CN1954073A - Modified Bouganin proteins, cytotoxins and methods and uses thereof - Google Patents
Modified Bouganin proteins, cytotoxins and methods and uses thereof Download PDFInfo
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Abstract
Modified forms of bouganin protein are provided having biological activity and a reduced propensity to activate human T cells as compared to the non-modified bouganin protein. Also provided are T-cell epitope peptides of bouganin, and modified T-cell epitope peptides of bouganin, which have a reduced propensity to activate human T cells as compared to the non-modified T-cell epitope peptide. Also provided are cytotoxins that comprise a ligand which binds to cancer cells, and is attached to modified bouganin proteins. Also provided are methods of inhibiting or destroying mammalian cancer cells using the cytotoxins and pharmaceutical compositions for treating human cancer.
Description
Invention field
The present invention relates to by modified bouganin albumen and comprising can be used as cancer therapeutic agent by the cytotoxin of modified protein.Exactly, thus by removing or changing the immunogenicity that the T-cell epitope reduces the bouganin toxin.
Background of invention
Have many such examples, the effectiveness of therapeutic protein is subject to the immune response of not expecting that this therapeutic protein is occurred.Several mouse monoclonal antibodies have shown the possibility as therapeutical agent in many human diseases environment, but they reply lost efficacy [Schroff, people such as R.W. (1985) Cancer Res. owing to inducing significant human antimouse antibody (HAMA) in case-specific
45: 879-885; Shawler, people such as D.L. (1985) J.Immunol.135:1530-1535].For monoclonal antibody, developed many technology and replied to attempt to reduce HAMA that [WO 89/09622; EP 0239400; EP 0438310, WO 91/06667].These recombinant DNA method usually reduce the mouse genetic information in the final antibody construction thing and improve people's genetic information in the final construction simultaneously.However, in some cases, " humanization " antibody of gained has still caused immunne response [Issacs J.D. (1990) Sem.Immunol. in patient's body
2: 449,456; Rebello, people such as P.R. (1999) Transplantation
68: 1417-1420].
The key of induce immune response is that exist in protein can be by presenting the peptide that stimulates the T cytoactive to the II class MHC molecule, promptly so-called t cell epitope.Such T-cell epitope normally any can with II class MHC molecule bonded amino acid residue sequence." T-cell epitope " inferred when it combines with the MHC molecule can be by the epi-position of T-cell receptors (TCR) identification, thereby and it can be by combine the activation promotion T-cell response that causes these T cells with TCR at least in theory.
II class MHC molecule is the highly polymorphic protein of a group, and they play an important role in helper T-cell is selected and activated.Human leucocyte antigen (HLA) group DR (HLA-DR) is the main isotype of this histone matter; Yet isotype HLA-DQ carries out similar function with HLA-DP.In the human colony, individuality has two to four DR allelotrope, two DQ and two DP allelotrope.The structure of many DR molecules is analyzed clear, and they are rendered as open end peptide binding groove with many and protein hydrophobic residue (pocket residue) bonded hydrophobic pocket [people (1993) Nature such as Brown
364: 33; People such as Stern (1994) Nature
368: 215].The polymorphism that is used to distinguish allotype II quasi-molecule has been facilitated different peptide mating surfaces extremely various in the peptide binding groove, and has guaranteed its identification heterologous protein and initiate maximum flexibility ratio to the immunne response of pathogeny biology on population level.
The immunne response of therapeutic protein is presented approach by II class MHC peptide to be taken place.Exogenous protein was swallowed and was presented with the II class MHC molecule of DR, DQ or DP type through processing this moment.II class MHC molecule is expressed by special antigen presenting cell (APC), such as scavenger cell and dendritic cell etc.II class MHC peptide complex is by the combination of the similar T-cell receptors of T-cell surface, and with cross coupled such as other so specific coreceptor of CD4 molecule, can induce in the T-cell to present active state.This activation causes the release of the cytokine one-step activation of going forward side by side to produce antibody or activate the T-killer cell such as the lymphocyte of such other of B cell and form full cellullar immunologic response.
According to what WO98/52976, WO00/34317, WO02/069232, WO02/079232 and WO02/079415 recognized, it is the first step that the T-cell epitope is identified that epi-position is removed.In these instructions, remove the T-cell epitope of expectation by the aminoacid replacement that in target protein, carries out discretion.Except that computer technology, also have the method for the synthetic peptide of many mensuration in conjunction with the ability of II class MHC molecule.A kind of exemplary method has utilized the source of the allotypic B-clone of definite MHC as II class MHC mating surface, and can be applicable to II class MHC part and identify [people (1994) J.Immunol. such as Marshall K.W
152: 4946-4956; People such as O ' Sullivan (1990) J.Immunol.
145: 1799-1808; People (1997) J.Immunol such as Robadey C.
159: 3238-3246].Yet such technology is unsuitable for screening a plurality of potential epi-positions from the various MHC allotype of extreme, and they also can't verify the ability that a kind of binding peptide works as the T-cell epitope.
The technology of the soluble complex of exploitation reorganization MHC molecule and synthetic peptide also is used [Kern, people such as F. (1998) Nature Medicine gradually
4: 975-978; Kwok, people such as W.W. (2001) TRENDS in Immunol.
22: 583-588].These reagent and method be used for identifying be derived from whether people or laboratory animal peripheral blood sample exist can be in conjunction with the T-cell clone of specific MHC-peptide complex, and be suitable for screening a plurality of potential epi-positions from various MHC allotype extremely
The bioassay method of T-cell activation provides a kind of actual selection approach for reading test peptide/protein sequence excites the ability of immunne response.The example of these class methods comprises that people such as Petra is applied to T-cell proliferation detection method streptokinase, utilizes synthetic peptide to stimulate T-clone to carry out work [Petra, people such as A.M. (2002) J.Immunol. of epitope mapping afterwards
168: 155-161].Similarly, utilized the T-cell proliferation detection method of the proteic synthetic peptide of tetanus toxin successfully to define main immune epitope [people (1993) J.Immunol. such as Reece J.C. of this toxin
151: 6175-6184].WO99/53038 discloses a kind of method, in the method by utilizing isolating people's immunocyte subgroup, under the situation that has the synthetic peptide of purpose, promote their vitro differentiation and cultivation, and measure the propagation that any institute inductive is cultivated the T-cell, can measure the T-cell epitope in the test proteins.People such as Stickler have also described same technology [Stickler, people such as M.M. (2000) J.Immunotherapy
23: 654-660], in these two examples, all this method is applied to the T-cell epitope in the bacterial detection subtilisin.The cell separation technology of such Technology Need carefulness and the cell cultures of adding the various kinds of cell factor to be obtaining gratifying immunocyte subgroup (dendritic cell, CD4+ and/or CD8+T-cell), and be unprofitable to utilize the fast flux screening of a plurality of donor samples.
Recently, developed a kind of will based on overall T-cell proliferation detection method and when the protein of epi-position is removed in design on computers simulating peptide MHC in conjunction with the combined method that combines.WO?03/104803]。
As above address its result, gratifying is to identify and remove or reduce at least a kind ofly to have therapeutic value in general but have T-cell epitope on immunogenic peptide, polypeptide or the protein at first.
Summary of the invention
The objective of the invention is to overcome this practical problems: can cause the immunne response that causes producing with soluble proteins bonded host antibody because of therapeutic purpose import soluble protein in the human body.The present invention attempts by providing the bouganin albumen with the initiation immunne response tendency that weakens to solve this problem.According to method described herein, the inventor has identified and has comprised the zone of driving to the T-cell epitope of this proteic immunne response in the bouganin molecule.
The present invention relates to a kind of modified Bouganin proteins, this is modified Bouganin and is had the tendency of the initiation immunne response that weakens.In a kind of preferred implementation, this is modified Bouganin and is had the tendency of the activation T-cell that weakens, and modification has been passed through at the one or more amino-acid residues place of this quilt modification Bouganin in the T-cell epitope.This T-cell epitope is preferably selected from:
A) AKVDRKDLELGVYKL (epitope regions R1, SEQ ID NO:2),
B) LGVYKLEFSIEAIHG (epitope regions R2, SEQ ID NO:3); With
C) NGQEIAKFFLIVIQM (epitope regions R3, SEQ ID NO:4).
The invention still further relates to a kind of cytotoxin that comprises with modified Bouganin proteins bonded target part of the present invention.In one embodiment, this target part is a kind of and cancer cells bonded part.In another embodiment, this part is a kind of and cancer cells bonded antibody or antibody fragment.In a kind of specific implementations, this antibody recognition Ep-CAM or tumour-knot related antigen.In the most specific a kind of embodiment, the invention provides the cytotoxin of a kind of VB6-845 of comprising or VB6-011.
On the other hand, the invention provides a kind of inhibition or tumoricidal method, comprise to cancer cells and use cytotoxin of the present invention.
The invention still further relates to a kind of needs and usually treat method for cancer by use cell toxicant of the present invention to it according to animal.
Further, also provide a kind of preparation to be used for the treatment of the method for the medicine of the animal that suffers from cancer, may further comprise the steps: identify the T-cell epitope of bouganin, modify Bouganin thereby the one or more amino-acid residues preparations in the modification T-cell epitope have the quilt of the activation T-cell tendency that weakens; Preparation has a cytotoxin that is incorporated into cancer-binding partner of being modified Bouganin; With this cytotoxin is suspended in pharmaceutical acceptable carrier, thinner or vehicle.
Aspect another, the invention provides a kind of pharmaceutical composition that is used for the treatment of cancer, it comprises cytotoxin of the present invention and pharmaceutical acceptable carrier, thinner or vehicle.
Cytotoxin of the present invention, composition and method can be used for treating various multi-form cancers, such as colorectal carcinoma, mammary cancer, ovarian cancer, carcinoma of the pancreas, head and neck cancer, bladder cancer, gastrointestinal cancer, prostate cancer, minicell and non-small cell carcinoma, sarcomata, neurospongioma, T-and B-cell lymphoma.
The present invention also provides the proteic T-cell epitope peptide of Bouganin and of the present invention by modification T-cell epitope peptide.
According to following detailed description, further feature of the present invention and advantage will manifest gradually.Yet, be to be understood that, the specific embodiment of this detailed description and the explanation preferred embodiment for the present invention provides as just explaining, because various different variations and the modification that obtains in spirit and scope of the invention from this detailed description is conspicuous to those skilled in the art.
The accompanying drawing summary
The present invention will describe in conjunction with the following drawings:
The T-cell proliferation of the PBMC donor sample that three synthetic peptides of accompanying drawing 2 demonstrations are different with two detects.The name that has detected 1 μ M final concentration final concentration (panel A) and 5 μ M final concentrations (panel B) is called the peptide of DeI-41, DeI-44 and DeI-50.These peptides are derived from the immunogenicity zone of bouganin molecule, are included as the replacement of removing its immunogenicity and designing.
Accompanying drawing 3 expression VB6-845, a kind of have a Fab anti--Ep-CAM modified the Bouganin cytotoxin, wherein this de-bouganin (Bou156) is connected with the C-end of CH structural domain by a furin joint.The bicistronic mRNA unit of the former sequence of accompanying drawing 3A code displaying, accompanying drawing 3B shows the nucleic acid coding sequence (SEQ IDNO:15) and the aminoacid sequence (SEQ ID NO:16) of this former sequence, and accompanying drawing 3C shows the assembling VB6-845 albumen that does not contain the pelB sequence.
Accompanying drawing 4 shows the figure of expression vector pING3302.Utilize EcoRI and XhoI restriction site that the insertion among the embodiment is connected in 3302 carriers.
Accompanying drawing 5 shows that the contrast Fab that does not contain plant poison resists-the Ep-CAM construct de-bouganin (VB5-845).The two suitable model subelement of the former sequence of accompanying drawing 5A code displaying, accompanying drawing 5B shows the nucleic acid coding sequence (SEQ ID NO:17) and the aminoacid sequence (SEQID NO:18) of this former sequence, and accompanying drawing 5C shows the assembling VB5-845 albumen that does not contain the pelB sequence.
Accompanying drawing 7 shows the anti-Ep-CAM of Fab, the de-bouganin construct, and VB6-845-NVH-de-bouganin, wherein Bou156 is connected in V
HThe N-end of structural domain.The two suitable model subelement of the former sequence of accompanying drawing 7A code displaying, accompanying drawing 7B shows the nucleic acid coding sequence (SEQ IDNO:21) and the aminoacid sequence (SEQ ID NO:22) of this former sequence, and accompanying drawing 7C shows the assembling VB6-845-NVH-de-Bouganin albumen that does not contain the pelB sequence.
Accompanying drawing 10 shows the reactive result of study of flow cytometry.Accompanying drawing 10A shows VB6-845 (construct of accompanying drawing 3) and VB6-845-C
L-de-bouganin (construct of accompanying drawing 6) is result among the A-375 at Ep-CAM-positive cell line CAL 27 and OVCAR-3 and Ep-CAM-negative cells, and accompanying drawing 10B shows with VB6-845 (construct of accompanying drawing 3) and VB6-845-and sets toxalbumin (construct of accompanying drawing 14C) in vain and contrast the result of the same test that (PBS) done.
Accompanying drawing 11 shows as described in example 7 above-VB6-845 and Proxinium
TMThe figure of competitive assay result in the NIH:OVCAR-3 cell.
Accompanying drawing 12 is the figure that show the acellular detected result of embodiment 7.
Compare VB6-845 (construct of accompanying drawing 3), VB6-845-CL-de-bouganin (construct of accompanying drawing 6) and the Cytotoxic MTS cytotoxicity detected result of de-bouganin (Bou156) in CAL 27 (accompanying drawing 13A) and NIH:OVCAR3 (accompanying drawing 13B) cell among the accompanying drawing 13 demonstration embodiment 8.
Accompanying drawing 14A and B show among the embodiment 8 that VB6-845 relatively (construct accompanying drawing 3), VB6-845-set toxalbumin (construct accompanying drawing 14C) in vain and set the Cytotoxic MTS cytotoxicity detected result of toxalbumin in CAL 27 (accompanying drawing 14A) and NIH:OVCAR3 (accompanying drawing 14B) cell in vain.Accompanying drawing 14C show nucleic acid encoding sequence (SEQ ID NO:25) and VB6-845-set the aminoacid sequence (SEQ ID NO:26) of toxalbumin construct in vain.
Accompanying drawing 15 shows the nucleic acid coding sequence (SEQ ID NO:27) and the aminoacid sequence (SEQ ID NO:28) of the former sequence of VB6-011.
Accompanying drawing 16 shows the MTS cytotoxicity detected result of embodiment 9, the cytotoxicity of its VB6-011 in the MB-435S cell.
Detailed Description Of The Invention
The inventor has identified the T-cell epitope among the Bouganin, and has designed with having prepared and compared the modified Bouganin proteins with the activation people T cell tendency that weakens with non--modified Bouganin proteins.
(A)
Modified Bouganin proteins
The present invention relates to a kind of modified Bouganin proteins, wherein compared the initiation immunity that has weakened by modification-Bouganin albumen and reply with non-for obtaining, be preferably the T-cell response, tendency bouganin is modified. Ripe Bouganin albumen is to have 250 amino acid whose single chain polypeptides, and molecular weight is about people (2002) the EuR.J. Biochem. such as 26,200 Da[Den Hartog269: 1772-1779; United States Patent (USP) NO.6,680,296]. Bouganin separates 1 type ribosome inactivating protein (RIP) from plant Bougainvillea spectabilis Willd [people (1997) Planta such as Bolognesi203: 422-429]. These RIP from plant make the main rRNA depurination of cell, destroy ribosomes and cause that protein is synthetic to be ended and the RNA N-glucosides enzyme of cell death thus.
The amino acid sequence (take the single-letter representation) of ripe Bouganin albumen as:
YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKR
FVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLDKV
PTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKVDRKDLELGVYK
LEFSIEAIHGKTINGQEIAKFFLIVIQMVSEAARFKYIETEVVDRGLY
GSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPW
VVNKVSQISPDMGILKFKSSK[SEQ ID NO：1]。
Term " non--modified Bouganin proteins " means, without causing the Bouganin albumen that the modification of tendency is replied in immunity for reducing it. The sequence of wild type or non--modified Bouganin is shown among the SEQ ID NO:1. Yet, it will be understood by those skilled in the art that term " non--quilt is modified Bouganin " also comprises the modification to SEQ ID NO:1, causes the tendency that immunity is replied as long as such modification can not reduce it. The conserved amino acid that the example of the modification that can make SEQ ID NO:1 comprises fragments of peptides and do not reduce this protein immunity originality replaces.
Term " modified Bouganin proteins " means to compare the Bouganin albumen through modifying with non--modified Bouganin proteins (as mentioned above), and wherein said modification can reduce bouganin and cause the tendency that immunity is replied. Modified Bouganin proteins can also refer to the bouganin of disimmunity. " modified Bouganin proteins " can be through the full length sequence of modification or through modification non--the modified Bouganin proteins fragment. Compare with wild type bouganin sequence, " modified Bouganin proteins " also may comprise the change that other does not change the immune originality of this peptide. Modified Bouganin proteins preferably has with non--quilt is modified the identical biology activity of Bouganin.
Term used herein " tendency that the initiation immunity of reduction is replied " means, and modified Bouganin proteins has than non--quilt is modified the lower immune originality of Bouganin.
Term " immunne response " comprise cell and humoral immunoresponse(HI) the two.In a kind of preferred implementation, modified the tendency that Bouganin has the activation T-cell of reduction.
Term used herein " tendency of the activation people T-cell of reduction " means the tendency that modified Bouganin proteins has the activation people T-cell lower than non--modified Bouganin proteins.The enough methods known in the art of those skilled in the art's energy comprise this proteic stimulation index of assessment, and test is modified Bouganin and whether had the tendency of lower activation T-cell.
Term used herein " stimulation index " refers to be modified or non--modified Bouganin proteins activates the tolerance of human T-cell's ability.For example, can test and modified or ability that non--modified Bouganin proteins or its peptide are replied in the external people of exciting T-cell proliferation.The primitive man T-cell of taking from healthy donors when employing is finished the method for the type, and the inventor determines, in a kind of like this operation of detection method, being equal to or greater than 2.0 stimulation index is a tolerance of effectively inducing propagation.Stimulation index derives from propagation mark (if for example use that will record with test peptides usually
3Words that the H-thymus pyrimidine mixes are calculated the radioactivity of per minute) divided by the mark that in the cell of Contact test peptide not, records.
In one embodiment, the invention provides a kind of modified Bouganin proteins, this modified Bouganin proteins biologically active and have the activation human T-cell's lower tendency than non--modified Bouganin proteins.
In another embodiment, the invention provides a kind of modified Bouganin proteins, this modified Bouganin proteins has the activation human T-cell's lower than non--modified Bouganin proteins tendency and has the biological activity that is lower than non--modified Bouganin proteins.In another embodiment, the invention provides a kind of modified Bouganin proteins, this modified Bouganin proteins has the activation human T-cell's of reduction tendency and lifeless matter activity.Like this by modified protein can, for example, detect or tolerize subjects with comparing, be used for.
Term as used herein " biological activity " is meant and modified or non--and modified Bouganin proteins suppresses the ability of the protein synthesis on the rrna, and it can be measured by several different methods.Should be noted that still biologically active of modified Bouganin proteins, though that such activity is lower than is non--adorned proteic, however but it need have the detection of active of certain level.For example, modified or the biological activity of non--modified Bouganin proteins can be tested and appraised its N-glycosidase activity and measures, and especially have enough activity remarkable inhibition to protein translation is provided.During a kind of so suitable detection method is included in that acellular albumen is synthetic and detects test variant bouganin albumen with non--modified the activity that Bouganin compares.The DNA that transcribes/translate coupling mixture, coding reporter protein luciferase and non-modified and the grade dilution of modified Bouganin proteins is hatched jointly that will comprise methionine(Met).Can easily measure the luciferase level of being translated with luminescent counter after adding substrate reagent.The bouganin N-glycosidase activity that exists in luminous quantity of being measured and the reaction is inversely proportional to.The albumen such as inactivation Bouganin is provided usually, for example comprises Y70A and replace such negative control.
In a kind of preferred implementation, modified the one or more T-cell epitopes of Bouganin peptide in the Bouganin protein sequence to be modified.
Term " T-cell epitope " means, can in conjunction with the main histocompatibility complex of II type (MHC), can stimulate the T-cell and/or with the mixture of II class MHC in can be in conjunction with the aminoacid sequence of (do not need can measure activation) T-cell.
On the one hand, can use the group method that obtains containing through the modified Bouganin proteins of the T-cell epitope modified may further comprise the steps in the present invention:
(i) determine the aminoacid sequence of protein or its part;
(ii) by identifying one or more potential T-cell epitope in this protein amino acid sequence such as utilizing external or computer chip technology or bioassay method to measure the such method of peptide and combining of MHC molecule;
(iii) design has one or more amino acid whose new sequence variants through modifying that is arranged in the T-cell epitope of being identified, according to utilize external or computer chip technology or bioassay method to measure peptide determined with combining of MHC molecule, come to reduce basically or remove the activity of this T-cell epitope in this way.Construct such sequence variants epi-position in the mode of avoiding forming new potential T-cell epitope owing to this series jump, unless next modify so new potential T-cell epitope more by this way with activity basic or removal T-cell epitope;
(iv) make up such sequence variants and test described variant and have the variant of desired characteristic to identify one or more according to the recombinant technology of knowing by recombinant DNA technology; With
(v) optionally repeating step (ii) to (iv).
In one embodiment, by the amino-acid residue in arbitrary T-cell epitope in non--modified Bouganin proteins being replaced, adds or lacking implementation step (iii).In another embodiment, the method for preparing modified Bouganin proteins is carried out with reference to homologous protein sequence and/or computer model.
Identify (ii) that according to step potential T-cell epitope can finish according to the method that this area was described in the past.Suitable method is disclosed in WO 98/59244, WO 98/52976, WO00/34317, WO 02/069232, can be used for identifying the tendency that combines of bouganin derived peptide and II class MHC molecule.Be the identification of organism related peptides, the inventor has developed the method that the stripped people T-cell proliferation of a kind of exploiting of being used for detects.Confirmed that this method is a kind of very effective method, it is included in certain scheme overlapping bouganin derived peptide sequence is tested to scan and to test whole bouganin sequence.The ability that the synthetic peptide of test excites the propagation of the people T-cell of vitro culture to reply.The inventor implements the type method with the natural human T-cell of taking from healthy donors, establishes, and in a kind of like this operation of detection method, being equal to or greater than 2.0 stimulation index is a tolerance of effectively inducing propagation.Stimulation index derives from the propagation mark that will record with test peptides (if for example using the 3H-thymus pyrimidine to mix the radioactivity that calculates per minute) usually divided by the mark that records in the cell of Contact test peptide not.
Correspondingly, in research of the present invention, in the T-cell proliferation of making of the PBMC (peripheral blood lymphocytes) that takes from natural donor (sensitization that does not also promptly have known bouganin) detects, 89 synthetic 15-mer peptides (listing in table 1) have been used.Selected 20 parts of donor PBMC samples to reach optimal II class MHC allotype fraction of coverage.Passing through
3The H-thymus pyrimidine mixes to be assessed before the propagation, with each peptide three parts of culture moderate stimulations 7 days.It is two concentration that all peptides all dilute: 1 μ M and 5 μ M.Stimulation index (SI) is calculated as: mix cell
3The H amount is divided by what mix in the vacation stimulation contrast
3The H amount.
Present method has identified bouganin molecule immunogenic zone of tool in human body.Therefore, in a kind of specific implementations, modified Bouganin proteins is selected from following one or more amino-acid residues and obtains modifying in the T-cell epitope:
A) AKVDRKDLELGVYKL is called epitope regions R1 (SEQID NO:2) herein;
B) LGVYKLEFSIEAIHG is called epitope regions R2 (SEQ IDNO:3) herein; With
C) NGQEIAKFFLIVIQM is called epitope regions R3 (SEQ IDNO:4) herein
SI＞2 according to providing in two or more donor PBMC samples have identified these T-cell epitopes.Above-mentioned disclosed peptide sequence has provided and has made up the required key message of modified Bouganin proteins, these by modified protein in one or more these peptides weakened.
In one embodiment of the invention, modified Bouganin proteins of the present invention has been removed at least one T-cell epitope.In another embodiment, modified Bouganin proteins of the present invention has been removed one, two or three T-cell epitopes.The present invention has also considered a kind of modified Bouganin proteins, wherein preferably has 1 to 9 amino-acid residue to be modified in the T-cell epitope.In another embodiment, modified 1 to 5 amino-acid residue.Term as used herein " by modifying " means, and by replacing, add or lacking amino-acid residue is carried out being modified, and preferably by replacement, but Bouganin albumen has the stimulation human T-cell's who weakens tendency.In another embodiment, adorned albumen has biologic activity.More preferably, modified Bouganin proteins of the present invention is modified by replacing in specific arbitrary amino acid whose position corresponding to above-mentioned sequence (a) and (b) or (c).
One embodiment of the present invention comprise bouganin albumen, for these albumen, the II class MHC part that identifies in any one at epi-position R1～R3 have been carried out modifying so that can the allotypic quantity of bonded MHC in conjunction with disappearing or reducing this peptide.Can be by replacing, add or disappearance being come making in R1 to the R3 zone in conjunction with disappearing or reducing the amino acid that this peptide can bonded MHC allotype quantity and modify.
For removing the T-cell epitope, estimate that in peptide sequence can realize weakening substantially or remove the active appropriate location of T-cell epitope carries out aminoacid replacement.In practice, suitable location is to be incorporated into II class MHC in conjunction with the amino-acid residue in the ditch pocket in one embodiment.
In one embodiment, the combination of first pocket in the crack that the so-called P1 that is positioned at peptide or P1 anchor position are put is modified.Peptide P1 anchor residue combines the main determining factor that quality that the combination between ditch first pocket does mutually is regarded as the total binding avidity of whole peptide molecule with II class MHC.The suitable replacement of this position of peptide will the residue of this pocket carries out with more being not easy to hold into, for example, is substituted by a more hydrophilic residue.Also considered to be arranged in the peptide and be equal to the bonded amino-acid residue of MHC in conjunction with other pocket area in crack.
Being appreciated that single amino acids replacement, the disappearance in given potential T-cell epitope or adding is a kind of approach of preferred removal epi-position.Can consider that the combination in single epi-position modifies (also promptly replace, lack and adds), for example when each definition list interdigit overlaps each other, be particularly suited for employing and make up modification, as epitope regions R1 and R2 among the present invention overlapping 5 residues.In addition, can be similar to that II class MHC carries out in conjunction with the position of ditch " pocket residue " that single amino acids in the given epi-position is modified or single epi-position in combination modify, but be not any position in peptide sequence.Can utilize the technology based on chip known in the art to do modification with reference to homologous structure or structure method.All such modifications all fall in the scope of the invention.
In suc scheme, in one embodiment of the invention, the replacement in the epitope regions R1 is included in the variation of position V123, D127 and/or E129.Similarly, for epitope regions R2, this replacement in one embodiment is positioned at Y133.This residue falls into the overlap of R1 and R2, but the replacement of Y133 position is enough to remove the relevant II class MHC part of R2 and himself is not enough to remove the relevant II class MHC part of R1.For epitope regions R3, in one embodiment of the invention, be that residue E151 and/or I152 are replaced.
In all situations, all be that one or more alternative amino-acid residue is replaced.With the analysis revealed that the MHCII simulation software is done R1, amino-acid residue 123,127,129 and 131 is in conjunction with the Key residues of MHC II molecule in this epi-position.Residue 123 is preferred mutational sites in R1 district, because it is arranged in molecular surface, is variable away from avtive spot and at the RIP sequence alignment.Yet not all replacement all produces bioactive molecule, therefore need verify sudden change in biological activity assay.Therefore, for example in R1, replacing V123T, V123A and V123Q is preferred selectable replacement example.Find among the RIP that residue 131 is absolute conservative, so it might be unsuitable for sudden change.Residue 127 and 129 is not a high conservative, but finds to have only the residue of limited quantity that MHC II is combined with influence.Replacement group: D127G, D127A, E129Q and E129G also are preferred replacements.For R2, residue 133 is shown as removes possible candidate's residue of MHC II bonded, and its tangible surface alignment (determining according to model) adds that it is not that such fact of high conservative makes it become good candidate's residue of sudden change in RIP.Find that preferred optional to select generation be Y133N, Y133T, Y133A, Y133R, Y133D, Y133E, Y133Q, Y133G, Y133K, Y133H and Y133S.For R3, amino-acid residue 152,155 and 158 is accredited as MHC II bonded Key residues.Yet residue 155 and 158 is parts of one section high conservative hydrophobic sequence, hints that thus their sudden change can not produce bioactive molecule.Find that weak conserved residues is more possible candidate.For R3, replacement group: I152Q and I152A also are preferred replacements.
Correspondingly, the invention provides a kind of modified Bouganin proteins, bouganin wherein is at following X
1, X
2, X
3, X
4Or X
5In one or more position modified:
A) AKX
1DRKX
2LX
3LGVX
4KL (epitope regions R1, SEQ ID NO:5);
B) LGVX
4KLEFSIEAIHG (epitope regions R2, SEQ ID NO:6); With
C) NGQEX
5AKFFLIVIQM (epitope regions R3, SEQ ID NO:7)
X wherein
1To X
5Can be any amino acid.
In a kind of specific implementations, X
1Be T or A or Q; X
2Be G or A; X
3Be Q or G; X
4Be N or D or T or A or R or Q or E or G or H or K or S; And X
5Be Q or A (epitope regions R1, SEQ ID NO:8; Epitope regions R2, SEQ ID NO:9; Epitope regions R3, SEQ ID NO:10).
Study, consider in conjunction with simulation and from the structure of sequence homology analysis based on the MHC peptide of chip and take all factors into consideration according to the peptide epitopes mapping of making of exsomatizing T-cell detection, can edit and obtain a most preferred replacement group.At last, if some bioactive peptides are preferred, can detect this external activity that undertaken by modified protein that comprises one or more replacements.
Correspondingly, in another embodiment, the invention provides a kind of Bouganin peptide of being modified, it comprises aminoacid sequence:
YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDK
RFVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLD
KVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKX
1DRKX
2LX
3L
GVX
4KLEFSIEAIHGKTINGQEX
5AKFFLIVIQMVSEAARFKYIETE
VVDRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQ
LISPSNDPWVVNKVSQISPDMGILKFKSSK
X wherein
1To X
5Can be any amino acid (SEQ ID NO:11).
In a kind of preferred implementation, X
1Be T or A or Q; X
2Be G or A; X
3Be Q or G; X
4Be N or D or T or A or R or Q or E or G or H or K or S; And X
5Be Q or A (SEQ ID NO:12).
In a kind of specific implementations, modified Bouganin proteins comprises aminoacid sequence:
YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDK
RFVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLD
KVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAK
ADRK
ALELG
V
NNKLEFSIEAIHGKTINGQE
AAKFFLIVIQMVSEAARFKYIETEVV
DRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLIS
PSNDPWVVNKVSQISPDMGILKFKSSK(SEQ?ID?NO：13)。
In another embodiment, modified Bouganin proteins comprises aminoacid sequence:
YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDK
RFVLVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLD
KVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAK
ADRK
ALELG
V
QKLEFSIEAIHGKTINGQE
AAKFFLIVIQMVSEAARFKYIETEVV
DRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLIS
PSNDPWVVNKVSQISPDMGILKFKSSK(SEQ?ID?NO：14)。
The underscore residue is replaced by the residue from non--modified Bouganin proteins.
As those skilled in the art institute clearly, a plurality of selectable modification groups can realize removing unwanted epi-position this purpose.Yet institute's calling sequence will keep homology with protein disclosed herein to a great extent, and they fall into scope of the present invention thus.The tangible chemical equivalence thing of sequence disclosed by the invention also is considered and falls in the scope of the invention.Such Equivalent comprises the protein of finishing identical function in essentially identical mode substantially.
Modified Bouganin proteins of the present invention in another embodiment has 1,2,3,4,5 or more a plurality of amino acid modified in proteinic T-cell epitope.
In another embodiment, when testing, modified Bouganin proteins of the present invention compares the stimulation index that inspires reduction in the T-cell detection with non--modified Bouganin proteins.
In another embodiment of the invention, with the T-cell detection the proteic T-cell epitope of Bouganin is mapped, afterwards these epi-positions are modified so that modified Bouganin proteins inspires the stimulation index that is lower than non--modified Bouganin proteins when testing in the T-cell detection once more, preferably this stimulation index is less than 2.0.
It will be apparent to those skilled in the art that if modified Bouganin proteins have a basic reduction or do not have a biological activity, then it also needs to replace, add or disappearance is done further to recover the biological activity of modified Bouganin proteins by amino-acid residue.Yet, such that have a basic reduction or do not have bioactive modified Bouganin proteins still to be included in the scope of the present invention, and can perhaps be used for tolerization as the contrast in detecting.
In one embodiment, thus modified Bouganin and undergo mutation at 70 tyrosine residues place and produce a kind of inactivation bouganin.In a kind of specific implementations, tyrosine position 70 is replaced by L-Ala.In a kind of preferred implementation, modified Bouganin and had sequence:
YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDK
RFVLVDITTTSKKTVKVAIDVTDVAVVGYQDKWDGKDRAVFLD
KVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKVDRKDLELG
VYKLEFSIEAIHGKTINGQEIAKFFLIVIQMVSEAARFKYIETEVVD
RGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISP
SNDPWVVNKVSQISPDMGILKFKSSK[SEQ?ID?NO：129]。
Under planning of the present invention, thereby by sudden change epi-position being weakened to handle obtains no longer the sequence that can work as the T-cell epitope.Might realize the target sequence directed mutagenesis with recombinant DNA method, many such technology all are that what can obtain also is known in the art.In the practice, can prepare and test many modified Bouganin proteins for obtaining required immunity and functional character.The particularly important is, when protein sequence was modified, the change of being conceived can not introduced new immunogenicity epi-position.In the practice can by utilize any suitable method test once more conceive epi-position in the sequence and/or II class MHC part existence whether avoid the generation of this incident.
