CA2188812A1 - New methods and compounds for the selective modulation of antigen-specific t-cell responsiveness - Google Patents

Abstract

The present invention relates to new Fc.gamma.RII bridging compositions for impairing the capacity of antigen presenting cells (APCs) to stimulate the activation of antigen-specific T-cells, resulting in modulation of antigen-specific T-cell responsiveness. More particularly, said Fc.gamma.RII bridging agents are chosen from the group consisting of: aggregated human IgG molecules; aggregated Fc fragments of human IgG
molecules; a bivalent monoclonal antibody to the Fc.gamma.RII; a multivalent monoclonal antibody to the Fc.gamma.RII; a functionally active fragment of said bivalent or multivalent monoclonal antibody; a recombinant fusion protein of 2 or more human IgG Fc parts; liposome vesicles comprising any of the foregoing, provided that said Fc.gamma.RII composition prevents the expression of the co-stimulatory molecules B7-1/2 and/or down modulates the ICAM-3 molecule expression by these professional APCs. The present invention also relates to prophylactic and therapeutic methods and compositions to prevent or treat the rejection of solid organs, tissues and cells after transplantation; for inducing T-cell anergy or T-cell tolerance; for treating allergic diseases; or for the treatment of autoimmune diseases. The present invention also relates to Fc.gamma.RII bridged professional APCs prepared by bridging APCs with an Fc.gamma.RII bridging agent according to the present invention.

Description

woss~3273~ 21 8881 2 r~ 2 NEW METTTODS AND COMPOUNl)S FOR TIIE ~T~ cTIvE MODUL~TION OF
ANTIGEN-SPF,CIFIC T-CELL RT~,~SPC)NSIVF.Nh ~
.
Field of the Inv~ntinn The present invention relates to the new finding that the bridging of Fc7~ type 11 S receptors on ~uf~ iulldl antigen presenting cells (APCs) impaires the expression of the essential co-st I y molecules B7-112 and causes the down mn~ inn of the adhesionmolecule ICAM-3 expression, resulting in the modulation of antigen-specific T-cell ,u",i~ ". The B7 molecules on APCs provide the essential co-stimulatory signal that determines whether TcR/CD3 signaling after activation of T cells with the specific antigen lû leads to full T-cell activation or T cell anergy. The ICAM-3 molecule mediates cellular of T cells and other ly , ' ~ at sites of i,.n ...,.,. ~ and specific immune responses.
The present inYention also relates to l~lu~l-ykL~ ; and therapeutic methods to prevent or to treat allergic diseases.
The present invention further relates to the treatment of T-cell mediated ,. ~. lil.. ..
diseases.
The present invention also relates to ~ JIlyldl,Li~, and therapeutic methods to prevent or to treat the rejection of solid organs or cells after allogeneic or xenogeneic Lld~ ld~Ldliùl~.
kl~round art ~0 A. Fc-Receytors Fcy-receptors play important roles in several immllnnlogir~l processes such as ,o~,yLu~i~ of opsonized particulate antigens, clearance of immune complexes, antibody-mediated cellular ~;yLu~u~ ;iLy, production of i,.n~,.",,,l..ly mediators, and regulation of synthesis (Mellman et al., J. Cell. Biol. 96:887 (1983); Kurlander et al., J. Immunol. 133:855 (1984); Shen et al., J. Immunol. 137:945 (1986); Graziano and Fanger, wo 9513273~ 2 1 8 8 8 1 2 P~,IILI ~ ~012 J. Immuno~. 138:945 (1987); Zheng et al., Eur, J. Immunol. 23:2826 (1993)).
The Fc,v-receptors on human leukocytes can be divided in three major classes, based on their molecular mass, specificity and affinity~for IgG, cellular distribution, and reactivity with mQnrrlQn~lantibodies (VandeWinkel etal.,J. ieuk. Bio~. 49:511(1991); Ravetchand S Kinet, Annu. Rel~. Immunol. 9:457 (1991)). Fc yRI is a lligh affinity receptor for monomeric IgG, consists of three Ig-like extracellular domains and is expressed on monocytes and IFN- y activated neutrophils (lerino et al., J. Immuno~. 150:1794 (1993). Fc yRII consists of two Ig-like Pxtr:lrP~ r domains. This receptor binds monomeric IgG only with low affinity, but has a higll affinity for complexed IgG (lerino et al., J. Immuno~. 150:1794 (1993)). Fc ,vRII
is the most widely expressed FcR, it is present on monocytes, dendritic cells, granulocytes, platelets and B cells (Anderson and Looney, Immunology today 7:264 ( 1986); Nestle et al J. ~mmunol. 151:6535 (1993)). Fc y Rlll also contains two Ig-like extracellular domains and also binds monomeric IgG only with low affinity, but has a high afhnity for complexed IgG
(lerino et al.,J. Immunol. 150:1794 (1993)). F~RIII is expressed on neutrophils, Pncinrlrhilc, a group of cells referred tQ as L cells which include natural killer cells arld large granular Iymphocytes, and on II~,IUIUl1..,5..~ but not on monocytes (Anderson and LQoney, I~ rOIO~y oday 7:264 (1986)).
B. T-rPII ~rtivrfirl Activatiûn of T cells is the result of ligand-receptor interactions. The TcR/CD320 complex has two functions in antigen-induced activation: a recognition function in which a specific antigen is recogni~ed in the context of the appropriate MHC molecule, and a signalling function in which the recognition event is transmitted across the plasma membrane (Weiss and Imboden Adv. Immunol. 41:1 (1987)). However, to induce ~lulirr~la~iull and maturation into effector cells, T cells need a second signal in addition to the one mediated 25 by the TcR/CD3 complex. This co-stimulatory signal is normally provided by the cell surface of APCs (Springer et al. Annu. Rev. Immunol. 5:223 (1987)). Intercellular signaling after TcR/MHC-peptide interaction in the absence of the co-stilrnll y signal results in T-cell ,Liva~iOI~ in the form of clonal anergy (Mueller et al. Annu. Rev. Immuno~. 7:445 (1989)).
A number of accessory molecules present on the cell surface of T cells with known ligands 30 on the APCs have been implicated in providing the co-stimulatory signal in T-cell activation:

WO 9513273 t 2 1 8 8 8 ¦ 2 PCT/EP95102012 CD2 with its ligand CD58 (LFA-3), CDlla/CD18 (LFA-I) Witl1 CD54 (ICAM-I), CD28 with B7, and CD29/CD49d (VLA4) with VCAM-I (Selvaraj et al. Nature 326:400 (1987);
Springer Nature 346:425 (1990); Marlin and Springer Cell 51:813 (1987); Van Noesel et al. Nature 333:850 (1988); Linsley et al. Proc. Natl. Acad. Sci. USA 87:5031(1990);
Damle et al. Proc. Nat~. Acad. Sci. USA 88:6403 (1991)).
So far, the best candidate co- ' ' y signal that determines whether TcR-5tinn~ tinn leads to full T-cell activation, or to T-cell anergy, is generated by interaction of CD28 on the T cells with B7-1/2 on APCs. It has been dclllul~LI ' in vitro that cross-linking of the CD28 molecule can rescue mouse T-cell clones from becoming anergic (Harding et al. Nature 356:607 (1992)). In addition, it has been shown that B7-1 but not ICAM-I mediated co-stimulation of T cells can prevent the induction of alloan~igen-specific tolerance (Vassiliki et al. J. Exp. Med. 178:1753 (1993)).
Co-stimulation of T cells with mAb to the TcR/CD3 complex and CD28 results in greatly enhanced activation (Thompson et al. Proc. Natl. Acad. Sci. USA 86:1333 (1989)).
It has been 1' ' ' that cross-linking CD28 with mAb can be replaced by B7, a natural ligand for CD28 (Linsley et al. J. Exp. Med. 173:721 (1991); Gimmi et al. Proc.
Natl. Acad. Sci. USA 88:6575 (1991); De Boer et al. Eur. J. Immunol. 22:3071 (1992); Van Gool et al. J. Immunol. 150:3254 (1993)). The B7-CD28 interaction can result in a strong lululir~ld~iv~ (Linsley et al. J. Exp. Med. 173:721 (1991); Gimmi et al. Proc. Natl. Acad.
Sci. USA 88:6575 (1991); De Boer et al. Eur. J. Immunol. 22:3071 (1992)) as well as a cytolytic T-cell response (Van Gool et al. J. Immunol. 150:3254 (1993)). It has recently been U~.lllUll~ ' that there are at least two B7 molecules that can functionally interact with CD28 (Hathcock et al. Science 262:905 (1993); Freeman et al. Science 262:907 (1993);
Freeman et al. Science 262:909 (1993); Azuma et al. Nature 366:76 (1993)). B7-1, originally 25 named B7/BB I is a IIIUIIUIII~,. il, 1, , .~" II ~ Iyuu~JI ut~;ll with an apparent molecular mass of 45-65 KDa and is, like CD28, a member of the ;~ gl~b~.lin superfamily (Freeman et al. J. Immunol. 143: 2714 (1989)). The second B7 molecule, named B70 or B7-2, is a tldll~ l,.dl.e ~,ly~u~lu.~;ll with an apparent molecular mass of approximately 70 KDa and isalsoamemberofthe i~ bl~ ,r~ lily (Freemanetal. Science26~:909(1993):
Azuma et al. Nature 366:76 (1993)). It was initially reported that the expression of the B7-1 molecule was restricted to activated B cells and monocytes stimulated with IFN- y (Freeman et al. J. Immunol. 143: 2714 (1989); Freedman et al. Cell. Immunol. 137:429 (1991)). More WO 9513273~1 2 1 8 8 8 l 2 PCT/EP95/02012 recently . B7-1 expression has also been found on cultured peripheral blood dendritic cells (Young et al. J. Clin. ~nvest . 90:229 (1992)) and on in vitro activated T cells (Azuma et al.
J. Exp. Med. 177:845 (1993)). In vivo, the Bi-i molecule is constitutively expressed on dendritic cells, on monocyoes/l"d~ cs in illlldlllllldLuly lesions, on a subset of B cells S in and around germinal centers and, on T cells in chronical '' y lesions (Vand.".L,~"~l.e et al. Int. Immunol. 5:317 (1993); Barnaba et al. Eur. J. Immunol. 24:71 (1994)). The B7-2 molecule seems to have a very similar distribution pattern as B7-1, with the exception that induction of cell-surface expression seems to be faster (Freeman et al.
Science 262:909 (1993)) and that it seems to be present on freshly isolated monocytes (Azuma et al. Nature 366:76 (1993)).
C. T-cell An~r~y Incomplete activation of T cells in the absence of the co-stimulatory signals from the APC results in T-cell tolerance or anergy (Mueller et al., Annu. Rev. Immunol. 7:445 (1989)). T-cell anergy is ~,llUld~t~ by the fact that anergic T cells do not anymore 15 respond to normal activation signals, even in the presence of all the co-stimulatory signals.
It has been d~.,.,u.",l_ ' that cross-linking the CD28 molecule on mouse T cells can rescue these cells from becoming anergic when stimulated with chemically modifled APC, a situation that otherwise would result in anergy (Harding et al., Nature 356:607 (1992)). It has also been shown that B7-1 but not ICAM- I mediated co-stimulation of human T cells can 20 prevent the induction of alloantigen-specific tolerance (Vassiliki et al., J. Exp. Med.
178:1753 (1993)). In addition, it has been shown that the (,ulllb;llaliull of blocking B7-IICD28-CTLA4 with a human B7-1 specific mnnnrlon~l antibody and cyclosporin A can induce alloantigen-specific tolerance (Van Gool et al., J. Exp. Med. 179: (1994)), and that blocking B7-1 and B7-2/CD28-CTLA4 with a soluble CTLA4 fusion protein can induce25 hypo~ a of human T cells when stimulated with alloantigen (Tan et al., J. Exp.
Med. 177:165 (1993)). Thus the B7/CD28-CTLA4 interaction seems to play an important role in Ul,~llllilli..l; whether T-cell stimulation leads to full activation or T-cell anergy.
D.

