CN1835976B - Aglycosyl anti- CD154 (CD 40 ligand) antibodies and uses thereof - Google Patents

Abstract

The invention relates to aglycosyl anti-CD154 antibodies or antibody derivatives, characterized by a modification at the conserved N-linked site in the CH2 domains of the Fc portion of said antibody. The invention also relates to the treatment of immune response related diseases and inhibition of unwanted immune response with such aglycosylated anti-CD154 antibodies or antibody derivatives thereof.

Description

Sugar based anti-cd 154 (CD40 part) antibody and uses thereof

Technical field

The present invention relates to sugar based anti-cd 154 antibodies or its antibody derivatives, the interaction of its blocking-up CD154 and CD40 molecule.In addition, the invention provides the method for preparing sugar based anti-cd 154 antibodies and antibody derivatives.Antibody of the present invention and antibody derivatives can be used for treatment and prevention and unwanted immunoreation diseases associated, and the disease of CD154-CD40 interaction mediation.

Background technology

The generation of body fluid and cell-mediated immunity is the results of interaction of activatory helper cell and antigen presenting cell (" APCs ") and effector T cell.The activation of helper cell not only depends on the interaction of T cells with antigenic specificity acceptor (" TCR ") and its corresponding peptides-MHC part, also needs the collaborative combination and the activation [Salazar-Fontana, 2001] of various kinds of cell adhesion molecule and costimulatory molecules.

Important costimulatory molecules be CD154 (be also referred to as the CD40 part, CD40L, gp39, T-BAM, T-cell activation molecule, TRAP), it is in CD4 with activation dependency, instantaneous limited mode ⁺The II type transmembrane protein of T cell surface expression.CD154 in activation later on also at CD8 ⁺Inferior group T cell, basophilic cell, mastocyte, eosinophil, natural killer cell, the B cell, scavenger cell is expressed on dendritic cell and the thrombocyte.

Corresponding acceptor (counter-receptor) CD40 of CD154 is an I type membranin, and its constitutive character and popularity ground comprise APCs [Foy, 1996] at many cell type surface expressions.

Through a series of incidents of signal enabling that CD40 produces, cause carrying the cell activation and the best CD4 of CD40 acceptor by CD154 ⁺The T cell activation.More specifically, the interaction between CD154 and the CD40 promotes the B cytodifferentiation to become the cell and the memory B cell [Burkly, 2001] of secretory antibody.In addition, CD154-CD40 interacts and promotes cell-mediated immunity [Burkly, 2001] through the activation of scavenger cell and dendritic cell and the generation of nk cell and CTL.

The importance of CD154 in regulating body fluid and cell-mediated immunoreation has excited the interest [USP 5,474,771] of people to the therapeutic immunization regulating effect of the suppressor factor that utilizes this approach.Therefore, anti-cd 154 antibodies is treated albumen or gene therapy, sensibiligen, useful [USP 5,474,771 in the immunoreation model of autoimmunization and transplanting multiple to other; Burkly, 2001].

CD40-CD154 interacts important in the autoimmune disease of multiple test-induced, such as collagen-induced sacroiliitis, and tentative supersensitivity meningitis (" EAE "), ovaritis, colitis, drug-induced lupus nephritis.Particularly, inducing of disease can be blocked [Burkly, 2001] through the CD154 antagonist in all these models when the antigen administration.

Utilize anti-cd 154 antagonist blocking-up disease also in spontaneous autoimmune disease animal model, to see; Comprise insulin-dependent diabetes and lupus nephritis, and graft versus host disease, transplant; Pulmonary fibrosis and atheromatosis model [Burkly, 2001].

Although the glycosylation anti-cd 154 antibodies can be used for prevention and treatment panimmunity reacting phase related disorders, among some experimenters, utilize their the concurrent sometimes thromboembolism of treatment active [Biogen Press Release, 2001; IDEC Press Release, 2001].Although the mechanism of this spinoff is unknown, it possibly comprise that anti-cd 154 antibodies or its aggregate to the gathering (colligation) of FcgRIIa on the thrombocyte and CD154, cause unsuitable platelet activation.With other Fc γ acceptor and complement combine also can strengthen this effect.Therefore, the form of the anti-cd 154 antibodies of debond effector acceptor maybe be safer and/or more effective for therepic use.

The mechanism that anti-cd 154 antibodies suppresses immunologic function is more complicated compared with combines interaction with blocking-up and CD40 simply with CD154, in fact comprises the contribution of effect approach.For example, antibody-antigen combines to pass through the elimination of the zygotic induction activated T cell of Fc structural domain and Fc γ acceptor or complement component.Optional, antibody combines and can strengthen through forming the antibody support at the cell surface that carries Fc γ acceptor with CD154's.In addition, antibody and its action site promotes near interacting through Fc γ receptors bind.

At glycosylated antibodies, comprise in the anti-cd 154 antibodies, be attached to Fc dimer C _H2The glycan in the site that conservative N-connects in the structural domain is included in C _H2Between the structural domain, make saccharide residue and relative C _H2Specific amino acid residue contact [Jeffries, 1998] on the structural domain.The external interior research confirmation of the body removal C that utilizes multiple glycosylated antibodies _H2Glycan change Fc structure makes and significantly reduces [Nose, 1983 with Fc acceptor and complement proteins C1Q bonded antibody; Leatherbarrow, 1985; Tao, 1989; Lund, 1990; Dorai, 1991; Hand, 1992; Leader, 1991; Pound, 1993; Boyd, 1995].Research confirms that the effector functions of sugar based antibody reduces in the body.For example, sugar based is anti--CD8 antibody can not consume the cell [Isaacs, 1992] that carries CD8-in the mouse, and the sugar based anti-CD 3 antibodies can not inducing mouse or cytokine release [Boyd, 1995 of philtrum; Friend, 1999].

Although remove C _H2Glycan in the structural domain seems pairing effect device function has positive effect, and other function of antibody and physical property are constant.Particularly, the removal that shows glycan is not almost arrived not influence [Nose, 1983 to serum half life and antigen combination; Tao, 1989; Dorai, 1991; Hand, 1992; Hobbs, 1992].

The invention summary

In this invention, the Fc effector functions in the mechanism of anti-cd 154 antibodies effect is eliminated through utilizing anti-CD-154 antibody, and the Fc effector functions is through modifying Fc dimer C in the said antibody _H2The site that conservative N-connects in the structural domain reduces, and causes " sugar based " anti-cd 154 antibodies.The instance of said modification comprises Fc dimer C _H2The site mutation that conservative N-connects in the structural domain is removed and C _H2The glycan that the N-connection site is connected in the structural domain, and prevent glycosylation.

For whether the inhibiting mechanism that anti-cd 154 antibodies is described relies on its Fc effector interact, relatively anti-cd 154 antibodies and sugar based obverse thereof suppress the ability of several conditions through blocking-up CD154-CD40 interaction.This paper results reported has confirmed that the glycosylation form provide protection of sugar based form and anti-cd 154 antibodies of anti-cd 154 antibodies is suitable.

Reduce because sugar based anti-cd 154 antibodies of the present invention is characterised in that effector functions, these antibody especially can be used for existing among the experimenter of the active possibility of unwanted thromboembolism.In addition; The Fc effector functions of the reduction of sugar based anti-cd 154 antibodies can reduce or eliminate other possible spinoff of anti-cd 154 antibodies therapy, such as eliminating the activated T cell and through the Fc dependency activation of other cell mass or the monocyte/macrophage of abduction delivering CD154.

Particularly, the present invention provides the sugar based anti-cd 154 antibodies of identification CD154.More specifically, the present invention provides " sugar based hu5c8 " and mouse sugar based anti-cd 154 antibodies-i.e. " the sugar based muMR1 " of humanization sugar based anti-cd 154 antibodies-promptly.

In one embodiment of the invention; Sugar based hu5c8 antibody is from the preparation of NS0 sugar based hu5c8 clone, and this cell lies in and was deposited in American type culture collection (" ATCC "), 10801 University Blvd. on January 14th, 2003; Manassas; Virginia (preserving number PTA-4931), sugar based MR1 antibody is from the preparation of NS0 sugar based mouse MR1 clone, and this cell lies in and was deposited in ATCC (preserving number PTA-4934) on January 14th, 2003.

In one embodiment of the invention, the sugar based anti-cd 154 antibodies can suppress the interaction between CD154 and the CD40.

In another embodiment of the present invention, the sugar based anti-cd 154 antibodies can combine with CD154 with the activatory mode that the cell of CD40 is carried in direct or indirect blocking-up.

The present invention also provides immunoreactive method among the inhibition experimenter, comprises administration experimenter sugar based anti-cd 154 antibodies or its antibody derivatives, and wherein said antibody or antibody derivatives are with the activatory amount administration of the immunocyte among effective inhibition experimenter.

The present invention also provides treatment or prevention immunoreation dependence disease or the method for illness among the experimenter; Comprise and give said experimenter's sugar based anti-cd 154 antibodies or its antibody derivatives; Said immunoreation dependence disease or illness are treated or prevented to said antibody or antibody derivatives thus with the activatory amount administration of immunocyte among effective inhibition experimenter.

The accompanying drawing summary

Fig. 1 shows that sugar based hu5c8 monoclonal antibody (" mAb ") combines human CD 154 with glycosylation hu5c8mAb with identical relative affinity.Biotinylated hu5c8mAb and combining of cell surface CD154 and being competed of unlabelled glycosylation hu5c8mAb or sugar based hu5c8mAb by titre thing (titrations).Utilize the average fluorescent strength of the biotinylated antibody of streptavidin-PE detection to map with respect to unmarked AC.Four parametric line matches are plotted in together.

Fig. 2 shows that sugar based hu5c8mAb has impaired FcR binding ability.The assessment anti-cd 154 antibodies, i.e. sugar based hu5c8mAb is at huCD154 and Fc γ RI ⁺Cell (a) or huCD154 ⁺Chinese hamster ovary celI and Fc γ RIII ⁺Form the ability of bridge between the cell (b).Fluorescently-labeled Fc γ R ⁺Cell adds microtiter plate, wherein contains glycosylation hu5c8mAb or sugar based hu5c8mAb, and they combine with CD154 in advance.Bonded Fc γ R ⁺Cell detects through the relative fluorescence unit (" RFU ") that utilizes excitation/emission spectrum 485/530nm to measure in each hole.

Fig. 3 shows that glycosylation hu5c8mAb and the sugar based hu5c8mAb serum half life in macaque is identical.Glycosylation hu5c8mAb and the concentration of sugar based hu5c8mAb in serum of macaque are measured after the single 20mg/kg intravenous dosages giving.The average serum concentration of each treatment group is described as ± standard deviation (" SD ").

Fig. 4 shows that glycosylation hu5c8mAb and sugar based hu5c8mAb suppress the initial immunoreation to Toxoid,tetanus (" TT ").The mAb treatment group of macaque and the result of saline control group have been described in glycosylation hu5c8mAb research (solid mark) and sugar based hu5c8mAb research (hollow mark).

Fig. 5 shows that sugar based hu5c8mAb suppresses the follow-up immunoreation to TT.Described individual macaque to TT initial (solid mark) and follow-up (hollow mark) overall antibody response (E _AUC).The 1A treated animal was accepted salt solution before the follow-up TT of initial sum attacks.The 1B treated animal was accepted salt solution before initial TT attacks, before follow-up TT attacks, accept sugar based hu5c8mAb and attack.

Fig. 6 shows the pharmacokinetics of the chimeric MR1 of glycosylation mouse (" muMR1 ") and sugar based muMR1 antibody in the BALB/c mouse.Described the result of glycosylation muMR1 antibody (diamond symbols) and sugar based muMR1 antibody (square symbol).

Fig. 7 (A&B) shows SNF ₁In the mouse, the sugar based anti-cd 154 antibodies reduces the autoantibody reaction to strand (A) and double-stranded (B) DNA.Described the result of muMR1 antibody (diamond symbols) and sugar based muMR1 antibody (square symbol).Contrast muIgG2a antibody is shown as triangle symbol.

Fig. 8 shows that the sugar based anti-cd 154 antibodies reduces SNF ₁The development of mouse mesonephric glomerulus ephritis.Describe muIgG2a (contrast), the mixed structure of the mouse that muMR1 and sugar based muMR1 handle the branch that learns.

Fig. 9 shows that the sugar based anti-cd 154 antibodies delays the appearance of SNF1 mouse mesonephric glomerulus ephritis.The result who shows muMR1 antibody (diamond symbols) and sugar based muMR1 antibody (square symbol).Contrast muIgG2a antibody is shown as triangle symbol.

Figure 10 shows that the sugar based anti-cd 154 antibodies prevents the increase of serum creatinine in the SNF1 mouse.The result who shows muMR1 antibody (diamond symbols) and sugar based muMR1 antibody (square symbol).Contrast muIgG2a antibody is shown as triangle symbol.

Figure 11 shows that the sugar based anti-cd 154 antibodies delays SNF ₁The appearance of the blood urea nitrogen that increases in the mouse (" BUN ") level.The result who shows muMR1 antibody (diamond symbols) and sugar based muMR1 antibody (square symbol).Contrast muIgG2a antibody is shown as triangle symbol.

Figure 12 is shown in the mouse of isotype contrast P1.17 antibody treatment and compares, and tentative autoimmunization meningitis (" EAE ") symptom do not occur with the antibody of muMR1 antibody treatment.Described the result of muMR1 antibody (open circles) and P1.17 control antibodies (filled circles).

Figure 13 shows in the mouse with sugar based muMR1 antibody treatment that sugar based muMR1 antibody is identical with muMR1 antibody for the validity that suppresses the EAE clinical manifestation.The result is expressed as disabled score (MV+MV standard error-" SEM ") and the % initial weight (MV+SEM) of inducing the back fate with respect to disease.P1.17 is contrast Ig.

Figure 14 has described fasting blood glucose (" the FBG ") level in the rhesus monkey after the allochthonous pancreatic islets transplantation.Acute renal allograft rejection is defined as FBG＞100mg/dl.Shown the animal that sugar based hu5c8mAb (dotted line) and glycosylation hu5c8mAb (solid line) handle.

