CN1777622A - Modulation of the poliovirus receptor function - Google Patents

Abstract

The present invention relates to the identification, the isolation and the use of molecules interfering with the function(s) mediated by the poliovirus receptor (PVR) on cells. The molecules can be used for the treatment of cells having a metastatic potential, metastasis and cancer. Further methods are provided that are useful for identifying and isolating molecules which have the capacity to modulate PVR mediated adhesion or invasion potential of cells.

Description

The adjusting of the poliovirus receptor function

The present invention is that the CA81668 government-funded that utilizes health ministry to give is finished.Therefore, government enjoys certain right in the present invention.

Summary

The present invention relates to the molecule of poliovirus receptor (PVR) function that mediates on the interference cell evaluation, separate and uses thereof.This molecule can be used to treat cell, metastasis and the cancer of metastatic potential.The method that the present invention further provides helps to identify and separates the cell adhesion that can regulate the PVR mediation or the molecule of Invasion Potential.

Background of invention

The cell that malignant tumour comes off can migrate to new organization and form secondary tumor.The process that generates secondary tumor is called as transfer, and this is a complex process, and wherein the tumor cell transplantation site is away from primary tumor.((1986) Cancer Res.46 1-7) once proposed three step hypothesis to this transfer process: the first step is that tumour cell is by means of adhering to that cell surface receptor is realized to Liotta.And then the tumour cell that is anchored is secreted lytic enzyme or is induced the enzyme of secretory host cell degradable local host.The stromatolysis most probable occurs in highly to be located and approaches in the zone of described tumor cell surface.The 3rd step was that tumour cell shifts in the matrix district that enters via the proteolysis modification.Recently, research concentrates on the specific proteins of identifying that transfer involves, the basis that this can be used as diagnosis better or improves treatment plan.Mediate the interactional cell adhesion molecule of cell-cell or cell-matrix (CAM ' s) and be considered to participate in above-mentioned transfer process.Cell adhesion in the normal cell comprises the interaction between many cell surface proteins.Particularly, known adhesion interacts and comprises the interaction between the adhesion receptor outside described cell peripheral material and the born of the same parents.Be clear that equally day by day cell adhesion molecule finished more sophisticated functionss that may cause cell to obtain multiplication capacity and attack host tissue.The cell adhesion acceptor comprises such as the molecule of selecting albumen, calcium attachment proteins, integrin and immunoglobulin (Ig).

Furtherd investigate integrin receptor family already, and adhesion receptor α v β 3 integrins influence the tumour cell function as everyone knows, and participated in melanoma growth and transfer potentially.People such as Felding-Habermann (Clin Exp Metastasis (2002) 19, result of study 427-36) shows specific adhesion, invasion and attack and the migrate attribute of the expression of beta 2 integrin alpha v β 3 by the described tumour cell of support, thereby has promoted the metastatic phenotype in the human melanoma.Inhibition to the α v β 3 that expresses on the melanoma cells has significantly reduced the generation of shifting, and has further prolonged the survival time of animal.The ability that people's such as Haier result of study tumor cells showed adheres to extracellular matrix protein is to shift important factor (Haier et al (2002) J.Exp.Ther.Oncol.2 that forms, 237-45 and Haier et al. (1999) Br.J.of Cancer 80,1867-74).

Shift forming process and also depend on invasion by tumor cells power.What invasiveness was described is the potential of tumor cell migration, and has ignored extracellular matrix.This extracellular matrix is made up of the supramolecule aggregate of the connective tissue protein that comprises collagen protein, elastin, fibronectin, ln, glycoprotein and glycosaminoglycan.Usually growth and the composition to tissue has support and trophic function, also served as the physical barrier that can limit the normal cell migration.Although separating in the tumour enzymic digestion is having great mass of data to utilize aspect the reticular tissue, with tumour cell with less with the relevant information of the mechanism of the extracellular matrix protein mixture of penetration height complexity.

Goal of the invention

Therefore, an object of the present invention is to identify and separate can be preferably by means of the acceptor that mainly is expressed on the tumour cell, the material of the adhesion of adjusting cancer or transitional cell and/or invasion and attack behavior.

Inventor's of the present invention achievement is to have determined a kind of test design, comprises the multiple functional examination test of identifying and finally separating specific molecular, and the principal character that described specific molecular has is to regulate the adhesion and/or the invasion and attack behavior of cell.In other tests, the inventor describes the associated receptor molecule that participates in cell adhesion and invasion and attack behavior and be subjected to molecular regulation of the present invention, i.e. poliovirus receptor (PVR) and derivative and/or analogue in detail.

PVR is the transmembrane glycoprotein with N-terminus signal sequence, 3 outer immunoglobulin (Ig) (Ig) the spline structure territories of born of the same parents, membrane spaning domain and cytoplasmic tails.Also can be described to the member of specific immunoglobulins superfamily, the molecular size of this immunoglobulin superfamily is about 80kDa, its ad hoc structure comprises 3 Ig spline structure territories, particularly is an outmost V spline structure territory and subsequent two C2 spline structure territories.The optional title of PVR is CD155.

At first, PVR is found to be poliomyelitic pathogenic agent, i.e. the cell receptor of poliovirus.Studied quite particularly already interaction between poliovirus and the PVR (referring to, Koike et al. (1991) Proc.Nat.Acad.Sci.USA88 for example, 4104-08; Belnap et al. (2000) PNAS 97,73-78; Racaniello (1996) Structure 4,769-73; Racaniello (1996) Proc.Natl.Acad.Sci.USA 93,11378-81).

However, still there are a large amount of the unknowns in the physiologic function of PVR, and the biology importance of PVR has also just begun little by little to be illustrated.((2002) J.Biol.Chem.277, what 25697-700) discuss recently is that PVR/CD155 participates in having mediated the adhesion of cell and extracellular matrix molecules to people such as Solecki, and is just regulated in neuroectodermal tumors.There are two kinds of effective CD155 transcriptional in the analysis revealed of the transcriptional regulatory of CD155, and (activator-2 and nuclear are breathed the factor-1.Two kinds of transcription factors are all expressed between the growth period at CNS, and may guide expansionary CD155 to express.Exist in the overexpression explanation neuroectodermal tumors of CD155 in neuroectodermal tumors and can activate the generegulation approach that CD155 expresses once more.

People such as Mason ((2001) Gut 49, result of study 236-40) show the CD155 gene in colorectal carcinoma also by overexpression, and this overexpression starts from tumorigenic extremely in early days, and continues to late period.

People such as Lange ((2001) Virol.285,218-227) further reported relevant adherent mediation of (PRR) albumen pair cell of multiple poliomyelitis acceptor and specific C D155/ vitronectin and interacted, this interaction is seemingly necessary in the built-in true immune response of attentioning of the germinal center of secondary lymphoid tissue.

But the physiologic function of CD155/PVR is not understood as yet fully.Determine that proteic physiological role is to judge to disturb whether this protein function may be the prerequisite of the approach of treatment disease.What must sincerely remember is in physiological environment, for example refers to promptly that in the abiogenous tumour cell of patient, PVR is by overexpression.This overexpression may make the reaction difference of cell to stimulating, thereby can regulate and change physiology PVR function.

Correspondingly, a further target of the present invention provides the mode and the method for regulating the PVR function, thereby helps to understand better the effect of PVR.

In addition, another further purpose of the present invention is that development can suppress PVR mediation function, such as the pharmaceutical composition or the medicine of the adhesivity and/or the invasiveness of cell.

Summary of the invention

The present invention provides the molecule that can regulate the necessary PVR function of pair cell adhesion, transportation, invasion and attack and/or metastatic potential for the first time.These molecules are selected from little compound, oligonucleotide, peptide, oligopeptides, polypeptide, protein, antibody, antibody fragment, antiidiotypic antibody and/or biological conjugate.

One of above-mentioned molecule be characterised in that they all can be specifically be expressed in specific cells on PVR, CD155 or one or more born of the same parents of its any derivative or analogue in or ectodomain combine, described specific cells has participated in proliferative disease or imbalance, and has metastatic potential or derive from abiogenous tumour.

This specificity is in conjunction with itself and/or to the activation of mark that above-mentioned molecule connects or induce the adjusting that has started the PVR function.In other words, owing to blocking-up and/or the destruction to avtive spot among the PVR, the function of the influenced cell adhesion of its mediation, transportation or invasion and attack behavior is induced, is increased, is stablized, is strengthened, is stoped, is suppressed, is reduced and/or weakened.

In nothing that the molecule to the adhesivity that can suppress abiogenous tumour cell and invasiveness carries out partially in the screening, unexpectedly identify the specific antibody fragment that combines and have aminoacid sequence SEQ ID NO:3 or SEQ ID NO:4 with the ectodomain of PVR already, can be used as described instrumentality.

The present invention further comprises peptide, polypeptide, protein, antibody or the antibody fragment of have aminoacid sequence LWLRRD (SEQ ID NO:1) and/or WTGDFDY (SEQ ID NO:2), they all can be specifically in conjunction with the ectodomain of PVR and suppress the adhesion and/or the invasion and attack behavior of cell.

The invention further relates to the dna sequence dna (SEQ ID No.:5 or 6) that can be translated as SEQ ID NO:3 or SEQ ID NO:4, SEQ ID NO:5 or 6 dna sequence dna or homologous dna sequence dna, and have degenerated code but still can be translated as the dna sequence dna of above-mentioned aminoacid sequence.

A kind of further embodiment of the present invention relates to the nucleic acid molecule of coding any molecule of the present invention or polypeptide.

The present invention further provides the expression vector of above-mentioned any DNA sequence of encoding, and the host cell that contains this carrier.The present invention also further provides the pharmaceutical composition that comprises above-mentioned arbitrary amino acid sequence, dna sequence dna or carrier, biological conjugate or test kit.

The invention provides and can be used as medicament or medicament adjusting or suppress the PVR function and/or be used for production for treating or prevent the medicament of adhesion, transportation, invasion and attack and/or metastatic potential of abiogenous cancer cell or the above-mentioned molecule of medicine, the adhesivity of wherein said cancer cell, invasiveness and/or metastatic potential depend on PVR mediation function.

A kind of further embodiment of the present invention relates to the method for the part of identifying the adhesivity help to suppress abiogenous cancer cell or invasiveness, especially to can be in conjunction with the evaluation of the part of PVR ectodomain.The first step that this method comprises is to set up ligand library.The typical part that this library provides is little compound, oligonucleotide, peptide, oligopeptides, polypeptide, protein, antibody, antibody fragment, antiidiotypic antibody and/or biological conjugate.This library is used to screen the tumour-specific part subsequently, further checks these parts, can identify the part of having regulated described PVR function in the test that the present invention further provides.

A kind of further embodiment of the present invention provides some kinds can determine the adhesivity of abiogenous cancer cell and/or the diagnostic method of invasiveness and metastatic potential, comprise and separate abiogenous cancer cell, use described test determining the adhesivity or the invasiveness of this cell, and the result that compares with normal cell of analysis.

A kind of further embodiment of the present invention relates to the method for adhesion, transportation, invasion and attack and/or the metastatic potential for the treatment of or preventing patient's cells in vivo, and wherein this method comprises that the people to this treatment of needs uses molecule of the present invention, carrier and/or the pharmaceutical composition of significant quantity.

Detailed Description Of The Invention

For invention described herein can be understood more fully, hereinafter specifically describe and define.

Realize target of the present invention, the scheme of promptly regulating the adhesion, transportation of cell and/or invasion and attack behavior is that the feature by independent claim realizes.

The above-mentioned molecule of being differentiated by the present inventor had common characteristic already, and promptly they all can be specifically in conjunction with described PVR acceptor or its any derivative.Specificity is irrelevant with combining with physiological environment of described PVR or its any derivative in conjunction with molecule or the part of PVR of the present invention, thereby can occur on naturally occurring acceptor, separation acceptor and the post bind receptor.

Term used herein " PVR " comprises the poliovirus receptor relevant with the CD155 molecule of initial discovery.PVR used herein further comprises any member of known relevant PVR family, produce by alternative RNA splicing, or relevant homologous gene, be people PRR1, PRR2 and PRR3 gene transcription thing (Racaniello (1996) Proc.Natl.Acad.Sci.USA, Vol.93 pp 11378-81; Lange et al. (2001) Virol.285,218-227).Even complete associated receptor family described below, for example CD155, PRR1-PRR3 or their derivative, analogue or fragment still adopt abbreviation PVR.

Molecule of the present invention is in conjunction with in one or more born of the same parents of PVR or extracellular region territory or structural domain.The extracellular region of PVR is defined as the part of PVR albumen outside cytolemma, particularly is the outer amino acid ring of born of the same parents between amino acid 26 and the amino acid 854.More specifically, be V spline structure territory and 2 C2 spline structure territories.Therefore, one or more epi-positions of molecular specificity of the present invention ground identification PVR, or the epi-position of the conservative variant of listed PVR above.

Term used herein " epi-position " comprise any can the specificity binding domain-immunoglobulin or the albumen determinant of antibody fragment.The epi-position determinant is made up of amino acid, the molecule of aminosugar or the chemically reactive surface of other sugared side chain assembled such as exposing usually, and has specific Three Dimensions Structure usually, and specific charge characteristic.Should be understood that PVR is a glycoprotein, so, but aminoacid sequence on it and sugar-modified epi-position or the identification division that all is considered to the PVR extracellular region.

Molecule of the present invention is characterised in that another kind of functional definition, and promptly they all have the part of can be used as, for example be positioned at live or fixed cell on the ligand specificity in conjunction with the ability of PVR.The part of " specificity is in conjunction with PVR " described herein can be under the appointment buffer condition of embodiment with PVR bonded part.Dissociation constant between part and the PVR can be measured, for example by utilization be called as BIACORE system (referring to, for example Fivash et al.Curr OpinBiotechnol. (1998) 9,97-101), " combination specifically " is if just can be understood that to refer in standard conditions, for example 20 ℃, environmental stress and at suitable damping fluid, for example contain 20mM Tris, 100mM NaCl and 0,1mM EDTA and pH are when measuring in 7.0 the damping fluid, dissociation constant between part and the PVR is less than 10 μ M, preferably less than 1 μ m, more preferably less than 500,400,300,200,100,50,20nM, most preferably be 0,1nM-20nM.

In addition, molecule of the present invention has the physiologic function and/or the interactional ability of regulating PVR." the PVR function is regulated molecule " is the bioactive molecule that can regulate PVR, for example, be the mediation that adheres to the extracellular matrix structure of PVR express cell, the mediation that is the PVR express cell by normal adjacent or long distance tissue, organ and/or the transportation of extracellular matrix structure and/or be the mediation that the invasion and attack of PVR express cell enter normal adjacent or long distance tissue, organ or extracellular matrix structure.By measuring one or multinomial PVR index of biological activity,, can assess this to the bioactive adjusting of PVR such as PVR dependency invasiveness or PVR dependency adhesivity.These PVR indexs of biological activity can be assessed by external or in vivo test (referring to example).The active ability of molecules in inhibiting PVR is mainly by suppressing PVR inductive adhesivity, and compares with the adhesion of aggressive human cancer cell (for example sarcoma cell, breast cancer cell), thereby obtains assessment.

It is not the molecule that can be used as common protein function inhibitor that PVR function of the present invention is regulated molecule, as proteolytic enzyme, such as the denaturing agent of urea or Guanidinium hydrochloride, and heavy metal atom or with the small molecules (for example aldehyde or isocyanate) of biomolecules (lipid, protein, sugar) covalency and nonspecific reaction.The PVR function is regulated the feature of molecule-especially-be, and it regulates the ability of PVR function with specific concentrations, the function that described molecule under this specific concentrations can not suppress insulin receptor (for example, in anti-Tyrosine O-phosphate western blotting test, measure, referring to for example B.Cariou et al. (2002) J Biol Chem., 277,4845-52), or the function of acetylcholine receptor (for example, flow into determined by measuring Ca, referring to M.Montiel et al. (2001) Biochem Pharmacol.63,337-42.) or the function of B-CAM cell surface glycoprotein is (for example, by as Udani et al. (1998) J.Clin.Irnvest.101, the described method of the 2551st page of " flow cavity test " part of 2550-2558 is measured combining of hemoglobin A red corpuscle (AARBCs) and immobilization ln).Have only and same concentrations to regulate the PVR function, and other three kinds molecules that are mentioned the function of acceptor of not remarkably influenced are only the molecule that this patent adopts.

According to a kind of embodiment, described PVR function regulate molecule with ad hoc fashion in conjunction with PVR, preferred but be not exclusively in conjunction with born of the same parents' outside part of PVR, described ad hoc fashion be complete acceptor or its at least one or a plurality of active structure domain by described minute subcovering, and therefore be not useable for other part.Correspondingly, because this receptor or its active structure domain have been capped, so receptor-mediated function can't take place.

According to another embodiment, described PVR function regulate molecule with ad hoc fashion in conjunction with PVR, preferred but be not exclusively in conjunction with born of the same parents' outside part of PVR, described ad hoc fashion is that described acceptor has only one or more active structure domains by described molecule combination, and therefore can not with other ligand interaction.Correspondingly, can't induce any abiogenous receptor-mediated function.

