CA2203364A1 - Binding agents for treatment of inflammatory, autoimmune or allergic diseases - Google Patents

Abstract

Binding agents to CD11b, CD11c, CD21, CD23, a 70 to 85 KDa protein expressed on endothelial cells or a 115 KDa protein expressed on endothelial cells, can be useful in the treatment of inflammatory, autoimmune or allergic disease.

Description

W 096/12742 PCT~EP95/041lQ

BINDING AGENTS FOR TREATMENT OF INFLAMMATORY, AUTOIMMUNE OR ALLERGIC DISEASES

The present invention relates to particular binding agents which can be used in the treatment of iu ndlllll~ ~, autoimmune or allergic diseases.
CD23 (FC~RII) is a type ll molecule of the C-lectin family which also includes the Iymphocyte homing receplor (MEL-14) and the end~ll,elial leukocyte adhesion molecule-1 (ELAM-1). It is a low affinity receptor for IgE. In humans a variety of hael~,alopoietic cell types e,~ ss CD23 on their surface, including follia~
10 de, n~l ilic cells, B cells, T celis and " ,ac;, o~l ,ages. CD23 molecules are also found in soluble forms in biological fluids. Soluble CD23 (sCD23) molecules are formedby proteoly~ic cleavage of transmel ~ ~l,r~ne I ~ce,ulor~. CD23 has pl~iul~pi~
activities including me~ ion of cell adhesion, regulation of IgE and l,;~ld,.,i"e release, r~scue of B cells ~rom a,l.o~;losis and regulation of myeloid cell growth.
15 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 Immunol 8, 623-645 1990); Delespesse, G., ef al., Adv Immunol 49, 149-191 (1991); Bonnefoy, J.Y., et al., Curr Opin /mmunol 5, 944-947 (1 993)).
In~eased ex,u,~ssion of CD23 has been observed in a number of i"n~"""~lo~y diseases. CD23 has been identified in synovial biopsies from patients with ulllon.c synovitis, and sCD23 can be measured at conce"l,dlio"s exceeding the norrnal range in the serum and synovial fluid of patients with rheumatoid arthritis (Bansal, 25 A.S., Oliver, W., Marsh, M.N., P-""phrey, 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); Cllolllal-~L, P., Brioloay, J., Banchereau, J., & Miossec, P., Arthritis Rheum 86, 234-242 (1993); Bansal, A., et al., Clin Exp Immunol 89, 452-455 (1992); Re~o",~lv, R., & Newkirk, M.M., Clin Immunol Immunopathol 71, 156-163 (1994)). In addition, levels of serum sCD23 in rheumatoid a,lhrilis palienls are related to disease status and co,-elale with serum rheumatoid factor (Bansal, A.S., ef al., Cljn Exp Rheumatol 12, 281-285 (1994)). Pro-infla,l " "a~, ~ cytokines a~,uear to be particularly i, ,~,uu, lanl in S rheumatoid alllllilis, and a central role for TNF~c and IL-1~ in the destruction of arthritic joints has been pOSt~ t~d (Br~ " ~a~ ~, F.M., Chantry, D., Jackson, A, Maini, R., & Feldman, M., Lancef 2, 244-247 (1989); Gten,1a,1, F.M., Maini, R.M., &
Feldman M., Br J Rheumatol 31, 293-298 (1992)).

It has also been poshJl~ted that CD23-CD21 inler~ulions may play a role in the controi of IgE production (Flores-Romo L. et al., Science 261 1038-1041 ~1993);
Aubr,v ef a/., Nature 358, 505-507 (1992)).

CD11b and CD11c are adhesion molecules that participate in many cell-cell and cell-matrix interactions. CD11b/CD18 and CD11c/CD18 (an ~ soGi-l;oll of CD11b and CD18 and of CD11c and CD18 respectively) have been reported to bind several ligands, including CDS4, fibrinogen, factor X, LPS, Con A and zymosan (S~ yer~ T.A, Nature 346, 425434 (1990)). The role of these binding molecules is not however completely understood. CD11blCD18 and CD11c/CD18 are also known as MAC-1 and p150, 95 respectively. They are mer,l~e,~ of the ~2 integrin family (sometimes known as Leu-CAM, ie leucocyte cell adhesion molecules).
This family also includes LFA-1 amongst its me" ,be, ~ (also known as CD11 alCD18).

EP 0205405 purports to disclose Mabs to Iy"~ o-;yte cellular receptors for IgE
(FCER) cross reacting with human immunoglobulin E bi,1di~ ly factor (IgE-BF), and derivatives thereof.

W O 96/12742 PCT~EP951~4110 WO 93/04173 purports to disclose a polypeptide which is capable of binding to one of FCEL (low affinity IgE receptor FCERII) or FCEH (high affinity receptor FC~RI) but whic-h is suL,~la"lially ill~r-'~l of L,i.lding to the other of FCEL or FCEH. Tre~l"~en~ of an allergic disor(ler is alleged with a FCEL or FCEH spec~ic5 polypeptide (provided the FCEH s~eciric polypeptide is irl~pable of c~uss~ king FCEH and inducing histamine r~lease).

EP 0269728 purports to ~I sclcse Mabs to the human l~ll,uho~iyte IgE receptor.

EP 0259585 purports to ~Ji;,close Mabs recognising a surface l~cef~or for IgE
(FC~R) on human B Iymphocytes.

WO 93/02108 purports to i ~;sclose pl il l~alised antibodies for therapeutic use.

The presenl inventors have sul,ulisil,yly discovered that binding agents to CD21, CD11 b, CD11 c, to a 70~5 KDa protein e~, ~ssed on e"doU ,elial cells, or to a 115 KDa protein exlJ,~ssed on er,doU,elial cells can be of utility in the treatment or prophylaxis of various ~lise~ses and in particular in the treatment or prophylaxis of al lhl ilis. Prior to the pr~senl invention no data has been presented which would support such a utility, ~lespile the pl ll ~lic~lion of a large number of papers in which CD21, CD11 b or CD11 c have been ~isa l.sse~

Accoldillg to the prese,1l invention, there is provided a binding agent to CD21,CD11b, CD11c, to an 70~5 KDa protein expressed on endothelial cells, or to a 115 KDa protein expressed on endothelial cells for use in the treatment or ~ prophylaxis of i, Indlllll~dlury, ~l ~c mlllune or allergic d;seases.

The binding agent may function by blocking the i"lerac~ion between the protein and a ligand which binds to it. In vitro assays e.g. radio-immune assays may be used to study such a blocking effect.

5 The binding agent may be in isol~c! form or as part of a ~ohdilll~
composiliGn. Desi,~bly it is in sterile form. Generally speaking a binding agentwhich is specific for CD21 CD11b CD11c to a 70~5 KDa protein e,~,uressed on endc,ll,elial cells (e.g. to a 76 kDa a 80 KDa or 85 KDa protein ex~,essed on endothelial cells) or to a 115 KDa protein ex~r~ssed on endothelial cells is useful 10 in the treatme, ,U,uruphylaxis disclosed.

Preferred 6inding agents include a, llil,odics r, ay" ,enls thereof or al liricial constructs co",~ ,i"y antibodies or fragments ll,e,eor or a,liricial constructs designed to mimic the binding of alll;l~o~-e5 or fragments thereof. Such bindingagents are r~iscllssed by Dougall etalin Tbtech 12 372-379 (1994).

Theyincludeco~, etea~lii-o~l;es F(ab')2fragments Fabr,~y",enls Fvf,ay,llellls ScFv fragments other rldy",e,ll~ CDR peptides and mimetics. These can be obtained/ prepared by those skilled in the art. For example enzyme digestion can20 be used to obtain F(ab')2 and Fab r,~g",e"ls (by subjecting an IgG to molecule to pepsin or papain cleavage respectively). R~rerences to "antibodies" in the following desui~lion should be taken to include all of the possibilities mentioned above.

