CN100509851C - Anti-alpha*beta* antibodies - Google Patents

Anti-alpha*beta* antibodies Download PDF

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CN100509851C
CN100509851C CNB200610103152XA CN200610103152A CN100509851C CN 100509851 C CN100509851 C CN 100509851C CN B200610103152X A CNB200610103152X A CN B200610103152XA CN 200610103152 A CN200610103152 A CN 200610103152A CN 100509851 C CN100509851 C CN 100509851C
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antibody
cell
mouse
seq
monoclonal antibody
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CN1908013A (en
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S·M·维欧雷特
P·H·维尼尔博
K·J·西蒙
D·舍帕德
D·R·利昂
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University of California
Biogen MA Inc
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University of California
Biogen Idec MA Inc
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Abstract

Monoclonal antibodies that specifically bind to M.96. Also included are methods of using these antibodies to treat mammals having or at risk of having alpha(v)beta(6)-mediated diseases, or to diagnose alpha(v)beta(6)-mediated diseases.

Description

Anti-α vβ 6Antibody
Invention field
The present invention relates to biology field, be specifically related to anti-α vβ 6Alpha 2 integrin antibodies.
Background of invention
Integrin is a cell surface receptor superfamily, and mediated cell-cell and cell-matrix adhere to.These protein be considered to grow and the tissue injury process in provide grappling and signal for growth, migration and the differentiation of cell.Integrin also with cell dedifferente and invade relevant, particularly lose their specialization form, and when becoming the cancer cells of transfer at cell.
The heterodimer protein that integrin is made up of the subunit-α and the β of two non-covalent connections.The binding specificity of integrin is by some the combination defineds in some and the 8 kinds of different beta chains in 18 kinds of different α chains.α vβ 6Integrin can be in conjunction with multiple aglucon, comprise fibronectin, cytotactin, vitronectin and the related peptides of identifying recently of hiding (LAP), be a kind of 278 amino acid whose peptides, it is as the proteic part of precursor TGF-β synthetic people such as (, Cell96 (3): 319-328 (1999)) Munger.In secretion process, LAP downcuts as the mature form of N end peptide from TGF-β, but still combines with TGF-β is non-covalent, thereby keeps latent state.This mixture can not combine with the TGF-beta receptor, so the abiology activity.α vβ 6Integrin can cause the release of LAP and the activation of TGF-β directly in conjunction with RGD motif contained in the LAP.Because α vβ 6Being converted into active condition with combining of LAP for TGF-β may be extremely important, and therefore blocking this combination can cause α vβ 6The TGF-β activated of mediation suppresses and relevant fibrotic disease Neo-Confucianism.
Summary of the invention
The present invention is based on anti-α vβ 6The discovery of high-affinity antibody and sign comprise the evaluation and the analysis of the middle key amino acid residue of complementary determining region (CDRs) of these antibody.
The present invention includes a kind of monoclonal antibody, its can (a) specificity in conjunction with α vβ 6(b) suppress α vβ 6With its part (as LAP, fibronectin, vitronectin and cytotactin) combine its IC 50Value is lower than the IC50 value (the open text WO 99/07405 of international patent application) of 10D5; (c) activation of blocking-up TGF-β; (d) contain α is provided vβ 6Some aminoacid sequence among the CDR of binding specificity (shown in Fig. 7 A and 7B); (e) specificity is in conjunction with β 6Subunit; And/or, discern α in the immunostaining as paraffin-embedded tissue (f) in the immunostaining program vβ 6
Have been found that and α vβ 6Bonded antibody can be classified as classes different on the biophysics and subclass.One antibody-like shows block ligand (as LAP) and α vβ 6Bonded ability (blocker).The subclass that this antibody-like can be relied on cationic blocker and not rely on cationic blocker by being further divided into.Some rely on cationic blocker and contain arginine-glycine-aspartic acid (RGD) peptide sequence, do not contain the RGD sequence and do not rely on cationic blocker.Another kind of antibody shows and α vβ 6The bonded ability, and can not block α vβ 6Combine (non-blocker) with part.
Therefore, in some embodiments of the present invention, antibody more of the present invention and α vβ 6Combination be to rely on divalent cation, and some is not rely on divalent cation.Exemplary positively charged ion is Ca 2+, Mg 2+And Mn 2+
In some embodiments, antibody comprises identical heavy chain and the light chain polypeptide sequence of antibody that produces with hybridoma 6.1A8,6.3G9,6.8G6,6.2B1,6.2B10,6.2A1,6.2E5,7.1G10,7.7G5 or 7.1C5.
In some embodiments, antibody comprises a heavy chain and/or a light chain, the complementary determining region of heavy chain (CDR) 1,2 and 3 respectively basic (promptly except some conservative propertys variations) is made up of SEQID NOs:1,4 and 7 sequence, and the CDRs 1,2 of this light chain and 3 is basic respectively to be made up of SEQ ID NOs:10,13 and 15 sequence.
In some embodiments, antibody comprises a heavy chain and/or a light chain, the CDRs1 of heavy chain, 2 and 3 is made up of SEQ ID NOs:3,5 and 8 sequence respectively substantially, and the CDRs1 of this light chain, 2 and 3 is made up of SEQ ID NOs:11,14 and 17 sequence respectively substantially.
In some embodiments, antibody comprises a heavy chain and/or a light chain, the CDRs 1,2 of heavy chain and 3 is made up of SEQ ID NOs:3,6 and 9 sequence respectively substantially, and the CDRs 1,2 of this light chain and 3 is made up of SEQ ID NOs:12,14 and 18 sequence respectively substantially.
In some embodiments, antibody comprises a heavy chain and/or a light chain, the CDRs 1,2 of heavy chain and 3 is made up of SEQ ID NOs:2,46 and 47 sequence respectively substantially, and the CDRs 1,2 of this light chain and 3 is made up of SEQ ID NOs:48,13 and 16 sequence respectively substantially.
In some embodiments, antibody comprises a heavy chain and/or a light chain, the CDRs 1,2 of heavy chain and 3 is made up of SEQ ID NOs:49,51 and 53 sequence respectively substantially, and the CDRs 1,2 of this light chain and 3 is made up of SEQ ID NOs:55,57 and 59 sequence respectively substantially.
In some embodiments, antibody comprises a heavy chain and/or a light chain, the CDRs 1,2 of heavy chain and 3 is made up of SEQ ID NOs:50,52 and 54 sequence respectively substantially, and the CDRs 1,2 of this light chain and 3 is made up of SEQ ID NOs:56,58 and 60 sequence respectively substantially.
In some embodiments, antibody comprises any one heavy chain variable domain sequence among SEQ ID NOs:19-36 and the 61-62.
In some embodiments, antibody comprises following heavy chain and light chain variable territory sequence respectively:
(1) SEQ ID NOs:19 and 37;
(2) SEQ ID NOs:20 or 21 and SEQ ID NO:38;
(3) SEQ ID NOs:22 and 43;
(4) SEQ ID NOs:23 and 44;
(5) SEQ ID NOs:24 and 45;
(6) SEQ ID NOs:25 or 26 and SEQ ID NOs:42;
(7) SEQ ID NOs27,28 or 29 and SEQ ID NOs:39;
(8) SEQ ID NOs:34 or 35 and SEQ ID NOs:40;
(9) SEQ ID NOs:36 and 41;
(10) SEQ ID NOs:61 and 63; Or
(11) SEQ ID NOs:62 and 64.
In some embodiments, but the antibody specificity in conjunction with α vβ 6, but do not suppress α vβ 6With combining of the related peptides of hiding (LAP).At least the α during some such antibody can be cut into slices with paraffin-embedded tissue vβ 6In conjunction with, therefore can be used in diagnostic uses.Exemplary antibody comprises 6.2A1 and 6.2E5.
The present invention also comprise can in conjunction with the antibody of the identical epi-position of above-mentioned any antibody.
The present invention also comprises the composition that contains one or more antibody of the present invention and pharmaceutically acceptable carrier.In some such compositions, antibody and cytotoxic agent (promptly influencing the material of the viability and/or the function of cell) are as toxin or radionuclide coupling.Antibody in these compositions can be to rely on cationic antibody.These compositions can be to suffering from or have trouble α vβ 6The object (for example Mammals, as the people) of the disease danger of mediation is used, with this disease of treatment (for example alleviating, alleviate, reduce, stop, postpone generation).The example of this class disease includes, but are not limited to: fibrosis (for example scleroderma, epulosis, hepatic fibrosis, pulmonary fibrosis and renal fibrosis); Psoriatic; Cancer (epithelial cancer for example; Oral cavity, skin, uterine cervix, ovary, pharynx, larynx, esophagus, lung, breast, kidney or colorectal carcinoma); Alport syndrome; Acute and chronic pulmonary, liver, kidney and other visceral injury; The sclerosis of lung, liver, kidney and other internal organ.The danger of suffering from these diseases may be because hereditary inducement; Some mode of life is as smoking and excessive drinking; The contact environment pollutent is as asbestos; Physiological maladies is as diabetes, hepatites virus infections (for example infection with hepatitis C virus), autoimmune disease; And therapeutic treatment, as radiotherapy.
The present invention comprises that also detection is from α in Mammals (for example people's) the tissue sample vβ 6Method, comprise making tissue sample contact antibody of the present invention, as 6.2A1 and 6.2E5.
The present invention also comprises the cell of hybridoma 6.1A8,6.2B10,6.3G9,6.8G6,6.2B1,6.2A1,6.2E5,7.1G10,7.7G5 and 7.1C5; The isolating nucleic acid that comprises sequence any among coding SEQ ID NOs:19-45 and the 61-64; The isolated polypeptide that comprises aminoacid sequence any among SEQ ID NOs:19-45 and the 61-64.
Antibody of the present invention is meant complete antibody, for example, contains the antibody of two heavy chains and two light chains, perhaps is meant the Fab of complete antibody, as Fab fragment, Fab ' fragment, F (ab ') 2Fragment or F (v) fragment.Antibody of the present invention can be murine antibody or its homologue, or complete people's antibody.Antibody of the present invention also can be humanized antibody, chimeric antibody or single-chain antibody.Antibody of the present invention can be any isotype and hypotype, and as IgA (for example IgA1 and IgA2), IgG (as IgG1, IgG2, IgG3 and IgG4), IgE, IgD, IgM, wherein the light chain of immunoglobulin (Ig) can be κ type or λ type.
In some embodiments, antibody of the present invention can contain sudden change (for example lack, replace or add) at one or more (for example 2,3,4,5 or 6) specific site place of heavy chain, make the effector function (for example ability of this antibodies Fc acceptor or complement factor) of this antibody change, and do not influence the antigen binding capacity of antibody.In the other embodiment, antibody of the present invention can contain sudden change at the amino-acid residue place as glycosylation site, makes glycosylation site eliminate.This antibody may have effector function or other undesirable function useful clinically, that reduce, and keeps its antigen-binding affinity.The sudden change of glycosylation site also has the technological development of being beneficial to (for example protein expression and purifying).In the other embodiment, heavy chain or light chain may contain the sudden change that improves avidity or usefulness.
Several fusion #6 and merge the #7 hybridoma according to the budapest treaty preservation at American type culture collection (ATCC; P.O.Box 1549, Manassas, and VA 20108, USA).Hybridoma clone 6.1A8,6.2B10,6.3G9,6.8G6 and 6.2B1 are in calendar year 2001 preservation on August 16, and preserving number is respectively ATCC PTA-3647 ,-3648 ,-3649 ,-3645 and-3646.Hybridoma clone 6.2A1,6.2E5,7.1G10,7.7G5 and 7.1C5 are in calendar year 2001 preservation on December 5, and preserving number is respectively ATCC PTA-3896 ,-3897 ,-3898 ,-3899 and-3900.Table 1 sees below.
Antibody of the present invention can be used for treatment by α vβ 6Combine any undesirable clinically illness or the disease (as described here) of mediation with part (as LAP and fibronectin).Because higher avidity or avidity, and with part bonded positively charged ion dependency or dependent/non-dependent, these antibody may be than previously known α vβ 6Antibody is more effective.
Except antibody of the present invention particularly the therepic use of blocker, the antibody of non-blocker class also can be used in diagnostic purpose, as is used for antigen capture mensuration, enzyme-linked immunosorbent assay (ELISA), immunohistochemical methods etc.
By as detailed below, accompanying drawing and claims, other features and advantages of the present invention will be conspicuous.
The accompanying drawing summary
Figure 1A and 1B are showed cell catch assay result's bar graphs, and this test is to measure multiple anti-α vβ 6Monoclonal antibody (" mAb ") is in conjunction with β 6The ability of the FDC-P1 cell of transfection (cell of untransfected in contrast).
Fig. 2 A is the figure that shows the ELISA measurement result, and this test is the anti-α that measures multiple purifying vβ 6" fusion 6 " monoclonal antibody is in conjunction with soluble recombined human α vβ 6(" hs α vβ 6") ability.These antibody are by the soluble human α with brachymemma vβ 6Immunity β 6-/-mouse produces.Numeral clone number in the legend.Corresponding clone's title sees Table 2.
Fig. 2 B is the figure that shows the ELISA measurement result, and this test is the anti-α that measures multiple purifying vβ 6The monoclonal antibody that " merges 7 " is in conjunction with solubility reorganization hs α vβ 6Ability.These antibody are by using β 6The immune β of the NIH 3T3 cell of transfection (merging #7) 6-/-mouse produces.
Fig. 3 A-F shows multiple anti-α vβ 6Monoclonal antibody and hs α vβ 6The dependent figure of the different positively charged ions of bonded.
Fig. 4 A and 4B show to merge #6 and merge the #7 monoclonal antibody to suppress vitamin H-hs α respectively vβ 6With LAP bonded figure.
