CA2203363A1 - Binding agents to cd23 - Google Patents

Abstract

Binding agents to CD23 useful in the treatment of inflammatory, autoimmune or allergic diseases.

Description

WO ~6/12741 . PCT/EP95/04109 The present invention relates to particular binding agents which can be used in the treatment of inflammatory, autoimmune or allergic diseases.

CD23 (FC~RII) is a type ll molecule of the C-lectin family which also includes the Iymphocyte homing receptor (MEL-14) and the endothelial leukocyte adhesion molecule-1 (ELAM-1). It is a low affinity receptor for IgE. In humans a variety of haematopoietic cell types express CD23 on their surface, including follicular dendritic cells, B cells, T cells and macrophages. CD23 molecules are also foundin soluble forms in biological fluids. Soluble CD23 (sCD23) molecules are formedby proteolytic cleavage of transmembrane receptors. CD23 has pleiotropic activities including mediation of cell adhesion, regulation of IgE and histaminerelease, rescue of B cells from apoptosis and regulation of myeloid cell growth.These functional activities are mediated through the binding to specific ligands of cell-associated CD23, or sCD23, the latter acting in a cytokine-like manner (Conrad, D. H., Annu Rev lmmunol 8, 6234~451 990); Delespesse, G., et al., Adv Immunol 49, 149-191 (1991); Bonnefoy, J.Y., et al., Curr Opin Immunol 5, 944-947 (1 993)) Increased expression of CD23 has been observed in a number of inflammatory diseases. CD23 has been identified in synovial biopsies from patients with chronic synovitis, and sCD23 can be measured at conce~ lions exceeding the normal range in the serum and synovial fluid o~ patients with rheumatoid arthritis (Bansal, A.S., Oliver, W., Marsh, M.N., Pumphrey, R.S., and Wilson, P.B., Immunology 79, 285-289 (1993); Hellen, E.A., Rowlands, D.C., Hansel, T.T., Kitas, G.D., and Crocker, J.J., Clin Pathol 44, 293-296 (1991); Chomarat, P., Brioloay, J., Banchereau, J., & Miossec, P., Arthritis Rheum 86, 234-242 (1993);Bansal, A., et al., Clin Exp Immunol 89, 452455 (1992); Re~o"~e,lv, R., &

wo 96/12741 PCT/EP95104109 Newkirk, M.M., Clin Immunol Immunopathol 7'1, 156-163 (1994)). In addition, levels of serum sCD23 in rheumatoid allillilis patients are related to disease status and correlate with serum rheumatoid factor (Bansal, A.S., et al., Clin Exp Rheumafol 12, 281-285 (1994)). Pro-i"n~" " "~lOIy cytokines appear to be particularly important in rheumatoid ~I Ll ll ilis, and a central role for TNF~ and IL-1,B in the destruction of ~Illllilic joints has been postulated (Br~l1llcu, F.M., Chantry, D., Jackson, A., Maini, R., & Feldman, M., Lancet 2, 244-247 (1989);
B~:rlll~ll, F.M., Maini, R.M., & Fekll"an M., BrJRheumatol31, 293-298 (1992)).

It has also been postulated that CD23-CD21 il,Ler~-;Lions may play a role in thecontrol of IgE production (Flores-Romo L. et al, Science 261 1038-1041 (1993);
Aubry et al., Nature 358, 505-507 (1992)).

CD11b and CD11c are adhesion molecules that pa,lici~le in many cell-cell and cell-matrix i"le, ~.;lions. CD11 b/CD18 and CD11 c/CD18 (an association of CD11b and CD18 and of CD11c and CD18 respectively) have been reported to bind several ligands, including CD54, fibrinogen, factor X, LPS, Con A and zymosan (Springer, T.A, Nature 346, 42~434 (1990)). The role of these binding molecules is not however completely understood. CD11 b/CD18 and CD11c/CD18 are also known as MAC-1 and p150, 95 respectively. They are members of the ,B2 integrin family (sometimes known as Leu-CAM, ie leucocyte cell adhesion molecules). This family also includes LFA-1 amongst its members (also known as CD11 a/CD18).

EP 0205405 purports to disclose Mabs to Iymphocyte cellular receptors for IgE
(FC~R) cross reacting with human immunoglobulin E binding factor (IgE-BF), and derivatives thereof.

W o 96112741 PCT~EP95104109 WO 93/04173 purports to disclose a polypeptide which is capable of binding to ~ one of FCEL (low affinity IgE receptor FC~RII) or FCEH (high affinity receptor FC~RI) but which is substantially i"capable of binding to the other of FCEL or FCEH. Treatment of an allergic disorder is alleged with a FCEL or FCEH specific polypeptide (provided the FCEH specific polypeptide is incapable of crosslinkingFCEH and inducing I ,ista" ,i, le release).

EP 0269728 purports to disclose Mabs to the human Iymphocyte IgE receptor.

EP 0259585 purports to disclose Mabs recognising a surface receptor for IgE
(FC~R) on human B Iymphocytes.

W O 93/02108 purports to disclose prill~lised antibodies for therapeutic use.

The present inventors have surprisingly discovered that binding agents to CD23 can be of utility in the treatment or prophylaxis of various diseases and in particular in the treatment or prophylaxis of arthritis. Prior to the present invention no data has been presented which would support such a utility, despite the pùblication of a large number of papers in which CD23 has been discussed.
Acconiil ,y to the present invention, there is provided a binding agent to CD23 for use in the treatment or prophylaxis of inflammatory, autoimmune or allergic diseases.

The binding agent may function by blocking the interaction between CD23 and a ligand which binds to it. In vitro assays e.g. radio-immune assays may be used to study such a blocking effect.

The binding agent may be in isolated form or as part of a pharmaceutical composition. Desirably it is in sterile form. Generally speaking a binding agentwhich is specific for CD23 is useful in the treatmenVprophylaxis disclosed.

The present inventors have del "o, ~ d that binding agents within the scope of the present invention work in vivo in treatment or prophylaxis of inflalllnl~lo~y or autoimmune diseases.

This demonstration is of great signihcance, given the fact that many of these diseases are difhcult or impossible to treat effectively, despite long standing research into their nature and causes. This is particularly the case in respect of al lhl iLis, which often affects people in middle age and can cause them to give up work prematurely. An effective treatment of a,ll,rilis has been a long standing goal of many research groups.
Preferred binding agents include antibodies, fragments thereof or artificial constructs co")prising antibodies or fragments thereof or artificial constructs designed to mimic the binding of antibodies or fragments thereof. Such binding agents are discussed by Dougall eta/in Tbtech 12, 372-379 (1994).
They include complete antibodies, F(ab')2 fragments, Fab fragments, Fv fragments, ScFv fragments, other r, ayl "e, ILs, CDR peptides and mimetics. These can be obtained/prepared by those skilled in the art. For example, enzyme digestion can be used to obtain F(ab')2 and Fab fragments (by subjecting an IgG
to molecule to pepsin or papain cleavage respectively). References to "antibodies" in the following description should be taken to include all of the possibilities mentioned above.

W 096/12741 PCT~P95104109 Recombinant antibodies may be used. The antibodies may be humanized; or chimaerised.