Detailed Description Of The Invention

For description of the invention can more be made much of, provide following detailed description.Only if definition is arranged in addition, used all technology are the same with the implication that one of ordinary skill in the art of the present invention understand with scientific terminology.Below describe illustrative methods and material, but those of similar methods described herein and material can be used for also that the present invention puts into practice and be tangible to those skilled in the art.

Various public publications among the application and reference are included in the square brackets.The full content of the disclosure of these public publications and reference is included among the application as a reference, more fully to describe the state in field according to the invention.The bibliography clause of these reference papers is found in the text or in experimental details part back and lists with the content of band numbering.If any conflict, be as the criterion with specification sheets.Said material, method and embodiment are just to giving an example rather than restriction.

The canonical reference document that the known recombinant DNA technology of those skilled in the art is described comprises Ausubel et al., Current Protocols In Molecular Biology, John Wiley & Sons, New York (1998 and Supplements to 2001); Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory Press, Plainview, New York (1989); Kaufman et al., Eds., Handbook Of Molecular And Cellular Methods In Biology And Medicine, CRC Press, Boca Raton (1995); McPherson, Ed., Directed Mutagenesis:A Practical Approach, IRL Press, Oxford (1991).

The canonical reference document that the conventional principle of the known immunology of those skilled in the art is described comprises: Harlow and Lane, Antibodies : A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1999); And Roitt et al., Immunology3d Ed.; Mosby-Year Book Europe Limited; London (1993) .Standard reference works setting forth the general principles of medicalphysiology and pharmacology known to those of skill in the art include:Fauci etal., Eds. Harrison ' s Principles Of Internal Medicine, 14th Ed., McGraw-HillCompanies, Inc. (1998).

Reagent of the present invention and method relate to utilizes sugar based antibody or its antibody derivatives to suppress immunoreation and treatment immunoreation inductive disease and illness--for example: autoimmune disease, allergy, transplant rejection; Inflammation; Graft versus host disease, fibrosis, and atherosclerosis.

More specifically, being used to treat specifically is human treatment's sugar based anti-cd 154 antibodies, comprises people's antibody, humanized antibody, chimeric antibody, polyclonal antibody and polymer antibody.

Antibody

Antibody is the gp of about MW 150kD, and its body fluid branch through vertebrate immune system responds to the inside and outside and comes the existence of molecule to produce.Function antibody or antibody derivatives can be discerned in vitro and in vivo and combine its specific antigens, and start any follow-up effect relevant with antibodies, for example comprise; Direct cell toxicant; The cell toxicant (" CDC ") that complement relies on, cell toxicant (" ADCC ") that antibody relies on and antibody generate.

In case combine with antigen, one or more in the immune many effector systems of antibody activation causes infection biological body and other to contain neutralization, destruction and the elimination of antigenic entity (for example cancer cells).

Although the natural antibody that exists is from single species, can be through the antibody transformed and antibody fragment from the animal of more than one species ,-for example, chimeric antibody.At present, the chimeric and mouse/non--people's primate antibody of mouse (mouse)/people produces, but the combination of other species also is possible.

In the embodiment, sugar based anti-cd 154 antibodies of the present invention is a chimeric antibody.Usually chimeric antibody comprises heavy chain and/or variable region of light chain, comprises the complementary determining region (" CDR ") and the framework residue of the species (being generally mouse) that the constant region with another species (being the people usually) merges.These gomphosis mouses/people's antibody contains the human amino acid sequence of difference about 75% and 25% mouse aminoacid sequence.The human sequence represents antibody constant region, and the mouse sequence is represented antibody variable region (and containing antigen binding site thus).

Utilizing this chimeric ultimate principle is to keep the antigen-specific of mouse antibodies but the immunogenicity (mouse antibodies will cause being directed against its immunoreation in the species except that mouse) that reduces mouse antibodies, and can in human therapy, utilize said mosaic thus.

In another specific embodiments, sugar based anti-cd 154 antibodies of the present invention comprises chimeric antibody, and it contains from a kind of framework region of antibody with from the CDR district of another kind of antibody.

More in the specific embodiments, sugar based anti-cd 154 antibodies of the present invention comprises chimeric antibody for another, and it contains the CDR district from different people antibody.

In another specific embodiments, sugar based anti-cd 154 antibodies of the present invention comprises chimeric antibody, and it contains the CDR district from least two kinds of different people antibody.

The method of above-mentioned all chimeric antibodies of preparation is [USP 5,807,715 well known by persons skilled in the art; Morrison, 1984; Sharon, 1984; Takeda, 1985].

In another embodiment of the present invention, the sugar based anti-cd 154 antibodies also comprises spirit long sourceization, humanized and people's antibody completely.Generally including from the mouse-anti body weight and/or the light chain CDR that are transplanted in non-human primates or the people's antibody V district framework with humanized antibody of the long sourceization of spirit also comprises human constant region [Riechmann, 1988 usually; Co, 1991; United States patents 6,054,297; 5,821,337; 5,770,196; 5,766,886; 5,821,123; 5,869,619; 6,180,377; 6,013,256; 5,693,761; With 6,180,370].

1. Humanized antibody

Humanized antibody is the antibody through recombinant DNA technology preparation, wherein unwanted human normal immunoglobulin light chain of conjugated antigen or heavy chain (the for example constant region of variable region and framework region) some or all amino acid be used to replace from homology non-human antibody's the light chain or the corresponding amino acid of heavy chain.For example, all has (1) people's antibody constant region on the heavy chain of the humanization version of given antigenic murine antibody and the light chain; (2) framework region of people's antibody variable region; (3) murine antibody CDR.In case of necessity, one or more residue in people's framework region can be changed into the residue of corresponding position in the murine antibody, to keep humanized antibody and antigenic binding affinity.This change is sometimes referred to as " reverse mutation ".Humanized antibody is compared with chimeric people's antibody, can not excite the immunoreation of philtrum usually, because the former contains the inhuman component of much less.The method for preparing humanized antibody is the known [European patents 239400 of antibody those skilled in the art; Jones, 1986; Riechmann, 1988; Verhoeyen, 1988; Queen, 1989; Orlandi, 1989; USP 6,180,370].

In one embodiment of the invention, humanized antibody is through being transplanted to mouse (with other non--people) CDR on people's antibody and producing.More specifically, can carry out as follows: the cDNA of (1) encoding heavy chain and variable region of light chain separates from hybridoma; (2) the variable region dna sequence dna comprises that CDR passes through order-checking and confirms; (3) DNA of coding CDR transfers to human antibody heavy chain and variable region of light chain respective regions through site-directed mutagenesis; (4) add the human constant region gene fragment of required isotype (for example, CH isotype 1, CL isotype k).At last, humanization heavy chain and light chain gene in the mammalian host cell (for example, CHO or NS0 cell) coexpression to produce the solubility humanized antibody.

Sometimes, CDR is directly transferred to people's framework and cause gained antibody forfeiture antigen-binding affinity.This is because in some homologous antibodies, amino-acid residue and CDR reaction influences the overall antigen-binding affinity of antibody thus in the specific framework region.In said situation, it is important introducing in the receptor antibody framework region with " reverse mutation ", so that keep the antigen-binding activity of homologous antibody.The domestic method of preparation reverse mutation is [Queen, 1989 well known by persons skilled in the art; Co, 1991; PCT patentapplication WO90/07861; Tempest, 1991].

2. People's antibody

In one embodiment of the invention, antibody and antibody derivatives are people's sugar based anti-cd 154 antibodies completely.

In the more concrete embodiment of the present invention, fully human antibodies utilizes antibody library [USP 6,300, the 064] preparation of external human spleen cell that excites [Boerner, 1991] and phage display.

In the more concrete embodiment of the present invention, fully human antibodies is through grand (repertoirecloning) [Persson, 1991, Cook; Huang and Stollar, 1991] preparation.In addition; USP 5,798,230 have described from human B cell and have prepared human monoclonal antibodies; The B cell that wherein produces people's antibody is through infecting immortalization with the Epstein-Barr virus or derivatives thereof of expressing eb nuclear antigen 2 (" EBNA2 "), and said EBNA2 is the required albumen of immortalization.The function of EBNA2 is closed subsequently, and causing antibody to generate increases.

Other method of preparation fully human antibodies comprises and utilizes the non-human animal, and its endogenous Ig site is inactivation, and for for human antibody heavy chain who resets and light chain gene, being genetically modified.Said transgenic animal can be used activated T cell or D1.1 albumen [USP 5,474,771; USP 6,331,433; USP 6455,044] immunity, hybridoma can generate from the B cell from said transgenic animal.The details of these methods is described in the art.See that for example various (Palo Alto, CA) public publication/patent comprise USP 5,789,650 with the GenPharm/Medarex that contains the transgenic mice of the little locus of people Ig (miniloci); (Fremont, CA) public publication/patent comprise USP 6 with the relevant various Abgenix of

mouse; 075,181,6; 150; 584 and 6,162,963; Green, 1997; Mendez, 1997; And various Kirin about " transomic " mouse (Japan) public publication/patent, comprise European patent 843961 and Tomizuka, 1997.

The generation of de-glycosylation antibody

The effector functions that exists two kinds of approach to reduce mAb at present keeps other valuable character of its Fc part simultaneously.A kind of method of modified antibodies is the amino acid [European patent 239400 that the effect binding interactions is participated on sudden change mAb surface; Jefferies, 1998].And some combinations that suddenly change probably will cause the suitable reduction of effector functions, show at present through the surface discontinuity body antibody that detects to keep the residue activity.The other problem of this method is that the amino acid change on mAb surface can excite immunogenicity.

The present invention relates to sugar based anti-cd 154 antibodies or antibody derivatives that effector functions reduces, it is characterized in that being positioned at said antibody Fc partial C _H2The modification in the site that conservative N-connects in the structural domain.

In one embodiment of the invention, said modification comprises that the sudden change that is positioned at the heavy chain glycosylation site is to prevent the glycosylation in this site.Therefore, in a preferred embodiment of the present invention, sugar based anti-cd 154 antibodies or antibody derivatives pass through sudden change heavy chain glycosylation site ,-promptly, also expression preparation in suitable host cell of sudden change N298Q (N297 utilizes Kabat EU numbering).For example, this sudden change can recommend to be used for Amersham-Pharmacia Biotech (Piscataway, NJ, the scheme realization of site-directed mutagenesis test kit USA) according to the manufacturer.The antibody of said sudden change can be in host cell (for example NS0 or Chinese hamster ovary celI) stably express purifying then.As an instance, purifying albumin A capable of using and gel permeation chromatography carry out.Those skilled in the art know that other is expressed and purification process also can use.

In another embodiment of the present invention, sugar based anti-cd 154 antibodies or antibody derivatives have the effector functions of reduction, wherein are positioned at the Fc partial C of said antibody or antibody derivatives _H2The modification in the site that the conservative N-in the structural domain connects comprises removes C _H2District's glycan ,-promptly, de-glycosylation.These sugar based anti-cd 154 antibodies can produce enzymatic de-glycosylation then through ordinary method.The deglycosylated method of the enzymatic of antibody is [Williams, 1973 well known by persons skilled in the art; Winkelhake &Nicolson, 1976].

In another embodiment of the present invention, de-glycosylation can realize through utilizing glycosylation inhibition tunicamycin (tunicamycin) [Nose & Wigzell, 1983].Be that said modification is to prevent in said antibody Fc portion C _H2The glycosylation in the site that the conservative N-in the structural domain connects.

In other embodiment of the present invention, the recombinant C D154 polypeptide cell or the cytolemma of said polypeptide (or contain) can be used as antigen to produce anti-cd 154 antibodies or antibody derivatives, and it is subsequently by de-glycosylation.Said antigen can mix or be connected in haptin and generate to increase antibody with adjuvant.

No matter whether the modification of sugar based antibody of the present invention or antibody derivatives is through above-mentioned site-directed mutagenesis and the preparation of enzymatic de-glycosylation method, and the basis that antibody generates is well known by persons skilled in the art.For example, the scheme of immune non-human mammal has been confirmed [Harlow, 1998 in this area; Coligan, 2001; Zola, 2000].

After the immunity, any routine techniques preparation capable of using of antibody of the present invention or antibody derivatives.See, for example, Howard, 2000; Harlow, 1998; Davis, 1995; Delves, 1997; Kenney, 1997.

In some embodiments of the present invention, host cell can be, for example; (1) bacterial cell, such as intestinal bacteria, crescent handle bacillus (Caulobacter crescentus); Streptomyces strain (Streptomyces species), and Salmonella typhimurium (salmonella typhimurium); (2) yeast cell, such as yeast saccharomyces cerevisiae, schizosaccharomyces pombe, pichia pastoris, pichia methanolica (Pichia methanolica); (3) insect cell line, such as from autumn mythimna separata (Spodoptera frugiperda) those-for example, Sf9 and Sf21 clone, and expresSF ^TMCell (Protein Sciences Corp., Meriden, CT, USA)-fruit bat S2 cell and Cells (Invitrogen, Carlsbad, CA, the semilooper (Trichoplusia) in USA); Or (4) mammalian cell.Common mammalian cell comprises COS1 and COS7 cell, Chinese hamster ovary (CHO) cell, NS0 myeloma cell, NIH 3T3 cell, 293 cells; The HEPG2 cell, HeLa cell, L cell, HeLa, MDCK; HEK293, WI38, mouse ES clone is (for example, from bacterial strain 129/SV, C57/BL6; DBA-1,129/SVJ), K562, Jurkat cell, and BW5147.Other useful mammal cell line is known and can be easily available from American type culture collection (" ATCC ") (Manassas; VA; USA) and Coriell CellRepositories (Camden; NJ, state-run general medical science research institute (National Institute ofGeneral Medical Sciences) USA) be people's heredity cell bank (NIGMS).These cell types only for representational be not exhaustive list.

In another embodiment of the present invention, sugar based anti-cd 154 antibodies or antibody derivatives are through the cell free translation preparation.