25 Recombinant antibodies may be used. The antibodies may be humanized; or chimaerised.

A typical prepa~alion of a h~lllldl ,ised antibody in which the CDRs are clerived from a dirr~rent species than the framework of the antibodrs variable domains is W O 96/12742 PCT~EPg5/04110 disclosed in EP-A~239400. The CDRs may be derived from a rat or mouse Illonoclonal a"li~o~y. The framework of the variable domains and the co,~sla"l do",ai. ,s of the altered a, ILibGdy may be derived from a human a- Ili~ody. Such a h~ anised antibody elicits a negliyil,le immune response when administered to a 5 human compared to the immune ,~s~onse mounted by a human against a rat or mouse anliL,o~Jy All~",alively the allli~ody may be a chimaeric antibody, for instance of the type desc, i~ed in WO 86/01533.
A d,i",aeric antibody accor~ling to WO 86/01~33 comp~ises an antigen binding region and a non-immunoglohlllin region. The antigen binding region is an antibody light chain variable domain or heavy chain variable don~ain Typically the chimaeric a"li~o~y co,np,ises both light and heavy chain variable domains.
1~ The non-immunoglobulin region is fused at its C-terrninus to the anligen 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 exf~,essecl by a gene. The two regions of the chimaeric antibody may be connected via a 20 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 (~:I,i",aeric) or CDRs (hu",~"ised).
r,i",a~i ing techniques may also be usedl such as those disclosed in 2~ WO93/021 08.

Other pr~rer,~d binding agents (apart from antiho~ies or derivatives thereofl are Factor X (i.e. Factor 10) Epstein Barr Virus or a part of Epstein Barr Virus.

o 96rl2742 PCTAEP95104110 Where intact viruses are used in medical treatment, they will generally be provided in non-virulent form. This can be achieved using techniques known to those skilled in the art, which include attenuation and muta~ esis.

As will be apprec- -'ed by those skilled in the art, where specific t..)~ agents are described herein, derivatives of such agents can also be used. The term "derivative" includes varianls of the agents desuibed, having one or more amino acid substitutions, deletions or i~,se, li~l Is relative to said agents, whilst still having the binding activity described. P,~r~bly these derivatives have substantial amino acid sequence identity with the binding agents specified.

The degree of amino acid sequence identity can be c~ ted using a program such as "bestfit" (Smith and W ~lellllctll, Advances in Applied Mathematics, 482-489 (1981)) to find the best segment of similarity between any two sequences.
The aiignment is based on ma,~in,isi"g the score a~l..3ved using a matrix of amino acid similarities, such as that desctiLed by Schwarz and Dayhof (1979) Atlas of Protein Sequence and Structure, Dayhof, M.O., Ed pp 353-358.

P, ~:r~r~bly the degree of sequence identity is at least 50% and more ~,rererctbly it is at least 75%. Sequence identities of at least 90% or of at least 95% are most preferred.

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 withoutst ~hst~ntially altering or adversely aflecting 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 canoften be s~ Ihstitl Ited for one another include:
phenyialanine, tyrosine and tr~,ulo~.lh au (amino acids having aro"~alic side chains);
~ Iysine, arginine and histidine (amino acids having basic side chains); aspa,l~te and glul~",ale (amino adds having acidic side chains); aspar;~y;"e and glutamine(amino acids having amide side chains) and cysteine and melZ ,io"i. ,e (amino acids having sulphur containing side chains). Thus the term "derivative" can also include a variant of an amino acid sequence co~ risi"g one or more such "co"se, ~ative" cl ,cnyes relative to said sequence.
The presen~ invention also includes r~dy,n~"l~ of the l~ di~ly agents or of the present invention or of derivatives ll,ereur vwhich stili have the binding activity described. P,~e"e~ r,~",e, IL~ 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 b:.,c;;ng agents of the ~r~se"~ invention are believed to be useful in the treatment or prophylaxis of several human ~lise~-ses including a,lh,ilis, lupus erytl ,e"~alosus, Masl ,i" lulos thyroiditis, multiple sclerosis,~Jiabeles, uveitis, de, l l lalilis, psoriasis, urticaria, nephrotic s~" nll on ,e, ylol "el ~lonephritis, in n~" " "~lory bowel ~ e~e, ulcerative colitis, Crohn's disease, Sjogren's syndrome, allergies, a~ll""a, rhinitis, e~e",a, GVH, COPD, insulitis, bronchitis(particularly ~1lo,1-~ b(o"cl,ilis) or didl.etes (particularly Type 1 diabetes). They may also be useful in studying the i"lelac1ions between CD23 and various ligandse.g. between CD23 and CD21, between CD23 and CD11b, between CD23 and CD11c, between CD23 and the ~roreC- ' 70 to 85 KDa endothelial cell protein (which may be an 80 or 85 KDa endoll ,elial cell protein) or between CD23 and a 115 KDa endothelial protein (which is believed to be related to the 70 to 85 KDaendothelial protein). One or more of the above inleraclions are believed to occur, in vivo. Antiho~lies or other binding agents which are ~r~hle of blocking these W O96112742 PCT~EP95/04110 inLer~iions are particularly ,~,l ere" ~d since it is believed that they may be especially suitable for reducing or alleviating cytokine mediated i"na""~dlory effects. They may also be useful against B-cell malignancies such as ~hlullic Iymphocytic leukaemia and hairy cell leukaemia.

Binding agents of the prt:se, ll invention are particularly applicable for use in the treatment or prophylaYis of rheumatoid a,Ll"ilis. Without being bound by theory the following possible ex~Jld"~lions are put forward:-In the rheumatoid ~IUllilis i~ med synovium m~u~pl1ages express both CD23 and the ,B2ill~eylinS CD11b and CD11c allowing possi~le homotypic interactions to take place in this tissue. In addilion diffusion of sol ~hle CD23 moleu~les through the synovium and their binding to the illley~ ligands is also possible.
Tl,ererore CD23-CD11b/CD11c i"lera~io,1s involving a positive activation loop could exist in vivo. If p,~:se"l in rheumatoid ~I ll llitis palienl~ it may explain some of the pathogenic ",ed Idl lisllls of ciisease exace,Ldlion and chronicity and would support the hypothesis that once localised to the joints ~"acr~phages themselvescan I I lai, llain and exdce, I-~le il In~, "" IdliOl I via a pathway involving CD23 molecules ~2-integrins CD11b and CD11c as well as pro-infla"""alory cytokines TNF oc IL-1,B and IL~.

The present inventors have found that CD23 L ~ding to CD11b and CD11c is blocked by Factor X since Factor X binds to CD11 b and CD11 c. Thus the present invention includes the use of Factor X or of a fragment thereof to block CD23 binding to CD11 b andlor CD11 c.

An alternative mechanism of action of anti CD23 therapy could involve the blocking of an IgE immune response.

W O96/12742 PCT~EP95104110 - In previously published work it has been shown that ~n vivo l,e~L~"e"l of rats with anti-CD23 anli~ody resulted in antigen-specific inhibition of IgE pro~ ~ction ,u, ubaL.ly by blocking the CD23-CD21 interaulio"s necsss~y for complete dirrer~nli~liol) of IgE~"""illed B cells (Flores-Romo et al., Science 261, 1038-1041 (1993)).

The ~,esent invention also includes binding agents to CD21 which block such a ,es~.,onse (e.g. the Epstein Barr virus or a part thereof).

Structurally the CD21 protein is cor"posed of an ext, i~cellular dor"~i" of 15 (Moore et al, Molecular clor,i"y of the cDNA encoding the Epstein Barr Virus C3d receptor (complement . ~ceplor type 2) of human B Iy" ~pl ,o~te Proc Natl Acad Sci USA 84:
9194 (1987)) or 16 (Weis et al, Structure of the human B l~ hû~;yte r~ceplor forC3d and the Epstein Barr Virus and reldled"ess to other members of the family ofC3/C4 bind;.ly ~,uteins JE~p Med 167: 1047 (1988)) repetitive units of 60 to 7 amino acids named short consensus repe~L~ (SCRs) followed by a transmemL"~"e do"~ain (24 amino acids) and an intracytoplas~ region of 34 amino acids. Using CD21 mutants bea,i"g deletions of extracytoplas",ic SCRs (Carel et al, Structural req~ ",e"ls for C3d g/Epstein Barr Virus receptor (CR21CD21) ligand L,i"-Ji"g i"le",alization and viral infection J Biol Chem 265:12293 (1990)) the present inventors have, ~ce, Illy 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 i, llt:l a~liu, "
involving ca, L,ohydrates on Asn 295 and 370. In contrast CD23 bi. ~di"~ to SCRs1-2 is a protein-protein i"l~r~c1ion (Aubry ef al, CD23 i"lera~ with a new functional extracytoplasmic domai" involving N-linked oligos~ a~ides on CD21 J Immunol 152: 5806 (1994)). The p,ese"l inventors have now tested the effect ofthe other ligands of CD21 (EBV C3d g and IFN~) on CD23 binding to CD21 and on the reg~ tion of Ig production in the presence of IL4. Only EBV particles andan 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 indicale that CD23 binding to the EBV-binding site on CD21 selectively reg~ tes human lg production in the presence of IL~.

