AU8018400A - Fcgammaria - Google Patents

Description

WO 01/26675 PCT/USOO/28321 FeyRIA This application claims priority from Provisional Application No. 60/159,096, filed October 13, 1999, the entire content of that application being incorporated herein by reference. 5 TECHNICAL FIELD The present invention relates, in general, to FcyRI and, in particular, to methods of modulating the signaling of the FcyRI-y-chain receptor complex and to compounds suitable for use in such methods. BACKGROUND 10 Fcy receptors play a central role in the handling of immune complexes, regulation of inflammatory responses, antibody secretion and T cell activity (Kimberly et al, Arthritis Rheum. 38:306-314 (1995), McKenzie et al. Curr. Opin. Hermatol. 5:16-21 (1998), Ravetch et al, Ann. Rev. Immunol. 16:421-432 (1998) and Sutterwala et al, J. Exp. Med. 188:217-222 (1998)). Common to each of 15 these functions is the initiation of tyrosine phosphorylation following receptor crosslinking (Daeron, Ann. Rev. Immunol. 15:203-234 (1997)) and the involvement of the y/( subunits leading to the view that Fc receptors serve redundant signaling functions. However, recent evidence suggests that these receptors are not redundant. For example, FcyRIIIa appears necessary for 20 initiating the Arthus inflammatory reaction (Hazenbos et al, Immunity 5:181-188 (1996), Syvestre et al, Immunity 5:387-390 (1996)), while FcyRIa and FcaRI can down regulate inflammatory responses by initiating the secretion of IL-10 and IL Ira respectively (Sutterwala et al, J. Exp. Med. 188:217-222 (1998), Wolf et al, - 1 - WO 01/26675 PCT/USOO/28321 Clin. Exp. Immunol. 105:537-543 (1996)). The basis for these differences are unknown. FcyRI is expressed on the cell surface in association with the y-chain (Ernst et al, Proc. Natl. Acad. Sci. USA 90:6023-6027 (1993), Scholl et al, Proc. 5 Natl. Acad. Sci. USA 90:8847-8850 (1993)). This association is not a prerequisite for transient receptor expression but is necessary for stable expression (Takai et al, Cell 76:510-529 (1994), van Vugt et al, Blood 87:3593-3599 (1996)). The y-chain cytoplasmic domain contains an immunoreceptor tyrosine activation motif (isoleucine-threonine-alanine-methionine-(ITAM) and current data suggest 10 that the y-chain cytoplasmic domain is both necessary and sufficient for FcyRIa induced functions (Indik et al, Exp. Hematol. 22:599-606 (1994), Davis et al, EMBO J. 14:432-441 (1995), Lowry et al, J. Exp. Med. 187:161-176 (1998)). Biochemical studies have shown that crosslinking of the FcyRIa-y-chain complex results in activation of a Src family kinase(s) and the tyrosine kinase p72syk 15 (McKenzie et al, Curr. Opin. Hermatol. 5:16-21 (1998), Daeron, Ann. Rev. Immunol. 15:203-234 (1997)). Activation of these kinases results in tyrosine phosphorylation of the y-chain and the initiation of a signaling cascade that can culminate in the induction of degranulation, phagocytosis, an oxidative burst, ADCC activity and the induction of gene transcription. The association between 20 FcyRIa and y-chain may also be important in the formation of a higher affinity receptor complex through the recruitment of two ligand binding chains to the y homodimer (Miller et al, J. Exp. Med. 183:2227-2233 (1996)). Unlike the y-chain, the cytoplasmic domain of FcyRI does not contain an ITAM or other tyrosine containing signaling motifs. Nonetheless, murine FcyRI 25 on J774 cells is constitutively phosphorylated on seine and, after phorbol myristate acetate (PMA) stimulation, the level of phosphorylation increases (Quilliam et al, Immunol. 78:358-363 (1993)). The cytoplasmic domain of FcyRI WO 01/26675 PCT/USOO/28321 may also associate with actin binding protein-280 (ABP-280, also known as non muscle filamin) in the absence of ligand (Ohta et al, Cell 67:275-282 (1991)). Receptor engagement by ligand apparently abrogates this association, although its functional significance is not clear. Furthermore, FcyRIa in the absence of the y 5 chain can signal for calcium in COS-1 cells and the transmission of this calcium signal requires the FcyRIa cytoplasmic domain (Indik et al, Immunobiology 185:183-192 (1992)). The present invention is based, at least in part, on the realization that the FcyRIa cytoplasmic domain serves to modify the signaling of the FcyRI-y-chain 10 receptor complex. This realization makes possible the identification of compounds that can be used to modulate the effects of such signaling. SUMMARY OF THE INVENTION The present invention relates to methods of modulating the signaling of the FcyRI-y-chain receptor complex and to compounds suitable for use in such 15 methods. Objects and advantages of the present invention will be clear from the description that follows. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1: Expression of human wild type (WT) and mutant (MIUT) 20 FcyRIa on the surface of stably transfected P388D1 cells. Cells were incubated with a saturating concentration of the anti-human FcyRIa mAb 22.2-FITC and analyzed by flow cytometry. Figures 2A and 2B: Receptor-specific phagocytosis by WT and MUT human FcyRIa. (Fig. 2A.) Quantitation of human FcyRIa-specific phagocytosis -3- WO 01/26675 PCT/USOO/28321 (E-22.2 F(ab') 2 , hatched bars, n=15) or H-2D -specific phagocytosis (E-3-25.4, solid bars, n=3) by WT and MUT human FcyRIa stable transfectants. As a control, parental non-transfected cells were analyzed. Phagocytosis was performed and quantitated by light microscopy. Data are expressed as the mean 5 phagocytic index ± SD. (Fig. 2B.). Kinetics of phagocytosis of E-22 F(ab') 2 by WT (0) and MUT (0) human FcyRIa P388D1 stable transfectants. Phagocytosis was performed and quantitated by flow cytometry. Data are presented from a single representative experiment (n=5). *, p<0.001, FcyRIa specific phagocytosis by MJT versus WT. 10 Figure 3: Receptor-specific endocytosis by WT and MUT human FcyRIa in P388D1 stable transfectants and of murine FcyRI in non-transfected P388D1 cells. Internalization of WT huFcyRIa (e), MUT human huFcyRIa (0) and murine FcyRI (A) after receptor-specific crosslinking was determined by flow cytometry. Data are presented as the mean + S.D. from a total of 8 experiments. 15 * p<0.01 (1 min) and p<0.005 (2 min and 5 min), WT versus MUT % internalized. Figures 4A and 4B. Differential sensitivity to pre-treatment with BAPTA. (Fig. 4A). Cells were pre-loaded with the intracellular Ca 2 * chelator BAPTA followed by the addition of E-22.2 F(ab') 2 to assess human FcyRIa-specific 20 phagocytosis in WT and MUT P388D I stable transfectants (n=8). WT (open bars) and MUT (hatched bars) P388D1 stable transfectants incubated with E-22.2 were prepared at maximal mAb conjugation ratios (prepared as in Figure 2A). Alternatively, WT P388D1 stable transfectants (solid bars) were incubated with E-22.2 prepared at lower conjugation ratio to match the quantitative level of 25 phagocytosis of the MUT huFcyRIa (Fig. 4B). As a control, FcyRIIa-specific phagocytosis in P388D1 cells transfected with human FcyRIIa (Edberg et al, J. -4- WO 01/26675 PCT/USOO/28321 Biol. Chem. 270:22301-22307 (1995)) was determined using E-IV.3 Fab (n=6). Data are expressed as the mean ± SD. *, p<0.01 relative to control (no BAPTA). N.D. = not done. Figure 5. IL-6 release, but not IL-1P release, requires the cytoplasmic 5 domain of FcyRIa. P388D1 stable transfectants were cultured for 8 hrs in tissue culture wells that had been pre-treated with F(ab') 2 GccM (XL) or mAb 22.2 F(ab') 2 + XL. Data are expressed as the mean pg/ml cytokine produced + SD (n=6). *, p<0.01 relative to the XL alone control. Figures 6A and 6B. Substitution of serines in the cytoplasmic domain of 10 FcyRI with alanines increases the phagocytic index of P388DI transfectants. Figure 7. Serine clusters (bolded) and neighboring charged residues (*) in the cytoplasmic domain of hFcyRIa are indicated. DETAILED DESCRIPTION OF THE INVENTION The present invention results, at least in part, from the demonstration that 15 the cytoplasmic domain FcyRIa alters the biological properties of the FcyRIa-y chain receptor complex. As shown in the Examples that follow, the cytoplasmic domain of FcyRIa directly contributes to the functional properties of the receptor complex. Deletion of the FcyRIa cytoplasmic domain leads to slower kinetics of receptor specific phagocytosis and endocytosis as well as lower total phagocytosis 20 despite identical levels of receptor expression. Deletion of the cytoplasmic domain also converts the phenotype of calcium independent FecyRIa specific phagocytosis to a calcium dependent phenotype. Finally, and particularly importantly in the context of the present invention, deletion of the cytoplasmic domain abrogates FcyRIa specific secretion of IL-6 (but does not affect -5- WO 01/26675 PCT/USOO/28321 production of IL-I3) and allows substantial levels of phagocytosis. These results demonstrate a functional role for the cytoplasmic domain of FecyRIa and provide a general model for understanding how multiple receptors that utilize the y-chain can generate diversity in function. 