CN1219882C - Method and combination for treating tumors based on ERBB-3 - Google Patents
Method and combination for treating tumors based on ERBB-3 Download PDFInfo
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- CN1219882C CN1219882C CNB02116259XA CN02116259A CN1219882C CN 1219882 C CN1219882 C CN 1219882C CN B02116259X A CNB02116259X A CN B02116259XA CN 02116259 A CN02116259 A CN 02116259A CN 1219882 C CN1219882 C CN 1219882C
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Abstract
The present invention relates to a composition for treating tumors of mammals and particularly human bodies and a method thereof. More specifically, the present invention provides a method for preventing, treating or delaying the tumors of the mammals by ErbB-3 protein, nucleic acid for coding the ErbB-3 protein, or the functional segments of the ErbB-3 protein and the nucleic acid. The present invention also provides separated nucleic acid for coding the ectodomain of the ErbB-3 protein, the functional segment of the separated nucleic acid, the basically purified ectodomain of the ErbB-3 protein, or the functional segment of the ectodomain, an antibody combined with one antigenic determinant position of the ectodomain of the ErbB-3 protein, or the functional segment of the antibody. The present invention further provides the ectodomain or the functional segment which comprises the ErbB-3 protein, the nucleic acid for coding the ErbB-3 protein, or a medical composition and/or a vaccine of the antibody combined with the ectodomain or the functional segments.
Description
Technical field
The present invention relates to be used for the treatment of composition and the method for Mammals, particularly human tumor.More particularly, be to the invention provides a kind of method of preventing, treat or delay mammal tumor with ErbB-3 albumen, the coding proteic nucleic acid of ErbB-3 or its function fragment.The present invention also provides the nucleic acid that separates the proteic ectodomain of coding ErbB-3, or its function fragment, basically the proteic ectodomain of the ErbB-3 of purifying, or its function fragment and can with an epitope bonded antibody of the proteic ectodomain of ErbB-3, or its function fragment.The present invention further provides the nucleic acid that comprises the proteic ectodomain of ErbB-3 or its function fragment or this extracellular protein of encoding and with the pharmaceutical composition of this extracellular protein bonded antibody or their function fragment or/and vaccine.
Background technology
Cancer is to cause human dead principal disease, and it is caused by physiological cell proliferation out of control.Physiological cell proliferation out of control has influenced the body normal physiological state, causes serious pathologic reaction, often causes death.Though the mankind are making great effort aspect cancer research and the treatment, cancer remains human main causes of death at present.The method that has multiple treatment cancer patient now comprises operation, radiation and chemotherapy.Because preceding two kinds of methods, i.e. operation can not be eliminated the intravital cancer cells of patient fully with radiotherapy, so the latter, and promptly chemotherapy combines or generally be used for individually the growth of control cancer cell with additive method.The anticancer compound that is used for patient normally target stops cancer cell multiplication or kills somatoblast.
When compound was poisonous to cancer cells, they also seriously influenced those normal somatoblasts essential for people's life usually.Therefore, cancer research main direction is exactly to seek the method that can specificity hinders cancer cells or kill cancer cell and do not influence normal cell propagation.Currently be starved of a kind of like this methods of treatment that is used for cancer patient.
ErbBs belongs to the receptor of first order protein tyrosine kinase.The cell signal of ErbB mediation plays key effect to fetal development and ripe organ dysfunction.On cell levels, the signal that ErbB is receptor-mediated cell proliferation, differentiation, migration and cellularstructure are organized again.The ErbB albumen that has four kinds of structural similitudies, i.e. ErbB-1, ErbB-2, ErbB-3 and ErbB-4.Urogastron (EGF) is can discern and in conjunction with one of several parts of last ErbB-1.ErbB-3 and ErbB-4 also can combine with several parts, comprise neuregulin-1 (NRG-1).The part of ErbB-2 is not also found so far.Yet ErbB-2 is as the composition of energy and ErbB-3, ErbB-4 or ErbB-1 formation heterodimer and plays a crucial role in neuregulin-1 (NRG-1) activated cell signaling.
Studies show that the animal body developmental defect that the ErbB-2 gene inactivation has caused and neuregulin-1 (NRG-1) gene inactivation is same in the body of assignment of genes gene mapping experiment.These two kinds of animals all show defective in cranial nerve ganglion and cardiac trabeculae growth.And the mouse of ErbB-3 or ErbB-4 gene inactivation has and the similar or eclipsed phenotype of mouse that has knocked out neuregulin-1 (NRG-1) or ErbB-2 gene.These data declarations ErbB-2, ErbB-3 and ErbB-4 all be in NRG-1 activated signal chains.
Except working in growth, people ErbB-2 gene increases in the broad variety human tumor of being everlasting and causes the ErbB-2 albumen overexpression of its coding.The oncogene point mutation that causes forming the ErbB-2 homodimer of discovering in early days of relevant ErbB-2 can cause significant phosphorylation conversely on the tyrosine residues of cell intracellular domain.Though do not find corresponding point mutation individuality in the human tumor of ErbB-2 gene overexpression, the incremental adjustments of ErbB-2 has caused the formation of homodimer, this has also increased the tyrosine residues phosphorylation of its cell intracellular domain.This process is a starting point that triggers the signal cascade amplification of cell transformation or growth by hypothesis, thereby has started the tumour generation.Yet exist and overthrow the evidence that the ErbB-2 homodimer has started tumorigenic hypothesis: i) some transform not influence by the ErbB-2 mutant pair cell that engineered method improves dimer and autophosphorylation degree.Ii) some combines with the ectodomain of ErbB-2 and imagines the growth effect that the antibody that promotes homodimer to form causes the tumour cell of ErbB-2 expression, and other then stop the growth of tumour cell.These data show that the homodimerization of ErbB-2 is not enough to produce short cancer effect of cell growth or cell transformation, and other conditions may relate to specific dimer direction or conformation, are necessary.
The ErbB-2 conduct is worked with the part of the heterodimer part of ErbB-3 or ErbB-4 receptors bind.Part, neuregulin-1 (NRG-1) has two independently receptor binding sites to be found: one has higher affinity with ErbB-3 or ErbB-4, and another has the low nonspecific affinity of all ErbB albumen.So the cell of expressing ErbB-3 or ErbB-4 and ErbB-2 can cause the heterodimer of ErbB-3 or ErbB-4 and ErbB-2 formation when being subjected to NRG-1 and influence.But when no part, whether ErbB-2 has affinity to other ErbB albumen is that unclear and such interaction may relate to the tumour generation.In the proteic acceptor of all ErbB, ErbB-3 is unique, because: i) the easy and ErbB-3 formation heterodimer of ErbB-2; Ii) the transformation efficiency that obtains when with ErbB-2 and ErbB-3 while transformant NIH3T3 transforms height separately than with ErbB-2; Iii) in the breast cancer cell relevant with the ErbB-2 overexpression, ErbB-3 also shows high expression level; Iv) ErbB-3 also obtains overexpression in the tumour cell from the ErbB-2 overexpression of ErbB-2 transgenic mice.
Many patents disclose ErbB-2 and/or the ErbB-3 relevant with tumour or cancer therapy with patent application.For example, WO 00/78347 discloses prevention or cell growth inhibiting, especially the method for growth of cancer cells, this method comprises the conformation that stops or reduce the heterodimer of ErbB-2/ErbB-3 in the formation of heterodimer of ErbB-2/ErbB-3 or the interference cell, medicament with stoping or reduce the conformation of the heterodimer of ErbB-2/ErbB-3 in the formation of heterodimer of ErbB-2/ErbB-3 or the interference cell stops or cell growth inhibiting thereby reach.U.S. Patent No. 5,578,482 relate to ErbB-2 part and its functional derivatives, and these materials possess and erbB-2 gene prod bonded characteristic.U.S. Patent No. 5,820,859 relate to a kind of method of expressing the erbB-3 receptor agents that is used for the treatment of of seeking.U.S. Patent No. 5,968,511 relate to ErbB-3 antibody.
In the present technique field, there is demand to the ErbB-3 of the more effective and/or higher price-performance ratio relevant with oncotherapy.The present invention relates to these one side and other related needs in present technique field.
Before this invention, it is new anticancer target that the existing invention (Chinese patent application 00137771.X) of the inventor proposes erbB-3, and the contriver discovers that the formed heterodimer of ErbB-2/ErbB-3 is the main mechanism of tumor development.The inventor of this application has systematically studied the ErbB-3 proteantigen that using gene engineering is expressed: DE3-1 is an escherichia coli expression albumen; B3 is the proteic antigen fragment of eukaryotic cell expression to specific tumour as effect and the method for tumor vaccine in cancers such as prevention and treatment human breast cancer.
Summary of the invention
In one aspect, the present invention relates to prevent, treat and delay the method for mammalian body tumour.Present method comprise to the Mammals animal take reach prevention, treat or delay tumour required or suitable, the ErbB-3 albumen of significant quantity, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or its function fragment, thereby produce the immune response of anti-described tumour, and then described tumour is prevented, is treated or delayed.
In yet another aspect, the present invention relates to isolating nucleic acid fragment, this isolating nucleic acid fragment comprises the proteic ectodomain of coding ErbB-3, or the nucleotide sequence of its function fragment, the proteic ectodomain of this ErbB-3 or its function fragment comprise the aminoacid sequence described in SEQ ID NO:2 (Fig. 5) or the SEQID NO:3 (Figure 11).
In yet another aspect, the present invention relates to the protein or the peptide of purifying basically, it comprises the proteic ectodomain of ErbB-3, or its function fragment, and the proteic ectodomain of this ErbB-3 or its function fragment comprise SEQ ID NO:2 or the described aminoacid sequence of SEQ ID NO:3.
In yet another aspect, the present invention relates to a kind of conjugate, this conjugate comprises: a) comprise the protein or the peptide of the proteic ectodomain of ErbB-3 or its function fragment, the proteic ectodomain of this ErbB-3 or its function fragment comprise SEQ ID NO:2 or the described aminoacid sequence of SEQ ID NO:3; B) with the proteic ectodomain of ErbB-3 or its function fragment directly or the promotor that is connected by joint, wherein promotor helps: i) affine isolated or purified conjugate; Ii) conjugate is adsorbed onto on the surface; Or iii) detect conjugate.
In yet another aspect, the present invention relates to a kind of antibody, the combining an of epitope of this antibody and the proteic ectodomain of ErbB-3 or its function fragment.The proteic ectodomain of this ErbB-3 or its function fragment comprise SEQ ID NO:2 or the described aminoacid sequence of SEQ ID NO:3.
The present invention also provides and has comprised the proteic ectodomain of ErbB-3 or its function fragment, or the nucleic acid of coding and with the pharmaceutical composition and/or the vaccine of this ectodomain bonded antibody and function fragment thereof.
The accompanying drawing summary
Fig. 1 represents B3 cDNA sequence (SEQ ID NO:4).
Fig. 2 illustrates the Restriction Enzyme digestion situation of B3 plasmid.Swimming lane 1:1kb ladder (NEB).Swimming lane 2-9: with BamHI/XbaI to the digestion of the DNA property identified.The clone on swimming lane 5, every other clone is correct clone.Swimming lane 10: the pCDNA3 carrier is digested separately with BamHI/XbaI.
Fig. 3 illustrates the structure of B3 plasmid.
Fig. 4 illustrates separation and/or purifying and SDS-PAGE analysis B3 albumen.Swimming lane 1-4:BSA contrast, the corresponding application of sample amount of every swimming lane is 10ug, 5ug, 3ug, 1ug.Swimming lane 5: protein standard, 7708S, NEB.Swimming lane 6-7:COS7 inductive B3 protein expression.
Fig. 5 represents the aminoacid sequence (SEQ ID NO:2) of B3.
Fig. 6 represents DE3-1 cDNA sequence (SEQ ID NO:5).
Fig. 7 represents the structure of DE3-1 plasmid.
Fig. 8 illustrates the digestion with restriction enzyme situation of DE3-1 plasmid.Swimming lane 1: the pGEX4T-1 that digests the DE3-1 that recombinated with BamHI/XhoI.Swimming lane 2: the pET32a that digests the DE3-1 that recombinated with BamHI/XhoI.Swimming lane 3:1kb ladder (NEB).
Fig. 9 illustrates the SDS-PAGE analytical results that DE3-1 expresses.Swimming lane 1: before inducing.Swimming lane 2: after inducing.Swimming lane 3: inclusion body.Swimming lane 4: the supernatant after the supersound process.