According to another embodiment, described PVR function regulate molecule with ad hoc fashion in conjunction with PVR, preferred but be not exclusively in conjunction with born of the same parents' outside part of PVR, described ad hoc fashion is that described molecule is combined near the active structure domain, and therefore disturbs and/or destroyed the combination of other part.Correspondingly, receptor-mediated function or adjusted receptor-mediated function only takes place can't take place.

According to another embodiment, described PVR function regulate molecule with ad hoc fashion in conjunction with PVR, preferred but be not exclusively in conjunction with born of the same parents' outside part of PVR, described ad hoc fashion is that described molecule resets the ectodomain of described acceptor by combination, and therefore disturbs and/or destroyed the combination of other part.Correspondingly, receptor-mediated function or adjusted receptor-mediated function only takes place can't take place.

According to another embodiment, described PVR function regulate molecule with ad hoc fashion in conjunction with PVR, preferred but be not exclusively in conjunction with born of the same parents' outside part of PVR, described ad hoc fashion has been described molecule by decohesion one or more active structure domains of described acceptor, perhaps or even whole acceptor, and therefore disturb and/or destroyed the combination of other part.Correspondingly, can't induce receptor-mediated function or only induce adjusted receptor-mediated function.

According to another embodiment, described PVR function regulate molecule with ad hoc fashion in conjunction with PVR, preferred but be not exclusively in conjunction with born of the same parents' outside part of PVR, described ad hoc fashion is described molecule by in conjunction with activation and strengthened above-mentioned receptor-mediated function.

According to another embodiment, described PVR function regulate molecule with ad hoc fashion in conjunction with PVR, preferred but be not exclusively in conjunction with born of the same parents' outside part of PVR, described ad hoc fashion be described molecule by in conjunction with promoted molecule and described acceptor or its one or more active structure domains further combined with.In this case, receptor-mediated function is enhanced, promotes and/or delays time.

The present invention further provides by screening originally or immune library, but preferred phage display library is to identify the method for specificity in conjunction with the part in the extracellular region territory of PVR, wherein said part can influence the biological function of PVR, and such as adhesivity and invasiveness, this method comprises the steps:

A) phage library of part is contacted with cancer cell;

B) above-mentioned cancer cell and bonded part thereof are separated with the part in conjunction with this cell not;

C) remove the phage of the above-mentioned cell of non-specific binding, for example by adopting buffered detergent solution this cell of washing under described cell can the cracked condition;

D) wash-out and above-mentioned cell bonded phage; And

E) determine the identity of the part that above-mentioned wash-out bacteriophage is showed;

F) utilize biochemistry or biological test to check above-mentioned part to disturb the ability of PVR function.

The identity of having showed the phage of part that step e) obtains can be definite by following method, for example, when this part is antibody or antibody fragment, DNA to this part of encoding checks order, perhaps phage that the commodity ligand library has by the situation of grid location or numbering in, then by measuring grid position or the numbering of this phage.This grid position or numbering can show the identity of the part of described phage display.

After step d) is finished, the phage library enrichment can be in conjunction with the phage of PVR.These PVR can finally be differentiated by all multi-methods known in the art in conjunction with phage.Phage can be separated, forms single clone, can utilize the part that is labeled in the PVR protein of mark or the PVR protein, for example has at least the peptide of 7 amino acid longs to survey this phage clone in the PVR extracellular region.Be accredited as the PVR-binding substances with this probe bonded clone.Also can utilize purifying PVR protein or reorganization PVR affinity purification phage.

Alternatively, when described part for example is, when antibody or antibody fragment, can from complete enrichment storehouse, will encode this antibody or antibody fragment open reading-frame (ORF) again time cloning advance in the expression vector, this antibody or antibody fragment can be expressed among the clone of another kind of host cell subsequently, and the host cell clone of having carried the particular expression carrier can for example be passed through, the method of the relevant phage clone of above-mentioned evaluation, embodiment 2 and 3 described methods or the affinity purification method that utilizes reorganization PVR to carry out are identified, but wherein said particular expression carrier contains the coding specificity in conjunction with the antibody of PVR or the nucleic acid of antibody fragment.

A unique advantage of this method is that the accessible part that has obtained the PVR extracellular region has specific part, because initial screening step utilizes intact cell to carry out, and intact cell provides the accessible part of PVR extracellular region for the phage combination.

In another kind of preferred embodiment of the present invention, described method comprises the step that another utilizes reorganization PVR protein to screen.

This method comprises step e) and other step of change:

E) isolating phage is contacted with reorganization PVR;

F) adopt buffering stain remover and/or high level salt solution to wash this PVR; And

G) elution of bound the phage of PVR; And

H) determine the identity of the part that above-mentioned wash-out bacteriophage is showed.

In certain embodiments, being expressed in part on the above-mentioned phage comprises and is selected from scFv, dsFv, Fab ', Fab, F (ab ') ₂, one of Fv, single domain antibody (DABs) and double antibody antibody or antibody fragment, more specifically be antibody or the antibody fragment that is selected from one of scFv, dsFv, Fv, single domain antibody or double antibody, also to more specifically be antibody or the antibody fragment that is selected from one of scFv, single domain antibody or double antibody, scFv more preferably also.

The phage that helps phage display can be that all can be by the phages that cultivate to obtain, and these phages can be accepted gene engineering and can be at its surface display exogenous ligand.Smith et al. (1985) Science, 228,1315-17 discloses display technique of bacteriophage, 91/17271 display technique of bacteriophage that discloses antibody of WO.

Step c) and f) " stain remover " that adopted refer to detergent solution, preferred buffered detergent solution, and can be that concentration is 0.001-0.5%, be preferably the Tween of 0.01-0.1%." high salt " in the step f) refers to high level salt solution, preferred buffered high level salt solution, and its ionic strength is 10mM-1M, is preferably 20-500mM 50-350mM more preferably, also 80-250mM more preferably.Typical case useful negatively charged ion be, for example, chlorion, citrate ion, phosphate anion, phosphoric acid hydrogen radical ion or borate ion.Typical case useful positively charged ion be, for example, sodium ion, potassium ion, lithium ion, calcium ion or magnesium ion.

The pH that above-mentioned buffered soln typical case has is 7-8.For example, can adopt preferably contain 1-20%, more preferably contain 5-15%, also to more preferably contain the DMEM of about 10%FCS or PBS as buffer reagent.

By continuing 3-20 minute with 200-300g, preferred 5-10 minute gentle centrifugal, the separable cell that combines phage.By adopting pH is 0-2.5, is preferably 1-2.5, and more preferably the 2-100mM of 1.5-2.5 is preferably 4-50mM, and 5-20mM more preferably also will more preferably be about the glycine solution of 10mM, but wash-out combines the phage of cell and immobilization PVR simultaneously.

Can disturb or suppress the functional of described part, functional PVR of especially described antibody or antibody fragment can be as mentioned above, in vivo or obtain in the cell culture test identifying.Cell culture test comprises the test of determining the inhibition effect of part of the present invention in embodiment 8 and 9,9.1-9.3 or embodiment 11 and 12 described invasion and attack, migration or adherence test.Accompanying drawing provides the result of different tests.

This method advantageously will can be differentiated the part that has interference or suppress the PVR Functional Capability based on combining in conjunction with the screening step of cell surface and screening step based on functional examination.

In a kind of preferred embodiment, described part can suppress the biological function of PVR.For example, the biological function of PVR can be invasion and attack, adhesion, propagation or vasculogenesis.What invasion and attack or migration test were measured is the invasiveness of cell.Invasion and attack are tested, for example embodiment 8 and 9 in the application's book, 9.1 and 9.2 tests of carrying out, and what embodiment 9.3 described is migration test.Embodiment 11 and 12 in the application's book has described adherence test.

Term used herein " invasiveness " phalangeal cell migration is by other cellular layer or the migration ability by extracellular matrix.Can be by the Matrigel test assessment invasiveness described in the embodiment.The invasion and attack phalangeal cell of measuring according to the present invention arrives at the filter bottom surface between a certain incubation period.In the invasion and attack test, in the described a kind of invasion and attack test of embodiment, when the cell more than 40% arrived at the filter another side and forms bacterium colony in 6-12 hour, just abiogenous cancer cell is defined as aggressive.The control cells that only forms 5% bacterium colony in the identical time is defined as Non-Invasive.

Term used herein " adhesivity " phalangeal cell separate with its matrix of once relying growth, with unicellular (not with described solution in other cell directly contact) the solution form suspend again and cover once more its may adherent matrix on after, the ability of adhering to again.In the described test of embodiment, if the cell more than 40% is realized adhesion in 30-120 minute time period, just this cell is defined as adhesivity.In contrast, in the identical time period, only have 5% to realize that adherent cell is a control cells.

The process that term " vasculogenesis " phalangeal cell induces neovascularity to form in its vicinity.Vasculogenesis can be followed the growth of malignant tissue, thereby can be used as the characteristic of malignant cell.Malignant cell can have the characteristic that induction of vascular generates in other words.

Preferably, identify that by the inventive method cancer cell will be subjected to part, the biological function that suppresses of antibody or antibody fragment for example is such as adhesivity and invasiveness.

Term " inhibition " is defined in invasion and attack test mentioned above as the PVR function, be understood that with except not having molecule of the present invention, the negative control test that other test conditions is all identical is compared, function has reduced at least 20%, preferably 30%, more preferably 50%, also will be more preferably 60%.If the function decrement that is subjected to molecules influence of the present invention is less than 20%, more preferably less than 15%, also will be more preferably less than 10%, then this molecule is defined as having no significant effect the function of other three kinds of acceptors.

In addition, if can detect in test the adhesivity of abiogenous cancer cell and invasiveness decrement greater than 20%, preferably greater than 60%, polypeptide then of the present invention, antibody or antibody fragment have been considered to suppress the biological function of PVR.

In addition, with regard to the molecule of the genetic expression that suppresses PVR of the present invention, when measuring in the quantitative protein trace test that is being normalized to the beta tubulin level, when this molecule concentration in test is 10nM-100 μ m, preferably approximately 1 μ m, wherein the amount of PVR is to compare in the sample of two others unanimities, and one of them sample is when allowing molecules in inhibiting PVR of the present invention to express, this molecule is expressed PVR and has been reduced more than 50%, preferably more than 80%, also will be more preferably more than 90%, most preferably more than 95%.In identical test, molecule of the present invention can not reduce the amount of the beta tubulin that exists on each cell more than 20%, and this molecule can not reduce the relative level of insulin receptor and B-CAM cell surface glycoprotein more than 20%.

Utilize this method, the inventor provides the possibility of identifying and separating PVR function adjusting molecule subsequently first.In addition, by with can under the control of for example laser or light beam, derivative specific markers link to each other, the inventor can modify chosen molecule with ad hoc fashion, and this mode is that above-mentioned chosen molecule can suppress about 41%-55% (referring to Fig. 3) with the cell adhesion behavior of PVR mediation.In addition, compare with undressed cell, the cell invasion behavior of PVR mediation has been suppressed at least 25% (Fig. 2 a, 2b, 2c or 2d).

According to a kind of embodiment of the present invention, specificity is identified with the ability of regulating the PVR function in conjunction with the ability of PVR and/or isolating molecule is selected from little compound, oligonucleotide, peptide, oligopeptides, polypeptide, protein, antibody, antibody fragment, antiidiotypic antibody and/or biological conjugate because of having.

Used " the little compound " of the present invention refers to molecular weight between 50Da-10000Da, preferably between 100Da-4000Da, and the more preferably molecule between 150Da-2000Da or its physiology acceptable salt.In addition, with regard to little compound of the present invention, if this compound has reduced the adhesivity of nature invasive cancer cell more than 30%, preferably reduce more than 60%, do not influence cellular form thus, cell cycle progress (dna content by dyeing of iodate third ingot and facs analysis cell colony is determined), and do not improve the per-cent of the cell that shows apoptosis sign (being determined by the per-cent that adopts for example usually said tunnel test to measure the cell that has shown dna break) in the culture, then this compound is considered to regulate or suppressed the biological function of PVR.

" polypeptide " used herein refers to contain more than 10, preferably more than 20, most preferably more than 30, and is less than 10000, more preferably is less than 2500, most preferably is less than 1000 amino acid whose molecules.Also comprise the modification that contains low ratio or the polypeptide of alpha-non-natural amino acid.

For containing aminoacid sequence more than 10000 residues, hereinafter adopted term " protein ", for containing the aminoacid sequence that is less than 10 residues, then adopt term " peptide ".The modification that contains low ratio or the peptide or the protein of alpha-non-natural amino acid have also been comprised.

Term used herein " antibody " and " immunoglobulin (Ig) " refer to all immunology wedding agents, comprise polyclone and monoclonal antibody.Common antibody has a total core texture, i.e. two consistent light chains and two consistent heavy chains., a light chain and a corresponding connection of heavy chain, two heavy chains are connected with each other again.Light chain and heavy chain all contain repetition, the homology unit of series, and the length of per unit is about 110 amino-acid residues, and these units are folded in usually said immunoglobulin (Ig) (Ig) structural domain independently.For making anti-antigenic antibody have unlimited and high specificity, and make structure have relevant diversity, the N-end of light chain and heavy chain has comprised usually said variable (V) district, distinguishes to distinguish over constant (C) more conservative in each chain rest part.Each V district contains three and is also referred to as " complementarity-determining region " hypervariable region (CDR), because these sequences have formed a three-dimensional surface complementary surface with conjugated antigen.The CDRs of antibody determines its specificity and the part that contacts with ligands specific in the above-mentioned molecule.CDRs is the most variable part in the described molecule, thereby makes these molecules have diversity.In human IgG, they structurally are defined as amino acid 26-38 (CDR-H1), the 51-70 (CDR-L2) of amino acid 24-41 (CDR-L1), the 50-57 (CDR-L2) of light chain and 90-101 (CDR-L3) and heavy chain and 100-125 (CDR-H3) (referring to Kabat et al. (1987) 4th edn USDepartment of Health and Human Services, Public Health Service, NIH, Bethesda).The CDR district of antibody fragment can be determined by antibody fragment and described human IgG are compared by those skilled in the art, for example utilize the NCBI program that to move " Blast ", two sequences are contrasted mutually, identify the amino acid of the antibody fragment corresponding with the CDRs of human IgG.

Antibody of the present invention also may be selected from the immunoglobulin (Ig) of modification, for example one of antibody, CDR grafted antibody or the humanized antibody that generates by chemistry or recombination method, site-directed mutagenesis antibody.

In a kind of further embodiment, molecule of the present invention is an antibody fragment, particularly is single chain antibody fragments (scFv), dsFv, Fab ', Fab, F (ab ') 2, Fv, single domain antibody and/or double antibody.

Used term " antibody fragment " refers to have all fragments of the antibody molecule of antigen binding domain, and this term comprises the antibody fragment such as scFv, dsFv, Fab ', Fab, F (ab ') 2, Fv, single domain antibody (DABs), double antibody etc.It is known in the art (referring to Kabat et al. (1991) J.Immunol.147,1709-19) preparing and utilizing multiple construct and segmental technology based on antibody.

" single chain antibody fragments " (scFv) contains the variable domains (VH) of heavy chain of antibody and the variable domains (VL) of light chain, and wherein these structural domains all appear in the wall scroll polypeptide chain.Usually, the scFv polypeptide contains between VH and VL structural domain further and can make scFv form the peptide linker of antigen in conjunction with required ideal structure.

" Fv " fragment is the minimum antibody fragment that has kept complete antigen binding site." dsFv " is that Fv is stablized in curing." F (ab ') 2 " fragment is to contain two segmental divalence fragments of Fab that are connected hinge area by disulfide linkage." Fab " fragment is a Fab, contains fully and the VH and the CH1 structural domain paired light chain of heavy chain." Fab " fragment is reductive F (ab ') 2 fragments.

" single domain antibody " is only to have the antibody of protein chain that one (rather than two) only derive from a structural domain of described antibody structure (DAB).DABs has opened up a discovery, and promptly for some antibody, almost molecule combines its target antigen (Davies et al. (1996) Protein Eng.9:531-537) to the part of antibody molecule as complete.

" double antibody (diabodies) " is divalence or bi-specific antibody, VH wherein and VL structural domain are expressed on the wall scroll polypeptide chain, to such an extent as to but utilized paired joint between too short two structural domains that can not make on the same chain, thereby force the complementary structure territory pairing on these structural domains and another chain, and form two antigen binding sites (Holliger et al. (1993) Proc.Natl.Acad.Sci.USA, 90,6444-6448).

In another embodiment, molecule of the present invention is polyclone or monoclonal antibody, and in another embodiment, described antibody is behaved or humanized antibody.In another embodiment, the light chain that described antibody may comprise is one or two scFv antibody fragment of the present invention.In another embodiment, described antibody is anti-people PVR binding antibody, this antibody may be selected from the immunoglobulin (Ig) of modification, for example by the antibody or the humanized antibody of chemistry or recombination method preparation, has kept the site-directed mutagenesis antibody to the identical avidity of PVR basically.