A typical ~r~pa,~lion of a humanised antibody in which the CDRs are derived from a different species than the framework of the antibody's variable do~ains is disclosed in EP-A-0239400. The CDRs may be derived from a rat or mouse monoclonal antibody. The framework of the variable domains and the constant don)~ s of the altered antibody may be derived from a human antibody. Such a humanised antibody elicits a negligible immune response when administered to a human compared to the immune response mounted by a human against a rat or mouse antibody.

Alternatively the antibody may be a chimaeric anlibod)/ for instance of the typedescribed in WO 86/01533.
A chimaeric antibody according to WO 86/01533 co~,urises an antigen binding region and a non-immunoglobulin region. The antigen binding region is an antibody light chain variable domain or heavy chain variable domain. Typically the chimaeric antibody co"~prises both light and heavy chain variable don,ai"s.
The non-immunoglobulin region is fused at its C-terminus to the antigen binding region. The non-immunoglobulin region is typically a non-immunoglobulin protein and may be an enzyme region a region derived from a protein having known binding specificity from a protein toxin or indeed from any protein expressed by a gene. The two regions of the chimaeric antibody may be connected via a cleavable linker sequence.

The antibody may be a human IgG such as IgG1 IgG2 IgG3 IgG4; IgM; IgA; IgE
or IgD carrying rat or mouse variable regions (chimaeric) or CDRs (humanised).

P, i" ~ ,g techniques may also be used, such as those disclosed in WO93/021 08.

As will be appreciated by those skilled in the art, where specihc binding agentsare described herein, derivatives of such agents can also be used. The terrn "derivative" includes variants of the agents described, having one or more aminoacid substitutions, deletions or insertions relative to said agents, whilst still having the binding activity described. P~erer~bly these derivatives have substantial amino acid sequence identity with the binding agents specified.
The degree of amino acid sequence identity can be c~lc~ ted using a program such as "besffit" (Smith and W~le"nall, Advances in Applied Mathematics, 482-4~9 (1981)) to find the best segment of silllilalily between any two sequences.
The alignment is based on maximising the score achieved using a matrix of amino acid similarities, such as that described by Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M.O., Ed pp 353-358.

Pl~rerably the degree of sequence identity is at least 50% and more prererably it is at least 75%. Sequence identities of at least 90% or of at least 95% are mostpreferred.

It will nevertheless be appreciated by the skilled person that high degrees of sequence identity are not necessarily required since various amino acids may often be substituted for other amino acids which have similar properties withoutsubstantially altering or adversely affecting certain properties of a protein. These are sometimes referred to as "conservative" amino acid changes. Thus the amino acids glycine, valine, leucine or isoleucine can often be substituted for one another (amino acids having aliphatic hydroxyl side chains). Other amino acids which can often be substituted for one another include:

CA 02203363 l997-04-22 wo 96Jl2741 PCTAEP95/04109 phenylalarline, tyrosine and tryptophan (amino acids having aromatic side chains); Iysine, arginine and histidine (amino acids having basic side chains);
aspartate and glutamate (amino acids having acidic side chains); asparagine and glutamine (amino acids having amide side chains) and cysteine and methionine (amino acids having sulphur containing side chains). Thus the term "derivative"
can also include a variant of an amino acid sequence cornprising one or more sùch "conservative" changes relative to said sequence.

The presel1L invention also includes fragments of the binding agents or of the present invention or of derivatives thereof which still have the binding activity described. Preferred fragn1ents are at least ten amino acids long, but they may be longer (e.g. up to 50 or up to 100 amino acids long).

The binding agents of the present invention are believed to be useful in the I,e~Lmenl or prophylaxis of several human diseases including arthritis, lupus erythematosus, Mashimotos thyroiditis, multiple sclerosis,diabetes, uveitis, dermatitis, psoriasis, urticaria, nephrotic s~/"d, o" ,e, glomerulonephritis, il irla" ". Iatory bowel disease, ulcerative colitis, Crohn's disease, Sjogren'ssyndrome, allergies, asthma, rhinitis, eczema, GVH, COPD, insulitis, bronchitis (particularly chronic bronchitis) or diabetes (particularly Type 1 diabetes).

They may also be useful in studying the i"lerd~;lions between CD23 and various ligands e.g. between CD23 and CD21, between CD23 and CD11b, between CD23 and CD11c, between CD23 and a 70 to 85 KDa endothelial cell protein (which may be a 76, 80 or 85 KDa endothelial cell protein) or between CD23 and a 115 KDa endothelial protein (which is believed to be related to the 70 to 85KDa endothelial protein). One or more of the above interactions are believed to occur, in vivo. Antibodies or other binding agents which are capable of blocking these interactions are particularly prer~" ~d since it is believed that they may be especially suitable for reducing or alleviating cytokine mediated infl~r~ al~ry effects. They may also be useful against B~ell malignancies such as chronic Iymphocytic leukaemia and hairy cell leukaemia.

Binding agents of the p,~se"l invention are particularly applicable for use in the treatment or prophyla~is of rheumatoid ~l ll lliLis. Without being bound by theory the following possible explanations are put forward:-ln the rheumatoid arthritis inflamed synovium macrophages express both CD23 and the ,B2 integrins CD11b and CD11c allowing possible homotypic interactions to take place in this tissue. In addition diffusion of soluble CD23 molecules through the synovium and their binding to the integrin ligands is also possible.Therefore CD23-CD11b/CD11c interactions involving a positive activation loop could exist in vlvo. If present in rheumatoid a~ L hl ilis patients it may explain some of the pathogenic mechanisms of disease exacerbation and ~ ~, u~ licily and would support the hypothesis that once localised to the joints macrophages themselves can maintain and exacerbate inflar"" ,~lion via a pathway involving CD23 molecules ~2-integrins CD11 b and CD11 c as well as pro-inflammatory cytokines TNF oc IL-1 ~ and IL~.
An alternative mechanism of action of anti CD23 therapy could involve the blocking of an IgE immune response.

In previously published work it has been shown that in vivo treatment of rats with anti-CD23 antibody resulted in antigen-specific inhibition of IgE production probably by blocking the CD23-CD21 interactions necessary for complete differentiation of IgE-commilted B cells (Flores-Romo et al., Science 261 1038-1041 (1993)).

W O96112741 PCT~EP~5/04109 The present invention also includes binding agents to CD23 which block such a response.

