CN1169735A - CD 23 binding agent - Google Patents

Abstract

The present invention relates to agents that bind CD23 and that can be used in the treatment of inflammatory, autoimmune or allergic diseases.

Description

The CD23 wedding agent

The present invention relates to can be used for treating struvite, the special wedding agent of autoimmunization or allergic disorder.

CD (FC ε RII) is the II type molecule of a kind of C-lectin family, and this family also comprises lymphocyte homing (homing) acceptor (MEL-14) and endothelial leukocyte adhesion molecule-1 (ELAM-1).It is the acceptor that IgE is had low affinity.The mankind, polytype hematopoietic cell comprises the folliculus dendritic cell at their surface expression CD23, B cell, T cell and scavenger cell.The CD23 molecule also is found with soluble form and is present in the biological fluid.Solubility CD23 (sCD23) molecule is cut generation by the proteolytic enzyme enzyme to transmembrane receptor.CD23 has multi-effective active, comprises that mediated cell adheres to, and regulates the release of IgE and histamine, saves the B cell from apoptosis, regulates the growth of medullary cell.The ligands specific of these functional activities by CD23 relevant with cell or sCD23 be in conjunction with mediating, and the latter is with the mode that is similar to cytokine work (Conard, D.H immunology yearbook (Annu RevImmunol 8,623-645 1990); Delespesse, G. etc., senior immunology 49,149-191 (1991); Bonnefby, J.Y. etc., immunology latest developments (Curr OpinImmunol) 5,944-947 (1933)).

The expression of observing CD23 in many diseases associated with inflammation increases.In the patient's of chronic synovitis synovia examination of living tissue, identify CD23, in rheumatoid arthritis patient's serum and synovia, can measure the sCD23 of paranormal concentration.(Bansal, A.S., Oliver, W.Marsh, M.N.Pumphrey, R.S. and Wilson, P.B., immunology 79,285-289 (1973); Hellen, E.A., Rowlands, D.C., Hansel, T.T., Kitas, G.D., and Crocker, J.J., clinical pathology 44,293-296 (1991); Chomarat, P., Brioloay, J., Banchereau, J., and Miossec, D., Arthritis and Rheumatism (ArthritisRheum) 86,234-242 (1993); Bansal, A., etc., clinical experiment immunology 89,452-455 (1992); Rezonzew, R., and Newkirk, M.M., clinical immunopathology 71,156-163 (1994).In addition, the sCD23 level in rheumatoid arthritis patient's the serum is relevant with the situation of disease, relevant with serum rheumatoid factor (SRF) (Bansal, A.S., etc., clinical experiment similar rheumatism 12,281-285 (1994)).Pro-inflammatory cytokine be it seems in rheumatoid arthritis particularly important.And thought that TNF-α and IL-1 β suffer from play an important role in the arthritic joint (Brennan, F.M., Chantry, D., Jackson, A., Maini, R. and Feldman, M., lancet 2,244-247 (1989) in damage; Brennan, F.M., Maini, R.M. and Feldman M., Britain's rheumatology magazine 31,293-298 (1992)).

Also think the interaction of CD23-CD21 may in regulation and control IgE produces, work (Flores-Romo L etc., science 261 1038-1041 (1993); Aubry etc., nature 358,505-507 (1992)).

CD11b and CD11c participate in many cells and cell and cell and the interactional adhesion molecule of matrix.CD11b/CD18 and CD11c/CD18 (be respectively combining of CD11b and CD18, CD11c combines with CD18's) are in the news in conjunction with many parts, comprise CD54, Fibrinogen, factor X, LPS, concanavalin A and zymosan (Springer, T.A., nature 346,425-434 (1990)).Yet the effect of these binding molecules imperfectly understands.CD11b/CD18 and CD11c/CD18 also are hereinafter referred to as MAC-1 and p150,95.They are members's (being sometimes referred to as Leu-CAM, i.e. leukocyte adhesion molecule) of β 2 integral protein families.This family also comprises the LFA-1 (being also referred to as CD11a/CD18) among its member.

EP 0205405 claims and discloses and the monoclonal antibody of the lymphocyte receptor of the IgE (FC ε R) of human normal immunoglobulin E binding factor (IgE-BF) cross reaction and its derivative.

WO 93/04173 claims and discloses a peptide species that this polypeptide can combine with one of FCEL (low affinity IgE acceptor FC ε RII) or FCEH (high-affinity receptor FC ε RI), but can not combine with other FCEL or FCEH basically.That the abnormalism treatment of diseases is concluded is relevant with FCEL or FCEH specific polypeptide (prerequisite is that the FCEH specific polypeptide is can not be with FCEH crosslinked and induce histamine release).

EP 0269728 claims the monoclonal antibody that discloses human lymphocyte IgE acceptor.

EP 0259585 claims the monoclonal antibody that discloses the IgE surface receptor on the identification human B lymphocyte.

WO 93/02108 claims (primatised) antibody that discloses the Primates source that is used for the treatment of purposes.

The wedding agent that the inventor is surprised to find CD23 can be used for treatment or prevention various diseases, especially treatment or prevention sacroiliitis.Before the present invention, there is not evidence to support this purposes, although disclose the document of a large amount of discussion CD23.

According to the present invention, provide to be used for the treatment of or preventing inflammation the CD23 wedding agent of autoimmunization or allergic disorder.

This combination can by blocking-up CD23 with and the interaction of its bonded part play a role.Analyzed in vitro, for example radioimmunoassay can be used to study this barrier effect.

Wedding agent can be unpack format or as part of pharmaceutical compositions, needed is that it is a sterile form, and as a rule the specific wedding agent of CD23 can be used in the disclosed disease of treatment/prevention.

The inventor confirmed also that wedding agent is within the scope of the present invention treated in vivo or preventing inflammation or autoimmune disease in work.

Although for a long time its feature and reason are studied, under the condition that many these diseases are difficult to maybe can not effectively treat, this discovery is extremely important.This is especially for causing damage in the middle age to the people usually, and the sacroiliitis that can cause them to walk off from one's job prematurely is like this.Arthritic effective treatment has been become the target that many researchists remain unchanged for a long period of time.

Preferred wedding agent comprises antibody, its fragment or comprise antibody or its segmental artificial constructed thing or design are used for analog antibody or the artificial constructed thing of its segmental bonded.These wedding agents by Dougall etc. at Tibtech 12, discuss among the 372-379 (1994).

They comprise complete antibody, F (ab ') ₂Fragment, Fab fragment, Fv fragment, ScFv fragment, other fragment, CDR peptide and stand-in.These can be obtained by those skilled in the art/prepare.For example, available enzyme digests and obtains F (ab ') ₂With Fab fragment (by allowing the IgG molecule carry out stomach en-or papain enzymolysis respectively).The scope of " antibody " in describing below should be believed to comprise above-mentioned all possibilities.Can use recombinant antibodies.Antibody can be humanized, or chimeric.