Figure 10 illustrates the proteic separation of DE3-1 and/or purifying and SDS-PAGE analysis.Swimming lane 1: whole protein.Swimming lane 2-8: through the elutriant of NTA histidine mark affinity column.
Figure 11 represents the aminoacid sequence (SEQ ID NO:3) of DE3-1
Figure 12 illustrates the influence of different vaccines to the sickness rate of FVB/N transgenic mice.
Figure 13 illustrates the influence (5 week) of different pharmaceutical to the mouse tumor growth.
Figure 14 illustrates the inhibiting rate (5 week) of different pharmaceutical to tumor growth.
Figure 15 illustrates the influence (5 week) of DE3-1 to the mouse breast cancer growth.
Figure 16 illustrates the inhibiting rate (5 week) of DE3-1 to tumor growth.
Figure 17 illustrates B2 and B3 cross immunity originality experiment (B3 albumen bag quilt).
Figure 18 illustrates B2 and B3 cross immunity originality experiment (B2 albumen bag quilt).
Embodiments of the present invention
In order to disclose clearly rather than, the detailed description of inventing to be divided into following trifle in order to limit.
A. lexical or textual analysis
Unless other definition is arranged, technology and scientific terminology have the same implication that the common skill personnel with the technical field of the invention generally understand as used herein for all.Here all patents of reference, application, disclosed application and other publications all are incorporated herein by reference.If the definition that this section is set forth with this with reference in patent, application, disclosed application and other publications definition of setting forth when opposite or inconsistent, the definition that this section is set forth is better than the definition of other reference.
Used at this, the meaning of " " is " at least one " or " one or more than one ".
Used at this, " tumour " is meant abnormal new growth, so comprise optimum and malignant tumour.Be different from hyperplasia, after lacking primitive stimulus, tumor proliferation can be proceeded.
Used at this, " cancer " is meant the general name by the disease due to the malignant tumour of any kind.
At this, " virulent " is used for tumour, is meant primary tumor, and it has the ability of transfer, promptly lost growth control and position control simultaneously.
Used at this, " erb " refers to two kinds of oncogenes, and erb A and erb B are with erythroblastosis virus relevant (a kind of acute convertibility retrovirus).
Used at this, " immune response " is meant in response to the immune effect change of antigenic organism; In the vertebrates body, this process may relate to antibody and produce, the immunity of inducing cell mediation, the formation of complement activation or immunoglobulin (Ig) tolerance.
Used at this, " immune response toughener " is meant the material of energy enhancement antigen effect in causing immune response.
Used at this, " vaccine " is meant any composition that is used for the active immunity prevention.Vaccine can be used for the treatment of disease on therapeutics, or in advance or infect the back and stop advancing of disease or reduce severity of disease.Typical vaccine include but not limited to this, dead thalline or the viable bacteria body of attenuated strain (mutation or mutant), perhaps microorganism, fungi, plant, protozoon or the metazoal derivative or the product of preparation virulent strain." vaccine " also comprises based on the proteins/peptides of vaccine and nucleic acid/oligonucleotide.
Used at this, " anti-tumor agents (using with the anticancer agent exchange) " is meant any medicament that is used for antineoplaston.This comprise any separately or with other compound use can alleviate, reduce, improve, prevent or maintain do not have clinical symptom or with tumour the medicament of relevant diagnostic markers, and these medicaments can be used in this method that provides, unite use and composition.Anti-tumor agents comprises, but be not limited only to this, anti-angiogenic agent, alkylating agent, antimetabolite, certain natural products, platinum coordination complex, amerantrone, substituted ureas, methylhydrazine derivative, adrenal cortex inhibitor, certain hormone and antagonist, anticancer saccharan and certain medicinal herbs extract be Chinese herbal medicine extract for example.
Used at this, " antineoplaston " is meant that any design is used for the treatment of tumour or treatment for cancer method by what alleviate or improve its symptom.The generation of prophylaxis of tumours or cancer or the treatment that alleviates its severity are also paid attention to.
Used at this, " anti-tumor agents (or anticancer agent) or antineoplaston " do not comprise ErbB-3 albumen or its function fragment or the coding proteic nucleic acid of ErbB-3 or its function fragment, perhaps they are used for the treatment of, but are included as all medicaments and the treatment modalities that certain mode that has known to those skilled in the art is improved tumour or cancer symptoms.
Used at this, " effective level that is used for the treatment of the compound of specified disease " is meant to be enough to improve, or reduces the consumption with the symptom of this disease-related in some way.Such consumption can be used as single dose or according to its effective taking dose of prescription.This consumption possibility cure diseases, but typical situation is to take in order to improve disease symptoms.For reaching the doing well,improving of expectation, may need to take continuously.
Used at this, the meaning of " treatment " is the method that the symptom of any state, discomfort or disease is improved or have useful variation.Treatment comprises that also wherein any pharmacological agent of composition is used.
At this, be meant any alleviating by taking the symptom " improvement " that specific pharmacological agent composition reaches specific discomfort, persistent or of short duration no matter be permanent or temporary transient, need only this alleviate can give the credit to or with take composition relation arranged.
Used at this, " antibody " is used with its wideest intended scope, as long as the many specificitys antibody that specifically comprises complete monoclonal antibody, polyclonal antibody, forms from least two kinds of complete antibodies is bi-specific antibody and show the antibody fragment of desired biological activity for example.For example, antibody can be IgM, IgG, for example IgG1, IgG2, IgG3 or IgG4, IgD, IgA or IgE.
Used at this, " antibody fragment " comprises the part of complete antibody, normally the antigen binding domain of complete antibody or variable region.The example of antibody fragment comprises Fab, Fab ', F (ab ')
2And Fv fragment; Binary; The single-chain antibody molecule; And the many specificitys antibody that forms by antibody fragment.
Used at this, " monoclonal antibody " is meant the antibody of the antibody population of homology in essence, and just, the single antibody of forming antibody population is identical, unless the few spontaneous mutation of contingent quantity.Monoclonal antibody also is highly single-minded, only at an antigen site.Further, with the prepared product difference that typically comprises at traditional antibody (polyclonal antibody) of the different antibodies of different determinants (epitope), each monoclonal antibody is only at the single determinant on the antigen.Except its specificity, monoclonal antibody also has an advantage, and promptly they are to cultivate synthetic by hybridoma, can not polluted by other immunoglobulin (Ig)s.
Used at this, " polyclonal antibody " is meant the situation just as in animal body, the antibody that is produced by several bone-marrow-derived lymphocytes clones.Typically refer to and appear at the intravital antibody of immune animal, and monoclonal antibody is usually by the product of the mono-clonal bone-marrow-derived lymphocyte of vitro culture.
Used at this, " hybridoma " is meant hybrid cell, and wherein tumour cell constitutes one of initiating cell.Typical hybridoma is the hybrid cell by the suitable myeloma cell line of T lymphocyte or bone-marrow-derived lymphocyte and generation monoclonal antibody.
Used at this, " humanized antibody " be meant through modifying and contain the antibody of " people's " aminoacid sequence, so that do not cause immune response when being used in human body.The method for preparing this antibody is known.For example, changing the hybridoma of expressing monoclonal antibody with recombinant DNA technology makes it express the antibody of non-amino acid sequences based on people's antibody.Existing computer program is used to distinguish such zone.
Used at this, " recombination method production " is meant the production method of using the recombinant nucleic acid method.Present method is with the well-known coded proteinic molecular biology method of expression of nucleic acid with cloning.
Used at this, when being meant two nucleic acid molecule, the meaning is that two nucleotide sequences can be hybridized " complementary ", preferably be lower than 25%, be more preferably and be lower than 15%, even to be more preferably being to be lower than 5%, most preferably is not have the mistake pairing at relative Nucleotide place.Preferably under stringent condition, these two molecular hybridizations.
Used at this, the wrong pairing of decision percentile " severity of hybridization " following regulation:
High severity: 0.1xSSPE, 0.1%SDS, 65 ℃;
Medium severity: 0.2xSSPE, 0.1%SDS, 50 ℃; (also referring to appropriate severity)
Low severity: 1.0xSSPE, 0.1%SDS, 50 ℃;
Be interpreted as using damping fluid, salt and the temperature of equity, can obtain suitable severity.
Used at this, " carrier (or plasmid) " is meant and is used for discontinuous element that the foreign DNA transfered cell is expressed or duplicated.Select and use this kind carrier in this technician's technical scope, to be widely known by the people.Expression vector comprise can expressible dna carrier, its DNA effectively with regulating and controlling sequence, for example promoter region links together.Regulating and controlling sequence can influence the segmental expression of this segment DNA.So expression vector is meant that recombinant DNA or RNA constitute thing, for example plasmid, phage, recombinant virus or other carriers, in a single day they import in the appropriate host cell, causes the DNA that clones to express.Suitable expression vector is fully aware of to those skilled in the art, and comprises the carrier that can duplicate and keep free state or be integrated into the carrier of host cell gene group in eukaryotic cell and/or prokaryotic cell prokaryocyte.
Used at this, " promoter region or promoter element " is meant transcription DNA or the RNA fragment of controlling with its DNA that effectively is connected or RNA.Promoter region comprises is enough to allow the particular sequence of RNA polymerase identification, combination and transcription initiation.This part of promoter region is meant promotor.And promoter region also comprises regulates RNA polymerase identification, combination and transcription initiation active sequence.These sequences can be the cis acting factors or trans-acting factor responded.According to the character of regulating, promotor can be composing type or adjustment type.Consider that being used for procaryotic typical promotor comprises phage t7 and T3 promotor and similar promotor.
Used at this, " effectively connect and effectively in conjunction with " be meant Nucleotide effect sequence and regulate sequence, for example promotor, enhanser, transcribe and translation termination site and other signal sequences and DNA between functional relationship.For example, make single-minded identification, combination and transcribe the RNA polymerase of this segment DNA can transcribing from initial this segment DNA of promotor for the effective physics and functional relationship that is meant between DNA and promotor that be connected of DNA and promotor, this relation.For ease of optimization expression and/or in-vitro transcription, be necessary to remove, increase or change clone's 5 ' not translator units, no matter so that eliminate unnecessary, potential inappropriate substituting translation initiation codon or other be transcribe or translation skill on disturb or reduce the sequence of expressing.Additive method is right after 5 of initiator codon ' insertion with the ribosome bind site consensus sequence in addition, can strengthen expression.(reference example, Kozak, journal of biological chemistry (J.Biol.Chem.),
266: 19867-19870 (1991).The hope of this correction (or needs) can be decided by experience.
Used at this, " protein bound sequence " is meant to possess extensively at other albumen or peptide sequence the albumen of the single-minded binding ability of one histone or peptide sequence or specific protein or peptide sequence or peptide.
Used at this, " epitope label " is meant that it helps subsequently to the albumen of " epitope mark " or the biological chemistry and the immune analysis of peptide corresponding to the short sequence of the amino-acid residue of epitope." epitope label " is to realize by additional " epitope label " sequence on the albumen coded sequence of suitable expression vector.The albumen of " epitope mark " can use the narrow spectrum antibody of height that is produced by label to carry out affinity purification.
Used at this, " albumin A or Protein G " be meant can with the Fc of many IgG isomorph zone bonded albumen.Albumin A or Protein G typically are present in the cell walls of some bacterial strain of staphylococcus.Should in albumin A or Protein G, comprise and not influence its active conserved amino acid alternative sequence really.
Used at this, " Nucleotide binding sequence " is meant to possess extensively at nucleotide sequence, the albumen or the peptide sequence of the single-minded binding ability of one group of nucleotide sequence or specific nucleotide sequence.
Used at this, " contaminated with lipid sequence " is meant to possess extensively at lipid, the albumen or the peptide sequence of the single-minded binding ability of one group of lipid or specific lipids material.
Used at this, " polysaccharide binding sequence " is meant to possess extensively at polysaccharide, the albumen or the peptide sequence of the single-minded binding ability of one group of polysaccharide or specific polysaccharide material.
Used at this, " melts combine sequence " is meant to possess extensively at metal ion, the albumen or the peptide sequence of one group of metal ion or the single-minded binding ability of special metal ionic.