In another embodiment of the present invention, sugar based anti-cd 154 antibodies or antibody derivatives produce in the bio-reactor of the cell that contains expressing antibodies, to promote scale operation.

In another embodiment of the present invention, sugar based anti-cd 154 antibodies or antibody derivatives are in transgene mammal (for example, the goat of expressing antibodies in milk; Cow, and sheep) the middle generation, to promote the extensive generation [USP 5 of sugar based anti-cd 154 antibodies; 827,690; Pollock, 1999].

As stated, sugar based anti-cd 154 antibodies of the present invention or antibody derivatives can prepare in protokaryon and eukaryotic cell.The present invention also provides the cell of expressing antibody of the present invention thus, comprises hybridoma, the B cell, and plasmocyte, and recombinant modified is to express the host cell of antibody of the present invention.

Except other consideration (some of them are described at preceding text), can select host cell with the proteic ability of the CD154 of required mode expression processing according to host cell strain.Except sugar basedization, the posttranslational modification of said polypeptide includes but not limited to, acetylize; Carboxylation, phosphorylation, fatization (lipidation); And acidylate, one aspect of the present invention provides sugar based anti-cd 154 antibodies or the antibody derivatives with one or more these posttranslational modifications.

Antibody modification

During administration, antibody is removed from circulation usually fast, and shows relatively short pharmaceutical activity thus.Therefore, need a large amount of relatively antibody of frequent injection to keep the validity of Antybody therapy.

In one embodiment of the invention, sugar based anti-cd 154 antibodies or antibody derivatives can be modified (that is, being connected with other part) with the integrity of increase antibody and prolong its volume lifetime.For example, sugar based anti-cd 154 antibodies of the present invention or antibody derivatives can be modified the part that can increase stability to comprise, thereby prolong the antibody serum half life.

In some embodiments of the present invention, the sugar based anti-cd 154 antibodies through with water-soluble polymers such as polyoxyethylene glycol, the multipolymer of polyoxyethylene glycol and W 166; CMC 99.5, VISOSE, Z 150PH; Polyvinylpyrrolidone--based or polyproline is covalently bound and modified-all these are compared without the albumen of modifying with corresponding through the albumen of modifying; After intravenous injection, in blood, show longer in fact half life [Abuchowski, 1981; Anderson, 1992; Newmark, 1982; Katre, 1987].

Antibody modification also can increase the solvability of albumen in the aqueous solution, eliminates and assembles, and increases proteic physics and chemicalstability, reduces proteic immunogenicity and antigenicity greatly.As a result, in the desired body biological activity can through with compare without the albumen of modifying, with lower frequency or to realize than the said polymkeric substance of low dosage administration-protein affixture.

In some embodiments of the present invention, said sugar based anti-cd 154 antibodies obtains modifying through come mark with the certification mark thing, this affinity tag for example, radioactive isotope, enzyme, dyestuff or vitamin H.

In some embodiments of the present invention, sugar based anti-cd 154 antibodies or antibody derivatives obtain modifying through being coupled to therapeutical agent, and this therapeutical agent for example; Emitting isotope or radionuclide (for example, 111In or 90Y), toxin moiety is (for example; Toxoid,tetanus or ricin); Toxoid or chemotherapeutics [USP 6,307,026].

In some embodiments of the present invention, sugar based anti-cd 154 antibodies or antibody derivatives are through obtaining modifying with the developer coupling.Developer for example can comprise that mark property part (for example, vitamin H, the fluorescence part, the radioactivity part, histidine mark or other are peptide-labeled) is used for facilitation and separates or detect.

Antibody derivatives

The invention still further relates to sugar based anti-cd 154 antibodies verivate.All these above-mentioned method and medicaments about the sugar based anti-cd 154 antibodies all are used for preparing sugar based anti-cd 154 antibodies verivate of the present invention.

In some embodiments of the present invention, sugar based anti-cd 154 antibodies verivate comprises different aggressiveness antibody complex body and antibody fusions, such as bi-specific antibody, and half dimer (hemidimeric) antibody, multivalent antibody (that is tetravalent antibody) and single-chain antibody.Half homodimeric antibody partly is made up of Fc part and a Fab.Single-chain antibody is made up of the variable region that the albumen spacer in the single protein chain connects.

In some embodiments of the present invention; Sugar based anti-cd 154 antibodies verivate of the present invention also comprises the protein that contains one or more light chain immunoglobulin and/or heavy chain; Such as monomer and the homology or the heteromultimers (for example dimer and tripolymer) of these chains, wherein these chains are optional by disulfide bonding or crosslinked.These antibody derivatives can combine with one or more antigen.

According to another embodiment, the present invention includes the sugar based Fab of complete antibody, such as Fab, Fab ', F (ab ') 2 and F (v) antibody fragment.In another embodiment, the present invention includes the Fab of complete antibody, such as Fab, Fab ', F (ab ') 2 and F (v) antibody fragment.

Clone

The present invention also provides the clone that produces the disclosed sugar based anti-cd 154 antibodies of this paper.A kind of such clone, it produces sugar based hu5c8 antibody, is preserved in ATCC on January 14th, 2003 according to the regulation of the budapest treaty that is used for patented procedure of international endorsement; 10801 UniversityBlvd.; Manassas, Virginia, 20110-2209; U.S.A., ATCC preserving number PTA-4931.Second kind of such clone, it produces mosaic type mouse sugar based mu5c8 antibody, is preserved in ATCC on January 14th, 2003 according to the regulation of the budapest treaty that is used for patented procedure of international endorsement; 10801 University Blvd.; Manassas, Virginia, 20110-2209; U.S.A., ATCC preserving number PTA-4934.

Treat-ment

In one embodiment of the invention, sugar based anti-cd 154 antibodies or its antibody derivatives or comprise the pharmaceutical composition of said antibody or antibody derivatives can suppress the immunoreation among the experimenter.Said antibody, antibody derivatives or pharmaceutical composition give the experimenter with effective inhibitory amount.

Antibody, " effective inhibitory amount " of antibody derivatives or pharmaceutical composition are the interactional any amounts of CD154-CD40 that effectively suppresses among its experimenter who gives.The method of measuring " amount of suppression " is well known by persons skilled in the art and depends on following factor, includes but not limited to: related experimenter's type, experimenter's size, the therapeutical agent of being sent.

In the specific embodiments of the present invention, the sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives can combine with the CD154 protein molecular.

In the specific embodiments of the present invention; Said sugar based anti-cd 154 antibodies; Antibody derivatives or contain said antibody or antibody derivatives pharmaceutical composition can with the CD154 protein binding, the sugar based hu5c8 specificity that the latter and ATCC clone PTA-4931 produce combines.

In the specific embodiments of the present invention; Said sugar based anti-cd 154 antibodies; Antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives can combine with the CD154 epi-position; Said epi-position combines with the sugar based hu5c8 specificity that ATCC clone PTA-4931 produces, and wherein said sugar based anti-cd 154 antibodies or antibody derivatives are characterised in that the sudden change (utilizing EU Kabat to be numbered N297) of N298Q.

In the specific embodiments of the present invention, said sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives not with the effector receptors bind.The present invention another more in the specific embodiments; Said sugar based anti-cd 154 antibodies; Antibody derivatives or contain said antibody or antibody derivatives pharmaceutical composition can with the CD154 protein binding; The sugar based hu5c8 specificity that the latter and ATCC clone PTA-4931 produce combines, and wherein said sugar based anti-cd 154 antibodies or antibody derivatives or pharmaceutical composition not with the effector receptors bind.

In the specific embodiments of the present invention, said sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives do not cause thrombosis.The present invention another more in the specific embodiments; Said sugar based anti-cd 154 antibodies; Antibody derivatives or contain said antibody or antibody derivatives pharmaceutical composition can with the CD154 protein binding; The sugar based hu5c8 specificity that the latter and ATCC clone PTA-4931 produce combines, and wherein said sugar based anti-cd 154 antibodies or antibody derivatives or pharmaceutical composition do not cause thrombosis.

In another specific embodiments of the present invention, said sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives can interact and suppress immunoreation through suppressing CD154-CD40.

In one embodiment of the invention, said sugar based anti-cd 154 antibodies, antibody derivatives or contain the pharmaceutical composition of said antibody or antibody derivatives can inflammation-inhibiting.Be the object of the invention, red, swollen, hot and pain that Inflammatory response is characterised in that is as the consequence of trichangiectasia and oedema and phagocytic leukocyte migration.Some instances of Inflammatory response comprise: sacroiliitis, and contact dermatitis, ultra-IgE syndrome, inflammatory bowel, allergic asthma and the Inflammatory response [Gallin, 1989] of the special property sent out.More arthritic instances comprise: rheumatoid arthritis, non-rheumatoid inflammatory arthritis, sacroiliitis relevant with Lyme disease and inflammatory osteoarthritis.Some instances of the special property sent out inflammatory diseases comprise: psoriatic and systemic lupus erythematous.

In one embodiment of the invention, said sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives can suppress the repulsive interaction of organ transplant recipient.

The present invention another more in the specific embodiments; Said sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives can suppress the repulsive interaction of the acceptor of heart, kidney, liver, skin, islet cells and marrow.

In one embodiment of the invention, said sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives can suppress the graft versus host disease in the acceptor.

In one embodiment of the invention, said sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives can suppress the anaphylaxis-for example among the experimenter, hay fever or to the allergy of penicillium mould or other medicines.

In one embodiment of the invention, said sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives can suppress to suffer from the autoimmune response among the experimenter of autoimmune disease.The instance of autoimmune disease includes, but not limited to rheumatoid arthritis, myasthenia gravis; Systemic lupus erythematous, Graves is sick, idiopathic thrombocytopenic purpura, hemolytic anemia; Mellitus, inflammatory bowel, clone disease, multiple sclerosis; Psoriatic and drug-induced autoimmune disease ,-for example, drug-induced lupus.

In one embodiment of the invention, said sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives can suppress to suffer from from the autoimmune response among the experimenter of the autoimmune response of infection.

In one embodiment of the invention; Said sugar based anti-cd 154 antibodies; Antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives can suppress to suffer from the syndrome from Reiter, and joint of vertebral column is scorching, Lyme disease; HIV infects, the autoimmune response among the experimenter of syphilis or autoimmune response lungy.

In one embodiment of the invention, said sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives can suppress the fibrosis among the experimenter.

More Fibrotic instances comprise: pulmonary fibrosis or fibrotic conditions.Some instances of pulmonary fibrosis comprise: be secondary to the pulmonary fibrosis of adult respiratory distress syndrome, drug-induced pulmonary fibrosis, idiopathic pulmonary fibrosis, or hypersensitivity pneumonitis (hypersensitivity pneumonitis).Some instances of fibrotic conditions comprise: hepatitis C; Hepatitis B; Liver cirrhosis; Be secondary to the liver cirrhosis of toxin injury; Be secondary to the liver cirrhosis of medicine; Be secondary to the liver cirrhosis of virus infection; And the liver cirrhosis that is secondary to autoimmune disease.

In one embodiment of the invention, said sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives can suppress gastrointestinal illness.Some instances of gastrointestinal illness comprise: dyskinesia of esophagus, inflammatory bowel and scleroderma.

In one embodiment of the invention, said sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives can suppress vascular disease.Some instances of vascular disease comprise: atherosclerosis or reperfusion injury.

In one embodiment of the invention; Said sugar based anti-cd 154 antibodies, antibody derivatives or contain the pharmaceutical composition of said antibody or antibody derivatives can suppressor T cell cancer (for example T HTLV or lymphoma) the propagation of T cell tumour cell among the patient.Said sugar based anti-cd 154 antibodies or antibody derivatives or pharmaceutical composition can be with the amount administration experimenters of T cell tumour cell proliferation among effective inhibition experimenter.

In one embodiment of the invention, said sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives can suppress the virus infection of HTLVI virus to experimenter T cell.Said sugar based anti-cd 154 antibodies, antibody derivatives or pharmaceutical composition can give the experimenter with the amount of effective inhibition virus infection.

In one embodiment of the invention; Said sugar based anti-cd 154 antibodies; Antibody derivatives or contain the pharmaceutical composition of said antibody or antibody derivatives can video picture tumour cell or neoplastic cell among the experimenter, it expresses the albumen of the sugar based hu5c8 specific recognition that ATCC clone PTA-4931 produces.The tumour cell among the video picture experimenter or the method for neoplastic cell may further comprise the steps: form under the condition of complex body at the albumen that allows antibody or antibody derivatives and tumour cell or neoplastic cell surface; Give the sugar based anti-cd 154 antibodies of experimenter's significant quantity; Antibody derivatives, or pharmaceutical composition; Antibody/the protein complexes of any formation of video picture or antibody derivatives/complex body, any tumour cell or the neoplastic cell among the video picture experimenter thus.

In one embodiment of the invention; Said sugar based anti-cd 154 antibodies; Antibody derivatives or the pharmaceutical composition that contains said antibody or antibody derivatives can detect tumour cell or the existence of vegetation cell among the experimenter, and it expresses the albumen of the sugar based hu5c8 specific recognition that ATCC clone PTA-4931 produces.The method that tumour cell among the detection experimenter or vegetation cell exist may further comprise the steps: allowing antibody or antibody derivatives and albumen to form under the condition of complex body; Give the sugar based anti-cd 154 antibodies of experimenter's significant quantity; Antibody derivatives, or pharmaceutical composition; Remove any unconjugated developer from the experimenter; With the antibody/protein complexes or the antibody derivatives/complex body that detect to form, the existence of said mixture shows the existence of tumour cell and vegetation cell among the experimenter.

Pharmaceutical composition

The present invention provides pharmaceutical composition, and it comprises sugar based anti-cd 154 antibodies described herein or antibody derivatives.

In one embodiment of the invention, said pharmaceutical composition comprises one or more sugar based anti-cd 154 antibodies or antibody derivatives.

In another embodiment of the present invention, said pharmaceutical composition also comprises pharmaceutically acceptable carrier, adjuvant, delivery vector, damping fluid or stablizer.

