CN103201287A

CN103201287A - Antibody molecules to oncogenic isoforms of fibroblast growth factor receptor-2 and uses thereof

Info

Publication number: CN103201287A
Application number: CN2011800389508A
Authority: CN
Inventors: 常小迦; 乌尔里克·S.·斯施瓦楚拉格; 凯瑟琳·简·图纳尔
Original assignee: ATTOGEN Inc
Current assignee: ATTOGEN Inc
Priority date: 2010-08-12
Filing date: 2011-08-12
Publication date: 2013-07-10
Anticipated expiration: 2031-08-12
Also published as: WO2012021841A3; EP2603521A4; WO2012021841A2; EP2603521A2; CN103201287B

Abstract

Antibody molecules that specifically bind to one or more isoforms expressed and/or associated with oncogenic phenotypes in a hyperproliferative cell (e.g., a cancerous or tumor cell) are disclosed. The isoform-binding antibody molecules can be used to treat, prevent and/or diagnose cancerous conditions and/or disorders. Methods of using the isoform-binding molecules to selectively detect oncogenic isoforms, to reduce the activity and/or induce the killing of a hyperproliferative cell expressing an oncogenic isoform in vitro, ex vivo or in vivo are also disclosed. Diagnostic and/or screening methods and kits for evaluating the function or expression of an oncogenic isoform are also disclosed.

Description

Antibody molecule of the carcinogenic isomer of fibroblast growth factor acceptor 2 and uses thereof

The cross reference of related application

The application requires the right of priority of the U.S. Provisional Application series number 61/373,072 of submission on August 12nd, 2010, and its content is all brought into herein in this mode by reference.

Sequence table

The application comprises the sequence table of submitting to ASCII fromat by EFS-Web, and brings among the application in full its its by reference at this.Above-mentioned ASCII copy creating was on August 12nd, 2011, and name is called A20497WO.txt, and size is 301,554 bytes.

Government-funded

At least part of use of work described herein from the contract number of the national health research institute (NIH) under the United States Government is

The fund of 1R43CA137929-01 carries out.Therefore, United States Government can enjoy certain right to the present invention.

Background technology

Although medical research has obtained numerous progress, in the U.S., cancer remains a main cause of death.The traditional mode of clinical care, as excision, radiation and chemotherapy has very big mortality, especially solid tumor.Failure occurs and be because infantile tumour slow in reacting to treatment, or reason is in original position regeneration and/or shift and recur.The cause of disease of cancer, diagnosis and excision still are the gonglions of medical research and development.

Because in most of the cases, the probability of alleviating cancer fully improves greatly by early diagnosis, and this is desirable, because the doctor can identify tumor as early as possible.Recognize that based on the variation of genetic expression cancer cells is desirable, because the variation of genetic expression may take place before histology changes, the histology variation differentiates malignant cell from normal cell.Use can be identified the biomarker of these changes in gene expression, and when changing appearred in genetic expression, people can identify cancer cells or preceding cancer cells, thereby can provide targeted therapy effectively to the individuality of benefiting from adjuvant therapy.But can carry out in early days the cancer of various ways, fast, accurately the development of the method that detects and detection reagent composition continues the challenge medical circle.Therefore, the important problem of cancer therapy still is suitable detection and the prognosis of excision that makes treatment processing or cancer.

For example, prostate cancer (CaP) is one of the most general malignant tumour of the male sex, and its incidence increases day by day.In 2007, only just have about 218,900 male sex to be diagnosed out prostate cancer in the U.S., and about 27,050 die from this disease.Although the early diagnosis of prostate gland malignant tumour has obtained major progress by the PSA horizontal survey, the patient of about 10% new diagnosis has the sign of CaP in local late period and 5% patient and long-range transfer (people such as Draisma, (2003) J.Natl.Cancer Inst.95:868-878 have occurred when diagnosing; People such as Thompson, (2003) N.Engl.J.Med.349,215-224; People such as Makinen, (2003) Clin.Cancer Res.9,2435-2439). cure Sex therapy to local late period CaP be effectively (people such as Bolla, (2002) Lancet360,103-106; People such as Messing, (1999) N.Engl.J.Med.341,1781-1788; People such as D'Amico, (2004) J.Am.Med.Assoc.292,821-827).On the contrary, have the patient of long-range transfer sign the non-constant of prognosis (people such as Cheville, (2002) Cancer95,1028-1036).Metastases is the main causes of death relevant with prostate cancer.Hormonal resistance prostate cancer (HRPC) is an example of intrusion type prostate cancer.

In case prostate cancer shifts, only there is limited treatment pattern at present.For example, whole body therapeutic is only for the various forms of androgen-deprivation.Though Most patients shows preliminary clinical improvements, inevitable androgen independence cell development almost.Endocrine therapy is palliative therapy, is not curing property.1,387 have can be by imaging (for example, bone or CT scan) detect in patient's research of metastatic disease, early stage hormonotherapy (namely, the development of androgen independence) after, the middle bit time of target disease progress (not comprising biochemistry/PSA progress) is 16-48 month people such as (, (1998) NEJM339:1036-42) Eisenberger.28-52 month (Eisenberger M.A. wait people (1998) supra.) of total median survival interval for beginning from hormonotherapy among these patients.

After the androgen independence cell development, do not have effective standard care, and median survival interval be the 9-12 month (Vollmer, R.T. wait the people. (1999) Clin Can Res5:831-7; People such as Hudes G., (1997) Proc Am Soc Clin Oncol16:316a (abstract); People such as Pienta K.J., (1994)/Clin Oncol12 (10): 2005-12; People such as Pienta K.J.. (1997) Urology50:401-7; People such as Tannock I.F., (1996)/Clin Oncol14:1756-65; People such as Kantoff P.W., (1996)/.Clin.Oncol.15 (Suppl): 25:110-25).In this age group, cytotoxicity chemotherapy tolerance difference and generally be considered to invalid and/or unpractical.In addition, prostate cancer is the agent of comparison anticytotoxin.Therefore, chemotherapy regimen does not show tangible existence benefit in this patient colony.In view of the shortcoming of existing treatment and diagnosis, still need to improve for prevention, treatment and/or diagnosing cancer are as the target mode of prostate cancer.

Summary of the invention

Feature of the present invention is at least in part for suppressing or reduce the isomer protein specific inhibitor of one or more isomer protein related activity, wherein said isomer specific inhibitor comprises, but be not limited to binding molecule (also referring to " isomer binding molecule " at this), described binding molecule and one or more isomer (for example nucleic acid of isomer polypeptide or this isomer polypeptide of encoding) specific effect, for example, in conjunction with, described isomer is from for example, alternative splicing, frameshit, one or more generations in translation and/or the translation back event, thus different transcribing or translation product caused.In one embodiment, this isomer specific inhibitor specificity is bonded to that carcinogenic or malignant phenotype expresses and/or relevant one or more isomer (referreding to herein as " carcinogenic isomer "), and/or suppresses that carcinogenic or malignant phenotype expresses and/or the activity of relevant one or more isomer (referreding to herein as " carcinogenic isomer ").For example, this isomer specific inhibitor may be carcinogenic isomer binding molecule, for example, antibody molecule or nucleic acid inhibitor, described antibody molecule or nucleic acid inhibitor specificity and one or more carcinogenic isomer (for example nucleic acid of carcinogenic isomer polypeptide or the carcinogenic isomer polypeptide of encoding) effect, for example, in conjunction with.In another embodiment, this isomer specific inhibitor is solvable receptor polypeptides or its fusion form that reduces or suppress one or more isomer (for example carcinogenic isomer) related activity, or peptide or its functional variant.

This carcinogenic isomer may be from for example, at cell for example in the height proliferative cell (for example cancer or tumour cell), the montage of various expression of proto-oncogenes product selectivity, frameshit produces in translation and/or the translation back event.Isomer binding molecule specificity described here is bonded to these carcinogenic isomer, and basic debond is to the proto-oncogene of the isomer of deriving.In certain embodiments, carcinogenic isomer (for example carcinogenic FGFR2 isomer III c) specific effect of isomer binding molecule (for example antibody molecule) and fibroblast growth factor acceptor 2 (FGFR2), for example, in conjunction with.In other embodiments, isomer binding molecule specificity described here is bonded to fibroblast growth factor acceptor 1 (FGFR1) (for example carcinogenic FGFR1L), RON receptor tyrosine kinase (c-met be correlated with Tyrosylprotein kinase) (for example lacking the carcinogenic RON receptor tyrosine kinase of exon 5 and 6), KIT receptor tyrosine kinase (for example lacking the carcinogenic KIT receptor tyrosine kinase of exons 1 1), platelet-derived growth factor (PDGF) (for example lacking the carcinogenic PDGF isomer of exon 6), or the carcinogenic isomer of pdgf receptor α (for example lacking the carcinogenic pdgf receptor α of

exon

7 and 8).Therefore, specificity is bonded at the binding molecule of this carcinogenic isomer that provides and can expresses relevant cancer or tumour cell with carcinogenic isomer for identification.

Therefore, the invention provides isomer specific inhibitor (antibody molecule for example, soluble receptors polypeptide and fusion form thereof, peptide and functional varient thereof and nucleic acid inhibitor), its pharmaceutical composition, and the nucleic acid for preparing this isomer binding molecule, recombinant expression vector and host cell.In certain embodiments, this isomer specific inhibitor optionally is bonded to and/or reduces, and suppresses or block the interaction of carcinogenic isomer and part or co-receptor, thereby reduces or the inhibition carcinogenic activity.In certain embodiments, isomer specific inhibitor and isomer (for example FGFR2-III c) competition is in conjunction with cognate ligand (for example FGF8b).In other embodiments, the isomer specific inhibitor is as negative dominance rival, for example is combined with isomer but do not produce the negative dominance rival of signal in the cell.In other embodiments, the isomer binding molecule can make optionally for example cancer or tumour cell of target height proliferative cell of cytotoxic agent or cytostatics.Isomer specific inhibitor described here can be used for handling, prevention and/or diagnosis cancer or pernicious symptom and/or disease, such as (former in cancer or tumour, recurrence or transfer), include but not limited to prostate gland, bladder, pancreas, mammary gland, ovary, brain (glioblastoma multiforme) and gastrointestinal cancer.Detect carcinogenic isomer with isomer binding molecule of the present invention, external to be reduced in, in the junctor or the height proliferative cell of the carcinogenic isomer of expression in vivo active and/or the method for killing the height proliferative cell be also contained among the present invention.Also announced for assessment of the function of carcinogenic isomer or diagnosis and/or screening method and the reagent of expression.

Therefore, on the one hand, feature of the present invention is isomer specific inhibitor (for example, antibody molecule, soluble receptors polypeptide and fusion form thereof), this isomer specific inhibitor and one or more isomer polypeptide or its fragment interact or more preferably specificity be combined.Typical isomer binding molecule is with high-affinity, for example with at least about 10 ⁷M ^-1, typically about 10 ⁸M ^-1And more typically about 10 ⁹M ^-1To 10 ¹⁰M ^-1Or higher affinity constant is bonded to one or more isomer polypeptide or its fragment, and reduces and/or suppress the activity of one or more isomer, for example the carcinogenic isomer in height hyperplasia (for example cancer or pernicious) cell and/or the tissue.For example, alternative and the specificity of binding molecule reduces or suppresses to be selected from one or more carcinogenic isomer related activity in following: (i) binding partner or co-receptor (for example, FGF part, for example FGF8b, FGF2, FGF17 or FGF18 to FGFR2 isomer III c); (ii) receptor dimerizationization (for example, FGFR2 isomer III c dimerization); (iii) isomer signal transduction, for example, FGFR2 isomer III c signal transduction; (iv) height hyperplasia (for example cancer or tumour) cell proliferation, growth and/or survival, for example, by inducing height proliferative cell apoptosis; And/or (the v) vasculogenesis of tumour and/or vascularization.In certain embodiments, this inhibitor may be directly in height hyperplasia (for example cancer or pernicious) cell and/or tissue, directly play a role (for example, directly inducing cell is committed suiside or apoptosis).In other embodiments, this inhibitor can play a role by acting on contiguous cell, for example, and the cell of height hyperplasia (for example, cancer or pernicious) cell and/or tissue periphery.For example, this inhibitor may reduce vasculogenesis and/or the vascularization of tumor tissues.

In one embodiment, the isomer binding molecule is for being bonded to Mammals, people for example, the antibody molecule of isomer polypeptide or its fragment.For example, this antibody molecule is bonded to the height proliferative cell, and for example cancer or tumour cell are expressed and/or relevant isomer polypeptide or fragment.For example, this antibody molecule specificity is bonded to epi-position, for example be positioned at or mainly at the height proliferative cell, as cancer or tumour cell, linear epitope or the conformational epitope of surface expression.In an embodiment, the epi-position of antibody molecule identification is the height proliferative disease, for example cancer or malignant disease express or relevant.For example, the epi-position of antibody molecule identification is that exon sequence or exon sequence are expressed or relevant, that described exon sequence is mainly expressed by one or more cancers or tumour cell or disease or relevant; Described epi-position may be located at the bonding land between two exons that significantly combine in one or more cancers or tumour cell or the disease, for example: because the use of the interior Exon deletion of framework or alternative splicing exon.Typical isomer polypeptide or the fragment of isomer binding molecule identification of the present invention include but not limited to FGFR2, FGFR1, RON receptor tyrosine kinase, KIT receptor tyrosine kinase, the carcinogenic isomer of PDGF and PDGF-acceptor α.

In one embodiment, described antibody molecule is bonded to isomer, for example FGFR2, for example carcinogenic isomer of people FGFR2.Described antibody molecule can specificity be bonded to FGFR2 isomer III c or its fragment, for example, basic debond is to other non-carcinogenic isomer of FGF acceptor, such as other alternative splicing variant of FGFR2 (for example, FGFR2 III b (SEQ ID NO:21), FGFR2 isomer 4 (SEQ ID NO:22), FGFR2 isomer 7 (SEQ ID NO:23), FGFR2 isomer 9 (SEQ ID NO:24), FGFR2 isomer 10 (SEQ ID NO:25), FGFR2 isomer 11 (SEQ ID NO:26), FGFR2 isomer 12 (SEQ ID NO:27), FGFR2 isomer 13 (SEQ ID NO:28), FGFR2 isomer 14 (SEQ ID NO:29), FGFR2 isomer 15 (SEQ ID NO:30), FGFR isomer 17 (SEQ ID NO:31), FGFR2 isomer 18 (SEQ ID NO:52) or FGFR2 isomer 19 (SEQ ID NO:53)).For example, described antibody molecule preferentially is bonded to FGFR2 isomer III c or its fragment, but basic debond is to (for example having showed with FGFR2 isomer III b and be lower than 10%, 8%, 5%, 4%, 3%, 2%, 1% cross reactivity) FGFR2 isomer III b, for example, about 314 to 351 amino acid of people FGFR2 isomer III b

(HSGINSSNAEVLALFNVTEADAGEYICKVSNYIGQANQ; SEQ ID NO:56); Amino acid (the HSGINSSNAEVLALF that people FGFR2 isomer III is b314 to 328; SEQ ID NO:57); Or the amino acid (CKVSNYIGQANQ of b340 to 351 of people FGFR2 isomer III; SEQ ID NO:58).In those embodiment, described antibody molecule specificity is bonded at least one or a plurality of amino acid (for example at least one epi-position) of the alternative splicing form that is positioned at the exon III, for example from about 301 to 360 amino acid of FGFR2-III c (SEQ ID NO:2); The amino acid that FGFR2-III c is about 314 to 324 (AAGVNTTDKEI, SEQ ID NO:4); The amino acid that FGFR2-III c is about 328 to 337 (YIRNVTFEDA, SEQ ID NO:6); The amino acid of about 350 to the 353of positions of FGFR2-III c (ISFH, SEQ ID NO:8); The amino acid of the about 314-353 of FGFR2 III c position

(AAGVNTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFH (SEP ID NO:84)); Or

The amino acid that FGFR2-III c is about 341 to 353 (TCLAGNS IGIS FH (SEQ ID NO:86), or SEQ ID NOs:1,3,5,7,83 or 85 nucleotide sequence coded aminoacid sequences; Or amino acid essentially identical with it or nucleotide sequence.In one embodiment, anti-FGFR2-III c antibody molecule is bonded to the 1st the L-Ala of SEQ ID NO:84, the 2nd L-Ala, the 4th Xie Ansuan, the 6th Threonine, the 7th Threonine, the 8th aspartic acid, the 9th Methionin, the 11st Isoleucine, the 12nd L-glutamic acid, the 13rd Xie Ansuan, the 15th tyrosine, the 16th Isoleucine, the 17th arginine, the 21st phenylalanine, the 22nd L-glutamic acid, the 28th Threonine, the 30th leucine, the 31st L-Ala, the 32nd glycine, the 34th Serine, the 37th Isoleucine, the 38th Serine, the 39th phenylalanine, or the 40th one in hyte propylhomoserin, two, three, four, five, six, seven, eight, nine, ten, ten two, 14,16,18,20,22 or more (with

AAG VN TTDKE IEVL YIRNVT FEDAGEY TC LAGN SIG ISFH(SEP ID NO:84)) the amino-acid residue correspondence that highlights); Or the 1st Threonine of SEQ ID NO:86, the 3rd leucine, the 4th L-Ala, the 5th glycine, the 7th Serine, the 10th Isoleucine, the 11st Serine, in the 12nd phenylalanine or the 13rd hyte propylhomoserin one or more (with TC LAGN SIG ISFHThe amino-acid residue correspondence that SEQ ID NO:86 highlights).

Embodiment 8-15 discloses typical anti-FGFR2-III c antibody molecule within the scope of the invention.In one embodiment, anti-FGFR2-III c antibody molecule is bonded to, for example, bind selectively to Mammals, for example, people FGFR2-III c(for example, about 301 to 360 the amino acid whose epi-position of people FGFR2-III c extracellular domain FGFR2-III c (SEQ ID NO:2) that is positioned at, or with its at least 85%, 90%, 95%, 99% or more identical sequence).In certain embodiments, described anti-FGFR2-III c antibody molecule is bonded to and is selected from: the residue of the 314-353 position of (epi-position that for example comprises amino-acid residue) people FGFR2-III c (AAGVNTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFH (SEQ ID NO:84)) or amino acid TCLAGNSIGISFH (SEQ ID NO:86), or one or more amino-acid residues of its modified forms (for example fragment or its replacement (for example conservative the replacement) form).In one embodiment, described anti-FGFR2-III c antibody molecule is bonded to synthetic on the extracellular domain of people FGFR2-III c, reorganization or natural epi-position.In certain embodiments, described anti-FGFR2-III c antibody molecule is bonded to and is present in prostate cancer cell, for example Human Prostate Cancer Cells (for example, the DU145 PC-3), or the natural FGFR2-III c of rat prostate cancerous cell line (for example, AT3B-1 rat prostate cancerous cell line).In one embodiment, anti-FGFR2-III c antibody molecule preferentially is bonded to FGFR2 isomer III c or its fragment, but basic debond is to (for example showing with FGFR2 isomer III b and be lower than 10%, 8%, 5%, 4%, 3%, 2%, 1% cross reactivity) FGFR2 isomer III b, the extracellular domain of people FGFR2 isomer III b (for example, about 314 to 351 amino acid of people FGFR2 isomer III b for example

(HSGINSSNAEVLALFNVTEADAGEYICKVSNYIGQANQ; SEQ ID NO:56); About 314 to 328 the amino acid (HSGINSSNAEVLALF of people FGFR2 isomer III b; SEQ ID NO:57); About 340 to 351 the amino acid (CKVSNYIGQANQ of people FGFR2 isomer III b; SEQ ID NO:58)).

In one embodiment, described anti-FGFR2-III c antibody molecule showed and preferentially has been bonded to people FGFR2-III c, as Figure 24 A-24B, and 25A-25B, 27A-27B, 28,29A-29F, 30A-30F, any monoclonal antibody shown in 32-39 and the 41-44.For example, described anti-FGFR2-III c antibody molecule has been showed clone B7 (also referring to " Atto-MuMab-03 " at this), C5, D2, D10, E3, E8, F3, F10 or the G9 as Figure 24 A-24B or 32; Or any people of Figure 25 A-25B, 27A-27B, 28,29A-29F, 30A-30F, 37-39 and/or 41-44 to clone 1 (also referring to " Atto-HuMab-01 " at this), 2,6 (also referring to " Atto-HuMab-06 " at this), 7 or 8 (also referring to " Atto-HuMab-08 " at this) same or analogous in conjunction with selectivity.In other embodiments, anti-FGFR2-III c antibody molecule showed as Atto-MuMab-01 or Atto-MuMab-02 (Figure 33,34A-34B, 35 and 36A-36B shown in VH and VL) same or similar in conjunction with selectivity.In another embodiment, described anti-FGFR2-III c antibody molecule has been showed the A-25B as Figure 25,27A-27B, 28-29,30A-30F, the people of 37-39 and/or 41-44

clones

1,6,8 (be respectively Atto-HuMab-01, Atto-HuMab-06, Atto-HuMab-08) same or analogous in conjunction with selectivity.In one embodiment, described FGFR2-III c antibody molecule specificity is bonded to people FGFR2-III c and competition inhibition second antibody molecule is bonded to FGFR2-III c, wherein the second antibody molecule may be for being selected from, for example: Atto-MuMab-01, Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06 or Atto-HuMab-08, antibody molecule.

Figure 28 has showed the aminoacid sequence comparison of people scFv clone 6 (Atto-HuMab-06) and clone 8 (Atto-HuMab-08).The site of the complementary determining region of light chain and variable region of heavy chain (CDRs) draws underscore and is labeled as VL-CDR-1 respectively, and-2 and-3 and VH-CDR-1 ,-2 and-3.

Figure 38 has showed the aminoacid sequence comparison of people scFv clone-1 (Atto-HuMab-01) and clone-6 (Atto-HuMab-06).The site of the complementary determining region of light chain and variable region of heavy chain (CDRs) draws underscore and is labeled as VL-CDR-1 respectively, and-2 and-3 and VH-CDR-1 ,-2 and-3.

Figure 39 has showed the aminoacid sequence comparison of people scFv clone-1 (Atto-HuMab-01) and clone-8 (Atto-HuMab-08).The site of the complementary determining region of light chain and variable region of heavy chain (CDRs) draws underscore and is labeled as VL-CDR-1 respectively, and-2 and-3 and VH-CDR-1 ,-2 and-3.

Figure 29 A-29B has showed Nucleotide and the aminoacid sequence of the full length coding region of people scFv clone's 8 (Atto-HuMab-08) variable region of light chain and variable region of heavy chain respectively.Figure 29 C-29D has showed Nucleotide and the aminoacid sequence of people scFv clone's 8 (Atto-HuMab-08) variable region of light chain respectively.Figure 29 E-29F has showed Nucleotide and the aminoacid sequence of people scFv clone's 8 (Atto-HuMab-08) variable region of heavy chain respectively.

Figure 30 A-30B has showed Nucleotide and the aminoacid sequence of the full length coding region of people scFv clone's 6 (Atto-HuMab-06) variable region of light chain and variable region of heavy chain respectively.Figure 30 C-30D has showed Nucleotide and the aminoacid sequence of people scFv clone's 6 (Atto-HuMab-06) variable region of light chain respectively.Figure 30 E-30F has showed Nucleotide and the aminoacid sequence of people scFv clone's 6 (Atto-HuMab-06) variable region of heavy chain respectively.The related locus of complementary determining region (CDRs) draws underscore in earlier figures.

Figure 35 has showed the light chain of Atto-MuMab-02 and the aminoacid sequence of variable region of heavy chain (being respectively SEQ ID NO:90 and 88).Figure 35 has pointed out the relevant position of the CDRs in heavy chain and the variable region of light chain.Figure 34 A and 34B have showed the nucleotide sequence (being respectively SEQ ID NO:87 and 89) of coding Atto-MuMab-02 heavy chain and variable region of light chain respectively.

Figure 37 A-37B has showed Nucleotide and the aminoacid sequence (SEQ ID NOs:190-191) of the full length coding region of people scFv clone's 1 (Atto-HuMab-01) variable region of light chain and variable region of heavy chain respectively.Figure 38-39 has showed the light chain variable region amino acid sequence (amino acid of the 1-111 position of the aminoacid sequence above corresponding is before connector area) of people scFv clone 1 (Atto-HuMab-01) respectively.Figure 38-39 has showed the aminoacid sequence (amino acid of the 133-252 position of the aminoacid sequence above corresponding is after connector area) of people scFv clone's 1 (Atto-HuMab-01) variable region of heavy chain.The relevant position of complementary determining region (CDRs) draws underscore and mark in earlier figures.Figure 40 is description Atto-HuMab-01 ,-06, and the table of the comparison between-08 CDR district (heavy chain and light chain CDRs).

In other embodiments, described anti-FGFR2-III c antibody molecule has: Figure 24 A-24B or 32 clone B7

(Atto-MuMab-03), C5, D2, D10, E3, E8, F3, F10 or G9, or Figure 25 A-25B, 27A-27B, 28-29,30A-30F, the people of 37-39 and/or 41-44 clones 1 (Atto-HuMab-01), 2,6 (Atto-HuMab-06), among 7 or 8 (Atto-HuMab-08) any one, and particularly, antibody A tto-MuMab-01, Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06 or Atto-HuMab-08, in one or more biological properties of any clone, described biological property includes but not limited to:

(i) described antibody molecule is bonded to the same or analogous epi-position on the people FGFR2-III c, as antibody A tto-MuMab-01, Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06 or Atto-HuMab-08 (for example anti-FGFR2-III c antibody molecule and monoclonal antibody Atto-MuMab-01, Atto-MuMab-02

Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06, or Atto-HuMab-08 compete combination);

(ii) be bonded to FGFR2 III c 314-353 position

At least one amino-acid residue of (AAGVNTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFH_ (SEQ ID NO:84)); Or at least one amino-acid residue of the TCLAGNSIGISFH of people FGFR2-III c (SEQ ID NO:86);

(iii) preferentially be bonded to and be present in people FGFR2-III c, but not at least one amino-acid residue that is present in people FGFR2-III b, (for example: the 1st the L-Ala that is bonded to SEQ ID NO:84, the 2nd L-Ala, the 4th Xie Ansuan, the 6th Threonine, the 7th Threonine, the 8th aspartic acid, the 9th Methionin, the 11st Isoleucine, the 12nd L-glutamic acid, the 13rd Xie Ansuan, the 15th tyrosine, the 16th Isoleucine, the 17th arginine, the 21st phenylalanine, the 22nd L-glutamic acid, the 28th Threonine, the 30th leucine, the 31st L-Ala, the 32nd glycine, the 34th Serine, the 37th Isoleucine, the 38th Serine, the 39th phenylalanine, or the 40th one in hyte propylhomoserin, two, three, four, five, six, seven, eight, nine, ten, 12,14,16,18,20,22 or more (with AAG VN TTDKE IEVL YIRNVT FEDAGEY TC LAGN SIG ISFH(SEP ID NO:84)) the amino-acid residue correspondence that highlights); Or the 1st Threonine of SEQ ID NO:86, the 3rd leucine, the 4th L-Ala, the 5th glycine, the 7th Serine, the 10th Isoleucine, the 11st Serine, in the 12nd phenylalanine or the 13rd hyte propylhomoserin one or more (with TC LAGN SIG ISFHThe amino-acid residue correspondence that SEQ ID NO:86 highlights);

(iv) be bonded to reorganization, synthetic or natural human FGFR2-III c;

(v) be bonded to the natural FGFR2-III c that is present on prostate cancer cell or the liver cancer cell, for example Human Prostate Cancer Cells is (for example: the DU145 PC-3) or hepatic cell line HepG2 for described prostate cancer cell or liver cancer cell;

(vi) showed and monoclonal antibody Atto-MuMab-01, Atto-MuMab-02, Atto-MuMab-03,

Atto-HuMab-01, Atto-HuMab-06 or Atto-HuMab-08 be same or analogous, be bonded to FGFR2-III c in conjunction with selectivity;

(vii) showed and monoclonal antibody Atto-MuMab-01, Atto-MuMab-02, Atto-MuMab-03,

Atto-HuMab-01, the same or analogous binding affinity of Atto-HuMab-06 or Atto-HuMab-08; And/or

(viii) showed and monoclonal antibody Atto-MuMab-01, Atto-MuMab-02, Atto-MuMab-03,

Atto-HuMab-01, the same or analogous binding kinetics of Atto-HuMab-06 or Atto-HuMab-08.

In one embodiment, described anti-FGFR2-III c antibody molecule is monoclonal antibody Atto-MuMab-01,

Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06 or Atto-HuMab-08.

In one embodiment, described anti-FGFR2-III c antibody molecule is with high-affinity, for example: to be lower than 10 ^-7M, 10 ^-8, 10 ^-9, 10 ^-10, 10 ^-11M or better Kd are bonded to FGFR2-III c.In other embodiments, described anti-FGFR2-III c antibody or its fragment can be with at least about 10 ^-8, 10 ^-9, 10 ^-10, 10 ^-11M or better IC50 reduce one or more

FGFR2-III c-related activity.

In other embodiments, described anti-FGFR2-III c antibody and fragment thereof and people FGFR2-III c are so that (Surface plasmon resonance SPR) definitely is lower than 1x10 at surface plasma resonance ^-4s ^-1To 1x10 ^-6s ^-1K _QffOr as surface plasma resonance determine 10 ³To 10 ⁷M ^-1S ^-1Between k _OnThe kinetics combination of scope.The avidity of anti-FGFR2-III c or its fragment and binding kinetics energy usefulness, biological example sensing technology (BIACORE) test.

In one embodiment, described anti-FGFR2-III c antibody molecule is complete antibody or its fragment (Fab for example, F (ab') ₂, Fv, or strand Fv fragment (scFv)).In certain embodiments, described anti-FGFR2-III c antibody molecule is monoclonal antibody or the antibody with monospecific.Described anti-FGFR2-III c antibody molecule also may be the people, and the people source is chimeric, camel, shark, or the antibody molecule of external generation.In one embodiment, described anti-FGFR2-III c antibody molecule is the humanization antibody molecule.The heavy chain of described anti-FGFR2-III c antibody molecule and light chain may (for example antibody may comprise at least one for total length, and preferred two complete heavy chains, and at least one, and preferred two complete light chains), may comprise that maybe Fab (for example: Fab, F (ab') ₂, Fv, strand Fv fragment, single domain antibody, binary antibody (dAb), divalence or bi-specific antibody or its fragment, its single domain antibody variant, or camel antibody).In other embodiments, described antibody molecule has CH (Fc), and described CH is selected from: IgG1 for example, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, the CH of IgD and IgE; Especially, be selected from: for example, IgG1, IgG2, the CH of IgG3 and IgG4, more particularly, IgG1 and IgG2 () CH for example: human IgG1 or IgG2.In one embodiment, described CH is the human IgG1.In another embodiment, described anti-FGFR2-III c has constant region of light chain, and described constant region of light chain is selected from: for example, and κ or λ, the constant region of light chain of preferred κ (for example, people κ).In one embodiment, constant region is changed, for example, and sudden change, thereby the character of modifying anti-FGFR2-III c antibody molecule (for example, increases or reduces Fc receptors bind, antibody glycosylation, the quantity of cysteine residues, effector cell function, or in the compensate function one or more).For example, at 296 (M to Y) of SEQ ID NO:55,298 (S to T), 300 (T to E), 477 (H to K) and 478 (N to F) are change Fc receptors bind thereby the position suddenlys change.

In certain embodiments, described anti-FGFR2-III c is covalently bound to cell surface protein (for example, phage) or solubility secreted form (for example, scFv or its fusions).In other embodiments, described anti-FGFR2-III c secretes with strand Fv or merges to Fc constant region (for example, forming strand or the duplex structure that human IgG1 Fc merges).

In other embodiments, described anti-FGFR2-III c antibody molecule comprises at least one antigen binding domain, for example: the variable region, described variable region is from being selected from: Atto-MuMab-01 for example, Atto-MuMab-02, Atto-MuMab-03,

Atto-HuMab-01, Atto-HuMab-06, or Atto-HuMab-08, antibody.In another embodiment, described anti-FGFR2-III c antibody molecule comprises at least one from antibody, two, three or four variable regions, described antibody is selected from: for example, and Atto-MuMab-01, Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01

Atto-HuMab-06, or Atto-HuMab-08.In another embodiment, described anti-FGFR2-III c antibody molecule comprises at least one or two variable region of heavy chain from antibody, and described antibody is selected from: for example, and Atto-MuMab-01,

Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06, or Atto-HuMab-08.In other embodiments, described anti-FGFR2-III c antibody molecule comprises at least one or two variable region of light chain from antibody, and described antibody is selected from: for example, Atto-MuMab-01, Atto-MuMab-02, Atto-MuMab-03,

Atto-HuMab-01, Atto-HuMab-06, or Atto-HuMab-08.In another embodiment, described anti-FGFR2-III c antibody molecule comprises at least one from antibody heavy chain variable region, two or three complementary determining regions (CDRs), or especially at least from the amino acid of those CDRs that contact FGFR2-III c, described antibody is selected from: for example, Atto-MuMab-01, Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06, or Atto-HuMab-08.In another embodiment, described anti-FGFR2-III c antibody molecule comprises at least one from antibody chain variable region, two or three complementary determining regions (CDRs), or especially at least from the amino acid of those CDRs of contact FGFR2-III c, described antibody is selected from: for example, and Atto-MuMab-01, Atto-MuMab-02, Atto-MuMab-03

Atto-HuMab-01, Atto-HuMab-06, or Atto-HuMab-08.In another embodiment, described

Anti-FGFR2-III c antibody molecule comprises at least one from heavy chain of antibody and variable region of light chain, two, three, four, five, or six complementary determining regions (CDRs), described antibody is selected from: for example, Atto-MuMab-01, Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06, or Atto-HuMab-08.

In one embodiment, described anti-FGFR2-III c antibody molecule comprises from Atto-MuMab-01 Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06, or all six CDRs or closely-related CDRs of Atto-HuMab-08, for example, identical or have at least one amino acid change, but no more than two, three, or four changes are (for example, replace, disappearance, or insert, for example, CDRs conservative the replacement).Selectively, this protein may comprise any CDR described here.

In another embodiment, described anti-FGFR2-III c antibody molecule comprises at least one from antibody heavy chain variable region, two, or three Chothia hypervariable region rings, or especially at least from the amino acid of those hypervariable region rings of contact FGFR2-III c, described antibody is selected from: Atto-MuMab-01 for example, Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06, or Atto-HuMab-08.In another embodiment, described anti-FGFR2-III c antibody molecule comprises at least one from antibody chain variable region, two, or three hypervariable region rings, or comprise from contact at least

The amino acid of those hypervariable region rings of FGFR2-III c, described antibody is selected from: Atto-MuMab-01 for example,

Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06, or Atto-HuMab-08.In another embodiment, described anti-FGFR2-III c antibody molecule comprises at least one from heavy chain of antibody and variable region of light chain, two, three, four, five, or six hypervariable region rings, described antibody is selected from: Atto-MuMab-01 for example, Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06, or Atto-HuMab-08.In one embodiment, described anti-FGFR2-III c antibody molecule comprises from FGFR2-III c, all six hypervariable region rings, or closely-related hypervariable region ring, for example, identical or have at least one amino acid change, but no more than two, three, or four changes (for example, replace, disappearance, or insert for example, conservative replacement) the hypervariable region ring.Selectively, described anti-FGFR2-III c antibody molecule may comprise any hypervariable region described here ring.

In another embodiment, described anti-FGFR2-III c antibody molecule comprises at least one, two, or three hypervariable region rings, described hypervariable region ring has and Atto-MuMab-01, Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06, or the identical standard construction of hypervariable region ring of Atto-HuMab-08 correspondence, for example, with

Atto-MuMab-01, Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06, or the heavy chain of Atto-HuMab-08 and/or variable region of light chain encircle 1 and/or encircle 2 identical standard constructions at least.Reference for example, is used for describing the people such as Chothia of hypervariable region ring standard construction, (1992) J.Mol.Biol.227:799-817; People such as Tomlinson, (1992) J.Mol.Biol.227:776-798.These structures can be determined by browse the form of describing in these reference.

In one embodiment, the light chain of described anti-FGFR2-III c antibody molecule or weight chain variable framework are (for example, comprise FR1 at least, FR2, FR3, and the district of FR4 selectively) can be selected from: (a) comprise from least 80%, 85%, 87% of people's light chain or variable region of heavy chain framework, 90%, 92%, 93%, 95%, 97%, the light chain of 98% or preferred 100% amino-acid residue or variable region of heavy chain framework are for example from the ripe antibody of people, the light chain of people's embryonal system sequence or the common sequence of people or variable region of heavy chain framework; (b) comprise from people's light chain or variable region of heavy chain framework from 20% to 80%, 40% to 60%, 60% to 90%, or the light chain of 70% to 95% amino-acid residue or variable region of heavy chain framework, for example from the ripe antibody of people, the light chain of people's embryonal system sequence or the common sequence of people or variable region of heavy chain framework; (c) inhuman framework (for example, rodent framework); Or (d) modified inhuman framework, for example remove antigen or cytotoxin factor of determination, for example, go immunity, or the part humanization.In one embodiment, described light chain or weight chain variable framework region (FR1, FR2 and/or FR3) particularly comprise the framework at least 70,75,80,85 with the VH fragment of people's germline gene, 87,88,90,92,94,95,96,97,98,99% identical or identical light chain or weight chain variable framework sequence.

In one embodiment, heavy chain or the variable region of light chain of described anti-FGFR2-III c antibody molecule comprise aminoacid sequence, described aminoacid sequence and antibody described here, Atto-MuMab-01 for example, Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06, or Atto-HuMab-08, variable region at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, 99% or more consistent; Or with antibody described here, Atto-MuMab-01 for example, Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06, or Atto-HuMab-08, the variable region at least 1 or 5 residue arranged but be lower than the difference of 40,30,20 or 10 residues.

In one embodiment, heavy chain or the variable region of light chain of described anti-FGFR2-III c antibody molecule comprise aminoacid sequence, described aminoacid sequence is by nucleotide sequence described here, or with nucleotide sequence described here (for example, the nucleotide sequence of concrete nucleotide sequence or the aminoacid sequence described here of encoding) or its complementary sequence, low rigorous condition described here for example, in rigorous condition, the nucleic acid encoding of hybridizing under high other hybridization conditions of rigorous conditioned disjunction.

In another embodiment, described anti-FGFR2-III c antibody molecule comprises at least one, two, and three or four antigen binding domains, for example, the variable region, described antigen binding domain has Figure 28,29B, 29D, 29F, 30B, 30D, 30F, 35,36A, 36B, 37A, 38, or 39 aminoacid sequences of describing, or sequence essentially identical with it (for example with it at least about 85%, 90%, 95%, 99% or more consistent, or and Figure 28,29B, 29D, 29F, 30B, 30D, 30F, 35,36A, 36B, 37A, 38, or 39 sequences no more than 1,2 of showing, the sequence of 5,10 or 15 amino-acid residue differences) in another embodiment, described anti-FGFR2-III c antibody molecule comprises VH and/or VL territory, described VH and/or VL territory are by having Figure 29 A, 29C, 29E, 30A, 30C, 30E, 34A, 34B, or the nucleotide sequence of 37B description, or sequence essentially identical with it (for example, with it at least about 85%, 90%, 95%, 99% or more consistent sequence, or with Figure 29 A, 29C, 29E, 30A, 30C, 30E, 34A, 34B, or sequence shown in 37 has no more than 3,6,15,30, or 45 Nucleotide difference) nucleic acid encoding.

In another embodiment, described anti-FGFR2-III c antibody molecule comprises at least one that comes from variable region of heavy chain, two or three CDRs, and described variable region of heavy chain has as Figure 28,29F, 30F, 35, or the aminoacid sequence of 38-40 description, or the sequence of homology basic with it (for example, with it at least about 85%, 90%, 95%, 99% or more consistent, and/or have one, two, three, or more replacement, insert, or disappearance, for example conservative sequence that replaces).In another embodiment, described anti-FGFR2-III c antibody molecule comprises at least one that comes from variable region of light chain, two or three CDRs, and described variable region of light chain has as Figure 28,29D, 30D, 35, or the aminoacid sequence of 38-40 description, or the sequence of homology basic with it (for example, with it at least about 85%, 90%, 95%, 99% or more consistent, and/or have one, two, three, or more replacement, insert, or disappearance, for example conservative sequence that replaces).In another embodiment, described anti-FGFR2-III c antibody molecule comprises at least one that comes from heavy chain and variable region of light chain, two, three, four, five or six CDRs, described heavy chain and variable region of light chain have as Figure 28,29D, 29F, 30D, 30F, 35, or the aminoacid sequence described of 38-40, or the sequence of homology basic with it (for example, with it at least about 85%, 90%, 95%, 99% or more consistent, and/or have one, two, three, or more replacement, insert, or disappearance, for example conservative sequence that replaces).

In one embodiment, described anti-FGFR2-III c antibody molecule comprises at least one that comes from variable region of heavy chain, two or three CDRs, described variable region of heavy chain has Atto-HuMab-01 (for example, SEQ ID NOs:147-149, corresponding CDRs1-3 respectively), Atto-HuMab-06 (for example, SEQ ID NOs:147,158,159, distinguish corresponding CDRs1-3), or Atto-HuMab-08 (for example, SEQ ID NOs:147-149, the corresponding CDRs1-3 of difference) aminoacid sequence, as shown in Figure 40, or the sequence of homology basic with it (for example, with it at least about 85%, 90%, 95%, 99% or more consistent, and/or have one, two, three, or more replacement, insert, or disappearance, for example conservative sequence that replaces).In one embodiment, described anti-FGFR2-III c antibody molecule comprises at least one that comes from variable region of light chain, two or three CDRs, described variable region of light chain has Atto-HuMab-01 (for example, SEQ ID NOs:144-146, corresponding CDRs1-3 respectively), Atto-HuMab-06 (for example, SEQ ID NOs:155-157, corresponding CDRs1-3 respectively), or Atto-HuMab-08 (for example, SEQ ID NOs:155,156,146, the corresponding CDRs1-3 of difference) aminoacid sequence, as shown in Figure 40, or the sequence of homology basic with it (for example, with it at least about 85%, 90%, 95%, 99% is identical or more consistent, and/or have one, two, three, or more replacement, insert, or disappearance, for example conservative sequence that replaces).

In another embodiment, described anti-FGFR2-III c antibody molecule comprises at least one from variable region of heavy chain, two or three CDRs, described variable region of heavy chain have the SSYAIS (SEQ ID NO:147) that is selected from following aminoacid sequence: CDR1, or the RIIPI X of CDR2 ₁G X ₂ANYAQKFQGR (SEQ ID NO:153), wherein X ₁Be F or L, and X ₂Be T or I, or the RDX of CDR3 ₁X ₂X ₃W X ₄X ₅X ₆FDX ₇(SEQ ID NO:154), wherein X ₁Be R or P, X ₂Be W or L, X ₃Be D or L, X ₄Be N or S, X ₅Be D or disappearance, X ₆Be A or Y, and X ₇Be I or Y.

In another embodiment, described anti-FGFR2-III c antibody molecule comprises at least one from variable region of light chain, two or three CDRs, and described variable region of light chain has the SGSSSNIG that is selected from following aminoacid sequence: CDR1

X ₁NX ₂VX ₃(SEQ ID NO:150), wherein X ₁Be S or N, X ₂Be T or Y, and X ₃Be S or N, or the X of CDR2 ₁NNX ₂RPSGX ₃(SEQ ID NO:151), wherein X ₁Be S or D, X ₂Be Q or K, and X ₃Be V or I, or the X of CDR3 ₁X ₂WDX ₃SLX ₄X ₅VV (SEQ ID NO:152), wherein X ₁Be A or G, X ₂Be A or T, X ₃Be D or S, X ₄Be N or S, and X ₅Be G or A.

In another embodiment, described anti-FGFR2-III c antibody molecule comprises:

(i) from variable region of heavy chain at least one, two or three CDRs, described variable region of heavy chain have the SSYAIS (SEQ ID NO:147) that is selected from following aminoacid sequence: CDR1, or the RIIPIX of CDR2 ₁GX ₂ANYAQKFQGR (SEQ ID NO:153), wherein X ₁Be F or L, and X ₂Be T or I, or the RD X of CDR3 ₁X ₂X ₃WX ₄X ₅X ₆FDX ₇(SEQ ID NO:154), wherein X ₁Be R or P, X ₂Be W or L, X ₃Be D or L, X ₄Be N or S, X ₅Be D or disappearance, X ₆Be A or Y, and X ₇Be I or Y; And

(ii) from variable region of light chain at least one, two or three CDRs, described variable region of light chain have the SGSSSNIG X that is selected from following aminoacid sequence: CDR1 ₁N X ₂V X ₃(SEQ ID NO:150), wherein X ₁Be S or N, X ₂Be T or Y, and X ₃Be S or N, or the X of CDR2 ₁NN X ₂RPSGX ₃(SEQ ID NO:151), wherein X ₁Be S or D, X ₂Be Q or K, and X ₃Be V or I, or the X of CDR3 ₁X ₂WDX ₃SLX ₄X ₅VV (SEQ ID NO:152), wherein X ₁Be A or G, X ₂Be A or T, X ₃Be D or S, X ₄Be N or S, and X ₅Be G or A.

In one embodiment, described anti-FGFR2-III c antibody molecule comprises described here, for example described six CDRs of Figure 40.Feature of the present invention also is to comprise the nucleic acid of nucleotide sequence of the CDRs of encoding heavy chain and variable region of light chain and described anti-FGFR2-III c antibody molecule, as said.For example, the coding respectively that is characterized as of the present invention

The heavy chain of anti-FGFR2-III c antibody molecule and first or second nucleic acid of variable region of light chain, described anti-FGFR2-III c antibody molecule is selected from: for example, Atto-MuMab-01, Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-HuMab-06, or one or more among the Atto-HuMab-08, as said.For example, described nucleic acid can comprise the A as Figure 29,29C, 29E, 30A, 30C, 30E, 34A, 34B, or the nucleotide sequence of 37B description, or with the sequence of its basically identical (for example, with its at least about 85%, 90%, 95%, 99% is identical or more consistent, or with Figure 29 A, 29C, 29E, 30A, 30C, 30E, 34A, 34B, or the sequence shown in the 37B has no more than 3,6,15,30, or the sequence of 45 Nucleotide differences).In certain embodiments, described nucleic acid comprises that coding comes from least one of variable region of heavy chain, the nucleotide sequence of two or three CDRs, and described variable region of heavy chain has as Figure 28,29F, 30F, 35, or the aminoacid sequence of 38-40 description, or the sequence of homology basic with it (for example, with it at least about 85%, 90%, 95%, 99% is identical or more consistent, and/or have one, two, three, or more replacement, insert, or disappearance, for example conservative sequence that replaces).In another embodiment, described nucleic acid comprises that coding comes from least one of variable region of light chain, the nucleotide sequence of two or three CDRs, and described variable region of light chain has as Figure 28,29D, 30D, 35, or the aminoacid sequence of 38-40 description, or the sequence of homology basic with it (for example, with it at least about 85%, 90%, 95%, 99% is identical or more consistent, and/or have one, two, three, or more replacement, insert, or disappearance, for example conservative sequence that replaces).In another embodiment, described nucleic acid comprises that coding comes from least one of heavy chain and variable region of light chain, two, three, four, the nucleotide sequence of five or six CDRs, described heavy chain and variable region of light chain have as Figure 28,29D, 29F, 30D, 30F, 35, or the aminoacid sequence described of 38-40, or the sequence of homology basic with it (for example, with it at least about 85%, 90%, 95%, 99% is identical or more consistent, and/or have one, two, three, or more replacement, insert, or disappearance, for example conservative sequence that replaces).

On the other hand, the application is characterised in that host cell and the carrier that contains nucleic acid described here.Described nucleic acid may be present in single carrier or divide in other carrier, and described isolated vectors is present in identical host cell or divides in other host cell.

