CN1835976A - Aglycosyl anti- CD154 (CD 40 ligand) antibodies and uses thereof - Google Patents

Aglycosyl anti- CD154 (CD 40 ligand) antibodies and uses thereof Download PDF

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CN1835976A
CN1835976A CNA2004800232319A CN200480023231A CN1835976A CN 1835976 A CN1835976 A CN 1835976A CN A2004800232319 A CNA2004800232319 A CN A2004800232319A CN 200480023231 A CN200480023231 A CN 200480023231A CN 1835976 A CN1835976 A CN 1835976A
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antibody
antibodies
sugar based
based anti
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CN1835976B (en
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弗雷德里克·R·泰勒
克里斯托弗·D·本杰明
琳达·C·伯克利
埃伦·A·加伯
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Than Ao Gen MA company
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Biogen Idec MA Inc
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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 immune response 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 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 is activating later on also at CD8 +Subgroup 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].
By 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] by the activation of scavenger cell and dendritic cell and the generation of natural killer cell and cytotoxic T cell.
The importance of CD154 in regulating body fluid and cell-mediated immune response has excited the interest [United States Patent (USP) 5,474,771] of people to the therapeutic immunization regulating effect of the inhibitor that utilizes this approach.Therefore, anti-cd 154 antibodies is treated albumen or gene therapy, anaphylactogen, useful [United States Patent (USP) 5,474,771 in the immune response 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] by 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 activity of treatment [Biogen Press Release, 2001; IDEC Press Release, 2001].Although the mechanism of this side effect is unknown, it may comprise that anti-cd 154 antibodies or its aggregate to the gathering (colligation) of FcgRIIa on the thrombocyte and CD154, cause unsuitable platelet activation.Can strengthen this effect with combining also of other Fc γ acceptor and complement.Therefore, the form of the anti-cd 154 antibodies of debond effector acceptor may be safer and/or more effective for therepic use.
The mechanism that anti-cd 154 antibodies suppresses immunologic function is come more complicatedly compared with combine interaction with blocking-up and CD40 simply with CD154, in fact comprises the contribution of effect approach.For example, antibody-antigen is in conjunction with the elimination that can pass through the zygotic induction activated T cell of Fc structural domain and Fc γ acceptor or complement component.Optional, antibody can strengthen by forming the antibody support at the cell surface that carries Fc γ acceptor with combining of CD154.In addition, antibody and its action site promotes near interacting by 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.Studies confirm that in the external body that utilizes multiple glycosylated antibodies and remove C 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].The effector functions that studies confirm that sugar based antibody in the body reduces.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 release of cytokines [Boyd, 1995 of philtrum; Friend, 1999].
Although remove C H2Glycan in the structural domain seems pairing effect device function 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 by utilizing anti-CD-154 antibody, and the Fc effector functions is by modifying Fc dimer C in the described antibody H2The site that conservative N-connects in the structural domain reduces, and causes " sugar based " anti-cd 154 antibodies.The example of described 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 corresponding part thereof suppress the ability of several conditions by blocking-up CD154-CD40 interaction.This paper results reported has confirmed that the glycosylation form provide protection of the sugar based form of anti-cd 154 antibodies and 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 side effect of anti-cd 154 antibodies therapy, such as eliminating the activated T cell and activating through other cell mass of abduction delivering CD154 or the Fc dependency of monocyte/macrophage.
Particularly, the invention provides the sugar based anti-cd 154 antibodies of identification CD154.More specifically, the 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 prepares from NS0 sugar based hu5c8 clone, this cell lies in and was deposited in American type culture collection (" ATCC ") on January 14th, 2003,10801 University Blvd., Manassas, Virginia (preserving number PTA-4931), sugar based MR1 antibody is from the preparation of NSO 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 in 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 provide treatment or prevention among the experimenter the immune response dependence disease or the method for illness, comprise and give described experimenter's sugar based anti-cd 154 antibodies or its antibody derivatives, described immune response dependence disease or illness are treated or prevented to described antibody or antibody derivatives thus with the activatory amount administration of immunocyte among effective inhibition experimenter.
The accompanying drawing summary
Fig. 1 show sugar based hu5c8 monoclonal antibody (" mAb ") and glycosylation hu5c8mAb with identical relative affinity in conjunction with human CD 154.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 antibody concentration.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 is pre-.Bonded Fc γ R +Cell detects by 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 immune response 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 immune response 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 initial and follow-up TT attacks.The 1B treated animal was accepted salt solution before initial TT attacks, accepted sugar based hu5c8mAb and attack before follow-up TT attacks.
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﹠amp; B) show in the SNF1 mouse that 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 that in the mouse of using sugar based muMR1 antibody treatment, sugar based muMR1 antibody is identical with muMR1 antibody for the validity that suppresses the EAE clinical manifestation.The result is expressed as with respect to disease and induces the back disabled score (mean value+mean value standard error-" SEM ") of fate and % initial weight (mean value+SEM).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 be more fully understood, provide following detailed description. Unless otherwise defined, used all technology are the same with the implication that those skilled in the art of the invention understand with scientific terminology. Below describe exemplary method and material, but those of similar methods described herein and material also can be used for the present invention's practice and be obvious 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, with the state in more abundant description field of the present invention. The bibliography clause of these reference papers is found in the text or after the experimental details part and lists with the content of band numbering. If any conflict, be as the criterion with specification. Described 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 Harbor Laboratory Press，Cold Spring Harbor，N.Y.(1999)；and Roitt et al.， Immunology，3d Ed.，Mosby-Year Book Europe Limited，London(1993). Standard reference works setting forth the general principles of medical physiology and pharmacology known to those of skill in the art include：Fauci et al.，Eds.， Harrison’s Principles Of Internal Medicine，14th EdMcGraw-Hill Compames，Inc.(1998)。
Reagent of the present invention and method relate to utilizing without glycosyl antibody or its antibody derivatives and suppress disease that immune response and treatment immune response induce and illness-for example: autoimmunity disease, allergy, graft rejection, inflammation, graft versus host disease, fibrillatable, and atherosclerotic.
More specifically, be used for the treatment of human treatment specifically without the glycosyl anti-cd 154 antibodies, comprise people's antibody, humanized antibody, chimeric antibody, polyclonal antibody and polymer antibody.
Antibody
Antibody is the glycoprotein of about MW 150kD, and its body fluid branch by vertebrate immune system responds to the inside and outside and comes the existence of molecule to produce. Function antibody or antibody derivatives can be identified and in vitro and in vivo in conjunction with its specific antigen, and start any and antibody in conjunction with relevant follow-up effect, for example comprise, direct cell toxicant, the cell toxicant of Complement Dependent (" CDC "), the cell toxicant (" ADCC ") that antibody relies on, and antibody generates.
In case be combined 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 the entity (for example cancer cell) of antigen.
Although the natural antibody that exists is from single species, antibody through transforming and antibody fragment can be from the animals 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 the immune response at it in the species except that mouse) that reduces mouse antibodies, and can utilize described mosaic thus in human therapy.
