C1q AND COLLECTIN RECEPTOR

Abstract: The present invention concerns novel uses of the cC1q Receptor (cC1qR) binding domain and inhibitors thereof.

Description: Clq and Collectin Receptor The present invention concerns novel uses of the Clq and collectin Receptor (cClqR) binding domain and inhibitors thereof. The C 1 q binding domain within cC 1 qR (cC 1 q receptor) has previously been identified (Stuart, G.R. et al, 1996, FEBS Letters, 397: 245-249 and references therein). The cClq receptor is also be referred to as the Clq receptor, which also binds collectins. The cClqR homologue Calreticulin (CaR/CRT) has also been identified and shown to have very high sequence homoiogy, and as such reference to cClqR is considered to also be reference to CRT and vice versa unless otherwise stated. Certain functionality has been attributed to cC lqR upon its activation by the binding of Clq, namely immunological responses such as phagocytosis, enhanced cytokine and antibody production and antibody-dependent cell cytotoxicity. cClqR is also known to bind the collectin proteins SP-A, MBL, CL43 and conglutinin. However, the exact nature of cClqR has not yet been determined nor its structure identified. Sequence analysis does not identify it as being part of a known class of cell-surface receptors. The present inventors have now found that the Clq binding domain of cClqR is in fact a CUB (Complement Ubiquitin) domain, and as such certain previously unknown functionality can be attributed to cClqR and inhibitors of same. CUB domains are well known (see for example Day, A.J. et al., 1993, Behring Inst. Mitt., 93: 31-40; Thiel, S. et al, 1997, Nature, 386: 506-510; Arlaud, G.J. et al, 1993, Behring Inst. Mitt., 93: 189-195). Thus according to the present invention there is provided the use of a cC lqR binding domain as a CUB domain. Thus the presentinvention may provide the use of a cClqR binding domain in the manufacture of a medicament to effect CUB domain functionality. Since the cClqR binding domain is both a collectin and ClqR binding domain, the term "cClqR binding domain" also encompasses Clq binding domains. Sequence analysis of cClqR shows little primary homoiogy with known CUB domains, but the cClqR binding domain does in fact have 6 to 7 consensus residues (out of a total of about 100) with CUB and this provides the cClqR binding domain (and thus cClqR) with CUB domain functionality. The Clq binding domain may form part of an existing molecule, for example cC lqR or it may form part or the whole of a novel molecule, for example a molecule comprising a recombinant cClqR binding domain. The cClqR binding domain may bind a site comprising five collagen repeats (Gly-X-Y triplets) (Malhotra, R. et al., Biochem. J., 293: 15-19). Also provided according to the present invention is the use of an inhibitor of the cClqR binding domain to inhibit CUB functionality. Such an inhibitor may of course be any molecule or other chemical agent which is capable of inhibiting the activation of the cClq receptor. Examples of such inhibitors include recombinant cClqR binding domains which competitively inhibit the binding of Clq to cClqR and thereby inhibit the activation of the cClq receptor. Such competitive inhibitors (a) inhibit Clq and mannose binding lectin mediated activation of complement and (b) bind to the part of the collagenous region of Clq where covalent binding to ss-amyloid occurs in Alzheimer's disease. Also provided according to the present invention is a method of manufacture of a medicament to effect CUB domain functionality, characterised in the use of a cClqR binding domain. Also provided according to the present invention is a method of manufacture of a medicament for inhibiting CUB domain functionality, characterised in the use of an inhibitor of the cClqR binding domain. The identification of the cC 1 qR binding domain as a CUB domain provides a wide range of previously unidentified functionality for the cC 1 q receptor and inhibitors thereof. Inhibition of the cClqR