5 The present invention therefore includes within its scope the use of Epstein Barr Virus or of a part thereof to block the binding of CD23 to CD21. A prere,-~d part of the Epstein Barr Virus is the gp350/gp220 glycoprotein or a fragment thereof.
All~ dli-/ely an unglycosylated form o~ this gl~/w~rut~ i l or of a rl~y"~enl thereof may be used.
Again without being bound by theory it is b~ d that the ~r~:se"l invention allows effective treatments to be achieved by su~.press;ng the de novo synthesis of pro~ na~"",dlorycytokines.

15 This cGIlllasls with previous uses of allliLJod:~s simply to directly neutralise the cytokine molecules already pr~se"l in il ,na" ,ed tissues.

It should also be noted that there are speculative publications in the art listing large numbers of a"liL,odies as well as large numbers of possible ~lise~Ses which 20 the antibodies are said to be useful in tlealilly but not providing any soundevidence or data for most of the possible com~:. ,alions. One such public~lion is WO93/02108 which is primarily directed to the production of particular c;l,i",aeric antibodies.

The ,u,esenl invention is clearly distinguished from such publications by providing binding agents to particular molecules 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 rli~e~es including non-lgE me~ t~d dise~-ces- They may be used in the treatment and propylaxis of ulcerative colitis. They may aiso be used in the L, eal" ,enl and prophylaxis of Crohn s disease.

The binding agents of the ~ seul invention may be used alone or in colllLJ;.lalion with immunosul,pressive agents such as ~ roids cyclospo, i" or dl .lil ~0~ ?5 such as an anti-iy" Ipl ,ocyte antibody or more p~erel ably with a toler~"ce-inducing anti-autoimmune or anti-iu n~" ", IdlOl~r agent such as a CD4+T cell il Ihibilil ,9 agent e.g.
an anti-CD4 antibody (prererdbly a ~ hing or non-depleting a"libody) an anti-CD8 ~ ibody a TNF anlas~ooisl e.g. an anti-TNF antibody or TNF i"l ,ibilor e.g.
soluble TNF receplor or agents such as NSAlDs.

The binding agent will usually be sur)plie~l as part of a sterile plldllll~cel~ic~y z~ccelJl-l)le co~ osilio". This ~JI Idlll l~r~lIfjr~l COI I Iposilior) may be in any suitable forrn depending upon the desi,ed ",ell.od of adln;n;~ lg it to a patient. It maybe provided in unit ~os~ge forrn and may be provided as part of a kit. Such a kit would normally (although not necessa~ ily) include instructions for use.

20 Binding agent ad" ~ini~ lions are generdlly given pal ~l IL~rdlly for exampleintravenously intram~l~y~- ly or sub-cutaneously. The binding agents are generally given by injection or by infusion. For this purpose a binding agent isformulated in a plldlll ,~cel~ic~l composition conlai"i ,9 a pl Idl11 l~r~e uti~lly acce~,table carrier or diluent. Any applu~lidLe carrier or diluent may be used for 25 example isotonic 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.

wo s6t12742 PCT/EP95/04110 They may be given orally or nasaily by means of a spray, especially for treatment of resp;, ~loly ~Jiso, ~e, ~.

They may be formulated as ueams or ointments, especially for use in ll ~3aLil 19 skin 5 ~lisorcler~.

They may be fommulated as drops, or the like, for admini~ to the eye, for use in treating disor~Jer-~; such as vernal conjunctivitis.

For i"; ~hle solutions, exo;~ie,)ls which may be used include, for example, water, alcohols, polyols, glycerine, and vegetable oils.

The pl,a""aceutical composilions may con~;n preserving agents, solubilising agents, sl~hilisillg agents, wetting agents, emulsifiers, swcctcners, colourants, odourants, salts (SUbSldl ,ces of the present invention may ll ,e" ,selves be provided in the form of a phall~cel~ lly accepldble salt), buffers, coating agents or anlioxida"ls. They may also conlai., other ll ,er~peutically active agents.

Suitable dos~ges of the substance of the presenl invention will vary, depending upon factors such as the disease or d;so, der to be treated, the route of admini~ lion and the age and weight of the individual to be treated. Without being bound by any particular dos~ges, it is believed that for inslance for ,c,~,~"le,~l adlllillisl,~lio,l, a daily dos~ge of from 0.01 to 50 mg/kg of a binding agent of the present invention (usually present as part of a pha""aceutical composition as indicated above) may be suitable for treating a typical adult. More suitably the dose might be 0.05 to 10 mg/kg, such as 0 1 to 2 mg/kg.

WO g6/12742 PCTI'EP95~04~0 This dosage may be repeated as often as approuriaLe. Typically adminisl.alio"
may be 1 to 7 times a weeks. If side effects develop the amount and/or frequencyof the ~lo.s~ge can be re~ ce~

5 A typical unit dose for i"cc ,I,ordlio,l into a ~ul ,a""aceutical coll~posilio" would thus be at least 1 mg of binding agent suitably 1 to 1000 mg.

The pr~3sel ,l invention includes within its scope an assay for determining whether or not a particular agent which binds to CD21 CD11 b CD11 c or to a 70~ or 115 10 KDa protein e~r~ssed on ~"dolhelial cells may be useful in the l,~cl~"enl of an illlldlllllldtoly ~ i ",.,une or alleryic ~I;se~se cc,-,~ i.,y. ~Jel~llll;.lLI5~ whether or not the agent is ~p~hlQ of blochlng the i nlardcliol) between CD23 and CD11 b orthe inlt:la~iGIl between CD23 and CD11c or the i"te,acliu" between CD23 and CD21 or the i"le,d-tio" between CD23 and a 70-85 KDa or 115 KDa protein 15 e~iesse~ on e, Icloll ,~lial cells.

This assay can be used for screening co"~pounds or molecules by using ce!l linese~,u,~ssi"g the ap~.,o~,iale moleu~'es r~e~e,ably CD11b is used in these assays as CD11b/CD18 and CD11c is used as CD11c;/CD18. CD11blCD18 and 20 CD11 c/CD18 can be co-e~r~ssed on cell surface.

Any a,op,u~.riale assay technique can be used e.g. protein-non protein assays (e.g. assaying the i"lerd~lion of l.rotei. ,s with ~;he~ .2's or sugars) protein-protein assays or protein-cell assays.
The present invention will now be described by way of example only with rt:rer~"ce to the acco" ,panying draw,. ,y:" wherein:

W O 96112742 PCT~EP95/04110 FIGURE 1a illustrates CD23-liposomes binding to CD14 positive blood ~ono"-lclear cells;

FIGURE 1b illL~dles various CD23 affinity purified p~ute~s on SDS-5 PAGE gels;

FIGURE 2 illuslldles the perce,ll~e inhibition of CD23-liposome binding to activated blood monocytes obla;l ,ed using certain ~ ~ lonoclo~ lal antibodies;

FIGURE 3 ill~ dles the binding of CD23 li~Joso",es to various transfected cells;

FIGURE 4 illu~dl~s the effect of various suL,~lances on CD2~CD11b and CD23-CD11 c il ,lerdclion FIGURE 5 ill~ls~dles the effects on nitrite prod~ction and oxidative burst in monocytes ~used by CD23 binding to CD11 b and CD11 c; and FIGURE 6 illu~ll ales that the b;, Idir ly of l~cc lllLil ~a"l CD23 to CD11b and20 GD11 c specifically i"creases cytokine production by monocytes.

FIGURE 7a iilusl,~les the inhibition of CD23-liposome binding to RPMI
8226 cells by various CD21 ligands.

FIGURE 7b illustrates the inhibition of IL~ induced IgE and IgG4 production by an EBV peptide binding to CD21.

FIGURE 7c illusl,ales the modulation of immunoglobulin production by an EBV peptide binding to CD21.

W O 96/12742 P~~ 9~104110 ~ FIGURE 7d illustrates the absence of i"hibilion of IgE production with a C3 ,ues~lide bi,ldiny to CD21.

FIGURE 8 illustrates the inlli~ilion of the binding of CD23 liposon~es to certain e, ldoll ,elial cells due to the ,ul ~ :s~"ce of an anti-CD23 MAb.