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 [United States Patent (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 that wherein some or all amino acid of unwanted human normal immunoglobulin light chain of conjugated antigen or heavy chain (for example constant region of variable region and framework region) are used to replace from homology non-human antibody's the light chain or the corresponding amino acid of heavy chain by the antibody of recombinant DNA technology preparation.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 immune response 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; United States Patent (USP) 6,180,370].
In one embodiment of the invention, humanized antibody is by being transplanted to mouse (with other non--people) CDR on people's antibody and producing.More specifically, can followingly carry out: 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 determines; (3) DNA of coding CDR transfers to human antibody heavy chain and variable region of light chain respective regions by 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 NSO 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 described 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 common method of preparation reverse mutation is [Queen, 1989 well known by persons skilled in the art; Co, 1991; PCT patentapplication WO 90/07861; 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.
More specifically in the embodiment, fully human antibodies utilizes antibody library [United States Patent (USP) 6,300, the 064] preparation of external human spleen cell that excites [Boerner, 1991] and phage display in the present invention.
More specifically in the embodiment, fully human antibodies is by grand (repertoirecloning) [Persson, 1991, Cook in the present invention; Huang and Stollar, 1991] preparation.In addition, United States Patent (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 by infecting immortalization with the Epstein-Barr virus or derivatives thereof of expressing eb nuclear antigen 2 (" EBNA2 "), and described 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.Described transgenic animal can be used activated T cell or D1.1 albumen [United States Patent (USP) 5,474,771; United States Patent (USP) 6,331,433; United States Patent (USP) 6455,044] immunity, hybridoma can generate from the B cell from described transgenic animal.The details of these methods is described in the art.See that for example various (Palo Alto, CA) public publication/patent comprise United States Patent (USP) 5,789,650 with the GenPharm/Medarex that contains the transgenic mice of the little locus of people Ig (miniloci); The various Abgenixs relevant with XENOMOUSE  mouse (Fremont, CA) public publication/patent comprise United States Patent (USP) 6,075,181,6,150,584 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 keeps other valuable character of its Fc part simultaneously at present.A kind of method of modified antibodies is the amino acid [European patent 239400 that sudden change mAb surface participates in the effect binding interactions; 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 described antibody Fc partial C H2The modification in the site that conservative N-connects in the structural domain.
In one embodiment of the invention, described 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 described sudden change can be in host cell (for example NSO or Chinese hamster ovary celI) stably express purifying then.As an example, purifying can utilize albumin A and gel permeation chromatography to 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 described 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 by ordinary method.The deglycosylated method of the enzymatic of antibody is [Williams, 1973 well known by persons skilled in the art; Winkelhake ﹠amp; Nicolson, 1976].
In another embodiment of the present invention, de-glycosylation can be by utilizing glycosylation inhibition tunicamycin (tunicamycin) [Nose ﹠amp; Wigzell, 1983] realize.Be that described modification is to prevent in described 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 described polypeptide (or contain) can be used as antigen to produce anti-cd 154 antibodies or antibody derivatives, and it is subsequently by de-glycosylation.Described antigen can mix or be connected in haptens and generate to increase antibody with adjuvant.
No matter whether the modification of sugar based antibody of the present invention or antibody derivatives is by 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 determined [Harlow, 1998 in this area; Coligan, 2001; Zola, 2000].
After the immunity, antibody of the present invention or antibody derivatives can utilize any routine techniques preparation.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 expresSFTM cell (Protein Sciences Corp., Meriden, CT, USA)-fruit bat S2 cell and Hihg Five  Cells (Invitrogen, Carlsbad, CA, USA) semilooper in (Trichoplusia); Or (4) mammalian cell.Common mammalian cell comprises COS1 and COS7 cell, Chinese hamster ovary (CHO) cell, NSO myeloma cell, NIH 3T3 cell, 293 cells, 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 limit list.
In another embodiment of the present invention, sugar based anti-cd 154 antibodies or antibody derivatives prepare by cell free translation.
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 [United States Patent (USP) 5 of sugar based anti-cd 154 antibodies, 827,690; Pollock, 1999].
As mentioned above, 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 above being described), 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 described 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 parts) 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 by with water-soluble polymers such as polyoxyethylene glycol, the multipolymer of polyoxyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone--based or polyproline is covalently bound and compared with corresponding not modified albumen by modification-all these modified albumen, 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 chemical stability, reduces proteic immunogenicity and antigenicity greatly.As a result, biological activity can be by comparing with not modified albumen, with lower frequency or to realize than the described polymkeric substance of low dosage administration-protein affixture in the desired body.
In some embodiments of the present invention, described sugar based anti-cd 154 antibodies obtains modifying by come mark with the certification mark thing, this marker 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 by being coupled to therapeutical agent, 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 [United States Patent (USP) 6,307,026].
In some embodiments of the present invention, sugar based anti-cd 154 antibodies or antibody derivatives are by obtaining modifying with the developer coupling.Developer for example can comprise that mark 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 derivative.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 derivative of the present invention.
In some embodiments of the present invention, sugar based anti-cd 154 antibodies derivative 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 derivative 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 sugar based anti-cd 154 antibodies disclosed herein.A kind of such clone, it produces sugar based hu5c8 antibody, be 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, be 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.
Methods of treatment
In one embodiment of the invention, sugar based anti-cd 154 antibodies or its antibody derivatives or comprise the pharmaceutical composition of described antibody or antibody derivatives can suppress the immune response among the experimenter.Described 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 described antibody or antibody derivatives can combine with the CD154 protein molecular.
In the specific embodiments of the present invention, described sugar based anti-cd 154 antibodies, antibody derivatives or contain described 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, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described antibody or antibody derivatives can combine with the CD154 epi-position, described 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, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described antibody or antibody derivatives not with the effector receptors bind.The present invention another more in the specific embodiments, described sugar based anti-cd 154 antibodies, antibody derivatives or contain described 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, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described antibody or antibody derivatives do not cause thrombosis.The present invention another more in the specific embodiments, described sugar based anti-cd 154 antibodies, antibody derivatives or contain described 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, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described antibody or antibody derivatives can interact and suppress immune response by suppressing CD154-CD40.
In one embodiment of the invention, described sugar based anti-cd 154 antibodies, antibody derivatives or contain the pharmaceutical composition of described antibody or antibody derivatives can inflammation-inhibiting.Be purpose of the present invention, red, swollen, hot and pain that Inflammatory response is characterised in that is as the consequence of telangiectasis and oedema and phagocytic leukocyte migration.Some examples of Inflammatory response comprise: sacroiliitis, and contact dermatitis, super-IgE syndrome, inflammatory bowel, allergic asthma and the Inflammatory response [Gallin, 1989] of the special property sent out.More arthritic examples comprise: rheumatoid arthritis, non-rheumatoid inflammatory arthritis, sacroiliitis relevant with Lyme disease and inflammatory osteoarthritis.Some examples of the special property sent out inflammatory diseases comprise: psoriatic and systemic lupus erythematous.