By, the epi-position of the FGFR2-III c of the one or more identifications among Atto-MuMab-01 or the Atto-MuMab-02 for example, for example people FGFR2-III c has feature.In one embodiment, the epi-position of anti-FGFR2-III c antibody molecule comprises at least one amino-acid residue of FGFR2 III c314-353 position (AAGVNTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFH (SEP ID NO:84)), or at least one amino-acid residue of TCLAGNS IGIS FH (SEQ ID NO:86).In one embodiment, people FGFR2-III c epi-position comprises the 1st the L-Ala of SEQ ID NO:84, the 2nd L-Ala, the 4th Xie Ansuan, the 6th Threonine, the 7th Threonine, the 8th aspartic acid, the 9th Methionin, the 11st Isoleucine, the 12nd L-glutamic acid, the 13rd Xie Ansuan, the 15th tyrosine, the 16th Isoleucine, the 17th arginine, the 21st phenylalanine, the 22nd L-glutamic acid, the 28th Threonine, the 30th leucine, the 31st L-Ala, the 32nd glycine, the 34th Serine, the 37th Isoleucine, the 38th Serine, the 39th phenylalanine, or the 40th one in hyte propylhomoserin, two, three, four, five, six, seven, eight, nine, ten, ten two, 14,16,18,20,22 or more (with

In an embodiment, described antibody molecule suppresses, and reduces or the described isomer that neutralizes, for example one or more activity of the carcinogenic isomer in height hyperplasia (for example, cancer or tumour) cell and/or the tissue.For example, described antibody molecule is alternative and reduce specifically or suppress to be selected from one or more carcinogenic isomer related activity in following: (i) binding partner or co-receptor (for example, FGF part, (FGF8b for example, FGF2, FGF17 or FGF18) be bonded to FGFR2 isomer III c); (ii) receptor dimerizationization (for example, FGFR2 isomer III c dimerization); (iii) receptor signal transduction, for example, FGFR2 isomer III c signal transduction; (iv) height hyperplasia (for example cancer or tumour) cell proliferation, growth and/or survival, for example, by inducing height proliferative cell apoptosis; And/or (the v) vasculogenesis of tumour and/or vascularization.In certain embodiments, this antibody molecule is bonded to one or more cytotoxic agents or cytostatics or group, medicine for example, send the compound of radiation, the molecule of plant, fungi or bacterial origin, or bioprotein (for example, proteotoxin) or particle (for example recombinant virus particle of recombinating by viral capsid proteins).The conjugated antibodies molecule be bonded to be positioned at that one or more cancers or tumour cell or disease are significantly expressed or relevant exon sequence or the epi-position on the bonding land (for example epi-position described here) after, conjugated antibodies molecular selectivity ground target or conveying cytotoxic agent or cytostatics are to height hyperplasia (for example, cancer or tumour) cell and/or tissue.In other embodiments, thereby described antibody molecule can use separately with uncombined form and pass through, for example, antibody dependent cellular is committed suiside machine-processed, (for example reduce the height hyperplasia such as the cytolysis of complement-mediated and/or the cell suicide of effector cell's mediation, cancer or tumour) cell and/or tissue activity (for example, cell growth or propagation) and/or kill height hyperplasia (for example, cancer or tumour) cell and/or tissue.In other embodiments, described antibody molecule can disturb in the cell and interact, for example, isomer, carcinogenic isomer for example, with the combination of homoreceptor or part, thus the activity that reduces or hinder height hyperplasia (for example, cancer or tumour) cell and/or tissue.For example, the described antibody molecule that optionally is bonded to the exon III c of FGFR2 can reduce or suppress FGFR2 isomer III c and its part, for example: FGF8b, FGF2, FGF17 or FGF18, in one or more interactions, thereby reduce FGFR2 isomer Illc express cell propagation and/or survival.

It should be understood that, described antibody molecule described here, solubility or fusion rotein, polypeptide can functionally be connected or derive (for example by chemical coupling with the nucleic acid inhibitor, gene fusion, non-covalent combination or other) one or more other molecular entity, such as antibody (for example, dual specific or multi-specificity antibody), toxin, radio isotope, cytotoxic agent or cytostatics, mark etc.For example, described antibody molecule described here, solubility or fusion rotein, polypeptide and nucleic acid inhibitor can be bonded to mark, such as fluorescent mark, biological activity enzyme labelling, radio isotope (for example, isotopic ion), the nucleus magnetic resonance activity mark, luminescent marking, or chromophore.In other embodiments, described antibody molecule described here, solubility or fusion rotein and polypeptide can be bonded to therapeutical agent, for example, cytotoxicity part, for example molecule of medicine, radio isotope, plant, fungi or bacterial origin, or bioprotein (for example, proteotoxin), or particle (for example recombinant virus particle of recombinating by viral capsid proteins), or its mixture.Described therapeutical agent can be pharmacological activation or other reagent in the cell, such as the short-range radiation body, comprises, for example short distance high-energy alpha emitter described here.In some preferred embodiments, described antibody molecule described here, solubility or fusion rotein and polypeptide can be bonded to the molecule (or derivatives thereof) of plant or bacterial origin; Ansamitocin for example, Taxan, calicheamicin.Radio isotope can for α-, β-or gamma emitter or β-and gamma emitter.As the radio isotope of therapeutical agent comprise yttrium ( ⁹⁰Y), lutetium ( ¹⁷⁷Lu), actinium ( ²²⁵Ac), praseodymium, astatine ( ²¹¹At), rhenium ( ¹⁸⁶Re), bismuth ( ²¹²Bi or ²¹³Bi), rhodium ( ¹⁸⁸Rh).With marking, for example be used for diagnosis, radio isotope comprise, iodine ( ¹³¹I or ¹²⁵I), indium ( ¹¹¹In), technetium ( ⁹⁹MTc), phosphorus ( ³²P), carbon ( ¹⁴C), and tritium ( ³H).Described antibody molecule described here, solubility or fusion rotein and polypeptide also can be connected to other antibody, thereby form, for example dual specific or multi-specificity antibody.

On the other hand, the invention provides, composition, for example, and pharmaceutical composition and at least a isomer specific inhibitor described here, described pharmaceutical composition comprises the medicinal carrier of accepting, vehicle or stablizer.In one embodiment, described isomer specific inhibitor is bonded to mark or therapeutical agent.In one embodiment, described composition, for example pharmaceutical composition comprises the two or more combination in the aforementioned isomer specific inhibitor, or different antibody molecules.For example, composition for example, comprises the pharmaceutical composition of isomer specific inhibitor described here, with other growth factor receptor inhibitors, such as at FGF1-23, FGF acceptor 1-4, VEGF, EGF or EGF acceptor, PSMA, or Her-2/neu, etc. antibody, in conjunction with.Isomer specific inhibitor and medicine, for example therapeutical agent (for example, cytotoxic drugs or cytostatic medicament, for example, DM1, calicheamicin, Taxan, topoisomerase enzyme inhibitor, or immunomodulator, for example, IL-1,2,4,6 or 12, interferon alpha or γ, or immune cell growth factor are such as GM-CSF) combination also within the scope of the invention.

Feature of the present invention also be the to encode nucleotide sequence of isomer binding molecule described here.For example, the invention is characterized in first and second nucleic acid of the heavy chain of the modification of the antibody molecule described here of encoding respectively and variable region of light chain.In other embodiments, the invention provides coding soluble receptors described here, fusions, the nucleotide sequence of polypeptide and functional analogue thereof.On the other hand, the invention is characterized in host cell and the carrier that comprises nucleic acid of the present invention.Described host cell may be eukaryotic cell, mammalian cell for example, and insect cell, yeast cell, or prokaryotic cell prokaryocyte, for example, intestinal bacteria (E.coli).For example mammalian cell may be cultured cells or clone.Typical Mammals comprise lymphocyte series (for example, NSO), Chinese hamster ovary cell (CHO), the COS cell, ovocyte and come from the cells of transgenic animal, for example, mammary gland upper epidermis cell.For example, the encode nucleic acid of isomer binding molecule described here can be expressed in transgenic animal.In one embodiment, described nucleic acid is located under the control of tissue-specific promoter's (for example mammary gland-specific promotor), and described antibody produces in transgenic animal.For example, described isomer binding molecule is secreted in the milk of transgenic animal, such as, transgenic cattle, pig, horse, sheep, goat or rodent.

On the one hand, the invention is characterized in, a kind of method for preparing isomer binding antibody molecule, described isomer binding antibody molecular specificity is bonded to isomer (for example, carcinogenic isomer) polypeptide.Described method comprises: isomer specific antigens (antigen that for example, comprises at least a portion of epi-position described here) is provided; Obtain the antibody molecule that specificity is bonded to the isomer polypeptide; And whether specificity (for example is bonded to the isomer polypeptide to assess antibody molecule, when there is one or more epi-position described here in assessment, whether the combination between described antibody molecule and the described isomer polypeptide descends), or assess described antibody molecule and regulating, for example suppress the effect of isomer (for example carcinogenic isomer) polypeptide.Described method can further comprise makes experimental subjects, and for example, people or non-human animal take described antibody molecule.

Isomer specificity epitope described here, for example the epi-position of Fen Liing is also contained among the present invention.Described epi-position may for isomer linearity or described () conformation albumen for example, carcinogenic isomer, for example about 2 to 80, about 4 to 75, about 5 to 70, about 10 to 60, about 10 to 50, about 10 to 40, about 10 to 30, about 10 to 20 amino-acid residues.In certain embodiments, described epi-position comprises, or the aminoacid sequence of the bonding land between two exons that comprises, described exon one or more cancers or tumour cell or disease express or relevant isomer protein in combine significantly, for example, because the use that the interior Exon deletion of framework or selectivity are sheared exon.For example.Described epi-position can contain or comprise the aminoacid sequence consistent with the selectivity shear-form of exon III, for example, from about 301 to 360 amino acid of FGFR2-III c (SEQ ID NO:2), amino acid (the AAGVNTTDKEI that FGFR2-III c is about 314 to 324, SEQ ID NO:4), amino acid (the YIRNVTFEDA that FGFR2-III c is about 328 to 337, SEQ ID NO:6), amino acid (the ISFH that FGFR2-III c is about 350 to 353, SEQ ID NO:8), or by SEQ ID NOs:1,3,5 or 7 nucleotide sequence coded amino acid, or with amino acid or the nucleotide sequence of its basically identical.In other embodiments, FGFR2-III c for example, people FGFR2-III c, epi-position may comprise or comprise FGFR2 III c314-353 position

At least one amino-acid residue of (AAGVNTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFH (SEQ ID NO:84)), or at least one amino-acid residue of TCLAGNS IGIS FH (SEQ ID NO:86).In one embodiment, people FGFR2-III c epi-position comprises the 1st the L-Ala of SEQ ID NO:84, the 2nd L-Ala, the 4th Xie Ansuan, the 6th Threonine, the 7th Threonine, the 8th aspartic acid, the 9th Methionin, the 11st Isoleucine, the 12nd L-glutamic acid, the 13rd Xie Ansuan, the 15th tyrosine, the 16th Isoleucine, the 17th arginine, the 21st phenylalanine, the 22nd L-glutamic acid, the 28th Threonine, the 30th leucine, the 31st L-Ala, the 32nd glycine, the 34th Serine, the 37th Isoleucine, the 38th Serine, the 39th phenylalanine, or the 40th one in hyte propylhomoserin, two, three, four, five, six, seven, eight, nine, ten, ten two, 14,16,18,20,22 or more (with

Feature of the present invention also is a kind of reduction height proliferative cell, for example, cancer or tumour cell (for example, are expressed carcinogenic isomer, such as FGFR2-III C described here and FGFR1, RON, Κ IT, cancer or the tumour cell of the Exon deletion isomer of PDGF and PDGFR-α) active (for example, cell growth or propagation), or induce the commit suiside method of (for example, apoptosis-induced) of height proliferative cell.Described method comprises with one or more isomer specific inhibitors described here to be enough to reduce isomer, the for example expression of carcinogenic isomer or active amount contact height proliferative cell, or the cell of close height proliferative cell (for example, vascular cell), thereby reduce the activity of height proliferative cell, or kill the height proliferative cell.Isomer specific inhibitor described here can with in conjunction with or uncombined form be used alone as monotherapy or be combined with one or more therapeutical agents, thereby kill the height proliferative cell or reduce the highly activity of proliferative cell, for example suppress the growth of height proliferative cell.

In an embodiment, described isomer binding molecule is the antibody molecule that specificity is bonded to FGFR2-III c, for example, specificity is bonded to the aminoacid sequence consistent with the selectivity shear-form of exon III, for example from about 301 to 360 amino acid of FGFR2-III C (SEQ ID NO:2), amino acid (the AAGVNTTDKEI that FGFR2-III c is about 314 to 324, SEQ ID NO:4), amino acid (the YIRNVTFEDA that FGFR2-III c is about 328 to 337, SEQ ID NO:6), amino acid (the ISFH that FGFR2-III c is about 350 to 353, SEQ ID NO:8), or by SEQ ID NOs:1,3,5 or 7 nucleotide sequence coded amino acid, or with amino acid or the Nucleotide of its basically identical.In such embodiments, described height proliferative cell is for coming from prostate gland, mammary gland, pancreas, ovary, brain (glioblastoma multiforme), cancer of the stomach, squamous cell lung carcinoma, nonsmall-cell lung cancer, thyroid carcinoma, carcinoma of endometrium, hematopoietic system cancer, and skeletal diseases are such as craniofacial dysostosis 1, Crewe ancestor syndromes (Crouzon syndrome), luxuriant and rich with fragrance Buddhist syndromes (Pfeiffer syndrome), Jackson-Wei Si syndromes (Jackson-Weiss syndrome) and A Peier syndromes (Apert syndrome), cancer or tumour cell.

Described method for example can be used for the cell at external or indirect culturing in vivo.For example, (for example, cancer or tumour cell are (for example for the height proliferative cell, prostatic, kidney, urothelial (for example, bladder), testis, ovary, mammary gland, colon, rectum, lung (for example, nonsmall-cell lung cancer), liver, brain, neural (for example, neuroendocrine), neuroglial (for example, glioblastoma multiforme), pancreas, melanoma (for example, malignant melanoma), or soft tissue sarcoma's cancer or metastatic cell)) can in in-vitro culture medium, cultivate, and contact procedure can realize by increasing isomer binding molecule to substratum.Alternatively, described method can be carried out at the height proliferative cell that is present in experimental subjects, as the part of (for example, treatment or prophylactic) scheme in the body.

Method of the present invention can be used for, for example, it is described here by experimental subjects is taken, be enough to treat or prevent the amount of these diseases, the treatment of isomer specific inhibitor or prevention height proliferative disease, for example, prostate gland, mammary gland, pancreas and brain (glioblastoma multiforme) cancer (former, recurrence or transfer).In one embodiment, described cancer is gland cancer or prostate cancer and/or tumor of testis.For example, described cancer is anti-hormone or intractable prostate cancer.In one embodiment, described cancer is anti-male sex hormone or the intractable prostate cancer relevant with FGFR2-III c high expression level.For example, described cancer with respect to reference value (has for example been showed, non-cancer prostata tissue) high level or FGFR2-III c albumen or the mRNA that expresses, selectively, follow the decline (for example, the level of surface epithelial cell adhesion molecule (Ep-CAM) or expression descend and/or the increase of mesenchymal cell mark) of one or more epithelium marks.In certain embodiments, described cancer is metastatic carcinoma, and described metastatic carcinoma has been showed the circulating tumor cell that high-caliber prostate gland derives (for example, the circulation FGFR2 III c that derives of prostate gland expresses prostate tumor cells).Method and composition described here is particularly useful to the treatment metastasis relevant with prostate cancer.In certain embodiments, the patient will carry out prostatectomy, chemotherapy, or in other antineoplaston one or more, and main or unique target is metastasis, for example, the transfer in marrow or lymphoglandula.

In other embodiments, the described cancer with isomer specific inhibitor treatment described here includes, but not limited to solid tumor, soft tissue neoplasm, and metastasis.The example of solid tumor comprises malignant tumour, for example, and the sarcoma of various tracts, gland cancer and cancer influence lung such as those, mammary gland, lymph, (for example, the colon) of stomach and intestine, sexual organ and urogenital tract (for example, kidney, urothelial, the bladder cell), pharynx, CNS(is for example, brain, neural or neurogliocyte), skin is (for example, melanoma), and pancreas, and comprise malignant tumour, such as, most of colorectal carcinomas, the rectum cancer, renal cell carcinoma, liver cancer, lung nonsmall-cell lung cancer, the gland cancer of carcinoma of small intestine and the esophageal carcinoma.Method and composition described here is particularly useful to the treatment metastasis relevant with above-mentioned cancer.In certain embodiments, described patient will organize excision, chemotherapy, or in other anticancer therapy one or more, and main or unique target is metastasis, for example, the transfer in marrow or lymphoglandula.For example, the expression of the carcinogenic isomer of FGFR2-III c or active decline can be used for preventing and/or treating the intractable prostate cancer of hormone, mammary cancer, bladder cancer, the cancer of thyroid carcinoma or other form.

In one embodiment, treat described experimental subjects with prevention height proliferative disease, for example, height proliferative disease described here.Described experimental subjects may be Mammals, for example, and primate, preferably, senior primate, for example, people's (for example, have maybe may have for example patient of prostate cancer disease of height proliferative disease described here).In one embodiment, described experimental subjects is the patient (patient who for example, suffers from recurrence or metastatic prostate cancer) with prostate cancer.Described subjects can be the people with described disease risks, for example, described disease had relevant worried object, for example, one or more objects of suffering from described disease among its grand parents/grand parents, father and mother, uncle/uncle (mother's brother) or aunt/one's mother's sister, siblings or the child, or the object with inherited character relevant with described disease risks.In one embodiment, described experimental subjects may be for Symptomatic or asymptomatic.For example, described experimental subjects may be subjected to Symptomatic or asymptomatic prostate cancer, for example suffers from anti-hormone or intractable prostate cancer.In certain embodiments, described experimental subjects suffers from metastatic prostate cancer.In some experiments, described experimental subjects has the high-caliber prostate gland circulating tumor cell (for example, prostate gland derive circulation FGFR2 III c express prostate tumor cells) of deriving.In other embodiments, the cancer that described experimental subjects has one or more normal levels is prostate cancer for example, mark.For example; described experimental subjects has the prostate specific antigen (PSA) of normal level; prostate specific membrane antigen (PSMA); prostate stem cell antigen (PSCA); androgen receptor (AR), chromogranin, synaptophysin; MIB-1, and/or α-formyl radical coenzyme A racemase (AMACR).

Experimental subjects can be capapie (for example, oral, administered parenterally, subcutaneous administration, intravenous injection, rectal administration, muscle administration, intraperitoneal administration, nose administration, percutaneous dosing or by suck or the chamber in insert), partly, or by applying to mucous membrane, such as nose, throat and segmental bronchus are taken isomer specific inhibitor described here.

Method of the present invention, for example, the method for the treatment of or prevention, the step that can further comprise the monitoring experiment object, for example, below change one or more in (increase or descend): the tumour size, the level of cancer mark (for example, the patient's of prostate cancer FGFR2 III c level or expression, the level of the FGFR2 III c express cell that the circulation prostate gland is derived, epithelial cell mark (Ep-CAM), the FGF part (for example, FGF8), stroma cell derivative factor (SDFa), VEGF(for example, VEGF121), the mesenchymal cell mark, PSA, PSMA, PSCA, AR, chromogranin, synaptophysin, MIB-1, AMACR, alkaline phosphatase, and/or serum oxyphorase; The speed that new focus occurs, for example, in bone scanning; The appearance of new disease related symptom; Or the size of soft mass, for example, reduce or stabilization; Quality of life, disease-related amount of pain for example, for example, ostalgia; Or any other clinical effectiveness correlation parameter.Described experimental subjects can be monitored in following one or more stages: before the treatment beginning, and during the treatment, or after having carried out one or more treatment elements.Monitoring can be used for assessing the needs of further treating or treating in addition with other reagent with identical isomer binding molecule.Usually, the decline of one or more parameters mentioned above is symbols of experimental subjects symptom improvement, although the improvement of the symptom of experimental subjects may be followed the raising of serum hemoglobin level.

Method of the present invention can further comprise analysis from the step of nucleic acid or the protein of experimental subjects, for example, analyzes the genotype of experimental subjects.In one embodiment, the analysis of encoding isomer, the upstream of for example carcinogenic isomer, and/or isomer signal transduction or the nucleic acid of downstream components, for example, incitant or supressor in the extracellular of isomer or the cell.This analysis can be used for, for example, estimate optionally treatment, for example, concrete dosage, transport model, delivery time, the content of assisting therapy, for example be combined with second reagent and take, well-formedness, or between above-mentioned optional treatment, select, or determine possible drug reaction phenotype or the genotype of experimental subjects widely.Can be in any stage for the treatment of, but preferably take isomer specific inhibitor analysis of nucleic acids or protein before, thereby be identified for suitable isomer specific inhibitor dosage and the treatment plan (for example, the consumption of each treatment or the frequency for the treatment of) of experimental subjects preventing disease or treatment disease.

Isomer specific inhibitor (for example isomer specificity combinating reagent) can uncombined form use separately, thereby pass through, for example antibody dependent cellular is committed suiside machine-processed, such as the cell suicide of the cytolysis of complement-mediated and/or effector cell's mediation, reduce isomer and express the active of height hyperplasia or cancer cells or induce isomer to express the suicide of height hyperplasia or cancer cells.In other embodiments, described isomer specific inhibitor can be bonded to substrate, for example cytotoxic agent or group are (for example, medicine, send the compound of radiation, the molecule of plant, fungi or bacterial origin, or bioprotein is (for example, or the particle recombinant virus particle of viral capsid proteins reorganization (for example by) proteotoxin).For example, the isomer specific inhibitor can be bonded to radio isotope, such as α-, β-or gamma emitter or β-and gamma emitter.Radioisotopic example comprise iodine ( ¹³¹I or ¹²⁵I), yttrium ( ⁹⁰Y), lutetium ( ¹⁷⁷Lu), actinium ( ²²⁵Ac), praseodymium or bismuth ( ²¹²Bi or ²¹³Bi).Optionally, described isomer binding molecule can be bonded to bioprotein, the molecule of plant or bacterial origin (or derivatives thereof), for example ansamitocin (for example, maytansinol or DM1), and Taxan (for example, taxol or taxotere), calicheamicin.Described ansamitocin can for, for example, maytansinol or maytansinol analogue.The example of maytansinol analogue comprises (for example having the property of modification aromatic nucleus, C-19-dechlorination base, the C-20-demethoxy, the C-20-acyloxy) and at other position (for example, C-9-CH, the C-14-alkoxyl-methyl, C-14-methylol or acetoxy-methyl, C-15-hydroxyl/acyloxy, C-15-methoxyl group, C-18-N-demethyl 4,5-deoxidation) those of modification is arranged.For example, U.S. Patent number 6,333,410 have described maytansinol or maytansinol analogue, quote its content as a reference at this.Calicheamicin can for, for example, bromine mixture calicheamicin (for example, α, beta, gamma, bromine mixture), Surgidine calicheamicin (for example, α, beta, gamma, Surgidine), or its analogue and imitated thing.Bromine mixture calicheamicin comprises α ₁-BR, α ₂-BR, α ₃-BR, α ₄-BR, β ₁-BR, β ₂-BR and γ ₁-BR.The Surgidine calicheamicin comprises α ₁-I, α ₂-I, α ₃-I, β ₁-Ι, β ₂-Ι, δ ₁-Ι and γ ₁-BR.For example, the U.S. Patent number of announcing November 13 nineteen ninety 4,970, the U.S. Patent number 5,264 that on November 23rd, 198,1993 announced, the U.S. Patent number 5,550 that on August 27th, 586,1996 announced, the U.S. Patent number 5,712 that on January 27th, 246,1998 announced, the U.S. Patent number 5,714 that on February 3rd, 374 and 1998 announced, 586 have described calicheamicin and mutant thereof, and analogue and imitated thing are quoted its content as a reference at this.Maytansinol can be bonded to antibody, described antibody uses, for example, N-succinimido 3-(2-pyridine disulfide group) propionic ester (being also referred to as N-succinimido 4-(2-pyridine disulfide group) valeric acid or SPP), 4-succinimido-carbonyl oxygen base-a-(2-pyridine disulfide group) toluene (SMPT), N-succinimido 3-(2-pyridine disulfide group) butyric acid (SDPB), 2-imido thia pentane, or S-acetyl succinyl oxide.

Method and composition of the present invention can be used in combination with other therapeutic modality.In one embodiment, method of the present invention comprises that experimental subjects is taken is described here, effectively treats or prevent the amount of described disease, in conjunction with the isomer specific inhibitor of cytotoxic agent.Described binding molecule and cytotoxic agent can simultaneously or be taken in succession.In other embodiments, method and composition of the present invention is used in combination with operation and/or radiative process.In other embodiments, described method can with immunomodulator, for example, IL-1,2,4,6 or 12, or interferon alpha or γ, or immune cell growth factor, such as GM-CSF, be used in combination.The typical cytotoxic agent of taking of being combined with the isomer specific inhibitor comprises anti-microtubule agent, topoisomerase enzyme inhibitor, metabolic antagonist, mitotic inhibitor, alkylating agent, intercalator, can disturb the reagent of signal transduction pathway, promotes reagent and the radiation of apoptosis.

In prostatosis, prostate cancer for example, treatment in, the isomer specific inhibitor can with existing therapeutic modality, for example, prostatectomy (local or radical cure), radiotherapy, hormonotherapy, male sex hormone ablation and cytotoxicity chemotherapy are used in combination.Usually, hormonotherapy is for reducing patient's male sex hormone level, and may comprise and (for example take luteinizing hormone-releasing hormone (LHRH) analogue or stimulant, leuprorelin acetate (Lupron), Zoladex (Zoladex), Leuprolide (leuprolide), buserelin (buserelin), and antagonist (for example, abarelix (Abarelix)) or goserelin (goserelin)).Non-sterol androgen antagonist, for example, flutamide (flutamide), bicalutamide (bicalutimade), or Nilutamide (nilutamide), also can be used for hormonotherapy, and sterol androgen antagonist (for example, cyproterone acetate or Magace), oestrogenic hormon (for example, stilboestrol), surgical operation castrating

Secondary or three grades of hormone regulating and controllings are (for example, comprise reflunomide (for example, hydrocortisone (hydrocortisone), prednisone (prednisone) or dexamethasone (dexamethasone),) first KETOKONAZOL (ketoconazole), and/or aminoglutethimide (aminogluthethimide)), 5 alpha reductase inhibitors (for example, finasteride (finisteride)), Chinese medicine preparation (for example, PC-SPES), pituitectomy, and suprarenalectomy.In addition, hormonotherapy can be carried out off and on, or adopts in conjunction with above-mentioned any treatment, for example is used in combination Leuprolide and flutamide and carries out.

Isomer specific inhibitor and other treatment mode can be used in combination or use in succession.Described isomer specific inhibitor and other therapeutic modality can be in the disease activation phase, or alleviate or take during the minimizing stage in that disease is active.Above-mentioned isomer specific inhibitor and other therapeutic modality can in the time for the treatment of, after the treatment, or be taken during disease alleviates before treatment.

On the other hand, the invention is characterized in for detection of (for example being present in vitro samples, biological sample, serum for example, seminal fluid or urine, or biopsy, for example, from height hyperplasia or carninomatosis kitchen range) in isomer (for example carcinogenic isomer described here) polypeptide or gene expression product.Topic is stated method and be can be used for assessment (for example, to the disease of experimental subjects described here, for example height hyperplasia or cancer disease carried out monitor therapy or progress, diagnosis and/or by stages).Described method comprises: (i) allowing that isomer binding molecule and described polypeptide or gene expression product take place under the interactional situation, with isomer binding molecule described here (for example antibody molecule) contact sample (and optionally, standard model, for example control sample); And the formation that (ii) detects the mixture between isomer binding molecule and the sample (and optionally, standard model, for example control sample).The formation of mixture is the symbol that polypeptide or gene expression product exist, and can indicate suitability and the demand for the treatment of described here.For example, with respect to standard model, control sample for example, the statistical significant change that mixture forms in the sample is isomer in the sample, for example, the symbol that carcinogenic isomer exists.In certain embodiments, described method comprises use more than a kind of isomer binding molecule, for example, is bonded to identical carcinogenic isomer (for example, FGFR2 isomer III c), or two kinds of antibody molecules of the different epi-positions on the different carcinogenic isomer.For example, described method may comprise immunohistochemistry, immunocytochemistry, FACS, the compound magnetic bead of antibody molecule, elisa assay, round pcr (for example, RT-PCR), for example, as described in the appended embodiment.

On the other hand, the present invention is by isomer (for example carcinogenic isomer described here) polypeptide in a kind of detection bodies or the method for gene expression product (for example in-vivo imaging of experimental subjects).Described method is used in the experimental subjects, Mammals for example, primate for example, for example human (for example, to disease described here, for example height hyperplasia or cancer disease are carried out monitor therapy or progress, diagnosis and/or by stages) assessment.Described method comprises: i) allowing that isomer binding molecule and polypeptide or gene expression product take place under the interactional situation, making experimental subjects take isomer binding molecule (for example antibody molecule described here); And the formation that (ii) detects the mixture between isomer binding molecule and polypeptide or the gene expression product.With respect to standard model, the bottom line of control sample or experimental subjects for example, the statistical significant change of the formation of mixture is the symbol that polypeptide or gene expression product exist in the sample.

In other embodiments, provide a kind of appraisal procedure (for example, to height hyperplasia or the cancer disease of experimental subjects described here, monitor therapy or progress, the diagnosis and/or the stage).Described method comprises: (i) identification suffers from described disease, may suffer from the experimental subjects of described disease; (ii) obtain to be subjected to the tissue of described sickness influence or the sample of cell; (iii) allowing that described binding molecule and isomer polypeptide or product take place under the interactional situation, with isomer binding molecule described here, for example antibody molecule described here contacts described sample or control sample; And the formation that (iv) detects mixture.With respect to standard model, control sample for example, the statistical significant change of the formation of mixture is the symbol in described disease or described stage.

Typically, thus in vivo with the in-vitro diagnosis method in the isomer binding molecule that uses be conducive in conjunction with wedding agent or not in conjunction with the detection of wedding agent with detectable material mark directly or indirectly.Suitable detectable substance comprises various bioactive enzymes, prothetic group, fluorescent material, luminescent material, paramagnetic (for example, the nucleus magnetic resonance activity) material, and radio active material.In certain embodiments, the isomer binding molecule is bonded to isotopic ion, for example, indium ( ¹¹¹In), iodine ( ¹³¹I or ¹²⁵I), yttrium ( ⁹⁰Y), lutetium ( ¹⁷⁷Lu), actinium ( ²²⁵Ac), bismuth ( ²¹²Bi or ²¹³Bi), sulphur ( ³⁵S), carbon ( ¹⁴C), and tritium ( ³H), rhodium ( ¹⁸⁸Rh), technetium ( ⁹⁹MTc), praseodymium, phosphorus ( ³²P).

Detection/diagnostic method described here can further comprise the step of monitoring experiment object, for example, below in one or more variations (increase or descend): the tumour size, the level of cancer markers (for example, the patient's of prostate cancer FGFR2 III c level or expression, the level of the FGFR2 III c express cell that the circulation prostate gland is derived, epithelial cell mark (Ep-CAM), the FGF part (for example, FGF8), matrix derivative factor α (SDF α), VEGF(for example, VEGF121), the mesenchymal cell mark, PSA, PSMA, PSCA, AR, chromogranin, synaptophysin, MIB-1, AMACR, alkaline phosphatase, and/or serum oxyphorase), the speed that new focus occurs, for example, in bone scanning, the appearance of new disease related symptom, or the size of soft mass, for example, reduce or stabilization, quality of life, disease-related amount of pain for example, for example, ostalgia, or any other clinical effectiveness correlation parameter.Can be in the following described experimental subjects of interim monitoring when one or more: before the treatment beginning, during the treatment, carried out one or more treatment elements after.Monitoring can be used for assessing the needs of further treating or treating in addition with other reagent with identical isomer binding molecule.Usually, the decline of one or more parameters mentioned above is symbols of the symptom improvement of experimental subjects, although the improvement of the symptom of experimental subjects may be followed the raising of serum hemoglobin level.

On the other hand, the invention is characterized in diagnosis or therapeutical agent box, described diagnosis or therapeutical agent box comprise diagnosis or the therapeutical agent box of isomer specific inhibitor described here and working instructions.

Article " one " refers to one (kind) or more than the object of the grammatical of one (kind) (for example, at least one (kind)) as used herein.

As used herein term " or " mean term " and/or " and with term " and/or " alternately use, unless context particularly points out.

Term " protein " and " polypeptide " alternately use.

" approximately " and " approaching " generally means under the situation of given measurement character or accuracy, the acceptable degree of the error of the quantity that records.The typical degree of error is at percent 20(% of the scope of set-point or value) in, typically, in 10%, and more typically, in 5%.

Quote all announcements referred in this, patent application, the full text of patent and other reference is as a reference.

Further feature of the present invention, purpose and advantage are clearer in the description of specification, drawings and the claims.

Description of drawings

Fig. 1 has described the isomer structure of FGFR2 receptor tyrosine kinase.Above: isomer III b expresses in the normal prostatic epithelial cell.Below: isomer III c expresses in the intractable prostate cancer of hormone.TM=strides film; The AB=acidic region; I, II or III=Ig sample ring I, II or III.

Fig. 2 has described the sequence alignment of III c isomer (SEQ ID NO:2) and III b isomer (SEQ ID NO:65).

Fig. 3 A has described the aminoacid sequence (SEQ ID NO:19) of people FGFR2 III c.

Fig. 3 B has described the nucleotide sequence (SEQ ID NO:20) of people FGFR2 III c.

Fig. 4 A has described the nucleotide sequence (SEQ ID NO:1) of FGFR2Exon-III c.

Fig. 4 B has described the nucleotide sequence (SEQ ID NO:64) of FGFR2Exon-III b.

Fig. 5 A has described aminoacid sequence (SEQ ID NO:4) and the nucleotide sequence (SEQ ID NO:3) of peptide III c-314.

Fig. 5 B has described aminoacid sequence (SEQ ID NO:6) and the nucleotide sequence (SEQ ID NO:5) of peptide III c-328.

Fig. 5 C has described aminoacid sequence (SEQ ID NO:8) and the nucleotide sequence (SEQ ID NO:7) of peptide III c-350.

Fig. 6 A has described III b (Loop3-C) fragment: the aminoacid sequence of amino acid 314-351 (SEQ ID NO:56) and nucleotide sequence (SEQ ID NO:60).

Fig. 6 B has described III b epi-position: the aminoacid sequence of amino acid 314-328 (SEQ ID NO:57) and nucleotide sequence (SEQ ID NO:61).

Fig. 6 C III b epi-position: the aminoacid sequence of amino acid 340-351 (SEQ ID NO:58) and nucleotide sequence (SEQ ID NO:62).

Fig. 7 has described the isomer structure of FGFR1.

The aminoacid sequence of the FGFR1L epitope sequences that Fig. 8 has described in the bonding part (SEQ ID NO:9) and nucleotide sequence (SEQ ID NO:10).

Fig. 9 has described aminoacid sequence (SEQ ID NO:11) and the nucleotide sequence (SEQ ID NO:12) of the RONA160 epi-position of the bonding land between exon 4 and exon 7.

Figure 10 has described aminoacid sequence (SEQ ID NO:13) and the nucleotide sequence (SEQ ID NO:14) of the epitope that designs for antibody target KIT isomer.

Figure 11 has described nucleotide sequence (SEQ ID NO:15) and the aminoacid sequence (SEQ ID NO:16) of the epi-position that designs for antibody target PDGF isomer.

Figure 12 has described nucleotide sequence (SEQ ID NO:17) and the aminoacid sequence (SEQ ID NO:18) of the epi-position of PDGFR-αYi Gouti.

Figure 13 A has described the structure of solubility FGFR2 III c-Fc fusion rotein.

Figure 13 B has described the nucleotide sequence (SEQ ID NO:54) of solubility FGFR2 III c-Fc fusion rotein.

Figure 13 C has described the aminoacid sequence (SEQ ID NO:55) of solubility FGFR2 III c-Fc fusion rotein.Signal peptide is corresponding to the amino acid/11 to 21. of SEQ ID NO:55

Figure 13 D has described the Western blot that the SDS-PAGE of the CHO stable cell lines of the recombination fusion protein of expressing solubility FGFR2 III c-Fc analyzes.

Figure 13 E has described the histogram that soluble receptors FGFR2 β-ECD is combined with the FGF8b part.

Figure 13 F has described the linear graph that mouse-anti Atto-MuMab-03 suppresses the GF8b part that FGFR2 III c is bonded to.

Figure 14 has described the sequence alignment from FGFR2 receptor immunoglobulin sample ring 3 zones of people and rat.The C end of ring 3 is by the exon 8 that produces III c (shown in the black matrix) (the 6-53 position residue of SEQ ID NO:2 and SEQ ID NO:67) and produces; Perhaps by the exon 9 that produces III b (italic) (the 6-51 position residue of SEQ ID NO:65 and SEQ ID NO:68) and generation.People and rat sequence are 100% consistent in these zones.

Figure 15 has described the dual-target strategy of FGFR2 acceptor.The outer ligand-binding site point of the born of the same parents of antibody A b-1 receptor targeted; And tyrosine-kinase prime field in TKI (for example RO4383596 or Pazopanib) the target born of the same parents.

Figure 16 has described the PCR primer that is used for analyzing FGFR2 III c and III b isomer.

Figure 17 A has described the aminoacid sequence (SEQ ID NO:32) of people FGFR2 gene.

Figure 17 B-17C has described aminoacid sequence (SEQ ID NO:21) and the nucleotide sequence (SEQ ID NO:63) of people FGFR2 III b respectively.

Figure 17 D – 17O has described the aminoacid sequence (SEQ ID NO:22) of people FGFR2 isomer 4 respectively, the aminoacid sequence of isomer 7 (SEQ ID NO:23), the aminoacid sequence of isomer 9 (SEQ ID NO:24), the aminoacid sequence of isomer 10 (SEQ ID NO:25), the aminoacid sequence of isomer 11 (SEQ ID NO:26), the aminoacid sequence of isomer 12 (SEQ ID NO:27), the aminoacid sequence of isomer 13 (SEQ ID NO:28), the aminoacid sequence of isomer 14 (SEQ ID NO:29), the aminoacid sequence of isomer 15 (SEQ ID NO:30), the aminoacid sequence of isomer 17 (SEQ ID NO:31), the aminoacid sequence (SEQ ID NO:53) of the aminoacid sequence of isomer 18 (SEQ ID NO:52) and isomer 19.Figure 18 A has described the amino acid (SEQ ID NO:33) of people FGFR1 gene.

Figure 18 B-18H has described the aminoacid sequence (SEQ ID NO:38) of people FGFR1 isomer 1 respectively, the aminoacid sequence of isomer 4 (SEQ ID NO:39), the aminoacid sequence of isomer 14 (SEQ ID NO:40), the aminoacid sequence of isomer 16 (SEQ ID NO:41), the aminoacid sequence of isomer 17 (SEQ ID NO:42), the aminoacid sequence (SEQ ID NO:44) of the aminoacid sequence of isomer 3 (SEQ ID NO:43) and isomer 18.

Figure 19 A has described the aminoacid sequence (SEQ ID NO:34) of people RON gene.

Figure 19 B has described the aminoacid sequence (SEQ ID NO:45) of the non-cancer RON isomer of people.

Figure 20 A has described the aminoacid sequence (SEQ ID NO:35) of people KIT gene.

Figure 20 B has described the aminoacid sequence (SEQ ID NO:46) of the people KIT variant that has lacked exons 11.

Figure 20 C has described the aminoacid sequence (SEQ ID NO:47) of people's total length KIT.

Figure 21 A has described the aminoacid sequence (SEQ ID NO:36) of people PDGF gene.

Figure 21 B has described the aminoacid sequence (SEQ ID NO:48) of people PDGF isomer 2.

Figure 21 C has described the aminoacid sequence (SEQ ID NO:49) of people's total length PDGF.

Figure 22 A has described the aminoacid sequence (SEQ ID NO:37) of people PDGFR-α gene.

Figure 22 B has described the aminoacid sequence (SEQ ID NO:50) of people PDGFR-αYi Gouti 1.

Figure 22 C has described the aminoacid sequence (SEQ ID NO:51) of the people PDGFR-αYi Gouti that has lacked exon 7-8.

Figure 23 has described the aminoacid sequence (SEQ ID NO:66) of people FGF8.

Figure 24 A compares for having described with being bonded to FGFR2 III b, monoclonal antibody clone B7, and C5, D2, D10, E3, E8, F3, F10 and G9 bind selectively to the histogram of FGFR2 III c-Fc.Clone B7 refers to " Atto-MuMab-03 " at this.Figure 24 B compares for having described with being bonded to FGFR2 III b, and the monoclonal antibody designated clone is bonded to (comprising Atto-MuMab-03) OD value and the standard deviation table of FGFR2 III c-Fc.

Figure 25 A compares for having described with being bonded to the contrast of uncorrelated people's Protalbinic acid (OA) and people's ferritin (Fer), and people's antibody scFv clone 1-8 group binds selectively to the histogram in ring 3 districts of the FGFR2 III c isomer that contains 128 amino acid (235-353 amino acids).

Figure 25 B compares for having described with being bonded to uncorrelated OA and Fer contrast, and people's antibody scFv clone group is bonded to OD value and the standard deviation table of 128 amino acid whose fragments of FGFR2 III c (mFc).

Figure 26 is the picture at the people antibody scFv clone's different with 30 kinds of FGFR2 III c dna fingerprint.

Figure 27 A be people's antibody scFv clonal selection of having described appointment be bonded to FGFR2 isomer III c contrast be bonded to FGFR2 III b histogram.

Figure 27 B is that the people scFv clonal selection of having described appointment is bonded to OD value and the standard deviation table that isomer III c contrast is bonded to FGFR2 III b.

Figure 28 has described the aminoacid sequence comparison of people scFv clone 6 (SEQ ID NO:160) and clone 8 (SEQ ID NO:161).There is underscore the position of the complementarity determining region of light chain and variable region of heavy chain (CDRs) and is labeled as VL-CDR-1 respectively, and-2 and-3, and VH-CDR-1 ,-2 and-3.Joint sequence is added with shade.Clone 6 and clone 8 also refer to " Atto-HuMab-06 " and " Atto-HuMab-08 " respectively at this.

Figure 29 A-29B has described nucleotide sequence (SEQ ID NO:162) and the aminoacid sequence (SEQ ID NO:161) of the full length coding region of people scFv clone's 8 (Atto-HuMab-08) variable region of light chain and variable region of heavy chain respectively.

Figure 29 C-29D has described nucleotide sequence (SEQ ID NO:164) and the aminoacid sequence (SEQ ID NO:163) of people scFv clone's 8 (Atto-HuMab-08) variable region of light chain respectively.(SEQ ID NOS:184-186 is the nucleotide sequence of corresponding CDRs1-3 respectively for the CDR sequence; SEQ ID NOS:155,156 and 146 aminoacid sequences of corresponding CDRs1-3 respectively) underscore is arranged.

Figure 29 E-29F has described nucleotide sequence (SEQ ID NO:166) and the aminoacid sequence (SEQ ID NO:165) of people scFv clone's 8 (Atto-HuMab-08) variable region of heavy chain respectively.(SEQ ID NOS:187-189 is the nucleotide sequence of corresponding CDRs 1-3 respectively for the CDR sequence; The aminoacid sequence of the corresponding CDRs1-3 of SEQ ID NOS:147-149 difference) underscore is arranged.

Figure 30 A-30B has described nucleotide sequence (SEQ ID NO:167) and the aminoacid sequence (SEQ ID NO:160) of the full length coding region of people scFv clone's 6 (Atto-HuMab-06) variable region of light chain and variable region of heavy chain respectively.

Figure 30 C-30D has described nucleotide sequence (SEQ ID NO:169) and the aminoacid sequence (SEQ ID NO:168) of scFv clone's 6 (Atto-HuMab-06) variable region of light chain respectively.(SEQ ID NOS:178-180 is the nucleotide sequence of corresponding CDRs1-3 respectively for the CDR sequence; The aminoacid sequence of the corresponding CDRs1-3 of SEQ ID NOS:155-157 difference) underscore is arranged.

Figure 30 E-30F has described nucleotide sequence (SEQ ID NO:171) and the aminoacid sequence (SEQ ID NO:170) of scFv clone's 6 (Atto-HuMab-06) variable region of heavy chain respectively.(SEQ ID NOS:181-183 is the nucleotide sequence of corresponding CDRs 1-3 respectively for the CDR sequence; SEQ ID NOS:147,158 and 159 aminoacid sequences of corresponding CDRs1-3 respectively) underscore is arranged.

Figure 31 is the table of having described the library screening pnagus medius clone rate of recovery.

Figure 32 is for describing the picture that monoclonal antibody Atto-MuMab-03 is bonded to the western blotting (Western blot) of endogenous FGFR2 III c in the tumour cell.

Figure 33 has described western blotting, and this Western blotting has showed that mAb Atto-MuMab-01 is bonded to the endogenous FGFR2 III c albumen among the prostate cancer cell line DU145.Band 1: from the DU145 cell lysates of 50,000 cells; Band 2: from the mAb IP of the lysate of 500,000 DU145 cells; Band 3: blank; Band 4:FGFR2 III c-Fc (100ng) over against photograph.

Figure 34 A has described nucleotide sequence (the SEQ ID NO:87 of Atto-MuMab-02 variable region of heavy chain; 358bp).Nucleotide sequence and the framework region of CDRs (SEQ ID NOS:91, the corresponding CDRs1-3 of 93 and 95 difference) are showed with underscore and italic respectively.

Figure 34 B has described nucleotide sequence (the SEQ ID NO:89 of Atto-MuMab-02 variable region of light chain; 324bp).Nucleotide sequence and the framework region of CDRs (SEQ ID NOS:97, the corresponding CDRs1-3 of 99 and 101 difference) are showed with underscore and italic respectively.

Figure 35 has described aminoacid sequence (the SEQ ID NO:90 of Atto-MuMab-02 variable region of light chain; 21-LC) and aminoacid sequence (the SEQ ID NO:88 of variable region of heavy chain; 21-HC).SEQ ID NOS:98, the corresponding VL CDRs1-3 of 100 and 102 difference.SEQ ID NOS:92, the corresponding VH CDRs1-3 of 94 and 96 difference.

Figure 36 A has described the aminoacid sequence comparison of Atto-MuMab-02 and mouse immuning ball protein μ chain.21HC：

Atto-MuMab-02 immunoglobulin heavy chain variable region (SEQ ID NO:88); Experimental subjects: immunoglobulin mu chain [house mouse] (SEQ ID NO:142; GenBank:AAA88255.1).

Figure 36 B has described the aminoacid sequence comparison of Atto-MuMab-02 variable region of light chain and mouse-anti human melanoma light chain immunoglobulin.21LC:Atto-MuMab-02 variable region of light chain (the 1-106 position residue of SEQ ID NO:90); Experimental subjects: anti-human melanoma light chain immunoglobulin [house mouse] (SEQ ID NO:143; GenBank:AA049727.1).

Figure 37 A-37B has described aminoacid sequence (SEQ ID NO:190) and the nucleotide sequence (SEQ ID NO:191) of the full length coding region of people scFv clone's 1 (scFv-1 also is expressed as " Atto-HuMab-01 " at this) variable region of light chain and variable region of heavy chain respectively.Joint draws shade.The CDR sequence draws underscore, and SEQ ID NOS:144-146 is the aminoacid sequence of corresponding VL CDRs1-3 respectively.SEQ ID NOS:147-149 is the aminoacid sequence of corresponding VH CDRs1-3 respectively.SEQ ID NOS:172-174 is the nucleotide sequence of corresponding VL CDRs1-3 respectively.SEQ ID NOS:175-177 is the nucleotide sequence of corresponding VH CDRs1-3 respectively.