B. use ErbB-3 to prevent, treat or delay the method for tumour
From an aspect, the present invention relates to prevent, treat and delay the method for mammalian body tumour.This method comprise to animal take reach prevention, treat or delay tumour required or suitable, the ErbB-3 albumen of significant quantity, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or its function fragment, thereby produce immune response, and then described tumour is prevented, is treated or delayed at described tumour.
Method of the present invention can be used for any mammal tumor prevention, treat or delay, for example mouse, home mouse, rabbit, cat, dog, pig, milk cow, ox, sheep, goat, horse, monkey and other inhuman primates.Preferably, method of the present invention can be used for human tumor prevention, treat or delay.
Any can initiation immunoreactive suitable ErbB-3 albumen occurs at the tumour that will treat, prevent or delay, or its function fragment, and the proteic nucleic acid of ErbB-3 of perhaps encoding, or its function fragment can both be used for present method.The immune response that ErbB-3 causes may be a cell levels, and body fluid level or both have both at the same time.For example, U.S. Patent No. 5,820,859 disclosed ErbB-3 albumen, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or its function fragment can be used for present method.In other example, derive from the ErbB-3 albumen of home mouse ErbB-3, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or its function fragment (GenBank Accession No.U29339; With Hellyer etc., gene (Gene),
165 (2): 279-284 (1995)), Fugu rubripes ErbB-3 (GenBank Accession No.AF056116; With Gellner and Brenner, genome research (Genome Res.),
9 (3): 251-258 (1999)), people ErbB-3 (GenBank Accession No.M29366; With Kraus etc., institute of NAS periodical (Proc.Natl.Acad.Sci.U.S.A.),
86: 9193-9197 (1989)) can be used in present method.Preferably, from the ErbB-3 albumen of people's ErbB-3, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or its function fragment is used for present method.Exist conservative nucleotide sequence to replace but not seriously influence its active any ErbB-3 albumen, or its function fragment can be used for present method.
In preferred embodiments, use the proteic ectodomain of ErbB-3 or its function fragment of significant quantity, the nucleic acid of the proteic ectodomain of ErbB-3 of perhaps encoding or its function fragment.In another preferred embodiment, use the ErbB-3 albumen that significant quantity comprises that the described aminoacid sequence of SEQ ID NO:1 is formed.Still in an other preferred embodiment, use the proteic ectodomain of ErbB-3 or its function fragment that significant quantity comprises that SEQ ID NO:2 or the described aminoacid sequence of SEQ ID NO:3 are formed.
Present method further comprises to administration immune response toughener.The immune response toughener can used ErbB-3 albumen, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or before its function fragment, simultaneously or carry out subsequently.Typical immunostimulant comprise bacille Calmette-Guerin vaccine (BCG) (Ratliff, Eur.Urol.,
2: 17-21 (1992)), Corynebacterium (Lillehojet al., Avian Dis.,
37 (3): 731-40 (1993)), brucella abortus extract (Brucellaabortus extract), dextran, LEVAMISOLE HCL, tilorone, enzyme, avirulent virus, polysaccharide, the medicinal herbs extract is Chinese herbal medicine extract for example.
ErbB-3 albumen, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or use prescription, dosage and the route of administration of its function fragment during especially as pharmaceutical composition, can be determined according to the method known to the present technique field.(referring to, for example, Lei Mingdun: the pharmaceutics science with put into practice (Remington:The Science and Practice of Pharmacy), AlfonsoR.Gennaro (editor), Mack press, in April, 1997; Therapeutic peptide and albumen: prescription, processing and transfer system (Therapeutic Peptides and Proteins:Formulation, Processing, and Delivery Systems), Banga, 1999; With the development (Pharmaceutical Formulation Development of Peptides andProteins) of peptide and albumen pharmaceutical formulation, Hovgaard and Frkjr (editor), Taylor ﹠amp; Francis company, 2000; The medical use of liposome (Medical Applications of Liposomes), Lasic and Papahadjopoulos (editor), Elsevier Science, 1998; Gene therapy study course (Textbookof Gene Therapy), Jain, Hogrefe ﹠amp; Huber press, 1998; Adenovirus: the basic biology of gene therapy (Adenoviruses:Basic Biology to Gene Therapy), 15 volumes, Seth, Landes Bioscience, 1999; The medicinal design of bio-pharmaceuticals and development (Biopharmaceutical Drug Design and Development), Wu-Pong and Rojanasakul (editor), Humana press, 1999; Blood vessel on the therapeutics takes place: to clinical (Therapeutic Angiogenesis:From Basic Science to theClinic), 28 roll up Dole etc. (editor), Springer-Verlag New York, 1999 from basic science).ErbB-3 albumen, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or its function fragment can be mixed be used for oral, rectal administration, local application, the inhalation medication, oral cavity medicine (for example hypogloeeis), injecting drug use is (for example, subcutaneous, intramuscular, intracutaneous, vein), percutaneous dosing or other route of administration that is fit to.Under any given situation, only route of administration will depend on character and the severity and the used specific ErbB-3 albumen of the situation of will treating, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or the character of its function fragment.
ErbB-3 albumen, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or its function fragment can be used for mammiferous any suitable position.Preferably, ErbB-3 albumen, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or its function fragment is used for the original position administration of tumour, promptly is used in tumorigenic place or its contiguous position.And preferably, present method further is included in tumorigenic original position and uses the immune response toughener.
ErbB-3 albumen, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or its function fragment can use separately.Alternatively and preferred mode be ErbB-3 albumen, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or acceptable carrier or vehicle use jointly in its function fragment and the pharmacological agent.Any suitable pharmacological agent acceptable carrier or vehicle can be used for present method.(referring to, for example, Lei Mingdun: the pharmaceutics science with put into practice (Remington:The Science and Practice of Pharmacy), Alfonso R.Gennaro (editor), Mack press, in April, 1997).
Present method can be used separately.Perhaps, present method can with other anti-tumor method, for example radiotherapy, chemotherapy and operative treatment are united use.Present method also can be united use with other anti-tumor agents.Other antineoplaston or medicament can be used in ErbB-3 albumen, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or before the use of its function fragment, among or afterwards.For embodiment, ErbB-3 albumen, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or its function fragment can use jointly with anti-tumor agents.
Any suitable anti-tumor agents can be used for present method.Typical anti-tumor agents comprise anti-angiogenic agent (referring to, for example, Auerbach and Auerbach, Pharmacol.Ther.,
63 (3): 265-311 (1994)), alkylating agent, antimetabolite, certain natural products, platinum coordination complex, amerantrone, substituted ureas, methylhydrazine derivative, adrenal cortex inhibitor, certain hormone, antagonist, oncogene inhibitor, tumor suppression subbase because of or albumen, antioncogene antibody, antioncogene antisense oligonucleotide, anticancer cell-surface antigens antibody and other anti-tumor vaccine.
Coding ErbB-3 proteic nucleic acid, or its function fragment, or any tumor suppression subbase is because of being used with the form of naked DNA, complex body DNA, cDNA, plasmid DNA, RNA or other mixture composition as gene transfer system.In another embodiment, the proteic nucleic acid of coding ErbB-3, or its function fragment, or the tumor suppression subbase is because of being contained in the virus vector.Any virus vector that is suitable for gene therapy can be united in the use at this and used.For example, adenovirus carrier (U.S. Patent No. 5,869,305), monkey disease poisonous carrier (U.S. Patent No. 5,962,274), condition rf human immune deficiency C-type virus C carrier (U.S. Patent No. 5,888,767), retrovirus, simian virus-40, herpes simplex virus amplicon vector and vaccinia virus vector can use.And these genes can change the non-virus carrier system over to, liposome for example, and in liposome, lipid protects DNA or other biological substance not oxidated in agglomeration process.
Present method can be used for the treatment of, prevents or delay any suitable tumour or cancer.Preferably, present method is used for the treatment of, prevents or the interaction that delays any suitable wherein ErbB-2 and ErbB-3 is the crucial paathogenic factor or the tumour or the cancer of the factor of reinforcement.For example, present method can be used for the treatment of, prevention or delay suprarenal gland, anus, auditory nerve, bile canaliculus, bladder, bone, brain, breast, bruccal, central nervous system, uterine neck, colon, ear, uterine endometrium, oesophagus, eyes, eyelid, uterine tube, gi tract, incidence, heart, kidney, larynx, liver, lung, lower jaw, the lower jaw external condyle, upper jaw bone, mouth, nasopharynx, nose, the oral cavity, ovary, pancreas, the parotid gland, penis, auricle, hypophysis, prostate gland, rectum, retina, sialisterium, skin, small intestine, spinal cord, stomach, testis, Tiroidina, tonsil, urethra, vagina, eighth cranial nerve and vaginal orifice tumour.Preferably, present method can be used for the treatment of, prevents or delay breast, ovary, stomach, prostate gland, colon and lung's cancer.More preferably, present method is used for the treatment of, prevents or delays breast cancer.
According to the present invention, no matter separately or with other medicament, carrier or vehicle to unite the ErbB-3 albumen of use, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or its function fragment, can system be used for any route of administration, such as injection in the cavernous body, subcutaneous injection, intravenous injection, intramuscular injection, intradermal injection, oral or local application.Present method can be used the injection formulations with the sanitas that contains interpolation of unit dosage form, ampoule injection or multi-dose container.This prescription can adopt the suspension liquid in oil or the water quality vehicle, solution or emulsion form and can comprise prescription reagent, for example suspension agent, stablizer and/or dispersion agent.Activeconstituents also can be a powdery, so as before use with the carrier, aseptic water or other solvent that does not contain thermal source that are fit to.Local application of the present invention can adopt foam, gelinite, frost, ointment, transdermal ointment or ointment.
May be used for pharmacological agent acceptable composition of the present invention and method and be included in, but be not limited only to United States Patent(USP) Nos. 5,736,154; 6,197,801 B1; 5,741,511; 5,886,039; 5,941,868; 6,258,374 B1; With 5,686, in 102.
The big young pathbreaker of treatment or prevention Chinese traditional medicine therapeutic dose changes with the severity and the route of administration of the situation of treatment.Dosage and dose frequency also will change according to patient self age, body weight, disease condition and reaction.
Please remember how and when the doctor in charge should know because drug toxicity or disadvantageous effect stop, interruption is maybe adjusted to low dosage with treatment.On the contrary, the doctor how and when should know because clinical effect deficiency (having got rid of the toxicity side effect) is adjusted to high level with treatment.
Any suitable route of administration can be used.Dosage form comprises tablet, lozenge, cachet, dispersion, suspension liquid, solution, capsule, ointment and similar form.Reference, Lei Mingdunshi pharmacological agent science.
In actual applications, no matter separately or with other medicament to unite the ErbB-3 albumen of use, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or its function fragment, can according to conventional medicament treat hybrid technology as activeconstituents with form tight interpolation mixture such as the pharmacological agent carrier or the vehicle of beta-cyclodextrin and 2-hydroxyl-propyl group-beta-cyclodextrin.Carrier is according to the expectation use-pattern, and part or injecting drug use are taked multiple formulation forms.When preparation is used for the composition of injection, for example intravenous injection or intravenous infusion, identical pharmacy medium can use, and water, ethylene glycol, oil, damping fluid, sugar, sanitas, liposome and other have the medium known to the people of present technique field technical ability.The example of injectable composition includes, but not limited to glucose, normal physiological salts solution or other solution of 5%w/v.No matter unite use separately or with other medicament, treat the ErbB-3 albumen of administration, its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or the total dose of its function fragment can contain volume at a bottle and uses from 1 milliliter to 2000 milliliters intravenous fluid.According to used total dose, the diluted liquid volume will change to some extent.
The present invention also provides the test kit that carries out treatment plan of the present invention.This test kit comprise place one or more containers no matter separately or with other medicament unite the ErbB-3 albumen of the treatment significant quantity of use, or its function fragment, the proteic nucleic acid of ErbB-3 of perhaps encoding, or its function fragment.Preferred medicament forms is and stroke-physiological saline solution that the acceptable sterile liquid is united use in glucose solution or buffer soln or other pharmacy.Composition also can freeze-drying or drying; In this case, test kit further can optionally comprise the pharmaceutically acceptable solution that is stored in the container, and preferred sterile solution forms injection solution to reassemble into mixture.Typical pharmaceutically acceptable solution is physiological saline and glucose solution.