Modified Bouganin proteins of the present invention can also comprise or be used for to obtain or design " peptide mimics "." peptide mimics " is that the structure that is used as the peptide surrogate in intermolecular mutual work (is summarized referring to people such as Morgan (1989) Ann.Reports Med.Chem.
24: 243-252).Peptide mimics comprises the 26S Proteasome Structure and Function feature that may comprise or may not comprise amino acid and/or peptide bond but keep peptide of the present invention, comprises the activation human T-cell's of biological activity and reduction tendency, composite structure.Peptide mimics also comprises peptide, oligopeptides (people (1972) Proc.Natl.Acad such as Simon, Sci USA89:9367).
Can be according to the amino acid whose system of L-being replaced, replaces and come the designed peptide stand-in by the information of replacing the peptide bond acquisition with amino key system with the side chain of the group that has the different electrical property features of tool by D-amino acid.Can also import the positional configuration restriction and come definite active configuration requirement candidate's peptide mimics.These stand-in comprise that amino key of isostere or D-amino acid are to stablize or to promote reverse configuration and auxiliary stable molecule.The annular amino acid analogue can be used for retraining amino-acid residue in specific configuration states.These stand-in also can comprise the stand-in of the proteinic secondary structure of the present invention.These structures can be simulated the three-dimensional orientation of amino-acid residue in the into proteinic known secondary configuration.Can also use N-substituted amino acid oligopeptides to intend peptide, also can will intend peptide as the structural motif that produces recruit's Chemical Diversity library.
Can prepare molecule of the present invention by any in the several method, but most preferably adopt conventional recombinant technology to finish.With the derive polynucleotide (DNA) of the arbitrary preferred protein sequence of coding of protein sequence provided herein and information is a kind of relative directly method.For example can utilize the computer software instrument,, realize such as DNSstar software cover [DNAstar Inc, Madison, WI, USA] or similar software.The dna sequence dna of any can encode preferred polypeptide of the present invention or its remarkable homologue like this is interpreted as embodiments of the present invention.
As an overall plan, the gene of arbitrary modified Bouganin proteins sequence of encoding can synthesize by gene and prepare and it is cloned into suitable expression vector.To express successively and import host cell and selected and cultured cells.Purifying protein of the present invention from substratum, and be made into the preparation of being used for the treatment of property administration.As selectable form, by for example utilizing the RNA that makes from Bougainvillea spectabilis Willd roots of plants tissue to obtain wild-type bouganin gene order according to cDNA clone strategy.Wild type gene can be used as the sudden change template and is used to make up preferred variant sequence.In this, utilize people such as Higuchi [people (1988) Nucleic Acids Res. such as Higuchi
16: 7351] " overlapping extension PCR " strategy of describing is very easily, although other method and system also is easy to use.
Can assess the proteinic biological activity of the present invention by many modes equally.In one embodiment, use the N-glycosidase activity, especially, identify and modified the Bouganin molecule with being enough to that protein translation is produced the activity that significantly suppresses.A kind of so suitable detection be included in the cell-free protein synthesis system test modified Bouganin proteins with non--modified the activity that Bouganin compares.To comprise methionine(Met), coding reporter protein matter luciferase DNA and non--modified and the coupling connection mixture of transcribing/translate of the serial dilution thing of modified Bouganin proteins advances cultivation altogether.Add substrate reagent and can easily survey the luciferase level of being translated with luminometer.The bouganin N-glycosidase activity that exists in the measured luminous and reaction is inversely proportional to.Provide and for example comprise Y70A to replace such inactivation Bouganin albumen be conventional as negative control.
Can realize the structure of preferred and active bouganin molecule by recombinant DNA technology, this comprises the bouganin molecule that merges with purpose antibody or other target part.The method of purifying and operation recombinant protein comprises that fusion rotein is well known in the art.Required technology has fully been described in the document, for example, " Molecular Cloning:A Laboratory Manual ", second edition (people such as Sambrook, 1989); " Oligonucleotide Synthesis " (M.J.Gait, ed., 1984); " Animal Cell Culture " (R.I.Freshney, ed., 1987); " Methods inEnzymology " (Academic Press, Inc.); " Handbook of ExperimentalImmunology " (D.M.Weir﹠amp; C.C.Blackwell, eds.); " Gene TransferVectors for Mammalian Cell " (J.M.Miller﹠amp; M.P.Calos, eds., 1987); " Current Protocols in Molecular Biology " (people such as F.M.Ausubel, eds., 1987); " PCR:The Polymerase Chain Reaction ", (people such as Mullis, eds., 1994); " Current Protocols in Immunology " (people such as J.E.Coligan, eds., 1991).
Can prepare protein of the present invention and peptide with recombinant DNA method.Can also utilize the technology of knowing in the protein chemistry such as solid phase synthesis (Merrifield, 1964, J.Am.Chem.Assoc.85:2149-2154) synthetic (Houbenweyl or in homogeneity liquid, 1987, Methodsof Organic Chemistry, ed.E.Wansch, 15I and II volume, Thieme Stuttgart) prepares protein of the present invention by chemosynthesis.
The present invention also provides and comprises code book invention modified Bouganin proteins or peptide sequence, the as herein described proteinic sequence of optimized encoding such as SEQ ID NO:13 or SEQ ID NO:14, the nucleic acid molecule of separation and purification.
Term used herein " separation and purification " refers to, by the acellular substantially material of recombinant DNA technology generation or the nucleic acid of substratum, by precursor or other compound of chemosynthesis.The nucleic acid of " separation and purification " does not contain the sequence (yet promptly being positioned at nucleic acid 5 ' and 3 ' terminal sequence) of its native sequences both sides substantially yet.
Term as used herein " nucleic acid " refers to the nucleotide sequence or the nucleoside monomers that are connected to form by natural base, sugar and intersugar (skeleton).This term also comprise have a similar functions comprise that non-natural is monomeric is modified or be substituted sequence or its part.Nucleotide sequence of the present invention can be Yeast Nucleic Acid (RNA) or thymus nucleic acid (DNA), can comprise the natural base that comprises VITAMIN B4, guanine, cytosine(Cyt), thymus pyrimidine and uridylic.Sequence can also comprise such as drifting down the Yellow River bright, xanthoglobulin, the 2-aminoadenine, the 6-methyl, the 2-propyl group, with other alkyl VITAMIN B4,5-halogen uridylic, 5-halogen cytosine(Cyt), the 6-azauracil, the 6-nitrogen cytosine, with the 6-azathymine, pseudouracil, the 4-thiouracil, 8-halogen VITAMIN B4, the 8-aminoadenine, 8-sulphur VITAMIN B4,8-sulphur-alkyl VITAMIN B4,8-hydroxyadenine and other 8-substituted adenines, 8-halogen guanine, the amino guanine of 8-, the 8-Tioguanine, 8-sulfane base guanine, 8-hydroxyl guanine and other 8-9 substituted guanine, other nitrogen and deaza uridylic, thymus pyrimidine deoxidation nuclear, cytosine(Cyt), VITAMIN B4, or guanine, 5-trifluoromethyl uridylic and 5-three flucytosines such by modified base.
In one embodiment, the nucleic acid molecule of purifies and separates comprises the sequence of coded protein of the present invention and peptide, and preferred SEQ ID NO:13 or SEQ ID NO:14 comprise:
(a) nucleotide sequence, wherein T also can be U;
(b) with (a) complementary nucleotide sequence sequence;
(c) with (a) or (b) homologous nucleotide sequence sequence;
(d) under stringent condition with (a) at least 15 bases, the fragment of preferred 20 to 30 bases of (a) to (c) of (c) hybridization; Or
(e) because genetic code degeneracy and on codon, be different from the nucleotide sequence molecule of nucleotide sequence arbitrary in (a) to (c).
In addition, be appreciated that the present invention includes and comprise nucleotide sequence molecule and the fragment thereof that has the nucleotide sequence of basic sequence homology with the nucleotide sequence of code book invention protein and peptide.Term " sequence with basic sequence homology " means those nucleotide sequence sequences with the slight or inessential variation that is different from these sequences,, produces the proteinic sequence of function equivalence in essentially identical mode that is.These variations can be facilitated positional mutation or structural modification.
Nucleotide sequence with basic sequence homology comprises that the nucleotide sequence with code book invention protein and peptide has at least 80%, the nucleotide sequence of preferred 90% identity.
Another aspect of the present invention is provided under the hybridization conditions, preferably under stringent condition, can and have the fragment of at least 15 bases with the nucleic acid molecule of nucleotide sequence molecular hybridization of the present invention.Promote that the suitable stringent condition of DNA hybridization is well known by persons skilled in the art, perhaps can be at Current Protocols in Molecular Biology, John Wiley﹠amp; Sons, N.Y. (1989), 6.3.1-6.3.6 finds.For example, can use following condition: 6.0 * sodium chloride/sodium citrate (SSC) under about 45 ℃, wash with 2.0 * SSC in 50 ℃ afterwards.Can select tight degree according to employed condition in the washing step.For example, the salt concn in the washing step can be selected from 50 ℃ of high tight degree of about 0.2 * SSC down.In addition, the temperature in the washing step can be in about 65 ℃ high stringent condition.
Correspondingly, can be integrated in the suitable expression vector of guaranteeing this protein and peptide good representation according to the nucleotide sequence molecule of the present invention that means known in the art will have code book invention protein and a peptide.Possible expression vector includes but not limited to clay, plasmid or adorned virus (for example, replication defective retrovirus, adenovirus and adeno associated virus), as long as this carrier is compatible with employed host cell.This expression means " to be suitable for the carrier of transformed host cell ", and this expression vector comprises nucleotide sequence molecule of the present invention and the regulating and controlling sequence of selecting according to the employed host cell that is used to express that links to each other with nucleotide sequence molecule operability." operability links to each other " means this nucleotide sequence and links to each other with regulating and controlling sequence in the mode that allows this nucleotide sequence to express.
The present invention considers to comprise the recombinant expression vector of the present invention of transcribing and translate necessary regulating and controlling sequence of nucleotide sequence of the present invention or its fragment and the protein sequence that inserts thus.Suitable regulating and controlling sequence can be from multiple source, (for example comprise bacterium, fungi or virogene, referring to Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, the regulating and controlling sequence of describing among the San Diego, CA (1990)).Selection to suitable regulating and controlling sequence depends on selected host cell, and can easily be finished by those of ordinary skills.The example of such regulating and controlling sequence comprises: transcripting promoter and enhanser or RNA polymerase binding sequence, comprise the ribosome binding sequence of translation initiation signal.In addition, according to selected host and employed carrier, can in expression vector, be integrated into and bring other such sequence of sequence of transcribing inducibility such as replication orgin, additional DNA restriction site, enhanser with for sequence.Be appreciated that required regulating and controlling sequence can be provided by natural protein and/or its flank region.
Recombinant expression vector of the present invention can also include be beneficial to transform with recombinant molecule of the present invention or transfection the selective marker selected of host cell.The example of selectable marker gene comprises the gene of the protein that brings certain drug resistance, beta-galactosidase enzymes, paraxin acyltransferase or the Photinus pyralis LUC of encoding ratio as G418 and Totomycin.By monitoring transcribing of selectable marker gene such as the variation of the so proteinic concentration of selective marker of beta-galactosidase enzymes, paraxin acyltransferase or Photinus pyralis LUC.If the selectable marker gene coding is given the protein of transformant antibiotics resistance such as hygromycin resistance, then can select G418.The cell that is integrated with selectable marker gene will be survived, and other cell is with death.This is can be with the expression of recombinant expression vector of the present invention visual and can detect, and especially measures sudden change for expressing and the effect of phenotype.Be appreciated that selective marker can import on the carrier that is independent of the purpose nucleotide sequence.
Recombinant expression vector can also comprise the gene that coding merges part, thereby this fusion part can improve the expression of recombinant protein, the purifying that improves the solubleness of recombinant protein and help the target recombinant protein as the part of affinity purification.For example, thus can add proteolytic enzyme cutting site in the target recombinant protein allows behind the fusion rotein purifying recombinant protein can be separated from merge part.
Thereby recombinant expression vector can be imported host cell and produce transformed host cells.Term " transformed host cells " is intended to comprise with expression vector of the present invention and transforms or the protokaryon and the eukaryotic cell of transfection.Term " usefulness ... conversion ", " using ... transfection ", " conversion " and " transfection " are intended to comprise that nucleotide sequence (for example carrier) is by one of many possibility technology known in the art importing in cell.Prokaryotic cell prokaryocyte can pass through, and for example, the conversion of click or calcium chloride mediation transforms.Can nucleotide sequence be imported mammalian cell by transfection, fat transfection, click or the such routine techniques of microinjection such as calcium phosphate or calcium chloride co-precipitation, the mediation of DEAE-dextran.At people such as Sambrook (Molecular Cloning:A Laboratory Manual, 2ndEdition, Cold Spring Harbor Laboratory press (1989)) and can find in other such laboratory textbook and transform or the proper method of transfection host cell.
Suitable host cells comprises various protokaryons and eukaryotic host cell.For example, can in the cell such, express protein of the present invention such as intestinal bacteria, insect cell (utilizing baculovirus), yeast cell or mammalian cell.At Goeddel, Gene ExpressionTechnology:Methods in Enzymology 185, Academic Press, San Diego can find other suitable host cell among the CA (1991).
If the modes of emplacement of nucleotide sequence makes it relevant on function with another nucleotide sequence, then this nucleotide sequence is " can be operatively connected ".For example, participate in the preceding albumen of polypeptide excretory if the DNA of presequence or secretion homing sequence is expressed as, then it is that operability links to each other with polypeptid DNA; If promotor or enhanser influence transcribing of sequence, then it is that operability links to each other with encoding sequence; Perhaps the position of ribosome bind site helps translation, and then it is that operability links to each other with encoding sequence.In a word, " operability link to each other " means connected dna sequence dna is contiguous, and for the secretion homing sequence, be close to and in same reading frame.Yet enhanser must not be contiguous.Can finish connection by connection at the restriction site of routine.If there is not such site, can use the synthetic oligonucleotide joint according to conventional practice so.
In some embodiments, expression vector comprises the such nucleotide sequence of being modified Bouganin of coding: they have the potential t cell epitope that reduces quantity, and are connected with the expression control sequenc operability.In different embodiments, expression vector comprises nucleotide sequence or its degeneracy variant of code book invention protein or peptide, will comprise at least and suitable RIP coding structure territory of expressing control and the described nucleotide sequence of selecting the sequence operability to link to each other.Degeneracy about polynucleotide refers to a fact of generally being familiar with like this: have many amino acid to specify by surpassing a kind of codon in the genetic code.Code degeneracy makes by 20 kinds of different amino acid of 64 kinds of possibility triplet sequence encodings of four kinds of different bases that constitute DNA.
Term as used herein " RIP coding structure territory " or " ribosome inactivating protein coding structure territory " mean gives bouganin bioactive functional domain.
Nucleotide sequence molecule of the present invention can also pass through the standard technique chemosynthesis.The whole bag of tricks of chemosynthesis polydeoxyribonucleotide is known, comprises full automatic solid phase synthesis in can the commercial dna synthesizer that obtains, for example peptide synthetic (referring to for example, people such as Itakura, United States Patent (USP) 4 598 049; People such as Carnthers, United States Patent (USP) 4 458 066; And Itakura, United States Patent (USP) 4 401 796 and 4 373 071).
The present invention also provides coding to comprise the nucleotide sequence of new protein of the present invention and selected albumen or the proteic fusion rotein of selective marker.
Another aspect of the present invention is the culturing cell that comprises at least a above-mentioned carrier.
Another aspect of the present invention is modified the method for Bouganin, is included under the condition that allows to be expressed from expression vector by modification Bouganin and cultivates above-mentioned cell and purifying bouganin from cell.
(B)
Modified the Bouganin cytotoxin:
As described above, thus bouganin makes the main ribosome-RNA(rRNA) depurination of cell cause protein synthesis to stop and the I type ribosome inactivating protein (RIP) of necrocytosis.Therefore, of the present inventionly modified Bouganin and be can be used for preparing cytotoxin.The cytotoxin that comprises modified Bouganin proteins is preferable over the cytotoxin that comprises non--modified Bouganin proteins, because the former has lower immunogenicity and is comparatively unlikely destroyed by immunity system before reaching the target thing.
Correspondingly, the present invention also provides cytotoxin, the target part that it comprises (a) modified Bouganin proteins of the present invention and (b) is attached to (a).
For convenient the introduction used term " modified Bouganin proteins of the present invention ", it comprises any or all of modified Bouganin proteins described herein, such as the modified Bouganin proteins described in above-mentioned part (A) and drawings and Examples.
Term as used herein " target part " refers to modified Bouganin proteins is transported to material, method or the technology of target cell.This target part is a kind of antibody in one embodiment.This target part may be a kind of liposome in one embodiment.This liposome can be connected in antibody in one embodiment.This target part is a kind of can the guiding and the mutual protein of doing of particular target cell generation particular combination in another embodiment.Such protein portion includes the various polypeptide ligands of specific cells surface receptor, comprise thus many cytokines, too or polypeptide hormone and other biological response regulate material.Main example comprises such as vascular endothelial growth factor, Urogastron, heregulin, interleukin, Interferon, rabbit, tumour necrosis factor and other protein and glycoprotein molecule.Can consider the fusion rotein of bouganin of the present invention and these or other molecule, can comprise at N-end or C-end direction according to the protein ligands structural domain and be modified the Bouganin part.Can will directly target part be linked to each other with protein of the present invention or be connected by joint.In one embodiment, joint is too joint or chemical joint.Similarly, also can consider the chemically crosslinked of purifying part and modified Bouganin proteins, this also within the scope of the present invention.
In a kind of preferred implementation, the invention provides cytotoxin, it comprises (a) modified Bouganin proteins of the present invention, (b) be attached to (a) can with cancer cells bonded part.
Part can be can with any molecule of cancer cells bonded, include but not limited to protein.In one embodiment, part is antibody or the antibody fragment that can discern the cancer cells surface.
Correspondingly, cytotoxin of the present invention can be used for treating various forms of cancers, such as colorectal carcinoma, mammary cancer, ovarian cancer, carcinoma of the pancreas, head and neck cancer, bladder cancer, gastrointestinal cancer, prostate cancer, minicell and nonsmall-cell lung cancer, sarcoma, neurospongioma, T-and B-cell lymphoma.
In one embodiment, the cancer cells binding partner comprises and the complete immunoglobulin molecules of cancer cells bonded.If the cancer cells binding partner is antibody or its fragment, cytotoxin is preferably immunotoxin.In another embodiment, the cancer cells binding partner is Fab, Fab ', scFv, single domain antibody fragment or by the segmental dimer of the stable Fv of disulfide linkage.In another embodiment, anticancrin comprises variable heavy chain, variable light chain, Fab, Fab ', scFv, single domain antibody fragment or by the stable Fv fragment of disulfide linkage.The part of cancer cells binding partner can be derived from one or more species, preferably comprises the part that is derived from the people, most preferably be complete the people's or humanized.Designedly be beneficial to purifying and also can be included in or be added in the cancer cells bound fraction with the zone of toxin coupling connection.
In a kind of specific implementations, cancer cells binding partner identification Ep-CAM.Ep-CAM (epithelial cell adhesion molecule, be also known as 17-1A, KSA, EGP-2 and GA733-2) be a kind of in many solid tumor, comprise lung cancer, mammary cancer, ovarian cancer, colorectal carcinoma and neck flakey cell sarcoma, camber express but in most normal epithelial tissues weak expression transmembrane protein.
Correspondingly, in one embodiment, the invention provides a kind of Ep-CAM-targeted-and modified the Bouganin cytotoxin, it comprises: (a) compare the modified Bouganin proteins of the activation T-cell tendency with reduction with non--modified Bouganin proteins, what (b) be attached to (a) can be in conjunction with the part of the Ep-CAM on the cancer cells (such as antibody or antibody fragment.
In a kind of specific implementations, cytotoxin comprises: the modified Bouganin proteins of (a) comparing the activation T-cell tendency with reduction with non--modified Bouganin proteins, (b) be attached to the humanized antibody or the antibody fragment of (a), it can combine and comprise from complementary determining region (CDR) sequence of Moc-31 antibody with people Ep-CAM extracellular domain.
Suitable Ep-CAM-targeted-according to the present invention is modified Bouganin and is comprised, is not limited to, and VB6-845 and variant thereof comprise other list of selective binding Ep-CAM or other cytotoxin and variant thereof of double-stranded immunoglobulin (Ig).Term as used herein " VB6-845 " means, comprise with by the bouganin of modified forms, the cytotoxin of the Fab of anti--Ep-CAM scFv antibody that Bou 156 (SEQ ID NO:13) is continuous.The aminoacid sequence of VB6-845 links to each other with nucleic acid and is shown in accompanying drawing 3B (being respectively SEQ ID NO:16 and SEQ ID NO:15).
In another embodiment, the cancer cells binding partner be identified in special discovery on the neoplastic cell and on normal cell undiscovered tumor associated antigen.In a kind of preferred implementation, part is the antibody with tumor associated antigen.The identification of antitumor related antigen antibodies specific derives from the cancer cells of various cancers but the normal non-cancerous cells of nonrecognition.
Therefore in another embodiment, the invention provides a kind of cytotoxin, it comprises: the modified Bouganin proteins of (a) comparing the activation T-cell tendency with reduction with non--modified Bouganin proteins; (b) be attached to (a) can with the tumor associated antigen bonded part (such as antibody or antibody fragment) on the cancer cells.
Suitable tumor associated antigen target according to the present invention is modified Bouganin and is comprised, is not limited to VB6-011 and variant thereof, other strand of selective binding tumor associated antigen or other cytotoxin of double-stranded immunoglobulin (Ig) or its variant.Term as used herein " VB6-011 " means, comprise with by the bouganin of modified forms, the cytotoxin of the Fab of the H11 human monoclonal antibodies that Bou 156 (SEQ ID NO:13) is continuous.H11 antibody is made hybridoma and is obtained by 64 years old male cancer peripheral blood of patients lymphocyte and human myeloma cell line are merged.Hybridoma NBGM1/H11 produces IgMk, it is engineered to the Fab antibody formation once more forms VB6-011 (seeing the preparation of H11 antibody-secreting hybridoma among US 6 207 153 or the WO 97/44461 for details).The aminoacid sequence of VB6-011 and nucleotide sequence are shown in accompanying drawing 15 (being respectively SEQ ID NO:28 and SEQ ID NO:27).
In a kind of specific non-limiting embodiment, cytotoxin comprises VB6-845 (accompanying drawing 3B, SEQ ID No.16) or VB6-011 (accompanying drawing 15, SEQ ID NO:28).In other non-limiting embodiment, cytotoxin comprises the variant of VB6-845 or VB6-011.
In conjunction with Ep-CAM epi-position or combination and the basic similarly Ep-CAM epi-position of epi-position that VB6-845 combines, this variant can competitive inhibition VB6-845 and Ep-CAM bonded at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% under physiological condition equally for the VB6-845 variant.The VB6-845 variant comprises the quilt identical with VB6-845 and modifies Bouganin, perhaps can comprise with different the present invention and be modified Bouganin.In the non-limiting embodiment of another kind, cytotoxin comprises the Ep-CAM-bound fraction that contains MOC31 variable region or its variant.In another embodiment, cytotoxin comprises and contains 4D5MOCB[C2] or the Ep-CAM-bound fraction of its part.Under physiological condition by with reference MOC31 or 4D5MOCB antibody competition, combining of any and Ep-CAM in these cytotoxins can be reduced at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.
VB6-011 variant and VB6-011 in conjunction with identical tumor associated antigen epi-position or in conjunction with and the basic similarly tumor associated antigen epi-position of VB6-011 institute bonded, under the physiological condition, this variant can competitive inhibition VB6-011 with tumor associated antigen combine at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.The VB6-011 variant can comprise the quilt identical with VB6-011 and modify Bouganin, maybe can comprise different the present invention and be modified Bouganin.In the non-limiting embodiment of another kind, cytotoxin comprises the tumor associated antigen bound fraction that contains H11 monoclonal antibody, H11 Fab or its variant.Under physiological condition by making in these cytotoxins any be reduced by at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% with combining of VB6-011 with reference H11 antibody competition.
In a kind of preferred implementation, according to the binding affinity of the Ep-CAM-bound fraction of standard test technical measurement or tumor associated antigen bound fraction low at least four orders of magnitude of binding affinity than VB6-845 or VB6-011, preferably at least three orders of magnitude more preferably hang down two orders of magnitude.In a kind of non-limiting embodiment, under physiological condition, the Ep-CAM-bound fraction can competitively be blocked known resisting-Ep-CAM antibody, such as but not limited to PANOREX or MT201, with Ep-CAM combine at least 0.1%, 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.In a kind of non-limiting embodiment, under physiological condition, the tumor associated antigen bound fraction can competitively be blocked known antitumor related antigen antibody, such as but not limited to H11, with tumor associated antigen combine at least 0.1%, 1%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%.
It will be understood by those skilled in the art that and to identify the specific residue of decision.Term " specificity decision residue ", be also referred to as " SDR ", finger-type becomes the residue of the partial antibody determinant of antibody, especially CDR residue, the single replacement of doing with L-Ala that does not rely on any other sudden change can reduce antibody at least 10 times of epi-positions, preferred at least 100 times, more preferably at least 1000 times binding affinity.The importance of residue for the ability of antibodies epi-position has been emphasized in the forfeiture of avidity.Referring to for example, people such as Tamura, 2000, " Structural correlates of an anticarcinomaantibody:identification of specificity-determining residues (SDRs) anddevelopment of a minimally immunogenic antibody variant by retention ofSDRs only ", J.Immunol.164 (3): 1432-1441.
Can estimate single or multiple sudden changes simultaneously in conjunction with active influence, especially to the influence of binding affinity, thereby assessment particular series amino acid is in conjunction with the importance of doing mutually (for example, light chain or heavy chain CDR2 contribute bonded).Thereby can also assess the contribution of when estimating separately, estimating single amino acids to the effect of certain amino acid mutation.Such assessment can be passed through, for example, by external saturated scanning (referring to for example, United States Patent (USP) NO.6 180 341; People such as Hilton, 1996, " Saturation mutagenesis of the WSXWS motif of theerythropoietin receptor ", J Biol Chem.271:4699-4708) and site directed mutation (referring to for example, Cunningham and Wells, 1989, " High-resolution epi-position mappingof hGH-receptor interactions by alanine-scanning mutagenesis, " Science244:1081-1085; People such as Bass, 1991, " A systematic mutational analysis ofhormone-binding determinants in the human growth hormone receptor, " ProcNatl Acad Sci.USA 88:4498-4502) finishes.In L-Ala scanning mutating technology, a plurality of residues place in molecule imports the single alanine sudden change, thereby the biological activity of test gained molecule is identified the key amino acid residue for molecular activity.
Can also pass through basis, for example, nucleus magnetic resonance, crystallization, electron diffraction or photoaffinity labeling, the structure of determining is carried out physical analysis and in conjunction with the contact site amino acid of inferring being suddenlyd change (referring to for example, people such as de Vos, 1992, " Human growth hormone and extracellular domain of its receptor:crystal structure of the complex ", Science 255:306-312; People such as Smith, 1992, " Human interleukin 4.Thesolution structure of a four-helix bundle protein ", J Mol Biol.224:899-904; People such as Wlodaver, 1992, " Crystal structure of human recombinant interleukin-4at 2.25 A resolution ", FEBS Lett.309:59-64) identify ligand-receptor or other biological site of doing mutually.In addition, can estimate the importance of specific single amino acids or aminoacid sequence by the aminoacid sequence that compares related polypeptide or similar binding site.
In addition, it will be understood by those skilled in the art that the raising of affinity (avidity) can compensate the reduction of avidity (affinity).Cytotoxin is to the tolerance of Ep-CAM-bound fraction to the bonding strength of Ep-CAM to the affinity of cancer cells acceptor.Functional bonding strength between Ep-CAM and Ep-CAM-bound fraction is represented the total intensity of all affine keys, and therefore single composition can be with relatively low avidity combination, and still the polymer of such composition then can confirm effective biological effect.In fact, the multiple combination of Ep-CAM-binding site and Ep-CAM epi-position can have more proof than additional biological effect, and also, the polyvalent advantage is that the equilibrium constant may be many orders of magnitude.
Similarly, cytotoxin is the tolerance of tumor associated antigen bound fraction and tumor associated antigen bonding strength to the affinity of cancer cells acceptor, and this combination has a plurality of binding sites.The functional bonding strength of tumor associated antigen and tumor associated antigen bound fraction is represented the total intensity of all affine keys, and therefore single composition can be with the low-affinity combination, and still the polymer of such composition can prove effective biological effect.In fact, the tumor associated antigen binding site more has explanation of force with the multiple mutual work of tumor associated antigen epi-position than additional biological activity, and also, the polyvalent advantage is that the equilibrium constant may be many orders of magnitude.
In a kind of non-limiting embodiment, the Ep-CAM-bound fraction has the basic similarly structure with 4D5MocB.This basic similarly structure can be carried out feature description with reference to the epi-position figure of the binding site that reflects cytotoxin Ep-CAM-bound fraction and Ep-CAM molecule.In the non-limiting embodiment of another kind, can produce the epi-position figure of tumor associated antigen bound fraction, this basic similarly structure can be carried out feature description with reference to the epi-position figure of the binding site that reflects cytotoxin tumor associated antigen bound fraction and tumor associated antigen molecule.
Cytotoxin of the present invention can utilize the technology of knowing in the protein chemistry to prepare by chemosynthesis, such as solid phase synthesis (Merrifield, J.Am.Chem.Assoc.85:2149-2154 (1964)) synthetic (Houbenweyl or in homogeneity liquid, Methods of OrganicChemistry, ed.E.Wansch, 15I and II volume, Thieme, Stuttgart (1987)).
In one embodiment, it all is protein that cancer binding partner and quilt are modified Bouganin, can join with technology coupling well known in the art.There is hundreds of linking agent to join this two kinds of protein by coupling.(referring to for example " Chemistry of Protein Conjugation and Crosslinking ".1991，Shans?Wong，CRC?Press，Ann?Arbor)。Usually can select linking agent according to reactive functional groups obtainable on part or the toxin or that inserted.In addition, if there is not reactive group, then can use can photoactivation linking agent.In particular case, be preferably in and comprise introns between part and toxin.Linking agent known in the art comprises with difunctional dose: glutaraldehyde, dimethyl adipimidate and Bis (di azobenzidine) and assorted difunctional dose: mMaleimidobenzoyl-N-Hydroxysuccinimide and Sulfo-m Maleimidobenzoyl-N-Hydroxysuccinimide.
Can also utilize recombinant DNA technology to prepare part-bouganin toxin fusion rotein.At this moment, the dna sequence dna of coding cancer binding partner and the dna sequence dna of coding modified Bouganin proteins are merged, thereby produce chimeric dna molecule.This gomphosis DNA array transfection is advanced to express the host cell of part-bouganin fusion rotein.Can from cell culture, reclaim and purified fusion protein with technology known in the art.
Can such as Ep-CAM and the such pair cell surface protein of tumor associated antigen specific antibody be arranged with the routine techniques preparation.Can be used in the peptide based immunogens form immunity Mammals that causes antibody response in the Mammals, (for example mouse, hamster or rabbit).The technology of giving peptide based immunogens comprises with carrier puts together or other technology well known in the art.For example, peptide can be used existing under the situation of adjuvant.Can monitor immunologic process by the antibody titers of surveying in blood plasma or the serum.Standard ELISA or other immunoassay procedure and assessed antibody horizontal originally as antigenic immunity.After the immunity, antiserum(antisera) can be obtained, if necessary, polyclonal antibody can be from serum, separated.
For producing single grand antibody, can be from by the animal of immunity results antibody produced cell (lymphocyte), and it is merged with the myeloma cell by standard body cytogamy method, thus make these cells not extremely and produce hybridoma.Such technology is known in the art, (for example hybridoma technology is at first by Kohler and Milstein (Nature 256:495-497 (1975) exploitation) and such as people B-quadroma technology (people such as Kozbor, Immunol.Today 4:72 (1983)), produce the EBV-hybridoma technology (people such as Cole of human monoclonal antibodies, Monoclonal Antibodies in Cancer Therapy Allen R., Bliss, Inc., pages 77-96 (1985)), with other such technology of screening combinatorial antibody library people such as (, Science 246:1275 (1989)) Huse.For producing the antibody that reacts with peptide specific, can carry out the immunochemistry screening to hybridoma, and separate monoclonal antibody.
Term used in the present invention " antibody " be intended to comprise monoclonal antibody and polyclonal antibody, also can with a kind of antibody fragment (for example Fab and F (ab ') 2, and single-chain antibody (scFv)) and chimeric antibody of cell surface composition specific reaction.Can utilize routine techniques with antibody fragmentization, screen the available fragment in the same manner as described above.For example, 2 fragments can be by producing with pepsin antibody for F (ab ').Gained F (ab ') thus 2 fragments can produce Fab ' fragment through handling to reduce disulfide linkage.Single-chain antibody combines with the antigen binding domain of antibody on a stable folding polypeptide chain.Single-chain antibody can produce by recombinant technology.
The chimeric antibody derivative promptly, is also considered within the scope of the present invention with non-human animal variable region and human constant region bonded antibody molecule.The chimeric antibody molecule can comprise, for example, and from the antigen binding domains and the human constant region of the antibody of mouse, rat or other species.The preparation of available ordinary method comprise the immune globulin variable region of recognizing cells surface antigen chimeric antibody (referring to, for example, people such as Morrison, Proc.Natl Acad.Sci.U.S.A.81:6851 (1985); People such as Takeda, Nature 314:452 (1985), people such as Cabilly, United States Patent (USP) 4 816 567; People such as Boss, United States Patent (USP) 4 816 397; People such as Tanaguchi, European patent 171 496; European patent 173,494, Hong Kong patent 2177096B).The expection chimeric antibody has lower immunogenicity than corresponding non-chimeric antibody in the people.Can be by people such as Pluckthun, the method that WO 00/61635 describes is stablized chimeric antibody.