In another embodiment, polypeptide behaviour antibody of the present invention is selected from one of IgA, IgD, IgE, IgG and IgM, and especially IgG and IgM more specifically are IgG1, IgG2a, IgG2b, IgG3, IgG4.

By utilizing suitable molecule of the present invention, especially described antibody fragment or antibody further may prepare antiidiotypic antibody of the present invention.This anti--idiotype antibody can utilize well-known hybridoma technology preparation (Kohler et al. (1975) Nature, 256:495).Antiidiotypic antibody is the antibody that can discern the unique determinant that exists on the another kind of antibody.These determinants are arranged in the hypervariable region of described antibody.Thereby this zone combines defined epitope and makes antibody have specificity just.Antiidiotypic antibody can be by with the described polypeptide of being paid close attention to, and especially described antibody fragment of being paid close attention to or antibody make animal immune and prepared.This immune animal will discern and reply the idiotypic determinant of this immune antibody, and generate the antibody of anti-these idiotypic determinants.By utilizing antiidiotypic antibody, might identify and to express other hybridoma with the specific monoclonal antibody of identical epi-position.Antiidiotypic antibody of the present invention can be used to screen antibody and check this antibody whether to have the binding specificity the same with molecule of the present invention and functional.

But it also is possible utilizing the monoclonal antibody of antiidiotype technology preparation mimic epitopes.For example, be produced the anti-idiotype monoclonal antibodies that is used for anti-first kind of monoclonal antibody and should have binding domains in the hypervariable region, this by the epi-position of described first kind of antibodies " as ".Therefore, this anti-idiotype monoclonal antibodies can be used to immunization, because this anti-idiotype monoclonal antibodies binding domains has served as antigen effectively.

In another embodiment, molecule of the present invention discriminably with another kind of protein, can with self form polymeric solid substrate (for example pearl), can further strengthen the effector molecule that maybe can change the PVR express cell or raise immunocyte and covalently or non-covalently combine and/or coupling toxic cytotoxic agent, cytostatic agent, the prodrug of being decided cell by target.All these binding substancess all are " biological conjugates " of the present invention.

Term of the present invention " biological conjugate " comprises molecule of the present invention, and antibody fragment especially of the present invention or antibody together with one or more cytotoxicity parts, utilize multiple bifunctional protein coupling agent preparation.Some examples of this reagent are the dual-function derivatives of N-succinimido 3-(2-pyridyl two sulphur)-propionic ester (SPDP), imines ester; such as dimethyl adipimide ester HC1, such as the Acibenzolar of two succinimido suberates, such as the aldehyde of glutaraldehyde, such as the double azido compound of Histidine (R-azido benzoyl base) hexanediamine, such as the dual azepine derivatives of two-(R-diazobenzene formyl radical) quadrol, such as 2; the vulcabond of 6-toluene-2,4-diisocyanate; and such as 1; 5-two fluoro-2, the dual-active fluorine cpd of 4-dinitrobenzene.The method that helps to prepare biological conjugate is well known by persons skilled in the art, specifically described Chemistry:Reactions in March ' s Advanced Organic, Mechanisms and Structure, 5th Edition, Wiley-Interscience or Bioconjugate Techniques, Ed.Greg Hermanson is among the Academic Press.

Can include, but are not limited to daunorubicin, taxol, Zorubicin, methotrexate, 5FU, vinblastine, dactinomycin, the inferior second glycosides of table mortar, cisplatin, Zorubicin, genistein and rrna inhibitor (for example Trichosanthin) or various bacteria toxin (for example Pseudomonas exotoxin, staphylococcal protein A) with the preferred cell toxic agents that biological conjugate of the present invention is connected.

In one embodiment, thereby this molecule is to regulate the molecule or the oligonucleotide of PVR function by the genetic expression that suppresses PVR, and for example antisense oligonucleotide or interference double-stranded RNA s (siRNAs) maybe can make cell generate the carrier of this siRNAs.In addition, this molecular specificity ground is in conjunction with the PVR that has expressed and suppress the PVR function relevant with adhesivity, invasiveness and/or the metastatic potential of cell.

Antisense oligonucleotide is well known in the art, be the method that reduces the genetic expression of specific gene (referring to for example, Probst J.C. (2000) Methods 22 (3): 271-281; HeasmanJ. (2002) Dev.Biol.243 (2): 209-214; Castanotto et al. (2000) Methods Enzymol.313:401-420, and Jen and Gewirtz (2000) Stem Cells.18 (5): 307-319).Recently, have found that the effective also inhibition specifically of short dsrna s has the expression of the mRNA of complementary exon sequence.This phenomenon is called as RNA and disturbs (RNAi).These double-stranded RNAs s is called as siRNAs, be siRNA s, have and contain at least 18, the collochore (this pairing double stranded region makes its target have the mRNA of complementary exon sequence surely) of preferred at least 19 Nucleotide, and has 20-25 Nucleotide, the length of preferred 21-23 Nucleotide (Elbashir et al. (2001) Nature 411,428-429).These siRNAs can be by method chemosynthesis well known in the art, and can obtain by the commercial channel (referring to for example at people's such as above-mentioned Elbashir report and Elbashiret al., (2002) Methods 26, the supplier who provides among the 199-213), perhaps transcribe acquisition (referring to Yu et al. (2002) PNAS99,6047-6052) by utilizing suitable dna profiling to carry out T7.They can be sent to pass cell type widely, and by for example, synthetic two RNA chains make their annealing and transfectional cells, wherein need to suppress the genetic expression (referring to people's such as people such as above-mentioned Elbashir and Yu report) of some gene.Elbashir et al., (2002) Methods 26,199-213 have described the concrete grammar of using siRNAs.

It is the transfection plasmid that another kind influences RNA interferential method, the siRNA sample hairpin RNA s that makes cell generate siRNAs or also work in RNAi.This can be achieved by utilizing the plasmid that justice and antisense strand are arranged that has siRNA simultaneously, this siRNA is subjected to Mammals, the control of the U6 promotor of preferred people or mouse, this promotor can make two strands transcribe in transfected cell, a part plasmid in transfected cell, anneal formation functional double-stranded siRNAs (Miyagishi andTaira (2002) Nature Biotech.19,497-500).Alternatively, the RNA by the U6 promoter transcription can be by the siRNA stem district that contains 19 base pairs and two structure ring and U by the antisense strand end _1-4The siRNA sample hairpin RNA s that the chain that 3 ' overhang links together is formed (Paul et al. (2002) Nature Biotech.19,505-508).Alternatively, RNA by the U6 promoter transcription can be short hairpin RNA s, this RNA is transcribed into little interim RNAs, the bob folder precursor that promptly contains about 70 Nucleotide, and processed advance its transcribe among the intracellular activation in the place siRNAs (Paddison et al. (2002) Genes andDevelopment 16,948-958).All these methods based on plasmid transfection all have common ground, and promptly they make cell generate siRNAs, and then have suppressed to have the gene in specific exon district, at least 18 base pairing district complementations that Nucleotide is long among this exon district and the described siRNA.Should be understood that also to have other method that can influence the existence of siRNAs in cell, can produce the ideal effect that suppresses the PVR function, and belong to category of the present invention.

In another embodiment, molecule of the present invention has been labeled detectable label.Term " mark " or " being labeled " refer to mixing of detectable label or following substances respectively, for example, by mix fluorophore-, chromophoric group-or radiolabeled amino acid, perhaps make fluorophore-, chromophoric group-or radio-labeling adhere to polypeptide, perhaps adhere to and can be contained the enzymatic activity that the detected part of fluorescently-labeled another kind of tagged molecule maybe can detect by optics or colorimetry.The example of this two step detection architecture is well-known vitamin H-avidin systems.The method that multiple labeling polypeptide and glycoprotein are arranged is known in the art, and can be employed (Lobl et al. (1988) Anal.Biochem., 170,502-511).Particularly, the example of detectable label of the present invention has radio isotope, chromophoric group, fluorophore, enzyme or radio isotope.

According to another embodiment, molecule of the present invention is labeled and/or chemically modified has and can induce mark, thereby has improved biologic activity.Inducing of this chemical labeling can be activated by temperature rising, induced with laser or other any high energy ripple.According to a kind of further embodiment of the present invention, described molecule is for example had by chemical labeling, and above-mentioned one or more chromogenic agents have improved the biologic activity of part in conjunction with chromophoric group auxiliary laser passivation (CALI).

The principle of CALI is based on photochemically reactive local initiation that can cause generating short-lived reactant, has optionally modified target molecule conversely, and has made its functionally inactive.High specific and the part (for example antibody, antibody fragment, small molecules) of unrestraint is labeled suitable chromophoric group (for example Victoria Green WPB, fluorescein, methylenum coeruleum, Yihong).Behind target molecule (for example protein) and the described ligand forming compound, adopt the light (laser or visible light) of suitable wavelength to shine this mixture to excite chromophoric group.This excites to have caused and can start the photochemical reaction that generates short-lived reactant (for example hydroxyl or hyperergy oxygen species).These reactants around it generates site among a small circle in modified described protein.Because the life-span is short, the mobilizable distance of reactant is very short.Therefore, the modification of amino-acid residue in the described protein is occurred in the place of approaching described ligand-binding site point.Described destruction is confined to the scope of 15-40 , just less than two proteinic mean distances in the cell, be about 80 , (suppose that average cytoplasmic protein concentration is 300mg/ml, the protein mean size is 50kDa) guaranteed the high spatial resolution for described process.When the binding site of described part near described proteinic critical function structural domain or within it the time, induced the modification of generation to cause described proteinic permanent deactivation.This proteinic functionally inactive is to measure in suitable reading in the test, and obtains assessment in the disease-related physiologic function category such as cell invasion, cell adhesion, cell signaling or apoptosis.Therefore, these parts can be used to regulate the effect of inhibition part.

In one embodiment, when adopting adherence test or invasion and attack tests (referring to embodiment) when testing, with people PVR bonded modified polypeptide of the present invention or biological conjugate the adhesivity and/or the invasiveness of human cancer cell reduced 20-60%, or preferred 30-55%, 40-50% or even at least 60%.

Further example is, even the modified antibodies of anti-specified protein does not show inhibit feature when not carrying out CALI, still can optionally suppress the function of this specified protein usually behind CALI.When the binding site of described part near described proteinic critical function structural domain or within it the time, these are induced the modification of generation to cause described proteinic permanent deactivation.This proteinic functionally inactive is to measure in suitable reading in the test, and obtains assessment in the disease-related physiologic function category such as cell invasion, cell adhesion, cell signaling or apoptosis.The inventor confirms that CALI can will have specificity and the part of unrestraint is converted into blocking-up reagent (referring to embodiment).

According to a kind of further embodiment of the present invention, the cell of expressing CD155, PVR or its any derivative preferably belongs to proliferative imbalance or the part of disease or the proliferative cell of participating.For example, this cell is a cancer cell, is derived from former or the cell of metastatic tumo(u)r or the cell that trace shifts.In addition, proliferative cell of the present invention is the cell with metastatic potential, for example enters or invade the cell that the ready state of remote tissue is strengthened.In another embodiment of the invention, the cell of expression PVR, CD 155 or their any derivative produces or derives from abiogenous tumour or cancer.

" producing or derive from the cell of abiogenous cancer " used herein refers to not transfected in the laboratory, transduction or the cell of crossing by the alternate manner genetic modification.This cell does not contain any artificial DNA sequence, for example is found to exist only in other species, and usually is not present in carrier or dna sequence dna in the species that abiogenous cancer cell originates.But, abiogenous cancer cell may comprise usually the sequence in the undiscovered species that are present in its source, condition is that these sequences occur because of individual esoteric sudden change, virus infection and/or chosen process in described abiogenous cancer cell source, and/or during this abiogenous cancer cell of cultured continuously, obtain.

" metastatic tumo(u)r " used herein comprises the tumour that is positioned on former the position can shifting and is positioned at the metastatic tumour at secondary position.This metastatic tumo(u)r can derive from any organ or tissue, such as intestines, liver, the chest of brain, central nervous system, lung, stomach, lower position, prostate gland, kidney or pancreas etc.

" metastatic potential " used herein refers to that tumour cell forms the ability (transfer) of new tumour at the position of the primary tumo(u)r in its source of long distance.Metastatic potential can be measured by following method, for example is about to 1 * 10 ⁶Individual injection cell advances the lateral tail vein of nude mouse, and for example injecting back 2 months tumor nodule quantity in definite lung, for example, as Huang et al (1996) Oncogene13, among the 2339-2347 the 2346th page " tumor cell injection " part or Radinsky et al. (1994) Oncogene 9:1877-1883 in the 1882nd page " generation of animal and tumour " and the described method of " to the tissue chemical analysis of calcification matrix " part.In this test, form more than 3 in lung, preferably more than 8, more preferably the clone more than 20 tumor nodules is considered to have transitivity.

" trace shift " refers to that tumour cell less than the gathering of 2mm, only can detect by Histological method usually.

According to a kind of further embodiment, proliferative cell of the present invention derives from such as following selected cancers cell type.

Preferably also preferably selected " the cancer cell type " of treatment is selected from astrocytoma according to the present invention according to the method for the invention screening, fibrosarcoma, myxosarcoma, liposarcoma, oligodendroglioma, ependymoma, medulloblastoma, original neuroectodermal tumors (PNET), chondrosarcoma, osteosarcoma, ductal pancreatic adenocarcinoma, little and maxicell adenocarcinoma of lung, chordoma, angiosarcoma, endotheliosarcoma, squamous cell carcinoma, the bronchovesicular cancer, epithelium gland cancer, and their hepatic metastases, lymphangiosarcoma, lymphangioendothelial sarcoma, hepatoma, melanoma, cholangiocarcinoma, synovioma, mesothelioma, the You Wenshi knurl, rhabdosarcoma, colorectal carcinoma, the matrix cells cancer, sweat gland carcinoma, papillary carcinoma, sebaceous carcinoma, papillary carcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, cholangiocarcinoma, choriocarcinoma, spermocytoma, embryonal carcinoma, wilms' tumor, testicular tumor, prostate gland, breast, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic tumor, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, leukemia, multiple myeloma, the WaldenstromShi macroglobulinemia, weigh and light chain disease, and uterine cervix, the gland cancer in uterus and epithelial ovarian cancer, the transitional cell carcinoma of bladder, B and t cell lymphoma (nodositas and dispersivity) plasmoma, acute and chronic leukemia, soft tissue sarcoma, leiomyosarcoma or other have the tumour cell that high PVR expresses arbitrarily.

According to a kind of further embodiment, and shown in the illustration result of a kind of test design as described in this paper and the example, isolated scFv, or had aminoacid sequence LWLRRD (SEQ ID No.:1) or WTGDFDY (SEQ ID No.:2) and specificity in conjunction with PVR and regulate the CDR of its function with aminoacid sequence SEQ ID No.:3, SEQ ID No.:4, SEQ ID No.:69 or SEQ ID No.:70.

In another embodiment of the invention, molecule of the present invention, polypeptide and/or biological conjugate be used to differentiate other can be in shaker test specificity in conjunction with the molecule of people PVR.These methods need make with reference to anti--PVR molecule or antibody fragment and contact with the target material that contains described PVR structural domain in existing under the situation of supposition competitor test-wedding agent.This contact procedure is carried out under given conditions, and this condition is fit to form mixture with reference to antibody fragment and described target material testing existing under the situation of wedding agent.Exist under the situation of test wedding agent, described is detected as the index of the specific binding activity of this test wedding agent and PVR with reference to formation mixture between antibody fragment and the target material.This screening method for example helps the screening of high-throughput ground, other antibody library or antibody fragment library, antisense oligonucleotide library or peptide and small molecules library, but characterize the molecule of other specificity with discriminated union in conjunction with PVR.Competition is measured by special test, and the tested antibody fragment in this test or other wedding agent have suppressed to combine with reference to the specificity of antibody fragment with the target material that contains described PVR structural domain basically.This is confirmable, for example by measure described with reference to antibody fragment at the supposition competitor, but promptly be fit to that the molecule in conjunction with PVR of specificity under the condition that mixture forms exists and the shortage situation under with the combining of the target material that contains the PVR structural domain.The competition of many types is known in conjunction with test and can be applied to of the present inventionly by routine that as US 4,376, the example in 110 is described.This test typically comprise utilize the target material contain the PVR structural domain (for example purifying PVR or express the antigenic clone of PVR), specificity in conjunction with the unmarked molecule of PVR and mark with reference to antibody fragment or other wedding agent.Competition suppresses to be determined with described target material bonded labelled amount by being determined under the PVR specific binding molecules existence condition.Usually, the excessive existence of PVR specific binding molecules.The PVR specific binding molecules of being differentiated by above-mentioned competition experiments (" competitive wedding agent ") comprises can be with described with reference to antibody fragment institute bonded epi-position or binding site bonded antibody, antibody fragment, peptide, antisense oligonucleotide, small molecules and other wedding agent, and with contiguous epi-position or binding site bonded PVR specific binding molecules basically with reference to epi-position that antibody fragment combines.Preferably, competitive wedding agent of the present invention is during excessive the existence, combines with the specificity of selected target material with reference to antibody fragment and suppressed at least 10%, preferably at least 25%, more preferably at least 50%, also will be more preferably 75%-90% or higher at least.Except polypeptide of the present invention, modified antibodies fragment especially of the present invention or modified antibodies, also can use can specificity natural or artificial part, peptide, antisense or other small molecules of guiding people PVR.