Figure 38 has described the aminoacid sequence comparison of people scFv clone 1 (scFv-1, SEQ ID NO:142) and clone 6 (scFv-6, SEQ ID NO:160).The position of the complementary determining region of light chain and variable region of heavy chain (CDRs) draws underscore and is labeled as VL-CDR-1 respectively, and-2 and-3, and VH-CDR-1 ,-2 and-3.Joint sequence draws shade.Clone 1 and clone 6 also are expressed as " Atto-HuMab-01 " and " Atto-HuMab-06 " respectively at this.

Figure 39 has described people scFv clone 1 (scFv-1, SEQ ID NO:142) and clone 8 (scFv-8; SEQ ID NO:161) aminoacid sequence comparison.The position of the complementary determining region of light chain and variable region of heavy chain (CDRs) draws underscore and is labeled as VL-CDR-1 respectively, and-2 and-3, and VH-CDR-1 ,-2 and-3.Joint sequence draws shade.Clone 1 and clone 6 also are expressed as " Atto-HuMab-01 " and " Atto-HuMab-06 " respectively at this.

Figure 40 is for having described people scFv clone scFv-1, the CDR district homology contrast between scFv-6 and the scFv-8.Unique amino-acid residue draws underscore.The amino-acid residue that has sequence homology between two among three clones is represented with italic.The amino-acid residue of homology is with conventional font representation between all three clones.

Figure 41 has described people scFv clone 1 (scFv-1) and has been bonded to the painted image of immunocytochemistry (ICC) that FGFR2 III c expresses Chinese hamster ovary celI.

The Fc that Figure 42 has described by definite people scFv clone 1 (dcFv-01) of fluorescence activated cell isolation technique (FACS) merges the graphic representation of being combined with FGFR2 III c expression Chinese hamster ovary celI.

Figure 43 has described the painted image of immunocytochemistry (ICC).The painted people scFv that showed of described immunocytochemistry (ICC) clones the Fc fusion of 1 (dcFv-01) and the combination of rat prostate cancerous cell line AT3B-1.

Figure 44 has described by the fluorescence activated cell isolation technique and has determined that the Fc of people scFv clone 1 (dcFv-01) of (FACS) merges the image of being combined with rat prostate cancerous cell line AT3B-1.

Specific embodiment

The present invention provides the isomer specific inhibitor at least in part, and this isomer specific inhibitor suppresses or reduces one or more isomer related activity.In certain embodiments, this isomer (for example, polypeptide or nucleic acid isomer) is expressed by carcinogenic or malignant phenotype (representing with carcinogenic isomer at this) and/or is relevant with carcinogenic or malignant phenotype (representing with carcinogenic isomer at this).For example, this isomer can be from for example, alternative splicing, and frameshit, the one or more generations in translation and/or the translation back event, thus cause different transcribing or translation product.In one embodiment, described isomer specific inhibitor is the isomer binding molecule, for example antibody molecule, or nucleic acid inhibitor.In another embodiment, described isomer specific inhibitor is soluble receptors polypeptide and fusion form thereof, or peptide and functional variant thereof.For example, described isomer specific inhibitor can be carcinogenic isomer binding molecule, for example, antibody molecule or nucleic acid inhibitor, this antibody molecule or nucleic acid inhibitor specificity and one or more carcinogenic isomer are (for example, the nucleic acid of carcinogenic isomer polypeptide or the carcinogenic isomer polypeptide of encoding) interact, for example, in conjunction with.In another embodiment, described isomer specific inhibitor is soluble receptors polypeptide or its fusion form that reduces or suppress one or more isomer (for example, carcinogenic isomer) related activity, or peptide or its functional variant.In an embodiment, described soluble receptors or fusions reduce or suppress the interaction of (for example, competition ground suppresses) isomer (for example, carcinogenic isomer) polypeptide and its cognate ligand and acceptor.

Described carcinogenic isomer can be from for example cell, for example various expression of proto-oncogenes product selectivity montage in the high proliferative cell (for example cancer or tumour cell), and frameshit produces in translation and/or the translation back event.Described isomer binding molecule described here is bonded to these carcinogenic isomer, but basic debond is to the dominant non-carcinogenic sequence of the proto-oncogene that derives described isomer.

In the context of protein or polypeptide, refer to can be from alternative splicing for term " isomer " as used herein, frameshit, the polymer of amino acid of any length of deriving in translation and/or the translation back event.The alternative splicing event is included in the intramolecular one or more selectivity exons of given RNA (part of the gene of coded protein) thereby combination is produced the process (during transcribing) of different mRNA by identical gene.Each this mRNA is called as " gene transcripts ".Usually, because the cell or tissue specificity causes different exon combinations, several different mRNA transcripts of individual gene codified.For example, the various ways of fibroblast growth factor acceptor 1-3 (FGFR1-3) is considered to be produced by the alternative splicing of mRNA.The montage event frequently that relates to FGFR1 and 2 produces acceptor, and described acceptor comprises three immunoglobulin (Ig)s (ig) territory, is commonly called αYi Gouti, or only immunoglobulin (Ig) II (Ig II) and Ig III, is called as beta isomer.Described αYi Gouti is identified as FGFR3 and FGFR4.Be referred to herein as FGFR III b and FGFR2 III c, the FGF acceptor that has a selectivity Ig III territory by the montage event of the FGFR1-3 of the C end in the Ig III territory that relates to two selectivity exons codings that repel mutually of being derived by the FGFR2 gene produce (referring to, Galzie, Z. wait the people, (1997) Biochem.Cell.Biol.75:669-685; Burke, people such as D. (1998) Trends Biochem Sci23:59-62).FGFR2-III c uses selectivity exon III, and the sequence of described exon III coding is different with the sequence of FGFR2-III b coding.Other reason/source of alternative splicing comprises frameshit (that is, rrna is translated as different groups with three codons in the mRNA/ transcript) or changes translation initiation or closed position, causes false intron to be retained in the mRNA transcript.Therefore different body tissues is relevant with the alternative splicing event with some diseases, and produces different protein in different tissues or ill tissue.

" carcinogenic isomer " refers to may be by alternative splicing, frameshit, and one or more any protein of deriving in translation and/or the translation back event, polypeptide, mRNA, or cDNA, its existence or abnormal level are relevant with cancer or malignant phenotype.For example, with reference value, for example tissue or the cell of the contrast of non-disease are compared, and find to be subjected to the abnormal level in the cell of tissue derived of sickness influence.May be protein isomer with unusual high level expression, the expression of variation and the appearance of cancer and/or progress are related.Carcinogenic isomer also may be the expression product of mutator gene or heritable variation, the cause of disease of this mutator gene or heritable variation gene pairs cancer bear direct liability or with linkage disequilibrium to responsible other gene of the cause of disease of cancer in.Typical carcinogenic isomer includes but not limited to, FGFR2(for example, carcinogenic FGFR2 isomer III c), FGFR1(carcinogenic FGFR1L for example), the RON receptor tyrosine kinase (for example, disappearance exon 5 and 6 carcinogenic RON receptor tyrosine kinase), KIT receptor tyrosine kinase (for example, the carcinogenic KIT receptor tyrosine kinase of disappearance exons 1 1) and pdgf receptor α (for example lacking the carcinogenic pdgf receptor α of exon 7 and 8)

Simply, " non-carcinogenic isomer " or " non-carcinogenic proto-oncogene " refers to the main protein of finding in non-cancer cells or tissue, polypeptide, mRNA or cDNA.These isomer and proto-oncogene may reach in pernicious state of an illness following table, but not relevant with the malignant phenotype usually.

The compositions and methods of the invention comprise polypeptide and nucleic acid, described polypeptide and nucleic acid have specific sequence or to its basically identical or similar sequence, for example with the identical or more consistent sequence of specific sequence at least 85%, 90%, 95%.In the context of aminoacid sequence, as used herein term " basically identical " or " basic identical " refer to first aminoacid sequence comprise with second aminoacid sequence in the amino acid i of comparison) consistent, or ii) conservative replacement, enough or minimum amino acid, thereby make first and second aminoacid sequences can have identical structural domain and/or identical functions activity.For example, comprise with reference sequences at least about 85%, 90%.91%, the amino acid in 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical ordinary construction territory, SEQ ID NO:2 for example, SEQ ID NO:4 or SEQ ID NO:6, for basically identical.

In the context of Nucleotide, as used herein term " basically identical " or " basic identical " refer to first aminoacid sequence comprise with second nucleotide sequence in the nucleic acid i of comparison) consistent/identical, or ii) conservative replacement, enough or minimum nucleic acid residue, thereby the polypeptide that makes first and second nucleic acid sequence encodings have the general function activity, or coding ordinary construction polypeptide domain or have the polypeptide of general function polypeptide active.For example, with reference sequences at least about 85%, 90%.91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical nucleotide sequence, for example, SEQ ID NO:1,3 or 5 is essentially identical.

Term " functional variant " refers to have with the aminoacid sequence of the sequence basically identical that generates naturally, or nucleotide sequence coded by basically identical, and can have one or more active polypeptide of the sequence that nature generates.

The calculating of the homology between the sequence or sequence identity (at this, this term can be used alternatingly) is undertaken by following.

For determining the per-cent consistence of two aminoacid sequences or two nucleotide sequences, for optimum compares purpose, aligned sequences (for example, for the optimum comparison, in one or two in first and second amino acid of gap introducing or the nucleotide sequence, and for purpose relatively, non-homogeneous sequence can be left in the basket).In a preferred embodiment, the length of the reference sequences of comparing for purpose relatively be at least reference sequences length 30%, preferably at least 40%, more preferably at least 50%, 60%, and more preferably at least 70%, 80%, 90%, 100%.Then relatively at the amino acid sites of correspondence or amino-acid residue or the Nucleotide of nucleotide site.When the site of first sequence was occupied by the amino-acid residue of corresponding site unanimity on second sequence or Nucleotide, this molecule was consistent (amino acid or nucleic acid " consistence " are equal to amino acid or nucleic acid " homology " as used herein) in this site

Per-cent consistence between the two sequences is subjected to the influence of the quantity in the total consistent site of sequence, has considered to compare the quantity in the gap that needs introducing and the length in each gap for the optimum of two sequences.

The comparison of sequence and the per-cent between the two sequences are conforming to be determined and can finish with mathematical algorithm.In a preferred embodiment, determine two per-cent consistence between the aminoacid sequence with Needleman and Wunsch algorithm ((1970) J.Mol.Biol.48:444-453).Needleman and Wunsch algorithm have been included in the GAP program of GCG software package (referring to http://www.gcg.com), and the GAP program is used Blossum62 matrix or PAM250 matrix and

gap weight

16,14,12,10,8,6 or 4 and

length weight

1,2,3,4,5 or 6.In another preferred embodiment, determine two per-cent consistence between the nucleotide sequence with the GAP program of GCG software package (referring to http://www.gcg.com), the GAP program is used NWSgapdna.CMP matrix and

gap weight

40,50,60,70 or 80 and

length weight

1,2,3,4,5 or 6.Concrete preferred setting the (and the parameter that should use except specifying) of parameter is 12 as the gap point penalty, the gap extend point penalty be 4 and frameshit gap point penalty be 5 the Blossum62 matrix of giving a mark.

Article two, the per-cent consistence of amino acid or nucleotide sequence can be used E.Meyers and W.Miller ((1989) CABIOS, 4:11-17) algorithm is determined, E.Meyers and W.Miller algorithm be included in use PAM120 weight residue table, gap length point penalty be 12 and the gap point penalty be in 4 the ALIGN program (version 2 .0).

Nucleic acid described here and protein sequence can be used as " search sequence " thereby carry out the common data library searching, for example, identify other family member or correlated series.These search can be with people such as Altschul, and the NBLAST of (1990) J.Mol.Biol.215:403-10 and XBLAST program (version 2 .0) are carried out.The BLAST nucleotide search can be with the NBLAST program, and the nucleotide sequence with nucleic acid of the present invention (SEQ ID NO:1) molecule homology is carried out obtaining in score value=100, word length=12.BLAST albumen search can be with the XBLAST program, and the aminoacid sequence with protein molecule homology of the present invention is carried out obtaining in score value=50, word length=3.In order to obtain for the relatively gap comparison of purpose, the gap BLAST that can use people such as Altschul in (1997) Nucleic Acids Res.25:3389-3402, to describe.When using BLAST and gap BLAST, can use the default parameter of each program (for example XBLAST and NBLAST).Referring to http://www.ncbi.nlm.nih.gov.

As used herein term " low rigorous condition, in rigorous condition, the hybridization under the very high rigorous condition of high rigorous conditioned disjunction " hybridization and elution requirement described.Up-to-date experimental methods of molecular biology compilation (Current Protocols in Molecular Biology) John Wiley﹠amp; Sons, N.Y. (1989) has the guide that carries out hybridization on the 6.3.1-6.3.6, quote its content as a reference.Describe water method and nonaqueous phase method in this reference, can use a kind of in these two kinds of methods.At this, as follows in this concrete hybridization conditions that refers to: 1) low rigorous hybridization conditions is 6X sodium chloride/sodium citrate (SSC), 45 ° of C, and subsequently with 0.2X SSC, 0.1%SDS, at least 50 ° of twice of C wash-outs (eluting temperature can be increased to 55 ° of C under the low rigorous condition); 2) rigorous hybridization conditions is 6X SSC in, about 45 ° of C, subsequently with 0.2X SSC, 0.1%SDS 60 ° of C wash-outs once or more times; 3) high rigorous hybridization conditions is 6X SSC, about 45 ° of C, subsequently with 0.2X SSC, 0.1%SDS, 65 ° of C wash-outs once or more times; And more preferably 4) very high rigorous hybridization conditions is the 0.5M sodium phosphate, 7%SDS, 65 ° of C, subsequently with 0.2X SSC, 1%SDS, 65 ° of C wash-outs once or more times.Very high rigorous condition (4) is optimum condition and is the condition that should use except specifying.

It should be understood that molecule of the present invention may have the replacement of other conservative or non-essential amino acid, this does not have substantial influence on their function.

Term " amino acid " means that comprise nature no matter or synthetic, contains amino functional and acid function, and can be contained in all molecules in the polymer of amino acid that nature generates.Typical amino acid comprises the amino acid that nature generates, and analogue, derivative be with similar, has amino acid analogue and any steric isomer before of the side chain of variation.Term " amino acid " comprises D-or L-optically active isomer and simulating peptide as used herein.

" conserved amino acid replacement " is that the amino-acid residue that amino-acid residue is had a similar side chain is replaced.Defined the family of the amino-acid residue with similar side chain in this area.These families comprise having basic side chain (for example, Methionin, arginine, Histidine), acid side-chain (for example, aspartic acid, L-glutamic acid), uncharged polar side chain is (for example, glycine, l-asparagine acid, glutamine, Serine, Threonine, tyrosine, halfcystine), non-polar sidechain (for example, L-Ala, Xie Ansuan, leucine, Isoleucine, proline(Pro), phenylalanine, methionine(Met), tryptophane), β branched building block (for example, Threonine, Xie Ansuan, Isoleucine) and aromatic series side chain (for example, tyrosine, phenylalanine, tryptophane, Histidine).

Term " polypeptide ", " peptide " and " protein " (if strand) can be used alternatingly at this, refers to the polymer of amino acid of any length.This polymkeric substance may for linearity or branch, it may comprise the amino acid of modification, and it may be interrupted by non-amino acid.This term also may comprise modified aminoacid polymers, and for example, disulfide linkage forms, glycosylation, and lipidization, acetylize, phosphorylation, or other operation are such as being combined with the set of tags compound.Term " polypeptide " refers to the two or more amino acid that connect by the peptide bond between an amino acid whose α carboxyl and another the amino acid whose α amino as used herein.Polypeptide can separate by natural resources, can be the product that produces by recombinant technology from eucaryon or protokaryon host, or can be the product of building-up process.

Term " nucleic acid ", " nucleotide sequence ", " nucleotide sequence " or " polynucleotide sequence " and " polynucleotide " can be used alternatingly.It refers to the polymerized form of the Nucleotide of any length, deoxyribonucleotide or ribonucleotide or its analogue.Polynucleotide can be strand or two strands, and strand can be coding strand or non-coding (antisense) chain.Polynucleotide can have three-dimensional structure, and can carry out various known or unknown functions.Below be the non-limitative example of polynucleotide: the coding of gene or gene fragment or non-coding region, the site (site) that linkage analysis is determined, exon, intron, messenger RNA(mRNA) (mRNA), transfer RNA (tRNA), ribosome-RNA(rRNA), ribozyme, cDNA, recombination of polynucleotide, branch polynucleotide, plasmid, carrier, the separated DNA of any sequence, the RNA of the separation of any sequence, nucleic acid probe and primer.Polynucleotide may comprise the Nucleotide of modification, such as methylated nucleotide and nucleotide analog.If exist, can before or after the polymkeric substance assembling, carry out the modification of nucleotide structure.Nucleotide sequence can be interrupted by the non-nucleotide composition.Nucleotide, is further modified such as by after the set of tags compound is combined in polymerization.This nucleic acid can be recombination of polynucleotide, or genomic, and cDNA is semisynthetic, or is not the polynucleotide nature generation or that be connected to the synthetic origin of another non-polynucleotide of arranging naturally.

" oligonucleotide " refers to have and is less than about 100 Nucleotide, is less than the strand polynucleotide of about 75,50,25 or 10 Nucleotide." oligonucleotide " refers to oligomer or the polymkeric substance of Yeast Nucleic Acid (RNA) and thymus nucleic acid (DNA) or its analogue as used herein.This term comprises by the base that generates naturally, and the oligonucleotide that sugar and covalency nucleosides (skeleton) key are formed and have non-generation naturally has the oligonucleotide of the part of identity function.Because the character of wanting absorbs as enhanced cell, the nucleic acid target target avidity of raising and the stability in the presence of nuclease of raising, oligonucleotide this modification or that replace is better than the oligonucleotide of natural form usually.

Term " separation " or " separation " refer to the material that removes from its original or natural environment (for example, physical environment generates if it is nature) as used herein.For example, the polynucleotide of Sheng Chenging or the polypeptide that is present in the animal that lives do not separate naturally, but the identical polynucleotide or the polypeptide that separate by the some or all of coexistence materials of human intervention from natural system separate.This polynucleotide can be the part that the part of carrier and/or this polynucleotide or polypeptide can be composition, when described carrier or composition and non-ambient (this environment is the environment when occurring in nature is found described carrier or composition) a part of, then described polynucleotide or polypeptide remain separation.

All respects of the present invention describe in further detail hereinafter.Complementary definition runs through in full.

The polypeptide of carcinogenic isomer or its epi-position

The invention provides the polypeptide of carcinogenic isomer or its epi-position, or the sequence consistent with it roughly.Term " epi-position " or " epi-position fragment " refer to that the preferential also specificity of antibody molecule is combined in the zone on the antigen.Monoclonal antibody preferentially is bonded on the molecule defined epitope that can define at a molecular level.The epi-position of concrete albumen or isomer protein may be made up of the amino-acid residue of limited quantity, for example on albumen or isomer protein with 2-30 residue of linearity or nonlinear tissue.Epi-position by antibody recognition may for, for example across 2-30 amino acid whose small peptide of the bonding part of two territories of non-existent carcinogenic isomer in the normal isomer of albumen or two polypeptide fragments.Carcinogenic isomer may lack one or more exons for the RNA with respect to the normal protein of encoding or have the translation product of the RNA alternative splicing variant of extra exon.This epi-position may contain or comprise the NOs:10 at SEQ ID, any one 15-16 in 12,14 or 18,15-17,15-18,15-19,15-20,15-21,15-22,15-23,15-24,15-25,15-26,15-27,15-28, the residue of 15-29 or 15-30 position.In another embodiment, this epi-position may contain or comprise the NOs:10 at SEQ ID, any one 14-16 in 12,14 or 18,14-17,14-18,14-19,14-20,14-21,14-22,14-23,14-24,14-25,14-26,14-27,14-28, the residue of 14-29 or 14-30 position.In another embodiment, this epi-position may contain or comprise the NOs:10 at SEQ ID, any one 13-16 in 12,14 or 18,13-17,13-18,13-19,13-20,13-21,13-22,13-23,13-24,13-25,13-26,13-27,13-28, the residue of 13-29 or 13-30 position.In another embodiment, this epi-position may contain or comprise the NOs:10 at SEQ ID, any one 12-16 in 12,14 or 18,12-17,12-18,12-19,12-20,12-21,12-22,12-23,12-24,12-25,12-26,12-27,12-28, the residue of 12-29 or 12-30 position.In another embodiment, this epi-position may contain or comprise the NOs:10 at SEQ ID, any one 11-16 in 12,14 or 18,11-17,11-18,11-19,11-20,11-21,11-22,11-23,11-24,11-25,11-26,11-27,11-28, the residue of 11-29 or 11-30 position.In another embodiment, this epi-position may contain or comprise the NOs:10 at SEQ ID, any one 10-16 in 12,14 or 18,10-17,10-18,10-19,10-20,10-21,10-22,10-23,10-24,10-25,10-26,10-27,10-28, the residue of 10-29 or 10-30 position.In another embodiment, this epi-position may contain or comprise the NOs:10 at SEQ ID, any one 9-16 in 12,14 or 18,9-17,9-18,9-19,9-20,9-21,9-22,9-23,9-24,9-25,9-26,9-27,9-28, the residue of 9-29 or 9-30 position.In another embodiment, this epi-position may contain or comprise the NOs:10 at SEQ ID, any one 8-16 in 12,14 or 18,8-17,8-18,8-19,8-20,8-21,8-22,8-23,8-24,8-25,8-26,8-27,8-28, the residue of 8-29 or 8-30 position.In another embodiment, this epi-position may contain or comprise the NOs:10 at SEQ ID, any one 7-16 in 12,14 or 18,7-17,7-18,7-19,7-20,7-21,7-22,7-23,7-24,7-25,7-26,7-27,7-28, the residue of 7-29 or 7-30 position.In another embodiment, this epi-position may contain or comprise the NOs:10 at SEQ ID, any one 6-16 in 12,14 or 18,6-17,6-18,6-19,6-20,6-21,6-22,6-23,6-24,6-25,6-26,6-27,6-28, the residue of 6-29 or 6-30 position.In another embodiment, this epi-position may contain or comprise the NOs:10 at SEQ ID, any one 5-16 in 12,14 or 18,5-17,5-18,5-19,5-20,5-21,5-22,5-23,5-24,5-25,5-26,5-27,5-28, the residue of 5-29 or 5-30 position.In another embodiment, this epi-position may contain or comprise the NOs:10 at SEQ ID, any one 4-16 in 12,14 or 18,4-17,4-18,4-19,4-20,4-21,4-22,4-23,4-24,4-25,4-26,4-27,4-28, the residue of 4-29 or 4-30 position.In another embodiment, this epi-position may contain or comprise the NOs:10 at SEQ ID, any one 3-16 in 12,14 or 18,3-17,3-18,3-19,3-20,3-21,3-22,3-23,3-24,3-25,3-26,3-27,3-28, the residue of 3-29 or 3-30 position.In another embodiment, this epi-position may contain or comprise the NOs:10 at SEQ ID, any one 2-16 in 12,14 or 18,2-17,2-18,2-19,2-20,2-21,2-22,2-23,2-24,2-25,2-26,2-27,2-28, the residue of 2-29 or 2-30 position.In another embodiment, this epi-position may contain or comprise the NOs:10 at SEQ ID, any one 1-16 in 12,14 or 18,1-17,1-18,1-19,1-20,1-21,1-22,1-23,1-24,1-25,1-26,1-27,1-28, the residue of 1-29 or 1-30 position." epi-position " can be for generation of specificity in conjunction with the antibody of carcinogenic isomer (for example the basic identical proto-oncogene of debond derive non-carcinogenic isomer).

In one embodiment, the invention provides the isolated polypeptide of the carcinogenic isomer of people or its epi-position.In one embodiment, isomer or its epi-position are the carcinogenic form that is selected from following proto-oncogene: people FGFR2 (SEQ ID NO:32), people FGFR1 (SEQ ID NO:33), people RON receptor tyrosine kinase (SEQ ID NO:34), people KIT receptor tyrosine kinase (SEQ ID NO:35), people PDGF (SEQ ID NO:36) and people PDGFR-α (SEQ ID NO:37) or with the sequence of its basically identical.

In one embodiment, the invention provides the rat polypeptide of the separation of carcinogenic isomer or its epi-position.In one embodiment, the invention provides the mouse polypeptide of the separation of the carcinogenic isomer of people or its epi-position.In other embodiments, the isolated polypeptide of the carcinogenic isomer of people or its epi-position can derive from other species, includes but not limited to dog, pig, cavy and rabbit.

FGFR2

Fibroblast growth factor acceptor 2(FGFR2) is also referred to as bacterial expression kinases (BEK) in this area, keratinocyte growth factor acceptor (KGFR), JWS, CEK3, CFDl, ECTl, TK14, TK25, BFR-1, CD332, K-SAM and FLJ98662.FGFR2 is the member of fibroblast growth factor acceptor family and to acidity, alkalescence and/or keratinocyte growth factor have high-affinity.FGFR2 is relevant with the signal transduction that causes mitotic division and differentiation.FGFR2 sudden change and craniofacial dysostosis 1, Crewe ancestor syndromes (Crouzon syndrome), luxuriant and rich with fragrance Buddhist syndromes (Pfeiffer syndrome), Jackson-Wei Si syndromes (Jackson-Weiss syndrome) is relevant with A Peier syndromes (Apert syndrome).

For example, people such as Dionne, (1990) EMBO J.9:2685-2692 with people such as Miki, (1992) PNAS89:246-250 discloses Nucleotide and the protein sequence of people FGFR2.For example, people such as Miki, people such as (1991) Science251:72-75 and Mansukhani, (1992) PNAS89:3305-3309 discloses Nucleotide and the protein sequence of mouse FGFR2.The undressed forebody length of people FGFR2 is that about 821 amino acid and molecular weight are about 90310Da.The undressed forebody length of mouse FGFR2 is that about 821 amino acid and molecular weight are about 90310Da.

In one embodiment, the invention provides by the carcinogenic isomer of nucleic acid encoding or the isolated polypeptide of its epi-position, described nucleic acid comprises one section Nucleotide that the selectivity of the exon III of the nucleic acid that derives from coding FGFR2 is used.In one embodiment, when comparing with FGFR2 III b, the selectivity of exon III is used and is caused from the sequence variation in 301-360 amino acids zone.Therefore, in one embodiment, described polypeptide contains or comprises and is selected from SEQ NOs:2,4,6 and 8 sequence.In another embodiment, described polypeptide contains or comprises and is selected from SEQ NOs:1, the sequence of 3,5 and 7 nucleic acid encoding, or with its consistent sequence roughly.

FGFR1

Fibroblast growth factor acceptor 1(FGFR1) is also referred to as CEK, FLG, FLT2, KAL2, BFGFR, CD331, FGFBR, HBGFR, N-SAM and FLJ99988 in the art.FGFR2 is the member of fibroblast growth factor acceptor family and has high-affinity to acidity with fibroblast growth factor alkalescence.FGFR1 is relevant with the signal transduction that causes mitotic division and differentiation and participate in limbs and induce.

For example, people such as Isacchi, people such as Nucleic Acids Res.18:1906-1906 (1990) and Hou, Science251:665-668 (1991) discloses Nucleotide and the protein sequence of people FGFR1.For example, people such as Harada, Biochem.Biophys.Res.Commun.205:1057-1063 (1994) discloses Nucleotide and the protein sequence of mouse FGFR1.The undressed forebody length of people FGFR1 is that about 822 amino acid and molecular weight are about 90420Da.The undressed forebody length of mouse FGFR1 is that about 822 amino acid and molecular weight are about 90420Da.

FGFR1 sudden change and luxuriant and rich with fragrance Buddhist syndromes (Pfeiffer syndrome), Jackson-Wei Si syndromes (Jackson-Weiss syndrome), the Antley-Bixler syndromes, dyschondroplasia is relevant with autosomal dominant Kallmann syndromes 2.The chromosome aberration that relates to this gene is relevant with stem cell leukemia lymphoma syndromes with the stem cell myeloproliferative diseases.

In one embodiment, the invention provides by the carcinogenic isomer of nucleic acid encoding or the isolated polypeptide of its epi-position, described nucleic acid comprises that selectivity has lacked one section Nucleotide of

exon

7 and 8 in the nucleic acid that derives from coding FGFR1.In one embodiment, when comparing with the FGFR1 proto-oncogene,

exon

7 and 8 selectivity disappearance cause 105 amino acid whose disappearances.Therefore, in one embodiment, described polypeptide contains or comprises SEQ NO:10 sequence, or with the sequence of its basically identical.On the other hand, described polypeptide comprises the sequence by SEQ NO:9 nucleic acid sequence encoding, or with the sequence of its basically identical.

In another embodiment, described epi-position contains or comprises and the Ig-II of FGFR1L and the bonding land between the Ig-III, or the aminoacid sequence of its fragment unanimity, or by the nucleotide sequence of SEQ ID NO:9 or the aminoacid sequence of its fragment coding, or with amino acid or the nucleotide sequence of above-mentioned sequence basically identical.

The RON receptor tyrosine kinase

Macrophage-stimulating 1 acceptor (c-met be correlated with Tyrosylprotein kinase) (RON) is called as MST1R in this area, PTK8, CD136 and CDwl36.RON is macrophage stimulating protein (MSP) acceptor and has protein tyrosine kinase activity.It participates in epithelium, the development of bone and neuroendocrine derivative.Nucleotide and the protein sequence of people RON have been announced, people such as Ronsin C. for example, Oncogene8:1195-1202 (1993); With people such as Collesi C., Mol.Cell.Biol.16:5518-5526 (1996).Nucleotide and the protein sequence of mouse RON have been announced, people such as Iwama A. for example, Blood83:3160-3169 (1994); People such as Waltz S.E., Oncogene16:27-42 (1998); With people such as Persons D.A., Nat.Genet.23:159-165 (1999).The undressed forebody length of people RON is that about 1400 amino acid and molecular weight are about 152227Da.The undressed forebody length of mouse RON is that about 1378 amino acid and molecular weight are about 150538Da.

In one embodiment, the invention provides by the carcinogenic isomer of nucleic acid encoding or the isolated polypeptide of its epi-position, described nucleic acid comprises that selectivity in the nucleic acid that derives from coding RON receptor tyrosine kinase has lacked one section Nucleotide of exon 5 and 6.In one embodiment, when comparing with RON receptor tyrosine kinase proto-oncogene,

exon

5 and 6 selectivity disappearance cause in the extracellular domain disappearance in 109 amino acid whose frameworks.In one embodiment, described polypeptide contains or comprises

exon

4 and 7 fusions and and the living peptide sequence of buying property.Therefore, in one embodiment, SEQ NOs:12 sequence is formed or comprised to described polypeptide, or with the sequence of its basically identical.In another embodiment, sequence by SEQ NO:11 nucleic acid sequence encoding is formed or comprised to described polypeptide, or with the sequence of its basically identical.

In other embodiments, described epi-position contains or comprises, aminoacid sequence or its fragment consistent with the exon 4 of isomer RON △ 160 and the bonding land between the exon 7 (SEQ ID NO:12), or by SEQ ID NO:11 nucleotide sequence coded aminoacid sequence or its fragment, or with amino acid or the nucleotide sequence of above-mentioned sequence basically identical.

The KIT receptor tyrosine kinase

V-kit Hardy-Zuckerman4 cat sarcoma virus oncogene homologue (KIT) is PBT, SCFR, C-Kit and CDl17 also in this area.Encode people's homologue of former cancer base c-kit of KIT.KIT is the MGF(mast cell growth factor, is also referred to as STEM CELL FACTOR) 3 type transmembrane receptors.

Announced Nucleotide and the protein sequence of people KIT, people such as Yarden for example, EMBO is people such as (1987) and Giebel J.6:3341-3351, Oncogene7:2207-2217 (1992).Announced Nucleotide and the protein sequence of mouse KIT, people such as Qiu for example, EMBO is people such as (1988) and Rossi J.7:1003-1011, Dev.Biol.152:203-207 (1992).About 976 amino acid of the undressed forebody length of people KIT and the about 107360Da of molecular weight.About 10725 amino acid of the undressed forebody length of mouse KIT, and molecular weight is 150538Da.

KIT sudden change and gi tract mesenchymal neoplasm, the mastocyte disease, acute myelogenous leukemia is relevant with piebaldism.

In one embodiment, the invention provides by the carcinogenic isomer of nucleic acid encoding or the isolated polypeptide of its epi-position fragment, described nucleic acid comprises the one section Nucleotide that has been lacked the generation of exons 11 by selectivity in the nucleic acid of encoded K IT receptor tyrosine kinase.Therefore, in one embodiment, described polypeptide contain or comprise SEQ NO:14 sequence or with the sequence of its basically identical.In another embodiment, described polypeptide contains or comprises the sequence by the nucleic acid sequence encoding of SEQ NO:13, or with the sequence of its basically identical.

In another embodiment, described epi-position contains or comprises identical aminoacid sequence or its fragment in KIT bonding land between the

exons

10 and 12 with SEQ ID NO:14, or by nucleotide sequence coded aminoacid sequence or its fragment of SEQ ID NO:13, or with amino acid or the nucleotide sequence of above-mentioned sequence basically identical.

PDGF

Thr6 PDGF BB α polypeptide (PDGFA) is also referred to as PDGF1 and PDGF-A in this area.PDGFA is a member of Thr6 PDGF BB family.PDGFA be mesenchymal cell source cell mitotic factor and have the feature of eight cysteine modified.

Nucleotide and the protein sequence of people PDGFA have been announced, people such as Bonthron for example, people such as Proc.Natl.Acad.Sci.U.S.A.85:1492-1496 (1988) and Betsholtz, Nature320:695-699 (1986).Nucleotide and the protein sequence of mouse PDGFA have been announced, people such as Rorsman for example, people such as Growth Factors6:303-313 (1992) and Mercola, Dev.Biol.138:114-122 (1990).About 211 amino acid of the undressed forebody length of people PDGFA and the about 23210Da of molecular weight.About 211 amino acid of the undressed forebody length of mouse PDGFA, and molecular weight is 23210Da.

Oligodendrocyte has been showed in research with knock out mice, the cell defect of the interstitial glands in alveolar smooth muscle cell and the testis; Knock out mice is dead immediately in the embryo or after the birth.

In one embodiment, the invention provides by the carcinogenic isomer of nucleic acid encoding or the isolated polypeptide of its epi-position fragment, described nucleic acid comprises that the interior selectivity of the nucleic acid middle frame that derives from coding PDGF has lacked one section Nucleotide of exon 6.Therefore, in one embodiment, described polypeptide contain or comprise SEQ NO:16 sequence or with the sequence of its basically identical.In another embodiment, described polypeptide contain or comprise by the sequence of the nucleic acid sequence encoding of SEQ NO:15 or with the sequence of its basically identical.

In another embodiment, described epi-position contains or comprises consistent aminoacid sequence or its fragment in PDGF bonding land between the

exon

5 and 7 with SEQ ID NO:16, or by nucleotide sequence coded aminoacid sequence or its fragment of SEQ ID NO:15, or with amino acid or the nucleotide sequence of above-mentioned sequence basically identical.

PDGFR-α

Platelet derived growth factor receptor, α polypeptide (PDGFA) be also referred to as in this area CD140A, PDGFR2,

MGC74795 and Rhe-PDGFRA.PDGFA is the Thr6 PDGF BB family member cell surface tyrosine kinase receptor of having encoded.These somatomedins are the mitogen of the cell in mesenchymal cell source.

Nucleotide and the protein sequence of people PDGFA have been announced, people such as Bonthron for example, people such as Pwc.Natl.Acad.Sci.U.S.A.85:1492-1496 (1988) and Betsholtz, Nature320:695-699 (1986).Nucleotide and the protein sequence of mouse PDGFA have been announced, people such as Stiles for example, people such as Mol.Cell.Biol.10:6781-6784 (1990) and Carninci, Science309:1559-1563 (2005).About 1089 amino acid of the undressed forebody length of people PDGFA and the about 119790Da of molecular weight.About 1089 amino acid of the undressed forebody length of mouse PDGFA, and molecular weight is 119790Da.

Because middle chromosome deletion, the fusion of PDGFRA and FIP1L1 (FIP1LI-PDGFRA) is the reason of some cases of high eosinophilic granulocyte syndromes (HES).HES is rare disease in the blood system, and it has, and eosinophilic granulocyte continues to increase in marrow, eosinophilia, the feature of tissue infiltration and organ injury.

In one embodiment, the invention provides by the carcinogenic isomer of nucleic acid encoding or the isolated polypeptide of its epi-position, described nucleic acid comprises that selectivity in the nucleic acid that derives from coding PDGF α has lacked for example amino acid of 374-456 position of exon 7 and 8() one section Nucleotide.Therefore, in one embodiment, described polypeptide contain or comprise SEQ NO:18 sequence or with the sequence of its basically identical.In another embodiment, described polypeptide contain or comprise by the sequence of SEQ NO:17 nucleic acid sequence encoding or with the sequence of its basically identical.

In another embodiment, described epi-position contains or comprises consistent aminoacid sequence or its fragment in PDGF α bonding land between the

exon

6 and 9 with SEQ ID NO:18, or by SEQ ID NO:17 nucleotide sequence coded aminoacid sequence or its fragment, or with amino acid or the nucleotide sequence of above-mentioned sequence basically identical.

Optionally, the isolated polypeptide of carcinogenic isomer or its epi-position may by with the nucleic acid encoding of the nucleic acid basically identical of the carcinogenic isomer that provides at this or its epi-position fragment.Similarly, the isolated polypeptide of carcinogenic isomer or its epi-position may with the carcinogenic isomer that provides at this or its epi-position basically identical.

A kind of method for preparing carcinogenic isomer or its epi-position fragment

The carcinogenic isomer of polypeptide or its epi-position fragment can be separated from natural resources, maybe may be the product of chemosynthesis process, maybe can produce by recombinant technology from protokaryon or eucaryon host.

The present invention also provides the method for the carcinogenic isomer of preparation or its epi-position fragment, and described method is included under the situation of allowing carcinogenic isomer or its epi-position fragment expression and cultivates host cell; And separate carcinogenic isomer or its epi-position fragment, thereby prepare carcinogenic isomer or its epi-position fragment.In one embodiment, the invention provides the method for the carcinogenic isomer of a kind of people of preparation or its epi-position fragment.Process with the method for preparing polypeptide is well known to those skilled in the art.

The isomer specific inhibitor

The present invention provides at least in part, isomer specific inhibitor (antibody molecule for example, soluble receptors polypeptide and fusion form thereof, peptide and functional variant thereof, with the nucleic acid inhibitor), described isomer specific inhibitor suppresses and/or reduces one or more activity of isomer, or with one or more isomer polypeptide or its fragment, or the nucleic acid of encode one or more isomer polypeptide or its fragment reacts to each other or more preferably specificity combination.In one embodiment, described isomer specific inhibitor is the isomer binding molecule, for example antibody molecule, or nucleic acid inhibitor.In another embodiment, described isomer specific inhibitor is soluble receptors polypeptide and fusion form thereof, or peptide and functional variant thereof.In certain embodiments, described isomer binding molecule specificity is bonded to carcinogenic isomer polypeptide or its fragment, or the nucleic acid of encode one or more carcinogenic isomer polypeptide or its fragment.

Typical isomer specific inhibitor (for example isomer binding molecule) is with high-affinity, for example with at least about 10 ⁷M ^-1, typically about 10 ⁸M ^-1And more typically about 10 ⁹M ^-1To 10 ¹⁰M ^-1Or stronger affinity constant is bonded to one or more isomer polypeptide or its fragment, and reduces and/or suppress isomer, for example one or more activity of the carcinogenic isomer in height hyperplasia (for example cancer or pernicious) cell and/or the tissue.For example, one or more carcinogenic isomer related activity during isomer specific inhibitor alternative and specificity reduction or inhibition are selected from: (i) binding partner or co-receptor are (for example, the FGF part, (FGF8b for example, FGF2, FGF17 or FGF18) be bonded to FGFR2 isomer III c); (ii) receptor dimerizationization (for example, FGFR2 isomer III c homologous dimerizationization or FGFR2 isomer III c and other acceptor or acceptor isomer allos dimerization); (iii) isomer signal transduction, for example, FGFR2 isomer III c signal transduction; (iv) height hyperplasia (for example cancer or tumour) cell proliferation, growth and/or survival, for example, by inducing height proliferative cell apoptosis; And/or (the v) vasculogenesis of tumour and/or vascularization.

Term " specificity combination " refers to association reaction as used herein, and this association reaction is determined by the target (for example specific polypeptide or Nucleotide) that exists in class protein and other biology.Therefore, " specificity combination " to the binding molecule of carcinogenic isomer means this compound in conjunction with carcinogenic isomer of the present invention, but the non-carcinogenic isomer that debond to identical proto-oncogene is derived.One skilled in the art will recognize that, depend on used condition, for example, used target proteins concentration, salt and damping fluid, and other condition, described isomer binding molecule can be showed cross reactivity in various degree between carcinogenic and non-carcinogenic isomer.In certain embodiments, term " specificity in conjunction with " or " the specificity combination " refer to the isomer binding molecule with high-affinity, for example with at least about 10 ⁷M ^-1, typically about 10 ⁸M ^-1And more typically about 10 ⁹M ^-1To 10 ¹⁰M ^-1Or stronger affinity constant is bonded to one or more isomer polypeptide or its fragment, or the character of the nucleic acid of encode one or more isomer polypeptide or its fragment; And (2) with than at least 2 times of the avidity height that is bonded to the non-carcinogenic isomer, and 50 times, 100 times, 1000 times or higher avidity preferably are bonded to isomer.In certain embodiments, isomer binding molecule preferred combination is to carcinogenic isomer, but basic debond is to (for example, showed that relative thing with non-carcinogenic is lower than 0%, 8%, 5%, 4%, 3%, 2%, 1% cross reactivity) the relative thing of its non-carcinogenic.

Antibody molecule

In one embodiment, described isomer binding molecule is antibody molecule, and described antibody molecule is bonded to Mammals, for example, and people, isomer polypeptide or its fragment (Fab for example, F (ab') ₂, Fv, strand Fv fragment, or camel variant).For example, described antibody molecule is bonded to the height proliferative cell, and for example cancer or tumour cell are expressed and/or relevant isomer polypeptide or fragment.For example described antibody molecule specificity is bonded to epi-position, for example linearity or conformational epitope, (for example epi-position described here), described epi-position are positioned at or mainly are expressed in the height proliferative cell, for example cancer or tumour cell, the surface.In an embodiment, by the epi-position of antibody molecule identification at the height proliferative disease, for example express in cancer or the malignant disease or with the height proliferative disease, for example cancer or malignant disease are relevant.For example, the epi-position of described antibody molecule identification is expressed by exon sequence or is relevant with exon sequence, described exon sequence mainly in one or more cancers or tumour cell or disease expression or with one or more cancers or tumour cell or disease-related; Described epi-position may be located at the bonding land between two exons that significantly combine in one or more cancer cells or tumour cell or the disease, for example: because the use of the interior Exon deletion of framework or alternative splicing exon.The identification of isomer binding molecule, typical isomer polypeptide of the present invention or fragment include but not limited to FGFR2, FGFR1, RON receptor tyrosine kinase, KIT receptor tyrosine kinase, the carcinogenic isomer of PDGF and PDGF-acceptor α.In one embodiment, the carcinogenic isomer of the described antibody molecule combination carcinogenic isomer of behaving.In another embodiment, the polypeptide isomer of described antibody molecule combination is the carcinogenic isomer enumerated of table 1 or the polypeptide of its epi-position.

In one embodiment, described antibody molecule specificity is bonded to polypeptide, and described polypeptide comprises SEQ ID NO:2,4,6,8,10,12,14,16,18 aminoacid sequences of showing or with the sequence of its basically identical.In another embodiment, described antibody molecule specificity is bonded to SEQ ID NO:2, and 4,6, or 8 polypeptide FGFR2-III c isomer, but basic debond is to the polypeptide isomer of people FGFR2-III b.In another embodiment, described antibody molecule is bonded to, the people FGFR2 polypeptide of SEQ ID NO:19 for example, but basic debond to FGFR2-III b(for example, SEQ ID NO:21) or other isomer (for example SEQ ID NOs:22,23,24,25,26,27,28,29,30,31,52 and/or 53) of FGFR2.

In another embodiment, described antibody molecule specificity is bonded to for example isomer of people FGFR1 of FGFR1, for example, and carcinogenic isomer.For example, described antibody molecule specificity is bonded to isomer FGFR1L, described isomer FGFR1L has lacked about 105 amino acid between exon 7 and 8, corresponding with partial immunity sphaeroprotein territory II (Ig-II) and the part Ig-III of FGFR1, thereby forms the bonding land between II and III.For example, the antibody molecule preferred combination is to FGFR1L or its fragment, but basic debond is to (for example, showed with FGFR1 and be lower than 10%, 8%, 5%, 4%, 3%, 2%, 1% cross reactivity) FGFR1(for example, non-carcinogenic people FGFR1, FGFR1 isomer 4 (SEQ ID NO:39) for example, FGFR1 isomer 14 (SEQ ID NO:40), FGFR1 isomer 16 (SEQ ID NO:41), FGFR1 isomer 17 (SEQ ID NO:42), FGFR1 isomer 3 (SEQ ID NO:43), or FGFR1 isomer 18 (SEQ ID NO:44)).In these embodiments, described antibody molecule specificity is bonded at least one epi-position or its fragment of finding on the Ig-II of SEQ ID NO:10 and the bonding land between the Ig-III, or by SEQ ID NO:9 nucleotide sequence coded aminoacid sequence or its fragment, or with amino acid or the nucleotide sequence of above-mentioned sequence basically identical.

In other embodiments, described antibody molecule is bonded to isomer, for example, the RON receptor tyrosine kinase, people RON receptor tyrosine kinase for example, carcinogenic isomer.For example described antibody molecule specificity is bonded to isomer RON △ 160, has lacked the exon 5 of the extracellular domain of skipping RON and about 109 amino acid of 6 in described isomer RON △ 160 frameworks, thereby form the bonding land between exon 4 and 7.For example, described antibody molecule preferred combination is to RON △ 160 or its fragment, but basic debond is to (for example, having showed with the RON receptor tyrosine kinase and be lower than 10%, 8%, 5%, 4%, 3%, 2%, 1% cross reactivity) RON receptor tyrosine kinase (for example, non-carcinogenic people RON receptor tyrosine kinase, for example SEQ ID NO:45).In these embodiments, described antibody molecule specificity is bonded at least one epi-position or its fragment of finding on the exon 4 of SEQ ID NO:12 and the bonding land between the exon 7, or by SEQ ID NO:11 nucleotide sequence coded aminoacid sequence or its fragment, or with amino acid or the nucleotide sequence of above-mentioned sequence basically identical.

In another embodiment, described antibody molecule specificity is bonded to the KIT receptor tyrosine kinase, the isomer of people KIT receptor tyrosine kinase for example, for example, carcinogenic isomer.For example, described antibody molecule specificity is bonded to the KIT isomer, and described KIT isomer has lacked exons 11.For example, described antibody molecule preferred combination is to KIT isomer (SEQ ID NO:46) or its fragment of exons 11 disappearance, but basic debond (for example, showed with KIT and be lower than 10%, 8%, 5%, 4%, 3%, 2%, 1% cross reactivity) to KIT(for example, non-carcinogenic people KIT, for example, total length acceptor (SEQ ID NO:47)).In these embodiments, described antibody molecule specificity is bonded at least one epi-position or its fragment of finding on the exons 10 of SEQ ID NO:14 and the bonding land between the exons 12, or by SEQ ID NO:13 nucleotide sequence coded hint sequence or its fragment, or with amino acid or the nucleotide sequence of above-mentioned sequence basically identical.