Can further make mono-clonal or chimeric antibody humanization with cell surface composition specific reaction by producing the human constant region mosaic, variable region part wherein, especially the conservative frame zone of antigen binding domain is the people source, and to have only hypervariable region be inhuman source.Such immunoglobulin molecules can pass through methods known in the art (people such as Teng for example, Proc.Natl.Acad.Sci.U.S.A., 80:7308-7312 (1983); People such as Kozbor, Immunology Today 4:7279 (1983); People such as Olsson, Meth.Enzymol., open text WO92/06193 of 92:3-16 (1982) and PCT or EP 239,400) preparation.Humanized antibody can also by commercialization approach preparation (Scotgen Limited, 2 Holly Road, Twickenham, Middlesex, GreatBritain.).In addition, by reduce with the mono-clonal of cell surface composition specific reaction or chimeric antibody on potential T-cell epitope quantity its immunogenicity is reduced.
Can also produce and in the bacterium that has the cell surface composition, express by screening coding immunoglobulin gene expression library with the specific antibodies of cell surface composition specific reaction or antibody fragment or its part.For example, complete Fab fragment, VH district and Fv district can express in bacterium (referring to people such as for example Ward, Nature 341:544-546 (1989) with the phage expression library; People such as Huse, Science 246:1275-1281 (1989); With people such as McCafferty, Nature 348:552-554 (1990)).As selection, the SCID-hu mouse for example by the model of Genpharm exploitation, can be used for producing antibody and fragment thereof.
In all situations with modified Bouganin proteins and antibody sequence fusion, preferably use wherein or to stimulate the T cell or to combine the t cell epitope of T cell or the antibody sequence that sequence has been removed jointly with II class MHC molecule in conjunction with II class MHC molecule.
Another embodiment of the present invention, modified Bouganin proteins can but still can lead with a kind of non-antibody albumen and be connected with the specificity bonded protein of particular target cell.Such protein portion comprises the various polypeptide ligands that have the specific cells surface receptor, and comprises many cytokines, peptide and polypeptide hormone and other biological response modifier thus.Main example comprises such as vascular endothelial growth factor, Urogastron, heregulin, interleukin, Interferon, rabbit, tumour necrosis factor and other protein and the such protein of glycoprotein molecule.The fusion rotein of these molecules and other molecule and bouganin of the present invention can consider that also they can comprise the part by modification Bouganin at the N-of protein ligands structural domain end or C-end direction.Similarly, the chemically crosslinked of purifying part and modified Bouganin proteins also can be considered, and fall into scope of the present invention.
In another embodiment, modified Bouganin proteins of the present invention can be used as and comprises such as the such water-soluble polymers of hydroxypropyl Methacrylamide or the mixture of other polymkeric substance, and wherein modified Bouganin proteins is done with non-covalent the combination mutually with the polymkeric substance covalent attachment or with polymkeric substance.A kind of embodiment like this can also comprise with polymkeric substance bouganin mixture bonded such as antibody or the such antigen binding domains of antibody fragment.
(C)
Cytotoxic purposes
Modified Bouganin proteins of the present invention can be used for the mammalian cell that specificity suppresses or destroy the infection cancer.Allow RIP to enter cell and effective kill cancer cell, this is to have the cytotoxic advantage than the present invention of reduced immunogenicity.Therefore, this cytotoxin can be used for selectively targeted cancer cells.In case enter cancer cells, bouganin then can make main ribosome-RNA(rRNA) depurination, destroys rrna thus and causes protein synthesis to stop and necrocytosis.
Correspondingly, in one embodiment, the invention provides a kind of method that suppresses or destroy cancer cell, comprise to the cytotoxic animal of needs the present invention and use cytotoxin of the present invention.The present invention comprises that also cytotoxin of the present invention is used for suppressing or tumoricidal purposes.The present invention also comprises the purposes of cytotoxin of the present invention in preparation inhibition or tumoricidal medicine.This cytotoxin suppresses or destructive cancer cells type can be determined by the antigen-specific of its antibody moiety.
In another embodiment, the invention provides a kind of inhibition or tumoricidal method, comprise preparation cytotoxin of the present invention and use this cytotoxic step to cell.Cancer can be the cancer of any kind, includes but not limited to colorectal carcinoma, mammary cancer, ovarian cancer, carcinoma of the pancreas, head and neck cancer, bladder cancer, lung cancer, kidney, melanoma, gastrointestinal cancer, prostate cancer, minicell and nonsmall-cell lung cancer, sarcoma, neurospongioma and T-and B-cell lymphoma.
Can utilize the animal cancerous cell line easily to suppress or tumoricidal ability in vitro test cytotoxin selectivity of the present invention.It is passable that the cytotoxic selectivity of the present invention suppresses effect, for example, determines by the propagation that confirms the selectivity anticancer.
Can measure toxicity according to cell viability, for example can relatively be exposed to the vigor of cytotoxic normal and cancerous cells culture.Can assess cell viability with known technology, such as the blue detection method of repelling of tongue phenol.
In another example, many models can be used for the cytotoxicity of test cell toxin.Thompson, people such as E.W (Breast Cancer Res.Treatment 31:357-370 (1994)) have described a kind of basilar membrane (collagen protein, ln, fibronectin, Matrigel or gelatin) of invading reconstruct by the extracellular matrix enzymolysis and the tumour cell of the mediation of mensuration tumour cell and have come the model of external definite human breast cancer cell invasion.Other applicable cancer cells model comprises ovary oncocyte (Young, people such as T.N., the Gyneco.L.Oncol.62:89-99 (1996) of cultivation; Moore, D.H. wait the people, Gyneco.L.Oncol.65:78-82 (1997)), people's folliculus thyroid carcinoma cell (Demeure, M.J. wait the people, World J.Surg.16:770-776 (1992)), human melanoma (A-2058) and fibrosarcoma (HT-1080) clone (Mackay, A.R. wait people Lab.Invest.70:781-783 (1994)) and lung flakey (HS-24) and gland cancer (SB-3) clone (Spiess, E. wait the people, J.Histochem.Cytochem.42:917-929 (1994)).Comprise tumour transplatation and measure tumor growth in the athymia joint mouse and body built-in test system (Thompson, people such as E.W, the Breast CancerRes.Treatment 31:357-370 (1994) of transfer; Shi, people such as Y.E, Cancer Res.53:1409-1415 (1993)) also obtained describing.
The invention still further relates to the treatment method for cancer, comprise one or more cytotoxins of the present invention from significant quantity to the animal that needs are arranged that use.The present invention includes the purposes of cytotoxin treatment cancer of the present invention.The present invention also comprises the purposes of cytotoxin of the present invention in the medicine of preparation treatment cancer.
Term " animal " comprises all members of animal kingdom, comprising the people interior.
Term " treatment cancer " refers to the inhibition that cancer cells duplicates, cancer spreads (transfer), tumor growth, the minimizing of cancer cells quantity and tumor growth, cancer other reduction of pernicious level or the related indication improvement of cancer.
In a kind of preferred implementation, animal is the people.In another embodiment, cancer is selected from colorectal carcinoma, mammary cancer, ovarian cancer, carcinoma of the pancreas, head and neck cancer, bladder cancer, lung cancer, kidney, melanoma, gastrointestinal cancer, prostate cancer, minicell and nonsmall-cell lung cancer, sarcoma, neurospongioma and T-and B-cell lymphoma.
Clinical effectiveness with cytotoxin treatment cancer of the present invention is easy to by those skilled in the art, and for example the doctor determines.For example, the standard clinical experiment that is used to measure the cancer clinical marker can be the strong indicator of result of treatment.Such experiment can comprise, be not limited to physical examination, performance scales, disease marker, 12-lead ECG, tumor measurements, tissue biopsy, cytoscopy, cytology, calculating tumour maximum diameter, radiography, tumour digital imagery, vital signs, weight, adverse events record, period of infection evaluation, subsidiary medicine evaluation, pain evaluation, blood or serum chemistry, urinalysis, CT scan and pharmacokinetic analysis.In addition, comprise that the synergy of the composition therapy of cytotoxin and another cancer therapeutic agent can be by determining with the patient's who does not receive treatment comparative studies.
The removal of the criterion evaluation malignant tumour that can accept with those skilled in the art.Referring to for example, people such as Therasse, 2000, " New guidelines to evaluate the response totreatment in solid tumors; European Organization for Research and Treatmentof Cancer; National Cancer Institute of the United States, National CancerInstitute of Canada, " J Natl Cancer Inst.Feb 2; 92 (3): 205-16.
The effective dose of specific cells toxin construct depends on various factors, comprises that cancer types, tumour size, carcinoma stage, cytotoxin are to patient's toxicity, to target-specific and patient's age, body weight and the health of cancer cells.
Can be according to dosage and the concentration of cytotoxin in potting compound, comprise the cytotoxin of being modified Bouganin in the dabbling mode of i.v. minute to use to the timed interval.
In one embodiment, cytotoxin poured into 3 hours the timed interval.
In one embodiment, the effective dosage ranges of i.v. perfusion dosed cells toxin is about 1 to 100mg/kg/ agent.In other embodiments, this dosage range is about 2 to 5mg/kg/ agent.In specific implementations, this dosage can be at least about 2,4,8,13,20,28,40, the 50mg/kg/ agent.
In one embodiment, single-dose is used about 1,2,3,4,5 or 6 weeks approximately weekly.Single-dose can used continuously in week, perhaps as selecting, can skip one or how all.After this circulation, can begin next circulation after about 1,2,4,6 or 12 weeks.Treatment plan can comprise 1,2,3,4,5,6 or more circulations, about 1,2,4,6 or 12 weeks of each interbody spacer that circulates.
In another embodiment, be administered once in every month, administration about 1,2,3,4,5 or 6 is the moon continuously.After this circulation, about 1,2,4,6 or can begin next circulation after 12 months.Treatment plan comprises 1,2,3,4,5,6 or more circulations, each interbody spacer that circulates about 1,2,4,6 or 12 months.
At certain specific non-limiting embodiment, cytotoxic effective dose about 1 and the 50mg/kg/ tumour/between day, wherein the patient is administered once every day.About single-dose every day (skip alternatively one or many days), about 1,2,3,4,5,6 or 7 running days of administration.After this circulation, begin next circulation after about 1,2,3,4,5 or 6 weeks.Treatment plan can comprise 1,2,3,4,5,6 or more circulations, about 1,2,3,4,5 or 6 weeks of each interbody spacer that circulates.
Volume injected preferably is at least significant quantity, and it is the sufficient quantity for tumor type and/or position.The maximum volume of single-dose between about gross tumor volume 25% and 75% between, about 1/4th, 1/3rd or 3/4ths of for example estimated target gross tumor volume.In a kind of specific non-limiting embodiment, the maximum injection volume of single-dose is about 30% of gross tumor volume.
In another embodiment, the solution three hours that comprises 1 to 10mg cytotoxin/mL with the speed perfusion of 100cc per hour.Cytotoxin can be diluted in suitable physiological compatibile solution.
Effective dose with the another kind of cancerous swelling therapeutical agent of cytotoxin administration in the circulation also changes according to administering mode.One or more cancer therapeutic agent can be transported in the tumour, perhaps passes through other mode administration.Typically, chemotherapeutics is the whole body administration.Standard dose and treatment plan be known in the art (referring to for example, the latest edition of Merck Index and the Physician ' s Desk Reference; NCCN Practice Guidelines in Oncology)).
Cytotoxic combination treatment can make cancer or tumour become responsive to using other cancer therapeutic agent.Correspondingly, the present invention considers to be used to prevent, treat and/or prevent the combination treatment of cancer return, is included in the cytotoxin of using significant quantity before the cancer therapy machine that application dosage reduces, afterwards or simultaneously.For example, the initial stage treatment of carrying out with cytotoxin can improve cancer or tumour to using the susceptibility of cancer chemotherapeutic agent subsequently.This dosage is approaching or be lower than when using cancer therapeutic agent separately or the lower range of the standard dose that uses when not having cytotoxin.When using simultaneously, cytotoxin can be used respectively with cancer therapeutic agent, selectively, and by different administering modes.
In another embodiment, cytotoxin and at least a other immunotherapeutic agent combined administration.
In another embodiment, cytotoxin and radiation therapy plan combined administration.This therapy can also comprise surgical operation and/or chemotherapy.For example, cytotoxin can with radiotherapy and cisplatin (Platinol), Fluracil (5-FU, Adrucil), carboplatin (Paraplatin) and/or paclitaxel (Taxol) combined administration.Also can use lower radiation dose and/or more not frequent radiotherapy with the cytotoxin treatment, this can for example reduce the sickness rate of seriously having sore throat that hinders function of deglutition and cause weight loss or dehydration thus potentially.
In another embodiment, cytotoxin and one or more combination of cytokines administration, cytokine comprises, be not limited to, lymphokine, tumour necrosis factor, the tumour necrosis factor like cell factor, lymphotoxin, Interferon, rabbit, macrophage inflammatory protein matter, granulocyte monocyte G CFS, interleukin (including but not limited to il-1, interleukin-2, interleukin-6, il-1 2, interleukin-15, il-1 8) and variant thereof comprise its pharmaceutically acceptable salt.
In another embodiment, cytotoxin and cancer vaccine combination medicine-feeding, cancer vaccine include but not limited to the tumour tumour specific antigen of autogenous cell or tissue, non--autogenous cell or tissue, carcinomebryonic antigen, α-fetus albumen, human chorionic gonadotrophin, BCG living vaccine, melanoma cell pedigree albumen and sudden change.
In another embodiment, cytotoxin and hormonotherapy combination medicine-feeding.The hormonotherapy agent comprises, be not limited to, hormone agonist, hormone antagonist are (for example, Sch-13521, tamoxifen, leuprolide acetate (LUPRON)) and steroid (for example, dexamethasone, retinoid, Betamethasone Valerate, hydrocortisone, cortisone, prednisone, boldenone, glucocorticosteroid, mineralocorticoid, oestrogenic hormon, testosterone, progesterone).
In another embodiment, the gene therapy scheme combination of cytotoxin and treatment or preventing cancer is used.
In another embodiment, the cytotoxin of Ep-CAM-target and one or more improve the agent combination administration that Ep-CAM expresses in the purpose tumour cell.Preferred Ep-CAM expresses and is improved so that tumor cell surface has the Ep-CAM developed by molecule of bigger quantity.For example, this reagent can suppress the Ep-CAM antigen endocytosis of normal circulation.Such combined therapy can improve the cytotoxic independent clinical effectiveness of Ep-CAM-target, perhaps with other cancer therapeutic agent or radiotherapeutic clinical effectiveness.In specific non-limiting embodiment, the reagent that improves the expression of Ep-CAM in tumour cell is vinorelbine tartrate (Navelbine) and/or paclitax (Taxol).Referring to for example, people such as Thurmond, 2003, " Adenocarcinoma cellexposed in vitro to Navelbine or Taxol increase Ep-CAM expression through anovel mechanism. " Cancer Immunol Immunother.Jul; 52 (7): 429-37.
Combination treatment can improve cancer or tumour to the cytotoxin used and/or the susceptibility of other cancer therapeutic agent thus.By this way, thus might only need short treatment circulation to reduce the toxicity incident.Correspondingly, the invention provides the method for a kind of treatment or prevention, comprise cytotoxin from a short treatment round-robin significant quantity to the patient that needs are arranged and at least a other cancer therapeutic agent of using.Circulating continuancing time can change according to employed certain cancer treatment agent.The present invention also considers to continue or do not continue medication, and perhaps is divided into the day administration that several sections is used.It will be appreciated by those skilled in the art that the suitable circulating continuancing time of certain cancer treatment agent, the present invention considers the ideal treatment time of each cancer therapeutic agent is continued assessment.The specific guidance policy is well known by persons skilled in the art.Referring to for example, people such as Therasse, 2000, " New guidelines toevaluate the response to treatment in solid tumors.European Organization forResearch and Treatment of Cancer, National Cancer Institute of the UnitedStates, National Cancer Institute of Canada, " J Natl Cancer Inst.Feb 2; 92 (3): 205-16.
As selection, might need longer treatment circulation.Correspondingly, circulating continuancing time can change between about 10 to 56,12 to 48,14 to 28,16 to 24 or 18 to 20 days.Circulating continuancing time can change according to employed cancer therapeutic agent.
The present invention considers at least one circulation, preferably more than a circulation, during used a kind of cancer therapeutic agent or a series of therapeutical agent.It will be appreciated by those skilled in the art that suitable circulation sum and intercycle.Cycle number can be 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 21 circulation.The intercycle can be 1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20 or 21 day.The present invention considers the optimal treatment time-histories of each cytotoxin and other cancer therapeutic agent is continued assessment.
In another embodiment, provide a kind of method that the mammiferous medicine of cancer is suffered from treatment for preparing, comprise step: identify bouganin T-cell epitope with the activation T-cell tendency that weakens; Preparation has the cytotoxin of the present invention of one or more T-cell epitopes and this protein is suspended from pharmaceutical acceptable carrier, thinner or the vehicle.
The present invention also provides the treatment that comprises cytotoxin of the present invention and pharmaceutical acceptable carrier, thinner or vehicle to suffer from the mammiferous pharmaceutical composition of cancer.
Cytotoxin of the present invention can be mixed with the pharmaceutical composition of the biocompatible form of using to the experimenter that is suitable for vivo medicine-feeding." be suitable for the biocompatible form of vivo medicine-feeding " and mean the administration material form that result of treatment has surpassed any toxic effect.These materials can be administered to the live organism that comprises humans and animals.It is defined as the pharmaceutical composition of the present invention of administering therapeutic live vol, is being required dosage of result and the significant quantity under the time length of achieving the goal.For example, the therapeutic activity amount of material can be according to cause the such factors vary of ability that purpose is replied in individuality such as morbid state, age, sex, whose body weight and antibody.Thereby can make adjustment to dosage provides optimal treatment to reply.For example, can divide administration several times every day, perhaps the urgency level according to the treatment situation suitably reduces dosage.
Active substance can be by using such as injection (subcutaneous, intravenously, intramuscular etc.), oral administration, suction, percutaneous dosing (being coated with frost or cream etc. such as the part) or the such convenient manner of suppository.According to route of administration, active substance can be coated in the material to protect this compound to avoid enzyme, acid and other can make the effect of the natural condition of this compound inactivation.
The composition that this paper does to describe can can be accepted the method for compositions preparation to the pharmacy that the experimenter uses by known preparation, and active substance and the pharmaceutical acceptable carrier with significant quantity is combined into mixture thus.Appropriate carrier is described in, for example, Remington ' s PharmaceuticalSciences (Remington ' s Pharmaceutical Sciences, Mack PublishingCompany, Easton, Pa., USA 1985).Based on this, said composition comprises, though be not special restriction, with one or more pharmaceutical acceptable carrier, thinner, be included in have appropriate pH and with the isoosmotic damping fluid of physiological liquid in material.
This pharmaceutical composition can be used for treatment and comprises Mammals, and preferred people is in the cancer of interior animal.Expect that these compositions are particularly suited for treating colorectal carcinoma, mammary cancer, ovarian cancer, carcinoma of the pancreas, head and neck cancer, bladder cancer, lung cancer, kidney, melanoma, gastrointestinal cancer, prostate cancer, minicell and nonsmall-cell lung cancer, sarcoma, neurospongioma and T-and B-cell lymphoma.Cytotoxic dosage of being used and type depend on and are easy to monitor various factors in people's acceptor.Such factor comprises neoplastic pathology and severity (rank and stage).
The pharmaceutical composition that is fit to directly use comprises, be not limited to, lyophilized powder or water-based or non-aqueous aseptic parenteral solution or suspension, it can also further comprise antioxidant, damping fluid, fungistat and make the isoosmotic substantially solute of blood of said composition and purpose acceptor.Other composition that can be present in such composition comprises, for example, and water, ethanol, polyvalent alcohol, glycerol and vegetables oil.Interim injection liquid and suspension can make from sterilized powder, particle or tablet.Cytotoxin can, but be not limited thereto, before the patient uses, using with the form of the lyophilized powder of sterilized water or salts solution reprovision.
Pharmaceutical composition of the present invention can comprise pharmaceutical acceptable carrier.Suitable pharmaceutical acceptable carrier comprises that or not basic of bioactive validity of interference medicament composition is not chemically inert non-toxic composition.The example of suitable pharmaceutical carriers comprises, but be not limited to water, salts solution, glycerol solution, ethanol, N-(1 (2,3-dioleyloxy) propyl) N, N, N-trimethyl ammonium chloride (DOTMA), diolesylphosphotidyl-thanomin (DOPE) and liposome.Such composition should comprise the efficient compound of treatment, and the carrier of sufficient quantity, thereby obtains the form to the direct administration of patient.
In another embodiment, pharmaceutical composition comprises cytotoxin and one or more additional cancer therapeutic agent, is included in the pharmaceutical acceptable carrier alternatively.
Composition can be the pharmaceutically acceptable salt form, comprise, be not limited to, those use the salt that forms such as the free amino group that is derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic, tartrate etc., and those use the salt that forms such as the free carboxyl group that is derived from sodium, potassium, amine, calcium, ironic hydroxide, Isopropylamine, triethylamine, 2-ethylarninoethanol, Histidine, PROCAINE HCL, PHARMA GRADE etc.
The present invention relates to be modified Bouganin, comprise the segmental composition of such modified Bouganin proteins or modified Bouganin proteins and compositions relatedly should be considered as falling into the scope of the invention.A related example of this respect is to develop by peptide-mediated tolerance-induced strategy, wherein uses one or more disclosed peptides with immune therapeutic purpose to the patient.Correspondingly, synthetic peptide molecule for example comprises a kind of in all or part of multiple synthetic peptide arbitrary among above-mentioned epi-position district R1～R3.Such peptide is considered as embodiments of the present invention.
Another aspect of the invention relates to the method for being modified Bouganin combination treatment people.For to individual administration, any all should be made into preferably by modifying composition at least 80% pure and do not contain pyrogen and other pollutent.
The present invention also provides and comprises the cytotoxic test kit of significant quantity, alternatively, also comprises one or more other cancer therapeutic agent, and treats the specification sheets of cancer with it.
(D)
The T-cell epitope peptide
Another embodiment of the present invention is the T-cell epitope peptide.Among this embodiment, the T-cell epitope peptide can excite the stimulation index greater than 1.8 in the T-cell detection, and more preferably 2.0.T-cell epitope peptide of the present invention can combine with II class MHC.
In one embodiment of the invention, this T-cell epitope peptide comprises at least 9 continuous amino acid residues of R1, R2 or the arbitrary sequence of R3 (above-mentioned).In another embodiment, arbitrary amino acid identity that has greater than 90% among T-cell epitope peptide sequence and peptide sequence R1, R2 or the R3; More preferably arbitrary amino acid identity that has greater than 80% among T-cell epitope peptide and peptide sequence R1, R2 or the R3.
Term as used herein " peptide " is a kind of comprise two and compound of amino acids more.These amino acid link to each other by peptide bond (as hereinafter definition).Relate to 20 kinds of different natural amino acids in the biosynthesizing of peptide, any amount of these amino acid can link to each other with random order and form peptide chain or peptide ring.Naturally occurring amino acid related in the biosynthesizing of peptide is the L-configuration.Available L-amino acid, D-amino acid or have two kinds not the various amino acid combination of isomorphism type with the synthetic peptide of ordinary method preparation.Small peptide for example, has the small peptide that is less than ten amino acid units, is commonly referred to " oligopeptides ".Other peptide comprises a large amount of amino-acid residues, for example reaches 100 or more, is called " polypeptide ".According to routine, " polypeptide " is regarded as comprising three or more amino acid whose any peptides, and " oligopeptides " is regarded as the specific type of " weak point " usually.Therefore, as used herein, be appreciated that any " polypeptide " mentioned all comprises oligopeptides.In addition, any " peptide " mentioned all comprises polypeptide, oligopeptides and protein.Amino acid whose various different the arrangement forms different polypeptide and protein.The quantity of the polypeptide that can form---and quantity of the different proteins that can form thus---is unlimited in practice.
Another embodiment of the present invention is the purposes that T-cell epitope peptide of the present invention prepares modified Bouganin proteins of the present invention and modified the T-cell epitope peptide.
Another embodiment of the invention is modified the T-cell epitope peptide, thereby its process modification makes that being modified the T-cell epitope peptide is modified the T-cell epitope peptide and compare the tendency with the activation human T-cell who weakens with non--quilt.In one embodiment, improvement of the present invention T-cell epitope peptide comprises and makes this epitope peptide can excite when testing in the T-cell detection with the T-cell epitope peptide of non--improvement to compare the modification of lower stimulation index.
Improvement T-cell epitope peptide of the present invention in one embodiment has following sequence:
AKX1DRKX2LX3LGVX4KL
Wherein among X1, X2, X3 and the X4 at least a from non--modified by modification sequence, as follows:
X1 is T or A or Q;
X2 is G or A;
X3 is Q or G; With
X4 is N or D or T or A or R or Q or E or G or H or K or S (SEQ IDNO:8).
Improvement T-cell epitope peptide of the present invention in another embodiment has following sequence:
LGVX4KLEFSIEAIHG
Wherein X4 is N or D or T or A or R or Q or E or G or H or K or S (SEQID NO:9).
Improvement T-cell epitope peptide of the present invention in another embodiment has following sequence:
NGQEX5AKFFLIVIQM
Wherein X5 is Q or A (SEQ ID NO:10).
The present invention also provides the nucleotide sequence of coding T-cell epitope peptide of the present invention or improvement T-cell epitope peptide.
Provide the following drawings, sequence table and embodiment to help to understand the present invention.Be appreciated that and modify given method and do not deviate from spirit of the present invention.
Following non-limiting example is an illustration of the present invention:
Embodiment
Embodiment 1:With the method for naive people T-cell proliferation experiment to the epitope mapping among the bouganin
Peptide according to ripe Bouganin protein sequences of the described synthetic covering of people [ibid] such as Den Hartog.The length of each peptide is 15 amino acid, and overlapping 12 residues of peptide in succession.The sequence of these peptides and their numbering are shown in table 1.
With these peptides be used for the T-cell proliferation experiment from the PBMC (peripheral blood lymphocytes) of naive donor (also promptly not having known bouganin sensitization).20 parts of donor PBMC have been collected to obtain optimal II class MHC allotype fraction of coverage.The allotype fraction of coverage surpasses 85%.HLA-DR allotype is shown in Table 2.
Mix mensuration propagation according to 3H-thymus pyrimidine (3H-Thy) before, in three parts of cultures, stimulated PBMC 7 days with each single peptide.All peptides are all tested two different concentration (1 μ M and 5 μ M).Stimulation index (S.I.) stimulates the 3H incorporation in the control cells to calculate according to the 3H incorporation divided by vacation.
The dark yellow layer of the human blood that shelf time is no more than 12 hours is available from National Blood Service (Addenbrooks hospital, Cambridge, Britain).Ficoll-paque is available from AmershamPharmacia Biotech (Amersham, Britain).Be used to cultivate the serum-free AIM V substratum Gibco-BRL (Paisley, Britain) of the lymphocytic L-of the comprising L-glutamic acid of primary human, 50 μ g/ml Streptomycin sulphates, 10 μ g/ml gentamicins and 0.1% human serum albumin.Synthetic peptide is available from Eurosequence (Groningen, The Netherlands Holland) and Babraham Technix (Cambridge, Britain).
Obtain red blood corpuscle and white corpuscle centrifugal from blood plasma and thrombocyte, the separation by dark yellow layer being carried out gentleness.Remove and abandon and go up phase (comprising blood plasma and thrombocyte) most.Red blood corpuscle and white corpuscle are spread go up to 15ml ficoll-paque (Amersham Pharmacia, Amersham UK) before, with its in phosphate-buffered salt (PBS) with dilution in 1: 1.The condition of recommending according to manufacturer is carried out centrifugal, from serum+PBS/ficoll paque surface results PBMC.PBMC is mixed (1: 1) and centrifugal collection with PBS.Remove and abandon supernatant liquor, the PBMC piece is resuspended among the 50ml PBS.Once more cell centrifugation is become piece, abandon the PBS supernatant liquor.With 50ml AIM V substratum re-suspended cell, count this moment and assess vigor with the blue dye exclusion method of tongue phenol.The recentrifuge collecting cell also abandons supernatant.Re-suspended cell is with 3 * 10
7Every milliliter of cryopreservation.Preserving substratum is 90% (v/v) heat inactivation AB human serum (Sigma, Poole, Britain) and 10% (v/v) DMSO (Sigma, Poole, Britain).To modulated freezing container (Sigma), and place-70 ℃ to spend the night cell transfer.When need using,, in 37 ℃ of water-baths, make the cell quick-thawing with before cell transfer is to the AIM V substratum of 10ml preheating.
Is the PBMC in 2 * 105 every holes with protein and peptide antigen in 96 hole flat underside plate moderate stimulation density.Before 3H-Thy (Amersham-Pharmacia, Amersham, Britain) pulse, PBMC was cultivated 7 days down at 37 ℃.In the experiment of each donor, used two control peptides that shown immunogenic called after C-32 and C-49 in advance and potential whole protein non--recall antigen keyhole relative hemocyanin (KLH).C-32=is from the sequence PKYVKQNTLKLAT (SEQ ID NO:127) of homo agglutinin residue 307-319.C-49=is from the sequence KVVDQIKKISKPVQH (SEQ ID NO:128) of chlamydozoan HSP60.
Peptide is dissolved in DMSO to final concentration 10mM, afterwards these stock solutions is diluted in AIM V substratum 500 times (final concentration 20 μ M).Peptide is joined in flat 96 orifice plates, to final concentration be 1 and 5 μ M, volume 100 μ l.With the vigor of the blue dyeing of tongue phenol method of exclusion assessment fusion PBMC, afterwards that cell is resuspended to density 2 * 106 cells/ml, and 100 μ l (2 * 105PBMC/ hole) are transferred in each hole of containing peptide.Each peptide concentration has all been measured three hole cultures.Under 37 ℃, plate was cultivated 7 days containing under the moist environment of 5%CO2.Before cell harvesting is to the filter membrane sheet, with 1 μ Ci 3H-Thy/ hole to its pulse 18-21 hour.Measure the CPM value with Wallac microplate beta topplate counter (Perkin Elmer).With the stimulation index ecbatic, stimulation index derives from the propagation mark that will test peptides be measured (for example per minute radioactivity counting) divided by to the mark of the raji cell assay Raji of Contact test peptide not.
The analysis of above-mentioned experimental result disclosed have four kinds of t cell epitopes, they corresponding to the peptide in the proteinic ripe process zone 41,44 and 50 and undressed form in peptide 88.Because peptide 88 is not the part of mature peptide, therefore in the solution of the present invention, it is ignored.
For peptide 41 (called after epitope regions R1), there are four corresponding to the donor of this peptide; Donor 4,5,10 and 11.They are respectively 3.6,4.9,2.1 and 2.0 at the S.I. of 5 μ M.
For peptide 44 (called after epitope regions R2), there are two corresponding to the donor of this peptide; Donor 4 (S.I.=3.5) and 11 (S.I.=2.3).Adjacent peptide 43 and 45 since with peptide 44 have 12 amino acid whose overlapping, therefore induce the T cell proliferation of lower level.
For peptide 50, there are two corresponding to the donor of this peptide; Donor 4 (S.I.=2.9) and 14 (S.I.=2.0).Peptide 51 induces the T cell proliferation (S.I.＞1.9) of lower level in donor 14.
The types of organization of all PBMC samples is all with measuring (Dynal, Wirral, Britain) by the commercial reagent system that obtains.Program, standard auxiliary reagent and the agarose electrophoresis system recommended according to supplier finish mensuration.The allotype specificity of each corresponding donor sample is shown in table 2.
Embodiment 2:From Bougainvillea spectabilis clone bouganin
With ' program that total RNA separation system of SV and supplier (Promega, Southampton, Britain) provide is separated total RNA from Bougainvillea spectabilis leaf.Under liquid nitrogen, the fresh leaf tissue is ground to form fine powder, and get about 50mg tissue abrasion and be used for RNA and separate.By visual inspection RNA quality and quantity in 1% sepharose, and utilize " Access RT-PCR System " (Promega) and special primer OL1032 and the OL1033 bouganin gene that from total RNA, increase, the about 1 μ gRNA of each reaction use.Primer sequence is shown in following table 3.This reaction has produced a 1242bp fragment, comprises natural homing sequence and total length bouganin sequence.According to the test kit explanation this fragment cloning is advanced pGEM-T Easy carrier (Promega), and with its called after pBoul.By dna sequencing checking sequence.
With the pBoul plasmid is template, with the bouganin gene through PCR transform into pET21a (Novagen, the Nottingham, UK).With pelB (pectate lyase polygalacturonase?) homing sequence adds 5 ' end to, and the 6 * histidine-tagged sequence of will encoding is added bouganin encoding sequence 3 ' end to.The pelB homing sequence with primer OL1322 (having mixed a Ndel site) and primer OL1067 from carrier pPMI-his amplification come [Molloy, people such as P., (1995) J.Applied Bacteriology,
78: 359-365].The bouganin-his fragment increases from pBoul with OL1068 and OL1323 (having mixed a Notl site).With overlapping PCR the pelB homing sequence is integrated into bouganin-his fragment frame, and the gained fragment amplification is advanced pGEM-TEasy (Promega).After the sequence checking, the pelB-bouganin-his fragment is advanced the pET21a that digests through Ndel-Notl as the Ndel-Notl fragment cloning.Should clone called after pBou32.
Embodiment 3:Make up sudden change bouganin albumen
Data that utilization is provided by T-cell epitope drawing method and utilize the bonded software that can simulating peptide combines with human II class MHC between ditch have designed many kind quilt modification (sudden change) bouganin albumen.A kind of method in back has a detailed description [WO 02/069232] in other place.Make up variant gene, and tested the functionally active of mutain.Usually, at first make up and test each and only comprise " single mutation " albumen of an aminoacid replacement, activated gene by modified protein is made up prepare multiple replacement afterwards by modified protein.