The invention further relates to by the recombinant technology molecule of the present invention that increases, comprise antibody fragment, more specifically is the method for scFv of the present invention, dsFv, Fv, single domain antibody or double antibody.These technology are (Skerra et al. (1993), Curr.Opin.Immunol.5,256-62 well known in the art; Chadd et al. (2001), Curr.Opin.Biotechnol.12,188-94).

For example, code book invention polypeptide, especially the nucleotide sequence of antibody fragment or antibody (gene of the SEQ ID No.:1-4 that for example encodes) can be separated and the clone advance in one or more expression vectors, this carrier can be transformed advance to be used for to express the suitable host clone of recombinant polypeptide of the present invention.The expression of gene of polypeptide of the present invention of encoding improves the output of this polypeptide, also pass through amino acid replacement, disappearance, interpolation and other modification, for example humanization is modified in the variable and constant region of (Rapley (1995) Mol.Biotechnol.3:139-154) importing antibody fragment of the present invention or antibody, and there are not critical loss binding specificity or PVR block function, thereby realized to the routine of this polypeptide modify (Skerra et al. (1993) Curr.Opin.Immunol.5,256-262).

Therefore, a kind of further embodiment of the present invention comprises the nucleotide sequence that derives from isolated molecule of the present invention.In the further embodiment of another kind, the invention provides nucleotide sequence and the homologous DNA sequence shown in SEQ IDNo.:5, SEQ ID No.:6, SEQ ID No.:71 or the SEQ ID No.:72, or have degenerated code but still can translate into dna sequence dna with the consistent basically aminoacid sequence of SEQ IDNo.:1-4 or SEQ ID No.:69 or SEQ ID No.:70." amino acid sequence identity substantially " used herein refers at least 70%, preferably at least 75%, 80%, 85%, 90%, more preferably two contrast aminoacid sequences, especially contrast in whole amino acid of CDRs and only have 5, also to more preferably only there be 3, also 1 amino acid difference will be only arranged more preferably.

The present invention further relates to protokaryon or the carrier for expression of eukaryon that contains nucleic acid of the present invention in another embodiment.This carrier for example, plasmid, phagemid or clay.These expression vectors contain at least one promotor, at least one be used for translation initiation signal, at least one nucleotide sequence of the present invention and, the signal that is used for translation termination in the prokaryotic expression carrier situation, the situation of carrier for expression of eukaryon then are preferred for Transcription Termination and are used for other signal of polyadenylation.

The prokaryotic expression carrier example that is used at expression in escherichia coli for example is, US 4,952,496 described expression vectors based on the promotor that can be discerned by the T7 RNA polymerase, the example that is used for the carrier for expression of eukaryon of expressing at yeast saccharomyces cerevisiae then for example, carrier p426Met25 or 526GAL1 (Mummberg et al. (1994) Nucl.Acids Res., 22,5767-5768), for example be used at the carrier of insect cell inner expression, EP-B1-0 127 839 or EP-B1-0 549 721 described baculovirus vectors, the carrier that is used for expressing in mammalian cell is carrier Rc/CMV and Rc/RSV or SV40-carrier for example, these carriers are normally known, and can obtain by the commercial channel.

The increase molecular biology method and the transfection host cell of above-mentioned expression vector and the method for cultivating transfected host cell, and all be that those of skill in the art know by the condition that this transformed host cell generated and obtained polypeptide of the present invention.For example, nucleic acid molecule of the present invention can be cloned into (1989) in the described expression vector of people such as Sambrook (" Molecular cloning:a laboratory manual (molecular cloning: laboratory manual) " second edition, Cold Spring Harbor Laboratory Press) by suitable manner.

The invention further relates to the host cell that contains nucleic acid of the present invention and/or carrier, particularly, host cell wherein is the microorganism such as yeast or other fungi, intestinal bacteria, Bacillus subtilus or other bacterium.This host cell can also be the cell of high eucaryon origin, such as insect cell, the insect cell that preferred virus infects, the insect cell of baculovirus infection more preferably, or such as the mammalian cell of HeLa, COS, MDCK 293-EBNA1, NS0 or hybridoma.

The present invention further provides and generated and duplicated polypeptide of the present invention, the method of antibody fragment especially of the present invention, comprise and cultivating through containing the recombinant vectors microorganism transformed of DNA of the present invention, and from substratum, reclaim corresponding polypeptide of the present invention, antibody fragment especially of the present invention or contain the fusion rotein of this antibody fragment.

According to a kind of further embodiment of the present invention, PVR can be detected in the test of having adopted the molecule, biological conjugate or polypeptide, antibody or the antibody fragment that contain the arbitrary sequence among sequence list listed SEQ ID No.:1-4 or SEQ ID No.:69 or the SEQID No.:70 in the expression of cell surface.The sample of this test is taken from the subject, and for example biopsy specimen is taken from the tissue of suffering from proliferative disease under a cloud.Usually, this sample is processed by well-known method before testing.Applicable test comprises ELISA, RIA, EIA, western blotting molecule, immunohistology dyeing etc.According to adopted test method, can enzyme, fluorophore or the described antigen of labelled with radioisotope or antibody is (referring to for example Coligan et al. (1994) Current Protocols in Immunology, John Wiley ﹠amp; Sons Inc., New York, New York; And Frye et al. (1987) Oncogene 4,1153-1157).This test especially helps to identify to utilizing the patient of the treatment susceptible that the PVR conditioning agent carries out, because shown the PVR expression from their primary tumo(u)r or the cell in metastatic tumor source.

The present invention further provides the adhering method of regulating and determining abiogenous PVR expression cancer cell.This method has been measured the adhesivity of target, and for example cell is to other material, and for example another kind of cell, virus, complex biological are learned mixture, for example adhesivity of extracellular matrix or stratum basale.This method comprises the steps:

A. making cell and PVR function regulate molecule contacts;

Cancer cell is contacted with the ECM egg white layer under the condition that is fit to its growth;

C. analyze the adhesion of this cancer cell and ECM egg white layer;

D. randomly, will adhere to the induced mark activation of described molecule, and carry out step c) once more; And

E. determine to compare step c) and optional step d with undressed cell) in the per-cent of the cell that adheres to once more.

Term used herein " ECM egg white layer " is understood that the protein soln that one deck is half-dried, can cultivate the cancer cell that contacts with this egg white layer and make this egg white layer of invasive cancer cell attachment.Thickness that should " ECM egg white layer " is between 0, and preferred 0 between the 1mm-1mm, 3mm is thick.The proteic example of ECM is the material of extracellular matrix.Particularly, ECM albumen is selected from protein collagen albumen, sense of touch albumen, nidogen, vitronectin, fibronectin and ln.More specifically, ECM albumen is selected from type i collagen Protein S, IV collagen type, fibronectin and ln.

In a kind of preferred embodiment, the disturbing molecule of step a) is the molecule of specificity in conjunction with the outer epi-position of born of the same parents of PVR, it particularly is decorating molecule of the present invention, more specifically be modified antibodies of the present invention or adorned antibody fragment, also to more specifically be adorned antibody fragment, even also will more specifically be adorned scFv, dsFv, Fv, single domain antibody or double antibody.

In the another embodiment, the step a) of described method comprises siRNA or the siRNA sample hairpin RNA of the antisense oligonucleotide that utilizes anti-PVR, anti-PVR, maybe can make cell the carrier of the siRNA sample hairpin RNA of the siRNA of anti-PVR or anti-PVR occur.Especially preferred is to utilize the siRNA of anti-PVR or siRNA sample hairpin RNA maybe can make cell the carrier of the siRNA sample hairpin RNA of the siRNAs of anti-PVR or anti-PVR occur.

In this particular of the present invention, the genetic expression of described antisense oligonucleotide molecules in inhibiting PVR.Preferably, the molecule that suppresses the genetic expression of PVR can be the antisense oligonucleotide of anti-PVR, be commonly called the interference double-stranded RNA of the anti-PVR of siRNA (siRNA), as the siRNA sample hairpin RNA s of the anti-PVR function of gene expression inhibitor, maybe can make cell the carrier of the siRNA sample hairpin RNA of the siRNA of anti-PVR or anti-PVR occur.

In another kind of preferred embodiment, step d) is introduced in the described method, will be attached to the induced mark activation of PVR specific binding molecules of the present invention.If, for example connect and be labeled as chromophoric group, then may destroy the active structure domain that closes on of PVR, thereby and improve functional adjusting of PVR and/or inhibition by the activation of aforementioned CALI.Show only have the functional ability of appropriate regulation PVR before, for example only have the inhibition ability or even the molecule that only has a PVR binding ability can therefore become the potential conditioning agent of height or the inhibitor of PVR function.This method can quantize adhering adjusting or inhibition to this cancer cell by cancer cell is compared with not cell that activated molecule combine and/or undressed cell.

Thought already that the adhesion interaction between tumour cell and host cell or the extracellular matrix was the basic steps that shifts in the forming process.Therefore, utilizing the adhesion blocker is the method that is hopeful to stop transfer to the interference of tumor cell adhesion potential.Achievement of the present invention not only is to provide and can combines with PVR also as Lange et al. ((2000) Virology, 285,218-227) the described molecule that may have the inhibition ability, also be by complicated and combination screening, selection and optional induction method evaluation, separate and the molecule that can regulate or destroy the PVR function on the specific cells basically be provided, this specific cells derives from proliferative imbalance or disease and/or abiogenous cancer.The preferred adjusting is inhibition to the behavior of tumour associated adhesion, and further preferred the adjusting is that active structure domain by destroying PVR is to the prevention of tumour associated adhesion behavior.

But, the present invention also provides the activation of described adhesion behavior or enhancing, for example strengthens to selected immunocyte, such as the adhesion behavior of natural killer cell or specific synthetic ingredient, described specific synthetic ingredient is passable, for example extracts described tumour cell in hemodialysis.In a word, the invention provides can be by combining with active structure domain or with its destruction, effectively stops the tumour PVR function of being correlated with, i.e. the molecule of the adhesion behavior of cancer cell or through modifying or the molecule of mark for example.

Tumor cell migration enters and organizes also is important step in the transfer process.Can utilize stride this invasive procedure of endothelium model research (referring to Woodward et al. (2002) InyestOphthalmol Vis Sci 43,1708-14 and Vachula et al. (1992) Invasion Metastasis 12,66-81).Similarly stride the endothelium model in vitro tests method that further helps to identify above-mentioned molecule and the molecule that screens the invasive procedure that can regulate or suppress the PVR mediation is provided.

Therefore, the present invention further provides the method for regulating and determining the invasiveness of abiogenous PVR expression cancer cell.This method comprises the steps:

A. making described cell and PVR function regulate molecule contacts;

Described cancer cell is contacted with gel-like matrix under the condition that is fit to its growth; And

C. analyze of the migration of this cancer cell by described gel formation matrix;

D. randomly, step c) is also carried out in the induced mark activation that will adhere to described molecule once more; And

E. determine to compare step c) and optional step d with undressed cell) the middle cell per-cent that moves.

Term used herein " gel-like matrix " refers to the semisolid of water content at least 90%, allow to cultivate the cancer cell that contacts with this matrix, and allow the invasive cancer cell migration by 0,1mm-1mm, preferred 0, " gel-like matrix " layer of 3mm thickness, but do not allow the Non-Invasive cell migration.The example of this " gel-like matrix " be with protein in extracellular matrix and the similar material of carbohydrate composition, particularly be by commercial channel obtainable " Matrigel ".Particularly, should " gel-like matrix " contain the protein that is selected from one of protein IV collagen type, fibronectin and ln.More specifically, this gel-like matrix contains protein IV collagen type, fibronectin and ln.Preferred gel-like matrix contains protein IV collagen type, ln, sense of touch albumen, nidogen and heparan sulfate proteoglycan or IV collagen type, fibronectin, ln, nidogen, sense of touch albumen and vitronectin.

In a kind of preferred embodiment, the disturbing molecule of step a) is the molecule of specificity in conjunction with the outer epi-position of born of the same parents of PVR, it particularly is modified polypeptide of the present invention, more specifically be modified antibodies of the present invention or antibody fragment through modifying, also will more specifically be the antibody fragment through modifying, also will more specifically be scFv, dsFv, Fv, single domain antibody or double antibody through modifying.

In another kind of preferred embodiment, the step a) of described method comprises that the siRNA of the antisense oligonucleotide that utilizes anti-PVR, anti-PVR or siRNA sample hairpin RNA maybe can make cell the carrier of the siRNA sample hairpin RNA of the siRNA of above-mentioned anti-PVR or anti-PVR occur.

In another kind of preferred embodiment, step d) is introduced in the described method, will adhere to the induced mark activation of PVR specific binding molecules of the present invention.If, for example connect and be labeled as chromophoric group, then may destroy the contiguous active structure domain of PVR, thereby and strengthen functional adjusting of PVR and/or inhibition by the activation of CALI hereinafter described.Show only have the functional ability of appropriate regulation PVR before, for example only have the ability that suppresses a kind of function and do not suppress other function or even the molecule that only has a PVR binding ability can therefore become the potential conditioning agent of height or the inhibitor of PVR function.This method can quantize adjusting or inhibition to the invasiveness of this cancer cell by cancer cell is compared with not cell that activated molecule combine and/or undressed cell.The adjusting of the invasion and attack behavior to cancer cell of the present invention or suppress greater than 20%, greater than 40%, greater than 60% or even be preferred greater than 80% molecule.

Described method also can be selected the molecule the most of the present invention of suitable individual patient, to avoid any individual mismatch that can the interference treatment method.

The vasculogenesis test determination the angiopoietic ability of cell induction, this vascularization is a process of following malignant tissue growth usually.The vasculogenesis test can be as above-mentioned Kanda et al (2002) J.Natl.Cancer Inst.94, and the described method of 1311-9 is carried out.

This test is included in the method for identifying suitable molecule of the present invention on the one hand, this suitable molecule except that with also can regulate its function PVR combines.But, it also is very useful that these methods are separated with the method for identifying molecule of the present invention, because they provide the instrument with high selectivity, can screen the cancer cell (for example by examination of living tissue) in patient source, to select suitable molecule of the present invention, this molecule and patient's individual PVR reaction is best, thereby the adjusting that shows the cancer cell that this patient is originated is the most effective.Preferably, described patient should be originated adhesion, transportation or the invasion and attack behavior of tumour cell of the molecule of identifying in the in vitro tests that is used for successive treatment suppresses about 20%, preferred 40%, 50%, 60%, 80% or up to 90%.Therefore, successive treatment can pass through specific adjusted, to avoid in the individual deviation aspect the receptor-binding activity.

In making a kind of embodiment, the present invention relates to a kind of pharmaceutical composition, this pharmaceutical composition contains at least a of significant quantity, but be not defined as a kind of molecule of the present invention, it particularly is the little compound that to regulate the PVR function, antibody, antibody fragment or biological conjugate, or at least a molecule that suppresses the genetic expression of PVR, more specifically, molecule wherein is the antisense oligonucleotide of anti-PVR, the interference dsRNA of anti-PVR (siRNA) or siRNA sample hairpin RNA maybe can make cell the carrier of the siRNA sample hairpin RNA of the siRNA of anti-PVR or anti-PVR occur, and this pharmaceutical composition also makes up and contains pharmaceutical acceptable carrier and/or thinner.PVR function inhibition molecule also can be used as single active compound and is used as medicine or therapeutic composition.

" treatment significant quantity " refers to eliminate or to alleviate patient's tumor load, maybe can prevent, postpones or suppress the amount that shifts.This dosage should depend on many parameters, comprises character, medical history, patient's situation, the shared cytotoxic agent and the medication of possibility of tumour.Medication comprises injection (for example parenteral, subcutaneous, intravenously, intraperitoneal etc.), and with regard to this method, it is to be provided in the nontoxic pharmaceutical acceptable carrier that the PVR function suppresses molecule.

" pharmaceutical acceptable carrier " is often referred to suitable carrier and the thinner of selecting for the biologic activity that does not significantly weaken wedding agent (for example binding specificity, affinity or stability), such as water, salt solution, Ringer's solution, glucose solution, 5% human serum albumin, fixed oil, ethyl oleate or liposome.Can accept carrier may comprise the inertia of biocompatible or biology can absorb salt, buffer reagent, widow or polysaccharide, polymkeric substance, such as hyaluronic viscoelastic compound, viscous modifier, sanitas etc.In addition, pharmaceutical composition or prescription also can comprise the stablizer, thinner etc. of other carrier, adjuvant or nontoxic, no therapeutic, non-immunogenicity.The scope of typical doses can be about 0.01 to about 20mg/kg or more specifically be about 1 to about 10mg/kg.

Pharmaceutical composition can be used to treat and people PVR overexpression or the relevant illness of unconventionality expression, especially treats tumour, has the tumour that metastatic potential and/or trace shift, and especially derives from the metastatic tumo(u)r of above-mentioned cancer cell type.