In another embodiment, test kit of the present invention further comprises the entry needle of one piece of preferred sterile packed that is used for injectable composition or the alcohol liner of syringe and/or packing.Being suitable for doctor or patient uses the indicative explanation of this composition optionally to be included in above the test kit.
The proteic ectodomain of C.ErbB-3 or its function fragment, the nucleic acid of the proteic ectodomain of ErbB-3 of perhaps encoding or its function fragment
From another aspect, the present invention relates to isolating nucleic acid fragment, this isolating nucleic acid fragment under low, the high stringent condition of neutralization with coding ErbB-3 proteic ectodomain, or the nucleotide sequence of its function fragment or the hybridization of its complementary strand, the proteic ectodomain of this ErbB-3, or its function fragment is included in the aminoacid sequence described in SEQ ID NO:2 or the SEQ ID NO:3.
In a preferred embodiment, isolating nucleic acid fragment under high stringent condition with coding ErbB-3 proteic ectodomain, or the nucleotide sequence of its function fragment or the hybridization of its complementary strand, the proteic ectodomain of this ErbB-3, or its function fragment is included in the aminoacid sequence described in SEQ ID NO:2 or the SEQ ID NO:3.In the another one embodiment preferred, isolating nucleic acid fragment comprises the proteic ectodomain of coding ErbB-3, or the nucleotide sequence of its function fragment or its complementary strand, the proteic ectodomain of this ErbB-3, or its function fragment is included in the aminoacid sequence described in SEQ ID NO:2 or the SEQ ID NO:3.Still in the another one embodiment preferred, isolating nucleic acid fragment is included in the pairing nucleotide sequence of aminoacid sequence or its complementary strand of expression among SEQ ID NO:4 (Fig. 1) or the SEQ ID NO:5 (Fig. 6).
Isolating nucleic acid fragment can be any suitable form.For example, isolating nucleic acid fragment can comprise DNA, RNA, PNA or derivatives thereof.In addition, isolating nucleic acid fragment can comprise DNA and RNA or derivatives thereof.Isolating nucleic acid fragment can be single stranded form and be suitable for hybridization analysis.In addition, isolating nucleic acid fragment also can be double chain form and can sex change become single-stranded structure before hybridization analysis.
Isolating nucleic acid fragment can comprise any oligonucleotide or the nucleic acid chains that comprises genetic information coding and/or abiogenous structure.Isolating nucleic acid fragment can comprise non-natural structure, for example non-natural base, for example xanthoglobulin and xanthine, non-natural carbohydrate, for example 2 '-methoxyl group nucleic acid, or non-natural phosphodiester bond, for example methylphosphonate, even phosphide and peptide.
Isolating nucleic acid fragment can produce with any suitable method.For example, isolating nucleic acid fragment can by chemosynthesis (referring to, Ausubel (editor) molecular biology popular approach (CurrentProtocols in Molecular Biology), 2.11. the synthetic and purifying (Synthesisand purification of oligonucleotides) of oligonucleotide, John Wiley ﹠amp; Sons company (2000)), from nature material, separate, by recombination method produce or by aforesaid method in conjunction with producing.Preferably, produce isolating nucleic acid fragment with recombination method.
Isolating nucleic acid fragment can give mark because of various objectives, for example help to detect, purifying and/or with the adhering to of surface.Marker can be chemical, enzyme, immunogenicity, radioactive, fluorescigenic, luminous or FRET (fluorescence resonance energy transfer) mark (FRET).
The present invention also provides the plasmid that comprises above-mentioned nucleic acid fragment.The cell that comprises above-mentioned plasmid further is provided.Can use any suitable cell, for example, bacterial cell, yeast cell, fungal cell, vegetable cell, insect cell, zooblast and people's cell.
In yet another aspect, the present invention relates to produce the proteic ectodomain of ErbB-3, or the method for its function fragment, this method is included in the proteic ectodomain of cell expressing ErbB-3, or cultivate above-mentioned cell under the condition of its function fragment and reclaim the proteic ectodomain of ErbB-3 of expressing, or its function fragment.
In yet another aspect, the present invention relates to the protein or the peptide of purifying basically, it comprises the proteic ectodomain of ErbB-3, or its function fragment, comprises SEQ ID NO:2 or the described aminoacid sequence of SEQID NO:3.Can produce the proteic ectodomain of ErbB-3 with any suitable method, or its function fragment.For example, the proteic ectodomain of ErbB-3, or its function fragment can separate from nature material by chemosynthesis, with recombination method produce or by aforesaid method in conjunction with production.Preferably, produce the proteic ectodomain of ErbB-3 with recombination method, or its function fragment.
In yet another aspect, the present invention relates to a kind of conjugate, this conjugate comprises: a) protein or the peptide of the proteic ectodomain of a kind of ErbB-3 of comprising or its function fragment (comprising the aminoacid sequence described in SEQ ID NO:2 or the SEQID NO:3); B) with the proteic ectodomain of ErbB-3 or its function fragment directly or the promotor that is connected by joint, wherein promotor helps: i) affine isolated or purified conjugate; Ii) conjugate is adsorbed onto on the surface; Or iii) detect conjugate.This conjugate can be a kind of fusion rotein.When this conjugate was a kind of fusion rotein, promotor can also be i) Subcellular Localization sequence in cell after synthetic; The ii) sequence of enhancing immunity originality; Or the iii) tumour antigen of other protein.In addition, ErbB-3 albumen or its function fragment can be connected by enough additive methods with promotor.When conjugate was fusion rotein, the nucleic acid of coding conjugate was also provided.
Conjugate can produce with chemical coupling, for example connects by mercaptan, but preferably produces with recombination method as fusion rotein.In fusion rotein, peptide or its fragment are aminoterminal (N-end) or the carboxyl terminales (C-end) that is connected to the proteic ectodomain of ErbB-3 or its function fragment.In the chemical coupling thing, peptide or its fragment can be connected Anywhere, so that linked reaction is affected, and numerous peptides may occur or its fragment is connected to an ErbB-3 albumen, or its function fragment, or numerous on them.
Linked reaction can be known to those skilled in the art any method affect.As described below, linked reaction can be subjected to the chemical process influence, by covalent linkage, ionic linkage or other suitable keys.For example, can use the reagent and the method for disclosed linked reaction in WO 01/02600.
In certain embodiments, conjugate is a fusion rotein, and this conjugate can give isolated or purified by the albumen of fusion rotein or the affine absorption between peptide fragment and the affine adsorption component.Any isolated or purified fusion rotein that can be used in of affinity interaction.Affinity interaction as described herein, but is not limited only to this, is albumen/albumen, albumen/Nucleotide, albumen/lipid, albumen/polysaccharide or albumen/intermetallic interaction.
In other embodiments, conjugate can be adsorbed onto on the surface.More preferably, conjugate can be adsorbed onto on the surface by the affine absorption between the affine adsorption component of promotor on the conjugate and adsorption surface.Any absorption conjugate that can be used in of affinity interaction comprises albumen/albumen, albumen/Nucleotide, albumen/lipid, albumen/polysaccharide or albumen/intermetallic interaction.
In yet another aspect, the present invention relates to a kind of pharmaceutical composition, this pharmaceutical composition comprises an isolating nucleic acid fragment and pharmaceutically acceptable carrier or vehicle, this isolating nucleic acid fragment under low, the high stringent condition of neutralization with coding ErbB-3 proteic ectodomain, or the nucleotide sequence of its function fragment or the hybridization of its complementary strand, the proteic ectodomain of this ErbB-3, or its function fragment comprises the aminoacid sequence that SEQ ID NO:2 or SEQ ID NO:3 represent.Preferably, this isolating nucleic acid comprises the proteic ectodomain of coding ErbB-3, or its function fragment, comprises nucleotide sequence or its complementary strand of the aminoacid sequence that SEQ ID NO:2 or SEQ ID NO:3 represent.Pharmaceutical composition may further include immune response toughener and/or anti-tumor agents.Comprise above-mentioned isolating nucleic acid fragment separately or also provided with immune response toughener blended vaccine.
In yet another aspect, the present invention relates to a kind of pharmaceutical composition, this pharmaceutical composition comprises protein or peptide and a kind of pharmaceutically acceptable carrier or the vehicle of purifying basically, this protein or peptide comprise the proteic ectodomain of ErbB-3, or its function fragment, comprise the aminoacid sequence that SEQ IDNO:2 or SEQ ID NO:3 represent.Pharmaceutical composition may further include immune response toughener and/or anti-tumor agents.Comprise the protein of above-mentioned purifying basically or peptide separately or also provided with immune response toughener blended vaccine.
In yet another aspect, the present invention relates to a kind of antibody, the proteic ectodomain of this antibody and ErbB-3 or its function fragment comprise an epitope combination of the aminoacid sequence that SEQ ID NO:2 or SEQ ID NO:3 represent.Preferably, this antibody specificity ground and the proteic ectodomain of ErbB-3 or its function fragment comprise an epitope combination of the aminoacid sequence that SEQ ID NO:2 or SEQ ID NO:3 represent.
Antibody can be any suitable form.For example, antibody can be monoclonal, polyclonal, chimeric, strand, the people's or humanized antibody (for example, referring to, U.S. Patent No. 5,968,511).Multi-form antibody can prepare according to any means known in the art (for example, referring to, Coligan etc. (editor), immunology popular approach (Current Protocols inImmunology), John Wiley; Sons company (2000)).Comprise above-mentioned antibody separately or also provided with anti-tumor agents hybrid medicine composition and a kind of pharmaceutically acceptable carrier or vehicle.
Embodiment
Be the typical embodiment that only is used for illustration purpose below.
The present inventor finds that B3, DE3-1 are as effect and the method for tumor vaccine in cancers such as treatment human breast cancers.
The present inventor finds that B3 has significant reduction effect as tumor vaccine in the incidence to tumours such as human breast cancers.
The present inventor provides with B3 has the method for remarkable reduction incidence effect as tumor vaccine in high-risk crowds' such as prevention human breast cancer tumour.
The present inventor finds that B3, DE3-1 also have significant delayed action as tumor vaccine to the time of origin of tumours such as human breast cancer.
The present inventor finds that B3, DE3-1 have significant inhibitory effect as tumor vaccine to growth of tumor such as human breast cancers.
The present inventor provides a kind of method that suppresses growth of cancer cells such as mammary cancer.This method is to reply to form by body immunoreactive to realize.
Above-mentioned cell can be a tumour cell, more can be people's the breast cancer cell and the cancer cells of other ErbB-2/ErbB-3 high expression levels.
Realize aforesaid method according to the present invention, be the ErbB-3 proteantigen of expressing with a kind of using gene engineering: DE3-1 is an escherichia coli expression albumen; B3 is proteic antigen of eukaryotic cell expression or the ErbB-3 antigen that produces with additive method, and ErbB-3 antigen can be ErbB-3 molecule or part fragment.
Under a kind of typical situation, be used for treatment for cancer such as mammary cancer with the ErbB-3 vaccine that produces in every way of doses and can suppress growth of tumor.
Above-mentioned described cancer comprises mammary cancer, ovarian cancer, cancer of the stomach, prostate cancer, the rectum cancer and lung cancer etc.
In order more to be expressly understood aforementioned invention, below describe in detail.
One. experiment material and method
(1) preparation of B3 and DE3-1 vaccine
In this research, related vaccine comprises with outer albumen of ErbB-2 cytolemma and the outer protein part fragment of film, is called B2, SD32 here.The outer proteic part fragment of ErbB-3 cytolemma outskirt protein molecular and film is a laboratory sample, is called B3, DE3-1 here; More than four kinds of vaccines prepare by Zesheng Science and Technology Development Co Ltd, Shanghai.The preparation method of B3 and DE3-1 is as follows:
1.B3 preparation:
The B3 gene is the proteic cDNA sequence of ErbB-3 cytolemma outskirt of coding, sees Fig. 1; With the PCR method amplification, primer sequence is
Wherein italicized item is the flag sequence.
Goal gene is cloned in the pMD-18T carrier behind pcr amplification, transformant through enzyme cut and check order identify correct after (see figure 2), cut out with BamHI/SalI, be connected among the pCDNA3BamHI/xhoI.
The foundation of efficient expression engineering and screening: learn from else's experience PCR and enzyme are cut the engineering bacteria clone of evaluation, carry out 15%SDS-PAGE electrophoresis, thin layer scanning analysis, Western-blotting evaluation, obtain a strain through repeated screening and stablize the high-expression target proteins engineering bacteria.The B3 protein purification, affinitive layer purification is seen Fig. 4.The B3 purifying protein is through determined amino acid sequence and target protein, and aminoacid sequence is seen Fig. 5.