EXAMPLES
1 0 (In some of the following examples the terms "ip" "id" and "n" are used. These mean "intraperiLonea'" "intraderrnal" and "number of aninnals" respectively.) EXAMPLES 1~

Inte, a-tiu" between CD23 and CD11 b and between CD23 and CD11c Examples 1 to 6 and the accon~pa,-ying Figures (see later) illustrate the i"leraction of CD23 with CD11 b and/or CD11 c.

In these Examples full-length r~co",Li.,anl CD23 incorporated into fluorescent liposon,~s was shown to bind to COS cells lldllar~cled with cDNA er,cûdi"9 either CD11 b/CD18 or CD11 c/CD18 but not with lr~"srectdnl~ expr~ssi"g CD11 a/CD18.
The i"l~:r~ion between CD2~1i~Joso",es and CD11b/CD18 or CD11c/CD18-transfected COS cells was specifically inhibited by anl:i-CD11b or anti-CD11c respectively and by anti-CD23 monoclonal antibodies. The functional siy"irica"ceof this ligand pairing was de",onsll-~led by l,igy~ril)y CD11b and CD11c on monocytes with either recombinant CD23 or anti-CD11 b and anti-CD11 c monoclonal antibodies to cause a marked i~1~ease in nitrite (N02-) oxidative products (H2O2) and proinfla"""alor~ cytokines (IL-1~ IL~ and TNFa). These CD23-mediated activities were dec;r~ased by Fab r, ~yl ~ ~e~ ~ls of monoclonal CA 02203364 l997-04-22 O96J12742 PCT~EPg5/04110 al~libodies to CD11b CD11c and CD23. These results demonstrate that the surface adhesion moleu~es CD11b and CD11c are receplo~ for CD23 and that this novel ligand pairing reg~ ?S illIpOI~ activities of monocytes.

The following ~is~ ~ssion explains briefly the ex,ue, i" ,ental design and the rationale behind Examples 1 to 6 (which follow):-Total blood mononuclear cells were incubated with reco" L~. Ianl full-length CD23 inco~oraled into fluorescel~l liposo",es and analysed by flow cyto,nel,~ (PochonS. et a/. J. Exp. Med. 176 389-398 (1992)). A r~uliG" bound CD23-li~,oso",es (Example 1 Fig. 1a) which was then shown by double staining to consist of CD14-positive cells (i.e. n ~u"o~;ytes)~ To cûl ,ri" " that monocytes were ab!e to bind CD23-liposomes blood ~ononuclear cells were FACS-sorted into CD14-positive and CD14-negative pop~ ions (Example 1 Fig. 1a). CD23-liposomes were shown to bind only to the CD1~positive pop~ ion (Exdl~ 1 Fig. 1a). Since monocytes were found to e~r~ss neither ",er,~L,r~"e IgE nor CD21 (not shown) the known ligands for CD23 it was inve:jligaled whether monocytes ex~,ess a clirrer~nl ece~.lor for CD23. MonGu~/tes were Iysed and cell ext, d~ purified over an affinity column coupled with recornbi"a"l soluble CD23. SDS-PAGE and silver staining analysis ûf the eluted ",aLerial revealed bands of around 80 and 160 kDa MW
(Example 1 Fig. 1 b). Antibodies identifying antigens within this range of MW and reported to be expressed on monocytes were tested by FACS for their capaci4 to inhibit CD23-liposome binding to monocytes (E-xample 2 Fig. 2). Anti-CD11 b and anti-CD11 c monoclonal antibodies both inhibited CD23-liposome binding to monocytes with varying degrees of potency (Example 2 Fig. 2). Anti-CD13 anti-CD49d anti-CD21 (not expressed on monocytes) and anti-CD11 a (the third member of the ~2 integrin family of adhesion molecules) had no siyl ,ir,canl effect (Example2 Fig.2). Antibodiesagai~slMHCClassl Classll CD14andCD45 all of which highly expressed on monocytes were also tested for their effect on CD23-W O96112742 PCT~EP9S104110 liposon ,e binding. None however had any effect (not shown). Anti-CD18 ,nonoclonal antibody gave a partial inhibition of CD23 binding. This could be due eith~r to steric ll;ndlc,.ce or to the induction of a ~"ro""~lio"al cl,anye in the CD11b and CD11c mo!ecl~'es upon anti-CD18 I\/llab binding. The monocyte-derived proteins eluted from the CD23-affinity column were immul,o,e~ /e with anti-CD11 c (Example 1, Fig.1 b) and anti-CD11 c/CD18 antibodies (not shown).

To COI llil ll~ that the a chain of CD11 b/CD18 and CD11c/CD11 b were l~:ce~lo~ for CD23, full-length cDNAs encoding CD11 b and CD11 c were ll dl l~iel llly co-transfected with CD18 cDNA into COS cells. Tr~nsre~anls expressing CD11b/CD18 and CD11c/CD18 were both shown to bind CD23-li,uosomes, in l to ll ~n~re~,~, lls ex~ ssing CD11 a/CD18 (Example 3, Fig. 3). This might be explained by the higher degree of homology be~ween CD11 b and CD11 c when co,,,,u~red to their ~lo"~n;oyy to CD11a. The specificity of the i,ll~r~:iion was ~t~l I 101 I~ l dLed by in~ibilil ly CD23-liposome binding using anti-CD11 b, anti-CD11 c and anti-CD23 "lonoclo"al antibodies. The same results were obtained using BHK cells expressing CD11b/CD18 and CD11c/C!D18 (not sho~Jvn). As further proof of the spec~lcity of the CD23 inler~clion, activated blood monocytes from a Leukocyte Adhesion Der,cien~y patient, lacking ,B2 i,,leylill expression due a mutation in the gene encoding the ~ subunit were unable to bind CD23-liposomes (not shown). Together, these data demo,~ dle that CD23 i"lerd~ with CD11b and CD11 c on normal human " ,ono~tes and on lr~l ,sre.,l~nls.

CD11b and CD11c are adhesion molecules that participate in many cell-cell and cell-matrix i"lera.;lions. The examples show that CD11 b/CD18 and CD11 c/CD18 may exhibit an ~clrlitional 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 cdpacily of factor X to inhibit in a dose dependent manner CD23-liposome binding(Example 4, Fig 4) without affecting surface expression of CD11b or CD11c on monoc~/tes (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
deu~ases CD23 binding to Illo,lo~tes (Example 4 Fig. 4) by chelation of Ca2' which is necess~y to CD23 ~ y and/or by chelation of the divalent cations which are ,~ecessa~y for the ~indi, ly of ligal~ds to CD11 b and CD11c (Altieri D.C.
J. Imm~nol. 147, 1891 -1898 (1991)). CD23-CD11 b/CD11 c inlerac1ion seems to involve sugars but not sialic acid as observed by the c~p~rily of tunicamycin but not ne~,d",i,l:~ase to .~e~ase CD23 binding to monocytes. CD23 bears ekl,~oell~ rly a triplet of amino acids (Asp Gly Arg) (~kutani H. ef al. Ce/l 47, 867-885 (1986)) which in the reverse ori~ lion is a co,nm~n recoy"ilion site forthe illl~ylill receplo~:,. Thererore, the effect of a polyclonal antibody directed aga; ,sl this tripeptide was tested for its capaci~ to inhibit CD23 binding to ",o"o~ytes. No inlliL~iLi~ was observed, confirming the absence of inhibition obtained with fibrinogen (Example 4. Fig. 4). IgE whicn is binding in the lectindo"!ai.~ of CD23 partially inhibits CD23 binding to ",o"oc~/tes (Example 4 Fig. 4).
Those resul~s i"dicaLe that CD23 would seem to be acting as a C-type lectin recog"isi"g partly sugar and protein structures re",;"iscenl of what has been observed for CD23 i"lerac~ion with CD21 (Aubry J-P. et al. J. Immunol. 152, 5806-~813 (1994)).
To evaluate the functional siy,lirica"ce of the inler~ction of CD23 with CD11b or CD11c we investigated whether CD23-CD11b/CD11c i"le,~-tion could trigger monocytes to release proinflammatory mediators such as nitric oxide H202 and cytokines. Triggering of adherence-activated normal monocytes using recor"binanl soluble CD23 anti-CD11b or anti-CD11c antiho~ies increased the generation of N02- indicating the activation of the N0 pathway (Moncada S.
Palmer R.M.J. & Higgs E.A. Pharmacol. Rev. 43, 109-144 (1991)). The effect of CD23 on nitrite production was inhibited by Fab fragments of anti-CD23 ",onoclo"al antibodies and by nilroa,yi,li,le a specihc inhibitor of the N0 synthase W O96112742 PCTAEPg5/04110 pathway (Example 5 Fig. 5a). The oxidative burst was also shown to be reg~ t~d through CD11b and CD11c since recc""L,i"d"l soluble CD23, anti-CD11b and anti-CD11c monoclonal a,.li6Gdies all caused oxidation of hydluell,idine to ethidium ~u~,ide in ll~ no~;ytes (Example 5 Fig. 5b). This co~ ~rilll~S and extends the finding that anti-CD11b mo,loclonal antibodies induce an oxidative burst in monocytes (Trezini C. Schuepp B. Maly F.E. & Jungi T.W. Brit. J. Haematol. 77, 1~24 (1991)). CD23 ~inding to CD11 b and CD11c was ~ssoci 'ed with an early ~Jeciric Ca2' flux in blood monocytes (not shown).

