccggta?aa???????????????????????1242
<210>4
<211>414
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(414)
＜223〉anti-SM5-1 antibody
<220>4
Met?Thr?Val?Leu?Ala?Pro?Ala?Trp?Ser?Pro?Thr?Thr?Tyr?Leu?Leu?Leu
1???????????????5???????????????????10??????????????????15
Leu?Leu?Leu?Leu?Ser?Ser?Gly?Leu?Ser?Gly?Thr?Gln?Asp?Cys?Ser?Phe
20??????????????????25??????????????????30
Gln?His?Ser?Pro?Ile?Ser?Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu
35??????????????????40??????????????????45
Ser?Asp?Tyr?Leu?Leu?Gln?Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu
50??????????????????55??????????????????60
Gln?Asp?Glu?Glu?Leu?Cys?Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln
65??????????????????70??????????????????75??????????????????80
Arg?Trp?Met?Glu?Arg?Leu?Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly
85??????????????????90??????????????????95
Leu?Leu?Glu?Arg?Val?Asn?Thr?Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala
100?????????????????105?????????????????110
Phe?Gln?Pro?Pro?Pro?Ser?Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser
115?????????????????120?????????????????125
Arg?Leu?Leu?Gln?Glu?Thr?Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp
130?????????????????135?????????????????140
Ile?Thr?Arg?Gln?Asn?Phe?Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro
145?????????????????150?????????????????155?????????????????160
Asp?Ser?Ser?Thr?Leu?Pro?Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala
165?????????????????170?????????????????175
Thr?Ala?Pro?Thr?Ala?Pro?Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr
180?????????????????185?????????????????190
Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe
195?????????????????200?????????????????205
Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro
210?????????????????215?????????????????220
Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val
225?????????????????230?????????????????235?????????????????240
Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr
245?????????????????250?????????????????255
Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val
260?????????????????265?????????????????270
Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys
275?????????????????280?????????????????285
Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser
290?????????????????295?????????????????300
Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro
305?????????????????310?????????????????315?????????????????320
Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val
325?????????????????330?????????????????335
Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly
340?????????????????345?????????????????350
Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp
355?????????????????360?????????????????365
Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp
370?????????????????375?????????????????380
Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His
385?????????????????390?????????????????395?????????????????400
Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys
405?????????????????410
<210>5
<211>45
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(45)
＜223〉manual splice
<220>5
ggcggtggag?gctctggtgg?aggcggttca?ggaggcggtg?gatct?????????????????????45
<210>6
<211>15
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(15)
＜223〉manual splice encoding sequence
<220>6
Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
1???????????????5???????????????????10??????????????????15
<210>7
<211>426
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(426)
＜223〉anti-SM5-1 antibody nucleotide sequence
<220>7
atcgccgcca?ccatggaatg?gagttggata?tttctctttc?tcctgtcagg?aactgcaggt?????60
gtccactctg?aggtccagct?gcagcagtct?ggacctgagc?tggtaaagcc?tggggcttca????120
gtgaagatgt?cctgcaaggc?ttctggatac?acattcacta?gctatgttat?gcactgggtg????180
aagcagaagc?ctgggcaggg?ccttgactgg?attggatata?ttgttcctta?caatgatggc????240
actaagtaca?atgagaagtt?caaaggcaag?gccacactga?cttcaggcaa?atcctccagc????300
acagcctaca?tggagctcag?cagactgacc?tctgaggact?ctgcggtcta?ttattgtgtc????360
tacggtagta?ggtacgactg?gtatttagat?gtctggggcg?cagggaccac?ggtcaccgtc????420
tcctca???????????????????????????????????????????????????????????????426
<210>8
<211>138
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(138)
＜223〉anti-SM5-1 antibody
<220>8
Met?Glu?Trp?Ser?Trp?Ile?Phe?Leu?Phe?Leu?Leu?Ser?Gly?Thr?Ala?Gly
1???????????????5???????????????????10??????????????????15
Val?His?Ser?Glu?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?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?Val?Met?His?Trp?Val?Lys?Gln?Lys?Pro?Gly?Gln?Gly?Leu
50??????????????????55??????????????????60
Asp?Trp?Ile?Gly?Tyr?Ile?Val?Pro?Tyr?Asn?Asp?Gly?Thr?Lys?Tyr?Asn
65??????????????????70??????????????????75??????????????????80
Glu?Lys?Phe?Lys?Gly?Lys?Ala?Thr?Leu?Thr?Ser?Asp?Lys?Ser?Ser?Ser
85??????????????????90??????????????????95
Thr?Ala?Tyr?Met?Glu?Leu?Ser?Arg?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val
100?????????????????105?????????????????110
Tyr?Tyr?Cys?Val?Tyr?Gly?Ser?Arg?Tyr?Asp?Trp?Tyr?Leu?Asp?Val?Trp
115?????????????????120?????????????????125
Gly?Ala?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser
130?????????????????135
<210>9
<211>465
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(465)
＜223〉anti-SM5-1 antibody nucleotide sequence
<220>9
atcatcacca?gaacagctta?cgagcagacc?gccagacagc?tcacagggat?caagcttgcc?????60
gccaccatgg?aatcacagac?tcaggtcttc?ctctccctgc?tgctctgggt?atctggtacc????120
tgtgggaaca?ttatgatgac?acagtcgcca?tcatctctgg?ctgtgtctgc?aggagaaaag????180
gtcactatga?gctgtaagtc?cagtcaaagt?gttttataca?gttcaaatca?gaagaactac????240
ttggcctggt?accagcagaa?accagggcag?tctcctaaac?tgctgatcta?ctgggcatcc????300
actagggaat?ctggtgtccc?tgatcgcttc?acaggcagtg?gatctgggac?agattttact????360
cttaccatca?gcagtgtaca?agctgaagac?ctggcagttt?attactgtca?tcaatatttc????420
tcctcataca?cgttcggagg?ggggaccaag?ctggaaataa?agcgg????????????????????465
<210>10
<211>133
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(133)
＜223〉anti-SM5-1 antibody
<220>10
Met?Glu?Ser?Gln?Thr?Gln?Val?Phe?Leu?Ser?Leu?Leu?Leu?Trp?Val?Ser
1???????????????5???????????????????10??????????????????15
Gly?Thr?Cys?Gly?Asn?Ile?Met?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ala
20??????????????????25??????????????????30
Val?Ser?Ala?Gly?Glu?Lys?Val?Thr?Met?Ser?Cys?Lys?Ser?Ser?Gln?Ser
35??????????????????40??????????????????45
Val?Leu?Tyr?Ser?Ser?Asn?Gln?Lys?Asn?Tyr?Leu?Ala?Trp?Tyr?Gln?Gln
50??????????????????55??????????????????60
Lys?Pro?Gly?Gln?Ser?Pro?Lys?Leu?Leu?Ile?Tyr?Trp?Ala?Ser?Thr?Arg
65??????????????????70??????????????????75??????????????????80
Glu?Ser?Gly?Val?Pro?Asp?Arg?Phe?Thr?Gly?Ser?Gly?Ser?Gly?Thr?Asp
85??????????????????90??????????????????95
Phe?Thr?Leu?Thr?Ile?Ser?Ser?Val?Gln?Ala?Glu?Asp?Leu?Ala?Val?Tyr
100?????????????????105?????????????????110
Tyr?Cys?His?Gln?Tyr?Phe?Ser?Ser?Tyr?Thr?Phe?Gly?Gly?Gly?Thr?Lys
115?????????????????120?????????????????125
Leu?Glu?Ile?Lys?Arg
130
<210>11
<211>2021
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(2021)
＜223〉anti-SM5-1 antibody nucleotide sequence
<220>11
atcgccgcca?ccatggaatg?gagttggata?tttctctttc?tcctgtcagg?aactgcaggt??????60
gtccactctg?aggtccagct?gcagcagtct?ggacctgagc?tggtaaagcc?tggggcttca?????120
gtgaagatgt?cctgcaaggc?ttctggatac?acattcacta?gctatgttat?gcactgggtg?????180
aagcagaagc?ctgggcaggg?ccttgactgg?attggatata?ttgttcctta?caatgatggc?????240
actaagtaca?atgagaagtt?caaaggcaag?gccacactga?cttcagacaa?atcctccagc?????300
acagcctaca?tggagctcag?cagactgacc?tctgaggact?ctgcggtcta?ttattgtgtc?????360
tacggtagta?ggtacgactg?gtatttagat?gtctggggcg?cagggaccac?ggtcaccgtc?????420
tcctcagcta?gcaccaaggg?cccatcggtc?ttccccctgg?caccctcctc?caagagcacc?????480
tctgggggca?cagcggccct?gggctgcctg?gtcaaggact?acttccccga?accggtgacg?????540
gtgtcttgga?actcaggcgc?cctgaccagc?ggcgtgcaca?ccttcccggc?tgtcctacag?????600
tcctcaggac?tctactccct?cagcagcgtg?gtgaccgtgc?cctccagcag?cttgggcacc?????660
cagacctaca?tctgcaacgt?gaatcacaag?cccagcaaca?ccaaggtgga?caagaaagtt?????720
ggtgagaggc?cagcacaggg?agggagggtg?tctgctggaa?gcaggctcag?cgctcctgcc?????780
tggacgcatc?ccggctatgc?agccccagtc?cagggcagca?aggcaggccc?cgtctgcctc?????840
ttcacccgga?gcctctgccc?gccccactca?tgctcaggga?gagggtcttc?tggctttttc?????900
ccaggctctg?ggcaggcaca?ggctaggtgc?ccctaaccca?ggccctgcac?acaaaggggc?????960
aggtgctggg?ctcagacctg?ccaagagcca?tatccgggag?gaccctgccc?ctgacctaag????1020
cccaccccaa?aggccaaact?ctccactccc?tcagctcgga?caccttctct?cctcccagat????1080
tccagtaact?cccaatcttc?tctctgcaga?gcccaaatct?tgtgacaaaa?ctcacacatg????1140
cccaccgtgc?ccaggtaagc?cagcccaggc?ctcgccctcc?agctcaaggc?gggacaggtg????1200
ccctagagta?gcctgcatcc?agggacaggc?cccagccggg?tgctgacacg?tccacctcca????1260
tctcttcctc?agcacctgaa?ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac????1320
ccaaggacac?cctcatgatc?tcccggaccc?ctgaggtcac?atgcgtggtg?gtggacgtga????1380
gccacgaaga?ccctgaggtc?aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg????1440
ccaagacaaa?gccgcgggag?gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca????1500
ccgtcctgca?ccaggactgg?ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag????1560
ccctcccagc?ccccatcgag?aaaaccatct?ccaaagccaa?aggtgggacc?cgtggggtgc????1620
gagggccaca?tggacagagg?ccggctcggc?ccaccctctg?ccctgagagt?gaccgctgta????1680
ccaacctctg?tcctacaggg?cagccccgag?aaccacaggt?gtacaccctg?cccccatccc????1740
gggatgagct?gaccaagaac?caggtcagcc?tgacctgcct?ggtcaaaggc?ttctatccca????1800
gcgacatcgc?cgtggagtgg?gagagcaatg?ggcagccgga?gaacaactac?aagaccacgc????1860
ctcccgtgct?ggactccgac?ggctccttct?tcctctacag?caagctcacc?gtggacaaga????1920
gcaggtggca?gcaggggaac?gtcttctcat?gctccgtgat?gcatgaggct?ctgcacaacc????1980
actacacgca?gaagagcctc?tccctgtctc?ccggtaaatg?a????????????????????????2021
<210>12
<211>468
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(468)
＜223〉anti-SM5-1 antibody
<220>12
Met?Glu?Trp?Ser?Trp?Ile?Phe?Leu?Phe?Leu?Leu?Ser?Gly?Thr?Ala?Gly
1???????????????5???????????????????10??????????????????15
Val?His?Ser?Glu?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?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?Val?Met?His?Trp?Val?Lys?Gln?Lys?Pro?Gly?Gln?Gly?Leu
50??????????????????55??????????????????60
Asp?Trp?Ile?Gly?Tyr?Ile?Val?Pro?Tyr?Asn?Asp?Gly?Thr?Lys?Tyr?Asn
65??????????????????70??????????????????75??????????????????80
Glu?Lys?Phe?Lys?Gly?Lys?Ala?Thr?Leu?Thr?Ser?Asp?Lys?Ser?Ser?Ser
85??????????????????90??????????????????95
Thr?Ala?Tyr?Met?Glu?Leu?Ser?Arg?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val
100?????????????????105?????????????????110
Tyr?Tyr?Cys?Val?Tyr?Gly?Ser?Arg?Tyr?Asp?Trp?Tyr?Leu?Asp?Val?Trp
115?????????????????120?????????????????125
Gly?Ala?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro
130?????????????????135?????????????????140
Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr
145?????????????????150?????????????????155?????????????????160
Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr
165?????????????????170?????????????????175
Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro
180?????????????????185?????????????????190
Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr
195?????????????????200?????????????????205
Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn
210?????????????????215?????????????????220
His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser
225?????????????????230?????????????????235?????????????????240
Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu
245?????????????????250?????????????????255
Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu
260?????????????????265?????????????????270
Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser
275?????????????????200?????????????????285
His?Glu?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Asp?Gly?Val?Glu
290?????????????????295?????????????????300
Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr
305?????????????????310?????????????????315?????????????????320
Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn
325?????????????????330?????????????????335
Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro
340?????????????????345?????????????????350
Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln
355?????????????????360?????????????????365
Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val
370?????????????????375?????????????????380
Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val
385?????????????????390?????????????????395????????????????400
Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro
405?????????????????410?????????????????415
Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr
420?????????????????425?????????????????430
Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val
435?????????????????440?????????????????445
Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu
450?????????????????455?????????????????460
Ser?Pro?Gly?Lys
465
<210>13
<211>786
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(766)
＜223〉anti-SM5-1 antibody nucleotide sequence
<220>13
atcatcacca?gaacagctta?cgagcagacc?gccagacagc?tcacagggat?caagcttgcc?????60
gccaccatgg?aatcacagac?tcaggtcttc?ctctccctgc?tgctctgggt?atctggtacc????120
tgtgggaaca?ttatgatgac?acagtcgcca?tcatctctgg?ctgtgtctgc?aggagaaaag????180
gtcactatga?gctgtaagtc?cagtcaaagt?gttttataca?gttcaaatca?gaagaactac????240
ttggcctggt?accagcagaa?accagggcag?tctcctaaac?tgctgatcta?ctgggcatcc????300
actagggaat?ctggtgtccc?tggtcgcttc?acaggcagtg?gatctgggac?agattttact????360
cttaccatca?gcagtgtaca?agctgaagac?ctggcagttt?attactgtca?tcaatatttc????420
tcctcataca?cgttcggagg?ggggaccaag?ctggaaataa?agcggactgt?ggctgcacca????480
tctgtcttca?tcttcccgcc?atcttatgag?cagttgaaat?ctggaactgc?ctctgttgtg????540
tgcctgctga?ataacttcta?tcccagagag?gccaaagtac?agtggaaggt?ggataacgcc????600
ctccaatcgg?gtaactccca?ggagagtgtc?acagagcagg?acagcaagga?cagcacctac????660
agcctcagca?gcaccctgac?gctgagcaaa?gcagactacg?agaaacacaa?agtctacgcc????720
tgcgaagtca?cccatcaggg?cctgagctcg?cccgtcacaa?agagcttcaa?caggggagag????780
tgttag???????????????????????????????????????????????????????????????786
<210>14
<211>239
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(239)
＜223〉anti-SM5-1 antibody
<220>14
Met?Glu?Ser?Gln?Thr?Gln?Val?Phe?Leu?Ser?Leu?Leu?Leu?Trp?Val?Ser
1???????????????5???????????????????10??????????????????15
Gly?Thr?Cys?Gly?Asn?Ile?Met?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ala
20??????????????????25??????????????????30
Val?Ser?Ala?Gly?Glu?Lys?Val?Thr?Met?Ser?Cys?Lys?Ser?Ser?Gln?Ser
35??????????????????40??????????????????45
Val?Leu?Tyr?Ser?Ser?Asn?Gln?Lys?Asn?Tyr?Leu?Ala?Trp?Tyr?Gln?Gln
50??????????????????55??????????????????60
Lys?Pro?Gly?Gln?Ser?Pro?Lys?Leu?Leu?Ile?Tyr?Trp?Ala?Ser?Thr?Arg
65??????????????????70??????????????????75??????????????????80
Glu?Set?Gly?Val?Pro?Asp?Arg?Phe?Thr?Gly?Ser?Gly?Ser?Gly?Thr?Asp
85???????????????????90?????????????????95
Phe?Thr?Leu?Thr?Ile?Ser?Ser?Val?Gln?Ala?Glu?Asp?Leu?Ala?Val?Tyr
100?????????????????105?????????????????110
Tyr?Cys?His?Gln?Tyr?Phe?Ser?Ser?Tyr?Thr?Phe?Gly?Gly?Gly?Thr?Lys
115?????????????????120?????????????????125
Leu?Glu?Ile?Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro
130?????????????????135?????????????????140
Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu
145?????????????????150?????????????????155?????????????????160
Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp
165?????????????????170?????????????????175
Asn?Ala?Leu?Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp
180?????????????????185?????????????????190
Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys
195?????????????????200?????????????????205
Ala?Asp?Tyr?Glu?Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln
210?????????????????215?????????????????220
Gly?Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys
225?????????????????230?????????????????235
<210>15
<211>426
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(426)
＜223〉anti-SM5-1 antibody nucleotide sequence
<220>15
agagccgcca?ccatggattg?ggtgtggacc?ttgctattcc?tgttgtcagt?aactgcaggt?????60
gtccactccc?aggtgcagct?ggtgcagtct?ggcggtggag?tggtccagcc?cggccgcagc????120
ctgaggctgt?cctgcaaggc?atctggctac?accttcacca?gctacgtgat?gacatgggtg????180
cgccaagccc?ccggaaaggg?cctcgaatgg?attggctaca?ttgtgcctta?taatgacggt????240
actaagtaca?atgaaaagtt?caagggcaga?tttacaatat?caagtgacaa?gagcaagtca????300
accgcattcc?tccaaatgga?cagcttgcgt?ccagaggaca?ccgccgtata?ctattgtgtg????360
cgcggcagcc?gttacgactg?gtacttggac?tactggggcc?aaggcactcc?agtcaccgtc????420
tcctct???????????????????????????????????????????????????????????????426
<210>16
<211>138
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(138)
＜223〉anti-SM5-1 antibody
<220>16
Met?Asp?Trp?Val?Trp?Thr?Leu?Leu?Phe?Leu?Leu?Ser?Val?Thr?Ala?Gly
1???????????????5???????????????????10??????????????????15
Val?His?Ser?Gln?Val?Gln?Leu?Val?Gln?Ser?Gly?Gly?Gly?Val?Val?Gln
20??????????????????25??????????????????30
Pro?Gly?Arg?Ser?Leu?Arg?Leu?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe
35??????????????????40??????????????????45
Thr?Ser?Tyr?Val?Met?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu
50??????????????????55??????????????????60
Glu?Trp?Ile?Gly?Tyr?Ile?Val?Pro?Tyr?Asn?Asp?Gly?Thr?Lys?Tyr?Asn
65??????????????????70??????????????????75??????????????????80
Glu?Lys?Phe?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Ser?Asp?Lys?Ser?Lys?Ser
85??????????????????90??????????????????95
Thr?Ala?Phe?Leu?Gln?Met?Asp?Ser?Leu?Arg?Pro?Glu?Asp?Thr?Ala?Val
100?????????????????105?????????????????110
Tyr?Tyr?Cys?Ala?Arg?Gly?Ser?Arg?Tyr?Asp?Trp?Tyr?Leu?Asp?Tyr?Trp
115?????????????????120?????????????????125
Gly?Gln?Gly?Thr?Pro?Val?Thr?Val?Ser?Ser
130?????????????????135
<210>17
<211>465
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(465)
＜223〉anti-SM5-1 antibody nucleotide sequence
<220>17
gagcattacc?ggccatactc?accaccatcc?caggatatct?ctagaaagct?tgccgccacc?????60
atggattttc?aagtgcagat?tttcagcttc?ctgctaatca?gtgcttcagt?cataatgtcc????120
agaggaaaca?tcatgatgac?tcagagccca?tccagcttga?gcgcatcagt?aggcgaccgc????180
gtaacgatca?cttgcaaatc?ctctcagtca?gtattgtact?ccagcaacca?gaagaactac????240
ctggccggat?atcagcagac?tcccggcaaa?gccccaaagt?tgctgattta?ttgggcctcc????300
acgcgcgagt?ctggcgtgcc?atcacgcttt?agcggcagcg?ggtccggtac?agattacacg????360
tttaccatta?gcagtctgca?gcctgaggac?atagccacct?actactgtca?ccagtacttt????420
agttcctaca?cttttggcca?gggaactaaa?ctgcagatta?ctcga????????????????????465
<210>18
<211>135
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(135)
＜223〉anti-SM5-1 antibody
<220>18
Met?Asp?Phe?Gln?Val?Gln?Ile?Phe?Ser?Phe?Leu?Leu?Ile?Ser?Ala?Ser
1???????????????5???????????????????10??????????????????15
Val?Ile?Met?Ser?Arg?Gly?Asn?Ile?Met?Met?Thr?Gln?Ser?Pro?Ser?Ser
20??????????????????25??????????????????30
Leu?Ser?Ala?Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Lys?Ser?Ser
35??????????????????40??????????????????45
Gln?Ser?Val?Leu?Tyr?Ser?Ser?Asn?Gln?Lys?Asn?Tyr?Leu?Ala?Trp?Tyr
50??????????????????55??????????????????60
Gln?Gln?Thr?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Trp?Ala?Ser
65??????????????????70??????????????????75??????????????????80
Thr?Arg?Glu?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly
85??????????????????90??????????????????95
Thr?Asp?Tyr?Thr?Phe?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?Ile?Ala
100?????????????????105?????????????????110
Thr?Tyr?Tyr?Cys?His?Gln?Tyr?Phe?Ser?Ser?Tyr?Thr?Phe?Gly?Gln?Gly
115?????????????????120?????????????????125
Thr?Lys?Leu?Gln?Ile?Thr?Arg
130?????????????????135
<210>19
<211>2021
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(2021)
＜223〉anti-SM5-1 antibody nucleotide sequence
<220>19
agagccgcca?ccatggattg?ggtgtggacc?ttgctattcc?tgttgtcagt?aactgcaggt?????60
gtccactccc?aggtgcagct?ggtgcagtct?ggcggtggag?tggtccagcc?cggccgcagc????120
ctgaggctgt?cctgcaaggc?atctggctac?accttcacca?gctacgtgat?gacatgggtg????180
cgccaagccc?ccggaaaggg?cctcgaatgg?attggctaca?ttgtgcctta?taatgacggt????240
actaagtaca?atgaaaagtt?caagggcaga?tttacaatat?caagtgacaa?gagcaagtca????300
accgcattcc?tccaaatgga?cagcttgcgt?ccagaggaca?ccgccgtata?ctattgtgtg????360
cgcggcagcc?gttacgactg?gtacttggac?tactggggcc?aaggcactcc?agtcaccgtc????420
tcctctgcta?gcaccaaggg?cccatcggtc?ttccccctgg?caccctcctc?caagagcacc????480
tctgggggca?cagcggccct?gggctgcctg?gtcaaggact?acttccccga?accggtgacg????540
gtgtctggga?actcaggcac?cctgaccagc?ggcatgcaca?ccttcccggc?tgtcctacag????600
tcctcaggac?tctactccct?cagcagcgtg?gtgaccgtgc?cctccagcag?cttgggcacc????660
cagacctaca?tctgcaacgt?gaatcacaag?cccagcaaca?ccaaggtgga?caagaaagtt????720
ggtgagaggc?cagcacaggg?agggagggtg?tctgctggaa?gcaggctcag?cgctcctgcc????780
tggacgcatc?ccggctatgc?agccccagtc?cagggcagca?aggcaggccc?cgtctgcctc?????840
ttcacccgga?gcctctgccc?gccccactca?tgctcaggga?gagggtcttc?tggctttttc?????900