5 The present invention provides a method of preventing or treating inflammation comprising administering to a patient in need thereof an inhibitor of the proinflammatory activity of the FcyRIa cytoplasmic domain. More specifically, the invention provides a method of inhibiting the release of cytokines (e.g., IL-6 or other macrophage or granulocyte-derived interleukins, and TNF-a) 10 and other biologically active molecules (e.g., reactive oxygen species) from cells such as macrophages and granulocytes. The method of the present invention can be used to prevent or treat inflammation of any of a variety of tissues including the lung (in the case of, for example, asthmatics and patients suffering from ARDS), the joints (e.g., in the 15 case of patients suffering from arthritis), the gastrointestinal tract (e.g., in the case of patients suffering from inflammatory bowel disease) and the kidney (e.g., in the case of patients suffering from glomerulonephritis). Generally, the present methods can be used to prevent or treat any disease or disorder resulting from the proinflammatory effects of the cytoplasmic domain of FcyRI. 20 Compounds suitable for use in the present methods include agents that bind the FcyRI cytoplasmic domain and thereby inhibit its proinflammatory activity, agents that inhibit an interaction of the cytoplasmic domain with another cellular component(s), which interaction results, directly or indirectly, in cytokine release, and agents that otherwise inhibit the proinflammatory effect of the 25 FcyRIa cytoplasmic domain. The compounds can be peptides (e.g., peptide fragments of the cytoplasmic domain of FcyRIa), or mimetics thereof, or other nonpeptidic agents. In the case of peptides, the peptide itself can be introduced -6- WO 01/26675 PCTUSOO/28321 into target cells directly, for example, using liposomes. (See also approaches described in Science 26:1877 (1993) for administration of peptides modified so as to render them capable of crossing cellular lipid membranes.) Alternatively, a DNA sequence encoding the peptide can be introduced using gene therapy 5 protocols so that the peptide is produced intracellularly. The mode of administration will, of course, depend on the compound, the patient and the effect sought. For example, in the case of administration to the lung, the peptide, or encoding sequence, or non-peptidic agent can be administered by inhalation. In the case of administration to the joints, the 10 compound can be administered, for example, by direct injection into the joint. Generally, the compounds can be administered by any route (e.g., intravenously, orally or as per rectum) that will ensure that the amount that reaches the target site is sufficient to inhibit the proinflammatory effect of the cytoplasmic domain of FcyRIa. 15 Compounds suitable for use in the present invention can be identified using any of a variety of art-recognized techniques. Compound identification screens can be based on simple binding assays, for example. That is, test compounds can be contacted with the cytoplasmic domain of FcyRIa, or portion thereof, and the determination made as to whether or not binding of the test 20 compound to the cytoplasmic domain occurs, test compounds that bind the cytoplasmic domain being potential inhibitors of the proinflammatory effect of the cytoplasmic domain of FcyRIa. Compound screens can also be used that are based on the ability of a compound to effect cytokine release. For example, test compounds can be contacted with cells capable of releasing IL-6 and the amount 25 of IL-6 released in the presence and absence of the test compound determined. Cells suitable for use in such screens include cells that naturally express Fcy receptors and cells that do not. In the latter case, cells such as mouse fibroblast -7- WO 01/26675 PCTUSOO/28321 NIH3T3 cells transfected with a FcyRIa encoding sequence can be used. Other cell types can also be used. Compounds that inhibit cytokine release in such systems can be expected to be suitable for use in the present invention. In the context of the above screens, the test compounds can be labeled 5 with a detectable label or unlabeled. The cytoplasmic domain, which also can be labeled or unlabeled, can be present in isolation or in a cell, as appropriate depending on the nature of the screen. Either the test compound or the cytoplasmic domain, or portion thereof, can be bound to a solid support. Compounds identified using the above-described screens, or otherwise 10 identified, can be formulated as pharmaceutical compositions. Such compositions comprise the compound and a pharmaceutically acceptable carrier. The amount of the compound administered will depend on the compound, the patient and the effect sought. Optimum dosing can be readily determined by one skilled in the art. 15 The sequence of FcyRIa is disclosed in Porges et al, J. Clin. Invest. 