AU5695200A

AU5695200A - Induction of immune tolerance

Info

Publication number: AU5695200A
Application number: AU56952/00A
Authority: AU
Inventors: David John Roberts; Britta Christina Urban
Original assignee: Oxford University Innovation Ltd
Current assignee: Oxford University Innovation Ltd
Priority date: 1999-06-30
Filing date: 2000-06-30
Publication date: 2001-01-22
Also published as: CA2378477A1; WO2001002005A3; EP1196543A2; WO2001002005A2; GB9915311D0

Description

WO 01/02005 PCT/GB00/02546 INDUCTION OF IMMUNE TOLERANCE The invention relates to the field of immune suppression and, in particular, to the identification 5 of molecules which act as agonists of the cell surface receptors CD36, CD51 and thrombospondin receptors expressed on mammalian dendritic cells and other antigen-presenting cells, to ex vivo and in vivo uses of such molecules for inducing peripheral immune 10 tolerance in mammals, to identification of molecules which inhibit the state of immune tolerance induced in a human by the binding of red blood cells infected with the malarial parasite to dendritic cells and to in vivo uses of such molecules in treating malaria. 15 Dysfunction of the immune system has been shown to play a role in the initial development and further progression of many human diseases. Impaired immune function can result in inability to fight infection or to destroy malignant cells as they develop within the 20 body. Other diseases are caused because the immune system mounts an inappropriate response to a particular antigen. This inappropriate response might be to an external antigen resulting in atopic disease such as hay fever, asthma, eczema, coeliac disease and 25 the like or to the body's own antigens resulting in auto-immune disease. For example both the non-organ specific auto-immune diseases, such as systemic lupus erythromatosis and rheumatoid arthritis and the organ specific auto-immune diseases such as auto-immune 30 haemolytic anaemia and idiopathic thrombocytopenic purpura are associated with an inappropriate T-cell response to self-antigens. Other auto-immune diseases where the antigen has been defined include auto-immune connective tissue 35 syndromes, insulin dependent diabetes mellitus and auto-immune thyroid disease. Diseases where the antigen is less well defined include auto-immune skin WO 01/02005 PCT/GB00/02546 -2 diseases such as eczema, psoriasis, alopecia areata and vitiligo, auto-immune diseases of the gastro intestinal system such as inflammatory bowel disease and auto-immune hepatitis, auto-immune diseases of the 5 nervous system such as multiple sclerosis and myasthenis gravis and auto-immune diseases of the kidney such as glomerulonephritis. In view of the diseases associated with inappropriate immune response, particularly T-cell 10 response, it is highly desirable to develop pharmaceuticals which are able to damp down certain of the body's immune defence mechanisms in order to alleviate the distressing symptoms associated with these diseases. 15 As well as treatment of diseases specifically associated with a mal-function of the immune system, down-modulation of immune mechanisms is desirable in circumstances where a recipient is exposed to allo antigens or xeno-antigens for therapeutic purposes 20 such as recipients of allogeneic or xenogeneic transplants. An allogeneic response in the case of allogeneic bone marrow transplantation or donor lymphocyte infusion might be avoided if one could induce a state of peripheral immune tolerance against 25 donor cells in the recipient. Other examples of situations where down-modulation of immune mechanisms might be desirable include haemolytic disease of the new born, neo-natal allo immune thrombocytopenia or the therapeutic administration of antigenic substances 30 such as blood products e.g. factor VIII, or any other therapeutic or prophylactic agent likely to induce an unwanted cellular immune response. A cellular immune response is mediated by T lymphocytes which are activated by antigen presenting 35 cells, the most important of which are dendritic cells, which present antigen and activate memory T cells and naive T-cells. Dendritic cells become WO 01/02005 PCT/GB00/02546 -3 potent antigen-presenting cells when exposed to an immune stimulus and thereafter are described as "mature". Maturation confers enhanced ability to stimulate T-cells and a reduction in pinocytosis and 5 phagocytosis compared with immature cells. Furthermore, maturation is accompanied by enhanced cell surface expression of HLA Class I and class II molecules as well as adhesion molecules, including CD54 and co-stimulatory molecules such as CD80, CD86 10 and the cell-surface marker CD83. Maturation of dendritic cells is also accompanied by the secretion of cytokines such as TNFa and IL12p70. The secreted cytokines have an autokrine effect on dendritic cell maturation itself and parakrine effects on interacting 15 T-cells. Immature dendritic cells present the cell surface antigens CD36 and CD51 (a,) (part of the vibronectin receptor av0 3 ). CD36 and integrin heterodimers avP3 or aVf 5 can be cross-linked by the soluble bridging 20 molecule thrombospondin (TSP). Through studies of malarial infection the present inventors have discovered that dendritic cell maturation on exposure to an immune stimulus, for example, lipopolysaccharide (LPS), can be inhibited by molecules which bind to 25 CD36 or to CD51 or both via the bridging molecule TSP and which act as agonists thereto. This discovery is based on the inventors' initial observations that red blood cells infected with the material parasite Plasmodium falciparum adhere to 30 dendritic cells via CD36 and/or TSP/CD51 (see Figure 1) and are able to.inhibit the maturation thereof on exposure to LPS. Plasmodium falciparum is one of the most successful human pathogens for which virulence factors 35 remain poorly defined, although adhesion of infected erythrocytes to venular endothelium has been associated with some of the symptoms of severe WO 01/02005 PCT/GB00/02546 -4 disease. Immune responses are unable to prevent symptomatic infections throughout life and immunity to severe disease develops only slowly during childhood. Understanding the obstacles to the development of 5 protective immunity is crucial for rational approaches to prevent the disease. Specific immunity to malaria has been attributed to cytotoxic lymphocytes active against the liver stage of infection or to antibodies reacting against 10 blood stage antigens. Antigenic diversity, clonal antigenic variation and T-cell antagonism may contribute to evasion of the protective and parasiticidal host responses. Furthermore, it is known that Plasmodium 15 falciparum-infected erythrocytes adhere to endothelial cells and it has been widely assumed that this adhesion has evolved to mediate sequestration of parasites to endothelial cells in the peripheral tissues and so reduce their destruction by splenic 20 macrophages. The present inventors have now identified a further mechanism by which the malarial parasite prevents the infected host from mounting an effective immune response and preventing recurrence of the 25 disease. Specifically, the inventors have observed that human erythrocytes which are infected with Plasmodium falciparum are capable of adhering to human dendritic cells and that immature dendritic cells exposed to 30 infected erythrocytes are no longer able to mature into full antigen-presenting cells or to stimulate T cell proliferation, when subsequently exposed to an immune stimulus. However, this state of immune tolerance is not observed when the dendritic cells are 35 exposed to uninfected erythrocytes, uninfected erythrocyte lysate, infected erythrocyte lysate, parasite-conditioned medium or a crude pigment WO 01/02005 PCT/GB00/02546 -5 preparation derived from infected erythrocytes. Further, the effect is not observed when dendritic cells are exposed to erythrocytes infected with a Plasmodium falciparum strain T9/96 which is known not 5 to be able to adhere to endothelial cells (Gardner et al (1996) Proc. Natl. Acad. Sci. USA 93 pp 3503-3508). This particular strain is not able to induce expression on the surface of infected erythrocytes of the parasite-derived protein pf-EMP-1 which is known 10 to undergo clonal antigenic variation and is thought to be the mediator of adherence to endothelial cells. It has been reported that most parasite lines and clones adhere to the known cell-surface receptors CD36 and via TSP to CD51/61 (aIs 3 ). It is also known that 15 pf-EMP-1 can bind to CD36. (see WO 96/33736). The present inventors have now shown that CD36 and CD51 influence the process of dendritic cell maturation and that agonists thereof, including the malarial parasite derived protein pf-EMP-1, antibodies 20 specific for CD36 and CD51, negatively charged phospholipids and apoptotic cells, are able to inhibit dendritic cell maturation in response to an immune stimulus. These agonists reduce the ability of the dendritic cells to stimulate T-cell proliferation in 25 response to an antigen to a level which is lower than cells which have not been exposed to an immune stimulus at all. Thus, agonists of CD36 and CD51 can induce a state of immune tolerance. It follows that agonists of CD36 and CD51 would 30 be useful for the treatment of the types of autoimmune disease described above where an over-reaction of the host immune system is responsible for the symptoms. Further the inventors have found that dendritic cells may be treated by CD36 and/or CD51 agonists in vitro 35 together with an antigen specific to the immune response manifested in the auto-immune disease in question. Thus, tolerance may be induced to a WO 01/02005 PCT/GB00/02546 -6 specific antigen so that, when the dendritic cells are reintroduced into the host, further auto-immune reaction is avoided or substantially reduced. In addition CD36 and CD51 agonists are useful for 5 inducing a state of immune tolerance in both host and donor dendritic cells where bone marrow transplantation or lymphocyte infusion is contemplated. The feasibility of such treatment is demonstrated herein in vivo in mice. The ability to 10 inhibit maturation of dendritic cells can be demonstrated in vitro so that molecules which act as CD36 or CD51 agonists can be easily identified in a high throughput screening assay. As used herein the term "agonist" means a 15 composition, molecule, cell or a component thereof which induces the same response when interacting with a receptor as the naturally-occurring ligand for that receptor. In accordance with a first aspect the invention 20 provides a method of identifying a molecule which is an agonist of cell surface receptor CD36 and/or CD51 as expressed by mammalian dendritic cells which method comprises: 25 a) exposing immature mammalian dendritic cells to the molecule to be tested, b) exposing said immature dendritic cells to an immune stimulus and 30 c) determining the degree of maturation manifested by said dendritic cells, wherein impaired maturation in response to the immune 35 stimulus is an indication that said molecule under test is a CD36 and/or CD51 agonist. Preferably, the method is performed using human WO 01/02005 PCT/GB00/02546 -7 dendritic cells. As used herein the term dendritic cells means cells that present antigen to and activate lymphocytes and which are distinguished by their ability to activate, not only memory T-cells but also 5 naive T-cells. Dendritic cells for use in the method of the invention may be derived by cultivation of adherent peripheral blood mononuclear cells with the addition of Granulocyte-Macrophage Stimulating Factor and Interleukin-4 for about 6 to 10 days. Such 10 dendritic cells can be characterised by their level of expression of the cell-surface markers HLA Class I and II (high), CDll c (high), CD3 and CD19 (negative), CD14 (low) and CD86 (high). These markers distinguish them from B-cells which are positive for CD19, T-cells 15 which are positive for CD3 and macrophages which are CD14 high and CD86 low. (See Banchereau et al, (1998) Nature 392, 245-252). Antibodies to HLA Class I, HLA class II, CD14, CD3, CD19 and CD86 useful for identifying immature dendritic cells are commercially 20 available as indicated in Table 1 below. Dendritic cells which may be used in the method of the invention can also be derived directly from circulating peripheral blood mononuclear cells or by culture of CD34+ stem cells as described by Caux et al 25 (1996) J. Exp. Med. 184:695-706 and Arrishi et al (1999) Blood 93:2244-2256. There are various ways in which maturation of dendritic cells in response to an immune stimulus, may be measured. On maturation the dendritic cells become 30 potent antigen presenting cells. As aforesaid maturation is accompanied by enhanced cell surface expression of HLA Class I and II molecules such as HLA DR, adhesion molecules such as CD54 and co-stimulatory molecules such as CD40, CD80, CD86 and CD83 which is a 35 specific marker for mature dendritic cells. Thus, examination of the cell's antigen presenting ability, for example variety of antigens and/or level of WO 01/02005 PCT/GB00/02546 -8 expression, is one way of determining whether maturation has occurred or whether it has been inhibited by the test molecule. Preferably, following immune stimulation, the level of expression of the HLA 5 Class I and II molecules and/or adhesion molecules and/or co-stimulatory molecules is measured. In one embodiment maturation of dendritic cells is detected by measurement of the level of expression of two or more of the cell-surface antigens HLA DR, CD54, CD40, 10 CD83 and CD86 whose level of expression is particularly enhanced. Preferably, the level of expression of all of the above in response to an immune stimulus is measured. Optionally the expression level of CD80 may also be measured. 15 Methods by which the expression of a cell-surface antigen may be quantified are well-known to those skilled in the art. The commonly used method is to apply an antibody specific for the antigen in question to the antigen-presenting cells which has been 20 labelled to give a quantifiable detectable signal. Suitable labels are well-known to those skilled in the art and include radioactive labels, enzyme labels, fluorescent labels, metallic particles and the like. Antibodies suitable for carrying out the screening 25 method of the present invention, as well as a commercial source, are shown in Table 1 below: 30 35 WO 01/02005 PCT/GB00/02546 -9 TABLE 1 5 Antigen Antibody Source HLA DR BF-1 Serotec HAL Class 1 W32/6 ATCC HB-95 10 CD14 Tuk4 DAKO CD54 6.5B5 DAKO CD40 LOB7/6 Serotec CD80 BB1 or DAL 1 Serotec CD83 HB15a Serotec 15 CD86 BU63 Serotec CD3 OKT3 ATCC CRL-8001 CD19 HD37 DAKO CD36 clone 89 Serotec clone SMf Immunocontakttec 20 Serotec: 22 Bankside, Station Approach, Kidlington, Oxford, UK DAKO Ltd: 16 Manor Courtyard, Hughenden Avenue, High Wycombe, Bucks HP13 5RE Immunokontakt: Centro Nord-Sud, CH-6934 Bioggio, 25 Switzerland, Peprotec: 23 St. James Square, London SW9Y 4JH, UK, ATCC: 10801 University Boulevard, Manassas, VA 20110-2209; USA, Sigma: Sigma Alderich Company Ltd: Fancy Road, Poole, Dorset, BH12 4QH, UK, Schering-Plough: Schering-plough House, Shire Park, 30 Welwyn Garden City, Herts, AL7 1TW. As an alternative to measuring the level of cell surface antigen to determine whether or not dendritic cell maturation has occurred, it is possible to measure the cell's ability to induce T-cell 35 proliferation. This is inhibited by agonists of CD36 or CD51. Dendritic cells which have been exposed to the molecule to be tested and to an immune stimulus WO 01/02005 PCT/GB00/02546 - 10 may be exposed to T-cells, for example allogeneic lymphocytes in a mixed lymphocyte reaction (MLR)with the T-cell receptor. The T-cells respond by growing and dividing, something which can easily be measured 5 using methods well-known to one skilled in the art. For example, growth and division can be assessed visually using a light microscope to observe clumps of dividing cells. Alternatively, cell proliferation can be quantified using a suitably labelled metabolite, 10 for example tritiated thymidine, which is incorporated into the cell's DNA. A yet further alternative for determining the degree of dendritic cell maturation is to measure the level of secretion of cytokines such as TNFa, IL2p7O 15 or IL10. For example IL12p70 is secreted by mature cells but not by immature cells. The level of TNFa secretion is reduced in immature as opposed to mature cells. Kits are commercially available for detection 20 and quantitation of all of the above cytokines. (see Examples). Preferably, the levels of TNFa, IL12p70 and IL10 secretion are measured. In the screening method of the invention a variety of immune stimuli may be used. Suitable 25 examples are lipopolysaccharide (available from Sigma), TNFaY (available from Peprotec) and monocyte conditioned medium (MCM)the preparation of which is described by Romani et al (1996) J. Immunol. Methods, Sep 27; 196(2):137-51. Another suitable 30 immune stimulant is CD40L which is expressed from plasmids having the ATCC Accession No's 79812,79813,79814 or 79815. The plasmids may be expressed in mouse fibroblasts STO (ATCC-CRL-1503). It will be understood that TNFa would not be used 35 as both immune stimulant and indicator of cell maturation in the same assay. In a particular embodiment of the method of the WO 01/02005 PCT/GB00/02546 - 11 invention immature dendritic cells (about 106) are exposed in duplicate to various concentrations of the test molecule for about 3 to about 12 hours in a multiwell plate. The test compound is prepared in a 5 suitable diluent which is not toxic to the dendritic cells such as tissue culture medium, PBS, water or a suitable non-toxic organic solvent, if appropriate. The duplicate wells are subsequently exposed to LPS (about 500 ng/ml) or left untreated for about 48 10 hours. For each concentration of the compound and time of exposure, the surface expression of the molecules identified above is compared with the surface expression on immature dendritic cells exposed to the test compound as well as untreated immature 15 dendritic cells. The increase in cell surface expression is evaluated using indirect immunofluorescence and FACScan analysis. A compound is a candidate for further evaluation if the surface expression on dendritic cells of at least two cell 20 surface antigens is not increased by addition of the immune stimulant, LPS. Preferably, molecules identified as potential CD36 or CD5l agonists by the method of the invention will be subject to further evaluation. For example, 25 if surface expression of lineage-specific molecules has been used to determine the degree of maturation it would be usual to check whether the compound can also prevent immune-stimulated dendritic cells from inducing proliferation of T-cells and visa versa. The 30 ability of the molecule to vary cytokine secretion could also be tested. In addition direct binding of the candidate molecule to CD36, CD51 or TPS should also be confirmed. This latter confirmation may be easily obtained by applying a sample of the candidate 35 molecule to a purified sample of CD36, CD51 or TPS. Purified CD36 may be prepared as described by Tandon et al (1989) The Journal of Biological Chemistry, 264 WO 01/02005 PCT/GB00/02546 - 12 pp 7570-7575. Purified CD51 may be prepared as described by Smith et al, (1990), Journal of Biological Chemistry, 265, 11008-11013 and purified TSP may be prepared as described by Silverstein et al 5 (1985), Journal of Clinical Investigation, 75, pp 2065-2073. Tests to detect binding of the test molecule are conveniently carried out by immobilizing the CD36, CD51 or TSP to a solid surface, for example the 10 surface of a well of a microtitre plate. Methods of immobilization of protein molecules on such surfaces are well-known to those skilled in the art. The test molecule identified as a CD36 or CD51 agonist is then applied to the immobilized protein. Following removal 15 of unbound test molecule the presence of bound molecule is directly detected. This may be achieved in a number of ways depending on the chemical or biochemical characteristics of the test molecule. For example where the test molecule is a protein 20 it would be usual to detect binding with a labelled antibody to that protein. If the test molecule is a non-antigenic small molecular weight compound then the compound itself may be radioactively labelled for detection. 25 The molecule whose activity is to be tested in the method of the invention .may have any type of molecular structure. For example, it may be a protein, a peptide, an amino acid, DNA, RNA, PNA, a nucleotide or a nucleoside, or a low molecular weight 30 compound. It may be a molecule having known pharmacological or biochemical activity or a molecule with no such known activity and may be a novel molecule. The method of the invention is suitable for testing entire libraries of molecules, for example 35 libraries such as would be created by combinatorial chemistry. Indeed, all the embodiments of the screening method above may be adapted for an automated WO 01/02005 PCT/GB00/02546 - 13 high throughput compound screen. Using the method of the invention the present inventors are able to confirm that the Plasmodium falciparum derived protein pf-EMP-1 is an agonist of 5 both CD36 and CD51. In particular a fragment of pf EMP-1 known as CIDR/A4 which comprises the CD36 binding domain is an agonist of CD36. CIDR/A4 is described by Smith et al (1998) Molecular and Biochemical Parasitology, 97, pp 133-148 and comprises 10 amino acids 402 to 846 of pf-EMP-1 as shown in Figure 2. Antibodies which bind CD36 and CD51 have also been identified as having agonist activity and are capable of inhibiting the maturation of dendritic .15 cells. Thrombospondin is also an agonist of CD51. The present invention is also directed to any individual molecule identified as an agonist of CD36 or CD51 by the methods described herein. The assays of the invention have allowed the 20 inventors to make the further observation that apoptotic cells, the natural ligand of CD36, are also able to inhibit dendritic cell maturation in response to LPS. This is yet further evidence of the role of CD36 in modulating immune response. 25 In accordance with a second aspect the invention provides a pharmaceutical composition suitable for inducing immune tolerance in a mammal which comprises an agonist of the cell surface receptor CD36 and a pharmacologically acceptable carrier or diluent. The 30 CD36 agonist may be a molecule identified by the method described above. Agonists which are suitable for incorporation into a pharmaceutical composition in accordance with the invention for the treatment of humans include antibodies with an affinity for an 35 epitope of CD36, in particular an antibody which blocks the binding domain on CD36 for pf-EMP-1. Monoclonal antibodies specific for CD36 which are WO 01/02005 PCT/GB00/02546 - 14 designated "clone 89" and "clone SMG" and which are commercially available from Serotech or Immunocontact(details above) are suitable for use in the pharmaceutical compositions of the invention. 5 Other commercially available CD36 antibodies which may be included in pharmaceutical compositions are listed in Appendix 1. It is contemplated that compositions comprising antibodies bispecific against CD36 and CD51 will be useful for inhibiting dendritic cell 10 maturation. Other agonists suitable for inclusion in pharmaceutical compositions are all variants of the Plasmodum falciparium pf-EMP-1 or fragments of such proteins which comprise the binding domain for CD36. 