The general preparation of humanized antibody is disclosed among the EP-A-0239400, and wherein CDRs is from the kind that is different from the antibody variable region framework.CDRs can come from rat or mouse monoclonal antibody.The variable region of the antibody that changes and the framework of constant region can be from people's antibody.When being applied to man-hour, this antibody from the people caused with by the inappreciable immunne response of the immunne response of people Chinese People's Anti-Japanese Military and Political College mouse or mouse antibodies.

Replacedly, antibody can be chimeric antibody, for example is the sort of type of describing among the WO 86/01533.

Chimeric antibody according to WO 86/01533 comprises an antigen binding domain and a NIg district.Antigen binding domain is antibody chain variable region or variable region of heavy chain.Usually, chimeric antibody comprises light chain and variable region of heavy chain.At the C-end, NIg district and antigen binding domain merge.The NIg district is generally NIg protein, and can be the enzyme zone, and this zone is from the protein with known binding specificity, from any protein of proteotoxin or next free genetic expression.But two districts of chimeric antibody can link to each other by means of the joint sequence of enzymolysis.

Antibody can be human IgG, IgG1 for example, IgG2, IgG3, IgG4; IgM; IgA; IgE or have rat or IgD of mouse variable region (chimeric) or CDRs (humanized).

Also can adopt Primates sourceization (Primatizing) technology, for example be disclosed among the WO 93/02108 those.

It will be understood by those skilled in the art that the place of describing specific-binding agent at this paper, also can adopt the derivative of these wedding agents.Term " derivative " comprises having one or more aminoacid replacement bases, has disappearance or the varient of the described wedding agent that inserts with respect to described wedding agent, yet that these varients still have is described in conjunction with active.These derivatives preferably have and the essentially identical aminoacid sequence of described wedding agent.

The degree of amino acid sequence identity for example can use the method (Smith and Waterman, applied mathematics progress, 482-489 (1981)) of " best coincideing " to find the similar best fragment between any two sequences to calculate.This comparison is so that amino acid similarity matrix and the mark that obtains reach and be the basis to the maximum, and for example Schwarz and Dayhof (1979) are at protein sequence and structure atlas, and Dayhof.M.O. describes among the Ed pp 353-358.

The degree of sequence identity is preferably at least 50%, more preferably is at least 75%.At least 90% or at least 95% sequence identity is most preferred.

Those skilled in the art will also be understood that high-caliber sequence identity is optional, because many amino acid can be usually by other aminoacid replacement, these amino acid have similar character, basically do not change or proteinic some character is had a negative impact, these are called as " conservative " amino acid sometimes and change.So the amino acid glycine, Xie Ansuan, leucine or Isoleucine can often be substituted by another kind (amino acid with aliphatic hydroxide radical side chain).Can be substituted by alternative other amino acid comprises:

Phenylalanine, tyrosine and tryptophane (amino acid) with aromatic side chain; Methionin, arginine and Histidine (amino acid) with basic side chain; Aspartic acid and L-glutamic acid (amino acid) with acid side-chain; L-asparagine and glutamine (amino acid) and halfcystine and methionine(Met) (amino acid) with sulfur-containing side chain with amide side chains.Therefore term " derivative " also can comprise the varient that comprises one or more this aminoacid sequences that change for " guarding " with respect to described sequence.

The present invention also comprises still having described fragment or derivatives thereof in conjunction with active wedding agent of the present invention.Preferred fragment is at least 10 amino acid longs, but they can be longer (for example reach 50 or reach 100 amino acid).

Wedding agent of the present invention is considered to can be used for treating or preventing the disease of various human, and they comprise sacroiliitis, lupus erythematosus, the Mashimotos thyroiditis, multiple sclerosis, diabetes, uveitis, dermatitis, psoriasis, urticaria, nephrotic syndrome, glomerulonephritis, inflammatory bowel disease, ulcerative colitis, Crohn disease, Si Yegelunshi (Sjogren) syndromes, allergic disease, asthma, rhinitis, eczema, GVH, COPD, insulitis, bronchitis (especially chronic bronchitis) or diabetes (especially type i diabetes).

They also can be used for studying the interaction between CD23 and the various part, for example CD23 and CD21, CD23 and CDllb, CD23 and CDllc, (it may be 76 for CD23 and 70-85KD endothelial cell protein, 80 or the 85KD endothelial cell protein), or the interaction of CD23 and 115KD endothelial cell protein (it is considered to relevant with the 70-85KD endothelial cell protein).One or more above-mentioned interactions are considered to take place in vivo.Especially preferably can block these interactional antibody or other wedding agent, because they are considered to be particularly suited for to reduce or alleviate the struvite effect by cytokine mediated.They also can be used for anti-B cell malignancies, for example chronic lymphocytic leukemia and hair shape (hairy) chronic myeloid leukemia.

Wedding agent of the present invention is specially adapted to treatment or prevention rheumatoid arthritis.Be not bound by theory possible explanation below having proposed.

In the synovial membrane of rheumatoid arthritis inflammation, express CD23 and β ₂Integral protein CDllb and CDllc make possible homotype interact and take place in this tissue.In addition, solubility CD23 molecule also is possible with them with combining of integral protein part through the diffusion of synovial membrane.Therefore, the interaction that relates to the CD23-CD11b/CD11c of positive activation ring can be present in the body.In the rheumatoid arthritis people, in a single day its soluble some diseases worsens and becomes chronic pathological mechanisms, and will support this hypothesis, promptly concentrates on joint, and scavenger cell itself can be by relating to CD23 molecule, β ₂Integral protein CD11b and CD11c, and pro-inflammatory cytokine TNF-α, the approach of IL-1 β and IL-6 continues and worsens inflammation.

The another kind of mechanism of action of anti-CD23 treatment can comprise blocking-up IgE immunne response.

In the former disclosed document, shown the antigen-specific inhibition that the interior therapeutic of rat has been caused the IgE generation with anti-CD23 antibody, this may interact by the necessary CD23-CD21 of differentiation fully that blocks the B cell of being finalized the design by IgE and carry out (Flores-Roma etc., science 261,1038-1041 (1993)).

The present invention also comprises this CD23 wedding agent of replying of blocking-up.

On structure, CD21 albumen is by 15 (Moore etc., the molecular cloning (2 type complement receptor) of the cDNA of the Epstein-Barr virus C3d acceptor of coding human B lymphocyte, institute of NAS reports 84:9194 (1987)) or 16 (Weis etc., the structure of the human B lymphocyte acceptor of C3d and Epstein-Barr virus and with other member's of the conjugated protein family of C3/C4 dependency, The Journal of Experimental Medicine 167:1047 (1988)) tool 60-75 amino acid whose repeating unit formed, this repeating unit is called as short consensus repeat (SCRs), then is to stride film district (24 amino acid) and 34 amino acid whose kytoplasm inner regions.Use has the CD21 mutant (Carel etc. of the outer SCRs disappearance of kytoplasm, C3d, the combination of g/EB virus receptor (CR2/CD21) part, the structural requirement journal of biological chemistry 265:12293 (1990) of internalization and virus infection), inventor's recent findings CD23 combines with SCRs 5-8 and 1-2 on the CD21.CD23 and SCRs 5-8 are combined into an interaction that is similar to lectin, relate to the carbohydrate on l-asparagine 295 and 370.On the contrary, the interaction that is combined into protein-protein of CD23 and SCRs 1-2 (Aubry etc., CD23 interacts Journal of Immunology 152:5806 (1994) with the new function kytoplasm outskirt that passes through the oligosaccharides that N end is connected on comprising CD21).The inventor tested out at present with CD21 bonded CD23 on CD21 other part (EBV, C3d, g and IFN-α) effect and to when having IL-4 to the influence of the generation of regulating Ig.Only the Toplink in EBV particle and EBV source suppresses and CD21 bonded CD23.And the EBV-peptide optionally reduces the generation of IgE and IgG4 and the generation of increase IgM.These data show in the presence of IL-4, with the optionally generation of mediator Ig of EBV binding site bonded CD23 on the CD21.