In another embodiment, described antibody molecule specificity is bonded to isomer, for example, PDGF, for example, people PDGF, carcinogenic isomer.For example described antibody molecule specificity is bonded to the PDGF isomer, has lacked exon 6 in the described PDGF isomer framework.For example, described antibody molecule preferred combination is to PDGF isomer or its fragment of exon 6 disappearance, but basic debond is to (for example, showed with PDGF and be lower than 10%, 8%, 5%, 4%, 3%, 2%, 1% cross reactivity) PDGF(for example, non-carcinogenic people PDGF, for example, PDGF isomer 1 (SEQ ID NO:49)).In these embodiments, described antibody molecule specificity is bonded at least one epi-position or its fragment of finding on the bonding land between the

exon

5 and 7 of SEQ ID NO:16, or by nucleotide sequence coded aminoacid sequence or its fragment of SEQ ID NO:15, or with amino acid or the nucleotide sequence of above-mentioned sequence basically identical.

In another embodiment, described antibody molecule specificity is bonded to pdgf receptor α, for example, people's pdgf receptor α, isomer, for example, carcinogenic isomer.For example described antibody molecule specificity is bonded to the PDGF αYi Gouti, has lacked

exon

7 and 8 in the described PDGF αYi Gouti framework.For example, described antibody molecule preferred combination is to PDGF αYi Gouti (SEQ ID NO:51) or its fragment of exon 7/8 disappearances, but basic debond (for example, showed with PDGF α and be lower than 10%, 8%, 5%, 4%, 3%, 2%, 1% cross reactivity) to PDGF α (for example, non-carcinogenic people PDGF α, for example, PDGF αYi Gouti 1 (SEQ ID NO:50)).In these embodiments, described antibody molecule specificity is bonded at least one epi-position or its fragment of finding on the bonding land between the

exon

6 and 9 of SEQ ID NO:18, or by nucleotide sequence coded aminoacid sequence or its fragment of SEQ ID NO:17, or with amino acid or the nucleotide sequence of above-mentioned sequence basically identical.

Term " antibody molecule " refers to the protein that comprises at least one immune ball Variable Area sequence as used herein.The term antibody molecule comprises, for example, and total length, the fragment of the conjugated antigen of ripe antibody and antibody.For example, described antibody molecule can comprise heavily (H) chain variable region sequence (being abbreviated as VH at this) and light (L) chain variable region sequence (being abbreviated as VL at this).In another embodiment, antibody molecule comprises two weight (H) chain variable region sequences and two light (L) chain variable region sequences, thereby forms two antigen binding sites, such as, Fab, Fab', F (ab') ₂, Fc, Fd, Fd', Fv, and single-chain antibody (for example, scFv), the variable region single domain antibody, bivalent antibody (DAb) (divalence or dual specific) and chimeric (for example, humanized) antibody, these antibody can be by to whole antibody or the Antibody Preparation of synthesizing again with the DNA recombinant technology.The ability that these functional antibodies fragments maintenances are combined with other antigen of its branch or receptor-selective.Antibody and antibody fragment may come from any kind of antibody, include but not limited to IgG, IgA, and IgM, IgD, and IgE, and from any subclass (for example, IgG1, IgG2, IgG3, and IgG4) of antibody.Antibody of the present invention can be for monoclonal or polyclonal.Described antibody can be the people, and is humanized, and CDR-transplants, or the antibody of external generation.This antibody can have and is selected from, IgG1 for example, IgG2, IgG3, or the CH of IgG4.This antibody can have the light chain that is selected from κ or λ.

The example of antibodies fragment comprises: (ⅰ) Fab fragment, and the unit price fragment comprises VL, VH, CL and CH1 district; (ⅱ) F (ab') 2 fragments, divalence fragment, described divalence fragment are included in two Fab fragments that hinge area connects by disulfide linkage; (ⅲ) Fd fragment, described Fd fragment comprises VH and CHI district; (ⅳ) Fv fragment, described Fv fragment is made up of VL and the VH district of the single armed of antibody; (ⅴ) binary antibody (dAb) fragment, described binary antibody fragment is made up by the VH district; (ⅵ) camel or camelization variable region; (ⅶ) strand Fv (scFv), referring to people such as Bird, people such as (1988) Science242:423-426 and Huston, (1988) Proc.Natl.Acad.Sci.USA85:5879-5883); (ⅷ) single domain antibody.Described antibody fragment obtains with the common technology known to those skilled in the art, and described fragment screens to use in the mode identical with complete antibody.

Term " antibody " comprise complete molecule with and functional fragment.The constant region of antibody can change, for example, and sudden change, thereby the character of modified antibodies (for example increase or reduce: Fc receptors bind, antibody glycosylation, the quantity of cysteine residues, one or more in effector cell function or the complement function)

Antibody of the present invention also can be single domain antibody.Single domain antibody can comprise that its complementary determining region is the antibody of the part of single domain polypeptide.Example includes, but not limited to heavy chain antibody, the natural antibody that lacks light chain, and by the single domain antibody that conventional 4 chain antibodies are derived, genetic engineering antibody and single domain support are except the single domain antibody that those antibody are derived.Single domain antibody can be any antibody in this area or following any single domain antibody.Single domain antibody may be derived by any species, and described species include, but not limited to mouse, people, camel, white horse with a black mane sheep, fish, shark, goat, rabbit, ox.According to a further aspect in the invention, single domain antibody is that nature generates, and is called as the single domain antibody of the heavy chain antibody that lacks light chain.For example, in WO9404678 one literary composition, these single domain antibodies have been announced.For reason clearly, from heavy chain antibody, to derive, the described variable region that lacks light chain naturally is referred to herein as VHH or nano antibody so that it is distinguished from the conventional VH of four chain immunoglobulin (Ig)s.This VHH molecule may be from the camel species, for example, camel, the white horse with a black mane sheep, dromedary camel, alpaca and vigone derive in the antibody of generation.Other species that belong to except camel can prepare the heavy chain antibody that nature lacks light chain; These VHH also within the scope of the invention.

VH and VL district can be further divided into hypervariable region, are called as " complementary determining region ", by what guard more, are called as " framework region " differentiation (FR) and loose.The scope of framework region and CDRs is accurately determined (referring to Kabat by many methods, E.A., Deng people (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No.91-3242; Chothia, C. wait people (1987) J.Mol.Biol.196:901-917), and the AbM antibody prototype software of Oxford molecule uses the AbM definition, referring to, usually, for example, protein sequence and binding analysis (Ed.:Duebel, S. and Kontermann, the R. of the antibody variable region in the antibody genetic engineering laboratory manual (Antibody Engineering Lab Manual), Springer-Verlag, Heidelberg).Usually, unless otherwise indicated, use to give a definition: the AbM definition of the CDR1 of variable region of heavy chain and the Kabat definition of other CDRs.In addition, the specific embodiments of the invention of the description of Kabat or AbM CDRs also available Chothia hypervariable region ring are implemented.Each VH and VL typically comprise three CDR and four FR, arrange in the following sequence from N-terminal to C-terminal: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

" immunoglobulin variable domain sequence " refers to the aminoacid sequence that can form the immune globulin variable region structure as used herein.For example, described sequence may comprise all or part of of amino acid sequences that nature generates.For example, described sequence may maybe can not comprise one, and two or more N or C terminal amino acid maybe may comprise other change compatible with protein structure information.

Term " antigen binding site " refers to antibody molecule part or its epi-position that comprises the determinant that forms the contact surface that is bonded to the isomer polypeptide.With respect to protein (or protein analogue), antigen binding site comprises that typically one or more formation are bonded to (at least four amino acid or amino acid analog thing) ring of the contact surface of isomer polypeptide.Typically, the antigen binding site of antibody molecule comprises at least one or two CDR or more typically, at least three, and four, five or six CDR.

Term " monoclonal antibody " or " monoclonal antibody combination " refer to the preparation of the antibody molecule of individual molecule composition as used herein.Monoclonal antibody combination has been showed single binding specificity and to the avidity of defined epitope.Monoclonal antibody can be by hybridoma technology or method (for example, the recombination method) preparation by not utilizing hybridoma technology.

" effectively people " protein is the antibody response protein of human antimouse antibody (HAMA) reaction for example that can not cause inefficacy.

HAMA is problematic in many cases, if for example antibody molecule repeats to take, for example, is treating chronic or is recurring in the disease condition.Since rise in the serum the cleaning antibody rate (referring to, for example, people such as Saleh, Cancer Immunol.Immunother., 32:180-190 (1990)) simultaneously also because potential anaphylaxis (referring to, people such as LoBuglio for example, Hybridoma, 5:5117-5123 (1986)), that repeatedly antibody is taken is invalid potentially for HAMA reaction.

Described anti-isomer antibody can be polyclone or monoclonal antibody.In other embodiments, described antibody can for example prepare by phage display or by combined method by the reorganization preparation.

Known in the art for generation of antibody isomer antibody phage display and combined method (as, for example, people such as Ladner, U.S.Patent No.5,223,409; People such as Kang, International Publication No.WO92/18619; People such as Dower, International Publication No.WO91/17271; People such as Winter, International Publication WO92/20791; People such as Markland, International Publication No.WO92/15679; People such as Breitling, International Publication WO93/01288; People such as McCafferty, International Publication No.WO92/01047; People such as Garrard, International Publication No.WO92/09690; People such as Ladner, International Publication No.WO90/02809; People such as Fuchs, (1991) Bio/Technology9:1370-1372; People such as Hay, (1992) Hum Antibod Hybridomas3:81-85; People such as Huse, (1989) Science246:1275-1281; People such as Griffths, (1993) EMBO J12:725-734; People such as Hawkins, (1992) J Mol Biol226:889-896; People such as Clackson, (1991) Nature352:624-628; People such as Gram, (1992) PNAS89:3576-3580; People such as Garrad, (1991) Bio/Technology9:1373-1377; People such as Hoogenboom, (1991) Nuc Acid Res19:4133-4137; With people such as Barbas, (1991) PNAS88:7978-7982, described in, quote its full content as a reference at this).

In one embodiment, described anti-isomer antibody be human antibody (for example, the antibody that in through the mouse of genetic modification with the antibody of generation human normal immunoglobulin sequence, prepares), or the non-human antibody, for example, rodent (mouse or rat), goat, primate (for example, monkey), camel antibody.Preferably, the non-human antibody is rodent (mouse or rat antibody).The method for preparing rodent antibody known in the art.

Human monoclonal antibodies can be by carrier's immunoglobulin gene but not the transgenic mouse of mouse system produce.From these transgenic mouses, with the splenocyte of interested antigen immune for the preparation of hybridoma, the secretion of described hybridoma has special avidity to the epi-position from human protein people mAbs(referring to, for example, people's such as Wood International Application No. WO 91/00906; People's such as Kucherlapati PCT announces WO91/10741; People's such as Lonberg International Application No. WO 92/03918; People's such as Kay international application 92/03917; Lonberg, people such as N., 1994Nature368:856-859; Green, people such as L.L., 1994Nature Genet.7:13-21; Morrison, people such as S.L., 1994Proc.Natl.Acad.Sci.USA

81:6851-6855; People such as Bruggeman, 1993Year Immunol7:33-40; People such as Tuaillon, 1993PNAS90:3720-3724; People such as Bruggeman, 1991Eur J Immunol21:1323-1326).

Anti-isomer antibody can be in this antibody, and the part of its variable region or variable region is CDR for example, at inhuman organism, for example, the antibody that produces in rat or the mouse.Chimeric, CDR transplanting and humanized antibody are in the present invention.At inhuman organism for example, produce in rat or the mouse, for example modify at variable region framework or constant region then, thereby antigenic antibody of reduction people is in the present invention.

Chimeric antibody can be by recombinant DNA technology preparation known in the art.For example, coding mouse (or other species) thus the gene of monoclonal antibody molecule Fc constant region removes the district of coding mouse Fc with Restriction Enzyme digestion, and the equity part of this gene of replacement coding people Fc constant region (referring to people such as Robinson, international patent publications PCT/US86/02269; People such as Akira, european patent application 184,187; Taniguchi, M., european patent application 171,496; People such as Morrison, european patent application 173,494; People such as Neuberger, International Application No. WO 86/01533; People such as Cabilly, U.S. Patent number 4,816,567; People such as Cabilly, european patent application 125,023; People such as Better, (1988Science240:1041-1043); People such as Liu, (1987) PNAS84:3439-3443; People such as Liu, 1987, J.Immunol.139:3521-3526; People such as Sun, (1987) PNAS84:214-218; People such as Nishimura, 1987, Cane.Res.47:999-1005; People such as Wood, (1985) Nature314:446-449; With people such as Shaw, 1988, J.Natl Cancer Inst.80:1553-1559).

People source or CDR grafted antibody have at least one or two but usually have three all acceptor (heavy chain immunoglobulin and/or light chain) CDR that replace with donor CDR.Described antibody may be replaced by at least a portion of inhuman CDR or only number of C DR replaced by inhuman CDR.Only need to replace the quantity that humanized antibody need be bonded to the CDR of isomer.Preferably, donor is rodent antibody, for example, rat or mouse antibodies, and acceptor behaviour framework or people guard framework.Typically, provide the immunoglobulin (Ig) of CDR to be called " donor ", and provide the immunoglobulin (Ig) of framework to be called " acceptor ".In one embodiment, the donor immunity sphaeroprotein is inhuman (for example, rodent).The acceptor framework is (for example, the people) framework or the conservative framework that nature generates, or about with it 85%, or higher, preferred 90%, 95%, 99% or more consistent sequence.

As used herein term " conserved sequence " refer in the family of correlated series the sequence that the amino acid (or Nucleotide) of the most frequent appearance forms (referring to, Winnaker for example, From Genes to Clones (Verlagsgesellschaft, Weinheim, Germany1987)).In protein families, each position of conserved sequence by in this position of this family the amino acid of frequent appearance occupy.If two same frequent appearance of amino acid, conserved sequence can comprise any one." conserved sequence " refers to the framework region in conservative immunoglobulin sequences.

Antibody can pass through the methods known in the art humanization.Humanized antibody can be by replacing the Fv variable region sequences of not participating in the antigen combination directly with people Fv variable region equity sequence.Produce the general method of humanized antibody by Morrison, S.L., 1985, Science229:1202-1207; People such as Oi, people such as 1986, BioTechniques4:214 and Queen, US5,585,089, US5,693,761and US5,693,762 provide, and quote its full content as a reference at this.These methods comprise separation, handle and express coding from all or part of nucleotide sequence of at least one IgF v variable region of heavy chain or light chain.The source of these nucleic acid is known for a person skilled in the art, and, for example can from the hybridoma of preparation at the antibody of isomer, obtain.Recombinant DNA or its fragment of coding humanized antibody can be cloned in the suitable expression vector.

Humanization or CDR grafted antibody can be transplanted or CDR replaces preparation by CDR, and wherein one of immunoglobulin chain, two or all CDR can be replaced.Referring to, United States Patent (USP) 5,225,539; People such as Jones, 1986Nature321:552-525; People such as Verhoeyan, 1988Science239:1534; People such as Beidler, 1988J.Immunol.141:4053-4060; Winter US5,225,539, specially quote its full content as a reference at this.Winter has described the CDR implantation method, and described CDR implantation method can be used for preparing humanized antibody of the present invention (the British Patent Application GB2188638A that on March 26th, 1987 submitted to, Winter US5,225,539), specially quotes its content as a reference.

The humanized antibody of replacement, disappearance or increase specific amino acid equally within the scope of the invention.Preferred humanized antibody has aminoacid replacement at framework region, such as in order to improve the combination with antigen.For example, humanized antibody has the framework residue consistent with donor framework residue or another amino acid except acceptor framework residue.In order to produce such antibody, the acceptor framework residue of selected a spot of Humanized immunoglobulin chain can be replaced by corresponding donor amino acid.Preferred the position of substitution comprise near CDR or the amino-acid residue that can react to each other with CDR (referring to, US5 for example, 585,089).From donor, select amino acid whose standard at US5,585,089, US5 for example, 585,089 12-16 row, US5 for example, 585,08 12-16 row are quoted its content as a reference at this.Other technology of humanized antibody in being disclosed in the EP519596Al on December 23rd, 1992, people such as Padlan is described.

In one embodiment, antibody can pass through with pure anti-isomer antigen, or its fragment or epi-position, for example fragment described here, with antigen, the film immunity preparation that tissue is relevant, for example, the natural tissues preparation, whole cell, preferred viable cell, lysing cell or cell fission, for example film division.

Described anti-isomer antibody can be single-chain antibody.Single-chain antibody (scFV) can transform (referring to, Colcher for example, people such as D., (1999) Ann N Y Acad Sci880:263-80 and Reiter, Y. (1996) Clin Cancer Res2:245-52).Thereby described single-chain antibody can dimerization or the different epi-positions that produce identical target isomer protein white matter of multimerization have specific multivalent antibody.

In other embodiments, described antibody molecule has CH, and described CH is selected from, for example IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD, with the CH of IgE, especially, be selected from, for example, described (for example, people) IgG1, IgG2, the variable region of heavy chain of IgG3 and IgG4.In another embodiment, described antibody molecule has constant region of light chain, and described constant region of light chain is selected from, for example, and described (for example, people) κ and lambda light chain constant region.Constant region can change, for example, and sudden change, thereby the character of modified antibodies (for example, thereby increasing or reduce the Fc receptors bind, antibody glycosylation, cysteine residues quantity, effector cell function, and/or one or more in the complement function).In one embodiment, described antibody has: the effector function, and can complement-fixing.In other embodiments, described antibody does not have compensation effect cell or complement-fixing.In another embodiment, described antibody have reduce or ability be not bonded to the Fc acceptor.For example, isotype or isomer, fragment or other mutant do not support to be bonded to the Fc acceptor, and for example, it has Fc receptor binding domain mutagenesis or disappearance.

The method of change antibody constant region known in the art.The function that has change, for example, pairing effect factor part, such as the FcR on the cell, or the Cl composition of part, the antibody of avidity of change can be by replacing at least one amino-acid residue preparation in the constant region of described antibody with different residue (referring to, EP388 for example, 151Al, U.S. Patent number 5,624,821 and U.S. Patent number 5,648,260, quote its full content as a reference at this), the change of same type can be described, if described change is implemented on mouse or other species, immunoglobulin (Ig) will reduce or eliminate these functions.

Another functional molecular (for example another polypeptide or protein) can be derived or be connected to isomer specific inhibitor (for example, isomer binding molecule).The antibody molecule of " deriving " as used herein is modified antibody molecule.Deriving method includes but not limited to fluorophor, the radioactive nuleus thuja acid, and toxin, enzyme or affinity ligand are such as the increase of vitamin H.Therefore, antibody molecule of the present invention comprise antibody described here derive or other modified forms, comprise immune adhesion molecule.For example, antibody molecule can (pass through chemical coupling, gene fusion, non-covalent combination or other) functional one or more other molecular entities, for example another antibody (for example, bi-specific antibody or binary antibody) of being connected to, detectable reagent, cytotoxic agent, pharmaceutical agent, and/or can regulate protein or the polypeptide (such as Streptavidin core area or polyhistidine mark) of the combination of antibody or antibody moiety and other molecule.

The antibody molecule of deriving by one type of two or more (same type or dissimilar, for example, in order to produce bi-specific antibody) antibody linked preparation.Suitable crosslinking agent comprises that abnormal shape is difunctional or homotype is difunctional (for example, the double amber imide suberate) crosslinked, bifunctional crosslinked two different, as to be separated by the suitable space reactive groups (for example m maleimide phenylformic acid-N-succinimide ester) that have of this abnormal shape.These linking agents can be at Pierce Chemical Company, and Rockford finds in the III.

But the useful detection reagent with antibody molecule of the present invention can be derived (or mark) thereby be contained fluorescent chemicals, various enzymes, prothetic group, luminescent material, bioluminescent material sends the fluorescence metal atom, for example, europium (Eu) and other lanthanon and radio active material (as mentioned below).But typical fluorescence detection reagent comprises fluorescein, fluorescein isothiocyanate, rhodamine, 5-dimethylin-1-naphthalic sulfonic chloride, phycoerythrin etc.Antibody also available detectable enzyme is derived, alkaline phosphatase for example, horseradish peroxidase, beta galactosidase enzyme, acetylcholinesterase, glucose oxidase etc.When antibody was derived with detectable enzyme, it was by adding described enzyme for the preparation of the other reagent detection of detectable reactor product.For example ought have detectable reagent horseradish peroxidase, the interpolation of hydrogen peroxide and diaminobenzidine produces the color reaction product, and this color reaction product is detectable.Antibody molecule also available prothetic group (for example Streptavidin/vitamin H and avidin/biotin) is derived.For example, antibody can be derived and detects by the indirect measurement to avidin or Streptavidin combination by vitamin H.The example of suitable fluorescent material comprises Umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, the amino fluorescein (dichlorotriazinylamine fluorescein) of dichlorotriazine, dansyl chloride or phycoerythrin; Luminescent material for example comprise luminol,3-aminophthalic acid cyclic hydrazide, and the bioluminescence examples of material comprises luciferase, luciferin and photoprotein.

The antibody molecule that is labeled can be for example, and diagnostic and/or sample plot are used in many cases,, comprise that (ⅰ) separates predetermined antigens by standard technique such as affinity chromatography or immunoprecipitation; (ⅱ) adequacy and the mode of expressing for evaluating protein matter detects predetermined antigens; (for example, in product of cell lysis or the cell conditioned medium liquid) (ⅲ) monitors protein level in the tissue as the part of clinical trial process, for example, determines the effect of given treatment plan.

Anti-isomer antibody molecule can be bonded to other molecular entity, typically, and (for example, cytotoxic or cell suppresses) reagent or group of label or treatment.

Radio isotope can be used for diagnosis or treatment is used.The radio isotope that can be bonded to anti-PSMA includes, but are not limited to α, β, or gamma emitter, or β and gamma emitter.These radio isotope include, but are not limited to iodine ( ¹³¹I or ¹²⁵I), yttrium ( ⁹⁰Y), lutetium ( ¹⁷⁷Lu), actinium ( ²²⁵Ac), praseodymium, astatine ( ²¹¹At), rhenium ( ¹⁸⁶Re), bismuth ( ²¹²Bi or ²¹³Bi), indium ( ¹¹¹In), technetium ( ⁹⁹MTc), phosphorus ( ³²P), rhodium ( ¹⁸⁸Rh), sulphur ( ³⁵S), carbon ( ¹⁴C), tritium ( ³H), chromium ( ⁵¹Cr), chlorine ( ³⁶C1), cobalt ( ⁵⁷Co or ⁵⁸Co), iron (Fe), selenium (Se), gallium (Ga).As the radio isotope of therapeutical agent comprise yttrium ( ⁹⁰Y), lutetium ( ¹⁷⁷Lu), actinium ( ²²⁵Ac), praseodymium, astatine ( ²¹¹At), rhenium ( ¹⁸⁶Re), bismuth ( ²¹²Bi or ²¹³Bi) and rhodium ( ¹⁸⁸Rh).With marking, for example be used for diagnosis, radio isotope comprise, iodine ( ¹³¹I or ¹²⁵I), indium ( ¹¹¹In), technetium ( ⁹⁹MTc), phosphorus ( ³²P), carbon ( ¹⁴C), and tritium ( ³H), or above-mentioned enumerate one or more the treatment isotropic substances.

The invention provides the radiolabeled antibody molecule with and the method for mark.The method of traget antibody molecule is disclosed in one embodiment.Thereby described method comprises with sequestrant contact antibody molecule preparation conjugated antibodies.Described conjugated antibodies radio isotope, for example, ¹¹¹Indium, ⁹⁰Yttrium and ¹⁷⁷Lutetium, radio-labeling, thereby the antibody molecule of preparation mark.

Institute is mentioned above, and this antibody molecule is bonded to therapeutical agent.The therapeutic activity radio isotope is mentioned.The example of other therapeutical agent comprises taxol, cytochalasin B, Gramicidin D, ethidium bromide, ipecamine, mitomycin, etoposide (etoposide), teniposide (tenoposide), vincristine(VCR), vinealeucoblastine(VLB), colchicine, Streptomycin sulphate, daunorubicin, dihydroxyl anthracin diketone, mitoxantrone, Plicamycin, dactinomycin, boldenone, glucocorticosteroid, PROCAINE HCL, PHARMA GRADE, tetracaine, lignocaine, Proprasylyte (propranolol), tetracycline, ansamitocin, for example maytansinol is (referring to U.S. Patent number 5,208,020), CC-1065(is referring to U.S. Patent number 5,475,092,5,585,499,5,846,545) and analogue or homologue.Therapeutical agent (for example includes, but not limited to metabolic antagonist, Rheumatrex, Ismipur, 6-mercapto guanine, cytosine arabinoside, 5 FU 5 fluorouracil dacarbazine (5-fluorouracil decarbazine)), alkylating agent is (for example, mustargen, thioepa Chlorambucil, CC-1065, melphalan, carmustine (bsnu), and lomustine (CCNU), endoxan, busulfan, mitobronitol, streptozotocin, ametycin, and Platinol (DDP) cis-platinum), the anthracene nucleus class is (for example, daunorubicin (original name daunomycin) and Streptomycin sulphate), microbiotic (for example, gengshengmeisu (original name actinomycin), bleomycin (bleomycin), Plicamycin (mithramycin), and anthramycin (AMC)) and antimitotic reagent (for example, vincristine(VCR), vinealeucoblastine(VLB), taxol and ansamitocin).

Conjugate of the present invention can be used for modifying given biological respinse.Therapeutical agent is not to be interpreted as being limited to typical chemotherapeutic.For example, described therapeutical agent can be and has bioactive protein or the polypeptide of wanting.These protein comprise that for example, toxin is such as abrin, ricin A, Pseudomonas exotoxin, diphtheria toxin or its composition (for example the composition of Pseudomonas exotoxin is PE38); Protein, such as tumour necrosis factor, Interferon, rabbit, nerve growth factor, Thr6 PDGF BB, tissue fibrin's dissolving activator, or biological response modifier, for example, lymphokine, interleukin-11 (" IL-1 "), interleukin-22 (" IL-2 "), interleukin 6 (" IL-6 "), granulocyte-macrophage colony stimulating factor (" GM-CSF "), granulocyte colony-stimulating factor (" G-CSF "), or other somatomedin.Simply, described therapeutical agent can be virion, for example recombinant virus particle, described virion (for example by chemical joint) and anti-isomer antibodies of the present invention or fusion.

On the one hand, of the present inventionly be characterized as a kind of method that the target binding molecule is provided, described molecular specificity is bonded to the isomer acceptor.For example, described target binding molecule is antibody molecule.Described method comprises: the target proteins of at least a portion that comprises the non-human protein is provided, the corresponding part of this part and people's target proteins (with its at least 70,75,80,85,87,90,92,94,95,96,97,98% unanimity) homology, but have at least one amino acid (for example at least one, two, three, four, five, six, seven, eight or nine amino acid) difference; Obtain the antibody molecule that specificity is bonded to antigen; And the assessment binding reagents is in the effect of regulating aspect target proteins active.Described method can further comprise makes people's experimental subjects take described binding reagents (for example, antibody molecule) or derivative (for example antibody molecule in people source).

The invention provides the isolated nucleic acid molecule of the above-mentioned antibody molecule of coding, carrier, and host cell.Described nucleic acid molecule includes but not limited to RNA, genomic dna and cDNA.

Soluble receptors and fusions thereof

In other embodiments, total length or fragment that described isomer specific inhibitor is the isomer receptor protein, for example, the inhibition ligand binding domain of isomer receptor polypeptides.For example, the described isomer specific inhibitor soluble form that can be FGFR2 isomer III c acceptor (for example, comprises part (for example, FGF) soluble form of the Mammals of land (for example, people) FGFR2 isomer III c acceptor).For example the isomer specific inhibitor can comprise the amino acid (amino acid of 1-262 position comprises signal sequence for Figure 13 C, SEQ ID NO:55) of the about 1-262 of people FGFR2 isomer III c acceptor position; Or with the sequence of its basically identical.Optionally, described isomer specific inhibitor can comprise by the nucleotide sequence of about 1 to 786 Nucleotide of people FGFR2 isomer III c (Figure 13 B, the 1-786 Nucleotide of SEQ ID NO:54) amino acids coding, or with the amino acid of its basically identical.

" soluble form of FGFR2 isomer III c acceptor " or " soluble form of isomer receptor polypeptides " are the acceptor isomer as used herein, for example, can not make the FGFR2 isomer III c receptor polypeptides that self is anchored on the film.These soluble polypeptides comprise, for example, isomer receptor polypeptides described here, FGFR2 isomer III c receptor polypeptides for example, the film district of striding of its grappling polypeptide has lacked enough parts, or is modified and make that striding the film district does not have function.Typically, solubility isomer receptor polypeptides keeps being bonded to the isomer part, FGF part for example, ability.For example, the soluble fragments of FGFR2 isomer III c receptor polypeptides (for example, the fragment of FGFR2 isomer III c acceptor that comprises the extracellular domain of people FGFR2 isomer III c acceptor, comprise about 1 to 262 amino acid (Figure 13 C of people FGFR2 isomer III c acceptor, the amino acid of the 1-262 position of SEQ ID NO:55, comprise signal sequence), or with the aminoacid sequence of its basically identical.Solubility FGFR2 isomer III c receptor polypeptides can comprise in addition, for example, merges extremely second group, for example polypeptide (for example, immunoglobulin chain, GST, Lex-A or MBP peptide sequence).For example, fused protein can comprise at least one section of FGFR2 isomer III c receptor polypeptides, it can be in conjunction with the FGF part, fusion to the second group, for example, polypeptide (for example, immunoglobulin chain, Fc fragment, various isotype, comprise the CH of IgG1, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD and IgE).

The soluble form of isomer receptor polypeptides can use separately or (by chemical coupling, gene or polypeptide merge, non-covalent combination or other) functional second group that is connected to, immunoglobulin fc region for example, serum protein, polyoxyethylene glycol, GST, Lex-A or MBP peptide sequence." fusion rotein " refers to and comprises two or more combinations of handling as used herein, for example, and connection, group, for example protein of protein group.Typically, described group covalent attachment.Described group can be by gap or directly combination or connection of joint.

Fusion rotein can comprise in addition with first group solubility isomer acceptor for example, is engaged to the joint sequence of second group.For example, described fusion rotein can comprise peptide linker, and for example, length is about 4 to 20, more preferably, 5 to 10 amino acid whose peptide linkers, described peptide linker length is 8 amino acid.Each amino acid is selected from glycine, Serine, l-asparagine, Threonine and L-Ala in the peptide linker.Described peptide linker comprises glycine-Serine element.In other embodiments, described fusion rotein comprises peptide linker, and described peptide linker comprises the sequence with (Serine-Gly-Gly-Gly-glycine) y molecular formula, and wherein y is 1,2,3,4,5,6,7, or 8 (SEQ ID NOs:73-80).

For example, the soluble form of isomer receptor polypeptides can merge with the CH of various isotypes, and described isotype comprises IgG1, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE.For example, fusion rotein can comprise people FGFR2 isomer III c acceptor the extracellular domain sequence of its homology (or with) and, for example merge the chain to human normal immunoglobulin Fc, for example human IgG (for example, human IgG1 or human IgG2, or its mutant form).Thereby the Fc sequence can strengthen or reduce effector cell function, Fc receptors bind and/or complement activity in one or more propylhomoserin sudden changes.For example, described constant region is at 296 (M to Y) of SEQ ID NO:55,298 (S to T), 300 (T to E), 477 (H to K) and 478 (N to F) thus the position sudden change changes the Fc receptors bind.Figure 13 C (SEQ ID NO:55) has showed that a typical fusion rotein of about 1 to 262 amino acid (Figure 13 C, the 1-262 amino acids of SEQ ID NO:55) that comprises people FGFR2 isomer III c acceptor merges the Fc to the human IgG1 by arginine-Serine joint.

In other embodiments, be conducive to express thereby other aminoacid sequence can increase to N or the C end of fusion rotein, detect and/or isolated or purified.For example, fusion rotein can be connected to one or more other groups, GST for example, His6 label, (His-His-His-His-His-His; SEQ ID NO:81), FLAG label.For example, fusion rotein can be connected to gst fusion protein in addition, wherein said fusion rotein sequence merge to GST(namely, glutathione s-transferase) the C end of sequence.These fusion roteins can be conducive to the purifying of receptor fusion protein.

In one embodiment, described fusion rotein comprises allos signal sequence (for example, by receptor nucleic acids coding, non-existent peptide sequence in polypeptide) at its N end.For example, the signal sequence of acceptor itself is removable and use from the signal sequence of another protein and replace.In some host cells (for example Mammals host cell), can increase receptor expression and/or secretion by using the allos signal sequence.

Chimeric or fusion rotein of the present invention can be by the DNA recombinant technology preparation of standard.For example, the dna fragmentation of different peptide sequences of encoding combines in framework according to routine techniques, for example, by using flat terminal and sticking terminal the connection, thereby Restriction Enzyme digestion provides suitable end, fill up and be suitable sticky end, thereby alkaline phosphatase treatment is avoided undesired connection, and enzymatic connects.In another embodiment, described fusion gene can be synthetic by routine techniques, and described routine techniques comprises automatic dna synthesizer.Alternatively, use anchor primer between two continuous gene fragments, to produce complementary giving prominence to, described two continuous gene fragments annealing and amplification more subsequently, thus produce chimeric gene sequence, the pcr amplification of realization gene fragment (referring to, for example.People such as Ausube (eds.) Current Protocols in Molecular Biology, John Wiley﹠amp; Sons, 1992).In addition, at the commercial expression vector that obtains many fusion groups (for example, the Fc district of heavy chain immunoglobulin) of encoding.The nucleic acid of coding acceptor can be cloned into this expression vector, makes that merging group is connected to immunoglobulin (Ig) in framework.

In certain embodiments, the acceptor fusion polypeptide exists as oligomer, for example dimer or tripolymer.

In other embodiments, the this receptor peptide group is the receptor polypeptides of variation, the receptor polypeptides of described variation causes higher (with respect to not mutated sequence) in receptor sequence (wild-type) sudden change that generates naturally, makes described receptor polypeptides be bonded to the avidity of corresponding part.

In other embodiments, be conducive to express thereby additional aminoacid sequence can increase to N or the C end of fusion rotein, the handiness in space detects and/or isolated or purified.This second polypeptide is preferably soluble.In certain embodiments, the second polypeptide improves the transformation period (for example, the transformation period of serum) of the polypeptide that connects.In certain embodiments, described second polypeptide includes and is beneficial to the sequence that fusion polypeptide is combined with second polypeptide.In an embodiment, described second polypeptide comprises at least a portion of immunoglobulin polypeptides.The immunoglobulin (Ig) fusion polypeptide is well known in the art, and, for example, U.S. Patent number 5,516 is described in 964,5,225,538,5,428,130,5,514,582,5,714,147 and 5,455,165.For example, the soluble form of acceptor can merge the CH to various isotypes, and described isotype comprises IgG1, IgG2, IgG3, IgG4, IgM, IgAl, IgA2, IgD, and IgE.Typically, described fusion rotein can comprise the extracellular domain sequence of its homology (or with) of people's acceptor, and, for example merge extremely, human normal immunoglobulin Fc chain, for example, human IgG (for example, human IgG1 or human IgG2, or its mutant form).

Thereby the Fc sequence can reduce effector cell function, Fc receptors bind and/or complement activity in one or more amino acid sudden changes.The method that changes antibody constant region is known in this area.For example have the function of change, at the effect part, such as the FcR on the cell, or the antibody of the avidity of the change of the Cl composition of complement, at least one amino-acid residue preparation of constant portion that can be by replacing described antibody with different residues (referring to, EP388 for example, 151Al, U.S. Patent number 5,624,821 and U.S. Patent number 5,648,260).Can describe the change of same type, if described change is implemented on mouse or other species, immunoglobulin (Ig) will increase or reduce these functions.For example, by replacing specific residue at its side chain with the residue with suitable function, or by introducing charged functional group, such as L-glutamic acid or aspartic acid, or aromatic series non-polar residue, such as phenylalanine, tyrosine, tryptophane or L-Ala for example can change the Fc district of antibody and FcR(, Fc γ RI) or the affinity of Clq combination (referring to, for example U.S. Patent number 5,624, and 821).

In an embodiment, the second polypeptide has the effector function effector function still less than the Fc district of wild-type heavy chain immunoglobulin.The Fc effector function comprises, for example, and the Fc receptors bind, complement fixation(CF) and t cell depletion activity (referring to, for example, U.S. Patent number 6,136,310).Analysis t cell depletion activity, the method for Fc effector function and antibody stability is known in this area.In one embodiment, described second polypeptide has low-affinity or does not have detectable avidity the Fc acceptor.In an optional embodiment, described second polypeptide has low-affinity or does not have detectable avidity complement proteins Clq.In other embodiments, described second polypeptide has the effector cell function of increase, for example, increase and be bonded to Fc acceptor (for example, FcyRI, FcyRIIA, FcyRIIB, FcyRIIIA and FcRn acceptor), as, people such as Shields for example, (JBC, 276:6591-6604,2001) and U.S. Patent number 6, described in 737,056.

It should be understood that, described antibody molecule described here and soluble receptors or fusion rotein are (for example, pass through chemical coupling, gene fusion, non-covalent connection or other) can functionally be connected to one or more other molecular entities, for example antibody is (for example, dual specific or multi-specificity antibody), toxin, radio isotope, cytotoxic agent or cytostatics etc.

Peptide or its functional variant

In another embodiment, described isomer specific inhibitor comprises peptide or its functional variant (for example, functional analogue or derivatives thereof).

To refer to its aminoacid sequence identical with aminoacid sequence except described peptide for peptide " analogue " as used herein, can have to be higher than 10, typically is higher than 8, is higher than 6, be higher than 5, be higher than 4, be higher than 3, be higher than 2, or be higher than 1 amino acid whose insertion, disappearance and/or replacement.Typically, analogue peptide as described equally is bonded to identical biological acceptor, and has showed at least some biological activitys of described peptide.Described peptide may derive or be connected to another functional molecular (for example, another polypeptide or protein, for example carrier proteins) and/or pass through to increase fluorophor, the radioactive nuleus thuja acid, and toxin, enzyme, polyoxyethylene glycol (PEG), or affinity ligand is such as vitamin H.

Term " carrier proteins " is protein or peptide as used herein, described protein or peptide can improve the production of the antibody of protein, and described protein is the protein relevant with carrier proteins and/or the protein that can be used for detecting the protein relevant with carrier proteins.For the purpose of immunity, the big portion of many different carrier proteinss can be used for being coupled with peptide.Select to use which kind of carrier should be based on immunogenicity, whether solubility can realize with the suitable combination of carrier and use shaker test so that the antibody recognition target proteins.Two kinds of the most frequently used carriers are hemocyanin (KLH) and bovine serum albumin (BSA).Other two kinds comprise secretor type alkaline phosphatase (SEAP), horseradish peroxidase, luciferase, beta-galactosidase enzymes, IgG Fc (γ chain), Thiadiazolidine isomerase (GST), label and other enzyme of containing the poly Histidine, as β-Nei Xiananmei, other secretary protein or polypeptide.

The peptide of modification of the present invention, binding substances or compound comprise covalently bound to peptide or activity of proteins group.Selection can form the active group of stable covalent linkage with serum protein or polypeptide, for example, by with one or more amino groups, oh group, or the reactive group that reacts at serum protein or polypeptide of sulfenyl group.Typically, reactive group and a kind of amino group, oh group, or the sulfenyl group reacts at serum protein or polypeptide.Typically, reactive group and specific amino group, oh group, or the sulfenyl group reacts at serum protein or polypeptide.Binding substances of the present invention comprises the peptide of modification, and described peptide covalency is by reactive group and amino group, oh group, or the reaction of sulfenyl group on serum protein or peptide is connected to serum protein or peptide.Therefore, binding substances of the present invention comprises the peptide of modification, and in described peptide, the residue of described reactive group and serum protein or peptide form covalent linkage." residue of reactive group " or " reactive group residue " refers to by reactive group and other group as used herein, the protein that exists in peptide or the blood for example, between covalent linkage form the chemical structure that produces.Peptide in modification of the present invention, among the embodiment of binding substances or compound, reactive group is maleimide, this maleimide comprises and is selected from γ maleimide butyric acid amine (GMBA), maleimide propionic acid (MPA), N-hydroxy-succinamide (NHS), N-hydroxy thiosuccinimide (sulfo-NHS), maleimide yl benzoic acid succinimide ester (MBS) and γ maleimide butyric acid succinimide ester (GMBS).

Peptide of the present invention comprises the peptide linker group, and described peptide may be synthetic by solid phase or the liquid phase chemical method of peptide standard.Stewart and Young (1963) Solid Phase Peptide Synthesis, W.H.Freeman Co. (San Francisco), and Meienhofer (1973) Hormonal Proteins and Peptides, can find the solid phase technique summary among the Academic Press (New York).Classical liquid phase is synthetic referring to Schroder and Lupke, The Peptides, Vol.1, Academic Press (New York).

Usually, these methods comprise that continuity increases one or more amino acid or suitable protected amino acid is growing chain.Usually, first amino acid whose amino or carboxyl are by suitable blocking group protection.Protected amino is connected to the inert solid support or uses in solution by increase next amino acid in sequence, and described sequence has complementation (amino or carboxyl) group appropriate protection and that be fit to form peptide bond under certain condition.Blocking group removes and increases next amino acid (appropriate protection) from the amino-acid residue that increases newly then, carries out with this.After all amino acid of wanting connect into suitable sequence, the blocking group of any reservation (with any solid support) thus remove the peptide that provides final in succession or simultaneously.By this general procedure is simply modified; may increase more than an amino acid to growing chain at every turn; for example, by protected tripeptides (under the situation that does not make the chiral centre racemization) is connected to suitable protected dipeptides, thereby going to protect the back to form pentapeptide.

In certain embodiments, peptide class of the present invention is synthesized by amino and carboxy protective group as prodrug.Blocking group is the chemical part that the reactive group on the protection peptide class to avoid undesirable reaction.In one embodiment, modified peptides of the present invention synthesizes by one or more blocking groups, and described blocking group is used for division in vivo, makes the reactive group of modified peptides or group be exposed to serum protein after experimental subjects is taken the peptide class with this.

Term " amino-protecting group " refers to the N-terminal for the protection of amino acid or peptide class, or the amino of protection amino acid or peptide class to avoid undesirable reaction.At Greene (1981) Protective Groups in Organic Synthesis (John Wiley﹠amp; Amino-the blocking group of general use is disclosed Sons, New York), the content of this article at this with reference to introducing.In addition, for example by enzymic hydrolysis, can use in vivo the blocking group of division easily, expose amino for reacting with serum protein in vivo with this.

Term " carbonyl-protection base " refers to for the carboxylic acid protection ester or the amide group that stop or prevent carboxylic acid function's property.In (1981) the carbonyl-protection group is disclosed at Greene " Protective Groups in Organic Synthesis " pp.152-186, the content of this article at this with reference to introducing.In addition, carbonyl-protection base useful as drug precursor, therefore, for example division easily by enzymic hydrolysis in vivo of carbonyl-protection base exposes carbonyl with this, to react with serum protein in vivo.Those skilled in the art is familiar with this carbonyl-protection group very much, and these carbonyl-protection groups are widely used for the protection of carbonyl group in penicillin and the cynnematin field, as United States Patent (USP) 3; 840,556 and 3,719; described in 667, the content of this patent at this with reference to introducing.

Preferred carbonyl-protection peptide class of the present invention is that wherein protection carbonyl is low alkyl group, the peptide class of cycloalkyl or alkyl aryl, for example: methyl esters; ethyl ester, propyl ester, isopropyl ester; butyl ester, sec-butyl ester, isobutyl ester; n-pentyl ester; isopentyl ester, monooctyl ester, cyclohexyl ester and styroyl ester or acyl group oxyalkyl; ring acyl group alkoxyl group, aroyl oxyalkyl or aromatic yl alkyl carbonyl alkoxy ester.Preferred amido carboxyl blocking group is the low-grade alkyl amino carbonylic group.For example, aspartic acid is protected by acid-unstable group (for example, the tertiary butyl) at the C-end, and terminal by the unstable group of hydrogenation (for example: phenmethyl) be protected, go protection subsequently in synthetic at β-C-.

In certain embodiments, the functional varient of peptide class or peptide class is made of aminoacid sequence, or comprise aminoacid sequence, the bonding land of described aminoacid sequence between two exons, this exon mainly combines in one or more cancer cells or tumour cell or disease expression or proteins associated isomer, for example, because the use that the interior Exon deletion of framework or selectivity are sheared exon.In certain embodiments, the functional varient of peptide class or peptide class is made of aminoacid sequence, or comprise aminoacid sequence, the bonding land of described aminoacid sequence between two exons, this exon mainly combines in one or more cancer cells or tumour cell or disease expression or proteins associated isomer, for example, because the use that the interior Exon deletion of framework or selectivity are sheared exon.In one embodiment, the functional varient of peptide class or peptide class is made of aminoacid sequence, or comprise aminoacid sequence, described aminoacid sequence reaches or is less than 60 seed amino acids and (for example, reaches or be less than 50,40,30, and equal the selectivity shear-form of exon III 20,10 amino acid),, for example, from the about 301-360 amino acid of FGFR2-IIIc (SEQ ID NO:2), the about 314-324 amino acid of FGFR2-IIIc (AAGVNTTDKEI, SEQ ID NO:4), the about 328-337 amino acid of FGFR2-IIIC (YIRNVTFEDA, SEQ ID NO:6), the about 350-353 amino acid of FGFR2-IIIc (ISFH, SEQ ID NO:8); The about 314-353 amino acid of FGFR2-IIIc (AAGVNTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFH_ (SEQ ID NO:84)), or the about amino acid TCLAGNS of FGFR2-IIIc IGIS FH (SEQ ID NO:86), or by SEQ ID NOs1,3,5,7,83 or 85 nucleotide sequence coded aminoacid sequence, or the amino acid that roughly equates with it or nucleotide sequence.In another embodiment, the functional varient of peptide class or peptide class is made of aminoacid sequence, or comprise that aminoacid sequence, described amino acid reach or be less than 60 seed amino acids and (for example, reach or be less than 50,40,30,20,10 amino acid), and equal FGFRIL (SEQ ID NO:10) or the Ig-II of its fragment and the engaging zones between the Ig-III, or by the nucleotide sequence coded aminoacid sequence of SEQ ID NO:9 or its fragment; Or the amino acid that roughly equates with it or nucleotide sequence.In another embodiment, polypeptide class or its functional varient are made of aminoacid sequence, or comprise aminoacid sequence, described aminoacid sequence reaches or (for example is less than 60 seed amino acids, reach or be less than 50,40,30,20,10 seed amino acids), and be equal to the exon 4 of isomer RONA160 or its fragment and the engaging zones between the exon 7, or by the nucleotide sequence coded aminoacid sequence of SEQ ID NO:11 or its fragment, or the amino acid that roughly equates with it or nucleotide sequence.In another embodiment, peptide class or its functional varient are made of aminoacid sequence, or comprise aminoacid sequence, and described aminoacid sequence reaches or (for example is less than 60 seed amino acids, reach or be less than 50,40,30,20,10 seed amino acids), and be equal to the engaging zones of the KIT between the exons 10 and 12 of SEQ ID NO:14 or its fragment, or by the nucleotide sequence coded aminoacid sequence of SEQ ID NO:13 or its fragment, or the amino acid that roughly equates with it or nucleotide sequence.In another embodiment, peptide class or its functional varient are made of aminoacid sequence, or comprise aminoacid sequence, and described aminoacid sequence reaches or (for example is less than 60 seed amino acids, reach or be less than 50,40,30,20,10 seed amino acids), and be equal to the engaging zones of the PDGF between the exon 5 and 7 of SEQ ID NO:16 or its fragment, or by the nucleotide sequence coded aminoacid sequence of SEQ ID NO:15 or its fragment, or the amino acid that roughly equates with it or nucleotide sequence.In another embodiment, peptide class or its functional varient are made of aminoacid sequence, or comprise aminoacid sequence, and described aminoacid sequence reaches or is less than 60 seed amino acids (for example, reach or be less than 50,40,30,20,10 seed amino acids).And be equal to PDGFR – α engaging zones between the exon 6 and 9 of SEQ ID NO:18 or its fragment, or by the nucleotide sequence coded aminoacid sequence of SEQ ID NO:17 or its fragment, or the amino acid that roughly equates with it or nucleotide sequence.