Structurally, the CD21 protein is composed of an extracellular domain of 15 (Moore et a/, Molecular cloning of the cDNA encoding the Epstein Barr Virus C3d receptor (complement receptor type 2) of human B Iymphocyte, Proc Natl Acad Sci USA 84: 9194 (1987)) or 16 (Weis et a/, Structure of the human B Iymphocyte receptor for C3d and the Epstein Barr Virus and relatedness to other members of the family of C3/C4 binding proteins, J E)~p Med 167: 1047 (1988)) repetitive units of 60 to 75 amino acids, named short consensus repeats (SCRs), followed by a transmembrane don~ain (24 amino acids) and an intracytoplasmic region of 34 amino aci~s. Using CD21 mutants bearing deletions of extracytoplasmic SCRs (Carel et a/, Structural requirements for C3d,g/Epstein Barr Virus receptor (CR2/CD21) ligand binding, intemalization, and viral infection J Biol Chem 265:
12293 (1990)), the p~se~l inventors have recently found that CD23 binds to SCRs 5-8 and 1-2 on CD21. The binding of CD23 to SCRs 5-8 is a lectin-like ir~le,~ion, involving carbohydrates on Asn 295 and 370. In contrast, CD23 binding to SCRs 1-2 is a protein-protein i"ler~ion (Aubry ef a/, CD23 interacts with a new functional extracytoplasmic domain involving N-linked oiigosaccharides on CD21, J Immunol 152: 5806 (1994)). The present inventors have now tested the effect of the other ligands of CD21 (EBV, C3d,g and IFN oc) on CD23 binding to CD21 and on the regulation of lg production in the presence of IL~. Only EBV particles and an EBV-derived peptide were able to inhibit CD23 binding to CD21. Moreover, the EBV-peptide selectively decreased IgE
and IgG4 production and increased IgM production. These data indicate that CD23 binding to the EBV-binding site on CD21 selectively regulates human Ig production in the presence of IL4.

CA 02203363 l997-04-22 W 096/12741 PCT~P95/04109 Again without being bound by theory, it is believed that the present invention allows effective trecL~enls to be achieved by suppressing the de novo syntnesis of pro-inflaml l l~loly cytokines.

This co"ll~sl~ with previous uses of antibodies simply to directly neutralise the cytokine molecules already present in inflamed tissues It should also be noted that there are speculative publications in the art listing large numbers of antibodies as well as large numbers of possible diseases which the antibodies are said to be useful in treating, but not providing any sound evidence or data for most of the possible combinations. One such publication is WO93/02108 which is primarily directed to the production of particular chimaericar,liL,odies.

The present invention is clearly distinguished from such publications by providing . . .
binding agents to particular rnolecules which are clearly indicated to be of utility in the treatment or prophylaxis of certain diseases in view of the data and explanations provided herein.

Binding agents of this invention are also of particular use in the treatment or prophylaxis of allergic diseases, including non-lgE mediated diseases. They may be used in the treatment and propylaxis of ulcerative colitis. They may also be used in the treatment and prophylaxis of Crohn's disease.

The binding agents of the present invention may be used alone or in combination with immunosuppressive agents such as steroids, cyclosporin, or antibodies such as an anti-lymphocyte antibody or more pl ereraL)ly with a tolerance-inducing, anti-autoimmune or anti-inflammatory agent such as a CD4+T cell inhibiting agent e.g. an anti-CD4 antibody (prererably a blocking or non-depleting antibody), an W O 9~/12741 PCT~EPg5/04109 anti-CD8 antibody, a TNF a"lauo, lisL e.g. an anti-TNF antibody or TNF inhibitor- e.g. soluble TNF receptor, or agents such as NSAlDs.

The binding agent will usually be supplied as part of a sterile, pharmaceuticaliy acce~lable composition. This pharmaceutical composition may be in any suitable form, depending upon the desired method of ad~ eri~ lg it to a patient. It may be provided in unit dosage form and may be provided as part of a kit. Such a kitwould norrnally (although not necessarily) include instructions for use.

Binding agent ad"1inisl,~lions are generally given parenterally, for example intravenously, intramuscularly or sub-cutaneously. The binding agents are generally given by injection or by infusion. For this purpose a binding agent isformulated in a pharmaceutical composition containing a ph~l "laceutically acceptable carrier or diluent. Any appru~, idLe carrier or diluent may be used, for example isolu,lic saline solution. Stabilizers may be added such as a metal chelator to avoid copper-induced cleavage. A suitable chelator would be EDTA, DTPA or sodium citrate.

They may be given orally or nasally by means of a spray, especially for treatment of respiratory disor~ler~.

They may be formulated as creams or ointments, especially for use in treating skin disorders.

2~ They may be formulated as drops, or the like, for administration to the eye, for ~ use in treating disorders such as vernal conjunctivitis.

For injectable solutions, excipients which may be used include, for example, water, alcohols, polyols, glycerine, and vegetable oils.

wo 961l274l PCT/EP9~/04109 The pharrnaceutical con"~osilions may contain preserving agents, sol~ c;llg agents, stabilising agents, wetting agents, emulsifiers, sweelenel~, colourants,odourants, salts, bu~fers, coating agents or antioxid~, lL~. They rnay also contain other therapeutically active agents.

Suitable dosages of the substance of the present invention will vary, depending upon factors such as the disease or disorder to be treated, the route of administration and the age and weight of the individual to be treated. Without being bound by any particular dosages, it is believed that for instance for parenteral ad",inial,~lion, a daily dosage of from 0.01 to 50 mg/kg of a bindingagent of the present invention (usually present as part of a pl,~llllaceutical composition as in~ ted above) may be suitable for treating a typical adult.
More suitably the dose might be 0.05 to 10 mgA~g, such as 0.1 to 2 mg/kg.
This dosage may be repeated as often as appl~pli~le. Typically ad"~illi~l,~lio"
may be 1 to 7 times a weeks. If side effects develop the amount and/or frequencyof the dosage can be reduced.

A typical unit dose for incorporation into a pharmaceutical composition would thus be at least 1 mg of binding agent, suitably 1 to 1000mg.

The present invention includes within its scope an assay for detemmining whetheror not a particular agent which binds to CD23 may be useful in the treatment of an inflammatory, autoimmune or allergic disease comprising: determining whether or not the agent is capable of blocking the interaction between CD23 andCD11 b, or the interaction between CD23 and CD11 c, or the interaction between CD23 and CD21, or the i"lerac;Lion between CD23 and a 70 to 85 KDa (such as a 76 KDa, 85 KDa or 80 KDa) or a 115 KDa protein expressed on endothelial cells.

This assay can be used for ~aee,1il ,9 compounds or molecules by using cell lines e~,~ss;"g the appropriate molecules. Preferably CD11 b is used in these assays as CD11 b/CD18 and CD11 c is used as CD11c/CD18. CD11b/CD18 and CD11c/CD18 can be co-expressed on cell surface.

Any ap~)ro~ri~le assay technique can be used e.g. protein-non protein assays (e.g. assaying the interaction of proteins with chemicals or sugars) protein-protein assays or protein cell assays.

The present invention will now be described by way of eAdl ",,~le only with reference to the accompanying drawings; wherein:

1~ FIGURE 1 illustrates the effect of preventative treatment against arthritis on mice using an anti-CD23 antibody;

FIGURE 2 illusl,~les the effect of the treatment of mice with an anti-CD23 al1liL,ody in respect of established ~llhliLi~ where multiple treatments of the antibody are used;

FIGURE 3 illustrates the effect of the treatment of mice with an anti-CD23 antibody in respect of established arthritis where a single treatment is used;
FIGURES 4 a) and b) illustrate the effect of the treatment of mice with a monoclonal anti-CD23 antibody in respect of established arthritis where multiple treatments are used;

W O 96/12741 PCTA~P95/04109 FIGURES 4 c) and d) illustrate the effect of the treatment of mice with F(ab')2 and Fab fragments of a monoclonal anti-CD23 antibody in respect of established a, lhrilis where multiple treatments are used;

FIGURE 5a illustrates CD23-liposomes binding to CD14 positive blood mononuclear cells;

FIGURE 5b illustrates various CD23 affinity purihed proteins on SDS-PAGE gels;
FIGURE 6 illustrates the percentage inhibition of CD23-liposome binding to activated blood monocytes obtained using certain monoclonal antibodies;

FIGURE 7 illustrates the binding of CD23 liposomes to various transfected cells;

FIGURE 8 illustrates the effect of various substances on CD23-CD11b and CD23-CD1 1 c il ~le~ ~.;lion;
FIGURE 9 illustrates the effects on nitrite production and oxidative burst in monocytes caused by CD23 binding to CD11 b and CD11 c; and FIGURE 10 illustrates that the binding of recombinant CD23 to CD11b and CD11 c specifically increases cytokine production by monocytes.