90:2102 (1998) (see also Fig. 7). Certain aspects of the present invention are described in greater detail in non-limiting Examples that follow. EXAMPLE I 20 The Cytoplasmic Domain of Human FcyRIa Alters the Functional Properties of the FcyRI-y-Chain Receptor Complex Experimental Procedures Cell Culture and Reagents. The murine macrophage cell line P388D1 stably transfected with a cDNA encoding human FcyRIa or a mutant form of FcyRIa 25 containing a stop codon after the first amino acid of the cytoplasmic domain -8- WO 01/26675 PCT/USOO/28321 (K315'|E1Stop 315) were prepared as previously described (Indik et al, Exp. Hematol. 22:599-606 (1994)). P388D1 cells transfected with human FcyRIIa were previously described (Edberg et al, J. Biol. Chem. 270:22301-22307 (1995)). All cell lines were maintained as adherent cultures (Coming Tissue 5 Culture Dishes) in RPMI- 1640 as previously described (Edberg et al, J. Biol. Chem. 270:22301-22307 (1995)). All tissue culture reagents were from Gibco (Grand Island, NY). Human and mouse IgG were obtained from Sigma (St. Louis, MO). Mouse F(ab') 2 fragments and F(ab') 2 goat anti-mouse IgG (GaM) were obtained 10 from Jackson ImmunoResearch (West Grove, PA). F(ab') 2 fragments of the anti FcyRIa mAbs 22.2 and 32.2 were obtained from Medarex (Annandale, NJ). 1gM anti-H-2Dd (clone 3-25.4) was obtained from Pharmingen (San Diego, CA). The hybridoma line expressing the rat anti-murine FeyRII/FcyRIII mAb 2.4G2 was obtained from ATCC (Manassas, VA). All other reagents were from Sigma. 15 Quantitative huFcyRI expression was matched for cells expressing the wild type (WT) and the cytoplasmic domain deletion mutant (MUT) by fluorescence activated cell sorting using anti-FcyRI mAb 22.2-FITC (Medarex). A polyclonal anti-y-chain Ab was provided by Dr. Jean-Pierre Kinet (Letourneur et al, J. Immunol. 147:2652-2656 (1991)). In addition, polyclonal 20 anti-y-chain Abs were prepared in rabbits immunized with a C-terminal peptide sequence that is shared by both human and murine y-chain exactly as described (Letourneur et al, J. Immunol. 147:2652-2656 (1991)). Analysis of[Ca 2 *]. Fura-2 (Molecular Probes, Eugene, OR), a fluorescent dye 25 with spectral properties that change with the binding of free Ca 2 *, was used to measure changes in intracellular calcium concentrations as described (Edberg et al, J. Biol. Chem. 273:8071-8079 (1998)). P388D1 cells, adhered to 25mm -9- WO 01/26675 PCT/USOO/28321 diameter round glass coverslips at 5 x 105 cells/ml, were incubated at 37 0 C for 15 min with 2pM fura-2 AM. During the last 5 min, anti-FcyRIa mAb 22.2 F(ab') 2 was added. After incubation, the cells were washed once with modified PBS (PBS prepared with 5mM KCl and 5mM glucose) and then re-warmed to 37 0 C for 5 5 min in modified PBS plus 1.1mM Ca * and 1.6mM Mg 2 + prior to analysis. The coverslips were transferred to the stage of a Nikon Diaphot and the ratio of fluorescence emission of fura-2 was monitored. After establishment of a baseline, F(ab') 2 goat anti-mouse IgG was added at a final concentration of 35ptg/ml. Analysis was continued for an additional 5 min. 10 Quantitation of intracellular [Ca 2+] before and after treatment of cells with BAPTA-AM was performed using Indo-1 (Molecular Probes) in an SLM Spectrofluorometer (Spectronics Instruments, Rochester, NY) exactly as previously described (Edberg et al, J. Biol. Chem. 270:22301-22307 (1995), Edberg et al, J. Biol. Chem. 273:8071-8079 (1998)). 15 Endocytosis and Phagocytosis. Endocytosis of transfected huFcyRIa was determined by monitoring the disappearance of cell surface associated anti-FcyRI mAb 32.2 F(ab') 2 (Medarex) upon crosslinking with F(ab') 2 GaM (Odin et al, Science 254:1785-1788 (1991)). Similarly, endocytosis of murine FcyRIa on 20 non-transfected cells was determined using mIgG2a (Sigma) and F(ab') 2 GoM. Cells (50pl, 5 x 10 6 /ml) were incubated with a saturating concentration mAb for 15 min at 4'C. Following two washes in PBS/1% BSA, F(ab') 2 GaM was added and cell were incubated for an additional 15 min at 4'C. Cells were then placed at 37 0 C for varying periods of time, rapidly pelleted and washed with PBS/l% BSA 25 containing azide at 4'C. Remaining cell surface associated receptor was quantitated with FITC-conjugated F(ab')2 donkey anti-goat IgG by flow cytometry. - 10 - WO 01/26675 PCTUSOO/28321 Phagocytosis by transfected P388D1 cells was determined in an adherent assay system (Edberg et al, J. Biol. Chem. 270:22301-22307 (1995)). Biotinylated mAb 22.2 F(ab') 2 and biotinylated bovine erythrocytes (E) were prepared as previously described (Edberg et al, J. Biol. Chem. 270:22301-22307 5 (1995)). Biotinylated E were saturated with streptavidin and washed. The resulting E were coated with biotinylated mAb and the level of mAb binding was verified by flow cytometry. P388D1 cells, adhered to round glass coverslips at 5 x 105 cells/ml, were incubated with anti-FcyRIa mAb 22.2 F(ab') 2 coated E (E-22.2) in RPMI/20% 10 FCS (50gl at 5 x 10W E/ml) for 1 hr at 37'C. Alternatively, E coated with an lgM anti-H2Dd (Pharmingen) were used. Non-internalized E were lysed by brief immersion of the coverslip in dH 2 O followed by immersion in buffer. Phagocytosis was quantitated by light microscopy and expressed as a phagocytic index (number of E internalized per 100 P388D1 cells). 