Made up mutator gene by overlapping PCR method, this method utilizes the sudden change in " overlapping primer " that the mutating acid codon is introduced gene.This flow process is well known in the art, has a detailed description [Higuchi waits people (1900) Nucl.Acids Res.16:7351] in other place.Make up and tested 37 active single mutation of reservation function altogether by modified protein.In addition, in all experiments, also made up and tested a negative control that comprises the Y70A replacement by modified protein.In fact, have one to comprise two adjacent replacements (E151T and I152E) in these 37 " single mutation " in by modified protein, it also counts single mutation in this article.Replacement of being tested and corresponding activity value are shown in table 4.
The multiple replacement that has made up and tested 11 retentive activities altogether is by modified protein.Sudden change of being tested and corresponding activity value are shown in table 5.
Table 6 is described and is replaced by the sequence of modified protein.Table 7 is listed some particular sequences.
In all embodiments, all carry out protein purification and test according to the program that provides in following examples 4 and 5.
Embodiment 4:Proteic expression of Bouganin and purifying
According to manufacturer's explanation, plasmid pBou32 is transformed into (Novagen) competition cell of BL21 (DE3), and on the LB that comprises 50 μ g/ml Pyocianils (Invitrogen, Paisley, Britain) plate, select.Inoculate 5ml 2 * YT (Invitrogen) liquid nutrient medium with a fresh clone who obtains from this conversion, do not contain microbiotic, under 37 ℃ with the 250rpm shaking culture to OD600=1.5-2.0.Afterwards under room temperature with the centrifugal culture of 2500rpm 15 minutes, and cell is resuspended among fresh 2 * YT that 5ml is added with 1mM IPTG.With this culture under 30 ℃ with 300rpm shaking culture 1.5 hours, centrifugal collecting cell is abandoned supernatant.
Cell mass is resuspended among the 1ml PEB2 (50mM Tris-HCl pH8,20% sucrose, 1mg/ml N,O-Diacetylmuramidase, 1 * Complete Protease Inhibitor Tablet (Roche, Lewes, Britain)), mild stirring cultivation on ice 1 hour.4 ℃ of following 14000rpm eccentric cell fragments discard centrifugal.The gained supernatant is " pericentral siphon fraction ".With commerce obtainable " spincolumn ", pass through the nickel affinity column chromatography according to manufacturer's (Qiagen, Crawley, Britain) explanation and from the pericentral siphon fraction, separate Bouganin albumen.4 ℃ down with the cut-off ' Slide-A-Lyzer ' (Pierce, Chester, Britain) of 10000 molecular weight with gains confrontation 4 liters of phosphate buffered saline(PBS) (0.138M NaCl, 0.0027M KCl, pH7.4) dialysed overnight.After the dialysis, estimate protein concn with Micro BCA detection kit (Pierce), and with sample retention in-20 ℃.
Use based on the detection system of ELISA and further determine the Bouganin protein concentration.In brief, by two rats being carried out inherited immunity, produce the antiserum(antisera) (Genovac, Freiburg, Germany) of anti-bouganin with the plasmid of expressing bouganin.For carrying out ELISA, the bouganin that will recombinate is captured on the Ni-agar glycolyx plate by its His-tag, afterwards with rat anti serum and secondary HRP-link coupled anti--rat Fc antibody (Sigma, Poole, Britain) detects.As standard, that has used a large amount of preparations in each time measured also uses the quantitative wild-type bouganin of total protein detection method at expression in escherichia coli.
Embodiment 5:The bouganin determination of activity
By measure wild-type and modified (sudden change) bouganin albumen synthetic protein in the synthetic mensuration of cell-free protein aptitude tests their activity.
Being the 10 μ l TNT of the 12.5 μ l mixture of transcribing/translating combined mixture (Promega), 20 μ M methionine(Met)s, 120ng pT7 fluorescein enzyme dna (Promega) and WT and sudden change Bouganin protein series diluent with final volume cultivated one hour in 30 ℃, made reaction terminating (Promega) by adding 100 μ l " SteadyGlow " luciferase detection reagent afterwards.Measure uciferase activity with the Wallac luminometer.Activated Bouganin Protein Detection is reduced by surveying uciferase activity.At least 5 concentration of each modified Bouganin proteins test, each data point is established a repetition.Comprise the positive and negative control in each experiment.
The proteinic table 4 that the results are shown in of single mutation.The multiple mutation modified Bouganin proteins the results are shown in table 5.In each situation, results expression is with respect to the wild-type protein activity.All mensuration comprises that all has the inactivation sudden change Bouganin albumen that Y70A replaces.
In addition, also luciferase assay result mapping can be shown with respect to the % uciferase activity of contrast and the protein concn of the bouganin that is added.The example of such figure is shown in accompanying drawing 1, and it has described the result to two different multiple mutation bouganin protein determinations.
Embodiment 6:Measure the T-cell epitope disappearance of variant bouganin sequence.
Select the multiple modified protein of called after Bou156 for further testing by immunogenicity determining.This variant comprises and replaces V123A, D127A, Y133N and I152A.The immunogenicity test comprises that use can be used the test destructive viable cell that full Bouganin albumen is done, and therefore utilizes the synthetic peptide that comprises the replacement of being mixed among the variant Bou156 to finish these mensuration.The peptide of being tested is listed in table 8.Utilize the PBMC donor pond of 20 individualities to finish these mensuration according to the program described in the embodiment 1 (above-mentioned).These peptides with two different peptide final concentrations (1 μ M and 5 μ M) to each donor sample triplicate.
The result is expressed as the every donor sample of the every peptide of SI, and is shown in accompanying drawing 2.DeI-41 is peptide sequence AKADRKALELGVNKL (SEQ ID NO:29).Del-44 is peptide sequence LGVNKLEFSIEAIHG (SEQ ID NO:30).DeI-50 is peptide sequence NGQEAAKFFLIVIQM (SEQ ID NO:31).All adorned peptides all do not induce t cell response (S.I.＜2) in arbitrary donor.On the contrary, the immunogenicity control peptide has stimulated the T cell (S.I.＞2) of 6 donors.
Embodiment 7:VB6-845: the recombined engineering that is used for most desirably transporting the Ep-CAM-specificity Fab antibody of disimmunity bouganin (De-bouganin).
To present embodiment and embodiment 8, employed disimmunity bouganin is Bou156.
The cancer target cytotoxin is made up of the antibody variable region that links to each other with bacterium, fungi or plant poison.This research illustrates disimmunity bouganin construct of the present invention, comprise be connected in target part disimmunity bouganin have the immunogenicity of reduction, and still keep simultaneously their biological activity.Table 12 has proved the combining of tumour of Ep-CAM antibody and several types, and shows that thus it can be used in the cancer of these types of treatment.
Disimmunity Bouganin construct: the target part that the Ep-CAM that links to each other with de-bouganin points to
With VB5-845, the Fab version of anti--Ep-CAM scFv antibody, heredity is connected in the disimmunity form (de-bouganin) of bouganin, Bou156, a kind of effective, plant origin, I type ribosome inactivating protein (RIP) forms antibody-toxin construct VB6-845.Accompanying drawing 3 illustrates construct VB6-845.The two suitable anti-unit of accompanying drawing 3A diagram pro-VB6-845 band pelB homing sequence.Accompanying drawing 3B provides aminoacid sequence (SEQ ID NO:16) and nucleic acid coding sequence (SEQ ID NO:15).Accompanying drawing 3C diagram assembling VB6-845 albumen below will be described in greater detail.To the test specification of this construct, this construct has kept the specificity (Ep-CAM antibody) of its biological activity (cytotoxicity) and target part.
The Orientation orientation of disimmunity bouganin construct
For determining optimal antibody-de-bouganin orientation, the bicistronic mRNA that has produced, expressed several forms is expressed the unit, and the effectiveness of having tested them.
In various situations, clone the bicistronic mRNA unit into that pING3302 carrier (accompanying drawing 4) makes under its control that is in pectinose-derivable araBAD promotor, and it is transformed into E104 intestinal bacteria.When inducing, the existence of pelB homing sequence instructs the Fab-de-bouganin fusion rotein to secrete into culture supernatants.Can cut joint de-bouganin can be cut down from target part, and bring into play its biological activity.In one embodiment, this joint is the furin joint, also may suit although those skilled in the art can understand other cut joint.Preferred joint can be selected according to target-specific and environment.The preparation of construct sample and test are carried out according to following:
Accompanying drawing 3:VB6-845, wherein de-bouganin (Bou156) links to each other with the C-of CH structural domain is terminal by the furin joint.Accompanying drawing 3A shows the bicistronic mRNA unit of former sequence, and accompanying drawing 3B shows that the nucleic acid coding sequence (SEQ ID NO:15) of former sequence and aminoacid sequence (SEQ ID NO:16) and accompanying drawing 3C show the assembling VB6-845 albumen that does not contain the pelB sequence.
Accompanying drawing 5 shows that the contrast Fab that does not contain plant poison resists-the Ep-CAM construct de-bouganin (VB5-845).Accompanying drawing 5A shows the two suitable model subelement of former sequence, and accompanying drawing 5B shows the nucleic acid coding sequence (SEQ ID NO:17) and the aminoacid sequence (SEQ IDNO:18) of former sequence, and accompanying drawing 5C shows the VB5-845 albumen of being assembled that does not contain the pelB sequence.
Accompanying drawing 6 shows that Fab resists-Ep-CAM de-bouganin construct, VB6-845-CL-de-bouganin, and wherein Bou156 is connected in the C-end of CL structural domain.Accompanying drawing 6A shows the two along the model subelement of former sequence, accompanying drawing 6B shows the nucleic acid coding sequence (SEQ IDNO:19) and the aminoacid sequence (SEQ ID NO:20) of former sequence, and accompanying drawing 6C shows the assembling VB6-845-CL-de-Bouganin albumen that does not contain the pelB sequence.
Accompanying drawing 7 shows the anti-Ep-CAM of Fab, the de-bouganin construct, and VB6-845-NVH-de-bouganin, wherein Bou156 is connected in the N-end of VH structural domain.The two suitable model subelement of the former sequence of accompanying drawing 7A code displaying, accompanying drawing 7B shows the nucleic acid coding sequence (SEQ IDNO:21) and the aminoacid sequence (SEQ ID NO:22) of former sequence, and accompanying drawing 7C shows the assembling VB6-845-NVH-de-Bouganin albumen that does not contain the pelB sequence.
Accompanying drawing 8 shows that Fab resists-Ep-CAM de-bouganin construct, VB6-845-NVL-de-bouganin, and wherein Bou156 is connected in the N-end of VL structural domain.The two suitable model subelement of the former sequence of accompanying drawing 8A code displaying, accompanying drawing 8B shows the nucleic acid coding sequence (SEQ IDNO:23) and the aminoacid sequence (SEQ ID NO:24) of former sequence, and accompanying drawing 8C shows the assembling VB6-845-NVL-de-Bouganin albumen that does not contain the pelB sequence.
In one embodiment, the de-bouganin molecule links to each other with heavy chain or light chain C-end.Optimal configuration comprises that pelB homing sequence adjacent with the VH-CH structural domain and the terminal Histidine affinity tag of N-are as first unit.Back to back is the Unit second that comprises the pelB-VL-CL structural domain that links to each other with de-bouganin by the responsive joint of a proteolytic enzyme.(accompanying drawing 6) resets the construct that is positioned at the N-end for de-bouganin wherein, western blot analysis shows does not have detectable product, have only the terminal de-bouganin that connects of C-(accompanying drawing 3 and 6 construct) to produce a complete soluble protein (accompanying drawing 9) that has the good combination characteristic with the Ep-CAM-positive cell line, as shown in the reactivity experiment by Flow cytometry.In western blot analysis, accompanying drawing 9 is presented at laboratory level VB6-845 and the expression of VB6-845 CL-de-bouganin in the inductive E104 of institute cell conditioned medium liquid.Under non-inductive condition, with the supernatant liquor aliquot load sample of 16 microlitres in the SDS-PAGE acrylamide gel, and utilize rabbit polyclonal anti--4D5 antibody and after goat anti--rabbit (1/2000) or utilize goat Anti-Human κ-light chain-HRP antibody (1/1000) to make western blot analysis, verify the identity and the size of recombinant protein.Arrow indication total length VB6-845 (construct accompanying drawing 3) and VB6-845-CL-de-bouganin (construct accompanying drawing 6).Western blotting to non--inductive E104 culture supernatants shows no corresponding band, and this has proved the specificity (not shown) of antibody.
(the Ep-CAM-negative cells is that the reactive experimental result that A-375) compares Ep-CAM positive cell line CAL27 and NIH:OVCAR-3 is shown in accompanying drawing 10A with contrast for VB6-845 (accompanying drawing 3) and VB6-845-CL-de-bouganin (accompanying drawing 6).These results with another anti--Ep-CAM construct, VB6-845-sets toxalbumin in vain, the same reaction experiment of being done is suitable, has replaced de-bouganin (referring to the accompanying drawing 14C that shows its aminoacid sequence (SEQ ID NO:26) and nucleotide sequence (SEQ ID NO:25)) with the white tree of another vegetable poison toxalbumin in this construct.The reactive experimental result of doing with white tree toxalbumin construct is shown in accompanying drawing 10B.In molecule, add second de-bouganin structural domain and do not produce product with ideal orientation.
On ice with construct or the contrast and 0.45 * 10
6Cell cultures was finished the flow cytometry experiment in one hour.After the washing, with rabbit anti--bouganin (accompanying drawing 10A) or mouse anti-His label (accompanying drawing 10B) detect the cell surface that is combined with construct on ice.Washed cell, and sheep anti-mouse (IgG) (accompanying drawing 10B) that it sheep anti-rabbit igg (accompanying drawing 10A) of puting together with FITC-and FITC-put together cultivated on ice 30 minutes.Washed cell afterwards is resuspended in it and is used among PBS 5%FCS that comprises propidium iodide carrying out the antibodies assessment by flow cytometry.After cultivating, VB6-845 and VB6-845-CL-de-bouganin and A-375 do not detect the change of fluorescence intermediate value.On the contrary, use the Ep-CAM positive cell line, CAL27 and NIH:OVCAR-3 have observed the noticeable change (accompanying drawing 10A) of fluorescence intermediate value.As above-mentioned, the result of VB6-845 and the white result similar (accompanying drawing 10B) who sets the toxalbumin construct.
The Ep-CAM specificity
VB6-845 (construct accompanying drawing 3) and Proxinium
TM, Proxinium
TMBe a kind of scFv form of VB6-845, but it comprises false pseudomonas bacillus exotoxin A, competitiveness experimental results show that when VB6-845 is engineered in the Fab form its Ep-CAM specificity do not change (accompanying drawing 11).
Accompanying drawing 11 illustrates the flow cytometry result of competitive experiment, raise the gradually Proxinium of (concentration range is 0 to 100 μ g/mL not) of VB6-845 and the concentration of 1 and 10 μ g/mL
TMCultivate altogether with NIH:OVCAR-3 cell (Ep-CAM positive tumor cell system).Cultivate down after one hour for 4 ℃, washed cell, with the biotinylation rabbit anti--bouganin and detect bonded VB6-845 with streptavidin subsequently.The 4B5-PE that is used as negative control has done identical experiment.Reaction conditions is shown in accompanying drawing 11.
Render a service (biological activity)
In addition, acellular (accompanying drawing 12) and MTS (accompanying drawing 13A and B) measure proof, still keep its effectiveness behind de-bouganin and Fab fragment coupling connection.Finish the MTS assay method that is used to measure effectiveness with standard technique known in the art, it is described in greater detail in following examples 8.Use the Ep-CAM-positive cell line, CAL27 and NIH:OVCAR-3, the IC of VB6-845
50Be respectively 3 to 4nM and 2 to 3nM.For VB6-845-C
L-de-bouganin has measured it for the effectiveness of 1 to 2nM CAL27 and for the effectiveness of NIH:OVCAR-3.Comprising the segmental Fab of people's tumour target antibody that is connected with disimmunity bouganin anti--exploitation of Ep-CAM construct, should allow using of this medicine duplicated system, and can produce bigger clinical benefit thus.
The results of construct
Can from cell culture, separate construct with technology known in the art.For example, if the His label is positioned at the N-end of peptidic constructs, can use Ni2
+-chelating prize law purifying Fab-Bouganin albumen.For example can adopt following scheme.
Finish in 15L CHEMAP fermentation container with the TB substratum fed-batch fermentation of VB6-845 variant is cultivated.OD
600Be 20 o'clock (mid-log), culture induced with feed and the inductor that comprises 50% glycerol and 200g/l L-pectinose.Induced back 30 hours, and collected culture, centrifugal 30 minutes min of 8000rpm, and with CM sepharose and Metal-Charged chelating sepharose post and use size exclusion column purification VB6-845 variant afterwards.In brief, concentrated supernatant, and carry out diafiltration with respect to 20mM sodium phosphate pH6.9 ± 0.1.Afterwards the diafiltration concentrated supernatant is joined with on the 20mM sodium phosphate equilibrated CM sepharose post 25mM NaCl pH6.9 ± 0.1.Use the 20mM sodium phosphate, 25mM NaCl pH6.9 ± 0.1, washing column is used the 20mM sodium phosphate afterwards, and 150mM NaCl pH7.5 ± 0.1 is with bonded VB6-845 wash-out.Regulating CM sepharose eluate, to make it comprise final concentration be 0.25% Triton-X100, and this eluate is used for charged chelating sepharose post.Afterwards from 20mM sodium phosphate, 150mM NaCl, 0.25%triton-X100 pH7.5 ± 0.1, use 20mM sodium phosphate, 150mM NaCl pH7.5 ± 0.1 afterwards, then wash chelating sepharose post with the lavation buffer solution of 3 different concns successively with 20mM sodium phosphate, 150mMNaCl, 10mM imidazoles pH7.5 ± 0.1.Use 20mM sodium phosphate, 150mM NaCl, 250mM imidazoles pH7.5 ± 0.1 with the VB6-845 wash-out afterwards, and in the 2mL fraction, it is collected.For making purity＞80%, measured each fraction at A
280Absorption, and each fraction and collected material be applied on the size-exclusion column S200.In one embodiment, for improving lipidated protein and removing intracellular toxin, with collected SEC fraction 20mM NaPO
4, pH7.5 dilutes five times, and passes through to use 20mM NaPO with the speed of about 5ml/min
4, the fast fluidization tower of 25mM NaCl pH7.5 equilibrated Q-sepharose 15ml.After sample is crossed post, with 10CV level pad washing column, with washing lotion and the initial Q-sepharose liquid mixed collection of flowing through.With 30kDa MWCO film (Sartorius hydrosart membrane) effluent being concentrated ten times makes final concentration reach 7.5mg/ml.Adding final concentration afterwards is 0.1%Tween-80.The end product filtration sterilization is stored in-80 ℃.Analyze the employed sample of each step in this method with anti--4D5 antibody mediated immunity hybridization back with Western blotting.The blue dyeing checking of colloid purity.Western blot analysis and ELISA measure the expression level of VB6-845 variant.
Embodiment 8:With the reorganization Ep-CAM-specificity Fab antibody that disimmunity Bouganin (de-bouganin) heredity is connected, the function of VB6-845 and biological property.
Chemotherapeutics is a high cell toxicity reagent, and it usually represents the care standard in many solid tumor cancer therapy.No matter the rapid target somatoblast of the cytotoxic effect of these medicines is normally or tumour cell, therefore causes various clinical side effects.VB6-845 is the Fab antibody of the disimmunity form of a kind of bouganin that is connected in the plant origin toxin.Be different from the chemotherapeutics that lacks accurate tumour target-specific, VB6-845 only is limited to Ep-CAM-positive tumor target with its cytotoxic effect.In this research, measured flow cytometry and cytotoxicity effectiveness and selectivity with assessment VB6-845.
Flow cytometry
Employed tumor cell line is available from ATCC in this research, and breeds according to the recommendation of ATCC, except clone C-4I and the TOV-112D that grows in RPMI 1640 that adds 10%FCS or DMEM respectively.The rate of being paved with reaches 60-70%, vigor surpasses at 90% o'clock and collects tumour cell.People's normal breast epithelial cell (HMEC) is available from CAMBREX, and keeps in defined medium according to the program that CAMBREX provides.The rate of being paved with reaches 70%, vigor surpasses 90% o'clock collecting cell.
Test is from gynaecology's clone of uterine endometrium ovary and cervical cancer sign and combine (table 9) of VB6-845 on flow cytometer.To each clone (3 * 10
5Cell) adds 10 micrograms/mL VB6-845 in, and cultivated 2 hours in 4 ℃.A-375 and CAL27 are used separately as negative and the positive cell line contrast.After washing not binding substance off, be added in the mouse monoclonal that has diluted 800 times among the PBS that contains 10%FCS anti--(Amersham Pharmacia Cat#27471001), continues to cultivate 1 hour at 4 ℃ Histidine antibody.Be added in afterwards the FITC-mark that diluted 100 times among the PBS-10%FCS anti--(The Binding Site Cat#AF271), cultivated 30 minutes mouse IgG.At last, after dead cell is removed in propidium iodide dyeing, analysis of cells on FACS Calibur.
Cytotoxicity
The kill and wound level of VB6-845 in cell of listing in the fluidic cell research is shown in table 10, shows that this construct has kept the de-bouganin cellular cytoxicity activity of its antagonism Ep-CAM-positive cell line.This cytotoxicity comprises the toxin of a different plant origin with another, sets active quite (accompanying drawing 14) of the Fab VB6-845 variant of toxalbumin in vain.Accompanying drawing 14A has compared white tree toxalbumin, and Fab is anti--and Ep-CAM-sets toxalbumin construct (VB6-845-sets toxalbumin in vain) in vain and Fab resists-toxicity of Ep-CAM-de-bouganin (Bou156) construct (VB6-845) in CAL27 (accompanying drawing 14A) and NIH:OVCAR-3 cell (accompanying drawing 14B).Nucleic acid and aminoacid sequence that VB6-845-sets the toxalbumin construct in vain are shown in accompanying drawing 14C.
Specificity and selectivity (construct of accompanying drawing 3) for research VB6-845, VB6-845 (90% purity) and 17 kinds of chemotherapeutics (LKB Laboratories Inc.) have been tested to the Ep-CAM-positive (NIH:OVCAR-3) and Ep-CAM-feminine gender (HMEC, DAUDI, A-375) cytotoxicity (table 11) of clone.
Finishing MTS with standard technique known in the art detects.Specifically be that 50 microlitre cells are inoculated in every hole, and (2 * 104 cells/ml) were cultivated plate 2 hours under 37 ℃ in 5%CO2.In substratum, add the spiked drug (also being construct to betested or control) that 50 microlitre concentration raise gradually afterwards.Respectively with contain or not celliferous substratum as the positive and negative control.Plate was kept 5 days under 37 ℃, 5%CO2.In the time of the 5th day, (Promega Cat#G5430) assesses the inhibition of on cell proliferation by adding 20 microlitre MTS reagent.Plate is continued to cultivate 2 hours in 37 ℃, 5%CO2, read the OD value at 490nm place with plate reader spectrophotometer.Cut background value in the sample value that will obtain each concentration, the result represents with the percentage ratio of viable cell.Calculate the IC50 of each medicine pair cell system.
When with one group of standard chemotherapeutics, when measuring the cytotoxicity to NIH:OVCAR-3 (the positive ovarian sarcoma of a kind of Ep-CAM-), VB6-845 is than in 12 in these tested 17 kinds more effective (table 11).Although there are five kinds of chemotherapeutics to have more cytotoxicity, because they lack any cell-specific and kill and wound thereby demonstrate stronger toxicity.In five kinds that the are recommended chemotherapeutics (Paclitaxel, Carboplatin, Cisplatin, Doxorubicin and Topotecan) that are used for the treatment of ovarian cancer, have only two kinds (Paclitaxel and Topotecan) to have more cytotoxicity.Although VB6-845 has confirmed more effective dissolved cell activity 1 to 2nM, this effectively kills and wounds, and to only limit to the Ep-CAM-positive tumor cell be NIH:OVCAR-3.Although VB6-845 has showed some to Ep-CAM-negative cells system and killed and wounded, its cytotoxic effect hangs down 220 times and low at the most greater than 1000 times at least.Therefore VB6-845 has represented a kind of effective antibody targeted therapeutical agent that substitutes the chemotherapeutics that the profile with other low toxicity is used in combination, and has prospect in the treatment to many dissimilar solid tumor.
Embodiment 9:VB6-011: the recombined engineering that is used for most desirably transporting the tumor associated antigen specificity Fab antibody of disimmunity bouganin (De-bouganin).
The cancer target cytotoxin is made of the variable region that is connected in bacterium, fungi or plant poison.This research explanation, disimmunity bouganin construct of the present invention comprises the disimmunity bouganin that links to each other with target part, has the immunogenicity of reduction, and still keeps its biological activity simultaneously.Table 13 has proved the combining of tumour of antibodies to tumor-associated antigen (taa) and several types, and shows thus that it can be used for treating the cancer of these types.
Disimmunity Bouganin construct: the target of the tumor associated antigen guiding that links to each other with de-bouganin
Part
H11 antibody, with a kind of monoclonal antibody of discerning tumor associated antigen, heredity is connected in a kind of disimmunity form (de-bouganin) of bouganin, Bou156, a kind of effective, plant origin, I type ribosome inactivating protein (RIP), thus antibody-toxin construct VB6-011 formed.Accompanying drawing 15 show nucleic acid encoding sequence and aminoacid sequences.To the test specification of this construct, this construct has kept its biological activity (cytotoxicity).
Render a service (biological activity)
MTS detects confirmation, still keeps its effectiveness (accompanying drawing 16) behind de-bouganin and the Fab fragment coupling connection.The MTS that is used to measure effectiveness detects and finishes with this area standard technique, as detailed description among the embodiment 8.
Cytotoxicity
For research VB6-011 specificity and and selectivity, tested its cellular cytoxicity activity to the MB-435S cell.Finishing MTS with standard technique known in the art detects.Specifically be that 50 microlitre cells (2 * 10 are inoculated in every hole
4Cell/ml), under 37 ℃ with plate at 5%CO
2The middle cultivation 2 hours.In substratum, add the spiked drug (also being construct to be tested or control) that 50 microlitre concentration raise gradually afterwards.Respectively with contain or not celliferous substratum as the positive and negative control.With plate at 37 ℃, 5%CO
2Under kept 5 days.In the time of the 5th day, (Promega Cat#G5430) assesses the inhibition of on cell proliferation by adding 20 microlitre MTS reagent.
Plate is continued at 37 ℃, 5%CO
2The middle cultivation 2 hours read the OD value at 490nm place with plate readerspectrophotometer.Cut background value in the sample value that will obtain each concentration, the result represents with the percentage ratio of viable cell.The result shows, the IC of VB6-011
50Value is 350nM.
Although present invention is described with reference to the preferred embodiment of the present invention of thinking at present, should be appreciated that to the invention is not restricted to disclosed these examples.On the contrary, this invention is intended to cover various modifications in the spirit and scope of institute of the present invention claim and be equal to arrangement.
All open source literatures, patent, patent application all are incorporated herein by reference in full with it, and independently open source literature, patent and patent application are pointed out with it in full as a reference especially or separately as each.
Table 1
| Peptide # | First amino acid whose position | SEQ ID NO | Sequence | Peptide # | First amino acid whose position | SEQ ID NO | Sequence |
| 1 | 1 | 32 | YNTVSFNLGEAYEYP | 46 | 136 | 77 | EFSIEAIHGKTINGQ |
| 2 | 4 | 33 | VSFNLGEAYEYPTFI | 47 | 139 | 78 | IEAIHGKTINGQEIA |
| 3 | 7 | 34 | NLGEAYEYPTFIQDL | 48 | 142 | 79 | IHGKTINGQEIAKFF |
| 4 | 10 | 35 | EAYEYPTFIQDLRNE | 49 | 145 | 80 | KTINGQEIAKFFLIV |
| 5 | 13 | 36 | EYPTFIQDLRNELAK | 50 | 148 | 81 | NGQEIAKFFLIVIQM |
| 6 | 16 | 37 | TFIQDLRNELAKGTP | 51 | 151 | 82 | EIAKFFLIVIQMVSE |
| 7 | 19 | 38 | QDLRNELAKGTPVCQ | 52 | 154 | 83 | KFFLIVIQMVSEAAR |
| 8 | 22 | 39 | RNELAKGTPVCQLPV | 53 | 157 | 84 | LIVIQMVSEAARFKY |
| 9 | 25 | 40 | LAKGTPVCQLPVTLQ | 54 | 160 | 85 | IQMVSEAARFKYIET |
| 10 | 28 | 41 | GTPVCQLPVTLQTIA | 55 | 163 | 86 | VSEAARFKYIETEVV |
| 11 | 31 | 42 | VCQLPVTLQTIADDK | 56 | 166 | 87 | AARFKYIETEVVDRG |
| 12 | 34 | 43 | LPVTLQTIADDKRFV | 57 | 169 | 88 | FKYIETEVVDRGLYG |
| 13 | 37 | 44 | TLQTIADDKRFVLVD | 58 | 172 | 89 | IETEVVDRGLYGSFK |
| 14 | 40 | 45 | TIADDKRFVLVDITT | 59 | 175 | 90 | EVVDRGLYGSFKPNF |
| 15 | 43 | 46 | DDKRFVLVDITTTSK | 60 | 178 | 91 | DRGLYGSFKPNFKVL |
| 16 | 46 | 47 | RFVLVDITTTSKKTV | 61 | 181 | 92 | LYGSFKPNFKVLNLE |
| 17 | 49 | 48 | LVDITTTSKKTVKVA | 62 | 184 | 93 | SFKPNFKVLNLENNW |
| 18 | 52 | 49 | ITTTSKKTVKVAIDV | 63 | 187 | 94 | PNFKVLNLENNWGDI |
| 19 | 55 | 50 | TSKKTVKVAIDVTDV | 64 | 190 | 95 | KVLNLENNWGDISDA |
| 20 | 58 | 51 | KTVKVAIDVTDVYVV | 65 | 193 | 96 | NLENNWGDISDAIHK |
| 21 | 61 | 52 | KVAIDVTDVYVVGYQ | 66 | 196 | 97 | NNWGDISDAIHKSSP |
| 22 | 64 | 53 | IDVTDVYVVGYQDKW | 67 | 199 | 98 | GDISDAIHKSSPQCT |
| 23 | 67 | 54 | TDVYVVGYQDKWDGK | 68 | 202 | 99 | SDAIHKSSPQCTTIN |
| 24 | 70 | 55 | YVVGYQDKWDGKDRA | 69 | 205 | 100 | IHKSSPQCTTINPAL |
| 25 | 73 | 56 | GYQDKWDGKDRAVFL | 70 | 208 | 101 | SSPQCTTINPALQLI |
| 26 | 76 | 57 | DKWDGKDRAVFLDKV | 71 | 211 | 102 | QCTTINPALQLISPS |
| 27 | 79 | 58 | DGKDRAVFLDKVPTV | 72 | 214 | 103 | TINPALQLISPSNDP |
| 28 | 82 | 59 | DRAVFLDKVPTVATS | 73 | 217 | 104 | PALQLISPSNDPWVV |
| 29 | 85 | 60 | VFLDKVPTVATSKLF | 74 | 220 | 105 | QLISPSNDPWVVNKV |
| 30 | 88 | 61 | DKVPTVATSKLFPGV | 75 | 223 | 106 | SPSNDPWVVNKVSQI |
| 31 | 91 | 62 | PTVATSKLFPGVTNR | 76 | 226 | 107 | NDPWVVNKVSQISPD |
| 32 | 94 | 63 | ATSKLFPGVTNRVTL | 77 | 229 | 108 | WVVNKVSQISPDMGI |
| 33 | 97 | 64 | KLFPGVTNRVTLTFD | 78 | 232 | 109 | NKVSQISPDMGILKF |
| 34 | 100 | 65 | PGVTNRVTLTFDGSY | 79 | 235 | 110 | SQISPDMGILKFKSS |
| 35 | 103 | 66 | TNRVTLTFDGSYQKL | 80 | 238 | 111 | SPDMGILKFKSSKLT |
| 36 | 106 | 67 | VTLTFDGSYQKLVNA | 81 | 240 | 112 | MGILKFKSSKLTQFA |
| 37 | 109 | 68 | TFDGSYQKLVNAAKV | 82 | 243 | 113 | LKFKSSKLTQFATMI |
| 38 | 112 | 69 | GSYQKLVNAAKVDRK | 83 | 246 | 114 | KSSKLTQFATMIRSA |
| 39 | 115 | 70 | QKLVNAAKVDRKDLE | 84 | 249 | 115 | KLTQFATMIRSAIVE |
| 40 | 118 | 71 | VNAAKVDRKDLELGV | 85 | 252 | 116 | QFATMIRSAIVEDLD |
| 41 | 121 | 72 | AKVDRKDLELGVYKL | 86 | 255 | 117 | TMIRSAIVEDLDGDE |
| 42 | 124 | 73 | DRKDLELGVYKLEFS | 87 | 258 | 118 | RSAIVEDLDGDELEI |
| 43 | 127 | 74 | DLELGVYKLEFSIEA | 88 | 261 | 119 | IVEDLDGDELEILEP |
| 44 | 130 | 75 | LGVYKLEFSIEAIHG | 89 | 264 | 120 | DLDGDELEILEPNIA |
| 45 | 133 | 76 | YKLEFSIEAIHGKTI |
Bouganin sequence peptide.The underscore residue does not contain in mature peptide.