15 A particular example is the fragment CIDR/A4 described herein comprising amino acids 402 to 846 of pf-EMP-1. (Figure 2). Pharmaceutical compositions comprising a bispecific CD36 antibody and the CIDR/A4 fragment are 20 also contemplated in accordance with the invention. Yet another agonist suitable for inclusion in a pharmaceutical composition are negatively charged phospholipids such as phosphatidylserine containing liposomes which have also been shown to bind to CD36 25 and other cellular receptors of immune cells. Yet another agonist suitable for inclusion in a pharmaceutical composition are apoptotic cells. In a third of its aspects the invention provides a pharmaceutical composition suitable for inducing 30 peripheral immune tolerance in a mammal which comprises an agonist of the cell surface receptor CD51 as expressed by mammalian dendritic cells and a pharmacologically acceptable carrier or diluent. As with CD36 acceptable agonists are antibodies, 35 preferably monoclonal antibodies, directed against an epitope of CD51. Particularly suitable are antibodies blocking the binding domain of CD51 for the bridging SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 15 molecule TSP. Antibodies suitable for incorporation in a pharmaceutical composition in accordance with this aspect of the invention are commercially available and set out in Appendix 2. 5 Thrombospondin (TSP) is also suitable for incorporation into a pharmaceutical composition as a CD51 agonist. Preferably, such compositions also include the Plasmodium falciparum protein pf-EMP-1 or a fragment thereof incorporating the thrombospondin 10 binding domain of pf-EMP-1. As with CD36, negatively charged phospholipids such as phosphatidylserine are also suitable for incorporation as CD51 agonists in pharmaceutical compositions of the invention as well as apoptotic 15 cells. Pharmaceutical compositions in accordance with the second and third aspects of the invention are useful for the treatment of autoimmune diseases associated with inappropriate dendritic cell 20 maturation and T-cell proliferation such as systemic lupus erythromatosis, rheumatoid arthritis, autoimmune haemolytic anaemia or idiopathic thrombocytopenic purpura. Vehicles suitable for delivery of pharmaceutically active substances are known to those 25 skilled in the art, especially those for delivery of pharmaceutically active proteins. In accordance with a fourth aspect of the invention there is provided a method of treating mammalian dendritic cells in vitro to induce immune 30 tolerance therein which comprises exposing said cells to an agonist of cell surface receptors CD36 and/or CD51 as expressed on mammalian dendritic cells. The invention also relates to preparations of cells so treated. Suitable agonists are any of those agonists 35 described above or any molecule or substance identified by the screening method described herein. Treatment of dendritic cells ex-vivo with an WO 01/02005 PCT/GB00/02546 - 16 agonist of CD36 and/or CD51 is beneficial in many therapeutic applications as described hereinafter. For example, in the case of bone marrow transplantation or lymphocyte infusion recipient cells 5 removed from the body are treated with agonists as described above to induce a state of immune tolerance therein. The treated cells are then re-introduced to the body before or simultaneously with the donor cells and the risk of allogeneic reaction is thereby reduced 10 or eliminated. It is contemplated that dendritic cells of the donor may also be treated with a CD36 and/or CD51 agonist to induce immune tolerance. The donor may be allogeneic or xenogeneic. The present inventors have demonstrated that in 15 mice tolerance to foreign antigens can be achieved by exposure of dendritic cells from one mouse strain, ex vivo, to a CD51 agonist followed by introduction of the treated cells into another strain of mice. Thus such therapy is expected to be applicable to humans. 20 In addition the present inventors have shown using a fragment of the a-subunit of the human acetylcholine receptor that immature dendritic cells treated with a CD36 agonist can be "modulated" to induce tolerance against a specific antigen by 25 subsequent ex-vivo exposure to that antigen. Once reintroduced in vivo, immune response to that antigen is reduced or avoided. Thus, dendritic cells may be removed from a patient suffering from an auto-immune disease, for example, and exposed to a CD36 and/or 30 CD51 agonist and an antigenic molecule associated with the auto-immune disease in question and the dendritic cell preparation, with or without maturation, reintroduced into the patient. Alternatively, this method may be used to induce tolerance to a particular 35 allo or xeno-antigen or other therapeutic substance which is likely to induce an unwanted immune response, WO 01/02005 PCT/GB00/02546 - 17 such as a blood product like factor VIII. Thus, the invention includes preparations of dendritic cells tailored to the treatment of a particular auto-immune disease by exposure to an 5 agonist of CD36 and/or CD51 and the specific auto antigen associated with the disease and cell preparations tolerant to other antigens likely to generate an unwanted immune response. It follows from the inventor's observations 10 concerning inhibition of maturation of dendritic cells with agonists of CD36 and CD51 that a similar effect will be observed with agonists of thrombospondin receptors in general and with other antigen-presenting cells of the immune system which also express CD36 and 15 CD51 and thrombospondin receptors such as macrophages, B-lymphocytes and monocytes. Thus, in accordance with a fifth aspect the invention provides a method of identifying a molecule which is an agonist of cell surface receptors CD36 20 and/or CD51 and/or a thrombospondin receptor as expressed on antigen-presenting cells of the mammalian immune system which method comprises: a) exposing mammalian antigen-presenting cells to the 25 molecule to be tested, b) exposing said cells to an immune stimulus and c) determining the response to said immune stimulus by 30 said cells, wherein an impaired response compared to the response in the absence of said test molecule is an indication that said molecule under test is a CD36 and/or CD51 35 agonist or an agonist of a thrombospondin receptor. Preferably, the response that is measured is maturation of said antigen presenting cell. Such a WO 01/02005 PCT/GB00/02546 - 18 screening method may be carried out using the general methodology already described herein for dendritic cells. For example, monocytes can be purified from peripheral blood by adherence of PBMC to plastic 5 dishes. Non-adherent cells are removed and the adherent cells can be detached by incubation with EDTA in PBS. Contaminating lymphocytes are depleted with the aid of magnetic heads and antiCD3 and antiCD19 monoclonal antibodies. Macrophages may be generated 10 by culturing monocytes, which have been isolated as described above, in RPMI 1640 supplemented with M-CSF for six days. p-lyphocytes can be isolated from blood by virtue of their non-adherence to plastic petri dishes. The non-adherent cells are subjected to 15 depletion of contaminating monocytes and T-cells by exposure to magnetic heads and antiCD14 and antiCD3 monoclonal antibodies. Once isolated the antigen presenting cells are exposed to a substance to be tested for agonist 20 activity against CD36, CD51 or a thrombospondin receptor and the degree of activation of said cells is measured. As with dendritic cells, activation may be determined by measuring the levels of secretion of various cytokines, or by testing ability of said 25 antigen presenting cells to stimulate T-cell proliferation. In a preferred embodiment the increased expression of certain cell surface receptors is used as a measure of activation. In the case of monocytes and macrophages activation is accompanied by 30 an increase in surface expression of HLA-DR, CD54 and CD86 which is measured in the manner described above, preferably with the use of a monoclonal antibodies to HLA-DR, CD54 and CD86. B-cell activation is determined by measuring the level of cell surface 35 expression of HLA-DR, CD86 and CD40. The expression may be detected using antibodies to these cell surface receptors. Such as clone BF1, Serotech (HLA DR) clone WO 01/02005 PCT/GB00/02546 - 19 BU63, Serotech (CD86) and clone TRAP1 (Pharmingen) (CD40). The invention also further relates to uses of an agonist as identified above using said antigen 5 presenting cells for treatment of any of the auto immune diseases listed above and for inducing immune tolerance in said antigen presenting cells ex-vivo as well as to antigen-presenting cell preparations which have been treated with a CD36 and/or CD51 agonist 10 and/or thrombospondin receptor agonist and optionally an antigenic material. The invention also relates to pharmaceutical compositions comprising an agonist of a thrombospondin receptor, for example av 3 or av, 5 , with a 15 pharmaceutically acceptable carrier or diluent suitable agonists include antibodies to the thrombospondin binding domain of said receptor, for example any of the antibodies listed in Appendix 3. Other suitable agonists include negatively 20 charged phospholipids such as phosphatidylserine containing liposomes. It is a further conclusion from the work of the present inventors that agonists of P-integrin associated with the cell surface receptor CD51 as 25 expressed on the surface of antigen-presenting cells of the mammalian immune system, will also be useful for inducing immune tolerance. The invention in a sixth aspect thus, further relates to methods of identifying P-integrin agonists by any of the 30 procedures described above and to uses of P-integrin agonists, as defined above, for any of the medical uses which are described herein. In a seventh aspect the present invention further relates to uses of apoptotic cells as a medicament for 35 inducing immune tolerance in antigen-presenting cells, preferably dendritic cells and to pharmaceutical compositions comprising those cells in a suitable WO 01/02005 PCT/GB00/02546 - 20 carrier or diluent. Apoptotic cells are suitable for delivering tissue specific antigens including major and minor histocompatibility antigens to dendritic or other antigen-presenting cells. Delivering antigens 5 in this way allows delivery of unknown antigens or antigens where the class II restricted epitope(s) are not defined. The tissue origin of the apoptotic cell may be varied depending upon the application. For example, it is preferred for the apoptotic cell to be 10 of the same tissue type as any cell bearing an antigen to which tolerance is to be induced. In accordance with an eighth aspect the invention further relates to the use of negatively charged phospholipids for inducing immune tolerance in antigen 15 presenting cells. Said immune tolerance may be induced by treatment of said antigen presenting cells, for example dendritic cells, with said negatively charged phospholipid either ex-vivo by the methods described herein or by administration of the 20 phospholipid to a patient by any of the conventional administration routes known to those skilled in the art. A preferred form of composition is liposomes comprising the negatively charged phospholipid. A preferred phospholipid is phosphatidylserine. 25 Since the inventions of the present application were developed following the basic observation that Plasmodium falciparum infected erythrocytes adhere to dendritic cells and inhibit the maturation thereof it follows that molecules which block or inhibit such 30 adherence may be useful as pharmaceuticals in the clinical management of malaria, in particular molecules which inhibit adherence of parasite-infected erythrocytes to CD36 or TSP. Thus, in accordance with a ninth aspect of the 35 invention a method comprising the following steps is used to identify a molecule capable of preventing adherence of erythrocytes infected with a malarial WO 01/02005 PCT/GB00/02546 - 21 parasite to human dendritic cells: (a) exposing a purified preparation of CD36 or TSP to: (i) the molecule'to be tested and 5 (ii) parasitsed human erythrocytes either consecutively or simultaneously and (b) determining the level of adherence of said 10 parasitised erythrocytes to said CD36 or TSP wherein a reduction in the level of adherence to CD36 or TSP in the presence of the test molecule compared to the level of adherence in the absence of said test 15 molecule is an indication that said molecule is capable of preventing the adherence of erythrocytes infected with the malarial parasite to human dendritic cells. The erythrocytes may be infected with Plasmodium 20 falciparum or another Plasmodium species. Suitable falciparum strains include ITO/A4 or ITO/C24 which may be derived as described by Roberts et al (1992) Nature 357 pp 689-692 or Malayan Camp (MC) which may be obtained as described by Roberts et al (1985) Nature 25 318:64-66. A suitable format for carrying out a screening method as described above is to immobilize the purified CD36 or TSP onto a solid surface. Preferably, immobilization is secured by adsorption of 30 the protein molecules to a plastic surface such as a petri dish. Parasitised erythrocytes suspended in a suitable binding medium are added to the adsorbed CD36 or TSP and incubated for a period sufficient to allow adherence, for example, about 1 hour. Thereafter the 35 binding medium and any non-adhered erythrocytes are removed and a suitable erythrocyte stain for example, Giemsa, added to the petri dish. Adhered erythrocytes WO 01/02005 PCT/GB00/02546 - 22 may be quantified by counting under a light microscope. Alternatively, depending on the stain used, erythrocyte adherence may be quantified by spectrometry, fluorescence microcopy and the like. 5 In a tenth aspect the invention provides a method of identifying a molecule capable of preventing the adherence of red blood cells infected with a malarial parasite to human dendritic cells which comprises: 10 a) exposing immature human dendritic cells to the Plasmodium falciparum protein pf-EMP-1 or an active binding domain thereof in the presence or absence of the molecule to be tested, 15 b) exposing said immature dendritic cells to an immune stimulus and c) determining the degree of maturation manifested by said dendritic cells, 20 wherein any maturation of said dendritic cells in the presence of the test molecule over and above that manifested in the absence of said molecule is an indication that said molecule is capable of preventing 25 adherence of red blood cells infected with a malarial parasite to human dendritic cells. Maturation of dendritic cells may be measured by any of the methods already described herein. Suitable immune stimulants include LPS, TNFa, CD40L and 30 monocyte conditioned medium (MCM). Preferably the pf EMP-1 preparation for use in the method is that designated in pf-EMP-1 A4var as described by Smith et al (see before) and having the Genbank Accession No. L42244. The fragment CIDR/A4 may also be used. 35 In a further aspect the invention provides for use of molecules identified by the aforementioned methods which inhibit infected erythrocyte adherence WO 01/02005 PCT/GB00/02546 - 23 to dendritic cells in pharmaceutical compositions for the treatment of malarial infection. Based on the present inventors' observations it is further contemplated that a modified CIDR region of 5 the pf-EMP-1 A4 variant protein could be incorporated in a multisubunit vaccine against falciparum malaria. This would induce blocking antibodies against the CD36 binding domain of pf-EMP-1 variant proteins so that the immune responses against other proteins are not 10 inhibited. All documents cited in this application are incorporated herein by reference. The invention will now be further described with reference to the following Figures and Examples. 15 FIGURE 1 shows schematically the molecular basis for the binding of Plasmodium falciparum infected red blood cells to CD36 and TSP on the surface of dendritic cells; 20 FIGURE 2 shows the amino acid sequence of the pf-EMP-1 fragment CIDR/A4; FIGURE 3 shows the increase in surface expression of 25 dendritic cell marker antigens HLA DR, CD54, CD40, CD80, CD83 and CD86 following immune stimulation after exposure to (a) LPS matured dendritic cells, (b) dendritic cells matured with LPS, with and without prior exposure to RBC, (c) dendritic cells matured 30 with LPS with and without prior exposure to parasite lysate and (d) dendritic cells matured with LPS with and without prior exposure to intact ITO/A4 infected RBC; 35 FIGURE 4; (A) shows the absolute binding of erythrocytes infected with parasite lines ITO/A4, ITO/C24, MC and T9/96 to CD54, CD56, and TSP (a,c,e,g) WO 01/02005 PCT/GB00/02546 - 24 and (B) shows the increase in surface expression of LPS matured dendritic cells compared with dendritic cells exposed to the respective parasite line prior to maturation (b,d,f,h); 5 FIGURE 5 shows transmission electron micrographs illustrating the interaction of dendritic cells with (a) ITO/A4 infected erythrocytes and (d) non-adherent T9/96 infected erythrocytes; 10 FIGURE 6 shows dendritic cell stimulation of T-cell proliferation (a) induced by immature dendritic cells (M), LPS-matured dendritic cells (0) and dendritic cells co-cultivated with intact ITO/A4 infected 15 erythrocytes (V) prior to maturation, primary CD4+ T cell responses to parasite lysate (b) and to keyhole limpit haemocyanin (c) induced by LPS-matured autologous dendritic cells (EI,O) and autologous dendritic cells co-cultivated with intact ITO/A4 20 infected erythrocytes (E,S)prior to maturation; FIGURE 7 shows the effect of monoclonal antibodies against CD36 and CD51 on maturation of dendritic cells represented graphically as relative increase in 25 surface expression of dendritic cells matured with LPS compared with immature dendritic cells; FIGURE 8 shows the effect of monoclonal antibodies against CD36 and CD51 on dendritic cell maturation as 30 a FACscan output; FIGURE 9 shows further results of experiments with apoptotic cells (a) output of FACscan, (b) staining with potassium iodide to exclude dead cells, (c) 35 proliferation of allogenic T-cells stimulated by increasing numbers of immature dendritic cells, (+)LPS matured dendritic cells (A) or dendritic cells WO 01/02005 PCT/GB00/02546 - 25 exposed to apoptotic dendritic cells and then matured with LPS(N). FIGURE 10 shows the effect of apoptotic neutrophils on 5 the maturation of dendritic cells; FIGURE 11 shows results of a T-cell proliferation assay including antigen specific T-cell proliferation. (a) Proliferation of allogeneic T-cells. (b) 10 proliferation of KLH specific CD4+CD45RO- autologous T-cells (c, d) proliferation of the T-cell clone TB-2 specific for the human Acetylcholine Receptor a subunit in response to polypeptide (c) or peptide (d). Stimulator dendritic cells were treated as follows: 15 immature DC alone (X) or matured with LPS (0) ; dendritic cells exposed to irrelevant antibodies with (0) or without (0) antigen and then matured with LPS; dendritic cells exposed to antiCD36 antibody with (v) or without (V) antigen and then matured with LPS; 20 dendritic cells exposed to antiCD51 antibody with (A) or without (A) antigen and then matured with LPS; dendritic cells exposed to antiCD36 and antiCD51 antibody with (0) or without (*) antigen and then matured with LPS. 25 FIGURE 12 shows secretion of cytokines TNFa, IL12p70 and IL10 by dendritic cells exposed to an antiCD36 antibody- or to apoptotic dendritic cells and respective controls; 30 FIGURE 13 shows in vitro maturation of mouse dendritic cells following exposure to an antiCD51 antibody; and FIGURE 14 shows results from mouse popliteal lymph 35 node assay. EXAMPLE 1 WO 01/02005 PCT/GB00/02546 - 26 Generation of antigen-presenting cells (a) Dendritic cells Immature dendritic cells were derived from peripheral 5 human blood cells using standard procedures as described by Sallusto et al (1995) J. Exp. Med. 182 pp 389-400. Briefly, monocytes were cultivated in RPMI 1640 supplemented with 2mM Glutamine,50 pg/ml Kanamycin, 1% nonessential amino acids (GibcoBRL), 10% 10 human AB serum and 50 ng/ml of each IL-4 (specific activity >2x10 6 U/mg, PeproTech) and GM-CSF (specific activity > 1x10 7 U/mg, Schering-Plough) for 6 days. Between day six and day nine of the culture non adherent immature dendritic cells were harvested and 15 purified by depletion of contaminating lymphocytes with the aid of magnetic beads (Dynal) and anti-CD3 and anti-CD19 monoclonal antibodies (DAKO). (b) Monocytes 20 Monocytes were purified from peripheral blood by adherence of PBMC to plastic dishes for 2 hours. Non adherent cells were removed and the adherent cells layer washed 2 times with warm PBS. For further 25 purification, the adherent cells were detached by incubation with 2 mM EDTA in PBS for 20 min and contaminating lymphocytes depleted with the aid of magnetic beads (Dynal or Miltenyi) and anti-CD3 and anti-CD19 monoclonal antibodies (DAKO). 30 (c) Macrophages Monocytes isolated as described above were cultured in RPMI 1640 supplemented with 2 mM Glutamine, 50 pg/ ml 35 Kanamycin, 10% human AB serum and 50 ng/ml of M-CSF (specific activity > 2 x 106 U/mg, Peprotech) for 6 WO 01/02005 PCT/GB00/02546 - 27 days. (d) B-lymphocytes 5 B-lymphocytes were isolated from human blood according to standard procedures. Briefly, non-adherent PBMC were subjected to depletion of contaminating monocytes and T-cells with the aid of magnetic beads (Dynal or Miltenyi) and anti-CD14 and anti-CD3 monoclonal 10 antibodies (DAKO). B-cells were cultured in RPMI 1640 supplemented with 2 mM Glutamine, 50 pg/ ml Kanamycin, 1% to 10% human AB serum. (e) CD34+ Cells 15 CD34+ cells were isolated from PBMC with the aid of anti-CD34 antibody conjugated magnetic beads (Dynal or Miltenyi). CD34+ progenitor were then cultured in RPMI 1640 supplemented with 2 mM Glutamine, 50 pg/ ml 20 Kanamycin, 1% to 10% human AB serum and the following cytokines: 100 ng/ml of GM-CSF (Schering-Plough), 50 ng/ml TNFa and 50 ng/ml SCF (Peprotech) for 12 days. As an alternative, CD34+ cells could be expanded in the above mentioned medium but supplemented with 25 25 ng/ml FLT3-L , 10U/ml TPO, SCF 20 ng/ml (Peprotech) for up to 8 weeks and then induced to differentiate to dendritic cell by culture of a further 3 days in medium supplemented with 25 ng/ml GM-CSF and 25 ng/ml IL-4." 30 EXAMPLE 2 Maturation assay 35 (a) For maturation assays 1x10 6 purified dendritic cells were incubated in duplicate wells (a) with WO 01/02005 PCT/GB00/02546 - 28 100 ng/ml LPS, (b) with 100ng/ml LPS with or without prior exposure to 1x10 8 RBC, (c) with 100 ng/ml LPS with or without prior exposure to parasite lysate corresponding to 1x10 8 parasite infected RBC, (d) 100 5 ng/ml LPS with or without prior exposure to 1x10 8 intact ITO/A4 infected RBCs. Incubation with LPS (Salmonella typhimurium) was for a period of 48 hours. Maturation of the dendritic cells was measured using 10 monoclonal antibodies to the following human cell surface markers: CD3 clone OKT3, HLA A,B,C clone W32/6, CD14 clone Tuk4, CD54 clone 6.5B5, CD19 clone HD37 (DAKO): CD36 clone 89 (IgG1) or clone SMQ(IgM), CD80 clone BB1, CD40 clone LOB7/6, CD86 clone BU63, 15 HLA DR clone BF-1 (Serotec), CD83 clone HB15a (Zhou et al (1995) J. Imm. 154, pp3821-3835. .Staining of dendritic cells was performed as described by Zhou et al above and immunofluorescence analysed by FACScan (Becton Dickenson). All experiments were repeated at 20 least six times with dendritic cells obtained from different donors. Dead cells were excluded from analysis using Propidium Iodide. The results are shown on Figure 3. The relative increase of surface expression is expressed as the mean fluorescence 25 intensity (MFI) of matured dendritic cells over the MFI on immature dendritic cells. The results show that dendritic cell maturation is inhibited by the direct interaction with intact 30 infected erythrocytes and is not due to the secretion of inhibitory parasite products or a toxic effect of parasite debris. The differences in surface expression on dendritic 35 cells exposed to intact infected erythrocytes to dendritic cells alone are statistically significant for all markers with p<0.01 (Student t-test).