15 Treatment of cells with BAPTA-AM (Molecular Probes) to quench intracellular Ca 2 * levels was performed as previously described (Edberg et al, J. Biol. Chem. 270:22301-22307 (1995)). Briefly, coverslip adherent cells were incubated with varying concentrations of BAPTA-AM in RPMI/20% FCS for 30 min at 37'C followed by two washes. E-22.2 in RPMI/20% FCS were then added 20 and handled as described above. Controls included loading cells with the BAPTA AM solvent (1% DMSO) for the same period of time. The kinetics of transfected FcyRIa specific phagocytosis was performed using a flow cytometric based assay (Pricop et al, J. Immunol. Methods 205:55-65 (1997)). In this assay, the E-22.2 were labeled with the PKH26 Red Fluorescence 25 Cell linker Kit (Sigma). Transfected P388D1 cells were mixed in suspension with labeled E-22.2 at a ratio of 50:1 (E:P388D1) (both in RPMI/20% FCS), pelleted and incubated at 37'C for varying periods of time. At each time point, the supernatant was removed and non-internalized E were rapidly lysed in hypotonic - 11 - WO 01/26675 PCT/USOO/28321 saline for 30 sec followed by 3 washes in PBS/1% BSA at room temperature. Samples were analyzed immediately by flow cytometry. Results are expressed as a phagocytic capacity (mean fluorescence intensity of phagocytic cells with one or more internalized E x % of cells with one or more internalized E) as described 5 (Pricop et al, J. Immunol. Methods 205:55-65 (1997)). Cytokine Analysis. Cells were stimulated in 96 well tissue culture plates (Coming) with either PMA, surface absorbed rabbit IgG or surface absorbed F(ab') 2 GaM IgG + mAb 22.2 F(ab') 2 . Wells were coated with absorbed protein 10 (20p g/ml rabbit IgG or F(ab') 2 GaM) for 2 hrs at 37'C. For anti-FcyRl mAb 22.2 F(ab')2 stimulation, mAb at 20pg/ml was added to F(ab') 2 GaM coated wells for 1 hr at 37 0 C. Cells (1-2.5 x 105 cells/ml) were added to the wells and cultured for varying periods of time. Levels of murine cytokines in diluted culture supernatants were quantitated by ELISA. For IL-13 determination, recombinant 15 standard, capture ab (polyclonal rabbit Ab) and biotinylated detection and neutralization mAb (clone 1400.24.17) were obtained from Endogen (Woburn, MA). For IL-6 determination, recombinant standard, capture mAb (clone MP5-2 F3) and biotinylated detection mAb (clone MP5-32C 11) were obtained from Pharmingen. HRP-conjugated streptavidin (Jackson) and then TMB substrate 20 were added and the A 450 nm was determined. Flow CYtometry. Aliquots of cells at 5 x 106 cell/ml were incubated with saturating concentrations of primary mAb for 30 min at 4'C following by two washes. For indirect immunofluorescence, the cells were then incubated with 25 saturating concentrations of FITC-conjugated goat anti-mouse IgG F(ab') 2 at 4'C for another 30 min. After washing, the cells were analyzed immediately for - 12 - WO 01/26675 PCT/USOO/28321 immunofluorescence using a FACScan (Becton Dickinson Immunocytometry Systems, San Jose, CA). Statistical analysis. Analysis of flow cytometry listmode data was done using 5 CellQuest (Becton Dickinson Immunocytometry). Statistical comparisons were performed with the paired t-test. A probability of 0.05 was used to reject the null hypothesis that there is no difference between the samples. Results 10 Assembly of Fc yRIa receptor complexes To investigate the functional significance of the cytoplasmic domain of human FcyRIa, P388D I cells stably transfected with cDNA encoding the full length wild type FcyRIa (WT) or a cDNA encoding a cytoplasmic domain 15 deletion mutant form of FcyRIa (MUT) were studied. Transfected cell lines were sorted to generate clones with identical levels of receptor expression (Fig. 1) that were used in all subsequent studies. Human FcyRIa expressed on monocytes and the myclomonocytic cell line U937 non-covalently associates with the y-chain of the FceRI receptor complex 20 (Ernst et al, Proc. Natl. Acad. Sci. USA 90:6023-6027 (1993), Scholl et al, Proc. Natl. Acad. Sci. USA 90:8847-8850 (1993)). The transmembrane regions of FcyRIa and the y-chain mediate receptor complex assembly and twenty of the twenty-one amino acids in the transmembrane region are identical in murine and human y-chain with one conservative difference (I t2 V). 