Fig. 5 A-E shows that exemplary monoclonal antibody of the present invention suppresses β 6The FDC-P1 cell of transfection and LAP bonded figure.Fig. 5 A and 5B show the result who merges #6 antibody.Fig. 5 C-E shows the result who merges #7 antibody.
Fig. 6 A and 6B show to merge #6 and merge #7 antibody to suppress α respectively vβ 6The TGF-β activated figure of-mediation wherein uses PAI-1 luciferase reporter gene to measure the activation of monitoring TGF-β.
Fig. 7 A shows α vβ 6The aminoacid sequence of the heavy chain variable domain of monoclonal antibody 6.1A8,6.8G6 (subclone A and B), 7.7G5,6.2B1,6.3G9,6.2B10 (subclone A and B), 6.2G2,6.2A1,6.4B4 (subclone A, B and C), 7.10H2,7.9H5,7.4A3 (subclone A and B), 7.1C5 (subclone A and B) and 7.1G10.Antibody 6.1A8,6.8G6 and 7.7G5 and α vβ 6Combination be to rely on cationicly, antibody 6.2B1,6.2A1,6.3G9,6.2B10,6.4B4,7.1C5 and 7.1G10 do not rely on cationic (seeing below).Digitized representation amino-acid residue site in the bracket.CDR contains the amino acid whose little frame of italic and then represents the polymorphism of antibody specific in the difference clone in big frame.
Fig. 7 B shows α vβ 6The aminoacid sequence in the light chain variable territory of monoclonal antibody 6.1A8,6.8G6,6.4B4,6.2A1,7.1C5,7.1G10,6.2B10,7.7G5,6.2B1 and 6.3G9.
Fig. 8 is a scatter diagram, shows α in human breast carcinoma and the people's squamous cell carcinoma tissue slice vβ 6Expression.Health adult tissue only shows negligible α vβ 6Expression level.
Fig. 9 A and 9B are quafric curve figure, show two kinds of anti-α vβ 6Antibody 6.8G6 and 6.3G9 are respectively to solubility α vβ 6The solution binding affinity.
Figure 10 A and 10B are two bar graphs, and the monoclonal antibody of confirmation purifying is competed respectively with biotinylated 6.3G9 and biotinylated 6.8G6 and combined α vβ 6Ability.
Figure 11 is a bar graph, is presented to use anti-α vβ 6In the kidney of the UUO animal that mab treatment is handled, the painted per-cent of smooth muscle actin.
Figure 12 shows that tumour cell is fastened α according to facs analysis vβ 6Expression (figure right side), and monoclonal antibody 6.3G9 and 6.4B4 suppress (figure left side) to tumor cell line and LAP part bonded.
Figure 13 is a bar graph, confirms anti-α vβ 6Monoclonal antibody 6.3G9,6.8G6 and 6.4B4 suppress three kinds of tumor cell lines and LAP part bonded.The combination of monoclonal antibody is compared with the total binding of not adding the test sheet clonal antibody (TB) and with the non-specific binding (NSB) of independent BSA contrast.
Figure 14 A and 14B were presented in research phase of 33 days, anti-α vβ 6Monoclonal antibody 6.3G9 and 6.4B4 are respectively to subcutaneous effect of implanting the tumour of Detroit 562 cells generation.
Figure 15 A-C shows anti-α vβ 6Monoclonal antibody is to the effect of bleomycin inductive pulmonary fibrosis.(A) Antybody therapy that uses the 6.3G9 monoclonal antibody was monitored 30 days when using bleomycin on the 0th day; (B) use the Antybody therapy of 6.3G9 monoclonal antibody to begin after 15 days, monitored 30 days in the bleomycin treatment; (C) use the Antybody therapy of 6.3G9,6.8G6 and 6.4B4 monoclonal antibody to begin after 15 days, monitored 60 days that prolong in the bleomycin treatment.In Figure 15 A and 15B, the bar figure in left side represents μ g oxyproline/lung, and the increase percentage ratio of oxyproline is compared in the bar figure on right side demonstration with the mouse (no bleomycin) of brine treatment.In Figure 15 C, this figure shows the hydroxyproline content of each lung.
Detailed Description Of The Invention
The present invention has characterized for beta 2 integrin alphavβ 6The class of special antibody and subclass. At least one antibody-like (blocking agent) can be blocked αvβ 6With the combination of LAP or the activation of prevention TGF-β.
The several different methods for preparing antibody of the present invention has below been described. But known in this field do not have specifically described method to be also contained in the scope of invention at this. For example, antibody of the present invention also can enough phage displaying antibodies library identifies, such as Smith, and Science 228:1315-7 (1985); United States Patent (USP) 5,565,332,5,733,743,6,291,650 and 6,303,313 is described. Other antibody of the present invention can be prepared as follows: the heavy chain that will herein identify and irrelevant (noncognate) light chain (light chain of for example identifying by display technique of bacteriophage) phase coupling.
Inhuman hybridoma antibody
Monoclonal antibody of the present invention can produce by well-known hybridoma technology. For this reason, β6-/-animal (for example mouse, rat or rabbit) is with following material immunity: α purifying or thickvβ 6Goods are used coding for alphav、β 6Or the cell of the cDNA construct transfection of these two kinds of antigens, constitutive expression αvβ 6Cell, etc. Can be used as the protein of purifying, at protein, its protein fragments or peptide that cell is expressed, perhaps send these antigen as the viral vectors of naked DNA or encode this protein, protein fragments or peptide. Detect then anti-α in the serum of immune animalvβ 6The existence of antibody. From the animal of test positive, separate the B cell, prepare hybridoma with these B cells.
The antibody of screening hybridoma secretion, this is according to they specific binding αvβ 6(for example in conjunction with β6The cell of transfection, and not in conjunction with the parental cell of untransfected) ability and the feature of other any hope for example contain CDR consensus sequence likely, to be lower than known anti-αvβ 6The IC of antibody 10D550Value suppresses (perhaps for non-blocking agent, not suppressing) LAP and αvβ 6Combination, or suppress the activation of TGF-β.
The hybridoma of test positive is in cell is cultivated in nutrient medium under the condition of culture medium endocrine monoclonal antibody in the screening experiment. Collection condition hybridoma culture supernatant then, contained antibody in the purifying supernatant. In addition, the antibody of hope also can produce by the intraperitoneal injection hybridoma to non-immune animal (for example mouse). Hybridoma is in the abdominal cavity internal breeding, and secretory antibody accumulates and is ascites. Can with syringe sucking-off ascites from the abdominal cavity, collect antibody then.
Monoclonal antibody also can followingly produce: separate the cDNA of encoding antibody from the hybridoma of hope, with this cDNA transfection mammalian host cell (for example CHO or NSO cell), cultivate the host cell of transfection, reclaim antibody from culture medium.
Chimeric antibody
Monoclonal antibody of the present invention also can make up relevant (cognate) hybridoma (for example mouse, rat or rabbit) antibody by engineering and produce. For example, can change associated antibodies by recombinant DNA technology, so that the part or all of hinge area of heavy chain and/or light chain and/or constant region are replaced by the corresponding composition from the antibody of another one kind (for example people). Usually, the variable domain of engineered antibody is identical or basic identical with the variable domain of associated antibodies. This engineered antibody is called as chimeric antibody, and when when using as the individuality of the kind (for example people) in hinge area and/or constant region source, antigenicity is lower than associated antibodies. Prepare the method for chimeric antibody known in this field.
The chimeric antibody that the present invention includes can comprise a heavy chain variable domain and/or a light chain variable territory, heavy chain variable domain contain with SEQ ID NOs:19-36 in the sequence of any one identical (or basic identical), the light chain variable territory contain with SEQ ID NOs:37-45 in the sequence of any one identical (or basic identical).
Preferred human constant region comprises the constant region that derives from IgG1 and IgG4.
Fully human antibodies
Monoclonal antibody of the present invention also comprises fully human antibodies. They can prepare with the human spleen cell of external immunity (prime), and such as people such as Boerner, J.Immunol.147:86-95 (1991) is described, and perhaps with the preparation of phage displaying antibody library, such as United States Patent (USP) 6,300,064 is described.
Some other method of producing fully human antibodies comprises that use contains the endogenous Ig locus of deactivation and is genetically modified non-human animal for the human antibody heavy chain who does not reset and light chain gene. These transgenic animals can be used αvβ 6Immunity, the origin B cell that comes from them prepares hybridoma then. These methods are described in following document, for example: about the open text/patent (for example, the Lonberg United States Patent (USP) 5,789,650) of the many pieces of GenPharm/Medarex (Palo Alto, CA) of the transgenic mice that contains people Ig mini gene seat; About the many pieces of Abgenix (Fremont, CA) of XEMOMICE open text/patent (for example, Kucherlapati United States Patent (USP) 6,075,181,6,150,584 and 6,162,963; The people such as Green, Nature Genetics 7:13-21 (1994); With the people such as Mendez, 15 (2): 146-56 (1997); Text/patent (for example, the people such as EP 843 961 and Tomizuka, Nature Genetics 16:133-1443 (1997)) is disclosed with the many pieces of Kirin (Japan) about " transomic " mouse.
Humanized antibody
Monoclonal antibody of the present invention also comprises the relevant anti-α of the humanization form that derives from other kindvβ 6Antibody. Humanized antibody is the antibody that produces by recombinant DNA technology, wherein uses antigen to come auto-correlation non-human antibody's light chain and the corresponding amino acid of heavy chain in conjunction with some or all amino acid (for example constant region of variable domain and framework region) displacement of unwanted human immunoglobulin(HIg) light chain or heavy chain. For example, the constant region that on its heavy chain and light chain, all contains (1) people's antibody for a kind of humanization mouse-anti body of specific antigen; (2) from the framework region of the variable domain of people's antibody; (3) from the CDR of mouse-anti body. In case of necessity, the one or more residues in people's framework region can be changed into the residue in the corresponding site of mouse-anti body, to keep the binding affinity of humanized antibody and antigen. This change is called as " back mutation " sometimes. Humanized antibody causes immunoreactive possibility and is usually less than chimeric people's antibody in human body, because the former contains quite few inhuman composition.
The method for preparing humanized antibody is described in following document, for example: Winter EP 239 400; The people such as Jones, Nature 321:522-525 (1986); The people such as Riechmann, Nature 332:323-327 (1988); The people such as Verhoeyen, Science239:1534-1536 (1988); The people such as Queen, Proc.Nat.Acad.Sci.USA 86:10029 (1989); United States Patent (USP) 6,180,370; With the people such as Orlandi, Proc.Nat.Acad.Sci.USA 86:3833 (1989). Mouse (or other is inhuman) CDR is to the common following realization of the transplanting on people's antibody. The cDNA that from hybridoma, separates encoding heavy chain and light chain variable territory. Variable domain comprises that the dna sequence dna of CDR is measured by order-checking. The DNA of coding CDR transfers to the respective area of human antibody heavy chain or light chain variable domain encoding sequence by direct mutagenesis. Add then the human constant region genetic fragment (for example add γ 1 for CH, add κ for CL) of the isotype of wishing. Humanization heavy chain and light chain gene be coexpression in mammalian host cell (for example CHO or NSO cell), produces the solubility humanized antibody. Large-scale production for enhancing antibody; usually wish in the bioreactor that contains the antibody expression cell, to produce these humanized antibodies; perhaps produce in milk the transgene mammal (for example goat, cow or sheep) of expressing this antibody (referring to; for example United States Patent (USP) 5; 827,690).
Sometimes, CDR loses antigen-binding affinity to the antibody that the direct transfer of people's framework causes obtaining. This is because in some associated antibodies, and some amino acid in the framework region and CDR interact, thereby affects total antigen-binding affinity of antibody. In this case, in order to keep the antigen-binding activity of associated antibodies, it is very crucial introducing " back mutation " (the same) in accepting the framework region of antibody.
The commonsense method that forms back mutation is known in the art. For example, the people such as Queen (the same), the people such as Co, Proc.Nat.Acad.Sci.USA 88:2869-2873 (1991) and WO 90/07861 (Protein Design Labs Inc.) have described a kind of method that comprises two committed steps. At first, by the optimum protein matter sequence homology of Computer Analysis with relevant mouse-anti body V district framework, select people V framework region. Then, with the tertiary structure in computer simulation mouse V district, with demonstration may with the interactional framework amino acid residue of mouse CDR, on people's framework of the homology that then these mouse amino acid residues is added to.
For the method for this two steps, there are several standards to be used for designing humanized antibody. First standard be use from usually with the framework of the particular person immunoglobulin (Ig) of inhuman donor immunity globulin homology as people's acceptor, perhaps use the total framework from various human antibody. Second standard is if people's acceptor residue is unusual and the donor residue is that the human sequence is typical at the specific residue place of framework, then to use donor amino acid rather than acceptor amino acid. The 3rd standard is to use donor framework amino acid residue rather than acceptor amino acid residue in the position of next-door neighbour CDR.
Also can use a kind of diverse ways, such as Tempest, Biotechnology 9:266-271 (1991) is described. For this method, carry out CDR with the V district framework that derives from NEWM and REI heavy chain and light chain respectively and transplant, and do not introduce the mouse residue at all. An advantage using the method is that the three-dimensional structure of NEWM and REI variable region is known by X-ray crystallography, so the specificity of CDR and V district framework residue interacts and can easily simulate.
Other parts
Monoclonal antibody of the present invention also can comprise to realize the other parts of the function of wishing. For example, these antibody can comprise toxin moiety (for example tetanus toxoid or ricin) or radionuclide (for example111In or90Y), be used for the apl antibody target cell (referring to, for example United States Patent (USP) 6,307,026). These antibody can contain and are easy to the part (for example biotin, fluorescence part, radioactive segment, histidine tail or other peptide tag) of separating or detecting. These antibody also can contain a part that can prolong its serum half-life, for example polyethylene glycol (PEG) part.