ccaggctctg?ggcaggcaca?ggctaggtgc?ccctaaccca?ggccctgcac?acaaaggggc?????960
aggtgctggg?ctcagacctg?ccaagagcca?tatccgggag?gaccctgccc?ctgacctaag????1020
cccaccccaa?aggccaaact?ctccactccc?tcagctcgga?caccttctct?cctcccagar????1080
tccagtaact?cccaatcttc?tctctgcaga?gcccaaatct?tgtgacaaaa?ctcacacatg????1140
cccaccgtgc?ccaggtaagc?cagcccaggc?ctcgccctcc?agctcaaggc?gggacaggtg????1200
ccctagagta?gcctgcatcc?agggacaggc?cccagccggg?tgctgacacg?tccacctcca????1260
tctcttcctc?agcacctgaa?ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac????1320
ccaaggacac?cctcatgatc?tcccggaccc?ctgaggtcac?atgcgtggtg?gtggacgtga????1380
gccacgaaga?ccctgaggtc?aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg????1440
ccaagacaaa?gccgcgggag?gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca????1500
ccgtcctgca?ccaggactgg?ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag????1560
ccctcccagc?ccccatcgag?aaaaccatct?ccaaagccaa?aggtgggacc?cgtggggtgc????1620
gagggccaca?tggacagagg?ccggctcggc?ccaccctctg?ccctgagagt?gaccgctgta????1680
ccaacctctg?tcctacaggg?cagccccgag?aaccacaggt?gtacaccctg?cccccatccc????1740
gggatgagct?gaccaagaac?caggtcagcc?tgacctgcct?ggtcaaaggc?ttctatccca????1800
gcgacatcgc?cgtggagtgg?gagagcaatg?ggcagccgga?gaacaactac?aagaccacgc????1860
ctcccgtgct?ggactccgac?ggctccttct?tcctctacag?caagctcacc?gtggacaaga????1920
gcaggtggca?gcaggggaac?gtcttctcat?gctccgtgat?gcatgaggct?ctgcacaacc????1980
actacacgca?gaagagcctc?tccctgtctc?ccggtaaatg?a????????????????????????2021
<210>20
<211>468
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(468)
＜223〉anti-SM5-1 antibody
<220>20
Met?Asp?Trp?Val?Trp?Thr?Leu?Leu?Phe?Leu?Leu?Ser?Val?Thr?Ala?Gly
1???????????????5???????????????????10??????????????????15
Val?His?Ser?Gln?Val?Gln?Leu?Val?Gln?Ser?Gly?Gly?Gly?Val?Val?Gln
20??????????????????25??????????????????30
Pro?Gly?Arg?Ser?Leu?Arg?Leu?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe
35??????????????????40??????????????????45
Thr?Ser?Tyr?Val?Met?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu
50??????????????????55??????????????????60
Glu?Trp?Ile?Gly?Tyr?Ile?Val?Pro?Tyr?Asn?Asn?Gly?Thr?Lys?Tyr?Asn
65??????????????????70??????????????????75??????????????????80
Glu?Lys?Phe?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Ser?Asp?Lys?Ser?Lys?Ser
85??????????????????90??????????????????95
Thr?Ala?Phe?Leu?Gln?Met?Asp?Ser?Leu?Arg?Pro?Glu?Asp?Thr?Ala?Val
100?????????????????105?????????????????110
Tyr?Tyr?Cys?Ala?Arg?Gly?Ser?Arg?Tyr?Asp?Trp?Tyr?Leu?Asp?Tyr?Trp
115?????????????????120?????????????????125
Gly?Gln?Gly?Thr?Pro?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro
130?????????????????135?????????????????140
Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr
145?????????????????150?????????????????155?????????????????160
Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr
165?????????????????170?????????????????175
Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro
180?????????????????185?????????????????190
Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr
195?????????????????200?????????????????205
Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn
210?????????????????215?????????????????220
His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser
225?????????????????230?????????????????235?????????????????240
Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu
245?????????????????250?????????????????255
Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu
260?????????????????265?????????????????270
Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser
275?????????????????280?????????????????285
His?Glu?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Asp?Gly?Val?Glu
290?????????????????295?????????????????300
Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr
305?????????????????310?????????????????315?????????????????320
Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn
325?????????????????330?????????????????335
Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro
340?????????????????345?????????????????350
Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln
355?????????????????360?????????????????365
Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val
370?????????????????375?????????????????380
Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val
385?????????????????390?????????????????395?????????????????400
Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro
405?????????????????410?????????????????415
Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr
420?????????????????425?????????????????430
Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val
435?????????????????440?????????????????445
Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu
450?????????????????455?????????????????460
Ser?Pro?Gly?Lys
465
<210>21
<211>786
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(786)
＜223〉anti-SM5-1 antibody nucleotide sequence
<220>21
gagcattacc?ggccatactc?atcaccatcc?caggatatct?ctagaaagct?tgccgccacc?????60
atggattttc?aagtgcagat?tttcagcttc?ctgctaatca?gtgcttcagt?cataatgtcc????120
agaggaaaca?tcatgatgac?tcagagccca?tccagcttga?gcgcatcagt?aggcgaccgc????180
gtaacgatca?cttgcaaatc?ctctcagtca?gtattgtact?ccagcaacca?gaagaactac????240
ctggccggat?atcagcagac?tcccggcaaa?gccccaaagt?tgctgattta?ttgggcctcc????300
acgcgcgagt?ctggcgtgcc?atcacgcttt?agcggcagcg?ggtccggtac?agattacacg????360
tttaccatta?gcagtctgca?gcctgaggac?atagccacct?actactgtca?ccagtacttt????420
agttcctaca?cttttggcca?gggaactaaa?ctgcagatta?crcgaactgt?ggctcaacca????480
tctgtcttca?tcttcccgcc?atctgatgag?cagttgaaat?ctggaactgc?ctctgttgtg????540
tgcctgctga?ataacttcta?tcccagagag?gccaaagtac?agtggaaggt?ggataacgcc????600
ctccaatcgg?gtaactccca?ggagagtgtc?acagagcagg?acagcaagga?cagcacctac????660
agcctcagca?gcaccctgac?gctgagcaaa?gcagactacg?agaaacacaa?agtctacgcc????720
tgcgaagtca?cccatcaggg?cctgagctcg?cccgtcacaa?agagcttcaa?caggggagag????780
tgttag???????????????????????????????????????????????????????????????786
<210>22
<211>241
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(241)
＜223〉anti-SM5-1 antibody
<220>22
Met?Asp?Phe?Gln?Val?Gln?Ile?Phe?Ser?Phe?Leu?Leu?Ile?Ser?Ala?Ser
1???????????????5???????????????????10??????????????????15
Val?Ile?Met?Ser?Arg?Gly?Asn?Ile?Met?Met?Thr?Gln?Ser?Pro?Ser?Ser
20??????????????????25??????????????????30
Leu?Ser?Ala?Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Lys?Ser?Ser
35??????????????????40??????????????????45
Gln?Ser?Val?Leu?Tyr?Ser?Ser?Asn?Gln?Lys?Asn?Tyr?Leu?Ala?Trp?Tyr
50??????????????????55??????????????????60
Gln?Gln?Thr?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Trp?Ala?Ser
65??????????????????70??????????????????75??????????????????80
Thr?Arg?Glu?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly
85??????????????????90??????????????????95
Thr?Asp?Tyr?Thr?Phe?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?Ile?Ala
100?????????????????105?????????????????110
Thr?Tyr?Tyr?Cys?His?Gln?Tyr?Phe?Ser?Ser?Tyr?Thr?Phe?Gly?Gln?Gly
115?????????????????120?????????????????125
Thr?Lys?Leu?Gln?Ile?Thr?Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile
130?????????????????135?????????????????140
Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val
145?????????????????150?????????????????155?????????????????160
Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys
165?????????????????170?????????????????175
Val?Asp?Asn?Ala?Leu?Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu
180?????????????????185?????????????????190
Gln?Asp?Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu
195?????????????????200?????????????????205
Ser?Lys?Ala?Asp?Tyr?Glu?Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr
210?????????????????215?????????????????220
His?Gln?Gly?Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu
225?????????????????230?????????????????235?????????????????240
Cys
<210>23
<211>2489
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(2489)
＜223〉anti-p230 antibody fusion protein encoding sequence
<220>23
agagccgcca?ccatggattg?ggtgtggacc?ttgctattcc?tgttgtcagt?aactgcaggt??????60
gtccactccc?aggtgcagct?ggtgcagtct?ggcggtggag?tggtccagcc?cggccgcagc?????120
ctgaggctgt?cctgcaaggc?atctggctac?accttcacca?gctacgtgat?gacatgggtg?????180
cgccaagccc?ccggaaaggg?cctcgaatgg?attggctaca?ttgtgcctta?taatgacggt?????240
actaagtaca?atgaaaagtt?caagggcaga?tttacaatat?caagtgacaa?gagcaagtca?????300
accgcattcc?tccaaatgga?cagcttgcgt?ccagaggaca?ccgccgtata?ctattgtgtg?????360
cgcggcagcc?gttacgactg?gtacttggac?tactggggcc?aaggcactcc?agtcaccgtc?????420
tcctctgcta?gcaccaaggg?cccatcggtc?ttccccctgg?caccctcctc?caagagcacc?????480
tctgggggca?cagcggccct?gggctgcctg?gtcaaggact?acttccccga?accggtgacg?????540
gtgtcttgga?actcaggcgc?cctgaccagc?ggcgtgcaca?ccttcccggc?tgtcctacag?????600
tcctcaggac?tctactccct?cagcagcgtg?gtgaccgtgc?cctccagcag?cttgggcacc?????660
cagacctaca?tctgcaacgt?gaatcacaag?cccagcaaca?ccaaggtgga?caagaaagtt?????720
ggtgagaggc?cagcacaggg?agggagggtg?tctgctggaa?gcaggctcag?cgctcctgcc?????780
tggacgcatc?ccggctatgc?agccccagtc?cagggcagca?aggcaggccc?cgtctgcctc?????840
ttcacccgga?gcctctgccc?gccccactca?tgctcaggga?gagggtcttc?tggctttttc?????900
ccaggctctg?ggcaggcaca?ggctaggtgc?ccctaaccca?ggccctgcac?acaaaggggc?????960
aggtgctggg?ctcagacctg?ccaagagcca?tatccgggag?gaccctgccc?ctgacctaag????1020
cccaccccaa?aggccaaact?ctccactccc?tcagctcgga?caccttctct?cctcccagat????1080
tccagtaact?cccaatcttc?tctctgcaga?gcccaaatct?tgtgacaaaa?ctcacacatg????1140
cccaccgtgc?ccaggtaagc?cagcccaggc?ctcgccctcc?agctcaaggc?gggacaggtg????1200
ccctagagta?gcctgcatcc?agggacaggc?cccagccggg?tgctgacacg?tccacctcca????1260
tctcttcctc?agcacctgaa?ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac????1320
ccaaggacac?cctcatgatc?tcccggaccc?ctgaggtcac?atgcgtggtg?gtggacgtga????1380
gccacgaaga?ccctgaggtc?aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg????1440
ccaagacaaa?gccgcgggag?gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca????1500
ccgtcctgca?ccaggactgg?ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag????1560
ccctcccagc?ccccatcgag?aaaaccatct?ccaaagccaa?aggtgggacc?cgtggggtgc????1620
gagggccaca?tggacagagg?ccggctcggc?ccaccctctg?ccctgagagt?gaccgctgta????1680
ccaacctctg?tcctacaggg?cagccccgag?aaccacaggt?gtacaccctg?cccccatccc????1740
gggatgagct?gaccaagaac?caggtcagcc?tgacctgcct?ggtcaaaggc?ttctatccca????1800
gcgacatcgc?cgtggagtgg?gagagcaatg?ggcagccgga?gaacaactac?aagaccacgc????1860
ctcccgtgct?ggactccgac?ggctccttct?tcctctacag?caagctcacc?gtggacaaga????1920
gcaggtggca?gcaggggaac?gtcttctcat?gctccgtgat?gcatgaggct?ctgcacaacc????1980
actacacgca?gaagagcctc?tccctgtctc?ccggtaaaac?ccaggactgc?tccttccaac????2040
acagccccat?ctcctccgac?ttcgctgtca?aaatccgtga?gctgtctgac?tacctgcttc????2100
aagattaccc?agtcaccgtg?gcctccaacc?tgcaggacga?ggagctctgc?gggggcctct????2160
ggcggctggt?cctggcacag?cgctggatgg?agcggctcaa?gactgtcgct?gggtccaaga????2220
tgcaaggctt?gctggagcgc?gtgaacacgg?agatacactt?tgtcaccaaa?tgtgcctttc????2280
agcccccccc?cagctgtctt?cgcttcgtcc?agaccaacat?ctcccgcctc?ctgcaggaga????2340
cctccgagca?gctggtggcg?ctgaagccct?ggatcactcg?ccagaacttc?tcccggtgcc????2400
tggagctgca?gtgtcagccc?gactcctcaa?ccctgccacc?cccatggagt?ccccggcccc????2460
tggaggccac?agccccgaca?gccccgtga??????????????????????????????????????2489
<210>24
<211>624
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(624)
＜223〉anti-p230 antibody fusion protein
<220>24
Met?Asp?Trp?Val?Trp?Thr?Leu?Leu?Phe?Leu?Leu?Ser?Val?Thr?Ala?Gly
1???????????????5???????????????????10??????????????????15
Val?His?Ser?Gln?Val?Gln?Leu?Val?Gln?Ser?Gly?Gly?Gly?Val?Val?Gln
20??????????????????25??????????????????30
Pro?Gly?Arg?Ser?Leu?Arg?Leu?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe
35??????????????????40??????????????????45
Thr?Ser?Tyr?Val?Met?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu
50??????????????????55??????????????????60
Glu?Trp?Ile?Gly?Tyr?Ile?Val?Pro?Tyr?Asn?Asp?Gly?Thr?Lys?Tyr?Asn
65??????????????????70??????????????????75??????????????????80
Glu?Lys?Phe?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Ser?Asp?Lys?Ser?Lys?Ser
85??????????????????90??????????????????95
Thr?Ala?Phe?Leu?Gln?Met?Asp?Ser?Leu?Arg?Pro?Glu?Asp?Thr?Ala?Val
100?????????????????105?????????????????110
Tyr?Tyr?Cys?Ala?Arg?Gly?Ser?Arg?Tyr?Asp?Trp?Tyr?Leu?Asp?Tyr?Trp
115?????????????????120?????????????????125
Gly?Gln?Gly?Thr?Pro?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro
130?????????????????135?????????????????140
Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr
145?????????????????150?????????????????155?????????????????160
Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr
165?????????????????170?????????????????175
Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro
180?????????????????185?????????????????190
Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr
195?????????????????200?????????????????205
Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn
210?????????????????215?????????????????220
His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser
225?????????????????230?????????????????235?????????????????240
Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu
245?????????????????250?????????????????255
Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu
260?????????????????265?????????????????270
Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser
275?????????????????280?????????????????285
His?Glu?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Asp?Gly?Val?Glu
290?????????????????295?????????????????300
Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr
305?????????????????310?????????????????315?????????????????320
Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn
325?????????????????330?????????????????335
Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro
340?????????????????345?????????????????350
Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln
355?????????????????360?????????????????365
Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Arg?Glu?Leu?Thr?Lys?Asn?Gln?Val
370?????????????????375?????????????????380
Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val
385?????????????????390?????????????????395?????????????????400
Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro
405?????????????????410?????????????????415
Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr
420?????????????????425?????????????????430
Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val
435?????????????????440?????????????????445
Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu
450?????????????????455?????????????????460
Ser?Pro?Gly?Lys?Thr?Gln?Asp?Cys?Ser?Phe?Gln?His?Ser?Pro?Ile?Ser
465?????????????????470?????????????????475?????????????????480
Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu?Ser?Asp?Tyr?Leu?Leu?Gln
485?????????????????490?????????????????495
Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu?Gln?Asp?Glu?Glu?Leu?Cys
500?????????????????505?????????????????510
Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln?Arg?Trp?Met?Glu?Arg?Leu
515?????????????????520?????????????????525
Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly?Leu?Leu?Glu?Arg?Val?Asn
530?????????????????535?????????????????540
Thr?Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala?Phe?Gln?Pro?Pro?Pro?Ser
545?????????????????550?????????????????555?????????????????560
Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser?Arg?Leu?Leu?Gln?Glu?Thr
565?????????????????570?????????????????575
Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp?Ile?Thr?Arg?Gln?Asn?Phe
580?????????????????585?????????????????590
Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro?Asp?Ser?Ser?Thr?Leu?Pro
595?????????????????600?????????????????605
Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala?Thr?Ala?Pro?Thr?Ala?Pro
610?????????????????615?????????????????620
<210>25
<211>2534
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(2534)
＜223〉anti-p230 antibody fusion protein encoding sequence
<220>25
agagccgcca?ccatggattg?ggtgtggacc?ttgctattcc?tgttgtcagt?aactgcaggt?????60
gtccactccc?aggtgcagct?ggtgcagtct?ggcggtggag?tggtccagcc?cggccgcagc????120
ctgaggctgt?cctgcaaggc?atctggctac?accttcacca?gctacgtgat?gacatgggtg????180
cgccaagccc?ccggaaaggg?cctcgaatgg?attggctaca?ttgtgcctta?taatgacggt????240
actaagtaca?atgaaaagtt?caagggcaga?tttacaatat?caagtgacaa?gagcaagtca????300
accgcattcc?tccaaatgga?cagcttgcgt?ccagaggaca?ccgccgtata?ctattgtgtg????360
cgcggcagcc?gttacgactg?gtacttggac?tactggggcc?aaggcactcc?agtcaccgtc????420
tcctctgcta?gcaccaaggg?cccatcggtc?ttccccctgg?caccctcctc?caagagcacc????480
tctgggggca?cagcggccct?gggctgcctg?gtcaaggact?acttccccga?accggtgacg????540
gtgtcttgga?actcaggcgc?cctgaccagc?ggcgtgcaca?ccttcccggc?tgtcctacag????600
tcctcaggac?tctactccct?cagcagcgtg?gtgaccgtgc?cctccagcag?cttgggcacc????660
cagacctaca?tctgcaacgt?gaatcacaag?cccagcaaca?ccaaggtgga?caagaaagtt????720
ggtgagaggc?cagcacaggg?agggagggtg?tctgctggaa?gcaggctcag?cgctcctgcc????780
tggacgcatc?ccggctatgc?agccccagtc?cagggcagca?aggcaggccc?cgtctgcctc????840
ttcacccgga?gcctctgccc?gccccactca?tgctcaggga?gagggtcttc?tggctttttc????900
ccaggctctg?ggcaggcaca?ggctaggtgc?ccctaaccca?ggccctgcac?acaaaggggc????960
aggtgctggg?ctcagacctg?ccaagagcca?tatccgggag?gaccctgccc?ctgacctaag???1020
cccaccccaa?aggccaaact?ctccactccc?tcagctcgga?caccttctct?cctcccagat???1080
tccagtaact?cccaatcttc?tctctgcaga?gcccaaatct?tgtgacaaaa?ctcacacatg???1140
cccaccgtgc?ccaggtaagc?cagcccaggc?ctcgccctcc?agctcaaggc?gggacaggtg???1200
ccctagagta?gcctgcatcc?agggacaggc?cccagccggg?tgctgacacg?tccacctcca???1260
tctcttcctc?agcacctgaa?ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac????1320
ccaaggacac?cctcatgatc?tcccggaccc?ctgaggtcac?atgcgtggtg?gtggacgtga????1380
gccacgaaga?ccctgaggtc?aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg????1440
ccaagacaaa?gccgcgggag?gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca????1500
ccgtcctgca?ccaggactgg?ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag????1560
ccctcccagc?ccccatcgag?aaaaccatct?ccaaagccaa?aggtgggacc?cgtggggtgc????1620
gagggccaca?tggacagagg?ccggctcggc?ccaccctctg?ccctgagagt?gaccgctgta????1680
ccaacctctg?tcctacaggg?cagccccgag?aaccacaggt?gtacaccctg?cccccatccc????1740
gggatgagct?gaccaagaac?caggtcagcc?tgacctgcct?ggtcaaaggc?ttctatccca????1800
gcgacatcgc?cgtggagtgg?gagagcaatg?ggcagccgga?gaacaactac?aagaccacgc????1860
ctcccgtgct?ggactccgac?ggctccttct?tcctctacag?caagctcacc?gtggacaaga????1920
gcaggtggca?gcaggggaac?gtcttctcat?gctccgtgat?gcatgaggct?ctgcacaacc????1980
actacacgca?gaagagcctc?tccctgtctc?ccggtaaagg?cggtggaggc?tctggtggag????2040
gcggttcagg?aggcggtgga?tctacccagg?actgctcctt?ccaacacagc?cccatctcct????2100
ccgacttcgc?tgtcaaaatc?cgtgagctgt?ctgactacct?gcttcaagat?tacccagtca????2160
ccgtggcctc?caacctgcag?gacgaggagc?tctgcggggg?cctctggcgg?ctggtcctgg????2220
cacagcgctg?gatggagcgg?ctcaagactg?tcgctgggtc?caagatgcaa?ggcttgctgg????2280
agcgcgtgaa?cacggagata?cactttgtca?ccaaatgtgc?ctttcagccc?ccccccagct????2340
gtcttcgctt?cgtccagacc?aacatctccc?gcctcctgca?ggagacctcc?gagcagctgg????2400
tggcgctgaa?gccctggatc?actcgccaga?acttctcccg?gtgcctggag?ctgcagtgtc????2460
agcccgactc?ctcaaccctg?ccacccccat?ggagtccccg?gcccctggag?gccacagccc????2520
cgacagcccc?gtga??????????????????????????????????????????????????????2534
<210>26
<211>639
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(639)
＜223〉anti-p230 antibody fusion protein
<220>26
Met?Asp?Trp?Val?Trp?Thr?Leu?Leu?Phe?Leu?Leu?Ser?Val?Thr?Ala?Gly
1???????????????5???????????????????10??????????????????15
Val?His?Ser?Gln?Val?Gln?Leu?Val?Gln?Ser?Gly?Gly?Gly?Val?Val?Gln
20??????????????????25??????????????????30
Pro?Gly?Arg?Ser?Leu?Arg?Leu?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe
35??????????????????40??????????????????45
Thr?Ser?Tyr?Val?Met?His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu
50??????????????????55??????????????????60
Glu?Trp?Ile?Gly?Tyr?Ile?Val?Pro?Tyr?Asn?Asp?Gly?Thr?Lys?Tyr?Asn
65??????????????????70??????????????????75??????????????????80
Glu?Lys?Phc?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Ser?Asp?Lys?Ser?Lys?Ser
85??????????????????90??????????????????95
Thr?Ala?Phe?Leu?Gln?Met?Asp?Ser?Leu?Arg?Pro?Glu?Asp?Thr?Ala?Val
100?????????????????105?????????????????110
Tyr?Tyr?Cys?Ala?Arg?Gly?Ser?Arg?Tyr?Asp?Trp?Tyr?Leu?Asp?Tyr?Trp
115?????????????????120?????????????????125
Gly?Gln?Gly?Thr?Pro?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro
130?????????????????135?????????????????140
Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr
145?????????????????150?????????????????155?????????????????160
Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr
165?????????????????170?????????????????175
Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro
180?????????????????185?????????????????190
Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr
195?????????????????200?????????????????205
Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn
210?????????????????215?????????????????220
His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser
225?????????????????230?????????????????235?????????????????240
Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu
245?????????????????250?????????????????255
Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu
260?????????????????265?????????????????270
Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser
275?????????????????280?????????????????285
His?Glu?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Asp?Gly?Val?Glu
290?????????????????295?????????????????300
Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr
305?????????????????310?????????????????315?????????????????320
Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn
325?????????????????330?????????????????335
Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro
340?????????????????345?????????????????350
Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln
355?????????????????360?????????????????365
Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val
370?????????????????375?????????????????380
Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val
385?????????????????390?????????????????395?????????????????400
Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro
405?????????????410?????????????????????415
Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr
420?????????????????425?????????????????430
Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val
435?????????????????440?????????????????445
Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu
450?????????????????455?????????????????460
Ser?Pro?Gly?Lys?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly
465?????????????????470?????????????????475?????????????????480
Gly?Gly?Ser?Thr?Gln?Asp?Cys?Ser?Phe?Gln?His?Ser?Pro?Ile?Ser?Ser
485?????????????????490?????????????????495
Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu?Ser?Asp?Tyr?Leu?Leu?Gln?Asp
500?????????????????505?????????????????510
Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu?Gln?Asp?Glu?Glu?Leu?Cys?Gly
515?????????????????520?????????????????525
Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln?Arg?Trp?Met?Glu?Arg?Leu?Lys
530?????????????????535?????????????????540
Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly?Leu?Leu?Glu?Arg?Val?Asn?Thr
545?????????????????550?????????????????555?????????????????560
Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala?Phe?Gln?Pro?Pro?Pro?Ser?Cys
565?????????????????570?????????????????575
Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser?Arg?Leu?Leu?Gln?Glu?Thr?Ser
580?????????????????585?????????????????590
Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp?Ile?Thr?Arg?Gln?Asn?Phe?Ser
595?????????????????600?????????????????605
Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro?Asp?Ser?Ser?Thr?Leu?Pro?Pro
610?????????????????615?????????????????620
Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala?Thr?Ala?Pro?Thr?Ala?Pro
625?????????????????630?????????????????635
<210>27
<211>1986
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(1986)
＜223〉anti-p230 antibody fusion protein encoding sequence
<220>27
atgacagtgc?tggcgccagc?ctggagccca?acaacctatc?tcctcctgct?gctgctgctg??????60
agctcgggac?tcagtgggac?ccaggactgc?tccttccaac?acagccccat?ctcctccgac?????120
ttcgctgtca?aaatccgtga?gctgtctgac?tacctgcttc?aagattaccc?agtcaccgtg?????180
gcctccaacc?tgcaggacga?ggagctctgc?gggggcctct?ggcggctggt?cctggcacag?????240
cgctggatgg?agcggctcaa?gactgtcgct?gggtccaaga?tgcaaggctt?gctggagcgc?????300
gtgaacacgg?agatacactt?tgtcaccaaa?tgtgcctttc?agcccccccc?cagctgtctt?????360
cgcttcgtcc?agaccaacat?ctcccgcctc?ctgcaggaga?cctccgagca?gctggtggcg?????420
ctgaagccct?ggatcactcg?ccagaacttc?tcccggtgcc?tggagctgca?gtgtcagccc?????480
gactcctcaa?ccctgccacc?cccatggagt?ccccggcccc?tggaggccac?agccccgaca?????540
gccccggagc?ccaaatcttg?tgacaaaact?cacagatgcc?caccgtgccc?agcacctgaa?????600
ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac?ccaaggacac?cctcatgatc?????660
tcccggaccc?ctgaggtcac?atgctgggtg?gtggactgga?gccacgaaga?ccctgaggtc?????720
aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg?ccaagacaaa?gccgcgggag?????780
gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca?ccgtcctgca?ccaggactgg?????840
ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag?ccctcccagc?ccccatcgag?????900
aaaaccatct?ccaaagccaa?agggcagccc?cgagaaccac?aggtgtacac?cctgccccca?????960
tcccgggatg?agctgaccaa?gaaccaggtc?agcctgacct?gcctggtcaa?aggcttctat????1020
cccagcgaca?tcgccgtgga?gtgggagagc?aatgggcagc?cggagaacaa?ctacaagacc????1080
acgcctcccg?tgctggactc?cgacggctcc?ttcttcctct?acagcaagct?caccgtggac????1140
aagagcaggt?ggcagcaggg?gaacgtcttc?tcatgctccg?tgatgcatga?ggctctgcac????1200
aaccactaca?cgcagaagag?cctctccctg?tctcccggta?aacaggtgca?gctggtgcag????1260
tctggcggtg?gagtggtcca?gcccggccgc?agcctgaggc?tgtcctgcaa?ggcatctggc????1320
tacaccttca?ccagctacgt?gatgacatgg?gtgcgccaag?cccccggaaa?gggcctcgaa????1380
tggattggct?acattgtgcc?ttataatgac?ggtactaagt?acaatgaaaa?gttcaagggc????1440
agatttacaa?tatcaagtga?caagagcaag?tcaaccgcat?tcctccaaat?ggacagcttg????1500
cgtccagagg?acaccgccgt?atactattgt?gtgcgcggca?gccgttacga?ctggtacttg????1560
gactactggg?gccaaggcac?tccagtcacc?gtctcctctg?gcggtggagg?ctctggtgga????1620
ggcggttcag?gaggcggtgg?atctaacatc?atgatgactc?agagcccatc?cagcttgagc????1680
gcatcagtag?gcgaccgcgt?aacgatcact?tgcaaatcct?ctcagtcagt?attgtactcc????1740
agcaaccaga?agaactacct?ggccggatat?cagcagactc?ccggcaaagc?cccaaagttg????1800
ctgatttatt?gggcctccac?gcgcgagtct?ggcgtgccat?cacgctttag?cggcagcggg????1860
tccggtacag?attacacgtt?taccattagc?agtctgcagc?ctgaggacat?agccacctac????1920
tactgtcacc?agtactttag?ttcctacact?tttggccagg?gaactaaact?gcagattact????1980
cgatga???????????????????????????????????????????????????????????????1986
<210>28
<211>661
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(661)
＜223〉anti-p230 antibody fusion protein
<220>28
Met?Thr?Val?Leu?Ala?Pro?Ala?Trp?Ser?Pro?Thr?Thr?Tyr?Leu?Leu?Leu
1???????????????5???????????????????10??????????????????15
Leu?Leu?Leu?Leu?Ser?Ser?Gly?Leu?Ser?Gly?Thr?Gln?Asp?Cys?Ser?Phe
20??????????????????25??????????????????30
Gln?His?Ser?pro?Ile?Ser?Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu
35??????????????????40??????????????????45
Ser?Asp?Tyr?Leu?Leu?Gln?Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu
50??????????????????55??????????????????60
Gln?Asp?Glu?Glu?Leu?Cys?Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln
65??????????????????70??????????????????75??????????????????80
Arg?Trp?Met?Glu?Arg?Leu?Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly
85??????????????????90??????????????????95
Leu?Leu?Glu?Arg?Val?Asn?Thr?Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala
100?????????????????105?????????????????110
Phe?Gln?Pro?Pro?Pro?Ser?Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser
115?????????????????120?????????????????125
Arg?Leu?Leu?Gln?Glu?Thr?Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp
130?????????????????135?????????????????140
Ile?Thr?Arg?Gln?Asn?Phe?Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro
145?????????????????150?????????????????155?????????????????160
Asp?Ser?Ser?Thr?Leu?Pro?Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala
165?????????????????170?????????????????175
Thr?Ala?Pro?Thr?Ala?Pro?Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr
180?????????????????185?????????????????190
Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe
195?????????????????200?????????????????205
Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro
210?????????????????215?????????????????220
Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val
225?????????????????230?????????????????235?????????????????240
Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr
245?????????????????250?????????????????255
Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val
260?????????????????265?????????????????270
Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys
275?????????????????280?????????????????285
Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser
290?????????????????295?????????????????300
Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro
305?????????????????310?????????????????315?????????????????320
Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val
325?????????????????330?????????????????335
Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly
340?????????????????345?????????????????350
Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp
355?????????????????360?????????????????365
Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp
370?????????????????375?????????????????380
Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His
385?????????????????390?????????????????395?????????????????400
Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys?Gln?Val
405?????????????????410?????????????????415
Gln?Leu?Val?Gln?Ser?Gly?Gly?Gly?Val?Val?Gln?Pro?Gly?Arg?Ser?Leu
420?????????????????425?????????????????430
Arg?Leu?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Ser?Tyr?Val?Met
435?????????????????440?????????????????445
His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Ile?Gly?Tyr
450?????????????????455?????????????????460
Ile?Val?Pro?Tyr?Asn?Asp?Gly?Thr?Lys?Tyr?Asn?Glu?Lys?Phe?Lys?Gly
465?????????????????470?????????????????475?????????????????480
Arg?Phe?Thr?Ile?Ser?Ser?Asp?Lys?Ser?Lys?Ser?Thr?Ala?Phe?Leu?Gln
485?????????????????490?????????????????495
Met?Asp?Ser?Leu?Arg?Pro?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg
500?????????????????505?????????????????510
Gly?Ser?Arg?Tyr?Asp?Trp?Tyr?Leu?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Pro
515?????????????????520?????????????????525
Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly
530?????????????????535?????????????????540
Gly?Gly?Gly?Ser?Asn?Ile?Met?Tht?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser
545?????????????????550?????????????????555?????????????????560
Ala?Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Lys?Ser?Ser?Gln?Ser
565?????????????????570?????????????????575
Val?Leu?Tyr?Ser?Ser?Asn?Gln?Lys?Asn?Tyr?Leu?Ala?Trp?Tyr?Gln?Gln
580?????????????????585?????????????????590
Thr?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Trp?Ala?Ser?Thr?Arg
595?????????????????600?????????????????605
Glu?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Asp
610?????????????????615?????????????????620
Tyr?Thr?Phe?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?Ile?Ala?Thr?Tyr
625?????????????????630?????????????????635?????????????????640
Tyr?Cys?His?Gln?Tyr?Phe?Ser?Ser?Tyr?Thr?Phe?Gly?Gln?Gly?Thr?Lys
645?????????????????650?????????????????655
Leu?Gln?Ile?Thr?Arg
660
<210>29
<211>2489
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(2489)
＜223〉anti-p230 antibody fusion protein encoding sequence
<220>29
cttgccgcca?ccatggaatg?gagttggata?tttctctttc?tcctgtcagg?aactgcaggt?????60
gtccactctg?aggtccagct?gcagcagtct?ggacctgagc?tggtaaagcc?tggggcttca????120
gtgaagatgt?cctgcaaggc?ttctggatac?acattcacta?gctatgttat?gcactgggtg????180
aagcagaagc?ctgggcaggg?ccttgactgg?attggatata?ttgttcctta?caatgatggc????240
actaagtaca?atgagaagtt?caaaggcaag?gccacactga?cttcagacaa?atcctccagc????300
acagcctaca?tggagctcag?cagactgacc?tctgaggact?ctgcggtcta?ttattgtgtc????360
tacggtagta?ggtacgactg?gtatttagat?gtctggggcg?cagggaccac?ggtcaccgtc????420
tcctcagcta?gcaccaaggg?cccatcggtc?ttccccctgg?caccctcctc?caagagcacc????480
tctgggggca?cagcggccct?gggctgcctg?gtcaaggact?acttccccga?accggtgacg????540
gtgtcttgga?actcaggcgc?cctgaccagc?ggcgtgcaca?ccttcccggc?tgtcctacag????600
tcctcaggac?tctactccct?cagcagcgtg?gtgaccgtgc?cctccagcag?cttgggcacc????660
cagacctaca?tctgcaacgt?gaatcacaag?cccagcaaca?ccaaggtgga?caagaaagtt????720
ggtgagaggc?cagcacaggg?agggagggtg?tctgctggaa?gcaggctcag?cgctcctgcc????780
tggacgcatc?ccggctatgc?agccccagtc?cagggcagca?aggcaggccc?cgtctgcctc????840
ttcacccgga?gcctctgccc?gccccactca?tgctcaggga?gagggtcttc?tggctttttc????900
ccaggctctg?ggcaggcaca?ggctaggtgc?ccctaaccca?gaccctgcac?acaaaggggc????960
aggtgctggg?ctcagacctg?ccaagagcca?tatccgggag?gaccctgccc?ctgacctaag???1020
cccaccccaa?aggccaaact?ctccactccc?tcagctcgga?caccttctct?cctcccagat???1080
tccagtaact?cccaatcttc?tctctgcaga?gcccaaatct?tgtgacaaaa?ctcacacatg???1140
cccaccgtgc?ccaggtaagc?cagcccaggc?ctcgccctcc?agctcaaggc?gggacaggtg???1200
ccctagagta?gcctgcatcc?agggacaggc?cccagccggg?tgctgacacg?tccacctcca???1260
tctcttcctc?agcacctgaa?ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac???1320
ccaaggacac?cctcatgatc?tcccggaccc?ctgaggtcac?atgcgtggtg?gtggacgtga???1380
gccacgaaga?ccctgaggtc?aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg???1440
ccaagacaaa?gccgcgggag?gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca???1500
ccgtcctgca?ccaggactgg?ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag???1560
ccctcccagc?ccccatcgag?aaaaccatct?ccaaagccaa?aggtgggacc?cgtggggtgc???1620
gagggccaca?tggacagagg?ccggctcggc?ccaccctctg?ccctgagagt?gaccgctgta???1680
ccaacctctg?tcctacaggg?cagccccgag?aaccacaggt?gtacaccctg?cccccatccc???1740
gggatgagct?gaccaagaac?caggtcagcc?tgacctgcct?ggtcaaaggc?ttctatccca???1800
gcgacatcgc?cgtggagtgg?gagagcaatg?ggcagccgga?gaacaactac?aagaccacgc???1860
ctcccgtgct?ggactccgac?ggctccttct?tcctctacag?caagctcacc?gtggacaaga???1920
gcaggtggca?gcaggggaac?gtcttctcat?gctccgtgat?gcatgaggct?ctgcacaacc???1980
actacacgca?gaagagcctc?tccctgtctc?ccggtaaaac?ccaggactgc?tccttccaac???2040
acagccccat?ctcctccgac?ttcgctgtca?aaatccgtga?gctgtctgac?tacctgcttc???2100
aagattaccc?agtcaccgtg?gcctccaacc?tgcaggacga?ggagctctgc?gggggcctct???2160
ggcggctggt?cctggcacag?cgctggatgg?agcggctcaa?gactgtcgct?gggtccaaga???2220
tgcaaggctt?gctggagcgc?gtgaacacgg?agatacactt?tgtcaccaaa?tgtgcctttc???2280
agcccccccc?cagctgtctt?cgcttcgtcc?agaccaacat?ctcccgcctc?ctgcaggaga???2340
cctccgagca?gctggtggcg?ctgaagccct?ggatcactcg?ccagaacttc?tcccggtgcc???2400
tggagctgca?gtgtcagccc?gactcctcaa?ccctgccacc?cccatggagt?ccccggcccc???2460
tggaggccac?agccccgaca?gccccgtga?????????????????????????????????????2489
<210>30
<211>624
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(624)
＜223〉anti-p230 antibody fusion protein
<220>30
Met?Glu?Trp?Ser?Trp?Ile?Phe?Leu?Phe?Leu?Leu?Ser?Gly?Thr?Ala?Gly
1???????????????5???????????????????10??????????????????15
Val?His?Ser?Glu?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?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?Val?Met?His?Trp?Val?Lys?Gln?Lys?Pro?Gly?Gln?Gly?Leu
50??????????????????55??????????????????60
Asp?Trp?Ile?Gly?Tyr?Ile?Val?Pro?Tyr?Asn?Asp?Gly?Thr?Lys?Tyr?Asn
65??????????????????70??????????????????75??????????????????80
Glu?Lys?Phe?Lys?Gly?Lys?Ala?Thr?Leu?Thr?Ser?Asp?Lys?Ser?Ser?Ser
85??????????????????90??????????????????95
Thr?Ala?Tyr?Met?Glu?Leu?Ser?Arg?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val
100?????????????????105?????????????????110
Tyr?Tyr?Cys?Val?Tyr?Gly?Ser?Arg?Tyr?Asp?Trp?Tyr?Leu?Asp?Val?Trp
115?????????????????120?????????????????125
Gly?Ala?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro
130?????????????????135?????????????????140
Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr
145?????????????????150?????????????????155?????????????????160
Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr
165?????????????????170?????????????????175
Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro
180?????????????????185?????????????????190
Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr
195?????????????????200?????????????????205
Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn
210?????????????????215?????????????????220
His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser
225?????????????????230?????????????????235?????????????????240
Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu
245?????????????????250?????????????????255
Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu
260?????????????????265?????????????????270
Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser
275?????????????????280?????????????????285
His?Glu?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Asp?Gly?Val?Glu
290?????????????????295?????????????????300
Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr
305?????????????????310?????????????????315?????????????????320
Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn
325?????????????????330?????????????????335
Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro
340?????????????????345?????????????????350
Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln
355?????????????????360?????????????????365
Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val
370?????????????????375?????????????????380
Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val
385?????????????????390?????????????????395?????????????????400
Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro
405?????????????????4l0?????????????????415
Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr
420?????????????????425?????????????????430
Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val
435?????????????????440?????????????????445
Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu
450?????????????????455?????????????????460
Ser?Pro?Gly?Lys?Thr?Gln?Asp?Cys?Ser?Phe?Gln?His?Ser?Pro?Ile?Ser
465?????????????????470?????????????????475?????????????????480
Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu?Ser?Asp?Tyr?Leu?Leu?Gln
485?????????????????490?????????????????495
Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu?Gln?Asp?Glu?Glu?Leu?Cys
500?????????????????505?????????????????510
Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln?Arg?Trp?Met?Glu?Arg?Leu
515?????????????????520?????????????????525
Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly?Leu?Leu?Glu?Arg?Val?Asn
530?????????????????535?????????????????540
Thr?Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala?Phe?Gln?Pro?Pro?Pro?Ser
545?????????????????550?????????????????555?????????????????560
Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser?Arg?Leu?Leu?Gln?Glu?Thr
565?????????????????570?????????????????575
Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp?Ile?Thr?Arg?Gln?Asn?Phe
580?????????????????585?????????????????590
Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro?Asp?Ser?Ser?Thr?Leu?Pro
595?????????????????600?????????????????605
Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala?Thr?Ala?Pro?Thr?Ala?Pro
610?????????????????615?????????????????620
<210>31
<211>2534
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(2489)
＜223〉anti-p230 antibody fusion protein encoding sequence
<400>31
cttgccgcca?ccatggaatg?gagttggata?tttctctttc?tcctgtcagg?aactgcaggt?????60
gtccactctg?aggtccagct?gcagcagtct?ggacctgagc?tggtaaagcc?tggggcttca????120
gtgaagatgt?cctgcaaggc?ttctggatac?acattcacta?gctatgttat?gcactgggtg????180
aagcagaagc?ctgggcaggg?ccttgactgg?attggatata?ttgttcctta?caatgatggc????240
actaagtaca?atgagaagtt?caaaggcaag?gccacactga?cttcagacaa?atcctccagc????300
acagcctaca?tggagctcag?cagactgacc?tctgaggact?ctgcggtcta?ttattgtgtc????360
tacggtagta?ggtacgactg?gtatttagat?gtctggggcg?cagggaccac?ggtcaccgtc????420
tcctcagcta?gcaccaaggg?cccatcggtc?ttccccctgg?caccctcctc?caagagcacc????480
tctgggggca?cagcggccct?gggctgcctg?gtcaaggact?acttccccga?accggtgacg????540
gtgtcttgga?actcaggcgc?cctgaccagc?ggcgtgcaca?ccttcccggc?tgtcctacag????600
tcctcaggac?tctactccct?cagcagcgtg?gtgaccgtgc?cctccagcag?cttgggcacc????660
cagacctaca?tctgcaacgt?gaatcacaag?cccagcaaca?ccaaggtgga?caagaaagtt????720
ggtgagaggc?cagcacaggg?agggagggtg?tctgctggaa?gcaggctcag?cgctcctgcc????780
tggacgcatc?ccggctatgc?agccccagtc?cagggcagca?aggcaggccc?cgtctgcctc?????840
ttcacccgga?gcctctgccc?gccccactca?tgctcaggga?gagggtcttc?tggctttttc?????900
ccaggctctg?ggcaggcaca?ggctaggtgc?ccctaaccca?ggccctgcac?acaaaagggc?????960
aggtgctggg?ctcagacctg?ccaagagcca?tatccgggag?gaccctgccc?ctgacctaag????1020
cccaccccaa?aggccaaact?ctccactccc?tcagctcgga?caccttctct?cctcccagat????1080
tccagtaact?cccaatcttc?tctctgcaga?gcccaaatct?tgtgacaaaa?ctcacacatg????1140
cccaccgtgc?ccaggtaagc?cagcccaggc?ctcgccctcc?agctcaaggc?gggacaggtg????1200
ccctagagta?gcctgcatcc?agggacaggc?cccagccggg?tgctgacacg?tccacctcca????1260
tctcttcctc?agcacctgaa?ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac????1320
ccaaggacac?cctcatgatc?tcccggaccc?ctgaggtcac?atgcgtggtg?gtggacgtga????1380
gccacgaaga?ccctgaggtc?aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg????1440
ccaagacaaa?gccgcgggag?gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca????1500
ccgtcctgca?ccaggactgg?ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag????1560
ccctcccagc?ccccatcgag?aaaaccatct?ccaaagccaa?aggtgggacc?cgtggggtgc????1620
gagggccaca?tggacagagg?ccggctcggc?ccaccctctg?ccctgagagt?gaccgctgta????1680
ccaacctctg?tcctacaggg?cagccccgag?aaccacaggt?gtacaccctg?cccccatccc????1740