2.DE3-1 preparation
The pcr amplification goal gene is the outer proteic segment cDNA sequence (the cDNA sequence is from Genebank) of coding ErbB-3 film, and sequence is seen Fig. 6; The structure of expression plasmid: target gene fragment is cut out with BamHI/XhoI from pGEX4T-1 carrier (Phamacia company), connect among pET32a carrier (Novagen company) BamHI/XhoI, this albumen is expressed with the T7 promoters driven, N end and Trx.Tag, HisTag and S-Tag merge, and collection of illustrative plates is seen Fig. 7.The plasmid construction enzyme is cut checking and is seen Fig. 8.
The DE3-1 protein expression: change plasmid over to the BL21 bacterial strain, inoculating strain spends the night in 5ml LB+AP; Be inoculated at 1: 100 among the warm LB+AP, 37 ℃, 2.5-3hrs (OD=0.6); Induce 37 ℃ through IPTG, 3hrs or 30 ℃, 8hrs; 4 ℃ centrifugal, 6K, 10min; Remove supernatant, precipitation is put on ice; Suspend the back ultrasonication with PBS cold, that 1/20 bacteria liquid is long-pending; 4 ℃ centrifugal, and 12K, 10min can get the target protein (see figure 9) of a large amount of 34KD.The DE3-1 protein purification: DE3-1 albumen appears in the inclusion body, behind the 6M guanidine hydrochloride dissolution, carries out dialysis NTA-O damping fluid (Histag purification solution), and renaturation is all right.With Histag affinity chromatography column purification (available from betting office) Figure 10, purifying DE3-1 albumen is through determined amino acid sequence, and is consistent with the target protein sequence, aminoacid sequence Figure 11.
(2) the anti-knurl effect study of B3 and DE3-1
1.B3 and DE3-1 is to tumorigenic prophylactic effect
Select the 8-10 FVB/N commentaries on classics neu dna murine (available from Jackson Lab.USA) in age in week for use, animal is divided into five groups, every group 40, be respectively control group, B2, SD32, B3 and DE3-1 group, after using BSA, B2, SD32, B3 and DE3-1 and complete Freund's adjuvant (CFA, complete Freud ' s adjuvant is available from Sigma company) to mix respectively, per 20 days abdominal injections once, totally 7 times.BSA, B2, SD32, B3 and DE3-1 vaccine dose are respectively 10,5,10,5,10 μ g//times.Check incidence weekly one time.A situation arises to determine tumour, and carry out statistical study.
2.B3 to the immunotherapy of tumors effect
The transplanted tumor model is selected the FVB/N transgenic mice spontaneous tumor that detects neu albumen high expression level through immunohistochemical methods for use, gets about 1000mm
3Left and right sides tumor mass is worn into individual cells with nylon wire with tumor mass, and every FVB/N transgenic mice injection cell concentration is 5 * 10
6Individual, injection inoculation under the mammary gland.Inoculation back about 10-14 days, control group can touch (＞5mm) tumour occurs, modeling i.e. success.
Experiment is grouped into control group, does not do any processing; SD32 experimental group, B3 experimental group begin to treat with SD32 and B3 vaccine after inoculation 24 hours, respectively above-mentioned vaccine are adsorbed on the Al (OH) of 0.1mg/ml
3On, the subcutaneous multi-point injection of mouse; Whenever biweekly, totally three times; Medication for the third time finishes experiment after 14 days.Check incidence weekly one time, measure the tumour size weekly, with gross tumor volume (major diameter * minor axis with vernier caliper measurement
2/ 2) represent its size, and paint tumor growth curve.
Experiment end back taking-up tumour is weighed and is calculated inhibiting rate, inhibiting rate=[(control group tumor weight-experimental group tumor weight)/control group tumor weight] * 100
3. various dose DE3-1 studies the immunotherapy of tumors effect
Animal and transplanted tumor model production method: the same (B3 and DE3-1 vaccine are to immunotherapy of tumors effect research).It is that the transplanted tumor model does not add any processing, negative control group is injection histag albumen that animal is respectively the blank group; Positive controls is with Zorubicin (Shantou Mingzhi medicine company limited) treatment, and the DE3-1 experiment component is for being 5 μ g, 20 μ g, the processing of 80 μ g dosage.
Inoculate after 1 day positive controls mouse, abdominal injection Zorubicin, 2.2mg/Kg, continuous use 7 days; Negative control group mouse peritoneal injection histag albumen+Al (OH) 3; DE3-1 experimental group, DE3-1 vaccine are adsorbed on the Al (OH) of 0.1mg/ml
3On, immunotherapy is carried out in the subcutaneous multi-point injection immunity of mouse, per two all medications once, totally three times.End is tested in medication for the third time after 14 days.Check incidence weekly one time, measure the tumour size with vernier caliper measurement, with gross tumor volume (major diameter * minor axis
2/ 2) represent its size, and paint tumor growth curve, carry out statistical study.
Experiment end back taking-up tumour is weighed and is calculated inhibiting rate, and inhibiting rate=[(control group tumor weight-experimental group tumor weight)/control group tumor weight] * 100 carries out statistical study.
4.B2 and B3 cross immunity originality experiment
With B2 albumen and the immune FVB transgenic mice of B3 albumen difference, immunity was got blood after ten days, surveyed its antibody titers with the ELISA method.B2 and B3 with the 0.3ug/ hole wrap quilt respectively, measure B2 and B3 and the B2 and the B3 standard serum of dilution in 1: 1000 on each plate respectively, hatch 30 minutes at 37 ℃, the back is with 1%BSA sealing, adds with two anti-, develops the color 15 minutes with DAD, the Bio-Rad microplate reader, 450nm detects.
Two. experimental result and discussion
1.B3 and DE3-1 presses down, and knurl effect study experimental result sees Table 1, Figure 12
Table 1.B3 and DE3-1 vaccine press down knurl effect study experimental result
Number of packet (only) treatment dosage (μ g//time) tumour time of origin (week) tumour incidence (%)
Negative control group 40 BSA+CFA 10 19 37.5
B2 experimental group 40 B2+CFA 5 21 12.5
SD32 experimental group 40 SD32+CFA 10 22 10
B3 experimental group 40 B3+CFA 5 20 12.5
DE3-1 experimental group 40 DE3-1+CFA 10 23 35
This experiment purpose is whether to understand B3 and DE3-1 vaccine to the prophylactic effect that has to tumour.Selecting the FVB/N transgenic mice for use is that this transgenic mice changes in the mouse body at the rat wild-type neu cDNA of mouse mastopathy virus promoter control, makes neu albumen high expression level, spontaneous generation mammary cancer in 5-8 month, and incidence is about 50%.This transgenic mice pathogenic process and histological type are similar to the human breast cancer pathogenic process.Therefore, be applied to when clinical, may be more effective.Sample is 40 every group, selects so big sample size for use, and purpose is to guarantee that every group is sent out rate number＞10, makes it have statistical significance.The selection of dosage is according to the preliminary experiment result and fixed.
With BSA, B2, B3, SD32, DE3-1 difference immune transgenic mouse, from chart, since negative control group be 37.5% tumour generation tumour incidence was arranged in 19 weeks; And use SD32, and B3, the time that B2 group tumour begins to take place was respectively for the 21st, 22 and 20 weeks, and the incidence of tumour is respectively 10%, 12.5%, 12.5%, and SD32 is described, and B3, B2 vaccine have significant inhibitory effect (P＜0.025 to tumour; χ2Jian Yan), simultaneously, they make the time of origin of tumour that delay also be arranged.DE3-1 group is more late than control group tumour time of origin, but the tumour incidence is 35% to compare (P＞0.05 that do not have significant difference with control group; χ
2Check).
2.B3 and DE3-1 vaccine anti-tumour effect research experiment result
B3 vaccine anti-tumour effect research experiment the results are shown in Table two and Figure 13-14
Table 2.B3 and DE3-1 vaccine anti-tumour effect research experiment result
Packet transaction gross tumor volume (mm
3) tumor weight (g) inhibiting rate (%)
Negative control group histag albumen+Al (OH)
37849.8 ± 849.8 5.76 ± 0.55
SD32 experimental group SD32+Al (OH)
34246.5 ± 540.6 3.28 ± 0.36 46
B3 experimental group B3+Al (OH)
35271.8 ± 658.9 3.13 ± 0.33 33
The artificial effect of determining the B3 vaccine in oncotherapy of invention is used for the transplanted tumor model with the B3 vaccine and carries out immunotherapy research.
Table 2 and Figure 13-14 is the influences of different vaccines to the mouse tumor growth, shows that SD32, B3 are respectively the inhibiting rate of tumor growth: 46%, 33%, show that they all have remarkable restraining effect (P＜0.01 to growth of tumor; The t check).
3.DE3-1 vaccine anti-tumour effect research experiment result
For the DE3-1 that further determines various dose to the immunotherapy effect of tumor growth and be to use clinically to seek suitable therapeutic dose, experimental group is with 5 μ g, 20 μ g, 80 μ g//time immune mouse, experimental result sees Table 3 and Figure 15-16.
Table 3.DE3-1 vaccine anti-tumour effect experimental result
Number of packet is handled gross tumor volume (mm
3) tumor weight (g) inhibiting rate %
DE3-1 experimental group 8 80 μ g DE3-1+Al (OH)
34810.8 ± 460.5 3.658 ± 0.37 26.3
DE3-1 experimental group 8 20 μ g DE3-1+Al (OH)
34715.0 ± 434,8 3.455 ± 0.41 28.9
DE3-1 experimental group 85 μ g DE3-1+Al (OH)
35563.7 ± 600.6 3.687 ± 0.45 22.4
Substantially is consistent to the growth of tumor inhibiting rate with survey volume result from the DE3-1 various dose, and it is best that DE3-1 20 μ g dosage groups suppress effect, reaches about 28.9%.Experiment is taken out tumour after finishing to put to death mouse, weigh, and the statistical study positive controls, each dosage group and negative control and blank have marked difference (P＜0.001, t test).They are 5 μ g when the 6th week, experiment finished, 20 μ g, and 80 μ g dosage groups are respectively 26.3%, 22.4% and 28.9% to the inhibiting rate of tumour
4.B2 and B3 cross immunity originality experiment
The experiment of B2 and B3 cross immunity originality is whether to have cross immunity originality between the two for B2 and B3 albumen that understanding belongs to a family.The results are shown in Figure 17-18, B2 and B3 do not have cross immunity originality between the two as can be known by the result.
Three. brief summary
In this research, new generation vaccine B3 and DE3-1 that we have found to design based on a new anticancer target ErbB-3 has that prophylaxis of tumours is had an effect and tumour is had the immunotherapy effect and has wide application prospect.
In research in the past, in part gland cancer, exist the ErbB-2 acceptor of high expression level, the past think always their form homodimer and and cancer close contact arranged.Think that the ErbB-2 high expression level is the major cause that part gland cancer takes place, reason is 1) there is the amplification of ErbB-2 gene in ErbB-2 and causes the high expression level of ErbB-2,2 in tumour cells such as mammary cancer, ovarian cancer) high expression level of ErbB-2 caused the phosphorylation in its endocellular function district to influence the interaction of endocellular signal molecule Shc and ErbB-2; 3) with wild-type ErbB-2 transfection inoblast, cause cell transformation; 4) can strengthen the ErbB-2 varient that the ErbB-2 homodimer forms and also can strengthen its cell transformation vigor.
And the inventor finds that before this ErbB-3 is another the new anticancer target outside the ErbB-2, and the high expression level that the contriver illustrates the ErbB-2 acceptor causes the heterodimer of ErbB-2 acceptor and the formation of ErbB-3 acceptor to be only the reason that cancer takes place.The discovery of this target makes us that new anticancer method arranged again: with ErbB-3 cytolemma outskirt albumen is prevention and the treatment that anti-cancer vaccine is used for cancer, makes the incidence of mammary cancer reduce and the effect of inhibition tumor growth.