The state of an illness and animal model
Antibody of the present invention can be used for αvβ 6The treatment of the disease of mediation comprises prevention. For example, these antibody are by activation or the blocking-up α of blocking-up TGF-βvβ 6With the combination of other any part (such as fibronectin, vitronectin and tenascin), can be used for treating fibrillatable (such as pulmonary fibrosis, ALI, kidney fibrosis, liver fibrosis, Alport syndrome and chorionitis). The novelty of the method comprises: the activation of (1) blocking-up TGF-β, rather than the combination of TGF-β and its acceptor, (2) can locally suppress TGF-β (namely at αvβ 6Raise site), rather than whole body suppresses (3) inhibition αvβ 6Combination with part. Except fibrotic disease, antibody of the present invention also can be used for treating cancer or cancer metastasis (comprising tumor growth and invasion and attack), particularly epithelioma. An epitheliomatous subclass is squamous cell carcinoma, for example head and neck cancer, mouth, breast, lung, prostate, cervix, pharynx, colon, pancreas and oophoroma. We use new αvβ 6Monoclonal antibody studies confirm that αvβ 6High expressed in multiple epithelioma is particularly in the leading edge of tumour. These new antibodies also can be used in αvβ 6Other any disease of mediation comprises psoriasis.
Treatment of the present invention is all effective to the humans and animals object of suffering from these diseases. The animal target that the present invention is suitable for expands to domestic animal and the livestock that obtains as pet or for commercial object. Example comprises dog, cat, ox, horse, sheep, pig and goat.
The usefulness of antibody of the present invention can be checked with different animal models. The pulmonary fibrosis mice model comprises (people such as Pittet, J.Clin.Invest.107 (12): the 1537-1544 (2001) that bleomycin is induced; With the people such as Munger, the same) and the radiation pulmonary fibrosis of inducing people such as (, Rad.Res.140:347-355 (1994)) Franko. In the mouse that bleomycin is processed, αvβ 6Be expressed in the lung epithelial alveolar cell and increase. But β6Knock-out mice is protected, and exempts from damage and fibrillatable that bleomycin is induced.
The kidney fibrosis mouse model comprise COL4A3-/-mouse (referring to, such as people such as Cosgrove, Amer.J.Path.157:1649-1659 (2000)), the mouse (people such as Wang, the Kidney International 58:1797-1804 (2000) that have the damage that adriamycin induces; The people such as Deman, Nephrol Dial Transplant 16:147-150 (2001)), the db/db mouse (people such as Ziyadeh, PNAS USA 97:8015-8020 (2000)) and the Induced by Unilateral Ureteral Obstruction mouse of blocking people such as (, Lab Investigation 81:189A (2001)) Fogo. In all these models, mouse is developed into injury of kidney and fibrillatable, can make progress to be kidney failure. For COL4A3-/-mouse that mouse that mouse, adriamycin are processed and Induced by Unilateral Ureteral Obstruction block, α in the epithelial layer of the up tubule of its kidney and descending tubulevβ 6Raise. In multiple renal injury model, αvβ 6Expression also may increase.
Also can be in as standard body the anti-α of check in these animal models of growth and metastasis of tumours modelvβ 6Monoclonal antibody suppresses the ability of tumor growth, progress and transfer. Referring to, for example, the people such as Rockwell, J.Natl.Cancer Inst.49:735 (1972); The people such as Guy, Mol.Cell Biol.12:954 (1992); The people such as Wyckoff, Cancer Res.60:2504 (2000); With the people such as Oft, Curr.Biol.8:124 (1998). Important α in the cancervβ 6Part may comprise and shift relevant TGF-β (summary is seen the people such as Akhurst, Trends in Cell Biology 11:S44-S51 (2001)), fibronectin and vitronectin.
The diagnostic method check that the usefulness for the treatment of of the present invention can obtain with multiple comprises deposition, kidney function test, ultrasonic wave, magnetic resonance imaging (MRI) and the CT scan of the observation of physical examination, blood test, albuminuria mensuration, creatinine level and CrCl, pulmonary function test, plasma urea nitrogen (BUN) level, scar or fibrillatable damage and scoring, extracellular matrix (such as collagen, smooth muscle actin and fibronectin).
Pharmaceutical composition
Pharmaceutical composition of the present invention comprises one or more antibody of the present invention, or its pharmacy acceptable derivates, randomly contains any pharmaceutically acceptable carrier. Term " carrier " comprises known acceptable adjuvant and carrier as used herein.
According to the present invention, pharmaceutical composition can be the form of aseptic injection preparation, for example sterile water for injection or oil suspension. This suspension can be formulated with suitable dispersant, wetting agent and suspending agent according to technology well known in the art.
Pharmaceutical composition of the present invention is can be on demand oral, local, in the intravenous, subcutaneous, peritonaeum, in the muscle, in the marrow, in the joint, in the synovia, in the breastbone, in the sheath, in the liver or encephalic use, perhaps be in inflammation or tumor location local application. Pharmaceutical composition of the present invention also can be used by using such as sprayer, Diskus or metered dose inhaler suction.
The present invention's dosage and the close rate of antibody that can effectively produce the effect of hope depends on many factors, as the character of the disease for the treatment of, experimenter's body weight, therapeutic purposes, the concrete pharmaceutical composition of use and the doctor in charge's judgement. The about 100mg/kg body weight of about 0.001-every day, for example the dosage level of the active compound component of the about 50mg/kg body weight of about 0.1-every day is useful. For example, antibody of the present invention will be with 1-14 days interval, and to about 20mg/kg body weight/day, for example about 0.1mg/kg body weight/day is used to the dosage of about 10mg/kg body weight/day with about 0.01mg/kg body weight/day. In another embodiment, when using in the antibody peritonaeum, use with the dosage of about 0.3-1mg/kg body weight. In another embodiment, when the antibody intravenous is used, to use about the dosage of 5-12.5mg/kg body weight. In one embodiment, antibody compositions with effective generation at least the amount of the plasma antibody level of 1mg/ml use.
Diagnostic method
Antibody capable of the present invention is enough to be diagnosed and α vβ 6Expression level changes diseases associated.From experimenter's tissue sample,, can wait and detect by antigen capture mensuration, ELISA, the immunohistochemical methods mensuration of using these antibody as biopsy, humoral specimen or irrigating solution (as bronchoalveolar lavage fluid).From the tissue sample of normal individual in contrast.
Unless otherwise noted, enforcement of the present invention will be used cytobiology, cell cultures, molecular biology, microbiology, recombinant DNA, albumen chemistry and immunology routine techniques, and these technology belong to the technical ability of this area.These technology are described in the literature.Referring to, for example " molecular cloning: lab guide ", second edition people such as (compile) Sambrook, 1989; " oligonucleotide is synthetic " (M.J.Gait volume), 1984; Authorize people's such as Mullis United States Patent (USP) 4,683,195; " nucleic acid hybridization " (B.D.Hames and S.J.Higgins), 1984: " transcribe and translate " (B.D.Hames and S.J.Higgins), 1984: " animal cell culture " (R.I.Freshney volume), 1987; " fixed cell and enzyme ", IRL Press, 1986: " molecular cloning practical guide " (B.Perbal), 1984; " Enzymology method ", the 154th and 155 volumes people such as (compile) Wu, Academic Press, New York; " gene transfer vector that is used for mammalian cell " (J.H.Miller and M.P.Calos compile), 1987; " immuno-chemical method in cell and the molecular biology " (Mayer and Walker compile), 1987; " experiment immunization is learned handbook, I-IV volume (D.M.Weir and C.C.Blackwell compile), 1986; " operation of mice embryonic ", 1986.
Unless otherwise defined, as used herein all technology all the implication with those skilled in the art in the invention's common sense is identical with scientific terminology.Typical method and material are described hereinafter, but also can use in enforcement of the present invention to similar or suitable method described herein and material.The open text of all that mention and other reference are incorporated by reference in this text and examine herein.If conflict with this specification sheets, comprises definition, be as the criterion.Material, method and embodiment are illustrative, but not are intended to restriction.In this manual, word " comprises " or its tense changes and means and comprise described integral body or whole group, but does not get rid of other any integral body or whole group.
Embodiment
The following example is intended to illustrate method of the present invention and material.Usually run in the antibody field, well known to a person skilled in the art, the suitable modification and the change of described condition and parameter, within the spirit and scope of the present invention.
In the following example, β 6People such as-/-mouse such as Huang, the described generation of J.Cell Biol.133:921 (1996).Recombinant human LAP is available from R﹠amp; D Systems (Minneapolis, MN).Antibody 10D5 available from Chemicon (Temecula, CA).The L230 hybridoma is available from ATCC, and excretory antibody is by affinity chromatography purifying from the supernatant liquor of saturated culture on the fixed albumin A.The isotype somatotype of antibody carries out according to manufacturers instruction with ISOSTRIP test kit (Roche Diagnostics).β 6People such as the SW480 clone of transfection such as Weinacker, the described preparation of J.Biol.Chem.269:6940-6948 (1994).
Embodiment 1: β 6The generation of the stable cell lines of transfection
β 6The NIH 3T3 of transfection and the following generation of FDC-P1 cell: with the DNA construct electroporation parental cell line that contains total length mouse β 6cDNA and Xin Meisu selected marker.The following screening of the cell of stable transfection: passage cell is 14 days in containing the substratum of G418, separates the surperficial β that expresses highest level by fluorescence-activated cell sorting (FACS) subsequently 6Cell.The FDC-P1 cell of transfection is cultivated in DMEM, wherein replenished the 4mM L-glutaminate, be adjusted to and contain 1.5g/L sodium bicarbonate, 4.5g/L glucose and 1.0mM Sodium.alpha.-ketopropionate, 10% FBS, 2.5% mouse IL-3 cultivation additive and the active G418 of 1.5mg/ml.The NIH 3T3 cell of transfection is cultivated in the DMEM that has replenished the active G418 of 10% FBS, 2mM L-glutaminate, penicillin/streptomycin and 1mg/ml.
Embodiment 2: soluble human α vβ 6Purifying
α vβ 6The described purifying of protein Weinacker substantially as mentioned.Cultivate a kind of expression hs α vβ 6Chinese hamster ovary celI system, the supernatant liquor that centrifugal collection obtains.Use anti-α vAntibody L230 is by the affinitive layer purification integrin.The L230 of purifying and CNBr activated Sepharose 4B (Sigma) are crosslinked with the ratio of 4.8mg antibody/ml resin.α vβ 6Supernatant liquor is added on the L230 affinity column with the ratio of 0.5mg antibody/ml resin, washes post with following every kind of solution of 10 times of volumes: (1) 50mM Tris-Cl, pH7.5,1M NaCl, 1mM MgCl 2(2) 50mM Tris-Cl, pH7.5,50mM NaCl, 1mM MgCl 2(3) 10mM Na 3PO 4, pH7.0.Hs α vβ 6Use the 100mM glycine, pH2.5 is eluted to the 1M Na of 1:10 volume 3PO 4, in the pH8.0.Protein is to phosphoric acid buffer (PBS) dialysis, during change PBS several times, be stored in-20 ℃.
Embodiment 3: β 6The immunity of-/-mouse
β 6-/-mouse is injected at emulsive 25 μ g purifying recombination human alphas in the complete Freund's adjuvant (CFA) by intraperitoneal (IP) vβ 6And immunity, its volume ratio is 1:1, cumulative volume is 200 μ l.In addition, β 6-/-mouse is also by IP injection 4 * 10 6β 6The NIH 3T3 cell of transfection and immunity, these cells are resuspended to and have replenished 1mg/ml CaCl 2With 1mg/ml MgCl 2100 μ l PBS in, same mouse is also injected 100 μ l CFA in adjacent regions.After 2 weeks of beginning immunity and 4 weeks, with identical reagent booster immunization mouse similarly, difference is to use incomplete Freund's adjuvant replaced C FA.Mouse is strengthened blood sampling after 7 days the last time, according to the recombinant human alpha of serum and purifying vβ 6Or and β 6Anti-β is measured in the combination of cells transfected 6Titre.For recombinant human alpha with purifying vβ 6Mice immunized was had a rest mouse 3 months, used and the immunity once more of ImmunEasy (Qiagen) blended same antigen.Merging separating spleen preceding 3 days, by IP and intravenous injection, with the recombinant human alpha of 12.5 μ g purifying for hybridoma vβ 6The protein immunization mouse.Merging the same day, kill animals is taken out spleen, makes single cell suspension.But splenocyte becomes infinite multiplication by merging with the cytogamy mating partner of medicament selection.
Embodiment 4: the screening of hybridoma
Pass through β 6The immunity of-/-mouse produces two groups of antibody.One group of antibody is by the soluble human α with brachymemma vβ 6(merging #6) immunity produces.Another group antibody is by using mouse β 6The NIH 3T3 cell of transfection (merging #7) immunity produces.Anti-α vβ 6The screening of antibody is with carrying out with acellular combination and functional examination based on cell as described below.The initial screening of positive colony is based on the hs α with purifying vβ 6And β 6The combination of the people of transfection and mouse cell (cell of untransfected in contrast).Make the clonal expansion of selection, utilize the cell capture test to estimate whole culture and β once more 6(they are meant respectively merges 6 and merge 7 for the representative embodiment that Figure 1A and 1B show, prefix " 6. " or " 7. " of wherein having omitted the mAb title in the combination (embodiment 5b, the same) of transfection and cell untransfected; See table 2).Some antibody preference is in conjunction with β 6Cells transfected, and some all combines with cell untransfected with transfection, shows to have only the antibody of a subclass to have β 6Preference (Figure 1A and 1B).Further screening is based on the biotinylated hs α of antibody blocking vβ 6And β 6The mouse cell of transfection and LAP bonded ability.The clone who uses the FACS subclone to select, frozen before use.