Table 2
| The donor numbering | Donor is stored | Allotype | |
| 1 | BC63 | DRB1 *04，DRB1 *07，DRB4 *01 | |
| 2 | BC86 | DRB1
*04，DRB1
*15， |
|
| 3 | BC90 | DRB1 *07，DRB1 *15，DRB4 *01，DRB5 | |
| 4 | BC134 | DRB1
*01，DRB1
*03， |
|
| 5 | BC167 | DRB1 *01，DRB1 *07 and DRB4 *01 | |
| 6 | BC216 | DRB1 *14，DRB1 *15，DRB3，DRB5 | |
| 7 | BC217 | DRB1 *04，DRB1 *12，DRB3，DRB4 *01 | |
| 8 | BC233 | DRB1 *04，DRB1 *11 and DRB3, DRB4 *01 | |
| 9 | BC241 | DRB1 *07，DRB1 *11，DRB3，DRB4 *01 | |
| 10 | BC246 | DRB1 *01，DRB1 *13 and DRB3 | |
| 11 | BC262 | DRB1 *03，DRB1 *07，DRB3，DRB4 *01 | |
| 12 | BC292 | DRB1 *07，DRB1 *13，DRB3，DRB4 *01 | |
| 13 | BC293 | DRB1
*04， |
|
| 14 | BC231 | DRB1 *03 or DRB1 *03，DRB1 *13 and DRB3 | |
| 15 | BC301 | DRB1 *07，DRB1 *14，DRB3 | |
| 16 | BC326 | DRB1 *03，DRB1 *15，DRB3，DRB5 | |
| 17 | BC316 | DRB1 *13，DRB1 *15，DRB3，DRB5 | |
| 18 | BC321 | DRB1 *01，DRB1 *15，DRB5 | |
| 19 | BC382 | DRB1 *04，DRB1 *08，DRB4 *01 | |
| 20 | BC336 | DRB1 *01，DRB1 *11，DRB3 |
The MHC allotype of PBMC donor
Table 3
Primer SEQ ID NO sequence
OL1032?121?CATTACAAACGTCTACCAAGTTT
OL1033?122?TTACAAAAGTAGATAAGTAATGTG
OL1322?123?GATATACATATGAAATACCTATTGCCTACG
OL1067?124?TGACACAGTGTTGTACGCTGGTTGGGCAGCGAGTAA
OL1068?125?GCTGCCCAACCAGCGTACAACACTGTGTCATTTAAC
OL1323?126?CGAGTGCGGCCGCTCAATGGTGATGGTGATGGTGT
Make up employed primer sequence in the WT bouganin gene
Table 4
List constructed and test replaces the bouganin variant.
| Sudden change | Coding mutation | Activity in the luciferase assay * | Clone ID ** |
| Negative control | |||
| Y70A | TAT-GCT | -- | BouY70A |
| Epitope regions R1 (peptide 41) | |||
| V123T | GTG-ACG | +/- | Bou2 |
| V123A | GTG-GCT | ++ | Bou3 |
| V123D | GTG-GAT | -- | - |
| V123E | GTG-GAA | -- | - |
| V123G | GTG-GGC | -- | - |
| V123H | GTG-CAC | -- | - |
| V123K | GTG-AAG | -- | - |
| V123N | GTG-AAC | -- | - |
| V123P | GTG-CCT | -- | - |
| V123Q | GTG-CAA | ++ | Bou4 |
| V123R | GTG-AGA | -- | - |
| V123S | GTG-TCA | -- | - |
| D127G | GAT-GGC | ++ | Bou5 |
| D127A | GAT-GCT | ++ | Bou6 |
| E129K | GAA-AAG | -- | - |
| E129R | GAA-AGA | -- | - |
| E129Q | GAA-CAA | +/- | Bou7 |
| E129G | GAA-GGC | ++ | Bou8 |
| Epitope regions R2 (peptide 44) | |||
| Y133P | TAC-CCC | -- | - |
| Y133N | TAC-AAC | ++ | Bou9 |
| Y133T | TAC-ACA | ++ | Bou10 |
| Y133A | TAC-GCT | ++ | Bou11 |
| Y133R | TAC-AGA | ++ | Bou12 |
| Y133D | TAC-GAT | ++ | Bou13 |
| Y133E | TAC-GAA | +/- | Bou14 |
| Y133Q | TAC-CAA | ++ | Bou15 |
| Y133G | TAC-GGC | ++ | Bou16 |
| Y133H | TAC-CAC | ++ | Bou17 |
| Y133K | TAC-AAG | ++ | Bou18 |
| Y133S | TAC-TCA | ++ | Bou19 |
| Epitope regions R3 (peptide 50) | |||
| E151T?I152E | GAGATA-ACGGAA | -- | - |
| I152Q | ATA-CAA | ++ | Bou20 |
| I152A | ATA-GCA | ++ | Bou21 |
| I152E | ATA-GAA | -- | - |
| F155P | TTC-CCA | -- | - |
| F155H | TTC-CAC | -- | - |
| I158P | ATT-CCA | -- | - |
*Activity in the luciferase assay:
++=be equal to or higher than WT albumen.In active 2 times of the +=WT.In active 3 times of+/-=WT.--=be less than WT active 1/3rd.The WT=wild-type protein.
*Clone ID.Only to the active variant name of function is arranged.
Table 5
Constructed and test polysubstituted bouganin variant.
| Clone ID | Epitope regions R1 (peptide 41) | Epitope regions R2 (peptide 44) | Epitope regions R3 (peptide 51) | Activity in the luciferase assay * | |
| Bou143 | V123Q | Y133Q | I152Q | ++ | |
| Bou144 | V123A | Y133N | I152A | ++ | |
| Bou145 | V123A | Y133Q | I152A | ++ | |
| Bou146 | V123A | D127G | ++ | ||
| Bou147 | V123A | D127A | ++ | ||
| Bou148 | V123Q | D127G | ++ | ||
| Bou149 | V123Q | D127A | ++ | ||
| Bou150 | V123Q | E129G | + | ||
| Bou151 | V123A | E129G | + | ||
| Bou156 | V123A | D127A | Y133N | I152A | ++ |
| Bou157 | V123A | D127A | Y133Q | I152A | ++ |
* the activity in the luciferase assay:
++=be equal to or higher than WT albumen.In active 2 times of the +=WT.In active 3 times of+/-=WT.--=be less than WT active 1/3rd.The WT=wild-type protein.
Table 6
| Clone ID | Replace * | Protein |
| ?Bou32 | ?WT | SEQ?ID?No?1 |
| ?Bou156 | ?V123A、D127A、Y133N，I152A | SEQ?ID?No?13 |
| ?Bou157 | ?V123A、D127A、Y133Q，1152A | SEQ?ID?No?14 |
| ?Bou143 | ?V123Q，Y133Q，I152Q | |
| ?Bou144 | ?V123A，y133N，I152A | |
| ?Bou145 | ?V123A，Y133Q，I152A | |
| ?Bou146 | ?V123A，D127G | |
| ?Bou147 | ?V123A，D127A | |
| ?Bou148 | ?V123Q，D127G | |
| ?Bou149 | ?V123Q，D127A | |
| ?Bou150 | ?V123Q，E129G | |
| ?Bou151 | ?V123A，E129G | |
| ?Bou2 | ?V123T | |
| ?Bou3 | ?V123A | |
| ?Bou4 | ?V123Q | |
| ?Bou5 | ?D127G | |
| ?Bou6 | ?D127A | |
| ?Bou7 | ?E129Q | |
| ?Bou8 | ?E129G | |
| ?Bou9 | ?Y133N | |
| ?Bou10 | ?Y133T | |
| ?Bou11 | ?Y133A | |
| ?Bou12 | ?Y133R | |
| ?Bou13 | ?Y133D | |
| ?Bou14 | ?Y133E | |
| ?Bou15 | ?Y133Q | |
| ?Bou16 | ?Y133G | |
| ?Bou17 | ?Y133H | |
| ?Bou18 | ?Y133K | |
| ?Bou19 | ?Y133S | |
| ?Bou20 | ?I152Q | |
| ?Bou21 | ?I152A |
*Read the numbering of residue 1 beginning of frame from bouganin, got rid of PelB homing sequence included in most of constructs thus.
Table 7
| ?SEQ?ID?No?1 ?Protein ?YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFVLVDITTTSKKT ?VKVAIDVTDVY ?VVGYQDKWDGKDRAVFLDKVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKVDRK ?DLELGVYKLEFSIE ?AIHGKTINGQEIAKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVLNLENNWG ?DISDAIHKSSP ?QCTTINPALQLISPSNDPWVVNKVSQISPDMGILKFKSSK |
| ?SEQ?ID?No?13 ?Protein ?YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFVLVDITTTSKKT ?VKVAIDVTDVY ?VVGYQDKWDGKDRAVFLDKVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKADRK ?ALELGVNKLEFSIE ?AIHGKTINGQEAAKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVLNLENNW ?GDISDAIHKSSP ?QCTTINPALQLISPSNDPWVVNKVSQISPDMGILKFKSSK |
Table 8
Further tested enzyme is modified the Bouganin with WT in the T cell detection.
| The peptide numbering | First amino acid position in the Bouganin | Sequence * | SEQ?ID?NO |
| DEI-41 | 121-135 | AK ADRK ALELGV NKL | 29 |
| DeI-44 | 130-144 | LGV NKLEFSIEAIHG | 30 |
| DeI-50 | 149-163 | NGQE AAKFFLIVIQM | 31 |
*The residue that is substituted (sudden change) is represented with underscore.
Table 9
VB6-845 represents breed multiple with the result that combines of gynaecology clone with MF ± SEM. through flow cytometer
| Indication | Clone | VB6-845 (propagation multiple MF ± SEM) |
| Uterine endometrium | HEC-1-A | 42.3±0.9 |
| RL95-2 | 4.9±0.7 | |
| SK-UT-1 | 1.1±0.1 | |
| Ovary | NIH:OVCAR-3 | 33.6±6.0 |
| SK-OV-3 | 4.3±1.0 | |
| TOV-112G | 1.1±0.1 | |
| Uterine neck | HT-3 | 29.1±1.2 |
| C-4I | 6.8±0.6 | |
| C-33A | 1.1±0.0 | |
| Melanoma | A-375 | 1.1±0.1 |
Table 10
The cytotoxicity of the VB6-845-mediation by the MTS measuring
| Indication | Clone | IC 50NM VB6-845 70% purity |
| Uterine endometrium | HEC-1-A | 43 |
| KLE | ＞100 | |
| RL95-2 | 100 | |
| Ovary | NIH-OVCAR-3 | 3.4 |
| Caov-3 | 1.3 | |
| SK-OV-3 | ＞100 | |
| Uterine neck | MS751 | 0.43 |
| HT-3 | 23 | |
| ME-180 | 37 | |
| C-41 | 1.7 | |
| Melanoma | A-375 | ＞100 |
Table 11
VB6-845 compares the specificity and the selectivity of chemotherapeutics
| IC 50nM ?NIH:OVCAR- 3 A-375 DAUDI HMEC | |
| ?Paclitaxel ?Docetaxel ?Vincristine ?Vinblastine?Sulfate ?Topotecan | ＜10 -6 4.9×10 -6?＜10 -6 ＜10 -6 ＜10 -6 ＜10 -6 ＜10 -6 ＜10 -6 4.4×10 -6?＜10 -6 ＜10 -6 ＜10 -6 1.1×10 -6?＜10 -6 ＜10 -6 ＜10 -6 0.071 1.5 0.009 4.1 |
| ?VB6-845(90％ ?pure) | 1 ＞1000 ＞1000 220 |
| ?Doxorubicin ?Mitomycin?C ?Bleomycin?Sulfate ?Bleomycin?A5 ?Irinotecan ?Etoposide ?Methotrexate ?Chlorambucil ?Fluorouracil ?Cyclophosphamide ?Cisplatin ? | 3 2.8 16×10 -6 16 28 14 2.8 50 30 170 22 600 150 290 130 1000 180 900 190 1000 2l0 280 1.7 600 ＞1000 6 3.6 41 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 ＞1000 |
Oncocyte indication during table 12:VB6-845 is swollen
| Indication | N 1 | Combination (IgG) to |
| |
3 | 148.9 |
| Ovary | 2 | 84.1 |
| |
3 | 72.4 |
| Bladder | 14 | 59.6 |
| |
5 | 50.1 |
| |
3 | 37.5 |
| |
1 | 23.8 |
| |
3 | 16.4 |
| Neck | 2 | 11.4 |
| |
3 | 9.4 |
| |
3 | 5.5 |
| |
3 | 1.6 |
1N represents the quantity of the clone of being tested in each indication.
2Mean in each indication from the intermediate value fluorescence of all cells system propagation multiple control antibodies.
Table 13:VB6-011 Tumor Cell Indications
| INDICATIONS | N 1 | Binding?for?mAb?011 (IgG) 2 |
| | 3 | 16.9 |
| | 3 | 15.1 |
| | 3 | 14.0 |
| | 3 | 13.1 |
| Ovarian | 2 | 11.1 |
| | 3 | 8.7 |
| | 3 | 6.9 |
| Liver | 2 | 6.5 |
| | 3 | 4.2 |
| Head and Neck | 2 | 2.9 |
1N represents the quantity of the clone of being tested in each indication.
2The mean value that value representation calculates the sum of the average propagation multiple of control antibodies from the intermediate value fluorescence of all cells system from each indication.Null value means with respect to control activity does not have measurable activity.
＜110〉horse is repaiied the Bake
Mark Lewis-Francis J Ka Er
Ke Enhelunduomu
Ji Ennikexizieryou
Glenn Christopher MacDonald
Joycelyn grace Tevez Te Er
Denis George Sha Baosike
Nicholas Peter Lonard Ge Luowo
＜120〉modified Bouganin proteins, cytotoxin and method thereof and purposes
<130>10241-43
<140>
<141>
<150>US?60/554,580
<151>2004-03-19
<150>US?60/630,571
<151>2004-11-26
<160>129
<170>PatentIn?version?3.3
<210>1
<211>250
<212>PRT
<213>Bougainvillea?spectabilis?Wild
<400>1
Tyr?Asn?Thr?Val?Ser?Phe?Asn?Leu?Gly?Glu?Ala?Tyr?Glu?Tyr?Pro?Thr
1 5 10 15
Phe?Ile?Gln?Asp?Leu?Arg?Asn?Glu?Leu?Ala?Lys?Gly?Thr?Pro?Val?Cys
20 25 30
Gln?Leu?Pro?Val?Thr?Leu?Gln?Thr?Ile?Ala?Asp?Asp?Lys?Arg?Phe?Val
35 40 45
Leu?Val?Asp?Ile?Thr?Thr?Thr?Ser?Lys?Lys?Thr?Val?Lys?Val?Ala?Ile
50 55 60
Asp?Val?Thr?Asp?Val?Tyr?Val?Val?Gly?Tyr?Gln?Asp?Lys?Trp?Asp?Gly
65 70 75 80
Lys?Asp?Arg?Ala?Val?Phe?Leu?Asp?Lys?Val?Pro?Thr?Val?Ala?Thr?Ser
85 90 95
Lys?Leu?Phe?Pro?Gly?Val?Thr?Asn?Arg?Val?Thr?Leu?Thr?Phe?Asp?Gly
100 105 110
Ser?Tyr?Gln?Lys?Leu?Val?Asn?Ala?Ala?Lys?Val?Asp?Arg?Lys?Asp?Leu
115 120 125
Glu?Leu?Gly?Val?Tyr?Lys?Leu?Glu?Phe?Ser?Ile?Glu?Ala?Ile?His?Gly
130 135 140
Lys?Thr?Ile?Asn?Gly?Gln?Glu?Ile?Ala?Lys?Phe?Phe?Leu?Ile?Val?Ile
145 150 155 160
Gln?Met?Val?Ser?Glu?Ala?Ala?Arg?Phe?Lys?Tyr?Ile?Glu?Thr?Glu?Val
165 170 175
Val?Asp?Arg?Gly?Leu?Tyr?Gly?Ser?Phe?Lys?Pro?Asn?Phe?Lys?Val?Leu
180 185 190
Asn?Leu?Glu?Asn?Asn?Trp?Gly?Asp?Ile?Ser?Asp?Ala?Ile?His?Lys?Ser
195 200 205
Ser?Pro?Gln?Cys?Thr?Thr?Ile?Asn?Pro?Ala?Leu?Gln?Leu?Ile?Ser?Pro
210 215 220
Ser?Asn?Asp?Pro?Trp?Val?Val?Asn?Lys?Val?Ser?Gln?Ile?Ser?Pro?Asp
225 230 235 240
Met?Gly?Ile?Leu?Lys?Phe?Lys?Ser?Ser?Lys
245 250
<210>2
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>2
Ala?Lys?Val?Asp?Arg?Lys?Asp?Leu?Glu?Leu?Gly?Val?Tyr?Lys?Leu
1 5 10 15
<210>3
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>3
Leu?Gly?Val?Tyr?Lys?Leu?Glu?Phe?Ser?Ile?Glu?Ala?Ile?His?Gly
1 5 10 15
<210>4
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>4
Asn?Gly?Gln?Glu?Ile?Ala?Lys?Phe?Phe?Leu?Ile?Val?Ile?Gln?Met
1 5 10 15
<210>5
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<220>
＜221〉other features
<222>(3)..(3)
＜223〉Xaa can be any natural amino acid
<220>
＜221〉other features
<222>(7)..(7)
＜223〉Xaa can be any natural amino acid
<220>
＜221〉other features
<222>(9)..(9)
＜223〉Xaa can be any natural amino acid
<220>
＜221〉other features
<222>(13)..(13)
＜223〉Xaa can be any natural amino acid
<400>5
Ala?Lys?Xaa?Asp?Arg?Lys?Xaa?Leu?Xaa?Leu?Gly?Val?Xaa?Lys?Leu
1 5 10 15
<210>6
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<220>
＜221〉other features
<222>(4)..(4)
＜223〉Xaa can be any natural amino acid
<400>6
Leu?Gly?Val?Xaa?Lys?Leu?Glu?Phe?Ser?Ile?Glu?Ala?Ile?His?Gly
1 5 10 15
<210>7
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<220>
＜221〉other features
<222>(5)..(5)
＜223〉Xaa can be any natural amino acid
<400>7
Asn?Gly?Gln?Glu?Xaa?Ala?Lys?Phe?Phe?Leu?Ile?Val?Ile?Gln?Met
1 5 10 15
<210>8
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<220>
＜221〉other features
<222>(3)..(3)
＜223〉Xaa can be T or A or Q
<220>
＜221〉other features
<222>(7)..(7)
＜223〉Xaa can be G or A
<220>
＜221〉other features
<222>(9)..(9)
＜223〉Xaa can be Q or G
<220>
＜221〉other features
<222>(13)..(13)
＜223〉Xaa can be N or D or T or A or R or Q or E or G or H or K or S
<400>8
Ala?Lys?Xaa?Asp?Arg?Lys?Xaa?Leu?Xaa?Leu?Gly?Val?Xaa?Lys?Leu
1 5 10 15
<210>9
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<220>
＜221〉other features
<222>(4)..(4)
＜223〉Xaa can be N or D or T or A or R or Q or E or G or H or K or S
<400>9
Leu?Gly?Val?Xaa?Lys?Leu?Glu?Phe?Ser?Ile?Glu?Ala?Ile?His?Gly
1 5 10 15
<210>10
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<220>
＜221〉other features
<222>(5)..(5)
＜223〉Xaa can be Q or A
<400>10
Asn?Gly?Gln?Glu?Xaa?Ala?Lys?Phe?phe?Leu?Ile?Val?Ile?Gln?Met
1 5 10 15
<210>11
<211>250
<212>PRT
<213>Bougainvillea?spectabilis
<220>
＜221〉other features
<222>(123)..(123)
＜223〉Xaa can be any natural amino acid
<220>
＜221〉other features
<222>(127)..(127)
＜223〉Xaa can be any natural amino acid
<220>
＜221〉other features
<222>(129)..(129)
＜223〉Xaa can be any natural amino acid
<220>
＜221〉other features
<222>(133)..(133)
＜223〉Xaa can be any natural amino acid
<220>
＜221〉other features
<222>(152)..(152)
＜223〉Xaa can be any natural amino acid
<400>11
Tyr?Asn?Thr?Val?Ser?Phe?Asn?Leu?Gly?Glu?Ala?Tyr?Glu?Tyr?Pro?Thr
1 5 10 15
Phe?Ile?Gln?Asp?Leu?Arg?Asn?Glu?Leu?Ala?Lys?Gly?Thr?Pro?Val?Cys
20 25 30
Gln?Leu?Pro?Val?Thr?Leu?Gln?Thr?Ile?Ala?Asp?Asp?Lys?Arg?Phe?Val
35 40 45
Leu?Val?Asp?Ile?Thr?Thr?Thr?Ser?Lys?Lys?Thr?Val?Lys?Val?Ala?Ile
50 55 60
Asp?Val?Thr?Asp?Val?Tyr?Val?Val?Gly?Tyr?Gln?Asp?Lys?Trp?Asp?Gly
65 70 75 80
Lys?Asp?Arg?Ala?Val?Phe?Leu?Asp?Lys?Val?Pro?Thr?Val?Ala?Thr?Ser
85 90 95
Lys?Leu?Phe?Pro?Gly?Val?Thr?Asn?Arg?Val?Thr?Leu?Thr?Phe?Asp?Gly
100 105 110
Ser?Tyr?Gln?Lys?Leu?Val?Asn?Ala?Ala?Lys?Xaa?Asp?Arg?Lys?Xaa?Leu
115 120 125
Xaa?Leu?Gly?Val?Xaa?Lys?Leu?Glu?Phe?Ser?Ile?Glu?Ala?Ile?His?Gly
130 135 140
Lys?Thr?Ile?Asn?Gly?Gln?Glu?Xaa?Ala?Lys?Phe?Phe?Leu?Ile?Val?Ile
145 150 155 160
Gln?Met?Val?Ser?Glu?Ala?Ala?Arg?Phe?Lys?Tyr?Ile?Glu?Thr?Glu?Val
165 170 175
Val?Asp?Arg?Gly?Leu?Tyr?Gly?Ser?Phe?Lys?Pro?Asn?Phe?Lys?Val?Leu
180 185 190
Asn?Leu?Glu?Asn?Asn?Trp?Gly?Asp?Ile?Ser?Asp?Ala?Ile?His?Lys?Ser
195 200 205
Ser?Pro?Gln?Cys?Thr?Thr?Ile?Asn?Pro?Ala?Leu?Gln?Leu?Ile?Ser?Pro
210 215 220
Ser?Asn?Asp?Pro?Trp?Val?Val?Asn?Lys?Val?Ser?Gln?Ile?Ser?Pro?Asp
225 230 235 240
Met?Gly?Ile?Leu?Lys?Phe?Lys?Ser?Ser?Lys
245 250
<210>12
<211>250
<212>PRT
<213>Bougainvillea?spectabilis
<220>
＜221〉other features
<222>(123)..(123)
＜223〉Xaa can be T or A or Q
<220>
＜221〉other features
<222>(127)..(127)
＜223〉Xaa can be G or A
<220>
＜221〉other features
<222>(129)..(129)
＜223〉Xaa can be Q or G
<220>
＜221〉other features
<222>(133)..(133)
＜223〉Xaa can be N or D or T or A or R or Q or E or G or H or K or S
<220>
＜221〉other features
<222>(152)..(152)
＜223〉Xaa can be Q or A
<400>12
Tyr?Asn?Thr?Val?Ser?Phe?Asn?Leu?Gly?Glu?Ala?Tyr?Glu?Tyr?Pro?Thr
1 5 10 15
Phe?Ile?Gln?Asp?Leu?Arg?Asn?Glu?Leu?Ala?Lys?Gly?Thr?Pro?Val?Cys
20 25 30
Gln?Leu?Pro?Val?Thr?Leu?Gln?Thr?Ile?Ala?Asp?Asp?Lys?Arg?Phe?Val
35 40 45
Leu?Val?Asp?Ile?Thr?Thr?Thr?Ser?Lys?Lys?Thr?Val?Lys?Val?Ala?Ile
50 55 60
Asp?Val?Thr?Asp?Val?Tyr?Val?Val?Gly?Tyr?Gln?Asp?Lys?Trp?Asp?Gly
65 70 75 80
Lys?Asp?Arg?Ala?Val?phe?Leu?Asp?Lys?Val?Pro?Thr?Val?Ala?Thr?Ser
85 90 95
Lys?Leu?Phe?Pro?Gly?Val?Thr?Asn?Arg?Val?Thr?Leu?Thr?Phe?Asp?Gly
100 105 110
Ser?Tyr?Gln?Lys?Leu?Val?Asn?Ala?Ala?Lys?Xaa?Asp?Arg?Lys?Xaa?Leu
115 120 125
Xaa?Leu?Gly?Val?Xaa?Lys?Leu?Glu?Phe?Ser?Ile?Glu?Ala?Ile?His?Gly
130 135 140
Lys?Thr?Ile?Asn?Gly?Gln?Glu?Xaa?Ala?Lys?Phe?Phe?Leu?Ile?Val?Ile
145 150 155 160
Gln?Met?Val?Ser?Glu?Ala?Ala?Arg?Phe?Lys?Tyr?Ile?Glu?Thr?Glu?Val
165 170 175
Val?Asp?Arg?Gly?Leu?Tyr?Gly?Ser?phe?Lys?Pro?Asn?Phe?Lys?Val?Leu
180 185 190
Asn?Leu?Glu?Asn?Asn?Trp?Gly?Asp?Ile?Ser?Asp?Ala?Ile?His?Lys?Ser
195 200 205
Ser?Pro?Gln?Cys?Thr?Thr?Ile?Asn?Pro?Ala?Leu?Gln?Leu?Ile?Ser?pro
210 215 220
Ser?Asn?Asp?Pro?Trp?Val?Val?Asn?Lys?Val?Ser?Gln?Ile?Ser?Pro?Asp
225 230 235 240
Met?Gly?Ile?Leu?Lys?Phe?Lys?Ser?Ser?Lys
245 250
<210>13
<211>250
<212>PRT
<213>Bougainvillea?spectabilis
<400>13
Tyr?Asn?Thr?Val?Ser?Phe?Asn?Leu?Gly?Glu?Ala?Tyr?Glu?Tyr?Pro?Thr
1 5 10 15
phe?Ile?Gln?Asp?Leu?Arg?Asn?Glu?Leu?Ala?Lys?Gly?Thr?Pro?Val?Cys
20 25 30
Gln?Leu?Pro?Val?Thr?Leu?Gln?Thr?Ile?Ala?Asp?Asp?Lys?Arg?Phe?Val
35 40 45
Leu?Val?Asp?Ile?Thr?Thr?Thr?Ser?Lys?Lys?Thr?Val?Lys?Val?Ala?Ile
50 55 60
Asp?Val?Thr?Asp?Val?Tyr?Val?Val?Gly?Tyr?Gln?Asp?Lys?Trp?Asp?Gly
65 70 75 80
Lys?Asp?Arg?Ala?Val?Phe?Leu?Asp?Lys?Val?Pro?Thr?Val?Ala?Thr?Ser
85 90 95
Lys?Leu?Phe?Pro?Gly?Val?Thr?Asn?Arg?Val?Thr?Leu?Thr?Phe?Asp?Gly
100 105 110
Ser?Tyr?Gln?Lys?Leu?Val?Asn?Ala?Ala?Lys?Ala?Asp?Arg?Lys?Ala?Leu
115 120 125
Glu?Leu?Gly?Val?Asn?Lys?Leu?Glu?Phe?Ser?Ile?Glu?Ala?Ile?His?Gly
130 135 140
Lys?Thr?Ile?Asn?Gly?Gln?Glu?Ala?Ala?Lys?Phe?Phe?Leu?Ile?Val?Ile
145 150 155 160
Gln?Met?Val?Ser?Glu?Ala?Ala?Arg?Phe?Lys?Tyr?Ile?Glu?Thr?Glu?Val
165 170 175
Val?Asp?Arg?Gly?Leu?Tyr?Gly?Ser?Phe?Lys?Pro?Asn?Phe?Lys?Val?Leu
180 185 190
Asn?Leu?Glu?Asn?Asn?Trp?Gly?Asp?Ile?Ser?Asp?Ala?Ile?His?Lys?Ser
195 200 205
Ser?Pro?Gln?Cys?Thr?Thr?Ile?Asn?Pro?Ala?Leu?Gln?Leu?Ile?Ser?Pro
210 215 220
Ser?Asn?Asp?Pro?Trp?Val?Val?Asn?Lys?Val?Ser?Gln?Ile?Ser?Pro?Asp
225 230 235 240
Met?Gly?Ile?Leu?Lys?Phe?Lys?Ser?Ser?Lys
245 250
<210>14
<211>250
<212>PRT
<213>Bougainvillea?spectabilis
<400>14
Tyr?Asn?Thr?Val?Ser?Phe?Asn?Leu?Gly?Glu?Ala?Tyr?Glu?Tyr?Pro?Thr
1 5 10 15
Phe?Ile?Gln?Asp?Leu?Arg?Asn?Glu?Leu?Ala?Lys?Gly?Thr?Pro?Val?Cys
20 25 30
Gln?Leu?Pro?Val?Thr?Leu?Gln?Thr?Ile?Ala?Asp?Asp?Lys?Arg?Phe?Val
35 40 45
Leu?Val?Asp?Ile?Thr?Thr?Thr?Ser?Lys?Lys?Thr?Val?Lys?Val?Ala?Ile
50 55 60
Asp?Val?Thr?Asp?Val?Tyr?Val?Val?Gly?Tyr?Gln?Asp?Lys?Trp?Asp?Gly
65 70 75 80
Lys?Asp?Arg?Ala?Val?Phe?Leu?Asp?Lys?Val?Pro?Thr?Val?Ala?Thr?Ser
85 90 95
Lys?Leu?Phe?Pro?Gly?Val?Thr?Asn?Arg?Val?Thr?Leu?Thr?Phe?Asp?Gly
100 105 110
Ser?Tyr?Gln?Lys?Leu?Val?Asn?Ala?Ala?Lys?Ala?Asp?Arg?Lys?Ala?Leu
115 120 125
Glu?Leu?Gly?Val?Gln?Lys?Leu?Glu?Phe?Ser?Ile?Glu?Ala?Ile?His?Gly
130 135 140
Lys?Thr?Ile?Asn?Gly?Gln?Glu?Ala?Ala?Lys?Phe?Phe?Leu?Ile?Val?Ile
145 150 155 160
Gln?Met?Val?Ser?Glu?Ala?Ala?Arg?Phe?Lys?Tyr?Ile?Glu?Thr?Glu?Val
165 170 175
Val?Asp?Arg?Gly?Leu?Tyr?Gly?Ser?Phe?Lys?Pro?Asn?Phe?Lys?Val?Leu
180 185 190
Asn?Leu?Glu?Asn?Asn?Trp?Gly?Asp?Ile?Ser?Asp?Ala?Ile?His?Lys?Ser
195 200 205
Ser?Pro?Gln?Cys?Thr?Thr?Ile?Asn?Pro?Ala?Leu?Gln?Leu?Ile?Ser?Pro
210 215 220
Ser?Asn?Asp?Pro?Trp?Val?Val?Asn?Lys?Val?Ser?Gln?Ile?Ser?Pro?Asp
225 230 235 240
Met?Gly?Ile?Leu?Lys?Phe?Lys?Ser?Ser?Lys
245 250
<210>15
<211>2404
<212>DNA
＜213〉artificial sequence
<220>
<223>VB6-845
<400>15
gaattcctgc?aggtctatgg?aacgataaat?gcccatgaaa?attctatttc?aaggagacag 60
tcataatgaa?atacctattg?cctacggcag?ccgctggatt?gttattactc?gctgcccaac 120
cagcgatggc?ggaagtacag?ctggttcagt?ccggcccggg?tcttgttcaa?ccgggtggtt 180
ccgttcgtat?ctcttgcgct?gcttctggtt?acacgttcac?caactacggc?atgaactggg 240
tcaaacaggc?tccgggtaaa?ggcctggaat?ggatgggctg?gatcaacacc?tacaccggtg 300
aatccaccta?cgctgactcc?ttcaaaggtc?gcttcacttt?ctccctcgac?acaagtgcta 360
gtgctgcata?cctccaaatc?aactcgctgc?gtgcagagga?tacagcagtc?tattactgcg 420
cccgtttcgc?tatcaaaggt?gactactggg?gtcaaggcac?gctgctgacc?gtttcctcgg 480
ctagcaccaa?aggcccatcg?gtcttccccc?tggcaccctc?ctccaagagc?acctctgggg 540
gcacagcggc?cctgggctgc?ctggtcaagg?actacttccc?cgaaccggtg?acggtgtcgt 600
ggaactcagg?cgccctgacc?agcggcgtgc?acaccttccc?ggctgtccta?cagtcctcag 660
gactctactc?cctcagcagc?gtggtgaccg?tgccctccag?cagcttgggc?acccagacct 720
acatctgcaa?cgtgaatcac?aagcccagca?acaccaaggt?ggacaagaaa?gttgagccca 780
aatcttgtac?caggcacagg?cagcccagag?gctgggagca?gctctacaac?accgtgtcat 840
ttaaccttgg?agaagcttat?gagtacccca?cttttataca?agatttgcgc?aatgaattgg 900
ctaagggcac?accagtatgt?caacttccag?tgacactaca?aaccatagcc?gatgacaagc 960
gatttgttct?agttgatatc?actacgacct?cgaagaaaac?agttaaggtt?gctatagatg 1020
tgacagatgt?gtatgttgtg?ggttatcaag?acaaatggga?tggcaaagat?cgagctgttt 1080
tccttgacaa?ggttcctact?gttgcaacta?gtaaactttt?cccaggggtg?actaatcgtg 1140
taacgttaac?atttgatggc?agctatcaga?aacttgtgaa?tgctgccaaa?gctgatagaa 1200
aggctctcga?actgggggtt?aacaaattgg?aattttccat?tgaagcaatc?catggtaaaa 1260
cgataaatgg?tcaagaggca?gccaagttct?ttcttattgt?catccaaatg?gtttcagagg 1320
cagctcggtt?caaatatatt?gagactgagg?tggttgatag?aggattatat?ggatcattca 1380
aacctaattt?taaagtattg?aacttggaga?acaattgggg?cgacatctct?gatgccattc 1440
acaaatcatc?cccacaatgt?accactatta?atccggcact?tcagttgata?agcccctcaa 1500
atgacccatg?ggttgtaaat?aaagtgagtc?aaattagtcc?cgatatgggt?atccttaagt 1560
ttaaaagctc?caaatagtga?tctagagtcg?acctgcaggt?ctatggaacg?ataaatgccc 1620
atgaaaattc?tatttcaagg?agacagtcat?aatgaaatac?ctattgccta?cggcagccgc 1680
tggattgtta?ttactcgctg?cccaaccagc?gatggcgcac?catcatcacc?atcacgatat 1740
ccagatgacc?cagtccccgt?cctccctgag?tgcttctgtt?ggtgaccgtg?ttaccatcac 1800
ctgccgttcc?accaaatccc?tcctgcactc?caacggtatc?acctaccttt?attggtatca 1860
acagaaaccg?ggtaaagctc?cgaaacttct?gatctaccag?atgtccaacc?tggcttccgg 1920
tgttccgtct?cgtttctcca?gttctggttc?tggtaccgac?ttcaccctga?ccatctcttc 1980
tctgcagccg?gaagacttcg?ctacctacta?ctgcgctcag?aacctggaaa?tcccgcgtac 2040
cttcggtcag?ggtaccaaag?ttgaacttaa?gcgcactgtg?gctgcaccat?ctgtcttcat 2100
cttcccgcca?tctgatgagc?agttgaaatc?tggaactgcc?tctgttgtgt?gcctgctgaa 2160
taacttctat?cccagagagg?ccaaagtaca?gtggaaggtg?gataacgccc?tccaatcggg 2220
taactcccag?gagagtgtca?cagagcagga?cagcaaggac?agcacctaca?gcctcagcag 2280
caccctgacg?ctgagcaaag?cagactacga?gaaacacaaa?gtctacgcct?gcgaagtcac 2340
ccatcagggc?ctgagctcgc?ccgtcacaaa?gagcttcaac?aggggagagt?gttagtagct 2400
<210>16
<211>750
<212>PRT
＜213〉artificial sequence
<220>
<223>VB6-845
<400>16
Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala?Ala?Gly?Leu?Leu?Leu?Leu?Ala
1 5 10 15
Ala?Gln?Pro?Ala?Met?Ala?Glu?Val?Gln?Leu?Val?Gln?Ser?Gly?Pro?Gly
20 25 30
Leu?Val?Gln?Pro?Gly?Gly?Ser?Val?Arg?Ile?Ser?Cys?Ala?Ala?Ser?Gly
35 40 45
Tyr?Thr?Phe?Thr?Asn?Tyr?Gly?Met?Asn?Trp?Val?Lys?Gln?Ala?Pro?Gly
50 55 60
Lys?Gly?Leu?Glu?Trp?Met?Gly?Trp?Ile?Asn?Thr?Tyr?Thr?Gly?Glu?Ser
65 70 75 80