EXAMPLES

W O96112741 PCT~EP95/04109 (In some of the following examples the terms "ip", "id" and "n" are used. These mean "intraperitoneal", "i~ dermal" and "number of animals" respectively.) EXAMPLE 1 - Preventative Treatment of Mice a~ai. ,sl Arthritis usinq Anti-CD23 Antibody Male DBA~1 mice (8-12 weeks old) were sedated with 0.1 ml of a 1:10 dilution of Fentanyl/Fluanisol 'H~/~llullll' ip and injected intradermally at the base of the tail with 100 mg bovine type ll collagen (Cll) emulsified in Freund's complete adjuvant (Difco). On day 13 post-CII immunisation, test mice were treated with one single injection of rabbit anti-CD23 IgG purified by protein-A Sepharose (Bio-Processing, UK) (2 mg/mouse, ip) (n=16, ~ ~) . The purified anti-CD23 IgG co~ ius 3-5% specific antibody.

A detailed description of its production is given in Flores-Romo L. et al., Science 261 1038-1041 (1993). Briefly, a rabbit polyclonal antibody was raised to a truncated form of human CD23 ~o"~sponding to amino acids 150 to 321 of full length CD23 [Kikutani et a/., Cea 47 657 (1986)]. The truncated polypeptide was produced in E coli and purified from a washed pellet of E c~li by ion-exchange and gel rilLI~lio". It had a molecular weight of 25kD and after puliric~lion wasinjected into a rabbit. The resultant antiserum tested positive in both an ELISAand a protein immunoblot with reco",bi, lant human CD23. An IgG fraction was then isolated by protein A-Sepharose affinity chromatography. Control animals received protein-A purified IgG from normal rabbit serum (2 mglmouse) (n=17, ~- - - - -- ). Mice were inspected daily for development of clinical symptoms of arthritis. The severity of disease was scored on each paw using a scale from O = no sylllptoms to 3 = pronounced swelling, erythema and impairment of movement as described in Williams et a/., Proc. Nafl. Acad. Sci.
L/SA 89 9734-9788 (1992); limb recruitment was assessed by counting the number of affected paws. Groups were compared by statistical analysis using the two-tailed t-test in (a), and the non-parametric Mann-Whitney test in (b); ~0.05 >p>0.01,~0.01 >p>0.005,~0.005'p.

No dir~er~,)ce in disease onset was observed ("~e-lial1 day of onset + sem: 20 +0.7 in test group and 20 0.5 in the control group) or incidence of disease (100%arthritic mice in both groups), demo, lsll ~li"g that treatment with anti-CD23 antibody had no effect on disease induction at the time of T-cell proliferation and IgG anti coll~en antibody induction. However, the overall clinical scores were lower in mice treated with anti-CD23 antibody (Fig. 1a). Most notable was the marked suppression of limb recruitment in the anti-CD23 treated group (Fig.1 b).These results indicate a strong influence of the treatment on the long-term progression of the disease.

EXAMPLE 2 - Treatment of Established Arthritis (multiple treatments) Arthritis was induced as ~lis~l~se~ for Example 1 and miGe were monitored daily for development of visible inflammation. Tre~l"~e~ ~l was administered on first day of clinical inflammation (day 1) and two days later (day 3) on randomly divided three groups of mice. Test mice received protein-A purified anti-CD23 IgG (200 ~g/injection, n=6, ~ -- ----; 400 ,ug/injection, n=8 ~ ~; control mice received protein-A purified normal rabbit IgG (200 ~g/injection, n=15,.
~- - - - --). Severity of disease (Fig. 2a) was assessed as described in Example1. Inflammation was assessed by the incremental swelling from day 1 of the firstpaw to become arthritic measured using callipers (Proctest 2T. Kroeplin Langenmesstechnik). Groups were compared using the two-tailed t-test, ~0.05 p~0.01, ~0.01 ~p>0.005, ~0.0005>p.

Marked improvement in disease severity can be seen, which is dose related. The anti-inflammatory property of anti-CD23 IgG is clearly demonstrated by reduced paw swelling in the group of mice receiving antibody treatment.

W O96/12741 PCT~EP95/04109 EXAMPLE 3 - Treatment of Established Arthritis (single treatment) Experiment design was similar to Exampie 2 with the exception that mice were treated only once, on day 1 of arthritis, with 2 mg/mouse of protein-A purified anti-CD23 IgG (n=10, ~ ~) or purified normal rabbit IgG
(n=10, ~- - - --).Clinical scores and limb recruitment was assessed as for Example 1.

The significant effect on limb recruitment obtained after one injection on day 1demonsl~;3les that in terms of joint recruitment a single injection administeredwhen al ll ,rilis is already established is suFficient to protect against further disease progression (Fig. 3b).

EXAMPLE 4 - Treatment of E:jlablisl ,ed Arlhritis with Monocional Anti-CD23 Arthritic DBA/1 mice were obk.;. ,ed as described above for Example 1. On first sign of clinical disease mice were separated l~ndolllly in four groups and treated on days 1 and 3 with three doses of "lo"oclonal antibody to CD23 (B3B4, kindly donated by Professor D. Conrad, Richmond, Virginia, USA and obtainable from Pharmingen. It is ~lisal.ssed in J Immunol 138: 1845-1851 (1987)); B3B4 25 ~g/injection, n=4 O--------- O, B3B4 50 ~lg/injection, n=4; ~ ~ ; B3B4 100 llg/injection, n=5. ~ ~). Controi mice received PBS (n= 6 ~- - -----).
Clinical scores and increment in paw thickness from day 1 were assessed as described for Example 2.

Intraperitoneal injections of 50 ~9 of B3B4 monoclonal antibody were sufficient for effective therapy as shown by the significant decrease in ciinical scores and the number of affected paws obtained using this treatment regimen. In contrast, the disease severity of mice treated with 25 1l9 monoclonal antibody did not improve the positive therapeutic effect obtained with 50 ~19 B3B4 monoclonal wc~ 96112741 PCT/EPg5/04109 antibody (Fig. 4). The dose-related anti-i"nam" ~atory effect of B3B4 administration is illustratecl in Fig. 4b which shows that swelling of the arthritic paws did not increase from day 1 in the groups receiving ip injections of 50 or 100 llg ",onoclonal antibody B3B4. Similarly to what was observed with the other clinical measurements, doses of 25 ~g monoclonal anti-CD23, ad",i"i~er~d after disease onset, were sub-therapeutic and the increments in paw swelling were similar to controls (Fig. 4b). Likewise, a significant reduction in severity of the established arthritis was obtained after treatment with 50 ,ug affinity-purifiedpolyclonal anti-CD23 IgG (data not shown).
The improvement in clinical severity following treatment with anti-CD23 antibody(both monoclonal and polyclonal) was co"ri""ed by histological exa",i"dlion of the arthritic paws. Treated mice showed reduced severity of disease with less apparent destruction of cartilage and bone and a marked decrease in cellular infiltration of the sublining layer of the synovium. Moreover, the proportion ofseverely affec~ed joints was significantly lower in anti-CD23 antibody treated animals in comparison to control mice (0 % vs 94 %), whereas the proportion of joints maintaining nommal structure was significantly increased (80 % vs 0 %).

