CN1905898A - Anti-hydroxylase antibodies and uses thereof - Google Patents

Abstract

Antibodies, or antigen-binding portions thereof, to aspartyl (asparaginyl) beta-hydroxylase are provided. The anti-aspartyl (asparaginyl) beta-hydroxylase antibodies, or antigen-binding portions thereof, can modulate activity of aspartyl (asparaginyl) beta-hydroxylase.

Description

Anti-hydroxylase antibodies and use thereof

The U.S serial number that the application requires to obtain on April 19th, 2004 and application on November 14th, 2003 is the interests of two applications of 60/563,514 and 60/520,114.Therefore, the content of previous two applications is drawn in full be reference here.

The statement of relevant federal funding research

Work described here, the part that once obtained the reasearch funds (number is 9843342) from National Science Foundation is subsidized.Therefore, U.S. government can enjoy certain power in the present invention.

Technical field

The present invention relates to discern the antibody of aspartoyl (asparaginyl-) B-hydroxylase and use these antibody, for example detect aspartoyl (asparaginyl-) B-hydroxylase and/or regulate its active method.

Background knowledge

Hydroxylating (Jia etc., the Proc.Natl.Acad.Sci.USA of the beta carbon of special aspartic acid and aspartoyl residue in aspartoyl (asparaginyl-) B-hydroxylase (being abbreviated as AAH) catalysis numerous protein [analog that comprises extracellular matrix protein, low density lipoprotein, LDL (LDL) receptor, Notch analog and Notch part] the epidermal growth factor-like domain 91(15): 7227-7231,1994; Jia etc., J.Biol.Chem. 267(20): 14322-14327,1992; Gronke etc., Proc.Natl.Acad.Sci.USA 86(10): 3609-2613,1989).Member in the α-Tong Wuersuan dependency dioxygenase family that this enzyme of striding film is prolyl and lysyl hydroxylase.Finder's AAH (HAAH) overexpression in many people's cancer, these cancers comprise hepatocarcinoma, cancer of biliary duct and neuroectodermal tumors (Lavaissiere etc., J.Clin.Investig. 98: 1313-1323,1996; Sepe etc., Lab.Investig. 82(7): 81-891,2002).Express the discovery that increases cell mobility and survival relevant for the overexpression and being forced to of AAH in numerous tumors, show that AAH may play effect (Sepe etc., Lab.Investig. to vicious transformation in the body 82(7): 881-891,2002).

Summary of the invention

The present invention part based on us to can be in conjunction with the discovery of people's single-chain antibody of aspartoyl (asparaginyl-) B-hydroxylase (AAH).Therefore, the main specific antibody that is of this invention, what comprise complete antibody, poly antibody [for example: the tetrameric antibody of people G class (IgG)] and it can be with fragment or other variant of AAH albumen specific bond.These fragments and variant comprise the anti-AAH antibody of strand, poly, and (for example: the tetramer) fragment of anti-AAH antibody or part and other can be with the variants of AAH albumen specific bond.Any anti-AAH antibody, its fragment or variant can be handled by sudden change, for example by a kind of affinity maturation (affinity maturation) method.

Compositions of the present invention comprises that anti-AAH antibody (for example: human monoclonal antibodies), its fragment or variant, the pharmaceutical composition that contains these and test kit.Antibody, its fragment or variant can, but not necessary, suppress one or several AAH biological activitys (for example: external, the hydroxylating of the natural or suitable substrate that non-natural exists during cell or tissue is cultivated or in the body) and/or one or the cellular activity (for example: cell growth, propagation or mobility) regulated of several AAH.Because improper cell proliferation and mobility pathological abnormalities (as cancer) too active with AAH or overexpression accompanies occur, so the compositions among the present invention can be used for differentiating the unusual patient of this kind, its prognosis of assessment and/or treats the patient who suffers from this disease or the danger of this disease of development (for example: cancer or other are correlated with and AAH overexpression or too active) is arranged.

More specifically, characteristics of the present invention are to identify that the patient that is suitable for treating (for example: the method patient who carries the cell of undue active or overexpression AAH) and the treatment or the prevention method (for example: the bonded human monoclonal antibodies of specificity and HAAH or people's single-chain antibody (scFv)) for the treatment of this patient with the anti-AAH antibody (or its fragment or other variant) of effective dosage.The present invention also provides the relevant nucleic acid that is used to express antibody of the present invention, the carrier that contains those nucleic acid, the cell that contains those nucleic acid or carrier, comprise anti-AAH antibody and/or its fragment or other variant the medicine acceptable composition compound method, identify and/or estimate the method for the characteristic of anti-AAH antibody (as: anti-HAAH antibody) and/or its fragment or other variant, and the sophisticated method of affinity that makes anti-AAH antibody (as: the anti-HAAH antibody of people) and/or its fragment or other variant.

For ease of reading, we no longer reuse the phrase of " and/or its fragment or other variant " in " antibody " each back that occurs.Should be understood that, no matter where anti-AAH antibody is applicable to that the antibody fragment or the variant that are attached to any available degree with AAH albumen specifically also can be used for this place.When being mentioned to " AAH ", we also can use " AAH albumen ".Unless the opposite meaning is clear and definite, otherwise we exchange use " albumen " when mentioning two or more amino acid residues, " peptide " reaches " polypeptide ".Similarly, no matter where the nucleic acid of the anti-AAH antibody of coding is applicable to that the functional fragment of the anti-AAH antibody of encoding or the nucleic acid of other variant also can be used for this place; Where be applicable to that the cell that coding contains anti-AAH antibody fragment or other variant also can be used for this place no matter contain the cell of anti-AAH antibody; And so on.Anti-AAH antibody fragment also can occur with the wording of anti-AAH antibody " antigen-binding portion thereof ", because this fragment can be with the bonded part of AAH antigen-specific.When hereinafter mentioning fragment and variant, note that at this they comprise Fab, Fab ' and F (ab ') ₂Fragment, and comprise scFvs (for example, with the bonded people scFv of HAAH antigen-specific).

Anti-AAH antibody, its fragment or its other variant can be made up of the aminoacid sequence in the table among Figure 34-38, maybe can comprise these sequences.Alternate, anti-AAH antibody can by with the figure of table among the 34-38 in aminoacid sequence show that the sequence of homology to a certain degree forms, maybe can comprise these sequences.For example: antibody can contain the variable region of heavy chain (VH) that at least 80% (as: 85%, 90%, 95%, 98% or 100%) homology is arranged with one of VH sequence shown in Figure 34 and 35.Displaced, these antibody can be with Figure 36 in one of the scFv that shows the scFv of at least 80% (as: 85%, 90%, 95%, 98% or 100%) homology is arranged.Displaced, antibody, its fragment or other variant therefore can comprise with Figure 37 and 38 in the complementary determining region (CDRs) that shows the CDRs of at least 40% (as: 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95% or 98%) homology is arranged.Other fragment and variant see below.

The antibody cocoa comprises variable light chain (VL or VLC), and described light chain comprises first CDR that contains aminoacid sequence Ser-Gln-Ser/Asn-Val-Ser-Ser/His-(Xaa)-Tyr/His-Leu-Ala (SEQ IDNO:320) (or be made up of above-mentioned sequence); The twoth CDR that contains aminoacid sequence Asp-Val-Ala-Asn-Xaa-Ala-Ala (SEQ ID NO:321) (or form by above-mentioned sequence); The 3rd CDR that contains aminoacid sequence Gln-Gln-Arg-Ser-Gln-Trp-Pro-Gln (SEQ IDNO:322) (or form by above-mentioned sequence).Unless special mark, otherwise " Xaa " is any amino acid residue or do not have amino acid residue in these sequences and following sequence.Alternate (or in addition), antibody can comprise VHC, and VHC comprises that containing aminoacid sequence is first CDR of Tyr/His-Ala-Met-His/Gly (SEQ ID NO:323) and to contain aminoacid sequence be the twoth CDR of Tyr-Ala-Xaa-Ser-Val-Lys-Gly/Ser (SEQ ID NO:324).In other embodiment in other embodiment, antibody can comprise VLC, VLC contain comprise aminoacid sequence Ser-Gly-Ser-Ser-Asn-Ile-Gly/Glu-Ser-Asn-His/Tyr-Val-His/ Tyr (or form by above-mentioned sequence) CDR (for example: first CDR) (SEQ ID NO:325).Displaced (or in addition), antibody can comprise VHC, and VHC contains: the CDR that comprises aminoacid sequence Ser/Gly-Asp/Asn-Ser/Gly-Ala-Ala-Trp-Ser/Asn (SEQ IDNO:326) (or be made up of above-mentioned sequence) (for example: first CDR) and comprise the twoth CDR of aminoacid sequence Arg-Ile/Thr-Tyr/His-Tyr/His-Gly/Arg-Xaa-Lys/Arg-Trp/Arg-Trp/Arg-Asn-Asp/Gly-Tyr/His-Ala-Val/Ala-Pro/Ser-Val/Ala-Lys-Ser (SEQ ID NO:327) (or be made up of above-mentioned sequence).In other embodiment, antibody can comprise contain aminoacid sequence Asp-Val-Xaa-Xaa-Arg-Pro-Ser (SEQID NO:328) CDR (for example: variable light chain the twoth CDR).Alternate, antibody can contain aminoacid sequence be Leu-Phe/Leu-Ile/Val-His/Tyr-Lys/Arg-Xaa-Asn-Gln-Arg-Pro-Ser (SEQ ID NO:329) (or form by above-mentioned sequence) CDR (for example: the twoth CDR), and optional, the CDR that contains aminoacid sequence and be Ala-Trp-Asp-Asp-Ser (SEQ IDNO:330) (or be made up of above-mentioned sequence) is (for example: the 3rd CDR).For example, the 3rd CDR is made up of Ala-Ala-Trp-Asp-Asp-Ser-Leu-Arg-Gly-Tyr-Val (SEQ IDNO:51) sequence.Antibody also can comprise VHC, VHC comprise contain aminoacid sequence be Ser-Ser-Ser-Trp-Val-Val-Xaa-Phe-Asp/Gly (SEQ ID NO:331) (or form by above-mentioned sequence) CDR (for example: variable heavy chain the 3rd CDR) (for example, Thr-Gly-Tyr-Ser-Ser-Ser-Trp-Val-Val-Asn-Phe-Asp-Tyr (SEQ IDNO:96)).

No matter its sequence how, any antibody among the present invention can be monoclonal (for example: single special) or polyclonal antibody; Any antibody can be the people's or humanized; Any antibody can be the affinity maturing; Any antibody can be separable (as: antibody is purified to a certain degree from the animal that produces this antibody or cell).People's antibody comprises the variable region with ethnic group system (germline) immunoglobulin sequences and the antibody of constant region.These antibody can have all or part of of human immunoglobulin heavy chain, and light chain is all or part of.This is not that the antibody that means (people or inhuman) among the present invention must contain naturally occurring sequence.Antibody among the present invention, can comprise can't help ethnic group be the immunoglobulin nucleic acid sequence encoding amino acid residue (for example: external at random or site-specific sudden change).The sophisticated antibody of the antibody of relevant sudden change or affinity is further described below asking for an interview.

These antibody be not limited to those in distinctive mode with the bonded antibody of AAH, they can with the catalyst structure domain (for example, the catalyst structure domain of HAAH) of AAH in conjunction with, they can also combine the conformation that changes catalyst structure domain in some way or reduce its activity with AAH.The repressed degree of bonded AAH antigen active can be different.In the scope of the invention, useful antibody (or its fragment and other variant) comprises that delivering the back to patient (for example: the catalytic activity of HAAH) to those antibody of clinical significant degree or the degree that takes effect suppresses the AAH activity in analyzed in vitro.For example: antibody can suppress 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% (as: about 95%, 98% or 99%) of the enzymatic activity of AAH or HAAH.This inhibition can be with respect to contrasting, estimating with reference to specimen or standard substance.Alternate, anti-AAH antibody can not suppress the AAH activity with AAH albumen specific bond.Such antibody can be used for the patient that detects AAH and be used to differentiate the cell with overexpression AAH.Described authentication method can for example suppress AAH biological activity (as: catalytic activity) and carrying out before imposing anti-AAH antibody to patient.

Can be according to the ability that in cell culture, suppresses to suppress in growth of tumour cell or mobility and/or the body growth of tumour cell or transfer, further qualitative or estimate antibody of the present invention.Also can measure the affinity of antibody.For example: (for example: affinity HAAH) is equal to or less than 1 μ M to the antibody among the present invention to AAH albumen.Also can estimate the competition of antibody of the present invention and other antibody.For example: the antibody among the present invention (as, people or humanized antibody) can be attached to the combinative epitope of murine antibody with anti-AAH murine antibody (for example: monoclonal antibody FB50 or 15C7) competition.Very clear and definite, FB50 and 15C7 be not within category of the present invention.Also can estimate antibody of the present invention and the ability of clone's 11 antibody ((seeing Figure 34) described herein) competition with the epitope of clone's 11 antibodies.Effectively with the antibody of cloning 11 antibody competitions, within category of the present invention.

As described above, we can use term " specific combination " or " specifically in conjunction with ", mention the ability of antibody aspect following: (1) with effective affinity (for example: the affinity of at least 1 * 106M-1) be attached to AAH; Or (2) are to combine with AAH greater than (for example: Senior Two doubly at least) affinity to heterogenetic antigen; Or (3) (for example: indication or the symptom of improving patient's (object of suffering from cancer or other abnormal cellular proliferation etc.) that needs treat) affinity combines with AAH to be enough to obtain the clinical curative effect of wanting.

These antibody can comprise that one or several Fc domains are (for example: the Fc domain of γ isotype (IgG1) in resembling).We use term " isotype ", refer to the type (for example: IgM, IgA, IgE, IgD or IgG1) by the antibody of weight chain constant area gene coding on conventional meaning.Antibody of the present invention can be any isotype.Alternate, or in addition, these antibody can comprise a kind of labelling (for example: as a token of thing (marker) or report thing polypeptide of sequence, or help the polypeptide of antibody from its sequence purification that adheres to).The labelling that is fit to comprises FLAG label, histadine label, zymoprotein or fluorescin.Alternate, or these antibody can comprise toxin in addition.