Still be not bound by theory, think that the present invention can obtain effective treatment by the de novo synthesis that suppresses pro-inflammatory cytokine.

This is with to use in the past antibody only to be used for the cytokine molecule that direct inhibition has been present in the Inflamed tissue different.

Should also be noted that the publication that predictive is arranged has in the prior art been enumerated lot of antibodies and lot of antibodies is said to be the possible disease of the treatment that can be used for it, but do not provide any reliable evidence or data most of possible combinations.Such publication is WO93/02108, and it relates generally to the preparation of specific chimeric antibody.

The present invention obviously is different from the wedding agent that these publications have been to provide specific molecular, is clearly illustrated with this wedding agent of explanation according to data provided herein to can be used for treatment or prevent some disease.

Wedding agent of the present invention especially also can be used for treatment or prevention allergic disorder, comprises the disease of non-IgE mediation.They can be used to treatment and prevention of ulcerative colitis.They also can be used to treatment and pre-anti-caking section ileitis.

Wedding agent of the present invention can be separately or and immunosuppressor, steroid for example, S-Neoral or antibody, for example antilymphocyte antibody is used in combination together, or more preferably with induce the tolerance agent, anti-autoimmunization agent or antiphlogistic drug, CD4+T cytostatics for example is as anti-CD 4 antibodies (preferably blocking-up or non-disappearance antibody), anti-CD8 antibody, the TNF antagonist as anti-TNF antibodies or tnf inhibitor, uses together as the TNF acceptor or the NSAIDs agent of solubility.

Wedding agent is supplied with as the part of aseptic pharmaceutically acceptable composition usually.This pharmaceutical composition can be any suitable form, and it depends on the required mode of it being granted patient.It can unitary dose form provide, and a part that can be used as medicine box provides.This medicine box (though optional) usually comprises operation instruction.

Wedding agent is usually with administered parenterally, intravenously for example, intramuscular or subcutaneous.Wedding agent generally comes administration by injection or infusion.For this purpose, wedding agent is formulated into the pharmaceutical composition that contains acceptable carrier on the medicine or thinner.Can use any suitable carrier or thinner, for example wait and ooze salts solution.Can add stablizer, for example metal chelator is to avoid the degraded of copper inducible.Suitable sequestrant is EDTA, DTPA or Trisodium Citrate.

Especially for the treatment of respiratory disorder, can provide by spraying per os or nose.

Especially for the treatment of skin disorder, they can be made emulsion or ointment.

They can be made drops etc. to be added drop-wise in the eye and be used for the treatment of disease disease as the membranous conjunctivitis in spring.

For Injectable solution, spendable vehicle comprises for example water, ethanol, polyvalent alcohol, glycerine and vegetables oil.

Pharmaceutical composition can comprise sanitas, solvating agent, stablizer, wetting agent, emulsifying agent, sweetener, tinting material, flavouring agent, salt, damping fluid, Drug coating or antioxidant.They also can comprise other therapeutic activity agent.

The optimal dose of material of the present invention can change, and it depends on as the disease that will treat and disorder, factors such as age that route of administration and treatment are individual and body weight.Do not limited by any concrete dosage, for example think for administered parenterally, for the general grownup of treatment, every day, the wedding agent of the present invention (form with above-mentioned part of pharmaceutical compositions exists usually) of 0.01-50mg/kg may suit.Can be 0.05-10mg/kg than appropriate dosage, as 0.1-2mg/kg.

This dosage can repeat suitably regularly.Administration is typically 1-7 time weekly.If have side effects, can reduce dosage and/or administration frequency.

Therefore the general unitary dose that is incorporated in the pharmaceutical composition is at least the 1mg wedding agent, conveniently is 1-1000mg.

The present invention includes and determine whether can be used for treating struvite with CD23 bonded specific formulation, the analytical method of autoimmunization or allergic disorder, it comprises: determine that can said preparation block the interaction of CD23 and CD11b, or the interaction of CD23 and CD11c, or the interaction of CD23 and CD21, or albumen or the interaction between the 115KD albumen of expressing on the endotheliocyte of CD23 and 70-85KD (for example 76KD, 85KD or 80KD).

This analytical procedure can be used for SCREENED COMPOUND or molecule by using the clone of expressing suitable cell.With CD11b/CD18, CD11c is preferred in these analytical procedures with the form of CD11c/CD18.CD11b/CD18 and CD11c/CD18 can be at the cell surface coexpressions.

Can use any suitable analytical procedure, protein nonprotein analytical method (for example interaction of analysing protein and chemical substance or carbohydrate) for example, protein-protein analysis method or protein-cell analysis method.

The present invention is described by embodiment with reference to accompanying drawing; Wherein

Fig. 1 illustrates and uses the preventative-therapeutic effect of anti-CD23 antibody to mouse arthritis;

Fig. 2 illustrates the effect of the established sacroiliitis of mouse being treated with anti-CD23 antibody, wherein adopts the repeatedly treatment of antibody;

Fig. 3 illustrates the effect of the established sacroiliitis of mouse being treated with anti-CD23 antibody, wherein adopts seance;

Fig. 4 a) and b) effect of the established sacroiliitis of mouse being treated with monoclonal anti CD23 antibody is described, wherein adopt repeatedly treatment;

Fig. 4 c) and d) explanation is with the F (ab ') of monoclonal anti CD23 antibody ₂Effect with the Fab fragment is treated the established sacroiliitis of mouse wherein adopts repeatedly treatment;

Fig. 5 a explanation and CD14 positive blood monocyte bonded CD23 liposome;

The protein of the various CD23 affinity purifications on Fig. 5 b explanation SDS-PAGE;

Fig. 6 illustrates and suppresses CD23 liposome and the activatory blood mononuclear cell bonded percentage ratio that uses some monoclonal antibody to obtain;

Fig. 7 illustrates combining of CD23 liposome and various transfectional cells;

Fig. 8 illustrates the interactional influence of various materials to CD23-CD11b and CD23-CD11c;

Fig. 9 explanation in monocyte because CD23 combines the generation of the nitrite that causes and the influence of oxidisability release (burst) with CD11b and CD11c;

Figure 10 illustrates that recombinant C D23 has increased monocytic production of cytokines especially with combining of CD11b and CD11c.