WO 01/02005 PCT/GB00/02546 - 29 (b) Other antigen-presenting cells For maturation assays 1 x 10' purified monocytes or macrophages were incubated in duplicate wells with or 5 without LPS in the presence of medium alone, antibodies such as anti-CD36 or anti-CD51 or control antibodies, apoptotic cells or necrotic cells. Maturation was measured by the increase in surface expression of HLA-DR, CD54 and CD86 using the 10 antibodies and flow cytometry as described above. For maturation assays 1 x 106 purified B-cells were incubated in duplicate wells with or without LPS in the presence of medium alone, antibodies such as anti 15 CD36 or anti CD51 or control antibodies, apoptotic cells or necrotic cells. Activation was measured by the increase in surface expression of HLA DR (clone BF-1, Serotec), CD86 (clone BU63, Serotec) and CD40 Ligand (clone TRAP1, Pharmingen) and flow cytometry. 20 EXAMPLE 3 Cultivation of Plasmodium falciparum infected red blood cells 25 Laboratory strains of Plasmodium falciparum were cultured in human RBC as described by Trager et al (1976) Science. 193 pp673 to 675. The cytoadherent cell lines ITO/A4 and ITO/C24 were clones isolated by 30 manipulation from the IT04 line, which is derived from a parasite isolate from Ituxi in Brazil. The cytoadherent parasite line Malayan Camp (MC) and the non-adherent cell line T9/96 were both adapted to in vitro culture from parasites originally isolated from 35 Thailand., All cultures were free from mycoplasma contamination. Infected erythrocytes were purified WO 01/02005 PCT/GB00/02546 - 30 either by differential sedimentation in Plasmagel or through 65% Percoll both of which gave a yield of more than 90% infected erythrocytes. Examination of a thin film revealed that more than 90% of infected 5 erythrocyes were viable. Parasite lysate was obtained by three rounds of freezing and thawing of mature infected RBC. Parasite pigment was prepared as described by Schwarzer et al (1994) BR. J. Haematol. 88, pp 740

-

745 . Parasite conditioned medium was the 10 supernatant derived after culturing 1x10 8 purified infected erythrocytes in dendritic cell medium for 24 hours. All materials were from Sigma unless otherwise stated. 15 EXAMPLE 4 Binding of parasites to purified proteins Binding of parasitised RBCs to purified proteins was 20 measured as previously described by Craig et al (1997) Infect. Immun. 65, pp 4580-4585. Briefly, two microlitres of a solution of TSP (Gibco-BRL), purified CD36 or purified CD54 (ICAM-Fc) were adsorbed onto bacteriological, plastic plates. Mature erythrocytes 25 parasitised with P. falciparum strains (a) ITO/A4, (c) ITO/C24, (e) MC and (g) T9/96, were suspended in binding medium and added to each dish. The erythrocytes were allowed to settle and then resuspended by gentle rotation every 10 minutes for 1 30 hour. Non-adherent cells were removed, the remaining cells fixed and stained with Giemsa. Adherent parasitised cells were counted by light microscopy and the number of cells bound per square millimeter were corrected to binding at 2% haematocrit and 5% 35 parasitaemia. The results are shown in Figure 4A and confirm that like ITO/A4, ITO/C24 and MC are able to WO 01/02005 PCT/GB00/02546 - 31 adhere to CD36 and TSP. However, their adherence to CD54 was much reduced. T6/96 does not adhere to CD54, CD36 or TSP. 5 EXAMPLE 5 Effect of parasite strains on maturation A maturation assay as described in Example 2 was 10 carried out but exposing immature dendritic cells to erythrocytes infected with (b) ITO/A4, (d) ITO/C24, (f) MC and (h) T9/96. The results are shown in Figure 4B. While parasite lines MC and ITO/C24 inhibited the maturation of dendritic cells in a similar vein to 15 clone ITO/A4, the non-adherent line T9/96 did not inhibit maturation of dendritic cells even at a ratio of infected erythrocytes to dendritic cells of 100:1. EXAMPLE 6 20 Electron microscopy Adherence of ITO/A4 infected erythrocytes but not T9/96 infected erythrocytes to dendritic cells was 25 confirmed by electron microscopy. One million purified immature dendritic cells were incubated for 2 hours and for 12 hours with lx10 8 ITO/A4 infected RBC (a) or T9/96 infected (d) in 2 ml of dendritic cell medium , harvested and fixed with 2.5% 30 glutaraldehyde/cacodylate buffer. Cells were post fixed in osmium tetroxide, dehydrated and embedded in epoxy resin. Thin sections were stained with uranyl acetate and lead citrate prior to examination in a Joel 1200EX electron microscope. The number of 35 adherent and infected erythrocytes and the number of phagosomes containing pigment granules was counted in WO 01/02005 PCT/GB00/02546 - 32 each sample in thin sections of 100 randomly selected dendritic cells. Transmission electron micrographs are shown in Figure 5. 5 Note the cell processes partially enclosing infected erythrocytes (arrows in a) and the close apposition of the limiting membranes of the infected erythrocytes and dendritic cells particularly at the knobs (b, arrowhead). Within dendritic cell cytoplasm are 10 phagosomes containing characteristic pigment granules (c, arrows). N -dendritic cell nucleus, P - infected erythrocyte. Bars are 2 pm (a and d), 200 pm (b), 500 pm (c). 15 ITO/A4 infected erythrocytes were observed to be in intimate contact with immature dendritic cells with cytoplasmic processes partially enclosing the parasites (Fig. 5a). The plasmalemma of the infected erythrocytes was in close apposition to the limiting 20 membrane of the dendritic cell particularly at the site of knobs (Fig. 5b). A similar apposition between parasitised erythrocytes and host cells is seen between infected red blood cells and endothelial cells (Berendt et al (1994) Parasitology 1Q8 Suppl. 519-28). 25 In contrast, only a few infected erythrocytes of the T9/96 strain were associated with the dendritic cells (Fig. 5d). When quantified, ten times more ITO/A4 infected erythrocytes were found adherent to dendritic cells than T9/96 infected erythrocytes in 100 thin 30 sections of dendritic cells. Furthermore, ingestion of intact ITO/A4 infected erythrocytes by dendritic cells was not observed during this time. Nevertheless, phagocytosis of parasite debris as revealed by the number of phagosomes containing pigment granules (Fig. 35 5c) was similar for dendritic cells incubated with ITO/A4 or with T9/96.

WO 01/02005 PCT/GB00/02546 - 33 EXAMPLE 7 T-cell proliferation assays 5 Total-T-cells (allogeneic MLR) or CD4+ T cells (primary T-cell responses) were purified using a Cellect column (TCS). For the allogeneic MLR, dendritic cells were added in increasing numbers (156 to 10,000) to 1 x 105 T-cells in triplicate and 10 incubated for 5 days. T-cells were pulsed with 0.5 pCi 3H-thymidine/well for the last 18 hours of the culture. For primary T-cell responses, 1 x 106 dendritic cells were incubated with medium alone or with 1 x 108 infected erythrocytes for 18 h and then 15 pulsed with 10 pg/ml parasite-lysate or with 30 pg/ml keyhole limpet haemocyanin, respectively. The dendritic cells were purified by sedimentation through Lymphoprep" and 1 X 105 dendritic cells were culterd with 1.5 x 106 CD4+ T-cells from the same donor. From 20 day 4 to day 6 of culture, 50 pl aliquots were taken in triplicate and pulsed with 0.5 pCi 3 H-thymidine/well for 8 hours. (see Plebanski et al (1992) Immunol. 75 86-90). The results are shown in Figure 6. 25 Dendritic cells exposed to intact infected erythrocytes are poor stimulators of T-cell proliferation. Allogeneic T-cell proliferation (a) induced by immature dendritic cells (0), LPS-matured 30 dendritic cells (El) and dendritic cells co-cultivated with intact ITO/A4 infected erythrocytes (V) prior to maturation. Primary CD4+ T-cell responses to parasite-lysate (b) -and to keyhole limpet haemocyanin (c) induced by LPS-matured autologous dendritic cells 35 ([],0) and autologous dendritic cells co-cultivated with intact ITO/A4 infected erythrocytes (E,@) prior WO 01/02005 PCT/GB00/02546 - 34 to maturation. Data from one out of three independent experiments are shown. Dendritic cells matured after incubation with 5 uninfected RBC, a crude pigment preparation or a lysate of infected erythrocytes induced a similar degree of T-cell proliferation in a mixed leukocyte, reaction to that induced by control mature dendritic cells (data not shown). 10 However, dendritic cells incubated with LPS after exposure to intact infected erythrocytes from the parasite line ITO/A4 were strikingly less efficient in their induction of T-cell proliferation compared with 15 the T-cell proliferation induced by mature dendritic cells (Fig 6a). Furthermore, dendritic cells exposed to intact infected erythrocytes before maturation with LPS did not induce primary CD4+ T-cell responses to lysate of infected erythrocytes or to keyhole limpet 20 haemocyanin (Plebanski et al) (Fig 6, b,c). It is concluded that the maturation of dendritic cells and their subsequent ability to activate T-cells is profoundly inhibited by their interaction 25 with intact infected erythrocytes. Non-adherent parasite lines, parasite debris and crude pigment do not modulate dendritic cell function in this way. These studies provide one explanation for the clinical and experimental evidence of immune 30 dysregulation during malaria infection such as the impairment of the delayed-type hypersensitivity response to recall antigens and the antibody response to vaccines. 35 EXAMPLE 8 Maturation assay with monoclonal antibody WO 01/02005 PCT/GB00/02546 - 35 A maturation assay was carried out as described in Example 2 except that instead of infected erythrocytes the immature dendritic cells were exposed to monoclonal antibodies to CD36, CD51 or 5 both prior to immune stimulation with LPS. Specifically, 1x10 6 purified dendritic cells were incubated in duplicate wells without or with either 25 pg irrelevant IgM antibody, 25 pg irrelevant IgG1 antibody, 25 pg antiCD36 antibody, 25 pg antiCD51 10 antibody or a combination thereof for at least 3 hours. Thereafter, dendritic cells were matured with 100 ng/ml LPS (Salmonella typhimurium) for 48 hours or left untreated as a control. The monodonal antibodies tested were CD36 clone SMQ (Immunocontact) 15 and clone 89 (Serotec), CD51 clone 13C2 (Immunocontact), IgM isotope control clone MOPC, IgG isotope control clone MOPC (Sigma). The results of two experiments are shown in Figures 7 and 8 respectively. As will be apparent both CD36 and CD51 20 antibodies have the effect of inhibiting dendritic cell maturation in a similar manner to infected erythrocytes. EXAMPLE 9 25 Maturation assay with apoptotic cells *A maturation assay was carried out as described in Example 2 except that instead of infected 30 erythrocytes the immature dendritic cells were exposed to apoptotic cells prior to immune stimulation with LPS. Apoptotic or necrotic cells were derived from purified autologous dendritic cells, monocytes or neutrophils. Specifically for 35 maturation assays in the presence of apoptotic bodies 1x10 6 purified dendritic cells were incubated in WO 01/02005 PCT/GB00/02546 - 36 duplicate wells without or with either 2 x 106 autologous apoptotic or necrotic cells for 12 hours. Maturation was induced by the addition of LPS or TNFa as stated above. Apoptosis was induced by radiation 5 with a calibrated UV lamp at a dose of 2500 mJ/cm 2 and evaluated by staining with FITC-AnnexinV/Propidium Iodide according to manufacturers recommendations (Roche Diagnostics) 3 hours and 12 hours after UV radiation. Necrosis was induced by at least three 10 cycles of rapid freezing at -70'C and thawing at 37C. Thereafter, more then 90% of cells were permeable for trypan blue. The results are shown in Figure 9 as follows: 15 Apoptotic cells but not necrotic cells inhibit the maturation of dendritic cells. (a) Immature dendritic cells were left untreated, matured with LPS or exposed autologous apoptotic or necrotic dendritic cells prior to maturation with LPS and subsequently 20 stained with antibodies directed against surface marker and analysed by FACScan as indicated. (b) Dead Cells and especially apoptotic cells were efficiently excluded from analysis by gating on forward scatter and exclusion of cells positive for Propidium Iodide. 25 (c) Proliferation of allogeneic T-cells stimulated by increasing numbers of immature dendritic cells (*), LPS-matured cells (A) or dendritic cells exposed to apopotic dendritic cells and matured with LPS (U). The results of a further experiment with 30 apoptotic neutrophils shown in Figure 10. EXAMPLE 10 Antigen specific T-cell responses 35 The proliferative response of the CD4+ T-cell clone WO 01/02005 PCT/GB00/02546 - 37 TB-2 for the peptide 144-163 of the human acetylcholine receptor (Nagvekar N et al, J. Clin in est, 1998 101 (10) pp 2268-77) was analysed. T-cell proliferation was measured as described in Example 7. 5 For antigen-specific T-cell responses, 1 x 101 dendritic cells were incubated with medium alone or with antibodies as indicated and then pulsed for 6 h with 0.025 mM AChR o: 3-181 polypeptide before or 1 mM AChR a:144-163 peptide after maturation with LPS. 10 For antigen specific T-cell responses of the clone TB-2 increasing numbers of MHC class II matched dendritic cells were incubated with 3 x 104 T-cells for 72 h. Proliferation was measured in all assays by adding 0.5 mCi 3 H-thymidine/well for the last 8 hours 15 of the culture. The results are shown in Figure 11 (c) and (d). Exposure of dendritic cells to antiCD36, antiCD51 or both antibodies abolished their ability to induce 20 proliferation in the T-cell clone as compared to dendritic cells exposed to irrelevant antibodies (Fig 2c, d). The proliferation of the T-cell clone remained low even when the modulated dendritic cells were exogenously loaded with peptide thus excluding a 25 defect in antigen-uptake or antigen-processing due to the presence of antibodies. EXAMPLE 11 30 Cytokine production by dendritic cells Secretion of the cytokines TNF alpha, IL12 p70 and IL10 was measured in supernatants of dendritic cells treated with either anti-CD36 antibody or apoptotic 35 cells or in the respective controls before or after maturation with LPS for 24 hours using commercially WO 01/02005 PCT/GB00/02546 - 38 available ELISA kits. These kits can be obtained from R and D Systems, Europe Ltd. 4-10, The Quadrant, Barton Lane, Abingdon, Oxford, OX143 YS and BD Pharmingen, 10975, Torreyana Road, San Diego, CA 5 92121, USA. Maturation with LPS induced secretion of TNF-a by dendritic cells irrespective whether they were exposed to anti-CD36 antibodies or apoptotic cells although the 10 concentration of TNF-a was consistently slightly lower than in the respective controls. IL12 p70 was secreted by control dendritic cells matured with LPS whereas IL10 was secreted by dendritic cells exposed to anti CD36 antibodies or apoptotic cells. Of note, the 15 absolute amount of IL10 varied considerably between dendritic cells treated with anti-CD36 and dendritic cells exposed to apoptotic cells in response to LPS. It is possible that intact cells bind to more than one receptor thus modifying the cytokine secretion induced 20 by CD36 alone. However, we investigated whether secretion of IL10 had a role in inhibition of dendritic cell maturation due to ligation of CD36 by maturing dendritic cells exposed anti-CD36 antibodies or to apoptotic cells in the presence of blocking anti-IL10 25 antibodies. The inhibition of dendritic cell maturation was not reversed (data not shown) and is therefore independent of the secretion of IL10. EXAMPLE 12 30 Modulation of mouse dendritic cell maturation and function by antiCD51 antibodies in vitro and in vivo Rationale 35 Since human monocyte derived dendritic cells can be modulated in their maturation and function by a variety WO 01/02005 PCT/GB00/02546 - 39 of agents including antibodies binding to CD36 and or CD51, in this study we began to investigate whether a similar phenomenon could be observed in mouse dendritic cells. 5 Methods Generation of bone-marrow derived dendritic cells: Bone marrow from male Balb/c (H-2k ) mice was harvested and total cells were cultured in RPMI supplemented with 2 10 mM glutamine, 50 mg Kanamycin, 10 % FCS,10 ng/ml each murine recombinant GM-CSF and IL-4. On day two of culture half the medium was replaced with fresh medium supplemented with cytokines and on day four of culture non-adherent cells were harvested. 15 In vitro maturation of bone-marrow derived mouse dendritic cells: One million of bone marrow derived dendritic cells (approximately 50% total cells) in duplicate were exposed to either medium alone, 25 mg 20 isotpype control antibody or 25 mg antiCD51 antibody for 8 hours. Cells were subsequently exposed to 100 ng LPS for 48 hours or left alone as a control. Maturation of dendritic cells was analysed by double staining with FITC conjugated antibodies against CDllc and PE 25 conjugated antibodies directed against either CD40, CD54, CD86 or I-A and subsequent FACScan analysis. Analysis was performed on CDllc-FITC positive cells. Popliteal lymph node assay: Dendritic cells were 30 exposed to medium alone or to antiCD51 antibodies and then matured with LPS as described above. The cells were then harvested and washed four times in PBS in order to remove LPS. Cells were resuspended in 10% FCS/PBS at a concentration of 6 x 10 total cells/20 ml. 35 Groups of six male C3H3/HE (H-2d) mice were injected with 20 ml of PBS into the right footpad and LPS matured dendric cells into the left footpad, with 20 ml WO 01/02005 PCT/GB00/02546 - 40 of PBS into the right footpath and dendritic cells exposed to antiCD51 antibody prior to LPS maturation into the left footpad or with 20 ml of PBS into the right and the left footpad. After one week mice were 5 sacrificed and the popliteal lymphnodes were removed. The weight of the left and the right lymphnode of each mouse in all three groups were determined and the ratio of the weight of the left lymphnode to the weight of the right lymphnode was calculated. The mean and SE of 10 the ratio was determined for each group. Results Dendritic cells matured with LPS (DC LPS) increased the surface expression of the molecules CD40, CD54 and CD86 15 as compared to immature dendritic cells (DC). However, when dendritic cells were treated with antiCD51 antibodies prior to exposure to LPS (DC CD51 lps), the dendritic cells failed to mature and the expression of surface molecules remained at the level of immature 20 dendritic cells (DC) . The antibody itself had no effect on dendritic cell maturation (DC CD51) . The results are shown in Figure 13. When dendritic cells matured with LPS were injected 25 into the footpad of allogeneic C3H/HE mice, they induced a strong allogeneic T-cell response as measured by the increase in weight of the popliteal lymphnode of the right footpad compared to the popliteal lymphnode of the left footpad. By contrast, the increase in 30 weight of the right popliteal lymphnode over that of the left popliteal lymphnode was markedly reduced when dendritic cells were exposed to antiCD51 antibody prior to maturation with LPS. However, the overall increase in weight was still above that of popliteal lympnodes 35 of mice that had been injected with PBS in both footpads. The results are shown in Figure 14.