In others, the present invention is characterised in that: (for example: the isolated nucleic acid molecule of sequence anti-AAHScFv) comprise code book invention antibody; Contain code book invention antibody nucleotide sequence expression vector (for example: plasmid); And contain the host cell (for example: prokaryotic cell or eukaryotic cell, for example yeast, mammalian cell (for example Chinese hamster ovary cell (CHO) or tumor cell (as, myeloma cell))) of a class or number these nucleic acid molecules of class or expression vector.Anti-AAH ScFv (for example: IgGs) can be easy to be transformed into the anti-AAH antibody of poly.More specifically, the present invention is characterised in that the antibody that cell surface (for example: be illustrated on the yeast cells) is expressed.(for example: the recombinant expression carrier transfection is prepared antibody to host cell mentioning when preparing antibody with recombinant technique; From the reorganization, the combinatorial antibody library separation antibody; Animal (as mice) by human immunoglobulin gene's metastatic gene goes up separation antibody; Or anyly prepare antibody to the mode of other DNA sequence through the montage human immunoglobulin gene), we can use term " reorganization ".Recombinant antibodies comprise antibody that humanized or CDR transplants, chimeric antibody and external (for example: the antibody of Chan Shenging display technique of bacteriophage).These antibody can comprise from ethnic group being the antibody of immunoglobulin sequences constant region.

Feature of the present invention also is to contain one or more above-listed compositionss and directions for use (no matter its form; Whether be printing, that can listen or visual) test kit.For example: the test kit among the present invention can comprise anti-AAH antibody (freeze dried, conc forms or be suspended in physiology with the finite concentration (as: with the concentration that is suitable for carrying out diagnostic analysis or is administered into patient) that be fit to use accept in the diluent).Test kit also can comprise nucleic acid described herein, carrier and/or host cell.Optional, any test kit can comprise (as syringe needle, syringe, cotton ball soaked in alcohol and the binder) that is used for the anti-AAH antibody of administration or be used for the paraphernalia of diagnostic analysis (as: with the reagent that compares) among the present invention.

Method of the present invention comprises the method that AAH expresses of estimating in cell.Cell can be the cell of the too active or overexpression of the cell people, inhuman and/or AAH, can be cells in vivo or maintain cell in the tissue culture, can be carcinogenic (as: tumor cell), and can be the cell that obtains from any types of organization (lung, liver, colon, pancreas, prostate, ovary, bile duct, brain or mammary gland) basically.Cell can be complete or incomplete (for example: available tissue homogenate, available albumen by cell purification).Described method can comprise: provide anti-AAH antibody (any antibody described here is available), same cell (tissue homogenate or by cell in the albumen of purification) contacts grace time and detection antibody or its antigen-binding portion thereof (for example: by Western engram analysis, SABC or other detection method based on antibody) with antibody under allowing antibody and the bonded condition of the AAH of cellular expression.Qualitative and the quantitative assessment that these determination steps can provide relevant AAH to express, and can compare with the result and from the data that contrast or sample for reference (or standard substance) obtains.Can be with implementing such method effectively with the antibody of anti-AAH specific bond, and do not influence the activity of AAH.These methods can be used as the part whether the diagnosis patient is fit to the trial for the treatment of with AAH inhibition antibody among the present invention.

Method of the present invention comprises that also adjusting (for example: the inhibition) method of AAH protein active in the cell.As use above-mentioned evaluation methodology, cell can for the people, inhuman cell and/or AAH be too active or the cell of overexpression, it can be the cell in cells in vivo or the tissue culture, can be carcinogenic (as: tumor cell), and can be the cell (as the cell of lung, liver, colon, pancreas, prostate, ovary, bile duct, brain or mammary gland) that obtains from any types of organization.These methods can comprise the submission cell and with cellular exposure in antibody described here (its fragment or other variant) and be enough to regulate in (for example: suppress) cell the competent time of effect under the active condition of AAH.When cell is exposed to anti-AAH antibody in vivo, these methods can be described to treatment and suffer from disease () patient or occur danger () people's the method for example: the too active or overexpression of AAH, but do not detect tumor as yet or have other tumor indication of this disease for example: cancer or other improper cell proliferation.These methods can comprise that to patient's administration antibody described here (its fragment or other variant) amount of administration and time are enough to make antibody to suppress the propagation and the transfer of patient's cancerous cell.When Therapeutic Method is further described below, we notice that the compositions that contains anti-AAH antibody can be by topical (for example: to knub position or the tumor residual tissue place after by excision), also can be by whole body administration (for example: through intravenous injection).Patient can accept the anti-AAH antibody of monotype, also can accept the combination medicine-feeding of a plurality of antibody; Single antibody (or a plurality of antibody) can be with another reagent (for example: chemotherapeutics, analgesic or antiemetic) administering drug combinations.

Refer to that with the relevant term of treatment compositions of the present invention used or be administered into patient or from patient's cell.Compositions can be the nucleic acid molecules or the expression vector of anti-AAH antibody, the anti-AAH antibody of coding or the host cell of expressing anti-AAH antibody (above-mentioned any form can make up with acceptable diluent on the physiology).Compositions can exsomatize and be administered into from the experimenter, preferably needs the cell of patient's separation (for example taking out) of treatment.After treatment finished, cell can send back in the patient body.In addition, treatment can be preventative.For example: the patient that these antibody can be applied to the danger that develops into tumor (has very perfect dangerous indication, (for example tumor associated antigen, for example: the level of PSA and AAH itself)).Treatment can be the sort of treatment of curing or restoring patient, but the present invention is not limited to this.Method of the present invention can also make the indication symptom of cancer or the cancer-prone trend of patient alleviate, and relaxes, changes, improves, slows down or improve.

" the effectively therapeutic dose " of anti-AAH antibody is meant and treats disease (for example: cancer, for example tumor or other vegetation or abnormal development syndrome) patient's the sign or the amount of the required anti-AAH antibody of symptom effectively." effectively preventive dose " of anti-AAH antibody is meant (for example: the cancer) appearance of outbreak or recurrence or alleviate its indication or the amount of the required anti-AAH antibody of symptom to delay disease effectively.

The patient who is suitable for treating can describe below, and notice that here these patients suffer from or overexpression relevant any proliferative disorder too active with AAH.For example: patient can have the tumor of the lung, liver, colon, pancreas, prostate, ovary, bile duct, brain or the mammary gland that contain the AAH positive cell.

On the other hand, the present invention is characterised in that the method for identifying specificity and the bonded antibody of AAH.These methods comprise, for example: (a) provide antibody library (as: people's antibody, it can be scFvs); (b) allowing antibody to combine with polypeptide under the condition, AAH albumen or its fragment are contacted with the member in library; And (c) filter out and the bonded antibody of AAH albumen (or its AAH fragment).This method can further comprise makes selected antibody affinity maturation.Ripe available following method obtains, and for example: the nucleic acid of fallibility (Error-prone) polymerase chain reaction (PCR) method or encoding antibody for example uses the method for CDR reorganization or the reorganization of chain shuffling technology with library nucleic acid.Feature of the present invention also is to prepare the method for specificity and the bonded human monoclonal antibodies of AAH.These methods can comprise the following steps: to differentiate the antibody with AAH albumen specific bond; In cell, express the nucleotide sequence of encoding antibody; And by separating the antibody of expressing in the cell.We can be called production method with these methods, and the anti-AAH antibody for preparing by these methods is within the scope of the invention.

Based on different reasons, the antibody among the present invention is favourable.For example: when non-full length antibody was used, the fragment of antibody or variant can penetrate tumor easilier.When the people's or humanized antibody when being administered into patient, they can not stimulate undesired immunoreation consumingly as the non-human protein.Antibody among the present invention also can not produce other undesired side effect, maybe can't detect because HAAH hangs down in non-carcinogenic tissue expression amount very much; And, to compare with non-specific treatment, the antibody that is directed to HAAH is less to the toxicity of non-carcinogenic tissue.According to our understanding, we have made bonded people's antibody with HAAH first.

All are publication, patent application, patent, and other reference as mentioned herein, and are incorporated by reference in full.Further feature of the present invention and advantage will become easier to understand in following detailed Description Of The Invention and claims.

The accompanying drawing summary

Fig. 1 is described as screening and combination research, shows the schematic diagram of yeast cell surface fusion rotein with the yeast surface Display Technique.The antigen of also describing different epi-position labels among the figure and can be used for detecting.The fluorescent antigen that uses in the test described herein is the reorganization HAAH albumen that is equivalent to total length or only is equivalent to catalyst structure domain.

Fig. 2 is in scFv clone who is described in the three strain uniquenesses of expressing on the yeast cells and the experiment of hatching with fluorescence HAAH, a picture group of the fluorescence that detects by flow cytometer.

Fig. 3 is in the experiment of hatching of scFv clone who is described in the eight strain uniquenesses of expressing on the yeast cells and the fluorescently-labeled HAAH fragment that contains catalyst structure domain, a picture group of the fluorescence that detects by flow cytometer.

Fig. 4 is a picture group of describing the affinity mensuration of two kinds of anti-HAAH scFv antibody clonings.

Fig. 5 describes in the experiment that a scFv clone expressing on the yeast cells hatches with fluorescently-labeled HAAH catalyst structure domain or bonded total length HAAH, a picture group of the fluorescence that detects by flow cytometer.As scheme indication, the total length HAAH albumen of labelling is to use FB50IgG or 15C7IgG to detect.

Fig. 6 describes segmental 6 unique clones of solubility scFv and the bonded block diagram of HAAH catalyst structure domain.

Fig. 7 describes segmental 6 unique clones of solubility scFv and the bonded block diagram of total length HAAH.

Fig. 8 is described in segmental 3 the unique clones of scFv to be attached in the experiment of H640 tumor cell, a picture group of the fluorescence that detects by flow cytometer.

Fig. 9 is described in to lack antibody (first cylindricality) and have segmental 5 the unique clones of scFv, under the situation of 15C7 (Mus IgG), and the block diagram of active H640 tumor cell percentage ratio.

Figure 10 is described in from clone's sophisticated scFv fragment of affinity of 11 to be attached in the experiment of HAAH catalyst structure domain, the figure of the fluorescence that detects by flow cytometer.

Figure 11 is the description of the sophisticated clone's 11 of relevant affinity the segmental aminoacid sequence of scFv.With respect to clone 11, the change of the amino acid residue of appearance is shown as runic or has underscore.

Figure 12 be the scFv fragment that is described in original clone 11, first round sudden change clone 11 the scFv fragment and come from clone's the analysis experiment of first round sudden change one group of scattergram of the two class fluorescence that detect by flow cytometer.The fluorescence intensity that X-axis is described is corresponding to the segmental level of the scFv that shows on the yeast cells.The fluorescence intensity that Y-axis is described is corresponding to the level that is attached to cell HAAH.

Figure 13 describes in clone 11 the segmental analysis experiment of scFv of sudden change, one group of scattergram of the two class fluorescence that detect by flow cytometer.The fluorescence intensity that X-axis is described is corresponding to the segmental level of the scFv that shows on the yeast cells.The fluorescence intensity that Y-axis is described is corresponding to the level that is attached to cell HAAH.

Figure 14 is described in from the ripe scFv fragment of clone's affinity of 13 to be attached in the experiment of HAAH catalyst structure domain, the figure of the fluorescence that detects by flow cytometer.

Figure 15 is the sophisticated clone's 13 of relevant affinity a scFv fragment, the description of the aminoacid sequence of 13m1.With respect to clone 13, the change of the amino acid residue of appearance is shown as runic or has underscore.

Figure 16 be the clone 13 of the scFv fragment of describing original clone 13, first round sudden change the scFv fragment, and second take turns in the analysis experiment of clone scFv of sudden change one group of scattergram of the two class fluorescence that detect by flow cytometer.The fluorescence intensity that X-axis is described is corresponding to the segmental level of the scFv that shows on the yeast cells.The fluorescence intensity that Y-axis is described is corresponding to the level that is attached to cell HAAH.

Figure 17 is the relevant sketch map that carries the DNA plasmid in the restriction endonuclease site that is used for chain reorganization.

Figure 18 A is a table, lists wild type clone's variable heavy chain and variable light chain district, and by 5 clones of wild clone through chain reorganization generation.

Figure 18 B describes producing the analysis experiment of set of mutant clon through chain reorganization from wild clone 11, the scattergram of the two class fluorescence that detect by flow cytometer.The fluorescence intensity that X-axis is described is corresponding to the segmental level of the scFv that shows on the yeast cells.The fluorescence intensity that Y-axis is described is corresponding to the level that is attached to cell HAAH.

Figure 19 A-K describes in the clone's that chain reorganization produces analysis experiment, scattergram (Figure 19 A: clone LLm1 of the two class fluorescence that detect by flow cytometer; Figure 19 B: clone LLm3; Figure 19 C: clone LLm5; Figure 19 D: clone LLm6; Figure 19 E: clone LLm7; Figure 19 F: clone LLm8; Figure 19 G: clone LLm9; Figure 19 H: clone LLm11; Figure 19 I: clone LLm14; Figure 19 J: clone LLm15 (3); Figure 19 K: clone LLm20 (8)).The fluorescence intensity that X-axis is described is corresponding to the segmental level of the scFv that shows on the yeast cells.The fluorescence intensity that Y-axis is described is corresponding to the level that is attached to cell HAAH.

Figure 20 is the description of the aminoacid sequence in relevant wild type scFv clone and 11 clones' producing through chain reorganization CDR1, CDR2 and CDR3 zone.

Figure 21 is that relevant encoding wild type is cloned the receptor dna plasmid of 11 scFv and the people library insertion sequence sketch map of recombinating with the plasmid that is used for CDR reorganization.

Figure 22 A-D describes in the analysis experiment of reorganizing the clone who produces through chain, detects scattergram (Figure 22 A:CM1 of two class fluorescence by flow cytometer; Figure 22 B:CM2; Figure 22 C:CM3; Figure 22 D:CM4).The fluorescence intensity that X-axis is described is corresponding to the segmental level of the scFv that shows on the yeast cells.The fluorescence intensity that Y-axis is described is corresponding to the level that is attached to cell HAAH.