Since activated I I lau u,ul ~ayes are an impûrtant source of pl ~ ~na~ "" IdlOI~
cytokines we evaluated the effect of f~co"~binanl soluble CD23 and of anti-CD11 b and anti-CD11c Illolloclonal antibodies on the production of such cytcl~ es by monocytes. Reco~Lina,)lsolubleCD23 anti-CD11bandanti-CD11c~u~oclonal al~ G~ s were potent stimulators of IL-1 ~ IL6 and TNFa. Again the spe~;i;i-;il~/ of this induction was c~ell~o,)sl,~led by using Fab r,ay",e"l~ of anti-CD11b anti-CD11c and anti-CD23 "-onoclonal antibodies (C~dm:le 6 Fig. 6). Inler~li"yly IL-1 and TNFa were potent inducers of CD23-li~oso" ,e ~ :. ldi~ lg to monocytes (not shown) sl Iggesting a polenlial cytokine autocrine loop through CD11 b and CD11 c stimulation and reg~ on.

a) CD23-liuosomes bind to CD14 positive blood mononuclear cells (See Fig.
1a).
Blood ",o"onuclear cells were -il. ;ned with anti-CD14 monoclonal antibody (Becton Didcinson Erembodegem Belgium) followed by sheep FlTC-conjugated - F(ab )2 antibûdies to mouse IgG and IgM (Bioart Meudon France) both diluted in PBS 0.5% BSA and 0.0~~/o sodium ~ide prior to FACS-sorting (FACStar Plus Becton Dickinson) into CD14-positive and CD14-negative cell populations.
Se~.~raled cells were then stained with CD23-liposomes or control (glycopho~ A)-W O96112742 PCT~EP9S/04110 liposomes diiuted in 0.5% BSA, 0.1% sodium azide, 2 mM CaCI2, 140 mM NaCI, 20 mM Hepes, pH 7 and inc~ Ih~tAd for 2 h at 4~C (Pochon, S et al., J. Exp. Med.176 389-3~8 (1992)). Afterwashes, cells (5,000 events/con~i~ion) were analysed by FACS.

b) ALIpdl~:l)l molecular wei~ht of CD23-afFinitv purified blood monocYte Pruteil~s and immLi~o~ ea~ itvwith an anti-CD11c "~onoclon,al a,llibod~ (See Fig.
1b).
Lysates of blood ~ûno~tes were affinity purified on a CD23 co1umn, eluted proteins separaled on SDS-PAGE gels and lr~":,re,.~d onto nitrûcellulose. Mr markers are shown on the left. The gel was silver stained (left lane). Filters were inu~h~ted with either an isotype "l~lcl1ed a"libo~y (middle lane) or with an anti-CD11c monoclonal antibody (BU-15, right lane), then with horseradish peroxidase-COI 1,- I9::~t~d goat anti-mouse anliL,ody (Kpl; GaiU ~e, ~burg, Massa ;l ~u,setts).

Anti-CD1 1 b and anti-CD11 c monoclonal antibodies decrease CD23-li,uosorl ,e bindinq to activated blood "~or,ocvtes (See Fig 2).
Mo nocytes were en, i~;l ,ed from l l lol ~ûnudear cells by Ficoll and o\~e~ l IL
adherence to plastic in RPMI 1640 (Seromed, Berlin, Germany) suppleme, lled with2 mM glutamine and 10% heat-inactivated FCS (Flow Laboratories, Irvine, Scotland). Activated ~ûnoc,vtes were then inalhz~itecl with CD23-liposomes in the presence of ~'irr~renl monoclonal a"li60c';es (o~CD) or isotype~,atched c~illlols (CTRL) (Becton Dickinson), all tested at 10 ~lg/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 monoclor,al antibody 44 was fromSerotec, mûn.gran 1 was from Janssen (Beerse, Belgium), Leu-15 was from Becton Dickinson (Ere"lbodegem, Belgium) and (Bear-1) was from Sera-Lab Ltd (Sussex, GB) Anti-CD1 1c ~o,~oclonal antibody 3.9 was from Serotec, SL9 was W O96/12742 PCTAEP95~04110 from Sera-Lab and BU-15 was from The Binding Site (Birrningham, UK). Anti-- CD13 (SJ1D1), anti-CD18 (BL~), anti-CD23 (mAb25) and anti-CD49d (HP2.1) monoclonal antibodies were from Immunotech. Anti-CD21 ",onoclo"al anlibud~
BL13 was from Imm~"lolech, OKB7 from Ortho and BU-33 was obta;ned from Dr.
S MacLennan (Binnil,gl,~r" University, UK), HB-5 from ATCC, OKB7 from Ortho Diayllu~lios System Inc (Raritan, NJ). Anti-CD14, anti-CD3, anti-CD16 and anti-CD20 monoclonal anlibod;cs were from Becton-Dickinson Cells were analysed by FACS and mean fluor~sce~ ~ce i"Lensi~ (MFI) measured. Data of a representative experi~ ~ ~e~ ~l are presented. MFI of cells stained with control-liposomes was 6.5 and with CD23-liposomes was 84.5. Perl;el,l;~ye il~ io~
using ~l ilhl l l~lic linear MFI values is ~lc~ 'ed acco~ dil lg to the following formula:

[~CD23-lipo)]-[(CD23-lipo)+Mab]
% inhibition = MFI x 100 (CD23-lipo) CD23-liposomes bind to ac chains of CD11 b/CD18 and CD11 c/CD18 on recombinant transfectants (See Fig. 3). cDNAs coding for CD11a (Corbi, AL., Miller, L.J., O'Connor, K, Larson, R.S. & Springer, T.A. EMBO J. 6, 4023-4028 (1987)) was recloned in pCDNA1 (Invil,~g~,1, San Diego, CA). cDNA for CD11b (Corbi, A.L., Kishimoto, T.~, Miller, L.J. & Springer, T.A. J. Biol. Chem. 263, 12403-12411 (1988)) and CD18 (K~sl,i,nolo, T.K, O'Connor, K, Lee, A., Roberts, T.M. & Springer, T.A. Cell 48, 681-690 (1987)) were recloned in pCDM8 (Seed, B.,Nafure 329 840 842 (1987)). 20 ~Lg ~liquot~ of DNA were transfected in COS-7 cells (ATCC) by electroporation (260 V, 960 ,uFD) using a Gene Pulser device (Bio-Rad, Ridlmond, CA) and 0.4 cm cuvettes in 20 mM Hepes pH 7.4, 1~0 mM
NaCI. Co-l, ar,sre~ions of CD11 a; b or c with CD18 were performed in order to get expression of the ,B2 int~y, i"s at the cell surface. Controls were done with single WO g6112742 P~ /04110 chain ~ ;GIIS. 48 h after lldllar~iOII COS cells were stained with anti-CD11 a anti-CD11 b and anti~D11 c ")o"oclo"al antibodies or isotype-" ~aL~I ~ed noclo~ ,al antibodies (control) followed by FlTC-labelled sheep anti-mouse ~Illi~o~. Between 10 to 15% of the cells were shown to e~r~ss CD11a b c or CD18 by staining with the res~,ec1ive n~onoclonal an~ o~ s Prior to staining with CD23~ Jo~o",es CD18-positive ~an~recle~ COS cells were then FACS-sorted in order to in~ ase the ~,e, ~"l~ge of cells ~*,r~si"g ,B2 il lleyl il lS. CD11 a/CD18 CD11 b/CD18 and CD11 c/CD18 ~ dl ,sre. ta"ls were then inc~ ~h~ted with CD23-I;, osor"es (trace 2) or control (gl~copl~ri,) A)-lip~sG,nes (trace 1). The sf~eciri.;it~
of CD23 interd~tion with CD11b and CD11c was demon-;lr~lad by inhibition of CD23-liposome binding to CD11 b/CD18 and CD11 c/CD18 l, ansre~dnl:, using anti-CD11b (trace 4) anti-CD23 (trace 5) and anti-CD11c (trace 6) ",onoclo"al antibodies res,~e~ tively. No binding of CD23-li~oso" ,es was observed on CD11a/CD18 t,d"~reclc, 11~ and no effect of anti-CD11 a r"onoclo"al al l~ibody was found (trace 3).

