JP2004531210A - CD36 as heat shock protein receptor and use thereof - Google Patents

Abstract

The present invention relates to the use of the CD36 ("CD36") receptor as a heat shock protein receptor, cells expressing CD36 bound to HSP, and antibodies and other molecules that bind to the CD36-HSP complex. The present invention is directed to screening assays to identify compounds that interact with CD36 and modulate the interaction of CD36 with its ligands (such as HSPs), as well as compositions comprising CD36 receptor sequences, for immunological disorders, proliferation, It also relates to methods used for the diagnosis and treatment of sexual disorders and infectious diseases.

Description

[0001]
This application claims the benefit of United States Patent Application No. 60 / 238,865, filed October 6, 2000, which is hereby incorporated by reference in its entirety.
[0002]
1.preface
The present invention relates to the use of CD36 as a heat shock protein receptor, cells expressing CD36 bound to HSP, and antibodies and other molecules that bind to the CD36-HSP complex. The invention also includes screening assays to identify compounds that modulate the interaction of HSP with CD36, and CD36-receptor sequences for the diagnosis and treatment of immune disorders, proliferative disorders and infectious diseases. It also relates to the method of using the composition.
[0003]
2.Background of the Invention
2.1.Heat shock protein
Heat shock proteins (HSPs), also called stress proteins, were initially identified as proteins synthesized by cells in response to heat shock. Hsp is classified into five families (Hsp100, Hsp90, Hsp70, Hsp60 and smHsp) according to the molecular weight. Many members of these families have since been found to be induced in response to other stressful stimuli, including nutritional deficiencies, metabolic disorders, oxygen radicals, and infection by intracellular pathogens (Welch, May 1993, Scientific American 56-64; Young, 1990, Annu. Rev. Immunol. 8: 401-420; Craig, 1993, Science 260: 1902-1903; Gething et al., 1992, 5335-35; And Lindquist et al., 1998, Annu. Rev. Genetics 22: 631-677).
[0004]
Heat shock proteins are one of the most highly conserved proteins existing. For example, DnaK, Hsp70 from E. coli, has about 50% amino acid sequence identity with the Hsp70 protein from excorates (Bardwell et al., 1984, Proc. Natl. Acad. Sci. 81: 848-852). ). The Hsp60 and Hsp90 families also show similar high levels of intra-family conservation (Hickey et al., 1989, Mol. Cell. Biol. 9: 2615-2626; Jindal, 1989, Mol. Cell. Biol. 9: 2279-2283). . In addition, the Hsp60, Hsp70 and Hsp90 families have been found to consist of proteins that are sequence related to stress proteins (eg, have more than 35% amino acid identity), but whose expression levels are not altered by stress.
[0005]
Studies on cellular responses to heat shock and other physiological stresses indicate that HSPs are involved not only in cell protection against these adverse conditions, but also in essential biochemical and immunological processes in non-stressed cells. Indicated. HSPs perform different types of chaperone functions. For example, members of the Hsp70 family located in the cytoplasm, nucleus, mitochondria or endoplasmic reticulum (Lindquist et al., 1988, Ann. Rev. Genetics 22: 631-677) are involved in presenting antigen to cells of the immune system, and It is also involved in the transfer, folding and assembly of proteins in normal cells. HSPs can bind to a protein or peptide and release the bound protein or peptide in the presence of adenosine triphosphate (ATP) or low pH.
[0006]
2.2.HSP- Immunogenicity of peptide conjugate
Srivastava et al. Demonstrated an immune response to methylcholanthrene-induced sarcoma in inbred mice (1988, Immunol. Today 9: 78-83). In these studies, the molecules involved in the distinct immunogenicity of these tumors were found to be the 96 kDa glycoprotein (gp96) and the 84-86 kDa intracellular protein (Srivastava et al., 1986, Proc. Natl. Acad. Sci. USA 83: 3407-3411; Ullrich et al., 1986, Proc. Natl. Acad. Sci. USA 83: 3121-3125). Immunization of mice with gp96 or p84 / 86 isolated from a particular tumor immunizes mice against that particular tumor but not against antigenically distinct tumors. Isolation and characterization of the genes encoding gp96 and p84 / 86 revealed significant homology between them, with gp96 and p84 / 86 being the endoplasmic reticulum and cytosolic counterparts of the same heat shock protein, respectively. (Srivastava et al., 1988, Immunogenetics 28: 205-207; Srivastava et al., 1991, Curr. Top. Microbiol. Immunol. 167: 109-123). In addition, Hsp70 was shown to elicit immunity against the tumor from which it was isolated, but not against tumors that are antigenically different. However, peptide-depleted Hsp70 was found to lose its immunogenic activity (Udono and Srivastava, 1993, J. Exp. Med. 178: 1391-1396). These observations indicate that the heat shock protein is not immunogenic by itself, but rather forms a non-covalent complex with the antigen peptide, which elicits specific immunity to the antigen peptide. (Srivastava, 1993, Adv. Cancer Res. 62: 153-177; Udono et al., 1994, J. Immunol., 152: 5398-5403; Suto et al., 1995, Science, 269: 1585-1588).