According to α vβ 6Bonded specificity screening monoclonal antibody, this is based on them in conjunction with β 6The ability of the parental cell of cells transfected and debond untransfected.According to they can not with express alpha vβ 3, α vβ 8, α 5β 1, α vβ 1Or α 5β 1The clone combination, these monoclonal antibodies further are proved to be α vβ 6Rather than other α vIntegrin or non-specific integrin are (promptly with the non-α of part bonded that contains RGD vIntegrin) specific binding agents.JY, K562, SW480, NIH3T3 and FDCP1 clone comprising the cell and the untransfected of stable transfection.
List in the following Table 1 by the some of them antibody of ATCC preservation.
The hybridoma of table 1 preservation
The hybridoma clone ATCC number Preservation date
6.1A8 PTA-3647 August 16 calendar year 2001
6.2B10 PTA-3648 August 16 calendar year 2001
6.3G9 PTA-3649 August 16 calendar year 2001
6.8G6 PTA-3645 August 16 calendar year 2001
6.2B1 PTA-3646 August 16 calendar year 2001
6.2A1 PTA-3896 December 5 calendar year 2001
6.2E5 PTA-3897 December 5 calendar year 2001
7.1G10 PTA-3898 December 5 calendar year 2001
7.7G5 PTA-3899 December 5 calendar year 2001
7.1C5 PTA-3900 December 5 calendar year 2001
Embodiment 5: be used to the test screening and characterize
a.α vβ 6ELISA
One 96 hole microwell plate (Corning COSTAR EASY-WASH) is with 50 μ l/ holes, 50 μ g/ml hs α vβ 6Bag is spent the night under 4 ℃.The dull and stereotyped lavation buffer solution (0.1% TWEEN-20 is dissolved in PBS) of using in automatic washer washs 4 times.Add 180 μ l/ holes then and be dissolved in 3% BSA of TBS, 25 ℃ of following incubations 1 hour with the blocking-up non-specific binding.Flat board washs as mentioned above, adds (50 μ l/ hole) hybridoma supernatant liquor (being used for shaker test) or antibody purified (being used for characterizing) and is containing 1mg/ml BSA, 1mM CaCl 2With 1mM MgCl 2TBS in diluent.Dull and stereotyped 25 ℃ of following incubations 1 hour, washing is then with 50 μ l/ hole superoxide link coupled goat anti-mouse IgG+A+M antibody (Cappel) incubation 1 hour.With 3,3 ', 5,5 '-tetramethyl benzidine (TMB) detects bonded antibody.Represent in conjunction with the absorbancy of measuring with 450nm.
B. cell capture test
One 96 hole microwell plate is with 50 μ l/ hole second antibody (the anti-mouse IgG of donkey (JacksonImmunoresearch); 5 μ g/ml dilute in the 50mM of pH9.2 sodium bicarbonate) wrap down at 4 ℃ and spent the night.Dull and stereotyped with 100 μ l/ holes mensuration damping fluid (RPMI+2%BSA) washed twice, measure damping fluid with 100 μ l/ holes then and sealed 1 hour down at 37 ℃.For FDC-P1 cell and β 6The FDC-P1 cell of transfection, dull and stereotyped with anti-mouse Ig (Jackson ImmunoResearch; 20 μ g/ml) at room temperature sealed 10 minutes, with the non-specific Fc receptors bind (other cellular type is omitted) that reduces second antibody.When sealing is dull and stereotyped, cell in measuring damping fluid with 5 * 10 6The concentration of cell/ml is with 2 μ M fluorescence dye (fluorexon-AM, MolecularProbes) marks.At 37 ℃ of water-baths and incubation 15 minutes under the jolting gently, centrifugal collection is resuspended in the mensuration damping fluid to 5 * 10 in measuring damping fluid for cell and dyestuff 6Cell/ml.After the sealing step, discard damping fluid by patting flat board, in flat board, add the supernatant liquor or the antibody purified in 25 μ l/ holes.37 ℃ of following incubations 15 minutes, add the cell of 25 μ l/ hole marks afterwards, dull and stereotyped 37 ℃ of following incubations 1 hour.Dull and stereotyped with measuring damping fluid (100 μ l/ hole) washing 3-5 time, write down the dull and stereotyped fluorescence that captured cell is sent of going up.By the fluorescence (being the bonded cell) after comparing last washing step fluorescence (i.e. total cell of Tian Jiaing) before and washing, measure in conjunction with per-cent.
c.FACS
By the tryptic digestion collecting cell, with the PBS washing once, be resuspended to FACS damping fluid (1 * PBS, 2% FBS, 0.1% NaN then 3, 1mM CaCl 2With 1mM MgCl 2) in.0.2 * 10 5Cell then in the FACS damping fluid incubation on ice 1 hour, contain the hybridoma supernatant liquor in this damping fluid, cumulative volume is 100 μ l.Behind the incubation, cell is with ice-cold FACS damping fluid washed twice, is resuspended in the FACS damping fluid that 100 μ l contain 5 μ g/ml donkey anti-mouse IgG PE (JacksonImmunoResearch), incubation on ice 30 minutes.Cell is used ice-cold FACS damping fluid washed twice then, is resuspended in the 200 μ l FACS damping fluids.Monitor the combination of the second antibody of PE mark by flow cytometry.
D. vitamin H-hs α vβ 6With combining of LAP
96 hole microwell plates (Cornin COSTAR EASY-WASH) are used in the 0.3 μ g/ml recombinant human LAP (R﹠amp that dilutes among the PBS; D Systems, Cat.#246-LP) (50 μ l/ hole) wrapped down at 4 ℃ and spent the night.Remove bag by solution after, dull and stereotyped sealed 1 hour down at 25 ℃ with 180 μ l/ holes, 3% BSA/TBS.On other one 96 hole circle base plate, 60 μ l/ holes, 2 * stock solution (0.5 μ g/ml (1.25nM) vitamin H-α vβ 6, 2mM CaCl 2With 2mM MgCl 2, be dissolved in the TBS that contains 1mg/mlBSA) mix with 60 μ l/ hole hybridoma supernatant liquors (be used for screening) or 2 * stoste of antibody purified (also being dissolved in the TBS that contains 1mg/ml BSA), and 25 ℃ of following incubations 1 hour.The flat board of LAP bag quilt washs 4 times with lavation buffer solution (0.1% TWEEN-20 is dissolved in PBS) with automatic washer, afterwards with 100 μ l antibody-α vβ 6Mixture was transferred on the flat board, 25 ℃ of following incubations 1 hour.Flat board washs as mentioned above, with the 1:1000 diluent of 50 μ l/ hole extravidin-horseradish peroxidase things (Sigma) in TBS (1mg/ml BSA) 25 ℃ of following incubations 1 hour.Bonded protein detects with tmb substrate.
E. β 6The adhesion of-FDC-P1 cell and LAP
96 hole microwell plates (Cornin COSTAR EASY-WASH) with 50 μ l/ holes at the 50mM sodium bicarbonate, the 0.5 μ g/ml recombinant human LAP (R﹠amp that dilutes among the pH9.2; D Systems) bag is spent the night under 4 ℃.Dull and stereotyped with PBS (100 μ l/ hole) washed twice, sealed 1 hour down at 25 ℃ with the 1%BSA (100 μ l/ hole) that is dissolved in PBS.It is dull and stereotyped that (the TBS perfect solution adds 1mM CaCl with 100 μ l/ holes mensuration damping fluid 2With 1mM MgCl 2) washed twice.Then, in each hole of flat board, add 25 μ l hybridoma supernatant liquors (or antibody purified) and 25 μ l β 6-FDC-P1 cell (5 * 10 6Cell/ml uses fluorexon AM mark as mentioned above).Dull and stereotyped 25 ℃ of following incubations 1 hour, then with measuring damping fluid (100 μ l/ hole) washing 4-6 time.Be recorded in the dull and stereotyped fluorescence that captured cell is sent of going up.By the fluorescence (being the bonded cell) after comparing last washing step fluorescence (i.e. total cell of Tian Jiaing) before and washing, measure in conjunction with per-cent.
F.TGF-β biological assay
The TGF-β biological assay of Shi Yonging is people such as Abe herein, and the described mink pulmonary epithelial cells of Anal.Biochem.216:276-284 (1994) (MLEC) PAI-1 luciferase is cultivated a kind of variation of mensuration, wherein β altogether 6Cells transfected and report co-culture of cells are with monitoring α vβ 6Activation (Munger, the same) to TGF-β.This is a kind of quantitative biological assay to TGF-β, is based on the ability that it can induce Type 1 plasminogen activator inhibitor-1 (PAI-1) to express.In this was measured, the MLEC cell contained the PAI-1 promotor of the brachymemma of merging with Lampyridea luciferase reporter gene with a kind of expression construct stable transfection, this construct.The MLEC cell of transfection and active TGF-β (0.2 to〉30pM) contact and cause that luciferase activity dependent dose ground increases in the cell lysate.
In order to carry out this mensuration, the PAI-1-luciferase construct transfection of TMLC (the mink pulmonary epithelial cells is Mv 1 Lu) cell.Cells transfected is cultivated in the DMEM+10%FBS that contains L-Gln, Pen/Strep and 200 μ g/ml G418.Use integrin β 6SW480 the cell (" β of construct transfection 6-SW480 " or " SW480 β 6" cell) in containing the DMEM+10% FBS of L-Gln and Pen/Strep, cultivate.From containing the picking cell the bottle that shakes of PBS+5mM EDTA,,, be inoculated in 96 orifice plates with the hematimeter counting with PBS+0.5% BSA washing.SW480-β 6Cell in lavation buffer solution with 4 * 10 4The cells/well inoculation.Monoclonal anti body and function DMEM (serum-free) dilution is added to SW480-β 6In the cell, preincubation at room temperature 20 minutes.Then with 2 * 10 4It is 100 μ l that cells/well adds TMLC cell to final volume.Dull and stereotyped moist, be rich in CO 2Incubator in incubation 20 hours.Discard dull and stereotyped supernatant liquor, replace with 100 μ l PBS+1mM Ca 2+With 1mM Mg 2+Cell on the cracking flat board detects the luciferase activity level with light emitting-type reaction Packard LUCLITE test kit (#6016911) and TROPIX microwell plate photometer then.
Embodiment 6: purifying antibody
Select 8 to clone (representing) from the hybridomas clone of merging #6 (with prefix " 6. " expression) and 14 from the hybridoma that merges #7 and further amplify and characterize (table 2) with prefix " 7. ".
The small-scale culture (150ml) for preparing every kind of hybridoma, centrifugal collection supernatant liquor.Utilize albumin A affinity chromatography antibody purification from these supernatant liquors.For IgG 2aIsotype antibody directly is added to supernatant liquor albumin A Sepharose 4 Fast Flow (Amersham PharmaciaBiotech, AB, Uppsala, Sweden) and goes up (1ml deposition bed volume).Wash post with PBS, IgG fraction 25mM phosphoric acid, 100mM NaCl, pH2.8 are eluted to the 0.5MNa of 1:20 volume 3PO 4, among the pH8.6.For mouse IgG 1Antibody is adjusted to the 1.5M glycine with supernatant liquor before last sample, 3M NaCl, and pH8.9 uses 25mM Na before wash-out 3PO 4, 3M NaCl, pH8.6 washes post.Carry out external biological chemical characterization described herein with these goods.
Table 2 hybridoma clone's sign
Clone's title Clone number Isotype Blocker
6.1A8 2 IgG2a Y
6.2B10 10 IgG2a Y
6.3G9 25 IgG1 Y
6.4B4 30 IgG1 N
6.6B5 46 IgG1 N
6.8B4 55 IgG1 N
6.8G6 56 IgG1 Y
6.2B1 85 IgG1 Y
7.1C5 2 IgG2a Y
7.1G10 5 IgG2a Y
7.2A1 6 IgG2a Y
7.2F5 11 IgG2a Y
7.2H2 12 IgG2a Y
7.4A3 17 IgG2a Y
7.7G5 32 IgG1 Y
7.8H12 39 IgG2a Y
7.9D4 40 IgG2a N
7.9G8 41 IgG2a Y
7.9H5 43 IgG2a Y
7.10D7 44 IgG2a Y
7.10H2 46 IgG2a Y
*: blocker is defined as a kind of blocking-up α vβ 6With LAP bonded antibody, according to the hs α of part and purifying vβ 6Or with express β 6Cell in conjunction with measuring.
In order to use in animal model, the hybridoma clone is enlarged into the 2L substratum, cultivates for 4 weeks in LifecellCulture Bags-PL732 (Nexell, catalog number (Cat.No.) R4R2113).At first, pass through the ion-exchange step on the Q Sepharose (AmershamPharmacia) subsequently, the antibody that the purifying hybridoma produces by aforesaid albumin A affinity chromatography.With 2M Tris alkali the elutriant that the albumin A chromatographic step obtains is adjusted to pH8.6,10 times of dilute with waters are added to and use 10mM Na 3PO 4, 25mM NaCl is on the pH8.6 equilibrated Q Sepharose post (20mg protein/ml resin).Level pad with 5 times of column volumes is washed post, uses 25mM Na 3PO 4, 150mM NaCl, the protein of pH7.2 elution of bound.The protein of sterile filtration (0.45 μ m) wash-out is stored in-70 ℃ up to use.