In another embodiment, the present invention relates to intravital any proliferative imbalance of treatment or prevention patient or disease, the method of former or secondary tumors and transfer, this method comprises uses the PVR function inhibition molecule that pharmacy can be accepted contained specified quantitative in the composition, this amount can effectively be treated, promptly suppress, postpone or the adhesion of prevention PVR mediation and/or the transfer of invasion and attack, particularly, the genetic expression of molecules in inhibiting PVR wherein, more specifically, molecule wherein is the antisense oligonucleotide of anti-PVR, the interference dsRNA of anti-PVR (siRNA) or siRNA sample hairpin RNA maybe can make cell the carrier of the siRNA sample hairpin RNA of the siRNA of anti-PVR or anti-PVR occur.Alternatively, particularly, it can be can be in conjunction with the molecule of PVR extracellular region that the PVR function suppresses molecule, can be tested and appraised specificity is identified in conjunction with the method for the part of PVR extracellular region, more specifically, molecule wherein is little compound of the present invention or antibody or antibody fragment or modified polypeptide, or biological conjugate of the present invention, also will be more preferably wherein molecule be scFv or the modified antibodies that derives from this scFv of the present invention through modifying of the present invention.In addition, this molecule can be by chromophoric group, fluorophore, enzyme or labelled with radioisotope, can be by for example using chemical inducer or being exposed to the photochemistry activator and being induced in the body or activate.Treatment or the imbalance of prevention proliferative, cancer, metastatic tumo(u)r, trace transfer and/or the method that shifts can effectively reduce or suppress the adhesion and/or the invasion and attack of abiogenous cancer cell in the particular cancers cell, described particular cancers cell derives from the metastatic tumo(u)r of above-mentioned selected cancers cell type, because this method shows adhesivity and/or the invasiveness that the blocking-up of PVR function has been suppressed the cancer cell in above-mentioned selection type source.In a kind of preferred embodiment, treatments that the antibody fragment that adopts one or more processes to modify carries out the patient who has abiogenous PVR expression cancer cell according to the present invention cause or have caused the adhesion of people's sarcoma cell and/or the reduction or the inhibition of invasive ability.

" treatment metastatic tumo(u)r " used herein or " treatment trace shift " refer to molecule of the present invention with the single medicine form or with the other medicines applied in any combination, stablized, stoped, postponed or suppressed to have the cell of metastatic potential or the transfer of tumour.Stable disease or " no change " (NC) are described be at least 4 week the back do not have to shift and change or alleviate degree less than 50% or increase the weight of degree less than 25% the course of disease.Prevention for example can refer to, does not detect new transfer after the treatment beginning.This can make the patient through treatment compare with untreated patient, has had 2-3 times median and/or the survival time in 5 years.Delay can refer to after treatment beginning at least 8 weeks, 3 months, 6 months or even year in do not detect new transfer.Inhibition can refer to compare with undressed test group, has reduced at least 30%, 40%, 50%, 60%, 70%, 80% or even 90% via the mean sizes or the total quantity of the new transfer of the test group of molecular therapy of the present invention.The skilled practitioners of oncology is followed received transfer and is detected practice and diagnostic method, the method summarized of Harrisons Principles of Internal Medicine 15th ed2001 Mc Graw Hill for example can detect quantity, size and the prevalence rate of transfer.

According to a kind of further embodiment, methods of treatment of the present invention can make up with multiple therapy methods.In this article, the combination of described treatment and term " methods of treatment " refers to according to tumor type, patient's situation, other health problem and multiple factor, will utilize the PVR function to suppress methods of treatment and chemotherapy, surgery and radiotherapy applied in any combination that molecule carries out.

Therefore, a kind of embodiment of the present invention be utilize at least a can be in conjunction with the molecule of PVR extracellular region, this molecule can be tested and appraised specificity and be identified in conjunction with the method for the part of PVR extracellular region, more specifically, molecule wherein is little compound of the present invention or antibody or antibody fragment or modified polypeptide, or biological conjugate of the present invention, also will be more preferably, molecule wherein is scFv or the modified antibodies that derives from this scFv of the present invention through modifying of the present invention, and tagged molecule of the present invention, this tagged molecule can be induced in the body or be activated by using or use specific inductor, described specific inductor can be used for producing can treat or prevent the adhesion of abiogenous cancer cell, the medicine of invasion and attack and/or any metastatic potential, the adhesivity of wherein said cancer cell, invasiveness and/or metastatic potential are subjected to the influence of PVR function.

The present invention thereby the method for pharmaceutical compositions or medicine is provided comprises molecule of the present invention or part is mixed step in treatment, prevention or the diagnosis composition.This can be by for example, fixed part or modified ligand are mixed with pharmaceutical acceptable carrier known in the art and is achieved, and wherein the content of part is to treat significant quantity.

In another embodiment, present invention includes diagnostic kit.This test kit comprises at least a biological conjugate of the present invention and/or at least a tagged molecule or polypeptide and/or antibody fragment of the present invention or antibody, or their mark pattern, comprise in addition and carry out standard competition or necessary reagent of sandwich test and material.This diagnostic kit can be used to measure biological sample, especially the Invasion Potential of the biological sample of some cancer cell type.Test kit typically further comprises container.

The following example that comprises test and the result that obtains only is used for illustration, and the present invention is not construed as limiting.

The accompanying drawing summary

Figure 1 shows that the 8 μ m filters of painted HT1080 cell invasion by Matrigel bag quilt.37 ℃. incubation is quantitative fluorescence after 6 hours.Shown in data be n=3 hole mean value+/-SD.

Fig. 2 a is depicted as the restraining effect of scFv1 to the HT1080 cell invasion.Invasion and attack are to measure in the chemotaxis test that carry out in the cell migration pond that utilizes Matrigel bag quilt.These invasion and attack are measured in laser radiation (carrying out CALI) back.The HT1080 cell is used as contrast (left post) in the invasion and attack that lack under all inhibition molecule conditions.Molecule scFv1 has suppressed about 22% (p-value＜0.05) with the invasion and attack of HT1080 cell

Fig. 2 b is depicted as the anti-PVR antibody of commodity (D171), scFv3 and the scFv4 restraining effect to the HT1080 cell invasion.Invasion and attack are to measure in the chemotaxis test that carry out in the cell migration pond that utilizes Matrigel bag quilt.(black post represents not carry out CALI, and gray columns represents to have carried out CALI) measured in these invasion and attack before rayed and afterwards.The HT1080 cell is used as contrast (left post) in the invasion and attack that lack under all inhibition molecule (0) conditions.Molecule scFv3 and scFv4 have suppressed about 30% (p-value＜0.001) with the invasion and attack of HT1080 cell

Fig. 2 c is depicted as scFv3 and the scFv4 restraining effect to the MDA-MD231 cell invasion.Invasion and attack are to measure in the chemotaxis test that carry out in the cell migration pond that utilizes Matrigel bag quilt.(black post represents not carry out CALI, and gray columns represents to have carried out CALI) measured in these invasion and attack before rayed and afterwards.The HT1080 cell is used as contrast (left post) in the invasion and attack that lack under all inhibition molecule (0) conditions.ScFv3 and scFv4 have suppressed about 23% (p-value＜0.001) with the invasion and attack of MDA-MD231 cell

Fig. 2 d is depicted as scFv3* and the restraining effect of scFv4* to two kinds of different glioblastoma clones (U87MG cell=left side post group, A172=right side post group) migration.(black post represents not carry out CALI, and gray columns represents to have carried out CALI) measured in migration before rayed and afterwards.Cell is used as contrast (the left post of each group) in the migrations that lack under all inhibition molecule (contrast) conditions.Symbol " * " expression is cloned into the IgG form by Figure 10 with each scFv that numbering shown in Figure 11 is distinguished.

Figure 3 shows that scFv1 and scFv2 restraining effect to HT1080 cell adhesion type i collagen Protein S.This adhesion is measured in laser radiation (carrying out CALI) back.The HT1080 cell is used as contrast (left post) in the adhesions that lack under all inhibition molecule conditions.Molecule scFv1 with this adherence inhibition about 56%, molecule scFv2 has then suppressed 42% (p-value＜0.05).

Figure 4 shows that the facs analysis result.Illustration scFv1 and scFv2 the activity that combine with HT1080 cell (thick line) and contrast HS-27 cell (dotted line).Also illustration scFv1 and PC3-cell, KHOS-cell and hepatocellular the combination.

Figure 5 shows that the result of immunoprecipitation test.ScFv1 and scFv2 are with the split product incubation of HT1080 cell and contrast HS-27 cell.By SDS-PAGE and silver dyeing separating immune mixture.Diagram has been pointed out scFv band and PVR specific band.

Figure 6 shows that the MALDI-MS collection of illustrative plates of the peptide mixt that obtains from the band that utilizes the about 75kDa size of scFv1 immunoprecipitation gained.Two trypsinase autodigestion peaks that are denoted as T are used to internal calibration.Amount to 6 peaks that are labeled with asterisk and mate with PVR that (SwissProt, P15151), mass deviation is less than 10ppm.The coupling peptide accounts for described proteinic 18% (74/417 residue).

Figure 7 shows that carrier figure and the corresponding sequence (SEQ ID No.:7) of scFv display carrier pXP10

Figure 8 shows that carrier figure and the corresponding sequence (SEQ ID NO.:8) of scFv expression vector pXP14

Figure 9 shows that aminoacid sequence (SEQ ID NO.:3) and the nucleotide sequence (SEQ ID NO.:5) of scFv1

Figure 10 shows that aminoacid sequence (SEQ ID NO.:4) and the nucleotide sequence (SEQ ID NO.:6) of scFv2

Figure 11 shows that aminoacid sequence (SEQ ID NO.:69) and the nucleotide sequence (SEQ ID NO.:71) of scFv3

Figure 12 shows that aminoacid sequence (SEQ ID NO.:70) and the nucleotide sequence (SEQ ID NO.:72) of scFv4

Figure 13 shows that the principle of chromophoric group auxiliary laser passivation (CALI)

Figure 14 shows that the sequence of primer SEQ ID No.:9-52,63-68.

Embodiment

Embodiment 1: the structure in immune library

Adopt 2 * 10 ⁷Individual Paraformaldehyde 96 fixed HT1080 cell (human fibrosarcoma cell system; ATCC, CCL-121) two BALB/c mouse of intradermal immunization inoculation.Behind the initial immunity, duplicate injection is 2 times in 39 days, puts to death mouse, and separating spleen also is chilled in the liquid nitrogen.

From two parts of spleen prepared products, respectively take out half, utilize RNeasy Midi test kit (QIAGEN #75142) and separate total RNA with reference to the described method of manufacturer.RNA concentration and purity are determined by sex change formaldehyde gel and photometry.

Utilize Superscript ^TMThe RNA of II test kit (GibcoBRL Life Technologies#18064-014) and 8.9 μ g prepared fresh and 10pmol primer mixture (IgG1-c (SEQ ID NO.:63), IgG2a-c (SEQ ID NO.:64), IgG2b-c (SEQ ID NO.:65), IgG3-c (SEQ ID NO.:66), VLL-c (SEQ ID NO.:67), VLK-c (SEQ ID NO.:68)) synthesize cDNA.These primer annealings are to the RNA of IgG heavy chain (VH) gene of encode κ and λ family and light chain (VL) gene.The VH gene is to utilize 9 kinds of forward primers (M-VH1 (SEQ ID NO.:9), M-VH2 (SEQ ID NO.:10), M-VH3 (SEQ ID NO.:11), M-VH4 (SEQ ID NO.:12), M-VH5 (SEQ ID NO.:13), M-VH6 (SEQ ID NO.:14), M-VH7 (SEQ ID NO.:15), M-VH8 (SEQ ID NO.:16), M-VH9 (SEQ ID NO.:17)) and the reverse primer in 4 kinds of unrestricted sites (M-JH1 (SEQ ID NO.:18), M-JH2 (SEQ ID NO.:19), M-JH3 (SEQ ID NO.:20), M-JH4 (SEQ ID NO.:21)) 36 kinds of individual combinations are got by 1 μ l cDNA amplification by PCR.VL is that the primer mixture (M-VK1 (SEQ ID NO.:22), M-VK2 (SEQ ID NO.:23), M-VK3 (SEQ ID NO.:24), M-VK4 (SEQ ID NO.:25), M-VL1 (SEQ ID NO.:26), M-JK1 (SEQ ID NO.:27), M-JK2 (SEQ ID NO.:28), M-JK3 (SEQ ID NO.:29), M-JL1 (SEQ ID NO.:30)) that utilizes a kind of unrestricted site gets by pcr amplification.Through gel-purified (QIAquick GelExtraction Kit, #28706) behind the PCR product, utilize 9 kinds of forward primers (MVH1 SfiI (SEQ ID NO.:31), MVH2 SfiI (SEQ ID NO.:32), MVH3 SfiI (SEQ ID NO.:33), MVH4 SfiI (SEQ ID NO.:34), MVH5 SfiI (SEQ ID NO.:35), MVH6 SfiI (SEQ ID NO.:36), MVH7 SfiI (SEQ ID NO.:37), MVH8 SfiI (SEQ ID NO.:38), MVH9 SfiI (SEQ ID NO.:39)) and 4 kinds of reverse primer (M-JH1SalI (SEQ ID NO.:40) with the restriction site that is used for VH, M-JH2 SalI (SEQ ID NO.:41), M-JH3 SalI (SEQ ID NO.:42), M-JH4 SalI (SEQ ID NO.:43)) individuality combination and a kind of primer mixture (M-VK1 ApaLI (SEQ ID NO.:44) with the restriction site that is used for VL, M-VK2 ApaLI (SEQ ID NO.:45), M-VK3 ApaLI (SEQ ID NO.:46), M-VK4 ApaLI (SEQ ID NO.:47), M-VL1 ApaLI (SEQ ID NO.:48), M-JK1 NotI (SEQ ID NO.:49), M-JK2 NotI (SEQ ID NO.:50), M-JK3 NotI (SEQ ID NO.:51), M-JL1 NotI (SEQ ID NO.:52)) once more with its amplification.Gel-purified (QIAquick GelExtraction Kit, #28706) behind the PCR product, the restriction site SfiI/SalI that is used for VH clones into Vector for Phage Display pXP10 (SEQ ID No.:7) with the restriction site ApaLI/NotI that is used for VL with it.To connect the mixture transfection by electroporation and advance among the intestinal bacteria TG-1, obtaining size is 10 ⁷Individual library of expressing the independent cloning (phage) of different single chain antibody fragments (scFv).

Embodiment 2: to the selection of tumor cell specific scFv (selection of carrying out based on fixed cell)

Following selection can be expressed the phage that tumour cell is had the scFv of high-affinity: utilize 0.05%EDTA results HT1080 cell, be fixed with Paraformaldehyde 96, be diluted to 1 * 10 in PBS ⁷Individual cell/ml, and be fixed on the hole of the crosslinked plate of 96 hole UV (Corning Costar).Employing is dissolved in 5% skim-milk among the PBS (MPBS), and (#70166 Fluka) seals the hole of the crosslinked plate of this UV.With MPBS under 25 ℃ of conditions with 10 ¹²Phage library/10 of cfu (colony-forming unit) ⁶The pre-sealing of individual cell 1 hour, and descended incubation 1.5 hours with cell in room temperature (RT) subsequently.With PBS+0.05%Tween-20 with the hole of the crosslinked plate of this UV washing 6 times after, again with PBS washing 6 times.By adding the 10mM glycine of pH2.2, and with the 1M Tris/HCl neutralization of pH7.4, wash-out is the bonded phage.Typically, select in the first round in wash-out 10 ³-10 ⁶Therefore cfu, compares with the primary repertoire, and the diversity of the repertoire of enrichment has reduced.By the e. coli tg1 of infestation index's growth, amplification contains the eluate of the repertoire of enrichment.Selection contains the intestinal bacteria of phagemid, and makes them under 30 ℃ of conditions, and (o/n) growth of spending the night on the LB agar plate that is supplemented with 100 μ g/ml penbritins and 1% glucose is with propagation.After this step, perhaps with the repertoire of enrichment as polyclone set amplification, and be used for further selecting with repetitive mode, till the convergence that realizes the expection characteristic, or screen by spatial isolation and in the mono-clonal level.The phage particle that is used for the next round selection prepares by following step, promptly adopt helper phage VCS-M13 (Stratagene, La Jolla, CA) the exponential growth culture of superingection previous round selection, and make this culture grow overnight in the 2xTY that is being supplemented with 100 μ g/ml penbritins (amp) and 50 μ g/ml kantlex (kan) under 20 ℃ of conditions.Utilize 0.5M NaCl/4%PEG-6000, from clarifying bacterium supernatant liquor, be settled out and select the phage of obtaining, and it is suspended among the PBS once more.After carrying out taking turns selection, as method as described in the embodiment 3 at the enterprising row filter of mono-clonal level.