[0007]
Non-covalent conjugates of HSPs and peptides purified from cancer cells can be used to treat and prevent cancer, see PCT Publication No. WO 96/10411 dated April 11, 1996, and March 1997. WO 97/1001, issued on April 20, 1998 (US Pat. No. 5,750,119 issued on Apr. 12, 1998, and US Pat. No. 5,837, issued Nov. 17, 1998). 251 (each incorporated by reference herein in its entirety). For example, the isolation and purification of stress protein-peptide complexes from pathogen-infected cells has been described, including infection of pathogens such as viruses and infections caused by other intracellular pathogens such as bacteria, protozoa, fungi and parasites. It can be used for treatment and prophylaxis (see, for example, PCT Publication No. WO 95/24923 dated September 21, 1995). Immunogenic stress protein-peptide conjugates can also be prepared by in vitro conjugation of stress proteins with antigenic peptides, using such conjugates for the treatment and prevention of cancer and infectious diseases. Are described in PCT Publication No. WO 97/10000 dated March 20, 1997 (US Pat. No. 6,030,618 issued Feb. 29, 2000). The use of stress protein-peptide complexes to sensitize antigen-presenting cells for use in adoptive immunotherapy in vitro has been described in PCT Publication No. WO 97/10002, March 20, 1997 (November 1999). See also U.S. Pat. No. 5,985,270, issued on Mar. 16, 2016).
[0008]
2.3.CD36
CD36 is a member of the class B scavenger receptor family and is expressed primarily in capillary vascular endothelial cells, mammary secretory epithelial cells, differentiated adipocytes, B cells, macrophages and several types of tumor cells. CD36 is thought to play a role in platelet adhesion and aggregation, phagocytosis of apoptotic cells, and long-chain fatty acid metabolism. CD36 is located on the plasma membrane in a microdomain called caveolae, which has been implicated in cell trafficking and signaling pathways. Structurally, CD36 has a large (463 aa) extracellular domain, a single transmembrane domain, and a short cytoplasmic tail containing a putative tyrosine kinase docking site. Amino acid residues # 184-204 of CD36 constitute the hydrophobic stretch of the receptor most likely associated with the plasma membrane. Anti-CD36 alloantibodies recognize a highly antigenic structure at amino acids 155-183, known as the immunodominant domain.
[0009]
CD36 is expressed on both monocytes and megakaryocytic lineages and is upregulated during differentiation. Monocyte expression of CD36 is regulated through adhesion to M-CSF and IL-4, and activated endothelial cells (Yesner et al., 1996, Arterioscler. Thromb. Vasc. Biol. 16: 1019-1025; Huh. Et al., 1995, J. Biol. Chem. 270: 6267-6271). Expression of mouse or human CD36 in CD36 deficient cells results in specific and high affinity binding of oxidized LDL, followed by LDL internalization and degradation (Endemann et al., 1993, J. Biol. Chem. 268: 11811-11816). Navazo et al., 1996, Arterioscler. Thromb. Vasc. Biol. 16: 1033-1039). CD36 also binds long-chain fatty acids, and its expression has been shown to be up-regulated in endothelial cells in tissues involved in fatty acid transport and metabolism (Greenwalt et al., 1995, J. Clin. Invest. 96: 1382-1388).
[0010]
CD36 deficiency in humans is found in about 3% of the Japanese population and 0.3% of Caucasians. Affected people are phenotypically normal, but some subjects have poor ability to bind to and internalize oxidized LDL (Nozaki et al., 1995, J. Clin. Invest. 96). : 1859-1865).