gggatgagct?gaccaagaac?caggtcagcc?tgacctgcct?ggtcaaaggc?ttctatccca????1800
gcgacatcgc?cgtggagtgg?gagagcaatg?ggcagccgga?gaacaactac?aagaccacgc????1860
ctcccgtgct?ggactccgac?ggctccttct?tcctctacag?caagctcacc?gtggacaaga????1920
gcaggtggca?gcaggggaac?gtcttctcat?gctccgtgat?gcatgaggct?ctgcacaacc????1980
actacacgca?gaagagcctc?tccctgtctc?ccggtaaagg?cggtggaggc?tctggtggag????2040
gcggttcagg?aggcggtgga?tctacccagg?actgctcctt?ccaacacagc?cccatctcct????2100
ccgacttcgc?tgtcaaaatc?cgtgagctgt?ctgactacct?gcttcaagat?tacccagtca????2160
ccgtggcctc?caacctgcag?gacgaggagc?tctgcggggg?cctctggcgg?ctggtcctgg????2220
cacagcgctg?gatggagcgg?ctcaagactg?tcgctgggtc?caagatgcaa?ggcttgctgg????2280
agcgcgtgaa?cacggagata?cactttgtca?ccaaatgtgc?ctttcagccc?ccccccagct????2340
gtcttcgctt?cgtccagacc?aacatctccc?gcctcctgca?ggagacctcc?gagcagctgg????2400
tggcgctgaa?gccctggatc?actcgccaga?acttctcccg?gtgcctggag?ctgcagtgtc????2460
agcccgactc?ctcaaccctg?ccacccccat?ggagtccccg?gcccctggag?gccacagccc????2520
cgacagcccc?gtga??????????????????????????????????????????????????????2534
<210>32
<211>639
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(639)
＜223〉anti-p230 antibody fusion protein
<400>32
Met?Glu?Trp?Ser?Trp?Ile?Phe?Leu?Phe?Leu?Leu?Ser?Gly?Thr?Ala?Gly
1???????????????5???????????????????10??????????????????15
Val?His?Ser?Glu?Val?Gln?Leu?Gln?Gln?Ser?Gly?Pro?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?Val?Met?His?Trp?Val?Lys?Gln?Lys?Pro?Gly?Gln?Gly?Leu
50??????????????????55??????????????????60
Asp?Trp?Ile?Gly?Tyr?Ile?Val?Pro?Tyr?Asn?Asp?Gly?Thr?Lys?Tyr?Asn
65??????????????????70??????????????????75??????????????????80
Glu?Lys?Phe?Lys?Gly?Lys?Ala?Thr?Leu?Thr?Ser?Asp?Lys?Ser?Ser?Ser
85??????????????????90??????????????????95
Thr?Ala?Tyr?Met?GLu?Leu?Ser?Arg?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val
100?????????????????105?????????????????110
Tyr?Tyr?Cys?Val?Tyr?Gly?Ser?Arg?Tyr?Asp?Trp?Tyr?Leu?Asp?Val?Trp
115?????????????????120?????????????????125
Gly?Ala?Gly?Thr?Thr?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro
130?????????????????135?????????????????140
Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr
145?????????????????150?????????????????155?????????????????160
Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr
165?????????????????170?????????????????175
Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro
180?????????????????185?????????????????190
Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr
195?????????????????200?????????????????205
Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn
210?????????????????215?????????????????220
His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser
225?????????????????230?????????????????235?????????????????240
Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu
245?????????????????250?????????????????255
Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu
260?????????????????265?????????????????270
Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser
275?????????????????280?????????????????285
His?Glu?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?Asp?Gly?Val?Glu
290?????????????????295?????????????????300
Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr
305?????????????????310?????????????????315?????????????????320
Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn
325?????????????????330?????????????????335
Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro
340?????????????????345?????????????????350
Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln
355?????????????????360?????????????????365
Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val
370?????????????????375????????????????380
Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val
385?????????????????390?????????????????395?????????????????400
Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro
405?????????????????410?????????????????415
Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr
420?????????????????425?????????????????430
Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val
435?????????????????440?????????????????445
Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu
450?????????????????455?????????????????460
Ser?Pro?Gly?Lys?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly
465?????????????????470?????????????????475?????????????????480
Gly?Gly?Ser?Thr?Gln?Asp?Cys?Ser?Phe?Gln?His?Ser?Pro?Ile?Ser?Ser
485?????????????????490?????????????????495
Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu?Ser?Asp?Tyr?Leu?Leu?Gln?Asp
500?????????????????505?????????????????510
Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu?Gln?Asp?Glu?Glu?Leu?Cys?Gly
515?????????????????520?????????????????525
Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln?Arg?Trp?Met?Glu?Arg?Leu?Lys
530?????????????????535?????????????????540
Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly?Leu?Leu?Glu?Arg?Val?Asn?Thr
545?????????????????550?????????????????555?????????????????560
Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala?Phe?Gln?Pro?Pro?Pro?Ser?Cys
565?????????????????570?????????????????575
Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser?Arg?Leu?Leu?Gln?Glu?Thr?Ser
580?????????????????585?????????????????590
Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp?Ile?Thr?Arg?Gln?Asn?Phe?Ser
595?????????????????600?????????????????605
Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro?Asp?Ser?Ser?Thr?Leu?Pro?Pro
610?????????????????615?????????????????620
Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala?Thr?Ala?Pro?Thr?Ala?Pro
625?????????????????630?????????????????635
<210>33
<211>1986
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(1986)
＜223〉anti-p230 antibody fusion protein encoding sequence
<220>33
atgacagtgc?tggcgccagc?ctggagccca?acaacctatc?tcctcctgct?gctgctgctg?????60
agctcgggac?tcagtgggac?ccaggactgc?tccttccaac?acagccccat?ctcctccgac????120
ttcgctgtca?aaatccgtga?gctgtctgac?tacctgcttc?aagattaccc?agtcaccgtg????180
gcctccaacc?tgcaggacga?ggagctctgc?gggggcctct?ggcggctggt?cctggcacag????240
cgctggatgg?agcggctcaa?gactgtcgct?gggtccaaga?tgcaagactt?gctggagcgc????300
gtgaacacgg?agatacactt?tgtcaccaaa?tgtgcctttc?agcccccccc?cagctgtctt????360
cgcttcgtcc?agaccaacat?ctcccgcctc?ctgcaggaga?cctccgagca?gctggtggcg????420
ctgaagccct?ggatcactcg?ccagaacttc?tcccggtgcc?tggagctgca?gtgtcagccc????480
gactcctcaa?ccctgccacc?cccatggagt?ccccggcccc?tggaggccac?agccccgaca????540
gccccggagc?ccaaatcttg?tgacaaaact?cacacatgcc?caccgtgccc?agcacctgaa????600
ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac?ccaaggacac?cctcatgatc????660
tcccggaccc?ctgaggtcac?aggcgtggtg?gtggacgtga?gccacgaaga?ccctgaggtc????720
aagttcaact?ggtacgtgga?cgacgtggag?gtgcataatg?ccaagacaaa?gccgcgggag????780
gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca?ccgtcctgca?ccaggactgg????840
ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag?ccctcccagc?ccccatcgag????900
aaaaccatct?ccaaagccaa?agggcagccc?cgagaaccac?aggtgtacac?cctgccccca????960
tcccgggatg?agctgaccaa?gaaccaggtc?agcctgacct?gcctggtcaa?aggcttctat???1020
cccagcgaca?tcgccgtgga?gtgggagagc?aatgggcagc?cggagaacaa?ctacaagacc???1080
acgcctcccg?tgctggactc?cgacggctcc?ttcttcctct?acagcaagct?caccgtggac???1140
aagagcaggt?ggcagcaggg?gaacgtcttc?tcatgctccg?tgatgcatga?ggctctgcac???1200
aaccactaca?cgcagaagag?cctctccctg?tctcccggta?aagaggtcca?gctgcagcag???1260
tctggacctg?agctggtaaa?gcctggggct?tcagtgaaga?tgtcctgcaa?ggcttctgga???1320
tacacattca?ctagctatgt?tatgcactgg?gtgaagcaga?agcctgggca?gggccttgac???1380
tggattggat?atattgttcc?ttacaatgat?ggcactaagt?acaatgagaa?gttcaaaggc???1440
aaggccacac?tgacttcaga?caaatcctcc?agcacagcct?acatggagct?cagcagactg???1500
acctctgagg?actctgcggt?ctattattgt?gtctacggta?gtaggtacga?ctggtattta???1560
gatgtctggg?gcgcagggac?cacggtcacc?gtctcctcag?gcggtggagg?ctctggtgga???1620
ggcggttcag?gaggcggtgg?atctaacatt?atgatgacac?agtcgccatc?atctctggct???1680
gtgtctgcag?gagaaaaggt?cactatgagc?tgtaagtcca?gtcaaagtgt?tttatacagt???1740
tcaaatcaga?agaactactt?ggcctggtac?cagcagaaac?cagggcagtc?tcctaaactg???1800
ctgatctact?gggcatccac?tagggaatct?ggtgtccctg?atcgcttcac?aggcagtgga???1860
tctgggacag?attttactct?taccatcagc?agtgtacaag?ctgaagacct?ggcagtttat???1920
tactgtcatc?aatatttctc?ctcatacacg?ttcggagggg?ggaccaagct?ggaaataaag???1980
cggtga??????????????????????????????????????????????????????????????1986
<210>34
<211>661
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(661)
＜223〉anti-p230 antibody fusion protein
<220>34
Met?Thr?Val?Leu?Ala?Pro?Ala?Trp?Ser?Pro?Thr?Thr?Tyr?Leu?Leu?Leu
1???????????????5???????????????????10??????????????????15
Leu?Leu?Leu?Leu?Ser?Ser?Gly?Leu?Ser?Gly?Thr?Gln?Asp?Cys?Ser?Phe
20??????????????????25??????????????????30
Gln?His?Ser?Pro?Ile?Ser?Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu
35??????????????????40??????????????????45
Ser?Asp?Tyr?Leu?Leu?Gln?Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu
50??????????????????55??????????????????60
Gln?Asp?Glu?Glu?Leu?Cys?Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln
65??????????????????70??????????????????75??????????????????80
Arg?Trp?Met?Glu?Arg?Leu?Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly
85??????????????????90??????????????????95
Leu?Leu?Glu?Arg?Val?Asn?Thr?Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala
100?????????????????105?????????????????110
Phe?Gln?Pro?Pro?Pro?Ser?Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser
115?????????????????120?????????????????125
Arg?Leu?Leu?Gln?Glu?Thr?Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp
130?????????????????135?????????????????140
Ile?Thr?Arg?Gln?Asn?Phe?Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro
145?????????????????150?????????????????155?????????????????160
Asp?Ser?Ser?Thr?Leu?Pro?Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala
165?????????????????170?????????????????175
Thr?Ala?Pro?Thr?Ala?Pro?Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr
180?????????????????185?????????????????190
Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe
195?????????????????200?????????????????205
Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro
210?????????????????215?????????????????220
Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val
225?????????????????230?????????????????235?????????????????240
Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr
245?????????????????250?????????????????255
Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val
260?????????????????265?????????????????270
Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys
275?????????????????280?????????????????285
Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser
290?????????????????295?????????????????300
Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro
305?????????????????310?????????????????315?????????????????320
Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val
325?????????????????330?????????????????335
Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly
340?????????????????345?????????????????350
Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp
355?????????????????360?????????????????365
Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp
370?????????????????375?????????????????380
Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His
385?????????????????390?????????????????395?????????????????400
Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys?Glu?Val
405?????????????????410?????????????????415
Gln?Leu?Gln?Gln?Ser?Gly?Pro?Glu?Leu?Val?Lys?Pro?Gly?Ala?Ser?Val
420?????????????????425?????????????????430
Lys?Met?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Ser?Tyr?Val?Met
435?????????????????440?????????????????445
His?Trp?Val?Lys?Gln?Lys?Pro?Gly?Gln?Gly?Leu?Asp?Trp?Ile?Gly?Tyr
450?????????????????455?????????????????460
Ile?Val?Pro?Tyr?Asn?Asp?Gly?Thr?Lys?Tyr?Asn?Glu?Lys?Phe?Lys?Gly
465?????????????????470?????????????????475?????????????????480
Lys?Ala?Thr?Leu?Thr?Ser?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr?Met?Glu
485?????????????????490?????????????????495
Leu?Ser?Arg?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys?Val?Tyr
500?????????????????505?????????????????510
Gly?Ser?Arg?Tyr?Asp?Trp?Tyr?Leu?Asp?Val?Trp?Gly?Ala?Gly?Thr?Thr
515?????????????????520?????????????????525
Val?Thr?Val?Ser?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly
530?????????????????535?????????????????540
Gly?Gly?Gly?Ser?Asn?Ile?Met?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ala
545?????????????????550?????????????????555?????????????????560
Val?Ser?Ala?Gly?Glu?Lys?Val?Thr?Met?Ser?Cys?Lys?Ser?Ser?Gln?Ser
565?????????????????570?????????????????575
Val?Leu?Tyr?Ser?Ser?Asn?Gln?Lys?Asn?Tyr?Leu?Ala?Trp?Tyr?Gln?Gln
580?????????????????585?????????????????590
Lys?Pro?Gly?Gln?Ser?Pro?Lys?Leu?Leu?Ile?Tyr?Trp?Ala?Ser?Thr?Arg
595?????????????????600?????????????????605
Glu?Ser?Gly?Val?Pro?Asp?Arg?Phe?Thr?Gly?Ser?Gly?Ser?Gly?Thr?Asp
610?????????????????615?????????????????620
Phe?Thr?Leu?Thr?Ile?Ser?Ser?Val?Gln?Ala?Glu?Asp?Leu?Ala?Val?Tyr
625?????????????????630?????????????????635?????????????????640
Tyr?Cys?His?Gln?Tyr?Phe?Ser?Ser?Tyr?Thr?Phe?Gly?Gly?Gly?Thr?Lys
645?????????????????650?????????????????655
Leu?Glu?Ile?Lys?Arg
660
<210>35
<211>426
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(428)
＜223〉anti-CD20 antibodies encoding sequence
<220>35
gccaccatgg?gattcagcag?gatctttctc?ttcctcctgt?cagtaactac?aggtgtccac?????60
tcccaggtac?aactacagca?gcctggggct?gagctggtga?agcctggggc?ctcagtgaag????120
atgtcctgca?aggcttctgg?ctacacattt?accagttaca?atatgcactg?ggtaaagcag????180
acacctggtc?ggggcctgga?atggattgga?gctatttatc?caggaaatgg?tgatacttcc????240
tacaatcaga?agttcaaggg?caaggccaca?ctgactgcag?acaaatcctc?cagcacagcc????300
tacatgcagc?tcagcagcct?gacatctgaa?gactctgcgg?tctattactg?tgcaagatcg????360
acttactacg?gcggtgactg?gtacttcaat?gtctggggcg?cagggaccac?ggtcaccgtc????420
tctgca???????????????????????????????????????????????????????????????426
<210>36
<211>140
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(140)
＜223〉anti-CD20 antibodies
<220>36
Met?Gly?Phe?Ser?Arg?Ile?Phe?Leu?Phe?Leu?Leu?Ser?Val?Thr?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
130?????????????????135?????????????????140
<210>37
<211>390
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(390)
＜223〉anti-CD20 antibodies encoding sequence
<220>37
accatggatt?ttcaagtgca?gattttcagc?ttcctgctaa?tcagtgcttc?agtcataatg?????60
tccagaggac?aaattgttct?ctcccagtct?ccagcaatcc?tgtctgcatc?tccaggggag????120
aaggtcacaa?tgacttgcag?ggccagctca?agtgtaagtt?acatccactg?gttccagcag????180
aagccaggat?cctcccccaa?accctggatt?tatgccacat?ccaacctggc?ttctggagtc????240
cctgttcgct?tcagtggcag?tgggtctggg?acctcttact?ctctcacaat?cagtagagtg????300
gaggctgaag?atgctgccac?ttattactgc?cagcagtgga?ctagtaaccc?acccacgttc????360
ggtggtggga?ccaagctgga?gatcaaacga?????????????????????????????????????390
<210>38
<211>129
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(129)
＜223〉anti-CD20 antibodies
<220>38
Met?Asp?Phe?Gln?Val?Gln?Ile?Phe?Ser?Phe?Leu?Leu?Ile?Ser?Ala?Ser
1???????????????5???????????????????10??????????????????15
Val?Ile?Met?Ser?Arg?Gly?Gln?Ile?Val?Leu?Ser?Gln?Ser?Pro?Ala?Ile
20??????????????????25??????????????????30
Leu?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr?Cys?Arg?Ala?Ser
35??????????????????40??????????????????45
Ser?Ser?Val?Ser?Tyr?Ile?His?Trp?Phe?Gln?Gln?Lys?Pro?Gly?Ser?Ser
50??????????????????55??????????????????60
Pro?Lys?Pro?Trp?Ile?Tyr?Ala?Thr?Ser?Asn?Leu?Ala?Ser?Gly?Val?Pro
65??????????????????70??????????????????75??????????????????80
Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu?Thr?1le
85??????????????????90??????????????????95
Ser?Arg?Val?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Trp
100?????????????????105?????????????????110
Thr?Ser?Asn?Pro?Pro?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys
115?????????????????120?????????????????125
Arg
<210>39
<211>2021
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(2021)
＜223〉anti-CD20 antibodies encoding sequence
<220>39
gccaccatgg?gattcagcag?gatctttctc?ttcctcctgt?cagtaactac?aggtgtccac??????60
tcccaggtac?aactacagca?gcctggggct?gagctggtag?agcctggggc?ctcagtgaag?????120
atgtcctgca?aggcttctgg?ctacacattt?accagttaca?atatgcactg?ggtaaagcag?????180
acacctggtc?ggggcctgga?atggattgga?gctatttatc?caggaaatgg?tgatacttcc?????240
tacaatcaga?agttcaaggg?caaggccaca?ctgactgcag?acaaatcctc?cagcacagcc?????300
tacatgcagc?tcagcagcct?gacatctgaa?gactctgcgg?tctattactg?tgcaagatcg?????360
acttactacg?gcggtgactg?gtacttcaat?gtctggggcg?cagggaccac?ggtcaccgtc?????420
tctgcagcta?gcaccaaggg?cccatcggtc?ttccccctgg?caccctcctc?caagagcacc?????480
tctgggggca?cagcggccct?gggctgcctg?gtcaaggact?acttccccga?accggtgacg?????540
gtgtcttgga?actcaggcgc?cctgaccagc?ggcgtgcaca?ccttcccggc?tgtcctacag?????600
tcctcaggac?tctactccct?cagcagcgtg?gtgaccgtgc?cctccagcag?cttgggcacc?????660
cagacctaca?tctgcaacgt?gaatcacaag?cccagcaaca?ccaaggtgga?caagaaagtt?????720
ggtgagaggc?cagcacaggg?agggagggtg?tctgctggaa?gcaggctctc?cgctcctgcc?????780
tggacgcatc?ccggctatgc?agccccagtc?cagggcagca?aggcaggccc?cgtctgcctc?????840
ttcacccgga?gcctctgccc?gccccactca?tgctcaggga?gagggtcttc?tggctttttc?????900
ccaggctctg?ggcaggcaca?ggctaggtgc?ccctaaccca?ggccctgcac?acaaaggggc?????960
aggtgctggg?ctcagacctg?ccaagagcca?tatccgggag?gaccctgccc?ctgacctaag????1020
cccaccccaa?aggccaaact?ctccactccc?tcagctcgga?caccttctct?cctcccagat????1080
tccagtaact?cccaatcttc?tctctgcaga?gcccaaatct?tgtgacaaaa?ctcacacatg????1140
cccaccgtgc?ccaggtaagc?cagcccaggc?ctcgccctcc?agctcaaggc?gggacaggtg????1200
ccctagagta?gcctgcatcc?agggacaggc?cccagccggg?tgctgacacg?tccacctcca????1260
tctcttcctc?agcacctgaa?ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac????1320
ccaaggacac?cctcatgatc?tcccggaccc?ctgaggtcac?atgcgtggtg?gtggacgtga????1380
gccacgaaga?ccctgaggtc?aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg????1440
ccaagacaaa?gccgcgggag?gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca????1500
ccgtcctgca?ccaggactgg?ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag????1560
ccctcccagc?ccccatcgag?aaaaccatct?ccaaagccaa?aggtgggacc?cgtggggtgc????1620
gagggccaca?tggacagagg?ccggctcggc?ccaccctctg?ccctgagagt?gaccgctgta????1690
ccaacctctg?tcctacaggg?cagccccgag?aaccacaggt?gtacaccctg?cccccatccc????1740
gggatgagct?gaccaagaac?caggtcagcc?tgacctgcct?ggtcaaaggc?ttctatccca????1800
gcgacatcgc?cgtggagtgg?gagagcaatg?ggcagccgga?gaacaactac?aagaccacgc????1860
ctcccgtgct?ggactccgac?ggctccttct?tcctctacag?caagctcacc?gtggacaaga????1920
gcaggtggca?gcaggggaac?gtcttctcat?gctccgtgat?gcatgaggct?ctgcacaacc????1980
actacacgca?gaagagcctc?tccctgtctc?ccggtaaatg?a????????????????????????2021
<210>40
<211>470
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(470)
＜223〉anti-CD20 antibodies
<220>40
Met?Gly?Phe?Ser?Arg?Ile?Phe?Leu?Phe?Leu?Leu?Ser?Val?Thr?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?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?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?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn
370?????????????????375?????????????????380
Gln?Val?Ser?Leu?Thr?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>41
<211>711
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(711)
＜223〉anti-CD20 antibodies encoding sequence
<220>41
accatggatt?ttcaagtgca?gattttcagc?ttcctgctaa?tcagtgcttc?agtcataatg?????60
tccagaggac?aaattgttct?ctcccagtct?ccagcaatcc?tgtctgcatc?tccaggggag????120
aaggtcacaa?tgacttgcag?ggccagctca?agtgtaagtt?acatccactg?gttccagcag????180
aagccaggat?cctcccccaa?accctggatt?tatgccacat?ccaacctggc?ttctggagtc????240
cctgttcgct?tcagtggcag?tgggtctggg?acctcttact?ctctcacaat?cagtagagtg????300
gaggctgaag?atgctgccac?ttattactgc?cagcagtgga?ctagtaaccc?acccacgttc????360
ggtggtggga?ccaagctgga?gatcaaacga?actgtggctg?caccatctgt?cttcatcttc????420
ccgccatctg?atgagcagtt?gaaatctgga?actgcctctg?ttgtgtgcct?gctgaataac????480
ttctatccca?gagaggccaa?agtacagtgg?aaggtggata?acgccctcca?atcgggtaac????540
tcccaggaga?gtgtcacaga?gcaggacagc?aaggacagca?cctacagcct?cagcagcacc????600
ctgacgctga?gcaaagcaga?ctacgagaaa?cacaaagtct?acgcctgcga?agtcacccat????660
cagggcctga?gctcgcccgt?cacaaagagc?ttcaacaggg?gagagtgtta?g?????????????711
<210>42
<211>235
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(235)
＜223〉anti-CD20 antibodies
<220>42
Met?Asp?Phe?Gln?Val?Gln?Ile?Phe?Ser?Phe?Leu?Leu?Ile?Ser?Ala?Ser
1???????????????5???????????????????10??????????????????15
Val?Ile?Met?Ser?Arg?Gly?Gln?Ile?Val?Leu?Ser?Gln?Ser?Pro?Ala?Ile
20??????????????????25??????????????????30
Leu?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr?Cys?Arg?Ala?Ser
35??????????????????40??????????????????45
Ser?Ser?Val?Ser?Tyr?Ile?His?Trp?Phe?Gln?Gln?Lys?Pro?Gly?Ser?Ser
50??????????????????55??????????????????60
Pro?Lys?Pro?Trp?Ile?Tyr?Ala?Thr?Ser?Asn?Leu?Ala?Ser?Gly?Val?Pro
65??????????????????70??????????????????75??????????????????80
Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu?Thr?Ile
85??????????????????90??????????????????95
Ser?Arg?Val?Glu?Ala?Glu?Asp?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Trp
100?????????????????105?????????????????110
Thr?Ser?Asn?Pro?Pro?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys
115?????????????????120?????????????????125
Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu
130?????????????????135?????????????????140
Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe
145?????????????????150?????????????????155?????????????????160
Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln
165?????????????????170?????????????????175
Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser
180?????????????????185?????????????????190
Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr?Glu
195?????????????????200?????????????????205
Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser?Ser
210?????????????????215?????????????????220
Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys
225?????????????????230?????????????????235
<210>43
<211>2489
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(2489)
＜223〉anti-CD20 antibodies fusion rotein encoding sequence
<220>43
gccaccatgg?gattcagcag?gatctttctc?ttcctcctgt?cagtaactac?aggtgtccac?????60
tcccaggtac?aactacagca?gcctggggct?gagctggtga?agcctggggc?ctcagtgaag????120
atgtcctgca?aggcttctgg?ctacacattt?accaattaca?atatgcactg?ggtaaagcag????180
acacctggtc?ggggcctgga?atggattgga?gctatttatc?caggaaatgg?tgatacttcc????240
tacaatcaga?agttcaaggg?caaggccaca?ctgactgcag?acaaatcctc?cagcacagcc????300
tacatgcagc?tcagcagcct?gacatctgaa?gactctgcgg?tctattactg?tgcaagatcg????360
acttactacg?gcggtgactg?gtacttcaat?gtctggggcg?cagggaccac?ggtcaccgtc????420
tctgcagcta?gcaccaaggg?cccatcggtc?ttccccctgg?caccctcctc?caagagcacc????480
tctgggggca?cagcggccct?gggctgcctg?gtcaaggact?acttccccga?accggtgacg????540
gtgtcttgga?actcaggcgc?cctgaccagc?ggcgtgcaca?ccttcccggc?tgtcctacag????600
tcctcaggac?tctactccct?cagcagcgtg?gtgaccgtgc?cctccagcag?cttgggcacc????660
cagacctaca?tctgcaacgt?gaatcacaag?cccagcaaca?ccaaggtgga?caagaaagtt????720
ggtgagaggc?cagcacaggg?agggagggtg?tctgctggaa?gcaggctcag?cgctcctgcc????780
tggacgcatc?ccggctatgc?agccccagtc?cagggcagca?aggcaggccc?cgtctgcctc????840
ttcacccgga?gcctctgccc?gccccactca?tgctcaggga?gagggtcttc?tggctttttc????900
ccaggctctg?ggcaggcaca?ggctaggtgc?ccctaaccca?ggccctgcac?acaaaggggc????960
aggtgctggg?ctcagacctg?ccaagagcca?tatccgggag?gaccctgccc?ctgacctaag???1020
cccaccccaa?aggccaaact?ctccactccc?tcagctcgga?caccttctct?cctcccagat???1080
tccagtaact?cccaatcttc?tctctgcaga?gcccaaatct?tgtgacaaaa?ctcacacatg???1140
cccaccgtgc?ccaggtaagc?cagcccaggc?ctcgccctcc?agctcaaggc?gggacaggtg???1200
ccctagagta?gcctgcatcc?aaggacaggc?cccagccggg?tgctgacacg?tccacctcca???1260
tctcttcctc?agcacctgaa?ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac???1320
ccaaggacac?cctcatgatc?tcccggaccc?ctgaggtcac?aggcgtggtg?gtggacgtga???1380
gccacgaaga?ccctgaggtc?aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg???1440
ccaagacaaa?gccgcgggag?gagcagtaca?acagcacgta?ccggatggtc?tgcgtcctca???1500
ccgtcctgca?ccaggactgg?ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag???1560
ccctcccagc?ccccatcgag?aaaaccatct?ccaaagccaa?aggtgggacc?cgtggggtgc???1620
gagggccaca?tggacagagg?ccggctcggc?ccaccctctg?ccctgagagt?gaccgctgta???1680
ccaacctctg?tcctacgggg?cagccccgag?aaccacaggt?gtacaccctg?cccccatccc???1740
gggatgagct?gaccaagaac?caggtcagcc?tgacctgcct?ggtcaaaggc?ttctatccca???1800
gcgacatcgc?cgtggagtgg?gagagcaatg?ggcagccgga?gaacaactac?aagaccacgc???1860
ctcccgtgct?ggactccgac?ggctccttct?tcctctacag?caagctcacc?gtggacaaga???1920
gcaggtggca?gcaggggaac?gtcttctcat?gctccgtgat?gcatgaggct?ctgcacaacc???1980
actacacgca?gaagagcctc?tccctgtctc?ccggtaaaac?ccaggactgc?tccttccaac???2040
acagccccat?ctcctccgac?ttcgctgtca?aaatccgtga?gctgtctgac?tacctgcttc???2100
aagattaccc?agtcaccgtg?gcctccaacc?tgcaggacga?ggagctctgc?gggggcctct???2160
ggcggctggt?cctggcacag?cgctggatgg?agcggctcaa?gactgtcgct?gggtccaaga????2220
tgcaaggctt?gctggagcgc?gtgaacacgg?agatacactt?tgtcaccaaa?tgtgcctttc????2280
agcccccccc?cagctgtctt?cgcttcgtcc?agaccaacat?ctcccgcctc?ctgcaggaga????2340
cctccgagca?gctggtggcg?ctgaagccct?ggatcactcg?ccagaacttc?tcccggtgcc????2400
tggagctgca?gtgtcagccc?gactcctcaa?ccctgccacc?cccatggagt?ccccggcccc????2460
tggaggccac?agccccgaca?gccccgtga??????????????????????????????????????2489
<210>44
<211>626
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(626)
＜223〉anti-CD20 antibodies fusion rotein encoding sequence
<220>44
Met?Gly?Phe?Ser?Arg?Ile?Phe?Leu?Phe?Leu?Leu?Ser?Val?Thr?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?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?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?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn
370?????????????????375?????????????????380
Gln?Val?Ser?Leu?Thr?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?????????????????450
Ser?Leu?Ser?Pro?Gly?Lys?Thr?Gln?Asp?Cys?Ser?Phe?Gln?His?Ser?Pro
465?????????????????470?????????????????475?????????????????480
Ile?Ser?Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu?Ser?Asp?Tyr?Leu
485?????????????????490?????????????????495
Leu?Gln?Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu?Gln?Asp?Glu?Glu
500?????????????????505?????????????????510
Leu?Cys?Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln?Arg?Trp?Met?Glu
515?????????????????520?????????????????525
Arg?Leu?Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly?Leu?Leu?Glu?Arg
530?????????????????535?????????????????540
Val?Asn?Thr?Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala?Phe?Gln?Pro?Pro
545?????????????????550?????????????????555?????????????????560
Pro?Ser?Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser?Arg?Leu?Leu?Gln
565?????????????????570?????????????????575
Glu?Thr?Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp?Ile?Thr?Arg?Gln
580?????????????????585?????????????????590
Asn?Phe?Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro?Asp?Ser?Ser?Thr
595?????????????????600?????????????????605
Leu?Pro?Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala?Thr?Ala?Pro?Thr
610?????????????????615?????????????????620
Ala?Pro
625
<210>45
<211>2534
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(2534)
＜223〉anti-CD20 antibodies fusion rotein
<400>45
gccaccatgg?gattcagcag?gatctttctc?ttcctcctgt?cagtaactac?aggtgtccac??????60
tcccaggtac?aactacagca?gcctggggct?gagctggtga?agcctggggc?ctcagtgaag?????120
atgtcctgca?aggcttctgg?ctacacattt?accagttaca?atatgcactg?ggtaaagcag?????180
acacctggtc?ggggcctgga?atggattgga?gctatttatc?caggaaatgg?tgatacttcc?????240
tacaatcaga?agttcaaggg?caaggccaca?ctgactgcag?acaaatcctc?cagcacagcc?????300
tacathcagc?tcagcagcct?gacatctgaa?gactctgcgg?tctattactg?tgcaagatcg?????360
acttactacg?gcggtgactg?gtacttcaat?gtctggggcg?cagggaccac?ggtcaccgtc?????420
tctgcagcta?gcaccaaggg?cccatcggtc?ttccccctgg?caccctcctc?caagagcacc?????480
tctgggggca?cagcggccct?gggctgcctg?gtcaaggact?acttccccga?accggtgacg?????540
gtgtcttgga?actcaggcgc?cctgaccagc?ggcgtgcaca?ccttcccggc?tgtcctacag?????600
tcctcaggac?tctactccct?cagcagcgtg?gtgaccgtgc?cctccagcag?cttgggcacc?????660
cagacctaca?tctgcaacgt?gaatcacaag?cccagcaaca?ccaaggtgga?caagaaagtt?????720
ggtgagaggc?cagcacaggg?agggagggtg?tctgctggaa?gcaggctcag?cgctcctgcc?????780
tggacgcatc?ccggctatgc?agccccagtc?cagggcagca?aggcaggccc?cgtctgcctc?????840
ttcacccgga?gcctctgccc?gccccactca?tgctcaggga?gagggtcttc?tggctttttc?????900
ccaggctctg?ggcaggcaca?ggctaggtgc?ccctaaccca?ggccctgcac?acaaaggggc?????960
aggtgctggg?ctcagacctg?ccaagagcca?tatccgggag?gaccctgccc?ctgacctaag????1020
cccaccccaa?aggccaaact?ctccactccc?tcagctcgga?caccttctct?cctcccagat????1080
tccagtaact?cccaatcttc?tctctgcaga?gcccaaatct?tgtgacaaaa?ctcacacatg????1140
cccaccgtgc?ccaggtaagc?cagcccaggc?ctcgccctcc?agctcaaggc?gggacaggtg????1200
ccctagagta?gcctgcatcc?agggacaggc?cccagccggg?tgctgacacg?tccacctcca????1260
tctcttcctc?agcacctgaa?ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac????1320
ccaaggacac?cctcatggtc?tcccggaccc?ctgaggtcac?atgcgtggtg?gtggacgtga????1380
gccacgaaga?ccctgaggtc?aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg????1440
ccaagacaaa?gccgcgggag?gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca????1500
ccgtcctgca?ccaggactgg?ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag????1560
ccctcccagc?ccccatcgag?aaaaccatct?ccaaagccaa?aggtgggacc?cgtggggtgc????1620
gagggccaca?tggacagagg?caggctcggc?ccaccctctg?ccctgagagt?gaccgctgta????1680
ccaacctctg?tcctacaggg?cagccccgag?aaccacaggt?gtacaccctg?cccccatccc????1740
gggatgagct?gaccaagaac?caggtcagcc?tgacctgcct?ggtcaaaggc?ttctatccca????1800
gcgacatcgc?cgtggagtgg?gagagcaatg?ggcagccgga?gaacaactac?aagaccacgc????1860
ctcccgtgct?ggactccgac?ggctccttct?tcctctacag?caagctcacc?gtggacaaga????1920
gcaggtggca?gcaggggaac?gtcttctcat?gctccgtgat?gcatgaggct?ctgcacaacc????1980
actacacgca?gaagagcctc?tccctgtctc?ccggtaaagg?cggtggaggc?tctggtggag????2040
gcggttcagg?aggcggtgga?tctacccagg?actgctcctt?ccaacacagc?cccatctcct????2100
ccgacttcgc?tgtcaaaatc?cgtgagctgt?ctgactacct?gcttcaagat?tacccagtca????2160
ccgtggcctc?caacctgcag?gacgaggagc?tctgcggggg?cctctggcgg?ctggtcctgg????2220
cacagcgctg?gatggagcgg?ctcaagactg?tcgctgggtc?caagatgcaa?ggcttgctgg????2280
agcgcgtgaa?cacggagata?cactttgtca?ccaaatgtgc?ctttcagccc?ccccccagct????2340
gtcttcgctt?cgtccagacc?aacatctccc?gcctcctgca?ggagacctcc?gagcagctgg????2400
tggcgctgaa?gccctggatc?actcgccaga?acttctcccg?gtgcctggag?ctgcagtgtc????2460
agcccgactc?ctcaaccctg?ccacccccat?ggagtccccg?gcccctggag?gccacagccc????2520
cgacagcccc?gtga??????????????????????????????????????????????????????2534
<210>46
<211>641
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(641)
＜223〉anti-CD20 antibodies fusion rotein encoding sequence
<400>46
Met?Gly?Phe?Ser?Arg?Ile?Phe?Leu?Phe?Leu?Leu?Ser?Val?Thr?Thr?Gly
1???????????????5???????????????????10??????????????????15
Val?His?Ser?Gln?Val?Gln?Leu?Gln?Gln?Pro?Gly?Ala?GIu?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?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp?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?Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn
370?????????????????375?????????????????380
Gln?Val?Ser?Leu?Thr?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?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
465?????????????????470?????????????????475?????????????????480
Gly?Gly?Gly?Gly?Ser?Thr?Gln?Asp?Cys?Ser?Phe?Gln?His?Ser?Pro?Ile
485?????????????????490?????????????????495
Ser?Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu?Ser?Asp?Tyr?Leu?Leu
500?????????????????505?????????????????510
Gln?Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu?Gln?Asp?Glu?Glu?Leu
515?????????????????520?????????????????525
Cys?Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln?Arg?Trp?Met?Glu?Arg
530?????????????????535?????????????????540
Leu?Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly?Leu?Leu?Glu?Arg?Val
545?????????????????550?????????????????555?????????????????560
Asn?Thr?Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala?Phe?Gln?Pro?Pro?Pro
565?????????????????570?????????????????575
Ser?Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser?Arg?Leu?Leu?Gln?Glu
580?????????????????585?????????????????590
Thr?Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp?Ile?Thr?Arg?Gln?Asn
595?????????????????600?????????????????605
Phe?Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro?Asp?Ser?Ser?Thr?Leu
610?????????????????615?????????????????620
Pro?Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala?Thr?Ala?Pro?Thr?Ala
625?????????????????630?????????????????635?????????????????640
Pro
<210>47
<211>1974
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(1976)
＜223〉anti-CD20 antibodies fusion rotein encoding sequence
<220>47
atgacagtgc?tggcgccagc?ctggagccca?acaacctatc?tcctcctgct?gctgctgctg??????60
agctcgggac?tcagtgggac?ccaggactgc?tccttccaac?aaagccccat?ctcctccgac?????120
ttcgctgtca?aaatccgtga?gctgtctgac?tacctgcttc?aagattaccc?agtcaccgtg?????180
gcctccaacc?tgcaggacga?ggagctctgc?gggggcctct?ggcggctggt?cctggcacag?????240
cgctggatgg?agcggctcaa?gactgtcgct?gggtccaaga?tgcaaggctt?gctggagcgc?????300
gtgaacacgg?agatacactt?tgtcaccaaa?tgtgcctttc?agcccccccc?cagctgtctt?????360
cgcttcgtcc?agaccaacat?ctcccgcctc?ctgcaggaga?cctccgagca?gctggtggcg?????420
ctgaagccct?ggatcactcg?ccagaacttc?tcccggtgcc?tggagctgca?gtgtcagccc?????480
gactcctcaa?ccctgccacc?cccatggagt?ccccggcccc?tggaggccac?agccccgaca?????540
gccccggagc?ccaaatcttg?tgacaaaact?cacacatgcc?caccgtgccc?agcacctgaa?????600
ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac?ccaaggacac?cctcatgatc?????660
tcccggaccc?ctgaggtcac?atgcgtggtg?gtggacgtga?gccacgaaga?ccctgaggtc?????720
aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg?ccaagacaaa?gccgcgggag?????780
gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca?ccgtcctgca?ccaggactgg?????840
ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag?ccctcccagc?ccccatcgag?????900
aaaaccatct?ccaaagccaa?agggcagccc?cgagaaccac?aggtgtacac?cctgccccca?????960
tcccgggatg?agctgaccaa?gaaccaggtc?agcctgacct?gcctggtcaa?aggcttctat????1020
cccagcgaca?tcgccgtgga?gtgggagagc?aatgggcagc?cggagaacaa?ctacaagacc????1080
acgcctcccg?tgctggactc?cgacggctcc?ttcttcctct?acagcaagct?caccgtggac????1140
aagagcaggt?ggcagcaggg?gaacgtcttc?tcatgctccg?tgatacatga?ggctctgcac????1200
aaccactaca?cgcagaagag?cctctccctg?tctcccggta?aacaggtaca?actacagcag????1260
cctggggctg?agctggtgaa?gcctggggcc?tcagtgaaga?tgtcctgcaa?ggcttctggc????1320
tacacattta?ccagttacaa?tatgcactgg?gtaaagcaga?cacctggtcg?gggcctggaa????1380
tggattggag?ctatttatcc?aggaaatggt?gatacttcct?acaatcagaa?gttcaagggc????1440
aaggccacac?tgactgcaga?caaatcctcc?agcacagcct?acatgcagct?cagcagcctg????1500
acatctgaag?actctgcggt?ctattactgt?gcaagatcga?cttactacgg?cggtgactgg????1560
tacttcaatg?tctggggcgc?agggaccacg?gtcaccgtct?ctgcaggcgg?tggaggctct????1620
ggtggaggcg?gttcaggagg?cggtggatct?caaattgttc?tctcccagtc?tccagcaatc????1680
ctgtctgcat?ctccagggga?gaaggtcaca?atgacttgca?gggccagctc?aagtgtaagt????1740
tacatccact?ggttccagca?gaagccagga?tcctccccca?aaccctggat?ttatgccaca????1800
tccaacctgg?cttctggagt?ccctgttcgc?ttcagtggca?gtgggtctgg?gacctcttac????1860
tctctcacaa?tcagtagagt?ggaggctgaa?gatgctgcca?cttattactg?ccagcagtgg????1920
actagtaacc?cacccacgtt?cggtggtggg?accaagctgg?agatcaaacg?atga??????????1974
<210>48
<211>657
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(657)
＜223〉anti-CD20 antibodies fusion rotein
<220>48
Met?Thr?Val?Leu?Ala?Pro?Ala?Trp?Ser?Pro?Thr?Thr?Tyr?Leu?Leu?Leu
1???????????????5???????????????????10??????????????????15
Leu?Leu?Leu?Leu?Ser?Ser?Gly?Leu?Ser?Gly?Thr?Gln?Asp?Cys?Ser?Phe
20??????????????????25??????????????????30
Gln?His?Ser?Pro?Ile?Ser?Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu
35??????????????????40??????????????????45
Ser?Asp?Tyr?Leu?Leu?Gln?Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu
50??????????????????55??????????????????60
Gln?Asp?Glu?Glu?Leu?Cys?Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln
65??????????????????70??????????????????75??????????????????80
Arg?Trp?Met?Glu?Arg?Leu?Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?GIy
85??????????????????90??????????????????95
Leu?Leu?Glu?Arg?Val?Asn?Thr?Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala
100?????????????????105?????????????????110
Phe?Gln?Pro?Pro?Pro?Ser?Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser
115?????????????????120?????????????????125
Arg?Leu?Leu?Gln?Glu?Thr?Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp
130?????????????????135?????????????????140
Ile?Thr?Arg?Gln?Asn?Phe?Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro
145?????????????????150?????????????????155?????????????????160
Asp?Ser?Ser?Thr?Leu?Pro?Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala
165?????????????????170?????????????????175
7hr?Ala?Pro?Thr?Ala?Pro?Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr
180?????????????????185?????????????????190
Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe
195?????????????????200?????????????????205
Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro
210?????????????????215?????????????????220
Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val
225?????????????????230?????????????????235?????????????????240
Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr
245?????????????????250?????????????????255
Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val
260?????????????????265?????????????????270
Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys
275?????????????????280?????????????????285
Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser
290?????????????????295?????????????????300
Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro
305?????????????????310?????????????????315?????????????????320
Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val
325?????????????????330?????????????????335
Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly
340?????????????????345?????????????????350
Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp
355?????????????????360?????????????????365
Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp
370?????????????????375?????????????????380
Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His
385?????????????????390?????????????????395?????????????????400
Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys?Gln?Val
405?????????????????410?????????????????415
Gln?Leu?Gln?Gln?Pro?Gly?Ala?Glu?Leu?Val?Lys?Pro?Gly?Ala?Ser?Val
420?????????????????425?????????????????430
Lys?Met?Ser?Cys?Lys?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Ser?Tyr?Asn?Met
435?????????????????440?????????????????445
His?Trp?Val?Lys?Gln?Thr?Pro?Gly?Arg?Gly?Leu?Glu?Trp?Ile?Gly?Ala
450?????????????????455?????????????????460
Ile?Tyr?Pro?Gly?Asn?Gly?Asp?Thr?Ser?Tyr?Asn?Gln?Lys?Phe?Lys?Gly
465?????????????????470?????????????????475?????????????????480
Lys?Ala?Thr?Leu?Thr?Ala?Asp?Lys?Ser?Ser?Ser?Thr?Ala?Tyr?Met?Gln
485?????????????????490?????????????????495
Leu?Ser?Ser?Leu?Thr?Ser?Glu?Asp?Ser?Ala?Val?Tyr?Tyr?Cys?Ala?Arg
500?????????????????505?????????????????510
Ser?Thr?Tyr?Tyr?Gly?Gly?Asp?Trp?Tyr?Phe?Asn?Val?Trp?Gly?Ala?Gly
515?????????????????520?????????????????525
Thr?Thr?Val?Thr?Val?Ser?Ala?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly
530?????????????????535?????????????????540
Ser?Gly?Gly?Gly?Gly?Ser?Gln?Ile?Val?Leu?Ser?Gln?Ser?Pro?Ala?Ile
545?????????????????550?????????????????555?????????????????560
Leu?Ser?Ala?Ser?Pro?Gly?Glu?Lys?Val?Thr?Met?Thr?Cys?Arg?Ala?Ser
565?????????????????570?????????????????575
Ser?Ser?Val?Ser?Tyr?Ile?His?Trp?Phe?Gln?Gln?Lys?Pro?Gly?Ser?Ser
580?????????????????585?????????????????590
Pro?Lys?Pro?Trp?Ile?Tyr?Ala?Thr?Ser?Asn?Leu?Ala?Ser?Gly?Val?Pro
595?????????????????600?????????????????605
Val?Arg?Phe?Ser?Gly?Ser?Gly?Ser?Gly?Thr?Ser?Tyr?Ser?Leu?Thr?Ile
610?????????????????615?????????????????620
Ser?Arg?Val?Glu?Ala?Glu?Ala?Ala?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?Trp
625?????????????????630?????????????????635?????????????????640
Thr?Ser?Asn?Pro?Pro?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Leu?Glu?Ile?Lys
645?????????????????650?????????????????655
Arg
<210>49
<211>426
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(426)
＜223〉anti-Her2 antibody coding sequence
<220>49
atggattttc?aggtgcagat?tttcagcttc?ctgctaatca?gtgcctcagt?cataatatcc?????60
agaggagagg?ttcagctggt?ggagtctggc?ggtggcctgg?tgcagccagg?gggctcactc????120
cgtttgtcct?gtgcagcttc?tggcttcaac?attaaagaca?cctatataca?ctgggtgcgt????180
caggccccgg?gtaagggcct?ggaatgggtt?gcaaggattt?atcctacgaa?tggttatact????240
agatatgccg?atagcgtcaa?gggccgtttc?actataagcg?cagacacatc?caaaaacaca????300
gcctacctgc?agatgaacag?cctgcgtgct?gaggacactg?ccgtctatta?ttgttcraga????360
tggggagggg?acggcttcta?tgctatggac?tactggggtc?aaggaaccct?ggtcaccgtc????420
tcctcg???????????????????????????????????????????????????????????????426
<210>50
<211>142
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(142)
＜223〉anti-Her2 antibody coding sequence
<220>50
Met?Asp?Phe?Gln?Val?Gln?Ile?Phe?Ser?Phe?Leu?Leu?Ile?Ser?Ala?Ser
1???????????????5???????????????????10??????????????????15
Val?Ile?Ile?Ser?Arg?Gly?Glu?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly
20??????????????????25??????????????????30
Leu?Val?Gln?Pro?Gly?Gly?Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly
35??????????????????40??????????????????45
Phe?Asn?Ile?Lys?Asp?Thr?Tyr?Ile?His?Trp?Val?Arg?Gln?Ala?Pro?Gly
50??????????????????55??????????????????60
Lys?Gly?Leu?Glu?Trp?Val?Ala?Arg?Ile?Tyr?Pro?Thr?Asn?Gly?Tyr?Thr
65??????????????????70??????????????????75??????????????????80
Arg?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Ala?Asp?Thr
85??????????????????90??????????????????95