This is demonstrated in Table 1 below:
Table 1: Histopatholo~Y of paws: Mice were treated and ioints processed as in Fiq. 4. Histoloqical ~re~ a~dlions were scored as described in methodolo~y.

-W O96/12741 PCT~EP95/04109 T.~:dl.. ~.... l. Norrnal Mild Moderate Severe Total .. . .
(no of:mic~ (%) ~ ~%) . (%) (%) number , . . . . .
of joints examined anti-CD23 80 8 12 0 59 (n=6) controls 0 0 6 94 69 (n=6) This table shows a co",pa,ison of histological preparations in respect of jointstaken from mice treated with anti-CD23 or from control mice. The mice had been treated as described above in relation to Fig 4.

Hi~lu~Lhology was ~-ssessed as follows:

The first limb to become arthritic was removed post-l "o, leh l7 fixed in 10% (wt/vol) buffered formalin and decalcified in EDTA in buffered formalin (5.5%). The paws were then embedded in par~rrin, sectioned and stained with haematoxylin and eosin. Histological preparations (3 sections/paw) were scored using the following criteria: mild + minimal synovitis, cartilage loss and bone erosions limited to dis~;~eLe foci; ll~oder~Le = synovitis and erosions present but joint architecture intact; severe = extensive synovitis and erosions with disruption of join architecture. All the joints present in each section were assessed and the percentage presenting normal, mild, moderate or severe scores was determined.
Inflammation was assessed by the increment in paw thickness relative to the thickness of the paw on the first day of treatment, measured using calipers (Proctest 2T, Kroeplin Langenmesstechnik).

Further evidence to support specificity of this treatment was obtained by treating arthritic mice with Fab and F(ab')2 r~cy"~e,1ls of B3B4 monoclonal ~IlLi~Jo~ly.
Although less potent than the intact IgG molecule, Fab and F(ab')2 rlay,llenl~ of B3B4 were still effective thus demo~ g that the Fc portion of the antibody is not compulsory for the activity (Table 2). Moreover, a"liLo~ies to CD72 and B220 molecules, both highly expressed on B Iymphocytes, showed little to no therapeutic activity (Table 2). In addition comparison with antibodies to TNF oc(Williams et al Proc Natl Acad Sci USA 36: 9784-9788 (1992)) and CD5 (Plater-Zyberk et al Clin Exp ImmLmol 98: 442~147 (1994)) showed that in our experimental conditions, anti-CD23 antibody treatment was the most effective (Table 2).

Table 2: Comparison in the response to dirrel~nl treatment re~imen: Mice were immunised with type ll collagen and treated therapeutically on day 1 and 3 of clinical ~llhlilis by i.p. injection of monoclonal antibodies. The decrease i~
clinical scores on day 5 was calculated as a perce,.lage of the isotype control used in each experiment.

T~dl."enl Decrease in clinical sco. ~s Mab Clone dose Isotype (%) CD23 B3B4 50 ,ug x 2 Rat IgG2a 74 75 ~19 x 2 Rat F(ab)2 52 150 ,ug x 2 Rat Fab 56 TNF oc1 F3F3D4 50 ,ug x 2 Rat IgM 63 CD5 TIB 104 200 ~9 x 2 Rat IgG2a 30 B220 TIB 146 200 ,ug x 2 Rat IgM 5 CD72 TIB 165 200 ~9 x 2 Mouse IgG2b o W O96fl2741 PCTAEP95/04109 Further data in respect of monoclonal antibodies and fragments thereof are provided in Figures 4c and 4d. Those data are based upon the following protocol:

Male 8-12 weeks old DBA/1 mice injected id with 100 ~19 bovine type ll collagen emulsified in complete Freund's adjuvant.

On first sign of clinical ~ise~se (+ 3 weeks later) mice are injected ip with the following prepar~lions of monoclonal antibodies, (injections on day 1 and day 3) B3B4 whole IgG 2a 50 ,ug/injection ip n = 8 Fab of B3B4 150 ~Lg/injection ip n = 6 F(ab')2 of B3B4 75 ~lg/injection ip n = 7 M6 control IgG2a 50 ,ug/injection ip n = 7 Mice are inspected daily and severity of clinical disease of each paw is scored (max/paw = 3; max/mouse = 12). The results for the scoring of clinical disease are shown in Figure 4c.

The swelling of the first arthritic paw is followed using calipers. The results for the scoring of paw swelling are shown in Figure 4d.

Mice are killed on day 10 of arthritis, the first paw to become arthritic is sectioned, fixed and decalcified for histopathological examination.
Interaction between CD23 and CD11 b and between CD23 and CD11 c Without wishing to be bound by theory, since it is not fully understood how anti-CD23 antibodies achieve the surprising results disclosed above, it is possible that =
CA 02203363 l997-04-22 W O96/12741 PCT~EP95/04109 this may be due to the interaction of CD23 with CD11 b andlor CD11 c. Examples 5 to 10 and the acco"~ ying Figures (see later) iliustrate this.
In these Examples, full-length ,~:coml~ a"t CD23 incorporated into fluo,~:scel~tliposomes was shown to bind to COS cells transfected with cDNA encoding either CD11b/CD18 or CD11c/CD18 but not with transfectants expressing CD11a/CD18. The interaction between CD23-liposomes and CD11b/CD18 or CD11c/CD18-transfected COS cells was specifically inhibited by anti-CD11b or anti-CD11 c, respectively, and by anti-CD23 monoclonal antibodies. The functional significance of this ligand pairing was demonstrated by triggering CD11b and CD11c on monocytes with either recombinant CD23 or anti-CD11b and anti-CD11c monoclonal ~libo-lies to cause a marked inuease in nitrite (N02-), oxidative products (H2O2) and proinflammatory cytokines (IL-1,~, IL~ andTNFa). These CD23-mediated activities were decreased by Fab fragments of monoclonal antibodies to CD11b, CD11c and CD23. These results ~iemo~ L, ~Le that the surface adhesion molecules CD11b and CD11c ar~ recepl~s for CD23 and that this novel ligand pairing regulates important activities of monocytes.