In one embodiment of the invention, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described antibody or antibody derivatives can suppress the repulsive interaction of organ transplant recipient.
The present invention another more in the specific embodiments, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described 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, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described antibody or antibody derivatives can suppress the graft versus host disease in the acceptor.
In one embodiment of the invention, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described antibody or antibody derivatives can suppress the anaphylaxis-for example among the experimenter, hay fever or to the allergy of penicillin or other medicines.
In one embodiment of the invention, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described antibody or antibody derivatives can suppress to suffer from the autoimmune response among the experimenter of autoimmune disease.The example of autoimmune disease includes, but not limited to rheumatoid arthritis, myasthenia gravis, systemic lupus erythematous, Graves disease, idiopathic thrombocytopenic purpura, hemolytic anemia, diabetes, inflammatory bowel, clone disease, multiple sclerosis, psoriatic and drug-induced autoimmune disease,-for example, drug-induced lupus.
In one embodiment of the invention, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described antibody or antibody derivatives can suppress to suffer from from the autoimmune response among the experimenter of the autoimmune response of infectious diseases.
In one embodiment of the invention, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described antibody or antibody derivatives can suppress to suffer from the syndrome from Reiter, the joint of vertebral column inflammation, Lyme disease, HIV infects, the autoimmune response among the experimenter of syphilis or autoimmune response lungy.
In one embodiment of the invention, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described antibody or antibody derivatives can suppress the fibrosis among the experimenter.
More Fibrotic examples comprise: pulmonary fibrosis or fibrotic conditions.Some examples 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 examples 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, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described antibody or antibody derivatives can suppress gastrointestinal illness.Some examples of gastrointestinal illness comprise: dyskinesia of esophagus, inflammatory bowel and scleroderma.
In one embodiment of the invention, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described antibody or antibody derivatives can suppress vascular disease.Some examples of vascular disease comprise: atherosclerosis or reperfusion injury.
In one embodiment of the invention, described sugar based anti-cd 154 antibodies, antibody derivatives or contain the pharmaceutical composition of described antibody or antibody derivatives can suppressor T cell cancer (for example T chronic myeloid leukemia or lymphoma) the propagation of T cell tumour cell among the patient.Described 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, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described antibody or antibody derivatives can suppress the virus infection of HTLVI virus to experimenter T cell.Described 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, described sugar based anti-cd 154 antibodies, antibody derivatives or contain the pharmaceutical composition of described 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 at the albumen that allows antibody or antibody derivatives and tumour cell or neoplastic cell surface under the condition of complex body, 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, described sugar based anti-cd 154 antibodies, antibody derivatives or the pharmaceutical composition that contains described antibody or antibody derivatives can detect the tumour cell among the experimenter or the existence of vegetation cell, 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 described mixture shows the existence of tumour cell and vegetation cell among the experimenter.
Pharmaceutical composition
The invention provides pharmaceutical composition, it comprises sugar based anti-cd 154 antibodies described herein or antibody derivatives.
In one embodiment of the invention, described pharmaceutical composition comprises one or more sugar based anti-cd 154 antibodies or antibody derivatives.
In another embodiment of the present invention, described pharmaceutical composition also comprises pharmaceutically acceptable carrier, adjuvant, delivery vector, damping fluid or stablizer.
More specifically in the embodiment, described pharmaceutically acceptable carrier is the salt solution of phosphate buffered in the present invention, physiological saline, and water, Citrate trianion/sucrose/Tween preparaton and emulsion-for example, oil/aqueous emulsion.
In one embodiment of the invention, thereby described pharmaceutical composition can reduce and prevents proteic host immune response little sending in by bag apparatus.Described antibody or antibody derivatives also can film such as liposome in the form of little tunicaization send.
In one embodiment of the invention, described 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 technology utilization known in the art dispersion agent, wetting agent and suspension agent are prepared.
In one embodiment of the invention, described pharmaceutical composition can be oral, and part or intravenously are sent.
The present invention another more in the specific embodiments, for oral administration, described pharmaceutical composition is formulated in suitable capsule, tablet is in aqueous suspensions or the solution.The oral administration of described composition 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, polyvinylpyrrolidone (Povidone TM), Vltra tears, sucrose, starch and ethyl cellulose.Spendable lubricant comprises Magnesium Stearate, stearic acid, silicone fluid, talcum, paraffin, oil, and silica gel.
The present invention another more in the specific embodiments, for topical application, described pharmaceutical composition can be formulated in the suitable ointment.Some examples 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%, described carrier is such as drug cream matrix.
In one embodiment of the invention, inhalation compositions and transdermal drug composition also can prepare easily.
In one embodiment of the invention, the liquid preparation of the pharmaceutical composition for oral administration for preparing in water or other aqueous carrier can contain multiple suspension agent, such as methylcellulose gum, alginate, tragacanth, pectin, kelgin, carrageenin (carrageenan), Sudan Gum-arabic, polyvinylpyrrolidone, 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 described pharmaceutical composition and powder formulation can prepare by the ordinary method that sucks in the mammiferous lung to be treated.
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, dimethyl formamide, ethyl lactate, ethyl-carbonate, isopropyl myristate, ethanol, polyvalent alcohol-be glycerine, propylene glycol, liquid macrogol, etc.In some embodiments, described composition comprises Citrate trianion/sucrose/tween carrier.For intravenous injection, the water-soluble version of composition can pass through the drip-injection method administration, and the pharmaceutical preparation that contains anti-mycotic agent and the acceptable preparation of physiology thus is by infusion.The acceptable vehicle of physiology can comprise, for example 5% glucose, 0.9% salt solution, Ringer's solution and other proper excipient.The suitable insoluble form of described composition can be used as suspension preparation and the administration in hydrated matrix or the acceptable oil matrix, the ethyl oleate of the ester of described matrix such as longer chain fatty acid-for example.
In one embodiment of the invention, described 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 preparation has been determined [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 described pharmaceutical composition.
In some embodiments of the present invention, described pharmaceutical composition also comprises inhibitive ability of immunity or immune regulative compound.For example, described inhibitive ability of immunity or immune regulative compound can be one of following material: the medicament that interrupts T cell co-stimulatory signal by CD28; Interrupt the medicament of calcineurin signal, reflunomide, the antiproliferative pharmaceutical agent, with expressed protein specificity bonded antibody on the immunocyte surface, described 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, described 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, described 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 in the mode that any medical science is accepted.Be purpose of the present 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, described sugar based anti-cd 154 antibodies, antibody derivatives and pharmaceutical composition can by 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, described sugar based anti-cd 154 antibodies, antibody derivatives and pharmaceutical composition can be oral by 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 of the present 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, described sugar based anti-cd 154 antibodies, and antibody derivatives and pharmaceutical composition can be by using emulsifiable paste, ointment etc. through the topical experimenter.
In other embodiment of the present invention, sugar based anti-cd 154 antibodies of the present invention, antibody derivatives and pharmaceutical composition can be by utilizing spraying gun, and Diskus or metered-dose inhaler suck and administration.