Just be based on and causing ErbB-2 relevant research to take place with kinds of tumors to the ErbB-2 high expression level, and be the immense success of target Humanized monoclonal antibodies-herceptin at ErbB-2, yet because ErbB-2 and ErbB-4 acceptor are at myocardial cell's coexpression, cause the formation of ErbB-2 acceptor and ErbB-4 acceptor heterodimer, this heterodimer is extremely important to normal configuration and the function of keeping the myocardial cell, therefore may destroy the myocardial cell at the anticarcinogen of ErbB-2 acceptor and cause heart failure, then not have this side effect at the anticarcinogen of ErbB-3 acceptor.Thereby ErbB-3 is extremely important as the application method of specific tumour vaccine in prevention such as mammary cancer, ovarian cancer, cancer of the stomach, prostate cancer, the rectum cancer and lung cancer and treatment.
The foregoing description only is used for illustrative purpose, is not in order to limit scope of the present invention.To multiple variation recited above is possible.Because the modifications and variations to the above embodiments are conspicuous to those skilled in the art, so the present invention only is subjected to the scope restriction of appending claims.
Sequence table
＜110〉Zesheng Science and Technology Development Co Ltd, Shanghai
＜120〉based on the method and composition that is used for oncotherapy of ERBB-3
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<170>FastSEQ?for?Windows?Version?4.0
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Met?Arg?Ala?Asn?Asp?Ala?Leu?Gln?Val?Leu?Gly?Leu?Leu?Phe?Ser?Leu
1 5 10 15
Ala?Arg?Gly?Ser?Glu?Val?Gly?Asn?Ser?Gln?Ala?Val?Cys?Pro?Gly?Thr
20 25 30
Leu?Asn?Gly?Leu?Ser?Val?Thr?Gly?Asp?Ala?Glu?Asn?Gln?Tyr?Gln?Thr
35 40 45
Leu?Tyr?Lys?Leu?Tyr?Glu?Arg?Cys?Glu?Val?Val?Met?Gly?Asn?Leu?Glu
50 55 60
Ile?Val?Leu?Thr?Gly?His?Asn?Ala?Asp?Leu?Ser?Phe?Leu?Gln?Trp?Ile
65 70 75 80
Arg?Glu?Val?Thr?Gly?Tyr?Val?Leu?Val?Ala?Met?Asn?Glu?Phe?Ser?Thr
85 90 95
Leu?Pro?Leu?Pro?Asn?Leu?Arg?Val?Val?Arg?Gly?Thr?Gln?Val?Tyr?Asp
100 105 110
Gly?Lys?Phe?Ala?Ile?Phe?Val?Met?Leu?Asn?Tyr?Asn?Thr?Asn?Ser?Ser
115 120 125
His?Ala?Leu?Arg?Gln?Leu?Arg?Leu?Thr?Gln?Leu?Thr?Glu?Ile?Leu?Ser
130 135 140
Gly?Gly?Val?Tyr?Ile?Glu?Lys?Asn?Asp?Lys?Leu?Cys?His?Met?Asp?Thr
145 150 155 160
Ile?Asp?Trp?Arg?Asp?Ile?Val?Arg?Asp?Arg?Asp?Ala?Glu?Ile?Val?Val
165 170 175
Lys?Asp?Asn?Gly?Arg?Ser?Cys?Pro?Pro?Cys?His?Glu?Val?Cys?Lys?Gly
180 185 190
Arg?Cys?Trp?Gly?Pro?Gly?Ser?Glu?Asp?Cys?Gln?Thr?Leu?Thr?Lys?Thr
195 200 205
Ile?Cys?Ala?Pro?Gln?Cys?Asn?Gly?His?Cys?Phe?Gly?Pro?Asn?Pro?Asn
210 215 220
Gln?Cys?Cys?His?Asp?Glu?Cys?Ala?Gly?Gly?Cys?Ser?Gly?Pro?Gln?Asp
225 230 235 240
Thr?Asp?Cys?Phe?Ala?Cys?Arg?His?Phe?Asn?Asp?Ser?Gly?Ala?Cys?Val
245 250 255
Pro?Arg?Cys?Pro?Gln?Pro?Leu?Val?Tyr?Asn?Lys?Leu?Thr?Phe?Gln?Leu
260 265 270
Glu?Pro?Asn?Pro?His?Thr?Lys?Tyr?Gln?Tyr?Gly?Gly?Val?Cys?Val?Ala
275 280 285
Ser?Cys?Pro?His?Asn?Phe?Val?Val?Asp?Gln?Thr?Ser?Cys?Val?Arg?Ala
290 295 300
Cys?Pro?Pro?Asp?Lys?Met?Glu?Val?Asp?Lys?Asn?Gly?Leu?Lys?Met?Cys
305 310 315 320
Glu?Pro?Cys?Gly?Gly?Leu?Cys?Pro?Lys?Ala?Cys?Glu?Gly?Thr?Gly?Ser
325 330 335
Gly?Ser?Arg?Phe?Gln?Thr?Val?Asp?Ser?Ser?Asn?Ile?Asp?Gly?Phe?Val
340 345 350
Asn?Cys?Thr?Lys?Ile?Leu?Gly?Asn?Leu?Asp?Phe?Leu?Ile?Thr?Gly?Leu
355 360 365
Asn?Gly?Asp?Pro?Trp?His?Lys?Ile?Pro?Ala?Leu?Asp?Pro?Glu?Lys?Leu
370 375 380
Asn?Val?Phe?Arg?Thr?Val?Arg?Glu?Ile?Thr?Gly?Tyr?Leu?Asn?Ile?Gln
385 390 395 400
Ser?Trp?Pro?Pro?His?Met?His?Asn?Phe?Ser?Val?Phe?Ser?Asn?Leu?Thr
405 410 415
Thr?Ile?Gly?Gly?Arg?Ser?Leu?Tyr?Asn?Arg?Gly?Phe?Ser?Leu?Leu?Ile
420 425 430
Met?Lys?Asn?Leu?Asn?Val?Thr?Ser?Leu?Gly?Phe?Arg?Ser?Leu?Lys?Glu
435 440 445
Ile?Ser?Ala?Gly?Arg?Ile?Tyr?Ile?Ser?Ala?Asn?Arg?Gln?Leu?Cys?Tyr
450 455 460
His?His?Ser?Leu?Asn?Trp?Thr?Lys?Val?Leu?Arg?Gly?Pro?Thr?Glu?Glu
465 470 475 480
Arg?Leu?Asp?Ile?Lys?His?Asn?Arg?Pro?Arg?Arg?Asp?Cys?Val?Ala?Glu
485 490 495
Gly?Lys?Val?Cys?Asp?Pro?Leu?Cys?Ser?Ser?Gly?Gly?Cys?Trp?Gly?Pro
500 505 510
Gly?Pro?Gly?Gln?Cys?Leu?Ser?Cys?Arg?Asn?Tyr?Ser?Arg?Gly?Gly?Val
515 520 525
Cys?Val?Thr?His?Cys?Asn?Phe?Leu?Asn?Gly?Glu?Pro?Arg?Glu?Phe?Ala
530 535 540
His?Glu?Ala?Glu?Cys?Phe?Ser?Cys?His?Pro?Glu?Cys?Gln?Pro?Met?Glu
545 550 555 560
Gly?Thr?Ala?Thr?Cys?Asn?Gly?Ser?Gly?Ser?Asp?Thr?Cys?Ala?Gln?Cys
565 570 575
Ala?His?Phe?Arg?Asp?Gly?Pro?His?Cys?Val?Ser?Ser?Cys?Pro?His?Gly
580 585 590
Val?Leu?Gly?Ala?Lys?Gly?Pro?Ile?Tyr?Lys?Tyr?Pro?Asp?Val?Gln?Asn
595 600 605
Glu?Cys?Arg?Pro?Cys?His?Glu?Asn?Cys?Thr?Gln?Gly?Cys?Lys?Gly?Pro
610 615 620
Glu?Leu?Gln?Asp?Cys?Leu?Gly?Gln?Thr?Leu?Val?Leu?Ile?Gly?Lys?Thr
625 630 635 640
His?Leu?Thr?Met?Ala?Leu?Thr?Val?Ile?Ala?Gly?Leu?Val?Val?Ile?Phe
645 650 655
Met?Met?Leu?Gly?Gly?Thr?Phe?Leu?Tyr?Trp?Arg?Gly?Arg?Arg?Ile?Gln
660 665 670
Asn?Lys?Arg?Ala?Met?Arg?Arg?Tyr?Leu?Glu?Arg?Gly?Glu?Ser?Ile?Glu
675 680 685
Pro?Leu?Asp?Pro?Ser?Glu?Lys?Ala?Asn?Lys?Val?Leu?Ala?Arg?Ile?Phe
690 695 700
Lys?Glu?Thr?Glu?Leu?Arg?Lys?Leu?Lys?Val?Leu?Gly?Ser?Gly?Val?Phe
705 710 715 720
Gly?Thr?Val?His?Lys?Gly?Val?Trp?Ile?Pro?Glu?Gly?Glu?Ser?Ile?Lys
725 730 735
Ile?Pro?Val?Cys?Ile?Lys?Val?Ile?Glu?Asp?Lys?Ser?Gly?Arg?Gln?Ser
740 745 750
Phe?Gln?Ala?Val?Thr?Asp?His?Met?Leu?Ala?Ile?Gly?Ser?Leu?Asp?His
755 760 765
Ala?His?Ile?Val?Arg?Leu?Leu?Gly?Leu?Cys?Pro?Gly?Ser?Ser?Leu?Gln
770 775 780
Leu?Val?Thr?Gln?Tyr?Leu?Pro?Leu?Gly?Ser?Leu?Leu?Asp?His?Val?Arg
785 790 795 800
Gln?His?Arg?Gly?Ala?Leu?Gly?Pro?Gln?Leu?Leu?Leu?Asn?Trp?Gly?Val
805 810 815
Gln?Ile?Ala?Lys?Gly?Met?Tyr?Tyr?Leu?Glu?Glu?His?Gly?Met?Val?His
820 825 830
Arg?Asn?Leu?Ala?Ala?Arg?Asn?Val?Leu?Leu?Lys?Ser?Pro?Ser?Gln?Val
835 840 845
Gln?Val?Ala?Asp?Phe?Gly?Val?Ala?Asp?Leu?Leu?Pro?Pro?Asp?Asp?Lys
850 855 860
Gln?Leu?Leu?Tyr?Ser?Glu?Ala?Lys?Thr?Pro?Ile?Lys?Trp?Met?Ala?Leu
865 870 875 880
Glu?Ser?Ile?His?Phe?Gly?Lys?Tyr?Thr?His?Gln?Ser?Asp?Val?Trp?Ser
885 890 895
Tyr?Gly?Val?Thr?Val?Trp?Glu?Leu?Met?Thr?Phe?Gly?Ala?Glu?Pro?Tyr
900 905 910
Ala?Gly?Leu?Arg?Leu?Ala?Glu?Val?Pro?Asp?Leu?Leu?Glu?Lys?Gly?Glu
915 920 925
Arg?Leu?Ala?Gln?Pro?Gln?Ile?Cys?Thr?Ile?Asp?Val?Tyr?Met?Val?Met
930 935 940
Val?Lys?Cys?Trp?Met?Ile?Asp?Glu?Asn?Ile?Arg?Pro?Thr?Phe?Lys?Glu
945 950 955 960
Leu?Ala?Asn?Glu?Phe?Thr?Arg?Met?Ala?Arg?Asp?Pro?Pro?Arg?Tyr?Leu
965 970 975
Val?Ile?Lys?Arg?Glu?Ser?Gly?Pro?Gly?Ile?Ala?Pro?Gly?Pro?Glu?Pro
980 985 990
His?Gly?Leu?Thr?Asn?Lys?Lys?Leu?Glu?Glu?Val?Glu?Leu?Glu?Pro?Glu
995 1000 1005
Leu?Asp?Leu?Asp?Leu?Asp?Leu?Glu?Ala?Glu?Glu?Asp?Asn?Leu?Ala?Thr
1010 1015 1020
Thr?Thr?Leu?Gly?Ser?Ala?Leu?Ser?Leu?Pro?Val?Gly?Thr?Leu?Asn?Arg
1025 1030 1035 1040
Pro?Arg?Gly?Ser?Gln?Ser?Leu?Leu?Ser?Pro?Ser?Ser?Gly?Tyr?Met?Pro
1045 1050 1055
Met?Asn?Gln?Gly?Asn?Leu?Gly?Glu?Ser?Cys?Gln?Glu?Ser?Ala?Val?Ser
1060 1065 1070
Gly?Ser?Ser?Glu?Arg?Cys?Pro?Arg?Pro?Val?Ser?Leu?His?Pro?Met?Pro
1075 1080 1085
Arg?Gly?Cys?Leu?Ala?Ser?Glu?Ser?Ser?Glu?Gly?His?Val?Thr?Gly?Ser
1090 1095 1100
Glu?Ala?Glu?Leu?Gln?Glu?Lys?Val?Ser?Met?Cys?Arg?Ser?Arg?Ser?Arg
1105 1110 1115 1120
Ser?Arg?Ser?Pro?Arg?Pro?Arg?Gly?Asp?Ser?Ala?Tyr?His?Ser?Gln?Arg
1125 1130 1135
His?Ser?Leu?Leu?Thr?Pro?Val?Thr?Pro?Leu?Ser?Pro?Pro?Gly?Leu?Glu
1140 1145 1150
Glu?Glu?Asp?Val?Asn?Gly?Tyr?Val?Met?Pro?Asp?Thr?His?Leu?Lys?Gly
1155 1160 1165
Thr?Pro?Ser?Ser?Arg?Glu?Gly?Thr?Leu?Ser?Ser?Val?Gly?Leu?Ser?Ser
1170 1175 1180
Val?Leu?Gly?Thr?Glu?Glu?Glu?Asp?Glu?Asp?Glu?Glu?Tyr?Glu?Tyr?Met
1185 1190 1195 1200
Asn?Arg?Arg?Arg?Arg?His?Ser?Pro?Pro?His?Pro?Pro?Arg?Pro?Ser?Ser
1205 1210 1215
Leu?Glu?Glu?Leu?Gly?Tyr?Glu?Tyr?Met?Asp?Val?Gly?Ser?Asp?Leu?Ser
1220 1225 1230
Ala?Ser?Leu?Gly?Ser?Thr?Gln?Ser?Cys?Pro?Leu?His?Pro?Val?Pro?Ile
1235 1240 1245
Met?Pro?Thr?Ala?Gly?Thr?Thr?Pro?Asp?Glu?Asp?Tyr?Glu?Tyr?Met?Asn
1250 1255 1260
Arg?Gln?Arg?Asp?Gly?Gly?Gly?Pro?Gly?Gly?Asp?Tyr?Ala?Ala?Met?Gly
1265 1270 1275 1280