Figure 23 is the description of the aminoacid sequence in relevant wild type scFv clone and 4 clones' producing through chain reorganization CDR1, CDR2 and CDR3 zone.

Figure 24 be describe the segmental concentration of anti-HAAH scFv sift out with respect to anti-HAAH in conjunction with active figure.

Figure 25 describes the sketch map that the scFv fragment changes into total length IgG antibody.

Figure 26 A-B describes with the anti-HAAH IgG of enzyme linked immunosorbent assay wild type antibody with the bonded figure of HAAH.The working concentration of every kind of antibody marks in coordinate figure below.

Figure 27 describes with the anti-HAAH IgG of cells were tested by flow cytometry wild type antibody with the bonded figure of H460 tumor cell.The working concentration of antibody is labeled on the coordinate figure.

Figure 28 describes the anti-HAAH IgG that sifts out with cells were tested by flow cytometry to suddenly change antibody with the bonded coordinate figure of H460 tumor cell.The working concentration of antibody is labeled on the coordinate figure.

Figure 29 A describes 6-22 IgG antibody concentration with respect to the bonded figure of H460 cell (with fluoremetry).

Figure 29 B describes 6-22 IgG antibody concentration with respect to the bonded figure of FOCUS cell (with fluoremetry).

Figure 30 A describes CDRm4 IgG antibody concentration with respect to the bonded figure of H460 cell (with fluoremetry).

Figure 30 B describes CDRm4 IgG antibody concentration with respect to the bonded figure of FOCUS cell (with fluoremetry).

Figure 31 is described under shortage or the situation of existence from the competition of 6-22 IgG antibody, and scFv 6-22 is with the bonded figure of H460 cell.

Figure 32 A is described in to lack or exist under the situation of CDRm4 IgG antibody competition, and HAAH is attached to the figure that expresses on the segmental yeast of different scFv.

Figure 32 B is described in to lack or exist under the situation of LLm11 IgG antibody competition, and HAAH is attached to the figure that expresses on the segmental yeast of different scFv.

Figure 33 A is that description HAAH is attached to the figure on the yeast that shows scFv CDRm4 and second filial generation sudden change scFvC4m18.The dissociation constant mark in the drawings.

Figure 33 B is the figure that is described in the reveal competence of the first generation and second filial generation scFv sudden change on the yeast.

Figure 34 comprises to can be used for maybe can being included in anti-AAH antibody, its fragment or other becomes the table of intravital aminoacid sequence.

Figure 35 is the table that comprises and can be used for maybe can being included in anti-AAH antibody, its fragment or other variant aminoacid sequence of (for example: anti-HAAH affinity is sophisticated, the antibody regions of sudden change).

Figure 36 comprises (for example: the table of aminoacid sequence anti-HAAH scFv antibody) to can be used for maybe can being included in anti-AAH antibody, its fragment or other variant.

Figure 37 comprises can be included in anti-AAH antibody, its fragment or other becomes the table of intravital aminoacid sequence (anti-AAH antibody CDRs).

Figure 38 can be included in the chart that anti-AAH antibody, its fragment or other become intravital aminoacid sequence (affinity is sophisticated, the anti-HAAH antibody CDRs of sudden change).

Detailed Description Of The Invention

Aspartoyl (asparaginyl-) B-hydroxylase (being abbreviated as AAH) is the KG dependence dioxygenase of a high conservative, this enzymatic numerous protein, comprise extracellular matrix albumen, low-density lipoprotein (LDL) acceptor, Notch analog and Notch part analog, the epidermal growth factor-like domain in hydroxylating (Jia etc., Proc.Natl.Acad.Sci.USA after the translation of beta carbon of special aspartic acid and aspartoyl residue91(15): 7227-7231, 1994; Jia etc., J.Biol.Chem.267(20): 14322-14327,1992; Gronke etc., Proc.Natl.Acad.Sci.USA86(10): 3609-2613,1989). Finder's AAH (HAAH) overexpression in many cancers of people, these cancers comprise hepatocellular carcinoma, cholangiocarcinoma And neuroderm tumour (Lavaissiere etc., J.Clin.Investig.98: 1313-1323,1996; Sepe etc., Lab.Investig.82(7): 881-891,2002). Relevant for AAH in numerous tumours In overexpression and the expression of the reinforcement discovery that increases cell mobility and survival, show AAH Contribute to vicious transformation (Sepe etc., Lab.Investig. in the body82(7): 881-891,2002). Relevant Use the invention of HAAH diagnosis and treatment cancer in following patent, to describe (Radosevich, U.S. Patent No. 6,166,176; Radosevich, U.S. Patent No. 6,727,080; Wands etc., U.S. Patent No. 6,783,758; Wands etc., U.S. Patent No. 6,797,696; Wands etc., U.S. Patent No. 6,812,206; Wands etc., U.S. Patent No. 6,815,415).

The present invention relates to have with the antibody of the binding specificity of the part of AAH or AAH and Its antigen-binding portion thereof. The present invention is special, and the people's monoclonal of being combined with the AAH specificity of setting forth is anti-Body. In one embodiment, antibody or its antigen-binding portion thereof are to people's AAH (HAAH) tool Specificity is arranged. The antibody that suppresses one or more functional characteristics of AAH is at model of the present invention Within enclosing, no matter this function be enzymatic activity (for example: hydroxylase activity), still show cell water Flat function (for example: promote Tumor Cell Migration). Therefore, for example, anti-AAH antibody can press down The antibody of Tumor Cell Migration processed. That substitute or in addition, the antibody among the present invention also can suppress (to subtract Less or prevent) AAH and natural part, for example: as extracellular matrix albumen, contain EGF The interaction of the albumen in spline structure territory. For (for example: special combination) people's monoclonal of AAH Antibody can suppress the function that AAH mediates, and comprises through hydroxylating and regulates the substrate activity. Preferably, Antibody and its antigen-binding portion thereof are to be higher than 1 * 10⁶M ^-1Affinity in conjunction with AAH.

The amino acid sequence of HAAH can be at GenBank^In with enter number I38423 (GI: 7433245) find. At present, those skilled in the art can be easily at GenBank^Or other This sequence is found in the source. The membrane spaning domain of HAAH is specified and is present in GenBank^Sequence 341-374 amino acid between. The extracellular of this molecule (or chamber) partial response is in carboxyl terminal. Anti-AAH antibody, (the example no matter total length whether, will interact with the fragment of AAH and this albumen Such as combination) (for example: anti-HAAH antibody can be attached to HAAH). This antibody can be incorporated into AAH Or the epi-position of the fragment of total length AAH albumen (for example: conformation or linear epi-position). Work as exposure When the sex change solvent, comformational epitope can be lost usually.

Antibody: the antibody among the present invention can present different configurations. For example: antibody can be The tetramer (antibody has two heavy chains and two light chains) also can be single-chain antibody. Therefore, this Antibody in bright comprise have one or two variable region of heavy chain and have one or two light chain can Become the albumen in district. It is certainly complementary that but variable region of heavy chain and variable region of light chain Further Division are called after The hypervariable region in fixed district (CDRs) and called after skeleton district (FRs), scatter high conservative region therebetween The territory. The degree of CDR and FR be defined (referring to Kabat, E.A., etc.Sequences of Proteins of Immunological Interest, the 5th edition, U.S.Department of Health and Human Services, NIH Publication No.91-3242,1991 and Chothia, etc., J. Mol.Biol.196: 901-917,1987, these draw as a reference at this). In the present invention Antibody when comprising one or more variable region of heavy chain and/or one or more variable region of light chain, every Variable region of heavy chain and variable region of light chain can comprise by three complementary determining regions and four frameworks The district, according to the order from the amino terminal to the carboxyl terminal, be arranged as FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

The chain of the variable region of heavy chain of antibody or variable region of light chain among the present invention can further comprise heavily Chain or constant region of light chain all or part of. In one embodiment, two immune balls of weight are arranged In the tetramer antibody of protein chain and two light immunoglobulin chains, heavily reach light immunoglobulin chain By interconnecting such as disulfide bond. CH comprises three domain: CH1, CH2 And CH3. Constant region of light chain comprises domain a: CL. Heavy chain and variable region of light chain contain can Binding structural domain with AI. The common mediate antibody of the constant region of antibody is attached to the place Main tissue or the factor comprise cell different in the immune system (such as the effector cell) and classical complement system First composition (C1q) in the system]. Term " antibody " comprises type I gA, IgG, IgE, IgD IgM with and hypotype (as: IgG₁、IgG ₂、IgG ₃And IgG₄) complete immune globulin In vain, its light chain can be κ or λ type.

Antibody: is referred to as " immunoglobulin (Ig) " (substantially by one of immunoglobulin gene coding The albumen that individual or a plurality of polypeptide form. Perhaps anti-AAH antibody also be referred to as anti-among the present invention The AAH immunoglobulin (Ig), and can contain people's one or more immunoglobulin genes coding Sequence). Know others immunoglobulin gene, comprised κ, λ, α (IgA₁And IgA₂)、 γ(IgG ₁、IgG ₂、IgG ₃、IgG ₄), δ, ε and μ constant region gene, and countless Immunoglobulin variable district gene. Total length immunoglobulin (Ig) " light chain " (about 25kDa and 214 Amino acid), be by being positioned at amino terminal variable region gene (about 110 amino acid) and being positioned at carboxyl terminal κ or λ constant region gene code. Similarly, total length heavy chain immunoglobulin (about 50kDa And 446 amino acid), above-mentioned by variable region gene (about 116 amino acid) and one other () The constant region gene, for example, γ, (about 330 amino acid of encoding) coding. Resisting among the present invention Body or immunoglobulin (Ig) can comprise people or inhuman CDRs (further describing at this). Immune globulin White skeleton can be the people, humanized or inhuman, for example, is modified to reduce the people The skeleton of the antigenicity Muridae in the body; Or for example be total to homotactic synthetic skeleton.

" antigen-binding portion thereof " this term of antibody (or is called for short " antibody moiety " as used herein Or " part "), relate to specificity and a kind of AAH (as, HAAH) one of the antibody of combination one Divide, for example, contain one or more non-total lengths but can be with the immunity of the special combination of AAH The molecule of globulin chain. Wrap in " its fragment (or antigen-binding portion thereof) or its variant " this term The example of the bound fraction of drawing together comprises: (i) Fab fragment, and namely by VLC, VHC, CL and CH1 The unit price fragment that consists of; (ii) F (ab ')₂Fragment is at two Fabs of hinge region by disulfide bridge connects The divalence fragment that fragment forms; (iii) the Fd fragment that is consisted of by VHC and CH1 domain; (iv) The Fv fragment that is consisted of by antibody single armed VLC and VHC domain; (v) consisted of by the VHC domain DAb fragment (Ward etc., Nature341: 544-546,1989); (vi) have enough specificitys In conjunction with skeleton, such as the complementary determining region of the separation of the antigen-binding portion thereof of variable region (CDR). Use the restructuring method can make variable region of light chain by synthetic joint antigen-binding portion thereof with The antigen-binding portion thereof of variable region of heavy chain, for example: two domains of Fv fragment, VLC and VHC Be merged into single protein chain, wherein VLC and VHC zone pairing formation monovalent molecule (is called as ScFv (scFv) is asked for an interview: the Science such as Bird242: 423-426,1988 and Huston etc., Proc.Natl.Acad.Sci.USA 85:5879-5883,1988). Such single-chain antibody is also wrapped Be contained in the term of " antigen-binding portion thereof " of antibody. At present, use those skilled in the art The technology of known routine can obtain the part of these antibody; And, can use and measure The mode that the practicality of complete antibody is identical is screened these parts. Available papain cracking four Polymers antibody obtains the Fab fragment; Also available pepsin cracking, and acquisition Fab ' and F (ab ') 2 Fragment.

As used herein, term " people's antibody " comprises that skeleton structure residue is corresponding to the people's Kind system and CDRs come from any antibody of V (D) J restructuring and somatic mutation. Yet, People's antibody also can comprise and not be that ethnic group is the amino acid residue (example of immunoglobulin (Ig) nucleic acid sequence encoding As: at random external or sudden change that mutation site-specific is introduced). Existing demonstration, the variable base of people Somatic mutation because of in vivo causes the sudden change of framework residue (to see Nat.Immunol.2: 537, 2001). Although there is the skeleton sudden change to occur, such antibody still is called " people's ", to indicate it The source. Mouse antibody variable territory is also contained somatic mutation and (is seen Sem. in framework residue Immunol8: 159,1996). Like this, the transgenic mice that contains human immunoglobulin(HIg) (Ig) site The antibody that produces is referred to as the antibody of " total man ", usually although they have average 4.5 (its scope is 1-8 in following document, sees Nat Genet.1997 Feb in the skeleton sudden change; 15 (2): 146-56). Therefore, acceptable use shows, is sequence based on kind but do not have logical Cross for example antibody variable territory of the skeleton sudden change of the interior somatic mutation method introducing of body, ordered " people's " by name. Like this, with people's antibody of AAH combination (for example, the present invention includes specifically HAAH is although these antibody contain (as the sudden change among the FR) sudden change] and fragment or variant are (for example, Contain people's antibody VLC of polymerization and the single-chain antibody of VHC). For example, the people's antibody among the present invention 1-8 skeleton sudden change (such as 2,4,6 or 8 displacements, insertion or disappearance) can be arranged. Preferably, first The sequence of beginning people antibody is the sequence of ethnic group system.

Make as described here HAAH is had specific people's single-chain antibody. In concrete reality Execute in the scheme, the invention provides HAAH is had specificity and is attached to antibody described herein (for example: derivative clone's 11 codings or the ripe clone 11 who produces of the affinity) table of combination The position. Can be by (for example: thin with HAAH being attached to HAAH with EGF spline structure territory competition Born of the same parents, for example HAAH transfectant or H460 tumour cell) ability, identify and with in this description The antibody that combines of the overlapping epi-position of epi-position of antibody combination. The binding site of anti-AAH antibody, Can be in the catalyst structure domain of HAAH.