WO 01/02005 PCT/GB00/02546 - 41 EXAMPLE 13 Modulation of human dendritic cells ex vivo 5 Agonists of CD36, CD51, thrombospondin receptors or -integrin may be used to modulate human immune response in patients with unwanted and/or harmful allo- or auto-immune responses. For such a method of treatment immature human dendritic cells which are 10 defined and identifiable as described herein and with the ability to phagocytose are derived from preparations of human peripheral blood. Specifically, the dendritic cells are derived from CD34+ stem cells or monocytes isolated from human 15 peripheral blood by the method described in Example 1. To the preparation of cells suspended in appropriate medium, for example RPMI as defined herein, an agonist of C36, CD51, thrombospondin receptor or p-integrin is added. The relative 20 concentration of agonist to cells is adjusted depending on the nature of the agonist used. For example, if the agonist is a monoclonal antibody, about 25 pg antibody to about 106 dendritic cells is appropriate at a concentration of 25 pg Ml~' The 25 cells are treated for between 3 and 24 hours. Depending on the particular application cells are returned to the individual from which they were originally derived or administered to another individual. Adminitration may be by intravenous 30 infusion, by inhalation or by sub-cutaneous or intramuscular injection. Administration of dendritic cells to a human following the method described above will give rise to a generalized immune suppressive effect which will 35 be useful, in a number of situations, for example in the prevention of rejection of allografts and WO 01/02005 PCT/GB00/02546 - 42 xenografts or for treatment of disease suspected of having an auto-immune basis but for which the auto antigen is not known. However, the method described above may be 5 modified to produce dendritic cells which are tolerant to a specific antigen. In this case the dendritic cell preparation is exposed to an antigen against which tolerance is to be induced as well as to the CD36, CD51, thrombospondrin receptor or P 10 integrin agonist. The cells may be exposed to antigenic material, before, after or simultaneously with the aforesaid agonist molecule. The antigenic material may be linked to, fused to or otherwise associated with said agonist molecule. Exposure to 15 the antigenic material is for about 6 to about 24 hours with or without an immune stimulant, then the cells are reintroduced to the patient as described above. The ability to induce tolerance to a specific 20 antigen, for example an allo, xeno or auto-antigen allows a great many therapeutic applications. For example tolerance can be induced in respect of the following antigens: 25 to major or minor histocompatibility antigens of a recipient of a bone marrow transplant (to modulate graft versus host disease in bone marrow transplantation in dendritic cells from the bone marrow donor) or 30 to donor major or minor histocompatibility antigens in dendritic cells of recipients of solid organ transplant or 35 to antigens to which there is pathological immune response causing auto-immune diseases for example: components of autologous red blood cells to modulate WO 01/02005 PCT/GB00/02546 - 43 the immune response in patients with auto-immune hemolytic anaemia components of autologous platelets in patients with auto-immune thrombocytopeniacomponents of beta islet 5 cells of the pancreas in patients with insulin dependent diabetes mellitus components of other endocrine organs in patients with other organ specific auto-immune diseases components of the acetylcholine receptor in patients 10 with myasthenia gravis other antigens or apoptotic cells containing antigens causing harmful or pathological immune responses in other auto-immune diseases 15 to antigens to which there is pathological immune response causing atopic or allergic diseases, for example antigens eliciting an immune response in hay fever, asthma, eczema or coeliac disease 20 to antigens to which there is pathological immune response that may be defined in other diseases for example in non-organ specific immune diseases (systemic lupus erythematosis or rheumatoid arthritis) or other immune mediated arthritis or 25 other connective tissue diseases in inflammatory bowel disease in auto-immune hepatitis in multiple sclerosis or in other auto-immune disease 30 to allo-antigens to which there is a harmful or pathological immune response for example components of red blood cells in haemolytic disease of the newborn or in previously transfused patients 35 components of platelets for example in neo-natal allo immune thrombocytopenia or in conditions where there is an allo-immune response to transfused platelets WO 01/02005 PCT/GB00/02546 - 44 other blood products or substitutes for example Factor VIII in haemophilia patients other synthesized or manufactured or naturally occurring products or substances 5 Although the treatment of humans with dendritic cells is described in this example it will be appreciated that other antigen-presenting cells, such as macrophages, monocytes or P-lymphocytes could be 10 used for treatment with an agonist of CD36, CD51, thrombospondin receptor or p-integrin and optionally an antigenic material. Specific tolerance can be introduced in such cells for use in any of the applications listed above. 15 Example 14 Preparation of phosphatidylserine liposome compositions and their therapeutic uses. 20 Liposomes containing phosphatidylserine or other negatively charged phospholipids, with or without additional targeting molecules, induce general immune unresponsiveness. Liposomes encapsulating antigens and phospholipids with or without additional 25 targeting molecules induce antigen specific immune unresponsiveness. Liposomes are prepared as described by Coradini et al, Anticancer Research 1998 18 177-182. In brief clean glass tubes are coated with 2 micromolar of 30 mixtures of phosphatidylcholine and phosphatidylserine, other negatively charged phospholipids or other phospholipids including cholesterol and/or cholesterol ester dissolved in chloroform. The solvent is evaporated under nitrogen 35 gas and the tubes incubated in a vacuum for 45 minutes. Sterilised phosphate buffered saline WO 01/02005 PCT/GB00/02546 - 45 (unmodified liposomes) or containing the antigen(s) to which unresponsiveness will be induced with or without molecules allowing targeting of the liposomes to CD36 and/or CD51 and/or beta-integrins and/or 5 other receptors of apoptotic cells or other molecules expressed on the surface of antigen presenting cells, is added to the lipid shell. Suitable targeting molecules are monoclonal antibodies to the respective receptors or fragments of the P. falciparum 10 erythrocyte membrane protein-1 that bound to CD36 and/or thrombospondin. The tubes are shaken at high' speed for 5 minutes and separated from free fatty acid by ultracentrifugation at 100,000g for 60 minutes. Targeting molecules (see above) may be 15 covalently or non-covalently attached to the surface of liposomes. The liposomes are filtered through a 0.22 micrometer filter. Encapsulation of antigens and targeting molecules can also be achieved by freeze-thawing or dehydration/rehydration or by 20 reverse phase evaporation (Monnard PA et al, Biochem. Biophys. Acta 1997 1329 39-50) or by other published methods of preparing liposomes. Liposomes prepared as described above would be added to 1 x 106 isolated immature dendritic cells or 25 to other antigen presenting cells at a concentration of 25 micrograms per ml. The maturation and function of the dendritic cells or other antigen presenting cells is assessed as previously described. The liposomes containing phosphatidylserine (with or 30 without targeting molecules) is used to treat dendritic cells or other antigen presenting cells ex vivo or for systemic treatment.