Ala?Cys?Pro?Ala?Ser?Glu?Gln?Gly?Tyr?Glu?Glu?Met?Arg?Ala?Phe?Gln
1285 1290 1295
Gly?Pro?Gly?His?Gln?Ala?Pro?His?Val?His?Tyr?Ala?Arg?Leu?Lys?Thr
1300 1305 1310
Leu?Arg?Ser?Leu?Glu?Ala?Thr?Asp?Ser?Ala?Phe?Asp?Asn?Pro?Asp?Tyr
1315 1320 1325
Trp?His?Ser?Arg?Leu?Phe?Pro?Lys?Ala?Asn?Ala?Gln?Arg?Thr
1330 1335 1340
<210>2
<211>640
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>2
Met?Arg?Ala?Asn?Asp?Ala?Leu?Gln?Val?Leu?Gly?Leu?Leu?Phe?Ser?Leu
1 5 10 15
Ala?Arg?Gly?Ser?Glu?Val?Gly?Asn?Ser?Gln?Ala?Val?Cys?Pro?Gly?Thr
20 25 30
Leu?Asn?Gly?Leu?Ser?Val?Thr?Gly?Asp?Ala?Glu?Asn?Gln?Tyr?Gln?Thr
35 40 45
Leu?Tyr?Lys?Leu?Tyr?Glu?Arg?Cys?Glu?Val?Val?Met?Gly?Asn?Leu?Glu
50 55 60
Ile?Val?Leu?Thr?Gly?His?Asn?Ala?Asp?Leu?Ser?Phe?Leu?Gln?Trp?Ile
65 70 75 80
Arg?Glu?Val?Thr?Gly?Tyr?Val?Leu?Val?Ala?Met?Asn?Glu?Phe?Ser?Thr
85 90 95
Leu?Pro?Leu?Pro?Asn?Lau?Arg?Val?Val?Arg?Gly?Thr?Gln?Val?Tyr?Asp
100 105 110
Gly?Lys?Phe?Ala?Ile?Phe?Val?Met?Leu?Asn?Tyr?Asn?Thr?Asn?Ser?Ser
115 120 125
His?Ala?Leu?Arg?Gln?Leu?Arg?Lau?Thr?Gln?Leu?Thr?Glu?Ile?Leu?Ser
130 135 140
Gly?Gly?Val?Tyr?Ile?Glu?Lys?Asn?Asp?Lys?Leu?Cys?His?Met?Asp?Thr
145 150 155 160
Ile?Asp?Trp?Arg?Asp?Ile?Val?Arg?Asp?Arg?Asp?Ala?Glu?Ile?Val?Val
165 170 175
Lys?Asp?Asn?Gly?Arg?Ser?Cys?Pro?Pro?Cys?His?Glu?Val?Cys?Lys?Gly
180 185 190
Arg?Cys?Trp?Gly?Pro?Gly?Ser?Glu?Asp?Cys?Gln?Thr?Leu?Thr?Lys?Thr
195 200 205
Ile?Cys?Ala?Pro?Gln?Cys?Asn?Gly?His?Cys?Phe?Gly?Pro?Asn?Pro?Asn
210 215 220
Gln?Cys?Cys?His?Asp?Glu?Cys?Ala?Gly?Gly?Cys?Ser?Gly?Pro?Gln?Asp
225 230 235 240
Thr?Asp?Cys?Phe?Ala?Cys?Arg?His?Phe?Asn?Asp?Ser?Gly?Ala?Cys?Val
245 250 255
Pro?Arg?Cys?Pro?Gln?Pro?Leu?Val?Tyr?Asn?Lys?Leu?Thr?Phe?Gln?Leu
260 265 270
Glu?Pro?Asn?Pro?His?Thr?Lys?Tyr?Gln?Tyr?Gly?Gly?Val?Cys?Val?Ala
275 280 285
Ser?Cys?Pro?His?Asn?Phe?Val?Val?Asp?Gln?Thr?Ser?Cys?Val?Arg?Ala
290 295 300
Cys?Pro?Pro?Asp?Lys?Met?Glu?Val?Asp?Lys?Asn?Gly?Leu?Lys?Met?Cys
305 310 315 320
Glu?Pro?Cys?Gly?Gly?Leu?Cys?Pro?Lys?Ala?Cys?Glu?Gly?Thr?Gly?Ser
325 330 335
Gly?Ser?Arg?Phe?Gln?Thr?Val?Asp?Ser?Ser?Asn?Ile?Asp?Gly?Phe?Val
340 345 350
Asn?Cys?Thr?Lys?Ile?Leu?Gly?Asn?Leu?Asp?Phe?Leu?Ile?Thr?Gly?Leu
355 360 365
Asn?Gly?Asp?Pro?Trp?His?Lys?Ile?Pro?Ala?Leu?Asp?Pro?Glu?Lys?Leu
370 375 380
Asn?Val?Phe?Arg?Thr?Val?Arg?Glu?Ile?Thr?Gly?Tyr?Leu?Asn?Ile?Gln
385 390 395 400
Ser?Trp?Pro?Pro?His?Met?His?Asn?Phe?Ser?Val?Phe?Ser?Asn?Leu?Thr
405 410 415
Thr?Ile?Gly?Gly?Arg?Ser?Leu?Tyr?Asn?Arg?Gly?Phe?Ser?Leu?Leu?Ile
420 425 430
Met?Lys?Asn?Leu?Asn?Val?Thr?Ser?Leu?Gly?Phe?Arg?Ser?Leu?Lys?Glu
435 440 445
Ile?Ser?Ala?Gly?Arg?Ile?Tyr?Ile?Ser?Ala?Asn?Arg?Gln?Leu?Cys?Tyr
450 455 460
His?His?Ser?Leu?Asn?Trp?Thr?Lys?Val?Leu?Arg?Gly?Pro?Thr?Glu?Glu
465 470 475 480
Arg?Leu?Asp?Ile?Lys?His?Asn?Arg?Pro?Arg?Arg?Asp?Cys?Val?Ala?Glu
485 490 495
Gly?Lys?Val?Cys?Asp?Pro?Leu?Cys?Ser?Ser?Gly?Gly?Cys?Trp?Gly?Pro
500 505 510
Gly?Pro?Gly?Gln?Cys?Leu?Ser?Cys?Arg?Asn?Tyr?Ser?Arg?Gly?Gly?Val
515 520 525
Cys?Val?Thr?His?Cys?Asn?Phe?Leu?Asn?Gly?Glu?Pro?Arg?Glu?Phe?Ala
530 535 540
His?Glu?Ala?Glu?Cys?Phe?Ser?Cys?His?Pro?Glu?Cys?Gln?Pro?Met?Glu
545 550 555 560
Gly?Thr?Ala?Thr?Cys?Asn?Gly?Ser?Gly?Ser?Asp?Thr?Cys?Ala?Gln?Cys
565 570 575
Ala?His?Phe?Arg?Asp?Gly?Pro?His?Cys?Val?Ser?Ser?Cys?Pro?His?Gly
580 585 590
Val?Leu?Gly?Ala?Lys?Gly?Pro?Ile?Tyr?Lys?Tyr?Pro?Asp?Val?Gln?Asn
595 600 605
Glu?Cys?Arg?Pro?Cys?His?Glu?Asn?Cys?Thr?Gln?Gly?Cys?Lys?Gly?Pro
610 615 620
Glu?Leu?Gln?Asp?Cys?Leu?Gly?Gln?Thr?Leu?Val?Leu?Ile?Gly?Lys?Thr
625 630 635 640
<210>3
<211>190
<212>PRT
＜213〉homo sapiens (Homo sapiens)
<400>3
Met?Arg?Ala?Asn?Asp?Ala?Leu?Gln?Val?Leu?Gly?Leu?Leu?Phe?Ser?Leu
1 5 10 15
Ala?Arg?Gly?Ser?Glu?Val?Gly?Asn?Ser?Gln?Ala?Val?Cys?Pro?Gly?Thr
20 25 30
Leu?Asn?Gly?Leu?Ser?Val?Thr?Gly?Asp?Ala?Glu?Asn?Gln?Tyr?Gln?Thr
35 40 45
Leu?Tyr?Lys?Leu?Tyr?Glu?Arg?Cys?Glu?Val?Val?Met?Gly?Asn?Leu?Glu
50 55 60
Ile?Val?Leu?Thr?Gly?His?Asn?Ala?Asp?Leu?Ser?Phe?Leu?Gln?Trp?Ile
65 70 75 80
Arg?Glu?Val?Thr?Gly?Tyr?Val?Leu?Val?Ala?Met?Asn?Glu?Phe?Ser?Thr
85 90 95
Leu?Pro?Leu?Pro?Asn?Leu?Arg?Val?Val?Arg?Gly?Thr?Gln?Val?Tyr?Asp
100 105 110
Gly?Lys?Phe?Ala?Ile?Phe?Val?Met?Leu?Asn?Tyr?Asn?Thr?Asn?Ser?Ser
115 120 125
His?Ala?Leu?Arg?Gln?Leu?Arg?Leu?Thr?Gln?Leu?Thr?Glu?Ile?Leu?Ser
130 135 140
Gly?Gly?Val?Tyr?Ile?Glu?Lys?Asn?Asp?Lys?Leu?Cys?His?Met?Asp?Thr
145 150 155 160
Ile?Asp?Trp?Arg?Asp?Ile?Val?Arg?Asp?Arg?Asp?Ala?Glu?Ile?Val?Val
165 170 175
Lys?Asp?Asn?Gly?Arg?Ser?Cys?Pro?Pro?Cys?His?Glu?Val?Cys
180 185 190
<210>4
<211>1914
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>4
agggcgaacg?acgctctgca?ggtgctgggc?ttgcttttca?gcctggcccg?gggctccgag 60
gtgggcaact?ctcaggcagt?gtgtcctggg?actctgaatg?gcctgagtgt?gaccggcgat 120
gctgagaacc?aataccagac?actgtacaag?ctctacgaga?ggtgtgaggt?ggtgatgggg 180
aaccttgaga?ttgtgctcac?gggacacaat?gccgacctct?ccttcctgca?gtggattcga 240
gaagtgacag?gctatgtcct?cgtggccatg?aatgaattct?ctactctacc?attgcccaac 300
ctccgcgtgg?tgcgagggac?ccaggtctac?gatgggaagt?ttgccatctt?cgtcatgttg 360
aactataaca?ccaactccag?ccacgctctg?cgccagctcc?gcttgactca?gctcaccgag 420
attctgtcag?ggggtgttta?tattgagaag?aacgataagc?tttgtcacat?ggacacaatt 480
gactggaggg?acatcgtgag?ggaccgagat?gctgagatag?tggtgaagga?caatggcaga 540
agctgtcccc?cctgtcatga?ggtttgcaag?gggcgatgct?ggggtcctgg?atcagaagac 600
tgccagacat?tgaccaagac?catctgtgct?cctcagtgta?atggtcactg?ctttgggccc 660
aaccccaacc?agtgctgcca?tgatgagtgt?gccgggggct?gctcaggccc?tcaggacaca 720
gactgctttg?cctgccggca?cttcaatgac?agtggagcct?gtgtacctcg?ctgtccacag 780
cctcttgtct?acaacaagct?aactttccag?ctggaaccca?atccccacac?caagtatcag 840
tatggaggag?tttgtgtagc?cagctgtccc?cataactttg?tggtggatca?aacatcctgt 900
gtcagggcct?gtcctcctga?caagatggaa?gtagataaaa?atgggctcaa?gatgtgtgag 960
ccttgtgggg?gactatgtcc?caaagcctgt?gagggaacag?gctctgggag?ccgcttccag 1020
actgtggact?cgagcaacat?tgatggattt?gtgaactgca?ccaagatcct?gggcaacctg 1080
gactttctga?tcaccggcct?caatggagac?ccctggcaca?agatccctgc?cctggaccca 1140
gagaagctca?atgtcttccg?gacagtacgg?gagatcacag?gttacctgaa?catccagtcc 1200
tggccgcccc?acatgcacaa?cttcagtgtt?ttttccaatt?tgacaaccat?tggaggcaga 1260
agcctctaca?accggggctt?ctcattgttg?atcatgaaga?acttgaatgt?cacatctctg 1320
ggcttccgat?ccctgaagga?aattagtgct?gggcgtatct?atataagtgc?caataggcag 1380
ctctgctacc?accactcttt?gaactggacc?aaggtgcttc?gggggcctac?ggaagagcga 1440
ctagacatca?agcataatcg?gccgcgcaga?gactgcgtgg?cagagggcaa?agtgtgtgac 1500
ccactgtgct?cctctggggg?atgctggggc?ccaggccctg?gtcagtgctt?gtcctgtcga 1560
aattatagcc?gaggaggtgt?ctgtgtgacc?cactgcaact?ttctgaatgg?ggagcctcga 1620
gaatttgccc?atgaggccga?atgcttctcc?tgccacccgg?aatgccaacc?catggagggc 1680
actgccacat?gcaatggctc?gggctctgat?acttgtgctc?aatgtgccca?ttttcgagat 1740
gggccccact?gtgtgagcag?ctgcccccat?ggagtcctag?gtgccaaggg?cccaatctac 1800
aagtacccag?atgttcagaa?tgaatgtcgg?ccctgccatg?agaactgcac?ccaggggtgt 1860
aaaggaccag?agcttcaaga?ctgtttagga?caaacactgg?tgctgatcgg?caaa 1914
<210>5
<211>475
<212>DNA
＜213〉homo sapiens (Homo sapiens)
<400>5
gatcctgtcc?tgggactctg?aatggcctga?gtgtgaccgg?cgatgctgag?aaccaatacc 60
agacactgta?caagctctac?gagaggtgtg?aggtggtgat?ggggaacctt?gagattgtgc 120
tcacgggaca?caatgccgac?ctctccttcc?tgcagtggat?tcgagaagtg?acaggctatg 180
tcctcgtggc?catgaatgaa?ttctctactc?taccattgcc?caacctccgc?gtggtgcgag 240
ggacccaggt?ctacgatggg?aagtttgcca?tcttcgtcat?gttgaactat?aacaccaact 300
ccagccacgc?tctgcgccag?ctccgcttga?ctcagctcac?cgagattctg?tcagggggtg 360
tttatattga?gaagaacgat?aagctttgtc?acatggacac?aattgactgg?agggacatcg 420
tgagggaccg?agatgctgag?atagtggtga?aggacaatgg?cagaagctga?ctcga 475
<210>6
<211>19
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>6
tctgcggagt?catgagggc 19
<210>7
<211>48
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>7
tgtgaccacg?actagccgtt?tctgatgttc?ctgctactgc?tgttcact 48
<210>8
<211>25
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>8
tctagagatt?ttctgcggag?tcatg 25
<210>9
<211>15
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>9
gacgacgacg?acaag 15