binding domain prevents (i.e. inhibits) complement activation via the classical and lectin pathways and provides therapeutic potential in all such diseases in which complement activation is involved in the initiation and maintenance of inflammation, for example myocardial infarction, brain ischemia (stroke), gut ischemia, rheumatoid arthritis, systemic lupus erythematosus, burns and immune complex nephritis. The cClqR binding domain may also be used to inhibit the binding of β-Amyloid to Clq, thereby inhibiting the formation of amyloid plaques in Alzheimers disease (Velazquez, P. etal, 1997, Nat. Med., 3(1): 77-79) . Additional CUB functionality includes the ability to bind carbohydrate domains of molecules, for example of collagens, and to cause opsonisation. Thus the present invention also provides the use of an inhibitor of the cClqR binding domain according to the present invention in the manufacture of a medicament for the treatment of complement activation involved in the initiation and maintenance of inflammation, for example for the treatment of myocardial infarction, brain ischemia (stroke), gut ischemia, rheumatoid arthritis, systemic lupus erythematosus, burns, or immune complex nephritis. Also provided is the use of an inhibitor of the ClqR binding domain according to the present invention in the manufacture of a medicament for the treatment of amyloid plaques in Alzheimers disease. The present invention also provides the advantage of achieving the desired effecting or inhibiting of CUB domain functionality through the use of a low molecular weight molecule. Various cClqR binding domains have been identified by the present inventors, namely those of humans (SEQ ID NO: 1), mice (SEQ ID NO: 2) (Mus musculus C57 black) and rats (SEQ ID NO: 3) (Rattus norvegicus whistar). Thus the cC lqR binding domain may have the sequence of any one of SEQ ID NOs: 1-3. Obviously, the sequence may be partially modified to retain CUB domain functionality yet have a sequence which is different from the one from which it was derived, i.e. one of SEQ ID NOs: 1-3, and the present invention encompasses the use of such partially modified domains. Partial modification may, for example, be by way of addition, deletion or substitution of amino acid residues. Substitutions may be conserved substitutions. Hence the partially modified molecule may be a homologue of the molecules from which it was derived. It may, for example, have at least 40% homoiogy with the molecule from which it was derived. It may for example have at least 50, 60, 70, 80, 90 or 95% homoiogy with the molecule from which it was derived. An example of a homologue is an allelic mutant. Also provided according to the present invention is a method of treatment of the human or animal body comprising the use of a cClqR binding domain or an inhibitor thereof according to the present invention. The method may be a method for effecting (in the case of a cC 1 qR binding domain) or the inhibiting (in the case of a cClqR binding domain inhibitor) CUB domain functionality. The invention will be further apparent from the following description, with reference to the several figures of the accompanying drawings, which show, by way of example only, forms of cClqR binding domain. Of the figures: Figure 1 shows the amino acid sequence of CRT (SEQ ID NO: 7). Amino acid sequences were deduced from the nucieotide sequence of human CRT (McCauliffe, D.P. et al, 1990, J. Clin. Invest., 85: 1379-1391.). The signal sequence residues are shown in lower case. The N- (italics), P- and C- (italics) domains are indicated. The CUB-domain is underlined. Domain constructs were expressed as thioredoxin fusion products; Figure 2 shows binding of Clq to Calreticulin domains. Solid-phase bound domains, with appropriate controls, were incubated with radioiodinated Clq. Binding levels of four separate experiments, at saturation, are shown, calculated as % (bound/loaded). These percentages were then standardised against the results for cClqR. Y-axis shows % relative binding. X-axis shows immobilised proteins (left to right): ClqR, N-domain, S-domain, P-domain, C-domain and BSA; Figure 3 shows binding of CUB-domain to immobilised Clq. Serial dilutions of radiolabelled CUB-domain were bound to immobilised Clq and BSA. After extensive washing, bound radioactivity was measured as described in Experimental section (below). Y-axis shows cpl bound. X-axis shows cpm loaded. Upper line (solid, with open squares) is Clq. Lower line (dashed, with open circles) is BSA; Figure 4 shows the inhibition of CUB-domain-Clq interaction by collectins, Clq and Clq collagen tails. Constant levels of radiolabelled CUB-domain were pre- incubated with serial dilutions of unlabelled Clq, Clq tails and collectin proteins. The incubation mixture was bound to, and eluted from, solid phase Clq. Solid line with open squares is Clq; dashed line with diamonds is SP-A; dashed line with open triangles is MBL; solid line with solid circles is SP-D; dashed line with solid squaresis CL43; dashed line with open squares is Clq tails; solid line with open circles is BSA; and Figure 5 shows the inhibition of the classical pathway of complement activation by addition of the recombinant CUB domain of cClqR. Haemolytic assays were used to determine the concentration-dependence of the inhibition of the classical activation pathway of complement by the recombinant CUB domain, the N-domain and the P-domain of cClqR. Y-axis shows % inhibition. X-axis hows μg/ml. Line with solid circles is CUB-domain. Line with solid squares is P-domain. Line with solid hexagons is N-domain. Line with open circles is BSA. EXPERIMENTAL The following experiments demonstrate the activity of the Clq binding domain of cClqR and that the Clq binding domain of cClqR is in fact a CUB and show (see Figure 5) the inhibition of the classical pathway of complement activation by addition of the recombinant CUB domain of cClqR. The sequence listing includes: the DNA sequence (SEQ ID NO: 4) and derived amino acid sequence (SEQ ID NO: 1) of human calreticulin; the DNA sequence (SEQ ID NO: 5) and derived amino acid sequence (SEQ ID NO: 2) of mouse calreticulin; the DNA sequence (SEQ ID NO: 6) and derived amino acid sequence (SEQ ID NO: 3) of rat calreticulin; and the amino acid sequence of CRT (SEQ ID NO: 7). Amino acid sequences were deduced from the nucieotide sequence of human CRT (McCauliffe, D.P. et al., 1990, J. Clin. Invest., 85: 1379-1391.). The signal sequence residues of SEQ ID NO: 7 are residues 1-17. The N-domain (shown in italics) is residues 18-196; the P- (residues 197-308) and C- (residues 309-417) (italics) domains are indicated. The CUB- domain is residues 162-283 (underlined). Domain constructs were expressed as thioredoxin fusion products. Recombinant CRT domains to be tested for Clq and collectin binding function were produced from a cDNA clone (phCRT-1) isolated from a human umbilical vein endothelial cell cDNA library (Stuart, G.R. et al, 1996, Exp. Lung Res.,22: 467-487; Stuart, G.R. et al., 1996, FEBS Lett., 397: 245-249). These domains, as described below, are based upon structural predictions for the molecule and have previously been used to localise CRT function within the molecule. The amino-terminal N-domain contains the binding regions for PDI (Baksh, S. et al, 1995, J. Biol. Chem, 270(52): 31338-31344), Zn 2+ (Baksh, S. et al, 1995, FEBS Lett., 376(1-2): 53-57) and integrins (Leung- Hagesteijn, C.Y. et al., 1994, J. Cell Sci., 107 (Pt 3): 589-600). The proline-rich central P-domain contains the high affinity Ca 2+ binding site (Baksh, S. and Michalak, M., 1991, J. Biol. Chem., 266: 21458-21465) and the lectin site (D. Williams, cited in Krause, K.H. and Michalak, M., 1997, Cell, 88(4): 439-443) within two sets of highly conserved repeats. The acidic C-domain contains the ER-retention terminal KDEL signal (McCauliffe, D.P. et al., 1990, J. Clin. Invest, 85: 1379-1391) and the low affinity Ca 2+ binding site (Baksh, S. and Michalak, M., 1991, J. Biol. Chem., 266,: 