In the more concrete embodiment of the present invention, said pharmaceutically acceptable carrier is the salt solution of phosphate-buffered, saline water, and water, Citrate trianion/sucrose/Tween preparaton and emulsion-for example, oil/aqueous emulsion.

In one embodiment of the invention, thereby said pharmaceutical composition can reduce and prevents proteic host immune response little sending in by bag apparatus.Said antibody or antibody derivatives also can film such as liposome in the form of little tunicaization send.

In one embodiment of the invention, said pharmaceutical composition can be the form of aseptic injection prepared product, for example sterile injectable water-based or oiliness suspension.This suspension can be suitable according to techniques make use known in the art dispersion agent, wetting agent and suspension agent are prepared.

In one embodiment of the invention, said pharmaceutical composition taking orally, part or intravenously are sent.

The present invention another more in the specific embodiments, for oral administration, said pharmaceutical composition is formulated in suitable capsule, tablet is in aqueous suspensions or the solution.The oral administration of said compsn can contain appropriate carrier or vehicle with solid preparation, such as W-Gum, and gel, lactose, Sudan Gum-arabic, sucrose, Microcrystalline Cellulose, kaolin, N.F,USP MANNITOL, Lin Suanergai, sodium-chlor, or alginic acid.Spendable disintegrating agent includes, but not limited to Microcrystalline Cellulose, W-Gum, sodium starch glycollate, and alginic acid.Spendable tablet binder comprises Sudan Gum-arabic, methylcellulose gum, Xylo-Mucine, Vinylpyrrolidone polymer (Povidone ^TM), Vltra tears, sucrose, starch and TKK 021.Spendable lubricant comprises Magnesium Stearate, Triple Pressed Stearic Acid, silicone fluid, talcum, paraffin, oil, and silica gel.

The present invention another more in the specific embodiments, for topical application, said pharmaceutical composition can be formulated in the suitable ointment.Some instances of the composite preparation of topical application comprise: drops, and tincture, lotion, emulsifiable paste, solution and ointment wherein contain active ingredient and various balustrade and carrier.

In one embodiment of the invention, local semi-solid ointment formulation is usually included in the about 1-20% of concentration in the carrier ,-for example, and the active ingredient of 5-10%, said carrier is such as drug cream matrix.

In one embodiment of the invention, inhalation compositions and transdermal drug compsn also can prepare easily.

In one embodiment of the invention, the liquid preparation of the pharmaceutical composition for oral administration that in water or other aqueous carrier, prepares can contain multiple suspension agent, such as methylcellulose gum; Alginate, tragacanth, pectin; Kelgin, X 5189 (carrageenan), Sudan Gum-arabic; Vinylpyrrolidone polymer, and polyoxyethylene glycol.The liquid preparation of pharmaceutical composition of the present invention also can comprise solution, emulsion, and syrup or elixir, it contains the wetting agent with active compound, sweeting agent and tinting material and seasonings.The various liquid of said pharmaceutical composition and powder formulation can wait that the ordinary method of treating in the mammiferous lung prepares through suction.

In one embodiment of the invention, the liquid preparation of medicinal composition for injections can comprise various carriers, such as vegetables oil, and N,N-DIMETHYLACETAMIDE, N; Ethyl lactate, ethyl-carbonate, isopropyl myristate, ethanol; Polyvalent alcohol-be glycerine, Ucar 35, liquid macrogol, etc.In some embodiments, said compsn comprises Citrate trianion/sucrose/tween carrier.For intravenous injection, the water-soluble version of compsn can pass through the drip-injection method administration, and the pharmaceutical prepn that contains anti-mycotic agent and the acceptable preparation of physiology thus is by infusion.The acceptable vehicle of physiology can comprise, 5% glucose for example, 0.9% salt solution, Ringer's solution and other proper excipient.The suitable insoluble form of said compsn can be used as suspension preparation and the administration in hydrated matrix or the acceptable oil matrix, the OE of the ester of said matrix such as longer chain fatty acid-for example.

In one embodiment of the invention, said pharmaceutical composition is included in sugar based anti-cd 154 antibodies or its antibody derivatives of the about 0.1-90% weight (such as 1-20% or 1-10%) in pharmaceutically acceptable carrier.

In one embodiment of the invention, the optimized percentage of antibody or antibody derivatives is according to the required result of treatment in preparation itself and concrete pathology and the associated treatment scheme and different in every kind of pharmaceutical composition.Pharmaceutical prepn has been confirmed [Gennaro, 2000 fully in this area; Ansel, 1999; Kibbe, 2000].The known ordinary method of medical field those of skill in the art can be used for giving the experimenter with said pharmaceutical composition.

In some embodiments of the present invention, said pharmaceutical composition also comprises inhibitive ability of immunity or immune regulative compound.For example, said inhibitive ability of immunity or immune regulative compound can be one of following material: the medicament that interrupts T cell co-stimulatory signal through CD28; Interrupt the medicament of calcineurin signal, reflunomide, the antiproliferative pharmaceutical agent, with expressed protein specificity bonded antibody on the immunocyte surface, said immunocyte includes but not limited to CD45; CD2, IL2R, CD4, CD8 and RANK FcR, B7; CTLA4, TNF, LT γ, and VLA-4.

In some embodiments of the present invention, said inhibitive ability of immunity and immune regulative compound are tacrolimus (tacrolimus), sirolimus (sirolimus); Mycophenlate mofetil (mycophenolate mofetil); Mizorubine, Gusperimus (deoxyspergualin), brequinar sodium (brequinar sodium); Leflunomide (leflunomide), Wyeth-Ayerst Laboratories (rapamycin) or azaspirane.

In other embodiment of the present invention, antibody, antibody derivatives or the pharmaceutical composition that comprises them can be included in container separately, in packing or the divider or as the part of test kit, said test kit has label and administration explanation.

Administration and route of delivery

Sugar based anti-cd 154 antibodies or its antibody derivatives and pharmaceutical composition of the present invention can give the experimenter with the mode that any medical science is accepted.Be the object of the invention, " administration " refers to administration antibody well known by persons skilled in the art, any standard method of antibody derivatives and pharmaceutical composition, and should not be limited to embodiment provided by the invention.

In some embodiments of the present invention; Said sugar based anti-cd 154 antibodies, antibody derivatives and pharmaceutical composition can through use standard method in intravenously, subcutaneous, intraperitoneal, intramuscular, marrow, Intraventricular, in epidural, in the intra-arterial, blood vessel, in the intraarticular, synovial membrane, in the breastbone, in the sheath, in the liver, in the spinal cord, in the tumour, in the brain, in the intestines, in the lung, in the mucous membrane, intrauterine, hypogloeeis or in the inflammation site or the tumor growth site carry out local injection and come administration.

In some embodiments of the present invention, said sugar based anti-cd 154 antibodies, antibody derivatives and pharmaceutical composition can be oral through comprising, intranasal, through eye, per rectum or partial approach give the experimenter.

Another is more in the specific embodiments, sugar based anti-cd 154 antibodies according to the invention, and antibody derivatives and pharmaceutical composition can be with the form oral administration administration experimenters of capsule, tablet, aqueous suspensions or solution.

Another is more in the specific embodiments, said sugar based anti-cd 154 antibodies, and antibody derivatives and pharmaceutical composition can be through using emulsifiable paste, ointment etc. through the topical experimenter.

In other embodiment of the present invention, sugar based anti-cd 154 antibodies according to the invention, antibody derivatives and pharmaceutical composition can be through utilizing spraying gun, and Diskus or metered-dose inhaler suck and administration.

In other embodiment of the present invention; Said sugar based anti-cd 154 antibodies; Antibody derivatives and pharmaceutical composition can give the experimenter in the following manner: sustained release administration; Such as the reservoir formula injection of direct applied degradable implant in surgical procedure, or with among infusion pump or the biocompatibility sustained release implants implantation experimenter.

Another is more in the specific embodiments; Sugar based anti-cd 154 antibodies according to the invention; Antibody derivatives and pharmaceutical composition can be through route of administration (the injectable depot route) administrations of injectable reservoir; Such as utilizing 1,3, or 6 months reservoir formula injectable or biodegradable material and method.

Another is more in the specific embodiments; Sugar based anti-cd 154 antibodies according to the invention; Antibody derivatives and pharmaceutical composition can be through containing antibody; The transdermal patch of antibody derivatives and pharmaceutical composition gives experimenter's skin and comes the administration experimenter, and makes said patch and experimenter's skin exposure, common every subsides 1-5 hour.

In other embodiment of the present invention, said sugar based anti-cd 154 antibodies, antibody derivatives and pharmaceutical composition can be with every kg body weight of any dosage and the acceptable any dose frequency administration experimenters of medical science.Acceptable dosage comprise about 0.01 with the scope of 200mg/kg experimenter's body weight.

In other embodiment, sugar based anti-cd 154 antibodies according to the invention, antibody derivatives and pharmaceutical composition can be to arrive interval repeat administration every other month every day.

In one embodiment of the invention, said sugar based anti-cd 154 antibodies, but antibody derivatives and pharmaceutical composition such as a plurality of dosage of needs administration every day are to obtain total required per daily dose.The validity of treat-ment can be assessed through monitoring experimenter's known sign or disease symptoms.

For all embodiments of the present invention; Effectively produce the sugar based anti-cd 154 antibodies according to the invention of required effect, the dosage of its antibody derivatives and pharmaceutical composition and administration frequency will depend on multiple factor, such as the character of waiting to treat disease; Experimenter's size; Therapeutic purpose, used concrete pharmaceutical composition, and treatment doctor's judgement.

Sugar based according to the invention resists-CDl54 antibody; Its antibody derivatives and pharmaceutical composition can be used as single dosage needle to specific indication administration; Such as preventing the experimenter to having exposed the antigenic immunoreation of short period, said antigen is such as the exogenous antigen of administration in one day treatment.The instance of said treatment comprises administation of combination antibody of the present invention or antibody derivatives and therapeutical agent, for example, and antigenicity medicine (antigenic pharmaceutical), sensibiligen or blood products, or gene therapy vector.In the long-standing indication of antigen; Such as in the immunoreation of control to the antigenicity medicine of the tissue transplanted or long term administration; The administration at interval of antibody of the present invention or antibody derivatives or pharmaceutical composition continues long period of medically indicating, all one's life from a couple of days or several weeks to the experimenter.

In one embodiment of the invention, can be animal through the experimenter of aforesaid method treatment.Preferred said animal is a Mammals.The mammiferous instance that can be treated includes, but not limited to the people, non-human primates, rodents (comprising rat, mouse, hamster and cavy), cow, horse, sheep, goat, pig, dog, and cat.Preferably, said Mammals is the people.

The present invention can better understand based on following examples.Yet it only is of the present invention giving an example that those skilled in the art will understand the concrete grammar and the result that are discussed easily, as specifically described in the present invention's embodiment subsequently.

Experimental details

Embodiment

For example clear method of the present invention of following examples and product.Suitable change and transformation to said condition and parameter are common in biology field, and are that those skilled in the art are conspicuous, and they within the spirit and scope of the present invention.

Embodiment 1: sugar based hu5c8 production of antibodies and assessment

The generation of sugar based hu5c8mAb and expression

For reducing the effector functions of hu5c8mAb, the sugar based form is through changing heavy chain C _H2The Asn site that classical N connects in the structural domain produces for the Gln residue.

Competition combines test to confirm to compare with glycosylation hu5c8mAb, and sugar based hu5c8mAb combines the ability of cell surface CD154 not change (Fig. 1).

Utilize bridge joint test form (bridging assay format), at the external test effector functions.Hu5c8mAb compares with glycosylation, and sugar based hu5c8mAb and Fc γ RI relative combines to weaken 25 times (Fig. 2 A).When reaching the concentration of 5mg/ml, can not confirm that sugar based hu5c8mAb combines with the remnants of Fc γ RIII, and normal glycosylation hu5c8mAb shows that in identical test form EC50 is 50ng/ml (Fig. 2 B).

The pharmacokinetics of sugar based hu5c8mAb in the macaque

After giving single dose 20mg/kg hu5c8mAb and sugar based hu5c8mAb; Serum-concentration-time diagram to from two independent experiments carries out pharmacokinetic analysis; Two compartment models of said analysis and utilization (compartment model), it has one-level clearance rate constant (first order elimination rateconstant) (WinNolin Professional Software v3.1, Pharsight Corp.; Cary, NC).Fig. 3 contains pharmacokinetics figure, and Fig. 1 contains the average pharmacokinetic parameter of hu5c8mAb and sugar based hu5c8mAb.The removing of hu5c8mAb and volume distributed median are bigger slightly than sugar based hu5c8mAb.

Table 1

Hu5c8 or sugar based hu5c8 single dose intravenously with 20mg/kg

The administration macaque ^ALater average pharmacokinetic parameter

^AData presentation is mathematical mean ± standard deviation, n=3 for the hu5c8 treatment group, n=4 for sugar based hu5c8. ^BMaximum serum-concentration, ^CThe System Cleaning rate, ^DThe steady-state distribution volume, ^EFinal serum half life.

Method-embodiment 1

1. Antibody generatesThe selection of hu5c8mAb, clone and humanization is former describes.See Lederman respectively, 1992 and Karpusas, 2001.The hu5c8mAb hybridoma can be available from ATCC (HB10916).Utilize Amersham-Pharmacia Biotech (Piscataway, NJ, test kit USA); Scheme according to manufacturer's recommendation; Eliminate mutagenesis through unique site, in glycosylation hu5c8mAb, carry out heavy chain glycosylation site sudden change N298Q (N297 utilizes the EU numbering).The sugar based hu5c8 that produces stably express in NS0 myeloma cell also passes through albumin A and gel permeation chromatography purifying.The clone that produces sugar based hu5c8 antibody derives from ATCC (PTA-4931).SDS-PAGE shows the tetramer that the disulfide linkage of said albumen formation expection is connected with analytical gel permeation chromatography.