Thr?Tyr?Ala?Asp?Ser?Phe?Lys?Gly?Arg?Phe?Thr?Phe?Ser?Leu?Asp?Thr
85 90 95
Ser?Ala?Ser?Ala?Ala?Tyr?Leu?Gln?Ile?Asn?Ser?Leu?Arg?Ala?Glu?Asp
100 105 110
Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Phe?Ala?Ile?Lys?Gly?Asp?Tyr?Trp
115 120 125
Gly?Gln?Gly?Thr?Leu?Leu?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?Thr?Arg?His?Arg?Gln?Pro?Arg?Gly?Trp?Glu?Gln?Leu?Tyr?Asn?Thr
245 250 255
Val?Ser?Phe?Asn?Leu?Gly?Glu?Ala?Tyr?Glu?Tyr?Pro?Thr?Phe?Ile?Gln
260 265 270
Asp?Leu?Arg?Asn?Glu?Leu?Ala?Lys?Gly?Thr?Pro?Val?Cys?Gln?Leu?Pro
275 280 285
Val?Thr?Leu?Gln?Thr?Ile?Ala?Asp?Asp?Lys?Arg?Phe?Val?Leu?Val?Asp
290 295 300
Ile?Thr?Thr?Thr?Ser?Lys?Lys?Thr?Val?Lys?Val?Ala?Ile?Asp?Val?Thr
305 310 315 320
Asp?Val?Tyr?Val?Val?Gly?Tyr?Gln?Asp?Lys?Trp?Asp?Gly?Lys?Asp?Arg
325 330 335
Ala?Val?Phe?Leu?Asp?Lys?Val?Pro?Thr?Val?Ala?Thr?Ser?Lys?Leu?Phe
340 345 350
Pro?Gly?Val?Thr?Asn?Arg?Val?Thr?Leu?Thr?Phe?Asp?Gly?Ser?Tyr?Gln
355 360 365
Lys?Leu?Val?Asn?Ala?Ala?Lys?Ala?Asp?Arg?Lys?Ala?Leu?Glu?Leu?Gly
370 375 380
Val?Asn?Lys?Leu?Glu?Phe?Ser?Ile?Glu?Ala?Ile?His?Gly?Lys?Thr?Ile
385 390 395 400
Asn?Gly?Gln?Glu?Ala?Ala?Lys?Phe?Phe?Leu?Ile?Val?Ile?Gln?Met?Val
405 410 415
Ser?Glu?Ala?Ala?Arg?Phe?Lys?Tyr?Ile?Glu?Thr?Glu?Val?Val?Asp?Arg
420 425 430
Gly?Leu?Tyr?Gly?Ser?Phe?Lys?Pro?Asn?Phe?Lys?Val?Leu?Asn?Leu?Glu
435 440 445
Asn?Asn?Trp?Gly?Asp?Ile?Ser?Asp?Ala?Ile?His?Lys?Ser?Ser?Pro?Gln
450 455 460
Cys?Thr?Thr?Ile?Asn?Pro?Ala?Leu?Gln?Leu?Ile?Ser?Pro?Ser?Asn?Asp
465 470 475 480
Pro?Trp?Val?Val?Asn?Lys?Val?Ser?Gln?Ile?Ser?Pro?Asp?Met?Gly?Ile
485 490 495
Leu?Lys?Phe?Lys?Ser?Ser?Lys?Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala
500 505 510
Ala?Gly?Leu?Leu?Leu?Leu?Ala?Ala?Gln?Pro?Ala?Met?Ala?His?His?His
515 520 525
His?His?His?Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala
530 535 540
Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ser?Thr?Lys?Ser?Leu
545 550 555 560
Leu?His?Ser?Asn?Gly?Ile?Thr?Tyr?Leu?Tyr?Trp?Tyr?Gln?Gln?Lys?Pro
565 570 575
Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Gln?Met?Ser?Asn?Leu?Ala?Ser
580 585 590
Gly?Val?Pro?Ser?Arg?Phe?Ser?Ser?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr
595 600 605
Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys
610 615 620
Ala?Gln?Asn?Leu?Glu?Ile?Pro?Arg?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val
625 630 635 640
Glu?Leu?Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro
645 650 655
Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu
660 665 670
Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn
675 680 685
Ala?Leu?Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser
690 695 700
Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala
705 710 715 720
Asp?Tyr?Glu?Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly
725 730 735
Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys
740 745 750
<210>17
<211>1618
<212>DNA
＜213〉artificial sequence
<220>
<223>VB5-845
<400>17
gaattcctgc?aggtctatgg?aacgataaat?gcccatgaaa?attctatttc?aaggagacag 60
tcataatgaa?atacctattg?cctacggcag?ccgctggatt?gttattactc?gctgcccaac 120
cagcgatggc?ggaagtacag?ctggttcagt?ccggcccggg?tcttgttcaa?ccgggtggtt 180
ccgttcgtat?ctcttgcgct?gcttctggtt?acacgttcac?caactacggc?atgaactggg 240
tcaaacaggc?tccgggtaaa?ggcctggaat?ggatgggctg?gatcaacacc?tacaccggtg 300
aatccaccta?cgctgactcc?ttcaaaggtc?gcttcacttt?ctccctcgac?acaagtgcta 360
gtgctgcata?cctccaaatc?aactcgctgc?gtgcagagga?tacagcagtc?tattactgcg 420
cccgtttcgc?tatcaaaggt?gactactggg?gtcaaggcac?gctgctgacc?gtttcctcgg 480
ctagcaccaa?aggcccatcg?gtcttccccc?tggcaccctc?ctccaagagc?acctctgggg 540
gcacagcggc?cctgggctgc?ctggtcaagg?actacttccc?cgaaccggtg?acggtgtcgt 600
ggaactcagg?cgccctgacc?agcggcgtgc?acaccttccc?ggctgtccta?cagtcctcag 660
gactctactc?cctcagcagc?gtggtgaccg?tgccctccag?cagcttgggc?acccagacct 720
acatctgcaa?cgtgaatcac?aagcccagca?acaccaaggt?ggacaagaaa?gttgagccca 780
aatcttgtta?gtgatctaga?gtcgacctgc?aggtctatgg?aacgataaat?gcccatgaaa 840
attctatttc?aaggagacag?tcataatgaa?atacctattg?cctacggcag?ccgctggatt 900
gttattactc?gctgcccaac?cagcgatggc?gcaccatcat?caccatcacg?atatccagat 960
gacccagtcc?ccgtcctccc?tgagtgcttc?tgttggtgac?cgtgttacca?tcacctgccg 1020
ttccaccaaa?tccctcctgc?actccaacgg?tatcacctac?ctttattggt?atcaacagaa 1080
accgggtaaa?gctccgaaac?ttctgatcta?ccagatgtcc?aacctggctt?ccggtgttcc 1140
gtctcgtttc?tccagttctg?gttctggtac?cgacttcacc?ctgaccatct?cttctctgca 1200
gccggaagac?ttcgctacct?actactgcgc?tcagaacctg?gaaatcccgc?gtaccttcgg 1260
tcagggtacc?aaagttgaac?ttaagcgcac?tgtggctgca?ccatctgtct?tcatcttccc 1320
gccatctgat?gagcagttga?aatctggaac?tgcctctgtt?gtgtgcctgc?tgaataactt 1380
ctatcccaga?gaggccaaag?tacagtggaa?ggtggataac?gccctccaat?cgggtaactc 1440
ccaggagagt?gtcacagagc?aggacagcaa?ggacagcacc?tacagcctca?gcagcaccct 1500
gacgctgagc?aaagcagact?acgagaaaca?caaagtctac?gcctgcgaag?tcacccatca 1560
gggcctgagc?tcgcccgtca?caaagagctt?caacagggga?gagtgttagt?agctcgag 1618
<210>18
<211>488
<212>PRT
＜213〉artificial sequence
<220>
<223>VB5-845
<400>18
Met?Lys?Tyr?Leu?Leu?pro?Thr?Ala?Ala?Ala?Gly?Leu?Leu?Leu?Leu?Ala
1 5 10 15
Ala?Gln?Pro?Ala?Met?Ala?Glu?Val?Gln?Leu?Val?Gln?Ser?Gly?pro?Gly
20 25 30
Leu?Val?Gln?Pro?Gly?Gly?Ser?Val?Arg?Ile?Ser?Cys?Ala?Ala?Ser?Gly
35 40 45
Tyr?Thr?Phe?Thr?Asn?Tyr?Gly?Met?Asn?Trp?Val?Lys?Gln?Ala?Pro?Gly
50 55 60
Lys?Gly?Leu?Glu?Trp?Met?Gly?Trp?Ile?Asn?Thr?Tyr?Thr?Gly?Glu?Ser
65 70 75 80
Thr?Tyr?Ala?Asp?Ser?Phe?Lys?Gly?Arg?Phe?Thr?Phe?Ser?Leu?Asp?Thr
85 90 95
Ser?Ala?Ser?Ala?Ala?Tyr?Leu?Gln?Ile?Asn?Ser?Leu?Arg?Ala?Glu?Asp
100 105 110
Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Phe?Ala?Ile?Lys?Gly?Asp?Tyr?Trp
115 120 125
Gly?Gln?Gly?Thr?Leu?Leu?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?Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala?Ala?Gly?Leu?Leu?Leu?Leu
245 250 255
Ala?Ala?Gln?Pro?Ala?Met?Ala?His?His?His?His?His?His?Asp?Ile?Gln
260 265 270
Met?Thr?Gln?Ser?pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly?Asp?Arg?Val
275 280 285
Thr?Ile?Thr?Cys?Arg?Ser?Thr?Lys?Ser?Leu?Leu?His?Ser?Asn?Gly?Ile
290 295 300
Thr?Tyr?Leu?Tyr?Trp?Tyr?Gln?Gln?Lys?pro?Gly?Lys?Ala?Pro?Lys?Leu
305 310 315 320
Leu?Ile?Tyr?Gln?Met?Ser?Asn?Leu?Ala?Ser?Gly?Val?Pro?Ser?Arg?Phe
325 330 335
Ser?Ser?Ser?Gly?Ser?Gly?Thr?Asp?phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu
340 345 350
Gln?Pro?Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Ala?Gln?Asn?Leu?Glu?Ile
355 360 365
Pro?Arg?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Leu?Lys?Arg?Thr?Val
370 375 380
Ala?Ala?Pro?Ser?Val?Phe?Ile?phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys
385 390 395 400
Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg
405 410 415
Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser?Gly?Asn
420 425 430
Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser?Thr?Tyr?Ser
435 440 445
Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr?Glu?Lys?His?Lys
450 455 460
Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser?Ser?Pro?Val?Thr
465 470 475 480
Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys
485
<210>19
<211>2404
<212>DNA
＜213〉artificial sequence
<220>
<223>VB6-845-CL-de-bouganin
<400>19
gaattcctgc?aggtctatgg?aacgataaat?gcccatgaaa?attctatttc?aaggagacag 60
tcataatgaa?atacctattg?cctacggcag?ccgctggatt?gttattactc?gctgcccaac 120
cagcgatggc?gcaccatcat?caccatcacg?aagtacagct?ggttcagtcc?ggcccgggtc 180
ttgttcaacc?gggtggttcc?gttcgtatct?cttgcgctgc?ttctggttac?acgttcacca 240
actacggcat?gaactgggtc?aaacaggctc?cgggtaaagg?cctggaatgg?atgggctgga 300
tcaacaccta?caccggtgaa?tccacctacg?ctgactcctt?caaaggtcgc?ttcactttct 360
ccctcgacac?aagtgctagt?gctgcatacc?tccaaatcaa?ctcgctgcgt?gcagaggata 420
cagcagtcta?ttactgcgcc?cgtttcgcta?tcaaaggtga?ctactggggt?caaggcacgc 480
tgctgaccgt?ttcctcggct?agcaccaaag?gcccatcggt?cttccccctg?gcaccctcct 540
ccaagagcac?ctctgggggc?acagcggccc?tgggctgcct?ggtcaaggac?tacttccccg 600
aaccggtgac?ggtgtcgtgg?aactcaggcg?ccctgaccag?cggcgtgcac?accttcccgg 660
ctgtcctaca?gtcctcagga?ctctactccc?tcagcagcgt?ggtgaccgtg?ccctccagca 720
gcttgggcac?ccagacctac?atctgcaacg?tgaatcacaa?gcccagcaac?accaaggtgg 780
acaagaaagt?tgagcccaaa?tcttgttagt?gatctagagt?cgacctgcag?gtctatggaa 840
cgataaatgc?ccatgaaaat?tctatttcaa?ggagacagtc?ataatgaaat?acctattgcc 900
tacggcagcc?gctggattgt?tattactcgc?tgcccaacca?gcgatggcgg?atatccagat 960
gacccagtcc?ccgtcctccc?tgagtgcttc?tgttggtgac?cgtgttacca?tcacctgccg 1020
ttccaccaaa?tccctcctgc?actccaacgg?tatcacctac?ctttattggt?atcaacagaa 1080
accgggtaaa?gctccgaaac?ttctgatcta?ccagatgtcc?aacctggctt?ccggtgttcc 1140
gtctcgtttc?tccagttctg?gttctggtac?cgacttcacc?ctgaccatct?cttctctgca 1200
gccggaagac?ttcgctacct?actactgcgc?tcagaacctg?gaaatcccgc?gtaccttcgg 1260
tcagggtacc?aaagttgaac?ttaagcgcac?tgtggctgca?ccatctgtct?tcatcttccc 1320
gccatctgat?gagcagttga?aatctggaac?tgcctctgtt?gtgtgcctgc?tgaataactt 1380
ctatcccaga?gaggccaaag?tacagtggaa?ggtggataac?gccctccaat?cgggtaactc 1440
ccaggagagt?gtcacagagc?aggacagcaa?ggacagcacc?tacagcctca?gcagcaccct 1500
gacgctgagc?aaagcagact?acgagaaaca?caaagtctac?gcctgcgaag?tcacccatca 1560
gggcctgagc?tcgcccgtca?caaagagctt?caacagggga?gagtgtacca?ggcacaggca 1620
gcccagaggc?tgggagcagc?tctacaacac?cgtgtcattt?aaccttggag?aagcttatga 1680
gtaccccact?tttatacaag?atttgcgcaa?tgaattggct?aagggcacac?cagtatgtca 1740
acttccagtg?acactacaaa?ccatagccga?tgacaagcga?tttgttctag?ttgatatcac 1800
tacgacctcg?aagaaaacag?ttaaggttgc?tatagatgtg?acagatgtgt?atgttgtggg 1860
ttatcaagac?aaatgggatg?gcaaagatcg?agctgttttc?cttgacaagg?ttcctactgt 1920
tgcaactagt?aaacttttcc?caggggtgac?taatcgtgta?acgttaacat?ttgatggcag 1980
ctatcagaaa?cttgtgaatg?ctgccaaagc?tgatagaaag?gctctcgaac?tgggggttaa 2040
caaattggaa?ttttccattg?aagcaatcca?tggtaaaacg?ataaatggtc?aagaggcagc 2100
caagttcttt?cttattgtca?tccaaatggt?ttcagaggca?gctcggttca?aatatattga 2160
gactgaggtg?gttgatagag?gattatatgg?atcattcaaa?cctaatttta?aagtattgaa 2220
cttggagaac?aattggggcg?acatctctga?tgccattcac?aaatcatccc?cacaatgtac 2280
cactattaat?ccggcacttc?agttgataag?cccctcaaat?gacccatggg?ttgtaaataa 2340
agtgagtcaa?attagtcccg?atatgggtat?ccttaagttt?aaaagctcca?aatagtagct 2400
<210>20
<211>750
<212>PRT
＜213〉artificial sequence
<220>
<223>VB6-845-CL-de-bouganin
<400>20
Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala?Ala?Gly?Leu?Leu?Leu?Leu?Ala
1 5 10 15
Ala?Gln?Pro?Ala?Met?Ala?His?His?His?His?His?His?Glu?Val?Gln?Leu
20 25 30
Val?Gln?Ser?Gly?pro?Gly?Leu?Val?Gln?Pro?Gly?Gly?Ser?Val?Arg?Ile
35 40 45
Ser?Cys?Ala?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Asn?Tyr?Gly?Met?Asn?Trp
50 55 60
Val?Lys?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Met?Gly?Trp?Ile?Asn
65 70 75 80
Thr?Tyr?Thr?Gly?Glu?Ser?Thr?Tyr?Ala?Asp?Ser?Phe?Lys?Gly?Arg?Phe
85 90 95
Thr?Phe?Ser?Leu?Asp?Thr?Ser?Ala?Ser?Ala?Ala?Tyr?Leu?Gln?Ile?Asn
100 105 110
Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Phe?Ala
115 120 125
Ile?Lys?Gly?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Leu?Thr?Val?Ser?Ser
130 135 140
Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val Phe?Pro?Leu?Ala?pro?Ser?Ser?Lys
145 150 155 160
Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr
165 170 175
Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser
180 185 190
Gly?Val?His?Thr?Phe?pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser
195 200 205
Leu?Ser?Ser?Val?Val?Thr?Val?pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr
210 215 220
Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?pro?Ser?Asn?Thr?Lys?Val?Asp?Lys
225 230 235 240
Lys?Val?Glu?pro?Lys?Ser?Cys?Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala
245 250 255
Ala?Gly?Leu?Leu?Leu?Leu?Ala?Ala?Gln?Pro?Ala?Met?Ala?Asp?Ile?Gln
260 265 270
Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala?Ser?Val?Gly?Asp?Arg?Val
275 280 285
Thr?Ile?Thr?Cys?Arg?Ser?Thr?Lys?Ser?Leu?Leu?His?Ser?Asn?Gly?Ile
290 295 300
Thr?Tyr?Leu?Tyr?Trp?Tyr?Gln?Gln?Lys?Pro?Gly?Lys?Ala?Pro?Lys?Leu
305 310 315 320
Leu?Ile?Tyr?Gln?Met?Ser?Asn?Leu?Ala?Ser?Gly?Val?pro?Ser?Arg?phe
325 330 335
Ser?Ser?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Ser?Leu
340 345 350
Gln?Pro?Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys?Ala?Gln?Asn?Leu?Glu?Ile
355 360 365
Pro?Arg?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val?Glu?Leu?Lys?Arg?Thr?Val
370 375 380
Ala?Ala?pro?Ser?Val?Phe?Ile?Phe?Pro?pro?Ser?Asp?Glu?Gln?Leu?Lys
385 390 395 400
Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg
405 410 415
Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser?Gly?Asn
420 425 430
Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser?Thr?Tyr?Ser
435 440 445
Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr?Glu?Lys?His?Lys
450 455 460
Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser?Ser?Pro?Val?Thr
465 470 475 480
Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys?Thr?Arg?His?Arg?Gln?Pro?Arg?Gly
485 490 495
Trp?Glu?Gln?Leu?Tyr?Asn?Thr?Val?Ser?phe?Asn?Leu?Gly?Glu?Ala?Tyr
500 505 510
Glu?Tyr?Pro?Thr?Phe?Ile?Gln?Asp?Leu?Arg?Asn?Glu?Leu?Ala?Lys?Gly
515 520 525
Thr?Pro?Val?Cys?Gln?Leu?Pro?Val?Thr?Leu?Gln?Thr?Ile?Ala?Asp?Asp
530 535 540
Lys?Arg?phe?Val?Leu?Val?Asp?Ile?Thr?Thr?Thr?Ser?Lys?Lys?Thr?Val
545 550 555 560
Lys?Val?Ala?Ile?Asp?Val?Thr?Asp?Val?Tyr?Val?Val?Gly?Tyr?Gln?Asp
565 570 575
Lys?Trp?Asp?Gly?Lys?Asp?Arg?Ala?Val Phe?Leu?Asp?Lys?Val Pro?Thr
580 585 590
Val?Ala?Thr?Ser?Lys?Leu?phe?Pro?Gly?Val?Thr?Asn?Arg?Val?Thr?Leu
595 600 605
Thr?Phe?Asp?Gly?Ser?Tyr?Gln?Lys?Leu?Val?Asn?Ala?Ala?Lys?Ala?Asp
610 615 620
Arg?Lys?Ala?Leu?Glu?Leu?Gly?Val?Asn?Lys?Leu?Glu?Phe?Ser?Ile?Glu
625 630 635 640
Ala?Ile?His?Gly?Lys?Thr?Ile?Asn?Gly?Gln?Glu?Ala?Ala?Lys?Phe?phe
645 650 655
Leu?Ile?Val?Ile?Gln?Met?Val?Ser?Glu?Ala?Ala?Arg?Phe?Lys?Tyr?Ile
660 665 670
Glu?Thr?Glu?Val?Val?Asp?Arg?Gly?Leu?Tyr?Gly?Ser?Phe?Lys?Pro?Asn
675 680 685
Phe?Lys?Val?Leu?Asn?Leu?Glu?Asn?Asn?Trp?Gly?Asp?Ile?Ser?Asp?Ala
690 695 700
Ile?His?Lys?Ser?Ser?Pro?Gln?Cys?Thr?Thr?Ile?Asn?Pro?Ala?Leu?Gln
705 710 715 720
Leu?Ile?Ser?pro?Ser?Asn?Asp?Pro?Trp?Val?Val?Asn?Lys?Val?Ser?Gln
725 730 735
Ile?Ser?Pro?Asp?Met?Gly?Ile?Leu?Lys?phe?Lys?Ser?Ser?Lys
740 745 750
<210>21
<211>2404
<212>DNA
＜213〉artificial sequence
<220>
<223>VB6-845-NVH-de-bouganin
<400>21
gaattcctgc?aggtctatgg?aacgataaat?gcccatgaaa?attctatttc?aaggagacag 60
tcataatgaa?atacctattg?cctacggcag?ccgctggatt?gttattactc?gctgcccaac 120
cagcgatggc?gtacaacacc?gtgtcattta?accttggaga?agcttatgag?taccccactt 180
ttatacaaga?tttgcgcaat?gaattggcta?agggcacacc?agtatgtcaa?cttccagtga 240
cactacaaac?catagccgat?gacaagcgat?ttgttctagt?tgatatcact?acgacctcga 300
agaaaacagt?taaggttgct?atagatgtga?cagatgtgta?tgttgtgggt?tatcaagaca 360
aatgggatgg?caaagatcga?gctgttttcc?ttgacaaggt?tcctactgtt?gcaactagta 420
aacttttccc?aggggtgact?aatcgtgtaa?cgttaacatt?tgatggcagc?tatcagaaac 480
ttgtgaatgc?tgccaaagct?gatagaaagg?ctctcgaact?gggggttaac?aaattggaat 540
tttccattga?agcaatccat?ggtaaaacga?taaatggtca?agaggcagcc?aagttctttc 600
ttattgtcat?ccaaatggtt?tcagaggcag?ctcggttcaa?atatattgag?actgaggtgg 660
ttgatagagg?attatatgga?tcattcaaac?ctaattttaa?agtattgaac?ttggagaaca 720
attggggcga?catctctgat?gccattcaca?aatcatcccc?acaatgtacc?actattaatc 780
cggcacttca?gttgataagc?ccctcaaatg?acccatgggt?tgtaaataaa?gtgagtcaaa 840
ttagtcccga?tatgggtatc?cttaagttta?aaagctccaa?aaccaggcac?aggcagccca 900
gaggctggga?gcagctcgaa?gtacagctgg?ttcagtccgg?cccgggtctt?gttcaaccgg 960
gtggttccgt?tcgtatctct?tgcgctgctt?ctggttacac?gttcaccaac?tacggcatga 1020
actgggtcaa?acaggctccg?ggtaaaggcc?tggaatggat?gggctggatc?aacacctaca 1080
ccggtgaatc?cacctacgct?gactccttca?aaggtcgctt?cactttctcc?ctcgacacaa 1140
gtgctagtgc?tgcatacctc?caaatcaact?cgctgcgtgc?agaggataca?gcagtctatt 1200
actgcgcccg?tttcgctatc?aaaggtgact?actggggtca?aggcacgctg?ctgaccgttt 1260
cctcggctag?caccaaaggc?ccatcggtct?tccccctggc?accctcctcc?aagagcacct 1320
ctgggggcac?agcggccctg?ggctgcctgg?tcaaggacta?cttccccgaa?ccggtgacgg 1380
tgtcgtggaa?ctcaggcgcc?ctgaccagcg?gcgtgcacac?cttcccggct?gtcctacagt 1440
cctcaggact?ctactccctc?agcagcgtgg?tgaccgtgcc?ctccagcagc?ttgggcaccc 1500
agacctacat?ctgcaacgtg?aatcacaagc?ccagcaacac?caaggtggac?aagaaagttg 1560
agcccaaatc?ttgttagtga?tctagagtcg?acctgcaggt?ctatggaacg?ataaatgccc 1620
atgaaaattc?tatttcaagg?agacagtcat?aatgaaatac?ctattgccta?cggcagccgc 1680
tggattgtta?ttactcgctg?cccaaccagc?gatggcgcac?catcatcacc?atcacgatat 1740
ccagatgacc?cagtccccgt?cctccctgag?tgcttctgtt?ggtgaccgtg?ttaccatcac 1800
ctgccgttcc?accaaatccc?tcctgcactc?caacggtatc?acctaccttt?attggtatca 1860
acagaaaccg?ggtaaagctc?cgaaacttct?gatctaccag?atgtccaacc?tggcttccgg 1920
tgttccgtct?cgtttctcca?gttctggttc?tggtaccgac?ttcaccctga?ccatctcttc 1980
tctgcagccg?gaagacttcg?ctacctacta?ctgcgctcag?aacctggaaa?tcccgcgtac 2040
cttcggtcag?ggtaccaaag?ttgaacttaa?gcgcactgtg?gctgcaccat?ctgtcttcat 2100
cttcccgcca?tctgatgagc?agttgaaatc?tggaactgcc?tctgttgtgt?gcctgctgaa 2160
taacttctat?cccagagagg?ccaaagtaca?gtggaaggtg?gataacgccc?tccaatcggg 2220
taactcccag?gagagtgtca?cagagcagga?cagcaaggac?agcacctaca?gcctcagcag 2280
caccctgacg?ctgagcaaag?cagactacga?gaaacacaaa?gtctacgcct?gcgaagtcac 2340
ccatcagggc?ctgagctcgc?ccgtcacaaa?gagcttcaac?aggggagagt?gttagtagct 2400
<210>22
<211>750
<212>PRT
＜213〉artificial sequence
<220>
<223>VB6-845-NVH-de-bouganin
<400>22
Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala?Ala?Gly?Leu?Leu?Leu?Leu?Ala
1 5 10 15
Ala?Gln?Pro?Ala?Met?Ala?Tyr?Asn?Thr?Val?Ser?Phe?Asn?Leu?Gly?Glu
20 25 30
Ala?Tyr?Glu?Tyr?Pro?Thr?Phe?Ile?Gln?Asp?Leu?Arg?Asn?Glu?Leu?Ala
35 40 45
Lys?Gly?Thr?Pro?Val?Cys?Gln?Leu?Pro?Val?Thr?Leu?Gln?Thr?Ile?Ala
50 55 60
Asp?Asp?Lys?Arg?Phe?Val?Leu?Val?Asp?Ile?Thr?Thr?Thr?Ser?Lys?Lys
65 70 75 80
Thr?Val?Lys?Val?Ala?Ile?Asp?Val?Thr?Asp?Val?Tyr?Val?Val?Gly?Tyr
85 90 95
Gln?Asp?Lys?Trp?Asp?Gly?Lys?Asp?Arg?Ala?Val?phe?Leu?Asp?Lys?Val
100 105 110
Pro?Thr?Val?Ala?Thr?Ser?Lys?Leu?Phe?Pro?Gly?Val?Thr?Asn?Arg?Val
115 120 125
Thr?Leu?Thr?Phe?Asp?Gly?Ser?Tyr?Gln?Lys?Leu?Val?Asn?Ala?Ala?Lys
130 135 140
Ala?Asp?Arg?Lys?Ala?Leu?Glu?Leu?Gly?Val?Asn?Lys?Leu?Glu?Phe?Ser
145 150 155 160
Ile?Glu?Ala?Ile?His?Gly?Lys?Thr?Ile?Asn?Gly?Gln?Glu?Ala?Ala?Lys
165 170 175
Phe?phe?Leu?Ile?Val?Ile?Gln?Met?Val?Ser?Glu?Ala?Ala?Arg?Phe?Lys
180 185 190
Tyr?Ile?Glu?Thr?Glu?Val?Val?Asp?Arg?Gly?Leu?Tyr?Gly?Ser?Phe?Lys
195 200 205
Pro?Asn?Phe?Lys?Val?Leu?Asn?Leu?Glu?Asn?Asn?Trp?Gly?Asp?Ile?Ser
210 215 220
Asp?Ala?Ile?His?Lys?Ser?Ser?Pro?Gln?Cys?Thr?Thr?Ile?Asn?Pro?Ala
225 230 235 240
Leu?Gln?Leu?Ile?Ser?Pro?Ser?Asn?Asp?pro?Trp?Val?Val?Asn?Lys?Val
245 250 255
Ser?Gln?Ile?Ser?pro?Asp?Met?Gly?Ile?Leu?Lys?Phe?Lys?Ser?Ser?Lys
260 265 270
Thr?Arg?His?Arg?Gln?Pro?Arg?Gly?Trp?Glu?Gln?Leu?Glu?Val?Gln?Leu
275 280 285
Val?Gln?Ser?Gly?Pro?Gly?Leu?Val?Gln?Pro?Gly?Gly?Ser?Val?Arg?Ile
290 295 300
Ser?Cys?Ala?Ala?Ser?Gly?Tyr?Thr?Phe?Thr?Asn?Tyr?Gly?Met?Asn?Trp
305 310 315 320
Val?Lys?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Met?Gly?Trp?Ile?Asn
325 330 335
Thr?Tyr?Thr?Gly?Glu?Ser?Thr?Tyr?Ala?Asp?Ser?phe?Lys?Gly?Arg?Phe
340 345 350
Thr?Phe?Ser?Leu?Asp?Thr?Ser?Ala?Ser?Ala?Ala?Tyr?Leu?Gln?Ile?Asn
355 360 365
Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Phe?Ala
370 375 380
Ile?Lys?Gly?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Leu?Thr?Val?Ser?Ser
385 390 395 400
Ala?Ser?Thr?Lys?Gly?pro?Ser?Val?phe?pro?Leu?Ala?Pro?Ser?Ser?Lys
405 410 415
Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr
420 425 430
Phe?pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser
435 440 445
Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser
450 455 460
Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr
465 470 475 480
Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys
485 490 495
Lys?Val?Glu?Pro?Lys?Ser?Cys?Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala
500 505 510
Ala?Gly?Leu?Leu?Leu?Leu?Ala?Ala?Gln?Pro?Ala?Met?Ala?His?His?His
515 520 525
His?His?His?Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala
530 535 540
Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ser?Thr?Lys?Ser?Leu
545 550 555 560
Leu?His?Ser?Asn?Gly?Ile?Thr?Tyr?Leu?Tyr?Trp?Tyr?Gln?Gln?Lys?Pro
565 570 575
Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Gln?Met?Ser?Asn?Leu?Ala?Ser
580 585 590
Gly?Val?Pro?Ser?Arg?Phe?Ser?Ser?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr
595 600 605
Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys
610 615 620
Ala?Gln?Asn?Leu?Glu?Ile?Pro?Arg?Thr?phe?Gly?Gln?Gly?Thr?Lys?Val
625 630 635 640
Glu?Leu?Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?phe?Ile?Phe?Pro?Pro
645 650 655
Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu
660 665 670
Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn
675 680 685
Ala?Leu?Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser
690 695 700
Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala
705 710 715 720
Asp?Tyr?Glu?Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly
725 730 735
Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys
740 745 750
<210>23
<211>2404
<212>DNA
＜213〉artificial sequence
<220>
<223>VB6-845-NVL-de-bouganin
<400>23
gaattcctgc?aggtctatgg?aacgataaat?gcccatgaaa?attctatttc?aaggagacag 60
tcataatgaa?atacctattg?cctacggcag?ccgctggatt?gttattactc?gctgcccaac 120
cagcgatggc?gcaccatcat?caccatcacg?aagtacagct?ggttcagtcc?ggcccgggtc 180
ttgttcaacc?gggtggttcc?gttcgtatct?cttgcgctgc?ttctggttac?acgttcacca 240
actacggcat?gaactgggtc?aaacaggctc?cgggtaaagg?cctggaatgg?atgggctgga 300
tcaacaccta?caccggtgaa?tccacctacg?ctgactcctt?caaaggtcgc?ttcactttct 360
ccctcgacac?aagtgctagt?gctgcatacc?tccaaatcaa?ctcgctgcgt?gcagaggata 420
cagcagtcta?ttactgcgcc?cgtttcgcta?tcaaaggtga?ctactggggt?caaggcacgc 480
tgctgaccgt?ttcctcggct?agcaccaaag?gcccatcggt?cttccccctg?gcaccctcct 540
ccaagagcac?ctctgggggc?acagcggccc?tgggctgcct?ggtcaaggac?tacttccccg 600
aaccggtgac?ggtgtcgtgg?aactcaggcg?ccctgaccag?cggcgtgcac?accttcccgg 660
ctgtcctaca?gtcctcagga?ctctactccc?tcagcagcgt?ggtgaccgtg?ccctccagca 720
gcttgggcac?ccagacctac?atctgcaacg?tgaatcacaa?gcccagcaac?accaaggtgg 780
acaagaaagt?tgagcccaaa?tcttgttagt?gatctagagt?cgacctgcag?gtctatggaa 840
cgataaatgc?ccatgaaaat?tctatttcaa?ggagacagtc?ataatgaaat?acctattgcc 900
tacggcagcc?gctggattgt?tattactcgc?tgcccaacca?gcgatggcgt?acaacaccgt 960
gtcatttaac?cttggagaag?cttatgagta?ccccactttt?atacaagatt?tgcgcaatga 1020