Structural cl ,ara~;lerisalion of CD23 CD11 b. CD11 c inter~;lio" (See Fig. 4).
(a) Involvement of suqars and divalent cations.
Purified activated blood monocytes were ll ~aled or not with tunicamycin (10 ~lglml) for 48 h or with neuraminidase (0.1 U/ml; both from Boel"inger Ma"ul,ei.n Mal " ,hein Germany) for 45 min. Cells were then ina lh~ted with CD23-liposor"esor control-liposomes in the absence or ,),ese"ce of EDTA (5 mM; top left panel) Ca2' or Mn2' (1 to 10 mM; top right panel).
(b) Factor X does inhibit CD23 bindin~ to ",~noc~/tes. Purified activated blood IIGI locytes were inalh~ted with CD23-li~ oso",es in absence or presence of factor X (0.1 to 10 U/ml; Sigma) (bottom left panel) ri~rinogen (50 ug/ml; Sigma) purified t:co,nbi,la,1l ICAM-1 (prod~ced in our laboratory) LPS (1 ~lg/ml; Sigma) human serum o,l~sollised-zymosan (1 mg/ml; Sigma) IgE (~0 ~lglml; The Binding Site - Birmingham) or polyclonal antibody to RGD peptide (1/~00 ATCC) (bottom right panel). Cells were analysed by FACS and MFI measured. Per~enl~e iul ,i~ilion was calc~ t~d as for Exdn ipl~ 2.

Recorl Ibi, lal ll CD23 by bindin~ to CD11 b and CD11 c specificallv inueases a the nitrite Product and b the oxidative burst bv monocvtes.
Monocytes were incl Ih~ted a for 4 days at 37~C or b ovemight in the absence or presence of reco~ I IL;I ~a~ ll sol- Ihle CD23 (Graber P. et a/., J. Immunol. Mefhods 149 215-226 (1992)) (50 ng/ml) anti-CD11a (clone 25.3) anti-CD11b (clone 44) anti-CD1 1c (clone BU-15) Illonoclo, ~al al llibod.~s (all at 10 ~lg/ml).

To assess the amount of NO produced (which is shown in Fig. 5a) the culture s~l~elr~ la were assayed for the stable end products of NO NO2 and NO3-accor~J;.,g to Green etal., Annu. Rev. ImmLmol. 2199-218 (1984). The s~Jecifiuily of CD23-me~ cl inaease of NO2- production was de~o~ cled by illhibiliûll of NO2- production by Fab fragments of anti-CD23 monoclonal cnli~oclies (mab25) (tested at 10 ~g/ml) and by inhibition with r,il, ual ~;"i"e (N-Arg at 1 mM) (Sigma).
Activated ",onocytes were ina~h~t~d with hyd,oell,:dine (Molecular probes Eugene OR) (0.3 llg/ml) for 30 min at 37~C (Rothe G. et al., J. Leukoc. Biol. 47440448 (1990)) and analysed by FACS. Pe~ uenlage inuease in red fluo~ ~:sce~ ~ceof stimulated monocytes is shown in com~a~ison to u"l,e~led monocytes (See Fig.
25 5b). Monocytes which had Ul ,der~ol ~e an oxidative burst shown an increase of red fluorescence signals co~l~,uared to ~ lad monocytes reflecting oxidation of hydroethidine to ethidium bromide (Lacal P.M. et al., Biochem. J. 268 707-712 (1990)). MFI values of "~o"oc~/tes alone were 159+/-10. Mean+/-SD values of 6 expe, i"~e, lls are ~l ~senled. Con A which is known to induce a respil ~loly burst in W O96112742 PCTA~P95104110 monocytes, was used as a positive control. The specificity of the CD23 interaction with CD11b and CD11c was der"onsl~ted by illil;,JiliGn of CD23-"~edidled increase of H2O2 production by Fab r~cyll~ellls of anti-CD11b (clone 44), anti-CD11 c (clone BU-15) and anti-CD23 (mAb25) monoclonal antibodies (tested at 10 5 ~Lg/ml).

Bindinq of I ~cor, ILi~ CD23 to CD11 b and CD11 c specihcally increases cytokine production bY ~ ~o"o.,./tes (See Fig. 6).
Mo"oc~tes were inulh~ted ovemight at 37~C in the aL,sence or p,~se"ce of r~cor"b;nanl soluble CD23 (Graber P. et aL, J. Immunol. ~S3lll0ds 149 215-226 (1992)) (50 ng/ml), anti-CD11a (cione 25.3), anti-CD11b (clone 44), anti-CD11c (clone BU-15), anti-CD23 (mAb 25 - this anlil,ody is available from Immunotech. It is ~isu ~ssed in published EUI UPeal1 Patent A~ liol l EP-A-0269728) rllonoclonal antibodies, Con A (Sigma) (all at 10 ~glml), LPS (1 ng/ml) (Sigma) or PMA (5 ng/ml) (Calbiochem, La Jolla, CA). Cylo:~ines were measured in the culture supei "al~l IL by specific ELISA. The ELlSA's limit of sensitivity is 0.05 ng/ml for IL-1~ (Ferrua ef al., J. Imm~mol. ~1~1flo~s 114 41~8 (1988)) 0.01 ng/ml for TNFa (Medgenix, Biotechnie, Rungis, F) and <0.01 ng/ml for IL~ (Manie et a/., Eur. Cyfok~ne Nefw. 4 51-56 (1993)). The s~e~iri~ily of CD23 inle~d~;lion with CD11 b and CD11c was demon~L,aled by il ll lil,ilion of CD23-me~ ted increase ofcytokine production by Fab fragments of anti-CD11b (clone 4), anti-CD11c (clone BU-15) and anti-CD23 (rnAb25) monoclonal antibodies (tested at 10 ~lg/ml).
Mean+/-SD values of 4 expe, i" ,e"l~ are ~ senled.

The following materials and methods were used in this example:

Cell Lines O 96/lZ742 Tonsil or blood mononuclear cells were separated into T and B cell subpopulations by rosetting with sheep red blood cells.

The B cell line RPMI 8226 was obl~ined from the American Type Culture Collection (ATCC, Rockville, MD) and cultured in RPMI 1640 complete medium.

Peptides and CD21 ligands Two peptides, from gp350 of EBV and C3, known to bind to CD21 (Servis ef a/, C3 synthetic peptides support growth of human CR2-positive Iymphobl~.stoid B cells, J
Immunol 142: 2207 (1989)) were synthesized. PepEBV (TGEDPGFFNVEIC-NH2) was produced on an ABI 431A synthesizer using FastMoc chemistry and PepC3 (GKQLYNVEATSYAC-NH2) was obtained from Neosystem (Sl, asbourg, France).
Aggregated C3d,g was prepar~d as des~i~ed previously (Carel ef al (1990) supra). Sucrose gradient purified EBV was obtained from Advanced Biotecllnologies (Columbia, ML) and IFN a was o~L~:ned from Sigma (St Louis, MO).

Liposome pl ~,a. dlion CD23-liposomes were made as previousiy described (Pochon et a/, Del l lol l~ lioof a second ligand for the low affinity ,~ceplor for immunoglobulin E (CD23) using reco"lbina"l CD23 reconstitut~cl into fluorescenl liposomes, J Exp Med 176: 389 (1992)) using 10 ~lmoles of the synthetic ~hospllolipiis POPC (Avanti Polarlipids Inc. Alabaster, AL) mixed with 50 nmoles of fluorescent dye DiO18 (Molecular Probes, Eugene, OR) and then dialyzed aS~ai,lsl HEPES buffer together with purified recol,lbil1ant CD23 or with glycophorin A (0.2 ~moles each) referred to as control protein.