These anti-AAH antibody can be monoclonal, also to be polyclone. Describe anti-at this Body and antigen-binding portion thereof thereof, in the composition and therapy that can be used for treating, the diagnosis composition And differentiate or suppress in the analysis of reagent of AAH albumen in the method and at needs. The present invention Comprise and can be used for treatment (comprising prevention) or diagnosis (for example: specifically disease or illness, such as cancer) Antibody or its antigen-binding portion thereof, and make usefulness with antibody or its antigen-binding portion thereof Medicine in treatment disease described here or illness.

Single-chain antibody and antibody chimeric, humanized or that CDR transplants, and comprise to come From the chimeric or humanized single-chain antibody of system of the same race not, also be included in the present invention and term In " antibody ". Available conventional art with the different piece chemical bond of these antibody to together or The different piece of these antibody is prepared into the polypeptide of adjacency with genetic engineering technology. For example: compile Code nucleic acid chimeric, humanized chain can be expressed to produce the polypeptide of adjacency. See: Cabilly Deng, U.S. Patent No. 4,816,567; Cabilly etc., European patent No.0,125,023 B1; Boss etc., U.S. Patent No. 4,816,397; Boss etc., European patent No.0,120,694 B1; Neuberger, M.S. etc., WO 86/01533; Neuberger, M.S. etc., European patent No. 0,194,276 B1; Winner, U.S. Patent No. 5,225,539 and Winter, European patent No. 0,239,400 B1. The antibody that relevant CDR transplants is seen Newman etc., BioTechnology10: 1455-1460,1992; Relevant single-chain antibody is seen Ladner etc., U.S. Patent No. 4,946,778 and Bird, R.E. etc., Science242：423-426，1988。

In addition, comprise chimeric, humanized, CDR antibody that transplant or single-chain antibody Antigen-binding portion thereof also can be manufactured and be comprised within the category of the present invention. Resisting of antibody Former bound fraction keeps at least a binding function of its original full length antibody. Preferred antigen Bound fraction (for example: special to AAH keeps antigen binding function corresponding to full length antibody The property). Function fragment can keep full length antibody and suppress the one or more of distinctive merits of AAH Can, for example: the hydroxylase activity of AAH. For example, function fragment can suppress the EGF spline structure The hydroxylating in territory. The guarantor who forms repetitive sequence in different albumen is contained in these EGF spline structure territories Keep motif, described albumen such as clotting factor, extracellular matrix albumen, LDL receptor, The analog of Notch analog or Notch part. Any AAH substrate comprises and just having described Those all can be used for estimating the analysis of anti-AAH antibody. The substrate that is used for the AAH analysis of example, Comprise EGF-IX1_1H[gronke etc., Proc Natl Acad Sci USA86(10)：3609-13， 1989]，EGF-X _1H(Gronke etc., J.Biol.Chem.265: 8558-8565,1990) and EGF-Asn (Gronke etc., J.Biol.Chem265: 8558-8565,1990; Wang etc., J. Biol.Chem.266：14004-14010，1991)。

For example: can with antibody moiety or its fragment of AAH combination, comprise Fv, Fab, Fab ' And F (ab ') 2 fragments. Can produce such part through enzymatic lysis or restructuring technology. For example wooden Melon protease or pepsin cracking can produce respectively Fab or F (ab ') 2 fragments. Also can be from antibody The form of the different brachymemma of gene produces antibody, one or more termination codons in the described gene Son has been introduced in the upstream of natural termination site. F (ab ') the 2 heavy chains part of for example encoding Mosaic gene can be designed to comprise the DNA order of coding CH1 domain and heavy chain hinge region Row.

The invention provides chimeric antibody, available genetic engineering technology is prepared into the many of connection with antibody (DNA of the chimeric antibody protein part of for example encoding can be expressed to produce the many of a kind of connection to peptide Peptide chain). Chimeric antibody example is among the present invention, comprises CDR (for example, in this description One or more CDR of antibody) and from the skeleton of another light chain of antibody and/or heavy chain (for example the people originates in the district; Have or do not have the antibody that CDR that skeleton changes transplants) one or many Individual antibody chain. In one embodiment, inosculating antibody physical efficiency and mouse 15C7 or FB50 monoclonal are anti-The body competition is attached to HAAH. The antigen binding domain of chimeric antibody can derive from described here anti-Body clone (for example: clone 11 or clone 11 mutant, for example: contain clone 11 light chain CDR1, CDR2 and CDR3 and the chimeric antibody that contains clone 11 heavy chain CDR1, CDR2 and CDR3). The single-chain antibody chimeric or CDR transplants also comprises humanized immunoglobulin (Ig). Referring to: Cabilly etc., U.S. Patent No. 4,816,567; Cabilly etc., European patent No. 0,125,023 B1; Queen etc., European patent No.0,451,216 B1; Boss etc., the U.S. is special Sharp No.4,816,397; Boss etc., European patent No.0,120,694 B1; Neuberger, M.S. Deng, WO 86/01533; Neuberger, M.S. etc., European patent No.0,194,276 B1; Winner, U.S. Patent No. 5,225,539 Winter, European patent No.0,239,400 B1; Padlan, E.A. etc., European patent Application No.0,519,596 A1. Relevant strand is anti-Body, also visible Ladner etc., U.S. Patent No. 4,946,778; Huston, U.S. Patent No. 5,476,786 and Bird, R.E. etc., Science242：423-426，1988。

Can use technology synthetic and/or restructuring nucleic acid to prepare the gene of the chimeric chain of coding expectation (as, cDNA), prepare chimeric antibody. Change the encoding antibody chain with PCR sudden change method The dna sequence dna nucleic acid that can rebuild the coding variable region (such as, DNA) sequence, for example use For the production of the method for humanized antibody (ask for an interview: Kanunan, etc., Nucl.Acids Res.17: 5404,1989; Sato, etc., Cancer Research53: 851-856,1993; Daugherty, etc., Nucleic Acids Res.19(9)：2471-2476，1991；Lewis and Crowe，Gene  101: 297-302,1991). Use these or other method of being fit to, also can easily prepare variant. In one embodiment, clone's variable region can be suddenlyd change, and coding has the special of expectation The sequence of property variant can be selected that (Tathagata is from the bacteriophage library; Referring to: Krebber etc., U.S. State patent No.5,514,548; Hoogenboom etc., WO 93/06213, is published in 1993 4 The moon 1).

Other preparation or the appropriate methodology that separates anti-AAH antibody are, for example: depend on that immunity can the complete storehouse of people's antibody transgenic animals (as, method mouse) (referring to: Jakobovits etc., Proc.Natl.Acad.Sci.USA90: 2551-2555,1993; Jakobovits etc., Nature362: 255-258,1993; Lonberg etc., U.S. Patent No. 5,545,806; Suran etc., U.S. State patent No.5,545,807).

Can and screen the member of being combined with AAH in the library through expressing recombinant antibody in the library, Differentiate the antibody that specificity is combined with AAH. By for example PCR sudden change, chain reorganization or CDR Shuffling technology and combination make these antibody affinities with one or more screenings circulations described here Maturing further strengthens the antibody of selection thus to the affinity of AAH. Other method, Also can be used for producing anti-AAH antibody. For example: anti-AAH antibody can produce by immune animal Give birth to. Many methods that are described are arranged, for the preparation of the antigen of immunity usefulness and from the animal system of immunity Standby anti-AAH antibody (referring to: Kohler etc., Nature256：495-497，1975；Kohler and Milstein，Eur，J.Immunol. 6: 511-519,1976; Milstein etc., Nature266: 550-552,1977; Koprowski etc., U.S. Patent No. 4,172,124; Harlow, E.and D.Lane, 1988, Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press:Cold Spring Harbor, N.Y.); Current Protocols In Molecular Biology, Vol.2 (Supplement 27, and Summer ' 94), Ausubel, F.M. Deng, Eds., (John Wiley ﹠ Sons:New York, N.Y.), Chapter 11,1991]. Usually, The permanent cell line (such as myeloma cell line) that is fit to and the cell that produces antibody are merged mutually, can Generate hybridoma. Produce the cell of antibody, preferred splenocyte or LNC can be from immunity Animal obtain. Fused cell (hybridoma), available selective condition of culture and limited dilution cloning Separated. Available suitable analysis (such as ELISA) is selected generation and is had the specificity of expectation The cell of antibody.

Antibody or its antigen-binding portion thereof can comprise for example Weight variable sequence, and this district is with following One of sequence has 80% (as: 85%, 90%, 95%, 98% or 100%) homology at least. This A little sequences are: SEQ ID NO:1, and SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO: 25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:119, SEQ ID NO:121, SEQ ID NO:123, SEQ ID NO:125, SEQ ID NO:127, SEQ ID NO:129, SEQ ID NO:131, SEQ ID NO:133, SEQ ID NO:135, SEQ ID NO:137, SEQ ID NO:139, SEQ ID NO:141, SEQ ID NO:143, SEQ ID NO:145, SEQ ID NO:147, SEQ ID NO:254, SEQ ID NO:256, SEQ ID NO:258, SEQ ID NO:260, SEQ ID NO:262, SEQ ID NO:264, SEQ ID NO:266, SEQ ID NO:268, and SEQ ID NO:270.

Antibody or its antigen-binding portion thereof can comprise, variable light chain district for example, and this district is with lower One of row sequence has 80% (as: 85%, 90%, 95%, 98% or 100%) homology at least. These sequences are: SEQ ID NO:2, and SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:30, SEQ ID NO:120, SEQ ID NO:122, SEQ ID NO:124, SEQ ID NO:126, SEQ ID NO:128, SEQ ID NO:130, SEQ ID NO:132, SEQ ID NO:134, SEQ ID NO:136, SEQ ID NO:138, SEQ ID NO:140, SEQ ID NO:142, SEQ ID NO:144, SEQ ID NO:146, SEQ ID NO:148, SEQ ID NO:255, SEQ ID NO:257, SEQ ID NO:259, SEQ ID NO:261, SEQ ID NO:263, SEQ ID NO:265, SEQ ID NO:267, SEQ ID NO:269, and SEQ ID NO:271.

，，，SEQ ID NO：1 80％(：85％、90％、95％、98％100％)， SEQ ID NO：280％(：85％、90％、95％、98％100％) ；SEQ ID NO：380％(：85％、90％、95％、 98％100％)，SEQ ID NO：480％(： 85％、90％、95％、98％100％)；SEQ ID NO： 580％(：85％、90％、95％、98％100％)， SEQ ID NO：680％(：85％、90％、95％、98％100％) ；SEQ ID NO：780％(：85％、90％、95％、 98％100％)，SEQ ID NO：880％(： 85％、90％、95％、98％100％)；SEQ ID NO： 980％(：85％、90％、95％、98％100％) ，SEQ ID NO：1080％(：85％、90％、95％、98％100％) ；SEQ ID NO：1180％(：85％、90％、 95％、98％100％)，SEQ ID NO：12 80％(：85％、90％、95％、98％100％)；SEQ ID NO：1380％(：85％、90％、95％、98％100％) ，SEQ ID NO：1480％(：85％、90％、95％、 98％100％)；SEQ ID NO：1580％(： 85％、90％、95％、98％100％)，SEQ ID NO： 1680％(：85％、90％、95％、98％100％) ；SEQ ID NO：1780％(：85％、90％、95％、 98％100％)，SEQ ID NO：1880％(：85％、90％、 95％、98％100％)；SEQ ID NO：19 80％(：85％、90％、95％、98％100％)， SEQ ID NO：2080％(：85％、90％、95％、98％100％) ；SEQ ID NO：2180％(：85％、90％、95％、 98％100％)，SEQ ID NO：22 80％(：85％、90％、95％、98％100％)；SEQ ID NO：2380％(：85％、90％、95％、98％100％) ，SEQ ID NO：2480％(：85％、90％、95％、 98％100％)；SEQ ID NO：2580％(： 85％、90％、95％、98％100％)，SEQ ID NO： 2680％(：85％、90％、95％、98％100％) ；SEQ ID NO：2780％(：85％、90％、95％、98％100％) ，SEQ ID NO：2880％(：85％、90％、 95％、98％100％)；SEQ ID NO：29 80％(：85％、90％、95％、98％100％)， SEQ ID NO：3080％(：85％、90％、95％、98％100％) ；SEQ ID NO：11980％(：85％、90％、95％、 98％100％)，SEQ ID NO：120 80％(：85％、90％、95％、98％100％)；SEQ ID NO：12180％(：85％、90％、95％、98％100％) ，SEQ ID NO：12280％(：85％、90％、95％、 98％100％)；SEQ ID NO：123 80％(：85％、90％、95％、98％100％)， SEQ ID NO：12480％(：85％、90％、95％、98％100％) ；SEQ ID NO：12580％(：85％、90％、95％、 98％100％)，SEQ ID NO：126 80％(：85％、90％、95％、98％100％)；SEQ ID NO：12780％(：85％、90％、95％、98％100％) ，SEQ ID NO：12880％(：85％、90％、95％、 98％100％)；SEQ ID NO：129 80％(：85％、90％、95％、98％100％)， SEQ ID NO：13080％(：85％、90％、95％、98％100％) ；SEQ ID NO：13180％(：85％、90％、95％、 98％100％)，SEQ ID NO：132 80％(：85％、90％、95％、98％100％)；SEQ ID NO：13380％(：85％、90％、95％、98％100％) ，SEQ ID NO：13480％(：85％、90％、95％、 98％100％)；SEQ ID NO：135 80％(：85％、90％、95％、98％100％)， SEQ ID NO：13680％(：85％、90％、95％、98％100％) ；SEQ ID NO：13780％(：85％、90％、95％、 98％100％)，SEQ ID NO：138 80％(：85％、90％、95％、98％100％)；SEQ ID NO：13980％(：85％、90％、95％、98％100％) ，SEQ ID NO：14080％(：85％、90％、95％、 98％100％)；SEQ ID NO：141 80％(：85％、90％、95％、98％100％)， SEQ ID NO：14280％(：85％、90％、95％、98％100％) ；SEQ ID NO：14380％(：85％、90％、95％、 98％100％)，SEQ ID NO：144 80％(：85％、90％、95％、98％100％)；SEQ ID NO：14580％(：85％、90％、95％、98％100％) ，SEQ ID NO：14680％(：85％、90％、95％、 98％100％)；SEQ ID NO：147 80％(：85％、90％、95％、98％100％)， SEQ ID NO：14880％(：85％、90％、95％、98％100％) ；SEQ ID NO：25480％(：85％、90％、95％、 98％100％)，SEQ ID NO：255 80％(：85％、90％、95％、98％100％)；SEQ ID NO：25680％(：85％、90％、95％、98％100％) ，SEQ ID NO：25780％(：85％、90％、95％、 98％100％)；SEQ ID NO：258 80％(：85％、90％、95％、98％100％)， SEQ ID NO：25980％(：85％、90％、95％、98％100％) ；SEQ ID NO：26080％(：85％、90％、95％、 98％100％)，SEQ ID NO：261 80％(：85％、90％、95％、98％100％)；SEQ ID NO：26280％(：85％、90％、95％、98％100％) ，SEQ ID NO：26380％(：85％、90％、95％、 98％100％)；SEQ ID NO：264 80％(：85％、90％、95％、98％100％)， SEQ ID NO：26580％(：85％、90％、95％、98％100％) ；SEQ ID NO：26680％(：85％、90％、 95％、98％100％)，SEQ ID NO：267 80％(：85％、90％、95％、98％100％)； SEQ ID NO：26880％(：85％、90％、95％、98％100％) ，SEQ ID NO：26980％(：85％、90％、 95％、98％100％)；SEQ ID NO：270 80％(：85％、90％、95％、98％100％)， SEQ ID NO：27180％(：85％、90％、95％、98％100％) 。

In different embodiments, antibody or its antigen-binding portion thereof can comprise complementary determining Fixed district (CDRs), this district and one of SEQ ID NOs:46-81 or 281-313 have at least 40% (as: 40%, 60%, 80%, 85%, 90%, 95%, 98% or 100%) homology.