wo ss/3t73~ 2 ~ 8 8 8 1 2 P~ l t~nl2 Atopic allergic diseases, i.e. asthma, allergic rhinitis, atopic dermatitis, and food and drug allergy, affect at least 20% of the population in tlle in~l.lc~iAli7P~ world and are an important cause of morbidity, and for asthma mortality. Tlle initial factors involved in the allergic reaction are allergen-specific antibodies of the IgE type alld mast cells, which express 5 high affinity receptors for IgE and which are widely distributed il~ tissues that form our protective barrier against the outside such as mucosae. When an antigen (the allergen) enters the human body, binding to and bridging of receptor-bound IgE on tl1e mast cells results in activation of these cells. This activation will result in the release of histamine that triggers an immediate response (within 20 minutes), as well as the release of soluble factors that 10 initiate a local inflAmm ~ry response. This results in the influx of infl y cells that give ~ rise to the late phase (6-24 hours) of tlle allergic response and to the chronici..n~ seenatplacesofrepeatedexposuretoallergen.Thislatephasereaction is characterized by the occurence of eosinophils.
Thus allergy is "I-al~,tl,..~,;l by immediate and late phase lly~Jr~ ity reactions 15 to allergerls. These immune reactions are associated with elevated serum levels of allergen-specificlgEandto~..,~il.l.~l.ili~,respectively.BothlgEproductionandeosinophilproduction are controlled by T helper (Th) Iymphocytes and evidence is ~ that the aberrant immllnn~ Zll Lll~lldl.i~li~ of atopic allergy can be explained by the hyperactivation of a particular subset of Th cells (Th2).
Helper T cells regulate immune responses via cytokines that they produce upon ~,o~lliLi~ of specific antigen presented by antigen presenting cells (APC). Individual Th cells (clones) can be ~ d on the basis of the cytokine secretion profile and hence their function (Mosmann et al. Ann Rev l~ununol. 7:145 (1989)). In response to various antigens, Th cells produce many cytokines sim~lnlnP~llcly (type 0 profile). However, in response to jnrrArpll~ r micro-organisms the production of cytokines of tlle Th cells is biased to high leYels of interferon (IFN)- y and low levels of interleukin (IL)-4 and IL-5 (type I
cytokine profile, Thl). Such a Thl response is protective because IFN-~t stimulates the intracellular killing of microbes by phagocytic cells. In contrast, in response to certain helminth types the Th cell response is biased to low levels of IFN- y and high levels of IL4 and Ik5 (type 2 cytokine profile, Th2). IL-4 induces B cells to secrete IgE, provided the level of secreted IFN- y is low, whereas IL-5 promotes the production of eosinophils in the bone marrow. In such cases, the Th2 response is protective, because both IgE and ...... _ ... ..... ... . .. . _ . , . _ . . . . . .. . .. _ . _ _ . _ Wo 9513273.J 2 1 8 8 8 1 2 6 PCT/EP95/02012 eosinophils are considered to contribute to tlle expellillg of tlle helmin~hs.
In vitro studies witll allergen-specific Tll cell clones prepared from peripheral blood of atopic asthma and eczema patients and from control individuals showed that eosinophilia and elevated levels of allergen-specific IgE are causally related with the occurrence and activation of allergen-specific Th2 cells (Wierenga et al. J. Immunol. 144:4651 (1991);
Romagnani l~nmunol. Today 13:379 (1993)). Tlle origin of the dominance of these Tl12 cells in atopic allergy is unknown. There is ~ evidence that type I and 2 profiles result from modulation of cytokine production (Trinchieri Imunol. Today 14:335 (1993); Snijdewint et al. J. Immunol. 150:5321 (1993)). Clearly, soluble factors secreted by APC during 10 antigen-~ .LdLiuare important. APC-derived factors include IL-12 and pr~c~gl:~nfiin (PGE)-2, that skew T cell cytokine production towards Thl and Tl12 profiles. respectively.
Thus, a low IL-12/PGE-2 production ratio in APC will results in IL-4 dominated T cell responses, whereas a high IL-12/PGE-2 production ratio wi~l result in IFN- y-dominated T
cell responses.
1"."." ~ y, which presently consists of the 5~ l, U~ ad~ L~d~iul~ of increasing doses of intact or chemically modified allergens, has shown to result in a down-regulation of allergen-induced T-cell proliferation and release of cytokines and histamine-releasing factors by the allergen-specific T cells (Varney et al., J. Clin. Invest. 92:644 (1993); Secrist et al., J. ~p. Med. 178:2123 (1993)). The exact mechanism of this classic iml~ iS not known, although it has been reported that this therapy induces a rise in blocking IgG4 antibodies (Van der Zee et al., J. Immunol. 137:3566 (1986); Reid et al., J. Allergy Clin. Immunol. 78:590 (1986); Bousquet et al., J. A~lergy Clin. Immunol. 99:43 ( 1991)) . Other reports have ~ ' ;I that CD8 + suppressor T cells are generated, which modulate allergen-specific IgE production (Rocklin et al., N. Engl. J. Med. 302:1213 (1980)).
More recently, attempts to specifically inactivate allergen-specific T cells have been undertaken using specific peptides of allergens. The basis for this ,l"", "l~l,. .~y is the ub~lvdtioll that peptides, when given to T cells in the absence of APCs in vitro will result in the antigen-specific ill~ iY~iUII (anergy) of the T cells recognizing the particular peptides (Higgins et al., J. Allergy Clin. Immunol. 90:749 (1992)). S~ ",~ injection of a specific peptide from the major allergen of cat hair and dander Fel-dl, in mice previously immunized with whole Fel-dl in Freund's complete wO ss~3273~ 2 1 8 8 8 1 2 -- 12 adjuvant resulted in tolerization of T cells (Brinner et al., Proc. Natl. Acad. Sci. USA
90:7608 (1993)) It is hypothesized that the rnec~anism of this illA~,~iVdLiUII is based on the ~"~ r", " of the T-cell receptor by the antigen in the absence of the A~,UlU,UlidL~ co-stimulatory signal (O'Hehir et al., Annu Rev ~mmunol. 9:67 (1991)). This co-stimulatory S signal is normally provided by the interaction of the B7 molecule on the APCs with the CD28 molecule of the T cells (Harding et al. Nature 356:607 (1992); Vassiliki et al. J. E~p.
Med. 178:1753 (1993)).
E. Autoimmune ~icP~cPc A number of studies indicate that r~lctimlllAtion through CD28 ligation might be the 10 initiating event in Al~ y. The potential of both a primary signal via the TcR and B7 as a rncfimlllAfrlry signal for the generation of il"l..u~le diabetes has cleariy been proven withtransgenicmice(Guerderetal.,/mmunity 1:155(1994);Harlanetal.,PNA591:3137 (1994)). In these studies, it is lly,uuLl~ that tolerance to peripheral antigens is induced by triggering the TcR in the absence of essentiai roctimlllAt(lry signals. Mice expressing both B7 and a high level of primary antigens (MHC molecules or viral y,ly~u~lu~;.la) on pancreatic beta cells developed dU~U;IlllllU.I., diabetes. The critical role of the absence of B7-mediated ~ ;"."IAl ;.". in the induction and IIIA;II.I .IAIII P of tolerance to peripheral antigens, and of the B7-mediated signalling in the breakdown of T-cell non-responsiveness, cdusing dU~UillllllUIli~y, was obvious.
The role of the B7-CD28 interaction in the chronic activation state of T cells, which have been implicated in dU~U' diseases, has been strongly suggested in various studies.
Using ' ' I techniques, strong B7 expression has been found in lesions of -1;1"-".'1... diseases, such as lI~C~ ' arthritis and psoriasis. FUILII~IIIUI~ it has been ~iPrn( I thdt blocking B7-CD28 interaction could block I(IAIII;IIO~iY production and 25 ,UlUlUll~A~iull of life in a murine model of: ...IIIIUI~ disease that closely resembles systemic lupus ~Iy~ u~, in humans (Finck et al., Science 265:1225 (1994)).
F. Transplant Rejection lUI~UIII~A~jIJjIj~Y for the h;~lu~ ility antigens, both major (MHC) and minor wo 9513273~ 2 1 8 8 8 1 2 r ~ ~ r ~o ~ 2 antigens, is the cause for graft rejeclion. Botl1 CD4+ llelper T cells (Th) and CD8+
cyto~oxic T cells (CTL) are involved in the rejection process. Activation of T cells after ~l~ul~,~llallL~liu~l is the result of ligand-receptor interactions, wllen tlle TcR/CD3 complex recognizes its specific alloantigen in tlle context of tl1e appropriate MHC molecule. To induce 5 proliferation and maturation into effector cells, T cells need a second signal in addition to the one mediated by the TcR/CD3 complex. Intercellular signaling after TcR/MHC-peptide interaction in the absence of the c~ctimlll y signal results in T-cell inactivation irl the form of clonal anergy (Mueller et al. Annu. Rel~. Imntunol. 7:445 (1989)). It has been d~ OIlaLl~Ltd that blocking B7-1/2, when combined with a donor-specific cell transfusion, 10 can prevent the rejection of MHC-~ cardiac allografts in a rat model (Lin et al.
J. E~p. Med 178:1801 (1993)). In addition, it has been demonstrated that co-stimulation of T cell by cross-linking the CD28 molecule is resistant to the inhibitory activity of the il""...l~ ive drug ~y~,lua~olill A (June et al. Intntunol. Today 11:211 (1990)). This demonstrates the importance of the B7/CD28 interaction in the rejection of transplants.
,AI~.C QF TTTF INVENTION ~
Activation of a certain type of Fc-receptors is reported to occur only when several Fc regions within an antigen-antibody complex c~ cly bind to several Fc-receptors, causjng them to be cross-linked (US Patent No. 4,753,927). Such Fc-receptor cross-linking by several Fc regions appears to be the critical signal required to activate certain types of Fc-20 receptors. The activation of Iylllpllo~y,, via Fc-receptors is generally viewed as a strong pro-i~,ll.l"",.,.ll"y event. Inhibition of the immune system functions is reported by these authors to occur if an active site peptide binds to and blocks such an Fc-receptor, and thus prevents Fc regions within an antigen-antibody complex from binding to tlle receptor. thus blocking Fc-receptor activation.
Although it is generally believed that preventing signal ~l~lladul,Liull via Fc-receptors is beneficial during various types of i"n"""" ~ )Iy reactions, tlle present invention d~ Iall that induction of specific signal Ll~ d~l~,LiOIl via Fc-receptor bridging is able ,, woss~3~73.l 2 1 8 8 8 1 2 r~ n12 to modulate T-cell responsiveness and can tllus be viewed as an anti~ nA~ ly action.
It is a primary aim of the present invention to provide new therapeutic and/or u~ yla~ uses for Fc~RII (CD32) bridging ~u~ OaiLiulL~ for treating T-cell mediated diseases, more ~auLi~uldl~y new therapeutic uses for modulation of antigen-specific T-cell . CatJolla i ~ .,.. ~,~ .
More particularly, it is an aim of the present invention to provide new ~lu~ yla~,Li~
and therapeutic uses for Fc yRII (CD32) bridging CullllJuailiulla for treating allergic diseases.
More particularly, it is an aim of the present invention to provide new therapeutic and/or ~lu~ ldu~i-, uses for Fc~yRII (CD32) bridging ~ n",~ for treating dU~UillllllUllC
10 diseases.
More particularly, it is an aim of the present invention to provide new therapeutic and/or ~JIulJllyla. Li~ uses for Fc7~RII (CD32) bridging compositions for preventing rejection of solid orgarls, tissues or cells after tr:~n~rl~n~ nn More ~al~iuulally, it is an aim of the present invention to provide new therapeutic 15 and/or L~lu~Jllyla~,Liu uses for Fc~yRII (CD32) bridging agentsluulll~uaiLiul~ for in vitro bridging ûf graft dûnor l..ur ~ APCs.
It is ,ualLi~,ulauly also an aim of the present inventiûn to provide and use new Fc~RII
bridging ~ which are ullaldut~,li~;l by their specific capacity to impaire the activation of antigen-presenting cells (APCs) to stimulate the activation of antigen-specific 20 T-cells, resulting in mn~ ion of T-cell responsiveness.
It is a further aim ûf the present invention to provide and use Fc~yRII bridgingwhich are ~llduduLcli~d in that they either (i) prevent the up-regulation (or prevent tbe expression or down-modulate the expression) of the co-stimulatory B7 molecules on IJlurc,:du..al APCs, and/or, (ii) they down-modulate (or impaire tlle expression of or 25 prevent the up-regulatiûn of) the adhesion molecule ICAM-3 on ,u,~,f."~iu,.al APCs.
It is a further aim of the present invention to provide methods for preparing such new FcyRII bridging .~,,,,1,,,~;~;,,..~
It is another aim of the present invention to provide methods for the IJlC~aldLiUI~ of new therapeutic and/or ,u~ul~llyla~,Lic l~ n~ comprising any of said FcyRII bridging 30 agents as an active principle.
It is also an aim of the present invention to provide and use Fc-yRII bridged donor ur~ iu.~al APCs, or therapeutic .","I"~;l;o"~ comprising the same, for preventing or ....... ..... .. . . . . . ... , . .. . .. . .. . .. ... .. . . . _ .. _ .. . . .. . .. ..

WO 9513273~l 2 1 8 8 ~ 1 2 PCT/EP9~/02012 treating the rejection of donor grafts or transplants.
It is also an aim of the present invention to provide a method for preparing said Fc~RIr bridged donor professional APCs.
It is finally an aim of the present invention to provide an in vitro method for screening S for or selecting new Fc7~RII bridging agents or compositions beillg ~ a~ t~ d in that they impaire the expression of B7 molecules and/or ICAM-3 molecules on professional APC's, resulting in modulation of T-cell responsiveness.
All the aims of the present invention are acllieved by the specific ~I-.budil..~ll., of the invention as detailed below.
DET~II Fn DFSCRIPTION OF THF INV~I~TIOI`I
The present invention is based in essence on the new finding that bridging several FcyRII (CD32) molecules on profesional antigen presenting cells (APCs) prevents the up-regulation of tdle essential co- ' y molecules B7-1/2 and causes the down rnn~llllq~inn of the adhesion molecule ICAM-3, resulting in the modulation of antigen-specific T cell Ulllt~l~ul~i~
It should be evident to the reader that by means of this new finding, the skilled man is put in the ability to screen for or select any new Fc~yRII bridging agent or in",~
designed to modulate antigen-specific T-cell responsiveness~ or any associated therapeutic use as detailed below.
The present invention thus relaoes more p.ll~iuukuly to an FcyRII (CD32) bridging rnmrn~irinn ~,lldld~;L~ ,d in that it impaires (for impairing) the capacity of antigen presenting cells (APCs) to stimulate the activation of antigen-specific T-cells, resulting in mn~ lq~inn of T-cell l-"lvul~
The expression "Fc yRII (CD32) bridging" within the context of the present invention refers to the fact that incubation of professional APCs carrying the Fc yRII (CD32) with a suitable bridging agent results in the prevention of the up-regulation of the essential co-y molecules B7-1/2 by these professioncLl APCs and/or results in the down mn~ qrinn of the adhesion molecule ICAM-3 by these ~lur~iUlldl APCs. The suitable bridging agents according to the present invention are listed below. Most preferably several Fc~RII (CD32) molecules are bridged by several Fc regions.
:

wossl3273~ 21 8 8 8 1 2 r~ 12 The terms "bridging" or "bridged" used in tl~e present invention have the same meaning as "cross-linking" or "cross-linked". The term cross-linking is preferably not used further in the ~ ;r~ , to avoid confusion with the strict meaning of the term "cross-linking" to couple 2 or more protein molecules together, for instance to obtain a state of S a~ ,aLiul, of the bridging âgent as is explained further.
The expression "prevention of the up-regulation of B7- 1/2 molecules " on ~1l urt~ ~iul~dl APC's refers to the impaired expression of B7-112 molecules as detailed in the Examples section upon applying certain Fc-yRII bridging agents. Tlle expression of B7-1/2 molecules may be measured by analyses such as shown in the Examples section (e.g. FACS analysis) 10 or by means of any other technique known in the art or as indicated below.
The expression "down-modulation of ICAM-3" refers to the impaired expression of ICAM-3 molecules upon applying certain FcyRII bridging agents or methods such asdescribed in the Examples section or as indicaoed below. The expression of ICAM-3 may be measured by analysis methods such as shown in the Examples section, as indicated below or 15 by any other technique known in the art.
The term "~luC~iul~l APCs" is reviewed below.
The terms ~Fc yRII (CD32)", "B7-1/2" and "ICAM-3" are reviewed above.
The FcyRII bridging agents/~...,.l..,~ili..,.~ of tlle present invention aim upon binding to the Fc7/RII on ,ulurt~iu--dl APCs and activating this receptor in such a way that the up-20 regulation of B7-1/2 co-s~il~LldLuly molecules is prevented and/or that of the adhesion molecule ICAM-3 is down-modulated resulting in an impaired capacity of the APCs to induce T-cell activation and thus resulting in modulation of T-cell responsiveness. Such Fc~yRII
bridging Cu~ n~iLiull~ according to the present invention should comprise at least one of the bridging agents as an active principle, with said agents being chosen from the group 25 consisting of:
- aggregated human IgG molecules;
- aggregaoed Fc fragments of human IgG molecules;
- a bivalent mnnrrlnn~31 antibody to the Fc-yRII;
- a multivalent rnnn~rlrn~l antibody to the Fc~RII;
30 - any functionally active fragment of said bivalent or multivalent l,..".n--l.. '1 antibody to the Fc yRII;
- a ~c~Cu.l~bil~d~L fusion protein of 2 or more human IgG Fc parts;