[0011]
CD36 has also been shown to play a role in cytoadherence to erythrocytes infected with Plasmodium falciparum. After infection, P. falciparum is sequestered within the microvasculature, which prevents clearance in the spleen and promotes bacterial survival. CD36 binds to the PfEMP1 protein produced by P. falciparum in infected erythrocytes. This binding can induce an oxidative burst of monocyte and platelet activation (Okenhouse et al., 1989, J. Clin. Invest. 84: 468-475; Huang et al., 1991, Proc. Natl. Acad. Sci. ScL USA 88: 7844-7848).
[0012]
2.4.GP96
Major histocompatibility complex (MHC) molecules present antigen on the cell surface of antigen presenting cells. The cytotoxic T lymphocytes (CTL) then recognize the MHC molecules and their related peptides and kill the target cells. Antigens are processed by two different antigen processing pathways, depending on whether their source is intracellular or extracellular. Intracellular or endogenous protein antigens (ie, antigens synthesized within antigen presenting cells) are presented to CD8 + cytotoxic T lymphocytes by MHC class I (MHC I) molecules. On the other hand, extracellularly or exogenously synthesized antigenic determinants are presented by MHC class II molecules to CD4 + T cells on the cell surface of “specialized” or “professional” APCs (eg, macrophages). (See Fundamental Immunology, WE Paul (eds.), New York: Raven Press, 1984 in general). This compartmentalization of the antigen processing pathway is important to prevent tissue destruction, otherwise tissue destruction can occur during the immune response due to shedding of adjacent cell MHC I antigens.
[0013]
The heat shock protein gp96 chaperones various peptides depending on the source from which gp96 is isolated (for an overview, see Srivastava et al., 1998, Immunity 8: 657-665). Tumor-derived gp96 carries tumor antigenic peptides (Ishii et al., 1999, J. Immunology 162: 1303-1309); gp96 preparations derived from virus-infected cells carry viral epitopes (Suto and Srivastava, 1995, Science). 269: 1585-1588; Nieland et al., 1998, Proc. Natl. Acad. Sci. USA 95: 1800-1805), gp96 preparations derived from cells transfected with a model antigen such as ovalbumin or β-galactosidase have the corresponding (Arnold et al., 1995, J. Exp. Med. 182: 885-889; Breloer et al., 1998, Eur. J. Immunol. 28: 016-1021). The association of gp96 with the peptide occurs in vivo (Menoret and Srivastava, 1999, Biochem. Biophys. Research Commun. 262: 813-818). The Gp96-peptide complex, whether isolated from cells (Tamura et al., 1997, Science 278: 117-120) or reconstituted in vitro (Blachele et al., 1997, J. Exp. Med. 186: 1183-1406) is an excellent immunogen and is widely used to elicit CD8 + T cell responses specific for gp96-chaperone antigen peptides.
[0014]
The ability of the gp96-peptide complex to elicit an immune response depends on the transport of the peptide to MHC class I molecules of antigen presenting cells (Suto and Srivastava, 1995, supra). Endogenous synthetic antigens chaperone by gp96 in the endoplasmic reticulum [ER] can prime antigen-specific CD8 + T cells (or MHC I-restricted CTL) in vivo; this priming of CD8 + T cells is a macrophage Need. However, the process by which an exogenously introduced gp96-peptide complex elicits an antigen-specific CD8 + T cell response is not fully understood due to the lack of an established pathway for translocation of extracellular antigens to the class I presentation machinery. It has not been. Nevertheless, antigenic peptides of extracellular origin associated with HSPs are somehow salvaged by macrophages, channeled into the intrinsic pathway, presented by MHC I molecules, and recognized by CD8 + lymphocytes (Suto and Srivastava, 1995, supra; Blachere et al., 1997, J. Exp. Med. 186: 1315-22).