25 - 13 - WO 01/26675 PCT/USOO/28321 The huFc yRIa CY domain alters the magnitude and kinetics of Fc yRIa internalization While devoid of tyrosine residues, the a-chain of murine FcyRIa has been shown to be phosphorylated on seine and/or threonine residues (Quilliam et al, 5 Immunol. 78:358-363 (1993)) and human FcyRIa has been shown to bind to ABP under some conditions (Ohta et al, Cell 67:275-282 (1991)). Furthermore, FcyRIa in the absence of the y-chain can signal for calcium in COS-1 cells and the transmission of this calcium signal requires the FcyRIa cytoplasmic domain (Indik et al, Immunobiology 185:183-192 (1992)). Accordingly, the possibility 10 that the cytoplasmic domain of huFcyRIa might contribute to the functional properties of the receptor complex was considered. Using E coated with the anti human FcyRIa mAb 22.2 F(ab') 2 , both WT and MUT huFeyRIa mediated receptor specific phagocytosis (Fig. 2). However, the WT construct consistently displayed a higher phagocytic index despite identical levels of receptor expression 15 (Fig. 2). There was no internalization of E-22.2 by parental non-transfected P388DI cells and no phagocytosis of E coated with an IgM anti-H-2Dd mAb (clone 3-25.4) by any cell type (Fig. 2A), despite comparable binding of the E-3 25.4 probe to the transfected cells when compared to E-22.2. Phagocytosis by WT huFcyRIa also displayed more rapid kinetics 20 (Fig. 2B). Similarly, while both WT and MUT huFcyRIa were capable of endocytosis, endocytosis by the WT receptor was more rapid than that mediated by the MUT receptor (Fig. 3). Endocytosis of endogenous murine FcyRIa, assessed on non-transfected P388D I cells, was indistinguishable from the transfected huFcyRIa. Since the WT and MUT cell lines were matched for 25 receptor expression, the differences in phagocytic capacity and the more rapid kinetics of phagocytosis and endocytosis by WT FeyRI provide the first evidence - 14 - WO 01/26675 PCT/USO0/28321 that the cytoplasmic domain of FeyRIa, in association with the y-chain, can affect receptor function. The CY domain of the a-chain determines Ca 2 * sensitivity of Fc yRIa 5 phagocytosis Through the use of chimeric and mutant receptors, the ITAM has been shown to be both necessary and sufficient for FcyR phagocytosis and the FcyR Ca 2 * transient (Davis et al, EMBO J. 14:432-441 (1995), Lowry et al, J. Exp. Med. 187:161-176 (1998), Edberg et al, J. Biol. Chem. 270:22301-22307 (1995), 10 Daeron et al, J. Immunol. 152:783-792 (1994), Mitchell et al, Blood 84:1753 1759 (1994), Park et al, Proc. Natl. Acad. Sci. USA 92:7381-7385 (1995)). The functional importance of the Ca2+ transient has been demonstrated with huFcyRIIa which incorporates an ITAM directly in the cytoplasmic domain and requires elevations in intracellular Ca 2+to mediate phagocytosis (Edberg et al, J. 15 Biol. Chem. 270:22301-22307 (1995)). In contrast, FcyRIa/y-chain specific phagocytosis is independent of the receptor-induced Ca2+ transient (Edberg et al, J. Biol. Chem. 270:22301-22307 (1995)), and the possibility that the cytoplasmic domain of FcyRIa confers a Ca2+ independent phenotype on FcyRIa specific phagocytosis was considered. When intracellular Ca 2 levels were quenched with 20 BAPTA (resulting in [Ca 2 *] = 57±9.3 nM with 20tM BAPTA treatment), receptor specific phagocytosis induced by the WT huFcyRIa was unaltered (Fig. 4A), as previously shown for FcyRI on human monocytes. In contrast, receptor specific phagocytosis induced by the MUT huFcyRla was blocked by pre-treatment of the cells with BAPTA in a dose dependent manner (Fig. 4A). 25 Since the absolute level of MUT huFcyRla phagocytosis is lower than WT huFcyRIa, the possibility that the BAPTA sensitivity might be related to the quantitative level of phagocytosis was considered. Accordingly, phagocytosis by - 15 - WO 01/26675 PCT/USOO/28321 the WT huFcyRIa was performed with E-22.2 prepared with a lower mAb conjugation level resulting in a phagocytic index of 65.1±13.2 compared to a PI of 70.4+6.2 for MUT huFcyRIa and E-22.2 prepared at the maximal conjugation level. WT huFcyRI insensitivity to BAPTA was maintained under these reduced 5 phagocytic conditions (Fig. 4A) indicating that the Ca 2 + insensitivity is a property of the cytoplasmic domain of huFcyRIa. As an additional control, FcyRIIa specific phagocytosis by P388D1 cells expressing full length huFcyRIIa (with a phagocytic index of 167±32.6) was also shown to be blocked by pretreatment of the cells with BAPTA (Fig. 4B), as previously reported (Edberg et al, J. Biol. 10 Chem. 270:22301-22307 (1995)). Importantly, both WT and MUT huFcyRIa receptor complexes induced indistinguishable Ca 2 * transients when crosslinked with anti-receptor mAb. Thus, WT huFcyRIa engages a Ca 2 + insensitive phagocytic pathway while MUT huFcyRla with the associated y-chain engages a Ca2 sensitive phagocytic pathway. These results provide additional evidence that 15 the cytoplasmic domain of the ligand binding a-chain of huFcyRla alters functional properties of y-chain ITAM dependent functions. Requirement for the Fc yRIa a-chain for the induction of IL-6 secretion In addition to its role in internalization, FcyRIa can also modulate the 20 immune response through the induction of cytokine secretion. In particular, activation of monocytes/macrophages by FcyRIa can result in the secretion of IL 6 and IL-1p (Krutmann et al, J. Immunol. 