Embodiment 7: the sign of antibody purified
To following aspect quantitatively characterizing antibody purified (table 2, the same): they measure in (the same) (1) in conjunction with hs α at MLEC vβ 6, (2) are in conjunction with β 6The SW480 of transfection and FDC-P1 cell, (3) suppress vitamin H-α vβ 6Combine with LAP, the FDC-P1 cell that (4) suppress β 6 transfections combines with LAP and (5) blocking-up α vβ 6The TGF-β activated ability of-mediation.The relative efficiency of every kind of mensuration and known α vβ 6Antibody 10D5 (people such as Huang, J.Cell Sci.111:2189 (1998)) compares, in some cases, with anti-α v-antibody L230 compares.To merge #7 antibody in order characterizing, to merge #6 antibody 6.8G6 also as positive control.
At 1mM Ca 2+With 1mM Mg 2+Exist an initial combination experiment (embodiment 5a, the same) of carrying out down to show the antibody and the hs α of most purified vβ 6In conjunction with (Fig. 2 A and 2B).Yet be not observe combination unexpectedly for 10D5 and clone 7.2F5 and 7.10D7.An experiment subsequently shows that the combination of 10D5 (Fig. 3 E), 7.2F5 and 7.10D7 is only by Ca 2+/ Mg 2+Support, but 1mM is MnCl more weakly 2Stronger.In these new clones, having three (6.1A8 (Fig. 3 A), 7.7G5 and 6.8G6 (Fig. 3 C)) to show needs divalent cation, but Ca 2+/ Mg 2+Attitude and Mn +-combined is not observed difference.
Remaining clone shows does not need divalent cation, promptly can combine (Fig. 3 B, 3D and 3F) with antigen in the presence of 10mM EDTA.Facs analysis can with β 6The NIH 3T3 cell of transfection or SW480 cell bonded antibody show a kind of similar pattern, and difference is in this article, and 10D5 is at Ca 2+/ Mg 2+And Mn 2+Combination in the same manner during attitude.With solubility α vβ 6Bonded need may be different from the α with cell surface expression vβ 6Bonded needs, because protein conformation or avidity effect difference.
These results suggest have 3 different classes of β at least in this group 6Blocking antibody.Wherein a class (10D5) can be distinguished Ca 2+/ Mg 2+With Mn 2+Condition.Another kind of (comprising 6.1A8,7.7G5 and 6.8G6) needs positively charged ion, but can not distinguish Ca 2+/ Mg 2+With Mn 2+Last class (comprises anti-α vAntibody L230,6.2B10,6.3G9 (Fig. 3 B) and 6.2B1 (Fig. 3 D), 7.1C5 and 7.1G10) be do not rely on cationic.
Estimate antibody purified then and suppress α vβ 6The interactional ability of-LAP.In the cell-less measurement of embodiment 5d mentioned above, antibody 6.1A8,6.2B1,6.3G9 and 6.8G6 show the IC that is lower than 10D5 50Value (Fig. 4 A; Table 3).6.2B10 show higher IC 50, suppress (Fig. 4 A) fully but still produce.6.4B4 only the display part suppresses, and 6.6B5 and 6.8B4 do not show inhibition (Fig. 4 A).Utilize same mensuration system, antibody 7.1C5,7.1G10,7.2A1,7.4A3,7.7G5,7.9G8,7.9H5 and 7.10H2 show the IC that is lower than 10D5 50Value (Fig. 4 B; Table 3).Antibody 7.2F5,7.2H2 and 7.8H12 show IC much at one or higher 50Value, and still produce inhibition (Fig. 4 B) fully.
Above observing similar trend in the described raji cell assay Raji of embodiment 5e, exception be 6.1A8,6.2B10 and 7.9D4, the usefulness of their pair cells is lower than proteinic usefulness (Fig. 5 A-E to purifying; Table 3).
In a word, these results show, but we have successfully produced specificity inhibition people and mouse α vβ 6With the interactional antibody of LAP.Some such antibody with high-affinity in conjunction with α vβ 6(apparent Kd ' s 〉=0.3nM measures by flow cytometry) suppresses β 6Cells transfected combines IC with LAP's 50〉=0.05nM (8ng/mL), and stop α vβ 6The activation of the TGF-β 1 of mediation, IC 50〉=0.58nM (87ng/mL).
At last, utilize PAI-1/ luciferase reporter gene to measure (embodiment 5f, the same) and estimate antibody purified blocking-up α vβ 6The TGF-β activated ability of mediation.6.3G9,6.8G6,6.2B1,7.1G10 and 7.7G5 can suppress α once more vβ 6The activation of the TGF-β of mediation, IC 50Value is lower than 10D5, and remaining antibody seems validity significantly lower (Fig. 6 A and 6B in this mensuration; Table 3).Therefore, blocking-up α vβ 6With the interactional ability of LAP and relevant in vitro inhibition TGF-β activated ability.
Table 3 hybridoma clone's sign
Figure C200610103152D00261
*: data are from different experiments.
*: do not detect.
* *: all experiments that table 3 is summed up are all at 1mM Ca 2+With 1mM Mg 2+Carry out under existing.
* * *: the antibody of representing with boldface type is antibody 10D5 of the prior art and L230, and to α vβ 6The new antibodies that extra high inhibition ability is arranged.
Then, utilize a kind of kinetics eliminating test (KinExA) mensuration 6.3G9 and 6.8G6 to solubility α vβ 6Solution avidity.A series of diluents (1 * 10 of solubility integrin -8M to 2.4 * 10 -12M) with 1 * 10 -10M antibody incubation 3 hours.(Boise Idaho), makes these samples by the polymethylmethacrylate pearl with integrin bag quilt for SapidyneInstruments, Inc. to use the KinExA instrument then.For 6.8G6, comprise 1mM CaCl in incubation and the mensuration damping fluid 2With 1mM MgCl 2The content of bonded and free antibody is measured with the anti-mouse second antibody of Cy5-mark.Carry out conic fitting with KinExA software, to obtain each interactional dissociation constant (Kd).Kd ' the s that measures with this method is, 6.3G9 is 15.6pM, and 6.8G6 is 22.8pM (Fig. 9 A and 9B).Therefore, these two kinds of antibody are to α vβ 6High avidity is all arranged.
We have further identified the anti-α of " activation " state that can discern integrin vβ 6The classification of antibody.α vβ 6Two kinds of possible states of activation are arranged.First kind of state, activated integrin are defined as that its part is had higher avidity.At activatory positively charged ion such as 1mM MnCl 2Exist down, show the enhancing that combines with integrin for the special antibody of this state of activation.Compare 1mM Mn Cl by flow cytometry 2With 1mM MgCl 2Combination degree in (non-activated positively charged ion) shows some α described herein vβ 6Antibody comprises 6.1A8 and 6.6B5, at MnCl 2Exist and show remarkable enhanced combination down.
For α vβ 6Second kind of state of activation, this integrin can activate the TGF-β that hides as mentioned above.Prepare to express the α of brachymemma vβ 6A kind of clone (SW480 (β 6-770T)).In TMLC luciferase assay people such as (, the same) Munger, this clone can be in conjunction with LAP, but can not activate TGF-β.Therefore, can with total length β 6The SW480 cell of transfection in conjunction with, but not with the transfectional cell bonded antibody of 770T brachymemma, for the α that can activate TGF-β vβ 6Form is special.Antibody 7.8B3 and 7.8C9 satisfy this standard.
Embodiment 8: according to the epitope mapping of antibody competition
Also the monoclonal antibody that has detected purifying with the ELISA form combines biotinylation α with the 6.8G6 competition vβ 6Ability.In this was measured, the 6.8G6 bag was by on elisa plate, to containing each 1mM Ca 2+And Mg 2+Damping fluid in add competition antibody and biotinylation α vβ 6Mixture.The bonded integrin detects with the extravidin-HRP conjugate, according to competitive antibody blocking-up bonded ability it is marked.Except 6.2B10 (a kind of weak blocker), all common blockers (table 2) all show and can compete (table 4) to some extent with 6.8G6.These data acknowledgements, these common blockers can be in conjunction with or eclipsed epi-position identical with 6.8G6.
Table 4 is according to the epitope mapping of antibody competition
Clone common blocker? competition with 6.8G6
6.2A1 N-6.4B4 N-6.6B5 N-6.8B4 N-7.9D4 Y/N-10D5 Y ++ L230 Y ++ 6.1A8 Y ++ 6.2B10 Y (weak)-6.3G9 Y+6.8G6 Y ++ 6.2B1 Y ++ 7.1C5 Y +++7.1G10 Y +++7.2A1 Y ++ 7.2F5 Y ++ 7.2H2 Y ++ 7.4A3 Y ++ 7.7G5 Y ++ 7.8H12 Y ++ 7.9G8 Y ++ 7.9H5 Y ++ 7.10D7 Y+7.10H2 Y ++
The monoclonal antibody of utilizing ELISA to detect purifying combines α with biotinylation 6.3G9 or biotinylation 6.8G6 competition vβ 6Ability.In this is measured, unlabelled α vβ 6Bag is by on elisa plate, to containing each 1mM Ca2 +And Mg 2+Damping fluid in add the mixture of competition antibody and biotinylated antibody.Utilize the neutravidin-HRP conjugate to detect the bonded biotinylated antibody.Data show that the strongest blocking antibody (for example 6.2B1,7.1C5 and 7.1G10) combines α with 6.3G9 and 6.8G6 competition vβ 6(table 4.1, Figure 10 A and 10B).Antibody 6.1A8 and 7.7G5 show lower competitiveness, and this may be because they are to α vβ 6Avidity lower.In this is measured, non-blocking antibody or anti-α vAntibody L230 does not show with 6.3G9 or 6.8G6 any competition.These results show, α vβ 6Special blocking antibody can in conjunction with α vβ 6Identical or eclipsed epi-position.
Table 4.1 is according to the epitope mapping of antibody competition
Clone common blocker? competition 6.3G9 competition with biotinylation and biotinylation 6.8G6
6.3G9 Y +++ +++6.2B1 Y +++ +++6.8G6 Y +++ ++7.1C5 Y +++ +++7.1G10 Y +++ +++7.7G5 Y ++ +6.1A8 Y ++ +6.2A1 N - -6.2E5 N - -6.2G2 N - -6.4B4 N - -7.8B3 N - -7.8C9 N - -L230 Y - -
Embodiment 9:CDR sequence
As people's such as Coligan (writing) " modern immunological method ", Wiley, Media, PA (2001) is described, utilizes the cDNA of the monoclonal antibody of standard technique separation and some purifying that checks order.The aminoacid sequence of inferring shows in Fig. 7 A and 7B.
The aminoacid sequence (dash indication notch) of following heavy chain CDR than higher affinity wedding agent 6.8G6,6.1A8,6.2B1,6.3G9 and 6.2A1 and non-blocker 6.2G2.
CDR1
6.8G6 SYTFTDYAMH(SEQ?ID?NO：1)
6.1A8 SYTFTDYTMH(SEQ?ID?NO：2)
6.2B1 GFTFSRYVMS(SEQ?ID?NO：3)
6.3G9 GFTFSRYVMS(SEQ?ID?NO：3)
6.2A1 GYDFNNDLIE(SEQ?ID?NO：49)
6.2G2 GYAFTNYLIE(SEQ?ID?NO：50)
CDR2
6.8G6 VISTYYGNTNYNQKFKG(SEQ?ID?NO：4)
6.1A8 VIDTYYGKTNYNQKFEG(SEQ?ID?NO：46)
6.2B1 SISSG-GSTYYPDSVKG(SEQ?ID?NO：5)
6.3G9 SISSG-GRMYYPDTVKG(SEQ?ID?NO：6)
6.2A1 VINPGSGRTNYNEKFKG(SEQ?ID?NO：51)
6.2G2 VISPGSGIINYNEKFKG(SEQ?ID?NO：52)
CDR3
6.8G6 GGLRRGDRPSLRYAMDY(SEQ?ID?NO：7)
6.1A8 GGFRRGDRPSLRYAMDS(SEQ?ID?NO：47)
6.2B1 GAIYDG-----YYVFAY(SEQ?ID?NO：8)
6.3G9 GSIYDG-----YYVFPY(SEQ?ID?NO：9)
6.2A1 IYYGPH-----SYAMDY(SEQ?ID?NO：53)
6.2G2 ID-YSG-----PYAVDD(SEQ?ID?NO：54)
In SEQ ID NO:7, represent that with " R " (the 12nd residue) that black matrix is represented it has polymorphism, for example can be Q.
The aminoacid sequence of the light chain CDR of these 4 kinds of high-affinity wedding agents of following comparison and non-blocker 6.2G2.
CDR1
6.8G6 RASQSVSTSS-YSYMY(SEQ?ID?NO：10)
6.1A8 RASQSVSIST-YSYIH(SEQ?ID?NO：48)
6.2B1 SASSSVSSS----YLY(SEQ?ID?NO：11)
6.3G9 SANSSVSSS----YLY(SEQ?ID?NO：12)
6.2A1 KASLDVRTAVA (SEQ?ID?NO：55)
6.2G2 KASQAVNTAVA (SEQ?ID?NO：56)
CDR2
6.8G6 YASNLES(SEQ?ID?NO：13)
6.1A8 YASNLES(SEQ?ID?NO：13)
6.2B1 STSNLAS(SEQ?ID?NO：14)
6.3G9 STSNLAS(SEQ?ID?NO：14)
6.2A1 SASYRYT(SEQ?ID?NO：57)
6.2G2 SASYQYT(SEQ?ID?NO：58)
CDR3
6.8G6 QHNWEIPFT(SEQ?ID?NO：15)
6.1A8 QHSWEIPYT(SEQ?ID?NO：16)
6.2B1 HQWSSYPPT(SEQ?ID?NO：17)
6.3G9 HQWSTYPPT(SEQ?ID?NO：18)
6.2A1 QQHYGIPWT(SEQ?ID?NO：59)
6.2G2 QHHYGVPWT(SEQ?ID?NO：60)
Shown in Fig. 7 A and 7B, as if the monoclonal antibody (for example 6.1A8 and 6.8G6) that belongs to the classification that relies on divalent cation contains very similarly aminoacid sequence in CDR, and the monoclonal antibody (for example 6.2B1 and 6.3G9) that does not rely on divalent cation contains other one group of motif at its CDR.