2. CD154 combines testTo huCD154 ⁺(Dr.Leonard Chess, Columbia University present also can derive from ATCC (CRL-10915) and carry out combining test based on the competition of FACS the D1.1 cell.0.1mg/ml combine and the titrate (titration) of hu5c8mAb and sugar based hu5c8mAb of biotinylated hu5c8 mAb and cell surface CD154 are competed.(BD-PharMingen San Diego, CA USA) detect cell bonded biotinylation hu5c8mAb with streptavidin-phycoerythrobilin (PE).RA can be from the IC of four parametric line matches ₅₀Value is inferred.

3. CD154-Fc γ R bridge joint is measuredThe utilization of Fc γ R binding affinity forms antigen based on antibody and carries the test determination (seeing below) of the ability of " bridge joint " between the cell of Fc γ R.The test of Fc γ RI (CD64) bridge joint is through the solvable human CD 154 (Biogen that recombinates with 1mg/ml; Karpusas, 1995) PBS solution encapsulates 96 hole Maxisorb elisa plate (Nalge-Nunc Rochester, NY at 4 ℃; USA) spend the night, seal with the PBS solution of 1%BSA subsequently and carry out.The titrate of hu5c8mAb (glycosylation and sugar based) combines 30 minutes with CD154 at 37 ℃ subsequently, washs this plate, measures fluorescently-labeled U937 (CD64 ⁺) combination of cell.The U937 cell is containing 10%FBS, and 10mM HEPES grows in the RPMI substratum of L-glutaminate and penicillin/streptomycin, with division in 1: 2, and is measuring front activating one day with 1000 units/ml IFN γ to increase Fc γ RI expression.

The test of Fc γ RIII (CD16) bridge joint utilizes and grows in (the Corning LifeSciences Acton of 96 hole tissue culturing plates; MA; USA) individual layer Chinese hamster ovary (CHO) cell (Biogen) of the expression CD154 in carries out, and measures with CD16 (Dana Farber Institute, Boston; MA, USA present) the mAb dependency of the fluorescently-labeled Jurkat cell of transfection combines.CHO-CD154 ⁺Cell is with 1x10 ⁵Cell/ml is inoculated in 96 orifice plates, contain 10% the dialysis FBS, the 100nM methotrexate, L-glutaminate, and penicillin/streptomycin (all reagent are all from Gibco-BRL Rockville, and MD grows into the full end among α-MEM USA).Be grown in and contain 10%FBS, 400mg/ml Geneticin (Geneticin), 10mM HEPES, Sodium.alpha.-ketopropionate, L-glutaminate, and the CD16 among the RPMI of penicillin/streptomycin (all reagent derive from Gibco-BRL) ⁺The Jurkat cell is carrying out division in 1: 2 before the said mensuration.

In experiment for Fc γ RI and Fc γ RIII acceptor; The cell that carries the Fc acceptor with 2 '; 7 '-(MolecularProbes Eugene, OR is USA) 37 ℃ of marks 20 minutes for two-(2-carboxy ethyl)-5-(with-6)-Fluoresceincarboxylic acid acetoxy-methyl ester (BCECF-AM).Excessive BCECF-AM, 1 * 10 are removed in washing ⁵The cell of mark is incubated 30 minutes at 37 ℃ in mensuration.Unconjugated Fc γ R ⁺Cell is removed for several times through washing, and (MilliporeCorporation Bedford, MA read plate, excitation wavelength 485nm, emission wavelength 530nm on USA) at Cytofluor 2350Fluorescent Microplate Reader.

Embodiment 2: sugar based hu5c8 antibody suppresses the follow-up humoral response of initial sum

Suppress in the macaque the antigenic initial humoral immune reaction of Toxoid,tetanus (TT)

Sugar based hu5c8mAb and the glycosylation hu5c8mAb (according to embodiment 1 preparation) of 20mg/kg of single dose of respectively doing for oneself suppresses the ability assessment in dividing other experiment to the initial antibodies reaction of TT.Compare with the control group of brine treatment, administration sugar based hu5c8mAb or glycosylation hu5c8mAb cause overall initial immunoreation (E _AUC) reduce by 70% and 77% respectively.Fig. 4 illustrates TT antibody tiring in whole 42 days, shows that sugar based hu5c8mAb is similar with glycosylation hu5c8mAb to the inhibition degree of initial humoral response, but its Fc γ R binding ability reduces.

The immunogenicity of humanized mAbs is another measurement index of their validity in this non-human primates model.Have in the animals that four are handled with single dose 20mg/kg sugar based hu5c8mAb three medicine after serum is removed soon; Anti--hu5c8 the antibody that low titre in about the 82nd day, occurred, the inhibition consistent (data not shown) of the humoral response when having sugar based mAb.

As another measurement index that initial reaction suppresses, remarkable minimizing is compared in the existence of germinal center (" GC ") in the animal of inguinal lymph nodes biopsy demonstration sugar based hu5c8mAb treatment with contrast.In the animal of treatment, GC is rarely found and very little, occupies below 20% of cortex.Control animal has a plurality of GC, and moderate is to tangible reactive secondary folliculus.The moderate of observed lymphoglandula is consistent to glycosylation hu5c8mAb observed (data not shown) with in the past to the severe hypoplasia.

With the not overall change of hematologic parameter after single dose 20mg/kg glycosylation or the sugar based hu5c8mAb administration macaque, TLC does not obviously change.In addition, the CD4/CD8T cell proportion is constant, shows CD4 widely ⁺(data not shown) do not appear in t cell depletion.

Suppress in the macaque the antigenic follow-up humoral response of Toxoid,tetanus (TT)

Single dose 20mg/kg sugar based hu5c8mAb suppresses the ability of follow-up humoral response and assesses through giving 8 saline control animals with TT attack for the second time, has normal initial reaction in the former described sugar based hu5c8mAb conceptual phase of said animal.Before TT attacks for the second time, there are 4 to accept 20mg/kg sugar based hu5c8mAb (group 1B) in these animals, accept salt solution (group 1A) for four.

Follow-uply immunoreactively be characterised in that beginning is very fast and stronger than initial immunoreactive degree.Calculate the degree (E of the follow-up reacting phase of single animal for initial reaction _AUCFollow-up/E _AUCInitially) (table 2).Fig. 5 shows the follow-up individual immunity reaction of overall initial sum.There is notable difference in the immunoreation degree that it should be noted that individual animals.On average; It is high 6.5 times to this antigenic initial reaction than them that follow-up TT in the saline control (group 1A) attacks the overall antibody response that produces, and it is only higher 2.0 times than their initial reaction on average to accept the overall follow-up antibody response of animal (group 1B) generation of sugar based hu5c8mAb.Therefore, administration sugar based hu5c8mAb compares with saline control, and the former causes the degree of follow-up antibody response to reduce by 70%.

Table 2

In the macaque to the follow-up immunoreation (E of the overall initial sum of Toxoid,tetanus _AUC)

^AThe 1A treated animal was accepted salt solution before the follow-up TT of initial sum attacks.The 1B treated animal was accepted salt solution before initial TT attacks, before follow-up TT attacks, accept sugar based hu5c8. ^BThe degree of follow-up reaction is through follow-up E _AUCWith initial E _AUCThe ratio value representation.

Method-embodiment 2

1. Humoral immune reaction to TTSame macaque among utilization and the embodiment 1 carries out two independently researchs in macaque.Collect to remain on room temperature, analyze getting blood day from the serum sample of each research and the whole blood that will be used for immunophenotyping.

This sugar based hu5c8mAb research comprises two treatment group.Group 1 comprised 4 male and four jennies, and it accepted salt solution and as untreated contrast at first day.Group 2 comprises two male and two jennies, and it accepted single dose 20mg/kg sugar based hu5c8mAb (as stated) at first day through intravenously.Handle after 4 hours, all animal via intramusculars (IM) are accepted the TT that 5Lf (flocculating of limiting the quantity of dosage (limes flocculating dose)) absorbs.(post-dosing) selected date blood sampling of the 190th day before and after handling in first day and after administration.Got the lymph node biopsy sample at the 15th day.

Glycosylation hu5c8mAb research comprises five groups of treatment group, and every group contains three female samples.First day, first winding received salt solution (untreated contrast), second to five group of 0.2,1,5 or 20mg/kg glycosylation hu5c8mAb that accepts single dose respectively, and it can derive from ATCC (CRL-10915).Handle after 4 hours the TT that the 5Lf of all animals received single IM injections absorbs.The 42nd day the selected date before and after handling in first day and after administration is from all group blood samplings.For allowing the comparability of these two independent studies, selected serum sample compares in anti-TT ELISA experiment side by side.

First TT attacked the back the 230th day in above-mentioned sugar based research, and control group 1 is divided into two groups.1A is as undressed contrast for group, and group 1B handles assessment with sugar based hu5c8mAb, and it suppresses follow-up immunoreactive ability.Animal is handled as follows: group 1B accepted single dose 20mg/kg sugar based hu5c8mAb at first day through intravenously.Group 1A accepted the salt solution of isopyknic phosphate-buffered through intravenously at first day.Handled the TT that the 5Lf of all animals received IM dosage absorbs back four hours.The 85th day selected date blood sampling before and after handling in first day and after administration.

2. The assessment immunoreationUtilize non-compartment analysis (noncompartmental analysis) assessment immunoreation.The immune parameter that calculates comprises maximum valence value (Emax), reaches this peaked time (tmax), and is administered into the antigenic overall antibody response (EAUC (0-last)) of last sampling time point to being given from antigen.Two compartment models that utilization has an initial elimination factor constant carry out pharmacokinetic analysis (WinNolin Professional Software V3.1, Pharsight Corp., Cary, NC, USA).The pharmacokinetic parameter of confirming comprises maximum serum-concentration (Cmax), System Cleaning speed (Cl), the final half life (t1/2) of steady-state distribution volume (Vss) and antibody.Statistical analysis comprises mathematical mean, and standard deviation and geometrical mean are utilized Microsoft Excel version 5.0 softwares (Microsoft Corp., Redmond, WA, USA) calculating.

3. The immunophenotyping of macaqueUtilize double-colored whole blood dyeing scheme to carry out the lymphocyte immunity somatotype with facs analysis then.In brief; The whole blood of 100 μ l EDTA-being handled in room temperature is cloned 2H7 (BD-Pharmingen San Diego CA with one of the following combination of the mAb of mark room temperature insulation 20min:CD20-FITC; USA) and CD2-PE clone RPA-2.10 (BD-Pharmingen), CD3-FITC clone SP-34 (BD-Pharmingen) clones OKT4 (Ortho Diagnostic Systems Raritan, NJ with CD4-PE; USA); Or CD3-FITC and CD8-PE clone DK-25 (Dako Corporation Carpinteria, CA, USA).Red corpuscle is with 2ml 1XFACS cracked solution (Becton-Dickinson Franklin Lakes, NJ, USA) cracking.Lymphocyte is fixed with 1% Paraformaldehyde 96, and analyzes with the FACScan (Becton-Dickinson) that is equipped with Cellquest software.The total lymphocyte number is confirmed through the number sum of all CD2 and CD20 positive cell.The B cell is accredited as the CD20 positive cell.The inferior group of T cell is accredited as CD3 and CD4 or the CD3 and the two positives of CD8.Total lymphocyte, B cell, CD4 ⁺T cell and CD8 ⁺The T cell is expressed as lymphocyte and analyzes the positive cell percentage in the door.Calculate the CD4/CD8 ratio of every group of data.

4. The ELISA. of hu5c8mAb pharmacokineticsElisa plate (Nalge-NuncRochester, NY, USA) with 5 μ g/ml recombinate solvable human CD 154 (Biogen, see also Karpusas1995) in PBS, 4 ℃ encapsulate and spend the night and block with 2% donkey serum.(Jackson?ImmunoResearchLaboratories?West?Grove，PA，USA-Catalog#017-000-121)。The typical curve of the hu5c8 of serum serial dilution and 8-500ng/ml makes in 1 hour process of room temperature insulation.Bonded hu5c8mAb utilizes donkey Anti-Human IgG horseradish peroxidase (HRP), and (JacksonImmunoResearch Laboratories West Grove, PA USA) detect; Use 3 then; 3 ', 5,5 '-TMB (" TMB ") substrate reagent box (Pierce Biotechnology Rockland; IL USA) shows.(Molecular Devices Sunnyvale, CA USA) reads plate to utilize Spectromax to read the plate appearance at 450nm.With Softmax Pro software (Molecular Devices) linear portion of four parametric line matches of standard substance is arrived in the contrary match (back-fit) of the serum of dilution.

5. Measure the ELISA of anti--hu5c8 antibody responseElisa plate (Corning-Costar) with 1 μ g/ml sugar based hu5c8mAb (above-mentioned) in bicarbonate buffer pH9.6,4 ℃ of incubated overnight and seal with 1%BSA.The serial dilution of serum of macaque is incubated 1.5h in room temperature.Bonded is anti--and hu5c8mAb utilizes the biotinylated hu5c8mAb of 100ng/ml to use streptavidin-HRP (Pierce Biotechnology) to detect then, uses tmb substrate test kit (PierceBiotechnology) to show then.Utilize Spectromax to read plate appearance (Molecular Devices) at 450nm and read plate.Antibody titer is defined as the inverse of the high dilution of value (prebleed value)＞0.100O.D. unit of produce surpassing before the bloodletting.

6. The ELISA of anti--TT reactionElisa plate (Corning-Costar) with 5ug/ml TT (Massachusetts Public Health Biologic Laboratories Boston, MA, USA) among the bicarbonate buffer pH9.6,4 ℃ of incubated overnight.The serum of macaque of serial dilution adds the plate of sealing, places 2 hours in room temperature.Bonded is anti--TT antibody with rabbit anti--(Cappel-OrganonTeknika Durham, NC USA) detect monkey IgG-HRP, use tmb substrate test kit (PierceBiotechnology) to show then.(Molecular DevicesSunnyvale, CA USA) read plate to utilize Spectromax to read the plate appearance at 450nm.(Molecular Devices) produces four parametric line matches for the serum sample of every kind of serial dilution with Softmax Pro software.Antibody titer is defined as and produces the dilution inverse that surpasses the preceding value 0.100O.D. unit of bloodletting.