In different embodiments, antibody or its fragment or variant comprise the complementary determining region (CDRs) that has at least 40% (as: 40%, 60%, 80%, 85%, 90%, 95%, 98% or 100%) homology with one of SEQ ID NOs:46-51,54,56,82-117,164-250 or 314-319.Antibody, its fragment or other variant also maybe can comprise: have the variable heavy chain district of 80% homology at least with SEQ ID NO:1, and have the variable light chain district of 80% homology at least with SEQ ID NO:2; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:3, and have the variable light chain district of 80% homology at least with SEQID NO:4; Have 80% homology variable heavy chain district at least with SEQ ID NO:5, and have the variable light chain of 80% homology district at least with SEQ ID NO:6; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:7, and have the variable light chain district of 80% homology at least with SEQ ID NO:8; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:9, and have the variable light chain district of 80% homology at least with SEQ ID NO:10; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:11, and have the variable light chain district of 80% homology at least with SEQ ID NO:12; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:13, and have the variable light chain district of 80% homology at least with SEQ ID NO:14; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:15, and have the variable light chain district of 80% homology at least with SEQ ID NO:16; Have the variable light chain district of 80% homology at least with SEQ ID NO:17, and have the variable light chain district of 80% homology at least with SEQ ID NO:18; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:19, and have the variable light chain district of 80% homology at least with SEQ ID NO:20; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:21, and have the variable light chain district of 80% homology at least with SEQ ID NO:22; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:23, and have the variable light chain district of 80% homology at least with SEQ ID NO:24; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:25, and have the variable light chain district of 80% homology at least with SEQ ID NO:26; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:27, and have the variable light chain district of 80% homology at least with SEQ ID NO:28; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:29, and have the variable light chain district of 80% homology at least with SEQ ID NO:30; With SEQ ID NO:119 have at least 80% homology the variable heavy chain district, and have the variable light chain district of 80% homology at least with SEQ IDNO:120; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:121, and have the variable light chain district of 80% homology at least with SEQ ID NO:122; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:123, and have the variable light chain district of 80% homology at least with SEQ ID NO:124; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:125, and EQ ID NO:126 have the variable light chain district and the S of 80% homology at least; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:127, and have the variable light chain district of 80% homology at least with SEQ ID NO:128; Have the variable heavy chain district of 80% homology at least with SEQ IDNO:129, and have the variable light chain district of 80% homology at least with SEQ ID NO:130; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:131, and have the variable light chain district of 80% homology at least with SEQ ID NO:132; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:133, and have the variable light chain district of 80% homology at least with SEQ ID NO:134; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:135, and have the variable light chain of 80% homology district at least with SEQ ID NO:136; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:137, and have the variable light chain of 80% homology district at least with SEQ IDNO:138; Have 80% homology variable heavy chain district at least with SEQ ID NO:139, reach the variable light chain district that has 80% homology with SEQ ID NO:140 at least; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:141, and have the variable light chain district of 80% homology at least with SEQ IDNO:142; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:143, and have the variable light chain district of 80% homology at least with SEQ ID NO:144; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:145, and have the variable light chain district of 80% homology at least with SEQ ID NO:146; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:147, and have the variable light chain district of 80% homology at least with SEQ ID NO:148; Have 80% homology variable heavy chain district at least with SEQ ID NO:254, reach the variable light chain district that has 80% homology with SEQ ID NO:255 at least; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:256, and have the variable light chain district of 80% homology at least with SEQ ID NO:257; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:258, and have the variable light chain of 80% homology district at least with SEQ ID NO:259; Have the variable heavy chain district of 80% homology at least with SEQ IDNO:260, and have the variable light chain of 80% homology district at least with SEQ ID NO:261; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:262, and have the variable light chain district of 80% homology at least with SEQ ID NO:263; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:264, and have the variable light chain district of 80% homology at least with SEQ ID NO:265; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:266, and have the variable light chain district of 80% homology at least with SEQ ID NO:267; Have the variable heavy chain district of 80% homology at least with SEQ ID NO:268, and have the variable light chain district of 80% homology at least with SEQID NO:269; Or have the variable heavy chain district of 80% homology at least, and has the variable light chain district of 80% homology at least with SEQ ID NO:271 with SEQ ID NO:270.

As used herein, term " basically identical " (or " basic homology ") refers to first aminoacid or nucleotide sequences, (for example: have similar side chain contain the identical or amino acid residue that is equal to of q.s or nucleoside with the twoth aminoacid or nucleotide sequence, for example, conservative amino acid replacement), first and the twoth aminoacid or nucleotide sequences have similar activity like this.With antibody is example, and the twoth antibody has the specificity same with first antibody, and has at least 50% affinity of first antibody.

Can calculate " homology " or " homogeneity " between two sequences with following method.Be to realize the purpose of best comparison, (for example: be best alignment, can or introduce vacancy in two first and the twoth aminoacid or nucleic acid simultaneously at one with sequence alignment; And, for the benefit of compare, can ignore non-homogeneous sequence).In different embodiments, be used for comparison and the length of the canonical sequence that aligns is 50% of this sequence length at least.Then, relatively be in the amino acid residue or the nucleotide of corresponding amino acid position or nucleic acid position.When position in first sequence by the twoth sequence in the same amino acid residue of relevant position or nucleotide when occupying, then molecule is same in this position.Homogeneity percentage ratio between two sequences is that the quantity with consistent position total between sequence becomes, and also the quantity of vacancy and the length of each vacancy will be taken into account, and these vacancies are the required introducings of the best alignment of two sequences.

Can use mathematical algorithm, finish relatively reaching of sequence and measure two sequence homology percentage ratios.Use Needleman and Wunsch (J.Mol.Biol.48:444-453,1970) algorithm, measure the percentage homology of two aminoacid sequences, this rule has been integrated into the GAP program of GCG software kit, here use BLOSUM 62 rating matrixs (scoring matrix), vacancy punishment 12 minutes, vacancy prolong (gap extend) punishment 4 minutes, and framework moves punishment 5 minutes.

As used herein, term " is hybridized under basic, normal, high or high stringent condition " and has been described hybridization and washing condition.Can be at Current Protocols in Molecular Biology (JohnWiley ﹠amp; Sons, N.Y.6.3.1.-6.3.6., 1989) find the guidance of carrying out hybridization in the book, and be introduced into reference here.Described two kinds of methods of water and non-water in the document, any one can use.Here the concrete hybridization conditions of mentioning is as follows: 1) low stringent hybridization condition, be in 6x sodium chloride/sodium citrate (SSC) solution, react under about 45 ℃ of conditions, be accompanied by with 0.2xSSC, 0.1% SDS is twice flushing of at least 50 ℃ (for low stringent hybridization condition, the flushing temperature can be brought up to 55 ℃); 2) in stringent hybridization condition, be in 6x sodium chloride/sodium citrate (SSC) solution, react under about 45 ℃ of conditions, be accompanied by with 0.2xSSC, 0.1% SDS 60 ℃ one or repeatedly the flushing; 3) high stringent hybridization condition is in 6x sodium chloride/sodium citrate (SSC) solution, reacts under about 45 ℃ of conditions, be accompanied by with 0.2xSSC, 0.1% SDS 65 ℃ one or repeatedly the flushing; 4) high stringent hybridization condition is at the 0.5x sodium phosphate, in the 7%SDS solution, reacts under about 65 ℃ of conditions, be accompanied by with 0.2xSSC, 1% SDS 65 ℃ one or repeatedly the flushing.

Be to be understood that, antibody among the present invention and antigen-binding site thereof, (" nonessential " amino acid residue is to be produced by the change of wild type peptide sequence can to have other displacement conservative or non essential amino acid, bonding agent for example, antibody for example, what had does not change biologic activity basically, and " essential " amino acid whose change then can produce the change of biologic activity).

A special displacement is accepted, that is to say, to for example not producing adverse influence in conjunction with active such biological characteristics, can by Bowie etc. (Science, 247: 1306-1310,1990) method described measures." conservative amino acid replacement " means, with (originally) amino acid residue that has the amino acid residue of similar side chain to replace.Amino acid residue family with similar side chain, the existing definition in this area.These families comprise: the aminoacid (as: lysine, arginine, histidine) with basic side chain; Aminoacid (as: aspartic acid, glutamic acid) with acid side-chain; Aminoacid (as: agedoite acid, glutamate, Glu, serine, threonine, tyrosine, cysteine) with uncharged polar side chain; Aminoacid (as: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan) with non-polar sidechain; Aminoacid (as: threonine, valine, histidine) with the ramose side chain of β; Aminoacid (as: tyrosine, phenylalanine, tryptophan, histidine) with aromatic side chain.

As described here, the antibody among the present invention can suppress the AAH hydroxylase activity, and/or can suppress the relevant function of hydroxylase, for example cell mobility.Describe below can be used for estimating to the active inhibition of AAH and with the diverse ways of the inhibition of this active correlation function.

Binding analysis

" mammal AAH albumen " used herein refer to naturally occurring or endogenic mammal AAH albumen and have with the albumen of naturally occurring or endogenic mammal AAH albumen same acid sequence (for example: recombiant protein).Correspondingly, here Ding Yi this term (going back) comprises the polymorphism of mammal AAH or allele variant, other isoform (for example: produce through alternative splicing or other cell processes) and aforesaid modification or non-modified forms (for example: glycosylated, nonglycosylated).AAH albumen can be isolating and/or the albumen of reorganization (comprising the synthetic albumen that produces).Naturally occurring or endogenic mammal AAH can be from the source of natural generation AAH (for example: tumor cell) obtain or isolate.

AAH proteic " functional variant " comprises, functional fragment, functional mutain and/or functional fusion rotein (for example: through the albumen of sudden change and/or recombinant technique generation).Usually AAH protein fragments or part comprise having with respect to sophisticated mammal AAH aminoacid, and those albumen (as: N-terminal, C-terminal or inner disappearance) of (one or more) disappearance are arranged.

Usually, the AAH protein mutant comprises, through inserting, lack and/or replace the proteic natural or artificial variant of AAH that amino acid residue one or more adjacency or non-adjacent produces.Such sudden change can be at conservative region for example, also at non-conservative region, extracellular, Cytoplasm or stride the film district.

AAH proteic " functional fragment or part ", " functional sudden change " and/or " functional fusion rotein ", refer to have the albumen or the polypeptide of the isolating of at least a functional characteristic of AAH albumen described here and/or reorganization, described functional characteristic is hydroxylase activity for example.

Comprise isolating and/or the mammal AAH of reorganization or the compositions of its part, can keep under the bonded condition being suitable for; Receptor contacts with antibody to be tested, detects or measure this combination.In one embodiment, receptor protein can be expressed in the stable or of short duration cells transfected at the cell of expressing AAH or with the nucleotide sequence construction that contains coding AAH or its part.Cell maintains under the condition that is suitable for expression of receptor.Be suitable for bonded condition (as: in the binding buffer liquid that is being fit to), cell contacts with antibody; Detect combination by standard technique then.For measuring combination, can decide bonded degree (for example: compare with respect to suitable contrast with there not being the background under the antibody situation; With the binding ratio of second antibody (that is standard substance); With antibody with the binding ratio of non-transfected cells).The cell fraction that contains AAH, for example the film fraction can be used for substituting full cell.

In one embodiment, come traget antibody, measure antibody by the certification mark thing with suitable label (for example: fluorescent labeling, isotopic labeling, enzyme labelling).In another embodiment, the second antibody with labelling detects bonded antibody.Can estimate bonded specificity with competition or the method for replacing.For example: use unlabelled antibody or part as competitor.

Also can be used for differentiating in conjunction with inhibition analysis and combine with AAH and suppress for example bonded antibody in EGF spline structure territory of other chemical compounds.For example binding analysis can detect or measure when having antibody to exist the combination of part and compares, with the bonded minimizing in EGF spline structure territory (antibody does not exist under the situation).AAH can contact simultaneously with the albumen and the antibody that contain EGF spline structure territory, also can contact in succession with any order with them.The minimizing of protein binding degree when antibody exists is indicating antibody to suppress combination.For example the combination in EGF spline structure territory can be lowered or eliminate.

Still there is other method to differentiate and the existence of the bonded antibody of AAH that for example other suitable binding analysis maybe can be monitored the active incident that starts of AAH, as the method for cytometaplasia or cell mobility.

Should be appreciated that can be with the inhibition effect of estimating antibody among the present invention in conjunction with inhibition analysis.To bonded competition, also can estimate in this way between two antibody.The antibody that in this way identifies can further be estimated to conclude whether they suppress other function of AAH and/or assess their treatment practicality after combination.