Peptide class or its functional varient can for example use solid phase synthesis technique to reconfigure or be synthetic, and it is at least a or optionally that this isomer-specific inhibitor can comprise, two kinds or above above-mentioned peptide class or its varient.For example, selectively by catenation sequence, two kinds or above peptide class or any combination of peptide class varient are set.This peptide class can be functionally and one or more other molecular entity, for example: carrier (for example: immunoglobulin fc region, serum albumin, polyoxyethylene glycol, GST, Lex-A or MBP peptide sequence) (for example connect, pass through Chemical bond, gene fusion, non-covalent combination or other side), to strengthen the peptide class stability in the body.The peptide class is optionally for example passed through, and adding the chemoproection group increases the interior peptide class stability of body.

Polyoxyethylene glycol

For increasing transformation period of pharmacy albumen and/or reduce the widely used technology of immunogenicity institute and comprise and add suitable pharmacology acceptable polymer, for example, poly-(ethylene glycol) (PEG) or derivatives thereof (for example, methoxyl group gathers (ethylene glycol) or mPEG).In general, can use the suitable form of any polyoxyethylene glycol, for example, be fit to the polyoxyethylene glycol of usefulness in the antibody molecule field; About this document the Nat.Biotechnol. of Chapman, 54,531-545 (2002) are arranged; The Adv.Drug Deliv.Rev.54 of Veronese and Harris, 453-456 (2003), the Nat.Rev.Drug.Discov. of Harris and Chess, 2, (2003) and WO04/060965.The different reagent of polyoxyethylene glycol albumen can commercially obtain equally, and for example, from Nektar Therapeutics, the USA place obtains.

Preferably, especially can use the fixed point polyoxyethylene glycol by cysteine residues (for example: Yang et al., Protein Engineering, 16,10,761-770 (2003)).For example, for this reason, PEG can be connected to the cysteine residues that exists naturally in the isomer specific inhibitor, this inhibitor can be introduced one or more cysteine residues suitably and be connected with PEG through modifying, comprise that perhaps the next aminoacid sequence that engages with PEG of one or more cysteine residues can be fused to N-and/or the C-end of inhibitor of the present invention, these technology that all are to use those skilled in the art to be familiar with realize.

Preferably, for the isomer specific inhibitor, use molecular weight greater than 5000, for example greater than 10,000 less than 200,000, as less than 100,000; For example at 20,000-80, the PEG in 000 scope.

For polyoxyethylene glycol, be noted that generally speaking the present invention comprises any SDAM molecule at one or more amino acid position adding polyoxyethylene glycol equally, preferably, its mode makes polyoxyethylene glycol (1) increase the transformation period in the body; Or (2) reduce immunogenicity; Or (3) or the favourable characteristics that provide one or more other polyoxyethylene glycol itself to be familiar with; (4) substantially do not influence the affinity (for example, do not reduce by 90% described affinity, preferably do not reduce greater than 50%, and do not reduce greater than 10%, do not determine that as suitable article for example following examples are described) of SDAB molecule; Or (4) do not influence any other desirable properties of isomer-specific inhibitor.No matter suitable PEG-group and the method that is connected them are specific or unspecific, all are familiar with to those skilled in the art.

Be used for the suitable external member of this polyoxyethylene glycol and reagent can for example (CA USA) locates to obtain from Nektar.In addition, general less preferred modification comprises that N-connects or O-connects glycosylation, and is general as translation and/or posttranslational modification altogether, depends on for the host cell of expressing the isomer specific inhibitor.

The nucleic acid binding molecule

In another embodiment, and isomer-specific inhibitor (for example: the expression of inhibition nucleic acid encoding isomer isomer-binding molecule), for example: carcinogenic isomer (for example, carcinogenic isomer described herein).The embodiment of this isomer-binding molecule comprises nucleic acid molecule, for example, and antisense molecule, ribozyme, RNAi is hybridized into the nucleic acid encoding isomer, for example: the triple helix molecule of carcinogenic isomer, or the transcriptional regulatory zone, and stop or the minimizing isomer, for example: the mRNA of carcinogenic isomer expresses.In one embodiment, the nucleic acid binding molecule that can suppress carcinogenic isomer expression is antisense oligonucleotide, and this antisense oligonucleotide can be hybridized into specially to the knurl isomer.

The sequence that should be appreciated that antisense compounds does not in the art need 100% to replenish its target nucleic acid, with special this sequence of hybridizing into.When compound was combined the target dna of having intervened normal function or RNA with target dna or RNA sequence, antisense compounds was hybridized into target dna or RNA sequence specially.This intervention should cause the loss of effectiveness, and need there be enough additional degree needing under the situation of particular combination avoiding, the non-special combination of antisense compounds and non-target sequence, this situation is under the interior chemical examination of body or the treatment treatment condition, and under the situation of external chemical examination, be in the physiological condition under the situation about chemically examining.

The Antisensedigonucleotsequence sequence that can hybridize into carcinogenic isomer specially can be identified by normal experiment.In one embodiment, this Antisensedigonucleotsequence sequence can ad hoc be provided into by the nucleotide sequence that provides at this, for example: be selected from SEQ ID NOs:2,4,6,8,10,12,14,16 and 18 sequence coded polypeptide.In another embodiment, Antisensedigonucleotsequence sequence can be hybridized into nucleic acid specially, and described nucleic acid comprises SEQ ID NO:1,3,5,7,9,11,13,15 or 17 sequence.

In another embodiment, can suppress the compound that carcinogenic isomer expresses is that RNAi makes up.In one embodiment RNAi makes up can hybridize into nucleotide sequence described herein specially, for example, is selected from and comprises SEQ ID NOs:2,4,6,8,10,12,14,16 and 18 or the sequence coded polypeptide that roughly equates with it.

This antisense oligonucleotide and RNA make up and can be used for suppressing specially the expression of carcinogenic polypeptide isomer, and can not suppress from same proto-oncogene and next non-carcinogenic polypeptide isomer.Use this technology, can study the specific function of each carcinogenic polypeptide isomer.In addition, translation oligonucleotide and RNAi make up and can be used for disease treatment.

This antisense oligonucleotide is short relatively nucleic acid, and this short relatively nucleic acid replenishes the coding strand (positive-sense strand) of the mRNA of encode specific protein matter.Though antisense oligonucleotide is generally the RNA base, they still can be the DNA base.In addition, antisense oligonucleotide is usually through modifying to increase their stability.For example, see Antisense Technology in Methods in Enzymology, Vols.313-314, ed.by Phillips, Abelson and Simon, Academic Press, 1999.

This oligonucleotide can be DNA or RNA, or their chimeric mixture or derivative, or their modification version, strand or two strands.This oligonucleotide can be in the alkali part, sugar moieties, or phosphate backbone modifies, and for example, improves its stability, crossability etc.This oligonucleotide can comprise other additional group, for example, peptide class (for example being used for the target host cell acceptor), or help (to ask for an interview at the reagent that cytolemma transmits, for example: Letsinger et al., Proc.Natl.Acad.Sci.U.S.A.86:6553-56 (1989); Lemaitre et al., Proc.Natl.Acad.Sci.U.S.A.84:648-52 (1987); International Patent Publication No.WO88/09810), or blood-brain barrier (is asked for an interview, for example: International Patent Publication No.WO89/10134), hybridization-trigger division reagent (to ask for an interview, for example: Krol et al., BioTechniques6:958-76 (1988)) or insert reagent, (ask for an interview, for example: Zon, Pharm.Res.5:539-49 (1988)).Therefore, this oligonucleotide can with other molecule coupling.Antisense oligonucleotide can comprise the skeleton of neutral similar peptide equally.This molecule is called peptide nucleic acid(PNA) (PNA)-oligomer, and as Perry-O'Keefe et al., Proc.Natl.Acad.Sci.U.S.A.93:14670 (1996) and in Eglom et al. is described in the Nature365:566 (1993).

Oligonucleotide of the present invention can synthesize by the standard method that this area is familiar with, for example, (for example can be from Biosearch Technologies by the use automatic dna synthesizer, Inc. (Novato, Calif.), Applied Biosystems (Foster City is Calif.) with other local commercial acquisition).For example, phosphorothioate oligonucleotide can synthesize by the method for Stein et al. (Nucl.Acids Res.16:3209 (1988)), and the methyl-phosphorous acid oligonucleotide can support (Sarin et al., Proc.Natl.Acad.Sci.U.S.A.85:7448-51 (1988)) by the glass, polymer that uses the control hole) prepare.

Based on above-mentioned explanation, the selection of suitable oligonucleotide can be undertaken by those skilled in the art easily.Nucleic acid for given encode specific protein matter, those skilled in the art can design the antisense oligonucleotide in conjunction with this albumen, and test these oligonucleotide, and in vivo or these oligonucleotide of living systems build-in test, with determine their in conjunction with and the degraded of mediating the mRNA of encode specific protein matter.In order to design special combination and to mediate the antisense oligonucleotide that specified protein is degraded, the sequence of being confirmed by oligonucleotide can be unique or roughly unique for this specific protein.For example, for the design of oligonucleotide of special affirmation and degraded customizing messages, on protein frequent repeating sequences can and nonideal selection.Those skilled in the art can design a kind of oligonucleotide, and sequence and the nucleotide sequence of this oligonucleotide compared, determining this sequence for being specific for specific protein, or roughly specific, this nucleotide sequence places the obtainable database of the public.

Many many methods that antisense DNA or RNA transferred to cell have been developed at present, for example, antisense molecule can directly inject tissue location, maybe will be for the desirable cell of target (for example, with the antisense molecule that peptide class or antibody are connected, this peptide class or antibody specifically are combined with the acceptor of expressing on the target molecule surface or antigen) the modification antisense molecule take for the experimental subjects whole body.Ask for an interview, for example: Antisense Technology in Methods in Enzymology, Vols.313-314, ed.by Phillips, Abelson and Simon, Academic Press, 1999.

RNAi makes up and comprises the double-stranded RNA that can stop target gene to be expressed specially, " RNA interference " or " RNAi " term are used in plant and the viewed phenomenon of worm at first, and its double center chain-RNA (dsRNA) stops genetic expression in the mode of specific and back-transcribe.As if under the situation that is not subjected to any particular theory restriction, RNAi comprises the mRNA degraded; Yet its biological chemistry mechanism becomes the field of enlivening of experiment.

Said, term " dsRNA " refers to the siRNA molecule, or other comprises double-stranded characteristic, and can be treated as the RNA molecule of siRNA in the cell, for example: the hairpin RNA part.

Said, term " RNAi structure " is the gene term in the whole specification sheets, and it comprises RNA interfering s (siRNAs), hairpin RNA s and other RNA kind, and these RNA kinds can divide in vivo, to form siRNAs.RNAi is structured in this and comprises expression vector (also referring to the RNAi expression vector) equally, and this expression vector can cause transcribing of the interior dsRNAs of formation cell or hairpin RNA s, and/or produces in vivo transcribing of siRNAs.

" RNAi expression vector " (dsRNA-encode plastid) (being called " dsRNA-coding " plastid equally) refers to that this RNAi expression vector produces the siRNA part in the cell of construction expression for the reproducible nucleic acid construct of expressing (transcribing) RNA.This carrier comprises the transcriptional units by following (1) (2) and (3) assembling, (1) genetic constitution, this genetic constitution has regulating and controlling effect in genetic expression, for example, activate son, operon, or enhanser, and be connected with (2) " coding " sequence effectively, should " coding " sequence through transcribing produce two-two RNA parts of chain RNA(in cell annealing with formation siRNA, or single hairpin RNA, this hairpin RNA can be processed into siRNA), and (3) suitable transcription initiation and terminator sequence.

The selection that activates son and other regulation and control composition generally changes according to host cell.In general, the expression vector of recombinant DNA technology effectiveness is generally " plastid " form, and it refers to circular double-stranded DNA ring, and their carrier format is not limited to karyomit(e).In specification sheets of the present invention, " plastid " and " carrier " uses convertibly, because plastid is the most common form of carrier.Yet, the objective of the invention is to comprise this other form of expression vector, described other form is as equivalent function, and is that this area is familiar with after this.The method of chemically modified RNA molecule make up applicable to modifying RNAi (ask for an interview, for example, Heidenreich et al., Nucleic Acids Res.25:776-80 (1997); Wilson et al., J.Mol.Recog.7:89-98 (1994); Chen et al, Nucleic Acids Res.23:2661-68 (1995); Hirschbein et al, Antisense Nucleic Acid Drug Dev.7:55-61 (1997)).As an illustration, the skeleton of RNAi structure can be used thiophosphoric acid, phosphoramidate, phosphorodithioate, chimeric methylphosphonate-phosphide-fat, peptide nucleic acid(PNA), comprise oligomer or sugar-modified-5-proyl-pyrimidine (for example, the ribonucleoside that 2'-replaces, a-configuration).

This pair-chain structure can be by single from mending the RNA chain or two complementary RNA chains form.The formation of RNA duplex can be in cell or the extracellular cause.This RNA can some amount introduces, and this makes provides and delivers each cell at least one copies.Useful equally for specific application when lower dosage, the more multiple doses of double-stranded material (for example: each cell has 5,10 at least, and 100,500 or 1000 are copied) can produce more effective inhibition.Suppress for sequence specific because corresponding to the nucleotide sequence in RNA duplex district with gene inhibition as target.

This siRNA molecule can obtain with the multiple technologies that those skilled in the art are familiar with.For example, can come chemosynthesis by the technology that this area is familiar with, or reorganization produces.For example, Duan justice and sense-rna oligomer can synthesize and anneal to be formed on double-stranded RNA structure (Caplen et al, Proc.Natl.Acad.Sci.U.S.A., the 98:9742-47 (2001) of the 2-Nucleotide of every end suspension; Elbashir et al., EMBO J., 20:6877-88 (2001)) Elbashir et al., EMBO J., 20:6877-88 (2001)).These double-stranded siRNA structures can directly be introduced cell subsequently by the selection of following passive absorption or delivery system.

A kind of intracellular method at this carcinogenic isomer that provides that suppresses is provided equally in the present invention, and this method comprises with the compound that can suppress carcinogenic isomer expression comes exposing cell.The inhibition that carcinogenic isomer is expressed can be useful on preventing and/or treating of cancer.The inhibition that the carcinogenic isomer of FGFR2-IIIc is expressed can be used for preventing and/or treating the intractable prostate cancer of hormone, mammary cancer, bladder cancer, the cancer of thyroid carcinoma or other form.The inhibition that FGFR1L expresses can be used for preventing and/or treating carcinoma of the pancreas, prostate cancer, or the cancer of other form.The inhibition that RON receptor tyrosine kinase Δ 160 isomer are expressed can be used for preventing and/or treating the transitivity rectum cancer, mammary cancer, ovarian cancer, lung cancer, bladder cancer, or the cancer of other form.The inhibition that the carcinogenic isomer of KIT receptor tyrosine kinase is expressed can be used for preventing and/or the cancer .PDGFR-a isomer for the treatment of gastrointestinal stromal tumor (GISTs) or other form can be used for preventing and/or treating the cancer of the brain, glioblastoma multiforme, prostate cancer, bone shifts, GIST, or the cancer of other form.

In one embodiment, this method is carried out external.In another embodiment, this method is carried out in vivo.These methods can expressed relevant research with carcinogenic isomer, and diagnosis and treatment aspect are used.Under study for action, can use these methods, for example: the mechanism of action that the carcinogenic isomer of the present invention is described.

Pharmacy composite and external member

On the other hand, the invention provides a kind of composition, for example, pharmaceutically acceptable composition, said composition comprises isomer-special inhibitor described herein, prepares with pharmaceutically acceptable carrier.Said, " pharmaceutically acceptable carrier " comprises any and all solvents, and dispersion medium waits to blend the absorption delay agent, etc.They can be compatible for physiology, and this carrier is applicable to vein, and muscle is subcutaneous, parenteral, rectum or epidermis administration (for example: by injection or instillation).

Composition of the present invention can be different forms.Comprise, for example: liquid, semi-solid and solid preparation form, (for example: injectable and indissoluble solution), dispersion agent or suspension, liposome and suppository.Its preferred form depends on the application in required pattern of taking and the treatment.Typical preferred composition is injectable or indissoluble solution.The preference pattern of administration is administered parenterally (for example, vein, subcutaneous, intraperitoneal, muscle).In a preferred embodiment, antibody is by instiling or injecting administration.In another preferred embodiment, this antibody comes administration by muscle or subcutaneous injection.

Term described herein " administered parenterally " and " take in the stomach other places " refer to the pattern of taking, rather than general stomach and topical by injection, and comprise without limitation: vein, muscle, artery is in the sheath, in the capsule, in the frame, in the heart, intraperitoneal, through tracheae, subcutaneous, under the epidermis, intraarticular is under the capsule, under the arachnoid membrane, in the backbone, exterior dura and breastbone inner injection and instillation.

Therapeutic composition generally should be sterilization, and stable under production and storage condition.Said composition can be mixed with solution, microemulsion, and dispersion agent, liposome, or other are applicable to the ordered structure of high antibody concentration.Sterile injectable solution can make by the active compound in the suitable solvent of incorporating the above-mentioned a kind of composition enumerated of being dissolved in of desired number or multiple composition combination into (that is: antibody or antibody moiety).As required, then carry out filter-sterilized.In general, dispersion agent makes by active compound being incorporated into the sterilization carrier, and this sterilization carrier comprises basic dispersion medium and above-mentioned other required composition of enumerating.Under the sterilization powder situation of the Injectable solution that preparation is sterilized, the preferred method of preparation is vacuum-drying and lyophilize, it has produced the activeconstituents powder and has added the desirable from aforesaid its sterilization-filtering solution of other, can pass through, for example: use the coating as Yelkin TTS, if dispersion agent then passes through the required particle size of maintenance, and by using tensio-active agent to keep the proper flow of solution.The lasting absorption of Injectable composition can realize that the reagent that this delay absorbs for example is: Monostearate and gelatin by comprising the reagent that postpones to absorb in the composition.

Isomer-specific inhibitor of the present invention can carry out administration by the different methods of this area, although in the field of many treatments, the optimization approach/pattern of administration is injection or instillation.For example, antibody molecule is by with less than 10mg/min, and the speed that preferably is less than or equal to 5mg/min is carried out intravenous injection, to reach about 1-100mg/m ², be preferably about 5-50mg/m ², about 7-25mg/m ², 10mg/m more preferably ²As skilled in the art to understand, the approach of administration and/or mode change along with desirable effect.In certain embodiments, active compound can prepare with the carrier that this compound of protection is avoided discharging fast, and the prescription that this carrier for example discharges for control comprises: bury and fill out agent, through the delivery system of skin patch and the microcapsule of packing into.Can use biology decomposable, biocompatible polymkeric substance, for example, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and poly(lactic acid).Have and manyly occur or be familiar with by those skilled in the art about the method for preparing this prescription.See: for example: Sustained and Controlled Release Drug Delivery Systems, J.R.Robinson, ed., Marcel Dekker, Inc., New York, 1978.

In certain embodiments, specific inhibitor isomer of the present invention can be oral, for example, and with inert diluent or absorbable edible carrier.This compound (when needing and other composition) can wrap into hard equally or soft shell capsule in, be collapsed into tablet, or directly incorporate in the diet of experimental subjects.For oral administration, this compound can use together with vehicle, and with absorbable tablet, sucks tablet, lozenge, and capsule, panacea, suspension, syrup, forms such as thin slice are used.Except by taking compound of the present invention with the mode of administered parenterally, can coat the material that prevents this compound inactivation, or take with this compound.The medical treatment device that therapeutic composition can be familiar with this area is equally taken.

Can adjust dosage regimen provides the ideal effect of optimization (for example, result for the treatment of).For example, can take single bolus, along with the time is taken several dosage that separate, or according to the treatment situation eager needs reduce or increase dosage in proportion.For the unification of the easy and dosage taken, it is very favourable preparing parenteral composition with the form of dosage device.Dosage device refers to the unit that physics disperses as used herein, and the unit that this physics disperses is applicable to is used as the dosage unit that object to be treated is arranged.Each unit comprises the active compound of predetermined amount, and the quantity of this active compound can produce desirable result for the treatment of together with required pharmacy carrier.Dosage unit form of the present invention explanation can by or directly depend on the unique features of (a) active compound and the particular treatment effect that will reach, and (b) develop this inherent limitations that is used for the treatment of the active compound of individual susceptibility in this area.

In one embodiment, the unrestricted scope of the antibody of the significant quantity that is used for the treatment of or prevents or antibody moiety is 0.1-20mg/kg, 1-10mg/kg more preferably, isomer-specific inhibitor passes through less than 10mg/min, the speed that more preferably is less than or equal to 5mg/min is carried out intravenous injection, to reach about 1 to 100mg/m ²Dosage, be preferably about 5-50mg/m ², about 7-25mg/m ², about 10mg/m more preferably ²Be noted that dosage can be along with the type of the situation that will alleviate and severity and change.For any specific object, further understand the given dose system and can come along with the time is adjusted along with the personnel's of individual need and composition administration or management administration professional judgement.And dosage range described herein only is example, is not scope or application for the claimed composition of restriction.

Pharmacy composite of the present invention can comprise antibody or the antibody moiety of " treatment significant quantity " or " prevention significant quantity "." treatment significant quantity " refers to obtain the effective dose of desirable treatment result in the time cycle of necessity.The antibody of modifying or the treatment significant quantity of antibody fragment can be according to for example, individual morbid state, and the age, sex, and body weight, and antibody or antibody moiety lean out the ability of ideal effect and change in individuality.Treatment significant quantity when the treatment significant quantity is similarly the treatment advantageous effects above toxicity any in the antibody of modifying or the antibody fragment or harmful effect.With respect to untreated object, " treatment significant quantity " preferably suppressed measurable parameter, and for example, growth of tumor speed suppresses at least about 20%, more preferably at least about 40%, and more preferably at least about 60%, and more preferably at least 80%.Compound suppresses measurable parameter, for example: the ability of cancer can be evaluated in the animal model system of predict human tumor function, alternately, the character of composition can be evaluated by the ability that detection compound suppresses, and this vitro inhibition by analysis is familiar with by those skilled in the art.

" prevention significant quantity " referred to reach the effective dose of desirable preventive effect in the cycle time of necessity.Usually, owing to used preventive dose before the commitment of disease, this prevention significant quantity can be less than the treatment significant quantity.

Be the external member that comprises the special inhibitor isomer equally, within the scope of the invention.

This external member comprises one or more other elements, comprising: operation instruction; Other reagent, for example: mark, therapeutical agent, or reagent is used for chelating, or conversely, antibody is connected to labeling or treatment reagent, or the radiological protection composition; Device or other material for the preparation of the antibody of taking; Pharmaceutically acceptable carrier; And the material taken of device and experimental subjects.Operation instruction can comprise isomer-binding molecule external, for example, and in sample, as: the explanation of the diagnostic use in cancer or prostatosis patient's living tissue or cell and the body.This explanation can comprise the explanation of the treatment application that has the suggestion consumption and/or take mode, for example, has among the patient of cancer or prostatosis.Other explanation can comprise antibody is connected to sequestrant, mark or treatment reagent, or be used for purifying coupling antibody, and as: from the explanation of unreacted conjugation composition.As mentioned above, this external member can comprise mark, for example any mark described herein.As mentioned above, this external member can comprise treatment reagent, for example: treatment reagent described herein.This external member can comprise reagent, is used for mark or treatment reagent chelating, or is attached to antibody conversely, for example: reagent described herein.For example, macrocyclic chelants is preferably and comprises 1,4,7,10-tetraazacyclododecanand-N, N', N ", N " ', 4-tetraacethyl (DOTA).This DOTA can be used as independent one-tenth and assigns to provide, or DOTA(or other sequestrant or coupling agent) can provide afterwards with antibodies.Other coupling agent, for example: N-maloyl imines (NHS) can be provided for sequestrant, for example: DOTA is bonded to antibody.In some applications, this antibody can react with other composition, and this other composition for example is: sequestrant or mark, or treatment reagent, for example: radio isotope, as: yttrium or gold-plating.In this case, this external member can comprise the container of one or more realization responses, or the device of separating.For example: chromatographic column is used for finished product is separated with starting raw material or reaction intermediate.

This external member also can comprise the reagent that at least one is extra, for example: diagnosis or treatment reagent, for example: diagnose as described here or treatment reagent, and/or one or more extra isomer-specific inhibitors, prepare to carry out suitable preparation with one or more separated drug.

This external member also can comprise radio-protector.Isotopic radiation essence, for example: known ⁹⁰Yttrium ( ⁹⁰Y).For fear of radiolysis, can comprise radio-protector, for example: in reaction buffer, as long as these radio-protectors are gentle, the meaning is the effect that they can not suppress or be unfavorable on the contrary labeled reactant, for example is isotropic substance, as: ⁹⁰The labeled reactant of Y and antibody.

Prescription damping fluid of the present invention can comprise radio-protector, and for example: human serum albumin (HSA) or ascorbate salt, it is because yttrium or other radionuclide minimize radiolysis.Other radio-protector is that this area is familiar with, and can be used for prescription damping fluid of the present invention equally, that is: free-radical scavengers (phenol, sulphite, gsh, halfcystine, gentisinic acid, nicotinic acid, ascorbyl palmitate HOP (: 0) H ₂I glycerine, rongalite, Na ₂S ₂O, Na ₂S ₂O ₃, and SO ₂Deng).

Preferred external member is carried out labelled with radioisotope sequestrant coupling protein or peptide class by the radio isotope of taking treatment to the patient.This external member comprises that (i) is equipped with the bottle of sequestrant coupling antibody, (ii) comprises the bottle for prescription damping fluid stable and that radiolabelled antibody is taken to the patient, and the explanation of (iii) carrying out the radio-labeling step.This external member provides as advising in the explanation, under mild conditions sequestrant-coupling antibody is exposed to radio isotope or its salt with the time of q.s.Produced and had enough purity, the radiolabelled antibody of specific activity and specificity combination.This radio-labeling can be diluted to suitable concentration, for example: and in the damping fluid of configuration, and through maybe need not directly taking to the patient through further purifying.This sequestrant-coupling antibody can freeze-drying form provide.

Purposes of the present invention

Isomer-specific inhibitor of the present invention has the diagnosis in external and the body, and treatment and prophylactic effect.For example, these binding molecules can be administered to the cell in the cultivation, for example: and in external or body, or in object, for example: in the body, with treatment, prevent and/or diagnose different diseases, for example: cancer (prostate gland and non-prostate cancer).As described here, term " object " comprises the mankind and non-human animal, and preferred human animal comprises having with isomer-express cell, and for example the improper function of cancer cells or prostatic cell is the disease human patients of feature.Term of the present invention " non-human animal " refers to comprise all vertebratess, for example: and Mammals and nonmammalian, for example: the non-human primate, sheep, enough, ox, chicken, Amphibians, Reptilia etc.

In one embodiment, experimental subjects is human subjects.Alternately, this object can be the Mammals of expression isomer-class antigen, wherein isomer-specific inhibitor of the present invention and this isomer-class antigenic cross-reaction.Isomer-specific inhibitor of the present invention can be taken to human subjects, is used for the treatment of purpose (following discussion).In addition, isomer-specific inhibitor can give be expressed the non-human mammal of isomer-class antigen and be taken, and wherein modified antibodies and this isomer-class antigenic cross-reaction are for veterinary purpose or as the animal model of human diseases.About the latter, this animal model can be used for evaluating the result for the treatment of (for example: the test of dosage, and the time course of taking) of antibody of the present invention.

Therapeutic action

In one embodiment, the invention provides a kind for the treatment of, for example, remove or eliminate as: proliferative cell (for example: prostate cancer), or malignant tumour, non-prostatic cell, for example: the cell that in non--prostate gland solid tumor, finds, soft tissue neoplasm, or metastatic lesion is (for example, at kidney, urothelium (as: bladder), testis, colon, rectum, lung (as: non--small cell lung cancer), breast, liver, nerve is (for example: neuroendocrine), neuroglia (for example: glioblastoma multiforme), pancreas (for example: ductus pancreaticus) cancer and/or transfer, melanoma (for example: malignant melanoma), or soft tissue sarcoma).Method of the present invention comprises the step that the hyperplasia cell is combined with isomer-specific inhibitor described herein, and amount wherein is enough to treatment, for example: reduce actively, melt or kill this hyperplasia cell.

Cell during this object method can be used for cultivating, for example: in the external or body.For example, cancer or metastatic cell are (for example: prostate gland, kidney, urothelium, colon, rectum, lung, breast or liver, cancer or metastatic cell) can be in the external use culture medium culturing, and begin contact procedure by adding isomer-specific inhibitor to substratum.This method can be carried out at the cell (for example: cancer or metastatic cell) of experimental subjects, as the part of (for example: treatment or prevention) agreement in the body.For external embodiment, this contact procedure causes in experimental subjects, and be included in and cause and allow to suppress and/or reduce one or more isomer activity, or isomer-binding molecule is attached under the situation of cell and takes isomer-specific inhibitor to experimental subjects, treat with this, for example: kill or melt cell.

At this, term " cancer " refers to comprise all types of growth of cancer cells or oncogenic process, metastatic tissue or malignant conversioning cell, and no matter tissue or organ are histological type or stage of infecting.The embodiment of Cancerous disease comprises without limitation: solid tumor, soft-tissue tumor, and metastasis (metastases).The embodiment of solid tumor comprises: the malignant tumour of Different Organs system, for example sarcoma, lung squamous cancer and cancer.For example: the prostate gland of infection, lung, breast, lymph, stomach (for example: colon), and genitourinary tract (for example: kidney, epithelial cell), pharynx.Lung squamous cancer comprises malignant tumour, for example, and most colorectal carcinomas, the rectum cancer, renal cell carcinoma, liver cancer, the non-small cell carcinoma of lung, carcinoma of small intestine and esophagus cancer.Above-mentioned cancer metastasis venereal disease becomes and can treat with method and composition of the present invention equally and prevent.

Aforesaid method is very useful aspect treatment Different Organs system malignant tumour, this different organ for example: the lung of infection, breast, lymph, and stomach (for example: colon), bladder, genitourinary tract (for example: prostate gland), pharynx, and lung squamous cancer, these diseases comprise malignant tumour, most cancer for example, renal cell carcinoma, prostate cancer and/or tumour, lung's non-small cell carcinoma, carcinoma of small intestine and esophagus cancer.

The method of taking isomer-specific inhibitor of the present invention more than has been described.The suitable dose of the molecule that uses will depend on individual age, body weight and employed certain drug.The antibody molecule of this modification can be used as the competitive reagent of part combination, to suppress, reduces undesirable influence.

Isomer-specific inhibitor of the present invention can pass through self, or with second reagent, for example: cytotoxic drug, radio isotope, or protein are as proteotoxin or viral protein combination.This method comprises: give the object of needs treatment independent, or be combined to take isomer-specific inhibitor with cytotoxic drug.

Isomer-specific inhibitor of the present invention can be used for the treatment reagent of providing and delivering different, for example: the cytotoxicity part, as: medicine, provide injectivity isotope, plant molecular, bacterial origin, or bioprotein (for example: archon) or particle (for example: recombinant virus particle, as: pass through viral capsid proteins), and composition thereof.This treatment reagent can be intracellular reactive medicine or other reagent, for example: and the short-range radiation projector, for example, above-mentioned short distance, high energy α projector.In certain embodiments, isomer-specific inhibitor of the present invention can with plant molecular or bacterial origin (or derivatives thereof) combination, for example: maytenin.Maytenin is cytotoxic agent, and this cytotoxic agent starts cell by the unzipping that stops microtubule formation and existing microtubule and eliminates.Compare with cancer therapy drug, approximately have 100-1000 cytotoxin doubly, this cancer therapy drug for example is: Zorubicin, and methotrexate, vinca alkaloids, these medicines are generally clinical use.Alternately, isomer-binding molecule can with Taxan, calicheamycin, proteasome inhibitor or topoisomerase enzyme inhibitor combination.[(lR)-3-methyl-l-[[(2S)-l-oxygen-3-phenyl-2-[(3-sulfydryl ethanoyl) amino] propyl group] amino] butyl] boric acid is a kind of suitable proteasome inhibitor.N, two [2-(9-toluphenazine-l-carboxamido) the ethyl]-l of N'-, the 2-quadrol is a kind of topoisomerase enzyme inhibitor.

The toxin of enzymatic activity and the example of fragment thereof have diphtheria toxin A fragment, the non-binding active fragments of diphtheria toxin, exotoxin A (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, the plain A chain of capsule lotus, α-Sa Kelin, some tung oil tree albumen, some oleanolic acid albumen, pokeroot radicin (PAP, PAPII and PAP-S), momordica charantia inhibitor, curcin, crotin, soap matter bark of official magnolia inhibitor, gelonin, mitogillin, restrictocin, phenomycin and enomycin.In one embodiment, isomer binding molecule and maytansinoid coupling, this maytansinoid for example is: maytansinol (asking for an interview: U.S.Pat.No.5,208,020), CC-1065(asks for an interview: Pat.Nos.5,475,092,5,585,499,5,846,545).The step of active polypeptide of preparation enzyme has illustrated in WO84/03508 and WO85/03508, these two pieces of patents at this with reference to introducing.The embodiment of cytotoxin part can with antibody coupling, described antibody comprises: Zorubicin, Chlorambucil, daunomycin, methotrexate, neocarzinostatin and platinum.

In order to kill or the prostatic epithelium cancer cells that comes off, first isomer-binding molecule can with the prodrug coupling, this prodrug when with the prodrug activator near the time be activated.This prodrug activator and second isomer of the present invention-binding molecule coupling preferably is combined in the noncompetitive position of prostate specific membrane antigen molecule.Can determine whether two modified antibodies are attached to competitiveness or noncompetitive binding site by traditional competitive binding analysis.

Be applicable to that medicine-prodrug of the present invention is at Blakely etc., " ZD2767an Improved System for Antibody-directed Enzyme Prodrug Therapy That Results in Tumor Regressions in Colorectal Tumor Xenografts; " (1996) Cancer Research, have among the 56:3287-3292 illustrated, the content of this article at this with reference to introducing.

Alternately, isomer-binding molecule of the present invention can be combined with the high-energy radiation body, for example, radio isotope, as: 131I, gamma emitter, it can be positioned at knub position, causes the elimination of several cell radius, ask for an interview: for example: S.E.Order, " Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy ", Monoclonal Antibodies for Cancer Detection and Therapy, R.W.Baldwin et al. (eds.), pp303-316 (Academic Press1985), the content of this article at this with reference to introducing.Other suitable radio isotope comprises alpha emitter, for example: and Bi, ²¹³Bi and ²¹¹At, and beta emitter, for example: ¹⁸⁶Re and ⁹⁰Y estimates that radiotherapy is practical, because prostatic epithelium cancer cells and blood vessel endothelium cancer cells relative radioactivity sensitivity.In addition, ¹¹⁷Lu can be used as imaging and cytotoxic agent equally.

Use with ¹³¹I, ⁹⁰Y and ¹¹⁷The radioimmunotherapy of Lu mark (RIT) is in the fierce clinical study.The physical properties of these three kinds of radionuclides has remarkable difference, and therefore, for the radiation dose with maximum is distributed to tumor site, the selection of radionuclide can be extremely important. ⁹⁰The energy particle that Y is higher is of value to huge tumor, but is not to be necessary to little tumour, and particularly bone shifts (for example: prostate cancer is common). ¹³¹The relative low energy beta particle of I is then more satisfactory, but the dehalogenation of the iodine labeling molecule in the body is bigger shortcoming for internalization antibody.On the contrary, ¹⁷⁷Lu has the lower beta particle of energy, and it has only the 0.2-0.3mm scope, and relatively ⁹⁰Y provides and delivers lower radiation dose to marrow.In addition, because longer physical half life (with respect to ⁹⁰Y), the residence time of tumour is longer relatively.As a result, ¹⁷⁷The more high reactivity of Lu labelled reagent (more mCi amount) can be few relatively radiation dose to the marrow administration.Several parts of clinical studyes are arranged, have investigated in the different cancer for the treatment of, ¹⁷⁷The use of Lu traget antibody.(Mulligan?T?et?al,(1995)Clin?Cancer?Res.1:1447-1454;Meredith?R?F,et?al.(1996)J?Nucl?Med37:1491-1496;Alvarez?R?D,et?al,(1997)Gynecologic?Oncology65:94-101)。

But the same coupling of isomer-specific inhibitor of the present invention or be fused to the virus surface proteins place that occurs on the virionization.For example, isomer-binding molecule of the present invention can merge (for example, forming fusion rotein) to virus surface proteins.Alternately, chemically coupling of whole isomer-specific inhibitor (for example: connect by chemistry) is to virus surface proteins.Preferably, virus merges with the endocytosis film, for example, influenza virus, like this, virus is with isomer-specific inhibitor internalization, and infects isomer-express cell with this.This virus can be used as cell toxicant and usually carries out the gene processing.For example, the genetic expression poisonous to cell can be expressed or guide to virus, and for example: necrocytosis promotes gene.Preferably, this virus can be carried out virus replication.

Isomer-specific inhibitor of the present invention can directly be used in vivo, to eliminate antigen-express cell by cytotoxicity (ADCC) additional naturally or antibody dependent cellular.Isomer-specific inhibitor of the present invention also can use under the condition that complement exists equally, and this isomer-specific inhibitor has the complement binding site, for example: from the IgGl of conjugated complement ,-2, or-3 or the part of IgM.In one embodiment, cell in vitro quantity treatment can replenish by the serum that adds complement or comprise complement, and this cell quantity comprises the target cell with binding reagents of the present invention and suitable effector cell.The phagolysis of target cell can improve by conjugated complement albumen, and this target cell scribbles antibody or its fragment that the present invention modifies.In another embodiment, this target cell scribbles isomer-specific inhibitor of the present invention, and comes cytolysis by complement equally.

The present invention comprises a kind of killing or the method for cast-off cells equally, and described method comprises uses isomer-specific inhibitor of the present invention to be used for the disease that prevents that isomer is relevant.For example, these materials can be used for preventing or postponing development or the progress of prostate gland or other cancer.

The use that is used for the treatment of prostate gland and other treatment for cancer method of the present invention has many advantages.Because isomer-specific inhibitor of the present invention is only at targeted cancerous cells, being organized as of other is standby.As a result, safer with the treatment of this isomer-specific inhibitor, especially for aged patient.Estimating according to treatment of the present invention can be effective especially, because it directly is directed to marrow and lymphoglandula with isomer-specific inhibitor, majority is prostate cancer and other metastasis of cancer part herein.In addition, method of the present invention is specially adapted to treat prostate cancer, and is smaller because the knub position of prostate cancer tends to, and therefore destroyed by cytotoxic agent easily.Can effectively control with clinical parameter effectively according to treatment of the present invention, for example, under the situation of prostate cancer, one or more marks are selected from following: blood-serum P SA, PSMA, PSCA, AR, synaptophysin, MIB-1 and/or AMACR), and/or the case characteristics of patient's cancer, comprise the stage, the Gleason scoring is outside the capsule, seminal fluid, folliculus or have a liking for neural invasion and attack, the incisxal edge positive, the lymphoglandula of getting involved, disease-related pain, etc.Alternately, when these parameters needs to use these treatments if can be used for indicating.Provide dna vaccination equally at this, this dna vaccination comprises nucleotide sequence, the epi-position of this nucleotide sequence coded carcinogenic polypeptide isomer, and it is used for prevention or treatment cancer.This epi-position can be small peptide, and described small peptide is from the straight line of carcinogenic polypeptide isomer or the 10-15 amino-acid residue of non-rectilinear sequence.This epi-position is the binding site between the company's of leap exon preferably, this binding site is unique to specific polypeptide isomer, this polypeptide isomer is relevant with cancer, does not appear on the isomer protein that finds on the healthy tissues of normal experimental subjects or disease object.In certain embodiments, dna vaccination two or more epi-positions of encoding, this epi-position are from single isomer protein, or from the multiple protein isomer, and can in this combination, use, for example: be used for the indication of certain disease.The dna vaccination epi-position particular sequence of can encoding equally, for example: coding 10-15 amino acid is fused in the framework to carrier proteins, for example: serum albumin, SEAP or other secretion peptide or albumen.Dna vaccination can be as discussed further below for prevention or treatment disease.Typical dna vaccination comprises nucleotide sequence, and this is nucleotide sequence coded described herein, or at this sequence peptide of identifying.

In order to detect the effect of dna vaccination, this vaccine can be used for experimental animal model.Animal model be this area be familiar be used for multiple disease, for example: human tumor.In an illustrative embodiment, vaccinated animal can face the inoculation of human tumor before or after the inoculation dna vaccination.The protection of vaccine or the positively effect tumor weight of the minimizing of property animal by experiment embody.Be not subject in the mechanism of specific function, tumour-specificity vaccine can excite one or two immunity equipment, that is: cellular immunization and humoral immunization of health.

Conjoint therapy

Isomer-specific inhibitor of the present invention can use with other therapies.For example, this conjoint therapy comprises that composition of the present invention and one or more extra treatment reagent prepares jointly, and/or takes jointly, for example: one or more antitumor and anticancer agents, cytotoxin or cytostatics, hormonotherapy, vaccine, and/or other immunotherapy.In other embodiments, this isomer-specific inhibitor uses with other therapeutic modality, comprises operation, radiation, cryosurgery, and/or thermotherapy.This combined therapy advantageously utilize the treatment reagent taken than low dosage, avoid possible toxicity or the complication relevant with different monotherapies with this.

" associating " described herein takes, and means in the process of experimental subjects experience disease misery, the different treatment of provide and deliver two kinds (or more than).For example: after experimental subjects is diagnosed out disease, and before disease obtains medical treatment or eliminates, provide and deliver two kinds or above treatment.In certain embodiments, the dispensing of first kind for the treatment of was still carried out in second kind of when beginning treatment of dispensing, make have overlapping.This is sometimes referred to as " simultaneously " or " dispensing simultaneously ".In other embodiments, a kind of dispensing for the treatment of finished before another kind treatment beginning.In some embodiment of any one situation, this treatment because unite is taken and more effective.For example, second kind for the treatment of is more effective, as: can see equal curative effect with the second less treatment, or than the effect of not using first treatment to see, second treatment can reduce symptom to a greater degree, or similar situation also takes place in first treatment.In certain embodiments, dispensing makes the minimizing of symptom, or observed situation when not having another kind with the minimizing of other parameter of disease-related greater than only providing and delivering a kind for the treatment of.The effect of two kinds of treatments can be portions additive, whole additives, or greater than additive.This dispensing can make that the effect of the first treatment dispensing is for detecting when dispensing second treatment.

Isomer-specific inhibitor of the present invention can be taken with a kind of or unnecessary a kind of existing pattern, and with the treatment prostate cancer, and comprise without limitation: operation (for example: radical prostatectomy); Radiation therapy (for example: external radiotherapy, this radiation therapy comprises measurements of the chest, waist and hips, conformal radiation therapy, the scope of its radiation is designed to meet the volume of tissue to be treated; Interstitial irradiation therapy, it implants the seed that radiates compound with ultrasonic guidance, and external radiotherapy and interstitial irradiation therapy); Hormonal therapy, it (for example: reduce the treatment of serum testosterone concentration can use before and after radical prostatectomy or radiation, or inhibition testosterone activity, for example: short corpus luteum releasing hormone (LHRH) analogue, or stimulant (for example: leuprorelin acetate, acetic acid Gao She Rayleigh, bright dried meat Li Te, buserelin or goserelin) or antagonist is (for example: abarelix).Can in hormonal therapy, use nonsteroidal androgen antagonist, for example: Drogenil, bicalutimade or Nilutamide, and steroidal class androgen antagonist (for example: cyproterone acetate or melengestrol acetic ester), oestrogenic hormon (for example: stilboestrol),

The operation of the second or the 3rd hormone (for example: comprise that reflunomide (for example: hydrocortisone, prednisone or dexamethasone), the first KETOKONAZOL, and/or aminoglutethimide), the 5 inhibitor (for example: finasteride), Chinese herbal and crude drugs preparations (for example: PC-SPES), pituitectomy operation and suprarenalectomy).In addition, hormonal therapy can carry out or use any combination of aforesaid method to carry out off and on, for example: being used in combination of Leuprolide and Drogenil.

In certain embodiments, isomer-specific inhibitor of the present invention is used in combination with immunomodulator, and this immunomodulator for example is: IL-1, and 2,4,6, or 12, or interferon alpha or γ.For example: the antibodies with human constant region and IL-2 is estimated to increase monoclonal antibody efficient potentially.IL-2 will estimate to be used for enlarging reticuloendothelial system, to identify antigen-antibody complex by its effect at NK cell and scavenger cell.Therefore, by stimulating the NK cell discharging IFN, GM-CSF, and TNF, these cytokines will increase the cell surface density of Fc acceptor, and the phagocytic activity of these cells.Therefore, the effector of humoral immunization and cell dress equipment is improved the artificially.Net effect will improve the efficient of mab treatment, make to obtain maximum effect.The minority clinical trial is with IL-2 and monoclonal antibody combination (Albertini et al. (1997) Clin Cancer Res3:1277-1288; Frost et al. (1997) Cancer80:317-333; Kossman et al. (1999) Clin Cancer Res5:2748-2755).IL-2 can take by bolus or persistent instillation.Therefore, antibody of the present invention can be combined with IL-2 and be taken, so that the maximization of their treatment potential.

Diagnostic use

On the one hand, the invention provides a kind of diagnostic method for detection of the isomer existence, as external (biological sample is as the biopsy from cancerous tissue) or body interior (as experimenter's the living imaging) detection of isomer protein.Described method comprises: (i) sample is contacted with isomer binding molecule as herein described (anti--the FGFR2-IIIc antibody molecule), or take the isomer binding molecule to the experimenter; (selectively) (ii) with reference sample such as check sample (namely contrasting biological specimen, as blood plasma, tissue, biopsy) or contrast experimenter contact; (iii) detect the formation of complex compound between isomer binding molecule and sample or experimenter, check sample or the experimenter, wherein sample or experimenter show the existence of sample isomer with respect to the complex compound noticeable change statistically of check sample or experimenter's formation.The isomer binding molecule can directly or indirectly be marked with the material that can measure, can be convenient for measuring whether to be combined with antibody.As mentioned above, be fit to measurable material and comprise different enzymes, prothetic group, fluorescent substance, luminescent material or radioactive substance, more detailed description will be arranged hereinafter.

Term " sample " refers to the sample for detection of polypeptide, includes but not limited to cell, cell pyrolysis liquid, cell protein or film extract, body fluid or tissue samples.

Between isomer binding molecule or the isomer formation of complex compound can by measure or imagination isomer antigen whether with combination

Molecule has in conjunction with detecting.Can adopt traditional detection method such as enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA) or histogenic immunity groupization.