7. HematologyCollect the blood sample of EDTA potassium anti-freezing and analyzed following hematologic parameter in every month: the total leukocyte counting; Red blood cell count(RBC), HC, hematocrit; MCV (mean corpuscular volume); Mean cell hemoglobin (mean corpuscularhemoglobin), mean cell hemoglobin concentration, platelet count and blood smear assessment (comprising difference).

8. Lymph node biopsyInguinal lymph nodes is gathered from all animals in the 15th day of the initial TT repercussion study of sugar based hu5c8mAb.The cleaning lymph node tissue is embedded in the paraffin, and section and sealing are on glass slide.Slide glass dyes with h and E.Said slide glass is through the existence of inspectional analysis germinal center, and based on the frequency and the big or small qualitative assessment of germinal center.

Embodiment 3: sugar based muMR1 antibody suppresses systemic lupus erythematosus

Systemic lupus erythematous (" SLE ") is the autoimmune disease of spontaneous appearance, is mainly seen in the women, is characterised in that the generation of the rational anti-nuclear autoantibody of several diseases.In lupus nephritis, injury of the kidney comprises being deposited on the also formation of the immunocomplex of complement activation cascade reaction in the renal glomerulus that it causes glomerulonephritis mainly by cell and the common mediation of humoral immunization mechanism.Confirmed in the past that the antinuclear antibody that in people and mouse SLE, produces all was to be driven by the reaction of the homology between the colony of selected autoimmunization Th cell and B cell (cognate interaction).[Kalled?et?al.，1998]。

Former research confirms, for making a definite diagnosis (SWR x NZB) F1 (SNF1) mouse that suffers from lupus nephritis, with the hamster MR1 (haMR1) of anti-cd 154 mAb long-term treatment, can prolong lifetime and reduce the sickness rate of serious ephritis at 5.5 monthly age begin treatments.

The validity of two kinds of chimeric MR1mAbs of mouse in this model has been described in this embodiment.The chimeric MR1 of mouse (" muMR1 ") is made up of the original hamster heavy chain and the variable region of light chain that are blended in mouse IgG2a heavy chain and kappa constant region of light chain.The muMR1 of sugar based form is through the C of sudden change IgG2a Fc _H2The glycosylation site that N connects in the structural domain produces.Utilize this two kinds of antibody, the effect of assessment Fc glycosylation antagonism-CD154mAbs in lupus nephritis.

The result of this embodiment confirms, chimeric mAbs, and muMR1 and sugar based muMR1 have kept the ability that suppresses the autoantibody reaction.Particularly, be similar to wild-type, parent hamster MR1 compares with the mouse of handling with mouse IgG2a contrast mAb, and the antibody of two kinds of mouse sourceizations all keeps minimizing the mouse middle kidney inflammation of handling with them, fibrosis, sclerosis and vasculitic ability.

The pharmacokinetic analysis of the pharmacokinetics muMR1 of muMR1 and sugar based muMR1 and sugar based muMR1 antibody discloses, and two kinds of antibody all are presented at the identical power collection of illustrative plates (Fig. 6) in the BALB/c mouse serum.Specifically, the serum half life of these chimeric molecules in normal mouse be estimated as～and 9 days, similar hamster MR1 (data not shown).

The analysis of autoantibody reaction

Compare with control animal, greatly reduce autoantibody reaction (Fig. 7 A&B) dsDNA and ssDNA with the processing of muMR1 or sugar based muMR1.

Histologic analysis

Confirmation is analysed in the nephridial tissue credit, has 3 to suffer from serious latter stage nephropathy in five isotype control animals, is characterized as the various features described in the marking scheme.In the treatment group animal except only a few disease seriousness obviously lower.Difference between the animal that wild-type (n=11) and sugar based (n=12) muMR1 handle is very little, but the scoring of the synthetic disease of wild-type is low slightly.Fig. 8 has shown integrative organization's scoring of this group kidney.In all animals of handling with sugar based muMR1, great majority suffer from slight early stage renal glomerulus and change, and matter changes seldom or not obvious between tubule.Useful muMR1 (n=11) animal of handling slight early stage the change arranged, do not have that matter changes between obvious tubule.Obviously visible from these results, it is the Histological change of characteristic that muMR1 and sugar based muMR1 can effectively prevent with the lupus nephritis.

Lymph appearance enlarged degree carries out the histologic analysis assessment through each spleen to every treated animal in B and the T cellular regions.Identify that secondary follicle is difficult, prepare relevant artificial behavior outcome with freezing microtome section because exist.There is not obvious dependency between spleen lymph appearance district's enlarged degree and the kidney disease score.Yet, compare with the animal that muMR1 handles, in the animal that isotype contrast and sugar based muMR1-handle, the degree of lymphocyte sheath (PALS) expansion around the arteriole seems much obvious (data not shown).

Renal function is analyzed

Be the assessment renal function, measure proteinuria (PU) level of every animal, and serum creatinine and blood urea nitrogen (BUN).Chimeric muMR1mAbs postpones the outbreak of serious ephritis in the SNF1 animal, that kind shown in protein contnt in urinating through mensuration.When comparing with control animal, the PU value lower (Fig. 9) of the time point of the mouse that anti-cd 154 is handled between 6-9 month.Yet, at～10 monthly ages, the PU value of all groups indication renal failure (Fig. 9).These results suggest anti-cd 154 treatments postpone albuminuretic beginning.

Figure 10 shows the creatinine level in the SNF1 mice serum, and Figure 11 shows the BUN level in the SNF1 mice serum.Control animals is at the 7.25-10.5 monthly age, and its serum creatinine and BUN level raise.Otherwise, in whole research, keeping stable with the mouse of glycosylation muMR1 treatment, its serum creatinine or BUN do not have the rising or the reduction of essence.The animal of sugar based muMR1 treatment keeps the normal serum creatinine level to～9 monthly ages, observes slight increase at that time.Similarly, the BUN level in this group raise at 11 monthly ages.

Viability assessment

Anti-cd 154 mAb treatment prolongs the SNF1 survival time of mice.At 11 monthly ages, surpass the mouse survival of 90% treatment, and contrast only there is 56% survival.By 14th month, all control mice were all dead, but had 86% and 75% muMR1 and sugar based muMR1 mouse still to survive respectively.(data not shown)

All in all, these tests confirm to prolong with the survival time of muMR1 or sugar based muMR1 long-term treatment ephritis SNF1 mouse, and autoantibody produces and reduces, and the ephrosis development delays.The slightly different result that glycosylation and sugar based muMR1 obtain representes that the Fc glycosylation is less to the influence of the validity of anti-cd 154 mAb in the lupus.Therefore, sugar based anti-cd 154 mAb representative is to effective treatment of lupus nephritis.

Method-embodiment 3

1. MouseBALB/c, SWR and NZB mouse available from The Jackson Laboratory (BarHarbor, ME).(SWR x NZB) F1 (SNF1) heterozygote is fed under conventional stable breeding condition in the animal rearing device of Biogen.Female SNF1 mouse is used for all lupus researchs.BALB/c mouse is used for pharmacokinetics (" PK ") research.

2. AntibodyMR1 hybridoma (ATCC#CRL-2580) (it produces the Armenian hamster anti--mouse CD154mAb) available from American type culture collection (Rockville, MD).

3. RegimenAll injections are through giving through the intraperitoneal approach.Lupus research comprises the SNF1 group of the SNF1 mouse control group of accepting PI.17muIgG2a and the treatment of accepting one of inosculating antibody-CD154mAbs.Give single dose 500 μ g mAb in preceding 6 weeks once in a week, one time every month single injection 500 μ g stop up to animal dead or research then.Research when～5.5 monthly ages, was once collected serum sample animal in every month.For PK research, BALB/c mouse is accepted single dose 100 μ g muMR1 or sugar based muMR1, after 4 hours and the 1st, 2,4,7, and 9,11 and 14 days collection blood samples.

4. The ELISA assay methodFor detecting the chimeric MR1mAbs of serum, NUNC Maxisorp plate is recombinated solvable mouse CD154 4 ℃ of incubated overnight with 5 μ g/ml.Sealed said plate in second day, add the serum of dilution, add the anti--mouse IgG2a (Southern Biotech) of horseradish peroxidase then, and show with tmb substrate.Reaction is with 2N sulfuric acid termination, and (Molecular Devices reads plate on CA) to read the plate appearance at 450nm at Spectramax.Anti--single stranded DNA (ssDNA) and anti--double-stranded (" dsDNA ") ELISA utilize NUNC MaxiSorp plate to carry out.Said plate methylates with 10 μ g/ml at 4 ℃, and (Calbiochem Corp, La Jolla CA) encapsulate to spend the night and use 5 μ g/ml I level calf thymus DNAs then (SIGMA, St.Louis MO) encapsulated 2 hours at 25 ℃ BSA.Calf thymus DNA is used the SI nuclease digestion then before use with ultrasonic shearing.For anti--ssDNA assay method, DNA boiled 10 minutes, and is freezing subsequent use on ice then.After the sealing, with the serial dilution thing adding of serum sample and room temperature insulation 2 hours.(SIGMA, ST.LOUIS MO) detects autoantibody and (SIGMA, ST.LOUIS MO) show in the 1M diethanolamine buffer with p-nitrophenyl phosphate with goat anti-mouse IgG-SEAP.Read plate at 405nm, and obtain typical curve through Anti-DNA mAb205 or the mAb 5c6 that utilizes known quantity, said antibody is all special to ss-and dsDNA.Anti-DNA is tired and is defined as the dilution inverse above background 0.1OD unit.

5. HistologyThe paraffin-embedded tissue of kidney and spleen freezing microtome section and formalin fixed is with hematoxylin-eosin (" H&E ") dyeing inflammatory infiltration.Kidney is also used the gloomy three-color process of horse (Masson Trichrome) coloured fibreization and with acid (Periodic acid) Schiff of cycle (Periodic Acid Schiff stain) (" PAS ") basement membrane thickened that dyes that dyes.Painted tissue slice is through the veterinary pathologist scoring.The histopathology classification total points of lupus nephritis is based on renal glomerulus, the change of a matter and tubule.Rank 0 to 4 ⁺Based on being examined the affected per-cent of structure (being renal glomerulus, blood vessel etc.), specific as follows: 0, not obviously damage; 1 ⁺, the 1%-30% structural damage; 2 ⁺, the 30%-60% structural damage; 3 ⁺,＞60% structural damage arrives to a certain degree; 4 ⁺,＞60% structure is badly damaged.

6. The analysis of urine and serum(Bayer Corp., Tarrytown NY) monitor weekly to measure proteinuria (" PU ") with Albustix in the analysis of the urine of every mouse.The proteinuria level is marked as follows: 0.5 ⁺, 15-30mg/dl; 1 ⁺, 30mg/dl; 2 ⁺, 100mg/dl; 3 ⁺, 300mg/dl; 4 ⁺,＞2000mg/dl.In the whole research process, upward intermittently measure serum creatinine and blood urea nitrogen (BUN) to measure renal function and PU at COBAS chemical analyzer (Roche).

Embodiment 4: sugar based muMR1 antibody inhibition test systemic autoimmune property encephalomyelitis (EAE)

Sealing CD40 part can suppress EAE development [Samoilova, 1997] during immunity.In this embodiment, analyze the ability that muMR1 and sugar based muMR1 antibody suppress EAE.This embodiment has also assessed through anti-cd 154 mAb and has suppressed EAE whether with initiatively to suppress mechanism relevant, with and the degree that mediates by the interactional mechanism of the Fc dependency that depends on antibody.

The result of this embodiment has confirmed that sugar based muMR1mAb and wild-type glycosylation hamster MR1mAb are effectively same in the development of blocking-up clinical disease as the EAE inhibition.More specifically, all MR1mAbs suppress disease progression fully, as through average maximum score and average disease severity assessment, have only a mouse exception in the hamster MR1-treatment group.As if the potential mechanism of the provide protection of these results suggest anti-cd 154s mAbs antagonism EAE induce irrelevant with the T adjusting.As if they show that also the Fc dependency effector functions of mAb does not have bigger influence to clinical validity, but the potential mechanism of the inhibition in the EAE autoimmunization environment is had contribution.

Suppress clinical EAE with glycosylation muMR1

Figure 12 shows, compares with the mouse of isotype contrast P1.17 treatment, and the mouse of muMR1 treatment does not show disease symptoms after initial peptide immunity.The animal of muMR1 treatment 80 days with examining the interim clinical symptom (data not shown) that do not show.When mouse is used in complete Freund's adjuvant emulsive PLP139-151 when immunity again, the seriousness of clinical symptom increases in the animal of P1.17-treatment.After attacking once more, occurring EAE the mouse of treating with muMR1 in the 0th, 2 and 4 day, its seriousness is equal to the fs of the EAE in the mouse that P1.17-treats.These results confirm that anti-cd 154 mAb treatment does not cause initiatively suppressing.We have also studied 20 * 10 ⁶Whether the transfer of individual splenocyte can cause initial acceptor mouse (naive recipient mice) to subsequently active EAE inductive resistance, and situation is not (data not shown) like this.Said splenocyte collection is hung oneself, and EAE induces and treat the back mouse in 1 or 3 weeks with muMR1.

Suppress clinical EAE with sugar based muMR1

Figure 13 and table 3 confirm, during 3 dosage of administration, 200 μ g, compare with contrast Ig P1.17, and it is whole with the EAE clinical sign during examining that muMR1 and sugar based muMR1mAbs effectively suppress.In this respect, muMR1 and sugar based muMR1 antibody and hamster MR1 same effectively (data not shown).These antibody of administration low dosage do not demonstrate between the antibody and have bigger difference in the ability to that suppresses EAE.

Suppress the CNS inflammatory infiltration

Aspect the inflammatory infiltration of assessment antibody in suppressing cns (" CNS "), whether there are differences; 4-5 mouse is divided into one group; Different groups is with antibody (muMR1 and sugar based muMR1, or 200 μ g of 3 dosage and the P1.17 contrast Ig) treatment of different amounts, at the 16th day; When peaking, put to death these mouse with the disease activity in the mouse of isotype control antibodies treatment.