WO95/3273~ 21 8 8 ~1 2 r~ 2 - a liposomal vesicle containing any of the foregoing agents as detailed below.
A selected agent may be conflrmed as being a proper Fc yRII bridging agent according to the present invention by means of the following test system:
The amount of B7-1, B7-2 and/or ICAM-3 expressed is measured by any of the S techniques known in the art (such as ELISA or i~ ..ullUnUUlt~G~ (FACS) analysis in combination with suitable mnnnrlon~l antibodies or ligand antigens as described in the Examples section) on the cell surface of profesional al1tigen presentil1g cells (or APCs, e.g.
monocytes cultured in tlle presence of IFN-~ or GM-CSF) in the presence of the Fc~RII
bridging agent to be tested in cul~l~ual i~OII to appropriate control conditions andlor agents.
Stricktly speaking, the terms "agent" and "~:u~ v~iLiull" within the meaning of the present invention differ in that a ~.ùlll~ iLiull should comprise at least one FcR-bridging agent as an active principle in addition to other Uu~ UIlGlli(s). Tl~e preferred Cu~ v5iLiull or ru~l-ulcLiull of said ~u~ n~ of the invention are detailed extensively below.
The term "aggregated~ may mean aggregated by means of any chemical cross-linkingagent known in the art or aggregated by any other means such by immobilizing the IgG's or fragments thereof to a solid phase as is explained below and in the examples section.
"Mnnnrlnn~l antibodies to the FcyRII" refer to mnnnclon~ll antibodies which are specifically directed to the Fc~RII. These monoclonal antibodies can be prepared as described inillLtllldLiol~lapplicationwo88looos2(describingtheproductionofl~nnnrlon:llantibodies specifically directed to the Fc yRI) or Anderson et al. ((1986) J. Biol. Chem. 261:12856). ]t should, however, be stressed that the Fc yRII monoclonal antibodies according to the present invention should bind to a site on the Fc y type 1I receptor (CD32) such that it preventis the up-regulation of B7-1/2 and/or c~Luses the down-modulation of ICAM-3 by the antigen presenting cells incubated with said Fc~yRII ~ 1 antibodies. The selection of such 2~i mnnnrlnn~l antibodies can be performed by measuring the amount of B7-1, B7-2 and/or ICAM-3 produced by profesional antigen presenting cells (e.g. mol1ocytes cultured in the presence of IFN-~y or GM-CSF) in the presence of tlle monoclonal antibodies to be tested in Colll~ to control monnrlon:~l antibodies by any of the techniques known in the art (e.g.
by ELISA or i~lllllu~lurluult~ ,e analysis as described in the Examples section or as described above).
"Bivalent or multivalent 1111)111~ 1 antibodies to the Fc~ Rll", in particular of the IgA or IgM isotype, can be prepared as described in international application WO 88/00052 woss/3273~ 21 8881 2 r~l~ 12 where the :iU~ laL~lL~ of the hybridoma clones are directly screened for the production of specific antibodies of the IgM or IgA isotype using specific reagents. Alternatively, IgA
secreting hybridoma cells can be obtained from an IgG secreting hybridoma cell line by limiting dilution cloning and selection of Ig-isotype switch variants by methods known to 5 those skilled in the art. In addition, Fc y Rll-specific antibodies of the IgA or IgM isotype can be prepared using recombinant DNA technology by expressing the antigen-binding variable region of the said antibodies in a vector containing the cDNA encoding for the IgA
or IgM constant region by methods known to those skilled in the art. These constant regions can be of mouse origin, but more preferably are of primate or l~uman origin. Bivalent or 10 multivalent mnnnrlnn:~l antibodies to Fcy Rll or functionally active fragments (such as F(ab'), fragments) of such antibodies can also be prepared by conjugating antibodies with known coupling or cross-~inking agents such as protein A, carbodiimide, N-succinimidyl-2(2-pyridythio) propionate (SPDP) Karpovsky et al. (1984) J. Exp. Med. 160:1686; Liu et al (1985) Proc. Natl. Acad. Sci. U.S.A. 82:8648). Alternatively, such fragments can be 15 generated by, for instance, enzymatic digestion of the antibodies with papain, pepsin, or other proteases.
The expression "functionally active fragment of said bivalent or multivalent antbody to the Fc-yRII" may refer to an F(ab') fragment of said antibody or conjugated Fab fragments of said multivalent or bivalent ~nnnclnn:ll antibody(ies), provided that the resulting 20 antibody fragments have the effect of preventing the up-regulation of B7-1/2 and/or cause the down~ ;.. of ICAM-3 expression by the APCs incubated with said antibodies asdescribed above. Conjugated antibody fragments may be obtained as detailed below.
The expression "a liposome vesicle comprising any of the foregoing agents" refers more pal~il,ulally to liposome vesicles having Fc regions of any of the foregoing bridging 25 agents sticking out of the liposome. Liposome vesicles according to this aspect of the invention may be prepared by any method for preparing liposomes known to the man skilled in the art such as described by A. Gabizon et al., Cancer Research 42:4734 (1982); D.S.
Cafiso, Biochem Biop)lys Acta 649:129 (1981) and F. Szoka, Ann ReY Biophys Eng 9:467 (1980). Other drug delivery systems known in tl1e art may also be applicable and are 30 described in e.g. M.J. Poznansky et al., "Drug Delivery Systems" (R.L. Juliâno, ed., Oxford, N.Y., 1980), pp. 253-315: M.L. Poznansky, Pharm Revs 36:277 (1984).
The present invention furtner . , ' an Fc yRII bridging ~U~ JU~iLiUI~ as defined . . .
., _ _ _ .. ... _ .. .. ... ...

wo 95/32734 2 1 8 ~ 8 ~ 2 ~ /~1 ?1~12 above, further characterized ill that said Fc yRII bridging agent prevents the up-regulation (or impaires the expression) of B7-112 molecules by these APCs.
The present invention contemplates also any of the Fc-yRII bridging UU~ JU~iliUII as defined above, furtker characterized in that said FcyRII bridging agent~causes tl~e down S mn~ lAti-)n (or impaires the expression) of ICAM-3 molecules by tllese APCs.
According to a preferred e...l~odilll~..l, tke present invention relates to any UUlllUU~i~iOI~
as defined above, further Ull~l~,it;li...,d in that said composition comprises a specifc antigen or antigen-complex combined with an agent capable of bridging Fc~RII molecules as defined above.
The term "combined" refers to any type of combination known in the art, more iiuul~ly implies covalently attaching or cross-linking said antigen or antigen-complex to said bridging agent preferentially via ckemlcal cross-linking. Said antigen or antigen-complex may thus be attached or aggregated to said bridging agent by means of any technique known in the art using any type of attaching agent or cross-linking agent known in the art. Liposome 15 vesicles as bridging agents will preferably contain said antigen or antigen-complex inside of the liposome vesicle. Such vesicles may be prepared by any of the techniques known in tke art for liposome vesicle u~ liu~l.
It is proposed in the context of tke present l."~ i-" ,l of the invention, that combining a specific antigen or antigen-complex with an FcyRII bridging agent of tke 20 invention in vivo would result in UIU~ d~iUll of antigenic peptides to specific T cells by IJIU~ ' I APC's in the absence of the essential co-stimulatory molecules B7-1/2 or in the presence of reduced, of such molecules on tke surface of these APC's. This treatment will result in the modulation of only those T-cells able to recognize the specific antigen in question. This finding is of particular importance in the treatment of diseases 25 where T cells play a crucial role in pathology such as allergic and ,."I.,il"""",~ diseases.
More particularly, the present invention relates to an Fc yRII bridging ~ as defined above, further ulldud~i~liL~d in tkat said specific antigen or antigen-complex causes allergic diseases ~often referred to as allergen). Examples of such antigens that can act as allergens in humans are tke major allergen of cat hair and dander, house dust mite antigen, 30 pollen antigens, and bee venom, Tke present invention also relates to an Fc~RII bridging ~ as defined above, furtker ull~du~.,li~ in tkat said specific antigen is an alloantigen.

wo ss/3273.l 2 1 8 8 8 1 2 r~

The term "alloantigel~" refers to foreign MHC antigel1s, recognized by specific T cells and IC~I.UII~il,lC for the onset of transplant rejection.
In addition, the present invention relates to an Fc yRI I bridging :UllI~)U~iliUII as defined above, further ~,lldld~ liLe;l in that said specific antigen is an antigen causing the S autoimmune attack on the body's own tissue (often referred to as an A~ A~
According to yet another preferred ~mhrl~limPnt tbe present invention relates to any of the Lvl~ u~i~iUIls as defined above, further l,ll~lldl,L;~I iL.,d in that said Fc~yRII bridging agent consists of aggregated human IgG molecules or aggregated Fc fragments of human IgG
molecules.
According to yet another preferred ~ the present invention relates to any of the ~ as defined above, further ~lldl d~l iL~d in that said Fc yRII bridging agent consists of a bivalent or multivalent FcyRII specific monrlrl~n~l antibody, or functionally active fragments of said Fc~yRII specific mnnt~ lc nAI antibody.
According to yet another preferred embodiment, the present invention relates w any 15 ~u~ ;as defined above, further ~ udu~liL~d in that said FcyRII bridging agentconsists of a l~uu~billdllL fusion protein of two or more human IgG Fc parts.
For this purpose, the present invention also relates to a I ~ulllbill~ulL vector, particularly for cloning and/or expression, with said Icl,ulllbilldllL vector comprising a vector sequence, an ~J~JIU~JIidLt~ ,UlUkdlyULiU~ eukaryotic or viral promoter sequence followed by the ~0 nucleotide sequences comprising the nucleic acid sequence encoding such a l~;CUlllbill~lllL
fusion protein comprising at least two human IgG parts, and with said recombinant vector allowing the expression of said IrcOl~ lL protein as defined above in a IJ~ULllyULiC, or eukaryotic host or in living mammals when injected as naked DNA.
The term "vector~ may comprise a plasmid, a cosmid, a phage, or a vitus.
In order to carry out the expression of the polypeptides of the invention according to this aspect of the present invention in bacteria such as E. coli or in eukaryotic cells such as in S. cerevisiae, or in cultured vertebrate or invertebrate hosts such as insect cells, Chinese Hamster Ovary (CHO), COS, BHK, and MDCK cells, the following steps are carried out:
- IIAII~rIII IllA~ of an ~I.UI,llu!Jlid--, cellular host with a I~Cullll,:.ldllLvector, in which a nucleotide sequence coding for a fusion protein of two or more human IgG Fc parts has been irlserted under the control of the ~JUI UUI id~e regulatory elements, particularly a promoter wo ss/32~3~ 2 1 8 8 8 ~ 2 16 r~ 12 recognized by the polymerases of ~lle cellular host and, in the case of a prokaryotic host, an appropriate ribosome binding site (RBS), enabling the expression in said cellular llost of said nucleotide sequence. In the case of an eukaryotic host any artificial signal sequence or pre/pro sequence might be provided, or tl~e natural signal sequence might be employed - culture of said Lla..~ru,l..ed cellular host under conditions enabling the expression of said insert.
According to yet another preferred rl"l,c,~lil"r,.l the prese:lt invention relates to any 10 of the ~l~",~ "-~ as defined above, further ~,llald~ ;d in that said Fc yRII bridging agent consists of a liposome vesicle comprising at least one Fc~RII bridging agents with said agents being chosen from the group consisting of:
- aggregated human IgG molecules;
- aggregated Fc fragments of human IgG molecules;
15 - a bivalent ~ ~ ' I antibody to the Fc yRII:
- a multivalent mnnnrlnn~l antibody to the Fc~RII
- any functionally active fragment of said bivalent or multivalent ".."..,rl.,--~l antibody to the Fc~RII:
- a l~u,,.bi~dll~ fusion protein of 2 or more human IgG Fc parts;
- a liposome vesicle comprising any of the foregoing agents as detailed above.
For this purpose, the present invention also relates to metllods of preparing liposomes containing an Fc~yRII bridging agent as detailed above.
The present invention also rnn~n-rl any method for preparing any of the Fc-yRII
bridging ~.u l~ as defined above.
Examples of the ~ lldLiUII of some of the Fc~RII bridging agents and ~UIIIpU~i~iOI~
according to the present invention are disclosed above, in the Examples section or are within the knowledge of the man skilled in the art.
The present invention relates particularly to the therapeutic uses of any of the above-defined Fc~RII bridging ~,~".I,n~;l;""
It should be stressed that the term "therapy" or "~ " within the concept of the present inevntion is to be il~ ' as including "therapy" or "Ll~ " as such as well as "ululJII~lr~iS" or "IJ-u,ul~yla~Li~