WO 01/02005 PCT/GB00/02546 - 46 APPENDIX 1 DI is distributor SD is standard designation Other MABs are OKM5 Ortho Pharmaceutical Corporation OKM8 1001 US Highway 202 P.O. Box 250 Raritan, N.J. SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 47 cd 36 in HDB-DI HDB-NONDI http://www.atcc.org/cgi-bin/Sfgat...abase=local%2FHDB-NOND1&text=cd36 Your query was: cd36 The selected databases contain 18 documents matching your query: 1: 1013396 RE 1.CE>platelet 1.SN>CD36 1.a.CC>differentia 2: 1003558 RE 1.CE>platelet 1.U>cell membrane 1.SN>CD36 3: 1018253 RE 1.SN>CD36 1.a.CC>differentiation 1.b.CC>gly 4: 1020319 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 5: 1013397 RE 1.CE>platelet 1.SN>CD36 l.a.CC>differentia 6: 1020540 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 7: 1017636 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 8: 1022016 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 9: 1019865 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 10: 1024459 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 11: 1019119 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 1.MW 12: 1009963 RE 1.G>Homo sapiens 1.CN>human 1.CE>monocyte 13: 1016854 RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 14: 1023242 RE 1.G>Homo sapiens 1.CN>human 1.U>cell membr 15: 22825 RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 16: 1012440 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 17: 1012380 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 18: 1003358 RE 1.SN>ACTH 1.FS>N-terminal region 1.a.CC>ho SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 48 013396 RE 1.CE>platelet 1.SN>CD36 1.a.CC>differentia 1013396 RE 1.CE>platelet 1.SN>CD36 1.a.CC>differentiation AN 1013396 DI P>Medica DI 2382 Camino Vida Roble, Suite I DI Carlsbad, CA 92009 USA DI 1-619-438-1886 DE C>CLB/703 ;developer DE P>MON1118 ;distributor PD ;IgGl RE 1.CE>platelet l.SN>CD36 1.a.CC>differentiation RE 2.CE>monocyte 2.SN>CD36 2.a.CC>differentiation RE 3.CE>macrophage 3.SN>CD36 3.a.CC>differentiation RE 4.CE>platelet 4.SN>CD36 4.a.CC>differentiation AP ;frozen section SD MON1118 LD USA BAL EI DA>9303 CI ;catalog SN Synonym>CD36 1013396 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 49 003558 RE 1.CE>platelet 1.U>cell membrane 1.SN>CD36 1003558 RE 1.CE>platelet 1.U>cell membrane l.SN>CD36 AN 1003558 DI P>Biodesign International DI 105 York Street DI Kennebunkport, ME 04043 USA DI 1-207-985-1944 DE P>N42540M ;distributor PD ;IgGl RE 1.CE>platelet 1.U>cell membrane 1.SN>CD36 RE 1.a.CC>differentiation 1.b.CC>protein RE 1.c.CC>blood coagulation factor AV ;purified SD N42540M LD USA BAL EI DA>9002 CV>9007 CI ;catalog SN Synonym>CD36 1003558 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 50 018253 RE 1.SN>CD36 1.a.CC>differentiation 1.b.CC>gl 1018253 RE 1.SN>CD36 1.a.CC>differentiation 1.b.CC>glycoprotein AN 1018253 DI P>BioGenex Laboratories DI 4600 Norris Canyon Road DI San Ramon, CA 94583 USA DI 1-510-275-0550 DI 1-800-421-4149 (toll free USA) DE P>1E8 ;distributor DO G>Mus musculus CN>mouse RE 1.SN>CD36 1.a.CC>differentiation 1.b.CC>glycoprotein AV ;purified SD 1E8 LD USA JMJ EI DA>9602 CI ;catalog SN Synonym>CD36 1018253 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 51 020319 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 1020319 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 AN 1020319 DI P>Harlan Bioproducts for Science, Inc. DI P.O. Box 29176 DI Indianapolis, IN 46229-0176 DI 1-317-894-7536 DI 1-800-9-SCIENCE DE C>89 ;distributor DE P>MCA1214 ;distributor DO G>Mus musculus CN>mouse PD ;IgG2b RE 1.G>Homo sapiens l.CN>human l.SN>CD36 RE 1.a.CC>differentiation AP ;flow cytometry ;Western blot AV ;purified SD 89 SD MCA1214 LD USA CLB EI DA>9702 CI ;catalog SN Synonym>CD36 1020319 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 52 013397 RE 1.CE>platelet 1.SN>CD36 1.a.CC>differentia 1013397 RE 1.CE>platelet l.SN>CD36 l.a.CC>differentiation AN 1013397 DI P>Caltag Laboratories DI 1849 Bayshore Blvd. #200 DI Burlingame, CA 94010 DI 1-650-652-0468 DI 1-800-874-4007 DI 2.P>Medica DI 2382 Camino Vida Roble, Suite I DI Carlsbad, CA 92009 USA DI 1-619-438-1886 DE C>VM58 ;developer DE P>MON1143 ;distributor DE P>VM58 ;distributor DE 2.P>MON1143 ;distributor PD ;IgGl RE 1.CE>platelet 1.SN>CD36 1.a.CC>differentiation RE 2.CE>monocyte 2.SN>CD36 2.a.CC>differentiation RE 3.CE>macrophage 3.SN>CD36 3.a.CC>differentiation RE 4.CE>platelet 4.SN>CD36 4.a.CC>differentiation AP ;frozen section SD VM58 LD USA BAL EI DA>9303 CI ;catalog SN Synonym>CD36 1013397 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 53 020540 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 1020540 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 AN 1020540 DI P>Harlan Bioproducts for Science, Inc. DI P.O. Box 29176 DI Indianapolis, IN 46229-0176 DI 1-317-894-7536 DI 1-800-9-SCIENCE DE P>89 ;developer DE P>MCA1214 ;distributor DO G>Mus musculus CN>mouse PD ;IgG2b RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 RE 1.a.CC>differentiation AP ;flow cytometry ;Western blot AV ;purified SD 89 SD MCA1214 LD USA CLB EI DA>9702 CI ;catalog SN Synonym>CD36 1020540 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 54 117636 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 1017636 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 AN 1017636 DI P>Novocastra Laboratories Ltd. DI 24 Claremont Place DI Newcastle upon Tyne NE2 4AA, UK DI 44-0191 222 8550 DE P>NCL-CD36 ;distributor DE P>SMO ;distributor DO G>Mus musculus CN>mouse AS ;immunohistochemical staining RE 1.G>Homo sapiens 1.CN>human l.SN>CD36 RE l.a.CC>differentiation AP ;frozen section AV ;ascites SD NCLCD36 SD SMO LD USA BAL EI DA>9904 CI ;catalog SN Synonym>CD36 1017636 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 55 022016 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 1022016 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 AN 1022016 DI P>O.E.M. Concepts, Inc. DI 1889 Route 9, Bldg. 25, Unit 96 DI Toms River, NJ 08755 USA DI 1-732-341-3570 DE C>289-10930 ;distributor DE P>M2-L69 ;distributor DO G>Mus musculus CN>mouse RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 RE 1.a.CC>differentiation AP ;cell surface marker AV ;purified SD 28910930 SD M2L69 LD USA EJK EI DA>9712 CI ;catalog SN Synonym>CD36 1022016 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 56 019865 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 1019865 RE 1.G>Homo sapiens 1.CN>human l.SN>CD36 AN 1019865 DI P>Upstate Biotechnology, Inc. DI 199 Saranac Avenue DI Lake Placid, NY 12946 USA DI 1-617-890-8845 DI 1-800-233-3991 (toll free USA) (sales) DE P>05-287 ;distributor DO G>Mus musculus CN>mouse S>BALB/c O>spleen PD ;IgM RE l.G>Homo sapiens 1.CN>human 1.SN>CD36 RE l.a.CC>differentiation l.b.CC>protein AP ;immunocytochemistry AV ;ascites SD 05287 LD USA JMJ EI DA>9611 CI ;catalog SN Synonym>CD36 1019865 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 57 016854 RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1009963 RE 1.G>Homo sapiens 1.CN>human 1.CE>monocyte 1009963 RE 1.G>Homo sapiens 1.CN>human 1.CE>monocyte 1.SN>CD36 AN 1009963 DI P>Harlan Bioproducts for Science, Inc. DI P.O. Box 29176 DI Indianapolis, IN 46229-0176 DI 1-317-894-7536 DI 1-800-9-SCIENCE DI 2.P>Immunotech S.A. DI Departement commercial DI Luminy Case 915 DI 13288 Marseille Cedex 9, France DI 33-91-41-41-38 DI 430246 F IMMTECH DE C>Fa6-152 ;developer DE P>MCA 682 ;distributor DE 2.P>0765 ;distributor DE 2.P>0766 ;distributor DE 2.P>FA6.152 ;distributor DO G>Mus musculus CN>mouse PD ;IgG1 RE 1.G>Homo sapiens 1.CN>human 1.CE>monocyte 1.SN>CD36 RE 1.a.CC>differentiation RE 2.G>Homo sapiens 2.CN>human 2.CE>macrophage 2.SN>CD36 RE 2.a.CC>differentiation RE 3.G>Homo sapiens 3.CN>human 3.CE>platelet 3.SN>CD36 RE 3.a.CC>differentiation AV ;purified ;2.fluorescein conjugate ;2.purified SD 0765 SD 0766 SD FA6152 SD MCA682 LD USA BAL EI DA>9103 CV>9104 CI ;catalog SN Synonym>CD36 1009963 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 58 -023242 RE 1.G>Homo sapiens 1.CN>human 1.U>cell member 1023242 RE 1.G>Homo sapiens 1.CN>human 1.U>cell membrane l.SN>CD36 AN 1023242 SO Exp Cell Res 1992;198:85-92 SO J Exp Med 1990;171:1883-92 DI P>Lab Vision-NeoMarkers DI 47770 Westinghouse Drive DI Fremont, CA 94539 USA DI 1-800-828-1628 DE C>1A7 ;distributor DE P>MS-466-P ;distributor IM G>Homo sapiens CN>human CE>platelet SN>CD36 a.CC>differentiation IM b.CC>glycoprotein c.CC>receptor DO G>Mus musculus CN>mouse PD ;IgG2b ;kappa RE 1.G>Homo sapiens l.CN>human 1.U>cell membrane l.SN>CD36 RE 1.MW>88 kD l.a.CC>differentiation l.b.CC>glycoprotein RE 1.c.CC>receptor AP ;flow cytometry immunofluorescencee ;immunoprecipitation AP ;Western blot ;immunohistology ;gold labelling AB platelet GPIIIb, platelet glycoprotein IIIb, and OKM5-antigen. SD 1A7 SD MS466P LD USA MCM EI DA>9806 CI ;catalog SN Synonym>CD36 1023242 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 59 !825 RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 22825 RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet AN 22825 AU Kemshead J AD Imperial Cancer Research Technology; AD Sardinia House; AD Sardinia Street; AD London WC2A 3NL; AD UK; AD Tel 01 242 1136; AD TELEX 265107.TCRFG; AD FAX 01 831 4991 SO Br J Haematol 1984;57:621 DE P>M148 ;developer IM G>Homo sapiens l.CN>human PA>medulloblastoma a.CC>neoplasm PD ;IgGl RE l.G>Homo sapiens 1.CN>human l.CE>platelet RE l.U>cell surface l.SN>CD36 l.MW>l10-130 kD RE l.a.CC>differentiation RE 2.G>Homo sapiens 2.CN>human 2.PA>medulloblastoma RE 2.a.CC>neoplasm RE 3.G>Homo sapiens 3.CN>human 3.PA>neuroblastoma RE 3.a.CC>neoplasm RE 4.G>Homo sapiens 4.CN>human 4.PA>rhabdomyosarcoma RE 4.a.CC>neoplasm AP ;immunofluorescence ;immunoprecipitation AB in vivo imaging and therapy SD M148 LD EUR BD FI>EUR0003951 EUR901.TXT EI DA>8901 CV>8904 CI ;catalog SN Synonym>CD36 22825 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 60 1012380 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 1012380 RE 1.G>Homo sapiens 1.CN>human l.SN>CD36 AN 1012380 DI P>Biodesign International DI 105 York Street DI Kennebunkport, ME 04043 USA DI 1-207-985-1944 DI 2.P>Harlan Bioproducts for Science, Inc. DI P.O. Box 29176 DI Indianapolis, IN 46229-0176 DI 1-317-894-7536 DI 1-800-9-SCIENCE DI 3.P>Lampire Biological Laboratories DI P.O. Box 270 DI Pipersville, PA 18947 USA DI 1-215-795-2838 DI 4.P>Sigma Chemical Company DI P.O. Box 14508 DI St. Louis, MO 63178 9916 USA DI 1-800-325-3010 (toll free USA) DI 1-314-771-5750 DE C>SMO ;developer DE P>P54168M ;distributor DE P>SMO ;distributor DE 2.P>MCA-722F ;discontinued designation DE 2.P>MCA722 ;distributor DE 2.P>SMO ;distributor DE 3.P>LBL 268 ;distributor DE 3.P>SMO ;distributor DE 4.P>C 4679 ;distributor DE 4.P>F5898 ;distributor DE 4.P>P9312 ;distributor DE 4.P>R6395 ;distributor DE 4.P>SMO ;distributor DO G>Mus musculus CN>mouse PD ;IgM RE 1.G>Homo sapiens l.CN>human l.SN>CD36 RE l.a.CC>differentiation AV ;purified ;2.purified ;4.fluorescein conjugate AV ;4.phycoerythrin conjugate SD C4679 SD F5898 SD LBL268 SD MCA722 SD MCA722F SD P54168M SD P9312 SD R6395 SD SMO SD SMO LD USA BAL EI DA>9803 CV>9111 CI ;catalog SN Synonym>CD36 1012380 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 61 1003558 RE 1.CE>platelet 1.U>cell membrane 1.SN>CD36 1003558 RE 1.CE>platelet 1.U>cell membrane l.SN>CD36 AN 1003558 DI P>Biodesign International DI 105 York Street DI Kennebunkport, ME 04043 USA DI 1-207-985-1944 DE P>N42540M ;distributor PD ;IgG1 RE 1.CE>platelet 1.U>cell membrane l.SN>CD36 RE l.a.CC>differentiation 1.b.CC>protein RE 1.c.CC>blood coagulation factor AV ;purified SD N42540M LD USA BAL EI DA>9002 CV>9007 CI ;catalog SN Synonym>CD36 1003558 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 62 1024459 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 1024459 RE 1.G>Homo sapiens 1.CN>human l.SN>CD36 DI P>Biogenesis Ltd. DI 7 New Fields DI Stinsford Road DI Poole BH17 7NF, England DI UK DI 44-1202 660006 DE C>SM-phi IgM ;distributor DE P>2125-3607 ;distributor DO G>Mus musculus CN>mouse PD ;Ig RE l.G>Homo sapiens l.CN>human l.SN>CD36 RE l.a.CC>differentiation AP ; immunofluorescence AV ;fluorescein conjugate AB CD36 is also known as platelet GPIV, GPIV, platelet GPIIIb, GPIIIb, platelet AB glycoprotein IV, and FAT (rat). SD 21253607 SD SMPHIIGM LD USA MCM EI DA>9811 CI ;catalog SN Synonym>CD36 1024459 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 63 1016854 RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1016854 RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet AN 1016854 DI P>PharMingen DI 10975 Torreyana Road DI San Diego, CA 92121 USA DI 1-619-677-7737 DI 1-800-848-6227 (toll free USA) DE P>CB38 ;distributor DO G>Mus musculus CN>mouse S>BALB/c PD ;IgM ;kappa RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet RE 1.U>cell membrane l.SN>CD36 1.MW>88 kD RE l.a.CC>differentiation 1.b.CC>glycoprotein AP ;flow cytometry ;immunoprecipitation AV ;fluorescein conjugate ;purified SD CB38 LD USA JMJ EI DA>9504 CI ;catalog SN Synonym>CD36 1016854 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 64 1012440 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 1012440 RE l.G>Homo sapiens 1.CN>human l.SN>CD36 AN 1012440 DI P>BioSource International DI 820 Flynn Roa DI Camarillo, CA 93012 USA DI 1-800-242-0607(toll free USA) DI 1-805-987-0086 DI 2.P>Cymbus Bioscience Limited DI 2 Venture Road DI Chilworth Research Center DI Southampton, Hampshire SO1 7NS UK DI 44-703-767178 DI 3.P>Roche Molecular Biochemicals DI formerly Boehringer Mannheim GmbH DI Sandhofer Strasse 116 DI D-68305 Mannheim Germany DI 49-621-759 8577 DE C>SMO ;developer DE P>AHS3601 ;distributor DE P>AHS3608 ;distributor DE P>CS-CD36-FI ;discontinued designation DE P>CS-CD36-UN ;discontinued designation DE P>SMO ;distributor DE 2.P>CBL 168 ;distributor DE 2.P>SMO ;distributor DE 3.P>1441 230 ;discontinued designation DE 3.P>1441 264 ;distributor DE 3.P>SMO ;distributor RE l.G>Homo sapiens l.CN>human l.SN>CD36 RE l.a.CC>differentiation AV ;fluorescein conjugate ;3.purified SD 1441230 SD 1441264 SD AHS3601 SD AHS3608 SD CBL168 SD CSCD36FI SD CSCD36UN SD SMO LD USA BAL EI DA>9709 CV>9111 CI ;catalog SN Synonym>CD36 1012440 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 65 1003358 RE 1.SN>ACTH 1.FS>N-terminal region 1.a.CC>ho 1003358 RE 1.SN>ACTH 1.FS>N-terminal region 1.a.CC>hormone AN 1003358 DI P>Biodesign International DI 105 York Street DI Kennebunkport, ME 04043 USA DI 1-207-985-1944 DI 2.P>Cymbus Bioscience Limited DI 2 Venture Road DI Chilworth Research Center DI Southampton, Hampshire SO1 7NS UK DI 44-703-767178 DE C>58 ;developer DE P>E54008M ;distributor RE 1.SN>ACTH 1.FS>N-terminal region l.a.CC>hormone AV ;purified AB CD36 is also known as GPIIIb, GPIV SD 58 SD E54008M LD USA BAL EI DA>9002 CV>9007 CI ;catalog SN Synonym>ACTH SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 66 1019119 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 1.MW 1019119 RE 1.G>Homo sapiens l.CN>human l.SN>CD36 1.MW>88 kD AN 1019119 SO J Cell Biol 1994;269:6011 SO J Cell Biol 1993;268:16179 DI P>Transduction Laboratories DI 133 Venture Ct., Suite 5 DI Lexington, Ky 40511-9923 DI 1-606-259-1550 DI 1-800-227-4063 DE P>73 ;distributor DE P>C23620 ;distributor IM G>Homo sapiens CN>human SN>CD36 FS>amino acids 70-242 IM a.CC>protein DO G>Mus musculus CN>mouse PD ;IgG2a RE 1.G>Homo sapiens 1.CN>human 1.SN>CD36 1.MW>88 kD RE l.a.CC>protein RE 2.G>Rattus norvegicus 2.CN>Norway rat 2.SN>CD36 RE 2.MW>88 kD 2.a.CC>protein RE 3.G>Gallus gallus 3.CN>chicken 3.SN>CD36 3.MW>88 kD RE 3.a.CC>protein AP ;Western blot ;immunofluorescence AV ;purified SD 73 SD C23620 LD USA JMJ EI DA>9901 CI ;catalog SN Synonym>CD36 1019119 SN Synonym>CD36 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GBOO/02546 -67 APPENDIX 2 DI is distributor SD is standard designation SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 68 1022961 RE 1.SN>CD51 1.a.CC>differentiation 1022961 RE 1.SN>CD51 1.a.CC>differentiation AN 1022961 DI P>Caltag Laboratories DI 1849 Bayshore Blvd. #200 DI Burlingame, CA 94010 DI 1-650-652-0468 DI 1-800-874-4007 DE C>NGX-IV/110 ;distributor DE P>MON1027 ;distributor RE 1.SN>CD51 1.a.CC>differentiation SD NGXIV110 LD USA MCM EI DA>9805 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 69 1022017 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51/61 c 1022017 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51/61 c AN 1022017 DI P>O.E.M. Concepts,. Inc DI 1889 Route 9, Bldg. 25, Unit 96 DI Toms River, NJ 08755 USA DI 1-732-341-3570 DE C>289-12336 ;distributor DE P>M2-L69 ;distributor DO G>Mus musculus CN>mouse RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51/61 complex RE 1.a.CC>differentiation AP ;cell surface marker AV ;purified AB Reactant#1: CD51/61 complex is also known as integrin alpha V beta 3. SD 28912336 SD M2L69 LD USA EJK EI DA>9712 CI ;catalog SN Synonym>CD51/61 complex 1022018 ****HB/HYBRID SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 70 1013413 RE 1.CE>platelet 1.SN>CD51 1.a.CC>differentiation 