Table 3: glycosylation is not that the retarding effect of anti-cd 154 is required

Antibody	Dosage (μ g)	N	Sickness rate	Average maximum score ± SD 1	Average accumulated score ± SD 1
						P1.17	3×200	14	13	2.4±0.8	32.1±32.5
muMR1	3×200	13	0	0±0 §	0±0 #
						muMR1	3×75	6	0	0±0 §	0.8±1.2 ##
muMR1	3×25	6	3	1.4±1.6 §§	39.3±53.1
						Sugar based MR1	3×200	14	0	0±0 §	0±0 #
Sugar based MR1	3×75	6	1	0.6±1.4 §	19.7±47.9
						Sugar based MR1	3×25	6	2	0.8±1.3 §	9±17.4 ＊

1. PD is presented among Fig. 1.For every independent mouse, maximum disabled score and cumulative score assessment respectively before calculating cell mean in the whole monitoring phase process.

§ compares p＜0.0005 with the mouse of P1.17 treatment; § § compares p＜0.05 with the mouse of P1.17 treatment.

# compares p=0.002 with the mouse of P1.17 treatment; ## compares p＜0.02 with the mouse of P1.17 treatment.

^*Compare p=0.05 with 25 μ g muMR1.

As shown in table 4, said antibody is in suppressing the PD early process, and the ability to that suppresses inflammatory infiltration progress among the CNS does not have difference.Because whether uncertain mouse subclinical activity occurs under the situation that lacks EAE sign, analyze CNS tissue from every group of 6-14 mouse in this test endpoint (the 58th day).

With opposite with the mouse of 200 μ g muMR1 treatment, the mouse of P1.17-treatment and the mouse of treating with 200 μ g sugar based MR1 show that slight inflammatory infiltration mainly is positioned at the evidence (table 5) of cerebellum.Yet, utilize the treatment of low dosage antibody to disclose sugar based MR1 similar with its glycosylation form (stronger than the provide protection of its glycosylation form sometimes).These results confirm that two kinds of antibody all can suppress the progress of clinical EAE on an equal basis.

Method-embodiment 4

1. EAE inducesFemale SJL mouse (age in 10-12 week, Harlan) be used in complete Freund's adjuvant (Difco, Detroit, MI) in emulsive 50 μ g PLP139-151 through subcutaneous immunity.After three days, to injecting bordetella pertussis (B.pertussis) organism (RIVM, Bilthoven, The Netherlands) of 109 heat killed in the mouse vein.The progress of EAE is through assessing body weight and the disabled monitoring of must assigning to every day.The score scope is from 0: asymptomatic, and 0.5: the forfeiture of tail nervous (tonus) part, 1: the tail anxiety completely loses, and 2: four limbs are weak; 2.5: local paralysis, 3: hind leg complete paralysis, 3.5: diaphragm and hind leg complete paralysis; Gatism, 4: dying, to 5: because EAE causes death.

Table 4: the influence (the 16th day) that anti-cd 154 soaks into CNS

Table 5: the influence (the 58th day) that anti-cd 154 soaks into CNS

2. Antibody generatesHamster is anti--and the heavy chain of mouse CD154mAb MR1 clones from the total RNA of hybridoma through RT-PCR with the variable region of light chain.The expression vector of the chimeric mAb of hamster/mouse is through utilizing the standard recombinant dna technology; Mouse IgG2a or mouse kappa constant region cDNAs (full length cDNA clone of the heavy chain of glycosylation hu5c8 and light chain from Anti-Human CD154mAb-promptly) are transformed respectively on the variable region of heavy chain and light chain and make up.Confirm that through flow cytometry and immunoprecipitation the CD154 that the chimeric MR1 mAb (being called muMR1) of transient expression has (recapitulate) hamster mAb combines character.

The chimeric MR1 of sugar based (being called aglyMR1) is built into through heavy chain being carried out the asparagine residue (in Kabat EU nomenclature, being N297) that site-directed mutagenesis changes in the glycosylation site that the N-of Fc connects and is glutamine residue.Structure contains the CMV-IE promoters driven type series connection that is useful on light chain immunoglobulin and heavy chain and transcribes box and glutamine synthetase gene as the stably express carrier of selective marker, is used for muMR1 and agly muMR1 IgG2a, kappa mAbs.Expression vector is transfected into the NS0 cell, through in the substratum that does not contain Stimulina, selecting the clone of separating stable.

MuMR1 and sugar based MR1 carry out size exclusion chromatography to remove aggregate at Sephacryl 300 through the avidity purifying then from bio-reactor cell conditioned medium liquid on the albumin A agarose.Chromatographic resin available from Amersham Pharmacia Biotech (Piscataway, NJ).Show that through SDS-PAGE mAbs purity＞95%, endotoxin analysis guarantee that these reagent are for the security of using in the body.In vitro tests finds that MuMR1 is identical with the relative affinity of sugar based MR1 pair cell surface muCD154, and they are in the intravital pharmacokinetics of BALB/c mouse half life identical (data not shown).According to the agreement of Biogen, mouse IgG2a isotype contrast mAb, P1.17 (ATCC#TIB-10) is that (Burlingame, CA) purifying is from the albumin A of ascites by Protos Immunoresearch.

3. Sending of anti-cd 154 antibodiesAt the 0th, 2 and 4 day, every mouse was accepted 200 μ l PBSi.p., wherein also contains following material: group 1=PBS (contrast); Group 2=200 μ g muMR1; Group 3=200 μ g hamster Ig (Ig contrast); The MR1 of group 4=200 μ g mouse sourceization; Group 5=200 μ g P1.17IgG2a contrast; Group 6=200 μ g sugar based muMR1.

In some tests, mouse was used in emulsive 50 μ gPLP139-151 immunity again in the complete Freund's adjuvant at the 80th day.

4. Disease assessmentThe mouse of weighing every day is in whole 56 days process, according to following points-scoring system monitoring clinical event (clinical activity): 0: asymptomatic; 0.5: the nervous part forfeiture of tail, 1: the tail anxiety completely loses, and 2: four limbs are weak; 2.5: local paralysis, 3: hind leg complete paralysis, 3.5: diaphragm and hind leg complete paralysis; Gatism, 4: dying, 5: because EAE causes death.

5. HistologyCerebral tissue of every mouse and spinal cord be fixing and embedding in paraffin in 10% Superlysoform.The 3-6 that obtains interval 100 μ m from every independent mouse opens spinal cord slice (4 μ m) and 6 brain sections (every comprises cerebellum, brain, brain stem, and subarachnoid space), and after with brazilwood extract dyeing, analyzes the degree of inflammatory infiltration.

Every independent section is according to following scoring: 0=does not have infiltration; The slight perivascular infiltration (every section is less than two place's inflammatory damages) that 1=is accidental; The many kitchen ranges of 2=, slight perivascular infiltration; The many kitchen ranges of 4=, serious perivascular infiltration is with the diffusion in essence.Based on the average case of all sections, mouse is categorized as nothing, accidental, moderate or serious the infiltration.

6. Statistical analysisThe result analyzes through One-Way ANOVA, utilizes the LSD post-hoc that upchecks to analyze then.P-value＜0.05 is considered to significantly.

Embodiment 5: sugar based hu5c8 antibody suppresses islet cell transplantation

Research in the past shows; With 20mg/kg induce/keep-rhesus monkey of the glycosylation hu5c8 of dosage treatment keeps kidney allograft thing function; And outbreak does not appear repelling during 6 months administration; And the untreated monkey of accepting the kidney allograft thing repelled their graft [Kirk, 1999] rapidly within 8 days.In the correlation test of the research group of Kirk, confirm that sugar based hu5c8 is invalid to the treatment transplant rejection.

Obviously opposite with the discovery of Kirk, result of the present invention has proved that in fact the hu5c8mAb of sugar based form can effectively treat the transplant rejection in other background.

Islet cells allograft in the rhesus monkey

Proved in the past that glycosylation hu5c8 made pancreas islet implant and keeps the survival of allograft to become possible [Kenyon, 1999].

Four 20mg/kg induce/rhesus monkey of Concept of Maintenance treatment after transplanting＞213,＞255,＞269 and＞obtained the Regular Insulin dependent/non-dependent in 341 days.The result of this research shows that the untreated contrast monkey of accepting the pancreas islet allograft showed acute cellular rejection at the 8th day, as lasting hyperglycemia and c peptide (product that endogenous insulin generates) the generation shortage that occurred by 11-14 days confirms (data not shown).

Opposite with untreated control animal, Figure 14 shows with the inducing of sugar based hu5c8/Concept of Maintenance (mentioned above) can successfully treat one of two rhesus monkeies.Although two monkeys have all experienced hyperglycemia and have repelled with initial at the 7th day; When insulinize monkey and lasting sugar based hu5c8 treatment; One of two monkeys show the partial function of allograft; As the 28th day (open diamonds) measured through the existence of c peptide, and remained to the 45th day.

These results suggest sugar based anti-cd 154s mAbs can be used for transplanting the regimen in the background.Yet, because the immunoreation in the migration process is very strong, promptly relating to body fluid, cell and inflammatory immunoreation might the sugar based anti-cd 154 antibodies be being united when sending the most effective with immunosuppressor and immunomodulatory compounds.For example, interrupt medicament, interrupt the medicament of calcineurin signal, reflunomide via the T cell co-stimulatory signal of CD28; Antiproliferative pharmaceutical agent and other specificity are combined in the proteinic antibody of immunocyte surface expression, include but not limited to CD45, CD2; IL2R, CD4, CD8 and RANK FcR, B7; CTLA4, TNF, LT β, and VLA-4.

Method-embodiment 5

1. Islet cells allotransplantation[Kenyon, 1999] are carried out in these researchs as stated.In brief, alloreactivity D-A rhesus monkey is to based on positive mixed lymphocyte culture reaction property selection.Acceptor was accepted (intraportal) allogene pancreatic islets transplantation in complete pancreas islet excision and the door at the 0th day.

2. Antybody therapyDosage is utilized in-1,0, and the keeping of 20mg/kg of the dosage of inducing and beginning in the 28th day in every month of 3,10 and 18 days 20mg/kg carried out.

3. The graft function assessmentPancreatic islets transplantation thing function is monitored through fasting and level of postprandial blood sugar every day.Islet failure (primary does not have function or acute cellular rejection) is defined as the c peptide that lacks fasting and stimulation and generates (endogenous insulin product).Primary does not have in the period that tissue that function is defined as transplanting can not be right after after transplanting and plays a role, and is characterized as and continues hyperglycemia and glycemic control is unstable.The acute cellular rejection outbreak is defined as fasting serum glucose＞100mg/dL, postprandial blood sugar＞150-175mg/dL.Overall graft function is kept the required exogenous insulin amount of euglycemia level through monitoring and is assessed.

Embodiment 6: sugar based hu5c8 (anti-cd 154) antibody prevents the purposes of the T-cell alloreactivity of anti-cd 154-mediation

The monoclonal antibody of CD154 on the target T cell display part prevents external and the interior alloreactivity of body.Yet whether the inhibition activity of not knowing anti-cd 154 mAb depends on that blocking-up pungency CD40-CD154 interacts or optional depending on directly sent the inhibition signal via CD154.

Block the effect of pungency CD40-CD154 interaction partners human T-cell's external alloreactivity for assessment; Said effect with stimulate CD154 (that is, directly send and suppress signal) opposite through CD154, with the not PBMC of fractional separation or the CD4+T cell of purifying; Exist solvable or be fixed in the humanized anti-cd 154 mAb (hu5c8 of culture hole with the irritation cell (CD40 positive cell) of allogene HLA mispairing; Biogen Inc., MA cultivates under condition USA) altogether.Most of blocking tests utilize solubility anti-cd 154 (sugar based hu5c8) variant of genetic modification to carry out, and the Fc effector functions of said variant reduces and itself and the crosslinked ability reduction of CD154 thus.The sugar based hu5c8 that is used for this embodiment is identical with the sugar based hu5c8mAb of full text description among the application.See; For example embodiment 1;

and sees above.Solvable but the non-anti-cd 154 antibodies that encapsulates suppresses former generation blended lymphocyte culture (" MLC "), and (40 ± 23%vs 3 ± 18% suppresses, n=4) middle allogene T cell proliferation.Similarly, the propagation of allogenic antigen T lymphocyte specific excites (utilizing solvable antibody) to be suppressed among the secondary MLC (n=5 test) through blocking with CD154, yet it can stimulate through CD154 (antibody that encapsulates) and increase.In addition, the stimulation of the anti-cd 154 antibodies that encapsulates increases the generation of allogenic antigen specific CTL effector strongly, and CTL does not receive the influence of CD154 blocking-up.

For whether the stimulating activity that detects anti-cd 154 needs B7-CD28 to stimulate interaction altogether, under the condition of this B7 of inhibition molecule of CTLA4-Ig and the existence of CD28 bonded molecule, carry out MLC.The propagation 75 ± 14% (n=3) and 64 ± 28% (n=6) that exist exciting under the CTLA4-Ig to suppress allogenic antigen T lymphocyte specific among the primary and secondary MLC respectively, and suppress CTL generation 48 ± 23% (n=2).The signal of the anti-cd 154 antibodies that encapsulates obviously increases elementary (58 ± 0.6%; N=2) and secondary (61 ± 49%; N=6) the remaining CD28-dependent/non-dependent propagation of allogenic antigen specific T-cells among the MLC, but and not exclusively eliminate the CTLA4-Ig-inductive and suppress.Yet CTLA4-Ig is stimulated (through the anti-cd 154 antibodies that encapsulates) to eliminate the retarding effect that CTL generates by CD154.Compare with the control cultures that contains or do not contain CTLA4-Ig, CD25, HLA-DR and the CD95 expression on alloreactivity T cell obviously increases through stimulating CD154 (n=2).