Flow cyl~ Qtly Liposo.,~e l,;.~ g assay O 96J12742 PCTAEPg5104110 Cells (105) were resu:"~e"ded in 50 1ll of the liposo~"e suspension, diluted 10 times in 0.5% BSA, 0.1% NaN3, 2 mM CaCI2, 140 mM NaCI, 20 mM Hepes, pH 7.0 and in~ Ih~tpd for 2 h at 4~C.

Cells were washed twice before analysis on a FACStar plus (Becton Dickinson, Ere",Locleggen, Belgium).

cG~ uelilion of CD23~ JosG..Ies with EBV, EBV peptide, IFN a, C3 peptide and C3d,g RPMI 8226 cells were co-ina Ih~ted with gl~;ophol i,1-liposomes or CD23-li~,oso",es and EBV (1x105 to 1x109 particles/ml), EBV peptide and C3 peptide (50 nM to 50 ~lM), aggregated C3d,g (4 ng/ml to 1 ~g/ml) and IFN oc (1000 U/ml) for 2 h at 4~C. Cells were analyzed as desc;, iL,ed above.

IL4-in~ efllg production assays Cells were incubated at 106/ml for 14 days in Iscove's medium enriched with l,~"sr~"i", bovine insulin, oleic acid, linoleic acid, palmitic acid, BSA (all from Sigma) and 10% FCS (Flow Laboralu~ies, Irvine, Scotland) as described by Claassen et a/ (A cell culture system that enhances mononuclear cells IgE
synthesis induced by reco"l~ na"l interleukin4, J Immunol Mefhods 126: 213 (1990)). Assays were performed using total PBMNC with IL4 alone (200 U/ml) or IL4 plus anti-CD40 (1 llg/ml) (Serotec Ud. Oxford, UK), or using purified tonsillar B cells with IL4 and anti-CD40. IgE, G, A and M were quantified by specific ELISA as previously desuibed (Bonnefoy et al, Inhibition of human interleukin4-induced IgE synthesis by a subset of anti-CD23/Fc epsilon Rll monoclonal antibodies, Eur J Immunol 20: 139 (1990)). IgG4 was measured by ELISA as follows. A mouse anti human IgG4 antibody (Southern Biotechnology, Bir, ~ yh~m) diluted at 10 llg/ml in bicar bol1ale buffer, pH 9.6 was coated ovemight in 96 well plates (100 ~ll/well). Saturation was then performed with PBS

W O 96/12742 PCTA~P95/0411Q

plus 1 % BSA (200 ~I/well) for 2 h at RT. Samples to be tested were diluted in PBS
~ plus 0.5% BSA and 0.1% Tween (100 ~I/well) and ina~h~ted ovemight at 4~C.
After washes with PBS plus Tween a peroxidate-labelled sheep anti human IgG4 antibody (Vital products St Louis MO) diluted 11~000 in PBSIBSA plus Tween was added for 1 h at RT. After washes with PBS plus Tween o-phenylene diamine (Si~ma) is added and the colo,i"~ellic rea~;liol~ was stopped with 2M
H2SO4. Plates were finally read at 492 nm.

The results obtained are ~iiscl ~ssed below:
Human CD21 has been previously described to be a rece,l~lor for the C3d g and jC3h proteins of the co~ . "ent system (Weis et a/ Ide, l~iric&lion of a 145 000 Mr mer,lbl-a"e protein as the C3d receptor (CR2) of human B l~")phoc~tes Proc Nafl Acad Sci USA 81: 881 (1984)) for the gp350/220 envelope gl~w~folei.l of EBV
(Nemerow et a/, identification of gp 350 as the viral glycc,~ ,tein medidlil,g "enl of [pslein Barr Virus (EBV) to the E13V/C3d r~ceplor of B cells:
sequence ho",clogy of gp350 and C3 complement fragment C3d J Virol 61: 1416 (1987); Tanner et al, Epstein Barr Virus gp 350/220 binding to the B Iymphocyte C3d receptor mediates adso".lion capping and endocytosis Cell 50: 203 (1987)) 20 and for IFN~ (Delcayre et a/, Epstein Barr Virus/complement C3d receptor is an i~ ~le, re, u~10C ~ ~ce~lùr EMBO J 10: 919 (1991)). We have therefore tested all these CD21 ligands for their ability to inhibit CD23-liposome binding to the CD21-expressing oells RPMI 8226 cells (Pochon ef a/ (1992) supra). Intact particles of EBV were able to inhibit CD23 binding to CD21 in a dose dependent manner.
25 This is shown in Figure 7a which illustrates the iulliL~ilioll of CD23-liposome ~i,ldillg to RPMI 8226 cells by some CD21 ligands. [RPMI 8226 cells were co-inulh~ted for 2 h with CD23-li"oso"~es or glycophorin-liposomes and various ~"ce"l~alions of EBV (pa,licles1ml) PepEBV and PepC3 (~lM). Per~"lage of inhibition is ~Ic U~ te~l as follows:

W O96112742 PCT~EP95/04110 (MFI CD23-L) - (MFI CD23-L I liqands) x 100 (MFI CD23-L) The MFI of glycol~h~,in-li,uoso,,,es was sul~t,~ted from the MFI of CD23-liposoh~es. Inset= FACS profiles of RPMI 8226 cells stained with gl~copl~o,i"-liposomes (traces 1) or CD23-liposomes (traces 3) alone or in presence of EBV
particles (top), PepEBV (medium) and PepC3 (bottom) (traces 2). Results are taken from a representative ex~e, i",~"l.]
Of the other CD21 li~ands tested for inhibition of CD23 liposome binding, only EBV decreased the binding of CD23. A complete inhibition of CD23 billcli. l9 wasobserved with EBV intact ~Jdl li-.les, although EBV is reported to bind to SCR 2 of CD21 and not to SCRs 5~ where CD23 binds to sugars in this latter region (Aubry et a/ (1994) supra). This complete inl~ibilion of CD23 Lindiny could be due to the size of the virus pallicl~ or to the fact that EBV may modify the collru""~liol1 of the CD21 molecule. în order to exclude that inl ,iLilion was due to steric hindrance by the virus pa, li~les, we then tested the effect of a peptide of gp350/220, known to bind to CD21 (Servis etal (1989) supra). This EBV peptide was able to inhibit CD23 binding to CD21 in a dose dependent ",a,~"er, with a maximum of 55%
inhibition (Fig 7a). These ex,ueri,,lenLs suggest that the EBV peplide binding is close to the CD23 binding site and partially blocks CD23 binding. These data co~ ,ri"~ our previous finding that CD23 does bind to CD21 (Aubry et a/, CD21 is a ligand for CD23 and reg~ teS IgE production, Nafur~ 358: 505 (1992)) and extend them by showing that SCR2 is probably a region interacting with CD23. In contrast, a C3 peptide co" es,oonding to the CD21 binding site on C3d (Servis et a/
(1989) supra) (Fig 7a), aggregated C3d,9 and IFN~ (data not shown) were unable to inhibit the binding of CD23 to RPMI 8226 cells. This suggests that SCRs 1 and3~, where C3 and IFN~ bind respectively, may not be involved in CD23 binding.

W O 96/~2742 PCTAEP9S/04110 ~ The EBV binding to CD21 does not require glycosylation of SCR2 of CD21 (Moore et a/ Inhibition of Epstein Barr Virus infection in vitro and in vivo by soluble CR2 (CD21) ~nlé il ,;, lg two short conser~s~ls repeats J Virol 67: 3559 (1991)).
5 Likewise, CD23 L.illdillg to SCR2 region is independent of sugars (Aubry et al(1994) supra). This is in line with our observation that a non~lycosylated synthetic peptide is able to de~;rease CD23 binding to CD21. Therefor~ CD23 binds to a binding site in SCR2 on CD21 that is close or ide"Lical to the EBV binding site which differs 1From the binding sites previously des~ ibed for C3d 9 and IFN~.
CD23 was previously shown to positively regulate IgE production by binding to CD21 on B cells (Aubry et al ~1992) supra). Based on the observation that an EBVpeptide blocked CD23 binding to CD21, we investigated the effect of this EBV
peptide on IgE pro~ tion. The EBV peplide was able to inhibit IL-4-induced IgE
15 pro~ ction in a dose~ependent ~ r"~er. This is shown in Figure 7b, which illustrates the inhibition of IL~induced IgE and IgG4 production by an EBV
peptide binding to CD21. [PBL or purified lo"sillar B cells (106/ml) were ina Ih~eli for 14 days with 200 U/ml of IL4 alone or in the presence of anti-CD40 antibody (1 ~g/ml) and i"~-easi"g co,-ce- lll dlions of EBV peptide. IgE and IgG4 were 20 measured by specific ELISA and mean values +/ SD of one representative expel imenl are presented (n-~)].