Measure the analysis of antibody activity

Under there was situation in ferrous iron, AAH was with the aspartic acid in proteic epidermal growth factor (EGF) spline structure territory or the beta carbon hydroxylating of aspartoyl residue.Contain conservative CX ₇CX ₄CX ₁₀CXCX ₈The EGF spline structure territory of C sequence is present in many different kinds in the albumen, for example: the analog of thrombin, extracellular matrix protein, low density lipoprotein receptor, Notch analog and Notch part.Can directly detect the hydroxylating of AAH substrate by the hydroxylase activity analysis, the hydroxylating that can also come indirect determination AAH substrate by the biological activity that for example uses detection hydroxylating downstream.

Whether the anti-AAH antibody that can analyze to measure the people can suppress the AAH activity, this analysis be level that hydroxyl is drawn in the enzyme reaction when test antibody is existed and the parallel reaction that does not have chemical compound comparison or with predetermined control value relatively.The hydroxylase analysis of standard is known, referring to, Lavaissiere etc. for example, J.Clin.Invest. 98: 1313-1323,1996; Jia etc., J.Biol.Chem. 267: 14322-14327,1992; Wang etc., J.Biol.Chem. 266: 14004-14010,1991; Or Gronke etc., J.Biol.Chem. 265: 8558-8565,1990.Can be in microtitration plate, with carbon dioxide ( ¹⁴CO ₂Catch analysis) measure hydroxylase activity (Zhang etc., Anal.Biochem. 271: 137-142,1999).

Can measure the active adjusting of AAH by the biochemistry influence of checking the hydroxylating downstream, described life influence comprises the influence to specific substrates and cell internal procedure.The active cell mobility that improves of AAH, propagation, survival and cell cycle progress.The AAH activity is suppressed and can measure by the reduction that detects one of above-mentioned these processes under the condition that antibody exists by antibody.

Use mobility for example to analyze analysis of cells mobility's adjusting.Usually, the suitable cell (as tumor cell) of mobility's analysis monitoring towards high-level chemical compound (as: somatomedin or polypeptide) from a surface of barrier towards another opposite surface orientation motion or migration enters or by barrier (as: filter).The barrier that filter membrane or filter are provided convenience, suitable like this cell enter or by filter toward the direction of high-level chemical compound, by a surface of barrier towards another opposite surperficial direction sense movement or move can be monitored.Can detect or measure that cell in the suitable containers is entered by a chamber or enter the inhibition situation of the migration in another chamber that separates through film and first chamber of containing test antibody by microporous membrane.Selection has the monitoring of being applicable to the reaction of chemical compound and the filter membrane that is fit in the suitable aperture of specificity migration, and for example such film can comprise celluloid, Merlon.The aperture of for example using is the 3-8 micron.

Be to estimate the inhibition of migration or migration, can use standard technique (as: microscope) to measure to pass through the filter postadhesion at the filter second surface and be accumulated in the cell quantity in second chamber.In one embodiment, come labeled cell with detectable labelling (as: radiosiotope, fluorescent labeling, antigen or epi-position labelling), exist or be not present under the situation in second chamber at antibody, estimate migration by mensuration absorption and film and/or the existence that is present in the labelling in second chamber with suitable method (as: detecting radioactivity, fluorescence, immunoassay).The inductive extent of migration of antibody agonist can (for example: the background migration of measuring during with shortage antibody relatively with respect to the contrast that is fit to; The extent of migration that causes with second chemical compound (as standard) compares; The migration of the non-transfected cells that causes with antibody is relatively) measure.When antibody existed, the extent of migration that is caused by chemical compound (as: calf serum) descended, and was to suppress active indication.

In one embodiment, cell is placed in the epicoele that Boyden chamber type is cultivated insert (cultrue insert) under the serum-free culture condition.For the migration stimulus object is provided, the culture medium that contains the 1-2% hyclone is put in cavity of resorption.About 4 hours of cultured cell occurs to allow migration.The cell in the quantitative upper and lower chamber of difference.Can use as ATPLite ^TM(Perkin Elmer; Document Sepe etc., Lab.Invest.82:881-891, describes mobility's analytical method in 2002) such ATP monitoring system, quantitatively living cells in each chamber.

The AAH overexpression is relevant with the propagation and the vicious transformation of cell.The method of cell characteristics that can be by measuring the cell malignant phenotype is analyzed the active inhibition of AAH, and described malignant phenotype for example transforms, do not rely on the cell proliferation with anchor and tumorigenicity in nude mice.Can estimate conversion (Copeland andCooper, Cell 16 (2): 347-56,1979) with AAH rotaring copolymering NIH 3 T 3 cell and the quantity of observing transforming focus.Can and contain 0.4% low melting point agarose complete medium with AAH transfectional cell, separation transfectant, it is layered on and contains on the 0.53% low melting point agarose culture medium bottom, and middle suspension culture transfectant is analyzed and do not relied on the cell proliferation that anchor.

Give nude mice with clone's (or tumor cell of expression AAH) injection of AAH transfection, analyze intravital tumorigenicity.In common analysis, about 1,000,000 to 10,000,000 cells of subcutaneous injection after the week to one month of growing, kill and take out tumor with animal and weigh.Can be in the animal of plantation AAH transfectant or tumor cell injection of antibodies, (for example: saline or non-specific antibody the cell growth fraction of) animal is measured antibody with accepting the contrast injection with the growth of the cell implanted.

In these are analyzed, have and suppress the efficient that active antibody can reduce cell growth, conversion and tumorigenicity.

Diagnosis and treatment are used

Antibody among the present invention can be used for multiple application, comprises research, diagnosis and treatment application.In one embodiment, come traget antibody with suitable labelling (for example: fluorescent labeling, chemiluminescent labeling, isotopic labeling, determinant or enzyme labelling).

The overexpression of HAAH is relevant with vicious transformation.Blocking-up or the inhibition active antibody of HAAH or its antigen-binding site can be used for suppressing transformation and/or diagnosis cell transformed.Therefore, the invention provides the relevant active method of AAH that suppresses to express the cell of AAH or its part, comprise with effective dose with AAH or the bonded antibody of AAH part or its antigen-binding site and cells contacting.Cell can be cell subgroup (as: tumor cell), and antibody or its antigen-binding site can be delivered in body and give the experimenter.The treatment of antibody or its antigen-binding site is used, and comprises prophylactic use (for example being used for tool is developed into the patient's of cancer danger treatment).

Antibody or its antigen-binding site, can with for example anticarcinogen administration simultaneously of one or more other therapeutic agent.The nonrestrictive example of anticarcinogen comprises: anti-microtubule agent, topoisomerase enzyme inhibitor, antimetabolite, mitotic inhibitor, alkylating agent, intercalator (intercalatingagent), interfering signal transduction by way of reagent, the reagent that promotes apoptosis (comprising cell death gene), radioactivity chemical compound and at the antibody (comprising naked antibody, immunotoxin and radiation conjugate) of other tumor associated antigens.The following example that the particular type anticarcinogen is provided: anti-tubulin/anti-microtubule, for example: paclitaxel, vincristine, vinblastine, vindesine, vinorelbine, taxotere; The topoisomerase I inhibitor, for example: hycamtin (topotecan), camptothecine, amycin, etoposide, mitoxantrone, rubidomycin,, idarubicin, teniposide, amsacrine, epirubicin, merbarone, piroxantrone hydrochlorate; Antimetabolite, for example: five fluorouracil (5-FU), methotrexate, Ismipur, 6-thioguanine, fludarabine phosphate, cytosine arabinoside, trimetrexate, gemcitabine, acivicin, alanosine .beta.-Pyrazofurin, N-phosphorus acetyl-altheine, pentostatin, 5-azacitidine, 5-azepine 2 '-deoxycytidine, arabinofuranosyladenine, cladribine, five floxuridines, FUDR, tiazofurine, N-[5-[N-(3,4-dihydro-2-methyl-4-oxygen quinazoline-6-ylmethyl)-N-methylamino]-the 2-thenoyl]-L glutamic acid; Alkylating agent, for example: cisplatin, NSC-241240, ametycin, Melphalan, tespamin, busulfan, chlorambucil, plicamycin, dacarbazine, ifosfamide, cyclophosphamide, chlormethine, uracil mustard, NSC-25154,4-ipomeano; The medicament that works through other mechanism, for example: dihydro lenperone, spiromustine and desipramine; Biological response modifier for example strengthens antitumor reaction, for example interferon; Apoptosis agent, for example actinomycin D and hormone antagonist, estrogen antagonist for example, for example tamoxifen or, for example androgen antagonist, for example 4 '-cyano group-3-(4-fluorophenyl sulfonyl)-2-hydroxy-2-methyl-3 '-(trifluoromethyl)-N-propionanilide].

Anti-AAH antibody also has diagnostic application value among the present invention.Anti-AAH antibody can be used for monitoring the growth and/or the transfer of in-vivo tumour, also can be used for the diagnostic indicator of disease stage.For the people, people's antibody right and wrong are immunogenic, and therefore, for mouse antibodies, people's antibody is more suitable in intravital diagnostic application.

Antibody labelling or cold or its antigen-binding site all can be used for diagnostic purpose.Usually, diagnostic analysis must be able to detect the complex that antibody or its part be attached to AAH and forms.Directly traget antibody or its part.Different labellings can be used, and includes but not limited to radionuclide, fluorescent whitening agent, enzyme, zymolyte, enzyme co-factor, enzyme inhibitor and part (for example: biotin, hapten or the like).Those skilled in the art will know that many suitable immune analysis methods (referring to for example United States Patent(USP) Nos. 3,817,827,3,850,752,3,901,654 and 4,098,876).Unlabelled antibody or fragment, can unite use with the reagent that (one or more plant) in addition can be used for of being fit to detected antibody, for example with first antibody (as, anti-idiotype antibody or other antibody special to unmarked immunoglobulin) or the antibody of the labelling of other reactant (as: protein A of labelling) that is fit to reaction.

The test kit that is used for detection of biological sample AAH also can be produced.Such test kit can comprise antibody or its antigen-binding site, and they can be incorporated into described receptor AAH albumen or its part, and comprises and be suitable for detecting one or more auxiliary reagents that complex that antibody or its part and AAH or its part form exists.Antibody compositions among the present invention can be a lyophilized form, but individualism or can with other special antibody of other epi-position are existed simultaneously.No matter labelling whether, the antibody in the test kit can be with additives composition (for example: buffer, as Tris, phosphate and carbonate, stabilizing agent, excipient, Biocide and/or the inert protein as the bovine serum albumin).For example, antibody can provide with the freeze-dried mixture form with the additives composition, and perhaps the additives composition also can provide separately, and is made up by user.When using can be with the bonded second antibody of monoclonal antibody the time, this antibody can be provided in test kit, for example be provided in the independent bottle or container.If there is second antibody, it normally is labeled, and can prepare according to the mode similar to above-mentioned antibody preparation.

Similarly, the present invention also relates to detect and/or the quantitative method of the expression of the part of AAH or enzyme in the cell, be suitable under the condition of antibodies, (for example: catalyst structure domain) bonded funtion part contacts, and the monitoring antibodies for the compositions that contains cell or its grade part (as: film level part) and antibody or itself and AAH or AAH part.Detect antibody, expression antibody is being indicated the existence of AAH with having formed complex between AAH or its part.Can measure antibody by the method for describing in " binding analysis " paragraph for example as mentioned combines with cell.Method can be used for detecting individual (for example: in the tumor biopsy specimen expression of AAH in) the cell.Can pass through for example expression of flow cytometer quantitative assessment tumor cell surface AAH, staining power is relevant with the progress or the danger of disease.

Therefore AAH is working aspect the mobility of cell, and anti-AAH antibody can be used for suppressing (reduce or prevent) tumor growth or transfer.Therefore, the antibody among the present invention, the also function that can in research and treatment application, be used to regulate AAH.Antibody for example described here can be used as inhibitor, suppresses (reduce or prevent) (a) combining of (for example proteic EGF spline structure territory) and AAH; (b) the receptor signal function that mediates by AAH; And/or (c) (for example: AAH or AAH by way of substrate) stimulatory function.Play the antibody of function of receptors inhibitor effect, can directly or indirectly block the combination of (for example: change) AAH by causing configuration.Therefore, the invention provides the method that suppresses cell mobility in the mammal (as: human patient), comprise that the bonded part of part with the antibody of effective dose or itself and AAH or AAH is administered into mammal.The medicable disease of Using such method comprises cancer, and can cause the improvement of morbid state.Medicable cancer includes but not limited to solid tumor, soft-tissue tumor and shifts infringement.The example of solid tumor comprises sarcoma, the cancer (carcinoma) of lung, mammary gland, lymph, gastrointestinal tract (as: colon) and urogenital tract (as: kidney, Urothelial Cell), liver, pharynx, prostate, ovary is involved as those by adenocarcinoma and Different Organs system, cancer of biliary duct and the adenocarcinoma that comprises for example most of colon cancer of malignant tumor, rectal cancer, renal cell carcinoma, hepatocarcinoma, nonsmall-cell lung cancer, carcinoma of small intestine, neural outer embryo tumor or the like.Method and composition treatment or prevention among above-mentioned also available the present invention of cancer metastasis infringement.

The method of theme of the present invention can be used for treating the malignant tumor of Different Organs system, for example those involve the malignant tumor of lung, mammary gland, lymph, gastrointestinal tract (as: colon) and urogenital tract, prostate, ovary, pharynx, and the adenocarcinoma that comprises malignant tumor, for example most of colon cancer, renal cell carcinoma, hepatocarcinoma, carcinoma of prostate and/or tumor of testis, nonsmall-cell lung cancer, carcinoma of small intestine and the esophageal carcinoma.The embodiment of medicable solid tumor comprises: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendothelial sarcoma, synovioma, mesothelioma, Ewing ' s tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, cancer of pancreas, breast carcinoma, ovarian cancer, carcinoma of prostate, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, syringocarcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchus source property cancer, renal cell carcinoma, hepatoma, cancer of biliary duct, choriocarcinoma, spermocytoma, the embryo cancer, Weir Ma Shi tumor, cervical cancer, tumor of testis, pulmonary carcinoma, small cell lung cancer, nonsmall-cell lung cancer, bladder cancer, epithelial cancer, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, Oligodendroglioma, meningioma, melanoma, neuroblastoma and retinoblastoma.