Serve as a mark the substituting of isomer binding molecule, the existence of the isomer in the sample can be analyzed by the competition immunization, and described competition immunization adopts standard substance and the cold isomer binding molecule that is marked with detectable substance.In this analysis, standard substance and the binding molecule of biological specimen, mark combine, and measure the amount of the mark standard substance that combine with the non-marked binding molecule again.The amount of the isomery scale of construction in the sample and the mark standard substance of being combined with binding molecule is inversely proportional to.

In another embodiment, the invention provides the method for the cancerous tissue of detection bodies internal (position) isomer expression.This method comprises: (i) experimenter's (as suffering from the carninomatosis people) takes the isomer binding molecule with the detectable conjugation; (ii) adopt the method that detects detectable in isomer expression tissue or the cell to detect the experimenter.

Among embodiment, above-mentioned can be antibody molecule with the binding molecule of the carcinogenic isomer specific combination of polypeptide therein.In another embodiment, described binding molecule is anti-FGFR2-IIIc antibody molecule herein.In one embodiment, have sequence number such as SEQ ID NO:2 with the antibody of polypeptide specific combination, 4,6,8,10,12,14,16,18 or the aminoacid sequence roughly the same with it.

It is very useful for cancer diagnosis whether the detection experimenter expresses carcinogenic isomer.Whether the detection experimenter expresses the carcinogenic isomer of FGFR2-IIIc can be used for diagnosing the intractable prostate cancer of hormone, mammary cancer, bladder cancer, thyroid carcinoma or other cancers.Whether the detection experimenter expresses FGFR1L can be used for diagnosing carcinoma of the pancreas, prostate cancer or other cancers.Whether the detection experimenter expresses RON receptor tyrosine kinase Δ 160 isomer can be used for the diagnosis of metastatic colorectal cancer, mammary cancer, ovarian cancer, lung cancer, bladder cancer or other cancers.Whether the carcinogenic isomer of expressing K IT receptor tyrosine can be used for diagnosing gastrointestinal stromal tumor (GIST) or other cancers to detect acceptor.Whether PDGF-B expression R-αYi Gouti cancer can be used for diagnosing the cancer of the brain, glioblastoma multiforme to detect the experimenter, prostate cancer, bone transfer, GIST or other cancers.

When not detecting the compound of being combined with carcinogenic polypeptide isomer in conspicuous level, diagnostic result is for negative.When detecting the compound of being combined with carcinogenic polypeptide isomer in conspicuous level, diagnostic result is for just.

According to the present invention, to the useful mark of diagnostic imaging can be radioactively labelled substance as

¹³¹I, ¹¹¹In, ¹²³I, ^99mTc, ³²P, ¹²⁵1, ³H, ¹⁴C and ^mRh, fluorescence labels such as fluorescein, rhodamine, nucleus magnetic resonance active label can be by the isotropic substance of the emission positron of positron emission computerized tomography (" PET ") scanner detection, chemoluminescence such as fluorescein, enzyme labelling thing such as peroxidase or Phosphoric acid esterases.Can adopt short-range radiation transmitter, for example can adopt short distance detection device probe, detect isotropic substance as transrectal probe.When uniting these isotropic substances of use and rectal detector probe, to detecting the recurrence of prostate gland nest, the pelvic lymph node pathology is particularly useful.The antibody that utilizes existing known technology these reagent marks can be modified.For example, Wensel and Meares (1983), radio-immuno-image and radioimmunotherapy, Elsevier N.Y. has put down in writing the technology of relevant radiolabelled antibody, at this as a reference.D.Colcher et al. and for example, (1986) Meth. enzymol .121:802-816, at this also as a reference.

Taking to the patient under the situation of radiolabeled modified antibodies, the antibody of modifying is concentrated at the tumor locus that carries with the antigen of modified antibodies reaction, can adopt existing known technology as nuclear radioscanning gamma camera or emission computed tomography is detected or " video picture " in vivo.See A.R.Bradwell etc., " progress of antibody video picture ", monoclonal antibody detects and cancer therapy and R.W.Baldwin etc., pp65785 (Science Press 1985), at this as a reference.

As selectable, as radiolabeled emission positron (as ^UC, ¹⁸F, ¹⁵0, ¹³N) can use the cross-section OCT of positron radiation the time, as be positioned at Brookhaven National Laboratory, called after Pet VI.

Fluorophor and chromophoric group mark modified antibodies can prepare by the known standard group of prior art.Because antibody and other protein adsorption light have the wavelength that is about 310nm, must be chosen to be at the fluorophor that has a large amount of absorptions above 310nm wavelength place, preferred 400nm place.Stryer (1968) science, 162:526 and Brand, L. etc. (1972), biological chemistry year looks back, and 41:843-868 has described multiple fluorophor and chromophoric group, at this as a reference.Can pass through as US patent Nos.3,940,475,4,289,747 and 4,376,110 described conventional procedures adopt fluorescent chromophore labelled isomer binding molecule, at this as a reference.

Aforesaid white dyes with some estimated performances is a ton dyestuff, it comprises and derives from 3,6-dihydroxyl-xanthic fluorescein of 9-phenyl hydrolysis and the cresamine and the rhodamine that derive from 3,6-diamino-9-phenyl hydrolysis xanthine and lissamine rhodamine B.9-oxygen-xanthic rhodamine of carboxyl phenyl hydrolysis and fluorescein derivative have 9-oxygen-carboxyl phenyl group.Have reaction coupling group such as amino, the fluorescent chemicals of isothiocyanate group such as fluorescein isothiocyanate and fluorescamine all is ready-made.The other fluorescent chemicals is naphthylamines (amino naphthalenes), and it has amino at α or β position.

In further embodiments, the present invention also provides the method for measuring dosage such as radiation dose, and its method is: when experimenter such as human body experimenter take isomer binding molecule with the radio isotope conjugation, detect different tissues.This method comprises: (i) experimenter takes the isomer binding molecule of foregoing labelled with radioisotope; (ii) in different time point determining different tissues such as the radioisotopic amount in pancreas, liver, kidney or the blood, in the body of some or all of radio isotope the experimenter, disappear; The (iii) total amount of the radiant matter that receives of each tissue of computational analysis.Measurement can be after predetermined time point be taken radiolabeled isomer binding molecule (0 day) as the experimenter carried out in the 1st, 2,3,5,7 and 12 day.The radiation concentration that exists in the given tissue to time integral, multiply by the given activity of radiation, can be used for calculating the dosage that given tissue receives.Use radio isotope such as gamma-radiation, ^mThe pharmacology information that the isomer binding molecule of In mark produces can be used for calculating same tissue from radio isotope different, that be not easy to measure such as beta-rays, ⁹⁰The projected dose that Y receives.

Pharmacology

As for prevention and methods for the treatment of, these methods can be rebuild especially or modify based on the knowledge of area of pharmacology." pharmacology " refers to gene engineering such as gene sequencing, statistical genetics, genetic expression, the application of expression analysis of the protein biomarker of medicine etc. on clinical development and the market herein.Referring to Eichelbaum, M. etc., (1996) Clin.Exp.Pharmaco.Physiol.23:983-985 and Linder, M.W. etc. (1997) Clin.Chem.43:254-266.The difference of metabolism therapy (changing the dosage of pharmacological activity medicine and the relation between the Plasma Concentration) may cause serious toxicity or treatment failure.As a rule, can distinguish two types pharmacological conditions.Change drug effect in the mode (change pharmaceutical activity) of the human body monofactor as the heredity condition, or change human body and act on the mode (change drug metabolism) of medicine as the monofactor of heredity condition.These pharmacological conditions take place with rare hereditary defect or the polymorphism form that exists naturally.More specifically, term refers to the gene to the patient is how to determine him to the research of the reaction of medicine (be " drug reaction phenotype " or " drug response genotype " as the patient).Therefore, another aspect of the present invention provides according to the drug response genotype of individuality and to the prevention of individuality or the modifying method of methods for the treatment of.

The research information of pharmacology genomics can be used for determining suitable dose and the treatment plan of prevention or methods for the treatment of.When these knowledge are applied to dosage or medicament selection aspect, can prevent untoward reaction or treatment failure, therefore, when a kind of method as treatment disorderly (foregoing cancer), when taking the therapeutic composition that comprises one or more isomer specific inhibitor or derivatives thereofs to the patient, can strengthen prevention or result for the treatment of.

In one embodiment, determining whether the experimenter takes pharmaceutical composition, as when comprising one or more isomer specific inhibitors, or derivatives thereof or selecting second dose composition, doctor or clinician may consider to use the knowledge that relevant pharmacogenomics research obtains.In another embodiment, when determining that the experimenter takes the dosage of foregoing pharmaceutical composition such as each treatment consumption or therapeutic frequency, doctor or clinician may consider to use these knowledge.

In another embodiment, doctor or clinician may determine to participate in the experimenter's of clinical trial the genotype of one or several gene locus, and wherein the experimenter shows disorder, foregoing cancer or prostate gland obstacle; The effect that comprises one or more isomer specific inhibitors or select second dose pharmaceutical composition is tested in the design clinical trial, and doctor or clinician attempt experimenter's genotype and its reaction to pharmaceutical composition are associated.

Utilize RT-PCR or PCR to detect the method for the carcinogenic isomer nucleic acid of coding

The present invention also provides the method for the nucleic acid that detects the carcinogenic isomer of coding, comprises that (a) obtains cDNA from the mRNA of suitable sample; (b) cDNA of amplification proto-oncogene, carcinogenic isomer or its epitope fragment correspondence; (c) DNA of known coded proto-oncogene, carcinogenic isomer or its epitope fragment and the cDNA of amplification are compared, wherein the existence of carcinogenic isomer shows the nucleic acid that detects the carcinogenic isomer of coding among Kuo Zeng the cDNA.

The present invention also provides a kind of detection method of nucleic acid of the carcinogenic isomer of encoding, and comprising: (a) with suitable sample and the above-mentioned compound contact that can be combined with the nucleic acid specificity of the carcinogenic isomer of coding; (b) whether detection has compound to be combined with nucleic acid, wherein has the existence of the compound of being combined with nucleic acid to show the nucleic acid that detects the carcinogenic isomer of coding in the sample.

Term " sample " refers to the sample for detection of nucleic acid, includes but not limited to nucleic acid extractive, tissue samples or the blood of human body of cell, cell pyrolysis liquid, cell.Body fluid includes but not limited to blood, blood plasma and saliva.Among embodiment, from the experimenter, obtain suitable sample therein.

The method of obtaining mRNA from suitable sample is prior art.Preparing the method for cDNA from mRNA, also is prior art as reverse transcription.

Here, " amplification " expression increases several copies of specific DNA fragments.Among embodiment, the amplification of cDNA adopts PCR (polymerase chain reaction) to carry out therein.

Among embodiment, the amplification of cDNA is to adopt the primer flank of the whole reading frame of the proto-oncogene of the carcinogenic isomer polypeptide of coding to carry out therein.In another embodiment, the amplification of cDNA is to adopt the primer two side portions of the nucleic acid of the carcinogenic isomer of coded polypeptide to carry out as exon.In another embodiment, the sequence hybridization of one or more primer and carcinogenic isomer, this sequence exists in the nucleic acid of the carcinogenic isomer of coding and lacks in the nucleic acid of coding non-carcinogenic isomer, and vice versa.In another embodiment, primer can be selected from SEQ ID NOs:1, a sequence hybridization in 3,5,7,9,11,13,15,17.In other embodiments, primer length can be 18-22 Nucleotide.

In one embodiment, relatively the cDNA of amplification and known coded proto-oncogene, carcinogenic isomer or its epitope cDNA fragment are by the sequence of cDNA and known comparing corresponding to proto-oncogene, carcinogenic isomer or its epitope fragments sequence with amplification.The existence of sequence or disappearance will show that carcinogenic isomer exists or do not exist in the extension increasing sequence.In another embodiment, relatively the cDNA of amplification and known coded proto-oncogene, carcinogenic isomer or its epitope cDNA fragment are that cDNA by will amplification and the size of known gene corresponding to proto-oncogene, carcinogenic isomer or its epitope fragment compare.The difference of size will show the carcinogenic isomer of dna encoding of amplification.

The present invention also provides the mensuration experimenter whether to express the method for carcinogenic isomer, comprising: (a) obtain cDNA from the mRNA of suitable experimenter's sample; (b) cDNA of amplification proto-oncogene, carcinogenic isomer or its epitope fragment correspondence; (c) DNA of known coded proto-oncogene, carcinogenic isomer or its epitope fragment and the cDNA of amplification are compared, wherein the existence of carcinogenic isomer shows experimenter's carcinogenic isomer of encoding among Kuo Zeng the cDNA.

Whether the said determination experimenter expresses in the method for carcinogenic isomer, and term " suitable sample " refers to comprise any sample of the experimenter of carcinogenic isomer.Include but not limited to body fluid and tissue samples.Body fluid includes but not limited to blood, blood plasma, urine and saliva.

Increasing, compare and detect the existence of cDNA can carry out as above-mentioned.

Nucleotide

As described herein, the present invention also provides the Nucleotide of the nucleotide sequence of the variable region of heavy chain, variable region of light chain and the CDRs that comprise the anti-FGFR2-IIIc antibody of encoding.For example, the invention provides variable region of heavy chain and variable region of light chain first Nucleotide and second Nucleotide of the anti-FGFR2-IIIc antibody molecule of encoding respectively, described anti-FGFR2-IIIc antibody molecule is selected from Atto-MuMab-01, Atto-MuMab-02, Atto-MuMab-03, Atto-HuMab-01, Atto-MuMab-06, one or more among the Atto-MuMab-08.Nucleotide comprises the A as Figure 29,29C, 29E, 30A, 30C, 30E, 34A (the SEQ ID NO:87 of variable region of heavy chain), 34B (the SEQ ID NO:89 of variable region of light chain), the nucleotide sequence shown in the 37B, or the sequence roughly the same with it (according to appointment 85%, 90%, 95%, 99% or the higher sequence of consistence), or with Figure 29 A, 29C, 29E, 30A, 30C, 30E, 34A (the SEQ ID NO:87 of variable region of heavy chain), 34B (the SEQ ID NO:89 of variable region of light chain), the nucleotide sequence shown in the 37B differs and is no more than 3,6,15,30, or 45 Nucleotide.

In certain embodiments, nucleic acid comprises that coding has as Figure 35 and (is respectively the SEQ ID NOs:92 of the CDRs1-3 of variable region of heavy chain, 94,96), 28,29F (being respectively the SEQ ID NOs:147-149 of the CDRs1-3 of variable region of heavy chain), 30F (is respectively the SEQ ID NOs:147 of the CDRs1-3 of variable region of heavy chain, 158,159), 38,39 (being respectively the SEQ ID NOs:147-149 of the CDRs1-3 of variable region of heavy chain), or 40, or homology basic with it (according to appointment 85%, 90%, 95%, 99% or consistence higher, and/or have one or more conservative sequences that substitute) at least one of variable region of heavy chain of aminoacid sequence, the nucleotide sequence of two or three CDRs.In further embodiments, nucleic acid comprises that coding has as Figure 35 and (is respectively the SEQ ID NOs:98 of the CDRs1-3 of variable region of light chain, 100,102), 28,29D (is respectively the SEQ ID NOs:155 of the CDRs1-3 of variable region of light chain, 156,146), 30D (being respectively the SEQ ID NOs:155-157 of the CDRs1-3 of variable region of light chain), 38,39 (being respectively the SEQ ID NOs:144-146 of the CDRs1-3 of variable region of light chain), or 40, or homology basic with it (according to appointment 85%, 90%, 95%, 99% or consistence higher, and/or have one or more conservative sequences that substitute) at least one of variable region of light chain of aminoacid sequence, the nucleotide sequence of two or three CDRs.In another embodiment, nucleic acid comprises that coding has as Figure 35 and (is respectively the SEQ ID NOs:92 of the CDRs1-3 of variable region of heavy chain, 94,96, be respectively the SEQ ID NOs:98 of the CDRs1-3 of variable region of light chain, 100,102), 28,29D (is respectively the SEQ ID NOs:155 of the CDRs1-3 of variable region of light chain, 156,146), 29F (being respectively the SEQ ID NOs:147-149 of the CDRs1-3 of variable region of heavy chain), 30D (being respectively the SEQ ID NOs:155-157 of the CDRs1-3 of variable region of light chain), 30F (is respectively the SEQ ID NOs:147 of the CDRs1-3 of variable region of heavy chain, 158,159), 38,39 (are respectively the SEQ ID NOs:144-146 of the CDRs1-3 of variable region of light chain, are respectively the SEQ ID NOs:147-149 of the CDRs1-3 of variable region of heavy chain), or 40, or homology basic with it (according to appointment 85%, 90%, 95%, 99% or consistence higher, and/or have one or more conservative sequences that substitute) the weight variable region of aminoacid sequence and at least one of variable region of light chain, two, three, four, the nucleotide sequence of five or six CDRs.

In certain embodiments, nucleic acid comprises that coding has as Figure 34 A and (is respectively the SEQ ID NOs:91 of the CDRs1-3 of variable region of heavy chain, 93,95), 29E (being respectively the SEQ ID NOs:187-189 of the CDRs1-3 of variable region of heavy chain), 30E (being respectively the SEQ ID NOs:181-183 of the CDRs1-3 of variable region of heavy chain), 37B (being respectively the SEQ ID NOs:175-177 of the CDRs1-3 of variable region of heavy chain), or homology basic with it is (as at least 85%, 90%, 95%, 99% or consistence higher, but or the sequence of hybridize under stringent condition) at least one of variable region of heavy chain of aminoacid sequence, the nucleotide sequence of two or three CDRs.In further embodiments, nucleic acid comprises that coding has as Figure 34 B and (is respectively the SEQ ID NOs:97 of the CDRs1-3 of variable region of light chain, 99,101), 29C (being respectively the SEQ ID NOs:184-186 of the CDRs1-3 of variable region of light chain), 30C (being respectively the SEQ ID NOs:178-180 of the CDRs1-3 of variable region of light chain), 37B (being respectively the SEQ ID NOs:172-174 of the CDRs1-3 of variable region of light chain), or homology basic with it (according to appointment 85%, 90%, 95%, 99% or consistence higher, but or the sequence of hybridize under stringent condition) at least one of variable region of light chain of aminoacid sequence, the nucleotide sequence of two or three CDRs.In another embodiment, nucleic acid comprises that coding has as Figure 34 A and (is respectively the SEQ ID NOs:91 of the CDRs1-3 of variable region of heavy chain, 93,95), 34B (is respectively the SEQ ID NOs:97 of the CDRs1-3 of variable region of light chain, 99,101), 29C (being respectively the SEQ ID NOs:184-186 of the CDRs1-3 of variable region of light chain), 29E (being respectively the SEQ ID NOs:187-189 of the CDRs1-3 of variable region of heavy chain), 30C (being respectively the SEQ ID NOs:178-180 of the CDRs1-3 of variable region of light chain), 30E (being respectively the SEQ ID NOs:181-183 of the CDRs1-3 of variable region of heavy chain), 37B (is respectively the SEQ ID NOs:175-177 of the CDRs1-3 of variable region of heavy chain, be respectively the SEQ ID NOs:172-174 of the CDRs1-3 of variable region of light chain), or homology basic with it (according to appointment 85%, 90%, 95%, 99% or consistence higher, but or the sequence of hybridize under stringent condition) the weight variable region of aminoacid sequence and at least one of variable region of light chain, two, three, four, the nucleotide sequence of five or six CDRs.

On the other hand, the invention provides host cell and the carrier that comprises above-mentioned nucleic acid.Nucleic acid can exist in the single carrier of same host cell or each host cell or each carrier, will describe in detail hereinafter.

In one of them embodiment of the present invention, the nucleic acid of the separation of the carcinogenic polypeptide isomer of encoding is provided, or its roughly the same sequence.

In one embodiment, the present invention also provides the nucleic acid of the separation of the polypeptide of the carcinogenic isomer of encoding or its epitope fragment.In one embodiment, the invention provides the nucleic acid of the separation of the coding polypeptide of human carcinogenic isomer or its epitope fragment.In one embodiment, the isomer of the carcinogenic form of the nucleic acid encoding proto-oncogene of described separation or its epitope fragment, described proto-oncogene is selected from FGFR2, FGFR1, RON receptor tyrosine kinase, KIT receptor tyrosine kinase, PDGF and PDGFR-α.

In one embodiment, the invention provides the nucleic acid of separation of the mouse polypeptide of coding carcinogenic isomer or its epitope fragment.In one embodiment, the invention provides the nucleic acid of separation of the mouse polypeptide of coding human carcinogenic isomer or its epitope fragment.In another embodiment, the nucleic acid of the separation of the human carcinogenic isomer of described coding or its epitope fragment comes from other kind, includes but not limited to dog, pig, cavy and rabbit.

FGFR2

In one embodiment of the invention, provide a kind of nucleic acid of separation, the carcinogenic polypeptide isomer of this nucleic acid encoding or its epi-position fragment, it comprises nucleotide segment, this nucleotide segment is used by the selectivity of the nucleic acid exon III of coding FGFR2 and produces.In one embodiment, the selectivity of exon III is used the sequence variations that causes the 301-360 amino acid region, and this zone amino acid is corresponding to FGFR2Illb.Therefore, on the one hand, this nucleic acid encoding polypeptide, described polypeptide comprise and are selected from one group of SEQ NO:2,4,6 and 8 sequence.On the other hand, this nucleic acid comprises that being selected from one group comprises EQ NO:1,3,5 and 7 sequence.

FGFR1

In another embodiment, the invention provides a kind of nucleic acid of separation, the carcinogenic polypeptide isomer of this nucleic acid encoding or its epi-position fragment, it comprises the nucleotide segment that is caused by FGFR1 exon 7 optionally and 8 disappearances.In one embodiment, when comparing with the FGFR1 proto-oncogene,

optionally exon

7 and 8 disappearances cause 105 aminoacid deletion for these.Therefore, on the one hand, the nucleic acid encoding polypeptide of this separation, described polypeptide comprises the sequence of SEQ NO:9.

The RON receptor tyrosine kinase

In another embodiment, the invention provides a kind of nucleic acid of separation, the polypeptide of the carcinogenic isomer of the nucleic acid encoding of this separation or its epi-position fragment, it comprises nucleotide segment, this nucleotide segment is by

optionally exon

5 and 6 disappearances of RON receptor tyrosine kinase produce.In one embodiment, this

optionally exon

5 and 6 disappearance cause when comparing with RON receptor tyrosine kinase proto-oncogene 109 amino acid at the in-frame deletion of extracellular region.In one aspect, the nucleic acid of this separation comprises

exon

4 and 7 side by side.Therefore, on the one hand, the nucleic acid encoding polypeptide of this separation, described polypeptide comprises the sequence of SEQ NO:12.On the other hand, the nucleic acid of this separation comprises the sequence of SEQ NO:11.

The KIT receptor tyrosine kinase

In another embodiment, the invention provides a kind of nucleic acid of separation, the polypeptide of the carcinogenic isomer of the nucleic acid encoding of this separation or its epi-position fragment, it comprises nucleotide segment, this nucleotide segment is produced by selectivity exons 11 disappearance of nucleic acid encoding KIT receptor tyrosine kinase.Therefore, on the one hand, the nucleic acid encoding polypeptide of this separation, described polypeptide comprise SEQ NO:14 sequence.On the other hand, this nucleic acid comprises SEQ NO:13 sequence.

PDGF

In another embodiment, the invention provides a kind of nucleic acid of separation, the polypeptide of the carcinogenic isomer of the nucleic acid encoding of this separation or its epi-position fragment, it comprises nucleotide segment, described nucleotide segment is by PDGF exon 6 in-frame deletions optionally and produce.Therefore, on the one hand, the nucleic acid encoding polypeptide of this separation, described polypeptide comprise SEQ NO:16 sequence.On the other hand, the nucleic acid of this separation comprises SEQ NO:15 sequence.

PDGFR-α

In another embodiment, the invention provides a kind of nucleic acid of separation, the polypeptide of the carcinogenic isomer of the nucleic acid encoding of this separation or its epi-position fragment, this polypeptide by alternative PDGFR-α exon 7 and 8(for example comprise: the nucleotide segment that causes of disappearance amino acid 374-456).Therefore, in one aspect, the nucleic acid encoding polypeptide of this separation, this polypeptide comprise SEQ NO:18 sequence.On the other hand, this nucleic acid comprises SEQ NO:17 sequence.

Alternately, the isolating nucleic acid of the polypeptide of this encode carcinogenic isomer or its epi-position fragment is by nucleic acid encoding, and this nucleic acid is substantially equal at this carcinogenic isomer or the nucleic acid of its epi-position fragment.In addition, the carcinogenic isomer of nucleic acid codified of separation or the polypeptide of its epi-position fragment, this polypeptide are substantially equal to carcinogenic isomer or its epi-position fragment.

The sequence (polypeptide or nucleic acid) of " roughly corresponding " another sequence can be and makes single amino acids or nucleotide substitution, disappearance and/or insert.In one embodiment, the sequence that is substantially equal to has 80% sequence identity.In another embodiment, roughly corresponding sequence has 90% sequence identity.In another embodiment, roughly corresponding sequence has 95% sequence identity.In another embodiment, roughly corresponding sequence has 97% sequence identity.In another embodiment, roughly corresponding sequence has 99% sequence identity.

In another embodiment, the carcinogenic isomer of nucleic acid encoding or its epi-position fragment, this carcinogenic isomer or its epi-position fragment are included in SEQ ID NOs:2,4,6,8,10,12,14,16,18,19, or the aminoacid sequence of explanation in 20, substitute but have conserved amino acid.In another embodiment, the carcinogenic isomer of nucleic acid encoding or its epi-position fragment, this carcinogenic isomer or its epi-position fragment comprise the NOs:2 with respect to SEQ ID, 4,6,8,10,12,14,16,18,19, or 20 1,2,3,4,5,6,7,8,9 or 10 conserved amino acid substitutes.The carcinogenic polypeptide of this nucleic acid encoding inserts variation, and this carcinogenic polypeptide inserts variation and comprises the NOs:2 with respect to SEQ ID, 4,6,8,10,12,14,16,18,19, or 20 11,12,13,14,15,16,17,18,19, or 20 conserved amino acids substitute.

The present invention provides the nucleic acid that separates equally, and the nucleic acid of this separation specifically is combined with the nucleic acid at this, or with can under highly strict condition, hybridize to nucleic acid described herein, or with its about equally the nucleic acid of sequence be combined.

The invention provides a kind of nucleic acid of separation, the nucleic acid of this separation can hybridize to the nucleic acid of the carcinogenic isomer of coding or its epi-position fragment under highly strict condition, and the nucleic acid of this carcinogenic isomer or its epi-position fragment comprises SEQ ID NO:2,4,6,8,10,12,14,16,18,19, or 20, or with its sequence about equally.The invention provides a kind of nucleic acid of separation, the nucleic acid of this separation can hybridize under highly strict condition and comprise that sequence is SEQ ID NO:1, and 3,5,7,9,11,13,15, or 17 or the nucleic acid of its fragment.

The present invention also provides a kind of nucleic acid of separation, the nucleic acid encoding of this separation carcinogenic isomer or its epi-position fragment, this nucleic acid at least 80% nucleic acid of carcinogenic isomer or its epi-position fragment that equals to encode wherein, wherein this coding nucleic acid carcinogenic or its epi-position fragment comprises nucleotide segment in the position of shearing the junction corresponding to selectivity, provides polypeptide carcinogenic when using in described position.In preferred embodiment, be not 80%, its percentage ratio is 85%, 90%, 95%, 97%, or 99%.

Nucleic acid described herein can indicate by detecting mark.This can detect mark and comprise without limitation: radioactively labelled substance, colorimetric marker, luminous marker, enzyme labelling thing and fluorescent marker.Radioactively labelled substance comprises without limitation: ³H, ¹⁴C, ³²P, ³³P, ³⁵S, ³⁶C1, ⁵¹Cr, ⁵⁷Co, ⁵⁹Co, ⁵⁹Fe, ⁹⁰Y, ¹²⁵I, ¹³1I and ¹⁸⁶Re.

Fluorescent marker comprises without limitation: fluorescein, rhodamine and auramine.

The colorimetric mark comprises without limitation: vitamin H and digoxigenin.The appropriate method that marker is added to nucleic acid can be used with Nucleotide of the present invention, and many these methods all are that this area is described familiar.

In addition, this is bought that nucleic acid with nucleic acid complementation disclosed herein is provided.Mean that by nucleotide sequence " homology " or " complementation " nucleic acid optionally hybridizes two strands or combination with the target nucleic acid sequence.For example, VITAMIN B4 and thymus pyrimidine complementation are because they form two hydrogen bonds.Similarly, guanine and cytosine(Cyt) complementation are because they form hydrogen bond.Can comprise the sequence that is shorter than or is longer than target sequence with the nucleotide sequence of target sequence homology, as long as this sequence meets illustrated function test requirement.

Those skilled in the art understands, when referring to the particular sequence table, this referring to comprises and is substantially equal to its complementary sequence, and illustrated slight sequence errors being arranged, single base changes, disappearance, the sequence that substitutes, like this, any this sequence variation is corresponding to nucleic acid encoding, and this polypeptide is relevant with relevant sequence table.

Carrier

The present invention provides carrier equally, and this carrier comprises Nucleotide, and this nucleotide coding is at this carcinogenic isomer that provides or the polypeptide of its epi-position.In one embodiment, this carrier comprises Nucleotide, and this nucleotide coding is at the polypeptide of this carcinogenic isomer that provides or its epi-position fragment.In one embodiment, this carrier comprises nucleotide sequence described herein.This carrier comprises without limitation: virus, plastid, clay, phage or yeast artificial chromosome (YAC).

According to the present invention, can use a plurality of carrier systems.For example, a class carrier utilizes the DNA composition, and this DNA composition is from animal virus, for example, bovine papilloma virus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (Rous sarcoma virus, MMTV or MOMLV) or SV40 virus, another kind of carrier utilizes the RNA composition, this RNA composition is from RNA viruses, for example, niche forest virus, Eastern equine encephalitis virus and arboviruses.

In addition, have and stable DNA is merged to their chromosomal cells can select by introducing one or more marks, these one or more marks are allowed the selection of transfection host cell.This mark can provide, and for example: prototropy is to the auxotroph main body, biocide resistance (for example: microbiotic), or anti-heavy metal, for example: copper, or analogue.Selectable marker gene can directly be connected with the dna sequence dna that will express, or introduces in the identical cell by concurrent transfer.The optimization that needs extra composition to be used for mRNA is equally synthesized.These compositions can comprise splicing signal, and transcripting promoter, enhanser, and termination signal.

In case comprise the expression vector of structure or dna sequence dna when expressing, but this expression vector transfection or introduce proper host cell.This can obtain by different technology, and for example, protoplastis merges, calcium phosphate precipitation, electroporation, retrovirus transduction, virus transfection, particle gun, lipofection or other traditional technology.Under the situation that protoplastis merges, cell is grown in medium, and screens according to suitable activity.

The genetic expression of the polypeptide of carcinogenic isomer or its epi-position fragment of encoding causes the polypeptide of carcinogenic isomer or its epi-position fragment to produce.

Based on the present invention, cultivate the transfectional cell of gained and be familiar with to those skilled in the art with method and the condition of the polypeptide that recovers carcinogenic isomer or its epi-position fragment, and can change or optimize along with specific expression vector and the mammalian host cell that adopts.

Cell

The present invention provides the host cell that comprises nucleic acid equally, the nucleic acid of the polypeptide of this nucleic acid encoding carcinogenic isomer described herein or its epi-position fragment.

In one embodiment, host cell is that gene is handled, to comprise the nucleic acid of the carcinogenic isomer of coding or its epi-position fragment.

In one embodiment, host cell is for handling by the gene that uses expression cassette.Term " expression cassette " refers to nucleotide sequence, and this nucleotide sequence can influence the genetic expression in the main body compatible with this sequence.This box can comprise enhanser, has or do not have the open reading frame of intron, and termination signal.

Can use the necessary of other equally or be useful on the factor that influence is expressed, for example: inducible promoter.

The present invention also provides the host cell that comprises above-mentioned carrier.

This cell can comprise without limitation: eukaryotic cell, bacterial cell, insect cell, or human cell.Suitable eukaryotic cell comprises without limitation: cytotoxin cell, HeLa cell, COS cell, Chinese hamster ovary celI, HEK293 cell, bhk cell and MDCK II cell.Suitable insect cell comprises without limitation: the sf9 cell.

Below help understand the present invention by unrestriced embodiment.

Embodiment

Embodiment 1: the isomer specificity epitope

Embodiment 1.1:FGFR2: isomer FGFR2-IIIc (SEQ ID NO:19)

Fibroblast growth factor (FGF) acceptor 2(FGFR2) isomer is mainly expressed in the intractable prostate cancer of hormone.The replacement of exon III is used in the different sequences of the Ig of extracellular domain class ring (Ig-like loop) generation, this for part in conjunction with extremely important.Isomer IIIb expresses in normal prostate epithelial cell.The malignant prostate cancerous cell transformation is the IIIc isomer, and its somatomedin such as FGF8b isomer to having high activity of conversion has high bonding force.

FGFR2-IIIc uses the exon III that replaces, the sequence that its coding is different with isomer FGFR2-IIIb.In the C-terminal district, half district of Ig-ring III, from 314 to 353 in amino acid, the FGFR2-IIIc isomer comprises and the nonhomologous sequence of IIIb isomer.The isomer structure of FGFR2 as shown in Figure 1.

The sequence alignment of IIIc and IIIb isomer (sequence alignment) has been showed the difference at the C-terminal of half of Ig ring III district.

The amino acid of FGFR2-IIIc (SEQ ID NO:19) and Nucleotide (SEQ ID NO:20) sequence are respectively shown in Fig. 3 A and 3B.

The Nucleotide of FGFR2 exon IIIc (SEQ ID NO:1) and amino acid (SEQ ID NO:2) sequence are respectively shown in Fig. 4 A and 2.The Nucleotide of FGFR2 exon IIIb (SEQ ID NO:64) and amino acid (SEQ ID NO:65) sequence are respectively shown in Fig. 4 B and 2.

Short peptide sequence also can be used as the epi-position that produces monoclonal antibody.The amino acid of IIIc-314 (SEQ ID NO:4) and Nucleotide (SEQ ID NO:3) sequence are shown in Fig. 5 A.The amino acid of IIIc-328 (SEQ ID NO:6) and Nucleotide (SEQ ID NO:5) sequence are shown in Fig. 5 B.The amino acid of IIIc-350 (SEQ ID NO:8) and Nucleotide (SEQ ID NO:7) sequence are shown in Fig. 5 C.IIIb(encircles 3-C ') fragment: sequence is as shown in Figure 6A for the amino acid of amino acid 314-351 (SEQ ID NO:56) and Nucleotide (SEQ ID NO:60).The IIIb epi-position: the amino acid of amino acid 314-328 (SEQ ID NO:57) and Nucleotide (SEQ ID NO:61) sequence are shown in Fig. 6 B.The IIIb epi-position: the amino acid of amino acid 340-351 (SEQ ID NO:58) and Nucleotide (SEQ ID NO:62) sequence are shown in Fig. 6 C.

Embodiment 1.2:FGFR1: isomer FGFR1L(disappearance Wai Xianzi7 ﹠amp; 8; 105 amino acid; Ig-II part and Ig-III part)

Fibroblast growth factor (FGF) acceptor 1(FGFR1) isomer structure as shown in Figure 7.Sequence as shown in Figure 8 to be positioned at the amino acid (SEQ ID NO:10) of the epi-position of tie point (junction) and Nucleotide (SEQ ID NO:9).

Embodiment 1.3:RON receptor tyrosine kinase: isomer RON △ 160

Macrophage-stimulating acceptor 1(RON) isomer is constitutively

activate.Skip exon

5 and 6 in-frame deletions at extracellular domain generation amino acid/11 09.

Epi-position is on the tie point between exon 4 and the exon 7.Sequence as shown in Figure 9 for the Nucleotide of this epi-position (SEQ ID NO:11) and amino acid (SEQ ID NO:12).

Embodiment 1.4:KIT receptor tyrosine kinase (disappearance on exon 11)

Most of gastrointestinal stromal tumor, GISTs has the oncogene sudden change at sarcoma virus oncogene homology (KIT) gene of v-kit Hardy-Zuckerman4 feline, and these main sudden changes that influence kinase whose nearly film territory are by exons 11 coding.Sequence as shown in figure 10 for the Nucleotide of this epi-position (SEQ ID NO:13) and amino acid (SEQ ID NO:14).

Embodiment 1.5:PDGF: the in-frame deletion of isomer 2(exon 6)

Thr6 PDGF BB (PDGF) isomer 2 has the in-frame deletion of exon 6.Sequence as shown in figure 11 for the Nucleotide of this epi-position (SEQ ID NO:15) and amino acid (SEQ ID NO:16).

Embodiment 1.6:PDGFR-α: δ-exon 7 and 8(amino acid 374-456)

Platelet derived growth factor receptor α (PDGFR-α) has the disappearance of exon 7 and 8.Sequence as shown in figure 12 for the Nucleotide of this epi-position (SEQ ID NO:17) and amino acid (SEQ ID NO:18).

Table 1. is used for the sequence of the epi-position of isomer specific antibody

Shadow zone=nucleotide sequence clear area=aminoacid sequence

The generation of embodiment 2:FGFR2 isomer specific antibody

From obtaining the nonspecific FGFR2 antibody of isomer on the market.Yet these antibody can not obviously be distinguished in the different isomerization body.In order to study that the isomer protein white matter distributes and to the effect of tumour and healthy tissues, designed be used for FGFR2-IIIc and IIIb(Fig. 1) antibody recognition isomer specific sequence.By common hybridoma technology, the manufacture order clonal antibody.Simply, respectively based on gene order, pcr amplification or the chemical synthesis coding sequence of SEQ ID NOs:19 and 63.Then dna fragmentation is cloned into commercial carrier for expression of eukaryon.This expression vector is used for the genetic immunization of 5 mouse, is used for every kind of antigen.The serum titer that ELISA records is used for and myelomatosis SP2/0 cytogamy greater than 40000 times immune mouse, to produce the hybridoma clone.

Avidity and specificity by ELISA and Western blotting screening monoclonal antibody.A plurality of monoclonal antibody clones of every kind of isomer further are characterized as binding specificity, avidity and the IC to the target acceptor ₅₀(50% concentration that suppresses).Acceptor is prepared as the total length membrane-bound receptor test of cell (be used for based on) or the soluble form (being used for the test based on ELISA) of the extracellular domain that merges with human IgG Fc.Select the positive monoclonal antibody clone of FGFR2IIIc, further study (i) based on following standard and do not detect cross reaction with the FGFR2IIIb isomer, (ii) based on EC ₅₀Value is to its nmole avidity of acceptor, (iii) the dyeing profile in tumor of prostate, other tumour and healthy tissues control group.Resist-FGFR2IIIb monoclonal antibody clone by similar Standard Selection, and be used as the contrast of experiment in vitro research, and dye for the IHC of healthy tissues and tumor tissues.

By ordinary method, can be with these monoclonal antibody human sourceizations.For example, the people source is anti--and FGFR2 isomer specific antibody can generate by the sequence that replaces the Fv variable region, do not participate in the antigen combination that is equal to sequence from people Fv variable region directly, as Morrison, S.L., 1985, Science229:1202-1207 and Oi et al., 1986, BioTechniques4:214 and Queen et al., US5,585,089, US5,693,761 and US5,693,762 is described, and its full content is by with reference to introducing herein.The people source is anti--and FGFR2 isomer specific antibody also can transplant or CDR replaces and produces by CDR, and wherein in the immunoglobulin chain, two or all CDRs are substituted, as U.S. patent 5,225,539; Jones et al., 1986Nature321:552-525; Verhoeyan et al., 1988Science239:1534; Beidler et al., 1988J.Immunol.141:4053-4060; Winter US5, described in 225,539, its full content is by with reference to introducing herein.

Described anti-FGFR2 isomer specific antibody also can produce by display technique of bacteriophage.Can produce anti-FGFR2 isomer specific antibody at display technique of bacteriophage known in the art.(described in following document: the United States Patent (USP) 5,223,409 of Ladner etc.; The International Patent Publication No. W of Kang etc. is the patent application of WO92/18619; The International Patent Publication No. W of Dower etc. is the patent application of WO91/17271; The International Patent Publication No. W of Winter etc. is the patent application of WO92/20791; The International Patent Publication No. W of Markland etc. is the patent application of WO92/15679; The International Patent Publication No. W of Breitling etc. is the patent application of WO93/01288; The International Patent Publication No. W of McCafferty etc. is the patent application of WO92/01047; The International Patent Publication No. W of Garrard etc. is the patent application of WO92/09690; The International Patent Publication No. W of Ladner etc. is the patent application of WO90/02809; Fuchs et al., (1991) Bio/Technology9:1370-1372; Hay et al., (1992) Hum Antibod Hybridomas3:81-85; Huse et al., (1989) Science246:1275-1281; Griffths et al., (1993) EMBO J12:725-734; Hawkins et al. (1992) J Mol BioI226:889-896; Clackson et al. (1991) Nature352:624-628; Gram et al., (1992) PNAS89:3576-3580; Garrad et al., (1991) Bio/Technology9:1373-1377; Hoogenboom et al., (1991) Nuc Acid Res19:4133-4137; And Barbas et al., (1991) PNAS88:7978-7982, its full content is by with reference to introducing herein).

Embodiment 3: the generation of solubility FGFR2IIIc-Fc acceptor

The extracellular domain of people FGFR2-β (IIIc) albumen (the Nucleotide 1-786 of SEQ ID NO:54) of dna sequence encoding is merged C-terminal Fc district into the human IgG1.These two gene fragments couple together by the 6 Nucleotide junctors (nucleotide linker) from restriction enzyme (Bgl-II), and this restriction enzyme produces two amino-acid residues, arginine and Serines.The complete sequence 491 amino acid whose polypeptide of encoding.Signal sequence is 21 amino acid, and therefore this chimeric maturation protein is 470 amino acid whose length.The molecular weight that calculates is 52.81 kilodaltons (kDa).

The structure of this fusion rotein as shown in FIG. 13A.

The Nucleotide of solubility FGFR2IIIc-Fc fusion rotein (SEQ ID NO:54) and amino acid (SEQ ID NO:55) sequence are respectively shown in Figure 13 B and 13C.

Fusion rotein is expressed by produce stable cell strain in the CHO host cell.This recombinant protein is solubility, and is secreted in substratum.By analyze SDS-PAGE under reductive condition, the migration of described recombinant protein is about 95kDa albumen, and general result is glycosylation (Figure 13 D).In Figure 13 D, band 7 has been showed molecular weight standard.(band 1-6 8-14) analyzes 13 clonal cell lines on trace.Conditioned medium (20 microlitres/band) from each clone is creeped at the SDS-PAGE gel, shifts then and carries out Western blotting (Western blot).This trace is by second antibody, the anti-human IgG coupling of goat alkaline

phosphatase staining.Band

3,10,11 and 14 has been showed the positive expression of Fc fusion rotein of counting FGFR2 β-ECD of 1D2,1F5,1F7 and 1F10 from stable clone.

Clone 1D2 is as the cell strain that produces the cell conditioned medium that is used for protein purification.After 72 hours cell cultures, the supernatant liquor of collecting cell conditioned medium.Use the Protein G agarose to flow (GE Healthcare, Life Sciences-Products) fast, according to dealer's method, by the fusion rotein of affinitive layer purification FGFR2 β-ECD.Shown in Figure 13 E, experimental results show that the functionally active of the soluble receptors of purifying by the part combination.Ligandin FGF8b is applied on the elisa plate with 2 mcg/ml, under 4 ℃, spends the night.With the PBS flushing paint sheet that contains 0.05% tween 20 (PBST), at room temperature it was blocked 2 hours in containing the PBST damping fluid of 2%BSA then.Subsequently, be that the preparation of fusion protein F GFR2 β-ECD of the purifying of 4 mcg/ml is bonded to the FGF8b that is coated on the described plate with concentration.By the adding second antibody, the anti-human IgG Fc of goat coupling horseradish peroxidase (HRP) and use

colour developing matrix

3,3 ', 5,5 '-tetramethyl benzidine (TMB) detects this combination.Detect the signal at absorbing wavelength 450nm place at the elisa plate readout instrument.

In Figure 13 E, two kinds of different preparations (preparation A and preparation B) of the solubility fusion receptors FGFR2c β-ECD of the Protein G purifying of 4 mcg/ml are added on the described plate 100 microlitres/hole.After at room temperature hatching 60 minutes, detect this combination with the anti-human IgG Fc-HRP of goat and TMB matrix.Detect the signal of 450nm at a plate readout instrument.The result shows that in conjunction with in testing, two kinds of preparations of the soluble receptors of purifying all demonstrate its part FGF8b similarly in conjunction with active at ELISA.

The experimental technique that Figure 13 E describes is used for measuring the isomer acceptor FGFR2IIIc of the inhibition activity anti-FGFR2IIIc of antibody is bonded to to(for) block ligand.FGF8b is the specificity growth factor part of a kind of isomer IIIc for acceptor FGFR2, does not detect it and is bonded to the IIIb isomer of same receptor (data not shown goes out; Zhang et al., (2006) J Biol Chem.281,

15694-15700）。

Be also referred to as " Atto-MuMab-03 " in order to prove in the mouse monoclonal antibody Mo-3B6(literary composition) the part combination of acceptor FGFR2IIIc capable of blocking, when described monoclonal antibody Atto-MuMab-03 exists or do not exist, carry out as the described part of Figure 13 E in conjunction with experiment.Before it being added into described part (FGF8b) paint sheet, with FGFR2IIIc-ECD-Fc preincubation Atto-MuMab-03.By the anti-human IgG Fc-HRP of second antibody coupling HRP(goat) in the presence of the Atto-MuMab-03 of different concns, or do not exist down, detect the combination of FGFR2IIIc and part.Shown in Figure 13 F, antibody A tto-MuMab-03 has showed on ligand-binding activity the specificity of acceptor FGFR2IIIc and the inhibition of concentration dependent.Negative control mouse antibodies, irrelevant mono-clonal clone, be called as 5D3 and be bonded to part for acceptor FGFR2IIIc and do not show tangible barrier effect.

The generation of embodiment 4:FGFR2IIIc peptide

The isomer specific peptide of FGFR2IIIc can produce by reorganization or the solid phase synthesis of standard.

For example, the peptide with the aminoacid sequence shown in table 1 and Fig. 6 A-6C can generate by corresponding nucleotide sequence being cloned on the embodiment 2 described expression vectors.

Perhaps, can come synthetic peptide by the standard method of solid phase or the reaction of liquid phase chemistry of peptides.The general introduction of this solid phase technique can be found in following document: Stewart and Young (1963) Solid Phase Peptide Synthesis, W.H.Freeman Co. (San Francisco), and Meienhofer (1973) Hormonal Proteins and Peptides, Academic Press (New York).Liquid phase for classics is synthetic, can be referring to Schroder and Lupke, and The Peptides, Vol.1, Academic Press (New York).Generally speaking, the amino acid of one or more amino acid or suitably protection can add the peptide chain of growth in succession.Subsequently the amino acid of described protection is connected to inert solid carrier or by be added in have additional group in the sequence (amino or carboxyl) next amino acid and in solution, utilize, the protection that this additional group can be suitable also is fit to form amido linkage under certain condition.This blocking group is removed from initiate amino-acid residue subsequently, and next amino acid (by suitable protection) is added into, etc.When all desirable amino acid have been connected to suitable sequence, remove any residual blocking group (and any solid carrier) successively or simultaneously, to obtain final peptide.Once a plurality of amino acid can be added growing chain, for example, after deprotection, by the tripeptides of protection is combined with the dipeptides of appropriate protection (not having under the situation at racemize center), to form pentapeptide.