145:1337-1342 (1990), Simms et al, J. Immunol. 147:265-272 (1991)). Accordingly, P388D1 expressing the WT and MUJT forms of huFcyRIa were stimulated with receptor specific mAb bound to 25 surface absorbed F(ab') 2 -GxM. Quantitation of IL-1p secretion after crosslinking of huFcyRIa demonstrated that both WT and MUT forms of the receptor were capable of eliciting comparable levels of secretion of this cytokine (Fig. 5). Cells - 16 - WO 01/26675 PCTIUSOO/28321 incubated in the presence of GaM alone were not stimulated to secrete IL-1@P above the baseline control. In contrast, crosslinking WT huFcyRIa, but not MUT FeyRIa, induced the secretion of IL-6 (8 hr time point). IL-6 production above baseline was detected at 24 hrs after MUT FcyRIa stimulation; however, 5 neutralization of endogenously produced IL-1 prevented this induction of IL-6 secretion by the MUT FcyRIa after 24 hr culture (253 ± 52 pg/mL and 75±21 pg/mL in the absence and presence of a neutralizing anti-IL-I P mAb). In contrast, neutralizing anti-IL-i I mAb did not abrogate the IL-6 induction observed at the 4 or 8 hr time points. No significant difference in the ability of 10 PMA (100ng/ml) or surface bound IgG, engaging endogenous murine FcyRIIa/FcyRIIIa and transfected human FcyRIa, to elicit IL-13 or IL-6 secretion was observed between the WT and MUT lines. These results document the requirement for the cc-chain of the FcyRIa receptor complex for the induction of the IL-6 response by the receptor complex and demonstrate that the pathways 15 leading to IL-6 secretion and IL-1p secretion are distinct. EXAMPLE II Effect of Substituting Serines in the Cytoplasmic Domain of FcyRIa with Alanines P388DI cells were transfected with constructs encoding wild type FcyRIa 20 or FcyRIa mutants bearing alanines in place of series in the cytoplasmic domain as indicated below: RI-SAS(400.1): The N-terminal two seine residues (Ser-328 and Ser 331) in the cytoplasmic domain of human FcyRl mutated to alanine (see Fig. 7). - 17 - WO 01/26675 PCT/USOO/28321 RI-SSA (400.1): The C-terminal two serines residues (Ser-339 and Ser 340) in the cytoplasmic domain of human FcyRI mutated to alanine (see Fig. 7). RI-dSdA (400.1): All four seine residues in the cytoplasmic domain of human FcyRI mutated to alanine (see Fig. 7). 5 The constructs described above have C-terminal Myc and poly-histidine epitope tag derived from pCDNA3.1 -MycHis vector (Invitrogen). All the constructs were expressed under the control of human beta-actin promoter (Gunning et al,. Proc Nati Acad Sci U S A 84:483 (1987)). For culture, DMEM 10 supplemented with L-glutamine (3 mM), penicillin (100 units/ml), streptomycin (100 y g/ml), 2-ME (50 sM) and 10% FCS was used. The phagocytic index of the transfectants is given in Fig. 6 (the "controls" are P388D1 cells transfected with wild type FcyRI while the "experimentals" are P388D1 cells transfected, as indicated, with one of the above mutant forms of 15 FcyRI. The results from multiple experiments are shown. The data presented in Fig. 6 resulted from experiments in which the phagocytic index was determined using sheep erythrocytes coated with anti-sheep erythrocyte rabbit antibodies (designated "EA" in Fig. 6A) which engage all the Fc receptors present or using sheep erythrocytes coated with "E22.2 Fab" (see Fig. 6B), which engage only 20 human FcyRI. The data indicate that mutation of the serines in the cytoplasmic domain of FcyRI increases phagocytosis generally and FcyRI mediated phagocytosis in particular. While not wishing to be bound by theory, it is believed that the cytoplasmic domain serines may bind a protein that leads to decreased FcyRI 25 signaling, and elimination of the series leads to increasing signaling. * * - 18 - WO 01/26675 PCT/USOO/28321 All documents cited above are hereby incorporated in their entirety by reference. One skilled in the art will appreciate from a reading of this disclosure that various changes in form and detail can be made without departing from the true 5 scope of the invention. 10 15 20 25 - 19 -