Be suitable for patient, can be described as and suffer from cancer or " cancer " with anti-AAH Antybody therapy described herein.Those skilled in the art are identified as the malignant tumor of epithelium or the malignant tumor of endocrine tissue with cancer, comprise respiratory system carcinoma, gastrointestinal system cancer, genitourinary system carcinoma, carcinoma of testis, breast carcinoma, carcinoma of prostate, hormonal system cancer and melanoma.The example of cancer comprises those cancers that formed by cervix uteri, lung, prostate, mammary gland, head and neck, colon and ovary tissue.Term also comprises carcinosarcoma, as the malignant tumor by the organizational composition of carcinous and sarcoma." adenocarcinoma ", be mentioned to the cancer that comes from gland tissue or wherein tumor cell form the cancer of discernible gland structure.Term " sarcoma " is identified as it by those skilled in the art and is called the malignant tumor in matter source.

Method of the present invention also can be used for suppressing, and for example comes from the propagation of the hypertrophy/oncocyte of the such hematopoietic origin of bone marrow, lymph, erythron or its precursor.For example, the present invention expects the different types of bone marrow lesion of treatment, include but not limited to acute before marrow leukemia (APML), acute myelogenous leukemia (AML) and chronic lymphocytic leukemia (CML) (see Vaickus, Crit Rev.in Oncol./Hemotol. 11: 267-97,1991 summary).The lymph malignant tumor of available this method treatment includes but not limited to acute lymphoblast leukemia (ALL); It comprises B cell line and T cell line ALL, chronic lymphocytic leukemia (CLL), prolymphocyte leukemia (PLL), hairy cell leukemia (HLL) and Walden Si Telunshi macroglobulinemia.The malignant lymphoma of other form of Therapeutic Method expection treatment of the present invention includes but not limited to hodgkin's lymphomas and variant thereof, lymphoma peripheral T cell, adult T chronic myeloid leukemia/lymphoma (ATL), cutaneous T cell lymphoma (CTCL), big granulocyte Lymphocytic leukemia (LGF) and lymphogranulomatosis.

Administering mode

According to this method, one or more antibody or its antigen-binding site can be through suitable by way of being administered into the host separately or with another kind medication combined (before, simultaneously or afterwards).Antibody for example of the present invention also can be united use with other monoclonal or polyclonal antibody or other chemotherapy.

The antibody of effective dose (that is: one or more antibody or fragment) is by administration.Effective dose is the dosage that is enough to obtain the desired therapeutic effect under the situation of administration, thus the amount that for example is enough to suppress the AAH function and suppresses tumor cell.

Different medicine-feeding ways is possible, include but not limited to parenteral (as: intravenous, intra-arterial, intramuscular, subcutaneous injection), oral, food, part, suction (as: in the bronchus, intranasal or oral cavity suck, the intranasal drop), be according to disease to be treated or disease and decide.The medication that other is fit to also can comprise and can fill device that add or biodegradable and release polymer device again.Pharmaceutical composition described here, a part that also can be used as therapeutic alliance is with other preparation administration.

Be used for the antibody of administration and the dosage form (as: solution, emulsion, capsule) of part thereof, according to select administration by way of and different.The suitable pharmaceutical composition that contains antibody or its antigen-binding site for the treatment of administration can prepare in acceptable media of physiology or carrier.Also can use the mixture of antibody and/or its part.The suitable carriers that is used for solution or emulsion comprises solution for example aqueous or alcohol/aqueous, emulsion or suspension, comprises saline and buffer medium.Parenteral vehicle can comprise sodium chloride solution, woods Ge Shi dextrose, dextrose and sodium chloride, newborn acidifying woods Ge Shi oil or fixed oil.Many suitable aqueous carriers are known for a person skilled in the art, comprise water, buffered water, buffered saline, polyol (for example: glycerol, propylene glycol, liquid polyethylene glycol), dextrose solution and glycine.Intravenous vehicle can comprise that the fill-in of different additives, antiseptic or fluidic, trophism or electrolyte (sees: Remington ' s Pharmaceutical Science, 16 ^ThEdition, Mack, Ed.1980).According to the needs near physiological condition, compositions can be chosen wantonly and contain acceptable auxiliary substance on the materia medica, for example: pH regulator agent and buffer agent, and the toxicity regulator as sodium acetate, sodium chloride, potassium chloride, calcium chloride and sodium lactate.The lyophilizing of secundum legem reaches dissolving technology again, and antibody can be stored by lyophilizing and with appropriate carriers it be dissolved before using again.The optium concentration of active component can determine according to method well known to those skilled in the art empirically in the vehicle of selecting, and also depends on the final pharmaceutical dosage form of expectation.To inhalation, the compound dissolution and the suitable allotter of packing into can be come administration (as: aerosol apparatus (atomizer or nebulizer) or pressurization aerosol dispenser.

Embodiment

To in following embodiment, the present invention further be described.These embodiment are not the restriction to the invention category of setting forth in the claim.

The library screening of embodiment 1 anti-HAAH antibody

With yeast surface Display Technique (Boder and Wittrup, Nat Biotechnol. 15(6): 553-7,1997), screen inmature people's strand Fv library, to obtain the Fv fragment of anti-HAAH.According to (Nature Biotech. such as Feldhaus 21: 163-170,2003) method described prepares this library.Briefly, clone people's antibody variable gene with round pcr from obtainable spleen and lymph node poly (A) mRNA that compiles from 58 adults of commerce.The primer of IgG, IgM, κ and λ is used for the synthetic of the first chain cDNA.Make up VH and VL library respectively, fit together through overlap extension PCR afterwards and form strand Fv (scFv) form.After this, with scFv library sub-clone, so that it is expressed as the Aga2p fusant at yeast surface.Fig. 1 has schematically described the Aga2p-scFv fusion rotein.In yeast, expressed about 10 ⁹The segmental library of individual scFv.

According to Boder and Wittrup[Biotechnol Prog. 14(1): 55-62, the 1998 method screening libraries of describing are to obtain to be attached to the HAAH of the recombinant forms that comprises extracellular domain.Briefly, analyze the yeast cells of the expression Aga2p-scFv fusant that is attached to fluorescence HAAH by flow cytometer (FACS).Reaching analysis again by cell divide selects in conjunction with the person.In 300nM HAAH, bonded 6 to take turns screening, identify the clone of 16 uniquenesses.Fig. 2 has described by the clone of 3 uniquenesses of facs analysis and combining of HAAH.Fig. 3 has described by the clone of 8 uniquenesses of flow cytometry analysis and combining of HAAH catalyst structure domain.Among these clones, 8 catalyst structure domains that are attached to HAAH.

The combination of embodiment 2 anti-HAAH antibody is active

Measure the affinity of different scFv antibody to HAAH by carrying out titration, the unmarked HAAH with variable concentrations in the described titration comes labelling to show the cell of specific scFv, changes the concentration of labelling HAAH then and passes through flow cytometer certification mark intensity.Fig. 4 has described 2 representational anti-HAAH scFv is cloned into affinity.The affinity to HAAH that clone 5-2 shows is about 500nM.The affinity to HAAH of clone 6-12 performance is about 150nM.

One can combine with total length HAAH but can not with the bonded antibody cloning of HAAH catalyst structure domain-clone 27, separated.Fig. 5 has described this scFv clone and HAAH catalyst structure domain and the bonded flow cytometry analysis of total length HAAH.

Embodiment 3 combines with the anti-HAAH scFv of solubility fragment

Be attached to the clone of catalyst structure domain with the formal representation of solubility, and analyze it in conjunction with active and other biologic activity.Analysis is attached to 6 unique clones' of HAAH catalyst structure domain (Fig. 6) and total length HAAH (Fig. 7) solubility scFv fragment.The scFv fragment exists with 1-2 μ M.During these were analyzed, clone 11 had shown the highest non-specific bond level.

Next, estimated the combination of 3 unique scFv clones to the H460 human lung carcinoma cell of expressing HAAH.Under about 1 μ M, detect and decide the scFv fragment.Fig. 8 has described these result of experiment.In these were analyzed, clone 11 scFv had also shown the combination of top level.Clone 12 is also coupled to the H460 cell.

Embodiment 4 suppresses the activeness of cell by the anti-HAAH scFv of solubility fragment

Detected solubility scFv clone and a mice IgG of 5 uniquenesses, 15C7 is to the inhibition (Fig. 9) of H460 cell mobility.Place aperture size to be about in the serum-free medium of 8 microns filter bowls in cell.Filter bowl is placed the culture medium that contains serum, and cultured cell allows migration to pass through filter.After cultivation, with violet staining and with the cell quantity of the every side of microscope and blood counting chamber counting filter.When lacking antibody, 60% H460 cell is to move.In the mobility analyzed, antibody concentration was 2 μ M.15C7 has blocked 100% mobility.When clone 11 scFv existed, only about 10% cell was to move. Clone 10 and 13 also shows the inhibition of pair cell motion appropriateness.Also analyze inhibition by clone's 11 cell growth.Clone 11 does not show when concentration is 2 μ M and suppresses H460 growth (data not shown).

Embodiment 5 makes the segmental affinity maturation of anti-HAAH scFv through error-prone PCR

Then, sudden change coding is in conjunction with the nucleic acid of the scFv of HAAH and reselect its associativity, with in the antibody of external generation " affinity maturation ".According to (J.Mol.Biol. such as Zaccolo 255: 589-603,1996) and (J.Mol.Biol. such as Zaccolo 285: 775-783,1999) description, use nucleoside analog to accept error-prone PCR and suddenly change by the DNA that makes scFv.The library of (preceding affinity selection) has 3 * 10 after the initial sudden change ⁶Individual member.Literal arts after sudden change is isolated the clone of selection, to containing the cloning and sequencing of total length open reading frame.With respect to the sequence of input, come among clone 11 the clone, the par that aminoacid sequence changes in 266 aminoacid altogether is 7 ± 8.

Heavily express the nucleic acid of sudden change with yeast surface demonstration (technology), and use flow cytometer to screen its associativity as described.Coding is separated in conjunction with person's DNA, sudden change again, express and screen or the like totally 6 circulations again.In 6 circulations 5 are to finish under the condition that reduces HAAH concentration (start from 1 μ M, terminate in 200mM) in succession.Be attached to the clone who detects antibody for exhausting, wherein one to take turns be to finish with detection antibody under the situation of no HAAH existence.After this sorting step, with respect to list entries, the par that changes from aminoacid sequence among 7 clones of clone 11 is 23 ± 16.Compare with 4 sudden changes among the original clone (is sequence with respect to kind), the scope of planting sudden change quantity in the system coding skeleton zone is 8 to 35.

Analyze combine (Figure 10) of improved clone's 11 scFv mutants and HAAH catalyst structure domain (0.5 μ M).Figure 11 has described this improved clone's aminoacid sequence.Be revealed as boldface type and added underscore with respect to primary clone's 11 amino acid whose changes.

As mentioned above by the HAAH combination of flow cytometry analysis from clone's 11 mutants of continuous sudden change circulation generation.The expression of parallel analysis scFv on yeast cell surface.Figure 12 has described these result of experiment.As showing among Figure 12, compare with initial clone 11, the mutant (the 1st cycle mutant body) of generation has shown the HAAH combination of higher level after 4 take turns screening.Compare with initial clone 11, a clone, 11m1-2 has also showed consistent, the combination of higher level.The combination that 11 mutants are cloned in relevant other affinity maturing also shows among Figure 13 again.The dissociation yield constant that the best of measuring is improved clone (as: 11m1-2) is 220 ± 60nM, has increased by two orders of magnitude than its initial nonmutationed scFv that derives the source.

From 5 among 7 affinity maturing clones of clone 11, the cysteine mutation of (according to the Kabat numbering) is arginine or tyrosine on heavy chain H22 position.This cysteine forms the disulfide bond in the domain.Have 3 among 5 clones of this sudden change, perhaps an extra cysteine is arranged in heavy chain skeleton district 3 (FW3) or at light chain complementary determining region 3 (CDR3).Do not contain among 2 clones of H22 cysteine mutation, one does not have extra cysteine.Another is in heavy chain complementary determining region 3 ring portions, from 4 amino acid residue places of H22 cysteine, and tyrosine is replaced by cysteine.Frequent cysteine mutation can show that the lax of heavy chain structure can promote combination.

Also carry out the affinity maturing to cloning 13.Figure 14 shows that clone 13 mutant 13m1 are attached to the catalyst structure domain of HAAH.13m1 is produced by 5 sudden change/screening circulations.Figure 15 describes the aminoacid sequence of this mutant.Be shown as runic and underscore with respect to initial clone's 13 amino acid whose changes.As to cloning as described in 11, analyze the combination of clone's 13 continuous mutants.As shown in figure 16, with respect to initial clone 13, clone 13m1 and the clone 12m2-3 that is produced by clone 13m1 have shown the combination that improves.

The reorganization of embodiment 6 chains is used for the segmental affinity maturation of anti-HAAH scFv

Chain reorganization is a kind of mutating technology, the wherein complete antibody chain or the part of chain and other chain reorganization.Be to produce scFv fragment, reorganize heavy chain from the inmature library that produces at the anti-HAAH light chain of wild type (keeping sequence of light chain, and connect it) and make up the library with different heavy chain to the anti-HAAH of HAAH high affinity.Specifically, as described in the image pattern 17, with NheI and BamHI restrictive diges-tion and by extracting heavy chain fragment in people's nature library, and the yeast that contains wild type scFv light chain that is connected to NheI-BamHI digestion shows on the plasmid.The initial library that produces through this method has about 1.2 * 10 ⁵Individual member.5 clones from initial library are checked order; They all have and the initial different heavy chain of heavy chain.Figure 18 A contains a form, has listed heavy chain and light chain among each clone.