attggctaag?ggcacaccag?tatgtcaact?tccagtgaca?ctacaaacca?tagccgatga 1080
caagcgattt?gttctagttg?atatcactac?gacctcgaag?aaaacagtta?aggttgctat 1140
agatgtgaca?gatgtgtatg?ttgtgggtta?tcaagacaaa?tgggatggca?aagatcgagc 1200
tgttttcctt?gacaaggttc?ctactgttgc?aactagtaaa?cttttcccag?gggtgactaa 1260
tcgtgtaacg?ttaacatttg?atggcagcta?tcagaaactt?gtgaatgctg?ccaaagctga 1320
tagaaaggct?ctcgaactgg?gggttaacaa?attggaattt?tccattgaag?caatccatgg 1380
taaaacgata?aatggtcaag?aggcagccaa?gttctttctt?attgtcatcc?aaatggtttc 1440
agaggcagct?cggttcaaat?atattgagac?tgaggtggtt?gatagaggat?tatatggatc 1500
attcaaacct?aattttaaag?tattgaactt?ggagaacaat?tggggcgaca?tctctgatgc 1560
cattcacaaa?tcatccccac?aatgtaccac?tattaatccg?gcacttcagt?tgataagccc 1620
ctcaaatgac?ccatgggttg?taaataaagt?gagtcaaatt?agtcccgata?tgggtatcct 1680
taagtttaaa?agctccaaaa?ccaggcacag?gcagcccaga?ggctgggagc?agctcgatat 1740
ccagatgacc?cagtccccgt?cctccctgag?tgcttctgtt?ggtgaccgtg?ttaccatcac 1800
ctgccgttcc?accaaatccc?tcctgcactc?caacggtatc?acctaccttt?attggtatca 1860
acagaaaccg?ggtaaagctc?cgaaacttct?gatctaccag?atgtccaacc?tggcttccgg 1920
tgttccgtct?cgtttctcca?gttctggttc?tggtaccgac?ttcaccctga?ccatctcttc 1980
tctgcagccg?gaagacttcg?ctacctacta?ctgcgctcag?aacctggaaa?tcccgcgtac 2040
cttcggtcag?ggtaccaaag?ttgaacttaa?gcgcactgtg?gctgcaccat?ctgtcttcat 2100
cttcccgcca?tctgatgagc?agttgaaatc?tggaactgcc?tctgttgtgt?gcctgctgaa 2160
taacttctat?cccagagagg?ccaaagtaca?gtggaaggtg?gataacgccc?tccaatcggg 2220
taactcccag?gagagtgtca?cagagcagga?cagcaaggac?agcacctaca?gcctcagcag 2280
caccctgacg?ctgagcaaag?cagactacga?gaaacacaaa?gtctacgcct?gcgaagtcac 2340
ccatcagggc?ctgagctcgc?ccgtcacaaa?gagcttcaac?aggggagagt?gttagtagct 2400
<210>24
<211>750
<212>PRT
＜213〉artificial sequence
<220>
<223>VB6-845-NVL-de-bouganin
<400>24
Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala?Ala?Gly?Leu?Leu?Leu?Leu?Ala
1 5 10 15
Ala?Gln?pro?Ala?Met?Ala?His?His?His?His?His?His?Glu?Val?Gln?Leu
20 25 30
Val?Gln?Ser?Gly?Pro?Gly?Leu?Val?Gln?Pro?Gly?Gly?Ser?Val?Arg?Ile
35 40 45
Ser?Cys?Ala?Ala?Ser?Gly?Tyr?Thr?phe?Thr?Asn?Tyr?Gly?Met?Asn?Trp
50 55 60
Val?Lys?Gln?Ala?Pro?Gly?Lys?Gly?Leu?Glu?Trp?Met?Gly?Trp?Ile?Asn
65 70 75 80
Thr?Tyr?Thr?Gly?Glu?Ser?Thr?Tyr?Ala?Asp?Ser?Phe?Lys?Gly?Arg?Phe
85 90 95
Thr?Phe?Ser?Leu?Asp?Thr?Ser?Ala?Ser?Ala?Ala?Tyr?Leu?Gln?Ile?Asn
100 105 110
Ser?Leu?Arg?Ala?Glu?Asp?Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Phe?Ala
115 120 125
Ile?Lys?Gly?Asp?Tyr?Trp?Gly?Gln?Gly?Thr?Leu?Leu?Thr?Val?Ser?Ser
130 135 140
Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?Phe?Pro?Leu?Ala?Pro?Ser?Ser?Lys
145 150 155 160
Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val?Lys?Asp?Tyr
165 170 175
Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala?Leu?Thr?Ser
180 185 190
Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly?Leu?Tyr?Ser
195 200 205
Leu?Ser?Ser?Val?Val?Thr?Val?Pro?Ser?Ser?Ser?Leu?Gly?Thr?Gln?Thr
210 215 220
Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys?Val?Asp?Lys
225 230 235 240
Lys?Val?Glu?Pro?Lys?ser?Cys?Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala
245 250 255
Ala?Gly?Leu?Leu?Leu?Leu?Ala?Ala?Gln?pro?Ala?Met?Ala?Tyr?Asn?Thr
260 265 270
Val?Ser?Phe?Asn?Leu?Gly?Glu?Ala?Tyr?Glu?Tyr?Pro?Thr?phe?Ile?Gln
275 280 285
Asp?Leu?Arg?Asn?Glu?Leu?Ala?Lys?Gly?Thr?Pro?Val?Cys?Gln?Leu?Pro
290 295 300
Val?Thr?Leu?Gln?Thr?Ile?Ala?Asp?Asp?Lys?Arg?Phe?Val?Leu?Val?Asp
305 310 315 320
Ile?Thr?Thr?Thr?Ser?Lys?Lys?Thr?Val?Lys?Val?Ala?Ile?Asp?Val?Thr
325 330 335
Asp?Val?Tyr?Val?Val?Gly?Tyr?Gln?Asp?Lys?Trp?Asp?Gly?Lys?Asp?Arg
340 345 350
Ala?Val?Phe?Leu?Asp?Lys?Val?Pro?Thr?Val?Ala?Thr?Ser?Lys?Leu?Phe
355 360 365
Pro?Gly?Val?Thr?Asn?Arg?Val?Thr?Leu?Thr?Phe?Asp?Gly?Ser?Tyr?Gln
370 375 380
Lys?Leu?Val?Asn?Ala?Ala?Lys?Ala?Asp?Arg?Lys?Ala?Leu?Glu?Leu?Gly
385 390 395 400
Val?Asn?Lys?Leu?Glu?Phe?Ser?Ile?Glu?Ala?Ile?His?Gly?Lys?Thr?Ile
405 410 415
Asn?Gly?Gln?Glu?Ala?Ala?Lys?Phe?Phe?Leu?Ile?Val?Ile?Gln?Met?Val
420 425 430
Ser?Glu?Ala?Ala?Arg?phe?Lys?Tyr?Ile?Glu?Thr?Glu?Val?Val?Asp?Arg
435 440 445
Gly?Leu?Tyr?Gly?Ser?Phe?Lys?Pro?Asn?Phe?Lys?Val?Leu?Asn?Leu?Glu
450 455 460
Asn?Asn?Trp?Gly?Asp?Ile?Ser?Asp?Ala?Ile?His?Lys?Ser?Ser?Pro?Gln
465 470 475 480
Cys?Thr?Thr?Ile?Asn?Pro?Ala?Leu?Gln?Leu?Ile?Ser?Pro?Ser?Asn?Asp
485 490 495
Pro?Trp?Val?Val?Asn?Lys?Val?Ser?Gln?Ile?Ser?Pro?Asp?Met?Gly?Ile
500 505 510
Leu?Lys?Phe?Lys?Ser?Ser?Lys?Thr?Arg?His?Arg?Gln?Pro?Arg?Gly?Trp
515 520 525
Glu?Gln?Leu?Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser?Ala
530 535 540
Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ser?Thr?Lys?Ser?Leu
545 550 555 560
Leu?His?Ser?Asn?Gly?Ile?Thr?Tyr?Leu?Tyr?Trp?Tyr?Gln?Gln?Lys?Pro
565 570 575
Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Gln?Met?Ser?Asn?Leu?Ala?Ser
580 585 590
Gly?Val?Pro?Ser?Arg?Phe?Ser?Ser?Ser?Gly?Ser?Gly?Thr?Asp?Phe?Thr
595 600 605
Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?Phe?Ala?Thr?Tyr?Tyr?Cys
610 615 620
Ala?Gln?Asn?Leu?Glu?Ile?Pro?Arg?Thr?Phe?Gly?Gln?Gly?Thr?Lys?Val
625 630 635 640
Glu?Leu?Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro
645 650 655
Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu
660 665 670
Asn?Asn?Phe?Tyr?Pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn
675 680 685
Ala?Leu?Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser
690 695 700
Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala
705 710 715 720
Asp?Tyr?Glu?Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly
725 730 735
Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser?Phe?Asn?Arg?Gly?Glu?Cys
740 745 750
<210>25
<211>2407
<212>DNA
＜213〉artificial sequence
<220>
＜223〉VB6-845-sets toxalbumin in vain
<400>25
gaattcctgc?aggtctatgg?aacgataaat?gcccatgaaa?attctatttc?aaggagacag 60
tcataatgaa?atacctattg?cctacggcag?ccgctggatt?gttattactc?gctgcccaac 120
cagcgatggc?ggaagtacag?ctggttcagt?ccggcccggg?tcttgttcaa?ccgggtggtt 180
ccgttcgtat?ctcttgcgct?gcttctggtt?acacgttcac?caactacggc?atgaactggg 240
tcaaacaggc?tccgggtaaa?ggcctggaat?ggatgggctg?gatcaacacc?tacaccggtg 300
aatccaccta?cgctgactcc?ttcaaaggtc?gcttcacttt?ctccctcgac?acaagtgcta 360
gtgctgcata?cctccaaatc?aactcgctgc?gtgcagagga?tacagcagtc?tattactgcg 420
cccgtttcgc?tatcaaaggt?gactactggg?gtcaaggcac?gctgctgacc?gtttcctcgg 480
ctagcaccaa?aggcccatcg?gtcttccccc?tggcaccctc?ctccaagagc?acctctgggg 540
gcacagcggc?cctgggctgc?ctggtcaagg?actacttccc?cgaaccggtg?acggtgtcgt 600
ggaactcagg?cgccctgacc?agcggcgtgc?acaccttccc?ggctgtccta?cagtcctcag 660
gactctactc?cctcagcagc?gtggtgaccg?tgccctccag?cagcttgggc?acccagacct 720
acatctgcaa?cgtgaatcac?aagcccagca?acaccaaggt?ggacaagaaa?gttgagccca 780
aatcttgtac?caggcacagg?cagcccagag?gctgggagca?gctcggcctg?gacaccgtga 840
gctttagcac?taaaggtgcc?acttatatta?cctacgtgaa?tttcttgaat?gagctacgag 900
ttaaattgaa?acccgaaggt?aacagccatg?gaatcccatt?gctgcgcaaa?aaatgtgatg 960
atcctggaaa?gtgtttcgtt?ttggtagcgc?tttcaaatga?caatggacag?ttggcggaaa 1020
tagctataga?tgttacaagt?gtttatgtgg?tgggctatca?agtaagaaac?agatcttact 1080
tctttaaaga?tgctccagat?gctgcttacg?aaggcctctt?caaaaacaca?attaaaacaa 1140
gacttcattt?tggcggcagc?tatccctcgc?tggaaggtga?gaaggcatat?agagagacaa 1200
cagacttggg?cattgaacca?ttaaggattg?gcatcaagaa?acttgatgaa?aatgcgatag 1260
acaattataa?accaacggag?atagctagtt?ctctattggt?tgttattcaa?atggtgtctg 1320
aagcagctcg?attcaccttt?attgagaacc?aaattagaaa?taactttcaa?cagagaatcc 1380
gcccgacgaa?taatacaatc?agccttgaga?ataaatgggg?taaactctcg?ttccagatcc 1440
ggacatcagg?tgcaaatgga?atgttttcgg?aggcagttga?attggaacgt?gcaaatggca 1500
aaaaatacta?tgtcaccgca?gttgatcaag?taaaacccaa?aatagcactc?ttgaagttcg 1560
tcgataaaga?tcctaaatag?tgatctagag?tcgacctgca?ggtctatgga?acgataaatg 1620
cccatgaaaa?ttctatttca?aggagacagt?cataatgaaa?tacctattgc?ctacggcagc 1680
cgctggattg?ttattactcg?ctgcccaacc?agcgatggcg?caccatcatc?accatcacga 1740
tatccagatg?acccagtccc?cgtcctccct?gagtgcttct?gttggtgacc?gtgttaccat 1800
cacctgccgt?tccaccaaat?ccctcctgca?ctccaacggt?atcacctacc?tttattggta 1860
tcaacagaaa?ccgggtaaag?ctccgaaact?tctgatctac?cagatgtcca?acctggcttc 1920
cggtgttccg?tctcgtttct?ccagttctgg?ttctggtacc?gacttcaccc?tgaccatctc 1980
ttctctgcag?ccggaagact?tcgctaccta?ctactgcgct?cagaacctgg?aaatcccgcg 2040
taccttcggt?cagggtacca?aagttgaact?taagcgcact?gtggctgcac?catctgtctt 2100
catcttcccg?ccatctgatg?agcagttgaa?atctggaact?gcctctgttg?tgtgcctgct 2160
gaataacttc?tatcccagag?aggccaaagt?acagtggaag?gtggataacg?ccctccaatc 2220
gggtaactcc?caggagagtg?tcacagagca?ggacagcaag?gacagcacct?acagcctcag 2280
cagcaccctg?acgctgagca?aagcagacta?cgagaaacac?aaagtctacg?cctgcgaagt 2340
cacccatcag?ggcctgagct?cgcccgtcac?aaagagcttc?aacaggggag?agtgttagta 2400
gctcgag 2407
<210>26
<211>751
<212>PRT
＜213〉artificial sequence
<220>
＜223〉VB6-845-sets toxalbumin in vain
<400>26
Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala?Ala?Gly?Leu?Leu?Leu?Leu?Ala
1 5 10 15
Ala?Gln?Pro?Ala?Met?Ala?Glu?Val?Gln?Leu?Val?Gln?Ser?Gly?Pro?Gly
20 25 30
Leu?Val?Gln?pro?Gly?Gly?Ser?Val?Arg?Ile?Ser?Cys?Ala?Ala?Ser?Gly
35 40 45
Tyr?Thr?Phe?Thr?Asn?Tyr?Gly?Met?Asn?Trp?Val?Lys?Gln?Ala?Pro?Gly
50 55 60
Lys?Gly?Leu?Glu?Trp?Met?Gly?Trp?Ile?Asn?Thr?Tyr?Thr?Gly?Glu?Ser
65 70 75 80
Thr?Tyr?Ala?Asp?Ser?Phe?Lys?Gly?Arg?Phe?Thr?phe?Ser?Leu?Asp?Thr
85 90 95
Ser?Ala?Ser?Ala?Ala?Tyr?Leu?Gln?Ile?Asn?Ser?Leu?Arg?Ala?Glu?Asp
100 105 110
Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Phe?Ala?Ile?Lys?Gly?Asp?Tyr?Trp
115 120 125
Gly?Gln?Gly?Thr?Leu?Leu?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?Thr?Arg?His?Arg?Gln?Pro?Arg?Gly?Trp?Glu?Gln?Leu?Gly?Leu?Asp
245 250 255
Thr?Val?Ser?Phe?Ser?Thr?Lys?Gly?Ala?Thr?Tyr?Ile?Thr?Tyr?Val?Asn
260 265 270
Phe?Leu?Asn?Glu?Leu?Arg?Val?Lys?Leu?Lys?Pro?Glu?Gly?Asn?Ser?His
275 280 285
Gly?Ile?Pro?Leu?Leu?Arg?Lys?Lys?Cys?Asp?Asp?Pro?Gly?Lys?Cys?Phe
290 295 300
Val?Leu?Val?Ala?Leu?Ser?Asn?Asp?Asn?Gly?Gln?Leu?Ala?Glu?Ile?Ala
305 310 315 320
Ile?Asp?Val?Thr?Ser?Val?Tyr?Val?Val?Gly?Tyr?Gln?Val?Arg?Asn?Arg
325 330 335
Ser?Tyr?Phe?Phe?Lys?Asp?Ala?Pro?Asp?Ala?Ala?Tyr?Glu?Gly?Leu?Phe
340 345 350
Lys?Asn?Thr?Ile?Lys?Thr?Arg?Leu?His?Phe?Gly?Gly?Ser?Tyr?Pro?Ser
355 360 365
Leu?Glu?Gly?Glu?Lys?Ala?Tyr?Arg?Glu?Thr?Thr?Asp?Leu?Gly?Ile?Glu
370 375 380
Pro?Leu?Arg?Ile?Gly?Ile?Lys?Lys?Leu?Asp?Glu?Asn?Ala?Ile?Asp?Asn
385 390 395 400
Tyr?Lys?Pro?Thr?Glu?Ile?Ala?Ser?Ser?Leu?Leu?Val?Val?Ile?Gln?Met
405 410 415
Val?Ser?Glu?Ala?Ala?Arg?Phe?Thr?Phe?Ile?Glu?Asn?Gln?Ile?Arg?Asn
420 425 430
Asn?Phe?Gln?Gln?Arg?Ile?Arg?Pro?Thr?Asn?Asn?Thr?Ile?Ser?Leu?Glu
435 440 445
Asn?Lys?Trp?Gly?Lys?Leu?Ser?Phe?Gln?Ile?Arg?Thr?Ser?Gly?Ala?Asn
450 455 460
Gly?Met?phe?Ser?Glu?Ala?Val?Glu?Leu?Glu?Arg?Ala?Asn?Gly?Lys?Lys
465 470 475 480
Tyr?Tyr?Val?Thr?Ala?Val?Asp?Gln?Val?Lys?Pro?Lys?Ile?Ala?Leu?Leu
485 490 495
Lys?Phe?Val?Asp?Lys?Asp?Pro?Lys?Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala
500 505 510
Ala?Ala?Gly?Leu?Leu?Leu?Leu?Ala?Ala?Gln?Pro?Ala?Met?Ala?His?His
515 520 525
His?His?His?His?Asp?Ile?Gln?Met?Thr?Gln?Ser?Pro?Ser?Ser?Leu?Ser
530 535 540
Ala?Ser?Val?Gly?Asp?Arg?Val?Thr?Ile?Thr?Cys?Arg?Ser?Thr?Lys?Ser
545 550 555 560
Leu?Leu?His?Ser?Asn?Gly?Ile?Thr?Tyr?Leu?Tyr?Trp?Tyr?Gln?Gln?Lys
565 570 575
Pro?Gly?Lys?Ala?Pro?Lys?Leu?Leu?Ile?Tyr?Gln?Met?Ser?Asn?Leu?Ala
580 585 590
Ser?Gly?Val?Pro?Ser?Arg?Phe?Ser?Ser?Ser?Gly?Ser?Gly?Thr?Asp?Phe
595 600 605
Thr?Leu?Thr?Ile?Ser?Ser?Leu?Gln?Pro?Glu?Asp?Phe?Ala?Thr?Tyr?Tyr
610 615 620
Cys?Ala?Gln?Asn?Leu?Glu?Ile?Pro?Arg?Thr?Phe?Gly?Gln?Gly?Thr?Lys
625 630 635 640
Val?Glu?Leu?Lys?Arg?Thr?Val?Ala?Ala?Pro?Ser?Val?phe?Ile?Phe?Pro
645 650 655
Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser?Gly?Thr?Ala?Ser?Val?Val?Cys?Leu
660 665 670
Leu?Asn?Asn?Phe?Tyr?pro?Arg?Glu?Ala?Lys?Val?Gln?Trp?Lys?Val?Asp
675 680 685
Asn?Ala?Leu?Gln?Ser?Gly?Asn?Ser?Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp
690 695 700
Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu?Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys
705 710 715 720
Ala?Asp?Tyr?Glu?Lys?His?Lys?Val?Tyr?Ala?Cys?Glu?Val?Thr?His?Gln
725 730 735
Gly?Leu?Ser?Ser?Pro?Val?Thr?Lys?Ser?phe?Asn?Arg?Gly?Glu?Cys
740 745 750
<210>27
<211>2431
<212>DNA
＜213〉artificial sequence
<220>
<223>VB6-011
<400>27
gaattcctgc?aggtctatgg?aacgataaat?gcccatgaaa?attctatttc?aaggagacag 60
tcataatgaa?atacctattg?cctacggcag?ccgctggatt?gttattactc?gctgcccaac 120
cagcgatggc?gcaggtgcag?ctggtggagt?ctgggggagg?cgtggtccag?cctgggaggt 180
ccctgagact?ctcctgtgca?gcctctggat?tccccttcag?aagctttgct?atgcactggg 240
tccgccaggc?tctaggcaag?gggctggagt?gggtggcagt?tatatcatat?gatggaagca 300
ctaaatacta?cgcagactcc?gtgaagggcc?gattcaccat?ctccagagac?acttccaaga 360
acacggtgta?tctaaaaatg?aacagcctga?gaactgagga?cacggctgtc?tattactgtg 420
cgagagatca?gagcctgttg?ggtgactatg?accactacta?cggtttggac?gtctggggca 480
aagggaccac?ggtcacggtc?tcttcagcta?gcaccaaagg?cccatcggtc?ttccccctgg 540
caccctcctc?caagagcacc?tctgggggca?cagcggccct?gggctgcctg?gtcaaggact 600
acttccccga?accggtgacg?gtgtcgtgga?actcaggcgc?cctgaccagc?ggcgtgcaca 660
ccttcccggc?tgtcctacag?tcctcaggac?tctactccct?cagcagcgtg?gtgaccgtgc 720
cctccagcag?cttgggcacc?cagacctaca?tctgcaacgt?gaatcacaag?cccagcaaca 780
ccaaggtgga?caagaaagtt?gagcccaaat?cttgtaccag?gcacaggcag?cccagaggct 840
gggagcagct?ctacaacacc?gtgtcattta?accttggaga?agcttatgag?taccccactt 900
ttatacaaga?tttgcgcaat?gaattggcta?agggcacacc?agtatgtcaa?cttccagtga 960
cactacaaac?catagccgat?gacaagcgat?ttgttctagt?tgatatcact?acgacctcga 1020
agaaaacagt?taaggttgct?atagatgtga?cagatgtgta?tgttgtgggt?tatcaagaca 1080
aatgggatgg?caaagatcga?gctgttttcc?ttgacaaggt?tcctactgtt?gcaactagta 1140
aacttttccc?aggggtgact?aatcgtgtaa?cgttaacatt?tgatggcagc?tatcagaaac 1200
ttgtgaatgc?tgccaaagct?gatagaaagg?ctctcgaact?gggggttaac?aaattggaat 1260
tttccattga?agcaatccat?ggtaaaacga?taaatggtca?agaggcagcc?aagttctttc 1320
ttattgtcat?ccaaatggtt?tcagaggcag?ctcggttcaa?atatattgag?actgaggtgg 1380
ttgatagagg?attatatgga?tcattcaaac?ctaattttaa?agtattgaac?ttggagaaca 1440
attggggcga?catctctgat?gccattcaca?aatcatcccc?acaatgtacc?actattaatc 1500
cggcacttca?gttgataagc?ccctcaaatg?acccatgggt?tgtaaataaa?gtgagtcaaa 1560
ttagtcccga?tatgggtatc?cttaagttta?aaagctccaa?atagtgatct?agagtcgacc 1620
tgcaggtcta?tggaacgata?aatgcccatg?aaaattctat?ttcaaggaga?cagtcataat 1680
gaaataccta?ttgcctacgg?cagccgctgg?attgttatta?ctcgctgccc?aaccagcgat 1740
ggcgcatcac?catcaccatc?acgatatcgt?gttgacgcag?tctccaggca?ccctgtcttt 1800
gtctccaggg?gaaagagcca?ccctctcctg?cagggccagt?cagagtgtta?gtagcagcta 1860
cttagcctgg?taccagcaga?aacctggcca?ggctcccagg?ctcctcatct?atggtgcatc 1920
caccagggcc?actggcatgc?cagacaggtt?cagtggcagt?gggtccggga?cagacttcac 1980
tctcaccatc?agtagactgg?agcctgaaga?ttttgcagtg?tattactgtc?agcagtatgg 2040
tagctcacct?cagacacctc?agatcacttt?cggcggaggg?accaaggtgg?agatcaaacg 2100
aactgtggct?gcaccatctg?tcttcatctt?cccgccatct?gatgagcagt?tgaaatctgg 2160
aactgcctct?gttgtgtgcc?tgctgaataa?cttctatccc?agagaggcca?aagtacagtg 2220
gaaggtggat?aacgccctcc?aatcgggtaa?ctcccaggag?agtgtcacag?agcaggacag 2280
caaggacagc?acctacagcc?tcagcagcac?cctgacgctg?agcaaagcag?actacgagaa 2340
acacaaagtc?tacgcctgcg?aagtcaccca?tcagggcctg?agctcgcccg?tcacaaagag 2400
cttcaacagg?ggagagtgtt?agtgactcga?g 2431
<210>28
<211>759
<212>PRT
＜213〉artificial sequence
<220>
<223>VB6-011
<400>28
Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala?Ala?Gly?Leu?Leu?Leu?Leu?Ala
1 5 10 15
Ala?Gln?Pro?Ala?Met?Ala?Gln?Val?Gln?Leu?Val?Glu?Ser?Gly?Gly?Gly
20 25 30
Val?Val?Gln?Pro?Gly?Arg?Ser?Leu?Arg?Leu?Ser?Cys?Ala?Ala?Ser?Gly
35 40 45
Phe?Pro?Phe?Arg?Ser?Phe?Ala?Met?His?Trp?Val?Arg?Gln?Ala?Leu?Gly
50 55 60
Lys?Gly?Leu?Glu?Trp?Val?Ala?Val?Ile?Ser?Tyr?Asp?Gly?Ser?Thr?Lys
65 70 75 80
Tyr?Tyr?Ala?Asp?Ser?Val?Lys?Gly?Arg?Phe?Thr?Ile?Ser?Arg?Asp?Thr
85 90 95
Ser?Lys?Asn?Thr?Val?Tyr?Leu?Lys?Met?Asn?Ser?Leu?Arg?Thr?Glu?Asp
100 105 110
Thr?Ala?Val?Tyr?Tyr?Cys?Ala?Arg?Asp?Gln?Ser?Leu?Leu?Gly?Asp?Tyr
115 120 125
Asp?His?Tyr?Tyr?Gly?Leu?Asp?Val?Trp?Gly?Lys?Gly?Thr?Thr?Val?Thr
130 135 140
Val?Ser?Ser?Ala?Ser?Thr?Lys?Gly?Pro?Ser?Val?phe?Pro?Leu?Ala?Pro
145 150 155 160
Ser?Ser?Lys?Ser?Thr?Ser?Gly?Gly?Thr?Ala?Ala?Leu?Gly?Cys?Leu?Val
165 170 175
Lys?Asp?Tyr?Phe?Pro?Glu?Pro?Val?Thr?Val?Ser?Trp?Asn?Ser?Gly?Ala
180 185 190
Leu?Thr?Ser?Gly?Val?His?Thr?Phe?Pro?Ala?Val?Leu?Gln?Ser?Ser?Gly
195 200 205
Leu?Tyr?Ser?Leu?Ser?Ser?Val?Val?Thr?Val?pro?Ser?Ser?Ser?Leu?Gly
210 215 220
Thr?Gln?Thr?Tyr?Ile?Cys?Asn?Val?Asn?His?Lys?Pro?Ser?Asn?Thr?Lys
225 230 235 240
Val?Asp?Lys?Lys?Val?Glu?Pro?Lys?Ser?Cys?Thr?Arg?His?Arg?Gln?pro
245 250 255
Arg?Gly?Trp?Glu?Gln?Leu?Tyr?Asn?Thr?Val?Ser?Phe?Asn?Leu?Gly?Glu
260 265 270
Ala?Tyr?Glu?Tyr?Pro?Thr?Phe?Ile?Gln?Asp?Leu?Arg?Asn?Glu?Leu?Ala
275 280 285
Lys?Gly?Thr?Pro?Val?Cys?Gln?Leu?Pro?Val?Thr?Leu?Gln?Thr?Ile?Ala
290 295 300
Asp?Asp?Lys?Arg?Phe?Val?Leu?Val?Asp?Ile?Thr?Thr?Thr?Ser?Lys?Lys
305 310 315 320
Thr?Val?Lys?Val?Ala?Ile?Asp?Val?Thr?Asp?Val?Tyr?Val?Val?Gly?Tyr
325 330 335
Gln?Asp?Lys?Trp?Asp?Gly?Lys?Asp?Arg?Ala?Val?Phe?Leu?Asp?Lys?Val
340 345 350
Pro?Thr?Val?Ala?Thr?Ser?Lys?Leu?Phe?Pro?Gly?Val?Thr?Asn?Arg?Val
355 360 365
Thr?Leu?Thr?Phe?Asp?Gly?Ser?Tyr?Gln?Lys?Leu?Val?Asn?Ala?Ala?Lys
370 375 380
Ala?Asp?Arg?Lys?Ala?Leu?Glu?Leu?Gly?Val?Asn?Lys?Leu?Glu?Phe?Ser
385 390 395 400
Ile?Glu?Ala?Ile?His?Gly?Lys?Thr?Ile?Asn?Gly?Gln?Glu?Ala?Ala?Lys
405 410 415
Phe?Phe?Leu?Ile?Val?Ile?Gln?Met?Val?Ser?Glu?Ala?Ala?Arg?Phe?Lys
420 425 430
Tyr?Ile?Glu?Thr?Glu?Val?Val?Asp?Arg?Gly?Leu?Tyr?Gly?Ser?Phe?Lys
435 440 445
Pro?Asn?Phe?Lys?Val?Leu?Asn?Leu?Glu?Asn?Asn?Trp?Gly?Asp?Ile?Ser
450 455 460
Asp?Ala?Ile?His?Lys?Ser?Ser?Pro?Gln?Cys?Thr?Thr?Ile?Asn?Pro?Ala
465 470 475 480
Leu?Gln?Leu?Ile?Ser?pro?Ser?Asn?Asp?Pro?Trp?Val?Val?Asn?Lys?Val
485 490 495
Ser?Gln?Ile?Ser?Pro?Asp?Met?Gly?Ile?Leu?Lys?Phe?Lys?Ser?Ser?Lys
500 505 510
Met?Lys?Tyr?Leu?Leu?Pro?Thr?Ala?Ala?Ala?Gly?Leu?Leu?Leu?Leu?Ala
515 520 525
Ala?Gln?Pro?Ala?Met?Ala?His?His?His?His?His?His?Asp?Ile?Val?Leu
530 535 540
Thr?Gln?Ser?pro?Gly?Thr?Leu?Ser?Leu?Ser?Pro?Gly?Glu?Arg?Ala?Thr
545 550 555 560
Leu?Ser?Cys?Arg?Ala?Ser?Gln?Ser?Val?Ser?Ser?Ser?Tyr?Leu?Ala?Trp
565 570 575
Tyr?Gln?Gln?Lys?Pro?Gly?Gln?Ala?Pro?Arg?Leu?Leu?Ile?Tyr?Gly?Ala
580 585 590
Ser?Thr?Arg?Ala?Thr?Gly?Met?Pro?Asp?Arg?phe?Ser?Gly?Ser?Gly?Ser
595 600 605
Gly?Thr?Asp?Phe?Thr?Leu?Thr?Ile?Ser?Arg?Leu?Glu?pro?Glu?Asp?Phe
610 615 620
Ala?Val?Tyr?Tyr?Cys?Gln?Gln?Tyr?Gly?Ser?Ser?Pro?Gln?Thr?Pro?Gln
625 630 635 640
Ile?Thr?Phe?Gly?Gly?Gly?Thr?Lys?Val?Glu?Ile?Lys?Arg?Thr?Val?Ala
645 650 655
Ala?Pro?Ser?Val?Phe?Ile?Phe?Pro?Pro?Ser?Asp?Glu?Gln?Leu?Lys?Ser
660 665 670
Gly?Thr?Ala?Ser?Val?Val?Cys?Leu?Leu?Asn?Asn?Phe?Tyr?Pro?Arg?Glu
675 680 685
Ala?Lys?Val?Gln?Trp?Lys?Val?Asp?Asn?Ala?Leu?Gln?Ser?Gly?Asn?Ser
690 695 700
Gln?Glu?Ser?Val?Thr?Glu?Gln?Asp?Ser?Lys?Asp?Ser?Thr?Tyr?Ser?Leu
705 710 715 720
Ser?Ser?Thr?Leu?Thr?Leu?Ser?Lys?Ala?Asp?Tyr?Glu?Lys?His?Lys?Val
725 730 735
Tyr?Ala?Cys?Glu?Val?Thr?His?Gln?Gly?Leu?Ser?Ser?Pro?Val?Thr?Lys
740 745 750
Ser?Phe?Asn?Arg?Gly?Glu?Cys
755
<210>29
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>29
Ala?Lys?Ala?Asp?Arg?Lys?Ala?Leu?Glu?Leu?Gly?Val?Asn?Lys?Leu
1 5 10 15
<210>30
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>30
Leu?Gly?Val?Asn?Lys?Leu?Glu?Phe?Ser?Ile?Glu?Ala?Ile?His?Gly
1 5 10 15
<210>31
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>31
Asn?Gly?Gln?Glu?Ala?Ala?Lys?Phe?Phe?Leu?Ile?Val?Ile?Gln?Met
1 5 10 15
<210>32
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>32
Tyr?Asn?Thr?Val?Ser?phe?Asn?Leu?Gly?Glu?Ala?Tyr?Glu?Tyr?Pro
1 5 10 15
<210>33
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>33
Val?Ser?Phe?Asn?Leu?Gly?Glu?Ala?Tyr?Glu?Tyr?Pro?Thr?Phe?Ile
1 5 10 15
<210>34
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>34
Asn?Leu?Gly?Glu?Ala?Tyr?Glu?Tyr?Pro?Thr?Phe?Ile?Gln?Asp?Leu
1 5 10 15
<210>35
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>35
Glu?Ala?Tyr?Glu?Tyr?Pro?Thr?Phe?Ile?Gln?Asp?Leu?Arg?Asn?Glu
1 5 10 15
<210>36
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>36
Glu?Tyr?Pro?Thr?Phe?Ile?Gln?Asp?Leu?Arg?Asn?Glu?Leu?Ala?Lys
1 5 10 15
<210>37
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>37
Thr?phe?Ile?Gln?Asp?Leu?Arg?Asn?Glu?Leu?Ala?Lys?Gly?Thr?Pro
1 5 10 15
<210>38
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>38
Gln?Asp?Leu?Arg?Asn?Glu?Leu?Ala?Lys?Gly?Thr?Pro?Val?Cys?Gln
1 5 10 15
<210>39
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>39
Arg?Asn?Glu?Leu?Ala?Lys?Gly?Thr?Pro?Val?Cys?Gln?Leu?Pro?Val
1 5 10 15
<210>40
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>40
Leu?Ala?Lys?Gly?Thr?Pro?Val?Cys?Gln?Leu?Pro?Val?Thr?Leu?Gln
1 5 10 15
<210>41
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>41
Gly?Thr?Pro?Val?Cys?Gln?Leu?Pro?Val?Thr?Leu?Gln?Thr?Ile?Ala
1 5 10 15
<210>42
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>42
Val?Cys?Gln?Leu?Pro?Val?Thr?Leu?Gln?Thr?Ile?Ala?Asp?Asp?Lys
1 5 10 15
<210>43
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>43
Leu?pro?Val?Thr?Leu?Gln?Thr?Ile?Ala?Asp?Asp?Lys?Arg?Phe?Val
1 5 10 15
<210>44
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>44
Thr?Leu?Gln?Thr?Ile?Ala?Asp?Asp?Lys?Arg?Phe?Val?Leu?Val?Asp
1 5 10 15
<210>45
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>45
Thr?Ile?Ala?Asp?Asp?Lys?Arg?Phe?Val?Leu?Val?Asp?Ile?Thr?Thr
1 5 10 15
<210>46
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>46
Asp?Asp?Lys?Arg?phe?Val?Leu?Val?Asp?Ile?Thr?Thr?Thr?Ser?Lys
1 5 10 15
<210>47
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>47
Arg?Phe?Val?Leu?Val?Asp?Ile?Thr?Thr?Thr?Ser?Lys?Lys?Thr?Val
1 5 10 15
<210>48
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>48
Leu?Val?Asp?Ile?Thr?Thr?Thr?Ser?Lys?Lys?Thr?Val?Lys?Val?Ala
1 5 10 15
<210>49
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>49
Ile?Thr?Thr?Thr?Ser?Lys?Lys?Thr?Val?Lys?Val?Ala?Ile?Asp?Val
1 5 10 15
<210>50
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>50
Thr?Ser?Lys?Lys?Thr?Val?Lys?Val?Ala?Ile?Asp?Val?Thr?Asp?Val
1 5 10 15
<210>51
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>51
Lys?Thr?Val?Lys?Val?Ala?Ile?Asp?Val?Thr?Asp?Val?Tyr?Val?Val
1 5 10 15
<210>52
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>52
Lys?Val?Ala?Ile?Asp?Val?Thr?Asp?Val?Tyr?Val?Val?Gly?Tyr?Gln
1 5 10 15
<210>53
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>53