Embodiment

(adopted term " ip " in certain embodiments, " id " and " n ".These refer to " intraperitoneal " respectively, " subcutaneous " and " animal number ".) embodiment 1: use the prophylactic treatment of anti-CD23 antibody to mouse arthritis

DBA/1 male mice (8-12 age in week) is made it calm with 1: 10 dilution Fentanyl/Fluanisol ' Hypnorm ' ip of 0.1ml, and the ox II collagen type (CII) that 100mg is emulsified in the Freund ' s Freund's complete adjuvant (Difco) in the bottom of its tail is injected to subcutaneous.After the CII immunization 13 days the time, (Bioprocessing, UK) the anti-CD23 IgG of the rabbit of purifying (2mg/ mouse, intraperitoneal) comes Processing Test mouse (animal number=16, ■-■) by protein-A agarose with a shot.The anti-CD23 IgG of purifying contains the 3-5% specific antibody.

, provide among science 261 1038-1041 (1993) by Flores-Eomo L etc. about its detailed description of preparation.Briefly, rabbit polyclonal antibody is prepared into the human CD 23 [Kikutani etc., cell 47657 (1986)] of the clipped form of the amino acid/11 50-321 that is equivalent to total length CD23.In intestinal bacteria, produce the polypeptide of brachymemma, and by ion-exchange and gel-filtration purifying from the colibacillary precipitation that is washed.It has the molecular weight of 25KD, is injected in the rabbit behind the purifying.Resulting antiserum(antisera) is tested as the positive in the protein immunoblotting of ELISA and use recombinant human CD23.Then separate the IgG fraction by a-protein-agarose affinity chromatography.Control animals obtain from the IgG (2mg/ mouse) of the protein-A purifying of normal rabbit serum (animal number n=17, ●-----●).Observe the development of mouse arthritis clinical symptom every day, use as Williams etc., institute of NAS reports the middle description of 89 9784-9788 (1992) asymptomatic to the obvious swelling of 3=from 0=, and erythema and dyskinetic standard are estimated the severity of the disease on every claw; The recovery of limbs is analyzed by the number of the claw that calculating is damaged.Statistical study by use two tail t-test in (a) and the test of the distribution free Mann-Whitney in (b) is relatively respectively organized; ^*0.05 〉=p＞0.01, ^*0.01 〉=p＞0.005, ^{* *}0.005 〉=p.

(the average fate+sem of outbreak: test group is 20+0.7 in seizure of disease, control group is 20+0.5) or disease incidence rate (in two groups 100% for sacroiliitis mouse) go up indifference, this explanation does not influence with the processing of anti-CD23 antibody the inducing with inducing of the former protein antibodies of IgG anticol of disease to T cell proliferation period.Yet in the mouse with anti-CD23 antibody treatment, all clinical scores are all low, and (Fig. 1 a).Be apparent that most the obvious inhibition (Fig. 1 b) that claw recovers in anti-CD23 treatment group.These results show the strong influence of treatment to the long-term progress of disease.Embodiment 2: to established arthritic treatment (repeatedly treatment)

Induce sacroiliitis as what embodiment 1 described, monitor every day mouse can macroscopic inflammation progress.First day (the 1st day) of clinical inflammation and after two days (the 3rd day) three groups of mouse of any division are treated.Test mice obtains anti-CD23IgG (200 μ g/ per injections, n=6, the ■ ..-of protein-A purifying ...-.. ■; 400 μ g/ per injections, n=8, ■-■; The normal rabbit igg of control group mice acquisition protein-A purifying (200 μ g/ per injections, n=15, ●-----●).(Fig. 2 a) for evaluation severity of disease as described in Example 1.Inflammation is used from the 1st day of first pawl to becoming and is used the arthritic swelling that increases gradually of kind of calliper to estimate (Proctest 2T.Kroeplin Langenmesstechnik).Use the t-that is positioned tail for twice to test relatively each group, ^*0.05 〉=p＞0.01, ^*0.01 〉=p＞0.005, ^{* *}0.0005 〉=p.

Can see the obvious improvement to disease seriousness, it is relevant with dosage.The anti-inflammatory character of anti-CD23 IgG is clearly confirmed by the swelling of the pawl of the mouse of reduction acquisition Antybody therapy.Embodiment 3: to established arthritic treatment (seance)

Experimental design is similar to embodiment 2, except mouse only at arthritic first day, with the anti-CD23 IgG of 2mg/ murine protein matter-A purifying (n=10, the ■-■) or the normal rabbit igg of purifying (n=10, ●-----●) handle once.The recovery of clinical score and claw is estimated as embodiment 1.

The positive effect of the claw recovery of injection back acquisition for the first time showed the recovery for the joint at the 1st day, and carrying out a shot when sacroiliitis forms is enough to protect further advancing of disease (Fig. 3 b).Embodiment 4: to established arthritic treatment with monoclonal anti CD23

Suffer from arthritic DBA/1 mouse as acquisition as described in the top embodiment 1.When first signal of clinical disease occurring, mouse is divided into four groups arbitrarily, at the 1st day and the 3rd day monoclonal antibody processing (B3B4 with the CD23 of three kinds of dosage, by D.Conrad, professor Richmond gives, Virginia, USA, and it can be buied from Pharmingen.In Journal of Immunology 138:1845-1851 (1987)) in it is discussed; B3B4 25 μ g/ injection, n=4 zero ... zero, B3B4 50 μ g/ injection, n=4; ■-----■; B3B4 100 μ g/ injection, n=5, ■-■).Control group mice accept PBS (n=6, ●-----●).Estimating as described in Example 2 since the 1st day the clinical score and the increase of pawl thickness.

As use shown in the number of the clinical score of the obvious reduction that this treatment plan obtains and affected pawl, be enough to effectively treat in peritoneal injection 50 μ g B3B4 monoclonal antibodies.On the contrary, the severity of disease of the mouse of handling with 25 μ g monoclonal antibodies is not improved the positive treatment effect (Fig. 4) that obtains with 50 μ gB3B4 monoclonal antibodies.Demonstrate in the group that obtains peritoneal injection 50 or 100 μ g monoclonal antibody B3B4, from the 1st day, the swelling of suffering from arthritic claw did not just have Fig. 4 b that increases that the anti-inflammatory effect relevant with dosage of using B3B4 has been described.With observed similar, after seizure of disease, use the dosage of 25 μ g monoclonal anti CD23 to be Asia treatment (sub-therapeutic), and the increase of claw swelling is similar to control group (Fig. 4 b) with other clinical measurement method.Equally, after anti-CD23 IgG treatment, obtained the obvious reduction (data are not listed) of established arthritic severity with the polyclone of 50 μ g affinity purifications.

Confirmed by the histological examination of suffering from arthritic claw in improvement with anti-CD23 antibody (mono-clonal and polyclone) treatment back clinical severity.The mouse of handling demonstrates severity of disease and reduces, and the cell submerged that has under less tangible cartilage and bone injury and the synovial membrane jacket layer obviously reduces.And the ratio in the joint that is had a strong impact in the animal of anti-CD23 antibody treatment is compared with control group mice and is obviously reduced (0% and 94%), and keeps the ratio in the joint of normal configuration significantly to increase (80% and 0%).

This is confirmed in following table 1.