Next, under the antigen concentration that reduces (by 800nM to 500nM), finish 6 and take turns flow cytometer and screen, better to sift out in conjunction with the person.Under the situation of no HAAH, finish other screening step, be attached to the clone who detects antibody with removal.Come from the scFv reveal competence (X-axis) of yeast cells of cell aggregation of sudden change and HAAH by flow cytometry analysis in conjunction with (Y-axis).Figure 18 B performance has the segmental clone of high-caliber high affinity scFv and is present in the set.

In 6 take turns screening after, measure 20 clones' sequence.By among the clone who checks order, 11 is unique, and each contains the heavy chain that is different from wild type scFv.Figure 19 A to K shows these clones' scFv reveal competence (X-axis) and HAAH combination (Y-axis).Select the clone for two, the dissociation constant of LLm11 and LLm13 is determined as 240 ± 70nM and 160 ± 50nM respectively, and this has improved two orders of magnitude than wild type.

The clone's who obtains with this technology frame sequence, with kind of the deviation minimum of pastern bone frame sequence (with the heavy chain kind is sequence compare produce average 4 ± 2 sudden changes), making them, existing immunogenicity might be littler for the clone with bigger quantity skeleton sudden change.Because all these clones have different heavy chain CDR3, its binding specificity is perhaps slightly different.Can have not homospecific a plurality of conjugates to same target position and easily screen second kind of desired characteristics, as: the combination to specific people's tissue minimizes.

Embodiment 7 uses CDR reorganization to make the segmental affinity maturation of anti-HAAH scFv

The third technology, CDR shuffling technology (being also referred to as the domain shuffling technology) are used to produce the scFv clone to the HAAH high affinity.For carrying out existing CDR reorganization, with acceptor carrier and multilinear insertion sequence cotransformation yeast cells.Acceptor carrier and insertion sequence are designed to, have homology sequence at its one or two end of 5 ' and 3 '.Homologous recombination between any site of these sites and genes of interest (as: antibody chain) produces " reorganization " product.Finish the further details of CDR reorganization, (Nucl.Acids Res. such as Swers 32(3): e36,2004) be described.

Clone 11 the segmental DNA of scFv with encoding wild type, reach the sequence that comes from people scFv library, carry out CDR and replace (Figure 21) as insertion sequence as acceptor carrier.Insertion sequence has the consistent light chain of light chain with clone's 11 light chains, three library insertion sequences separately contain corresponding to by HC FW1 to CDR1, by HC FW1 to CDR2 or by the zone of HC FW1 to CDR3, use the inmature heavy chain gene sequence displacement of the VH family identical with original clone.Contain by HC FW1 to CDR1 and, produce the receptor editor's who is similar to the interior natural B of the being present in cell of body sudden change by the library of HC FW1 to the CDR2 regional replacement.

Have by HC FW1 to CDR1, by HC FW1 to CDR2 or by HC FW1 to the library that CDR3 inserts, contain 1.1 * 10 respectively ⁴, 1.2 * 10 ⁴, 1.1 * 10 ⁴Individual member.For screening gathers these libraries.Be accompanied by the reduction of antigen concentration, HAAH concentration drops to 160nM by 500nM, finishes 6 by flow cytometer and takes turns screening.When no HAAH exists, carry out other screening step, be attached to the clone who detects antibody with removal.

Separate and check order 10 and clone, wherein 4 clones be uniqueness.Figure 22 A to D shows these clones' scFv reveal competence (X-axis) and HAAH combination (Y-axis).Figure 23 shows the aminoacid sequence of wild type clone and 4 plant mutants clone's CDR.In the sequence of 4 kinds of uniquenesses, 3 kinds of clones have the FW1 that replaces with inmature library sequence to the CDR3 zone.4 clones have the FW1 that replaces with inmature library sequence to the CDR2 zone.The dissociation constant of mutant is 1-2 μ M, exceeds order of magnitude of original wild type clone.The skeleton district of this mutant has only two amino acid positions and kind pastern bone frame district that difference is arranged.

Different clones' dissociation constant is determined.Figure 24 show each clone in conjunction with vs concentration.Each clone's who compares among the figure KD measured value is as follows:

Wild type clone 6-22:＞10 μ M

Clone LLm13:26 ± 8nM

Clone CM4:17 to 5nM

Clone C4m18:0.6 ± 0.1nM

Embodiment 8 is an IgG antibody with the scFv fragment expression of selecting

By isolated variable heavy chain and variable sequence of light chain being inserted into the expression vector that contains coding IgG heavy chain and constant region of light chain sequence, the vector expression total length IgG antibody that obtains, thus create total length IgG antibody (Figure 25) by the scFv fragment.In brief, segmental heavy chain of scFv and the sequence of light chain that increases respectively and select by round pcr.Then, (Pacific Northwest National Laboratory) in place is connected to the fragment that produces on the expression vector pPNL501.To carrier cloning, separation and the order-checking that produces, to determine to have inserted correct sequence.By standard method the carrier transfection is entered COS-7L cell (Invitrogen) then.These COS-7L cellular expression justacrines IgG that obtains is in culture medium.Separation and Culture liquid is by standard technique protein A resin purification IgG.

With the ELISA method and with express combining of HAAH tumor cell, measure specificity combination to total length IgG antibody.For measuring binding specificity, by the ELISA hole, make not commensurability IgG combination in different holes with HAAH or bovine serum albumin bag with ELISA.Finish detection with goat anti-human IgG peroxide enzyme conjugates and chemical luminous substrate.Figure 26 shows the experimental result of relevant IgG6-22,6-23,6-25 and 6-28.For detecting binding specificity, make not commensurability IgG antibody with the combination of H460 tumor cell, described cellular expression high level HAAH to HAAH expressing tumor cell.Use the anti-human IgG FITC of goat conjugate to detect combining of IgG antibody and cell by flow cytometer.Figure 27 shows the experimental result of relevant IgG 6-22 and 6-23; Figure 28 shows the experimental result of relevant IgG LLm11, LLm13, LLm15 and CDRm4.

Measured the dissociation constant of IgG 6-22 and CDRm4.Under 4 ℃ of conditions, the IgG that makes variable concentrations is with H460 or the combination of FOCUS cell.The FOCUS cell is the hepatocellular carcinoma cells system of also expressing HAAH.After the IgG combination, with the anti-human IgG phycoerythrin of goat conjugate labeled cell, and with flow cytometer detection fluorescence.Figure 29 shows that 6-22 IgG is in conjunction with vs. concentration; Figure 30 shows that CDRm4 IgG is in conjunction with vs. concentration.K _DMeasured value, show below:

H460 FOCUS

6-22?IgG：?1.3±0.2nM 1.1±0.2nM

CDRm4?IgG：1.0±0.1nM 0.7±0.1nM

Detect that anti-HAAH scFv fragment and deutero-IgG antibodies go up similar epi-position to HAAH whether to measure.Under the situation of 6-22IgG or contrast IgG existence, the scFv fragment 6-22 that makes the FLAG labelling is with the combination of H460 cell.By exist only in the FLAG label of scFv 6-22 with biotinylated mouse anti FLAG antibody labeling, measure the combination of labelling scFv 6-22 by being attached to streptavidin-phycoerythrin conjugate subsequently.IgG 6-22, rather than contrast IgG can combine with scFv 6-22 competition, represents equally with scFv 6-22, and the IgG form of 6-22 is attached to similar epi-position (Figure 31) on the HAAH.

For further proof scFv fragment and IgG antibodies, make to show that the segmental yeast cells of different scFv is with 15nM HAAH combination to similar HAAH epi-position under competitive CDRm4 IgG, LLm11 IgG or buffer individualism situation.As described in above-mentioned, detect HAAH and show combining of the segmental cell of scFv by flow cytometer.Result shown in figure 32 shows that IgG antibody has the competitiveness that is attached to HAAH, and then illustrates that IgG antibody and scFv fragment are attached to similar epi-position.

Embodiment 9 makes the segmental second filial generation affinity of anti-HAAH scFv maturation by fallibility PCR

ScFv carries out the affinity maturation to the CDRm4 mutant, to produce second filial generation sudden change C4m8 and C4m18.As described in above-mentioned, use nucleoside analog to suddenly change with realization with the DNA of fallibility pcr amplification coding CDRm4 scFv.As mentioned above, the nucleic acid of expressing sudden change again with the yeast surface Display Technique, and use flow cytometer screening combination.As mentioned above, finish and 4 take turns separation, sudden change again, express and screen the circulation of coding again in conjunction with person's DNA.Through the method, separate improved second filial generation scFv clone.

As mentioned above, analyze combining of the improved clone C4m8 scFv mutant be apparent on the yeast cells and HAAH.Compare with CDRm4, this improved sudden change no matter be in conjunction with, or, all improved about twice (Figure 33) aspect the demonstration.

Many embodiments of the present invention have been described.Yet, should be appreciated that under the situation that does not depart from spirit of the present invention and scope, can carry out different modifications.Therefore, other embodiment also within the scope of the claims.

Claims (35)

1. a compositions contains separative people's antibody or its fragment or other variant, and wherein antibody or its fragment or other variant can combine with aspartoyl (asparaginyl-) B-hydroxylase (AAH) specificity.

2. the compositions of claim 1, wherein other variant is single-chain antibody (scFv).

3. the compositions of claim 1, wherein antibody or its fragment or other variant combine with the catalyst structure domain of HAAH specifically.

4. the compositions of claim 1, wherein when antibody or its fragment or other variant were administered into patient, the enzymatic activity that suppresses HAAH was to clinical effective degree.

5. the compositions of claim 1, wherein when antibody or its fragment or other variant contacts with HAAH proliferative cell too active or overexpression, the propagation of inhibition cell.

6. the compositions of claim 1 is wherein when antibody or its fragment or other variant and HAAH is too active or during mobility's cells contacting of overexpression, suppress the mobility of cell.

7. the compositions of claim 1, wherein antibody or its fragment or other variant are equal to or less than about 1 μ M to the affinity of HAAH.

8. the compositions of claim 1, wherein antibody or its fragment or other variant suddenly change.

9. the compositions of claim 8 wherein by fallibility PCR, chain reorganization or CDR or domain reorganization, makes the sudden change of antibody or its fragment or other variant.

10. the compositions of claim 1, wherein antibody or its fragment or other variant contain with SEQ ID NOs.31-45,149-163 or 272-280 in the complementary determining region of any complementary determining region (CDR) with at least 40% homogeneity.

11. the compositions of claim 1, wherein antibody or its fragment or other variant contain the CDR that has at least 40% homogeneity with Figure 37 or any complementary determining region shown in Figure 38.

12. the compositions of claim 1, wherein antibody or its fragment or other variant are attached to the 11 bonded epi-positions by the clone with clone's 11 competitions effectively.

13. the compositions of claim 1, wherein antibody or its fragment or other variant contain the complementary determining region that comprises following aminoacid sequence: S-Q-S/N-V-S-S/H-(X)-Y/H-L-A; D-V-A-N-X-A-A; Q-Q-R-S-N-W-P-Q; Y/H-A-M-H/G; Y-A-X-S-V-K-G/S; S-G-S-S-S-N-I-G/E-S-N-H/Y-V-H/Y; S/G-D/N-S/G-A-A-W-S/N; R-I/T-Y/H-Y/H-G/R-X-K/R-W/R-Y/R-N-D/G-Y/H-A-V/A-P/S-V/A-K-S; D-V-X-X-R-P-S; L-F/L-I/V-H/Y-K/R-X-N-Q-R-P-S; A-W-D-D-S; Or S-S-S-W-V-V-X-F-D/G, wherein X is any amino acid residue or does not have amino acid residue.

14. the compositions of claim 2, wherein scFv contain with SEQ ID NOs.31-45,149-163 or 272-280 in any aminoacid sequence that at least 80% homogeneity is arranged.

15. the compositions of claim 1 also contains the medicine acceptable diluent.

16. the compositions of claim 1, wherein antibody or its fragment or other variant also contain label or toxin.

17. the compositions of claim 1, its compositions is made up of antibody or its fragment or other variant.

18. isolated nucleic acid molecule contains coding and the people's antibody of AAH specific bond or the sequence of its fragment or other variant.

19. contain the expression vector of the nucleic acid molecules of claim 18.

20. contain the host cell of the expression vector of claim 19.

21. the host cell of claim 20, wherein host cell is a yeast cells.

22. the host cell of claim 20, wherein host cell is a mammalian cell.

23. the host cell of claim 22, wherein mammalian cell is a tumor cell.

24. test kit contains compositions and the diagnosis or the treatment description of claim 1.

25. the active method of AAH in the adjusting cell, this method comprises:

Be enough to regulate in the cell under active time of AAH and condition, the compositions of cellular exposure to claim 1.

26. treatment suffers from and too enlivens or the method for the cancer patient that the AAH of overexpression is relevant, this method comprises with the dosage that is enough to suppress cancer cell metastasis in patient's body or propagation and time the compositions of claim 1 is administered into patient.

27. the method for claim 26, wherein compositions is by the whole body administration.

28. the method for claim 26, wherein cancer cell is the tumor cell of lung, liver, colon, pancreas, prostate, ovary, bile duct, brain or mammary gland.

29. differentiate and the method for the bonded specifically antibody of AAH or its fragment or other variant that this method comprises: the library is provided, and its member is antibody or its fragment or other variant;

Allowing under antibody or its fragment or the condition of other variant in conjunction with polypeptide, the polypeptide that will contain AAH or its domain contacts with the library member, thereby forms one or more complex; With

Identify and bonded one or more complex of AAH that each complex contains antibody or its fragment or other variant.

30. the method for claim 29, wherein antibody or its fragment or other variant are from the people.

31. the method for claim 29, wherein other variant is scFv.

32. the method for claim 29, its Chinese library is expressed on the surface of yeast cells.

33. the method for claim 29 also comprises the antibody of having identified of affinity maturing.

34. pass through antibody or its antigen-binding portion thereof of the method preparation of claim 30.

35. one kind prepares the method that specificity is attached to human monoclonal antibodies or its fragment or other variant of AAH, this method comprises:

Identify that specificity is attached to antibody or its fragment or other variant of AAH;

In cell, express the nucleotide sequence of encoding antibody or its fragment or other variant, with the antibody of preparation expression or fragment or other variant of expression; With

The antibody that purification is expressed or fragment or other variant of expression.

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