The screening of embodiment 3:scFv (screening of carrying out based on fixed cell)

For screening, with the gene of the selected scFv of the contained coding of Vector for Phage Display again time cloning advance among the expression vector pXP14 (SEQ ID No.:8).This carrier has guided and has merged the expression of the scFv that Strep-mark and E-mark are arranged, and does not contain filobactivirus gene-3.The expression vector of the e. coli tg1 that contains single bacterium colony source is grown in the separate wells of microtiter plate, makes each hole only contain a scFv clone.Under 30 ℃ of conditions, (#9297 TPP) is supplemented with in each hole among the 2xTY of 100 μ g/ml penbritins and 0.1% glucose, till OD600 is 0.7 at 96 hole microtiter plates to make bacterial growth.Utilize the IPTG abduction delivering of ultimate density, and spend the night in 25 ℃ of continuous expressions for 0.5mM.(#L-6876, Sigma) to ultimate density 50 μ g/ml, incubation is 1 hour under 25 ℃ of conditions, and with 3000xg centrifugal 15 minutes, prepares the clarification split product that contains strand Fv by adding hen's egg-white lysozyme.Screen before the ELISA, by adding equal-volume DMEM+10%FCS, with above-mentioned clarification split product sealing 1 hour.For screening ELISA, adopt 0.05%EDTA results HT1080 cell, fix with Paraformaldehyde 96, in PBS, be diluted to 1 * 10 ⁷Individual cell/ml, and be fixed in the hole of the crosslinked plate of 96 hole UV (Corning Costar).Adopt MPBS to seal each hole of the crosslinked plate of this UV, add the clarification split product of sealing that contains scFv and in 25 ℃ of incubations 1.5 hours.After adopting PBS+0.1%Tween-20 to wash this plate 2 times, again with PBS washing 1 time, with the α that combines HRP-E-mark (#27-9413-01, Pharmacia Biotech; The MPBS that utilization contains 0.1%Tween-20 was with dilution in its 1: 5000) incubation 1 hour together, utilize PBS+0.1%Tween-20 with its washing 3 times after, again with PBS washing 3 times, (#1 484 281, Roche) develop, and in the 370nm read output signal with POD.

Utilize above-mentioned ELISA screening method to check the clone of the positive colony and the anti-contrast human fibroblasts Hs-27 (ATCC CRL-1634) of anti-HT1080 cell once more, and they are kept in the frozen pipe of glycerine.In typical screening, individual clone screens to 2760 (30 * 92), with the clone that 5% positive clone is defined as providing background deduction signal＞0.1 that is combined with of HT1080 cell.Check 155 positive colonies to combine once more with the specificity of HT1080 cell, and compare with the Hs-27 control cells, 28% the male clone that is combined into is defined as at least 2 times the clone that available background deduction signal value based on HT1080 is based on the signal value of Hs-27 control cells.

Embodiment 4: to order-checking and extensive expression the thereof of fixed scFv

To the order-checking of scFv1-scFv4 and their gene is Sequiserve GmbH by Germany, Vaterstetten utilize primer pXP2 Seq2 (5 '-CCCCACGCGGTTCCAGC-3 ' (5 ' TACCTATTGCCTACGGC-3 ' (SEQ ID No.:54) carries out for (SEQID No.:53) and pXP2 Seq1.Accompanying drawing has shown their aminoacid sequence and nucleotide sequence.

To from the frozen pipe of glycerine, draw by the uniqueness clone that order-checking is identified and take out, and streak inoculation is incubated overnight in 30 ℃ to LB/Amp (100 μ g/ml)/1% agar glucose flat board.Single colony inoculation is advanced in 10ml LB/Amp/Glu (1%) substratum, make its grow overnight under 30 ℃ and 200rpm concussion condition.Second day morning, inoculation advance be supplemented with the 1L 2xTY substratum of 100 μ g/ml penbritins and 0.1% glucose in the 2L vertebra shape bottle before, overnight culture is placed on ice.This culture is that the IPTG of 0.1mM induces with ultimate density after growing to OD600 0.5-0.6 under 25 ℃ of concussion conditions.The fresh penbritin that adds 50 μ g/ml, and under 22 ℃ of concussion conditions, continue to be incubated overnight.Second day morning, centrifugal this culture is 15 minutes under 4 ℃, 5000xg condition, abandon supernatant liquor, and utilize pipette will precipitate carefully on ice to be suspended in again and contain the adequate proteins enzyme inhibitors (#1697498 is in 10ml precooling PBS-0.5M Na damping fluid Roche).Again suspend finish after, bacterial suspension is shifted in the akrodge centrifuge tube of 20ml into, (#L-6876 is Sigma) to ultimate density 50 μ g/ml, and in placing 1 hour on ice to add hen's egg-white lysozyme.Centrifugal cracked bacterium is 15 minutes under 4 ℃ and 20000xg condition, and supernatant liquor (split product) is shifted in the plastics tubing of 15ml.For carrying out affinity purification, by means of parallel protein purification system, (#2-1505-010 is IBA) on the post to the 1ml StrepTactin that crosses via the PBS-0.5M Na damping fluid balance of 10 times of column volumes (CV) with sample on the above-mentioned split product with the speed of 1ml/min.Adopt the PBS of 10CV to wash, and (#D-1411 Sigma) carry out wash-out, collects the 1ml elution fraction to adopt the PBS/5mM Desthiobiotin of 5CV.Detect this elution fraction at the UV280 wavelength, will contain proteinic elution fraction and mix, and (#UFC801024 Millipore) concentrates it with 4700xg to utilize super centrifugal filter device 10.000 MWCO of Amicon.

On via the 12%Bis-Tris SDS-PAGE gel of Coomassie blue stain, check the purity that concentrates scFv, and it is divided into aliquots containig with 20% glycerine-80 ℃ freezing preservation.

Embodiment 4.1: mouse scFv reconstruct enters chimeric IgG (people-mouse) and expresses

Mouse scFvs is made up of the variable light chain that links together by joint sequence and the sequence of heavy chain.This variable light chain and variable heavy chain utilize primer to be increased by PCR respectively, contain restriction site.These restriction sites also are present in the specific support, and this carrier contains the mouse constant domain that is suitable for described heavy and light chain.Adopt the variable domains of restriction enzyme cutting amplification, and it is cloned in the carrier that has into cut.Confirm that by order-checking sequence is correct.

Utilize 4 kinds of carriers, wherein a kind of carrier contains the mouse constant domain of the heavy chain that is useful on the IgG1 form.Second kind of carrier contains the mouse constant domain of the heavy chain that is useful on the IgG4 form, and other two kinds of carriers contain the mouse constant domain of λ and κ light chain respectively.Different restriction sites can make the cutting of these carriers is achieved with the variable domains in these carriers is connected.

For described chimeric IgGs is expressed in mammal cell line, above-mentioned carrier contains Epstein-Barr virus replication orgin (oriP sequence), can improve the intracellular transcriptional level at 293-EBNA-HEK, because EBNA albumen can cause duplicating of episomal vector.

The carrier that utilization is used for described heavy chain carries out cotransfection with the carrier that is used for described light chain, makes two chains of cell expressing, and realizes the assembling of IgG in endoplasmic reticulum.Subsequently, the IgG that has assembled is secreted in the substratum.What transfection method adopted is calcium phosphate transfection method, has wherein formed the precipitation of calcium phosphate and described DNA, and it is integrated in the cell.After the transfection, above-mentioned substratum is replaced by serum free medium.IgG of per 3 days results, every kind of IgG gets three duplicate samples.Sterile filtration supernatant liquor (substratum) and in 4 ℃ of preservations.

For IgG purification s, by means of A albumen agarose, according to the volume of supernatant liquor, perhaps by gravity flowage or by HPLC purifying supernatant liquor.For for the volume of 200ml, adopt the gravity flowage method.For this purification process of two types, all be with sample on the supernatant liquor on A albumen post, adopt the 50mM Tris damping fluid washing of pH7, and adopt the 0.1M Citrate trianion of the about 2-3 of pH to carry out wash-out.The 0.25M Tris of pH9 is added in the elution fraction, make its pH value be 5.5-6.0.According to the further purposes of IgGs, they are dialysed and in-80 ℃ or 4 ℃ of preservations with the PBS damping fluid.

Embodiment 5: at tumor cell specific bonded facs analysis

In order to check the ability of the anti-HT1080 scFv of purifying specificity in conjunction with target cell, we adopt HT1080 cell (ATCC CCL-121) and control cells is Hs-27 cell (10 ⁶Individual cell/ml) carry out fluorescence-activated cell sorter (FACS) to analyze (referring to Fig. 4).Also checked ScFv and KHOS cell (ATCC CRL-1544), PC-3 cell (ATCC CRL-1435), and CCL 13 (the DSMZ ACC-57 that contains Hela cell impurity) (is 10 ⁶The combination of individual cell/ml) compares with the HT1080 cell.Under 4 ℃ of conditions, incubation CellWash (BD (Becton, Dickinson and Company) cell #349524) and the pure scFv of 10 μ g/ml 20 minutes after the washing, utilize the anti-E-mark mab (Amersham #27-9412-01) of secondary FITC mark to detect bonded scFv ' s.Washing sample also utilizes Becton Dickinson FACSscan that it is analyzed.Fig. 4 (last figure) is depicted as the logarithm fluorescence intensity (FL1-H of the cell that reacts with scFv1 and scFv2; The x-axle) with relative cell quantity (counting; The y-axle) relation curve.Fine rule is represented control cells system (HS-27), and thick line is then represented the HT1080 cell.ScFv1 and scFv2 make tumor cell line painted specifically, and its signal intensity ratio control cells system is up to 10 times.Figure below of Fig. 4 is depicted as combining of scFv1 and PC3, KHOS and CCL 13, is compared to itself and the combining of HT1080 cell.ScFv1 can combine with whole these three kinds of clones, but compares these bonded degree lower (PC3, KHOS or CCL 13=dotted line) with the HT1080 cell.

Embodiment 6: the competitive analysis of being undertaken by FACS

For the ability of total PVR epi-position on the anti-PVR-scFv blocking-up of the check purifying target cell, with 0, the single cell suspension of 5mM EDTA/PBS results HT1080.Under 4 ℃ of conditions, at the CellWash that contains 10 μ g/ml scFv (BD, #349524) middle incubation about 1 * 10 ⁶ Individual cell 1 hour.With after the Cell Wash washing, add the FITC mark scFv of 10 μ g/ml, and in 4 ℃ of incubations 20 minutes.Utilize Becton Dickinson FACSscan to passing through and do not pass through other PVR wedding agent analyzing in conjunction with the signal of FITC mark scFvs of incubation in advance.

Embodiment 7: adopt the segmental mark of FITC antagonist

By following method, adopt fluorescein isothiocyanate (FITC) (Molecular Probes, Eugene, USA #F1906) mark scFv: the aliquots containig of the 10mg/ml solution that FITC is formed in dimethyl sulfoxide (DMSO) adds that (FITC: ratio solvent scFvl) was in the PBS/0.5M of pH 9.5 NaHCO with 30: 1 by 100 μ g scFv ₃In the formed solution.The incubation sample is 2 hours under the stirring at room condition, utilizes desalting column (2 Micro Spin G-25, Pharmacia27-5325-01) FITC of separated free.The mark ratio is determined by mass spectrometry and UV/VIS spectrography, can be calculated the FITC concentration of protein concn and the 494nm of 280nm thus.

Embodiment 8: be used to identify the invasion and attack test of inhibition molecule

Can adopt ChemoTx ^System (Neuro Probe Inc.#106-8, Gaithersburg) as disposable chemotaxis/cell migration pond, this system is 96 well format with 8 μ m filters, indentation polycarbonate material, the aperture is 5.7mm diameter/position.

To be diluted in 13 among the Dulbeccos PBS (Gibco #14040-091), (Matrigel is from being rich in the tumour of extracellular matrix protein to 3 μ l0.3mg/ml Matrigel, i.e. the lyotropy basilar membrane preparation that extracts in Engelbreth-Holm-Swarm (EHS) murine sarcoma.Its main component is a ln, secondly is collagen protein IV, heparan sulfate proteoglycan, sense of touch albumen and nidogen.It also contains TGF-α fibroblast growth factor, tissue plasminogen activator and the somatomedin of other spontaneous generation in the EHS tumour) (Becton Dickenson, BD #356234) is applied on above-mentioned 96 orifice plate B-H row's the membrane filter, with 0, among 05M HCl (Sigma #945-50) the dilution A row 1, the type i collagen Protein S of 2 μ g/ positions (Roche #10982929), and in moisture eliminator 20 ℃ be incubated overnight, make its gelation.The HT1080 cell grows to 70-80% and is paved with in the DMEM that is supplemented with GlutamaxI (862mg/1 (Gibco#31966-021)) and 10%FCS (Gibco #10270106).After washing this cell 2 times with DMEM/GlutamaxI/0.1%BSA (Sigma #A-7030),, and be diluted in DMEM/GlutamaxI/0.1%BSA with 1: 100 with Bisbenzimide H 33342 (Sigma #B-2261) original position mark, at 37 ℃ and 7,5%CO ₂Incubation is 15 minutes under the condition.With DMEM/GlutamaxI/0.1%BSA washed cell 2 times, add DMEM/GlutamaxI/0.1%BSA, in 37 ℃ and 7,5%CO ₂Incubation is 15 minutes under the condition, makes cellular-restoring.With no Ca ²⁺, Mg ²⁺(PBSGibco, 10010-015) behind the washed cell 2 times, make cell detachment with 0.5mM EDTA (Sigma #E8008), and collect with DulbeccosPBS/0.1%BSA/10mM Hepes (Gibco #15630-056), with DulbeccosPBS/0.1%BSA/10mM Hepes washing 2 times, cell suspension in DulbeccosPBS/0.1%BSA/10mM Hepes, is diluted to 6,7 * 10 with Dulbeccos PBS/0.1%BSA/10mM Hepes with it ⁶Individual cell/ml.On ice 1 hour ratio of incubation be 1: 16,7 * 10 ⁶Individual cell/ml and 40 μ g/ml are as the contrast scFv of the negative control that suppresses invasion and attack, and ratio be 1: 16,7 * 10 ⁶Individual cell/ml and HT1080 specificity scFv.With DMEM/GlutamaxI/0.1%BSA with cell dilution to 6,7 * 10 ⁵Behind individual cell/ml, HT1080 cell and HT1080 cell/scFv diluent are drawn three parts, be transferred to chemotactic pond (B-H row), density is 3,4 * 10 ⁴Individual cells/well, and in 37 ℃ and 7,5%CO ₂Incubation is 6 hours under the condition.With the DMEM/GlutamaxI that contains 5%FCS as the chemical attractant in several rows pond, back.Make 1 * 10 in the A of type i collagen Protein S bag quilt row chemotactic pond ⁴-4 * 10 ⁴The typical curve of individual cell/position.In several rows pond, back, use DMEM/GlutamaxI/0.1%BSA (cell does not move).Migrating cell does not scrape back (except A row's typical curve) from film top, utilize the excitation/emission wavelength of Fluostar Galaxy (bMG) microtitration card reader and 370/460nm, measure the fluorescence (Fig. 1) of migration by the cell of film (not migration in the situation of typical curve).

Embodiment 9: utilize CALI to identify the invasion and attack test of target

The principle of this example is consistent with example 8, but has utilized the scFv of FITC-mark and combine the CALI method in addition in the invasion and attack test.In the CALI method, scFv is marked with CALI can induce mark.

Can adopt ChemoTx ^System (Neuro Probe Inc.#106-8, Gaithersburg) as disposable chemotaxis/cell migration pond, this system is 96 well format with 8 μ m filters, indentation polycarbonate material, the aperture is 5.7mm diameter/position.