In other embodiment of the present invention, described 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 of the present invention, antibody derivatives and pharmaceutical composition can be by 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 of the present invention, antibody derivatives and pharmaceutical composition can be by containing antibody, the transdermal patch of antibody derivatives and pharmaceutical composition gives experimenter's skin and comes the administration experimenter, and make described patch and experimenter's skin contact, common every subsides 1-5 hour.
In other embodiment of the present invention, described 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 comprises about 0.01 and the scope of 200mg/kg experimenter's body weight.
In other embodiment, sugar based anti-cd 154 antibodies of the present invention, antibody derivatives and pharmaceutical composition can be to arrive interval repeat administration every other month every day.
In one embodiment of the invention, described 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 methods of treatment can be assessed by monitoring experimenter's known sign or disease symptoms.
For all embodiments of the present invention, effectively produce the sugar based anti-cd 154 antibodies of the present invention of required effect, the dosage of its antibody derivatives and pharmaceutical composition and administration frequency will depend on multiple factor, character such as disease to be treated, experimenter's size, therapeutic purpose, used concrete pharmaceutical composition, and treatment doctor's judgement.
Sugar based anti-cd 154 antibodies of the present invention, 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 immune response of short period, described antigen is such as the exogenous antigen of administration in one day treatment.The example of described treatment comprises Combined Preparation antibody of the present invention or antibody derivatives and therapeutical agent, for example, and antigenicity medicine (antigenic pharmaceutical), anaphylactogen or blood products, or gene therapy vector.In the long-standing indication of antigen, such as in the immune response 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 by the experimenter of aforesaid method treatment.Preferred described animal is a Mammals.The mammiferous example 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, described 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
Following examples are for example understood method of the present invention and product.Suitable change and transformation to described 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 hu5c8 mAb and expression
For reducing the effector functions of hu5c8 mAb, the sugar based form is by changing heavy chain C H2The Asn site that classical N connects in the structural domain produces for the Gln residue.
Competition has confirmed to compare with glycosylation hu5c8 mAb in conjunction with test, and sugar based hu5c8 mAb does not change (Fig. 1) in conjunction with the ability of cell surface CD154.
Utilize bridge joint test form (bridging assay format), at the external test effector functions.Compare with glycosylation hu5c8 mAb, sugar based hu5c8 mAb weakens 25 times (Fig. 2 A) with the relative combination of Fc γ RI.When reaching the concentration of 5mg/ml, can not confirm that sugar based hu5c8 mAb combines with the remnants of Fc γ RIII, and normal glycosylation hu5c8 mAb shows that in identical test form EC50 is 50ng/ml (Fig. 2 B).
The pharmacokinetics of sugar based hu5c8 mAb in the macaque
After giving single dose 20mg/kg hu5c8 mAb and sugar based hu5c8 mAb, serum-concentration-time diagram from two independent experiments is carried out pharmacokinetic analysis, two compartment models of described 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 hu5c8 mAb and sugar based hu5c8 mAb.The removing of hu5c8 mAb and volume distributed median are bigger slightly than sugar based hu5c8 mAb.
Table 1
Hu5c8 or sugar based hu5c8 single dose intravenously with 20mg/kg
The administration macaque ALater average pharmacokinetic parameter
Antibody Cmax B (μg/mL) CI C (mLhr/kg) Vss D (mL/kg) t 1/2 E (d)
Hu5c8 sugar based hu5c8 515(±16) 869(±360) 4.61(±0.70) 3.10(±1.10) 71(±10) 47(±11) 11.5(±2.5) 11.8(±3.0)
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 generates the selection of hu5c8 mAb, clone and humanization is former describes.See Lederman respectively, 1992 and Karpusas, 2001.Hu5c8 mAb hybridoma can be available from ATCC (HB10916).Utilize Amersham-Pharmacia Biotech (Piscataway, NJ, test kit USA), scheme according to manufacturer's recommendation, eliminate mutagenesis by unique site, in glycosylation hu5c8 mAb, carry out heavy chain glycosylation site sudden change N298Q (N297 utilizes the EU numbering).The sugar based hu5c8 that produces stably express and by albumin A and gel permeation chromatography purifying in NSO myeloma cell.The clone that produces sugar based hu5c8 antibody derives from ATCC (PTA-4931).SDS-PAGE shows the tetramer that the disulfide linkage of described albumen formation expection is connected with analytical gel permeation chromatography.
2.CD154 in conjunction with testing to huCD154 +(Dr.Leonard Chess, Columbia University present also can derive from ATCC (CRL-10915) and carry out competition based on FACS in conjunction with test the D1.1 cell.0.1mg/ml biotinylated hu5c8mAb competes with combining with the titrate (titration) of hu5c8mAb and sugar based hu5c8mAb of cell surface CD154.(BD-PharMingen San Diego, CA USA) detect cell bonded biotinylation hu5c8 mAb 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 measured the test determination (seeing below) that the utilization of Fc γ R binding affinity forms antigen based on antibody and carries the ability of " bridge joint " between the cell of Fc γ R.The test of Fc γ RI (CD64) bridge joint is by the solvable human CD 154 (Biogen that recombinates with 1mg/ml, Karpusas, 1995) PBS solution at 4 ℃ of bags by 96 hole Maxisorb elisa plate (Nalge-Nunc Rochester, NY, 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 measure and use CD16 (Dana Farber Institute, Boston, MA, USA present) the mAb dependency combination of the fluorescently-labeled Jurkat cell of transfection.CHO-CD154 +Cell is with 1 * 10 5Cell/ml is inoculated in 96 orifice plates, containing 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 described 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 by washing, and (MilliporeCorporation Bedford, MA read plate, excitation wavelength 485nm, emission wavelength 530nm on USA) at Cytofluor 2350 Fluorescent Microplate Reader.
Embodiment 2: sugar based hu5c8 antibody suppresses initial and follow-up humoral response
Suppress in the macaque the antigenic initial humoral immune reaction of Toxoid,tetanus (TT)
Respectively the do for oneself sugar based hu5c8 mAb of 20mg/kg of single dose and glycosylation hu5c8 mAb (according to embodiment 1 preparation) suppresses ability assessment in dividing other experiment to the initial antibodies reaction of TT.Compare with the control group of brine treatment, administration sugar based hu5c8 mAb or glycosylation hu5c8 mAb cause overall initial immune response (E AUC) reduce by 70% and 77% respectively.Fig. 4 illustrates TT antibody tiring in whole 42 days, shows that sugar based hu5c8 mAb is similar to glycosylation hu5c8 mAb 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 hu5c8 mAb 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, the existence of germinal center (" GC ") significantly reduces compared with the control in the animal of inguinal lymph nodes biopsy demonstration sugar based hu5c8 mAb treatment.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 to the severe hypoplasia with consistent to glycosylation hu5c8 mAb observed (data not shown) in the past.