Ser?Lys?Asn?Thr?Ala?Tyr?Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp
100?????????????????105?????????????????110
Thr?Ala?Val?Tyr?Tyr?Cys?Ser?Arg?Trp?Gly?Gly?Asp?Gly?Phe?Tyr?Ala
115?????????????????120?????????????????125
Met?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser
130?????????????????135?????????????????140
<210>51
<211>390
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(390)
＜223〉anti-Her2 antibody coding sequence
<220>51
atggattttc?aggtgcagat?tttcagcttc?ctgctaatca?gtgcctcagt?cataatatcc?????60
agaggagaca?tccagatgac?ccagtccccg?agctccctgt?ccgcctctgt?gggcgatagg????120
gttaccatca?cctgccgtgc?cagtcaggat?gtgaatactg?ctgtagcctg?gtatcaacag????180
aaaccaggaa?aagctccgaa?actactgatt?tactcggcat?ccttcctcta?ctctggagtc????240
ccttctcgct?tctctggctc?cagatctggg?acggatttca?ctctgaccat?cagcagtctg????300
cagccggaag?acttcgcaac?ttattactgt?cagcaacatt?atactactcc?tcccacgttc????360
ggacagggta?ccaaggtgga?gatcaaacgt?????????????????????????????????????390
<210>52
<211>130
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(130)
＜223〉anti-Her2 antibody
<220>52
Met?Asp?Phe?Gln?Val?Gln?Ile?Phe?Ser?Phe?Leu?Leu?Ile?Ser?Ala?Ser
1???????????????5???????????????????10??????????????????15
Val?Ile?Ile?Ser?Arg?Gly?Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser
20??????????????????25??????????????????30
Leu?Ser?Ala?Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser
35??????????????????40??????????????????45
Gln?Asp?Val?Asn?Thr?Ala?Val?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys
50??????????????????55??????????????????60
Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Ser?Ala?Ser?Phe?Leu?Tyr?Ser?Gly?Val
65??????????????????70??????????????????75??????????????????80
Pro?Ser?Arg?Phe?Ser?Gly?Ser?Arg?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr
85??????????????????90??????????????????95
Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln
100?????????????????105?????????????????110
His?Tyr?Thr?Thr?Pro?Pro?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile
115?????????????????120?????????????????125
Lys?Arg
130
<210>53
<211>2021
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_featute
<222>(1)..(2021)
＜223〉anti-Her2 antibody coding sequence
<220>53
atggattttc?aggtgcagat?tttcagcttc?ctgctaatca?gtgcctcagt?cataatatcc?????60
agaggagagg?ttcagctggt?ggagtctggc?ggtggcctgg?tgcagccagg?gggctcactc????120
cgtttgtcct?gtgcagcttc?tggcttcaac?attaaagaca?cctatataca?ctgggtgcgt????180
caggccccgg?gtaagggcct?ggaatgggtt?gcaaggattt?atcctacgaa?tggttatact????240
agatatgccg?atagcgtcaa?gggccgtttc?actataagcg?cagacacatc?caaaaacaca????300
gcctacctgc?agatgaacag?cctgcgtgct?gaggacactg?ccgtctatta?ttgttctaga????360
tggggagggg?acggcttcta?tgctatggac?tactggggtc?aaggaaccct?ggtcaccgtc????420
tcctcggcta?gcaccaaggg?cccatcggtc?ttccccctgg?caccctcctc?caagagcacc????480
tctgggggca?cagcggccct?gggctgcctg?gtcaaggact?acttccccga?accggtgacg????540
gtgtcttgga?actcaggcgc?cctgaccagc?ggcgtgcaca?ccttcccggc?tgtcctacag????600
tcctcaggac?tctactccct?cagcagcgtg?gtgaccgtgc?cctccagcag?crtgggcacc????660
cagacctaca?tctgcaacgt?gaatcacaag?cccagcaaca?ccaaggtgga?caagaaagtt????720
ggtgagaggc?cagcacaggg?gaggagggtg?tctgctggaa?gcaggctcag?cgctcctgcc?????780
tggacgcatc?ccggctatgc?agccccagtc?cagggcagca?aggcaggccc?cgtctgcctc?????840
ttcacccgga?gcctctgccc?gccccactca?tgctcaggga?gagggtcttc?tggctttttc?????900
ccaggctctg?ggcaggcaca?ggctaggtgc?ccctaaccca?ggccctgcac?acaaaggggc?????960
aggtgctggg?ctcagacctg?ccaagagcca?tatccgggag?gaccctgccc?ctgacctaag????1020
cccaccccaa?aggccaaact?ctccactccc?tcagctcgga?caccttctct?cctcccagat????1080
tccagtaact?cccaatcttc?tctctgcaga?gcccaaatct?tgtgacaaaa?ctcacacatg????1140
cccaccgtgc?ccaggtaagc?cagcccaggc?ctcgccctcc?agctcaaggc?gggacaggtg????1200
ccctagagta?gcctgcatcc?agggacaggc?cccagccggg?tgctgacacg?tccacctcca????1260
tctcttcctc?agcacctgaa?ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac????1320
ccaaggacac?cctcatgatc?tcccggaccc?ctgaggtcac?atgcgtggtg?gtggacgtga????1380
gccacgaaga?ccctgaggtc?aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg????1440
ccaagacaaa?gccgcgggag?gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca????1500
ccgtcctgca?ccaggactgg?ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag????1560
ccctcccagc?ccccatcgag?aaaaccatct?ccaaagccaa?aggtgggacc?cgtggggtgc????1620
gagggccaca?tggacagagg?ccggctcggc?ccaccctctg?ccctgagagt?gaccgctgta????1680
ccaacctctg?tcctacaggg?cagccccgag?aaccacaggt?gtacaccctg?cccccatccc????1740
gggatgagct?gaccaagaac?caggtcagcc?tgacctgcct?ggtcaaaggc?ttctatccca????1800
gcgacatcgc?cgtggagtgg?gagagcaatg?ggcagccgga?gaacaactac?aagaccacgc????1860
ctcccgtgct?ggactccgac?ggctccttct?tcctctacag?caagctcacc?gtggacaaga????1920
gcaggtggca?gcaggggaac?gtcttctcat?gctccgtgat?gcatgaggct?ctgcacaacc????1980
actacacgca?gaagagcctc?tccctgtctc?ccggtaaatg?a????????????????????????2021
<210>54
<211>472
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(472)
＜223〉anti-Her2 antibody coding sequence
<220>54
Met?Asp?Phe?Gln?Val?Gln?Ile?Phe?Ser?Phe?Leu?Leu?Ile?Ser?Ala?Ser
1???????????????5???????????????????10??????????????????15
Val?Ile?Ile?Ser?Arg?Gly?Glu?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly
20??????????????????25??????????????????30
Leu?Val?Gln?Pro?Gly?Gly?Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly
35??????????????????40??????????????????45
Phe?Asn?Ile?Lys?Asp?Thr?Tyr?Ile?His?Trp?Val?Arg?Gln?Ala?Pro?Gly
50??????????????????55??????????????????60
Lys?Gly?Leu?Glu?Trp?Val?Ala?Arg?Ile?Tyr?Pro?Thr?Asn?Gly?Tyr?Thr
65??????????????????70??????????????????75??????????????????80
Arg?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Ala?Asp?Thr
85??????????????????90??????????????????95
Ser?Lys?Asn?Thr?Ala?Tyr?Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp
100?????????????????105?????????????????110
Thr?Ala?Val?Tyr?Tyr?Cys?Ser?Arg?Trp?Gly?Gly?Asp?Gly?Phe?Tyr?Ala
115?????????????????120?????????????????125
Met?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser
130?????????????????135?????????????????140
Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr
145?????????????????150?????????????????155?????????????????160
Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro
165?????????????????170?????????????????175
Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val
180?????????????????185?????????????????190
His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser
195?????????????????200?????????????????205
Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile
210?????????????????215?????????????????220
Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val
225?????????????????230?????????????????235?????????????????240
Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala
245?????????????????250?????????????????255
Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro
260?????????????????265?????????????????270
Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val
275?????????????????280?????????????????285
Val?Asp?Val?Ser?His?Glu?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp
290?????????????????295?????????????????300
Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln
305?????????????????310?????????????????315?????????????????320
Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln
325?????????????????330?????????????????335
Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala
340?????????????????345?????????????????350
Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro
355?????????????????360?????????????????365
Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr
370?????????????????375?????????????????380
Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser
385?????????????????390?????????????????395?????????????????400
Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr
405?????????????????410?????????????????415
Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr
420?????????????????425?????????????????430
Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe
435?????????????????440?????????????????445
Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys
450?????????????????455?????????????????460
Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys
465?????????????????470
<210>55
<211>711
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(711)
＜223〉anti-Her2 antibody coding sequence
<220>55
atggattttc?aggtgcagat?tttcagcttc?ctgctaatca?gtgcctcagt?cataatatcc?????60
agaggagaca?tccagatgac?ccagtccccg?agctccctgt?ccgcctctgt?gggcgatagg????120
gttaccatca?cctgccgtgc?cagtcaggat?gtgaatactg?ctgtagcctg?gtatcaacag????180
aaaccaggaa?aagctccgaa?actactgatt?tactcggcat?ccttcctcta?ctctggagtc????240
ccttctcgct?tctctggctc?cagatctggg?acggatttca?ctctgaccat?cagcagtctg????300
cagccggaag?acttcgcaac?ttattactgt?cagcaacatt?atactactcc?tcccacgttc????360
ggacagggta?ccaaggtgga?gatcaaacgt?actgtggctg?caccatctgt?cttcatcttc????420
ccgccatctg?atgagcagtt?gaaatctgga?actgcctctg?ttgtgtgcct?gctgaataac????480
ttctatccca?gagaggccaa?attacagtgg?aaggtggata?acgccctcca?atcgggtaac????540
tcccaggaga?gtgtcacaga?gcaggacagc?aaggacagca?cctacagcct?cagcagcacc????600
ctgacgctga?gcaaagcaga?ctacgagaaa?cacaaagtct?acgcctgcga?agtcacccat????660
cagggcctga?gctcgcccgt?cacaaagagc?ttcaacaggg?gagagtgtta?g?????????????711
<210>56
<211>236
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(236)
＜223〉anti-Her2 antibody
<220>56
Met?Asp?Phe?Gln?Val?Gln?Ile?Phe?Ser?Phe?Leu?Leu?Ile?Ser?Ala?Ser
1???????????????5???????????????????10??????????????????15
Val?Ile?Ile?Ser?Arg?Gly?Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser
20??????????????????25??????????????????30
Leu?Ser?Ala?Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ala?Ser
35??????????????????40??????????????????45
Gln?Asp?Val?Asn?Thr?Ala?Val?Ala?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys
50?????????????????55?????????????????60
Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Ser?Ala?Ser?Phe?Leu?Tyr?Ser?Gly?Val
65??????????????????70??????????????????75??????????????????80
Pro?Ser?Arg?Phe?Ser?Gly?Ser?Arg?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr
85??????????????????90??????????????????95
Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Gln?Gln
100?????????????????105?????????????????110
His?Tyr?Thr?Thr?Pro?Pro?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Ile
115?????????????????120?????????????????125
Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp
130?????????????????135?????????????????140
Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn
145?????????????????150?????????????????155?????????????????160
Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu
165?????????????????170?????????????????175
Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp
180?????????????????185?????????????????190
Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr
195?????????????????200?????????????????205
Glu?Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser
210?????????????????215?????????????????220
Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys
225?????????????????230?????????????????235
<210>57
<211>2489
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(2489)
＜223〉anti-Her2 antibody fusion protein encoding sequence
<220>57
atggattttc?aggtgcagat?tttcagcttc?ctgctaatca?gtgcctcagt?cataatatcc??????60
agaggagagg?ttcagctggt?ggagtctggc?ggtggcctgg?tgcagccagg?gggctcactc?????120
cgtttgtcct?gtgcagcttc?tggcttcaac?attaaagaca?cctatataca?ctgggtgcgt?????180
caggccccgg?gtaagggcct?ggaatgggtt?gcaaggattt?atcctacgaa?tggttatact?????240
agatatgccg?atagcgtcaa?gggccgtttc?actataagcg?cagacacatc?caaaaacaca?????300
gcctacctgc?agatgaacag?cctgcgtgct?gaggacactg?ccgtctatta?ttgttctaga?????360
tggggagggg?acggcttcta?tgctatggac?tactggggtc?aaggaaccct?ggtcaccgtc?????420
tcctcggcta?gcaccaaggg?cccatcggtc?ttccccctgg?caccctcctc?caagagcacc?????480
tctgggggca?cagcggccct?gggctgcctg?gtcaaggact?acttccccga?accggtgacg?????540
gtgtcttgga?actcaggcgc?cctgaccagc?ggcgtgcaca?ccttcccggc?tgtcctacag?????600
tcctcaggac?tctactccct?cagcagcgtg?gtgaccgtgc?cctccagcag?cttgggcacc?????660
cagacctaca?tctgcaacgt?gaatcacaag?cccagcaaca?ccaaggtgga?caagaaagtt?????720
ggtgagaggc?cagcacaggg?agggagggtg?tctgctggaa?gcaggctcag?cgctcctgcc?????780
tggacgcatc?ccggctatgc?agccccagtc?cagggcagca?aggcaggccc?cgtctgcctc?????840
ttcacccgga?gcctctgccc?gccccactca?tgctcaggga?gagggtcttc?tggctttttc?????900
ccaggctctg?ggcaggcaca?ggctaggtgc?ccctaaccca?ggccctgcac?acaaaggggc?????960
aggtgctggg?ctcagacctg?ccaagagcca?tatccgggag?gaccctgccc?ctgacctaag????1020
cccaccccaa?aggccaaact?ctccactccc?tcagctcgga?caccttctct?cctcccagat????1080
tccagtaact?cccaatcttc?tctctgcaga?gcccaaatct?tgtgacaaaa?ctcacacatg????1140
cccaccgtgc?ccaggtaagc?cagcccaggc?ctcgccctcc?agctcaaggc?gggacaggtg????1200
ccctagagta?gcctgcatcc?agggacaggc?cccagccggg?tgctgacacg?tccacctcca????1260
tctcttcctc?agcacctgaa?ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac????1320
ccaaggacac?cctcatgatc?tcccggaccc?ctgaggtcac?atgcgtggtg?gtggacgtga????1380
gccacgaaga?ccctgaggtc?aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg????1440
ccaagacaaa?gccgcgggag?gagcagtaca?acagcactta?ccgggtggtc?tgcgtcctca????1500
ccgtcctgca?ccaggactgg?ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag????1560
ccctcccagc?ccccatcgag?aaaaccatct?ccaaagccaa?aggtgggacc?cgtggggtgc????1620
gagggccaca?tggacagagg?ccggctcggc?ccaccctctg?ccctgagagt?gaccgctgta????1680
ccaacctctg?tcctacaggg?cagccccgag?aaccacaggt?gtacaccctg?cccccatccc????1740
gggatgagct?gaccaagaac?caggtcagcc?tgacctgcct?ggtcaaaggc?ttctatccca????1800
gcgacatcgc?cgtggagtgg?gagagcaatg?ggcagccgga?gaacaactac?aagaccacgc????1860
ctcccgtgct?gaactccgac?ggctccttct?tcctctacag?caagctcacc?gtggacaaga????1920
gcaggtggca?gcaggggaac?gtcttctcat?gctccgtgat?gcatgaggct?ctgcacaacc????1980
actacacgca?gaagagcctc?tccctgtctc?ccgataaaac?ccaggactgc?tccttccaac????2040
acagccccat?ctcctccgac?ttcgctgtca?aaatccgtga?gctgtctgac?tacctgcttc????2100
aagattaccc?agtcaccgtg?gcctccaacc?tgcaggacga?ggagctctgc?gggggcctct????2160
ggcggctggt?cctggcacag?cgctggatgg?agcggctcaa?gactgtcgct?gggtccaaga????2220
tgcaaggctt?gctggagcgc?gtgaacacgg?agatacactt?tgtcaccaaa?tgtgcctttc????2280
agcccccccc?cagctgtctt?cgcttcgtcc?agaccaacat?ctcccgcctc?ctgcaggaga????2340
cctccgagca?gctggtggcg?ctgaagccct?ggatcactcg?ccagaacttc?tcccggtgcc????2400
tggagctgca?gtgtcagccc?gactcctcaa?ccctgccacc?cccatggagt?ccccggcccc????2460
tggaggccac?agccccgaca?gccccgtga??????????????????????????????????????2489
<210>58
<211>628
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(628)
＜223〉anti-Her2 antibody fusion protein
<220>58
Met?Asp?Phe?Gln?Val?Gln?Ile?Phe?Ser?Phe?Leu?Leu?Ile?Ser?Ala?Ser
1???????????????5???????????????????10??????????????????15
Val?Ile?Ile?Ser?Arg?Gly?Glu?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly
20??????????????????25??????????????????30
Leu?Val?Gln?Pro?Gly?Gly?Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly
35??????????????????40??????????????????45
Phe?Asn?Ile?Lys?Asp?Thr?Tyr?Ile?His?Trp?Val?Arg?Gln?Ala?Pro?Gly
50??????????????????55??????????????????60
Lys?Gly?Leu?Glu?Trp?Val?Ala?Arg?Ile?Tyr?Pro?Thr?Asn?Gly?Tyr?Thr
65??????????????????70??????????????????75??????????????????80
Arg?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Ala?Asp?Thr
85??????????????????90??????????????????95
Ser?Lys?Asn?Thr?Ala?Tyr?Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp
100?????????????????105?????????????????110
Thr?Ala?Val?Tyr?Tyr?Cys?Ser?Arg?Trp?Gly?Gly?Asp?Gly?Phe?Tyr?Ala
115?????????????????120?????????????????125
Met?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser
130?????????????????135?????????????????140
Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr
145?????????????????150?????????????????155?????????????????160
Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro
165?????????????????170?????????????????175
Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val
180?????????????????185?????????????????190
His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser
195?????????????????200?????????????????205
Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile
210?????????????????215?????????????????220
Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val
225?????????????????230?????????????????235?????????????????240
Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala
245?????????????????250?????????????????255
Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro
260?????????????????265?????????????????270
Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val
275?????????????????280?????????????????285
Val?Asp?Val?Ser?His?Glu?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp
290?????????????????295?????????????????300
Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln
305?????????????????310?????????????????315?????????????????320
Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln
325?????????????????330?????????????????335
Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala
340?????????????????345?????????????????350
Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro
355?????????????????360?????????????????365
Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr
370?????????????????375?????????????????380
Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser
385?????????????????390?????????????????395?????????????????400
Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?pro?Glu?Asn?Asn?Tyr
405?????????????????410?????????????????415
Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr
420?????????????????425?????????????????430
Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe
435?????????????????440?????????????????445
Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys
450?????????????????455?????????????????460
Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys?Thr?Gln?Asp?Cys?Ser?Phe?Gln?His
465?????????????????470?????????????????475?????????????????480
Ser?Pro?Ile?Ser?Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu?Ser?Asp
485?????????????????490?????????????????495
Tyr?Leu?Leu?Gln?Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu?Gln?Asp
500?????????????????505?????????????????510
Glu?Glu?Leu?Cys?Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln?Arg?Trp
515?????????????????520?????????????????525
Met?Glu?Arg?Leu?Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly?Leu?Leu
530?????????????????535?????????????????540
Glu?Arg?Val?Asn?Thr?Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala?Phe?Gln
545?????????????????550?????????????????555?????????????????560
Pro?Pro?Pro?Ser?Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser?Arg?Leu
565?????????????????570?????????????????575
Leu?Gln?Glu?Thr?Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp?Ile?Thr
580?????????????????585?????????????????590
Arg?Gln?Asn?Phe?Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro?Asp?Ser
595?????????????????600?????????????????605
Ser?Thr?Leu?Pro?Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala?Thr?Ala
610?????????????????615?????????????????620
Pro?Thr?Ala?Pro
625
<210>59
<211>2534
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(2534)
＜223〉anti-Her2 antibody fusion protein encoding sequence
<400>59
atggattttc?aggtgcagat?tttcagcttc?ctgctaatca?gtgcctcagt?cataatatcc?????60
agaggagagg?ttcagctggc?ggagtctggc?ggtggcctgg?tgcagccagg?gggctcactc????120
cgtttgtcct?gtgcagcttc?tggcttcaac?attaaagaca?cctatataca?ctgggtgcgt????180
caggccccgg?gtaagggcct?ggaatgggtt?gcaaggattt?atcctacgaa?tggttatact????240
agatatgccg?atagcgtcaa?gggccgtttc?actataagcg?cagacacatc?caaaaacaca????300
gcctacctgc?agatgaacag?cctgcgtgct?gaggacactg?ccgtctatta?ttgttctaga????360
tggggagggg?acggcttcta?tgctatggac?tactggggtc?aaggaaccct?ggtcaccgtc????420
tcctcggcta?gcaccaaggg?cccatcggtc?ttccccctgg?caccctcctc?caagagcacc????480
tctgggggca?cagcggccct?gggctgcctg?gtcaaggact?acttccccga?accggtgacg????540
gtgtcttgga?actcaggcgc?cctgaccagc?ggcgtgcaca?ccttcccggc?tgtcctacag????600
tcctcaggac?tctactccct?cagcagcgtg?gtgaccgtgc?cctccagcag?cttgggcacc????660
cagacctaca?tctgcaacgt?gaatcacaag?cccagcaaca?ccaaggtgga?caagaaagtt????720
ggtgagaggc?cagcacaggg?agggagggtg?tctgctggaa?gcaggctcag?cgctcctgcc????780
tggacgcatc?ccggctatgc?agccccagtc?cagggcagca?aggcaggccc?cgtctgcctc????840
ttcacccgga?gcctctgccc?gccccactca?tgctcaggga?gagggtcttc?tggctttttc????900
ccaggctctg?ggcaggcaca?ggctaggtgc?ccctaaccca?ggccctgcac?acaaaggggc????960
aggtgctggg?ctcagacctg?ccaagagcca?tatccgggag?gaccctgccc?ctgacctaag???1020
cccaccccaa?aggccaaact?ctccactccc?tcagctcgga?caccttctct?cctcccagat???1080
tccagtaact?cccaatcttc?tctctgcaga?gcccaaatct?tgtggcaaaa?ctcacacatg???1140
cccaccgtgc?ccaggtaagc?cagcccaggc?ctcgccctcc?agctcaaggc?gggacaggtg???1200
ccctagagta?gcctgcatcc?agggacaggc?cccagccggg?tgctgacacg?tccacctcca???1260
tctcttcctc?aacacctgaa?ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac???1320
ccaaggacac?cctcatgatc?tcccggaccc?ctgaggtcac?atgcgtggtg?gtggacgtga????1380