The following discussion explains briefly the experimental design and the rationale behind Examples 5 to 10 (which follow):-Total blood mononuclear cells were incubated with recombinant full-length CD23 incorporated into fluorescent liposomes and analysed by flow cytometry (Pochon, S. et a/. J. Exp. Med. 176, 389-398 (1992)). A fraction bound CD23-liposomes (Example 5, Fig. 5a) which was then shown by double staining to consist of CD14-positive celis (i.e. monocytes). To co"ri"ll that monocytes were able to bind CD23-liposomes, blood mononuclear cells were FACS-sorted into CD14-positive and CD14-negative populations (Example 5, Fig. 5a). CD23-liposomes were shown to bind only to the CD14-positive population (Example 5, Fig. 5a).
Since monocytes were found to express neither membrane IgE nor CD21 (not W O96/12741 PCT~EP95/04109 shown), the known ligands for CD23, it was investigated whether monocytes ~ express a dirr~re"l rece~ r for CD23. Monocytes were Iysed and cell extracts purified over an affinity cclumn coupled with recol~binant soluble CD23. SDS-PAGE and silver staining analysis of the eluted ",~l~rial revealed bands of around 80 and 160 kDa MW (Example 5, Fig. 5b). Antibodies identifying antigens within this range of MW and reported to be expressed on monocytes were tested by FACS for their cal~a~ily to inhibit CD23-liposome bi~di~y to monocytes (Example 6, Fig. 6). Anti-CD11 b and anti-CD11 c monoclonal antibodies both inhibited CD23-liposome binding to monocytes, with varying degrees of potency (Example 6, Fig. 6). Anti-CD13, anti-CD49d, anti-CD21 (not expressed on monocytes) and anti-CD11a (the third member of the ~2 integrin family of adhesion molecules) had no siy~lirical,l effect (E-xample 6, Fig 6).
Antibodies against MHC Class 1, Class ll, CD14 and CD45, all of which highly expressed on monocytes, were also tested for their effect on CD23-liposome 1~ binding.. None however had any effect (not shown). Anti-CD18 monoclonal antibody gave a partial inhibition of CD23 binding. This could be due either to steric hindrance or to the induction of a conrull,,~lional change in the CD11 b and CD1 1c molecules upon anti-CD18 Mab binding. The monocyte-derived proteins eluted from the CD23-affinity column were immunoreactive with anti-CD11 c (Example 5, Fig. ~b) and anti-CD11 c/CD18 antibodies (not shown).

To confirm that the a chain of CD1 1b/CD18 and CD1 1c/CD1 1 b were receptor~
for CD23, full-length cDNAs encoding CD11b and CD11c were transiently co-transfected with CD18 cDNA into COS cells. Transfectants expressing CD11b/CD18 and CD11clCD18 were both shown to bind CD23-liposomes, in contrast to tran~rectal1ts expressing CD11a/CD18 (Example 7, Fig. 7). This mightbe explained by the higher degree of homology between CD11b and CD11c when compared to their homology to CD11a. The specificity of the interaction was demonstrated by inhibiting CD23-liposome binding using anti-CD11b, anti-CD11c and anti-CD23 monoclonal antibodies. The same results were obtained using BHK cells expressing CD11b/CD18 and CD11c/CD18 (not shown). As further proof of the specihcity of the CD23 interaction, activated blood monocytes from a Leukocyte Adhesion Deficiency patient, lacking ,~2 integrin e,~ressiol I due a mutation in the gene encoding the ~ subunit were unable to bind CD23-liposomes (not shown). Together, these data del,lo,lsll~le that CD23 i,lter~s with CD11 b and CD11 c on normal human monocytes and on transfectants.

CD11 b and CD11 c are adhesion molecules that participate in many cell-cell and cell-matrix interactions. The examples showthat CD11b/CD18 and CD11c/CD18 may exhibit an additional adhesive function by virtue of their ability to bind CD23.
CD23 seems to identify an epitope close or identical to factor X as observed by the capacily of factor X to inhibit in a dose dependent manner CD23-liposome binding (~xample 8, Fig. 8) without affecting surface expression of CD11b or CD11c on monocytes (not shown). None of the other ligands tested had any effect. CD23 may be acting as a C-type lectin in its interaction with CD11b and CD11c. EDTA decreases CD23 bindins to monocytes (Example 8, Fig. 8) by chelation of Ca2+ which is necessary to CD23 binding and/or by chelation of the divalent caiions which are necessary for the binding of ligands to CD11b and CD11 c (Altieri, D.C. J. ImmLmol. 147, 1891 -1898 (1991)). CD23-CD11 b/CD11 c interaction seems to involve sugars, but not sialic acid, as observed by the capacity of tunicamycin, but not neuraminidase, to decrease CD23 binding to monocytes. CD23 bears extracellularly a triplet of amino acids (Asp, Gly, Arg) (Kikutani, H. ef al. Cell 47, 867-885 (1986)), which in the reverse orientation is a common recognition site for the integrin receptors. Therefore, the effect of a polyclonal antibody directed against this tripeptide was tested for its capacity to inhibit CD23 binding to monocytes. No inhibition was observed, conrill"illg the absence of inhibition obtained with fibrinogen (Example 8. Fig. 8). IgE which isbinding in the lectin domain of CD23, partially inhibits CD23 binding to monocytes (Example 8 Fig. 8). Those results indicate that CD23 would seem to be acting - as a C-type lectin recognising partly sugar and protein structures reminiscent of what has been observed for CD23 i"l~r~;iion with CD21 (Aubry J-P. et al. J.
Immunol. 152 5806-5813 (1994)).
To evaluate the functional significance of the interaction of CD23 with CD11 b or CD11c we investigated whether CD23-CD11b/CD11c interaction could trigger monocytes to release ,urui~ m~Lu~y mecJidlor~ such as nitric oxide H2O2 and cytokines. Triggering of adherence-activated norrnal monocytes using recombinant soluble CD23 anti-CD11b or anti-CD11c antibodies increased the generation of NO2- i"dicali,1g the activation of the NO pathway (Moncada S.
Palmer R.M.J.&Higgs E.A.Pharmacol.Rev. 43 109-144(1991)). Theef~ectof CD23 on nitrite production was inhibited by Fab fragments of arlti-CD23 monoclonal antibodies and by nitroarginine a specific inhibitor of the N0 synthase pathway (Example 9 Fig. 9a). The oxidative burst was also shown to be re~ ted through CD11b and CD11c since reco",L,i,1a"L soluble CD23 anti-CD11 b and anti-CD11 c monoclonal antihorlies all caused oxidation of hydroethidine to ethidium bromide in monocytes (Example 9 Fig. 9b). This conlilll~s and extends the finding that anti-CD11b monoclonal antibodies induce an oxidative burst in monocytes (Trezini C. Schuepp B. Maly F.E. & Jungi T.W. Brit J. Haematol. 77 16-24 (1991)). CD23 binding to CD11b and CD11c was associated with an early specific Ca2+ flux in blood monocytes (not shown).