1013413 RE 1.CE>platelet 1.SN>CD51 1.a.CC>differentiation AN 1013413 DI P>Medica DI 2382 Camino Vida Roble, Suite I DI Carlsbad, CA 92009 USA DI 1-619-438-1886 DE C>706 ;developer DE P>MON1130 ;distributor PD ;IgGl RE 1.CE>platelet 1.SN>CD51 l.a.CC>differentiation SD MON1130 LD USA BAL EI DA>9303 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 71 1024461 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 1024461 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 AN 1024461 DI P>Biogenesis Ltd. DI 7 New Fields DI Stinsford Road DI Poole BH17 7NF, England DI UK DI 44-1202 660006 DE C>13C2 ;distributor DE P>2125-5108 ;distributor DE P>2125-5114 ;distributor DE P>2125-5119 ;distributor DO G>Mus musculus CN>mouse PD ;Ig RE l.G>Homo sapiens 1.CN>human 1.SN>CD51 RE l.a.CC>differentiation AP ; immunofluorescence AV ;R-phycoerythrin conjugate ;fluorescein conjugate AB CD51 is also known as integrin alpha V subunit and vitronectin receptor AB alpha subunit. SD 13C2 SD 21255108 SD 21255114 SD 21255119 LD USA MCM EI DA>9811 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 72 1017037 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 1017037 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 DI P>Zymed Laboratories Inc. DI 458 Carlton Court DI South San Francisco, CA 94080 USA DI 1-800-874-4494 (toll free USA) DI 1-415-871-4494 DE P>07-5103 ;distributor DE P>NK1-M9 ;distributor DO G>Mus musculus CN>mouse S>BALB/c PD ;IgGl RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 RE l.a.CC>differentiation 1.b.CC>protein AP ;flow cytometry ;immunofluorescence AV ;purified SD 075103 SD NK1M9 LD USA JMJ EI DA>9708 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 73 1009962 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 1009962 RE l.G>Homo sapiens 1.CN>human l.SN>CD51 AN 1009962 DI P>Biodesign International DI 105 York Street DI Kennebunkport, ME 04043 USA DI 1-207-985-1944 DI 2.P>Harlan Bioproducts for Science, Inc. DI P.O. Box 29176 DI Indianapolis, IN 46229-0176 DI 1-317-894-7536 DI 1-800-9-SCIENCE DE C>AMF7 ;developer DE P>AMF7 ;distributor DE P>P42770M ;distributor DE 2.P>MCA 683 ;distributor DO G>Mus musculus CN>mouse PD ;IgGl RE 1.G>Homo sapiens 1.CN>human l.SN>CD51 RE l.a.CC>differentiation AV ;purified ;2.purified SD AMF7 SD MCA683 SD P42770M LD USA BAL EI DA>9103 CV>9104 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 74 1021411 RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1021411 RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1.SN>CD51 AN 1021411 DI P>Immunotech S.A. DI Departement commercial DI Luminy Case 915 DI 13288 Marseille Cedex 9, France DI 33-91-41-41-38 DI 430246 F IMMTECH DE C>69-6-5 ;distributor DE P>1603 ;distributor DO G>Mus musculus CN>mouse S>BALB/c O>spleen PD ;IgG2a RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1.SN>CD51 RE 1.a.CC>protein AV ;purified SD 1603 SD 6965 LD USA JMJ EI DA>9707 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 75 1015384 RE 1.G>Mus musculus 1.CN>mouse 1.SN>CD51 1015384 RE 1.G>Mus musculus 1.CN>mouse l.SN>CD51 AN 1015384 DI P>PharMingen DI 10975 Torreyana Road DI San Diego, CA 92121 USA DI 1-619-677-7737 DI 1-800-848-6227 (toll free USA) DE C>H9.2B8 ;developer DE P>01520D ;distributor DE P>01521D ;distributor DE P>01522D ;distributor DE P>01525B ;distributor DO G>Cricetulus sp. CN>hamster IP G>Mus musculus CN>mouse PD ;IgG RE 1.G>Mus musculus 1.CN>mouse l.SN>CD51 RE 1.a.CC>differentiation AP ;flow cytometry ;immunofluorescence AV ;biotin conjugate ;fluorescein conjugate AV ;phycoerythrin conjugate ;purified SD 01520D SD 01521D SD 01522D SD 01524D SD 01525B SD H92B8 LD USA BAL EI DA>9408 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 76 1023962 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 1023962 RE l.G>Homo sapiens 1.CN>human l.SN>CD51 AN 1023962 SO Cell 1992;69:11-25 DI P>Ancell Corporation DI 243 Third Street North DI P.O. Box 87 DI Bayport, MN 55003 USA DI 1-800-374-9523 (toll free USA) DI 1-612-439-0835 DE C>P2W7 ;distributor DE P>202-020 ;distributor DE P>202-030 ;distributor DE P>202-040 ;distributor DE P>202-050 ;distributor IM G>Homo sapiens CN>human O>eye PA>melanoma CD>V+B2 a.CC>neoplasm DO G>Mus musculus CN>mouse PD ;IgGl ;kappa RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 RE 1.a.CC>differentiation AP ;immunoprecipitation ;flow cytometry ;frozen section AV ;R-phycoerythrin conjugate ;biotin conjugate AV ;fluorescein conjugate ;purified SD 202020 SD 202030 SD 202040 SD 202050 SD P2W7 LD USA MCM EI DA>9808 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 77 1023559 RE 1.G>Mus musculus 1.CN>mouse 1.SN>integrin 1023559 RE 1.G>Mus musculus 1.CN>mouse l.SN>integrin alpha V SO Biochemistry 1990;29:10191 SO Exp Cell Res 1993;205:25 DI P>Upstate Biotechnology, Inc. DI 199 Saranac Avenue DI Lake Placid, NY 12946 USA DI 1-617-890-8845 DI 1-800-233-3991 (toll free USA) (sales) DE P>05-437 ;distributor DO G>Mus musculus CN>mouse RE l.G>Mus musculus l.CN>mouse l.SN>integrin alpha V RE l.MW>160 kD l.a.CC>differentiation l.b.CC>receptor AP ;Western blot ;immunoprecipitation ;immunohistochemistry AV ;ascites AB Reactant is also known as vitronectin receptor alpha subunit and CD51. SD 05437 LD USA MCM EI DA>9807 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 78 1023927 RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin 1023927 RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin alpha V AN 1023927 SO J Biol Chem 1994;269:6940 DI P>Chemicon International, Inc. DI 28835 Single Oak Dr. DI Temecula, CA 92590 USA DI 1-909-676-8080 DI 1-800-437-7500(toll free USA) DE C>P3G8 ;distributor DE P>MAB1953 ;distributor IM G>Homo sapiens CN>human O>lung PA>carcinoma a.CC>neoplasm DO G>Mus musculus CN>mouse PD ;IgGl RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin alpha V RE l.a.CC>differentiation l.b.CC>receptor AP ;immunocytology ;immunohistochemistry ;immunoprecipitation AP ;flow cytometry ;ELISA ;FACS AV ;purified AB Reactant is also known as CD51 and vitronectin receptor alpha subunit. AB Product reacts with all alpha V-containing integrin receptors. AB Product will react with some lymphoid cell lines (B cells), many carcinoma and AB melanoma cell lines and osteosarcomas. SD MAB1953 SD P3G8 LD USA MCM EI DA>9808 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 79 1015432 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 1.FS 1015432 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 1.FS>alpha subunit AN 1015432 DI P>Biodesign International DI 105 York Street DI Kennebunkport, ME 04043 USA DI 1-207-985-1944 DI 2.P>Caltag Laboratories DI 1849 Bayshore Blvd. #200 DI Burlingame, CA 94010 DI 1-650-652-0468 DI 1-800-874-4007 DI 3.P>Cymbus Bioscience Limited DI 2 Venture Road DI Chilworth Research Center DI Southampton, Hampshire SO1 7NS UK DI 44-703-767178 DI 4.P>Endogen Inc. DI 30 Commerce Way DI Woburn, MA 01801-1059 USA DI 1-781-937-0890 DI 5.P>Genosys Biotechnologies, Inc. DI 1442 Lake Front Circle, Suite 185 DI The Woodlands, TX 77380-3600 USA DI 1-713-363-3693 DI 1-800-234-5362 (toll free USA) DI 6.P>Harlan Bioproducts for Science, Inc. DI P.O. Box 29176 DI Indianapolis, IN 46229-0176 DI 1-317-894-7536 DI 1-800-9-SCIENCE DI 7.P>Lampire Biological Laboratories DI P.O. Box 270 DI Pipersville, PA 18947 USA DI 1-215-795-2838 DI 8.P>PharMingen DI 10975 Torreyana Road DI San Diego, CA 92121 USA DI 1-619-677-7737 DI 1-800-848-6227 (toll free USA) DI 9.P>T Cell Diagnostics, Inc. DI 6 Gill Street DI Woburn, MA 01801-1721 USA DI 1-800-624-4021 DI 1-617-937-9587 DE C>23C6 ;developer DE P>23C6 ;distributor DE P>P54490M ;distributor DE 2.P>23C6 ;distributor DE 2.P>MON1167 ;distributor DE 3.P>23C6 ;distributor DE 3.P>CBL490 ;distributor DE 4.P>23C6 ;distributor DE 4.P>MA-5100 ;distributor DE 5.P>23C6 ;distributor DE 5.P>AM-19-760 ;distributor DE 6.P>23C6 ;distributor DE 6.P>MCA-757 ;discontinued designation SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 80 DE 6.P>MCA757G ;distributor DE 7.P>23C6 ;distributor DE 7.P>LBL 590 ;distributor DE 8.P>23C6 ;distributor DE 8.P>31561A ;distributor DE 8.P>31564X ;distributor DE 9.P>23C6 ;distributor DE 9.P>IAlSO4 ;distributor DO G>Mus musculus CN>mouse PD ;IgGl RE 1.G>Homo sapiens 1.CN>human 1.SN>CD51 1.FS>alpha subunit RE 1.MW>125 kD 1.a.CC>differentiation AV ;purified ;4.purified ;6.purified ;8.fluorescein conjugate AV ;8.purified ;9.supernatant SD 23C6 SD 31561A SD 31564X SD AM19760 SD CBL490 SD IAlSO4 SD LBL590 SD MA5100 SD MCA757 SD MCA757G SD MON1167 SD P54490M LD USA BAL EI DA>9706 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 81 1011348 RE 1.G>Homo sapiens 1.CN>human 1.SN>vitronect 1011348 RE 1.G>Homo sapiens 1.CN>human 1.SN>vitronectin receptor AN 1011348 DI P>Chemicon International, Inc. DI 28835 Single Oak Dr. DI Temecula, CA 92590 USA DI 1-909-676-8080 DI 1-800-437-7500(toll free USA) DE P>CLB-706 ;distributor DE P>MAB1980 ;distributor RE 1.G>Homo sapiens 1.CN>human 1.SN>vitronectin receptor RE l.a.CC>receptor AV ;purified AB beta subunit of vitronectin receptor referred to as CD51 also AB Reactant#1: vitronectin receptor beta subunit syn. for CD51 SD CLB706 SD MAB1980 LD USA BAL EI DA>9107 CV>9108 CI ;catalog SN Synonym>vitronectin receptor SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GBOO/02546 - 82 APPENDIX 3 DI is distributor SD is standard designation SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 83 Beta 5 1019741 RE 1.G>Homo sapiens l.CN>human 1.SN>integrin beta 5 AN 1019741 DI P>Upstate Biotechnology, Inc. DI 199 Saranac Avenue DI Lake Placid, NY 12946 USA DI 1-617-890-8845 DI 1-800-233-3991 (toll free USA) (sales) DE C>B5-IVF2 ;distributor DE P>05-283 ;distributor DO G>Mus musculus CN>mouse S>BALB/c O>spleen PD ;IgGl RE l.G>Homo sapiens l.CN>human l.SN>integrin beta 5 RE l.a.CC>protein AP ;immunocytochemistry ;blocks cell adhesion AV ;ascites SD 05283 SD B5IVF2 LD USA JMJ EI DA>9611 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 84 Beta 3 1011332 RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin beta 3 AN 1011332 DI P>Chemicon International, Inc. DI 28835 Single Oak Dr. DI Temecula, CA 92590 USA DI 1-909-676-8080 DI 1-800-437-7500(toll free USA) DE P>MAB1974 ;distributor PD ;IgGl RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin beta 3 RE l.a.CC>receptor AV ;ascites SD MAB1974 LD USA BAL EI DA>9107 CV>9108 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 85 1014236 RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin beta 3 AN 1014236 AU Ylanne J SO Blood 1989;72:1478-86 SO Blood 1990;76:570-7 DI P>Biohit OY DI Verkkosaarenkatu 4 DI 00580 Helsinki, Finland DI 358-0-773-2900 DI 2.P>Harlan Bioproducts for Science, Inc. DI P.O. Box 29176 DI Indianapolis, IN 46229-0176 DI 1-317-894-7536 DI 1-800-9-SCIENCE DI 3.P>ICN Biomedicals DI Biomedical Research Products DI 3300 Hyland Avenue DI Costa Mesa, CA 92626 DI 1-800-854-0530 (toll free USA) DI 1-714-545-0100 DI 4.P>Locus Genex Oy DI Verkkosaarenkatu 4 DI 00580 Helsinki, Finland DI 358-9-773-861 DE C>BB10 ;developer DE P>M-9006000 ;distributor DE P>M-9006100 ;distributor DE 2.P>MCA-781 ;distributor DE 2.P>bblO ;distributor DE 3.P>69-323-1 ;distributor DE 3.P>69-323-2 ;distributor DE 3.P>BB10 ;distributor DE 4.P>BB10 ;distributor DE 4.P>M-9006000 ;distributor DE 4.P>M-9006100 ;distributor IM G>Homo sapiens CN>human SN>CD41 ;purified a.CC>differentiation RM ;in vivo DO G>Mus musculus CN>mouse S>BALB/c O>spleen IP G>Mus musculus CN>mouse PA>myeloma PD ;IgGl RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin beta 3 RE 1.a.CC>receptor RE 2.G>Homo sapiens 2.CN>human 2.SN>platelet GPIIIa RE 2.a.CC>blood coagulation factor AP ;immunoassay ;not paraffin section ;immunohistochemical staining AP ;immunoblotting AV ;purified ;2.purified ;3.purified ;4.purified SD 693231 SD 693232 SD BB10 SD M9006000 SD M9006100 SD MCA781 LD USA BAL SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GBOO/02546 - 86 EI DA>9711 SN Synonym>CD41 1014236 SN Synonym>CD41 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 -87 1014281 RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin beta 3 AN 1014281 DI P>Bio-Science Products AG DI Gerliswilstrasse 43 DI Postfach 1173 DI CH-6020 Emmenbrucke, Switzerland DI 41-555875 DE P>0121022 ;distributor RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin beta 3 RE 1.a.CC>receptor RE 2.G>Homo sapiens 2.CN>human 2.SN>CD41 RE 2.a.CC>differentiation AP ;immunoassay ;immunoblotting ;not paraffin section AP ;immunohistochemical staining LD USA BAL EI DA>9305 CI ;catalog SN Synonym>CD41 1014281 SN Synonym>CD41 SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 88 1019109 RE l.G>Homo sapiens l.CN>human l.SN>integrin beta 3 AN 1019109 SO J Cell Biol 1993;122:223 SO J Cell Biol 1993;121:689 DI P>Transduction Laboratories DI 133 Venture Ct., Suite 5 DI Lexington, Ky 40511-9923 DI 1-606-259-1550 DI 1-800-227-4063 DE P>26 ;distributor DE P>I19620 ;distributor IM G>Mus musculus CN>mouse SN>integrin beta 3 FS>amino acids 16-223 IM a.CC>protein DO G>Mus musculus CN>mouse PD ;IgM RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin beta 3 RE 1.MW>90 kD 1.a.CC>protein RE 2.G>Canis familiaris 2.CN>dog 2.SN>integrin beta 3 RE 2.MW>90 kD 2.a.CC>protein RE 3.G>Rattus norvegicus 3.CN>Norway rat 3.SN>integrin beta 3 RE 3.MW>90 kD 3.a.CC>protein RE 4.G>Mus musculus 4.CN>mouse 4.SN>integrin beta 3 RE 4.MW>90 kD 4.a.CC>protein RE 5.G>Gallus gallus 5.CN>chicken 5.SN>integrin beta 3 RE 5.MW>90 kD 5.a.CC>protein AP ;Western blot AV ;purified SD 26 SD 119620 LD USA JMJ EI DA>9705 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 89 1023930 RE l.G>Homo sapiens l.CN>human l.SN>integrin beta 3 AN 1023930 SO Cell 1986;45:269-80 DI P>Chemicon International, Inc. DI 28835 Single Oak Dr. DI Temecula, CA 92590 USA DI 1-909-676-8080 DI 1-800-437-7500(toll free USA) DE C>25E11 ;distributor DE P>MAB1957 ;distributor IM T>blood CE>mononuclear cell CS>activated DO G>Mus musculus CN>mouse PD ;IgG2a RE l.G>Homo sapiens l.CN>human l.SN>integrin beta 3 RE l.a.CC>differentiation 1.b.CC>receptor RE 2.SN>CD41/CD61 complex 2.a.CC>protein RE 3.G>Homo sapiens 3.CN>human 3.CE>monoblast 3.PA>leukemia RE 3.CD>U937 3.SN>CD41/CD61 complex 3.a.CC>protein AP ;Western blot ;immunocytology ;immunoprecipitation AP ;flow cytometry AV ;purified a.PM>protein A chromatography AB Integrin beta 3 is also known as CD61, GPIIIa, and vitronectin AB receptor beta chain. SD 25E11 SD MAB1957 LD USA MCM EI DA>9808 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 90 1024059 RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1.SN>CD61 AN 1024059 DI P>PharMingen DI 10975 Torreyana Road DI San Diego, CA 92121 USA DI 1-619-677-7737 DI 1-800-848-6227 (toll free USA) DE C>VI-PL2 ;distributor DE P>33821 ;distributor DE 2>33824 ;distributor DE P>33825 ;distributor DO G>Mus musculus CN>mouse PD ;IgGl ;kappa RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet l.SN>CD61 RE 1.MW>105 kD 1.a.CC>differentiation 1.b.CC>glycoprotein RE 2.G>Homo sapiens 2.CN>human 2.CE>megakaryocyte 2.SN>CD61 RE 2.MW>105 kD 2.a.CC>differentiation 2.b.CC>glycoprotein RE 3.G>Homo sapiens 3.CN>human 3.CE>osteoclast 3.SN>CD61 RE 3.MW>105 kD 3.a.CC>differentiation 3.b.CC>glycoprotein RE 4.G>Homo sapiens 4.CN>human 4.T>endothelium 4.SN>CD61 RE 4.MW>105 kD 4.a.CC>differentiation 4.b.CC>glycoprotein XR 1.G>Canis sp. 1.CN>dog 1.SN>CD61 l.a.CC>differentiation XR 1.b.CC>glycoprotein XR 2.G>Felis sp. 2.CN>cat 2.SN>CD61 2.a.CC>differentiation XR 2.b.CC>glycoprotein NR 1.G>Sus sp. 1.CN>swine l.SN>CD61 1.a.CC>differentiation NR 1.b.CC>glycoprotein AP ;acetone fixed ;frozen section AV ;R-phycoerythrin conjugate ;biotin conjugate AV ;fluorescein conjugate ;purified AB CD61 is also known as integrin beta 3 subunit. SD 33821 SD 33824 SD 33825 SD VIPL2 LD USA MCM EI DA>9809 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 91 1011347 RE 1.G>Homo sapiens 1.CN>human 1.SN>vitronectin receptor AN 1011347 DI P>Chemicon International, Inc. DI 28835 Single Oak Dr. DI Temecula, CA 92590 USA DI 1-909-676-8080 DI 1-800-437-7500(toll free USA) DE P>MAB1984 ;distributor RE 1.G>Homo sapiens 1.CN>human 1.SN>vitronectin receptor RE 1.a.CC>receptor AB Reactant#1 vitronectin receptor alpha subunit syn. for CD61 SD MAB1984 LD USA BAL EI DA>9107 CV>9108 CI -;catalog SN Synonym>vitronectin receptor SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 92 1012445 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD61 AN 1012445 DI P>Cymbus Bioscience Limited DI 2 Venture Road DI Chilworth Research Center DI Southampton, Hampshire SO1 7NS UK DI 44-703-767178 DE C>Thromb/1 ;developer DE P>CBL 458 ;discontinued designation RE 1.G>Homo sapiens 1.CN>human 1.SN>CD61 RE 1.a.CC>differentiation SD THROMB1 LD USA BAL EI DA>9110 CV>9111 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 93 1013416 RE 1.CE>platelet 1.SN>CD61 l.a.CC>differentiation AN 1013416 DI P>Caltag Laboratories DI 1849 Bayshore Blvd. #200 DI Burlingame, CA 94010 DI 1-650-652-0468 DI 1-800-874-4007 DI 2.P>Medica DI 2382 Camino Vida Roble, Suite I DI Carlsbad, CA 92009 USA DI 1-619-438-1886 DI 3.P>Sigma Chemical Company DI P.O. Box 