Embodiment 5: with fibroblast growth factor (FGF) acceptor 2(FGFR2 in the cancer before the prostatitis) isomer IIIc is the test of the antibody molecule of target

This embodiment has assessed in the prostate cancer pattern, the treatment feasibility of the antibody drug (anti-FGFR2-IlIc antibody) of antagonism FGFR2IlIc.The molecular target of described antibody, FCFR2 isomer IIIc, the growth of tumor with androgen independence is relevant with transfer.This method is based on the high expression level of this receptor at hormonal resistance prostate cancer (HRPC), and its key is to improve the intrusion behavior of tumour cell, and (epithelium transforms to a matter, EMT).The target that designs this isomer specific antibody medicine is that " bad isomer " with the FGFR2 acceptor on the tumour is target (targeting), but do not go pipe on the normal prostatic epithelium, have " good isomer " FGFR2-IIIb that suppresses the tumor growth function.

Cell strain

The androgen independence Human Prostate Cancer Cells DU145(ATCC that this research is used) and DU9479(Duke University) all be to show the good feature cell strain that transfer performance and androgen independence are grown.DU145 derives from the cancer (Stone et al. (1978) Int.J.Cancer21:274-281) that prostate cancer is transferred to brain.This cell strain has showed significant FGFR2-IIIc(Carstens et al., (1997) Oncogene15,3059-3065).It often is used to (Garrison et al., (2007) Cancer Res.67:11344-11352 in the Research of Animal Model for Study of tumor growth and vasculogenesis; Russel and Voeks (2003) Methods in Molecular Medicine ^TM: " Prostate Cancer:Methods and Protocols " Animal Models of Prostate Cancer.Page89-112).Other human prostate tumor line is as the PC-3(hormonal independent) or LNCaP(hormonal dependent, express IIIb) in a few thing, be used as contrast or negative control.DU9497 is another kind of androgen insensitivity post, comprise whole FGFR2IIIc isomer (Carstens et al., (1997) Oncogene15,3059-3065).

In the cell in vitro experiment, DU145 is fit to most of experiment, comprises the experiment of propagation and receptor activation.In conjunction with experiment, use the transfectional cell of express recombinant FGFR2IIIc target spot for part.

The anti-FGFR2IIIc of monoclonal antibody and IIIb are (being called " Ab-1 " or " Atto-MuMab-01 " and " Ab-2 " or " Atto-MuMab-02 " in the literary composition) of having produced and having described feature.Other biochemical reagents (FGFs) and immuno-chemical reagent (as phosphotyrosine antibody, Grb2, ERK1/2, the signaling molecule of STAT1 and SHP2) can be bought from different commercial supplier.

Use the hormonal independent tumor line, DU145 and DU9479 antagonist molecule carry out external and the body build-in test.Can carry out following experiment:

1) external activity and the celelular mechanism of test Ab-1

A. suppress receptor activation and send signal

B. block ligand in conjunction with or receptor dimerizationization

C. influence cell proliferation and apoptosis

2) the human prostata cancer of test Ab-1 is transplanted the interior effect of body of effluent (xenografts)

D. to suppressing the effect of tumor growth, tumor-blood-vessel growth

Exploitation has the monoclonal antibody Atto-MuMab-01 of dual-use function in this design.The binding mode of this antibody can comprise inhibit feature, namely blocks the activation of acceptor, and immunologic function, i.e. inducing cytotoxic T cytoactive.Atto-MuMab-01 is bonded to the isomer specific regions of the Ig class ring 3 of FGFR2IIIc acceptor.This zone relates to the specificity of part combination, (Shaun et al., (2006) Genes﹠amp of proving as former crystal structure analysis; Dev.20:185-198).Estimate antibody A tto-MuMab-01 part combination capable of blocking, therefore suppress receptor activation.The second, but the cellular immune function of described antibody human activin.This antibody is human IgG1's isomer (isotype), and can induce the strong immune response of the cytotoxicity (ADCC) of antibody dependent cellular, and/or the cytolysis of complement-mediated.Atto-MuMab-01 is designed on 333 in the amino acid from L-glutamic acid to L-Ala that is positioned at the Fc district, with the ADCC activity of the described antibody of further raising.Therefore, when described antibodies was to the FGFR2IIIc positive tumor cell, it can kill activity to increase effective tumour by the T cell of ADCC absorptive cell toxicity.

Therefore, Atto-MuMab-01 possesses powerful anti-tumor activity, simultaneously, can possess attractive security feature.Because it strictly is bonded to the IIIc positive tumor, it is the kill tumor cell optionally, and normal epithelium is not produced severe side effect (expressing the IIIb isomer).

Embodiment 5.1: be used for the proof test that FGFR2IIIc expresses

In far-ranging cancer cell strain, comprise and carry out the proof test that FGFR2IIIc expresses in prostate gland, bladder, lung (NSCLC) and the Tiroidina.Also study the expression of FGFR2IIIc by tissue distribution analysis (IHC dyeing).Carry out following research.

The proof specificity is bonded to prostate tumor cells, and the normal prostate gland that do not match (Tissue Arrays compliant with FDA, from US Biomax, Rockville, MD20849; Multi-Tumor Microarrays from Invitrogen).

With 30 kinds of organ-tissue arrays of IHC dyeing, with proof and health tissues no cross reaction (Tissue Arrays from US Biomax, Rockville, MD20849).

Embodiment 5.2: make up FGF8-SEAP

Carry out this structure, so that sensitive, non-marked part is in conjunction with experiment.Encoding sequence (SEQ ID NO:66) from cDNA template clone's FGF8b is carried out PCR, and insert the seap gene back in the commercially available expression vector.The junctor flexibly of ten amino acid GGGGSGGGGS (SEQ ID NO:59) is added in described two fragments, and its label is inserted the C end of described fusion rotein, so that protein purification.Gained fusion protein F GF8-SEAP can be easy to be produced in cell conditioned medium liquid and make secretor type, and it can be directly used in most experiment.For quantitative enzymic activity, the SEAP(commercialization of purifying) as standard, chemiluminescent matrix is used for measuring optical signal.SEAP is active directly relevant with the amount of described part FGF8b.

Embodiment 5.3: the in vitro study of celelular mechanism

The anticancrin medicine of having set up, for example Trastuzumab (the anti-Her2 acceptor that is used for breast cancer) and Erbitux (the anti-EGFR acceptor that is used for head and neck cancer) show anti-tumor activities by different mechanism.These mechanism comprise the blocking-up receptor signal, influence the ligand receptor combination, trigger apoptosis, and by ADCC or complement-mediated cracking inducing cell toxic action (Baselga et al., (2001) Semin Oncol.5Suppl16:4-11; Trauth et al. (1989) Science245:301; Yang et al. (1999) Cancer Res.59:1236).In this case, can study the anti-tumor activity of the prostate cancer cell of Ab-1 by several experiment in vitro, purpose provides the information of the drug cell mechanism of understanding in prostate cancer cell.

To understand antibody to the celelular mechanism of the effect of tumour cell in order providing, can to check three aspects of cell function.

A. the barrier effect of the FGF signal of antibody A b-1

Receptor activation experiment-dose-dependent inhibition: can check in the DU145 cell neutralization activity of the antibody of FGFR2 receptor activation.It is significant that known DU145 expresses FGFR1, FGFR2IIIc() and FGFR4(Coombes et al., (2000) Book " Endocrine Oncology ", Chapter12,237-253; Carstens et al., (1997) Oncogene15,3059-3065).FGFR2IIIc and FGF8 and FGF2 knot are associated with response, but isomer IIIb acceptor does not respond (Zhang et al., (2006) J Biol Chem.281:15694-15700) to these somatomedins.The activation of acceptor can be analyzed in the phosphorylation increase, can be by the western blot analysis of cell lysate (cell lysate) is analyzed.In some cases, be necessary by with the immunoprecipitation of described anti-acceptor FGFR2IIIc, the described acceptor of from full cell lysate, " leaving behind ".Analyze the gained immunoprecipitate at sds gel, use anti-phosphotyrosine antibody to carry out Western blotting subsequently.

In order to obtain IC ₅₀The dose-dependent inhibition curve of value, with FGF8 competition before, with or the antibody hatching DU145 that increases without concentration.The concentration range of antibody can determine by rule of thumb that this affinity of antibody and receptor expression level by concrete cell determines.Quantitative (for example densitometric scan) of acceptor that can be by phosphorylation set up antibody and suppressed curve, thereby analyzes by these, can derive the IC that the antibody of receptor activation suppresses ₅₀Value.

In addition, by analyzing signaling molecule or the effector of FGFR2, as Grb2, ERK1/2, p38 or STAT1, check the downstream signal event.These extra readings can be used for specified data.Receptor activation, and analysis of Phosphorylation, these results provide the information of the potential effectiveness of medicine.

It is active whether the provable antibody A b-1 of these data has neutralization.In theory, described antibody can be competed with the part that is bonded to described acceptor, perhaps its receptor dimerizationization capable of blocking.The two all can provide the identical reading that suppresses receptor activation and receptor signal.Below experiment is used for these problems of answer.

B. the effect of block ligand combination or receptor dimerizationization

Bao Dao FGFR2 crystal structure analysis in the past (Olsen et al., (2006) Genes﹠amp; Dev.20:185-198) show half of C ' end ring 3, by interchangeable exon 8 codings, participate in the specificity of the part combination of described acceptor.The ring 3 of IIIc isomer is bonded to FGF8, but the ring of IIIb 3 is bonded to FGF7.Yet report also claims the ring 2 of described acceptor also can promote the part combination.Therefore, be necessary to obtain by experiment direct evidence, prove that Ab-1 whether by being bonded to half the epi-position of C ' end of its ring 3, comes complete block ligand FGF8 to be bonded to its acceptor FGFR2IIIc.Following the carrying out of experiment that the antibody of part combination suppresses.

Respectively, another effect-antibodies to acceptor can influence the dimerization of acceptor, can detect receptor activation and send the prerequisite step of signal.Simultaneously, these molecular interaction analyses molecule mechanism of can be the binding mode of antibody provides detailed explanation.

Part is in conjunction with experiment

In order to estimate antibody to the inhibition of part combination, the transfection HEK293 that expresses described acceptor FGFR2-IIIc is used for the experiment of 96 orifice plates.Exploitation on-radiation and responsive luminous experiment come the combination of detector ligand and its acceptor.This experimental design (assay format) comprise use to inject secretor type alkaline phosphatase, FGF8-SEAP reorganization FGF8(as mentioned above).This experimental design makes ligand receptor binding events reading with instant enzyme by powerful luminous signal.In conjunction with condition, the concentration range of spendable FGF8-SEAP is 20pM to 5nM(Zhang et al., (2006) J Biol Chem.281:15694-15700 according to the part of former report).Adding concentration is the Vitrum AB of 10 μ g/ml, so that FGF8 is bonded to described acceptor.Chemiluminescent matrix by adding SEAP is (from Applied Biosystems's

Or from the PhosphaGLO of KPL ^TM), but the described receptors bind part of direct quantitative is measured (Luminoskan, Thermo Scientific) with microplate reader.

From competitive assay, obtain IC ₅₀Value, wherein antibody A b-1 and concentration are the cell preincubation of 1pM to 100nM.Then, part SEAP-FGF8 is added in this cell culture fluid.The dose-dependently of SEAP signal reduces the binding site that means on antibody and the part competition acceptor.

Statistical study--can be by have the S type tracing analysis statistics-binding curve of variable slope with nonlinear regression and fitting.Packet data address is on average+/-SD or SEM.

The receptor dimerization experiment

Known FGFs is bonded to their acceptor, to induce receptor dimerizationization.This can pass through chemical cross-linking agent, for example crosslinking aid S DP(succinyl phosphorons amino propyl acid ester) prove.Distinguish monomer and dimer acceptor based on the apparent molecular weight on the irreducibility sds gel, carry out Western blotting subsequently.Acceptor from untreated cell should exist with monomer (92Kda).The cell that FGF8 handles should be shown as main dimer (about 180Kda).

In order to check the whether dimerization of acceptor capable of blocking of antibody A b-1, with the transfectional cell (in 6 well culture plates) of expressing FGFR2IIIc and antibody 0, EC ₅₀With preincubation under the saturation concentration.Irrelevant antibody can be used as negative control.After the antibody preincubation, FGF8 is added in the cell, to induce the dimerization of acceptor.In cell, add chemical cross-linking agent DSP then, at room temperature additionally hatched 10 to 15 minutes.At last, the preparation cell lysate, and utilize anti--receptor antibody to carry out western blot analysis.Trace shows main acceptor monomer in stimulated cells not, and the dimer of increase (does not have Ab-1 to handle) in the FGF8 stimulated cells.Do not reduce dimeric amount in the FGF8 stimulated samples with the preincubation of negative control antibody.Compare the Ab-1 cell of handling and the cell of handling with negative control antibody after FGF8 induces, dimeric minimizing.These data provide Ab-1 antibody whether to block the evidence that receptor dimer forms.

C.Ab-1 on cell proliferation and apoptotic effect:

Can assess the antiproliferative effect of antibody A b-1.In addition, can analyze the short apoptosis effect of the tumour cell of antibody A b-1.

Proliferation experiment:

(Mosmann et al., (1983) J.lmmunol.Methods 65:55-63) at the several cell strains of 96 orifice plates analysis from prostate cancer, comprise PC-3, DU145 and LNCaP to use previously described MTT experiment.MTT is provided at the measurement of the mitochondrial dehydrogenase activity in the cell, thereby the indication of proliferative activity is provided.

The cell that is exponential growth can be sowed in the hole of 96 orifice plates by the 2000-3000 cell density.The AB-1 adding of nM scope is had in the hole of substratum, and hatched 48 hours.With MTT(1mg/ml) add in the described cell, hatched 2 hours down at 37 ℃.Lysis, and in the absorbancy of enzyme micro-plate reader mensuration dyestuff at the 600nm place.

Apoptotic assessment:

With method (Reid et al. as previously described, (1996) J lmmunol Methods, 192:43-54), use lipotropy dyestuff MC540 in conjunction with DNA dye Hoechest33342, can check apoptotic inducing action on the tumour cell of antibody A b-1.MC540 checks early stage apoptosis (being the conformational change on the plasma membrane).For the similar mode of proliferation experiment, use the antibody treatment tumour cell with aforementioned.Measure the variation of film by adding dyestuff MC540.In order further to estimate the biochemical change in the apoptotic cell, the applicant has checked the expression of the activated form of caspase-7 by Western blotting.Can buy anti--caspase-7 from Cell Signaling Technology.

Embodiment 5.4: in testing in the body, and the effect of the people's tumour transplatation of Ab-1 effluent

Can check the interior curative effect of tumour of the hormonal independent of the nude mice of Ab-1 with the DU145 graft.End points (endpoints) comprises gross tumor volume, weight, tumor vessel and shift index.Also can analyze other reading, as survival time, immune response and toxicological experiment.

D. in transplanting effluent to the blocking effect of tumor growth and/or tumor-blood-vessel growth

Check the mouse toxicity and the uncomfortable mark that participate in experiment in per two days, comprise weight, active degree, skin abnormality, dysentery and general appearance.

Can use subcutaneous (s.c.) tumour transplatation effluent model with cancer cells foundation before the DU145 prostatitis (Coombes et al., Book " Endocrine Oncology ", Edited by Stephen P.Ethier.Chapter12,237-253).Simply, with 5x10 ⁶The nude mice in age in tumor cell inoculation to 6 week, and tumour is grown up to 1cm ³(be 3-4 week for DU145).With 100mm ³The tumor fragment of volume migrates to mouse.Per 3 days, with slide calliper rule from externally measured growth of tumor.Tumor-bearing mice is divided into 3 groups, 10 every group.Available taxol treatment group 1 is as positive controls.The antibody A b-1 treatment group 2 of available 10mg/kg, 2 times weekly, intraperitoneal injection, continuous 5 weeks.Available media thing treatment group 3 is as negative control group.By externally measured monitoring growth of tumor.From eye socket clump taking heparin blood sample, be used for determining plasma A b-1 concentration.

After experiment finished, tumor resection was also weighed, and be fixed in the formalin.Collect following end-point data:

1. tumour weight in wet base (gram)

2. transfer-the lymphoglandula of the second position, lung, pancreas, spleen, kidney, suprarenal gland, diaphragm, bone and brain

3. to the immunohistochemical staining analysis of the fixed sample of the target spot FGFR2IIIc that expresses in tumour, activation/phosphorylation of FGFR2IIIc, and the accumulation (by IHC method use anti-people antibody staining) of Ab-1 on tumour

4. utilize anti--CD31 dyeing (Dako) assessment vascularity.Calculate positive vessels 5 different field

Endotheliocyte.

Statistical study: gross tumor volume is calculated as V=(L ²/ I)/2, wherein L and I represent bigger or less diameter of tumor.The end points measurement of tumour is that to restrain be the weight in wet base of unit.Utilize ANOVA to carry out statistical, to analyze the meaning between the different value.Carry out the regression analysis of slide calliper rule amount and weight in wet base.Integrated data with on average+/-SD or SEM report.P value＜0.005 is significant.Embodiment 5.5: selectable strategy

A. transplanted tumor research:

For transitivity HRPC, in disease progression, relate to complicated mechanism and a plurality of stage.The critical stage of mechanism that relates to the disease of FGFR2IIIc can be explored the anti-tumor activity of provable Ab-1 in the transplanted tumor model of having set up by Ab-1.This research can be used for determining the activity of monoclonal antibody in tumor model.Further research may need to use different tumor inoculation methods, as in-situ inoculating or intracardiac injection, thereby carries out dissection and analysis in the main phase of metastases.

Except the DU145 transplanted tumor, also can use other to have the CaP tumor line of target acceptor high expression level.

Perhaps, can use Deng Ning rat prostate cancer model and AT-3 hormonal independent cell strain.This model system has been widely used in studying the function/adjusting of FGFR2 isomer, and is considered to relevant (Sebastian et al., (2006) PNAS103:14116-14121 with people HRPC; Muh et al., (2002) JBC277:50143-50154; Carstens et al., (2000) MCB, 20:7388-7400).This method can be evaluated to determine monoclonal antibody, Ab-1 and Ab-2(are respectively anti-FGFR2IIIc and anti-FGFR2IIIb) with the cross reaction of rat receptor.At IIIc(Human:amino acids301-353of SEQ ID NO:2; Rat:SEQ ID NO:67) and IIIb(Human:amino acids301-351of SEQ ID NO:65; The aminoacid sequence of selectivity shear zone Rat:SEQ ID NO:68) (alternatively spliced region) is guarded (Figure 14) fully between people and rat.

B. in vitro study

Except the DU145 cell, the transfectional cell with low endogenous FGFR2IIIc expression can be used in vitro study.

Embodiment 5.6: other experiment

Other experiment comprises the immunization of test Ab-1 from vitro study, and in the tumour cell that monoclonal antibody was handled, the cell-mediated cytotoxicity of test T.In addition, can be to being used in combination antibody and FGFR selectivity tyrosine kinase inhibitor (TKIs), for example R04383596 or Pazopanib(be as shown in figure 15) two target strategies (dual targeted strategy) analyze.Particularly, the effect of the TKI drug-resistant tumor of research Ab-1 cell.This pair of target strategy shows the anti-tumor activity (Huang et al., (2004) Cancer Res.64:5355-5362) that increases in the EGFR target on cancer.Embodiment 6:FGFR2-IIIc is as the potential biomarker that is used for the prostate cancer circulating tumor cell

This embodiment is by histopathology method traditional, approved, by the existence of the FGFR2IIIc acceptor on the cell strain of the intractable prostate cancer of similar hormone in the CTC tests positive inspection patient peripheral blood cells.Utilize isomer Auele Specific Primer collection (primer sets) to confirm the tumour character of the cell positive that FGFR2IIIc expresses by PCR method.This result of study is identified tumour and is depended on patient's subgroup that FGFR2 isomer IIIc expresses with shifting, its, and the specificity of identifying the extra increase of the CTC test that utilizes the existing of Ep-CAM and ratify.

Particularly, this embodiment has estimated and has detected and enrich CTCs(or epithelium transform (ETM) to a matter a prostate tumor cells) feasibility, its expression has the carcinogenic acceptor FGFR2 isomer IIIc(FGFR2IIIc of isomer specific antibody).Initial focus on to take from patient's peripheral blood of suffering from known metastatic disease, the circulating cells that carries FGFR2IIIc is identified and is detected.In case set up positive detection and specific data, can in normal healthy controls group, Benign Prostatic Hypertrophy and patients with prostate cancer, proceed the technical optimization of detection sensitivity.

Below be the objectives of present embodiment:

1) research is derived from the existence of the positive CTCs of FGFR2IIIc of peripheral blood, and confirms that these cells are cancer cells.

2) abundant and separates FGFR2IIIc positive CTCs by immune purifying.

3) use exon specific PCR primer, analyze the existence of the CTC that determines to carry the FGFR2IIIc acceptor by RT-PCR.

This embodiment is FGFR2IIIc as the feasibility study of the effect of effective biomarker of identification prostate cancer CTCs, and CTCs is used for metastatic disease and the malignant tumour of the asymptomatic patients with prostate cancer of diagnosis.Further research concentrates on:

A. (the FGFR2-IIIc positive, for example DU145 PC3), optimizes described detection method by (spiked in) prostate tumor cells of diluting in the quantitative recovery peripheral blood sample.

B. in the TURP(of benign prostatic hyperplasia transurethral prostatectomy) reach before after, calculate before the prostate removal surgery and FGFR2IIIc positive cell in the peripheral blood of patients afterwards.

C. collect the mass data collection from the intractable patients with prostate cancer of asymptomatic and Symptomatic hormone, to determine diagnostic value and the predictor of this test.

Embodiment 6.1: the meaning that detects the test of circulating tumor cell

The metastatic cancer cell of in blood or lymph, propagating for " circulating tumor cell " (CTCs), the metastatic cancer cell of in marrow, propagating for " tumour cell of diffusion " (DTCs).CTCs and DTCs represent unique diagnosis and treatment target spot.Circulating tumor cell is very rare in the patient of nonmalignant disease, but but is present in (Allard et al. (2004) Clin Cancer Res.1O:6897-6904) in the various metastatic carcinomas with probability very widely.Some clinical studyes show that the assessment of CTCs can assist a physician and check and prediction cancer progression and estimate the metastatic carcinoma patient to reaction (Berrepoot et al., (2004) Ann Oncol.15:139-145 for the treatment of; Aquino et al., (2002) J.Chemother.14:412-416; Katoh et al., (2004) Anticancer Res.24:1421-1425).In castration resistivity prostate cancer (CRPC), the recent research for the treatment of back CTC counting and the relation of total lifetime (OS) is shown that CTC counting prediction OS is better than PSA decrement algorithm (de Bono et al., (2008) Clin Cancer Res.4 (19): 6302-9) at all time points.

Present CTC detection method is based on the epithelium marker, for example Ep-CAM may miss the positive circulating tumor cell of FGFR2-IIIc, because the expression of FGFR2IIIc on prostate cancer cell and the disappearance of epithelium marker and relevant (Moffa and Ethier (2007) the J Cell Physiol.210 (3): 720-31) of acquisition of mesenchymal cell marker.

Be used for independent or as follows in conjunction with the method classification several commonly used that detects CTCs:

I) molecular biology: RT-PCR(reverse transcription PCR for example)

Ii) immunochemistry: the magnetic bead of antibody coupling for example; Immunofluorescence microscopy; Flow cytometer (FACS) is analyzed

RT-PCR provides a kind of highly sensitive method to detect gene.But PCR detects viable cell, dead cell and free DNA, has potential false positive.The specificity of the target gene of amplification is the limiting factor of its diagnostic value or predictor.

It is believed that the tumour cell of finding in the androgen independence tumour that carries carcinogenic acceptor FGFR2IIIc isomer is respectively to the invasive growth of tumor, and propagate the transfer that causes in the organ, and the infection that blood flow causes is responsible for.Be the diagnosis of prostate cancer and selectivity step by stages by the FGFR2IIIc specific antibody identification prostate gland circulating tumor cell (CTCs) of deriving.After prostatectomy, in circulation, still exist these cells can show the development of metastatic disease.Therefore, the CTC that this embodiment shows detects can provide extra sensitivity and specificity, is used to the HPCR patient diagnosis to shift.

Embodiment 6.2: the CTC counting is to the prediction of total lifetime in prostate cancer

The CTC counting that obtains at baseline between known time-delay of obtaining by immune magnetic is than PSA level and change prediction total lifetime of disadvantageous result (de Bono et al., (2008) Clin Cancer Res.14 (19): 6302-9 more reliably; Danila et al., (2007) Clin Cancer Res.13 (23): 7053-8).In prostate cancer, help to understand the molecule mechanism of prostate cancer and metastatic disease as the detection of the FGFR2IIIc of the potential biomarker of CTCs.In addition, this analysis can be provided for the specificity experiment of present unacknowledged HRPC subgroup.

Embodiment 6.3: the immune magnetic purifying of the DU145 tumour cell that rises violently in normal blood

For purifying is diluted in DU145 tumour cell in the normal blood, carry out following method.

A. prepare immunomagnetic beads: with the PBS of FGFR2IIIc(at the 0.1mg/ml that contains 1%BSA) monoclonal antibody be fixedly on the magnetic bead that this magnetic bead and goat anti-mouse Fc(are from Becton Dickinson) by under 4 ℃, spending the night hatching and pre-coupling.

B. tumour cell mark-on dilution experiment (spiking experiment): the PC12(FGFR2IIIc feminine gender), the DU145(FGFR2IIIc positive) tumour cell uses the fluorescence dye fluorexon of active somatic cell (from Molecular Probes, Eugene, Oregon) preload was hatched 5 minutes down at 37 ℃.Labeled cell is diluted in the normal plasma cell of 7.5ml of following ratio: 1000 cells, 500 cells, 100 cells, 50 cells, 10 cells.These labeled cells are exposed to immunomagnetic beads, amplify or calculate by the flow cytometer facs analysis fluorocyte (recovered fluorescent cells) of recovery under fluorescent microscope by 20x.The good result that the immune magnetic of constant recovery rate proof FGFR2IIIc positive cell is selected.

Embodiment 6.4: to from the detection of the CTCs of patients with prostate cancer and abundant

Method and instrument (de Bono et al., (2008) Clin Cancer Res.14 (19): 6302-9) that this announces according to Veridex.By reported method before, detect the CTCs(Berrepoot et al. that carries FGFR2IIIc, (2004) Ann Oncol.15:139-145 with patient's blood sample; Aquino et al., (2002) JChemother.14:412-416; Katoh et al. (2004) Anticancer Res.24:1421-1425; Allard et al. (2004) Clin Cancer Res.10:6897-6904; De Bono et al., (2008) Clin Cancer Res.14 (19): 6302-9).Basically, blood sample is inhaled into 10ml EDTA evacuated collection (Becton Dickinson), adds cell sanitas (Berrepoot et al., (2004) Ann

Oncol.15:l39-145;Aquino?et?al.,(2002)J?Chemother.14:412-416;Katoh?et?al.(2004)Anticancer?Res.24:1421-1425;Allard?et?al.(2004)Clin?Cancer?Res.10:6897-6904;de?Bono?et?al.,(2008)Clin?Cancer?Res.14(19):6302-9）。After collecting, sample is kept at room temperature, and in 72 hours, handle.Cell is hatched with the anti-FGFR2IIIc that loads magnetic bead.Fluorescent core acid dye DAPI(4,2-diamidino-2-phenylindone dihydrochloride) for the dyeing karyocyte.Can carry out identification and the counting of the positive CTCs of FGFR2IIIc by cell overseer analyser, this cell overseer analyser is a kind of semi-automatic fluorescent base microscopic system, and it makes computer produce the reorganization of cell image.Circulating tumor cell is calculated as the karyocyte of expressing FGFR2IIIc.For the epithelial cell characteristic of definite CTCs that separates, with epithelial cell marker cytokeratin 19 double stainings, with another kind of fluorescence dye phycoerythrin (PE) mark.

The RT-PCR of embodiment 6.5:FGFR2IIIc isomer

For the specificity that the immune magnetic that proves the positive CTCs of FGFR2IIIc is selected, carry out the RT-PCR experiment, to confirm the expression of isomer FGFR2-IIIc in isolated cell.Can use RNeasy Mini test kit (Qiagen, Hilden, the Germany) isolation of RNA that comprises RNase-Free DNase Set.For reverse transcription, RNA is diluted in the RNase-free water of 15 μ l, hatched 5 minutes down at 65 ℃, and place on ice.Preparation contains the oligp(dT of 2 μ l) 15 primers (0.8 μ g/ μ l), the deoxynucleoside triphosphate of 2 μ l (5mM), 0.5 the RNAsin(40 unit of μ l/μ l), the Omniscript reversed transcriptive enzyme of 1 μ l (4.5 units/μ l), with the mixed solution of 7.5 μ l of the reverse transcription damping fluid (x10) of 2 μ l, and it is added among the RNA of described dilution.37 ℃ of down hatchings 1 hour, 95 ℃ of activity of destroying the Omniscript reversed transcriptive enzymes down 5 minutes, cDNA is stored in-20 ℃.Use IIIc exon Auele Specific Primer ((IIIc-F:

Aggttctcaaggccgccggtgt (SEQ ID NO:71) and IIIc-R:caaccatgcagagtgaaagga (SEQ ID NO:72); IIIb exon Auele Specific Primer (IIIb-F:ggttctcaagcactcgggga (SEQ ID NO:69) and IIIb-R:

Gccaggcagactggttggcc (SEQ ID NO:70)) carries out pcr amplification as a reference.Be used for pcr analysis the isomer Auele Specific Primer design as shown in figure 16.Tumour cell, the PC-3(IIIb positive) and the DU145(IIIc positive) can be used as the positive control of PCR experiment.Described PCR product should be shown as 140 base pair frequency bands on sepharose.

Embodiment 6.6: other experiment

Other experiment comprises optimizes this testing method and assessment/the be lifted at clinical correlation of the CTCs counting in HRPC patient's clinical effectiveness.Use biological statistical method to carry out data analysis and inquiry.

Embodiment 7:FGFR2IIIc is as biomarker, for detection of the intractable prostate cancer of hormone

This embodiment has described the foundation for detection of immunohistochemical staining (IHC) experiment of invasive, steroid-resistant prostate cancer.As disclosed, FGFR2 isomer IIIc is relevant with transfer with the androgen independence growth of tumor, and it is at the surface expression of prostate cancer tissue.

Several biomarkers have been developed for immunohistochemical methods (IHC) the dyeing experiment of prostate cancer.These comprise prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), prostate stem cell antigen (PSCA); androgen receptor (AR), chromogranin, synaptophysin; MIB-1, and α-formyl radical coenzyme A racemase (AMACR).These markers are not specific for transfering state or metastatic potential.The inspection of FGFR2 isomer IIIc and the IIIb expression in examination of living tissue and excision sample should provide extra information for the patient of hormone refractory disease and potential transfer.

Prostate cancer cell strain DU145 and LNCaP obtain from American type culture collection (ATCC) at first.These cell cultures use standard method to keep.

Prostate cancer and people's organization chip with normal prostate tissue chip of coupling can MD20849) obtain from U.S. Biomax(Rockville; Many oncogenes chip will be from Invitrogen(CA) obtain.These tissues should meet FDA and supervision requirement.Patient's clinical data, for example the pathology stage of Gleason scoring and disease is available.Patient's personal information is protected.

The IHC experiment can be used for the operation sample from radical prostatectomy or needle puncture biopsy (NBX) and transurethral prostatectomy (TURP).

The purpose of this embodiment comprises that (i) uses to be fixed in the paraffin and sets up the IHC experimental technique as the tumor cell line of cell precipitation; (ii) be used to assess from the tissue samples of patients with prostate cancer and Benign Prostatic Hypertrophy the effect of this IHC diagnostic method.

Carry out following experiment:

I. study the different isomer acceptor of two kinds of functions of FGFR2 in the prostate tumor cells strain, the DU145(IIIc positive) with the LNCaP(IIIb positive) in different expression.Proof mAbs is to specificity and the sensitivity of every kind of isomer being used for IHC and using.Set up the IHC method.

Ii. the 30 organ-tissue chips that dye, thus be used to the Biomax(Rockville from US, organization chip research IIIb MD20849) and the different tissue distribution of IIIc.

Iii. prostate cancer and benign prostatic hyperplasia (BPHs) respectively check about 20 examples, to differentiate tumour and non-cancer tissue (from Invitrogen, the tumour chip of CA).

Iv. analyze IHC dyeing and determine classification and dyeing pattern; For example positive, negative score, and the expression pattern (with pathology expert jointly work) of FGFR2IIIc in tumor tissues/cell.

V. explore the clinical correlation of biomarker and disease severity, and assessment uses blocking-up to shift the advantage of the target antibody medicine of disease.

This biomarker also can be combined with other IHC tissue markers and be used for the analysis of many biomarkers.

Embodiment 7.1:IHC dyeing process

MAbs ordinary stain method for the FGFR2 acceptor is described below.Can be each mAbs optimization experiment condition of anti-FGFR IIIc or IIIb.

Immunohistochemical methods with the paraffin-embedded tissue section

Antibody: monoclonal anti-FGFR2IIIc and anti--FGFR2IIIb antibody produce as mentioned above.Monoclonal anti-cytokeratin (Pan) clone AE1/AE3 antibody is from Zymed(San Francisco, and CA), the polyclone anti-PSA antibody is from Dako Cytomation.With peroxidase, ChemMate ^TM, DAKO Envision ^TMThe second antibody of detection kit coupling is from Dako Cytomation(Denmark).

Diaminobenzidine (DAB) can be used as chromogen, carries out Meyer's haematoxylin redyeing look subsequently.

I. Qie Pian preparation

From DU145 and LNCaP cell preparation cell precipitation, be fixed in 10% the formalin and spend the night, the method with routine is processed into Pathologic specimen then, to produce paraffin-embedded cell lump.Paraffin mass by commercial supplier prepares tissue slice.

II. deparaffinization

1. know all sections of mark with pencil, indicate antibody and extent of dilution.

2. deparaffnize and aquation as follows: in dimethylbenzene 5 minutes, 3 times; In 100% ethanol 5 minutes, 2 times; In 95% ethanol 5 minutes, 2 times; In 80% ethanol 5 minutes, once.

3. at room temperature will all cut into slices (sections) be positioned over endogenous blocking-up liquid (methyl alcohol+2% hydrogen peroxide) 20 minutes.

4. flushing section twice in deionized water, each 5 minutes.

5. flushing is cut into slices 2 times in the phosphate buffered saline (PBS) (PBS) of pH7.4, each 5 minutes.

III. block and dye

1. at room temperature use PBS/1% bovine serum albumin (PBA) to block all sections 1 hour.

2. will cut into slices and be diluted in PBA(2%) rabbit anteserum at room temperature hatched 30 minutes, to reduce the non-specific binding of antibody.In airtight humidity cabinet, hatch, to prevent that tissue slice is by dry air.

3. carefully shake off unnecessary antibody, and cover section with the mAb that is diluted among the PBA.The lid of changing humidity cabinet was also at room temperature hatched 1 hour, or the hatching of spending the night under 4 ℃.

4. in PBS, wash twice, each 5 minutes, careful shake.

5. carefully take out unnecessary PBS, and at room temperature, in humidity cabinet, cover section 30 minutes to 1 hour with the anti-mouse antibodies of HRP coupling rabbit that is diluted among the PBA.

6. flushing is cut into slices 2 times in PBS, and each 5 minutes, shake gently.

The scoring of immunohistochemical staining:

Veteran urological pathology man (specialist) can assess stained.With exploitation a kind of based on staining power in various degree with the methods of marking of the per-cent of cell dyeing.This assessment will be carried out in the double blinding mode.

Statistical study:

Can utilize the assessment of Fisher ' s rigorous examination and FGFR2 to express the single argument relevant with the Gleason scoring, and the clinical stage of androgen independence and progression.For all analyses, think that p＜0.05 is for statistically evident.

Embodiment 7.3: other experiment

Other experiment is included in the clinical development of AB-1 as the treatment of prostate cancer medicament, patient's selection and the effect of signature analysis.

To the biomarker express spectra that retrospective analysis and drafting have disease severity and clinical pathology mathematic(al) parameter that carries out from patient's bigger data set.

The generation of embodiment 8:FGFR2IIIc monoclonal antibody specific

40 amino acid FGFR2IIIc fragment (SEQ ID NO:84 with the dna structure coding; The amino-acid residue 314-353 of FGFR2IIIc) or synthetic FGFR2IIIc specific peptide (TCLAGNSIGISFH (SEQ ID NO:86) immunity, purifying Balb-c mouse, and make itself and KLH coupling.

In first method, merge 40 amino acid fragments (the amino-acid residue 313-352 of FGFR2 at the C-terminal of mouse IgG Fc; AAG VN TTDKE IEVL YIRNVT FEDAGEY TC LAGN SIG ISFH(SEQ ID NO:84)) as immunogen.Primary, unique residue among the letter representation isomer FGFR2 of underscore is with isomer FGFR2IIIb homology not.With 40 nucleotide sequence coded amino acid FGFR2IIIc fragment (SEQ ID NO:84; Amino-acid residue 313-352) insert from Invivogen, the carrier polyvinyl acetate (pVAC) of the improvement of San Diego CA, the result produces born of the same parents' film mating surface expression of 40 amino acid fragments in (EC) territory outward of FGFR2IIIc.The pVAC-40-amino acid carrier of improvement is used for dna immunization.It is expressed as the film anchorin.MFc-40 amino acid is as proteantigen.Its transfection CHO cell purifying from conditioned medium is secreted protein.

Following PCR design of primers for be used for clone FGFR2IIIc the 1242nd to 1361(nt) coding region of the nucleotide sequence of position:

Forward primer (comprising BamH I site): ATAGGATCCTTGCCGCCGGTGTTAAC (SEQ ID NO:103) reverse primer (comprising EcoR I site): GCGGAATTCGTGAAAGGATATCCC (SEQ ID NO:104)

In order to produce FGFR2IIIc(40aa)-mFc, with 40 amino acid fragments (amino-acid residue 314-353) in outer (EC) territory of nucleotide sequence coded FGFR2IIIc born of the same parents and mouse IgG1-Fc at the frame endomixis.Following PCR design of primers is carried out the PCR reaction for 40 amino acid to the 120bp fragment coding.Each Oligonucleolide primers sequence comprises restriction endonuclease sites and the 3-end is overhang.

The PCR primer:

Forward primer (comprising BamH I site):

ATA GGATCCTT?GCCGCCGGTGTTAAC(SEQ?ID?NO:105)

Reverse primer (comprising EcoR I site):

GCG GAATTC?GTGAAAGGATATCCC(SEQ?ID?NO:106)

In the second approach, the amino-acid residue 337-352(of FGFR2IIIc TC LAGN SIG ISFH; SEQ ID NO:86) epitope peptide and KHL coupling are as immunogen.The residue of uniqueness among the letter representation isomer FRFR2IIIc of first place and underscore, FGFR2IIIb is non-homogeneous with isomer.

In its antiserum(antisera), has immune mouse that height tires for separating of splenocyte.Splenocyte and cancer cell of bone marrow strain SP20/Ag-14(ATCC CRL-1581) merge, to produce hybridoma (Georges Kohler and Cesar Milstein1975) according to the method for having set up.The SP20/Ag-14 cell remains on the Eagle substratum of Dulbecco improvement, and in 10% the foetal calf serum.At first in nutrient solution, filter out positive hybridoma group (hybridoma populations), be used for positive being bonded at the FGFR2IIIc epitope peptide (TCLAGNSIGISFH (SEQ ID NO:86)) of standard ELISA with carrier proteins BSA coupling.Further detect positive colony, be used for being bonded to as following examples 9 described recombinant receptor albumen FGFR2IIIc-Fc.

In ELISA, antiserum(antisera) is diluted to 1:27000 from 1:1000, as shown in table 2 below.Use the combination that detects coating antigen onboard with the goat anti-mouse IgG of HRP coupling.

Table 2 has been showed the O.D value of detection signal.

Table 2. pair is from the ELISA test of the antiserum titre of epitope peptide immune mouse

In 96 well culture plates, obtain single crosses knurl clone by limiting dilution.Observe this mono-clonal at microscopically by visual inspection.Twice this process of repeated observation is until obtaining mono-clonal.

Utilize mouse antibodies isomer test kit, determine the isomer of individual monoclonal antibody according to the explanation (Sigma-Aldrich, Catalog number ISO-2) of manufacturers.

With the synthetic peptide of KLH or BSA coupling from GenScipt company, Piscataway, NJ. obtains.PCR primer and sequencing primer be from SigmaGenosys, Sigma-Aldrich, and St Louis, MO obtains.With the second antibody of horseradish peroxidase (HRP) or alkaline phosphatase (AP) coupling all available from Santa Cruz Biotechnology Inc., Santa Cruz, CA, or Sigma-Aldrich, St Louis, MO.

Conclusion: use the isomer acceptor of tire to having height (18000 times of dilutions) to produce immunoreactive epitope specificity peptide, and the antibody that the natural receptor FGFR2IIIc of epitope peptide antibody recognition produces carries out immunity to mouse.

Embodiment 9: the monoclonal antibody that binds selectively to FGFR2IIIc

Carry out the screening experiment of hybridoma, binding selectively to the mAbs of FGFR2IIIc, and the clone of elimination and isomer FGFR2IIIb cross reaction.Utilize FGFR2IIIc-Fc to carry out preliminary screening by elisa assay, with the hybridoma of selecting to be combined with the FGFR2IIIc isomer.In the ELISA screening second time, with the antibody that FGFR2IIIb-Fc identifies and eliminating is corresponding with the IIIb isomer.The human IgG1 is as negative control, with any antibody of removal with the Fc part cross reaction of human IgG.

The ELISA method is described below.(Maxisorb/NUNC, Roskilde are coated with 2 μ g/ml proteantigens in the 0.05M bicarbonate buffer (pH9.6) on Denmark), spend the night under 4 ℃ at polystyrene micropore plate.After the phosphate buffered saline (PBS) flushing that contains 0.05% polysorbas20, at room temperature plate was blocked 2 hours at 1% bovine serum albumin.The every hole of hybridoma is added 100 μ, 1 emiocytosis conditioned medium, and hatched 1 hour down at 37 ℃.Wash described hole then, utilize goat anti-mouse antibody (Santa Cruz Biotechnology, USA) the detection binding antibody of horseradish peroxidase coupling.

In another binding analysis based on ELISA, capture monoclonal antibody at the elisa plate that is applied in advance by the goat anti-mouse IgG of 5 μ g/ml.After washing or blocking step, will with the same FGFR2IIIc(128 amino acid that merges of alkaline phosphatase (people PRL3-AP))-AP and irrelevant reference protein be added on the plate, and 37 ℃ of hatchings 1 hour down.After flushing port, by adding the matrix that is used for alkaline phosphatase, pNPP(Sigma-Aldrich) detect conjugated antigen.By alkaline phosphatase and 128 amino acid FGFR2IIIc fragment (SEQ ID NO:107; The fusion in the C-terminal territory amino-acid residue 250-377 of FGFR2IIIc) and produce FGFR2IIIc(128 amino acid)-AP, and express from dna structure pATTO-FGFR2I1Ic (128aa)-AP.Template cDNA clone is from Open Biosystems(BC039243; Protein ID:AAH39243) locates to order.

The aminoacid sequence of described cloned sequence of encoding sequence of the 3rd Ig class ring that comprises FGFR2IIIc is as follows: (these 2 sites " R " and " T " are produced by the cloning site on the carrier):

RTERSPHRPI?LQAGLPANAS?TVVGGDVEFV?CKVYSDAQPH?IQWIKHVEKNGSKYGPDGLP?YLKVLKAAGV?NTTDKEIEVL?YIRNVTFEDA?GEYTCLAGNSIGISFHSAWL?TVLPAPGREK?EITASPDYLE(SEQ?ID?NO:82)

Design following PCR primer, be used for the nucleotide sequence of clones coding 128 amino acid FGFR2IIIc fragments:

Forward primer: GAGCGATCGCCTCACCGGCC (SEQ ID NO:108)

Reverse primer: CTCCAGGTAGTCTGGGGAAGCT (SEQ ID NO:109)

In another binding analysis based on ELISA, use FGFR2IIIc-Fc' and the FGFR2IIIb-Fc'(of 2mg/ml to purchase in R﹠amp; D Systems, Inc.Minneapolis, MN; Catalog number (Cat.No.): 665-FR and 684-FR) coating elisa plate (hatching of spending the night under 4 ℃).By at room temperature hatching 1 hour, the monoclonal antibody in the hybridoma conditioned medium (100ml) is bonded to the albumen of coating.(goat anti-mouse-HRP) detect combination adds matrix tetramethyl benzidine (TMB) subsequently to utilize HRP coupling second antibody.Measure the O.D. value that 450nm goes out.In 25 kinds of clones of screening, based on the selective binding of FGFR2IIIc isomer, select 4 kinds of

clones.Clone

4,9,16,21 shows the strong selective binding with FGFR2IIIc, perhaps has the combination (background level of binding) of background level, perhaps with the very weak combination of FGFR2IIIb isomer.Test is from the isomer of the monoclonal antibody of hybridoma cell clone-9 and-21, and all antibody are Muridae IgG2b.These two kinds of monoclonal antibodies are designed to Atto-MuMab-01 and Atto-MuMab-02 respectively.

Embodiment 10: the western blot analysis that is bonded to the antibody of FGFR2IIIc

Carry out western blot analysis, be bonded to the FGFR2 fusion rotein (Fc fusion rotein), FGFR2IIIc-Fc of soluble form or as purifying protein or as the specific antibody of the FGFR2IIIb-Fc of the Chinese hamster ovary celI secretory protein in the conditioned medium with detection.On 8% SDS-PAGE gel, albumen is carried out electrophoretic separation, and smear on the nitrocellulose filter (Amersham Pharmacia Biotech, Uppsala, Sweden).At 4 ℃, in phosphate buffered saline (PBS) with 5% the skimmed milk blocking-up non-specific binding of spending the night.Nitrocellulose filter and mAb Atto-MuMab-01 were at room temperature hatched 1 hour, at room temperature handled 1 hour with the goat anti-mouse IgG of horseradish peroxidase coupling subsequently.After the flushing several times, by interpolation chromogenic substrate DAB(DAKO, Carpinteria, Calif. USA) handles.

The positive control of the Fc fusion rotein of FGFR2IIIc ' and FGFR2IIIb ' (IIIb ' and IIIc ') is available from R﹠amp; D Systems, Inc.Minneapolis, MN; Catalog number (Cat.No.): 665-FR and 684-10FR.Prepare the fusion rotein of FGFR2IIIb (IIIb) and FGFR2IIIc (IIIc) by transfection CHO cell, as secretory protein.Shown in embodiment 3, by human IgG1-Fc(227 amino acid) with the amino-acid residue 1-262 of FGFR2IIIc(FGFR2IIIc) the carboxyl terminal frame endomixis in outer (EC) territory of born of the same parents produce FGFR2IIIc-Fc.Design following PCR primer, be used for clone FGFR2IIIc 64 to 786(nt) coding region of the nucleotide sequence of position.

Forward primer (comprising EcoR I site): AGAGAATTCGCGGCCCTCCTTCAGTTTAGT (SEQ ID NO:112)

Reverse primer (comprising the BglII site):

GTGAGATCTCTCCAGGTAGTCTGGGGAAGCT(SEQ?ID?NO:113)

By human IgG1-Fc(SEQ ID NO:110; 227 amino acid) with FGFR2IIIb(SEQ ID NO:114; The frame endomixis of the C-terminal in outer (EC) territory of the born of the same parents amino-acid residue 32-289 of FGFR2IIIb (SEQ ID NO:192)) produces FGFR2IIIb-Fc.Design following PCR primer, be used for clone SEQ ID NO:193 from 94 to 867(nt) coding region of the nucleotide sequence of position:

Forward primer (comprising EcoR I site): AGAGAATTCGCGGCCCTCCTTCAGTTTAGT (SEQ ID NO:115)

Reverse primer (comprising the Bgl11 site):

GTGAGATCTCTCCAGGTAGTCTGGGGAAGC(SEQ?ID?NO:116)

Be used for the dna profiling of pcr amplification of FGFR2IIIb available from Open Biosystems, Thermo Scientific, Huntsville AL (catalog number (Cat.No.) IHS1380-8840381).