Ile?Asp?Val?Thr?Asp?Val?Tyr?Val?Val?Gly?Tyr?Gln?Asp?Lys?Trp
1 5 10 15
<210>54
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>54
Thr?Asp?Val?Tyr?Val?Val?Gly?Tyr?Gln?Asp?Lys?Trp?Asp?Gly?Lys
1 5 10 15
<210>55
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>55
Tyr?Val?Val?Gly?Tyr?Gln?Asp?Lys?Trp?Asp?Gly?Lys?Asp?Arg?Ala
1 5 10 15
<210>56
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>56
Gly?Tyr?Gln?Asp?Lys?Trp?Asp?Gly?Lys?Asp?Arg?Ala?Val?Phe?Leu
1 5 10 15
<210>57
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>57
Asp?Lys?Trp?Asp?Gly?Lys?Asp?Arg?Ala?Val?Phe?Leu?Asp?Lys?Val
1 5 10 15
<210>58
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>58
Asp?Gly?Lys?Asp?Arg?Ala?Val?Phe?Leu?Asp?Lys?Val?Pro?Thr?Val
1 5 10 15
<210>59
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>59
Asp?Arg?Ala?Val?Phe?Leu?Asp?Lys?Val?Pro?Thr?Val?Ala?Thr?Ser
1 5 10 15
<210>60
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>60
Val?Phe?Leu?Asp?Lys?Val?Pro?Thr?Val?Ala?Thr?Ser?Lys?Leu?Phe
1 5 10 15
<210>61
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>61
Asp?Lys?Val?Pro?Thr?Val?Ala?Thr?Ser?Lys?Leu?Phe?Pro?Gly?Val
1 5 10 15
<210>62
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>62
Pro?Thr?Val?Ala?Thr?Ser?Lys?Leu?Phe?Pro?Gly?Val?Thr?Asn?Arg
1 5 10 15
<210>63
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>63
Ala?Thr?Ser?Lys?Leu?phe?Pro?Gly?Val?Thr?Asn?Arg?Val?Thr?Leu
1 5 10 15
<210>64
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>64
Lys?Leu?phe?pro?Gly?Val?Thr?Asn?Arg?Val?Thr?Leu?Thr?Phe?Asp
1 5 10 15
<210>65
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>65
Pro?Gly?Val?Thr?Asn?Arg?Val?Thr?Leu?Thr?Phe?Asp?Gly?Ser?Tyr
1 5 10 15
<210>66
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>66
Thr?Asn?Arg?Val?Thr?Leu?Thr?Phe?Asp?Gly?Ser?Tyr?Gln?Lys?Leu
1 5 10 15
<210>67
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>67
Val?Thr?Leu?Thr?Phe?Asp?Gly?Ser?Tyr?Gln?Lys?Leu?Val?Asn?Ala
1 5 10 15
<210>68
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>68
Thr?Phe?Asp?Gly?Ser?Tyr?Gln?Lys?Leu?Val?Asn?Ala?Ala?Lys?Val
1 5 10 15
<210>69
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>69
Gly?Ser?Tyr?Gln?Lys?Leu?Val?Asn?Ala?Ala?Lys?Val?Asp?Arg?Lys
1 5 10 15
<210>70
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>70
Gln?Lys?Leu?Val?Asn?Ala?Ala?Lys?Val?Asp?Arg?Lys?Asp?Leu?Glu
1 5 10 15
<210>71
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>71
Val?Asn?Ala?Ala?Lys?Val?Asp?Arg?Lys?Asp?Leu?Glu?Leu?Gly?Val
1 5 10 15
<210>72
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>72
Ala?Lys?Val?Asp?Arg?Lys?Asp?Leu?Glu?Leu?Gly?Val?Tyr?Lys?Leu
1 5 10 15
<210>73
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>73
Asp?Arg?Lys?Asp?Leu?Glu?Leu?Gly?Val?Tyr?Lys?Leu?Glu?Phe?Ser
1 5 10 15
<210>74
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>74
Asp?Leu?Glu?Leu?Gly?Val?Tyr?Lys?Leu?Glu?phe?Ser?Ile?Glu?Ala
1 5 10 15
<210>75
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>75
Leu?Gly?Val?Tyr?Lys?Leu?Glu?phe?Ser?Ile?Glu?Ala?Ile?His?Gly
1 5 10 15
<210>76
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>76
Tyr?Lys?Leu?Glu?Phe?Ser?Ile?Glu?Ala?Ile?His?Gly?Lys?Thr?Ile
1 5 10 15
<210>77
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>77
Glu?Phe?Ser?Ile?Glu?Ala?Ile?His?Gly?Lys?Thr?Ile?Asn?Gly?Gln
1 5 10 15
<210>78
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>78
Ile?Glu?Ala?Ile?His?Gly?Lys?Thr?Ile?Asn?Gly?Gln?Glu?Ile?Ala
1 5 10 15
<210>79
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>79
Ile?His?Gly?Lys?Thr?Ile?Asn?Gly?Gln?Glu?Ile?Ala?Lys?Phe?Phe
1 5 10 15
<210>80
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>80
Lys?Thr?Ile?Asn?Gly?Gln?Glu?Ile?Ala?Lys?Phe?Phe?Leu?Ile?Val
1 5 10 15
<210>81
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>81
Asn?Gly?Gln?Glu?Ile?Ala?Lys?phe?Phe?Leu?Ile?Val?Ile?Gln?Met
1 5 10 15
<210>82
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>82
Glu?Ile?Ala?Lys?Phe?Phe?Leu?Ile?Val?Ile?Gln?Met?Val?Ser?Glu
1 5 10 15
<210>83
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>83
Lys?Phe?Phe?Leu?Ile?Val?Ile?Gln?Met?Val?Ser?Glu?Ala?Ala?Arg
1 5 10 15
<210>84
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>84
Leu?Ile?Val?Ile?Gln?Met?Val?Ser?Glu?Ala?Ala?Arg?phe?Lys?Tyr
1 5 10 15
<210>85
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>85
Ile?Gln?Met?Val?Ser?Glu?Ala?Ala?Arg?Phe?Lys?Tyr?Ile?Glu?Thr
1 5 10 15
<210>86
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>86
Val?Ser?Glu?Ala?Ala?Arg?Phe?Lys?Tyr?Ile?Glu?Thr?Glu?Val?Val
1 5 10 15
<210>87
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>87
Ala?Ala?Arg?Phe?Lys?Tyr?Ile?Glu?Thr?Glu?Val?Val?Asp?Arg?Gly
1 5 10 15
<210>88
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>88
Phe?Lys?Tyr?Ile?Glu?Thr?Glu?Val?Val?Asp?Arg?Gly?Leu?Tyr?Gly
1 5 10 15
<210>89
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>89
Ile?Glu?Thr?Glu?Val?Val?Asp?Arg?Gly?Leu?Tyr?Gly?Ser?phe?Lys
1 5 10 15
<210>90
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>90
Glu?Val?Val?Asp?Arg?Gly?Leu?Tyr?Gly?Ser?phe?Lys?Pro?Asn?Phe
1 5 10 15
<210>91
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>91
Asp?Arg?Gly?Leu?Tyr?Gly?Ser?Phe?Lys?Pro?Asn?Phe?Lys?Val?Leu
1 5 10 15
<210>92
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>92
Leu?Tyr?Gly?Ser?Phe?Lys?Pro?Asn?Phe?Lys?Val?Leu?Asn?Leu?Glu
1 5 10 15
<210>93
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>93
Ser?Phe?Lys?Pro?Asn?Phe?Lys?Val?Leu?Asn?Leu?Glu?Asn?Asn?Trp
1 5 10 15
<210>94
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>94
Pro?Asn?Phe?Lys?Val?Leu?Asn?Leu?Glu?Asn?Asn?Trp?Gly?Asp?Ile
1 5 10 15
<210>95
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>95
Lys?Val?Leu?Asn?Leu?Glu?Asn?Asn?Trp?Gly?Asp?Ile?Ser?Asp?Ala
1 5 10 15
<210>96
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>96
Asn?Leu?Glu?Asn?Asn?Trp?Gly?Asp?Ile?Ser?Asp?Ala?Ile?His?Lys
1 5 10 15
<210>97
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>97
Asn?Asn?Trp?Gly?Asp?Ile?Ser?Asp?Ala?Ile?His?Lys?Ser?Ser?Pro
1 5 10 15
<210>98
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>98
Gly?Asp?Ile?Ser?Asp?Ala?Ile?His?Lys?Ser?Ser?Pro?Gln?Cys?Thr
1 5 10 15
<210>99
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>99
Ser?Asp?Ala?Ile?His?Lys?Ser?Ser?Pro?Gln?Cys?Thr?Thr?Ile?Asn
1 5 10 15
<210>100
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>100
Ile?His?Lys?Ser?Ser?Pro?Gln?Cys?Thr?Thr?Ile?Asn?Pro?Ala?Leu
1 5 10 15
<210>101
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>101
Ser?Ser?Pro?Gln?Cys?Thr?Thr?Ile?Asn?Pro?Ala?Leu?Gln?Leu?Ile
1 5 10 15
<210>102
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>102
Gln?Cys?Thr?Thr?Ile?Asn?pro?Ala?Leu?Gln?Leu?Ile?Ser?Pro?Ser
1 5 10 15
<210>103
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>103
Thr?Ile?Asn?Pro?Ala?Leu?Gln?Leu?Ile?Ser?Pro?Ser?Asn?Asp?Pro
1 5 10 15
<210>104
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>104
Pro?Ala?Leu?Gln?Leu?Ile?Ser?Pro?Ser?Asn?Asp?Pro?Trp?Val?Val
1 5 10 15
<210>105
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>105
Gln?Leu?Ile?Ser?Pro?Ser?Asn?Asp?Pro?Trp?Val?Val?Asn?Lys?Val
1 5 10 15
<210>106
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>106
Ser?Pro?Ser?Asn?Asp?Pro?Trp?Val?Val?Asn?Lys?Val?Ser?Gln?Ile
1 5 10 15
<210>107
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>107
Asn?Asp?Pro?Trp?Val?Val?Asn?Lys?Val?Ser?Gln?Ile?Ser?Pro?Asp
1 5 10 15
<210>108
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>108
Trp?Val?Val?Asn?Lys?Val?Ser?Gln?Ile?Ser?pro?Asp?Met?Gly?Ile
1 5 10 15
<210>109
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>109
Asn?Lys?Val?Ser?Gln?Ile?Ser?Pro?Asp?Met?Gly?Ile?Leu?Lys?Phe
1 5 10 15
<210>110
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>110
Ser?Gln?Ile?Ser?pro?Asp?Met?Gly?Ile?Leu?Lys?Phe?Lys?Ser?Ser
1 5 10 15
<210>111
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>111
Ser?Pro?Asp?Met?Gly?Ile?Leu?Lys?Phe?Lys?Ser?Ser?Lys?Leu?Thr
1 5 10 15
<210>112
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>112
Met?Gly?Ile?Leu?Lys?Phe?Lys?Ser?Ser?Lys?Leu?Thr?Gln?Phe?Ala
1 5 10 15
<210>113
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>113
Leu?Lys?Phe?Lys?Ser?Ser?Lys?Leu?Thr?Gln?Phe?Ala?Thr?Met?Ile
1 5 10 15
<210>114
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>114
Lys?Ser?Ser?Lys?Leu?Thr?Gln?Phe?Ala?Thr?Met?Ile?Arg?Ser?Ala
1 5 10 15
<210>115
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>115
Lys?Leu?Thr?Gln?Phe?Ala?Thr?Met?Ile?Arg?Ser?Ala?Ile?Val?Glu
1 5 10 15
<210>116
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>116
Gln?Phe?Ala?Thr?Met?Ile?Arg?Ser?Ala?Ile?Val?Glu?Asp?Leu?Asp
1 5 10 15
<210>117
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>117
Thr?Met?Ile?Arg?Ser?Ala?Ile?Val?Glu?Asp?Leu?Asp?Gly?Asp?Glu
1 5 10 15
<210>118
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>118
Arg?Ser?Ala?Ile?Val?Glu?Asp?Leu?Asp?Gly?Asp?Glu?Leu?Glu?Ile
1 5 10 15
<210>119
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>119
Ile?Val?Glu?Asp?Leu?Asp?Gly?Asp?Glu?Leu?Glu?Ile?Leu?Glu?Pro
1 5 10 15
<210>120
<211>15
<212>PRT
<213>Bougainvillea?spectabilis
<400>120
Asp?Leu?Asp?Gly?Asp?Glu?Leu?Glu?Ile?Leu?Glu?Pro?Asn?Ile?Ala
1 5 10 15
<210>121
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer OL1032
<400>121
cattacaaac?gtctaccaag?ttt 23
<210>122
<211>24
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer OL1033
<400>122
ttacaaaagt?agataagtaa?tgtg 24
<210>123
<211>30
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer OL1322
<400>123
gatatacata?tgaaatacct?attgcctacg 30
<210>124
<211>36
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer OL1067
<400>124
tgacacagtg?ttgtacgctg?gttgggcagc?gagtaa 36
<210>125
<211>36
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer OL1068
<400>125
gctgcccaac?cagcgtacaa?cactgtgtca?tttaac 36
<210>126
<211>35
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer OL1323
<400>126
cgagtgcggc?cgctcaatgg?tgatggtgat?ggtgt 35
<210>127
<211>13
<212>PRT
＜213〉influenza virus
<400>127
Pro?Lys?Tyr?Val?Lys?Gln?Asn?Thr?Leu?Lys?Leu?Ala?Thr
1 5 10
<210>128
<211>15
<212>PRT
＜213〉chlamydozoan sp.
<400>128
Lys?Val?Val?Asp?Gln?Ile?Lys?Lys?Ile?Ser?Lys?Pro?Val?Gln?His
1 5 10 15
<210>129
<211>250
<212>PRT
<213>Bougainvillea?spectabilis
<400>129
Tyr?Asn?Thr?Val?Ser?Phe?Asn?Leu?Gly?Glu?Ala?Tyr?Glu?Tyr?Pro?Thr
1 5 10 15
Phe?Ile?Gln?Asp?Leu?Arg?Asn?Glu?Leu?Ala?Lys?Gly?Thr?Pro?Val?Cys
20 25 30
Gln?Leu?Pro?Val?Thr?Leu?Gln?Thr?Ile?Ala?Asp?Asp?Lys?Arg?Phe?Val
35 40 45
Leu?Val?Asp?Ile?Thr?Thr?Thr?Ser?Lys?Lys?Thr?Val?Lys?Val?Ala?Ile
50 55 60
Asp?Val?Thr?Asp?Val?Ala?Val?Val?Gly?Tyr?Gln?Asp?Lys?Trp?Asp?Gly
65 70 75 80
Lys?Asp?Arg?Ala?Val?Phe?Leu?Asp?Lys?Val?Pro?Thr?Val?Ala?Thr?Ser
85 90 95
Lys?Leu?Phe?Pro?Gly?Val?Thr?Asn?Arg?Val?Thr?Leu?Thr?Phe?Asp?Gly
100 105 110
Ser?Tyr?Gln?Lys?Leu?Val?Asn?Ala?Ala?Lys?Val?Asp?Arg?Lys?Asp?Leu
115 120 125
Glu?Leu?Gly?Val?Tyr?Lys?Leu?Glu?Phe?Ser?Ile?Glu?Ala?Ile?His?Gly
130 135 140
Lys?Thr?Ile?Asn?Gly?Gln?Glu?Ile?Ala?Lys?Phe?Phe?Leu?Ile?Val?Ile
145 150 155 160
Gln?Met?Val?Ser?Glu?Ala?Ala?Arg?Phe?Lys?Tyr?Ile?Glu?Thr?Glu?Val
165 170 175
Val?Asp?Arg?Gly?Leu?Tyr?Gly?Ser?Phe?Lys?Pro?Asn?phe?Lys?Val?Leu
180 185 190
Asn?Leu?Glu?Asn?Asn?Trp?Gly?Asp?Ile?Ser?Asp?Ala?Ile?His?Lys?Ser
195 200 205
Ser?Pro?Gln?Cys?Thr?Thr?Ile?Asn?Pro?Ala?Leu?Gln?Leu?Ile?Ser?Pro
210 215 220
Ser?Asn?Asp?Pro?Trp?Val?Val?Asn?Lys?Val?Ser?Gln?Ile?Ser?Pro?Asp
225 230 235 240
Met?Gly?Ile?Leu?Lys?phe?Lys?Ser?Ser?Lys
245 250
Claims (34)
1. modified Bouganin proteins, the wherein said Bouganin that modified has the activate immunity that weakens and replys tendency.
2. modify Bouganin according to the quilt of claim 1, wherein said bouganin has the tendency of the activation T-cell that weakens and is modified at one or more amino-acid residues place of T-cell epitope.
3. modify Bouganin according to the quilt of claim 2, wherein said bouganin is modified at the one or more amino-acid residues place that is selected from following T-cell epitope:
D) AKVDRKDLELGVYKL (epitope regions R1, SEQ ID NO:2),
E) LGVYKLEFSIEAIHG (epitope regions R2, SEQ ID NO:3); With
F) NGQEIAKFFLIVIQM (epitope regions R3, SEQ ID NO:4)).
4. modify Bouganin according to the quilt of claim 3, wherein said bouganin is at following X
1, X
2, X
3, X
4Or X
5In a place or many places modified:
A) AKX
1DRKX
2LX
3LGVX
4KL (epitope regions R1, SEQ ID NO:5)
B) LGVX
4KLEFSIEAIHG (epitope regions R2, SEQ ID NO:6); With
C) NGQEX
5AKFFLIVIQM (epitope regions R3, SEQ ID NO:7)
X wherein
1To X
5Can be any amino acid.
5. modify Bouganin according to the quilt of claim 4, wherein:
X
1Be T or A or Q;
X
2Be G or A;
X
3Be Q or G;
X
4Be N or D or T or A or R or Q or E or G or H or K or S; With
X
5Be Q or A (SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10).
6. modify Bouganin according to the quilt of claim 1, wherein the aminoacid sequence of modified Bouganin proteins comprises:
YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFV
LVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLDKVPTVAT
SKLFPGVTNRVTLTFDGSYQKLVNAAKX
1DRKX2LX
3LGVX
4KLEFSIEAIHGKTINGQEX
5AKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPWVVNKVSQISPDMGILKFKSSK(SEQ?IDNO：11)
X wherein
1To X
5Can be any amino acid.
7. modify Bouganin according to the quilt of claim 6, wherein:
X
1Be T or A or Q;
X
2Be G or A;
X
3Be Q or G;
X
4Be N or D or T or A or R or Q or E or G or H or K or S; With
X
5Be Q or A (SEQ ID NO:12).
8. modify Bouganin according to the quilt of claim 1, wherein modified Bouganin proteins comprises following sequence:
YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFV
LVDITTTSKKTVKVAIDVTDVYVVGYQDKWDGKDRAVFLDKVPTVAT
SKLFPGVTNRVTLTFDGSYQLVNAAKADRKALELGVNKLEFSIEAIH
GKTINGQEAAKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVL
NLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPWVVNKVSQISPD
MGILKFKSSK(SEQ?ID?NO：13)。
9. a cytotoxin comprises
(a) according to modified Bouganin proteins arbitrary in the claim 1～8;
(b) be attached to the target part of (a).
10. a cytotoxin comprises
(a) according to modified Bouganin proteins arbitrary in the claim 1～8;
What (b) be attached to (a) can be in conjunction with the part of cancer cells.
11. the cytotoxin of claim 10, part wherein are and cancer cells bonded antibody or antibody fragment.
12. the cytotoxin of claim 11, antibody wherein or antibody fragment combine with the Ep-CAM on cancer cells surface.
13. the cytotoxin of claim 12 is humanized antibody or the antibody fragment that combines and comprise the complementary determining region sequence that is derived from MOC-31 antibody with the extracellular domain of people Ep-CAM with Ep-CAM bonded antibody or antibody fragment wherein.
14. the cytotoxin of claim 12, the variable region that wherein is attached to the cancer binding partner of modified Bouganin proteins is 4D5MOCB.
15. the cytotoxin of claim 14 comprises the aminoacid sequence shown in the SEQ ID NO:16.
16. the cytotoxin of claim 11, antibody wherein or antibody fragment are in conjunction with the tumor associated antigen on cancer cells surface.
17. the cytotoxin of claim 16 comprises the aminoacid sequence shown in the SEQ ID NO:28.
18. according to the purposes of cytotoxin arbitrary among the claim 9～17 in preparation inhibition or tumoricidal medicine.
19. the purposes of claim 18, cancer cells wherein are selected from colorectal carcinoma, mammary cancer, ovarian cancer, carcinoma of the pancreas, head and neck cancer, bladder cancer, lung cancer, kidney, melanoma, gastrointestinal cancer, prostate cancer, minicell and nonsmall-cell lung cancer, sarcoma, neurospongioma and T-and B-cell lymphoma.
20. according to the purposes of cytotoxin arbitrary among the claim 9～17 in the medicine of preparation treatment cancer.
21. the purposes of claim 20, cancer cells wherein are selected from colorectal carcinoma, mammary cancer, ovarian cancer, carcinoma of the pancreas, head and neck cancer, bladder cancer, lung cancer, kidney, melanoma, gastrointestinal cancer, prostate cancer, minicell and nonsmall-cell lung cancer, sarcoma, neurospongioma and T-and B-cell lymphoma.
22. comprise pharmaceutical composition according to cytotoxin arbitrary among the claim 9～17 and pharmaceutical acceptable carrier, thinner or vehicle.
23. the method for the medicine of cancer animal is suffered from the preparation treatment, comprises
(a) the T-cell epitope of evaluation bouganin;
(b) the one or more amino-acid residues in the T-cell epitope are modified the quilt modification Bouganin that has the activation T-cell tendency that weakens with preparation;
(c) preparation has the cytotoxin that is attached to the cancer binding partner of being modified Bouganin; With
(d) this cytotoxin is suspended from pharmaceutical acceptable carrier, thinner or the vehicle.
24. the method for claim 23, cancer wherein are selected from colorectal carcinoma, mammary cancer, ovarian cancer, carcinoma of the pancreas, head and neck cancer, bladder cancer, lung cancer, kidney, melanoma, gastrointestinal cancer, prostate cancer, minicell and nonsmall-cell lung cancer, sarcoma, neurospongioma and T-and B-cell lymphoma.
25. coding is according to the nucleic acid molecule of being modified Bouganin arbitrary in the claim 1～8.
26. coding is according to arbitrary by cytotoxic nucleic acid molecule in the claim 9～17.
27. have the T-cell epitope peptide of following sequence:
(a) AKVDRKDLELGVYKL (epitope regions R1, SEQ ID NO:2),
(b) LGVYKLEFSIEAIHG (epitope regions R2, SEQ ID NO:3); Or
(c) NGQEIAKFFLIVIQM (epitope regions R3, SEQ ID NO:4).
28. the T-cell epitope peptide of claim 27, it have comprise following by modification sequence:
AKX
1DRKX
2LX
3LGVX
4K
X wherein
1, X
2, X
3, and X
4In at least one from non--modified by modification sequence, such as following:
X
1Be T or A or Q;
X
2Be G or A;
X
3Be Q or G; With
X
4Be N or D or T or A or R or Q or E or G or H or K or S (SEQ IDNO:8).
29. the T-cell epitope peptide of claim 27, it have comprise following by modification sequence:
LGVX
4KLEFSIEAIHG
X wherein
4Be N or D or T or A or R or Q or E or G or H or K or S (SEQID NO:9).
30. the T-cell epitope peptide of claim 27, it have comprise following by modification sequence:
NGQEX
5AKFFLIVIQM
X wherein
5Be Q or A (SEQ ID NO:10).
31. coding is according to the nucleic acid molecule of T-cell epitope peptide arbitrary among the claim 27～30.
32. a modified Bouganin proteins, its tyrosine of the 70th is modified.
33. according to the modified Bouganin proteins of claim 32, tyrosine is wherein replaced by L-Ala.
34. according to the modified Bouganin proteins of claim 33, it has following sequence:
YNTVSFNLGEAYEYPTFIQDLRNELAKGTPVCQLPVTLQTIADDKRFVLVDITTTSKKTVKVAIDVTDVAVVGYQDKWDGKDRAVFLDKVPTVATSKLFPGVTNRVTLTFDGSYQKLVNAAKVDRKDLELGVYKLEFSIEAIHGKTINGQEIAKFFLIVIQMVSEAARFKYIETEVVDRGLYGSFKPNFKVLNLENNWGDISDAIHKSSPQCTTINPALQLISPSNDPWVVNKVSQISPDMGILKFKSSK[SEQ?ID?NO：129]。
Applications Claiming Priority (5)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US55458004P | 2004-03-19 | 2004-03-19 | |
| US60/554,580 | 2004-03-19 | ||
| US63057104P | 2004-11-26 | 2004-11-26 | |
| US60/630,571 | 2004-11-26 | ||
| PCT/CA2005/000410 WO2005090579A1 (en) | 2004-03-19 | 2005-03-18 | Modified bouganin proteins, cytotoxins and methods and uses thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1954073A true CN1954073A (en) | 2007-04-25 |
| CN1954073B CN1954073B (en) | 2013-01-16 |
Family
ID=38059728
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
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| Country | Link |
|---|---|
| CN (1) | CN1954073B (en) |
| ZA (1) | ZA200608696B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101671397B (en) * | 2008-10-09 | 2012-09-05 | 四川大学 | Anti-EGFR single-chain antibody fusion Gelonin recombination immunotoxin, preparation method and usage thereof |
| WO2013053080A1 (en) * | 2011-10-12 | 2013-04-18 | 中国人民解放军军事医学科学院生物工程研究所 | Anti-tumour fusion protein and coding gene and use thereof |
| CN105764925A (en) * | 2013-10-02 | 2016-07-13 | 维文蒂亚生物公司 | Anti-EpCAM antibodies and methods of use |
| CN112996904A (en) * | 2018-09-07 | 2021-06-18 | 南特生物公司 | Targeted IL-12 therapy and methods for stimulating HANK and NK92MI cells |
| CN116875520A (en) * | 2023-07-12 | 2023-10-13 | 吉林农业大学 | Lactic acid bacteria expressing ribosome inactivating protein and application thereof in resisting rotavirus |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6680295B1 (en) * | 1994-09-22 | 2004-01-20 | The Administrators Of The Tulane Educational Fund | Method and pharmaceutical composition for prevention and treatment of brain damage |
| ATE352559T1 (en) * | 1998-12-08 | 2007-02-15 | Biovation Ltd | METHOD FOR REDUCING THE IMMUNOGENICITY OF PROTEINS |
-
2005
- 2005-03-18 CN CN2005800159002A patent/CN1954073B/en active Active
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2006
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101671397B (en) * | 2008-10-09 | 2012-09-05 | 四川大学 | Anti-EGFR single-chain antibody fusion Gelonin recombination immunotoxin, preparation method and usage thereof |
| WO2013053080A1 (en) * | 2011-10-12 | 2013-04-18 | 中国人民解放军军事医学科学院生物工程研究所 | Anti-tumour fusion protein and coding gene and use thereof |
| CN105764925A (en) * | 2013-10-02 | 2016-07-13 | 维文蒂亚生物公司 | Anti-EpCAM antibodies and methods of use |
| CN112996904A (en) * | 2018-09-07 | 2021-06-18 | 南特生物公司 | Targeted IL-12 therapy and methods for stimulating HANK and NK92MI cells |
| CN116875520A (en) * | 2023-07-12 | 2023-10-13 | 吉林农业大学 | Lactic acid bacteria expressing ribosome inactivating protein and application thereof in resisting rotavirus |
Also Published As
| Publication number | Publication date |
|---|---|
| ZA200608696B (en) | 2008-05-28 |
| CN1954073B (en) | 2013-01-16 |
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