wo ss/3273~ 2 1 8 8 8 1 2 r~ 12 Target cells for treatment with the Fc~yRII bridgillg l~-Jl~ according to the present invention may include IJ-urt~iu,.al antigell presenting cells (APCs) such as human leukocytes, preferably III~IU,UI~ , monocytes, deDdritic cells~ Lallgerhans cells or B cells.
These tdrget cells may possibly be activated before or during treatment with e.g. IFN- y, or 5 GM-CSF. If desired, tdrget cells to be treated may be derived from Ll1e donor of a graft in transplantdtion .
The ~ulll~Ju~iLiulls of this invention will be administrated at a certdin l,UllC~,l.L,d~iu.. that is Ll~"d~ ULi~dlly effective for the envisaged treatment.
To accomplish this goal, the LUlll~U~iLiUII may be formulaLed using a variety of10 acceptdb~e excipients known in the art. Typically, the ~ u~ o~iLiu~ are administered by injection, either intravenously, illLldd~.~llldlly, illLldlll~.,UUldl or ~ lru~ y Methods to accomplish this ad~ L~dLiu.. are known to those of ordinary skill in the art. It may also be possible to obtain CUIlluu~iLiull~ which may be tOpicdlly or orally administered, or which may be capable of Lldllallli~iull across mucous Ill~.lllbldll~
Before dd~ LId~iull to patients, formulants may be added to Lhe ~ or bridging agents of the invention. A liquid rullll,da~iull is preferred. For example, Lhese formulants may include oils, polymers, vitdmins, ~ubollydl . amino acids, salts, buffers, albumin, surfactdnts, or bulking agents. Preferably cdrbohydrates include sugar or sugar alcohols such as mono, di, or ~)oly~dl ~,llal id~", or water soluble glucans. The ~d~l~lldl id~ or 20 glucans can include fructose, dextrose, lactose, glucose, mannose, sorbose, xylose, maltose, sucrose, dextran, pullulan, dextrin, alpha and betd cyclodextrin, soluble stdrch, llydlu~Lllyl starch and ~,~ubu~y ' yl~,cllulose, or mixtures thereof. Sucrose is most preferred. ~Sugar alcohol~ is defined as a C4 to C8 llydluwllJull having an -OH group and includes galactitol, inositol, mannitol, xylitol, sorbitol, glycerol, and arabitol. Mannitol is most preferred. These 25 sugars or sugar alcohols mentioned above may be used individually or in UUllll.~;.ldLiull. There is no fixed limit to amount used as long as the sugar or sugar alcohol is soluble in the aqueous ~n~l~dldLiull~ Preferably, the sugar or sugar alcohol ~o~ il,ll is between 1.0 w/v% and 7.0 w/v%, more preferably between 2.0 and 6.0 w/v%. Preferably amino acids include levorotary (L) forms of carnitine, arginine, and betdine: however, other amino acids 30 may be added. Preferred polymers include polyvinylpyrrolidone (PVP) wiLh an average molecular weight between 2,000 and 3,000, or polyethylene glycol (PEG) with an average molecular weight between 3,000 and 5,000. It is also preferred to use a buffer itl the ... ... ... , .. . : .. . . ... . . .... .. . . . .... .. ..... . . . _ . . . . _ w095/3273~ 2 1 8 8 8 ~ 2 PCTIEP95/02012 osiLiu~ to minimize pH changes in tlle solutioll before Iyopllilization or afterIr~ Most any physiological buffer may be used, but citrate, phosphate, succinate, and glutamate buffers or mixtures thereof are preferred. Most preferred is a citrate buffer.
Preferably, the ~UIl~ L,~Li~., is from 0.01 to 0.3-molar. Surfactant3 tllat can be added to the formulation are shown in EP Nos. 270,799 and 268,110.
Additionally, in case the ~J~ comprises antibodies as an active principle these can be chemically modified by covalent uO~ Liu~l to a polymer to increase its circulating half-life, for example. Preferred polymers, and methods to attach tllem to peptides, are shown in US Patent Nos. 4,766,106; 4,179,337; 4~495,285; alld 4,609,546 which are all hereby ill~,UllJUI ~Led by reference in their entireties. Preferred polymers are polyu~ ,l.ylated polyols and poly~,il.yl~..., glycol ~PEG). PEG is soluble in water at room l~,.,.l,~,.d~u,r and has the general formula: R(O-CHi-CH2)~O-R where R cal1 be hydrogen, or a protective group such as an alkyl or alkanol group. Preferably, tlle pretective group llas between I and 8 carbons, more preferably it is methyl. The symbol n is a positive integer, preferably between 1 and 1,000, more preferably between 2 and 500. The PEG has a preferred average molecular weight between 1000 and 40,000, more preferably between 2000 and 20,000, most preferably between 3,000 arld 12,000. Preferably, PEG has at least one hydroxy group, more preferably it is a terminal hydroxy group. It is this llydroxy group which is preferably activated to react with a free amino group on the inhibitor. However, it will be understood 20 that tlle type and amount of the reactive groups may be varied to achieve a covalently conjugated PEG/antibody of the present invention.
Water soluble polyu~ yl~L~d polyols are also useful ill the present inventîon. They include polyu~y~Lhy~ ~ sorbitol, polyu,~ y' ~ glucose, polyu~ lly' ' glycerol (POG), etc. POG is preferred. One reason is because the glycerol backbone of 25 polyu~y~,il-ylGL~d glycerol is the same backbone occurring naturally in, for example, animals and humans in mono-, di-, Lliglyl;~.id.,3. Therefore, this branching would not necessarily be seen as a foreign agent in the body. The POG has a preferred molecular weight in the same range as PEG. The structure for POG is shown in Knauf et al., 1988, J. I~io. Chem. 263:
15064-15070, and a discussion of POG/IL-2 conjugates is found In US Patent No. 4,766,106, 30 both of which are hereby ill~,Ul,UUI ' I by reference in their entireties.
After the liquid ~ ulll~cruLi~ UIIll~osiliu~ is prepared, it is preferably Iyophilized to prevent liP.~r~ inn and to preserve sterility. Methods for Iyophilizing liquid ~
~ ~ , ....

WO95/32731 2 1 8 8 8 1 2 P~l/r~

are known to those or ordinary skill In tbe art. Just prior tO use, tlle ~o...~uOiLiu., may be .~(.."~ . d with a sterile diluent (Ringer's solution, distilled water, or sterile saline, for example) which may include additional ingredients. Upon lCcul.Oii~u~iun, tlle romr~siti~n is preferably administered to subjects using those met]lods that are known to those skilled in 5 the art.
The dosage and mode of ad,l,il-i,udtiu,l will depend oll the therapeutic purpôse.
Generally, the f.~ iliu~lc are ~ cd so that aggregated IgG antibodies are given at a dose between l,~lg/kg and 20mglkg, more preferably between 20~g/kg and lOmg/kg, most preferably between I and 7mg/kg.
10 TherapywiththeFcyRllbridging-,.. ,l.. l~ili.Jl~ortheFcyRllbridgedcellsaccording to the present invention may be performed in uullju~ Liul~ witll any other known techniques or ( ...~.r.u~ for treatment of " y, allergic or dUlUillllllUIlc; diseases or for treatment of L,..~ rejection, such as for instance i."~ ta~,ive and anti-n ' y drugs (e.g. u.yclu~L)c~lillA (CsA), I.l.~lloLl- , prPrlnicnlonp~ riPY~mPth~cnnP, 15 FK506, rapamycin, ~;y~,lou~.~b.,lldOc inhibitors such as inrlnmPth~Ain and cul~iuu~liullo) ûr i,...."... ' ' y cytokines (such as IFN-7~, IL-I0 or IL-12).
TiLenewFcyRIIbridging~u.~.l...~;li....~accordingtothepresentinventionmayfurther also be used for any other human or animal treatment or diagnostic purpose for which they are possibly suited.
According to a pdl ~iCLlLII Iy preferred CIlllJOdilll~ . the present invention relates to the use of any of the Fc~yRII bridging ~--.,,I..I~il;-n~. as defined above as a lI.~ .., more particularly for treating T-cell mediated diseases, even more p~ iull;dlly for the IJIC~dUd~i of a .. ,.. 1;~,.. ,. for treating T-cell mediated diseases.
An Fc yRII bridging ~:ulll,uo~iliull to be used according to this aspect of the invention 25 comprises pdl~icukll~y new Fc~RII bridging agents or ~ n~ according to the above specified aspects of the present invention.
According to an even more preferred I .~.I.n.li,.,. ~/ tlle present invention relates to the use of any of the Fc~RII bridging uu~,uoOi~iullo as dehned above, for the m~ tinn ûf antigen-specihc T-cell ~,O,uu-~iv~"leS, more p~i~ul~,ly for the l~lC~Ud~iOII of a ,.. l;, ,.. :
30 for the Illodul~.Liu-l of antigen-specific T-cell Ic~ulbiv~
Alternatively, the present invention relates to tbe use of any of tlle Fc yRII bridging UOlllUUoi~iUllo as defined above, for inducing T-cell tolerance or anergy, more particularly for , ,, ... ,,, ... ,, .. , ,, , . _ ... .. ....... .. ... . .

wo ss/32~3~ 2 1 8 8 8 1 2 P~ 5~V~0l2 the ~lrlJdudiiol~ of a m~iirAm~ nt for inducing T-cell tolerance or anergy.
The terms "T-cell anergy" and "T-cell tolerance" are reviewed above.
The invention provides a method for tl1e induction of antigen-specific T-cell tolerance or T-cell anergy by modulating the co-stimulatory functioll of professional APCs by 5 providing to a subject in the need of SUCIl a treatment an effective amount of a specific antigen combined with an agent capable of bridging tlle FcyRII Witll at least one of the above-described functional effects of preventing the essential co-stimulatory molecules B7-1 and -2 from being up-regulated and by down-modulating the adhesion molecule ICAM-3.
Said Fc yRII-bridging agent is selected from the group consisting of: aggregated human IgG
10 molecules; aggregated Fc fragments of human IgCi molecules; a bivalent monoclonal antibody specific to the FcyRII; a functionally active fragment of the latter antibodies; a multivalent mnnnclnnAI antibody specific to the FcyRII: a l~,ulllbilldllL fusion protein of 2 human IgG
Fc parts; liposome vesicles containing at least one of the toregoing brdiging agents; all as detailed above.
15 FcyRII bridging agents or ~.. ~.I.. ~;Ii.. ~ to be used according to this particular aspect of the present invention may comprise known FcR bridging u,.l~ , agents or principles or in a preferred way specific Fc yRII bridging ~ Jal Liuulal ly aimed at in the present invention (resulting in the prevention of expression of B7 molecules and/or the down-mn~ inn of ICAM-3 molecules expression) as detailed above.
According to a more particuiar r,~ Ol~ , the present invention relates to the use of any of the Fc yRII bridging r~ as defined above for treating allergic diseases, more ~Jal~i~.ulally for the ~ aldliOII of a ~ for treating allergic diseases in humans, such as asthma, allergic rhinitis, atopic dermatitis. food and drug allergy.
The invention also provides a method for treating allergic diseases in humans, wherein said method comprises providing to a subject in the need of such a treatment an effective amount of a specific antigen combined with an agent capable of bridging Fc yRII molecules with the above-described functional effects of preventing the essential co- ' y molecu~es B7-1 and -2 from being up-regulated and by down-modulating the adhesion mo~ecule ICAM-3, wherein said Fc yRII-bridging agent is selected from the group consisting of: aggregated human IgG molecules; aggregated Fc fragments of human IgG molecules; a bivalent mnnnrlnnAI antibody specific to the FcyRII, a multivalent monoclonal antibody specific to the Fc~RII, or funtionally active fragments of the latter antibodies; a IC;~,Ulllbilldll~
_ _ _ _ _ _ _ _ _ -~' wossl3~73l 2 1 8 8 8 ~ 2 ~ ol~

fusion protein of 2 or more human IgCi Fc parts; liposomes containing any of the foregoing as detailed above, possibiy ill iulllbilldLiul~ with an allergen and otller i~ u~o~udulatory agents as detailed above.
Fc yRII bridging agents or . ~ to be used according to this par~icular aspect 5 of the present invention may comprise known FcR bridging agents or ~,1",1,n~i~i.."~ or in a preferred way, specific Fc^rRII bridging agents or . ulll~,o~iLiu..~ ~JalLiculdlly aimed at in the present invention (resulting in the prevention of B7-1/2 molecules expression and/or the down-motl~ tinn of ICAM-3 molecules expression) as detailed above.
According to another particular ~,~.l)ol~ the present invention relates to the use lû of any of the FcyRII bridging ~ as defined above, for preventing rejection ofsolid organs, tissues and cells after Llal~lJ' Ull~, more pdlLiculldlly for the IJlc~)dla~iol~
of a mPrlir:lmPnt for preventing rejection of solid organs, tissues or cells after transplantation in humarls.
The invention also provides a method for preventing rejection of solid organs, tissues 15 or cells after lldll~ulallLdliul~ in humans, wherein said method comprises providing to a subject in the need of such a treatment an effective amount of a specific alloantigen combined with an agent capable of bridging FcyRII molecules witll the above-described functional effects of preventing the essential co-stimulatory molecules B7-1 and -2 expression and by down-mn~ tin~ the adhesion molecule ICAM-3 expression, wherein said Fc~RII-bridging 20 agent is selected from the group consisting of: aggregated human IgG molecules; aggregated Fc fragments of human IgG molecules; a bivalent mnn~)clr,n~l antibody to the FcyRII; a ...~.'Li~..l~,..~ 1".~ ,1., 1ll,l ~l antibody to the Fc yRII, or functionally active fragments of the latter antibodies; a lc~ulllb;ld~L fusion protein of 2 or more human IgG Fc parts; liposomes containing any of foregoing as detailed above, possibly in Lolllbillà~iol~ with an alloantigen 25 and an i~ lc~ive or i" ~ n~l..l y agent as detailed above.
Fc yRII bridging agents or rr~mrnciri~mS to be used according to this particular aspect of the present invention may comprise known FcR bridging agents or principles or in a preferred way specific Fc~yRII bridging agents or I~.,,l,n~i~;..,,~ particularly aimed at in the present invention (resulting in the prevention of B7-1/2 molecules expression and/or the 30 down-,.,.~ ;.l.. of ICAM-3 molecules expression) as detailed above.
Target cells for treating in vitro or in vivo with the FcyRII bridging agents or~ulll~Jo~iLiu.l, according to the present aspect of the invention are preferably ,ulurc~iulldl ~ . _, . , ... . . . . . . .. . . .. .... .. _ .... ... . .. . . . .