<210>10
<211>16
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>10
gccatggctg?atatcg 16
<210>11
<211>23
<212>DNA
＜213〉artificial sequence
<220>
＜223〉primer
<400>11
gcaccaccac?caccaccact?gag 23
Claims (30)
1. isolating nucleic acid fragment, this isolating nucleic acid fragment has the coding proteic ectodomain of ErbB-3 or its segmental nucleotide sequence, and the proteic ectodomain of this ErbB-3 or its fragment have the aminoacid sequence described in SEQ ID NO:2 or the SEQ ID NO:3.
2. the described isolating nucleic acid fragment of claim 1, wherein said nucleic acid is DNA.
3. the described isolating nucleic acid fragment of claim 1, wherein said nucleic acid is RNA.
4. plasmid, this plasmid comprises the described nucleic acid fragment of claim 1.
5. cell, this cell comprises the described plasmid of claim 4.
6. the described cell of claim 5, it is selected from bacterial cell, yeast cell, fungal cell, vegetable cell, insect cell, zooblast and people's cell.
7. one kind produces the proteic ectodomain of ErbB-3 or its segmental method, and this method is included in and cultivates the described cell of claim 5 and recovery ErbB-3 albumen ectodomain or its fragment under the proteic ectodomain of cell expressing ErbB-3 or its segmental condition.
8. the albumen or the peptide of purifying basically, this albumen or peptide have the proteic ectodomain of ErbB-3 or its fragment, and the proteic ectodomain of this ErbB-3 or its fragment have the aminoacid sequence described in SEQID NO:2 or the SEQ ID NO:3.
9. conjugate, this conjugate comprises:
A) a kind of albumen or peptide have the proteic ectodomain of ErbB-3, or its fragment, and the proteic ectodomain of this ErbB-3 or its fragment have the aminoacid sequence described in SEQ ID NO:2 or the SEQ ID NO:3; With
B) a kind of and the proteic ectodomain of ErbB-3 or its fragment directly or the promotor that is connected by joint, wherein said promotor helps:
I) affine isolated or purified conjugate;
Ii) conjugate is adsorbed onto on the surface; Or
Iii) detect conjugate.
10. the described conjugate of claim 9, it is a kind of fusion rotein.
11. the described conjugate of claim 10, wherein when described conjugate was a kind of fusion rotein, promotor can also be i) Subcellular Localization sequence in cell after synthetic; The ii) sequence of enhancing immunity originality; Or the iii) tumour antigen of other protein.
12. a pharmaceutical composition, said composition comprise the described isolating nucleic acid fragment of claim 1 and pharmaceutically acceptable carrier and vehicle.
13. the described pharmaceutical composition of claim 12 further comprises immune response toughener and/or anti-tumor agents.
14. a pharmaceutical composition, said composition comprise albumen or peptide and the pharmaceutically acceptable carrier and the vehicle of the described purifying basically of claim 8.
15. the described pharmaceutical composition of claim 14 further comprises immune response toughener and/or anti-tumor agents.
16. an antibody, this antibody combines with the proteic ectodomain of ErbB-3 or its segmental epitope, and the proteic ectodomain of this ErbB-3 or its fragment have the aminoacid sequence of representing among SEQID NO:2 or the SEQ ID NO:3.
17. the described antibody of claim 16, it is polyclonal antibody or monoclonal antibody.
18. the described antibody of claim 16, it is people's antibody or humanized antibody.
19. a pharmaceutical composition, said composition comprise the described antibody of claim 16 and pharmaceutically acceptable carrier and vehicle.
20. the described pharmaceutical composition of claim 19 further comprises a kind of anti-tumor agents.
21. a vaccine, this vaccine comprise the described isolating nucleic acid fragment of claim 1.
22. the described vaccine of claim 21 further comprises the immune response toughener.
23. a vaccine, this vaccine comprise the albumen or the peptide of the purifying basically described in the claim 8.
24. the described vaccine of claim 23 further comprises the immune response toughener.
25. a test kit, this test kit comprise the operation instruction that the described isolating nucleic acid fragment of the claim 1 that places container and this isolating nucleic acid fragment are used for tumor prevention, treat and delay.
26. a test kit, this test kit comprise the albumen of the purifying basically described in the claim 8 that places container or peptide and this albumen of purifying or the operation instruction that peptide is used for tumor prevention, treats and delays basically.
27. a binding substances, this binding substances comprise described isolating nucleic acid fragment of claim 1 and anti-tumor agents.
28. the described binding substances of claim 27 further comprises pharmaceutically acceptable carrier and vehicle.
29. a binding substances, this binding substances comprise albumen or the peptide and the anti-tumor agents of the described purifying basically of claim 8.
30. the described binding substances of claim 29 further comprises pharmaceutically acceptable carrier and vehicle.
Priority Applications (11)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB02116259XA CN1219882C (en) | 2002-03-18 | 2002-03-26 | Method and combination for treating tumors based on ERBB-3 |
| PCT/CN2003/000217 WO2003080835A1 (en) | 2002-03-26 | 2003-03-26 | Erbb3 based methods and compositions for treating neoplasms |
| CA2480099A CA2480099C (en) | 2002-03-26 | 2003-03-26 | Erbb3 based methods and compositions for treating neoplasms |
| JP2003578561A JP4660094B2 (en) | 2002-03-26 | 2003-03-26 | ErbB3-based methods and compositions for treating neoplasms |
| US10/516,759 US7919098B2 (en) | 2002-03-26 | 2003-03-26 | ErbB-3 based methods and compositions for treating neoplasms |
| AU2003218600A AU2003218600C1 (en) | 2002-03-26 | 2003-03-26 | ERBB3 based methods and compositions for treating neoplasms |
| EP11178809.7A EP2400021B1 (en) | 2002-03-26 | 2003-03-26 | ErbB3 based methods and compositions for treating neoplasms |
| EP03711804.9A EP1495123B1 (en) | 2002-03-26 | 2003-03-26 | Erbb3 based methods and compositions for treating neoplasms |
| CNB038067625A CN100424175C (en) | 2002-03-26 | 2003-03-26 | Methods and compositions for treating neoplasms by ERBB3 |
| JP2010101148A JP5249282B2 (en) | 2002-03-26 | 2010-04-26 | ErbB3-based methods and compositions for treating neoplasms |
| US13/035,244 US20110229478A1 (en) | 2002-03-26 | 2011-02-25 | Erbb3 based methods and compositions for treating neoplasms |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN02107357 | 2002-03-18 | ||
| CN02107357.0 | 2002-03-18 | ||
| CNB02116259XA CN1219882C (en) | 2002-03-18 | 2002-03-26 | Method and combination for treating tumors based on ERBB-3 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1444992A CN1444992A (en) | 2003-10-01 |
| CN1219882C true CN1219882C (en) | 2005-09-21 |
Family
ID=28042606
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB02116259XA Expired - Fee Related CN1219882C (en) | 2002-03-18 | 2002-03-26 | Method and combination for treating tumors based on ERBB-3 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1219882C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AR056857A1 (en) | 2005-12-30 | 2007-10-24 | U3 Pharma Ag | DIRECTED ANTIBODIES TO HER-3 (RECEIVER OF THE HUMAN EPIDERMAL GROWTH FACTOR-3) AND ITS USES |
| CN101611150A (en) * | 2006-02-08 | 2009-12-23 | 利佳赛普特有限责任公司 | Bivalent erbb ligand binding molecules and preparation thereof and using method |
-
2002
- 2002-03-26 CN CNB02116259XA patent/CN1219882C/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN1444992A (en) | 2003-10-01 |
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