Since activated Illauupi~ages are an important source of proinfl~",n,alory cytokines we evaluated the effect of recombinant soluble CD23 and of anti-CD11 b and anti-CD11 c monoclonal antibodies on the production of such cytokines by monocytes. Recombinant soluble CD23 anti-CD11b and anti-CD11c monoclonal antibodies were potent stimulators of IL-1,~ IL6 and TNFa.
Again the specificity of this induction was der,)o"~ led by using Fab fragments of anti-CD11b anti-CD11c and anti-CD23 monoclonal antibodies (Example 10 CA 02203363 l997-04-22 W O96/12741 PCT~P95/04109 Fig. 10). Interestingly, IL-1 and TNFoc were potent inducers of CD23-liposome binding to monocytes (not shown), suggesting a potential cytokine autocrine loopthrough CD11 b and CD11 c stimulation and regulation.

a) CD23-liposomes bind to CD14-Positive blood Illono~ ~uclear cells (See Fig. Sa).
Blood mononuclear cells were stained with anti-CD14 Illollo~;lo~)~l antibody (Becton Dickinson, Erembodegem, Belgium) followed by sheep FlTC~o,.; Ig~ted F(ab')2 antibodies to mouse IgG and IgM (Bioart, Meudon, France), both diluted in PBS, 0.5% BSA and 0.05% sodium azide prior to FACS-sorting (FACStar Plus, Becton Dickinson) into CD14-positive and CD14-negative cell populations.
Separ~led cells were then stained with CD23-liposomes or control (gl~;ophori~l A)-liposomes diluted in 0.5% BSA, 0.1% sodium azide, 2 mM CaCI2, 140 mM
NaCI, 20 mM Hepes, pH 7 and incubated for 2 h at 4~C (Pochon, S. ~t al., J. Ej(p.
Med. 176 389-398 (1992)). After washes, cells (5,000 events/condition) were analysed by FACS.

b) Apparent molecular wei~ht of CD23-affinitv purified blood monocyte proteins and immunoreactivitywith an anti-CD11c monoclonal antibody (See Fig.
5b).
Lysates of blood monocytes were afhnity purified on a CD23-column, eluted proteins separated on SDS-PAGE gels and transferred onto nitrocellulose. Mr markers are shown on the left. The gel was silver stained (left lane). Filters were incubated with either an isotype matched antibody (middle lane) or with an anti-CD 11 c monoclonal antibody (BU-15, right lane), then with horseradish peroxidase-conjugated goat anti-mouse antibody (Kpl; Gaithersburg, Massachusetts).

E)CAMPLE 6 CA 02203363 l997-04-22 W ~96~12741 PCT~EP9~/04109 Anti-CD11b and anti-CD11c monoclonal antibodies decrease CD23-liPosome ~ bindin~ to activated blood monocvtes (See Fig.6).
Monocytes were en, ic~ ,ed from mononuclear cells by Ficoll and ove" .i~l ,l adherence to plastic in RPMI 1640 (Seromed, Berlin, Germany) supplemented with 2 mM glutamine and 10% heat-inactivated FCS (Flow Laboratories, Irvine, Scotland). Activated Illo~ ;ytes were then incubated with CD23-liposomes in the presence of dirrer~nl ",onoclollal antibodies (aCD) or isotype-ll,dlcl~ed controls (CTRL) (Becton Dickinson), all tested at 10 llg/ml. Anti-CD11 a monoclonal ~ antibodies 25.3 and B-B15 were obtained from Immunotech (Luminy, France) and Serotec (Oxford, UK), respectively. Anti-CD11 b monoclonal a"libody 44 was from Serotec, mon.gran 1 was from Janssen (Beerse, Belgium), Leu-15 was from Becton Dickinson (Erembodegem, Belgium) and (Bear-1) was from Sera-Lab Ltd (Sussex, GB). Anti-CD11c ",or,oclonal antibody 3.9 was from Serotec, SL9 was from Sera-Lab and BU-15 was from The Binding Site (Bi~ lyhalI~, UK). Anti-CD13 (SJ1D1), anti-CD18 (BL5), anti-CD23 (mAb25) and anti-CD49d (HP2.1) monoclonal anLi6O-lies were from Immunotech. Anti-CD21 monoclonal antibody BL13 was from Immunotech, OKB7 from Ortho and BU-33 was obtained from Dr.
MacLennan (Birmingham University, UK), HB-5 from ATCC, OKB7 from Ortho Diagnostics System Inc (Raritan, NJ). Anti-CD14, anti-CD3, anti-CD16 and anti-CD20 monoclonal antibodies were from Becton-Dickinson. Cells were analysed by FACS and mean fluorescence inLensily (MFI) measured. Data of a representative experiment are presented. MFI of cells stained with control-liposomes was 6.5 and with CD23-liposomes was 84.5. Percentage inhibition using ariLhr"eLic linear MFI values is calculated according to the following formula:

CA 02203363 l997-04-22 wo 96/12741 PCT~EP95/04109 [(CD23-lipo)]-[(CD23-lipo)+Mab]
% inhibition = MFI x 100 (CD23-lipo) CD23-liposomes bind to cc chains of CD11b/CD18 and CD11c/CD18 on recombinant transfectants (See Fig. 7). cDNAs coding for CD11a (Corbi, A.L., Miller, L.J., O'Connor, K, Larson, R.S. & Springer, T.A. EMBO J. 6, 4023~028 (1987)) was recloned in pCDNA1 (Invitrogen, San Diego, CA). cDNA for CD11 b (Corbi, A.L., Kishimoto, T.K., Miller, L.J. ~ Springer, T.A. J. Biol. Chem. 263,12403-12411 (1988)) and CD18 (Kisl,i,noLo, T.K., O'Connor, K., Lee, A., Roberts,T.M. & Springer, T.A. Cell 48, 681~90 (1987)) were recloned in pCDM8 (Seed, B., Nature 329 840 842 (1987)). 20 ~Lg aliquots of DNA were transfectsd in COS-7 cells (ATCC) by electroporation (260 V, 960 ,uFD) using a Gene Pulser device (Bio-Rad, Richmond, CA) and 0.4 cm cuvettes in 20 mM Hepes pH 7.4, 150 mM
NaCI. Co-transfections of CD11a, b or c with CD18 were performed in order to get expression of the ~2 integrins at the cell surface. Controls were done with single chain transfections. 48 h after transfection, COS cells were stained withanti-CD~1a, anti-CD11b and anti-CD11c monoclonal antibodies or isotype-matched monoclonal antibodies (control) followed by FlTC-labelled sheep anti-mouse antibody. Between 10 to 15% of the cells were shown to express CD11 a, b, c or CD18 by staining with the respective monoclonal antibodies. Prior to staining with CD23-liposomes, CD18-positive transfected COS cells were then FACS-sorted in order to increase the percentage of cells expressing ,~2 integrins.
CD11 a/CD18, CD11 b/CD18 and CD11 c/CD18 transfectants were then incubated with CD23-liposomes (trace 2) or controi (glycophorin A)-liposomes (trace 1).
The specificity of CD23 interaction with CD11 b and CD11 c was demonstrated by inhibition of CD23-liposome binding to CD11 b/CD18 and CD11 c/CD18 W O96/12741 . PCTAEP95/04109 transfectants using anti-CD11b (trace 4), anti-CD23 (trace 5) and anti-CD11c (trace 6) monoclonal antibodies, respectively. No binding of CD23-liposomes was observed on CD11 a/CD18 transfectants and no effect of anti-CD11 a monoclonal antibody was found (trace 3).