3~ 2 1 8 8 8 l 2 ~ A7nl2 APCs treated with any form of aggregated or immobilized human IgG-s, or aggregated Fc fragments of human IgG; or soluble human IgG's in tlle presence of antibodies to human IgG; or in the presence of bivalent or multivalent FcyRII monoclonal antibodies, or in the presence of functionally active fragments of the latter antibodies, or in the presence of a 5 l ~cv~ illalll fusion protein of 2 or more human IgG Fc parts, or in tlle presence of llposomes containing any of ~he foregoing.
The term "aggregated" may mean aggregated by means of any known chemical cross-linking agent.
The term "immobili~ed " may refer to any known immobilization method on any 10 known subtrate such as a microtiter plate, a membrane (e.g. nylon or nitrocellulose), a Illi~lU~ (bead) or inserted into a liposome vesicle. Prior to application to the membrane or fixation it may be convenient to modify the IgG in order to facilitate fixation or improve its binding efficiency to Fc-receptors.
According to yet another particular embodiment, the present invention relates to the 15 use of any of the Fc yRII bridging ~ as defined above for trcating dUlU;IlllllU.._ diseases, more ~dl~i~ukuly for the preparâtion of a I P~li.A. ..I for the treatment of dULvilllll-ullc diseases, such as thyroiditis, rheumatoid arthritis, systemic lupus ~IyL~l~lla~u~
(SLE), multiple sclerosis, .. v diabetes.
The invention also provides a method for the treatment of ~..I..i,..ll...ll~. diseases, 20 wherein said metbod comprises providing to a subject in the need of such a treatment an effective amount of a specific ~ - ,l ic,. . . combined with an agent capable of bridging Fc yRII
moleculcs with the above-described functional effects of preventing the essential co-y molecules B7-1 and -2 expression andlor by down-modulating the adhesion molecule ICAM-3 expression, wherein said Fc-yRII-bridging agent is selected from the group 25 consisting of: aggregated human IgG molecules; aggregated Fc fragments of hurnan IgG
molecules; a bivalent r~nrlrlonAl antibody to the Fc yRII; a multivalent mrln~rl-~n:ll antibody to the Fc~yRII; functionally active fragments of the latter antibodies; a I~I,UIllbilldll~ fusion protein of 2 or more human IgG Fc parts; liposomes containing any of the foregoing as detailed above, possibly in COIlli~ iul~ with an ~ -,..,l and an i"""...,..~..l.l"",,i~e or 30 ;~ c~ I.y agent as detailed above.
Fc yRII bridging agents or ~ to be used according to this particular aspect of the present invention may comprise known FcR bridging agents or principles or in a ~ wo 95/3273~ 2 1 8 8 8 1 2 r~ ,nl2 preferred way specific FcyRII bridging agents or l:U~ /O~i~iOI~ particularly aimed at ill the present invention (resulting in the prevention of B7-1/2 molecules expression and/or the down-m~ tinn of ICAM-3 molecules expressioll) as detailed above.
According to a special embodiment. the presel1t inventiol1 relates to a mP~ rnPnr comprising a composition as defined above.
According to a further r~ n~ the present invention relates to Fc yRII bridged professional APCs such as monocytes such as prepared by bridgillg ~/lUrt~iUlldl APCs such as monocytes with any of the Fc-yRII bridging ~UIIIIJU~iLiUlls as defined above.Tl1e invention also provides bridged professional APCs as defined above for use as a ~.PII;I ~lllrlll, particularly for preventing a condition as detailed below.
The invention also relates to a method for preparing SUCIl bridged APCs as detailed above.
T'tte invention also provides a method for preventing rejection of solid organs, tissues or cells after transplantation in humans, wl1erein said method comprises providing to a subject in the need of such a treatment an effective amount of alloantigen-expressing ~IUI~iU~ d APCs such as monocytes from the graft donor on which several FcyRII
molecules have been bridged with the above-described functional effects of preventing the essential Co-~LillluldLvly molecules B7-1 and -2 expression and by down-modulating the adhesion molecule ICAM-3 expression, wherein said Fc yRII-bridging is du~,u~llpli~ d by a method selected from the group consisting of: culturing the ~IUf~ iUlldl APCs (such as monocytes) in culture dishes coated with human IgG; culturing tlle APCs in culture dishes together with aggregated human IgG molecules or aggregated Fc fragments of human IgG
molecules; culturing the APCs in culture dishes in the presence of soluble l1uman IgG and an antibody to human IgG; culturing the APCs in culture dishes in the presence of a bivalent monoclonal anti60dy to the FcyRII; culturing the APCs in culture dishes in the presence of a multivalent mnnnrln~ antibody to the Fc~RII, or culturing the APCs in culture dishes in the presence of any functionally active fragment of tlle latter antibodies; culturing the APCs in culture dishes in tbe presence of a Ir,-,vlllbilldllL fusion protein of 2 or more human IgG
Fc parts; liposome vesicles comprising any of the foregoing as detailed above.
Fc yRII bridging agents or ~ n" ,l,n~ ",~ to be used according to this particular aspect of the present invention may comprise known FcR bridging agents or principles or in a preferred way specific Fc-yRII bridging ~UIIIIJU~iLiO~ JalLi-,ul~lly aimed at in the present ... .. .... .. . , _ WO9S/3273-l 2 1 8 88 1 2 ~ /P.I~ O12 invention (resultin~ in the prevention of B7~ molecules expression and/or the down-modulation of ICAM-3 molecules expression) as detdiled above.
The present invention also contemplates a tllerapeutic Lu~ ,o~iLiu-, comprising Fc yRII
bridged pluC~ iul~dl APCs as defined above.
According to another t;lllbOdil~ lL, the present invention relates to FcyRII bridged ~IIUr~iUlldl APCs as defined above, for use as a ~ Al....~r, more particularly a~"r.lil.A.". .,1 for treating or preventing any of the above-mentioned disease stdtes.
More particularly, the present inven~ion relates to tlle the use of FcyRII bridged plur~a~iulldl APCs as defined above for the pl~aldLiull of a mP~irArnPn~ for preventing rejection of solid organs, tissues or LIAI~ JnC in humans.
The present invention also relates to an in vitro method for screening for or selecting a new Fc~yRII bridging agent or, " I~I~r~ , witll said bridging agent or LUIlllJo~i~iUII being clldud~L~li,.~d as preventing the B7 molecules expression on professional APCs and/or down modulating ICAM-3 expression on IJIU~ iulldl APCs.
1~ A test system to screen for such molecules comprises an in vitro ~ ILdI set up in which ~luf.,~:,iulldl APCs (for instdnce monocytes) are incubated in the absence or presence of the molecule(s) to be tested as having possible Fcyll bridging capacities as detdiled above by any of the techniques known in the art (such as ELISA or lullulluul~ (FACS) andlysis as described in the ~xamples section) and measuring tlle amount of B7-1, B7-2 and/or ICAM-3 is measured.

~ woss/3273~ 2 l 8 8 8 ~ 2 r~l,~ 12 BRIEF DFscRlpTloN ~F TITF TARLFS ~NI) FIGU~FS
Table 1: ~Ir ' ' ' of cell surface antigens on monocytes after FcR bridging. Cell surface expression of various antigens was determined after overnight culture on plates coated witll human IgG or on un-coated plates as control. The relative expression is defined as the S expression after overnight culture on human IgG-coaoed plates compared to dhe expression after culture in un-coated plates. Strong increase is indicated by + + +, modest increase by + +, slight increase by +, strong decrease by ---, modest decrease by -- and slight decrease by -. No change in expression is indicated by o in the Table. Cell surface expression was measured by FACS analysis using specific monoclonal antibodies to the cell surface 10 molecules as described in dhe specific Examples. (*) designates that B7-1/2 expression was r P~PrminPd with dhe CTLA-41g fusion protein as described in the specific Examples.
Table 2: FcR briùging inlhibits ' ,. ~ ;fi, ".~' ~ ' of T cells. PBMC (106 cells/ml) were cultured in 96-well flat bottom culture plates pre-coaoed overnight either widh human serum albumin (HSA) or with human IgG (HGG). The following antigens were added IS to dhe culture: Tetanus toxoid (O.SLfu/ml), Varidase (lOOlU/ml), Tuberculin (SlU/ml), Cytomegalovirus antigen (CMV) (O.OllU/ml), Herpes simplex virus antigen (0.021U/ml), Varicella antigen (O.llU/ml), Mumps antigen (1/2000), Influenza virus antigen (1/1000), Candida albicans (O.S~g/ml). Afoer 6 days of culture, cells were pulsed for 8h wid~
I~Ci(3H)-Thymidine.Proliferativevaluesrepresentdheaverageoftriplicatewells. (++):The 20 following mitogens were used as controls: PHA (O.S~g/ml), ConA (S~g/ml), PWM
(O.SIlg/ml). Afoer 3 days of culture (6 days in dhe case of PWM), cells were pulsed for 8h widh l,uCi(3H)-Thymidine. Proliferative values represent the average of triplicaoe wells.
Table 3: Release of soluble ! , ~ C~ mediators after FcR bridging. Purified T cells (106 cells/ml) were cultured in the presence of syngeneic or allogeneic monocytes (106 25 cells/ml) as stimulator cells. Cultures were set up in normal plates, or plates precoaoed with human IgG or F(ab')~ fragments of human IgG. MLR ~U~ L~I~s were taken afoer 24h culture and tested for the presence of various cytokines. The results are Ic~)lr~ L~ILivc of one C,~ lL out of three.
, _, .. . . .. ... . .

w0 9s/:3273~ 2 1 8 8 8 1 2 ~ JIfV~OI2 (++): TNF-f~ was determined by ELISA with a detection limit of 10 pglml. (): IL-IO was measured by ELISA witl1 a detection limit of 5 pg/ml. (11) TGF-B was measured in a bioassay using the MVI Lu cell line. This bioassay has a detection limit of 50 pglml. (<) means below the detection limit of the specific bioassay.
5 Table 4: PGE2 ~.. ' ' by human monocytes after FcR bridging.
Purified T cells (106/ml) were cultured in the presence of syngeneic or allogeneic monocytes (1061ml) as stimulator cells. After 3 days of culture, cells were pulsed for 16h with 0.5 ~Ci(3H)-Tllymidine. Proliferative values represent the average of triplicate wells. (++): After 24h, ~u~ were recovered and analyzed for PGE2 content using an ELISA system.
(t): Cells were cultured in 96-well round bottom culture plates. (11) Cells were cultured in 96-well round bottom culture plates pre-coated with human IgG. (7r): as in ('1) but also the cycluu~y,~ ~., inhibitor. jl,rlfll),. ;l -r;.. (IOO~M) was added at tlle beginning of the culture.
Table 5: FcR bridging on monocytes from an Fc~RII-deficient individual results in inhibition of ~ ;r.~ of T cells. PBMC ( 106 cellslml) from an Fc7dl-deficient individual were cultured in 96-well flat bottom culture plates pre-coated overnight either with human serum albumin (HSA) or with human IgG (IgG). The following antigens were added to the culture: Tetanus toxoid (O.SLfulml). Varidase (lOOlUlml), Tuberculin (SlUlml), Cytomegalovirus antigen (CMV) (O.OllUlml), Herpes simplex virus antigen (0.021Ulml), Varicella antigen (O. IlU/ml), Mumps antigen (1/2000), Influenza virus antigen (1/1000), Candida albicans (0.5,uglml). After 6 days of culture, cells were pulsed for 8h with I~Ci(3H)-Thymidine. Proliferative values represent the average of triplicate wells. (++): The following mitogens were used as controls: PHA (0.5,~glml), ConA (5~glml), PWM
(0.5~glml). After 3 days of culture (6 days in the case of PWM), cells were pulsed for 8h with l~Ci(3H)-Thymidine. Proliferative values represent the average of triplicate wells.
Figure 1. ~1- ' ' ' of B7-1 expression on human monocytes. Monocytes (1061ml) were cultured on human IgG-coated dishes or with various cytokines for 24h and analyzed by FACS for B7-1 expression using mAb B7-24. A: as control monocytes were analysed directly after isolation; b: monocytes cultured 24h in medium; c: monocytes cultured 24h on human IgG-coated dishes; d: monocytes cultured 24h with IFN- y: e: monocytes cultured 24h wossr3273.l 2 ~ 8 8 8 ~ 2 PCTIEP95/02012 with TNF-~; f: monocytes cultured 24h with GM-CSF; g: monocytes cultured 24h with IL-2;
11: monocytes cultured 2411 with IL-4. Staining witll isotype-matched antibody is shown as control.
Figure 2. Mc ' ' ' of B7-1 expression on human monocytes by FcyRII bridging.
5 Monocytes (106/ml) were cultured for 24h in tlle presence of soluble human IgG (dotted line), rabbit anti-human IgG (solid line) or soluble human IgG and rabbit anti-human IgG
(dashed line). B7-1 expression was analyzed by FACS using mAB B7-24.
Figure 3. Effects oF FcR bridging on B7-1, CD40, HLA-DR and ICAM-I expression onI - yt.. when activated by IFN-l~ or GM-CSF. Monocytes (106/ml) were cultured for 24h in medium; in medium on human IgG-coated dishes; in the presence of GM-CSF; in the presence of IFN-y; on human IgG-coated dishes in the presence of IFN-y. After a 24h culture period, cells were recovered and analyzed by FACS. Staining with isotype-matched antibody is shown as control. The mon~ '( ' antibodies used are as detailed in the Materials and Methods.
Figure 4. FcR bridging modulates both B7-1 and B7-2 on monocytes. Monocytes (106/ml) were cultured ~il.11111;111. ~,"~ly on human IgG-coated dishes in the presence of IFN7, GM-CSF, or medium alone. After 24h, cells were recovered and analy~ed for B7- 1 and B7-2 expression by FACS analysis using the CTLA4-lg fusion protein. As control the staining with the secondary antibody only is shown. A: monocytes cultured 24h in medium; b: monocytes 20 cultured 24h on human IgG-coated dishes; c: monocytes cultured 24h with GM-CSF; d:
monocytes cultured 24h with GM-CSF on human IgG-coated dishes; e: monocytes cultured 24h with IFN-; f: monocytes cultured 24h with IFN- on human IgG-coated dishes.
Figure 5A. Effect of FcR bridging on rnonocytes on the activation of T cells in MLR.
Purified T cells (106/ml) were cultured in 96-well round bottom tissue culture plates (circles) 25 or plates pre-coated with human IgG (triangles) in the presence of various numbers of syngeneic (closed symbols) or allogeneic (open symbols) monocytes as stimulator ce~ls. A:
T-cell proliferation was measured after 3 days by [3H]-Thymidine illW~ dLi~ll and is expressed as the mean of three different ~ ,.. ;s + SD.