To be diluted in 13 among the Dulbeccos PBS (Gibco #14040-091), 3 μ l0.3mg/ml Matrigel (referring to example 8) are applied on above-mentioned 96 orifice plate B-H row's the membrane filter, with 0, among 05M HCl (Sigma #945-50) the dilution A row 1, the type i collagen Protein S of 2 μ g/ positions (Roche #10982929), and in moisture eliminator 20 ℃ be incubated overnight, make its gelation.The HT1080 cell grows to 70-80% and is paved with in the DMEM that is supplemented with GlutamaxI (862mg/l (Gibco#31966-021)) and 10%FCS (Gibco #10270106).After washing this cell 2 times with DMEM/GlutamaxI/0.1%BSA (Sigma #A-7030), with Bisbenzimide H 33342 (Sigma #B-2261) original position mark, and be diluted in DMEM/GlutamaxI/0.1%BSA with 1: 100, at 37 ℃ and 7, incubation is 15 minutes under the 5%CO2 condition.With DMEM/GlutamaxI/0.1%BSA washed cell 2 times, add DMEM/GlutamaxI/0.1%BSA, in 37 ℃ and 7,5%CO ₂Incubation is 15 minutes under the condition, makes cellular-restoring.With no Ca ²⁺, Mg ²⁺PBS (Gibco, 10010-015) behind the washed cell 2 times, make cell detachment with 0.5mM EDTA (Sigma #E8008), and collect with DulbeccosPBS/0.1%BSA/10mM Hepes (Gibco #15630-056), after DulbeccosPBS/0.1%BSA/10mM Hepes washing 2 times, cell suspension in DulbeccosPBS/0.1%BSA/10mM Hepes, and is diluted to 6,7 * 10 with Dulbeccos PBS/0.1%BSA/10mMHepes with it ⁶Individual cell/ml.On ice 1 hour ratio of incubation be 1: 16,7 * 10 ⁶Individual cell/ml and 40 μ g/ml suppress the anti-β integrin of the FITC-mark monoclonal antibody (JB1, Chemicon #MAB1963) of the contrast of invasion and attack after as CALI, and ratio be 1: 16,7 * 10 ⁶Individual cell/ml and HT1080 specificity FITC mark scFv.With 1,3 * 10 ⁵Individual HT1080 cells/well or HT1080 cell/scFv or Ab diluent are drawn three parts, shift two and have in the flat black 96-orifice plate of ultra-thin transparent special optical (Costar #3615).A plate is remained on dark place on ice, and another piece plate is then accepted the irradiation (0.5W, 30 seconds) of the continuous wave laser of 488nm on ice cube.With DMEM/GlutamaxI/0.1%BSA with cell dilution to 6,7 * 10 ⁵Behind individual cell/ml, the diluent of HT1080 cell and HT1080 cell/scFv is drawn three parts (accepting three parts and three parts of acceptance irradiation of irradiation), be transferred to chemotactic pond (B-H row), density is 3,4 * 10 ⁴Individual cells/well, and in 37 ℃ and 7, incubation is 6 hours under the 5%CO2 condition.With the DMEM/GlutamaxI that contains 5%FCS as the chemical attractant in several rows pond, back.Make 1 * 10 in the A of type i collagen Protein S bag quilt row chemotactic pond ⁴-4 * 10 ⁴The typical curve of individual cell/position.DMEM/GlutamaxI/0.1%BSA (cell does not move) is used in several rows pond in the back.Migrating cell does not scrape back (except A row's typical curve) from film top, utilize the excitation/emission wavelength of Fluostar Galaxy (bMG) microtitration card reader and 370/460nm, measure the fluorescence of migration by the cell of film (not migration in the situation of typical curve).The result of the invasion and attack test of carrying out under the laser radiation situation is shown in Fig. 2 a.ScFv1 will attack and suppress about 22%.

Embodiment 9.1: utilize CALI (adopting the light of elimination blue light) to identify the invasion and attack test of target

The principle of this example is consistent with example 9, but the irradiation sample is the 300W light of elimination blue light, but not laser radiation.

, shone 1 hour after 1 hour with 20 μ g/mL fluorescein isothiocyanate (FITC) traget antibody incubation HT-1080 cells by 300 watts of light of elimination blue light are other.Then analysis of cells 6 hours by the Matrigel bag by the invasiveness of porous-film (Neuro Probe Inc.).Histogram graph representation is normalized to the invasion and attack per-cent ± standard error under the no irradiation condition, and at least 2 each parallel 3 times independent experiments of expression.Left post among Fig. 2 b (0) is depicted as the invasion and attack without the cell of any antibody treatment, and D171 (NeoMarker #MS-465) is the monoclonal antibody by the obtainable direct anti-PVR/CD155 in commercial channel.These two kinds of scFV of scFv3 and scFv4 have all suppressed about 30% with the invasion and attack of HT1080 cell.Referring to Fig. 2 b.

Embodiment 9.2: the invasion and attack test that utilizes breast cancer cell to carry out

The principle of this example is consistent with example 9.1, but has utilized the MDA-MD231 breast cancer cell to replace the HT1080 cell.ScFv3 and scFv4 show that all the invasion and attack with the MDA-MD231 cell have suppressed about 23%.Referring to Fig. 2 c.

Embodiment 9.3: the migration test of utilizing spongioblast oncocyte (U87MG and A172 cell) to carry out

The principle of this example is consistent with example 9.1, but has utilized Beckton Dickinson plate to replace ChemoTx  system.These Beckton Dickinson plates comprise the embedded part of being made up of PET film and blue dyes (Fluoroblok), help to measure the fluorescence that only derives from top or bottom.25,000 cells (U87MG and A172) are contained in each hole.ScFv1 shows that the migration with the U87MG cell has suppressed about 15% (data not shown).ScFv1* and scFv2* demonstration are respectively 16% and 17% to the U87MG cell inhibiting, the A172 cell inhibiting is respectively 20% and 18%, and (symbol " * " refers to be cloned to I gG form by the corresponding scFv that Figure 10 and numbering shown in Figure 11 are distinguished, referring to Fig. 4 .1), the result is shown in Fig. 2 d.

The PVR that this example has further been studied different clones expresses and how to change, and related with migratory behaviour.The PVR expression level is by the immunoblotting assay of 4 glioblastomas (GBM) clone is determined that this expression level is used to analyze the migratory behaviour in the migration test subsequently.PVR is the highest in the intracellular expression of HT1080, and is medium at U87 and the intracellular expression of U251GBM, minimum at SNB19 and the intracellular expression of A172 GBM.Migratory behaviour in the middle of this expression level and the GBM clone is irrelevant, because the invasion and attack velocity ratio SNB19 (poor) of U87 (medium) and A172 (poor) or U251 (medium) are faster.(data not shown).As everyone knows, the HT1080 cell is by overexpression N-ras and immortalization, and U87 and U251 GBM cell are all had the ras activity of high composition level by report.Because these observationss are relevant with the PVR expression level, therefore, be interesting in the supposition that may play a role in some way aspect the adjusting PVR expression to ras.

Embodiment 10:MTS viability is measured

(MTS, Celltiter Aqueous one is Promega#G4000) to the conversion of first, to detect viable cell from tetrazole dyestuff MTS by measuring.HT1080 cell and HT1080 cell/scFv diluent (being obtained by the diluent for preparing in the above-mentioned adherence test) are drawn three parts, shift in the 96-orifice plate (black, ultra-thin transparent flat, special optical, Costar #3615), density is 3,4 * 10 ⁴Individual cells/well.10 μ l MTS are added each hole, and in 37 ℃ and 7,5%CO ₂Incubation is 1 hour under the condition.Utilize Fluostar Galaxy (bMG) microtitration card reader to measure the absorbancy of 492nm.For tested scFv1 and scFv2, do not find that the viability of pair cell has any influence.

Embodiment 11: be used to identify the segmental cell-matrix adherence test of inhibiting antibody

Under 4 ℃ of conditions, the bag of 21 holes in the 96-hole flat underside (Costar #3614) is spent the night to be selected from one of following stromatin, promptly be dissolved in type i collagen Protein S 1 μ g/ hole (Roche #10982929), IV collagen type 1 μ g/ hole (Rockland 009-001-106), fibronectin 1 μ g/ hole (SigmaF2518) and the ln 1 μ g/ hole (Roche 1243217) of Dulbeccos PBS (Gibco#14040-091) respectively.Simultaneously with 3 holes bags among the A row by with 2%BSA (Sigma #A-7030)/Dulbeccos PBS, as blank.Wash each hole 2 times with Dulbeccos PBS, sealed 1 hour with 2%BSA (Sigma #A-7030)/Dulbeccos PBS, and wash with Dulbeccos PBS in 37 ℃.Results HT1080 cell is with 2, after 5mM (ultimate density) fluorexon AM (the Molecular Probes C-3099) dyeing, with no CaCl ₂No MgCl ₂PBS (Gibco 10010-015) washing 2 times, and in damping fluid (0,5%BSA (Sigma #A-7030)+10mM Hepes+DMEM (Gibco31966-02)), be diluted to 1,5 * 10 ⁵/ ml.The HT1080 cell is mixed with 10 μ g/ml scFv, and in incubation on ice 30 minutes.Independent HT1080 cell and three parts of transfers of HT1080/scFv diluent absorption are advanced in 96 orifice plates, and density is 1,5 * 10 ⁴Individual cells/well, and in 37 ℃ and 7,5%CO ₂Incubation is 1 hour under the condition.With in addition washing 2 times of Dulbeccos PBS, not behind the adherent cell flush away, can based on A arrange in triplicate sample make with the DulbeccosPBS dilution obtained 3,7 * 10 ³-1,5 * 10 ⁴The typical curve in individual staining cell/hole.Each hole after the washing is full of 100 μ l Dulbeccos PBS, and utilizes the excitation/emission wavelength of Fluostar Galaxy (bMG) microtitration card reader and 485/520nm to measure the absorbancy of adherent cell and the absorbancy of typical curve.

Embodiment 12: utilize CALI to identify the cell-matrix adherence test of target

Under 4 ℃ of conditions, with the B-H round bag of 96 orifice plates (TPP #9296) (handling) via cell culture by type i collagen Protein S 1 μ g/ hole (Roche #10982929) to be dissolved in Dulbeccos PBS (Gibco #14040-091), A row's hole 10-12 then wraps by with 2%BSA (Sigma #A-7030)/Dulbeccos PBS, spends the night.Wash this plate with Dulbeccos PBS, and, B-H row and A row's hole 10-12 was sealed 1 hour, once more with Dulbeccos PBS washing with 2%BSA/Dulbeccos PBS in 37 ℃.The HT1080 cell grows to 70-80% and is paved with in the DMEM that is supplemented with GlutamaxI (862mg/1 (Gibco #31966-021)) and 10%FCS (Gibco#10270106).Wash this cell 2 times with DMEM/GlutamaxI/0.1%BSA (Sigma #A-7030), behind Bisbenzimide H 33342 (Sigma #B-2261) original position mark, ratio with 1: 100 is diluted among the DMEM/GlutamaxI/0.1%BSA, and in 37 ℃ and 7,5%CO ₂Incubation is 15 minutes under the condition.With DMEM/GlutamaxI/0.1%BSA washed cell 2 times, and add DMEM/GlutamaxI/0.1%BSA, in 37 ℃ and 7,5%CO ₂Incubation is 15 minutes under the condition, makes cellular-restoring.With no Ca ²⁺, Mg ²⁺PBS (Gibco, 10010-015) washing 2 times after, make cell detachment with 0.5mM EDTA (Sigma #E8008), collect with DulbeccosPBS/0.1%BSA/10mM Hepes (Gibco #15630-056), after DulbeccosPBS/0.1%BSA/10mM Hepes washing 2 times, make cell suspension in DulbeccosPBS/0.1%BSA/10mM Hepes, and it is diluted to 6,7 * 10 with Dulbeccos PBS/0.1%BSA/10mMHepes ⁶Individual cell/ml.On ice 1 hour ratio of incubation be 1: 16,7 * 10 ⁶Individual cell/ml and 40 μ g/ml suppress to adhere to the anti-beta 1 integrin monoclonal antibody of FITC-mark (JB1, Chemicon #MAB1963) of contrast after as CALI, and ratio be 1: 16,7 * 10 ⁶Individual cell/ml and HT1080 specificity FITC mark scFv.With 1,3 * 10 ⁵Individual HT1080 cells/well or HT1080 cell/scFv or Ab diluent are drawn three parts, shift two and have in the flat black 96-orifice plate of ultra-thin transparent special optical (Costar #3615).A plate is remained on dark place on ice, and another piece plate is then accepted the irradiation (0.5W, 30 seconds) of the continuous wave laser of 488nm on ice cube.With DMEM/GlutamaxI/0.1%BSA with cell dilution to 6,7 * 10 ⁵Behind individual cell/ml, the diluent of HT1080 cell and HT1080 cell/scFv is drawn three parts (accepting three parts and three parts of acceptance irradiation of irradiation), be transferred in the plate that wraps quilt and sealing.In A row's hole 10-12, add with 6,7 * 10 of DMEM/GlutamaxI/0.1%BSA dilution back acquisition ⁵Individual cell/ml contrasts as a setting.In 37 ℃ and 7,5%CO ₂This plate of incubation is 1 hour under the condition, and washs 2 times with Dulbeccos PBS, not the adherent cell flush away.At A round 1-9, do 1 * 10 ⁴-4 * 10 ⁴The typical curve of individual cells/well then adds 50 μ lDulbeccos PBS in other hole.Utilize the excitation/emission wavelength mensuration of Fluostar Galaxy (bMG) microtitration card reader and 370/460nm and the fluorescence of the cell of type i collagen Protein S adhesion (in the situation of typical curve, adhering to).ScFv1 with adherence inhibition 55.5%, scFv2 then with this adherence inhibition 42%.The result corresponding with these two kinds of scFv as shown in Figure 3.

Embodiment 13: immunoprecipitation

Make HT1080 and Hs-27 cell (10 ⁸) cracking contains 150mM NaCl, 1%Triton X-100 (v/v), protease inhibitor cocktail (50ml damping fluid 1 ball) (Boehringer Mannheim at 3ml, Cat.-No.1697498) and 100 μ M Pefablock (Roth, Cat.-No.A154.1) 50mM Tris-HCl is among the pH 8.0.Under 4 ℃ of conditions, split product with Streptactin-coupling magnetic bead incubation 2 hours in advance, and is used for immunoprecipitation with supernatant liquor.HT1080 specificity scFv (50 μ g/1mg cell extract) is added in the clarifying split product, in 4 ℃ of stirred samples 2 hours, be placed on the magnet, to collect the magnetic bead on the tube wall, with this magnetic bead washing 4 times, use the PBS+0.1%Tween damping fluid of 1ml volume, afterwards at every turn, carry out wash-out by 20 μ l 0.1M citrate solutions, mixture is separated with the streptactin magnetic bead with pH 3.1.Add 5 μ l1M Tris-HCl pH 8.0 immediately, with in and eluate.Separate this immunocomplex by SDS-PAGE, be used for MS after the silver dyeing and analyze.

ScFv1 and scFv2 all can catch a kind of protein, can make this protein about 70kDa molecular weight place on SDS-PAGE form a detectable band by silver dyeing.This band is found and only appears in the HT1080 cell extract, does not occur in Hs-27 cell (control cells), referring to Fig. 5.

Embodiment 14: the identification of proteins of being undertaken by mass spectroscopy

Under 37 ℃ of conditions, the gel strips band that is successively obtained by immunoprecipitation and SDS PAGE is carried out in the trypsinase glue digestion spend the night.Utilize 5% formic acid extraction peptide, and with ZipTip μ C18 (Millipore) with the peptide mixt desalination that obtains, at first carry out wash-out with the 30%ACN/0.1%TFA of 2 μ l after, use the 70%ACN/0.1%TFA wash-out of 2 μ l again.Two parts of elution fractions are merged, 1 peptide mixt that microlitre obtains is mixed with 1: 1 ratio with α-cyanogen-4-hydroxycinnamic acid solution (3mg/ml), cocrystallization on the stainless steel target of Teflon bag quilt, and utilize MALDI-TOF equipment to analyze, obtaining mass range is the peptide quality fingerprinting collection of illustrative plates (PMF) of m/z 800-3000.The PMF that obtains is used to study all clauses and subclauses relevant with the homo sapiens in NCBI and the SwissProt database.In all situations, only consider evaluation and specified protein coupling and mass deviation peptide less than 10ppm.

To with database in the 4-6 kind peptide of PVR coupling detect, the difference between measured value and the actual mass all the time+/-the 10ppm scope in.In some experiments, these 4 kinds of peptides are divided into 2 pairs observe, each to equal representatives owing to the methionine(Met) oxidation has or do not have+peptide of 16Da.When this peptide mixt is carried out de-glycosylation, observe another kind of peptide, this peptide contains Asn120, is explained to glycosylated in database.Collection of illustrative plates as shown in Figure 6.

Also measured another sample by nano ES-MSMS.4 kinds of peptides have been detected with this method.By CID (collision induced dissociation) general three kinds of fragmentations wherein, be used for MSMS and analyze.Obtain the sequence mark corresponding, and use it for database research with each fragment.These three kinds of peptides and known poliovirus receptor coupling, a kind of methionine(Met) wherein with oxidation.

Embodiment 15: the epitope mapping method

Epitope mapping can carry out according to one of following method:

Embodiment 15.1: " classics " epitope mapping

CDNA is used for being paid close attention to antigenic definition fragment as recombination fusion protein or protein expression, and in multiple test, it is surveyed such as Western blotting or ELISA.

Embodiment 15.2: display technique of bacteriophage

The technology of utilizing peptide phage display library at random to carry out epitope mapping is used for that by development cDNA is used for being paid close attention to antigenic little random fragment and clones the into phage albumen pIII of filobactivirus, and they are illustrated in surface (the Fack et al. of this phage, (1997) J.Immunol.Methods 7,43-52).In the operation that is called as " biological elutriation ", can utilize antibody capture epitope display phage.The segmental order-checking of the insertion of corresponding phage is provided some information of relevant described epi-position.This method is used to identify the conformational epitope.

Embodiment 15.3: the peptide screening technology

The basis of this technology is to utilize Fmoc chemistry synthetic immobilization peptide on the activation film.Amino acid solution is applied to this activation film, make on this film (activating this film) with PEG amino and use between the amino acid whose activated carboxyl and form peptide bond.After each specificity washing operation circulation, free amine group is carried out acetylize, deprotection and monitoring.Compare with the body internal protein is synthetic, film is synthetic progressively from C-to the N-end in conjunction with the oligopeptides chain.Contain the length that the oligopeptides of natural and modified amino acid can be synthesized and to reach 20 amino acid.After synthetic the finishing, this film of balance also seals nonspecific binding site.With after being paid close attention to antibody and carrying out incubation and several washing steps, utilize HRP bonded secondary antibodies and detect in conjunction with the ECL-system.According to the situation of antibody, film can be peeled off, regenerated and reuse reaches 10 times.The synthetic little overlapping oligopeptides of being paid close attention to antigenic complete amino acid sequence that comprised ideally on solid support.This method is used to identify the line style epi-position of amino acid levels.Also be used to quick mutation research.