Anti-α vβ 6The usefulness of monoclonal antibody and specificity are controlled by the amino-acid residue of fine difference may.For 6.1A8 and 6.8G6, the aminoacid sequence of variable domain is closely similar, contains 10 amino acid differences in heavy chain, wherein guards for 3, contains 11 amino acid differences in light chain.Yet these antibody have about 100 times activity difference in external test.Amino acid difference is dispersed in the whole variable domain of polypeptide chain, and these residues can work separately, perhaps with same chain or spouse's chain on the residue co-action, influence the usefulness of antibody.In heavy chain, the position of 7 residues makes that they may be closely near α vβ 6, perhaps with α vβ 6Combination in play active function.
In a large amount of integrin conjugated proteins (part), found a kind of RGD motif.This motif has shown the interaction that can mediate they and integrin by the binding pocket on the direct contact integrin.Because this is quite common in the integrin conjugated protein for RGD, the flank residue outside this motif works aspect the binding specificity certainly in that integrin-ligand interaction is had.In 6.1A8 and 6.8G6, such flank residue is positioned at 101 places, site of heavy chain, in CDR3.This amino-acid residue is positioned at RGD motif flank, may be positioned at the antigen recognition site place, influences bonded usefulness and specificity.
6.1A8 other different residues interior with the identical heavy chain CDR of 6.8G6 comprise site 33 (CDR1); Site 52,57 and 65 (CDR2); The residue of locating with site 115 (CDR3).Another one difference in the heavy chain is positioned at 4 places, site of framework 1, near the N end.By the folding CDR of this residue of crystallography model prediction near antibody, may be at α vβ 6In conjunction with in play an important role.6.1A8 and all the other 3 differences between the 6.8G6 are the conservative difference that site 20 (framework 1), 44 (frameworks 2) and 82 (frameworks 3) are located.
The aminoacid sequence that does not rely on cationic antibody also is the height homologous.They can be divided into two classes: with (being 6.2B1,6.3G9,7.10H2,7.9H5,7.1C5,7.1G10 and 7.4A3) of the antibody 6.8G6 competition that contains RGD; Not with it the competition (being 6.2A1,6.2B10 and 6.4B4).In the CDR3 of heavy chain, contain the FXY motif with a class of 6.8G6 competition, and a non-competing class does not contain.This species diversity prompting, the FXY motif is for mediation and α vβ 6Do not rely on cationic in conjunction with most important.In addition, this class contain FXY antibody may with α vβ 6On an epi-position combination, this epi-position and RGD binding pocket are overlapping, but different with it.Antibody 6.2B10 and 6.4B4 do not contain the FXY motif, are relatively poor α vβ 6Blocker.Their confirm can with α vβ 6The sample part combination of I territory, defined can with anti-α vβ 6The another one epi-position of antibodies.Ironically, monoclonal antibody 6.2A1 belongs to and does not rely on cationic classification, but not rely on cationic monoclonal antibody the same with other, do not contain the RGD sequence.
Monoclonal antibody 7.7G5 belongs to the cationic classification of dependence.Yet, the sequence of light chain of 7.7G5 with do not rely on cationic I territory binding antibody 6.2B10 height homology.7.7G5 heavy chain on CDR1, also be similar to and do not rely on cationic antibody.But its CDR2 and CDR3 more are similar to and rely on cationic classification.This discovery prompting, specific CDR makes antibody have specificity.This is correct especially for the CDR3 of heavy chain, and this may be because the height variability in this part of antibody.In fact, 3 kinds of 2 kinds and 9 kinds 7 kinds of not relying in the cationic antibody that rely in the cationic antibody contain heavy chain CDR3 sequence, and this sequence may be at α vβ 6Play an important role in the identification.As everyone knows, 7.7G5 lacks the RGD motif in its heavy chain CDR2, but contains the XGD motif.This XGD motif may work in the mode that is similar to RGD, and makes 7.7G5 have binding affinity/specificity.
Above-mentioned sequence checking and the appropriate design that is inferred as in view of the above provide the particular variable region amino acid sequence of particular combination character to provide the foundation.
Embodiment 10: diagnosis antibody
Can detect the α in paraffin-embedded tissue section or other tissue sample vβ 6The antibody of expressing can be used as diagnostic reagent.For example, these diagnostic tools can be used for detecting the α that raises in the tissue slice as cancer or Fibrotic indication vβ 6
In order to identify the α that can detect in the paraffin-embedded tissue vβ 6Antibody, our examination for the first time a series of β that are used in conjunction with the HPLC purifying 6The antibody of subunit.Can may discern the linear peptides epi-position in conjunction with the antibody of this subunit, therefore estimate in paraffin-embedded tissue, to have higher successful possibility.Use and mensuration α vβ 6Measure β with purifying in conjunction with described (the same) identical ELISA form 6The combination of subunit, difference are the dull and stereotyped fixedly β of purifying that goes up 6Integrin, rather than α vβ 6Albumen.Utilize this method, having identified in a large number can be in conjunction with the α of purifying vβ 6The β of albumen and purifying 6Fusion 6 antibody of subunit.Table 5 in seeing above wherein omits the prefix " 6. " of cloning title.
Table 5 can with the α of purifying vβ 6Or the β of purifying 6Subunit bonded antibody
Clone's title and α vβ 6In conjunction with the β of HPLC purifying 6The subunit combination
1A1 + 61A8 + -1A11 + +1E1 + +1E6 + +1H10 +/- -2A1 + +2A10 - -2B8 - -2B10 + -2C4 + +2C7 + -2E5 + +2G3 + -3A6 + -3B1 + -3B2 + +3B11 + +3C2 + +3D5 + -3D10 + +3F1 + -3G3 - -3G5 + -3G9 + -3H2 + -3H11 + -4A4 +/- +4B2 + -4B4 + -4B10 + -4D2 + -4E4 + +4G3 + -4G4 + -4H4 + +4H12 + -5A2 + -5B6 + +5D6 + +5D8 - -
Clone's title and α vβ 6In conjunction with the β of HPLC purifying 6The subunit combination
5G9 + +5G10 + +5H3 + +6B1 + +6B5 + +6C4 + -6D12 + +6E6 + -6E10 + -6G3 +/- -7C7 + +7E5 + -7F8 + -8B4 + +8G6 + -9B5 + +9B7 + +9B9 + -9B10 + -9D11 + +9E12 + +9F5 + +9F7 - -9G1 + -9H11 + -10A2 + -10A3 +/- -10A4 + -10A8 + -10A9 + -10B10 + -10D3 + -10D11 + -10E4 + +10F1 + -10F12 + -10G1 + -10G2 - -10H11 + +1A7 + -1D6 + -1D9 + -
Clone's title and α vβ 6In conjunction with the β of HPLC purifying 6The subunit combination
1F6 + -2B1 + -2D9 + +2G2 + +3D9 + +3E5 + -4C12 + -4E6 + +4G5 + -5A3 + -5B3 + -5B8 + +5B9 + -5F7 + -6C10 + +6D8 + -6F4 + -6G9 + -6H8 + +6H9 + -7A5 + +7A11 + -7E6 + +7G9 - +7H3 + -9A2 - +9A3 + +9A4 - -9A9 + -9D2 - -9D3 - -9E4 - -9F1 + -9F12 - -9G11 - -10B5 + -10B7 + -10B9 +/- -10C5 +/- -10C7 +/- -10D6 +/- -10E12 +/- -10F7 - -
As implied above, some antibody can with the β of purifying 6The subunit combination.They and sex change α vβ 6The bonded possibility is higher, thereby can be used in the α that detects in the paraffin-embedded tissue slice vβ 6Some antibody can be in conjunction with solubility α vβ 6, and debond β 6Subunit.This two antibody-like all be used for dyeing paraffin-embedded β of sex change 6The SW480 cell of transfection and the parental cell of untransfected, data are displayed in Table 6.
For paraffin-embedded tissue or the cell of dyeing, the sample slide glass is at first removed paraffin (de-paraffinized) by incubation in following solution: (1) dimethylbenzene, 5 minutes, twice; (2) 100% ethanol, 2 minutes, twice; (3) 95% ethanol, 2 minutes, twice; (4) 50% ethanol, 2 minutes, once; (5) distilled water, 2 minutes, once.Slide glass is containing 200ml methyl alcohol and 3ml 30% H then 2O 2Solution in incubation 15 minutes, to seal endogenous peroxidase.Slide glass PBS rinsing twice, each 2 minutes.Use stomach en-(Zymed 00-3009) to expose paraffin section on the slide glass then, 37 ℃ 5 minutes.Slide glass is used PBS rinsing twice again, each 2 minutes.Then, use vitamin H (Vector SP-2001 then with avidin earlier; Vector Laboratories, Burlingame, CA) sealing slide glass, at room temperature 10 minutes at every turn, washing as mentioned above between each incubation.Remove lock solution from slide glass, on slide glass, add first antibody (hybridoma culture supernatant) afterwards, under 4 ℃, be incubated overnight with PBS/0.1% BSA dilution.
Next day, slide glass is used the PBS rinsing as mentioned above.Simultaneously, be prepared as follows avidin-biotin composite-horseradish peroxidase solution (ABC reagent): 1ml PBS mixes with 20 μ l solution A (1:50) and 20 μ l solution B (1:50) from Vector Kit PK-6102; Mixture is incubation 30 minutes at room temperature before use.During this period, slide glass with contain 15 μ l/ml normal serums from the anti-mouse biotinylated antibody (1:200) of Vector Kit incubation 30 minutes at room temperature.Use PBS rinsing slide glass twice then, each 2 minutes.Add above-mentioned ABC reagent then on slide glass, at room temperature incubation is 30 minutes.Rinsing slide glass as mentioned above once more.On slide glass, add substrate (Vector SK-4100)---100 μ l DAB (3,3 '-diaminobenzidine), at room temperature incubation is 5 minutes.DAB is prepared as follows: to 5ml H 2Add 2 damping fluid stock solution, thorough mixing among the O; Add 4 DAB stock solution then, thorough mixing; Add 2 H then 2O 2Solution, thorough mixing.With flowing water rinsing slide glass 2 minutes.Secondly, the DAB signal of following all slide glasss of enhancing: use the 0.05M sodium bicarbonate, pH9.6 rinsing paraffin section 10 minutes; The damping fluid that trace is excessive; Adding I) AB strengthened solution 15 seconds; 1 minute termination reaction of the quick rinsing of water then.Slide glass dyeed 1 minute in Mayer ' s phenodin (a kind of nuclear counterstain) then.With flowing water rinsing slide glass 1 minute, submergence 1 minute in PBS then made phenodin become blueness.Use flowing water rinsing slide glass 1 minute again, dehydration, following clarification: be immersed in (1) 95% ethanol twice 1 minute; In (2) 100% ethanol 1 minute, twice; (3) in the dimethylbenzene 2 minutes, twice.Use permount on slide glass, to add cover glass then.
Results suggest, merge 6 antibody 1A1,2C4,3B2,3B11,5D6,5G9,5H3,6D12,7C7,9B5,9B7,9D11,9F5,10E4,10H11,6H8,7A5,7G9,9A3,2A1,2E5,4E4,4H4,8B4,2G2 and 4E6, they can both be in conjunction with the β of purifying 6Subunit (table 5), paraffin-embedded β really strongly can dye 6The SW480 cell of transfection, and the parental cell (table 6) of the untransfected that do not dye.
Table 6 can with paraffin-embedded SW480 cell bonded antibody
ID# clone's title and SW480/ β 6+ in conjunction with combining with SW480
1 1A1 +++ -2 1A8 + +3 1A11 ++ -4 1E1 + -5 1E6 ++ +7 2A1 +++ -10 2B10 - -11 2C4 +++ -12 2C7 - -13 2E5 +++ -17 3B2 +++ -18 3B11 ++ -25 3G9 - -30 4B4 + +33 4E4 +++ -35 4G4 - -36 4H4 +++ -39 5B6 + -40 5D6 +++ -42 5G9 +++ -43 5G10 - -44 5H3 +++ -46 6B5 - -48 6D12 +++ -52 7C7 ++ -54 7F8 - -55 8B4 +++ -56 8G6 - -57 9B5 ++ -58 9B7 ++ -61 9D11 ++ -62 9E12 + -63 9F5 ++ -68 10A3 - -75 10E4 +++ -80 10H11 ++ -85 2B1 + +87 2G2 +++ -91 4E6 +++ -95 5B8 - -
ID# clone's title and SW480/ β 6+ in conjunction with combining with SW480
98 6C10 + -102 6H8 ++ -104 7A5 +++ -107 7G9 +++ -110 9A3 +++ -
Embodiment 11: the diagnosis of cancer
α vβ 6In health adult tissue usually with negligible level to low expression level.Yet, α vβ 6Be expressed in damage, fibrosis and the cancer and raise (referring to, for example, people .J.Invest.Dermatology 117:67-73 (2001) such as Thomas; People such as Brunton, Neoplasia3:215-226 (2001); People such as Agrez, Int.J.Cancer 81:90-97 (1999); Breuss, J.Cell Science 108:2241-2251 (1995)).Therefore, for diagnosis of fibrosis, cancer and α vβ 6Other any disease that raises can be used the α that expresses with paraffin-embedded tissue in standard immunoassay group technology vβ 6Specificity bonded antibody test α vβ 6Expression.