WO 9513273~ 2 1 ~ g 8 ~ 2 PCT/EP95/02012 Figure 5B. Effect of FcR bridging on monocytes on the activation of T celis in MLR.
Purified T cells (106/ml) were cultured in 96-well round bottom tissue culture plates (circles) or plates pre-coated witlI human IgG (triangles) in tlle presellce of various numbers of syngeneic (closed symbols) or allogenelc (open symbols) monocytes as stimulator cells. IL-2 5 production by the T cells was measured after 24 hours and is expressed as the ~ulir~ld~b~e response of the CTLL bioassay.
Figure 6A. Blocking the B7-1/CD28 i.lt.. ' is not sufficier~t to block the activation of T cells in MLR. Purified T cells (lOh/ml) were cultured in tl1e presence of various numbers of monocytes as stimulator cells. Monocytes were added directly to the T cells 10 (circles) or after pre-incubation with an anti-B7 mAb (triangles). After 3 days of culture T-cell proliferation was measured by [3H]-Thymidine i..~,ul~uldlion and is expressed as the mean of three different ~ + SD.
Figure 6B. Blocking the B7-1/CD28 is not sufficient to block the activation of T cells in MLR. Purified T cells (106/ml) were cultured in tlle presence of various 15 numbers of allogeneic monocytes as stimulator cells. Monocytes were added directly to the T cells (circles) or after pre-incubation with an anti-B7 mAb (triangles). IL-2 production by the T cells was measured after 24 hours and is expressed as the proiiferative response of the CTLL bioassay.
hgure 7A. I , ~ of T-ceU ' ' ' ~ sapacity of monocytes after FcR bridging 20 with intact hurnan IgG j5, r' ~y mediated by Fcl~RII. Purified T cells (lOh/ml) were cultured in 96-well fiat bottom tissue culture plates pre-coated with F(ab'). fragments of human IgG (light bars) or plates pre-coated with intact human IgG (dark bars) in the presence of syngeneic monocytes which were pre-incubated with monoclonal antibodies to different Fc-receptors (anti-Fc yRI mAb 197, anti-FclRII mAb IV.3). T-cell proliferation was 25 measured after 6 days by (3H)-Thymidine ill~Ul~)UldLiUII. T-cell proliferation induced by PWM.

woss/3273~ 2 ~ 88 8 1 2 i~ 2 Figure 7B. I , , of T-cell ' ' ' y capacity of monocytes after FcR bridging with intact human IgG is r ~ mediated by Fc yRII. Purified T cells ( 106/ml) were cultured in 96-well flat bottom tissue culture plates pre-coated with F(ab'), fragments of lluman IgG (light bars) or plates pre-coated with intact human IgG (dark bars) in the presence S of syngeneic monocytes which were pre-incubated with mnnnclnn:~l antibodies to different Fc-receptors (anti-Fc yRI mAb 197, anti-Fc yRII mAb IV.3). T-cell proliferation was measured after 6 days by (3H)-Thymidine ill~ullJulaliùll. T-cell proliferation specific for the Varidase antigen.
F~gure 7C. I , ~ of T-cell ' "v capacity of monocytes after FcR bridging 10 with intact human IgG is ~ mediated by Fc~RII. Purified T cells (106/ml) werecultured in 96-well flat bottom tissue cultùre plates pre-coated with F(ab')~ fragments of human IgG (light bars) or plates pre-coated with intact human IgG (dark bars) in the presence of syngeneic monocytes which were pre-incubated with mnnnrlnn:~l antibodies to different Fc-receptors (anti-Fc~RI mAb 197, anti-Fc yRII mAb IV.3). T-cell proliferation was 15 measured after 6 days by (3H)-Thymidine illCUI~Ul.~liul~. T-cell proliferation specific for the Herpes antigen.
Figure 8. The impaired capacity of monocytes to stimulate antigen-specific T-cell activation mediated by FcR bridging with intact human IgG can be prevented by rnAb to Fc yRII. Purified T cells were cultured in 96-well flat bottom tissue culture plates pre-20 coated with human serum albumin (black bars and dotted bars) or plates pre-coated with intact human IgG (squared bars and hatched bars) with syngeneic monocytes in the absence (black bars and squared bars) or presence (dotted bars and hatched bars) of anti-FcRII mAb IV 3 T-c~ll roli~tion w~ ~easurtd afif r 6 days by '(H) ~hymidlm i~ n~

W095/3273~ 21 8881 2 r~ 2 FX ~ MPI ~
The present invention is based on the new finding that tlle bridging of low affinity IgG
receptor Fc yRll (CD32) molecules on monocytes specifically prevents the up-regulation of tlle essential co-stimulatory molecules B7-1/2 expression and results in the down modulation S of tile adhesion molecule ICAM-3 expression, with the functional ~u~la~ e of an impaired capacity of the monocytes to co-stimulate the a~ctivation of antigen-specific T cells. Such impaired co-stimulatory capacity of monocytes presenting specific antigen to T cel~s may lead to T-cell Ulu~,."Ju~lai~ or T-cell tolerance (Harding et al. Nature 356:607 (1992);
Vassiliki et al. J. Exp. Med. 178:1753 (1993)). This T-cell tolerance toward specific antigens 10 is valuable for certain clinical usages in treating T-cell mediated diseases. Although the importance of the B7 molecules in co-stimulation of T cells and the prevention of T-cell tolerance is known in the art and although it is also known that bridging of FcyRII results in the activation of monocytes resulting in the release of cytokines and other soluble mediators, nothing in the art relates to the finding that the specific bridging of the low 15 affinity IgG receptor Fc yRII (CD32) on monocytes very selectively prevents the up-regulation of the essentiai co- ' y molecules B7-1/2 expression and down-modulaoes tile ICAM-3 adhesion molecule expression. The observed modulation of the B7 molecules and the ICAM-3 molecule expression is a specific process in the cells which is mediaoed by specific mediators of intercellular signal lldll:~l.lU~.~iUII. This is ~l~ml ' by the fact that 20 treatment of monocytes with IFN- y or GM-CSF normally up-regulates the expression of such molecules as B7, HLA-DR, ICAM-I and CD40. In contrast, treatment of monocytes with IFN-~ or GM-CSF with ~o~rmi~ bridging of the FcyRII on monocytes prevents the up-regulation of the B7 molecules without affecting the up-regulation of HLA-DR and ICAM- 1.
Monocyoes are ~)lUrt;~iUlldl antigen presenting cells and Fc~RII bridging does not affect the 25 up-regulation of HLA-DR on these cells and should therefore not affect their capacity to present antigenic peptides in the context of the HLA-DR molecules. Fu. 1~,~. ..u, ~, it has been (' 'thatFcyRlliscapableofmediatingphagocytosisoflgG-opsonized~lyL;IIu.,y (Indik et al., J. Clin. Invest. 88:1766 (1991); Tuijnman et al., Blood 79 1651 (1992)) and has been shown to be active in .,I~du~ of human IgG immune complexes (Engelhardtet al., Ellr. J. Imm~nol. 21:2227 (1991)). It is therefore proposed that combining the . , . . , = _ . .

1-- wo9sl3273~ 2 1 888 1 2 r~ 2 presence of a specific antigen with an Fc~RII-bridging agent in vivo would result in dliUll of antigenic peptides to specific T cells in tlle absellce or reduced presence of the essential co-stimulatory molecules B7-1/2. Tllis treatment will result in modulation of ,vVll~ of only those T cells able to recognize tlle specific antigen in question. This 5 finding is of importance for the treatment of diseases where T cells play a crucial role in pathology such as allergic diseases,: diseases and transplant rejection. The mechanism by which this treatment results in T-cell inactivation is based on the fact that specific antigen presented aS bound to or in Uulll~ ld~iull Witll a Fc~ Rll-bridging agent, would result in the internalization of this antigen. Once internalized, the antigen will be 10 degraded into small peptides and these peptides will be presenoed in the context of class 11 MHC antigens on the cell surface of tbe APC. At tlle same time, proper bridging of Fc y Rll molecules on the APC prevents the up-regulation of B7 co-stimulatory molecules resulting in ~JI~CIlLd~iUII of tbe specific antigen to T cells in the absence of the essential co-stimulatory molecules.
In order to prevent transplant rejection, clinicians have ~ ed with injection of immune cells from the donor prior to the rrPn~p!s~nt~ti--n with the aim of inducing T-cell tolerance or T-cell anergy against the Pll ~i,, However, instead of .~ ll this procedure of oen resuloed in priming against the foreign ~lln~nti~f~nc This priming effect must have been the result of l ~es~ iiull of the alloantigen in the presence of the co- ' y 20 molecules B7-1 and B7-2. Given the present invention of specifically preventing the up-regulation of the B7 molecules, it is now possible to treat rransplant patients with monocyoes from the donor which have been treaoed with an Fc7~RII-bridging agent. This procedure will result in the induction of donor allodl~LiO~.I specific T-cell tolerance.
The following examples serve to illustrate the present invention, but are in no way 2~i to be il.~ ' as being limitative.
and ~ thf~
r 1 - - antibodies All mAbs were used as purifled i,l,,,,..l-..~l--'vvlinc The anti-B7 mAb B7-24 (IgG2a) and anti-CD40 mAb SD12 (IgG2b) have been previously described (De Boer et al. ~ur. J.
ImmunoL 22:3071 (1992)). The anti-lCAM-I mAb B-C14 was kindly provided by Dr. J.
....... . .
_ _ _ .. . . _ . , .. . . .. _ .. ..

Wo 9s/3273~ 2 1 8 8 8 1 2 32 r~~ tnl2 Wijdenes (Innotherapie Bessançon, France). T~le mAbs to HLA-DR (PE-labeled), CD20 (PEI-labeled), CD3 (FlTC-labeled),CD14 (FlTC-labeled) and the isotype control antibodies were purchased from Becton and Dickinson (E-~,.,Lod~g~, Belgium). The CTLA-4 human IgG
fusion protein was purified from the ~ulJ~ aLdllL of a cell line stably transfected Witll a cDNA
5 encoding the fusion protein. This cell line was a gift of Dr. A. Lanzavecchia (Basel Institute for Immunology, Basel, Switzerland). Goat anti-human (affinity isolated polyclonal goat F(ab')l anti-human IgG FlTC-labeled, Tago, CA, USA) was used as second reagent to reveal CTLA-4 IgG staining.
Isolation and culture of monocytes Buffy coats obtained after uylu~llvl~ of healthy donors were used to prepare monocyte cultures. Monnn~ r cell ~ were obtained after buoyant density C~IlLli[u~dLiull of buffy coats on Lymphoprep (Nycomed, Oslo, Norway). The monocyte-enriched, E-negative, fraction was separated from T Iymphocytes by standard roset formation with SRBC followed by Lymphoprep ' Monocytes were further enriched by the cold aggregation technique. Briefly the cell suspension was allowed to clump by low speed rotation at 4 C. Cell clumps were separated from the rest of the cells by .,~ ,d~iUII, this population was > 89% CD14+. In some c,~,u~ c~L~ monocytes were isolated by adherence to plastic dishes for 30-40 min at 37 'C in a 5 % CO2 ~tml . ~- ~. After incubation; the non-adherent cells were removed by repeated vigorous washing. The resulting cultures contained at least 80% monocytes and less than 20% of cells as revealed by phenotype analysis (FACS). Monocytes (IxlO6/ml) were cultured in RPMI 1640 s~ . . ;I with 10 % heat-inactivaKd fetal calf serum, non-essential amino acids, 100 IU/ml penicillin, 100 ~g/ml sLIq~lv~ cill, 10 mM L-glutamine, 2 mM sodium pyruvate and, 50 ,uM 2-111~
in the absence or presence of varlous stimuli or human Ic~vlllbilldllL cytokines. IFN- y (100 U/ml), GM-CSF (0.8 ~g/ml), TNF-o~ (1000 U/ml), IL-2 (50 U/ml) were added to monocyte cultures for a period of 24 h. Bridging of the FcR was achieved by culturing monocytes on Petri dishes that had been pre-coated for 30 min at 37 C with standard humarl IgG (y globulins), whole molecule or F(ab')i fragment as control (ChromPure human IG, Jækson Immuno Research, West Grove, PA) and extensively washed with PBS. After overnight culture the cells were recovered with a cell scrapper and counted by Trypan Blue exclusion.