Embodiment 16: utilize the proteic eliminating of RNAi pair cell PVR of anti-PVR mRNA

Following ribose oligonucleotide can be available from for example, Proligo (Hamburg, Germany, wWW.proligo.com), Pierce Chemical (part of Perbio Science, Rockford, Illinois, WWW.perbio.com) or other supplier of RNA oligonucleotide:

1A (5′-CCAGTCACTTGTCTGGAGCTT-3′)

(SEQ?ID?No.：55)，

2A (5′-GAGCTTGAAGAAGTGGGTATT-3′)

(SEQ?ID?No.：56)，

1B (5′-TGCTGGTGGC ATTACTGGTGC-3′)

(SEQ?ID?No.：57)，

2B (5′-GCTGTCCTGGCCACCCC GTGGAA-3′)

(SEQ?ID?No.：58)，

3A (5′-GGCGCGGAGCTGCGGAATGCCT-3′)

(SEQ?ID?No.：59)，

4A (5′-CCTCGCTGAGGATGTTCGGGTT-3′)

(SEQ?ID?No.：60)，

3B (5′-CCC GTGAACACAGCTGAGGTT-3′)

(SEQ?ID?No.：61)，

4B (5′-CAAGGTGGA CCCACGAGAGCTTT-3′)

(SEQ?ID?No.：62)，

5A (5’-CAACUUUAAUCUGCAACGUTT-3’)

(SEQ?ID?No.：73)，

5B (5’-TTGUUGAAAUUAGACGUUGCA-3’)

(SEQ?ID?No.：74).

Oligonucleotide 1A and 2A annealing form the directly siRNA of anti-described PVR mRNA, and oligonucleotide 1B and 2B annealing form the negative control of going up a pair of oligonucleotide, and the Nucleotide and the described PVR mRNA mismatch of line arranged down.Equally, the oligonucleotide 3A of line and the siRNA that 4A annealing forms directly anti-described PVR mRNA are arranged down, oligonucleotide 3B and 4B then anneal and form 3A/4A-right negative control, Nucleotide shown in the black matrix and described PVR mRNA mismatch.The target of 5A/B is 1094 of PVR mRNA sequence (GI 19923371).

Adopt Elbashir et al., the 203rd page of described scheme 2 makes oligonucleotide to 1A/2A, 1B/2B, 3A/4A, 3B/4A annealing among Methods (2002) 26:199-213.When studying with RNA, need pay special attention to, pollute oligonucleotide to avoid RNAse.The water of handling through DEPC is used to prepare buffer reagent.All the time have gloves on and pollute with the RNAse that avoids causing because of skin contact.But " Molecular Cloning:A Laboratory Manual " (1989) second edition the 7.3rd and 7.4 chapters that specify the people such as Sambrook that reference example such as ColdSpring Harbor Laboratory Press publish about the RNA-correlative study.

The HT1080 cell grows to about 90% and is paved with in the DMEM that is supplemented with Glutamax (Gibco# 31966-021) and 10%FCS (Gibco #10270106).Utilize above-mentioned annealed oligonucleotide preceding 24 hours to transfection, above-mentioned 90% cell that is paved with of results from the Tissue Culture Flask of 175ml for example uses 10ml trypsinase-EDTA solution (Life Technologies) with its trypsinized, gentle centrifugal and be suspended in again among the 10ml DMEM.The DMEM substratum of antibiotic-free dilutes this cell suspension at 1: 10 with containing 10%FCS, and is divided into the aliquots containig of 500 μ l, shifts in each hole of 24 well culture plates.Inoculate behind this cell 24 hours, obtain about 50% be paved with.

According to Elbashir et al., the 207th page of described scheme 5 of Methods (2002) 26,199-213 utilizes above-mentioned annealed oligonucleotide to the cell in each hole of transfection.

After carrying out 2 days incubation according to the 5th step of such scheme 5, gather in the crops the cell in each hole, and the cell in each hole processed, be used for western blot analysis, for example, with the hole (undressed cell) that derives from without the oligonucleotide transfection, via oligonucleotide to 1A/2A, 1B/2B (negative control 1/2), sample (the corresponding same cell quantity) application of sample in the hole of 3A/4A and 3B/4B (negative control 3/4) transfection is on acrylamide gel, after electrophoresis finishes, for example, utilize the semidry blotter device, western blotting to for example, on the soluble cotton, and is surveyed this nitrocellulose membrane with anti-PVR antibody.

Compare with undressed cell, the proteinic amount of detected PVR has reduced more than 75% in sample 1A/2A or 3A/4A.Compare with undressed cell, in negative control 1B/2B and 3B/4B, do not detect the remarkable reduction of PVR protein level.

Embodiment 17: the inhibition of the RNAi of anti-PVR to adhering to and attacking

The HT1080 cell grows to about 90% earthenware money box in the DMEM that is supplemented with Glutamax (Gibco #31966-021) and 10%FCS (Gibco #10270106).Utilize above-mentioned annealing oligonucleotide preceding 24 hours, the 90% cell trypsinized that is paved with that will from the 175ml Tissue Culture Flask, gather in the crops with 10ml trypsinase-EDTA solution (LifeTechnologies) to transfection.The DMEM substratum of antibiotic-free dilutes this cell suspension at 1: 10 with containing 10%FCS, and is divided into the aliquots containig of 500 μ l, shifts in each hole of 24 well culture plates.Inoculate behind this cell 24 hours, obtain about 50% be paved with.

According to Elbashir et al., the 207th page of described scheme 5 of Methods (2002) 26,199-213 utilizes above-mentioned annealing oligonucleotide to the cell in each hole of transfection.

After carrying out 2 days incubation according to the 5th step of such scheme 5, in 37 ℃ and 7, usefulness Bisbenzimide H 33342 (Sigma #B-2261) original position labeled cell is 15 minutes under the 5%CO2 condition.After the washing, make cell dissociation, it is suspended among 0.1%BSA (Sigma #A7030)/DMEM with 0.5mM EDTA/PBS (Sigma #E8008).Then with 0.1%BSA/DMEM with cell dilution to 6,7 * 10 ⁵Individual cell/ml adds example 8 described chemotactic ponds or example 11 described 96 orifice plates with the HT1080 cell.Then as definite invasiveness as described in the example 8, as definite adhesivity as described in the example 11.

Compare with undressed cell, adhesivity or the invasiveness measured at least one sample 1A/2A or 3A/4A have reduced more than 40%.Compare with undressed cell, in negative control 1B/2B and 3B/4B, do not detect the remarkable reduction of invasiveness.

Embodiment 17.1: the RNAi of anti-PVR is to the inhibition of migration

In similar test, the HT1080 cell grows to about 75% and is paved with in 6 well culture plates, utilizes Oligofectamine and 200nM 5A/B to carry out transfection.After 48 hours,, and use the EGTA/Versene harvested cell with the orange dye marker cell of cell tracker agent.Pair cell counts, and is adjusted to expection concentration to carry out migration test according to example 9.3.

Embodiment 18: at the immunohistochemistry scheme of paraffin slide glass

IHC is at LifeSpan BioSciences, Inc., and Seattle, USA carries out.Utilize slicing machine to obtain the tissue slice of 4-5 micron thickness.This tissue slice is swum in the water-bath, and be transferred on the slide glass.The slide glass dewaxing that utilizes dimethylbenzene and ethanol to make to contain paraffin section, rehydration, and it is implemented method of evaporation to extract target (DAKO reagent #S1700).The operation and the reagent that utilize DAKO automatic staining instrument and follow DAKO research and development carry out the IHC test.Particularly, with above-mentioned slide glass sealing 20 minutes (with DAKO serum-free protein encapsulant #X0909), applicating biotin-people's primary antibody, and in room temperature incubation 2 hours, this slide glass of rinsing in TBST.Application vector ABC-AP (AK-5000) reagent.With carrier Red (SK-5100) as substrate.Used antibody concentration is 200 μ g/ml.In the healthy tissues in suprarenal gland, bladder, brain, breast, colon, new, pancreas, placenta, prostate gland, skin, skeletal muscle, small intestine epithelium, spleen, stomach, thymus gland, Tiroidina or uterus, do not detect corresponding with scFv2* painted.Detect corresponding with scFv2* appropriate painted in rare germinal center cell in kidney, plasmocyte, liver, lung, ovary inner membrance, testis and tonsil.In the situation of cancer, painted be accredited as colorectal carcinoma 2, nonsmall-cell lung cancer 1, ovarian cancer 2, prostate cancer 4, carcinoma of the pancreas 7 and the renal cell carcinoma 4 from faint to appropriateness corresponding with scFv2*.

Embodiment 19: test by the FasL-mediated apoptosis that CALI carries out

Summary: chromophoric group fill-in light passivation (CALI) can be used to the protein (for example PVR) on the passivation HT1080 cell surface.Behind the CALI, utilize Fas part (FasL) (or in contrast) attack cells, with apoptosis-induced with buffer reagent.Compare with the attack of contrast buffer reagent, be measured to the raising of cysteine protease activity after 4 hours.Cysteine protease activity is only to utilize the fluorogenic substrate that just produces fluorescence after by active L-Cysteine HCL Anhydrous cracking to measure.If by the CALI passivation albumen relevant with apoptosis, after adding FasL apoptosis does not appear.

Handle the scFv1 inhibition L-Cysteine HCL Anhydrous activatory ability of identifying by FasL.Behind CALI, observe enhancing to the L-Cysteine HCL Anhydrous reaction of FasL_.This can make an explanation by passivation PVR.

Test design: the HT-1080 human fibrosarcoma cell is distributed in the microtiter plate (MTP), and use with fluorescein isothiocyanate (FITC) bonded scFv1 and carry out incubation.CALI utilizes this FITC-bonded scFv with guiding incandescence passivation specific protein.CALI utilizes the diffused light of elimination blue light to carry out.The MTP that handles with dark condition carries out controlled trial.Behind CALI, handle cell with inducer of apoptosis (FasL).Behind the short period of time incubation, (the preceding fluorogenic substrate with L-Cysteine HCL Anhydrous in the time of lysing cell carries out incubation for Roche, Cat-No.2-236-869) described method according to " homogeneous phase Caspase detection ".The amount of rhodamine-110 of dissociating cracked reflected the specificity of apoptosis early stage/mid-term labeling moiety Gelucystine protease activities.Cysteine protease activity utilizes automatic fluorescence MTP reader to measure.

Utilize with fluorescein bonded commodity anti--CALI that Fas antibody (UB2) carries out Fas acceptor (Fas) causes the function specificity forfeiture of Fas, blocked the signalling of FasL-mediation.Cysteine protease activity for scFv1 significantly reduces (inhibition greater than 20%).There is not the next no CALI influence of situation of any scFv.Sample is divided into three parts assesses, data are the representative data of three independent experiments.

Claims (19)

1. a specific specificity is in conjunction with at least one born of the same parents of CD155 (differentiation bunch 155), poliovirus receptor (PVR) or its any derivative or the molecule of ectodomain, and wherein this molecule has the ability of receptor-mediated adhesion, transportation and/or the invasion and attack behavior of regulating the cell of expressing CD155, PVR or its any derivative.

2. the described molecule of claim 1, wherein this molecule comprises little compound, oligonucleotide, peptide, oligopeptides, polypeptide, protein, antibody, antibody fragment, antiidiotypic antibody and/or biological conjugate.

3. claim 1 or 2 described molecules, wherein this molecule is and can detects and/or can induce mark physics to link to each other.

4. aforesaid right requires any described molecule among the 1-3, and wherein regulating power comprises adhesion, transportation and/or the invasion and attack behavior of inducing, increase, stablize, strengthen, stop, suppress, reduce, destroy and/or weaken by apoptosis the cell of expressing CD155, PVR or its any derivative.

5. aforesaid right requires any described molecule among the 1-4, and the cell of wherein expressing CD155, PVR or its any derivative has participated in proliferative disease or imbalance, has metastatic potential or derives from abiogenous tumour.

6. aforesaid right requires any described molecule among the 1-5, this molecule wherein, and preferred peptide, polypeptide, antibody, antibody fragment or biological conjugate contain aminoacid sequence LWLRRD (SEQID No.:1) and/or WTGDFDY (SEQ ID No.:2).

7. aforesaid right requires any described molecule among the 1-6, this molecule wherein, preferred peptide, polypeptide, antibody, antibody fragment or biological conjugate contain aminoacid sequence SEQ ID No.:3, SEQ ID No.:4, SEQ ID No.:69 or SEQ ID No.:70.

8. aforesaid right requires 6 or 7 described molecules, this molecule wherein, preferred peptide, polypeptide, antibody, antibody fragment or biological conjugate, contain dna sequence dna SEQ ID No.:5, SEQ IDNo.:6, SEQ ID No.:71 or SEQ ID No.:72, or homologous DNA sequence or have degenerated code but still can be translated as the dna sequence dna of the arbitrary amino acid sequence of SEQ ID No.:1-SEQ ID No.:4, SEQ ID No.:69 or SEQ ID No.:70.

9. aforesaid right requires any described molecule among the 1-5, and wherein this molecule is preferably oligonucleotide, especially contains the RNA that is selected from SEQ ID No.:55-62,73 or 74 sequence.

10. identify and separate the method for the described molecule of claim 1-9, comprise the steps:

A) phage library of part is contacted with cancer cell;

B) this cancer cell and bonded part thereof are separated with the part in conjunction with this cell not;

C) remove the phage of this cell of non-specific binding, for example by at described cell not under the cracked condition, with buffered detergent solution washed cell;

D) wash-out and described cell bonded phage; And

E) determine the identity of the part showed by above-mentioned wash-out bacteriophage;

E) utilize biochemistry or biological test to check this part to disturb the ability of PVR function.

The nucleic acid molecule of above-mentioned any one or some described molecules of claim or the part that can identify according to the described method of claim 10 11. encode.

12. contain the carrier of the described nucleic acid molecule of claim 11.

13. comprise the described molecule of claim 1-9, the molecule that can identify according to the described method of claim 10 or the pharmaceutical composition of part, the described nucleic acid molecule of claim 11, the described carrier of claim 12 and/or pharmaceutical acceptable carrier and/or thinner.

14. the described molecule of claim 1-9, the molecule that can identify according to the described method of claim 10 or the described pharmaceutical composition of part, the described nucleic acid molecule of claim 11, the described carrier of claim 12 and/or claim 13 are as the purposes of the medicine that is used to prevent and/or treat proliferative imbalance, cancer or transfer.

15. the method for the adhesion behavior of the cell of external adjusting expression CD155, PVR or its any derivative, this method comprises

A) this cell is contacted with any described molecule among the claim 1-9;

B) this cell is contacted under the condition that is fit to its growth with the ECM egg white layer;

C) adhesion of this cell and ECM egg white layer is analyzed;

D) randomly, after the molecule through chemically modified of the present invention is induced its biologic activity of raising, analyze the adhesion of this cell and ECM egg white layer; And

E) mensuration is compared unmodified molecule bonded cell described by the present invention and/or the per-cent that adheres to once more with inducing molecule bonded cell of having modified described by the present invention with undressed cell.

16. the method for the invasion and attack behavior of the cell of external adjusting expression CD155, PVR or its any derivative, this method comprises:

A) this cell is contacted with any described molecule among the claim 1-9;

B) this cell is contacted under the condition that is fit to its growth with gel-like matrix;

C) analyze of the migration of this cell by gel-like matrix;

F) randomly, after the molecule through chemically modified of the present invention is induced its biologic activity of raising, analyze of the migration of above-mentioned cell by described gel-like matrix; And

G) measure and to compare, unmodified molecule bonded cell described by the present invention and/or the per-cent of having modified with the cell migration of inducing molecule bonded described by the present invention with undressed cell.

17. the method for treatment or prevention proliferative imbalance or disease, cancer and/or transfer, this method comprise to the patient that needs are arranged use the described isolated molecule of claim 1-9 of specified quantitative, according to molecule or part, the described nucleic acid molecule of claim 11, the described carrier of claim 12 and/or the described pharmaceutical composition of claim 13 that claim 10 is identified, described specified quantitative is the amount that can effectively regulate the adhesion, transportation and/or the invasion and attack behavior that are subjected to CD155, PVR or its derivative mediated arbitrarily.

18. comprise the test kit of the described molecule of claim 1-9, the molecule that can identify according to the described method of claim 10 or part, the described nucleic acid molecule of claim 11, the described carrier of claim 12 and suitable test chamber.

19. detect and/or separate the method for the cell of expressing CD155, PVR or its any derivative, comprise the steps:

A) claim 6 or 7 described molecules are contacted with the cancer cell in patient source;

B) separate and molecule bonded cell of the present invention

C) separate and molecule bonded cell of the present invention; And

D) whether the described molecule of check detects and has selected to express the cell of PVR.

Images