Table 1: the histopathology of claw: handle mouse and the joint is handled as Fig. 4.Described in methodology, tissue sample is cut into slices.

Treatment (no mouse)	Normally (%)	Slightly (%)	Medium (%)	Seriously (%)	Counting of the joint of being checked
						Anti--CD23 (n=6)	????80	????8	????12	????0	????59
Contrast (n=6)	????0	????0	????6	????94	????69

This table has shown for the comparison from the tissue sample in mouse of handling with anti-CD23 or the joint of obtaining from control group mice.Handle mouse as the mode described above relevant with Fig. 4.

Histopathology is estimated as follows:

After death take off the foot that first begins to produce inflammation, in stuck-at-0% (weight/volume) the buffer-type formalin, and decalcification among the EDTA in buffer-type formalin (5.5%).Then with the claw paraffin embedding, section is with phenodin and eosin dyeing.Use following standard recording tissue sample (3/claw): slight+minimum synovitis, cartilage loss and bone erosion are confined to the dispersive point; Medium=synovitis and appearance are corroded, but articulation structure is complete; Serious=widely synovitis and have the erosion of articulation structure damage.Evaluation is present in all joints in each section, determines that representative is normal, and is slight, the percentage ratio of medium or serious record.The increase of the claw thickness by the claw thickness when handling first day comes assess inflammation, measures and uses slide calliper rule (Proctest 2T, Kroeplin Langenmesstechnik).

By Fab and F (ab ') with the B3B4 monoclonal antibody ₂Fragment is handled the arthritic mouse of trouble and has been obtained to support specific other evidence of this treatment.Though it is effective to be not so good as complete IgG molecule, the Fab of B3B4 and F (ab ') ₂Fragment is still effective, thereby the Fc part that has proved antibody is not to be activity necessary (table 2).And, show very little activity or do not have activity (table 2) at the CD72 of bone-marrow-derived lymphocyte camber expression and the antibody of B220 molecule.In addition, with TNF-α (Williams etc., institute of NAS reports 36:9784-9788 (1992)) and CD5 (Plater-Zyberk etc., clinical experiment immunology 98:442-447 (1994)) antibody comparison shows that under our experiment condition, and the treatment of anti-CD23 antibody is the most effectively (table 2).

Table 2: to the comparison of the reaction of different treatment methods:, carried out therapeutic treatment by the peritoneal injection monoclonal antibody at the 1st day and the 3rd day that the clinical arthritis symptom occurs with II collagen type immune mouse.The reduction of the 5th day clinical value is calculated by the percentage ratio that contrasts with the isotype that is used for each experiment.

????Mab	The clone	Therapeutic dose	Isotype	The minimizing of clinical score (%)
					????CD23	????B3B4	??50μg×2 ??75μg×2 ??150μg×2	??Rat?IgG2a ??Rat?F(ab) 2??Rat?Fab	????74 ????52 ????56
????TNF-α	????1F3F3D4	??50μg×2	??Rat?IgM	????63
					????CD5	????TIB104	??200μg×2	??Rat?IgG2a	????30
????B220	????TIB146	??200μg×2	??Rat?IgM	????5
					????CD72	????TIB165	??200μg×2	??Mouse?IgG2b	????0

Monoclonal antibody and its segmental other data are provided in Fig. 4 c and 4d.These data are with following method basis:

It is subcutaneous that the II collagen type that 100 μ g are emulsified in complete Freund ' s adjuvant is injected to 8-12 DBA/1 male mice in age in week.

(after ± three weeks) are injected to the preparation of following monoclonal antibody in the mouse peritoneum when the signal of clinical disease occurring for the first time, (the 1st day and injection in the 3rd day)

B3B4 is complete, and IgG2a 50 μ g/ are injected to intraperitoneal, n=8

The Fab 150 μ g/ of B3B4 are injected to intraperitoneal, n=6

The F of B3B4 (ab ') ₂75 μ g/ are injected to intraperitoneal, n=7

M6 contrast IgG2a 50 μ g/ are injected to intraperitoneal, n=7

Observe mouse every day, write down severity (maximum/claw=3 of the clinical disease of every claw; Maximum/mouse=12).The record of clinical disease the results are shown among Fig. 4 c.

Killed mouse in arthritic the 10th day trouble, first is suffered from arthritic claw section, fixing, decalcification is to be used for histopathological examination.The interaction of CD23 and CDllb and CD23 and CDllc

Being reluctant to be bound by theory, is how to obtain top disclosed unexpected result owing to do not understand anti-CD23 antibody fully, so this may be because CD23 and CDllb and/or CDllc results of interaction.Embodiment 5-10 and accompanying drawing (seeing below) have confirmed this point.

In these embodiments, be incorporated into total length recombinant C D23 in the fluorescent lipid body and be proved with the COS cell and combine, wherein this COS cell is with the cDNA transfection of coding CD11b/CD18 or CD11c/CD18, and need not express the transfectant transfection of CD11a/CD18.Interaction between the COS cell of CD23-liposome and CD11b/CD18 or CD11c/CD18 transfection is respectively by anti-CD11b or anti-CD11c and the inhibition of anti-CD23 monoclonal antibody specificity.By with CD11b and CD11c on recombinant C D23 or anti-CD11b and the anti-CD11c monoclonal antibody triggering monocyte, cause to produce nitrite (NO ₂), oxidation products (H ₂O ₂) and the remarkable increase of pro-inflammatory cytokine (IL-1 β, IL-6 and TNF α) and confirmed part paired functional importance.The activity of these CD23 mediations is by CD11b, and the Fab fragment of the monoclonal antibody of CD11c and CD23 reduces.These results show that surface adhesion molecule CD11b and CD11c are the acceptor of CD23, and monocytic important activity is regulated in this new part pairing.

Following description is briefly described experimental design and the ultimate principle of the embodiment 5-10 of back:

With the recombinant full-lenght CD23 incubation of whole blood monocyte in being incorporated into the fluorescent lipid body, with flow cytometry analysis (Pochon, S. etc., The Journal of Experimental Medicine, 176,389-398 (1992)).What then show with two dyeing forms (being monocyte) with CD23-liposome bonded part by the CD14 positive cell.In order to prove that monocyte can combine with the CD23-liposome, blood mononuclear cell is divided into positive and negative crowd of the CD14 of CD14 by FACS, and (embodiment 5, and Fig. 5 a).The CD23-liposome only is proved to be and combines with the positive group of CD14 that (embodiment 5, and Fig. 5 a).Neither express film IgE and also do not express the CD21 (not shown) that is known as the CD23 part owing to find monocyte, then the not isoacceptor whether monocyte is expressed CD23 is studied.Make the molten born of the same parents of monocyte, purifying cells extract on the affinity column of recombinant soluble CD23 is arranged in coupling.SDS-PAGE that eluate is carried out and silver-colored staining analysis demonstrate and are about 80 and the band (embodiment 5, Fig. 5 b) of 160KD molecular weight.Tested the antigen of discerning this molecular weight ranges by the FACS that detects their inhibition CD23-liposomes and monocyte bonded ability, and (embodiment 6, Fig. 6) to be reported in the antibody of expressing in the monocyte.(embodiment 6, Fig. 6) with monocytic the combination all suppressing the CD23-liposome in varying degrees for anti-CD11b and anti-CD11c monoclonal antibody.Anti-CD13, anti-CD49d, (embodiment 6, Fig. 6) not have obvious effects for anti-CD21 (not expressing in monocyte) and anti-CD11a (the 3rd members of the β 2 integral protein families of adhesion molecule).Also tested MHC I type, the II type, the antibody of CD14 and D45 is to the influence of CD23-liposome bonded, and wherein all these antibody are all expressed at the monocyte camber.Yet they do not have any effect (not shown).Anti-CD18 monoclonal antibody produces the CD23 bonded is partly suppressed.This can owing to steric hindrance or induce anti-CD18Mab in conjunction with in CD11b and the conformational change in the CD11c molecule.Protein and the anti-CD11c (embodiment 5, Fig. 5 b) and the anti-CD11c/CD18 antibody (not shown) generation immune response of the cells of monocytic origin of wash-out from the CD23 affinity column.