With the not overall change of hematologic parameter after single dose 20mg/kg glycosylation or the sugar based hu5c8 mAb administration macaque, total lymphocyte count does not obviously change.In addition, CD4/CD8 T 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 hu5c8 mAb suppresses the ability of follow-up humoral response and assesses by giving 8 saline control animals with TT attack for the second time, has normal initial reaction in the former described sugar based hu5c8 mAb conceptual phase of described animal.Before TT attacks for the second time, there are 4 to accept 20mg/kg sugar based hu5c8 mAb (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 overall initial and follow-up individual immunity reaction.There is notable difference in the immune response 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 hu5c8 mAb.Therefore, administration sugar based hu5c8 mAb 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 overall initial and follow-up immune response (E of Toxoid,tetanus AUC)
The treatment group Group 1A (salt solution-salt solution) A Group 1B (salt solution-sugar based hu5c8) B
Animal # 1A-1 1A-2 1A-3 1A-4 1B-1 1B-2 1B-3 1B-4
Initial E AUC (×10 5) 2.1 1.1 2.0 0.3 3.9 0.9 1.6 2.6
Follow-up E AUC (×10 5) 8.7 5.2 9.6 6.0 8.3 1.5 3.3 5.5
Intensity 4.2 4.8 4.8 18.8 2.1 1.6 2.1 2.2
The group average intensity 6.5 2.0
AThe 1A treated animal was accepted salt solution before initial and follow-up TT attacks.The 1B treated animal was accepted salt solution before initial TT attacks, accepted sugar based hu5c8 before follow-up TT attacks. BThe degree of follow-up reaction is by follow-up E AUCWith initial E AUCThe ratio value representation.
Method-embodiment 2
1. to same macaque among the humoral immune reaction utilization of TT and the embodiment 1, in macaque, carry out two independently researchs.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 hu5c8 mAb 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 mentioned above) 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 hu5c8 mAb research comprises five groups of treatment group, and every group contains three female samples.First day, first winding was subjected to salt solution (untreated contrast), second to five group of 0.2,1,5 or 20mg/kg glycosylation hu5c8 mAb 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 hu5c8 mAb, and it suppresses follow-up immunoreactive ability.The following processing of animal: group 1B accepted single dose 20mg/kg sugar based hu5c8 mAb 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 immune response utilizes non-compartment analysis (noncompartmental analysis) assessment immune response.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 determining 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 macaque utilizes 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) and CD4-PE clone OKT4 (Ortho Diagnostic Systems Raritan, NJ, USA), or CD3-FITC and CD8-PE clone DK-25 (Dako Corporation Carpinteria, CA, USA).Red corpuscle 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 determined by the number sum of all CD2 and CD20 positive cell.The B cell is accredited as the CD20 positive cell.T cell subgroup 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 interior positive cell percentage of door.Calculate the CD4/CD8 ratio of every group of data.
4.hu5c8 the ELISA. elisa plate (Nalge-NuncRochester of mAb pharmacokinetics, NY, USA) with 5 μ g/ml recombinate solvable human CD 154 (Biogen, seealso Karpusas1995) in PBS, 4 ℃ of bags are spent 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) (JacksonImmunoResearch Laboratories West Grove, PA, USA) detect, use 3 then, 3 ', 5,5 '-tetramethyl benzidine (" TMB ") substrate reagent box (Pierce Biotechnology Rockland, IL USA) shows.(Molecular Devices Sunnyvale, CA USA) reads plate to utilize Spectromax to read the plate instrument 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. the ELISA elisa plate (CorBing-Costar) of measuring anti--hu5c8 antibody response 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 hu5c8 mAb 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 instrument (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 elisa plate (Corning-Costar) of anti--TT reaction with 5 μ/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 instrument 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. hematology is collected the blood sample of EDTA potassium anti-freezing and was analyzed following hematologic parameter in every month: the total leukocyte counting, red blood cell count(RBC), hemoglobin concentration, hematocrit, MCV (mean corpuscular volume), mean cell hemoglobin (mean corpuscularhemoglobin), mean cell hemoglobin concentration, platelet count and blood smear assessment (comprising difference).
8. the 15th of the initial TT repercussion study of lymph node biopsy sugar based hu5c8 mAb the day, gather inguinal lymph nodes from all animals.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.Described slide glass is by 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 anti-nuclear of multiple pathologic autoantibody.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.Determined in the past that the antinuclear antibody that 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) in people and mouse SLE.[Kalled et al.，1998]。
Studies confirm that in the past 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 MR1 mAbs 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 by the C of sudden change IgG2a Fc H2The glycosylation site that N connects in the structural domain produces.Utilize this two kinds of antibody, assessment Fc glycosylation is to the effect of anti-cd 154 mAbs 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 is 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﹠amp dsDNA and ssDNA with the processing of muMR1 or sugar based muMR1; B).
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.From these results obviously as seen, muMR1 and sugar based muMR1 can effectively prevent with the lupus nephritis to be the Histological change of feature.
Lymph sample enlarged degree carries out the histologic analysis assessment by 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 sample 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, as by measure in the urine shown in the protein content like that.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 represents 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. mouse BALB/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.
Antibody MR1 hybridoma (ATCC#CRL-2580) (it produces the Armenian hamster anti--mouse CD154 mAb) available from American type culture collection (Rockville, MD).
3. all injections of treatment plan are by 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 chimeric anti-cd 154 mAbs.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, collects blood sample in 9,11 and 14 days.
4.ELISA assay method is for detecting the chimeric MR1 mAbs of serum, NUNC Maxisorp plate is recombinated solvable mouse CD154 4 ℃ of incubated overnight with 5 μ g/ml.Sealed described 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 stops with 2N sulfuric acid, and (Molecular Devices reads plate on CA) to read the plate instrument at 450nm at Spectramax.Anti--single stranded DNA (ssDNA) and anti--double-stranded (" dsDNA ") ELISA utilize NUNC MaxiSorp plate to carry out.Described plate 4 ℃ with 10 μ g/ml methylate BSA (Calbiochem Corp, La Jolla, CA) bag spent the night use then 5 μ g/ml I level calf thymus DNAs (SIGMA, St.Louis, MO) at 25 ℃ of bags by 2 hours.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 standby 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-alkaline phosphatase.Read plate at 405nm, and obtain typical curve by Anti-DNA mAb205 or the mAb 5c6 that utilizes known quantity, described antibody is all special to ss-and dsDNA.Anti-DNA is tired and is defined as dilution inverse above background 0.1 OD unit.
5. the paraffin-embedded tissue of histology kidney and spleen freezing microtome section and formalin fixed is with hematoxylin-eosin (" H﹠amp; E ") the dyeing inflammatory infiltration.Kidney is also used the gloomy three-color process of horse (Masson Trichrome) coloured fibreization and with cycle acidity (Periodic acid) Schiff dyeing (Periodic Acid Schiff stain) (" the PAS ") basement membrane thickened that dyes.Painted tissue slice is marked by veterinary pathologist.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. (Bayer Corp., Tarrytown NY) monitor weekly to measure proteinuria (" PU ") with Albustix in the analysis of the urine of every mouse of analysis of urine and serum.The scoring of proteinuria level is 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 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 by 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 MR1 mAbs suppress disease progression fully, as by 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.Occurring EAE the mouse for the treatment of with muMR1 in the 0th, 2 and 4 day after attacking once more, 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.Whether the transfer that we have also studied 20 * 106 splenocytes can cause initial acceptor mouse
Figure A20048002323100401
Recipient mice) to subsequently active EAE inductive resistance, situation is not (data not shown) like this.Described splenocyte collection is hung oneself, and EAE induces and treat the mouse in 1 or 3 weeks of back 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 muMR1 mAbs 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 aspect the abilities that suppresses EAE.