21458-21465). Previous studies have indicated that the Clq binding site lies across the intersection of the N and P-domains (Stuart, G.R. et al., 1996, FEBS Lett., 397: 245-249; Stuart, G.R. et al, 1997, Immunopharmacology, 3_8:73-80). Within this region we have identified and expressed a 123 amino acid region containing a putative Clr/Cls (also termed CUB) module (Day, A.J. et al., 1993, Behring Inst. Mitt., 93. : 31-40) based upon amino acid sequence alignments. We termed this segment the CUB-domain and show here that it contains the Clq and collectin-binding site of cClqR/CRT. Purification and Radio iodination of cC I qR, Clq and Collectins Native cClqR was purified from human U937 cells as previously described (Malhotra, R. et al., 1993, Immunology, 78: 341-348). cClqR and CUB-domain samples were iodinated by the Iodogen method (Fraker, P.J. and Speck, J.C. Jr., 1978, Biochem. Biophys. Res. Commun., 80: 849-857). C lq was purified as previously described (Reid, K.B.M., 1981, Methods in Enzymology, .80: 16-25) and radioiodinated as described by Bolton & Hunter (Bolton, A.E. and Hunter, W.M., 1973, Biochem. J., 133: 529-539) as this method of iodination causes less damage to large, oxidation-sensitive molecules such as Clq than the more frequently utilised Iodogen method (Stuart, G.R. et al, 1996, Exp. Lung Res.,22: 467-487). Radiolabelled proteins were stored at 4 °C. Clq collagen tails were prepared as described by Reid (Reid, K.B.M., 1976, Biochem. J., 155: 5-17). Collectins were purified as previously described (Malhotra, R. et al., 1990, J. Exp. Med., 172: 955-959; Holmskov, U. et al., 1995, Biochem. J., 305: 889-896). Prokaryotic expression of recombinant Calreticulin domains Given that CRT, Clr and Cls all interact with Clq, a sequence comparison was performed to investigate the structural basis for this interaction. A region that may correspond to a CUB module was identified in CRT and was analysed by multiple sequence alignment as described previously (Day, A.J. et al., 1993, Behring Inst. Mitt., 93: 31-40). This region, termed the S region (Cls-like (CUB) domain), spans the intersection of the N and P-domains (residues 160-283). A 1.9kb cDNA clone for CRT (phCRT-1) was isolated from a human umbilical vein endothelial cell library in the eukaryotic expression vector CDM8 (Aruffo, A. and Seed, B., 1987, PNAS USA, 84: 8573-8577). Sequence analysis revealed that phCRT-1 comprised the complete coding sequence for CRT with absolute identity to the previously published human CRT sequence (McCauliffe, D.P. et al, 1990, J. Clin. Invest., 85: 1379-1391). The Thiobond expression system was used to produce N, P, C and CUB-domains of cClqR/CRT (representing the N-terminal region, the proline-rich central region, the C- terminal region, and a region spanning the intersection of the N and P-domains (as described above) (Figure 1). The individual domains were expressed as thioredoxin fusion proteins in E. Coli using the plasmid pTrxfus (Invitrogen BV, Leek, Netherlands) as described previously (Stuart, G.R. et al., 1996, FEBS Lett., 397: 245-249; Stuart, G.R. et al, 1997, Immunopharmacology, 3 S:73-80). Samples were assayed for recombinant calreticulin domain expression by SDS-PAGE (Laemmli, U.K., 1970, Nature,227: 680-685) and by Western blotting with rabbit antisera to:(l) whole cClqR (raised against human cClqR purified from U937 cells (Malhotra, R. et al, 1993, Immunology, 78: 341-348); (2) CRT C-terminal region (raised against a GST fusion protein containing the final 18 residues of recombinant human CRT), and (3) CRT N-terminal region (raised against a GST fusion protein containing residues 7-18 of recombinant human CRT). Interaction of immobilised recombinant human cClqR/CRT domains with radiolabelled Clq Binding experiments with the cClqR/CRT domains were performed throughout in low salt (lOmM potassium phosphate, 0.5mM EDTA (pH 7.4)) in order to maximise the ionic interaction with Clq. Microtitre plates were coated with the N-, P-, C- and CUB-domains and with three