gccacgaaga?ccctgaggtc?aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg????1440
ccaagacaaa?gccgcgggag?gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca????1500
ccgtcctgca?ccaggactgg?ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag????1560
ccctcccagc?ccccatcgag?aaaaccatct?ccaaagccaa?aggtgggacc?cgtggggtgc????1620
gagggccaca?tggacagagg?ccggctcggc?ccaccctctg?ccctgagagt?gaccgctgta????1680
ccaacctctg?tcctacaggg?cagccccgag?aaccacaggt?gtacaccctg?cccccatccc????1740
gggatgagct?gaccaagaac?caggtcagcc?tgacctgcct?ggtcaaaggc?ttctatccca????1800
gcgacatcgc?cgtggagtgg?gagagcaatg?ggcagccgga?gaacaactac?aagaccacgc????1860
ctcccgtgct?ggactccgac?ggctccttct?tcctctacag?caagctcacc?gtggacaaga????1920
gcaggtggca?gcaggggaac?gtcttctcat?gctccgtgat?gcatgaggct?ctgcacaacc????1980
actacacgca?gaagagcctc?tccctgtctc?ccggtaaagg?cggtggaggc?tctggtggag????2040
gcggttcagg?aggcggtgga?tctacccagg?actgctcctt?ccaacacagc?cccatctcct????2100
ccgacttcgc?tgtcaaaatc?cgtgagctgt?ctgactacct?gcttcaagat?tacccagtca????2160
ccgtggcctc?caacctgcag?gacgaggagc?tctgcggggg?cctctggcgg?ctggtcctgg????2220
cacagcgctg?gatggagcgg?ctcaagactg?tcgctgggtc?caagatgcaa?ggcttgctgg????2280
agcgcgtgaa?cacggagata?cactttgtca?ccaaatgtgc?ctttcagccc?ccccccagct????2340
gtcttcgctt?cgtccagacc?aacatctccc?gcctcctgca?ggagacctcc?gagcagctgg????2400
tggcgctgaa?gccctggatc?actcgccaga?acttctcccg?gtgcctggag?ctgcagtgtc????2460
agcccgactc?ctcaaccctg?ccacccccat?ggagtccccg?gcccctggag?gccacagccc????2520
cgacagcccc?gtga??????????????????????????????????????????????????????2534
<210>60
<211>643
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(643)
＜223〉anti-Her2 antibody fusion protein
<400>60
Met?Asp?Phe?Gln?Val?Gln?Ile?Phe?Ser?Phe?Leu?Leu?Ile?Ser?Ala?Ser
1???????????????5???????????????????10??????????????????15
Val?Ile?Ile?Ser?Arg?Gly?Glu?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly
20??????????????????25??????????????????30
Leu?Val?Gln?Pro?Gly?Gly?Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly
35??????????????????40??????????????????45
Phe?Asn?Ile?Lys?Asp?Thr?Tyr?Ile?His?Trp?Val?Arg?Gln?Ala?Pro?Gly
50??????????????????55??????????????????60
Lys?Gly?Leu?Glu?Trp?Val?Ala?Arg?Ile?Tyr?Pro?Thr?Asn?Gly?Tyr?Thr
65??????????????????70??????????????????75??????????????????80
Arg?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Ala?Asp?Thr
85??????????????????90??????????????????95
Ser?Lys?Asn?Thr?Ala?Tyr?Leu?Gln?Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp
100?????????????????105?????????????????110
Thr?Ala?Val?Tyr?Tyr?Cys?Ser?Arg?Trp?Gly?Gly?Asp?Gly?Phe?Tyr?Ala
115?????????????????120?????????????????125
Met?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Val?Thr?Val?Ser?Ser?Ala?Ser
130?????????????????135?????????????????140
Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys?Ser?Thr
145?????????????????150?????????????????155?????????????????160
Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr?Phe?Pro
165?????????????????170?????????????????175
Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser?Gly?Val
180?????????????????185?????????????????190
His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser?Leu?Ser
195?????????????????200?????????????????205
Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr?Tyr?Ile
210?????????????????215?????????????????220
Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys?Lys?Val
225?????????????????230?????????????????235?????????????????240
Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr?Cys?Pro?Pro?Cys?Pro?Ala
245?????????????????250?????????????????255
Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro?Lys?Pro
260?????????????????265?????????????????270
Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys?Val?Val
275?????????????????280?????????????????285
Val?Asp?Val?Ser?His?Glu?Pro?Glu?Val?Lys?Phe?Asn?Trp?Tyr?Val?Asp
290?????????????????295?????????????????300
Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu?Glu?Gln
305?????????????????310?????????????????315?????????????????320
Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu?His?Gln
325?????????????????330?????????????????335
Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn?Lys?Ala
340?????????????????345?????????????????350
Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly?Gln?Pro
355?????????????????360?????????????????365
Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Arg?Asp?Glu?Leu?Thr
370?????????????????375?????????????????380
Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr?Pro?Ser
385?????????????????390?????????????????395?????????????????400
Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn?Asn?Tyr
405?????????????????410?????????????????415
Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe?Leu?Tyr
420?????????????????425?????????????????430
Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Gln?Gly?Asn?Val?Phe
435?????????????????440?????????????????445
Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr?Gln?Lys
450?????????????????455?????????????????460
Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly
465?????????????????470?????????????????475?????????????????480
Gly?Ser?Gly?Gly?Gly?Gly?Ser?Thr?Gln?Asp?Cys?Ser?Phe?Gln?His?Ser
485?????????????????490?????????????????495
Pro?Ile?Ser?Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu?Ser?Asp?Tyr
500?????????????????505?????????????????510
Leu?Leu?Gln?Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu?Gln?Asp?Glu
515?????????????????520?????????????????525
Glu?Leu?Cys?Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln?Arg?Trp?Met
530?????????????????535?????????????????540
Glu?Arg?Leu?Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly?Leu?Leu?Glu
545?????????????????550?????????????????555?????????????????560
Arg?Val?Asn?Thr?Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala?Phe?Gln?Pro
565?????????????????570?????????????????575
Pro?Pro?Ser?Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser?Arg?Leu?Leu
580?????????????????585?????????????????590
Gln?Glu?Thr?Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp?Ile?Thr?Arg
595?????????????????600?????????????????605
Gln?Asn?Phe?Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro?Asp?Ser?Ser
610?????????????????615?????????????????620
Thr?Leu?Pro?Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala?Thr?Ala?Pro
625?????????????????630?????????????????635?????????????????640
Thr?Ala?Pro
<210>61
<211>1998
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(1996)
＜223〉anti-Her2 antibody fusion protein encoding sequence
<220>61
atgacagtgc?tggcgccagc?ctggagccca?acaacctatc?tcctcctgct?gctgctgctg?????60
agctcgggac?tcagtgggac?ccaggactgc?tccttccaac?acagccccat?ctcctccgac????120
ttcgctgtca?aaatccgtga?gctgtctgac?tacctgcttc?aagattaccc?agtcaccgtg????180
gcctccaacc?tgcaggacga?ggagctctgc?gggggcctct?ggcggctggt?cctggcacag????240
cgctggatgg?agcggctcaa?gactgtcgct?gggtccaaga?tgcaaggctt?gctggagcgc????300
gtgaacacgg?agatacactt?tgtcaccaaa?tgtgcctttc?agcccccccc?cagctgtctt????360
cgcttcgtcc?agaccaacat?ctcccgcctc?ctgcaggaga?cctccgagca?gctggtggcg????420
ctgaagccct?ggatcactcg?ccagaacttc?tcccggtgcc?tggagctgca?gtgtcagccc????480
gactcctcaa?ccctgccacc?cccatggagt?ccccggcccc?tggaggccac?agccccgaca????540
gccccggagc?ccaaatcttg?tgacaaaact?cacacatgcc?caccgtgccc?agcacctgaa????600
ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac?ccaaggacac?cctcatgatc????660
tcccggaccc?ctgaggtcac?atgcgtggtg?gtggacgtga?gccacgaaga?ccctgaggtc????720
aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg?ccaagacaaa?gccgcgggag????780
gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca?ccgtcctgca?ccaggactgg????840
ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag?ccctcccagc?ccccatcgag????900
aaaaccatct?ccaaagccaa?agggcagccc?cgagaaccac?aggtgtacac?cctgccccca????960
tcccgggatg?agctgaccaa?gaaccaggtc?agcctgacct?gcctggtcaa?aggcttctat???1020
cccagcgaca?tcgccgtgga?gtgggagagc?aatgggcagc?cggagaacaa?ctacaagacc???1080
acgcctcccg?tgctggactc?cgacggctcc?ttcttcctct?acagcaagct?caccgtggac???1140
aagagcaggt?ggcagcaggg?gaacgtcttc?tcatgctccg?tgatgcatga?ggctctgcac???1200
aaccactaca?cgcagaagag?cctctccctg?tctcccggta?aagaggttca?gctggtggag???1260
tctggcggtg?gcctggtgca?gccagggggc?tcactccgtt?tgtcctgtgc?agcttctggc???1320
ttcaacatta?aagacaccta?tatacactgg?gtgcgtcagg?ccccgggtaa?gggcctggaa???1380
tgggttgcaa?ggatttatcc?tacgaatggt?tatactagat?atgccgatag?cgtcaagggc???1440
cgtttcacta?taagcgcaga?cacatccaaa?aacacagcct?acctgcagat?gaacagcctg???1500
cgtgctgagg?acactgccgt?ctattattgt?tctagatggg?gaggggacgg?cttctatgct???1560
atggactact?ggggtcaagg?aaccctggtc?accgtctcct?cggctagcac?caagggccca???1620
tcggtcggcg?gtggaggctc?tggtggaggc?ggttcaggag?gcggtggatc?tgacatccag???1680
atgacccagt?ccccgagctc?cctgtccgcc?tctgtgggcg?atagggttac?catcacctgc???1740
cgtgccagtc?aggatgtgaa?tactgctgta?gcctggtatc?aacagaaacc?aggaaaagct???1800
ccgaaactac?tgatttactc?ggcatccttc?ctctactctg?gagtcccttc?tcgcttctct???1860
ggctccagat?ctgggacgga?tttcactctg?accatcagca?gtctgcagcc?ggaagacttc???1920
gcaacttatt?actgtcagca?acattatact?actcctccca?cgttcggaca?gggtaccaag???1980
gtggagatca?aacgttga?????????????????????????????????????????????????1998
<210>62
<211>665
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(665)
＜223〉anti-Her2 antibody fusion protein
<220>62
Met?Thr?Val?Leu?Ala?Pro?Ala?Trp?Ser?Pro?Thr?Thr?Tyr?Leu?Leu?Leu
1???????????????5???????????????????10??????????????????15
Leu?Leu?Leu?Leu?Ser?Ser?Gly?Leu?Ser?Gly?Thr?Gln?Asp?Cys?Ser?Phe
20??????????????????25??????????????????30
Gln?His?Ser?Pro?Ile?Ser?Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu
35??????????????????40??????????????????45
Ser?Asp?Tyr?Leu?Leu?Gln?Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu
50??????????????????55??????????????????60
Gln?Asp?Glu?Glu?Leu?Cys?Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln
65?????????????????70??????????????????75??????????????????80
Arg?Trp?Met?Glu?Arg?Leu?Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly
85??????????????????90??????????????????95
Leu?Leu?Glu?Arg?Val?Asn?Thr?Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala
100?????????????????105?????????????????110
Phe?Gln?Pro?Pro?Pro?Ser?Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser
115?????????????????120?????????????????125
Arg?Leu?Leu?Gln?Glu?Thr?Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp
130?????????????????135?????????????????140
Ile?Thr?Arg?Gln?Asn?Phe?Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro
145?????????????????150?????????????????155?????????????????160
Asp?Ser?Ser?Thr?Leu?Pro?Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala
165?????????????????170?????????????????175
Thr?Ala?Pro?Thr?Ala?Pro?Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr
180?????????????????185?????????????????190
Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe
195?????????????????200?????????????????205
Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro
210?????????????????215?????????????????220
Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val
225?????????????????230?????????????????235?????????????????240
Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr
245?????????????????250?????????????????255
Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val
260?????????????????265?????????????????270
Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys
275?????????????????280?????????????????285
Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser
290?????????????????295?????????????????300
Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro
305?????????????????310?????????????????315?????????????????320
Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val
325?????????????????330?????????????????335
Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly
340?????????????????345?????????????????350
Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp
355?????????????????360?????????????????365
Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp
370?????????????????375?????????????????380
Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His
385?????????????????390?????????????????395?????????????????400
Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys?Glu?Val
405?????????????????410?????????????????415
Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly?Leu?Val?Gln?Pro?Gly?Gly?Ser?Leu
420?????????????????425?????????????????430
Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly?Phe?Asn?Ile?Lys?Asp?Thr?Tyr?Ile
435?????????????????440?????????????????445
His?Trp?Val?Arg?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Val?Ala?Arg
450?????????????????455?????????????????460
Ile?Tyr?Pro?Thr?Asn?Gly?Tyr?Thr?Arg?Tyr?Ala?Asp?Ser?Val?Lys?Gly
465?????????????????470?????????????????475?????????????????480
Arg?Phe?Thr?Ile?Ser?Ala?Asp?Thr?Ser?Lys?Asn?Thr?Ala?Tyr?Leu?Gln
485?????????????????490?????????????????495
Met?Asn?Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ser?Arg
500?????????????????505?????????????????510
Trp?Gly?Gly?Asp?Gly?Phe?Tyr?Ala?Met?Asp?Tyr?Trp?Gly?Gln?Gly?Thr
515?????????????????520?????????????????525
Leu?Val?Thr?Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Gly?Gly
530?????????????????535?????????????????540
Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Asp?Ile?Gln
545?????????????????550?????????????????555?????????????????560
Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly?Asp?Arg?Val
565?????????????????570?????????????????575
Thr?Ile?Thr?Cys?Arg?Ala?Ser?Gln?Asp?Val?Asn?Thr?Ala?Val?Ala?Trp
580?????????????????585?????????????????590
Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Ser?Ala
595?????????????????600?????????????????605
Ser?Phe?Leu?Tyr?Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Gly?Ser?Arg?Ser
610?????????????????615?????????????????620
Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?Phe
625?????????????????630?????????????????635?????????????????640
Ala?Thr?Tyr?Tyr?Cys?Gln?Gln?His?Tyr?Thr?Thr?Pro?Pro?Thr?Phe?Gly
645?????????????????650?????????????????655
Gln?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg
660?????????????????665
<210>63
<211>1098
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(1098)
＜223〉Trail fusion rotein encoding sequence
<220>63
atgacagtgc?tggcgccagc?ctggagccca?acaacctatc?tcctcctgct?gctgctgctg?????60
agctcgggac?tcagtgggac?ccaggactgc?tccttccaac?acagccccat?ctcctccgac????120
ttcgctgtca?aaatccgtga?gctgtctgac?tacctgcttc?aagattaccc?agtcaccgtg????180
gcctccaacc?tgcaggacga?ggagctctgc?gggggcctct?ggcggctggt?cctggcacag????240
cgctggatgg?agcggctcaa?gactgtcgct?gggtccaaga?tgcaaggctt?gctggagcgc????300
gtgaacacgg?agatacactt?tgtcaccaaa?tgtgcctttc?agcccccccc?cagctgtctt????360
cgcttcgtcc?agaccaacat?ctcccgcctc?ctgcaggaga?cctccgagca?gctggtggcg????420
ctgaagccct?ggatcactcg?ccagaacttc?tcccggtgcc?tggagctgca?gtgtcagccc????480
gactcctcaa?ccctgccacc?cccatggagt?ccccggcccc?tggaggccac?agccccgaca????540
gccccgggcg?gtggaggctc?tggtggaggc?ggttcaggag?gcggtggatc?tgtgagagaa????600
agaggtcctc?agagagtagc?agctcacata?actgggacca?gaggaagaag?caacacattg????660
tcttctccaa?actccaagaa?tgaaaaggct?ctgggccgca?aaataaactc?ctgggaatca????720
tcaaggagtg?ggcattcatt?cctgagcaac?ttgcacttga?ggaatggtga?actggtcatc????780
catgaaaaag?ggttttacta?catctattcc?caaacatact?ttcgatttca?ggaggaaata????840
aaagaaaaca?caaagaacga?caaacaaatg?gtccaatata?tttacaaata?cacaagttat????900
cctgacccta?tattgttgat?gaaaagtgct?agaaatagtt?gttggtctaa?agatgcagaa????960
tatggactct?attccatcta?tcaaggggga?atatttgagc?ttaaggaaaa?tgacagaatt???1020
tttgtttctg?taacaaatga?gcacttgata?gacatggacc?atgaagccag?tttttttggg???1080
gcctttttag?ttggctaa?????????????????????????????????????????????????1098
<210>64
<211>365
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(365)
＜223〉Trail fusion rotein
<220>64
Met?Thr?Val?Leu?Ala?Pro?Ala?Trp?Ser?Pro?Thr?Thr?Tyr?Leu?Leu?Leu
1???????????????5???????????????????10??????????????????15
Leu?Leu?Leu?Leu?Ser?Ser?Gly?Leu?Ser?Gly?Thr?Gln?Asp?Cys?Ser?Phe
20??????????????????25??????????????????30
Gln?His?Ser?Pro?Ile?Ser?Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu
35??????????????????40??????????????????45
Ser?Asp?Tyr?Leu?Leu?Gln?Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu
50??????????????????55??????????????????60
Gln?Asp?Glu?Glu?Leu?Cys?Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln
65??????????????????70??????????????????75??????????????????80
Arg?Trp?Met?Glu?Arg?Leu?Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly
85??????????????????90??????????????????95
Leu?Leu?Glu?Arg?Val?Asn?Thr?Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala
100?????????????????105?????????????????110
Phe?Gln?Pro?Pro?Pro?Ser?Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser
115?????????????????120?????????????????125
Arg?Leu?Leu?Gln?Glu?Thr?Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp
130?????????????????135?????????????????140
Ile?Thr?Arg?Gln?Asn?Phe?Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro
145?????????????????150?????????????????155?????????????????160
Asp?Ser?Ser?Thr?Leu?Pro?Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala
165?????????????????170?????????????????175
Thr?Ala?Pro?Thr?Ala?Pro?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
180?????????????????185?????????????????190
Gly?Gly?Gly?Gly?Ser?Val?Arg?Glu?Arg?Gly?Pro?Gln?Arg?Val?Ala?Ala
195?????????????????200?????????????????205
His?Ile?Thr?Gly?Thr?Arg?Gly?Arg?Ser?Asn?Thr?Leu?Ser?Ser?Pro?Asn
210?????????????????215?????????????????220
Ser?Lys?Asn?Glu?Lys?Ala?Leu?Gly?Arg?Lys?Ile?Asn?Ser?Trp?Glu?Ser
225?????????????????230?????????????????235?????????????????240
Ser?Arg?Ser?Gly?His?Ser?Phe?Leu?Ser?Asn?Leu?His?Leu?Arg?Asn?Gly
245?????????????????250?????????????????255
Glu?Leu?Val?Ile?His?Glu?Lys?Gly?Phe?Tyr?Tyr?Ile?Tyr?Ser?Gln?Thr
260?????????????????265?????????????????270
Tyr?Phe?Arg?Phe?Gln?Glu?Glu?Ile?Lys?Glu?Asn?Thr?Lys?Asn?Asp?Lys
275?????????????????280?????????????????285
Gln?Met?Val?Gln?Tyr?Ile?Tyr?Lys?Tyr?Thr?Ser?Tyr?Pro?Asp?Pro?Ile
290?????????????????295?????????????????300
Leu?Leu?Met?Lys?Ser?Ala?Arg?Asn?Ser?Cys?Trp?Ser?Lys?Asp?Ala?Glu
305?????????????????310?????????????????315?????????????????320
Tyr?Gly?Leu?Tyr?Ser?Ile?Tyr?Gln?Gly?Gly?Ile?Phe?Glu?Leu?Lys?Glu
325?????????????????330?????????????????335
Asn?Asp?Arg?Ile?Phe?Val?Ser?Val?Thr?Asn?Glu?His?Leu?Ile?Asp?Met
340?????????????????345?????????????????350
Asp?His?Glu?Ala?Ser?Phe?Phe?Gly?Ala?Phe?Leu?Val?Gly
355?????????????????360?????????????????365
<210>65
<211>1203
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(1203)
＜223〉Trail fusion rotein encoding sequence
<220>65
atgacagtgc?tggcgccagc?ctggagccca?acaacctatc?tcctcctgct?gctgctgctg?????60
agctcgggac?tcagtgggac?ccaggactgc?tccttccaac?acagccccat?ctcctccgac????120
ttcgctgtca?aaatccgtga?gctgtctgac?tacctgcttc?aagattaccc?agtcaccgtg????180
gcctccaacc?tgcaggacga?ggagctctgc?gggggcctct?ggcggctggt?cctggcacag????240
cgctggatgg?agcggctcaa?gactgtcgct?gggtccaaga?tgcaaggctt?gctggagcgc????300
gtgaacacgg?agatacactt?tgtcaccaaa?tgtgcctttc?agcccccccc?cagctgtctt????360
cgcttcgtcc?agaccaacat?ctcccgcctc?ctgcaggaga?cctccgagca?gctggtggcg????420
ctgaagccct?ggatcagtcg?ccagaacttc?tcccggtgcc?tggagctgca?gtgtcagccc????480
gactcctcaa?ccctgccacc?cccatggagt?ccccggcccc?tggaggccac?agccccgaca????540
gccccgatga?agcagatcga?ggacaaaatt?gaggaaatcc?tgtccaagat?ttaccacatc????600
gagaacgaga?tcgcccggat?taagaaactc?attggcgaga?cctctgagga?aaccatttct????660
acagttcaag?aaaagcaaca?aaatatttct?cccctagtga?gagaaagagg?tcctcagaga????720
gtagcagctc?acataactgg?gaccagagga?agaagcaaca?cattgtcttc?tccaaactcc????780
aagaatgaaa?aggctctggg?ccgcaaaata?aactcctgcg?aatcatcaag?gagtgggcat????840
tcattcctga?gcaacttgca?cttgaggaat?ggtgaactgg?tcatccatga?aaaagggttt????900
tactacatct?attcccaaac?atactttcga?tttcaggagg?aaataaaaga?aaacacaaag????960
aacgacaaac?aaatggtcca?atatatttac?aaatacacaa?gttatcctga?ccctatattg???1020
ttgatgaaaa?gtgctagaaa?tagttgttgg?tctaaagatg?cagaatatgg?actctattcc???1080
atctatcaag?ggggaatatt?tgagcttaag?gaaaatgaca?gaatttttgt?ttctgtaaca???1140
aatgagcact?tgatagacat?ggaccatgaa?gccagttttt?ttggggcctt?tttagttggc???1200
taa?????????????????????????????????????????????????????????????????1203
<210>66
<211>400
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(400)
＜223〉Trail fusion rotein
<220>66
Met?Thr?Val?Leu?Ala?Pro?Ala?Trp?Ser?Pro?Thr?Thr?Tyr?Leu?Leu?Leu
1???????????????5???????????????????10??????????????????15
Leu?Leu?Leu?Leu?Ser?Ser?Gly?Leu?Ser?Gly?Thr?Gln?Asp?Cys?Ser?Phe
20??????????????????25??????????????????30
Gln?His?Ser?Pro?Ile?Ser?Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu
35??????????????????40??????????????????45
Ser?Asp?Tyr?Leu?Leu?Gln?Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu
50??????????????????55??????????????????60