Suppress the CNS inflammatory infiltration
Aspect the inflammatory infiltration of assessment antibody in suppressing central nervous system (" CNS "), whether there are differences, 4-5 mouse is divided into one group, antibody (muMR1 and sugar based muMR1 that different groups is measured with difference, or 200 μ g of 3 dosage and P1.17 contrast Ig) treatment, 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. progression of disease 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, described antibody is in suppressing the progression of disease early process, and the ability aspect 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, the CNS that analyzes from every group of 6-14 mouse in this test endpoint (the 58th day) organizes.
With opposite with the mouse of 200 μ g muMR1 treatment, the mouse of P1.17-treatment and the mouse for the treatment of 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 to 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 induce female 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 dead bordetella pertussis (B.pertussis) organism (RIVM, Bilthoven, The Netherlands) of 109 heat kills in the mouse vein.The progress of EAE is by 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 anxiety (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
Antibody Dosage (μ g) The size of animal of suffering from infiltration
Do not have Accidental Moderate Severe
P1.17 3×200 0 3 1 1
muMR1 3×200 3 1 0 0
muMR1 3×75 3 2 0 0
muMR1 3×25 1 1 3 0
Sugar based MR1 3×200 5 0 0 0
Sugar based MR1 3×75 4 1 0 0
Sugar based MR1 3×25 2 1 2 0
Table 5: the influence (the 58th day) that anti-cd 154 soaks into CNS
Antibody Dosage (μ g) The size of animal of suffering from infiltration
Do not have Accidental Moderate Severe
The P1.17 isotype 200 3 8 3 0
muMR 200 12 1 0 0
muMR 75 3 3 0 0
muMR 25 0 5 1 0
Sugar based MR1 200 8 5 1 0
Sugar based MR1 75 4 2 0 0
Sugar based MR1 25 4 2 0 0
2. antibody generates hamster, and clone from the total RNA of hybridoma by RT-PCR the anti--heavy chain of mouse CD154mAb MR1 and the variable region of light chain.The expression vector of the chimeric mAb of hamster/mouse is by 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 by flow cytometry and immunoprecipitation the chimeric MR1mAb (being called muMR1) of transient expression has the CD154 of (recapitulate) hamster mAb in conjunction with character.
The chimeric MR1 of sugar based (being called aglyMR1) is built into by heavy chain being carried out the asparagine residue (being N297 in Kabat EU nomenclature) 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 NSO cell, by select the clone of separating stable in the substratum that does not contain glutamine.
MuMR1 and sugar based MR1 carry out size exclusion chromatography to remove aggregate at Sephacryl 300 by 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 by 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 antibodies at the 0th, 2 and 4 day, every mouse is 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. the disease assessment mouse of weighing every day, in whole 56 days process, monitor clinical event (clinical activity) according to following points-scoring system: 0: asymptomatic, 0.5: the nervous part forfeiture of tail, 1: the tail anxiety completely loses, 2: four limbs are weak, and 2.5: local paralysis, 3: the hind leg complete paralysis, 3.5: diaphragm and hind leg complete paralysis, gatism, 4: dying, 5: because EAE causes death.
5. cerebral tissue of every mouse of histology and spinal cord fixing and embedding in paraffin in 10% formalin.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. results of statistical analysis is analyzed by One-Way ANOVA, utilizes the LSD post-hoc that upchecks to analyze then.It is remarkable that P-value＜0.05 is considered to.
Embodiment 5: sugar based hu5c8 antibody suppresses islet cell transplantation
Studies show that in the past, 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 the lasting hyperglycemia that occurred by 11-14 days and c peptide (product that endogenous insulin generates) generate shortage confirm (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 initial the repulsion 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 by 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 treatment plan in the background.Yet, because the immune response in the migration process is very strong, promptly relating to body fluid, cell and inflammatory immune response 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 via the T cell co-stimulatory signal of CD28, reflunomide, 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. [Kenyon, 1999] are carried out in these researchs of islet cells allotransplantation as mentioned above.In brief, alloreactivity D-A rhesus monkey is to based on positive mixed lymphocyte culture reaction selection.Acceptor was accepted (intraportal) allogene pancreatic islets transplantation in complete pancreas islet excision and the door at the 0th day.
2. the Antybody therapy dosage utilizes-1, and the keeping of 20mg/kg of the dosage of inducing and beginning in the 28th day in every month of 0,3,10 and 18 days 20mg/kg carried out.
3. graft function assessment pancreatic islets transplantation thing function is monitored by 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 instability.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 by 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, described effect and stimulation CD154 are (promptly, directly send and suppress signal by CD154) opposite, 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 described 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 hu5c8 mAb of full text description among the application.See that for example embodiment 1,
Figure A20048002323100471
With
Figure A20048002323100472
See above.Solvable but anti-cd 154 antibodies non-bag quilt suppresses former generation blended lymphocyte culture (" MLC "), and (40 ± 23% vs 3 ± 18% suppress, 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) by blocking with CD154, yet it can stimulate by CD154 (antibody of bag quilt) and increase.In addition, the stimulation of the anti-cd 154 antibodies of bag quilt increases the generation of allogenic antigen specific CTL effector strongly, and CTL is not subjected to 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 of bag quilt 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 the retarding effect that CTLA4-Ig generates CTL is stimulated (by the anti-cd 154 antibodies of bag quilt) to eliminate 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 by 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 by solubility sugar based hu5c8 mAb 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 (68)

1. sugar based anti-cd 154 antibodies or antibody derivatives is characterized in that being positioned at described 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 or the antibody derivatives 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 or the antibody derivatives of claim 2, wherein said modification comprise sudden change N298Q (N297 utilizes EU Kabat numbering).
4. the sugar based anti-cd 154 antibodies or the antibody derivatives of claim 1, wherein said modification comprise removes C H2The structural domain glycan.
5. the sugar based anti-cd 154 antibodies or the antibody derivatives of claim 1, wherein said modification comprise and preventing at C H2The glycosylation of structural domain.
6. sugar based anti-cd 154 antibodies or the antibody derivatives of one of claim 1-5, wherein said sugar based anti-cd 154 antibodies or antibody derivatives not with the effect receptors bind.
7. sugar based anti-cd 154 antibodies or the antibody derivatives of one of claim 1-5, wherein said sugar based anti-cd 154 antibodies or antibody derivatives do not cause thrombosis.
8. sugar based anti-cd 154 antibodies or the antibody derivatives of one of claim 1-5, wherein said antibody is selected from: monoclonal antibody, polyclonal antibody, murine antibody, chimeric antibody, the antibody of spirit lengthization, the group of humanized antibody and fully human antibodies composition.