When our data presentation was fixed on the culture plate, anti-cd 154 antibodies can promote, rather than the blocking t cell alloreactivity.CD154 can not strengthen external T cell activation and favourable in vivo thus through solubility sugar based hu5c8mAb blocking-up; And based on these discoveries; No matter use separately or stimulate the molecule coupling of approach altogether with other blocking-up, it can effectively reduce allogenic antigen t cell responses in the body in the transplant experiment model.

It will be understood by those skilled in the art that and to carry out multiple change and modification to the preferred embodiment of the invention and do not depart from spirit of the present invention.Intention comprises all this changes within the scope of the present invention.

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Claims (69)

1. the sugar based anti-cd 154 antibodies is characterized in that being positioned at said antibody Fc partial C _H2The modification in the site that conservative N-connects in the structural domain.

2. the sugar based anti-cd 154 antibodies of claim 1, wherein said modification comprises the sudden change in the heavy chain glycosylation site, wherein said sudden change prevents the glycosylation in this site.

3. the sugar based anti-cd 154 antibodies of claim 2, wherein said modification comprise sudden change N298Q, and wherein the position of this sudden change N298Q is the N297 position that utilizes EU Kabat to number.

4. the sugar based anti-cd 154 antibodies of claim 1, wherein said modification comprise removes C _H2The structural domain glycan.

5. the sugar based anti-cd 154 antibodies of claim 1, wherein said modification comprise and preventing at C _H2The glycosylation of structural domain.

6. the sugar based anti-cd 154 antibodies of one of claim 1-5, wherein said sugar based anti-cd 154 antibodies not with the effect receptors bind.

7. the sugar based anti-cd 154 antibodies of one of claim 1-5, wherein said sugar based anti-cd 154 antibodies does not cause thrombosis.

8. the sugar based anti-cd 154 antibodies of one of claim 1-5, wherein said antibody is selected from: monoclonal antibody, polyclonal antibody, murine antibody, chimeric antibody, the antibody of the long sourceization of inhuman spirit, the group of humanized antibody and fully human antibodies composition.

9. the sugar based anti-cd 154 antibodies of one of claim 1-5, wherein said antibody is selected from: polymer antibody, the group that tetravalent antibody and bi-specific antibody are formed.

10. the sugar based anti-cd 154 antibodies of claim 9, wherein said polymer antibody is heterodimer antibody or half homodimeric antibody.

11. the sugar based anti-cd 154 antibodies of one of claim 1-5, wherein said antibody are the sugar based hu5c8 through the clone generation of ATCC preserving number PTA-4931.

12. the sugar based anti-cd 154 antibodies of one of claim 1-5, wherein said antibody is used the detectable label substance markers.

13. the sugar based anti-cd 154 antibodies of claim 12, wherein said detectable is a radioactive isotope, enzyme, dyestuff or vitamin H.

14. the sugar based anti-cd 154 antibodies of one of claim 1-5, wherein said antibody coupling is in therapeutical agent.

15. the sugar based anti-cd 154 antibodies of claim 14, wherein said therapeutical agent is a radionuclide, toxin, toxoid or chemotherapeutics.

16. the sugar based anti-cd 154 antibodies of claim 14, wherein said therapeutical agent is a ri.

17. the sugar based anti-cd 154 antibodies of one of claim 1-5, wherein said antibody coupling is in developer.

18. the sugar based anti-cd 154 antibodies of claim 17, wherein said developer is a mark part.

19. the sugar based anti-cd 154 antibodies of claim 17, wherein said developer is a vitamin H, the fluorescence part, and the radioactivity part, or peptide-labeled.

20. the sugar based anti-cd 154 antibodies of claim 18, wherein said peptide-labeled be histidine mark.

21. comprise the pharmaceutical composition of the sugar based anti-cd 154 antibodies of one of claim 1-5.

22. the said pharmaceutical composition of claim 21 also comprises pharmaceutically acceptable carrier.

23. the said pharmaceutical composition of claim 21 also comprises inhibitive ability of immunity and immune regulative compound.

24. produce the clone of the sugar based anti-cd 154 antibodies of one of claim 1-5.

25. the clone of claim 24, wherein said clone produces sugar based hu5c8, and the ATCC preserving number is PTA-4931.

26. said sugar based anti-cd 154 antibodies of one of claim 1-5 of effective inhibitory amount or the pharmaceutical composition that comprises said antibody suppress the purposes in the immunoreactive medicine of experimenter in preparation.

27. the purposes of claim 26, wherein said sugar based anti-cd 154 antibodies or pharmaceutical composition inhibition CD154 combine with CD40's.

28. the purposes of claim 26, wherein said sugar based anti-cd 154 antibodies or pharmaceutical composition can specificity combine can be by the protein of sugar based hu5c8 specific recognition, wherein sugar based hu5c8 produces through the clone of ATCC preserving number PTA-4931.

29. it is the sugar based hu5c8 specificity bonded epi-position of the clone generation of PTA-4931 that the purposes of claim 26, wherein said sugar based anti-cd 154 antibodies or pharmaceutical composition specificity combine with the ATCC preserving number.

30. said sugar based anti-cd 154 antibodies of one of claim 1-5 of significant quantity or the pharmaceutical composition that comprises said antibody suppress the purposes in the medicine of experimenter's Inflammatory response in preparation.

31. the purposes of claim 30, wherein said Inflammatory response is selected from: sacroiliitis, contact dermatitis, height-IgE syndrome, inflammatory bowel, the group that allergic asthma and Te Fa property inflammatory diseases are formed.

32. the purposes of claim 31, wherein said sacroiliitis is selected from: rheumatoid arthritis, non-rheumatoid inflammatory arthritis, the group that Lyme disease dependency sacroiliitis and inflammatory osteoarthritis are formed.

33. the purposes of claim 31, wherein said spy's property sent out inflammatory diseases are selected from the group of psoriatic and systemic lupus erythematous composition.

34. the said sugar based anti-cd 154 antibodies of one of claim 1-5 of significant quantity perhaps contains the purposes of pharmaceutical composition in the medicine of preparation inhibition experimenter transplant rejection of said antibody.

35. the purposes of claim 34, wherein said transplant rejection relates to the heart of transplanting, kidney, liver, skin, islet cells or marrow.

36. one of the claim 1-5 of inhibition significant quantity said sugar based anti-cd 154 antibodies perhaps contains the purposes of pharmaceutical composition in the medicine of preparation inhibition experimenter graft versus host disease of said antibody.

37. one of the claim 1-5 of inhibition significant quantity said sugar based anti-cd 154 antibodies perhaps contains the pharmaceutical composition of said antibody and suppresses the purposes in the anaphylactoid medicine of experimenter in preparation.

38. the group that the purposes of claim 37, wherein said anaphylaxis are selected from spring fever or the allergy of penicillium mould or other medicines is formed.

39. one of the claim 1-5 of inhibition significant quantity said sugar based anti-cd 154 antibodies perhaps contains the purposes of pharmaceutical composition in the medicine of preparation inhibition experimenter autoimmune response of said antibody.

40. the purposes of claim 39, wherein said autoimmune response is derived from infection.

41. the purposes of claim 39, wherein said autoimmune response is derived from Reiter syndrome, and joint of vertebral column is scorching, Lyme disease, and HIV infects, syphilis or tuberculosis.

42. the purposes of claim 39, wherein said autoimmune response is selected from rheumatoid arthritis, myasthenia gravis; Systemic lupus erythematous, Graves is sick, idiopathic thrombocytopenic purpura; Hemolytic anemia, mellitus, inflammatory bowel; Clone disease, multiple sclerosis, the group that psoriatic and drug-induced autoimmune disease are formed.

43. the purposes of claim 42, wherein said drug-induced autoimmune disease is drug-induced lupus.

44. one of the claim 1-5 of inhibition significant quantity said sugar based anti-cd 154 antibodies perhaps contains the pharmaceutical composition of said antibody and suppresses the purposes in the Fibrotic medicine of experimenter in preparation.

45. the purposes of claim 44, wherein said fibrosis are selected from the group of pulmonary fibrosis and fibrotic conditions composition.

46. the purposes of claim 45, wherein said pulmonary fibrosis is selected from the pulmonary fibrosis that is secondary to adult respiratory distress syndrome, drug-induced pulmonary fibrosis, the group that idiopathic pulmonary fibrosis and hypersensitivity pneumonitis are formed.

47. the purposes of claim 45, wherein said fibrotic conditions is selected from: hepatitis C; The group that hepatitis B and liver cirrhosis are formed.

48. the purposes of claim 47, wherein said liver cirrhosis are selected from the liver cirrhosis that is secondary to the toxin injury; Be secondary to the liver cirrhosis of medicine; Be secondary to the liver cirrhosis of virus infection; And the group that is secondary to the liver cirrhosis composition of autoimmune disease.

49. one of the claim 1-5 of inhibition significant quantity said sugar based anti-cd 154 antibodies perhaps contains the pharmaceutical composition of said antibody and suppresses the experimenter by the purposes in the medicine of T cell virus infection due to the HTLVI virus in preparation.

50. one of the claim 1-5 of inhibition significant quantity said sugar based anti-cd 154 antibodies perhaps contains the purposes of pharmaceutical composition in the medicine of preparation inhibition experimenter gastrointestinal illness of said antibody.

51. the purposes of claim 50, wherein said gastrointestinal illness is selected from: dyskinesia of esophagus, the group that inflammatory bowel and scleroderma are formed.

52. one of the claim 1-5 of inhibition significant quantity said sugar based anti-cd 154 antibodies perhaps contains the purposes of pharmaceutical composition in the medicine of preparation inhibition experimenter vascular disease of said antibody.

53. the purposes of claim 52, wherein said vascular disease are selected from: the group that atherosclerosis or reperfusion injury are formed.

54. the pharmaceutical composition that suppresses the said sugar based anti-cd 154 antibodies of one of claim 1-5 of significant quantity or contain said antibody suppresses to suffer from the purposes in the medicine of propagation of experimenter T cell tumour cell of T cell carcinoma in preparation.

55. the purposes of one of claim 26-54, wherein said sugar based anti-cd 154 antibodies is selected from: monoclonal antibody, polyclonal antibody, murine antibody, chimeric antibody, the antibody of the long sourceization of inhuman spirit, the group that humanized antibody and fully human antibodies are formed.

56. the purposes of one of claim 26-54, wherein said sugar based anti-cd 154 antibodies is selected from: polymer antibody, the group that tetravalent antibody and bi-specific antibody are formed.

57. the purposes of claim 56, wherein said polymer antibody are heterodimer antibody or half homodimeric antibody.

58. the purposes of one of claim 26-54, wherein said medicine be through in intravenously, subcutaneous, intraperitoneal, intramuscular, marrow, Intraventricular, in epidural, in the intra-arterial, blood vessel, in the intraarticular, synovial membrane, in the breastbone, in the sheath, in the liver, in the spinal cord, in the tumour, in the brain, in the intestines, in the lung, in the mucous membrane, intrauterine, hypogloeeis or in the inflammation site or the tumor growth site come the said experimenter's of administration medicine through local injection.

59. the purposes of one of claim 26-54, wherein said medicine are oral through being selected from, intranasal, and through eye, the said experimenter's of administration of the group that per rectum and topical are formed medicine.

60. the purposes of claim 59, wherein said medicine is with capsule, tablet, the said experimenter's of form administration of aqueous suspensions or solution medicine.

61. the purposes of claim 59, wherein said medicine are through using emulsifiable paste or the said experimenter's of ointment topical medicine.

62. the purposes of claim 59, wherein said medicine are through utilizing spraying gun, the sucker of Diskus or metering sucks and the said experimenter's of administration medicine.

63. the purposes of one of claim 26-54, wherein said medicine are the said experimenter's of administration through sustained release administration medicines.

64. the purposes of one of claim 26-54, wherein said medicine are the medicines with the said experimenter of therapeutical agent administration.

65. the purposes of one of claim 26-54, wherein said medicine are the medicines with immunomodulatory or the said experimenter of immunosuppressive compounds administration.

66. the purposes of claim 65, wherein said immunomodulatory or inhibitive ability of immunity compound are selected from:

(a) interruption is through the medicament of the T cell co-stimulatory signal of CD28;

(b) medicament of interruption calcineurin signal,

(c) reflunomide,

(d) antiproliferative pharmaceutical agent; With

(e) with expressed protein specificity bonded antibody on the immunocyte surface, said albumen is selected from one or more among CD45, CD2, IL2R, CD4, CD8 and RANK FcR, B7, CTLA4, TNF, LT β and the VLA-4.

67. the purposes of claim 65, wherein said inhibitive ability of immunity or immune regulative compound are selected from tacrolimus, sirolimus, mycophenlate mofetil, mizorubine, Gusperimus, brequinar sodium, leflunomide, the group that Wyeth-Ayerst Laboratories and azaspirane form.

68. the purposes in the preparation of the said pharmaceutical composition of the claim 21 of significant quantity video picture tumour cell and vegetation cell in being prepared in the experimenter; Said experimenter is the proteinic experimenter of expression by the sugar based hu5c8 institute specific recognition of the clone generation of ATCC preserving number PTA-4931, and said video picture may further comprise the steps:

(a) forming under the condition of complex body between the protein that allows antibody and tumour cell or vegetation cell surface, give said experimenter with the said pharmaceutical composition of the claim 21 of significant quantity; With

(b) make any antibody/protein complex body video picture of formation, make any tumour cell or vegetation cell imaging among the experimenter thus.

69. the said pharmaceutical composition of the claim 21 of significant quantity detects the purposes in the reagent of existence of tumour cell among the experimenter or vegetation cell in preparation; Said experimenter is the proteinic experimenter of expression by the sugar based hu5c8 institute specific recognition of the clone generation of ATCC preserving number PTA-4931, and said detection may further comprise the steps:

(a) forming under the condition of complex body between the protein that allows antibody and tumour cell or vegetation cell surface, give said experimenter with the said pharmaceutical composition of the claim 21 of significant quantity;

(b) remove any unconjugated developer from the experimenter; With

(c) any antibody/protein complex body that detect to form, the existence of said complex body show and have tumour cell or vegetation cell among the experimenter.

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