For the α chain that confirms CD11b/CD18 and CD11c/CD11b is the acceptor of CD23, with CD18 cDNA will encode CD11b and CD11c full-length cDNA temporarily transfection in the COS cell.Confirmed that the transfectant of expressing CD11b/CD18 and CD11c/CD18 all combines with the CD23-liposome, and opposite with the transfectant of expressing CD11a/CD18 (embodiment 7, Fig. 7).This can by when with they homology and the CD11b that obtains relatively the time of CD11a and CD11c between the homology of height obtain explaining.Interactional specificity is by using anti-CD11b, and anti-CD11c and anti-CD23 monoclonal antibody suppress the combination of CD23-liposome and confirmed.Use the bhk cell of expressing CD11b/CD18 and CD11c/CD18 to obtain identical (not shown) as a result.As the interactional specific further evidence of CD23, the activatory blood mononuclear cell of the expression of shortage β 2 integral proteins that cause from the leukocyte adhesion deficiency patient's because in the transgenation of coding β subunit can not be in conjunction with CD23-liposome (not shown).These data have proved that together CD23 and CD11b and CD11c interact in normal people's monocyte and transfectant.

CD11b and CD11c are for participating in many cell-cells and the interactional adhesion molecule of cell-matrix.These embodiment show that CD11b/CD18 and CD11c/CD18 may demonstrate other adhesion function because they can combine with CD23.As by in dosage dependence method, factor X suppresses the CD23-liposome in conjunction with (embodiment 8, and the ability of surface expression that Fig. 8) and does not influence CD11b in the monocyte or CD11c is viewed, and as if CD23 can discern or identical epi-position approaching with factor X.Other part of test is without any effect.CD23 may be equivalent to C-type lectin in the interaction of it and CD11b and CD11c.EDTA by chelating to CD23 in conjunction with essential Ca ²⁺(embodiment 8, Fig. 8) and/or chelating reduce CD23 with the necessary divalent cation of combining of CD11b and CD11c (Altieri, D.C. Journal of Immunology 147,1891-1898 (1991)) for part and combine with monocytic.As if as by tunicamycin, rather than neuraminidase reduces CD23 and combines observed arriving with monocytic, and the interaction of CD23-CD11b/CD11c relates to carbohydrate rather than sialic acid.CD23 have in the extracellular amino acid triplet (Asp, Gly, Arg) (Kikutani, H. etc., cell 47,867-885 (1986)), it is the corecognition position of integral protein acceptor in the other direction.Therefore, tested at the polyclonal antibody of this triplet suppressing the influence of CD23 and monocyte bonded ability.Do not observe restraining effect, (embodiment 8, Fig. 8) to have confirmed not exist the inhibition of using the Fibrinogen acquisition.(embodiment 8, Fig. 8) with monocytic the combination partly to suppress CD23 at the lectin district of CD23 bonded IgE.As if these results show that CD23 plays a part the C type lectin of part identification sugar and protein structure, this remembers for CD23 and CD23 interaction viewed (Aubry, Journal of Immunologies such as J-P 152,5806-5813 (1994)) people.

In order to estimate the interactional importance of CD23 and CD11b or CD11c, whether the interaction that we have tested CD23-CD11b/CD11c can discharge short struvite medium, routine nitrogen oxide, H by monokaryon ₂O ₂And cytokine.Use recombinant soluble CD23, the normal monocytic triggering of adhesion activatory that anti-CD11b or anti-CD11c antibody cause has increased the activatory NO that means the NO approach ₂Generation (Moncada, S., Palmer, R.M.J. and Higgs, E.A. pharmacology comment 43,109-144 (1991)).CD23 is suppressed by the nitro arginine of the specific inhibitor of the Fab fragment of anti-CD23 monoclonal antibody and NO synthetic enzyme approach to the effect of the generation of nitric acid that (embodiment 9, and Fig. 9 a).Because at monocyte recombinant soluble CD23, anti-CD11b and anti-CD11c monoclonal antibody all make hydrogenation second ingot be oxidized to ethidium bromide, it seems that oxidation release also regulated (embodiment 9, Fig. 9 b) by CD11b and CD11c.This confirmation and expanded in monocyte anti-CD11b monoclonal antibody and induced oxidation to discharge (Trezzini, C., Sch ü epp, B., Maly, F.E. and Jungi, T.W. Britain hematology magazine 77,16-24 (1991)).CD23 and CD11b and CD11c combine with blood mononuclear cell in early stage specific C a ²⁺Relevant (not shown) flows.

Because the activatory scavenger cell is the important source of pro-inflammatory cytokine, we have measured recombinant soluble CD23 and anti-CD11b and the anti-CD11c monoclonal antibody influence to this type of production of cytokines of monocyte.Recombinant soluble CD23, anti-CD11b and anti-CD11c monoclonal antibody are IL-1 β, the effective stimulon of IL6 and TNF α.And (embodiment 10, Figure 10) to pass through to use anti-CD11b, anti-CD11c and anti-CD23 monoclonal antibody to confirm this inductive specificity.Interesting is that IL-1 and TNF α are the effective inductor (not shown) of CD23-liposome and monocyte bonded, this means a possible cytokine autocrine loop through CD11b and CD11c stimulation and adjusting.Embodiment 5a) the CD23-liposome (is seen Fig. 5 a) with monocytic combination of CD14-positive blood

With blood mononuclear cell with anti-CD14 monoclonal antibody (Becton Dickinson, Erembodegem, Belgium) and then with the sheep FITC link coupled F (ab ') of anti-mouse IgG and IgM ₂Antibody staining (Bioart, Meudon, France), before FACS classification (FACStar Plus, Becton Dickinson) became the CD14 positive and CD14 negative cells group, these two kinds of antibody were diluted in PBS, in 0.5%BSA and 0.05% sodiumazide.Then isolated cells is diluted in 0.5%BSA with CD23-liposome or contrast (with glycophorin A)-liposome, 0.1% sodiumazide, 2mM CaCl ₂, 140mM NaCl, 20mM Hepes is among the pH7 and in (Pochon, S. etc., The Journal of Experimental Medicine 176 389-398 (the 1992)) dyeing in 2 hours of 4 ℃ of following incubations.After the washing, by facs analysis cell (5,000 events/conditions).B) the proteinic apparent molecular weight of the blood mononuclear cell of CD23-affinity purification and with the immunoreactivity (seeing Fig. 5 b) of anti-CD11c monoclonal antibody