9. sugar based anti-cd 154 antibodies or the antibody derivatives of one of claim 1-5, wherein said antibody is selected from: polymer antibody, heterodimer antibody, half homodimeric antibody, tetravalent antibody, bi-specific antibody, Fab, Fab ', Fab ' 2, F (v) antibody fragment, single-chain antibody, or derivatives thereof.
10. sugar based anti-cd 154 antibodies or the antibody derivatives of one of claim 1-5, wherein said antibody are the sugar based hu5c8 that the clone by ATCC preserving number PTA-4931 produces.
11. sugar based anti-cd 154 antibodies or the antibody derivatives of one of claim 1-5, wherein said antibody or antibody derivatives detectable label substance markers.
12. the sugar based anti-cd 154 antibodies or the antibody derivatives of claim 11, wherein said detectable is a radioactive isotope, enzyme, dyestuff or vitamin H.
13. sugar based anti-cd 154 antibodies or the antibody derivatives of one of claim 1-5, wherein said antibody or antibody derivatives are coupled to therapeutical agent.
14. the sugar based anti-cd 154 antibodies or the antibody derivatives of claim 13, wherein said therapeutical agent is a radio isotope, radionuclide, toxin, toxoid or chemotherapeutics.
15. sugar based anti-cd 154 antibodies or the antibody derivatives of one of claim 1-5, wherein said antibody or antibody derivatives are coupled to developer.
16. the sugar based anti-cd 154 antibodies or the antibody derivatives of claim 15, wherein said developer is a mark part.
17. the sugar based anti-cd 154 antibodies or the antibody derivatives of claim 15, wherein said developer is a vitamin H, fluorescence part, radioactivity part, histidine mark or peptide-labeled.
18. comprise the sugar based anti-cd 154 antibodies of one of claim 1-5 or the pharmaceutical composition of antibody derivatives.
19. the described pharmaceutical composition of claim 18 also comprises pharmaceutically acceptable carrier.
20. the described pharmaceutical composition of claim 18 also comprises inhibitive ability of immunity and immune regulative compound.
21. produce the sugar based anti-cd 154 antibodies of one of claim 1-5 or the clone of antibody derivatives.
22. the clone of claim 21, wherein said clone produces sugar based hu5c8 (ATCC preserving number PTA-4931).
23. suppress the immunoreactive method among the experimenter, one of its claim 1-5 by administration experimenter effective inhibitory amount described sugar based anti-cd 154 antibodies or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that comprises described antibody or antibody derivatives carry out.
24. the method for claim 23, wherein said sugar based anti-cd 154 antibodies, antibody derivatives or pharmaceutical composition inhibition CD154 combine with CD40's.
25. the method for claim 23, wherein said sugar based anti-cd 154 antibodies, antibody derivatives or pharmaceutical composition can specificity in conjunction with can be by the protein of sugar based hu5c8 specific recognition, sugar based hu5c8 produces by the clone of ATCC preserving number PTA-4931.
26. the method for claim 23, wherein said sugar based anti-cd 154 antibodies, antibody derivatives or combination of pharmaceutical composition specificity and sugar based hu5c8 (ATCC preserving number PTA-4931) specificity bonded epi-position.
27. suppress the method for Inflammatory response among the experimenter, described sugar based anti-cd 154 antibodies of one of claim 1-5 that it suppresses significant quantity by the administration experimenter or sugar based anti-cd 154 antibodies derivative, or the pharmaceutical composition that comprises described antibody or antibody derivatives carries out.
28. the method for claim 27, wherein said Inflammatory response is selected from: sacroiliitis, contact dermatitis, height-IgE syndrome, inflammatory bowel, the group that allergic asthma and Te Fa inflammatory diseases are formed.
29. the method for claim 28, wherein said sacroiliitis is selected from: rheumatoid arthritis, non-rheumatoid inflammatory arthritis, the group that Lyme disease dependency sacroiliitis and inflammatory osteoarthritis are formed.
30. the method for claim 28, wherein said spy's property sent out inflammatory diseases are selected from the group of psoriatic and systemic lupus erythematous composition.
31. suppress the method for the transplant rejection among the experimenter, described sugar based anti-cd 154 antibodies of one of claim 1-5 that it suppresses significant quantity by the administration experimenter or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that contains described antibody or antibody derivatives carry out.
32. the method for claim 31, transplant rejection wherein relates to the heart of transplanting, kidney, liver, skin, islet cells or marrow.
33. suppress the method for the graft versus host disease among the experimenter, described sugar based anti-cd 154 antibodies of one of claim 1-5 that it suppresses significant quantity by the administration experimenter or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that contains described antibody or antibody derivatives carry out.
34. suppress the method for the anaphylaxis among the experimenter, described sugar based anti-cd 154 antibodies of one of claim 1-5 that it suppresses significant quantity by the administration experimenter or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that contains described antibody or antibody derivatives carry out.
35. the group that the method for claim 34, wherein said anaphylaxis are selected from spring fever or the allergy of penicillin or other medicines is formed.
36. suppress the method for the autoimmune response among the experimenter, described sugar based anti-cd 154 antibodies of one of claim 1-5 that it suppresses significant quantity by the administration experimenter or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that contains described antibody or antibody derivatives carry out.
37. the method for claim 36, wherein said autoimmune response is derived from infectious diseases.
38. the method for claim 36, wherein said autoimmune response is derived from Reiter syndrome, the joint of vertebral column inflammation, and Lyme disease, HIV infects, syphilis or tuberculosis.
39. the method for claim 36, wherein said autoimmune response is selected from rheumatoid arthritis, myasthenia gravis, systemic lupus erythematous, the Graves disease, idiopathic thrombocytopenic purpura, hemolytic anemia, diabetes, inflammatory bowel, clone disease, multiple sclerosis, psoriatic and drug-induced autoimmune disease, the group that drug-induced lupus is formed.
40. suppress the Fibrotic method among the experimenter, described sugar based anti-cd 154 antibodies of one of claim 1-5 that it suppresses significant quantity by the administration experimenter or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that contains described antibody or antibody derivatives carry out.
41. the method for claim 40, wherein said fibrosis are selected from the group of pulmonary fibrosis and fibrotic conditions composition.
42. the method for claim 41, 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.
43. the method for claim 41, wherein said fibrotic conditions is selected from: 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 group that is secondary to the liver cirrhosis composition of autoimmune disease.
44. suppress the method for experimenter by the virus infection of the virogenetic T cell of HTLVI, described sugar based anti-cd 154 antibodies of one of claim 1-5 that it suppresses significant quantity by the administration experimenter or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that contains described antibody or antibody derivatives carry out.
45. suppress the method for the gastrointestinal illness among the experimenter, described sugar based anti-cd 154 antibodies of one of claim 1-5 that it suppresses significant quantity by the administration experimenter or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that contains described antibody or antibody derivatives carry out.