Gln?Asp?Glu?Glu?Leu?Cys?Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln
65??????????????????70??????????????????75??????????????????80
Arg?Trp?Met?Glu?Arg?Leu?Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly
85??????????????????90??????????????????95
Leu?Leu?Glu?Arg?Val?Asn?Thr?Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala
100?????????????????105?????????????????110
Phe?Gln?Pro?Pro?Pro?Ser?Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser
115?????????????????120?????????????????125
Arg?Leu?Leu?Gln?Glu?Thr?Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp
130?????????????????135?????????????????140
Ile?Thr?Arg?Gln?Asn?Phe?Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro
145?????????????????150?????????????????155?????????????????160
Asp?Ser?Ser?Thr?Leu?Pro?Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala
165?????????????????170?????????????????175
Thr?Ala?Pro?Thr?Ala?Pro?Met?Lys?Gln?Ile?Glu?Asp?Lys?Ile?Glu?Glu
180?????????????????185?????????????????190
Ile?Leu?Ser?Lys?Ile?Tyr?His?Ile?Glu?Asn?Glu?Ile?Ala?Arg?Ile?Lys
195?????????????????200?????????????????205
Lys?Leu?Ile?Gly?Glu?Thr?Ser?Glu?Glu?Thr?Ile?Ser?Thr?Val?Gln?Glu
210?????????????????215?????????????????220
Lys?Gln?Gln?Asn?Ile?Ser?Pro?Leu?Val?Arg?Glu?Arg?Gly?Pro?Gln?Arg
225?????????????????230?????????????????235?????????????????240
Val?Ala?Ala?His?Ile?Thr?Gly?Thr?Arg?Gly?Arg?Ser?Asn?Thr?Leu?Ser
245?????????????????250?????????????????255
Ser?Pro?Asn?Ser?Lys?Asn?Glu?Lys?Ala?Leu?Gly?Arg?Lys?Ile?Asn?Ser
260?????????????????265?????????????????270
Trp?Glu?Ser?Ser?Arg?Ser?Gly?His?Ser?Phe?Leu?Ser?Asn?Leu?His?Leu
275?????????????????280?????????????????285
Arg?Asn?Gly?Glu?Leu?Val?Ile?His?Glu?Lys?Gly?Phe?Tyr?Tyr?Ile?Tyr
290?????????????????295?????????????????300
Ser?Gln?Thr?Tyr?Phe?Arg?Phe?Gln?Glu?Glu?Ile?Lys?Glu?Asn?Thr?Lys
305?????????????????310?????????????????315?????????????????320
Asn?Asp?Lys?Gln?Met?Val?Gln?Tyr?Ile?Tyr?Lys?Tyr?Thr?Ser?Tyr?Pro
325?????????????????330?????????????????335
Asp?Pro?Ile?Leu?Leu?Met?Lys?Ser?Ala?Arg?Asn?Ser?Cys?Trp?Ser?Lys
340?????????????????345?????????????????350
Asp?Ala?Glu?Tyr?Gly?Leu?Tyr?Ser?Ile?Tyr?Gln?Gly?Gly?Ile?Phe?Glu
355?????????????????360?????????????????365
Leu?Lys?Glu?Asn?Asp?Arg?Ile?Phe?Val?Ser?Val?Thr?Asn?Glu?His?Leu
370?????????????????375?????????????????380
Ile?Asp?Met?Asp?His?Glu?Ala?Ser?Phe?Phe?Gly?Ala?Phe?Leu?Val?Gly
385?????????????????390?????????????????395?????????????????400
<210>67
<211>1749
<212>DNA
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(1749)
＜223〉Trail fusion rotein encoding sequence
<220>67
atgacagtgc?tggcgccagc?ctggagccca?acaacctatc?tcctcctgct?gctgctgctg?????60
agctcgggac?tcagtgggac?ccaggactgc?tccttccaac?acagccccat?ctcctccgac????120
ttcgctgtca?aaatccgtga?gctgtctgac?tacctgcttc?aagattaccc?agtcaccgtg????180
gcctccaacc?tgcaggacga?ggagctctgc?gggggcctct?ggcggctggt?cctggcacag????240
cgctggatgg?agcggctcaa?gactgtcgct?gggtccaaga?tgcaaggctt?gctggagcgc????300
gtgaacacgg?agatacactt?tgtcaccaaa?tgtgcctttc?agcccccccc?cagctgtctt????360
cgcttcgtcc?agaccaacat?ctcccgcctc?ctgcaggaga?cctccgagca?gctggtggcg????420
ctgaagccct?ggatcactcg?ccagaacttc?tcccggtgcc?tggagctgca?gtgtcagccc????480
gactcctcaa?ccctgccacc?cccatggagt?ccccggcccc?tggaggccac?agccccgaca????540
gccccggagc?ccaaatcttg?tgacaaaact?cacacatgcc?caccgtgccc?agcacctgaa????600
ctcctggggg?gaccgtcagt?cttcctcttc?cccccaaaac?ccaaggacac?cctcatgatc????660
tcccggaccc?ctgaggtcac?atgcgtggtg?gtggacgtga?gccacgaaga?ccctgaggtc????720
aagttcaact?ggtacgtgga?cggcgtggag?gtgcataatg?ccaagacaaa?gccgcgggag????780
gagcagtaca?acagcacgta?ccgggtggtc?tgcgtcctca?ccgtcctgca?ccaggactgg????840
ctgaatggca?aggagtacaa?gtgcaaggtc?tccaacaaag?ccctcccagc?ccccatcgag????900
aaaaccatct?ccaaagccaa?agggcagccc?cgagaaccac?aggtgtacac?cctgccccca????960
tcccgggatg?agctgaccaa?gaaccaggtc?agcctgacct?gcctggtcaa?aggcttctat???1020
cccagcgaca?tcgccgtgga?gtgggagagc?aatgggcagc?cggagaacaa?ctacaagacc???1080
acgcctcccg?tgctggactc?cgacggctcc?ttcttcctct?acagcaagct?caccgtggac???1140
aagagcaggt?ggcagcaggg?gaacgtcttc?tcatgctccg?tgatgcatga?ggctctgcac???1200
aaccactaca?cgcagaagag?cctctccctg?tctcccggta?aagtgagaga?aagaggtcct???1260
cagagagtag?cagctcacat?aactgggacc?agaggaagaa?gcaacacatt?gtcttctcca???1320
aactccaaga?atgaaaaggc?tctgggccgc?aaaataaact?cctgggaatc?atcaaggagt???1380
gggcattcat?tcctgagcaa?cttgcacttg?aggaatggtg?aactggtcat?ccatgaaaaa???1440
gggttttact?acatctattc?ccaaacatac?tttcgatttc?aggaggaaat?aaaagaaaac???1500
acaaagaacg?acaaacaaat?ggtccaatat?atttacaaat?acacaagtta?tcctgaccct???1560
atattgttga?tgaaaagtgc?tagaaatagt?tgttggtcta?aagatgcaga?atatggactc???1620
tattccatct?atcaaggggg?aatatttgag?cttaaggaaa?atgacagaat?ttttgtttct???1680
gtaacaaatg?agcacttgat?agacatggac?catgaagcca?gtttttttgg?ggccttttta???1740
gttggctaa???????????????????????????????????????????????????????????1749
<210>68
<211>582
<212>PRT
＜213〉artificial sequence
<220>
<221>misc_feature
<222>(1)..(582)
＜223〉Trail fusion rotein
<220>68
Met?Thr?Val?Leu?Ala?Pro?Ala?Trp?Ser?Pro?Tht?Thr?Tyr?Leu?Leu?Leu
1???????????????5???????????????????10??????????????????15
Leu?Leu?Leu?Leu?Ser?Ser?Gly?Leu?Ser?Gly?Thr?Gln?Asp?Cys?Ser?Phe
20??????????????????25??????????????????30
Gln?His?Ser?Pro?Ile?Ser?Ser?Asp?Phe?Ala?Val?Lys?Ile?Arg?Glu?Leu
35??????????????????40??????????????????45
Ser?Asp?Tyr?Leu?Leu?Gln?Asp?Tyr?Pro?Val?Thr?Val?Ala?Ser?Asn?Leu
50??????????????????55??????????????????60
Gln?Asp?Glu?Glu?Leu?Cys?Gly?Gly?Leu?Trp?Arg?Leu?Val?Leu?Ala?Gln
65??????????????????70??????????????????75??????????????????80
Arg?Trp?Met?Glu?Arg?Leu?Lys?Thr?Val?Ala?Gly?Ser?Lys?Met?Gln?Gly
85??????????????????90??????????????????95
Leu?Leu?Glu?Arg?Val?Asn?Thr?Glu?Ile?His?Phe?Val?Thr?Lys?Cys?Ala
100?????????????????105?????????????????110
Phe?Gln?Pro?Pro?Pro?Ser?Cys?Leu?Arg?Phe?Val?Gln?Thr?Asn?Ile?Ser
115?????????????????120?????????????????125
Arg?Leu?Leu?Gln?Glu?Thr?Ser?Glu?Gln?Leu?Val?Ala?Leu?Lys?Pro?Trp
130?????????????????135?????????????????140
Ile?Thr?Arg?Gln?Asn?Phe?Ser?Arg?Cys?Leu?Glu?Leu?Gln?Cys?Gln?Pro
145?????????????????150?????????????????155?????????????????160
Asp?Ser?Ser?Thr?Leu?Pro?Pro?Pro?Trp?Ser?Pro?Arg?Pro?Leu?Glu?Ala
165?????????????????170?????????????????175
Thr?Ala?Pro?Thr?Ala?Pro?Glu?Pro?Lys?Ser?Cys?Asp?Lys?Thr?His?Thr
180?????????????????185?????????????????190
Cys?Pro?Pro?Cys?Pro?Ala?Pro?Glu?Leu?Leu?Gly?Gly?Pro?Ser?Val?Phe
195?????????????????200?????????????????205
Leu?Phe?Pro?Pro?Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro
210?????????????????215?????????????????220
Glu?Val?Thr?Cys?Val?Val?Val?Asp?Val?Ser?His?Glu?Asp?Pro?Glu?Val
225?????????????????230?????????????????235?????????????????240
Lys?Phe?Asn?Trp?Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr
245?????????????????250?????????????????255
Lys?Pro?Arg?Glu?Glu?Gln?Tyr?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val
260?????????????????265?????????????????270
Leu?Thr?Val?Leu?His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys
275?????????????????280?????????????????285
Lys?Val?Ser?Asn?Lys?Ala?Leu?Pro?Ala?Pro?Ile?Glu?Lys?Thr?Ile?Ser
290?????????????????295?????????????????300
Lys?Ala?Lys?Gly?Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro
305?????????????????310?????????????????315?????????????????320
Ser?Arg?Asp?Glu?Leu?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val
325?????????????????330?????????????????335
Lys?Gly?Phe?Tyr?Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly
340?????????????????345?????????????????350
Gln?Pro?Glu?Asn?Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp
355?????????????????360?????????????????365
Gly?Ser?Phe?Phe?Leu?Tyr?Ser?Lys?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp
370?????????????????375?????????????????380
Gln?Gln?Gly?Asn?Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His
385?????????????????390?????????????????395?????????????????400
Asn?His?Tyr?Thr?Gln?Lys?Ser?Leu?Ser?Leu?Ser?Pro?Gly?Lys?Val?Arg
405?????????????????410?????????????????415
Glu?Arg?Gly?Pro?Gln?Arg?Val?Ala?Ala?His?Ile?Thr?Gly?Thr?Arg?Gly
420?????????????????425?????????????????430
Arg?Ser?Asn?Thr?Leu?Ser?Ser?Pro?Asn?Ser?Lys?Asn?Glu?Lys?Ala?Leu
435?????????????????440?????????????????445
Gly?Arg?Lys?Ile?Asn?Ser?Trp?Glu?Ser?Ser?Arg?Ser?Gly?His?Ser?Phe
450?????????????????455?????????????????460
Leu?Ser?Asn?Leu?His?Leu?Arg?Asn?Gly?Glu?Leu?Val?Ile?His?Glu?Lys
465?????????????????470?????????????????475?????????????????480
Gly?Phe?Tyr?Tyr?Ile?Tyr?Ser?Gln?Thr?Tyr?Phe?Arg?Phe?Gln?Glu?Glu
485?????????????????490?????????????????495
Ile?Lys?Glu?Asn?Thr?Lys?Asn?Asp?Lys?Gln?Met?Val?Gln?Tyr?Ile?Tyr
500?????????????????505?????????????????510
Lys?Tyr?Thr?Ser?Tyr?Pro?Asp?Pro?Ile?Leu?Leu?Met?Lys?Ser?Ala?Arg
515?????????????????520?????????????????525
Asn?Ser?Cys?Trp?Ser?Lys?Asp?Ala?Glu?Tyr?Gly?Leu?Tyr?Ser?Ile?Tyr
530?????????????????535?????????????????540
Gln?Gly?Gly?Ile?Phe?Glu?Leu?Lys?Glu?Asn?Asp?Arg?Ile?Phe?Val?Ser
545?????????????????550?????????????????555?????????????????560
Val?Thr?Asn?Glu?His?Leu?Ile?Asp?Met?Asp?His?Glu?Ala?Ser?Phe?Phe
565?????????????????570?????????????????575
Gly?Ala?Phe?Leu?Val?Gly
580

Claims (49)

1. a fusion rotein is characterized in that, described fusion rotein comprises first part and second section, and first part is Flt3 part or its bioactive fragment, and second section is albumen or the antitumor thing of polypeptide class.
2. fusion rotein as claimed in claim 1, wherein, antitumor thing can be to cause apoptotic molecule.
3. fusion rotein as claimed in claim 1, Flt3 part therein or its bioactive fragment can hemopoietic stem cell or progenitor cell proliferations.
4. fusion rotein as claimed in claim 1, wherein, Flt3 part or its bioactive fragment can stimulate bone marrow precursor, monocyte, scavenger cell, B cell and dendritic cell and NK cell proliferation.
5. fusion rotein as claimed in claim 1, wherein, Flt3 part or its bioactive fragment can derive from Mammals.
6. fusion rotein as claimed in claim 1, wherein, Flt3 part or its bioactive fragment can derive from the people.
7. fusion rotein as claimed in claim 1, wherein, Flt3 part or its bioactive fragment are soluble proteinss.
8. fusion rotein as claimed in claim 1, wherein, the Flt3 part comprises at least 100 amino acid and with aminoacid sequence shown in the SEQ IDNO:2 at least 40% homology is arranged.
9 fusion roteins as claimed in claim 1, wherein, the Flt3 part can with a kind of antibodies, but the polypeptide that this kind antibody specificity and aminoacid sequence shown in the SEQ ID NO:2 constitute combine, and the Flt3 part has fully kept its biologic activity.
10. fusion rotein as claimed in claim 1, wherein, the Flt3 part comprises aminoacid sequence shown in the SEQ ID NO:2.
11. fusion rotein as claimed in claim 1, wherein, the 28th to 128 amino acids sequence has at least 80% homology shown in aminoacid sequence that the Flt3 part comprises and the SEQ ID NO:2.
12. fusion rotein as claimed in claim 1, wherein, the aminoacid sequence of Flt3 part comprises the 28th to 128 amino acids shown in the SEQ ID NO:2.
13. fusion rotein as claimed in claim 1, wherein, the aminoacid sequence that the Flt3 part comprises can be selected from the 28th to 182 amino acids shown in the 28th to 160 amino acids shown in the SEQ IDNO:2 or the SEQ ID NO:2.
14. fusion rotein as claimed in claim 1, wherein, antitumor thing can be an antibody molecule.
15. fusion rotein as claimed in claim 14, wherein, antibody molecule can be the complete antibody molecule, Fab, and Fab ', F (ab ') 2, Fv, disome, fragment such as single-chain antibody or the multi-specificity antibody of forming by various antibody fragments.
16. fusion rotein as claimed in claim 14, wherein, antibody can be anti-p230 antibody, anti-CD20 antibodies, anti-Her2 antibody, anti-Her3 antibody, anti-Her4 antibody, anti-egfr antibodies or their bioactive fragment.
17. fusion rotein as claimed in claim 14, wherein, antibody can be people source or humanized antibody.
18. fusion rotein as claimed in claim 1, wherein, antitumor thing can be the Fas part, TNF, TRAIL, or their bioactive film outskirt fragment.
19. fusion rotein as claimed in claim 1, wherein, the Flt3 part can be positioned at the N end of fusion rotein.
20. fusion rotein as claimed in claim 1, wherein, the Flt3 part can be positioned at the C end of fusion rotein.
21. fusion rotein as claimed in claim 1, wherein, Flt3 part and antitumor thing can be connected peptide and separate.
22. fusion rotein as claimed in claim 1, wherein, connection peptides can be (Gly 4Ser) 3
23. fusion rotein as claimed in claim 1 comprises SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:44, SEQ ID NO:46, SEQ IDNO:48, SEQ ID NO:58, SEQ ID NO:60, SEQ ID NO:62, SEQ ID NO:64, aminoacid sequence shown in SEQ ID NO:66 or the SEQ ID NO:68.
24. an isolated nucleic acid molecule is characterized in that, the described bifunctional fusion proteins of its coding claim 1.
25. nucleic acid molecule as claimed in claim 24 comprises SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:43, SEQ ID NO:45, SEQ IDNO:47, SEQ ID NO:57, SEQ ID NO:59, SEQ ID NO:61, SEQ ID NO:63, nucleotide sequence shown in SEQ ID NO:65 or the SEQ ID NO:67.
26. an isolated nucleic acid molecule is characterized in that, it comprises and the described nucleic acid molecule complementary nucleotide sequence of claim 24.
27. a carrier is characterized in that, it contains the nucleic acid molecule of the described fusion rotein of coding claim 1.
28. carrier as claimed in claim 27 comprises and coding FLt3 part and the nucleic acid molecule of albumen or the antitumor thing of polypeptide and the expression regulation sequence that links to each other with its operability.
29. a reconstitution cell is characterized in that, it contains the described nucleic acid molecule of claim 24.
30. reconstitution cell as claimed in claim 29 is characterized in that, it is an eukaryotic cell.
31. reconstitution cell as claimed in claim 30 is characterized in that, it is CHO, COS or NSO cell.
32. a method that produces fusion rotein is characterized in that, it comprises how cultivating the reconstitution cell that contains the described nucleic acid molecule of claim 24, expresses the fusion rotein of coding and reclaims this fusion rotein with reconstitution cell so far.
33. method as claimed in claim 32 is characterized in that, it comprises separates and purified fusion protein.
34. the product of method as claimed in claim 32.
35. a pharmaceutical composition is characterized in that, it comprises the Flt3 part of effective dose and the fusion rotein of albumen or the antitumor thing composition of polypeptide class, and pharmaceutically acceptable carrier.
36. a scheme is characterized in that, it comprises Flt3 part and albumen or the fusion rotein of the antitumor thing composition of polypeptide class and the method for how to take described fusion rotein of effective dose.
37. comprising to needs treatments or the animal that meets the requirements, a method for the treatment of mammal tumor, this method take the fusion rotein that the Flt3 part of effective dose and albumen or the antitumor thing of polypeptide class are formed.
38. method as claimed in claim 37, wherein, Mammals can be the people.
39. method as claimed in claim 37, wherein, tumour can be melanoma, mammary cancer or liver cancer.
40. a pharmaceutical composition is characterized in that it comprises
A) fusion rotein of the antitumor thing composition of the Flt3 part of effective dose and albumen or polypeptide class.
B) anti-tumor agent of effective dose.
41. composition as claimed in claim 40, wherein, anti-tumor agent is a kind of treatment melanoma, the preparation of mammary cancer or liver cancer.
42. comprising to needs treatments or the animal that meets the requirements, a method for the treatment of mammal tumor, this method take the composition as claimed in claim 40 of effective dose.
43. the method by the caspase-3 cell death inducing is characterized in that, it comprises the fusion rotein to needs are induced or the cell that meets the requirements uses the Flt3 part of effective dose and albumen or the antitumor thing of polypeptide class to form.
44. method as claimed in claim 43, wherein, cell can be a mammalian cell.
45. method as claimed in claim 44, wherein, cell can be a mammalian tumor cell.
46. method as claimed in claim 43, wherein, cell can be in mammalian body.
47. a vaccine is characterized in that, it comprises the Flt3 part of effective dose and the fusion rotein of albumen or the antitumor thing composition of polypeptide class, and a kind of immunostimulant.
48. a method that excites anti tumor immune response in mammalian body is characterized in that, it comprises to needs treatments or the animal that meets the requirements takes the vaccine as claimed in claim 47 of effective dose.
49. one kind produces the lymphocytic method of tumour-specific, it is characterized in that it comprises to Mammals takes the Flt3 part of effective dose and the tumour-specific lymphocyte of fusion rotein to produce the tumour-specific lymphocyte and to produce from described Mammals recovery of albumen or the antitumor thing composition of polypeptide class.
CN 200310119930 2003-06-13 2003-11-25 Double functional fusion protein possessing antineoplastic functions, method for making same and use thereof Expired - Lifetime CN1297567C (en)

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CN 200310119930 CN1297567C (en) 2003-06-13 2003-11-25 Double functional fusion protein possessing antineoplastic functions, method for making same and use thereof
AU2004252465A AU2004252465A1 (en) 2003-06-13 2004-06-04 Preparation and application of anti-tumor bifunctional fusion proteins
EP04776301A EP1633398A4 (en) 2003-06-13 2004-06-04 Preparation and application of anti-tumor bifunctional fusion proteins
CA002528595A CA2528595A1 (en) 2003-06-13 2004-06-04 Preparation and application of anti-tumor bifunctional fusion proteins
JP2006533567A JP2007515946A (en) 2003-06-13 2004-06-04 Preparation and application of anti-tumor dual function fusion protein
PCT/US2004/017765 WO2005001048A2 (en) 2003-06-13 2004-06-04 Preparation and application of anti-tumor bifunctional fusion proteins
US11/004,639 US20050232931A1 (en) 2003-06-13 2004-12-02 Preparation and application of anti-tumor bifunctional fusion proteins
IL172243A IL172243A0 (en) 2003-06-13 2005-11-29 Preparation and application of anti-tumor bifunctional fusion proteins

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CN103370622A (en) * 2011-02-14 2013-10-23 埃特根有限公司 Method of diagnosing cancer and diagnosis kit using measurement of NK cell activity
WO2015054958A1 (en) * 2013-10-17 2015-04-23 上海张江生物技术有限公司 Anti-cd20-flex bifunctional fusion protein, and preparation method and use thereof
CN104628863A (en) * 2013-11-14 2015-05-20 中国医学科学院医药生物技术研究所 Double-target and double-warhead anti-tumor fusion protein as well as coding gene and application thereof
CN105343874A (en) * 2015-11-11 2016-02-24 固安鼎泰海规生物科技有限公司 Prostate cancer nucleic acid vaccine
CN108779167A (en) * 2015-10-28 2018-11-09 宾夕法尼亚州大学信托人 Intrathecal application adeno-associated virus vector is used for gene therapy

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103370622A (en) * 2011-02-14 2013-10-23 埃特根有限公司 Method of diagnosing cancer and diagnosis kit using measurement of NK cell activity
CN103370622B (en) * 2011-02-14 2018-02-13 埃特根有限公司 For measuring the method and kit of NK cytoactives
WO2015054958A1 (en) * 2013-10-17 2015-04-23 上海张江生物技术有限公司 Anti-cd20-flex bifunctional fusion protein, and preparation method and use thereof
CN104558194A (en) * 2013-10-17 2015-04-29 上海张江生物技术有限公司 Anti-CD20-Flex bi-functional fusion protein as well as preparation method and application thereof
AU2013403112B2 (en) * 2013-10-17 2017-05-04 Shanghai Biomabs Pharmaceuticals Co., Ltd Anti-CD20-Flex bifunctional fusion protein, and preparation method and use thereof
CN104558194B (en) * 2013-10-17 2018-04-27 泰州迈博太科药业有限公司 A kind of anti-CD20-Flex bifunctional fusion proteins, preparation method and the usage
CN104628863A (en) * 2013-11-14 2015-05-20 中国医学科学院医药生物技术研究所 Double-target and double-warhead anti-tumor fusion protein as well as coding gene and application thereof
CN108779167A (en) * 2015-10-28 2018-11-09 宾夕法尼亚州大学信托人 Intrathecal application adeno-associated virus vector is used for gene therapy
CN105343874A (en) * 2015-11-11 2016-02-24 固安鼎泰海规生物科技有限公司 Prostate cancer nucleic acid vaccine
CN113171448A (en) * 2015-11-11 2021-07-27 固安鼎泰海规生物科技有限公司 Prostate cancer nucleic acid vaccine

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