This effect was observed in T-cell dependent and also in T-cell independent IgE
production systems (Fig 7b) in which T-cell help is replaced by anti-CD40 Ab.
25 This collfilll~s our previous observation (Henchoz et al Stimulation of human IgE
production by a subset of anti-CD21 monoclonal a~llibudies: requirement of a co-signal to modulate ~llanS~ipls~ 81: 285 (1994)) that CD23-CD21 can regulate IgE productiûn even in absence of T-cells by a homotypic B-B cell i"lera.;lion since B cells can express both CD23 and CD21 molecules. Intact EBV has been W O 96112742 PCT~EPgS/04110 repoi led to provide the pe""issive signal for IgE switching (Thyphronitis et a/, IgE
secretion by Epstein-Barr virus infected puri~ied human B l~ IlJh~cytes is stimulated by interleukin 1 and sL.,upr~ssed by intelr~ru"-y, Proc Nafl Acad SciUSA 86: 5580 (1989)), like T~ells or CD40L. In co, Itlasl to the EBV particles, the 5 EBV ,~ ,lic3e is probably unable to uusslink ,~e,nlL"alle CD21 and is l~,e,erur~
unable to increase IgE production. The EBV peptide is rather inhibitory, de~ ~:asi, lg IgE production by preventing the CD23-CD21 il ~le~ cclio~ I.

Since IL4 is known to induce IgG4 as well as IgE (Lu~dylen et al, Interleukin4 induces sy~lU,esis of IgE and IgG4 in human B cells, Eur J Immunol 19: 1311 (1989)), we investigated the effect of the EBV peptide on IL4-induced IgG4 production. As shown in Fig 7b the EBV peptide was also able to inhibit IgG4 in a dose dependent manner. This observation s~l~gest.s that the CD23-CD21 il ,lerd~lion also controls IgG4 production.
The effect of the EBV peptide was then in\~e:,ligdtecl on the production of other Ig classes. No signihcant effect was found on polyclonal IgG and IgA production.
This is shown in Figure 7c, which illustrates the modulation of immunoglobulin production by an EBV peptide binding to CD21. [PBL or purified tonsillar B cells(106/ml) were incllh~ted for 14 days with 200 U/ml of IL-4 alone or in the presence of anti-CD40 anliL)ody (1 ~Lg/ml) and increasing concentrations of EBV peptide. Ig were measured by specihc ELISA and mean values +/- SD of one r~prtse"lali-/e experiment are presented (n=4)].

In contrast, IgM production was signihcantly increased in the presence of the EBV
peptide, especially when whole PBL were used (Fig 7c).

Not all CD21 ligands can regulate IgE/lgG4 production. A C3 peptide binding CD21 did not inhibit IgE and IgG4 production tnot shown). This is shown in Figure W O 96112742 PCT~EP95/04110 7d which illustrates the absence of inhibition of IgE production with a C3 peptide ~ bi~ l~il Iy to CD21. [Purihed tonsillar B cells (106/ml) were incubated for 14 days with 200 U/ml of IL-4 and anti-CD40 anlibody (1 ~g/ml) and increasing cG"ce"~l~lio"s of C3 peptide or EBV peptir~e !gE was measured by specific ELISA and mean values +/- SD of one representative experiment are pr~se. ~led (n--4)].

The C3 peptide did not inhibit CD23 binding (Fig 7a). These results highlight again the correlation between CD23-CD21 pairing and IgE/lgG4 production. IFN-a was not tested since it is already known that IFN~ inhibits IgE prodl ~ction (Pene 10 et a/, IgE production by normal human Iy",pho.;ytes is induced by IL4 and suppressed by a-il ,lei ren~n~ y-il ~te, r~, Ul " and prosP~ d;l ~ E2 Proc Natl Acad Sd USA 85: 8166 (1988)) although IFN~ had no effect on CD23 L~ i lg to CD21 (not shown).

15 In conclusion this study therefore shows that an EBV ~e,clide dec~t:ases CD23Linding to CD21 and selectively de~eases IgE and IgG4 production by human B
cells.

EXAMPLE 8 - CD23 binds to endothelial cells An endothelial celi line (LT2 Endolethium vol 2 p 191-201 1994) or purified human umbilical venule endothelial cells were ina~h~ted with CD23-liposomes (CD23L) or gl~copl ~ol i"e-liposor"es (L-Gly) as a control. Specificity of the binding was demonsllated by the inhibition of CD23-liposome binding by an anti-CD23 25 mAb (mAb25 = a-CD23). Cells were analysed by FACS and MFI measured.

The results are shown in Figure 8.

CA 02203364 l997-04-22 W O 96/12742 PcT/~9-/o1llo LT2 - derived prl.lei,)s were purified on a CD23 - affinity column. Two bands of1 15 and 76 K Da were identified.

Claims (16)

1. A binding agent to CD21, CD11b, CD11c, to a 70-85 KDa protein expressed on endothelial cells, or to a 115 KDa protein expressed on endothelial cells, for use in the treatment or prophylaxis of inflammatory, autoimmune or allergic diseases.

2. A binding agent according to claim 1, wherein the binding agent is an antibody, a fragment thereof, an artificial construct comprising an antibody or comprising a fragment thereof, a mimetic, or a derivative of any of these binding agents.

3. A binding agent according to claim 1 or 2 which is a humanised or chimaerised antibody.

4. A binding agent according to claim 1 which is Epstein Barr Virus, Factor X, apart of the Epstein Barr Virus (which may be in glycosylated or unglycosylated form), a fragment of Factor X, or a derivative of any of these binding agents.

5. A binding agent according to claim 4, which is the gp350/220 glycoprotein of Epstein Barr Virus, the corresponding protein in unglycosylated form, a fragment of the aforesaid glycoprotein or protein, or a derivative of any of these binding agents.

6. A binding agent according to claim 5 which is a peptide with the amino acid sequence TGEDPGFFNVEIC, a fragment thereof, or a derivative of said peptide or fragment.

7. A binding agent according to any preceding claim which blocks interaction between CD23 and ligands which bind to it in vivo.

8. A binding agent according to any preceding claim for use in the treatment of arthiritis, lupus erythematosus, systemic lupus erythematosus, Mashimotos thyroiditis, multiple sclerosis, diabetes, uveitis, dermatitis, psoriasis, urticaria, nephrotic syndrome, glomerulonephritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, Sjogren's syndrome, allergies, asthma, eczema, GVH, COPD, bronchitis, insulitis, rhinitis or diabetes.

9. A binding agent according to any of claims 1 to 7 for use in the treatment ofarthritis, allergis, ulcerative colitis or Crohn's disease.

10. A binding agent according to claims 9 for use in the treatment of rheumatoidarthritis.

11. The use of a binding agent to CD21, CD11b, CD11c, to a 70 to 85 KDa protein expressed on endothelial cells, or to a 115 KDa protein expressed on endothelial cells for the manufacture of a medicament for the treatment of arthritis, lupus erythematosus, systemic lupus erythematosus, Mashimotos thyroiditis, multiple sclerosis,diabetes, uveitis, dermatitis, psoriasis, urticaria, nephrotic syndrome, glomerulonephritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, Sjogren's syndrome, allergies, asthma, eczema, GVH, COPD, bronchitis, insulitis, rhinitis or diabetes.

12. The use of a binding agent according to claim 11 for the manufacture of a medicament for the treatment of arthritis, allergies, ulcerative colitis, or Crohn's disease.

13. The use of a binding agent according to claim 12 for the manufacture of a medicament for the treatment of rheumatoid arthritis.

14. A pharmaceutical composition comprising a binding agent according to any of claims 1 to 7 and a pharmaceutically acceptable carrier.

15. A binding agent according to claim 1 substantially as hereinbefore described.

16. A method of treating an inflammatory, autoimmune or allergic disease comprising administering to a pharmaceutically effective amount of a binding agent to CD21, CD11b, CD11c, to a 70 to 85 KDa protein expressed on endothelial cells, orto a 115 KDa protein expressed on endothelial cells to a patient.