Claims (17)

1. A method of preventing or treating an inflammatory 5 condition comprising administering to a patient in need of said prevention or treatment an agent that inhibits the proinflammatory activity of the cytoplasmic domain of FcyRIa in an amount sufficient to effect said prevention or treatment.

2. The method according to claim 1 wherein said 10 inflammatory condition is an inflammatory condition of a lung of said patient.

3. The method according to claim 2 wherein said inflammatory condition is asthma or ARDS.

4. The method according to claim 1 wherein said 15 inflammatory condition is an inflammatory condition of a joint of said patient.

5. The method according to claim 4 wherein said inflammatory condition is arthritis.

6. The method according to claim 1 wherein said 20 inflammatory condition is an inflammatory condition of the gastrointestinal tract of said patient.

7. The method according to claim 6 wherein said inflammatory condition is inflammatory bowel disease. -20- WO 01/26675 PCTUSOO/28321

8. The method according to claim 1 wherein said inflammatory condition is an inflammatory condition of a kidney said patient.

9. The method according to claim 8 wherein said 5 inflammatory condition is glomerulonephritis.

10. A method of inhibiting release of a cytokine from a cytokine-producing cell comprising contacting said cell with an agent that inhibits the proinflammatory activity of the cytoplasmic domain of FcyRIa in an amount sufficient to effect 10 said inhibition.

11. The method according to claim 10 wherein said cell is a granulocyte or macrophage.

12. The method according to claim 10 wherein said cytokine is IL-6 or TNF'l. 15

13. A method of inhibiting release of a reactive oxygen species from a cell comprising contacting said cell with an agent that inhibits the proinflammatory activity of the cytoplasmic domain of FcyRIa in an amount sufficient to effect said inhibition. 20

14. A method of screening a test compound for its potential as an inhibitor of a proinflammatory effect of the cytoplasmic domain of FcyRa comprising contacting the cytoplasmic domain of FcyRIa with said test compound and determining whether said test compound binds to said cytoplasmic domain of FcyRIa, - 21 - WO 01/26675 PCTUSOO/28321 wherein a test compound that binds to said cytoplasmic domain of FcyRIa is a potential inhibitor of said proinflammatory effect.

15. A method of screening a test compound for its potential as an inhibitor of a proinflammatory effect of the 5 cytoplasmic domain of FcyRa comprising contacting a cytokine producing cell with said test compound and measuring the level of cytokine released from said cell, relative to the level of cytokine released in the absence of said test compound, wherein a test compound that inhibits said cytokine release is a potential 10 inhibitor of said proinflammatory effect.

16. The method according to claim 15 wherein said cytokine is IL-6.

17. The method according to claim 15 wherein said cell produces a heterologous FcyRa. 15 - 22 -