As mentioned above, the β of some antibodies HPLC purifying of the present invention 6Integrin and paraffin-embedded and fixed β 6Cells transfected.Confirm also that in immunostaining these antibody can combine with typical squamous cell carcinoma and breast carcinoma tissue.Referring to, Fig. 8 for example, wherein monoclonal antibody 6.2A1 is used for showing the relative dyeing of paraffin-embedded breast cancer and squamous cell carcinoma.Therefore, these new antibodies can be used as diagnostic tool.
Embodiment 12: anti-α vβ 6The effect of blocking-up monoclonal antibody in the Alport mouse
Collagen protein 4A3 (COL4A3) knocks out the body inner model that (Alport) mouse has been set up as a kind of renal fibrosis, is used for detecting the result of treatment (the same) of pharmacological preparation.We have checked mAb6.8G6 (relying on cationic) and 6.3G9 (not relying on cationic) in the Alport mouse, determine whether they are suppressed at common fibrosis in the 7 week age Alport mouse.As implied above, find that these two kinds of antibody can suppress α in biological assay vβ 6With combining of LAP, and the activation of inhibition TGF-β.Antibody 1E6 is as negative control.
3 Alport mouse one weeks age in week one of 3 peritoneal injection following antibody: (1) 6.8G6,4mg/kg (7 mouse); (2) 6.3G9,4mg/kg (4 mouse); (3) 1E6,1mg/kg (6 mouse).Injected for 4 weeks continuously.Kill mouse then, get kidney.
Prepare paraffin-embedded kidney section as mentioned above, dyeing then detects smooth muscle actin---a kind of marker of sarcoplast and apposition in the renal fibrosis.We find, compare with the mouse that 1E6 handles, and in matter and the renal glomerulus district, smooth muscle actin dyeing significantly reduces between the kidney of the Alport mouse that mAb6.8G6 or 6.3G9 handle.
The painted point diagram of smooth muscle actin in the renal glomerulus of Figure 11 A and 11B demonstration Alport kidney and the matter district.Compare with the mouse that negative control 1E6 handles, smooth muscle actin dyeing significantly reduces in the kidney of the Alport mouse that 6.8G6 and 6.3G9 handle.
Embodiment 13: anti-α vβ 6The validity of mAb aspect the one-sided ureter obstruction of prevention inductive Nephrosclerosis
We use another renal fibrosis progress mouse model to check the anti-fibrosis usefulness of 6.8G6 and 6.3G9.In this mouse model, the side ureter of animal is caused one-sided ureter to block (UUO) by ligation.In mouse, UUO causes carrying out property Nephrosclerosis and does not cause short-term renal failure, because the kidney of Zu Saiing can not kept normal relatively renal function.The kidney that blocks experiences quick renal glomerulus fibrosis, and the kidney of Zu Saiing does not then experience the adaptability hypertrophy.
This research is by the quantitative anti-α of morphometry vβ 6Processing is to the influence of UUO inductive renal fibrosis.The 8-12 male C 57 BL mouse in age in week that does not contain virus antigen of use body weight 25.5 ± 0.2g (Jackson Laboratories, Bar Harbor, ME).Before beginning one's study, make mouse adapt to 7 days.Eat and sterilized water the standard mouse that adapts to and the experimental session mouse can arbitrarily obtain to shine.Regularly weigh in, as the part of animal health monitoring.The result shows, not mouse weight increase about 10% in the research phase in two weeks of operation of age-matched.It is about 9% that UUO mouse to the second a day body weight reduces, but the body weight that reduced by the 14th day is recovered gradually.This body weight changes pattern and handles irrelevant with treatment.
In order to bring out renal fibrosis, at ketamine: xylazine (100:10mg/kg is subcutaneous) anesthesia down, by the aseptic separation left ureter of laparotomy on the left of the center line.The obstruction 6-0 silk ligature of two tensions is placed the level that is positioned at the low utmost point (lower pole) of kidney on the ureter, between ligature, cut ureter.Stomach wall 4-0 Vicryl suture closure, skin 4-0 nylon closure.Animal is recovered on the heat pad, subcutaneously the 0th day and the 1st day give 0.05mg/kg buprenorphine, twice of every day.This program is from people such as Ma, Kidney Int.53 (4): 937-944 (1998).
Then mouse is divided into following study group:
Figure C200610103152D00421
*Number of animals
All animals all begin administered twice weekly the day before yesterday from performing the operation except the 5th group.
The carbon dioxide narcosis animal is used in after ligation the 10th day then, and dissects.In the UUO mouse, the remarkable swelling of renal plevis and ureter, liquid riddles on ligation place of obstruction.Between treatment group, the swelling degree is different with all the other nephridial tissue mass ranges.The 2nd group shows half of the swelling be about negative control group.The kidney color of ligation is greyish white.The offside kidney is scarlet, increases about 1/3rd.
Then, take out two kidneys (left side ligation, the right side is ligation not) of animal, by double crosscut in renal plevis center.Place the formalin of 10% neutral buffered to carry out fixing organization dyeing half of each kidney.Second half of each kidney places 15% sucrose, with being placed in 30% sucrose, carries out immunohistochemical staining.
At myofibroblast (smooth muscle actin)---Fibrotic a kind of mark, the kidney section of immunostaining formalin fixed.Be provided with the exposure of standardized illumination condition and digital camera and obtain image,, and calibrate according to criterion distance according to background correction.The image that obtains the adjacent visual field of containing whole left kidney segment from every animal carries out quantitatively.
Smooth muscle actin is expressed as the percentage ratio of total tissue area in the visual field of measurement.All cortex and the medullary substance tissue that comprise the section except nephridial papilla.
In a word, the mouse display fibersization of handling with 6.3G9 and 6.8G6 significantly reduces.
Embodiment 14: anti-α vβ 6The validity of the mouse pulmonary fibrosis that the blocking-up monoclonal antibody is brought out bleomycin
The mouse pulmonary fibrosis that bleomycin brings out has been established as pulmonary fibrosis model in a kind of body, is used for checking the result of treatment of pharmacological preparation.Inflammation is obvious usually after bleomycin is handled 5-15 days.129 is in the mouse, and the pulmonary fibrosis degree increases gradually, after can arriving bleomycin and handling 60 days.The matrix accumulation can detect about the 15th day usually.In this embodiment, since the 0th day or the 15th day, injection monoclonal antibody 6.3G9 in the pulmonary fibrosis mouse peritoneum that bleomycin is brought out with 4mg/kg/ time concentration, 3 times weekly.Brought out pulmonary fibrosis at the 0th day, method is to use the bleomycin of single dose in the tracheae, and it is dissolved in the 50 μ l Sterile Salines, and concentration is 0.03 unit/kg.The 30th day kill animals, estimate the pulmonary fibrosis degree.Antibody 1E6 is as negative control.
Collect the lung of every animal, people such as Munger is described as mentioned, measures the index of hydroxyproline content as the lung collagen deposition.Shown in Figure 15 A, began at the 0th day to handle the increase that significantly suppresses the lung hydroxyproline content that bleomycin brings out with 6.3G9.Importantly, effective when 6.3G9 handles at least as use at bleomycin beginning in back 15 days, collagen deposition begins at this moment.
We have also checked that with the bleomycin induce fibrosis scheme that prolongs (continuing 60 days) 6.3G9, the cationic 6.8G6 of dependence and non-blocking antibody 6.4B4 suppress the effect of higher degree pulmonary fibrosis.For this reason, we after using bleomycin 15 days (the 15th day) begins antibody treatment.Get lung then at the 60th day, detect hydroxyproline content.Shown in Figure 15 C, handle the remarkable fibrosis (compare with the animal of bleomycin and brine treatment, hydroxyproline content reduces more than 70%) that bleomycin brings out that suppresses with 6.8G6.Handle the trend that yet shows protection with 6.3G9, but these results there is not significance,statistical (Figure 15 C).
In a word, in the mouse that bleomycin is handled, rely on cationic and do not rely on cationic anti-α v β 6The blocking-up monoclonal antibody all reduces pulmonary fibrosis.And, even beginning just to begin antibody treatment after the generation up to fibrosis, this intervention is still effective.
Embodiment 15: α in people's development of psoriatic lesions vβ 6Rise
In order to determine α vβ 6Whether relevant with psoriatic, checked the damage of 5 psoriatics and 4 normal individuals and the α in the non-injured skin biopsy vβ 6Express.Utilize mAb 6.2A1 immunostaining, we find, compare α in the development of psoriatic lesions with normal control with psoriatic's non-injured skin vβ 6Expression significantly improves.Therefore, α in the development of psoriatic lesions vβ 6Rise prompting use anti-α vβ 6The diagnosis of antibody and treatment meaning.
Embodiment 16: α in the mouse that suffers from biliary tract and people's liver vβ 6Rise
As previously mentioned, α vβ 6Expression relevant with tissue injury.In this research, studied α in the mouse that damaged by biliary tract and the people's liver vβ 6Expression.
Bring out the liver injury of mouse by BDL.Referring to, for example, people such as George, PNAS96:12719-24 (1999); People such as George, Am J Pathol 156:115-24 (2000).Use mAb 6.2G2, we find α vβ 6Be expressed in behind the BDL significantly raise in the 9th, 14,16 day.
Similarly, detect by the immunohistochemical methods that uses mAb 6.2G2, people's liver section of suffering from the patient of biliary tract shows α vβ 6Up-regulated.For example, after from 1 acute cholestasis male patient of 44 years old, 1 transplanting of 59 years old, observe α in acute biliary obstruction male patient, 1 Biliary atresia male patient of 22 years old and 1 chronic obstruction of biliary tract male patient's of 24 years old the liver specimens vβ 6Express and improve.
In a word, these new anti-α vβ 6Antibody is for hepatopathy useful diagnosis and treatment tool.
Embodiment 17: α in the multiple human cancer vβ 6Rise
Beta 2 integrin alpha vβ 6In health adult tissue usually with negligible level to low expression level.The method of using antibody 6.2A1 and summarizing has herein been estimated the α of various human tumor tissues vβ 6Express.The result is presented at α in several people's epithelial cancers vβ 6The expression of integrin is significantly raised.Particularly, immunohistology shows, α vβ 6On edge, multiple epitheliomatous tumour island, express especially significantly.In order further to study α in the epithelial cancer cells vβ 6Expression, Detroit 562 cells (pharynx cancer) and SCC-14 cell (squamous cell carcinoma of tongue) and SW 480 β 6Cell (the same) passes through flow cytometry with 6.3G9 and 6.4B4 dyeing.
The right side of Figure 12 shows the α that different tumour cells are fastened vβ 6Express, with the combination expression of 6.3G9 in the fluorescence-activated cell sorting (FACS).The combination of 6.3G9 is represented at solid peak, and the independent background combination of second monoclonal antibody is represented at hollow peak.6.3G9 that the line chart display density in Figure 12 left side raises gradually or 6.4B4 suppress tumor cell line and LAP part bonded.Compare with 6.3G9,6.4B4 is α vβ 6With the remarkable lower 10 times of IC of inhibitor (for Detroit 562,〉of a kind of usefulness of LAP bonded 50For SW480B6,〉30 times of IC 50For SCC-14,〉100 times of IC 50).This is consistent with former in vitro results, shows that 6.3G9 is a kind of strong blocking-up monoclonal antibody, and 6.4B4 is a kind of weak blocking-up monoclonal antibody.These data also with the Detroit heteroplastic transplantation model in the negligible inhibition active consistent (Figure 14 B) of 6.4B4.Figure 13 further shows multiple anti-α vβ 6Monoclonal antibody suppresses tumor cell line is relative with the LAP bonded.6.3G9 the inhibition activity (with former all data consistents) suitable with the 6.8G6 demonstration, and 6.4B4 is α vβ 6With the remarkable lower inhibitor of a kind of usefulness of LAP bonded.
Embodiment 18: anti-α vβ 6The effect of blocking-up monoclonal antibody in the human tumor xenograft model
The immune deficiency animal (for example nude mice and SCID mouse) of having transplanted people's tumor xenogeneic graft has been established as a kind of useful body inner model, be used for checking carcinostatic agent result of treatment (referring to, for example, people such as van Weerden, Prostate 43 (4): 263-71 (2000); People such as Bankert, Front Biosci 7:c44-62 (2002)).Therefore, can in heteroplastic transplantation model, check the anti-α of blocking-up of the present invention in the body vβ 6Monoclonal antibody suppresses the ability of tumor growth.In this experiment, we have checked some new α in having transplanted the female nude mice of athymia that the people swallows cancer (Detroit 562 clones) heterograft vβ 6The ability of antibody inhibiting tumor growth.
For this reason, Detroit 562 cells (ATCC) are subculture in vitro separately in MEM (Eagle), contain 2mM L-glutaminate and Earle ' s BSS in this substratum, be adjusted to and contain 1.5g/L carbonic acid chlorine sodium, 0.1mM non-essential amino acid and 1.0mM Sodium.alpha.-ketopropionate and 10% foetal calf serum, do not contain microbiotic.About 5 * 10 6The right side side of body of the subcutaneous implantation nude mice of cell/0.2ml substratum (not containing serum).Begin to measure the tumour size after 3-4 days, last till that tumour reaches about 5mm (length) * 5mm (width).With the mouse randomization, detect antibody or contrast solution at the 1st day peritoneal injection, inject totally 33 days subsequently weekly 3 times.Detecting antibody and contrast solution is: (1) 6.3G9,1mg/kg, 10 mouse; (2) 6.3G9,4mg/kg, 10 mouse; (3) 6.3G9,10mg/kg, 10 mouse; (4) 6.4B4,1mg/kg, 10 mouse; (5) 6.4B4,4mg/kg, 10 mouse; (6) 6.4B4,10mg/kg, 10 mouse; (7) vehicle Control (PBS), every mouse of 0.2ml/, 30 mouse.In addition, 10 mouse subcutaneous injection 2mg/kg cis-platinums are contrasted as chemotherapy.Cis-platinum is injected at the 1st day to carry out, and injection in per then 2 days is once treated for 6 times altogether.When finishing in 33 days, measure the weight of animals and tumour size, estimate α by immunohistology vβ 6Expression, measure the anti-α of serum vβ 6Level.