With lysate affinity purification on the CD23-post of blood mononuclear cell, the protein of wash-out is separated on the SDS-PAGE gel, and is transferred on the nitrocellulose membrane.Molecular weight marker is shown in the left side.With gel silver dyeing (road on the left side).Be incubated together with the antibody (intermediary road) of filter and isotype coupling or with anti-CD11c monoclonal antibody (BU-15, the road on the right), then the goat anti-mouse antibody with horseradish peroxidase is incubated (Kpl; Gaithersburg, Massachusetts).Embodiment 6 anti-CD11b and anti-CD11c monoclonal antibody reduce the (see figure 6) that combines of CD23-liposome and activatory blood mononuclear cell

Adhere on the plastics among the RPMI 1640 (Seromed, Berlin, Germany) that is added with 2mM glutamine and 10% heat-inactivated FCS (Flow laboratory, Irvine, England) by Ficoll and spending the night and to come the enrichment monocyte.Then when having the contrast (CTRL) (Becton Dickinson) of different monoclonal antibody (α CD) or isotype coupling, the activatory monocyte is incubated with the CD23-liposome, all tests are 10 μ g/ml.Anti-CD11a monoclonal antibody 25.3 and B-B15 obtain from Immunotech (Luminy, France) and Serotec (Oxford, Britain) respectively.Anti-CD11b monoclonal antibody 44 is from Serotec, mon.gran 1 is from Janssen (Beerse, Belgium), and Leu-15 is from Becton Dickinson (Erembodegem, Belgian) and (Bear-1) from Sera laboratory company limited (Sussex, Great Britain).Anti-CD11c monoclonal antibody 3.9 is from Serotec, and SL9 is from the Sera laboratory, and BU-15 is from Binding Site (Birmingham, Britain).Anti-CD13 (SJ1D1), anti-CD18 (BL5), anti-CD23 (mAb25) come anti-CD49d (HP 2.1) monoclonal antibody from Immunotech.Anti-CD21 monoclonal antibody BL13 is from Immunotech, and OKB7 is from Ortho, and BU-33 is from Dr.MacLennan (University of Birmingham, Britain), and HB-5 is from ATCC, OKB7 from OrthoDiagnostics System Inc (Raritan, NJ).Anti-CD14, anti-CD13, anti-CD16 and anti-CD-20 monoclonal antibody are from Becton-Dickinson.The cell facs analysis is measured average fluorescent strength (MFI).Listing representative experimental data, is 6.5 with the MFI of the painted cell of contrast-liposome, and painted with the CD23-liposome is 84.5.Calculate the inhibition percentage ratio that uses the linear MFI value of arithmetic according to following formula: Embodiment 7CD23-liposome and reorganization in the transfectant CD11b/CD18 and the α chain of CD11c/CD18 combine (see figure 7)

With the cDNA (Corbi, A.L., Miller, L J., O ' Connor, K, Larson, R.S. and Springer, T.A.EMBO J.6,4023-4028 (1987)) of coding CD11a in pCDNA1 again the clone (Invitrogen, San Diego, CA).CDNA (Corbi, A.L., Kishimoto, T.K. with coding CD11b, Miller, L J. and Springer, T.A. journal of biological chemistry 263,12403-12411 (1988)) and the coding CD18 cDNA (Kishimoto, T.K., O ' Connor, K., Lee, A., Roberts, T.M. and Springer, T.A. cell 48,681-690 (1987)) in pCDM8, clones (Seed, B., nature 329 840-842 (1987)) again.By at 20mM Hepes pH 7.4, (electroporation CA) (260V, 960 μ FD) carries out transfection with the aliquots containig of 20 μ g DNA in the COS7 cell for Bio-Rad, Richmond to use the gene pulse device among the 150mMNaCl.Carry out CD11a, the cotransfection of b or c and CD18 so that β 2 integral proteins at cell surface expression.Contrast transfection with strand, transfection is after 48 hours, and with the anti-CD11a of COS cell, the monoclonal antibody (contrast) of anti-CD11b and anti-CD11c monoclonal antibody or isotype coupling dyes.Then the goat anti-mouse antibody with the FITC mark dyes.By with the dyeing of separately monoclonal antibody, the cell of 10-15% demonstrates has expressed CD11a, b, c or CD18.Before with the dyeing of CD23-liposome, the COS cell of the positive transfection of CD18-is carried out the FACS classification to increase the percentage ratio of the cell of expressing β 2 integral proteins.Then with CD11a/CD18, CD11b/CD18 and CD11c/CD18 transfectant are with CD23-liposome (trace 2) or contrast (glycophorin A)-liposome (trace 1) insulation.By using anti-CD11b (trace 4), anti-CD23 (trace 5) and anti-CD11c (trace 6) monoclonal antibody suppress CD23-liposome and CD11b/CD18 and combining of CD11c/CD18 transfectant respectively and have confirmed the interactional specificity of CD23 and CD11b and CD11c.On the CD11a/CD18 transfectant, do not observe the combination of CD23-liposome, do not find the effect of anti-CD11a monoclonal antibody.Embodiment 8CD23-CD11b, the participation of interactional structural characterization (see figure 8) (a) carbohydrate of CD11c and divalent cation

With the activatory blood mononuclear cell of purifying with or do not use tunicamycin (10 μ g/ml) handled 48 hours with or do not use neuraminidase (0.1U/ml, both are all from BoehringerMannheim, Mannhein, Germany) processing 45 minutes.Then there is or do not exist EDTA (5mM; The figure on the top left side), Ca ²⁺Or Mn ²⁺(1-10mM; The figure on top the right) time cell is incubated together with CD23-liposome contrast-liposome.(b) factor X inhibition CD23 combines with monocytic

There is not or exists factor X (0.1-10U/ml; Sigma) (figure on the following left side), Fibrinogen (50 μ g/ml; Sigma), the reorganization ICAM-1 (this prepared in laboratory) of purifying, LPS (1 μ g/ml; Sigma), the zymosan (1mg/ml that is regulated by human serum; Sigma), IgE (50 μ g/ml; The Binding Site, Birmingham) or the polyclonal antibody of rgd peptide (1/500, ATCC) when (figure on following the right), the activatory blood mononuclear cell of purifying is incubated with the CD23-liposome.Use the facs analysis cell, measure MFI.Calculate inhibition percentage ratio as embodiment 6.Embodiment 9 is by combining with CD11b and CD11c, and recombinant C D23 has increased the generation of monocyte a. nitrite and the release of b. oxide compound specifically.

There are not or exist recombinant soluble CD23 (Graber P. etc. under 37 ℃, immunological method magazine 149 215-226 (1992)) (50ng/ml), anti-CD11a (clone 25.3), anti-CD11b (clone 44), during anti-CD11c (clone BU-15) monoclonal antibody (being 10 μ g/ml), monocyte is cultivated a, and 4 days or b spend the night.