14508 DI St. Louis, MO 63178 9916 USA DI 1-800-325-3010 (toll free USA) DI 1-314-771-5750 DE C>BL-E6 ;developer DE P>BL-E6 ;distributor DE P>MHCD6101 ;distributor DE P>MHCD6101-4 ;distributor DE P>MHCD6115 ;distributor DE P>MHCD6115-4 ;distributor DE P>MON1051 ;distributor DE 2.P>MON1051 ;distributor DE 3.P>BL-E6 ;distributor DE 3.P>C4321 ;distributor DE 3.P>F7902 ;distributor DO G>Mus musculus CN>mouse PD ;IgGl RE 1.CE>platelet 1.SN>CD61 1.a.CC>differentiation RE 2.CE>megakaryocyte 2.SN>CD61 2.a.CC>differentiation AP ;frozen section AV ;biotin conjugate ;fluorescein conjugate AV ;3.fluorescein conjugate SD BLE6 SD C4321 SD F7902 SD MHCD6101 SD MHCD61014 SD MHCD6115 SD MHCD61154 SD MON1051 LD USA BAL EI DA>9803 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 94 1013417 RE 1.CE>platelet 1.SN>CD61 1.a.CC>differentiation AN 1013417 DI P>Caltag Laboratories DI 1849 Bayshore Blvd. #200 DI Burlingame, CA 94010 DI 1-650-652-0468 DI 1-800-874-4007 DI 2.P>Medica DI 2382 Camino Vida Roble, Suite I DI Carlsbad, CA 92009 USA DI 1-619-438-1886 DE C>CRC54 ;developer DE P>CRC54 ;distributor DE P>MON1147 ;distributor DE 2.P>MON1147 ;distributor PD ;IgGl RE 1.CE>platelet 1.SN>CD61 l.a.CC>differentiation RE 2.CE>megakaryocyte 2.SN>CD61 2.a.CC>differentiation AP ;frozen section SD MON1147 LD USA BAL EI DA>9303 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 -95 1014017 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD61 DI P>Diagast Laboratories DI 59, rue de Trevise-B.P. 2034 DI 59014 Lille Cedex, France DI 33-20-52-68-00 DI DIAGAST (042) 160716F DE P>16101V ;distributor DE P>16103A ;distributor DE P>16105E ;distributor PD ;IgG RE 1.G>Homo sapiens 1.CN>human 1.SN>CD61 RE l.a.CC>differentiation AV ;fluorescein conjugate ;phycoerythrin conjugate ;purified SD 16101V SD 16103A SD 16105E LD USA BAL EI DA>9304 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 96 1014618 RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1.SN>CD61 AN 1014618 DI P>BioSource International DI 820 Flynn Roa DI Camarillo, CA 93012 USA DI 1-800-242-0607(toll free USA) DI 1-805-987-0086 DI 2.P>Cymbus Bioscience Limited DI 2 Venture Road DI Chilworth Research Center DI Southampton, Hampshire S01 7NS UK DI 44-703-767178 DI 3.P>Endogen Inc. DI 30 Commerce Way DI Woburn, MA 01801-1059 USA DI 1-781-937-0890 DI 4.P>Genosys Biotechnologies, Inc. DI 1442 Lake Front Circle, Suite 185 DI The Woodlands, TX 77380-3600 USA DI 1-713-363-3693 DI 1-800-234-5362 (toll free USA) DI 5.P>Harlan Bioproducts for Science, Inc. DI P.O. Box 29176 DI Indianapolis, IN 46229-0176 DI 1-317-894-7536 DI 1-800-9-SCIENCE DI 6.P>Lampire Biological Laboratories DI P.O. Box 270 DI Pipersville, PA 18947 USA DI 1-215-795-2838 DI 7.P>Novocastra Laboratories Ltd. DI 24 Claremont Place DI Newcastle upon Tyne NE2 4AA, UK DI 44-0191 222 8550 DI 8.P>Southern Biotechnology Associates, Inc. DI P.O. Box 26221 DI Birmingham, AL 35260 USA DI 1-800-722-2255 (toll free USA) DI 1-205-945-1774 DI 9.P>T Cell Diagnostics, Inc. DI 6 Gill Street DI Woburn, MA 01801-1721 USA DI 1-800-624-4021 DI 1-617-937-9587 DE C>PM 6/13 ;developer DE P>AHS6101 ;distributor SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 97 DE P>AHS6107 ;distributor DE P>AHS6108 ;distributor DE P>CS-CD61-CF ;discontinued designation DE P>CS-CD61-FI ;discontinued designation DE P>CS-CD61-PE ;discontinued designation DE 2.P>CBL479 ;distributor DE 2.P>PM6/13 ;distributor DE 3.P>MA-6100 ;distributor DE 3.P>PM6/13 ;distributor DE 4.P>AM-19-705 ;distributor DE 4.P>PM6/13 ;distributor DE 5.P>MCA-728 ;distributor DE 5.P>MCA-728F ;distributor DE 5.P>MCA-728PE ;distributor DE 5.P>PM6/13 ;distributor DE 6.P>LBL 579 ;distributor DE 6.P>PM6/13 ;distributor DE 7.P>NCL-CD61 ;distributor DE 7.P>PM6/13 ;distributor DE 8.P>9470-01 ;distributor DE 8.P>9470-02 ;distributor DE 8.P>9470-08 ;distributor DE 8.P>PM6/13 ;distributor DE 9.P>IA1SO9 ;distributor DE 9.P>PM6/13 ;distributor DO G>Mus musculus CN>mouse PD ;IgGl RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1.SN>CD61 RE 1.MW>90 kD 1.a.CC>differentiation RE 2.G>Homo sapiens 2.CN>human 2.T>plasma 2.SN>CD61 RE 2.MW>90 kD 2.a.CC>differentiation RE 3.G>Homo sapiens 3.CN>human 3.T>plasma RE 3.PA>unspecified neoplasm 3.SN>CD61 3.MW>90 kD RE 3.a.CC>neoplasm 3.b.CC>differentiation AV ;fluorescein conjugate ;phycoerythrin conjugate ;purified AV ;3.purified ;5.fluorescein conjugate AV ;5.phycoerythrin conjugate ;5.purified ;8.biotin conjugate AV ;8.fluorescein conjugate ;8.purified ;9.purified SD 947001 SD 947002 SD 947008 SD AHS6101 SD AHS6107 SD AHS6108 SD AM19705 SD CBL479 SD CSCD61CF SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 98 SD CSCD61FI SD CSCD61PE SD IAlSO9 SD LBL579 SD MA6100 SD MCA728 SD MCA728F SD MCA728PE SD NCLCD61 SD PM613 LD USA BAL EI DA>9904 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 99 1017042 RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1.SN>CD61 AN 1017042 DI P>Becton Dickinson Immunocytometry Systems DI 2350 Qume Drive DI San Jose, CA 95131-1807 DI 1-800-223-8226 (toll free USA) DI 1-408-954-2347 DE P>348090 ;distributor DE P>348093 ;distributor DE P>559936 ;distributor DE P>RUU-PL7F12 ;distributor DO G>Mus musculus CN>mouse S>BALB/c PD ;IgGl ;kappa RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1.SN>CD61 RE 1.a.CC>differentiation 1.b.CC>protein AP ;immunofluorescence ;immunoprecipitation AV ;fluorescein conjugate ;purified SD 348090 SD 348093 SD 559936 SD RUUPL7F12 LD USA JMJ EI DA>9505 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 100 1017635 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD61 AN 1017635 DI P>Novocastra Laboratories Ltd. DI 24 Claremont Place DI Newcastle upon Tyne NE2 4AA, UK DI 44-0191 222 8550 DE P>NCL-CD61 ;distributor DE P>PM6/13 ;distributor DO G>Mus musculus CN>mouse AS ;immunohistochemical staining RE 1.G>Homo sapiens 1.CN>human 1.SN>CD61 RE l.a.CC>differentiation AP ;frozen section AV ;ascites SD NCLCD61 SD PM613 LD USA BAL EI DA>9904 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 101 1019745 RE 1.G>Homo sapiens 1.CN>human 1.SN>CD61 AN 1019745 DI P>Upstate Biotechnology, Inc. DI 199 Saranac Avenue DI Lake Placid, NY 12946 USA DI 1-617-890-8845 DI 1-800-233-3991 (toll free USA) (sales) DE P>05-275 ;distributor DO G>Mus musculus CN>mouse S>BALB/c O>spleen PD ;IgGl RE l.G>Homo sapiens 1.CN>human 1.SN>CD61 RE 1.a.CC>differentiation l.b.CC>protein AP ; immunocytochemistry AV ;ascites SD 05275 LD USA JMJ EI DA>9611 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 102 1020416 RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1.SN>CD61 AN 1020416 DI P>PanVera Corporation DI 545 Science Drive DI Madison, WI 53711 USA DI 1-800-791-1400 DI 1-608-233-9450 DE C>PL8-5 ;distributor DE P>TAK M068 ;distributor DO G>Mus musculus CN>mouse S>BALB/c O>spleen PD ;IgG RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1.SN>CD61 RE l.a.CC>differentiation 1.b.CC>protein AV ;purified SD PL85 SD TAKM068 LD USA JMJ EI DA>9702 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 103 1020417 RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1.SN>CD61 AN 1020417 DI P>PanVera Corporation DI 545 Science Drive DI Madison, WI 53711 USA DI 1-800-791-1400 DI 1-608-233-9450 DE C>PL11-7 ;distributor DE P>TAK M069 ;distributor DO G>Mus musculus CN>mouse S>BALB/c O>spleen PD ;IgG RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1.SN>CD61 RE 1.a.CC>differentiation 1.b.CC>protein AV ;purified SD PL117 SD TAKMO69 LD USA JMJ EI DA>9702 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 104 1021005 RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1.SN>CD61 AN 1021005 DI P>Cortex Biochem, Inc. DI 1933 Davis Street, Suite 321 DI San Leandro, CA 94577 USA DI 1-800-888-7713 (toll free USA) DI 1-510-568-3911(technical) DE P>CR1153 ;distributor RE 1.G>Homo sapiens 1.CN>human 1.CE>platelet 1.SN>CD61 RE 1.a.CC>differentiation RE 2.G>Homo sapiens 2.CN>human 2.CE>megakaryocyte 2.SN>CD61 RE 2.a.CC>differentiation LD USA CLB EI DA>9704 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 105 1011328 RE l.G>Homo sapiens 1.CN>human l.SN>integrin alpha V AN 1011328 DI P>Chemicon International, Inc. DI 28835 Single Oak Dr. DI Temecula, CA 92590 USA DI 1-909-676-8080 DI 1-800-437-7500(toll free USA) DI 2.P>Life Technologies, Inc. DI 8400 Helgerman Ct. DI P.O. Box 6009 DI Gaithersburg, MD 20884-9980 USA DI 1-301-840-8000 DI 1-800-828-6686 (Toll free USA) DE P>MAB1958 ;distributor DE P>VNR147 ;distributor DE 2.P>12084-018 ;distributor DE 2.P>VNR147 ;distributor PD ;IgGl RE l.G>Homo sapiens l.CN>human l.SN>integrin alpha V RE 1.a.CC>receptor AV ;ascites ;2.ascites SD 12084018 SD MAB1958 SD VNR147 LD USA BAL EI DA>9706 CV>9108 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 106 1011329 RE l.G>Homo sapiens l.CN>human l.SN>integrin alpha V AN 1011329 DI P>Chemicon International, Inc. DI 28835 Single Oak Dr. DI Temecula, CA 92590 USA DI 1-909-676-8080 DI 1-800-437-7500(toll free USA) DI 2.P>Life Technologies, Inc. DI 8400 Helgerman Ct. DI P.O. Box 6009 DI Gaithersburg, MD 20884-9980 USA DI 1-301-840-8000 DI 1-800-828-6686 (Toll free USA) DE P>MAB1960 ;distributor DE P>VNR139 ;distributor DE 2.P>12085-015 ;distributor DE 2.P>VNR139 ;distributor PD ;IgG1 RE l.G>Homo sapiens l.CN>human l.SN>integrin alpha V RE l.a.CC>receptor AV ;ascites ;2.ascites SD 12085015 SD MAB1960 SD VNR139 LD USA BAL EI DA>9706 CV>9108 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 107 1011842 RE 1.G>Homo sapiens 1.CN>human l.SN>integrin alpha V AN 1011842 DI P>Biogenesis Ltd. DI 7 New Fields DI Stinsford Road DI Poole BH17 7NF, England DI UK DI 44-1202 660006 DE C>1U4/1 ;developer DE P>5355-2505 ;distributor RE l.G>Homo sapiens l.CN>human 1.SN>integrin alpha V RE l.a.CC>protein AP ;immunoblotting AV ;ascites SD 1U41 SD 53552505 LD USA BAL EI DA>9109 CV>9110 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 108 1011843 RE 1.G>Homo sapiens 1.CN>human l.SN>integrin alpha V AN 1011843 DI P>Biogenesis Ltd. DI 7 New Fields DI Stinsford Road DI Poole BH17 7NF, England DI UK DI 44-1202 660006 DE C>1U3/0 ;developer DE P>5355-2515 ;distributor RE 1.G>Homo sapiens l.CN>human l.SN>integrin alpha V RE 1.a.CC>protein AP immunofluorescencee AV ;ascites SD 1U30 SD 53552515 LD USA BAL EI DA>9109 CV>9110 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 109 1012705 RE 1.G>Homo sapiens 1.CN>human l.SN>integrin alpha V AN 1012705 DI P>Chemicon International, Inc. DI 28835 Single Oak Dr. DI Temecula, CA 92590 USA DI 1-909-676-8080 DI 1-800-437-7500(toll free USA) DE P>LM142 ;distributor DE P>MAB1978 ;distributor PD ;IgG RE l.G>Homo sapiens 1.CN>human 1.SN>integrin alpha V RE l.a.CC>protein AV ;ascites SD LM142 SD MAB1978 LD USA BAL EI DA>9112 CV>9201 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 110 1013223 RE 1.SN>integrin alpha V 1.a.CC>protein AN 1013223 DI P>American Qualex International, Inc. DI 920-A Calle Negocio St. DI San Clemente, CA 92673 DI 1-714-521-3753 DI 1-800-772-1776 (toll free USA) DE P>M2580 ;distributor RE 1.SN>integrin alpha V 1.a.CC>protein SD M2580 LD USA BAL EI DA>9204 CV>9204 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 111 1013510 RE 1.G>Homo sapiens 1.CN>human 1.CE>endothelial cell AN 1013510 AU Freed E SO EMBO J 1989;8:2955 DI P>Calbiochem Novabiochem International DI P.O. Box 12087 DI La Jolla, CA 92039-2087 DI 1-800-854-3417(toll free USA) DI 1-619-450-9600 DE P>407281 ;distributor PD ;IgGl RE 1.G>Homo sapiens 1.CN>human l.CE>endothelial cell RE 1.SN>integrin alpha V 1.FS>type 1 1.a.CC>protein RE 2.G>Homo sapiens 2.CN>human 2.SN>vitronectin RE 2.a.CC>protein RE 3.G>Homo sapiens 3.CN>human 3.SN>fibrinogen RE 3.a.CC>protein RE 4.G>Homo sapiens 4.CN>human 4.SN>osteopontin RE 4.a.CC>protein RE 5.G>Homo sapiens 5.CN>human 5.SN>von Willebrand factor RE 5.a.CC>protein RE 6.G>Homo sapiens 6.CN>human 6.O>bone 6.SN>sialoprotein I RE 6.a.CC>protein AP ;ELISA ;immunofluorescence ;immunoprecipitation AV ;ascites ;lyophilized SD 407281 LD USA BAL EI DA>9303 CI ;catalog SN Synonym>fibrinogen SN Synonym>von Willebrand factor SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 112 1013511 RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin alpha V AN 1013511 DI P>Calbiochem Novabiochem International DI P.O. Box 12087 DI La Jolla, CA 92039-2087 DI 1-800-854-3417(toll free USA) DI 1-619-450-9600 DE P>407282 ;distributor PD ;IgGl RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin alpha V RE 1.FS>type 2 1.a.CC>protein RE 2.G>Homo sapiens 2.CN>human 2.SN>vitronectin RE 2.a.CC>protein RE 3.G>Homo sapiens 3.CN>human 3.SN>fibrinogen RE 3.a.CC>protein RE 4.G>Homo sapiens 4.CN>human 4.SN>osteopontin RE 4.a.CC>protein RE 5.G>Homo sapiens 5.CN>human 5.SN>von Willebrand factor RE 5.a.CC>protein RE 6.G>Homo sapiens 6.CN>human 6.0>bone 6.SN>sialoprotein I RE 6.a.CC>protein AP ;ELISA ;immunoblotting AV ;ascites ;lyophilized SD 407282 LD USA BAL EI DA>9303 CI ;catalog SN Synonym>fibrinogen SN Synonym>von Willebrand factor SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 113 1014225 RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin alpha V beta 5 AN 1014225 AU Wayner EA SO J Cell Biol 1991;113:919 DI P>Becton Dickinson Immunocytometry Systems DI 2350 Qume Drive DI San Jose, CA 95131-1807 DI 1-800-223-8226 (toll free USA) DI 1-408-954-2347 DI 2.P>Chemicon International, Inc. DI 28835 Single Oak Dr. DI Temecula, CA 92590 USA DI 1-909-676-8080 DI 1-800-437-7500(toll free USA) DI 3.P>Life Technologies, Inc. DI 8400 Helgerman Ct. DI P.O. Box 6009 DI Gaithersburg, MD 20884-9980 USA DI 1-301-840-8000 DI 1-800-828-6686 (Toll free USA) DI 4.P>Telios Pharmaceuticals, Inc. DI 4757 Nexus Centre Drive DI San Diego, CA 92121 USA DI 1-619-622-2650 DE C>P1F6 ;developer DE P>550045 ;distributor DE P>P1F6 ;distributor DE 2.P>MAB1961 ;distributor DE 2.P>PlF6 ;distributor DE 3.P>12078-010 ;distributor DE 3.P>P1F6 ;distributor DE 4.P>A035 ;distributor DO G>Mus musculus CN>mouse PD ;IgGl RE 1.G>Homo sapiens l.CN>human l.SN>integrin alpha V beta 5 RE 1.a.CC>receptor AP ;immunofluorescence ;immunoprecipitation AV ;2.ascites ;3.ascites ;4.ascites SD 12078010 SD 550045 SD A035 SD MAB1961 SD P1F6 LD USA BAL EI DA>9706 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 114 1014277 RE 1.G>Homo sapiens 1.CN>human l.CE>endothelial cell AN 1014277 DI P>Bio-Science Products AG DI Gerliswilstrasse 43 DI Postfach 1173 DI CH-6020 Emmenbrucke, Switzerland DI 41-555875 DE P>0121005 ;distributor RE l.G>Homo sapiens l.CN>human 1.CE>endothelial cell RE 1.SN>integrin alpha V l.a.CC>receptor RE 2.G>Homo sapiens 2.CN>human 2.PA>carcinoma RE 2.SN>integrin alpha V 2.a.CC>neoplasm 2.b.CC>receptor NR 1.G>Homo sapiens 1.CN>human l.SN>fibronectin receptor NR l.a.CC>receptor NR 2.G>Homo sapiens 2.CN>human 2.SN>CD41 NR 2.a.CC>differentiation AP ;ELISA immunofluorescencee ;not immunoblotting AP ;immunoprecipitation LD USA BAL EI DA>9305 CI ;catalog SN Synonym>CD41 1014277 SN Synonym>CD41 SN Synonym>fibronectin receptor SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 115 1014278 RE l.G>Homo sapiens l.CN>human l.CE>endothelial cell AN 1014278 DI P>Bio-Science Products AG DI Gerliswilstrasse 43 DI Postfach 1173 DI CH-6020 Emmenbrucke, Switzerland DI 41-555875 DE P>0121006 ;distributor AS ;immunoblot RE l.G>Homo sapiens l.CN>human l.CE>endothelial cell RE l.SN>integrin alpha V l.a.CC>receptor RE 2.G>Homo sapiens 2.CN>human 2.PA>carcinoma RE 2.SN>integrin alpha V 2.a.CC>neoplasm 2.b.CC>receptor AP ;immunoassay LD USA BAL EI DA>9305 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 116 1023559 RE l.G>Mus musculus l.CN>mouse l.SN>integrin alpha V SO Biochemistry 1990;29:10191 SO Exp Cell Res 1993;205:25 DI P>Upstate Biotechnology, Inc. DI 199 Saranac Avenue DI Lake Placid, NY 12946 USA DI 1-617-890-8845 DI 1-800-233-3991 (toll free USA) (sales) DE P>05-437 ;distributor DO G>Mus musculus CN>mouse RE l.G>Mus musculus l.CN>mouse l.SN>integrin alpha V RE l.MW>160 kD l.a.CC>differentiation l.b.CC>receptor AP ;Western blot ;immunoprecipitation ;immunohistochemistry AV ;ascites AB Reactant is also known as vitronectin receptor alpha subunit and CD51. SD 05437 LD USA MCM EI DA>9807 CI ;catalog SUBSTITUTE SHEET (RULE 26) WO 01/02005 PCT/GB00/02546 - 117 1023927 RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin alpha V AN 1023927 SO J Biol Chem 1994;269:6940 DI P>Chemicon International, Inc. DI 28835 Single Oak Dr. DI Temecula, CA 92590 USA DI 1-909-676-8080 DI 1-800-437-7500 (toll free USA) DE C>P3G8 ;distributor IM G>Homo sapiens CN>human O>lung PA>carcinoma a.CC>neoplasm DO G.Mus musculus CN>mouse PD ;IgGl RE 1.G>Homo sapiens 1.CN>human 1.SN>integrin alpha V RE l.a.CC>differentiation 1.b.CC>receptor AP ; immunocytology ; immunohistochemistry ; immunoprecipitation AP ;flow cytometry ;ELISA ;FACS AV ;purified AB Reactant is also known as CD51 and vitronectin receptor alpha subunit. AB Product reacts with all alpha V-containing integrin receptors. AB Product will react with some lymphoid cell lines (B cells), many AB carcinoma and melanoma cell lines and osteosarcomas. SD MAB1953 SD P3G8 LD USA MCM EI DA>9808 CI ;catalog SUBSTITUTE SHEET (RULE 26)