Immunohistology dyeing shows that the tumour cell of implantation is strong expression α in vivo vβ 6The tumor weight data prove that further blocking-up monoclonal antibody 6.3G9 effectively suppresses tumor growth (Figure 14 A) in all three concentration that detect.On the contrary, the monoclonal antibody 6.4B4 of weak blocking-up does not suppress tumor growth (Figure 14 B).
In a word, in the mouse of handling, blocking antibody suppresses tumor growth 40-50%.On the contrary, the anti-α of weak blocking-up vβ 6Antibody does not suppress tumor growth.
Embodiment 19: α vβ 6The internalization of antibody
For some clinical indication (as cancer) advantage is arranged by the antibody of cell internalizing because these antibody can with toxin, radioactive compound or other carcinostatic agent coupling, thereby the growth of selectivity target and anticancer.At SW480 β 6Studied anti-α in (the same) and the SCC-14 cell vβ 6Antibody is by the ability of internalization.
With cell 1:5 separately, be inoculated on the 4 Room slide glasss, at 37 ℃ and 5%CO 2Under be incubated overnight.Next day, dilution monoclonal antibody 6.8G6,6.1A8,6.3G9,7.1C5,6.4B4,10D5 and 8B3 to final concentration be 20 μ g/mL.These monoclonal antibodies or independent substratum are added in the suitable hole.The time of internalization was from 0 hour to 48 hours.The time point that comprises is 0,5,10,30 minute and 1,4,24,48 hour.Add second antibody (anti-mouse-Alexa 594) as negative control.By removing antibody and stopping internalization at each time point with damping fluid washed cell layer.Add wheat germ agglutinin-Alexa-488,18 ℃ 20 minutes, with the outer rim of green fluorescence staining cell.Behind the washed cell, add Cytofix/Cytoperm solution, 18 ℃ 20 minutes, fixing and saturatingization cell.Washed cell once more adds the anti-mouse-Alexa 594 of second antibody (red fluorescence), 18 ℃ 20 minutes, the mouse α of mark bonded or internalization vβ 6Antibody.Washed cell adds 2% paraformaldehyde and fixes then, checks with Laser Scanning Confocal Microscope.Use LeitzPlan-Apochromatic 63x (1.32 numerical apertures, air are exposed to the sun and soaked (ail immersion)) object lens (Leica) with the 2x digital focal length photographic images then.Each picture is represented an optic section, and it is from the middle section of the cell of observing internalization under all conditions.In nuclear, do not observe dyeing.
For relying on cationic monoclonal antibody (the simulation part that contains RGD),, observe internalization as 6.8G6 and 6.1A8.For not relying on cationic monoclonal antibody,, do not observe internalization as 6.3G9,7.1C5 and 6.4B4.
Sequence table
<110>BIOGEN，INC
THE?REGENTS?OFTHE?UNIVERSITY?OF?CALIFORNIA
＜120〉anti-α vβ 6Antibody
<130>A136PCT
<140>PCT/US03/08048
<141>2003-03-13
<150>60/364,991
<151>2002-03-13
<150>60/426,286
<151>2002-11-13
<160>64
<170>PatentIn?Ver.2.1
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<213>Homo?sapiens
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Figure C200610103152D00481
<210>2
<211>10
<212>PRT
<213>Homo?sapiens
<400>2
Figure C200610103152D00482
<210>3
<211>10
<212>PRT
<213>Homo?sapiens
<400>3
Figure C200610103152D00483
<210>4
<211>17
<212>PRT
<213>Homo?sapiens
<400>4
Figure C200610103152D00491
<210>5
<211>16
<212>PRT
<213>Homo?sapiens
<400>5
Figure C200610103152D00492
<210>6
<211>16
<212>PRT
<213>Homo?sapiens
<400>6
Figure C200610103152D00493
<210>7
<211>17
<212>PRT
<213>Homo?sapiens
<220>
<221>MOD_RES
<222>(12)
<223>Arg?or?Gln
<400>7
Figure C200610103152D00494
<210>8
<211>12
<212>PRT
<213>Homo?sapiens
<400>8
Figure C200610103152D00495
<210>9
<211>12
<212>PRT
<213>Homo?sapiens
<400>9
Figure C200610103152D00501
<210>10
<211>15
<212>PRT
<213>Homo?sapiens
<400>10
Figure C200610103152D00502
<210>11
<211>12
<212>PRT
<213>Homo?sapiens
<400>11
Figure C200610103152D00503
<210>12
<211>12
<212>PRT
<213>Homo?sapiens
<400>12
Figure C200610103152D00504
<210>13
<211>7
<212>PRT
<213>Homo?sapiens
<400>13
Figure C200610103152D00505
<210>14
<211>7
<212>PRT
<213>Homo?sapiens
<400>14
Figure C200610103152D00506
<210>15
<211>9
<212>PRT
<213>Homo?sapiens
<400>15
<210>16
<211>9
<212>PRT
<213>Homo?sapiens
<V400>16
Figure C200610103152D00512
<210>17
<211>9
<212>PRT
<213>Homo?sapiens
<400>17
Figure C200610103152D00513
<210>18
<211>9
<212>PRT
<213>Homo?sapiens
<400>18
Figure C200610103152D00514
D<210>19
<211>126
<212>PRT
<213>Homo?sapiens
<400>19
Figure C200610103152D00515
Figure C200610103152D00521
<210>20
<211>126
<212>PRT
<213>Homo?sapiens
<400>20
Figure C200610103152D00522
<210>21
<211>126
<212>PRT
<213>Homo?sapiens
<400>21
Figure C200610103152D00523
Figure C200610103152D00531
<210>22
<211>119
<212>PRT
<213>Homo?sapiens
<400>22
Figure C200610103152D00532
<210>23
<211>120
<212>PRT
<213>Homo?sapiens
<400>23
Figure C200610103152D00533
Figure C200610103152D00541
<210>24
<211>120
<212>PRT
<213>Homo?sapiens
<400>24
Figure C200610103152D00542
<210>25
<211>121
<212>PRT
<213>Homo?sapiens
<400>25
Figure C200610103152D00543
Figure C200610103152D00551
<210>26
<211>121
<212>PRT
<213>Homo?sapiens
<400>26
Figure C200610103152D00552
<210>27
<211>119
<212>PRT
<213>Homo?sapiens
<400>27
Figure C200610103152D00561
<210>28
<211>119
<212>PRT
<213>Homo?sapiens
<400>28
Figure C200610103152D00562
<210>29
<211>119
<212>PRT
<213>Homo?sapiens
<400>29
Figure C200610103152D00571
<210>30
<211>118
<212>PRT
<213>Homo?sapiens
<400>30
Figure C200610103152D00581
<210>31
<211>118
<212>PRT
<213>Homo?sapiens
<400>31
Figure C200610103152D00582
<210>32
<211>118
<212>PRT
<213>Homo?sapiens
<400>32
Figure C200610103152D00591
<210>33
<211>118
<212>PRT
<213>Homo?sapiens
<400>33
<210>34
<211>118
<212>PRT
<213>Homo?sapiens
<400>34
Figure C200610103152D00593
Figure C200610103152D00601
<210>35
<211>118
<212>PRT
<213>Homo?sapiens
<400>35
Figure C200610103152D00602
<210>36
<211>118
<212>PRT
<213>Homo?sapiens
<400>36
Figure C200610103152D00603
Figure C200610103152D00611
<210>37
<211>111
<212>PRT
<213>Homo?sapiens
<400>37
Figure C200610103152D00612
<210>38
<211>111
<212>PRT
<213>Homo?sapiens
<400>38
Figure C200610103152D00621
<210>39
<211>112
<212>PRT
<213>Homo?sapiens
<400>39
<210>40
<211>107
<212>PRT
<213>Homo?sapiens
<400>40
<210>41
<211>107
<212>PRT
<213>Homo?sapiens
<400>41
Figure C200610103152D00632
<210>42
<211>106
<212>PRT
<213>Homo?sapiens
<400>42
Figure C200610103152D00633
Figure C200610103152D00641
<210>43
<211>106
<212>PRT
<213>Homo?sapiens
<400>43
<210>44
<211>108
<212>PRT
<213>Homo?sapiens
<400>44
Figure C200610103152D00643
Figure C200610103152D00651
<210>45
<211>108
<212>PRT
<213>Homo?sapiens
<400>45
<210>46
<211>17
<212>PRT
<213>Homo?sapiens
<400>46
<210>47
<211>17
<212>PRT
<213>Homo?sapiens
<400>47
Figure C200610103152D00661
<210>48
<211>15
<212>PRT
<213>Homo?sapiens
<400>48
Figure C200610103152D00662
<210>49
<211>10
<212>PRT
<213>Homo?sapiens
<400>49
Figure C200610103152D00663
<210>50
<211>10
<212>PRT
<213>Homo?sapiens
<400>50
Figure C200610103152D00664
<210>51
<211>17
<212>PRT
<213>Homo?sapiens
<400>51
Figure C200610103152D00665
<210>52
<211>17
<212>PRT
<213>Homo?sapiens
<400>52
Figure C200610103152D00666
<210>53
<211>12
<212>PRT
<213>Homo?sapiens
<400>53
Figure C200610103152D00672
<210>54
<211>11
<212>PRT
<213>Homo?sapiens
<400>54
Figure C200610103152D00673
<210>55
<211>11
<212>PRT
<213>Homo?sapiens
<400>55
Figure C200610103152D00674
<210>56
<211>11
<212>PRT
<213>Homo?sapiens
<400>56
<210>57
<211>7
<212>PRT
<213>Homo?sapiens
<400>57
Figure C200610103152D00676
<210>58
<211>7
<212>PRT
<213>Homo?sapiens
<400>58
Figure C200610103152D00681
<210>59
<211>9
<212>PRT
<213>Homo?sapiens
<400>59
Figure C200610103152D00682
<210>60
<211>9
<212>PRT
<213>Homo?sapiens
<400>60
Figure C200610103152D00683
<210>61
<211>120
<212>PRT
<213>Homo?sapiens
<400>61
Figure C200610103152D00684
<210>62
<211>121
<212>PRT
<213>Homo?sapiens
<400>62
Figure C200610103152D00691
<210>63
<211>107
<212>PRT
<213>Homo?sapiens
<400>63
Figure C200610103152D00692
<210>64
<211>107
<212>PRT
<213>Homo?sapiens
<400>64

Claims (8)

1. monoclonal antibody, its can specificity in conjunction with α vβ 6, but do not suppress α vβ 6With combining of the related peptides LAP that hides; Wherein this antibody contains identical heavy chain and the light chain polypeptide sequence of antibody that produces with the hybridoma 6.2A1 of ATCC preserving number PTA-3896, or wherein this antibody contains identical heavy chain and the light chain polypeptide sequence of antibody that produces with the hybridoma 6.2E5 of ATCC preserving number PTA-3897.
2. the antibody of claim 1 is used for detecting the α of mammalian tissues sample in preparation vβ 6Preparation in purposes.
3. isolating nucleic acid, it contains the sequence of coded polypeptide SEQ ID NO:62, and the wherein said polypeptide that is encoded forms specificity in conjunction with α v β 6, but do not suppress α vβ 6Heavy chain variable domain with the bonded monoclonal antibody of the related peptides LAP that hides.
4. isolated polypeptide, it contains the aminoacid sequence of SEQ ID NO:62, and wherein said polypeptide forms specificity in conjunction with α vβ 6, but do not suppress α vβ 6Heavy chain variable domain with the bonded monoclonal antibody of the related peptides LAP that hides.
5. isolating nucleic acid, it contains the sequence of coded polypeptide SEQ ID NO:63, and the wherein said polypeptide that is encoded forms specificity in conjunction with α vβ 6, but do not suppress α vβ 6Light chain variable territory with the bonded monoclonal antibody of the related peptides LAP that hides.
6. isolated polypeptide, it contains the aminoacid sequence of SEQ ID NO:63, and wherein said polypeptide forms specificity in conjunction with α vβ 6, but do not suppress α vβ 6Light chain variable territory with the bonded monoclonal antibody of the related peptides LAP that hides.
7. the monoclonal antibody of claim 1, its hybridoma by ATCC preserving number PTA-3896 produces.
8. the monoclonal antibody of claim 1, its hybridoma by ATCC preserving number PTA-3897 produces.
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CN105017420A (en) * 2012-02-17 2015-11-04 西雅图基因公司 Antibodies to integrin [alpha]v[beta]6 and use of same to treat cancer

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0719859A1 (en) * 1994-12-20 1996-07-03 MERCK PATENT GmbH Anti-alpha V-integrin monoclonal antibody

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0719859A1 (en) * 1994-12-20 1996-07-03 MERCK PATENT GmbH Anti-alpha V-integrin monoclonal antibody

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105017420A (en) * 2012-02-17 2015-11-04 西雅图基因公司 Antibodies to integrin [alpha]v[beta]6 and use of same to treat cancer
CN105017420B (en) * 2012-02-17 2019-05-28 西雅图基因公司 Antibody for beta 2 integrin alpha v β 6 and use the antibodies for treating cancer

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