(be shown in Fig. 9 a), according to Green etc., the therapy among immunology yearbook 2 199-218 (1984) is measured the NO in the culture supernatants, NO for the amount of the NO that estimates generation ₂ ^-And NO ₃ ^-The amount of stable final product.Fab fragment by anti-CD23 monoclonal antibody (mab25) (recording concentration is 10 μ g/ml) suppresses NO ₂ ^-Generation and use nitro arginine (N-Arg is 1mM) inhibition (Sigma) to confirm the NO of CD23 mediation ₂ ^-The specificity of increase.

Under 37 ℃, (molecular probe, Eugene OR) are incubated 30 minutes (Rothe G. etc., white corpuscle biology magazine 47 440-448 (1990)) together, and use facs analysis with activatory monocyte and hydrogenation second ingot.Listed the percentage ratio (seeing Fig. 9 b) of the increase of the monocytic red fluorescence that the quilt compared with untreated monocyte stimulates.Compare with reflecting the untreated monocyte that hydrogenation second ingot is oxidized to ethidium bromide, experienced the increase (Lacal P.M. etc., journal of biological chemistry 268707-712 (1990)) that monocyte that oxidation discharges demonstrates the red fluorescence signal.Only there is monocytic MFI value to be 159+/-10.Listed the mean value of 6 experiments+/-value of SD.Be used as positive control by known concanavalin A of inducing the breathing in the monocyte to discharge.By anti-CD11b (clone 44), the H that the Fab fragment of anti-CD11c (clone BU-15) and anti-CD23 (mAb25) monoclonal antibody (concentration records when being 10 μ g/ml) produces to the CD23 mediation ₂O ₂The inhibition of the increase that produces has confirmed the interactional specificity of CD23 and CD11b and CD11c.The binding specificity ground of embodiment 10 recombinant C D23 and CD11b and CD11c increases monocytic production of cytokines (see figure 10)

At 37 ℃, there are not or exist recombinant soluble CD23 (Graber P. etc., immunological method magazine 149 215-226 (1992)) (50ng/ml), anti-CD11a (clone 25.3), anti-CD11b (clone 44), anti-CD11c (clone BU-15), (this antibody of mAb 25-can obtain from Immunotech anti-CD23.In disclosed European patent application EP-A-0269728, it is discussed) monoclonal antibody, concanavalin A (Sigma) (being 10 μ g/ml), LPS (1ng/ml) (Sigam) or during PMA (5ng/ml) (Calbiochem. La Jolla, Canada) with the monocyte incubated overnight.By the cytokine in the specific ELISA mensuration culture supernatants.The limit of ELISA susceptibility is 0.05ng/ml (Ferrua etc. for IL-1 β, immunological method magazine 114 41-48 (1988)), for TNF α is 0.01ng/ml (Medgenix, Biotechnie, Rungis, F), for IL-6 be＜0.01ng/ml (Manie etc., European cytokine network 451-56 (1993)).By anti-CD11b (clone 44), the Fab fragment (measuring at 10gg/ml) of anti-CD11c (clone BU-15) and anti-CD23 (mAb25) monoclonal antibody has confirmed the interactional specificity of CD23 and CD11b and CD11c to the inhibition of the increase of the cytokine generation of CD23 mediation.Listed the mean value of 4 experiments+/-value of SD.Embodiment 11

By in mouse, produce monoclonal antibody except the standard method of using usefulness lymphoglandula rather than splenocyte from colibacillary recombinant soluble human CD 23 (25KD, amino acid/11 50-321).At external these antibody blockings the activity of human CD 23.Embodiment 12 ulcerative colitiss with having no side effect with anti-CD23 Mab to 3 The monkey administration has caused the improvement of subjective ight soil score.

Dosage be per 4 days by intramuscular injection 1mg.Embodiment 13

Toxic action is not observed in any test at this report.

Claims (20)

1. one kind is used for the treatment of or preventing inflammation, the wedding agent of the CD23 of autoimmunization or allergic disorder.

2. according to the wedding agent of claim 1, wherein wedding agent is a kind of antibody, and its fragment comprises antibody or comprises its segmental artificial constructed thing, the stand-in of any these wedding agents or derivative.

3. according to the wedding agent of claim 1 or 2, it is humanized or chimeric antibody.

4. according to the wedding agent of aforementioned each claim, it block in vivo CD23 and and its bonded part between interaction.

5. according to the wedding agent of aforementioned arbitrary claim, be used for the treatment of sacroiliitis, lupus erythematosus, systemic lupus erythematous, Mashimotos thyroiditis, multiple sclerosis, diabetes, uveitis, dermatitis, psoriasis, urticaria, nephrotic syndrome, glomerulonephritis, inflammatory bowel disease, ulcerative colitis, Crohn disease, the Si Yegelunshi (syndromes of Sjogren ' s), allergic disease, asthma, eczema, GVH, COPD, bronchitis, insulitis, rhinitis or diabetes.

6. according to each wedding agent of claim 1-4, be used for the treatment of sacroiliitis, transformation reactions, ulcerative colitis or Crohn disease.

7. according to the wedding agent of claim 6, be used for the treatment of rheumatoid arthritis.

8.CD23 the application of wedding agent in the medicine of the following disease of preparation treatment: sacroiliitis, lupus erythematosus, systemic lupus erythematous, Mashimotos thyroiditis, multiple sclerosis, diabetes, uveitis, dermatitis, psoriasis, urticaria, nephrotic syndrome, glomerulonephritis, inflammatory bowel disease, ulcerative colitis, Crohn disease, the Si Yegelunshi (syndromes of Sjogren ' s), transformation reactions asthma, eczema, GVH, COPD, bronchitis, insulitis, rhinitis or diabetes.

9. the application of claim 8 is used for the treatment of sacroiliitis, transformation reactions, ulcerative colitis or Crohn disease.

10. the application of claim 9 is used for the treatment of rheumatoid arthritis.

11. a pharmaceutical composition comprises CD23 wedding agent and pharmaceutically acceptable carrier.

12., comprise the wedding agent of the unitary dose of 1mg at least according to the composition of claim 11.

13. according to the pharmaceutical composition of claim 11 or 12, wherein wedding agent is an antibody.

14. according to the pharmaceutical composition of claim 13, wherein antibody is humanized or chimeric antibody.

15. as described earlier in this article pharmaceutical composition basically according to claim 11.

16., use or pharmaceutical composition wherein wedding agent and immunosuppressor, tolerance-induced dose, anti-autoimmunization agent or antiphlogistic drug combination according to the wedding agent of aforementioned each claim.

17. according to the wedding agent of claim 16, use or pharmaceutical composition, wherein wedding agent combines with CD4+T cytostatics or TNF antagonist.

18. according to the wedding agent of claim 17, use or pharmaceutical composition, wherein wedding agent combines with anti-CD 4 antibodies.

19. as described earlier in this article wedding agent basically according to claim 1.

20. a treatment is struvite, the method for autoimmune disorder or allergic disorder comprises the CD23 wedding agent of using medicinal significant quantity to patient.

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