Embodiment 11: the anti--FGFR2IIIc antibody that is bonded to the endogenous protein in the tumour cell

This embodiment has showed anti-FGFR2IIIC antibody of the present invention and combination as the tumour cell of DU145 and HepG2 etc.By immunoprecipitation (IP) analysis of cells lysate, carry out Western blotting then.

DU145, a kind of Human Prostate Cancer Cells strain obtains (ATCC HTB-81) from ATCC.It derives from the human prostata cancer that is transferred to brain at first.This cell strain is bred in the foetal calf serum of Eagle ' s limit minimal medium and 10%.

Analyze HepG2 by immunoprecipitation (IP), hepatoma cell strain carries out Western blotting subsequently.

In order to carry out immunoprecipitation, as follows from the fresh inoculum preparation cell lysate of DU145 cell or HepG2 cell.Harvested cell, centrifugation cell, and it is resuspended in contain 50mM Tris(pH7.5), 150mM NaCl, 1%Triton-X100 is in the lysis buffer of 10mM DTT and protease inhibitor cocktail (PMSF, aprotinin, leupeptin, pepstatin).Cell was hatched 15 minutes under 10,000g, 4 ℃ of conditions centrifugal 10 minutes then in lysis buffer on ice.This cracking supernatant liquor by with Atto-Mu mAb-01 or Atto-Mu mAb-B7(literary composition in be also referred to as " Atto-mu Mab-03 ") 4 ℃ of hatchings of spending the night, be used for immunoprecipitation.Albumen-A agarose is used for the purifying immunocomplex, and this immunocomplex is separated by SDS PAGE subsequently, analyzes by Western blotting as described below then.

In order to carry out Western blotting, by SDS-PAGE analytic sample on 8% polyacrylamide gel, subsequently electrotransfection to nitrocellulose filter (Amersham Pharmacia Biotech, Uppsala, Sweden).In phosphate buffered saline (PBS), under 4 ℃, the blocking-up non-specific binding of spending the night of the skimmed milk with 5%.Then nitrocellulose filter and Atto-Mu mAb-01 or Atto-mu Mab-03 were at room temperature hatched 1 hour, at room temperature hatched 1 hour with the goat anti-mouse IgG of horseradish peroxidase subsequently.After the flushing, according to test kit (Amersham Pharmacia Biotech) the processing trace is described, with enhanced chemiluminescence (ECL).Outcome record is on X-ray film.

Figure 32 has described western blotting, has showed mAb ATTO-mu Mab-03 and hepatoma cell strain, the combination of the endogenous FGFR2IIIc among the HepG2.Band 1 and 2: cytosol and the cell membrane component of stablizing the FGFRIIIc acceptor of CHO expression from positive control.The cytosol of band 3 and 4:HepG2 and cell membrane component.Shown in figure 32, identify the apparent molecular weight band of 85KDa by ATTO-Mu mAb-B7.Therefore, monoclonal antibody ATTO-Mu mAb-B7 is bonded to the endogenous FGFR2IIIc in the tumour cell.

As shown in figure 33, identify the apparent molecular weight band of 110KDa by mAb Atto-Mu mAb-01.50KDa and 25KDa band are respectively IgG heavy chain and light chain, from the mAb that is used for IP.

Embodiment 12: the selective binding of the IIIc isomer of mouse monoclonal antibody clone and FGFR2

Serum titer is higher than 16,000 immune mouse for merging with myeloma cell SP2/0, to produce hybridoma.After HAT selects to grow 7-10 days in the substratum, utilize the antigen coating plate, for example 128aa-mFc or 40aa-mFc screen hybridoma by ELISA.Further test is used for IIIc or IIIb soluble receptor body protein, and the isomer of FGFR2IIIc β-ECD and FGFR2IIIb β-ECD is positive wedding agent (positive binders) optionally.Utilize goat anti-mouse IgG Fc-HRP test monoclonal antibody and the combination that applies soluble receptors onboard.Figure 24 A describes the bar graph with the FGFR2IIIc clone's of appointment strong binding signal, yet it is lower or not in conjunction with active to detect clone with the FGFR2IIIb isomer.In the figure, do not remove background signal (Figure 24 B).Clone B7 is referred to herein as " Atto-Mu-Mab03 ".

Embodiment 13: by screening phage display storehouse isolating human antibodies scFv

According to front Sblattero D.et al. (2000) Nature Biotechnology18,74-

Preparation described in 80 comprises about 1 to 10 * 10 ¹⁰People's phage display storehouse of phage antibody.In order to screen the storehouse for the isomer specific antibody, have the FGFR2IIIc epi-position and insert the fusion rotein of site (epitope inserts) (as described in embodiment 1.1) fragment as the bait in the biological elution process.Fusion rotein is structured in mouse IgG Fc merges in the trunk, for example contain 128 amino acid (amino acid 235-353), FGFR2IIIc isomer ring 3 districts of 120 amino acid (mFc) or 40 amino acid (amino acid 314-353) 40aa-mFc.

Utilization is coupled to immunity pipe (Nunc, Rochester, NY) fusion rotein spends the night under 4 micrograms/ml and carries out the selection of phage antibody, blocks in 2% skimmed milk phosphate buffered saline (PBS) (MPBS), and hatches 1-2 hour with phage antibody library (blocking in MPBS equally).Flushing after the first round comprises the flushing of 5 PBS flushings and 5 PBS-0.1% polysorbas20s.By in OD5500.5, adding 1ml DH5aF wash-out bacteriophage.After the wash-out, the amplification bacterium, and be further to select step to prepare phage.Flushing subsequently is stricter, after fourth round, by the reaction of ELISA test phage antibody.Figure 31 has showed every summary sheet of taking turns the phage that is bonded to target protein of enrichment.

In order to detect antibody binding specificity, identify the positive colony that is bonded to target protein, for example 128aa(mFc with phage E LISA).Utilize two kinds of incoherent albumen, people's ovalbumin (OA) and people's ferritin (Fer) check the non-specific binding of negative control albumen.Simply, after the biological elutriation of four-wheel and screening, pick out individual phage clone, in phage E LISA, test then.Figure 25 A has described the representative result's that 8 clones in conjunction with in testing that are coated in the FGFR2IIIc-128aa on the elisa plate are detected bar graphs.Use coating onboard people's ovalbumin (OA) and people's ferritin (Fer) as negative control albumen.These clones show the positive combination to the FGFR2IIIc-128aa antigen protein, and do not detect the combination with incoherent reference protein people ovalbumin (OA) and people's ferritin (Fer).

Also can carry out phage E LISA to the individual phage clone that is bonded to target isomer acceptor FGFR2IIIc for specificity detects.Utilize the ELISA form to carry out the combination test.Utilize soluble receptors FGFR2IIIc β-ECD of 2 micrograms/ml to apply elisa plate.Opposite (counter) isomer receptor fusion protein FGFR2IIIb β-CED with same concentrations is used for the cross reaction experiment.Ovalbumin (OA) is as negative control.Figure 27 A-27B has showed the solubility scFv antibody comparing result that combination is combined with FGFR2IIIb to FGFR2IIIc.With the coating elisa plate of FGFR2IIIc-hFc(2 microgram/ml) or FGFR2IIIb-hFc(2 microgram/ml).To be bonded to coating isomer acceptor onboard by the soluble antibody that the scFv clone makes.Ovalbumin (OA) is as negative control.Induce among the clone at 30, by the ELISA binding analysis, 8 clones have showed antigen FGFR2IIIc(128aa-mFc) activity.Test these clones to the selectivity of isomer FGFR2IIIc.Shown in Figure 27 A, 5 selective binding that showed isomer IIIc among 8 clones.3 in 8 can be bonded to isomer IIIc and IIIb.

In order to determine the different clone of phage antibody clone for having the different coding sequence, utilize dna fingerprinting analysis clone.Take turns the library screening from one, by 101 clones of pattern analysis of dna fingerprinting.These clones are carried out the pcr amplification in CDR district.Utilize CL-6B Micro-Column Separation PCR product.Utilize restriction enzyme Mva1 dna digestion product.Dna fragmentation shown in Figure 26 identifies 30 kinds of individual clones by different dna fingerprintings.

These positive colonies are carried out sequential analysis.By sequence being committed to V BASE19, and the feature of utilizing the different V genes of online information tool analysis.Figure 28 has described people scFv clone 6 and clone's 8 aminoacid sequence comparison.There is underscore the position of the complementarity determining region of light chain and variable region of heavy chain (CDRs) and is labeled as VL-CDR-1 respectively, and-2 and-3, and VH-CDR-1 ,-2 and-3.Catenation sequence is added with shade.Clone 6 and clone 8 also refer to " Atto-HuMab-06 " and " Atto-HuMab-08 " respectively at this.Data show that they are the different antibody clonings that have the different coding sequence in the CDR district.

Figure 29 A-29B has described nucleotide sequence and the aminoacid sequence of the full length coding region of people scFv clone's 8 (Atto-HuMab-08) variable region of light chain and variable region of heavy chain respectively.Catenation sequence is added with shade.

Figure 29 C-29D has described nucleotide sequence and the aminoacid sequence of people scFv clone's 8 (Atto-HuMab-08) variable region of light chain respectively.The CDR sequence has underscore.

Figure 29 E-29F has described nucleotide sequence and the aminoacid sequence of people scFv clone's 8 (Atto-HuMab-08) variable region of heavy chain respectively.The CDR sequence has underscore.

Figure 30 A-30B has described nucleotide sequence and the aminoacid sequence of the full length coding region of people scFv clone's 6 (Atto-HuMab-06) variable region of light chain and variable region of heavy chain respectively.Catenation sequence has shade.

Figure 30 C-30D has described nucleotide sequence and the aminoacid sequence of people scFv clone's 6 (Atto-HuMab-06) variable region of light chain respectively.The CDR sequence has underscore.

Figure 30 E-30F has described nucleotide sequence and the aminoacid sequence of scFv clone's 6 (Atto-HuMab-06) variable region of heavy chain respectively.The CDR sequence has underscore.

Carry out the expression of solubility scFv antibody, in the intestinal bacteria system, to prepare soluble antibody.Detection is by 30 kinds of solubility expressions of independently cloning of genetic fingerprint identification, and these 30 kinds of independent clonings influence the HB2151 strain by helper phage M13 and prepare.After 0.5mM IPTG induces, produce the secretor type soluble antibody of scFv.Utilize the purification by chromatography antibody of nickel post.By immunochemistry, immunofluorescence and immunohistochemistry technique, utilize these antibody purified samples in tumour cell and tumor sample, to carry out combination research.Test in conjunction with experiment with based on the receptor activation of cell by proliferation experiment, part, study the blocking-up activity of these scFv antibody.These antibody of research are to the activity in vivo of blocking-up tumor growth, intrusion and survival rate in mouse tumor transplanted tumor model.

Embodiment 14: the heavy chain of antibody A tto-MuMab-01 and Atto-MuMab-02 and the clone of variable region of light chain

Utilize MagMAX ^TM-96Total RNA extracts test kit (Ambion) to hybridoma (10 ⁵Cell) carrying out RNA separates.Utilize Superscript ^TM111 reversed transcriptive enzymes (Invitrogen) synthesize the first chain cDNA.Use following primer to carry out the synthetic of cDNA:

Cy2b:CGACTAGTCGACCAGGGATCCAGAGTTCCAAG(SEQ?ID?NO:117)

MKVL:GCGCCGTCTAGAATTAACACTCATTCCTGTTGAA(SEQ?ID?NO:118)

Utilize following primer, by the CDR encoding sequence of PCR method amplification heavy chain (VH) and light chain (VL):

The PCR primer of VH:

VHB1:SAGGTCCAGCTGCAGCAGYYTGG(SEQ?ID?NO:119)

VHB2:GAGGTTCAGCTGCAGCAGTCTGK(SEQ?ID?NO:120)

VHF1:GAGGAAACGGTGACCGTGGT(SEQ?ID?NO:121)

VHF2:GAGGAGACTGTGAGAGTGGT(SEQ?ID?NO:122)

VHF3:GCAGAGACAGTGACCAGAGT(SEQ?ID?NO:123)

VHF4:GAGGAGACGGTGACTGAGGT(SEQ?ID?NO:124)

The PCR primer of VL:

VLB1:GATGYTKTKVTGACCCAAACTCC(SEQ?ID?NO:125)

VLB2:GATATCCAGA?TGACACAGACTAC(SEQ?ID?NO:126)

VLB3:RACATTGTGCTGACMCAATCTCC(SEQ?ID?NO:127)

VLB4:SAAAWTGTKCTCWCCCAGTCTCC(SEQ?ID?NO:128)

VLB5:GAMATCMWGATGACCCARTCTCC(SEQ?ID?NO:129)

VLB6:RRCATTGTGATGACCCAGWCTCM(SEQ?ID?NO:130)

VLB7:GATATTGTGATRACBCAGGYTGM(SEQ?ID?NO:131)

VLB8:RAMATTDTGWTGWCACAGTCTAY(SEQ?ID?NO:132)

VLB9:GACATCCAGATGACWCARTCTYC(SEQ?ID?NO:133)

VLB10:GACATCCAGATGAMMCAGTCTCC(SEQ?ID?NO:134)

VLB11:GA?Y?ATYSTGMTRACRCAGTCTCC(SEQ?ID?NO:135)

VLB12:GACATTGTGATGACTCAGTCTCC(SEQ?ID?NO:136)

VLB13:GAAACAACTGTGACCCAGTCTCC(SEQ?ID?NO:137)

VLF1:ACGTTTGATTTCCAGCTTGG(SEQ?ID?NO:138)

VLF2:ACGTTTTATTTCCAGCTTGG(SEQ?ID?NO:139)

VLF3:ACGTTTTATTTCCAACTTTG(SEQ?ID?NO:140)

VLF4:ACGTTTCAGCTCCAGCTTGG(SEQ?ID?NO:141)

PCR method is as follows: 94 ℃ of following sex change 5 minutes, subsequently 94 ℃ following 1 minute, 63 ℃ following 30 seconds, 58 ℃ following 50 seconds, 72 ℃ following 1 minute, carry out 7 and take turns, then 94 ℃ following 1 minute, 63 ℃ following 30 seconds, 72 ℃ following 1 minute, at last 72 ℃ following 5 minutes, carry out 23 and take turns.

The dna fragmentation of pcr amplification be cloned on the pUCm-T carrier (Biomatik USA, L LC, Wilmington, DE, USA).

The nucleotide sequence of Atto-MuMab-02 heavy chain (SEQ ID NO:87) and light chain (SEQ ID NO:89) variable region is showed in respectively among Figure 34 A and the 34B.Atto-MuMab-02 heavy chain (SEQ ID NO:88) and light chain (SEQ ID NO:90) amino acid sequences are showed in Figure 35.Shown in Figure 36 A and 36B, Atto-MuMab-02 heavy chain immunoglobulin (SEQ ID NO:88) or the comparison of light chain (SEQ ID NO:90) amino acid sequences with mouse immunoglobulin genes storehouse have disclosed Atto-MuMab-02 variable region of heavy chain (SEQ ID NO:88) and mouse immuning ball protein mu chain variable region (SEQ ID NO:142 respectively; Gene pool: AAA88255.1) 91% homology, and variable region of light chain (SEQ ID NO:90) and anti-Humanmachine tumour immunoglobulin light chain variable region (EQ ID NO:143; Gene pool: AA049727.1) 92% homology shows that heavy chain and the sequence of light chain from AttoMuMab-02 is new sequence.

Embodiment 15: to clone and the analysis of people's antibody A TTO-HuMAb-01 of the IIIc isomer that binds selectively to FGFR2

The people's antibody A TTO-HuMAb-01 that binds selectively to the IIIc isomer of FGFR2 clones 1(scFv-l from initial people scFv).For example, embodiment 13 has described several strand people antibody clonings, scFv-1 for example, scFv-2, scFv-6, and scFv-8.Particularly, Figure 25 A-25B and 27A-27B have described scFv-1.The high homology in light chain and heavy chain VL-CDR or VH-CDR district has been showed in the analysis of the sequence alignment between the scFv antibody cloning among this embodiment.Studies show that further scFv-1 compares scFv-2, scFv-6, and scFv-8 has similar or better bonding properties and (for example, is bonded to target acceptor as shla molecule, and be bonded to the target acceptor that is expressed in cell surface), and better stably express and constitutional features.

As shown in this embodiment, in following antibody formation, built up and tested the scFv-1 clone: phage display scFv fragment, be called in the literary composition " the scFv-1(phage) "; The escherichia coli expression secretor type, the scFv soluble antibody as purifying is called " scFv-1 (sol) " in the text; Duplex structure as human IgG1 Fc merges is called " dcFv-01 " in the text; And complete human IgG molecule (in the frame of the conventional I gG1 structure with total length heavy chain and light chain), be called " ATTO-HuMAB-01 " in the text.

The analysis of the sequence alignment between the embodiment 15.1:scFv antibody cloning

Figure 37 A-37B has described amino acid and the nucleotide sequence of the full length coding region of the variable region of light chain of people scFv-1 and variable region of heavy chain respectively.Catenation sequence is shade.Complementary determining region (CDR) sequence has underscore.

The aminoacid sequence comparison of people scFv-1 and scFv-6 is showed among Figure 38.There is underscore the position of the CDRs of light chain and variable region of heavy chain, and is marked as VL-CDR-1 respectively, and-2 and-3, and VH-CDR-1 ,-2 and-3.The result of sequence alignment (sequence alignment) has disclosed 217/252 (86%) consistence between scFv-1 and scFv-6,232/252 (92%) similarity (positives), and 1/252 (0%) space (gaps) (mark=434 (1117), expectation=8e-127, method: form permutation adjustment (Compositional matrix) adjust).

The aminoacid sequence comparison of people scFv-1 and scFv-8 is showed among Figure 39.There is underscore the position of the complementary determining region of light chain and variable region of heavy chain (CDRs), and is marked as VL-CDR-1 respectively, and-2 and-3, and VH-CDR-1 ,-2 and-3.The result of sequence alignment has disclosed 240/252 (95%) consistence between scFv-1 and scFv-8,247/252 (98%) similarity, and 0/252 (0%) space (marking=491 (1264) expectation=6e-144, method: form the permutation adjustment).

The comparison reveals in the CDR district between the scFv antibody cloning (scFv-1, scFv-6, and scFv-8) is in Figure 40.As shown in figure 40, these scFv that are bonded to FGFR2IIIc are cloned in the sequence homology that high level is shared in light chain CDRs and heavy chain CDRs district.

Therefore, the high homology in the light chain that comprises VL-CDR or VH-CDR district and heavy chain has been showed in the analysis of the sequence alignment between the scFv antibody cloning.

Embodiment 15.2: the immunocytochemistry of the Chinese hamster ovary celI of transient transfection (ICC) dyeing

Utilize the Chinese hamster ovary celI of transient expression FGFR2-IIIb or FGFR2-IIIc, by the combination of immunocytochemistry (ICC) dyeing reference antibody scFv-1 couple cell surface receptor FGFR2IIIc.Simply, Chinese hamster ovary celI or simulation transfection, or have the transfection of expressing the structure of FGFR2-IIIb or FGFR2-IIIc with the N-terminal FLAG label that is positioned at extracellular domain.FGFR2-IIIb or FGFR2-IIIc show by the ICC dyeing of using anti-FLAG antibody in the expression of cell surface.Figure 41 has described the representative image of immunocytochemistry (ICC) dyeing.As shown in figure 41, people's antibody scFv of purifying-1(sol) especially FGFR2-IIIc is expressed Chinese hamster ovary celI dyeing, and FGFR2-IIIb-is not expressed Chinese hamster ovary celI dyeing.Embodiment 15.3: the fluorescence-activated cell sorting (FACS) to the dcFv-01 that is bonded to Chinese hamster ovary celI stably express surface receptor FGFR2-IIIc is analyzed

By the scFv-1 fragment is merged to the N-terminal of human IgG1 Fc, sdFv-1 is configured to double-stranded antibody with people's antibody cloning.Gained antibody is called dcFv-01.Check that by flow cytometer dcFv-01 is active with the combination of the cell of expressing surface receptor FGFR2-IIIc.Represent the data display of dyeing pattern in Figure 42.As shown in figure 42, the Chinese hamster ovary celI of dcFv-01 stained positive, Chinese hamster ovary celI stably express total length FGFR2-IIIc acceptor (middle figure).When using anti-FGFR2 antibody (R﹠amp; (left figure) also can be observed positive staining during d system) as positive control.Further experimental results show that the dcFv-01 FGFR2-III express cell that do not dye.

Embodiment 15.4: utilize the rat prostate JEG-3 of dcFv-01 AT3B-1 to carry out immunocytochemistry (ICC) dyeing

Merge the combination of antibody, dcFv-01 and rat prostate JEG-3 AT3B-1 by ICC chromoscopy Fc.Personnel selection IgF(negative control) or the AT3B-1 cell dyeing of dcFv-01.Figure 43 has described the representative image of immunocytochemistry (ICC) dyeing.As shown in figure 43, dcFv-01 is bonded to AT3B-1 cell (right figure), the AT3B-1 cell (middle graph) and human IgG does not dye.

Embodiment 15.5: the fluorescence-activated cell sorting (FACS) to the dcFv-01 that is bonded to rat prostate cell strain AT3B-1 is analyzed

The combination that checks the rat prostate cell strain of dcFv-01 AT3B-1 by flow cytometer is active.Representative dyeing mode data is showed among Figure 44.As shown in figure 44, dcFv-01 has showed 23% positive staining to the AT3B-1 cell mass.On the contrary, the negative control of AT3B-1 cell mass (not main antibody) and irrelevant contrast (human IgG-1) dyeing is respectively 11% and 12%.

Table 3.Atto-MuMab-02, Atto-HuMab-01, the gathering of Atto-HuMab-06 and Atto-HuMab-08 sequence

Introducing by reference

All open text, patent and the accession number of mentioning in the literary composition be by with reference to introducing its full content, just as each independent open text or patent particularly, the independent reference of passing through is introduced into.

Equivalent

Although specific embodiments of the invention have been discussed, above-mentioned explanation is illustrative, and nonrestrictive.On the basis of specification sheets and following claim, it is apparent to those skilled in the art that the present invention is made a lot of changes.Scope of the present invention should by claim, with and the four corner of equivalent, specification sheets and modification limit.

Claims

1. antibody molecule, this antibody molecule is bonded to human fibroblastic growth factor acceptor 2(FGFR2) hypotype III c or its fragment, and demonstrate with people FGFR2 hypotype III b and be lower than 10% cross reactivity.

2. antibody molecule according to claim 1, it is characterized in that, described antibody molecule optionally is bonded to the exon III amino acid sequence coded of FGFR2, the amino acid that described aminoacid sequence and FGFR2-III c are about 301 to 360 (SEQ ID NO:2), amino acid (the AAGVNTTDKEI that FGFR2-III c is about 314 to 324, SEQ ID NO:4), amino acid (the YIRNVTFEDA that FGFR2-III c is about 328 to 337, SEQ ID NO:6), the amino acid that FGFR2-III C is about 350 to 353 (ISFH, SEQ ID NO:8), (TCLAGNSIGISFH (SEQ ID NO:86) is corresponding for the amino-acid residue of the about 314-353 of FGFR2 III c position (AAGVNTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFH (SEQ ID NO:84)) or about 342 to 353 amino acid of FGFR2-III c.

3. antibody molecule according to claim 2 is characterized in that, described antibody molecule optionally is bonded to one or more in the and the following: the 1st the L-Ala of SEQ ID NO:84, the 2nd L-Ala, the 4th Xie Ansuan, the 6th Threonine, the 7th Threonine, the 8th aspartic acid, the 9th Methionin, the 11st Isoleucine, the 12nd L-glutamic acid, the 13rd Xie Ansuan, the 15th tyrosine, the 16th Isoleucine, the 17th arginine, the 21st phenylalanine, the 22nd L-glutamic acid, the 28th Threonine, the 30th leucine, the 31st L-Ala, the 32nd glycine, the 34th Serine, the 37th Isoleucine, the 38th Serine, the 39th phenylalanine or the 40th hyte propylhomoserin (with AAG VN TTDKE IEVL YIRNVT FEDAGEY TC LAGN SIG ISFHThe amino-acid residue correspondence that (SEQ ID NO:84) highlights); Or the 1st Threonine of SEQ ID NO:86, the 3rd leucine, the 4th L-Ala, the 5th glycine, the 7th Serine, the 10th Isoleucine, the 11st Serine, the 12nd phenylalanine or the 13rd hyte propylhomoserin (with TC LAGN SIG ISFH; The amino-acid residue correspondence that SEQ ID NO:86 highlights).

4. antibody molecule according to claim 3, it is characterized in that, the aminoacid sequence that described antibody molecule demonstrates with people FGFR2 hypotype III b is less than 10% cross reactivity, and the aminoacid sequence of described people FGFR2 hypotype III b is selected from: about 314 to 351 the amino acid (HSGINSSNAEVLALFNVTEADAGEYICKVSNYIGQANQ of people FGFR2 hypotype III b; SEQ ID NO:56), about 314 to 328 the amino acid (HSGINSSNAEVLALF of people FGFR2 hypotype III b; SEQ ID NO:57), about 340 to 351 the amino acid (CKVSNYIGQANQ of people FGFR2 hypotype III b; SEQ ID NO:58).

5. according to claim 2 described antibody molecules, it is characterized in that described antibody molecule has one or more biological properties that are selected from and the following:

(i) described antibody molecule and monoclonal antibody Atto-MuMab-01, Atto-MuMab-02 or Atto-MuMab-03 competition combination;

(ii) described antibody molecule and human monoclonal antibodies Atto-HuMab-01, Atto-HuMab-06 or Atto-HuMab-08 competition combination;

(iii) be bonded to 314 to 353 (at least one amino-acid residues of AAGVNTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFH (SEQ ID NO:84) of FGFR2 III c; Or be bonded at least one amino-acid residue of the TCLAGNSIGISFH (SEQ ID NO:86) of people FGFR2-III c;

(iv) mainly be bonded to and be present among the people FGFR2-III c, but be not at least one amino-acid residue that is present among the people FGFR2-III b, described at least one amino-acid residue is selected from one or more in the and the following: the 1st the L-Ala of SEQ ID NO:84, the 2nd L-Ala, the 4th Xie Ansuan, the 6th Threonine, the 7th Threonine, the 8th aspartic acid, the 9th Methionin, the 11st Isoleucine, the 12nd L-glutamic acid, the 13rd Xie Ansuan, the 15th tyrosine, the 16th Isoleucine, the 17th arginine, the 21st phenylalanine, the 22nd L-glutamic acid, the 28th Threonine, the 30th leucine, the 31st L-Ala, the 32nd glycine, the 34th Serine, the 37th Isoleucine, the 38th Serine, the 39th phenylalanine or the 40th hyte propylhomoserin (corresponding to AAG VN TTDKE IEVL YIRNVT FEDAGEY TC LAGN SIG ISFHThe amino-acid residue that highlights in (SEQ ID NO:84)); Or be selected from one or more in the and the following: the 1st Threonine of SEQ ID NO:86, the 3rd leucine, the 4th L-Ala, the 5th glycine, the 7th Serine, the 10th Isoleucine, the 11st Serine, the 12nd phenylalanine or the 13rd hyte propylhomoserin (corresponding to TC LAGN SIG ISFH; The amino-acid residue that highlights among the SEQ ID NO:86);

(v) be bonded to reorganization, synthetic or natural human FGFR2-III c;

(vi) be bonded to the natural FGFR2-III c that is present on the DU145 PC-3;

(vii) showed with monoclonal antibody Atto-MuMab-01, Atto-MuMab-02 or the same or analogous FGFR2-of the being bonded to III of Atto-MuMab-03 c in conjunction with selectivity;

(viii) showed with human monoclonal antibodies Atto-HuMab-01, Atto-HuMab-06 or the same or analogous people of the being bonded to FGFR2-of Atto-HuMab-08 III c in conjunction with selectivity;

(ix) showed binding affinity with monoclonal antibody Atto-MuMab-01, Atto-MuMab-02 or the same or analogous people of the being bonded to FGFR2-of Atto-MuMab-03 III c;

(x) showed binding affinity with human monoclonal antibodies Atto-HuMab-01, Atto-HuMab-06 or the same or analogous people of the being bonded to FGFR2-of Atto-HuMab-08 III c;

(xi) showed and monoclonal antibody Atto-MuMab-01, Atto-MuMab-02 or the same or analogous binding kinetics of Atto-MuMab-03; And/or

(xii) showed and human monoclonal antibodies Atto-HuMab-01, Atto-HuMab-06 or the same or analogous binding kinetics of Atto-HuMab-08.

6. antibody molecule according to claim 5 is characterized in that, described antibody molecule is human monoclonal antibodies Atto-HuMab-01, Atto-HuMab-06 or Atto-HuMab-08.

7. antibody molecule according to claim 5, it is characterized in that, described antibody molecule has variable region of heavy chain, described variable region of heavy chain comprises and comes from Atto-MuMab-02, Atto-HuMab-01, at least one complementary determining region (CDR) of the variable region of heavy chain of Atto-HuMab-06 or Atto-HuMab-08, wherein said at least one complementary determining region contains: be showed in Figure 35 respectively, 28,29F, 30F, 37A, 38,39 or 40 aminoacid sequence, or by being showed in Figure 34 A respectively, 29E, the aminoacid sequence that the CDR of 30E or 37B is nucleotide sequence coded, or at least 95% consistent or have an aminoacid replacement with it, the aminoacid sequence that inserts or lack.

8. antibody molecule according to claim 5, it is characterized in that, described antibody molecule has variable region of light chain, described variable region of light chain comprises respectively from Atto-MuMab-02, Atto-HuMab-01, at least one complementary determining region (CDR) of the variable region of light chain of Atto-HuMab-06 or Atto-HuMab-08, wherein said at least one CDR contains: be showed in Figure 35,28,29D, 30D, 37A, 38,39 or 40 aminoacid sequence, or by being showed in Figure 34 B respectively, 29C, the aminoacid sequence that the CDR of 30C or 37B is nucleotide sequence coded, or at least 95% consistent or have an aminoacid replacement with it, the aminoacid sequence that inserts or lack.

9. antibody molecule according to claim 5, it is characterized in that, described antibody molecule comprises and comes from Atto-MuMab-02, Atto-HuMab-01, six complementary determining regions of the heavy chain of Atto-HuMab-06 or Atto-HuMab-08 and all of variable region of light chain (CDR), wherein said six complementary determining regions (CDR) contain: be showed in Figure 35 respectively, 28,29D, 29F, 30D, 30F, 37A, 38, aminoacid sequence in 39 or 40, or by being showed in Figure 34 A, 34B, 29C, 29E, 30C, the aminoacid sequence that CDR among 30E or the 37B is nucleotide sequence coded, or at least 95% consistent or have an aminoacid replacement with it, the aminoacid sequence that inserts or lack.

10. antibody molecule according to claim 5, it is characterized in that, described antibody molecule comprises from least one of variable region of heavy chain, two or three CDR, described variable region of heavy chain has the SSYAIS (SEQ ID NO:147) that is selected from following aminoacid sequence: CDR1, or the RIIPIX of CDR2 ₁G X ₂ANYAQKFQGR, wherein X ₁Be F or L, and X ₂Be T or I (SEQ ID NO:153), or the RDX of CDR3 ₁X ₂X ₃W X ₄X ₅X ₆FDX ₇, X wherein ₁Be R or P, X ₂Be W or L, X ₃Be D or L, X ₄Be N or S, X ₅Be D or disappearance, X ₆Be A or Y, and X ₇Be I or Y (SEQ ID NO:154).

11. antibody molecule according to claim 5 is characterized in that, described antibody molecule comprises from least one of variable region of light chain, two or three CDR, and described variable region of light chain has the SGSSSNIG X that is selected from following aminoacid sequence: CDR1 ₁N X ₂V X ₃(SEQ ID NO:150), wherein X ₁Be S or N, X ₂Be T or Y, and X ₃Be S or N, or the X of CDR2 ₁NN X ₂RPSG X ₃, X wherein ₁Be S or DD, X ₂Be Q or K, and X ₃Be V or I (SEQ ID NO:151), or the X of CDR3 ₁X ₂WD X ₃SLX ₄X ₅VV, wherein X ₁Be A or G, X ₂Be A or T, X ₃Be D or S, X ₄Be N or S, and X ₅Be G or A (SEQ ID NO:152).

12. antibody molecule according to claim 5 is characterized in that, described antibody molecule comprises:

(i) from variable region of heavy chain at least one, two or three CDR, described variable region of heavy chain has the SSYAIS (SEQ ID NO:147) that is selected from following aminoacid sequence: CDR1, or the RIIPIX of CDR2 ₁G X ₂ANYAQKFQGR (SEQ ID NO:153), wherein X ₁Be F or L, and X ₂Be T or I, or the RD X of CDR3 ₁X ₂X ₃W X ₄X ₅X ₆FDX ₇, X wherein ₁Be R or P, X ₂Be W or L, X ₃Be D or L, X ₄Be N or S, X ₅Be D or disappearance, X ₆Be A or Y, and X ₇Be I or Y (SEQ ID NO:154); And

(ii) from variable region of light chain at least one, two or three CDRs, described variable region of light chain have the SGSSSNIG X that is selected from following aminoacid sequence: CDR1 ₁N X ₂V X ₃, X wherein ₁Be S or N, X ₂Be T or Y, and X ₃Be S or N (SEQ ID NO:150), or the X of CDR2 ₁NN X ₂RPSG X ₃, X wherein ₁Be S or DD, X ₂Be Q or K, and X ₃Be V or I (SEQ ID NO:151), or the X of CDR3 ₁X ₂WD X ₃SLX ₄X ₅VV, wherein X ₁Be A or G, X ₂Be A or T, X ₃Be D or S, X ₄Be N or S, and X ₅Be G or A (SEQ ID NO:152).

13. antibody molecule according to claim 5 is characterized in that, described antibody molecule comprises:

(i) from three CDR of variable region of heavy chain, described variable region of heavy chain has the SSYAIS (SEQ ID NO:147) that is selected from following aminoacid sequence: CDR1, the RIIPI X of CDR2 ₁G X ₂ANYAQKFQGR, wherein X ₁Be F or L, and X ₂RD X for T or I (SEQ ID NO:153) and CDR3 ₁X ₂X ₃W X ₄X ₅X ₆FDX ₇, X wherein ₁Be R or P, X ₂Be W or L, X ₃Be D or L, X ₄Be N or S, X ₅Be D or disappearance, X ₆Be A or Y, and X ₇Be I or Y (SEQ ID NO:154); And

(ii) from three CDR of variable region of light chain, described variable region of light chain has the SGSSSNIG X that is selected from following aminoacid sequence: CDR1 ₁N X ₂V X ₃, X wherein ₁Be S or N, X ₂Be T or Y, and X ₃Be S or N (SEQ ID NO:150), the X of CDR2 ₁NN X ₂RPSG X ₃, X wherein ₁Be S or DD, X ₂Be Q or K, and X ₃X for V or I (SEQID NO:151) and CDR3 ₁X ₂WD X ₃SLX ₄X ₅VV, wherein X ₁Be A or G, X ₂Be A or T, X ₃Be D or S, X ₄Be N or S, and X ₅Be G or A (SEQ ID NO:152).

14., it is characterized in that described antibody molecule further comprises one or more light chains or the weight chain variable framework that is selected from the and the following according to each described antibody molecule among the claim 5-13:

(a) light chain or weight chain variable framework, described light chain or weight chain variable framework comprise at least 85% amino-acid residue of people's light chain of coming from the ripe antibody of people, people's embryonal system sequence or people's consensus sequence or weight chain variable framework;

(b) light chain or weight chain variable framework, described light chain or weight chain variable framework comprise 20% to 85% amino-acid residue of people's light chain of coming from the ripe antibody of people, people's embryonal system sequence or people's consensus sequence or weight chain variable framework;

(c) inhuman framework; Or

(d) thus modifiedly remove antigenicity or Cytotoxic decision a small bundle of straw, etc. for silkworms to spin cocoons on, or the humanized inhuman framework of part.

15. antibody molecule according to claim 14, it is characterized in that, described antibody molecule comprises light chain and variable region of heavy chain, described light chain and variable region of heavy chain comprise and are showed in Figure 28,29B, 29D, 29F, 30B, 30D, 30F, 37A, 38 or 39 aminoacid sequence, or with its at least 85% consistent aminoacid sequence.

16. antibody molecule according to claim 14, it is characterized in that, antibody or its Fab of described antibody molecule behaviour, people source, chimeric, camel or external generation, described Fab are selected from one or more in the and the following: Fab, F (ab') ₂, Fv, strand Fv fragment, single domain antibody, binary antibody (dAb), two valency or bi-specific antibody or its fragment, its single domain antibody variant, or camel antibody.

17. antibody molecule according to claim 14, it is characterized in that, further comprise IgG1, IgG2, IgG3 and IgG4 people's CH, or its mutant form, wherein, described mutant form increases or reduces one or more in quantity, effector cell's effect or the complementary action of Fc receptors bind, antibody glycosylation, cysteine residues.

18. antibody molecule according to claim 17 is characterized in that, described antibody molecule further comprises the people's constant region of light chain that is selected from κ or lambda light chain constant region.

19. antibody molecule according to claim 14 is characterized in that, is connected to other on the described antibody molecule function and is selected from one or more molecular entities in antibody, toxin, radioisotope, cytotoxic agent or cytostatics and the label.

20. antibody molecule according to claim 19 is characterized in that, described cytotoxic agent or cytostatics are: medicine, radio isotope, the molecule of plant, fungi or bacterial origin, proteotoxin, recombinant virus coat protein, or above every mixture.

21. antibody molecule according to claim 19 is characterized in that, wherein one or more molecular entities are selected from maytansinol, Taxan, calicheamicin, radio isotope.

22. antibody molecule according to claim 19 is characterized in that, described radio isotope is selected from α, β or gamma emitter, or β and gamma emitter.

23. antibody molecule according to claim 22 is characterized in that, wherein said radio isotope be selected from yttrium ( ⁹⁰Y), lutetium ( ¹⁷⁷Lu), actinium ( ²²⁵Ac), praseodymium, astatine ( ²¹¹At), rhenium ( ¹⁸⁶Re), bismuth ( ²¹²Bi or ²¹³Bi), rhodium ( ¹⁸⁸Rh), iodine ( ¹³¹I or ¹²⁵I), indium ( ¹¹¹In), technetium ( ⁹⁹MTc), phosphorus ( ³²P), carbon ( ¹⁴C), and tritium ( ³H) one or more in.

24. a pharmaceutical composition, described pharmaceutical composition comprise according to any one described antibody molecule among the claim 5-13 and the medicinal carrier of accepting, vehicle or stablizer.

25. pharmaceutical composition according to claim 24, it is characterized in that described pharmaceutical composition further comprises with the next item down or multinomial combination: at the antibody of FGFs (1 to 23), FGF acceptor (1 to 4), VEGF, EGF or EGF acceptor, PSMA or Her-2/neu; Be selected from cytotoxic drugs or the cytostatic medicament of ansamitocin, calicheamicin, Taxan, topoisomerase enzyme inhibitor; Perhaps be selected from IL-1,2,4,6 or 12, interferon alpha or γ, or the immunomodulator of GM-CSF,

26. an isolating nucleic acid, heavy chain of antibody or the variable region of light chain of any one described antibody molecule among this isolating nucleic acid coding claim 5-13.

27. an isolating nucleic acid, heavy chain of antibody or the variable region of light chain of the described antibody molecule of this isolating nucleic acid coding claim 14.

28. nucleic acid according to claim 26 is characterized in that, described nucleic acid comprise the antibody heavy chain variable region that is showed in Figure 29 A, 29E, 30A, 30E, 34A or 37B nucleotide sequence or with its at least 85% identical aminoacid sequence.

29. nucleic acid according to claim 26 is characterized in that, described nucleic acid comprise the antibody chain variable region that is showed in Figure 29 A, 29C, 30A, 30C, 34B or 37B nucleotide sequence or with its at least 85% identical aminoacid sequence.

30. a host cell, described host cell comprise the described nucleic acid of claim 26.

31. a host cell, described host cell comprise the described nucleic acid of claim 28.

32. an expression vector, described expression vector comprise the described nucleic acid of claim 26.

33. an expression vector, described expression vector comprise the described nucleic acid of claim 28.

34. a method for preparing antibody molecule, described method are included in and cultivate the described host cell of claim 26 under the condition that is fit to genetic expression.

35. method that antibody molecule is provided, described antibody molecule specificity is bonded to people FGFR2-III c, described method comprises provides length to be up to 60 amino acid or people FGFR2-III c specificity epitope still less, and wherein said epi-position comprises amino-acid residue (AAGVNTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFH (SEQ ID NO:84)) or the amino acid TCLAGNSIGISFH (SEQ ID NO:86) of the 314-353 position of FGFR2 III c;

Obtain the antibody molecule that specificity is bonded to people FGFR2-III c polypeptide; And

Assessment one or more in the and the following:

(a) whether specificity is bonded to people FGFR2-III c polypeptide to the assessment antibody molecule;

(b) be evaluated under the situation that has one or more people FGFR2-III c specificity epitope, whether the combination between antibody molecule and the people FGFR2-III c polypeptide descends; Or

(c) the assessment antibody molecule is in the effect that regulate to suppress aspect people FGFR2-III c polypeptide active.

36. a people FGFR2-III c specificity epitope, described epi-position comprise amino-acid residue (AAGVNTTDKEIEVLYIRNVTFEDAGEYTCLAGNSIGISFH (SEQ ID NO:84)) or the amino acid TCLAGNSIGISFH (SEQ ID NO:86) of FGFR2 III c314-353 position.

37. one kind is reduced cell growth or propagation or induces cancer or the method for tumour cell suicide, the carcinogenic hypotype of described cancer or tumor cells expression FGFR2, described method comprises: with any one described antibody molecule among the claim 5-13, with the amount contact cancer of the activity of the carcinogenic hypotype of enough reductions or tumour cell or near the cell of cancer or tumour cell, thereby reduce cell growth or the propagation of cancer or tumour cell or induce cancer or tumour cell is committed suiside.

38. according to the described method of claim 37, it is characterized in that, any one described antibody molecule is conjugation or non-conjugated form among the claim 5-13, only be combined as monotherapy or with one or more therapeutical agents, thereby the cell that kills cancer or tumour cell or reduction cancer or tumour cell is grown or propagation.

39. according to the described method of claim 37, it is characterized in that described cancer or tumour cell are from prostate gland, testis, mammary gland, pancreas, ovary, bladder, stomach and intestine, squamous cell lung carcinoma, nonsmall-cell lung cancer, thyroid carcinoma, carcinoma of endometrium, hemopoietic system or the cancer of the brain (glioblastoma multiforme).

40. comprising, a method for the treatment of or prevent primary, recurrent or the metastatic cancer of prostate gland, testis, mammary gland, pancreas, bladder, stomach and intestine, squamous cell lung carcinoma, nonsmall-cell lung cancer, thyroid carcinoma, carcinoma of endometrium, hematopoietic system cancer or brain, described method allow experimental subjects take any one described antibody molecule among the claim 5-13 of the amount that can effectively treat or prevent these cancers.

41., it is characterized in that described cancer is gland cancer or prostate cancer or carcinoma of testis according to the described method of claim 40.

42., it is characterized in that described cancer is with FGFR2-III c expresses or high expression level is relevant anti-hormone or intractable prostate cancer according to the described method of claim 40.

43., it is characterized in that described experimental subjects is for accepting the patient of one or more therapeutic modalities in prostatectomy, chemotherapy or other antineoplastons according to the described method of claim 42.

44., it is characterized in that described experimental subjects is to suffer from the patient of recurrent or metastatic prostate cancer according to the described method of claim 40.

45., it is characterized in that described experimental subjects suffers from symptom or asymptomatic anti-hormone or intractable prostate cancer are arranged according to the described method of claim 40.

46. according to the described method of claim 40; it is characterized in that; one or more cancer marks in the described experimental subjects body are in abnormal level, and described cancer mark is selected from prostate specific antigen (PSA), prostate specific membrane antigen (PSMA); prostate stem cell antigen (PSCA); androgen receptor (AR), chromogranin, synaptophysin; MIB-1, and α-formyl radical coenzyme A racemase (AMACR).

47. according to the described method of claim 40, it is characterized in that, further comprise the one or more variations in the monitoring experiment object and the following: the tumour size; FGFR2 III c level or expression; The level of the FGFR2 III c express cell that the circulation prostate gland is derived; One or more epithelial cell mark (Ep-CAM) level or expression, FGF8, stroma cell derivative factor (SDF), VEGF121, mesenchymal cell mark, PSA, PSMA, PSCA, AR, chromogranin, synaptophysin, MIB-1, AMACR, alkaline phosphatase, or serum oxyphorase; The speed that new focus occurs; The appearance of new disease related symptom; Or the size of soft mass.

48. according to the described method of claim 47, it is characterized in that, before treatment beginning, during the treatment or the key element of one or more treatments implemented the back and monitored described experimental subjects.

49. according to the described method of claim 40, it is characterized in that, comprise that further analysis is from the step of nucleic acid or the albumen of described experimental subjects, thereby assess the content of suitable concrete dosage, mode of administration, administration time, assisting therapy, or determine possible drug reaction phenotype or the genotype of experimental subjects widely.

50. according to the described method of claim 49, it is characterized in that, further comprise the part or radical prostatectomy, radiotherapy, hormonotherapy, male sex hormone ablation and cytotoxicity chemotherapy in one or more.

51. method that in vitro samples, detects the existence of the carcinogenic hypotype of FGFR2 polypeptide, comprise: (i) allowing antibody molecule and described polypeptide to take place under the interactional situation, with any one described antibody molecule contact sample (and optionally, adopting standard model) among the claim 5-13; And the formation that (ii) detects the mixture between antibody molecule and the sample (and optionally, adopting standard model).

52., it is characterized in that described sample is the tumour cell of serum, seminal fluid, urine, blood transportation according to the described method of claim 51, or from the tissue sample of surgical operation or biopsy.

53., it is characterized in that with respect to standard model, the variation in the described sample in the formation of mixture is the symbol that has carcinogenic FGFR2 hypotype in this sample according to the described method of claim 51.

54. the method for detection of the existence of the carcinogenic hypotype of FGFR2 polypeptide in the body comprises: (i) allowing that hypotype binding molecule and polypeptide take place under the interactional situation, making experimental subjects take any one described antibody molecule among the claim 5-13; And the formation that (ii) detects the mixture between hypotype binding molecule and polypeptide or the gene expression product.

55. the cancer disease in the experimental subjects body diagnosed and/or by stages and the method that described cancer treatment of diseases or progress are monitored, comprising: (i) identification suffers from the experimental subjects that maybe may suffer from the cancer disease for one kind; (ii) obtain to be subjected to the tissue of cancer sickness influence or the sample of cell; (iii) allowing that antibody molecule and isoform polypeptide take place under the interactional situation, contact described sample or control sample with any one described antibody molecule among the claim 5-13; And the formation that (iv) detects mixture, wherein, antibody molecule is the symbol of cancer disease or cancer disease stage with respect to the increase of the formation of mixture between the standard model.

56., it is characterized in that directly or indirectly with detectable material mark, described detectable material is selected from described antibody molecule: organized enzyme, prothetic group, fluorescent material, luminescent material, paramagnetic material, and radio active material according to the described method of claim 55.