Structural characterisation of CD23-CD11 b, CD11 c interaction (See Fig. 8).
(a) Involvement of su~ars and divalent cations.
Purihed activated blood monocytes were treated or not with tunicamycin (10 ~Lg/ml) for 48 h or with neuraminidase (0.1 U/ml; both from Boehringer Mannheim,Mannhein, Germany) for 45 min. Cells were then incubated with CD23-liposomes or control-liposomes in the absence or presence of EDTA (5 mM; top left panel), Ca2+ or Mn2' (1 to 10 mM; top right panel).
(b) Factor X does inhibit CD23 bindinq to monocvtes. Purihed activated blood monocytes were incl~h~ted with CD23-liposomes in absence or presence of factor X (0.1 to 10 U/ml; Sigma) (bottom left panel), fibrinogen (50 ~Lg/ml; Sigma), purihed recombinant ICAM-1 (produced in our laboratory), LPS (1 ~lg/ml; Sigma), human serum opsonised-zymosan (1 mg/ml; Sigma), lgE (50 ~g/ml; The Binding Site, Birmingham) or polyclonal antibody to RGD peptide (1/500, ATCC) (bottom right panel). Cells were analysed by FACS and MFI measured. Percenlage inhibition was calculated as for Example 6.

Recombinant CD23 by bindinq to CD11 b and CD11 c specifically increases a~ the nitrite product and b. the oxidative burst bv monocvtes.
Monocytes were incubated a, for 4 days at 37~C or b, ovemight in the absence or presence of recombinant soluble CD23 (Graber P. et al., J. Immunol. Methods 149 215-226 (1992)) (50 ng/ml), anti-CD11 a (clone 25.3), anti-CD11 b (clone 44), anti-CD11 c (clone BU-15) monoclonal antibodies (all at 10,ug/ml).

To assess the amount of NO produced (which is shown in Fig. 9a), the cuiture supernatants were assayed for the stable end products of NO, NO2- and NO3 acco,~i"g to Green etal., Annu. Rev. Immunol. 2 199-218 (1984). The specificity of CD23-mediated increase of NO2- production was demonsl,~led by i"hiLi~ o~
NO2- production by Fab rr~yl~le~lls of anti-CD23 monoclonal antibodies (mab25) (tested at 10,ug/ml) and by inhibition with nil, ~,aryi"ine (N-Arg at 1 mM) (Sigma).

Activated monocytes were incubated with hydroethidine (Molecular probes, Eugene, OR) (0.3 ,ug/ml) for 30 min at 37OC (Rothe G. et a/., J. Leukoc. Biol. 47 440448 (1990)) and analysed by FACS. Percentage increase in red fluorescence of stimulated monocytes is shown in co" ,pal ison to untreated monocytes (See Fig. 9b). Monocytes which had undergone an oxidative burst shown an increase of red fluoresce"ce signals compared to ~r ~ led monocytes reflecting oxidation of hydroethidine to ethidium brc~ (Lacal P.M. et al., Biochem. J. 268 707-712 (1990)). MFI values of monocytes alone were 159+/-10. Mean+/-SD values of 6 experiments are presente~. Con A, which is known to induce a respiratory burst in monocytes, was used as a positive control. The specificity of the CD23 interaction with CD11 b and CD11 c was demonstrated by inhibition of CD23-mediated increase of H202 production by Fab fragments of anti-CD11 b (clone 44), anti-CD11 c (clone BU-15) and anti-CD23 (mAb25) monoclonal antibodies (tested at 10 ~g/ml).

Bindin~ of recombinant CD23 to CD11 b and CD11 c specifically increases cytokine production bv monocvtes (See Fig.10).
Monocytes were incubated ovemight at 37~C in the absence or presence of recombinant soluble CD23 (Graber P. et a/., J. Immunol. Methods 149 215-226 (1992)) (50 ng/ml), anti-CD11a (clone 25.3), anti-CD11b (clone 44), anti-CD11c (clone BU-15), anti-CD23 (mAb 2~ - this antibody is available from Immunotech.
- It is ~isa lssed in published European Patent Application EP-A-0269728) ",onoclonal antibodies, Con A (Sigma) (all at 10 ~g/ml), LPS (1 ng/ml) (Sigma) or PMA (5 ng/ml) (Calbiochem, La Jolla, CA). Cytokines were measured in the culture supelllal~1l by specific ELISA. The ELlSA's limit of sensitivity is 0.05ng/ml for IL-1~ (Ferrua et al., J. Immunol Methods 114 41~8 (1988)) 0.01 ng/ml for TNFa (M~d~~enix, Biotechnie, Rungis, F) and <0.01 ng/ml for IL~ (Manie ef a/.t Eur. Cytokine Nefw. 4 51-56 (1993)). The specificity of CD23 interaction with CD11 b and CD11 c was demonstrated by inhibition of CD23-mediated increase of cytokine production by Fab fragments of anti-CD11 b (clone 4), anti-CD11 c (clone BU-15) and anti-CD23 (mAb25) monoclonal antibodies (tested at 10 ,ug/ml).
Mean+l-SD values of 4 experi, l ,el1ts are presented.

Monoclonal ~r,libodies to recombinant E. coli derived soluble human CD23 (25 kD, amino acids 150-321) were raised in mice, by standard procedures except using Iymph nodes not spleen cells. These blocked the activities of human CD23 in vitro.

Ulcerative colitis 3 tamarin monkeys were dosed with anti-CD23 Mab without adverse effects, resulting in an improvement in subjective laecal score.

2~ The dosage was 1 mg every 4 days by intraml Isc~ 3r injection.

No toxic effects were observed in any tests reported here.

Claims (13)

Claims

1. The use of a binding agent to CD23 which blocks the interaction of CD23 with a ligand which binds to it in vivo for the manufacture of a medicament for the treatment of autoimmune disorders.

2. The use according to claim 1 in which the interaction between CD23 and CD21, CD11b or CD11c is blocked.

3. The use according to claim 1 or 2 for the treatment of rheumatoid arthritis.

4. The use according to claim 1 or 2 for the treatment of arthritis, lupus erythematosus, systemic lupus erythematosus, Mashimotos thyroiditis, multiple sclerosis, diabetes, uveitis, dermatitis, psoriarsis, urticaria, nephrotic syndrome, glomerulonephritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, Sjogren's syndrome, asthma, eczema, graft vs host disease, insulitis.

5. The use according to any of the preceding claims wherein the binding agent is an antibody, a fragment therof, an artificial construct comprising an antibody or comprising a fragment therof, a mimetic or a derivative of any of these binding agents.

6. The use according to claim 5 wherein the antibody is humanised or chimaerised.

7. A pharmaceutical composition comprising a unit dose of at least 1 mg, of a binding agent to CD23 which blocks the interaction of CD23 with a ligand which binds to it in vivo, and a pharmaceutically acceptable carrier.

8. A pharmaceutical composition according to claim 7 containing a unit dose of 1-1000mg.

9. A pharmaceutical composition according to claims 7 or 8 wherein the binding agent is an antibody.

10. A pharmaceutical composition according to claim 9 wherein the antibody is humanised or chimaerised.

11. A use or pharmaceutical composition according to any one of the preceding claims, wherein the binding agent is in combination with an immunosuppressive, tolerance inducing, anti-autoimmune or anti-inflammatory agent.

12. A use or pharmaceutical composition according to claim 11 wherein the binding agent is in combination with a CD4+T cell inhibiting agent or a TNF antagonist.

13. A use or pharmaceutical composition according to claim 12 wherein the binding agent is in combination with an anti-CD4 antibody.