~ wo gs/3273~ 2 1 8 8 8 1 2 1~l,~ _ ?~112 Pu.'~ of T cells Peripheral blood ",.~ ",~ cells (PBMC) were isolated from buffy coat by centrifugation on Lymphoprep (Nycomed, Oslo, Norway). T cells were further purihed by depletion of monocytes, B ceils and NK cells using Lympho-Kwilc T (One Lambda, Los S Ange~es, CA) according to the IllallurduLulcl~ protocol.
Mixed 1,~ J ~e cultures Purifled T cells (IxlO~/well) were cultured in 96-well round-bottom tissue culture plates (Falcon 307~, Becton Dickinson, Oxnard~ CA) ill the presence of various numbers of syngeneic or allogeneic monocytes as stimulator cells. After 3 days of culture, cells were pulsed for 16 h with 0.5 ~Ci [3H]-Thymidine (specific activity 5 Ci/mMol, Isotnrrhim Gdl~L~i~-P~,ylui~, France), after which the cells were harvested using an automated cell harvester. [3H]-Thymidine illCulL~uld~iu.l was determined with a liquid ~rintill~inn counter.
Proliferation of T cells were performed in triplicate wells. In inhibition c,.l,~,. illl~,llL~
monocytes were pre-incubated with mAb B7-24 (2 ~-g/ml per 106cells) or an isotype control mAb in complete RPMI for 30 min at 4 C before they were dispensed in 96 well round-bottom plates. Bridging the FcR of the monocytes during the MLR was achieved by using 96-wells round-bottom culture plates that had been pre-coated with human IgG, I mg/ml, in PBS for 30 min at 37 C and washed three times with cold PBS. Human igG F(ab')~
fragment coated plates were used as control.
A '' Peripheral blood ~ . I - cells (PBMC) isolated on a Ficoll-Hypaque gradient (density 1.077) (Pharmacia LKB, Uppsala, Sweden) were washed and Ir~ fl in complete medium, consisting of RPMI 1640 (Gibco,Paisley, Scotland) ~u~ ,J with penicillin, ~LIqJ~VIIIy~,ill, glutamine and 5% autologous plasma. They were cultured at a ~ ."1,~ " of lxlO6 cells per ml, in 96 well flat bottom culture plates (Falcon, Labware, Lincoln Park, NJ), pre-coated by incubation overnight either with human serum albumin (HSA) (B~ , Marburg, Germany) or with human ~-globulins (Cohn fraction 11) (Sigma, St Louis, MO), each at a ~n"~ ;."l of 10,ug/ml of phosphate buffered saline (PBS), and followed by extensive washing. The following antigens were used, each at an indicated final ~ ,ll which in preliminary ~ had been shown to induce ~-- .- .. - .. .. ... .. _ .. , ___ _ ~

wo 9sl3273~ 2 1 8 8 g 1 2 PcrlEPs5/020I2 optimal T cell u~ulir~,.aLiull. Tetanous toxoid (Wyetll, USA) 0.5 Lfu/ml; Varidase (Lederle, Belgium) lOOlU/ml; Tuberculin (Statens Serum Institute, Copenhagen, Danmark) SlU/ml; Cytomegalovirus antigen (~clllil~ .k~) O.OllU/ml: Herpes simplex virus antigen (Bel~ kc) 0.021U/ml; Varicella antigen (Behringwerke) O.lU/ml; Mumps antigen S (~(., 11 illf,~,.k~) 1/2000; Influenza virus antigen (Duphar, Belgium) 1/1000; Candida albicans (Haarlem, Allergenen Laboratorium, The Netherlands ) 0.5 ~g/ml. The following mitogens were also used for ~tim~ inn Phyi ' v~ (PHA) (Wellcome Diagnostics, Temple Hill Dartford, England) 0.5 llg/ml; Concanavalin A (ConA) (Sigma) 5 ,ug/ml; and Pokweed Mitogen (PWM) (Calbiochem, La Jolla, CA) 0.5 ~g/ml . Cells were cultured in a 5% C02 humidified atmosphere for 3 days (PHA, ConA) or 6 days (PWM, antigens). Eight hours after a I ~Ci (3H)-thymidine pulse (Amersham, R~ A~ re~ England), cells were harYested and processed for .1. ~. .",i., .l;l.n of (3H)-thymidine ill~UI~ulaiiUII in a liquid crintill~tinn counter.
Cytokine ~ ' MLR ~u~.,. Ila~all~ were taken after 24h culture and tested for the presence of various cytokines. IL-2 production was measured by bioassay using a subclone of the murine CTLL
cell line, using Ic~,ollllJillall~ human IL-2 as standard and is expressed as (3H)-thymidine illCullJold~iull of the CTLL cells. TNF-~ was determined by ELISA (Innotest hTNF-o~, I."..,,Ef .1. ,;I c, Belgium) with a detection limit of 10 pg/ml. IL-I0 was measured by ELISA
using two mAbs: B-NI0 as coating antibody and biu~illy~ ,J BTI0 as detecting antibody.
Rf wlllbil~ll~ IL-I0 was used as a standard, the detection limit of this ELISA is S pg/ml.
TGF-13 was measured in a bioassay using the MVI Lu cells line . This bioassay has a detection limit of 50 pg/ml.
Pl ' ' ` '' E2 Pl-). ~ 1;- E2 produced by monocytes during the MLR under various culture conditions was measured using an ELISA system (Boehringer Manheim, Brussels, Belgium).
To test the effect of the prn~r~g!~n~in synthesis on T cell activation, the cycluu~yL
inhibitor 1,~ . . ;1, ;,. (Sigma) (100 ,uM) was added at the beginning of the culture.

~ woss/3273J 2 l 8 8 8 1 2 r~~ ~?~1~2 FACS analysis For i~-ullullulllul~s~llcc analysis, purified monocytes (Ix106) were Ir~ fd in PBS containing I % FCS, 0.1 % NaN3 and 10 % normal rabbit serum. The cells were incubated at 4 C for 30 min to block FcR-binding sites. The cells were ,,~ y S incubaoed for 30 min at 4 C with primary mAbs of the d~ ULJI specificity, washed in PBS containing I % FCS and 0.1 % NaN3 and, incubated for an additional 30 min at 4 C with FlTC-labeled goat anti-mouse Ig (Sigma). The cells were analysis on a FACScan instrument (Becton Dickinson & Co, Mountain View, CA).
F '- 1 ~ of B7-1 expression on human monocytes It is known that B7-1 expression on monocytes ean be up-regulated by culture in the presence of IFN- y (Freedman et al. Cell. Immunol. 137:429 (1991)). Several cytokines were tested for their eapaeity to induce B7-1 expression on monocytes. In addition, it was tested wl1ether B7-1 expression could be induced by FeR bridging on monocytes. Primary human monocytes were isolated by the cold ~L~ Liu~l technique and examined for eell surfaee 15 expression of B7- 1 using flow cytometry. Figure I shows that freshly isolated monoeytes laek detectable B7-1 cell surface protein, when stained with the anti-B7-1 mAb B7-24. However, after 24 h in culture medium alone, low but significant amounts of B7-1 could be detected on their cell surface. Upon culture for 24 h, of the different eytokines tested, not only as previously reported in the literature IFN- y (Freedman et al. Cell. Immunol. 137:429 (1991)), 20 and GM-CSF markedly increased B7-1 expression. Il~ ly, FcR bridging by eulturing monocytes on human IgG-coated dishes seemed to prevent the ~uOIlLallCvu~ increase of B7-1 surface expression.
Down-mn(l~ inn of B7-1 expression on human monocytes could also be observed after incubation with human IgG in solution. However, binding of the Fc part oF human IgG
25 by itself was not enough to modulate the expression of B7-1 on the monocytes. Only when tlIe FcR-bound human IgG was ~ ly bridged by the addition of a rabit antiserum against human IgG, the down ~ inn of B7-1 could be achieved (Figure 2).

WO 95/3273~1 2 1 8 8 8 1 ~ PCT/EP95/02012 Examnle 2: Effects of FcR bridging on monocyte B7-1, CD40, HLA-DR and ICAM-1 expression when activated by IFN-y or GM-CSF
The experiment described above illustrates that FcR bridging on monocytes prevents cp~n~nrollc increase of B7-1 surface expression. It was tl~erefore also tested as described in tlle legend of Figure 3 and in the section Materials and Methods above. whether bridging of the FcR on monocytes could prevent up-regulation of B7-1, even in tlle presence of potent inducers such as IFN- y or GM-CSF. Monocytes were cultured overnight on human IgG-coated dishes before IFN- y or GM-CSF was added to tlle cultures. After an additional 24 1l culture period, the cells were recovered and analyzed by FACS for expression of different cell surface molecules. As shown in Figure 3, the IgG-treatment had little or no effect on the ~o~ )u~ expression level of the cell surface molecules HLA-DR, ICAM-I and CD40.
Interestingly, FcR bridging strongly inhibited the B7-1 and CD40 up-regulation by IFN- y or GM-CSF. Whereas only a modest effect was observed on the up-regulation of HLA-DR, and ICAM-I by IFN-y or GM-CSF.
The effects of FcR bridging on the expression of a number of other molecules on monocytes is ~u~ d in Table 1. Table I show that the relative expression levels of B7, ICAM-3 and CD14 show the strongest decrease. The expression of CDllb, CD16, CD48and CDw50 is only slightly decreased. The expression od CD40, CD43, CD55, CD58, CD59, ICAM-I and HLA-DR is slightly increased whereas CD44 a stronger up-regulation.
F ' 3: FcR bridging modulate both B7-1 and B7-2 on monocytes Recently it was reported in the literature that there are two different B7 molecules, named B7-1 and B7-2 (Freeman et al., Science 262:909 (1993)). The ~ described above were performed with a -' I antibody specific for the B7-1 molecule. The CTLA-4-lg fusion protein as described in the section Materials and Metdlods above, is known 25 to recognize both B7-1 and B7-2 on monocytes and otller antigen presenting cells (Freeman et al., Science 262:909 (1993)). It was therefore also tested, as described in the legend of Figure 4 and in the section Materials and Methods above, whether the expression of the B7-2 molecule on monocytes was influenced by FcR bridging. Figure 4 shows that when monocytes cultured on IgG-coated plates were analyzed for expression of both B7 molecules .

WO95/3273~l 2 ~ 8 ~ ~ ~ 2 PCT/}P95A12~1~Z

using a CTLA-4-lg fusion protein, il was found tllat this treatmel1t dowll-modulated both B7-I and B7-2.
F ' 4: Effect of FcR bridging on monocytes on the acti- ation of T cells in Mixed L,~ .' yt. Cultures S As bridging of FcR is able to prevent tlle up-regulation of the B7 moleculcs on monocytes, it was tested whether this treatment would influence their ' y capacity to activate resting T cells. Purified T cells were cultured in tlle presence of various numbers of syngeneic or allogeneic monocytes in 96-well round-bottom culture plates pre-coated with IgG, so that FcR bridging was persistent during the entire culture period and ~JlUlir~ldLiO.~
was measured by (3H)-Thymidine i.lcul~Jul~llioll~ In Figure 5a it is shown that monocyoes cultured on IgG-coated plates were much less potent in stimulating T-cell proliferation than untreated monocytes. A mean inhibition of 54 i 15 % was found in a total of 10 G~ L, using cells from different individuals. In addition tlle proliferative response of T cells to autologous monocytes was completely absent in the IgG-coated plates.
Cell proliferation measured by [3H]-Thymidine illuul,uuldLiOIl on day 4 is a delayed Ill~ul~ ,ntofAPC-Tcelli"..~l;l."~thathaveoccurredmuchearlierintheMLR.ltwas therefore also evaluated whether another parameter of T-cell activation, namely IL-2 secretion, was concordant with the proliferative activity under identical culture conditions.
We determined the content of IL-2 in the ~u~J.,I IlaL lllL~ of the MLR cultures using the CTLL
20 bioassay. Figure Sb shows that untreated monocytes have the stimlll y capacity to induce a strong IL-2 release in the MLR ~U~ LdllL~. In contrast, the ~u~ allL~ obtained with monocytes isolated and cultured in the presence of coated human IgG did not contain detectable IL-2 activity. These results indicate that bridging of FcR on monocytes greatly affects their capacity to stimulate resting T cells, both at tlIe level of proliferation and 25 Iymphokines secretion.
F. ' 5: Blocking the B7-1/CD28 ' ' ' is not sufficient to block the activation of T cells in Mixed L,~ Cultures In view of the importance of the B7-llCD28 interaction as a co-stimulatory signal the ... .. . . .. .. . . ... ... . .. . . . . .. .. .... . . . . . . .. .... .... ..... .

W09SB273.1 21 8881 2 38 P~_l/r.~ Atnl2 importance of this interaction for the regulation of alloantigen-mediated~T-cell proliferation and IL-2 production was measured. To this, purified T cells were stimulated witll allogeneic monocytes in the presence of mAb B7-24, which is capable to block ami-CD3-induced T-cell activation using mouse fibroblasts transfected witll human B7-1 (De Boer el al., ~ur. J.
S ImmunoI. 22:3071 (1992)). Figure 6 shows that addition of mAb B7-24 had little or no effect on the T-cell proliferation but ~ rl~,d,.~ly decreased the IL-2 release in au~cll~d~dll~a.
Although these results suggest the importance of the B7-1 molecule on the APC function of monocytes for Iylll~ okillc secretion, the blockillg of the B7-1/CD28 interaction could not account for the strong inhibition observed in the ~;Ap~ above, when using IgG-treated monocytes as APC. This means that for complete illllJdil~ of the co-stimulatory function of monocytes both B7-1 and B7-2 must be absent, which is achieved by the specific cross linking of FcR as described in this invention.
. ' 6: FcR bridging inhibits antigen-specific ".~ ' '` ' of T cells.
To determine the effect of FcR bridging on the APC function of monocytes to induce antigen-specific proliferation of T cells, purified T cells and monocytes obtained from healthy donors were stimulated as described above in the Materials and Methods section, with a number of different recall antigens. Table 2 shows that FcR bridging on monocytes strongly inhibits their capacity to induce a specific T-cell response to viral, bacterial and fungal antigens, whereas the treatment did not impair the capacity of the T cells to respond to mitogenic stimuli, all measured by (3H)-Thymidine ill~Ul~JUld~iUI~. These results indicate that specific proliferative responses of human T cells tO soluble antigen are markedly reduced or even abolished by specific bridging of FcR on the APC.
~, ' 7: The nmpaired capacity of APC to co-stiimulate T cells after FcR bridgingis not mediated by the induction of soluble i , . .,,:.. mediators FcR bridging on monocytes is known to deliver a very strong activation signal for the release of soluble mediators. It is also known that monocytes can produce potent soluble ;aaiv~ factors (Valitutti et al., Immunology 67:44 (198g); Paswell et al., J.
.