Claims

1. A method of treating mammalian dendritic cells in vitro to induce immune tolerance therein 5 which comprises exposing said cells to an agonist of the cell surface receptors CD36 and/or CD51 as expressed on mammalian dendritic cells.

2. A method as claimed in claim 1 wherein said 10 dendritic cells are human or mouse.

3. A method as claimed in claim 1 or 2 wherein said agonist is a molecule identified as such by any one of the methods of claims 15 to 32. 15

4. A method as claimed in claim 1 or 2 wherein said agonist is selected from: an antibody with an affinity for an epitope of CD36, an antibody with an affinity for an epitope of CD51, the Plasmodium 20 falciparum protein pf-EMP-1, a protein comprising the active binding domain of pf-EMP-1, thrombospondin, apoptotic cells or a negatively charged phospholipid.

5. A method as claimed in claim 1 or 2 which 25 comprises exposing said dendritic cells to two or more of the agonists of claim 4.

6. A method as claimed in claim 4 or claim 5 wherein the CD36 agonist any one of the antibodies 30 listed in Appendix 1.

7. A method as claimed in claim 4 or claim 5 wherein the CD51 agonist is any one of the antibodies listed in Appendix 2. 35

8. A method as claimed in claim 4 or claim 5 WO 01/02005 PCT/GB00/02546 - 119 wherein the pf-EMP-1 active binding domain comprises the amino acid sequence shown in Figure 2.

9. A method as claimed in any one of claims 40 5 to 47 wherein said cells are exposed to an antigenic material.

10. A method as claimed in claim 9 wherein said antigenic material is an auto-antigen associated with 10 a particular auto-immune disease.

11. A method as claimed in claim 9 or 10 wherein the cells so produced are subsequently matured by exposure to an immune stimulus. 15

12. A method as claimed in any of claims 1 to 11 wherein said dendritic cells are prepared from human peripheral blood and or derived from CD34+ stem cells or monocytes. 20

13. A dendritic cell preparation obtainable by the method of any of claims 1 to 12 for use as a medicament. 25

14. A dendritic cell preparation obtainable by the method of any of claims 1 to 12 for use in inducing peripheral immune tolerance in a human.

15. A method of identifying a molecule which is 30 an agonist of cell surface.receptor CD36 and/or CD51 as expressed by mammalian dendritic cells which method comprises: a) exposing immature mammalian dendritic cells to the molecule to be tested, 35 b) exposing said immature dendritic cells to an immune stimulus and WO 01/02005 PCT/GB00/02546 - 120 c) determining the degree of maturation manifested by said dendritic cells, wherein impaired maturation in response to the immune stimulus is an indication that said molecule 5 under test is a CD36 and/or CD51 agonist.

16. A method as claimed in claim 15 wherein said dendritic cells are human cells or mouse cells. 10

17. A method as claimed in claim 15 or 16. wherein maturation of said dendritic cells is determined by examining the antigen-presenting ability of said cells. 15

18. A method as claimed in any of claims 15 to 17 wherein maturation of said dendritic cells is determined by examining said cells for expression of at least one cell surface antigen whose level of expression is enhanced in response to an immune 20 stimulus.

19. A method as claimed in claim 18 wherein maturation of said dendritic cells is determined by measuring the level of expression of one or more of 25 the following panel of antigens: HLA DR, CD54, CD40, CD83 and CD86.

20. A method as claimed in claim 19 wherein said cells are also examined for expression of CD80. 30

21. A method as claimed in any one of claims 18 to 20 wherein the level of expression of said antigens is detected using a labelled antibody. 35

22. A method as claimed in 15 or 16 wherein maturation of said dendritic cells is determined by measuring said cells' ability to induce T-cell WO 01/02005 PCT/GB00/02546 - 121 proliferation.

23. The method of claim 15 or 16 wherein maturation of said dendritic cells is determined by 5 quantifying the level of cytokines secreted from said cells.

24. The method of claim 23 wherein the level of secretion TNFa, IL12P70 and IL10 is measured. 10

25. A method as claimed in any one of claims 15 to 24 wherein said immune stimulus is lipopolysaccharide, TNFa, CD40L or monocyte conditioned medium (MCM). 15

26. A method as claimed in any one of claims 15 to 25 wherein if said test molecule is found to be a potential agonist of CD36 and/or CD51 the method further comprises the step of exposing said molecule 20 to a purified sample of CD36 and/or CD51 and detecting any direct binding between said molecule and CD36 and/or CD51.

27. A method as claimed in claim 26 wherein said 25 purified CD36 or CD51 is immobilised to a solid surface.

28. A method as claimed in claim 26 or claim 27 wherein said molecule is labelled with a detectable 30 label.

29. A method as claimed in any of claims 26 to 28 which further comprises the step of exposing said molecule to a purified sample of avs 3 or aP 5 and 35 detecting any direct binding between said molecule and said as 3 or av 5 . WO 01/02005 PCT/GB00/02546 - 122

30. A method as claimed in any one of claims 26 to 29 which further comprises the step of exposing said molecule to a purified sample of thrombospondin and detecting any direct binding between said molecule 5 and thrombospondin.

31. A method as claimed in claim 29 or claim 30 wherein said molecule is labelled with a detectable label. 10

32. A method as claimed in any of claims 29 to 31 wherein said ap3, avA 5 or thrombospondin is immobilised to a solid surface. 15

33. A pharmaceutical composition suitable for inducing peripheral immune tolerance in a mammal which comprises an agonist of the cell surface receptor CD36 as expressed on mammalian dendritic cells and a pharmacologically acceptable carrier or diluent. 20

34. A composition as claimed in claim 33 which is suitable for inducing peripheral immune tolerance in a human wherein said CD36 agonist is selected from: an antibody with an affinity for an epitope of CD36, 25 the Plasmodium falciparum protein pf-EMP-1, a protein comprising the active binding domain of pf-EMP-1 for CD36 and/or thrombospondin, thrombospondin, apoptotic cells or a negatively-charged phospholipid. 30

35. A composition as claimed in claim 20 wherein said CD36 agonist is any one of the antibodies listed in Appendix 1.

36. A composition as claimed in claim 20 wherein 35 the pf-EMP-1 active binding domain comprises the amino acid sequence as shown in Figure 2. WO 01/02005 PCT/GB00/02546 - 123

37. A composition as claimed in claim 19 wherein said CD36 agonist is a molecule identified as such by any one of the methods of claims 15 to 32. 5

38. An agonist of the cell surface receptor CD36 as expressed on mammalian dendritic cells for use as a medicament.

39. An agonist for use as claimed in claim 38 10 wherein said medicament is used to induce a state of immune tolerance in a human.

40. An agonist for use as claimed in claim 38 or 39 which is suitable for treating a human and wherein 15 said CD36 agonist is selected from: an antibody with an affinity for an epitope of CD36, the Plasmodium falciparum protein pf-EMP-1, a protein comprising the active binding domain of pf-EMP-1 for CD36 or thrombospondin, thrombospondin, apoptotic cells or a 20 negatively charged phospholipid.

41. An agonist for use as claimed in claim 40 wherein said CD36 agonist is any one of the antibodies listed in Appendix 1. 25

42. An agonist for use as claimed in claim 40 wherein the pf-EMP-1 active binding domain comprises the amino acid sequence as shown in Figure 2. 30

43. A pharmaceutical composition suitable for inducing peripheral immune tolerance in a mammal which comprises an agonist of the cell surface receptor CD51 as expressed by mammalian dendritic cells and a pharmacologically acceptable carrier or diluent. 35

44. A composition as claimed in claim 43 suitable for inducing immune tolerance in a human WO 01/02005 PCT/GB00/02546 - 124 wherein said CD51 agonist is selected from: an antibody with an affinity for an epitope of CD51, thrombospondin, apoptotic cells or a negatively charged phospholipid. 5

45. A composition as claimed in claim 44 wherein said CD51 agonist is any one of the antibodies listed in Appendix 2. 10

- 46. A composition as claimed in claim 43 or 44 which comprises the Plasmodium falciparum protein pf EMP-1 or a protein comprising an active binding domain thereof and thrombospondin. 15

47. A composition as claimed in claim 46 wherein said active binding domain of pf-EMP-1 comprises the amino acid sequence shown in Figure 2.

48. A composition as claimed in claim 43 wherein 20 said CD51 agonist is a molecule identified as such by any one of the methods of claims 15 to 32.

49. An agonist of the cell surface receptor CD51 as expressed on mammalian dendritic cells for use as a 25 medicament.

50. An agonist for use as claimed in claim 49 wherein said medicament is used to induce a state of immune tolerance in a human. 30

51. An agonist for use as claimed in claim 49 or 50 which is suitable for administration to a human wherein said CD 51 agonist is selected from: an antibody with an affinity for an epitope of CD51, 35 thrombospondin, apoptotic cells or a negatively charged phospholipid. WO 01/02005 PCT/GB00/02546 - 125

52. An agonist for use as claimed in claim 51 wherein said CD51 agonist is any one of the antibodies listed in Appendix 2. 5

53. An agonist for use as claimed in claim 49 which is suitable for administration to a human and which comprises, in combination, the Plasmodium falciparum protein pf-EMP-1 or a protein comprising an active binding domain thereof and thrombospondin. 10

54. Use of a method comprising the following steps for identifying a molecule capable of preventing the adherence of red blood cells infected with a malarial parasite to human dendritic cells: 15 a) exposing a purified preparation of the human cell surface receptor CD36 to: I) the molecule to be tested and ii) parasitised human red blood cells, either consecutively or simultaneously and 20 b) determining the level of adherence of said parasitised red blood cells to CD36 wherein a reduction in the level of adherence in the presence of the test molecule compared to the level in the absence of said molecule is an 25 indication that said molecule is capable of preventing the adherence of red blood cells infected with the malarial parasite to human dendritic cells.

55. Use of a method comprising the following 30 steps for identifying a molecule capable of preventing the adherence of red blood cells infected with the malarial parasite to human dendritic cells: a) exposing a purified preparation of human thrombospondin to: 35 I) the molecule to be tested and ii) parasitised human red blood cells, either consecutively or simultaneously and WO 01/02005 PCT/GB00/02546 - 126 b) determining the level of adherence of said parasitised red blood cells to thrombospondin, wherein a reduction in the level of adherence to thrombospondin in the presence of the test molecule 5 compared to the level in the absence of said molecule is an indication that said molecule is capable of preventing the adherence of red blood cells infected with the malarial parasite to human dendritic cells. 10

56. Use of a method as claimed in claim 54 or claim 55 wherein said red blood cells are infected with Plasmodium falciparum.

57. Use of a method as claimed in claim 56 15 wherein the Plasmodium falciparum strain is ITO/A4, ITO/C24 or MC.

58. Use of a method as claimed in claim 54 wherein said CD36 is immobilised on a solid surface. 20

59. Use of a method as claimed in claim 55 wherein said thrombospondin is immobilised on a solid surface. 25

60. Use of a method as claimed in claim 58 or claim 59 wherein the level of adherence of said parasitised red blood cells to CD36 or thrombospondin is determined by the additional steps of: a) washing the immobilised CD36 or thrombospondin 30 to remove non-adhered red blood cells and b) applying a stain to said immobilised CD36 or thrombospondin which is specific for parasitised or non-parasitised red blood cells. 35

61. Use of a method as claimed in claim 60 wherein said stain is detectable by eye, by microscopy or by a spectrophotometric method. WO 01/02005 PCT/GB00/02546 - 127

62. Use of a method as claimed in claim 54 which comprises applying simultaneously or consecutively the method of claim 55. 5

63. A method of identifying a molecule capable of preventing the adherence of red blood cells infected with a malarial parasite to human dendritic cells which comprises: 10 a) exposing immature human dendritic cells to the Plasmodium falciparum protein pf-EMP-1 or an active binding domain thereof in the presence or absence of the molecule to be tested, b) exposing said immature dendritic cells to an 15 immune stimulus and c) determining the degree of maturation manifested by said dendritic cells, wherein any maturation of said dendritic cells in the presence of the test molecule over and above that 20 manifested in the absence of said molecule is an indication that said molecule is capable of preventing adherence of red blood cells infected with a malarial parasite to human dendritic cells. 25

64. A method as claimed in claim 63 wherein maturation of said dendritic cells is determined by examining the antigen-presenting ability of said cells. 30

65. A method as claimed in claim 63 or claim 64 wherein maturation of said dendritic cells is determined by examining said cells for expression of at least one cell surface antigen whose expression level is enhanced in response to an immune stimulus. 35

66. A method as claimed in claim 65 wherein maturation of said dendritic cells is determined by WO 01/02005 PCT/GB00/02546 - 128 measuring the level of expression of two or more of the following panel of antigens: HLA DR, CD54, CD40, CD83 and CD86. 5

67. A method as claimed in claim 66 wherein said cells are also examined for expression of CD80.

68. A method as claimed in any one of claims 65 to 67 wherein the level of expression of said antigen 10 is detected using a labelled antibody.

69. A method as claimed in claim 63 wherein maturation is determined by measuring said cells' ability to induce T-cell proliferation. 15

70. A method as claimed in claim 63 wherein maturation of said dendritic cells is determined by quantifying the level of cytokines secreted from said cells. 20

71. A method as claimed in claim 70 wherein the level of secretion of TNFa, IL12P70 and IL10 is measured. 25

72. A method as claimed in any one of claims 63 to 71 wherein said immune stimulus is lipopolysaccharide, TNF alpha, CD40L or monocyte conditioned medium (MCM). 30

73. A pharmaceutical composition useful for the treatment of malaria which comprises a molecule capable of preventing the adherence of red blood cells infected with the malarial parasite to human dendritic cells which has been identified by the method of any 35 one of claims 63 to 72 and a pharmacologically acceptable carrier or diluent. WO 01/02005 PCT/GB00/02546 - 129

74. A molecule identified as being capable of preventing the adherence of red blood cells infected with the malarial parasite to human dendritic cells by the method of any one of claims 63 to 72 for use in 5 the treatment of malaria.

75. A pharmaceutical composition useful for the treatment of malaria which comprises a molecule capable of preventing the adherence of red blood cells 10 infected with the malarial parasite to human dendritic cells which has been identified by the use of the method of any one of claims 54 to 62.

76. A molecule identified as being capable of 15 preventing the adherence of red blood cells infected with the malarial parasite to human dendritic cells by use of the method as claimed in any one of claims 54 to 62. 20

77. A method of identifying a molecule which is an agonist of cell surface receptors CD36 and/or CD51 and/or a receptor for thrombospondin as expressed on antigen-presenting cells of the mammalian immune system which method comprises: 25 a) exposing mammalian antigen-presenting cells to the molecule to be tested, b) exposing said cells to an immune stimulus and c) determining the response to said immune stimulus by said cells, 30 wherein an impaired response compared to the response in the absence of said test molecule is an indication that said molecule under test is a agonist of CD36 and/or CD51 and/or a thrombospondin receptor. 35

78. A method as claimed in claim 77 wherein said thrombospondin receptor is not CD47. WO 01/02005 PCT/GB00/02546 - 130

79. A method as claimed in claim 77 or claim 78 wherein said response is maturation of said antigen presenting cell. 5

80. A method as claimed in any one of claims 77 to 79 wherein said antigen-presenting cell of the immune system is selected from a dendritic cell, a macrophage, a B-lymphocyte or a monocyte. 10

81. A method as claimed in any of claims 77 to 80 which includes the features of any of claims 16 to 18 or 21 to 32.

82. A method of treating mammalian antigen 15 presenting cells in vitro to induce immune tolerance therein which comprises exposing said cells to an agonist of the cell surface receptor for thrombospondin and/or an agonist for the cell surface receptors CD36 and/or CD51. 20

83. A method as claimed in claim 82 wherein said antigen-presenting cell is human or mouse. 25

84. A method as claimed in claim 82 or 83 wherein said antigen presenting cell is selected from a dendritic cell, a macrophage, a B-lymphocyte or a monocyte. 30

85. A method as claimed in any one of claims 82 to 84 wherein said agonist is a molecule identified as such by the method of any one of claims 77 to 81.

86. A method as claimed in any one of claims 82 35 to 85 which includes the features of any one of claims 4 to 12. WO 01/02005 PCT/GB00/02546 - 131

87. A method as claimed in any one of claims 82 to 85 wherein said antigen-presenting cells are exposed to an antibody to an epitope of a thrombospondin receptor. 5

88. A method as claimed in claim 87 wherein said agonist is any one of the antibodies listed in Appendix 3. 10

89. A method as claimed in claim 87 or claim 88, which includes the features of any of claims 9 to 12.

90. An antigen-presenting cell preparation obtainable by the method of any one of claims 82 to 89 15 for use as a medicament.

91. As antigen-presenting cell preparation obtainable by the method of any one of claims 82 to 89 for use in inducing peripheral immune tolerance in a 20 human.

92. A method of identifying a molecule which is an agonist of a @-integrin associated with the cell surface receptor CD51 as expressed on antigen 25 presenting cells of the mammalian immune system which method comprises: a) exposing mammalian antigen-presenting cells to the molecule to be tested, b) exposing said cells to an immune stimulus and 30 c) determining the response to said immune stimulus by said cells, wherein an impaired response compared to the response in the absence of said test molecule is an indication that said molecule is an agonist of a P 35 integrin associated with the cell surface receptor CD51. WO 01/02005 PCT/GB00/02546 - 132

93. A method as claimed in claim 92 which includes the features of claim 78 or claim 79

94. A method as claimed in claim 92 to 93 which 5 further includes the features of any one of claims 16 to 25 or 29 to 32.

95. A method as claimed in any one of claims 92 to 94 wherein said @-integrin is P3 or P5. 10

96. A pharmaceutical composition suitable for inducing peripheral immune tolerance in a mammal which comprises an agonist of a @-integrin associated with the cell surface receptor CD51 as expressed on 15 mammalian antigen-presenting cells and a pharmacologically acceptable carrier or diluent.

97. A pharmaceutical composition as claimed in claim 96 wherein the @-integrin is P3 or P5. 20

98. A method of inducing a state of immune tolerance in antigen-presenting cells of the mammalian immune system which comprises exposing said cells ex vivo to an agonist of a P-integrin. 25

99. A method as claimed in claim 97 wherein said @-integrin is @3 or P5.

100. A method as claimed in claim 97 to 98 30 wherein said antigen presenting cells are human.

101. A method as claimed in claim 97 or 99 wherein said cells are exposed to an antigenic material. 35

102. A preparation of cells obtainable by the method as claimed in any one of claims 97 to 100 for WO 01/02005 PCT/GB00/02546 - 133 use as a medicament.

103. A preparation of cells obtainable by the method as claimed in any one of claims 97 to 100 for 5 use in inducing immune-tolerance in a human.

104. A pharmaceutical composition suitable for inducing peripheral immune tolerance in a mammal which comprises an agonist of a thrombospondin receptor and 10 a pharmacologically acceptable carrier or diluent.

105. A composition as claimed in claim 104 wherein said thrombospondin receptor is not CD47. 15

106. A preparation of apoptotic cells for use in inducing peripheral immune tolerance in a mammal.

107. A preparation as claimed in claim 106 wherein said mammal is a human. 20

108. A preparation comprising a negatively charged phospholipid for use in inducing peripheral immune tolerance in a mammal. 25

109. A preparation as claimed in claim 108 wherein said mammal is a human.

110. A preparation as claimed in claim 108 or 109 which comprises liposomes including a negatively 30 charged phospholipid.

111. A preparation as claimed any one of claims 108 to 110 wherein said negatively charged phospholipid is phosphatidylserine. 35

112. A method of inducing a state of immune tolerance in antigen-presenting cells of the mammalian WO 01/02005 PCT/GB00/02546 - 134 immune system which comprises exposing said cells ex vivo to a composition or preparation as claimed in any one of claims 104 to 111. 5

113. A method as claimed in claim 112 wherein said cells are exposed to an antigenic material.

114. A preparation of antigen-presenting cells obtainable by the method of claims 112 or 113. 10

115. A method of treating a human to induce peripheral immune tolerance therein comprising administering to said human a substance selected from the group consisting of: an agonist of CD36, an 15 agonist of CD51, an agonist of a thrombospondin receptor, an agonist of a @-integrin and a preparation of cells of any of claims 13, 14, 90, 91,102 or 114.