controls, cClqR, BSA and Thioredoxin, (8mg/ml in 35mM NaHCO3, 15mM Na2CO3 pH 9.6) for 2hr at 37 °C. Non-specific interactions were blocked by incubation with lOmM potassium phosphate, 0.5mMEDTApH 7.4 containing BSA (lOmg/ml). Any free -SH groups in the samples, due to the presence of the thioredoxin fusion protein, were blocked by a brief washing step using the phosphate buffer containing 2mM iodoacetamide. After washing, serial dilutions of radioiodinated Clq (in lOmM . potassium phosphate, 0.5mM EDTA, pH 7.4) were added to the wells and incubated for 2h at 37 °C. Wells were washed three times with phosphate buffer and bound radioactivity eluted with 100ml 4M NaOH and measured. Concentration dependent binding of radiolabelled CUB-domain to immobilised Clq Clq binds to the Fc regions oflgG. This property was utilised in order to correctly orient the Clq on microtitre plates. Breakable microtitre plates (Life Sciences International) were coated with rabbit Fc (5mg per well in 35mM NaHC0 3 , 15mM Na 2 CO 3 pH 9.6) . Non-specific sites were blocked as described above, and the wells were incubated with Clq (5 mg per well in lOmM potassium phosphate buffer). Certain wells were also coated with BSA as a negative control. After further washing, serial dilutions of radioiodinated CUB-domain (in lOmM potassium phosphate, 0.5mM EDTA, pH 7.4) were added to the wells and incubated for 2h at 37 °C. Wells were washed three times with the phosphate buffer and bound radioactivity in the individual wells measured. Competitive inhibition of the CUB-domain-Clq interaction by fluid phase Clq, collectins and Clq collagen tails Clq was immobilised onto Fc-coated microtitre plates as described above. Non-specific binding was blocked by incubation with lOmM potassium phosphate, 0.5mM EDTA (pH 7.4) containing BSA (lOmg/ml). Serial dilutions of the collectins (SP-A, MBL, SP-D, CL43), Clq, Clq tails and BSA (maximum quantity = 9mg/well) were prepared in lOmM potassium phosphate, 0.5mM EDTA (pH 7.4). Each dilution (100ml) was then incubated for lh at 37 °C with a constant level of radiolabelled CUB-domain and loaded onto the plate. Following 2 hours incubation at 37 °C, wells were extensively washed and bound radioactivity measured. Results Amino acid sequence alignments A region spanning the intersection of the N- and P-domains of CRT has previously been implicated in C lq binding (Stuart, G.R. et al, 1996, FEBS Lett., 397: 245-249; Stuart, G.R. et al, 1997, Immunopharmacology, 3£:73-80). Amino acid sequence alignment of a region within CRT (residues 160-283) showed little primary homoiogy to a CUB module, but the region is in fact a CUB module. Two CUB modules, together with an EGF module, form a binding region within Clr2Cls2 for the collagenous tails of Clq. cClqR competes with Clr2Cls2 for binding to Clq, implying a similarity in Clq binding sites on Clr, Cls and cClqR (Sobel, A.T. and Bokisch, V.A., in: Membrane receptors of lymphocytes (M. Seligman, FL Preud'homme, FM Kourilsky eds). North Holland Publishing Co., Amsterdam, pl51, 1975; Van den Berg, R.H. et al, 1995, Eur. J. Immunol, 25(8): 2206-2210). This segment, the CUB-domain (residues 162-283, see Figure 1), was tested for Clq binding. Interaction of recombinant human Calreticulin domains with Clq N-, P-, C-, and CUB-domains of human CRT were expressed as thioredoxin fusion proteins. Correct expression was verified by SDS-PAGE and Western blotting. Figure 2 summarises the results of four separate solid phase direct binding experiments. Significant binding to radioiodinated Clq was observed for cClqR, the CUB-domain, the P-domain, and, to a lesser extent, the N-domain. The C-domain showed no binding. Serial dilutions of radioiodinated CUB-domain were incubated with immobilised Clq and BSA (Figure 3). Concentration-dependent, saturable binding was observed to Clq but not to BSA. Competitive inhibition ofClq-CUB-domain interaction by Clq tails and Collectins. Clq was immobilised on microtitre plates by interaction with solid phase