-~ wossl3273~ 21 8 ~ 8 ~ 2 r~ ol2 Immunol. 123:115 (1979)). In order to determine whether the release of soluble im~ ,,,ive factors play a role in the T-cell un-responsiveness after FcR bridging on monocytes, the content of the MLR au~ dLd~ were analyzed by specific immuno assays or bioassays as described in detail in the section Materials and Methods above, for the S presence of the well known cytokines IL-I0, TGF-B and TNF-cY. As shown in Table 3, there was no significant release of TCF-B in MLR 5U,U~,I lldLdll~ when using IgG-treated monocytes as APC, whereas only small amounts of ILI0 wl1ere found. In contrast, very large amounts of TNF-lx were secreted in the MLR cultures following tlle FcR bridging. This TNF-o! was mainly produced by the monocytes, since control cultures of mol1ocytes only on IgG-coated 10 plates gave about the same TNF-~ production as in the presence of T cells. To assess the role of TNF-a as i,l", "..~ .",,~ive agent in our ~ llLdl system, we added recombinant human TNF-~Y to a MLR using untreated monocytes as APC. Under those culture conditions, we found no inhibition of the T-cell ~JlUIir~14Li~JIl or IL-2 release (data not shown), This ~ that TNF-o~ is not involved in tlle inhibition of T-cell responses 15 induced by the FcR bridging on monocytes.
Lipid mediators derived from cell membranes, such as the ~ -.LI~I..lill~, are known to be produced during T cell ~,~hd~ ldULi~ (Coquette et al., Eur. J. Pharmacol.
226:1 (1992)). Pl~ ;,r~ JdULil_ULIy ~ L~gldllllill E12 (PGE2), modulates the function of illlll~llllll~llllll.. .l ~ll cells by ~ulJ~JIta~illg T-cell and Ill~lUlJ~ function (Wong et al., J.
rmn~unol. 148:2118 (1992); Gallay et al., J. Immunol. 150:5086 (1993)). There are many reports tbat PGE2 I);O~YIIL}~ can be initiated by cytokines Sucll as IL-I or TNF-(x (Elliott et al., Growth-factors 6:15 (1992)). ~ , it has been ll J~ rd that FcR bridging on monocytes can result in the secretion of PGE2 (Finbloom et al., J. Immunol. 150:2382 (1993); Singh et al., J. Immunol. 151:2786 (1993)). Therefore the PGE2 content in ~u~ .L~u.~ of MLR cultures was determined using a specific immuno assay as described in the section Materials and Methods described above. Table 4 shows that PGE2 is released in large amounts only in the cultures on IgG-coated plates. In order to investigate the illhibitory effect of PGE2 on the T-cell activation during tlle MLR, tlle ~y~,lou~.yrv~ e inhibitor i~ldul~l~Lll~ was added at the beginning of the culture. Although il~lll...l. :1 - ;.~
30 effectively prevented PGE2 release, the T-cell ~JIUIi[~;ld~illll still remained inhibited. This fi~ that PGE2 was not responsible for the immune ~u~lt~ observed when monocytes are cultured on human IgG-coated plates.
, , , ,:, .. ,, ,, , .... .. , . ,, ., .,,,,, ., .,, ,------ --Wo gs/3273~ 2 1 8 8 8 1 2 pcrlEp95/02012 F l 9 The irnpaired capacib of APC to co-stilDulate T-cells after FcR bridging with Fcl/ RI-deficieDt monocytes To investigate which Fc~-receptors on the monocytes are involved in the down modulation of the B7 molecules and the resulting impaired co-stimulawry capacity, 5 monocytes from an individual known to have a mutation in the gene for tlle Fc~yRI, resulting in the absence of this molecule on the cell surface, were used to present several antigens as listed in Table 5. Table S shows that FcR-bridging on monocytes from an Fc y Rl-deficient indiYidual also strongly inhibits the capacity of these monocytes to induce a specific T-cell response to viral, bacterial and fungal antigens, whereas the treatment did not impair the 10 capacity of the T cells to respond to mitogenic stimuli. These results indicate that if Fc y Rl could mediate the down modulation of tl~e B7-molecules and the resulting impaired co-y capacity of monocytes, other Fc y-receptors can do so as well, li. ' 10: The impaired capacity of APC to co-stimulate T cells after FcR
bridging can be blocked by specific ' ' antibodies to Fc y RII
To further investigate which Fc7/-receptors on the monocytes are involved in thedown-- - ~ tif~n of the B7 molecules and the resulting impaired co-~ .ul4luly capacity, monocytes cultured on IgG-coated plated were stimulated with several antigens in the presence of specific m~nn(~ l antibodies directed to tlle different Fcy-receptors. The 20 proliferative capacity of T cells added to these cultures was measured by (3H)-Thymidine illCUI~uldLiull as described in detail in the Materials and Methods section above. Figure 7 shows that only a monoclonal antibody specific for the Fc y Rll can remove the effect of FcR
bridging on monocytes. Figure 8 shows that indeed for several antigens tested a monoclonal antibody to the Fc~y Rll completely prevents the illl,u~ of monocytes to stimulate T-cell 25 activation after FcR bridging. ~ ~

~ WO 9~i/3273~J 2 ~ 8 8 8 ~ 2 P.~ ,G2012 Table 1 Cell surface antigen Relative expression le~el after FcR
bridging CD 1 Ib CD3 1 o CD44 + +

CDw50 CDS8 +

ICAM-I +

B7-11~* ~~~
HLA-DR +
.

w0 95/3273~ 2 1 8 8 ~ 1 2 r ~ nl2 ~k~: FcR bridging inhibits antigen-specific 1~ . " of T cells~
Expt. I l~xpt. 2 HSA(I0 HGG(I HSA(I0 HGG(I
~g/ml) O~g/ml) ~g/ml) O~g/ml) Stimulus Mean Mean %inhib Mean Mean % inhib PWM++ 40,127 64,174 - 74,931 79,458 PHA+ + 76,658 73,960 - 106.241 93.462 5 ConA++ 55,817 60,883 - 75,715 70,921 Tetanus 76,370 12.100 84 93.067 73,012 75 Candida 38,007 9,904 74 97,209 2,904 97 Varidase 17,199 3,441 80 59,238 1,830 97 CMV 303 419 - 24,002 423 98 10 Herpes 25,793 3,127 88 84,844 2,268 97 Varicella 40,157 3,509 91 92,281 14,737 84 Mumps 14,472 1,142 92 23,150 631 97 Influenza 6,421 1,032 84 31,211 5,731 82 Tuberculin 10,346 1,135 89 44,987 1,019 98 2788~12 ~ WO9513273S P~,1/l~,J._. ~2 ~: Release of solubles i ~ ~it. mediators after FcR bridging Untr IgG- F(ab' eated treat ).-mon ed treat ocyte mon ed s ocyte mon s ocyte s IL- TGB TNF- IL- TGB TNF IL- TG TNF
10~ -B~ ++ 10 -B -~Y 10 B- ,~
(pg/ (pg/ (pg/m (pg/ (pg/ (pg/ (pg/ B (pg/
ml) ml) I) ml) ml) mi) ml) (pg ml) /ml ) Monocytes ~ < 183 < < 1974 < < 75 alone Syngeneic < < 183 30 < 1934 < < 84 Allogeneic < c 199 28 < 1591 < < 81 MLR

wo ssr3273~ 2 1 8 8 8 1 2 ~ 12 Table 4: PGE2 1,.. ' ' by human monocytes after FcR bridging~' Untreat IgG- IgG-ed treated treated monocy monocy monocy tes~ tesll tes +
indomet llacinr PGE2+ Prolifer PGE Prolifer PGE~ Prolifer + ation (pg/ml) atioll (pg/ml) ation (pg/ml) (CPM) (CPM) (CPM) Monocytes alone 1,810 2.945 14,100 468 1,834 426 Syngeneic MLR 1,632 9,036 9,410 2,184 2,156 1,999 Aliogeneic MLR I 2,179 70,504 10,300 47,423 1,623 40,030 5 Allogeneic MLR 11 2,534 75,687 11,000 53,699 1,644 47,270 -~ WO 95/3273.1 2 ~ 8 8 8 1 2 PCT/EP95/02012 Tablc 5: FcR bridging on monocytes from an Fc~l-deficient indi~idual rcsults in inhibition of antigen-specific s HSA( lO~g/ml) IgG (I O~g/ml) Stimulus Meall Mean PWM++ 42~643 18~768 PHA++ 74,193 81~101 Con A+~ 52~287 59~975 10 Tetanus 66~022 14,010 Candida 30,505 11,589 Varidase 14,979 7.690 Herpes 20,417 3,451 15 Varicella 34,952 4,054 Mumps 12,510 965 Influenza 6,090 800 ~ube~ulin 10,105 2 4al .

Claims (35)

1. Composition comprising at least one Fc.gamma.RII (CD32) bridging agent, said agent being characterized as impairing the capacity of antigen presenting cells (APCs) to stimulate the activation of antigen-specific T-cells, resulting in antigen-specific T-cell unresponsiveness, with said bridging agent being chosen from the group consisting of:
- aggregated human IgG molecules;
- aggregated Fc fragments of human IgG molecules;
- a bivalent monoclonal antibody to the Fc.gamma.RII;
- a multivalent monoclonal antibody to the Fc.gamma.RII;
- a functionally active fragment of said bivalent or multivalent monoclonal antibody;
- a recombinant fusion protein of 2 or more human IgG Fc parts; or, - a liposome vesicle comprising any of the foregoing bridging agents.

2. Composition according to claim 1, further characterized in that said Fc.gamma.RII bridging agent prevents the expression of B7-1/2 molecules by these APCs.

3. Composition according to any of claims 1 or 2, further characterized in that said Fc.gamma.RII bridging agent causes the down modulation of ICAM-3 molecules expression by these APCs.

4. Composition according to any of claims 1 to 3, wherein said Fc.gamma.RII bridging agent is contained in liposome vesicles.

5. Composition according to any of claims 1 or 4, further characterized in that said composition comprises a specific antigen or antigen-complex combined with said Fc.gamma.RII
bridging agent.

6. Composition according to claim 5, further characterized in that said specific antigen is a specific alloantigen, an autoantigen, or an allergen.

7. Composition according to any of claims 1 to 6, further characterized in that said Fc.gamma.RII bridging agent consists of aggregated human IgG molecules, or aggregated Fc containing fragments of human IgG molecules.

8. Composition according to any of claims 1 to 6, further characterized in that said Fc.gamma.RII bridging agent consists of a bivalent or multivalent Fc.gamma.RII specific monoclonal antibody, or a functionally active fragment of said antibody.

9. Composition according to any of claims 1 to 6, further characterized in that said Fc.gamma.RII bridging agent consists of a recombinant fusion protein of two or more human IgG
Fc parts.

10. Composition according to any of claims 1 to 6, further characterized in that said bridging agent consists of a liposome vesicle comprising any of the bridging agents of claim 1, further characterized in that the Fc regions of said bridging agent are on the outside of the liposome vesicle, and in case of a specific antigen or antigen-complex being added according to claim 5, said antigen or antigen-complex being on the inside of said liposome vesicle.

11. Method for preparing a composition according to any of claims 1 to 10

12. Composition according to any of claims 1 to 10 for use as a medicament.

13. Composition according to any of claims 1 to 10 for use in therapy or prophylaxis, more particularly for treating or preventing T-cell mediated diseases.

14. Composition according to claim 13 for modulation of antigen-specific T-cell responsiveness.

15. Composition according to claim 13 for treating allergic diseases.

16. Composition according to claim 13 for treating rejection of solid organs, tissues and cells after transplantation.

17. Composition according to claim 13 for the treatment of autoimmune diseases.

18. Composition according to claim 16 for inducing T-cell anergy.

19. Composition according to claim 16 for inducing T-cell tolerance.

20. Composition according to any of claims 12 to 19, with said composition also comprising immunosuppressive drugs and/or anti-inflammatory drugs such as cyclosporin A, cyclooxygenase inhibitors or corticosteroids.

21. Composition according to any of claims 12 to 19, with said composition also comprising immunomodulatory cytokines.

22. Use of a composition according to any of claims 1 to 21 for the preparation of a medicament for treating or preventing T-cell mediated diseases.

23. Use of a composition according to any of claims 1 to 21 for the preparation of a medicament for modulating antigen-specific T-cell responsiveness.

24. Use of a composition according to any of claims 1 to 21 for the preparation of a medicament for inducing T-cell anergy.

25. Use of a composition according to any of claims 1 to 21 for the preparation of a medicament for inducing T-cell tolerance

26. Use of a composition according to any of claims 1 to 21 for the preparation of a medicament for treating allergic diseases.

27. Use of a composition according to any of claims 1 to 21 for the preparation of a medicament for preventing or treating rejection of solid organs, tissues or cells after transplantation.

28. Use of a composition according to any of claims 1 to 21 for the preparation of a medicament for the treatment of autoimmune diseases.

29. Medicament comprising as an active principle a composition according to any of claims 1 to 21.

30. Fc.gamma.RII bridged professional antigen presenting cells obtainable by bridging professional antigen presenting cells with any of the Fc.gamma.RII bridging agents or compositions according to any of claims 1 to 21.

31. Therapeutic composition comprising Fc.gamma.RII bridged professional antigen presenting cells according to claim 30.

32. Fc.gamma.RII bridged professional antigen presenting cells according to claim 30 for use as a medicament, more particulraly for treating any of the diseases mentioned in claims 22 to 28.

33. Use of Fc.gamma.RII bridged professional antigen presenting cells according to claim 30 for the preparation of a medicament for treating any of the diseases mentioned in claims 22 to 28.

34. Method for producing Fc.gamma.RII bridged professional antigen presenting cells comprising the step of bridging antigen presenting cells with any of the Fc.gamma.RII bridging agents or according to any of claims 1 to 21.

35. Method for screening for new Fc.gamma.RII bridging agents, with said agent being characterized in that it prevents the B7 molecule expression on professional APCs and/or down-modulates the ICAM-3 expression on professional APCs comprising the step ofincubating professional APCs in the absence or presence of the possible Fc.gamma.RII bridging agent and measuring the amount of B7-1/2 and/or ICAM-3 expression.