46. the method for claim 45, wherein said gastrointestinal illness is selected from: dyskinesia of esophagus, the group that inflammatory bowel and scleroderma are formed.
47. suppress the method for the vascular disease among the experimenter, described sugar based anti-cd 154 antibodies of one of claim 1-5 that it suppresses significant quantity by the administration experimenter or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that contains described antibody or antibody derivatives carry out.
48. the method for claim 47, wherein said vascular disease are selected from: the group that atherosclerosis or reperfusion injury are formed.
49. suppress to suffer from the method for the propagation of the T cell tumour cell among the experimenter of T cell carcinoma, described sugar based anti-cd 154 antibodies of one of claim 1-5 that it suppresses significant quantity by the administration experimenter or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that contains described antibody or antibody derivatives carry out.
50. suppress the method for HTLVI virus to the virus infection of experimenter's T cell, described sugar based anti-cd 154 antibodies of one of claim 1-5 that it suppresses significant quantity by the administration experimenter or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that contains described antibody or antibody derivatives carry out.
51. the method for one of claim 23-50, wherein said sugar based anti-cd 154 antibodies or sugar based anti-cd 154 antibodies derivative are selected from: monoclonal antibody, polyclonal antibody, murine antibody, chimeric antibody, the antibody of the long sourceization of spirit, the group that humanized antibody and fully human antibodies are formed.
52. the method for one of claim 23-50, wherein said sugar based anti-cd 154 antibodies or sugar based anti-cd 154 antibodies derivative are selected from: polymer antibody, heterodimer antibody, half homodimeric antibody, tetravalent antibody, bi-specific antibody, Fab, Fab ', Fab ' 2, F (v) antibody fragment, single-chain antibody, or derivatives thereof.
53. the method for one of claim 23-50, wherein said sugar based anti-cd 154 antibodies or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that comprises described antibody or antibody derivatives are with the described experimenter of the acceptable any way administration of medical science.
54. the method for one of claim 23-50, wherein said sugar based anti-cd 154 antibodies or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that comprises described antibody or antibody derivatives by 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 described experimenter of administration through local injection.
55. the method for one of claim 23-50, wherein said sugar based anti-cd 154 antibodies or sugar based anti-cd 154 antibodies derivative or to comprise the pharmaceutical composition of described antibody or antibody derivatives oral by being selected from, intranasal, through eye, the described experimenter of administration of the group that per rectum and local approach are formed.
56. the method for claim 55, wherein said sugar based anti-cd 154 antibodies or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that comprises described antibody or antibody derivatives be with capsule, tablet, the described experimenter of the form administration of aqueous suspensions or solution.
57. the method for claim 55, wherein said sugar based anti-cd 154 antibodies or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that comprises described antibody or antibody derivatives be by using emulsifiable paste, ointment or the similar described experimenter of preparation topical.
58. the method for claim 55, wherein said sugar based anti-cd 154 antibodies or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that comprises described antibody or antibody derivatives are by utilizing spraying gun, and the sucker of Diskus or metering sucks and the described experimenter of administration.
59. the method for one of claim 23-50, wherein said sugar based anti-cd 154 antibodies or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that comprises described antibody or antibody derivatives are by sustained release administration and the described experimenter of administration.
60. the method for one of claim 23-50, wherein said sugar based anti-cd 154 antibodies or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that comprises described antibody or antibody derivatives are with the described experimenter of therapeutical agent administration.
61. the method for one of claim 23-50, wherein said sugar based anti-cd 154 antibodies or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that comprises described antibody or antibody derivatives are with the described experimenter of a plurality of dosed administrations every day.
62. the method for one of claim 23-50, wherein said sugar based anti-cd 154 antibodies or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that comprises described antibody or antibody derivatives are with from every day to every other month the described experimenter of interval repeat administration.
63. the method for one of claim 23-50, wherein said sugar based anti-cd 154 antibodies or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that comprises described antibody or antibody derivatives according to the medical science indication from a couple of days or several weeks to the experimenter described experimenter of long period administration in all one's life.
64. the method for one of claim 23-50, wherein said sugar based anti-cd 154 antibodies or sugar based anti-cd 154 antibodies derivative or the pharmaceutical composition that comprises described antibody or antibody derivatives are with immunomodulatory or the described experimenter of immunosuppressive compounds administration.
65. the method for claim 64, wherein said immunomodulatory or inhibitive ability of immunity compound are selected from:
(a) interruption is by 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, described immunocyte includes but not limited to CD45, CD2, IL2R, CD4, CD8 and RANK FcR, B7, CTLA4, TNF, LT, and VLA-4.
66. the method for claim 64, wherein said inhibitive ability of immunity and immune regulative compound are selected from tacrolimus, sirolimus, mycophenlate mofetil, mizorubine, Gusperimus, brequinar sodium, leflunomide, the group that Wyeth-Ayerst Laboratories or azaspirane form.
67. the method for video picture tumour cell and vegetation cell in the proteinic experimenter of the sugar based hu5c8 institute specific recognition that the clone of expressing by ATCC preserving number PTA-4931 produces may further comprise the steps:
(a) forming under the condition of complex body between the protein that allows antibody or antibody derivatives and tumour cell or vegetation cell surface, give the experimenter with the described pharmaceutical composition of the claim 18 of significant quantity; With
(b) make the antibody/protein complex body or the antibody derivatives/complex body video picture of formation, make any tumour cell or vegetation cell imaging among the experimenter thus.
68. detect the method for expression, may further comprise the steps by the existence of tumour cell or vegetation cell among the proteinic experimenter of the sugar based hu5c8 institute specific recognition of the clone generation of A TCC preserving number PTA-4931:
(a) forming under the condition of complex body between the protein that allows antibody or antibody derivatives and tumour cell or vegetation cell surface, give the experimenter with the described pharmaceutical composition of the claim 18 of significant quantity;
(b) remove any unconjugated developer from the experimenter; With
(c) any antibody/protein complex body and the antibody derivatives/complex body that detect to form, the existence of described complex body show and have tumour cell and vegetation cell among the experimenter.
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Publication number Priority date Publication date Assignee Title
CN104245732A (en) * 2011-10-13 2014-12-24 百时美施贵宝公司 Antibody polypeptides that antagonize cd40l
CN107949572A (en) * 2015-06-23 2018-04-20 首尔大学校产学协力团 CD154 Binding peptides and application thereof

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NZ502051A (en) * 1997-06-20 2001-04-27 Biogen Inc CD154 blockade therapy for therapeutic protein inhibitor syndrome

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104245732A (en) * 2011-10-13 2014-12-24 百时美施贵宝公司 Antibody polypeptides that antagonize cd40l
CN104245732B (en) * 2011-10-13 2020-04-07 百时美施贵宝公司 Antibody polypeptides antagonizing CD40L
CN107949572A (en) * 2015-06-23 2018-04-20 首尔大学校产学协力团 CD154 Binding peptides and application thereof
CN107949572B (en) * 2015-06-23 2021-10-26 首尔大学校产学协力团 CD154 binding polypeptides and uses thereof

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