Fc. Figure 4 shows the results of competitive inhibition of the CUB-domain-C 1 q interaction. As expected, native fluid-phase Clq demonstrates concentration-dependent inhibition. Clq tails also cause inhibition, indicating that the interaction of the CUB-domain with Clq is via the collagenous Clq tails. Inhibition studies with the collectin proteins demonstrated that SP-A, MBL and CL43 interact with the CUB-domain, via the same, or an overlapping binding site as Clq. SP-D and BSA did not inhibit the CUB-domain- Clq interaction. Competitive inhibition of Clq mediated complement activation, i.e. classical pathway activation. The ability of cClqR and fragments thereof to bind to Clq and interfere with binding of the Clq associated serine proteases Clr2Cls2 was determined by haemolysis assays. Sheep erythrocytes (E) and rabbit anti-sheep erythrocytes (A) were used to prepare EAClq cells as described by Borsos and Rapp (Borsos,T. and Rapp, H.J.,1967, J.Immunol., 99: 263-268) and de Bracco and Stroud (De Bracco, M.M.and Stroud, R.M„ 1971, J. Clin.Invest.50:838-848). EA and EAClq cells (100ml x 10 8 cells/ml) were incubated at 37 °C for lh with serial dilutions of C lq deficient serum, from a patient with homozygous Clq deficiency, in order to establish the minimum serum concentration required to cause complete cell lysis od EAClq cells. EAClq cells (lθθml)were then incubated for lh at 37 °C with increasing concentrations of the recombinant N-domain, P-domain and the CUB-domain of human cClqR and BSA as a control and after incubation, the extent of lysis established by the addition of cold DGVB ^ (1ml) (Mayer, 1961). After centrifugation (3000g, 10 min) the OD 412 of the supernatant was measured. Contois of 100% lysis comprise 100ml cells and 1.1 ml water. The results of this assay are shown in Figure 5. Hereby, the CUB domain of cClqR was shown to effectively inhibit C 1 q mediated complement activation. At higher concentrations, the P-domain and the N-domain (which contain parts of the CUB-domain) show inhibition at a lesser extent.

Claims: CLAIMS 1. The use of a cC 1 qR binding domain in the manufacture of a medicament to effect CUB domain functionality. 2. The use of a cClqR binding domain according to claim 1, the binding domain forming part of a Clq receptor. 3. The use of a cClqR binding domain according to either one of claims 1 or 2, the cClqR binding domain being recombinant. 4. The use of an inhibitor of the cC 1 qR binding domain in the manufacture of a medicament to inhibit CUB functionality. 5. The use of an inhibitor of the cC 1 qR binding domain according to claim 4, the inhibitor comprising a cClqR binding domain. 6. The use of an inhibitor of the cClqR binding domain according to either one of claims 4 or 5 in the manufacture of a medicament for the treatment of complement activation involved in the initiation and maintenance of inflammation. 7. The use of an inhibitor of the cC lqR binding domain according to claim 6 in the manufacture of a medicament for the treatment of myocardial infarction, brain ischemia (stroke), gut ischemia, rheumatoid arthritis, systemic lupus erythematosus, burns, or immune complex nephritis. 8. The use of an inhibitor of the cC lqR binding domain according to either one of claims 4 or 5 in the manufacture of a medicament for the treatment of amyloid plaques in Alzheimers disease. 9. The use of a cC 1 qR binding domain or inhibitor thereof according to any one of the preceding claims, the cClqR binding domain having the sequence of any one of SEQ ID NOs: 1-3 or a partially modified form thereof. 10. A method of treatment of the human or animal body comprising the use of a cClqR binding domain or an inhibitor thereof according to any one of the preceding claims.

Inventors: SCHWAEBLE WILHELM, SCHWAEBLE, WILHELM
Applicants: SCHWAEBLE WILHELM, UNIV LEICESTER, SCHWAEBLE, WILHELM, UNIVERSITY OF LEICESTER
Application year: 1998
Main classification: A61K38
IPC codes: A61K38, A61K45, A61P9, A61P13, A61P25, A61P29, A61P37, A61P43, C07K14