[0015]
Several models have been proposed to explain the delivery of extracellular peptides for antigen presentation. One proposal, known as the "direct transport" model, suggests that HSP chaperone peptides are transported to MHC I molecules on the cell surface of macrophages and presented to CD8 + T lymphocytes. Another suggestion is that soluble extracellular proteins can be transferred to the cytosol via constitutive macropinocytosis in myeloid-derived macrophages and dendritic cells (Norbury et al., 1997, Eur. J. Immunol. 27: 280-288). A further proposed mechanism is that HSPs are taken up by macrophage MHC class I molecules, which stimulate appropriate T cells (Srivastava et al., 1994, Immunogenetics 39: 93-98). Others have suggested that a novel intracellular transfer pathway may be involved in the transport of peptides from the extracellular medium into the lumen of the ER (Day et al., 1997, Proc. Natl. Acad. Sci. 94: 8064). -8069; Niccitta, 1998, Curr. Opin. In Immunol. 10: 103-109). Further suggested are: (a) an implicit pathway that directs the protein from the phagosome to the cytosol, where it enters the normal class I pathway; and (b) digests ingested substances in lysosomes. And involvement of phagocytic cells to release peptides onto the surface for class I molecules (Bevan, 1995, J. Exp. Med. 182: 639-41).
[0016]
Still others have proposed receptor-mediated pathways to deliver extracellular peptides to the cell surface of APCs for antigen presentation. The minimal amount of the gp96-chaperone antigen peptide required for immunization (Blachere et al., 1997, supra) and that the immunogenicity of the gp96-peptide complex is completely dependent on functional antigen presenting cells (APCs) (Udono et al., 1994, Proc. Natl. Acad. Sci. USA 91: 3077-3081), it has been proposed that APCs have a receptor for gp96 (Srivastava et al., 1988). 1994, Immunogenetics 39: 93-98). Such a receptor has recently been identified and determined as the α (2) macroglobulin receptor or CD91 (Binder et al., 2000, Nature Immunol. 1: 151-155). It has been proposed that gp96 carries peptides into cells via CD91 and transports these peptides to MHC class I molecules on dendritic cells and antigen presenting cells.
[0017]
Reagents useful for eliciting specific immunity with HSPs and HSP-peptide complexes by identifying and characterizing specific molecules involved in HSP-mediated antigen presentation of peptides and for developing novel diagnostic and therapeutic methods Skill may be obtained.
[0018]
Citation and discussion of the references herein shall not be construed as an admission that they are prior art to the present invention.
[0019]
3.Summary of the invention
The present invention relates to compositions and methods using CD36 as a heat shock protein receptor. The present invention is based, in part, on Applicants' discovery that CD36 is a cell surface receptor for heat shock proteins. In particular, Applicants have shown herein that the heat shock protein gp96 binds to CD36 expressing cells and initiates a signaling cascade to produce cytokines, chemokines and nitric oxide production.
[0020]
The present invention provides compositions comprising a complex of HSP and CD36, as well as antibodies and other molecules that bind to the CD36 complex. The present invention also encompasses methods of using CD36 as a heat shock protein receptor, methods of screening for compounds that modulate the interaction of HSP with CD36, and HSP-CD36 mediated processes and HSP-CD36 related disorders. / Methods of treating and detecting symptoms (such as autoimmune disorders, proliferative disorders and infectious diseases).
[0021]
The invention includes (a) contacting a test compound with a heat shock protein and CD36; and (b) measuring the level of CD36 activity or expression, wherein the level of activity or expression measured in (b) If the level differs from the level of CD36 activity in the absence of the test compound, then identifying a compound that modulates an HSP-CD36 mediated process; Provide a method. In one embodiment of the method, the compound identified is an antagonist that interferes with the interaction of heat shock protein with CD36, and (c) the level interferes with the interaction of heat shock protein with CD36. Determining if there is any more. In another embodiment, the test compound is an antibody specific for CD36. In another embodiment, the test compound is an antibody specific for a heat shock protein. In another embodiment, the test compound is a small molecule. In yet another embodiment, the test compound is a peptide. In another embodiment, the peptide comprises at least 5 contiguous amino acids of CD36. In yet another embodiment, the peptide comprises at least five consecutive amino acids of a heat shock protein sequence. In another embodiment, the compound is an agonist that enhances the interaction of heat shock protein with CD36. In another embodiment, the HSP-CD36 mediated process is an autoimmune disorder, a disease or disorder involving cell signaling, a disease or disorder with cytokine clearance or inflammation, a proliferative disorder, a viral disorder or other infectious disease. Affects hypercholesterolemia, Alzheimer's disease, diabetes, or osteoporosis.
[0022]
The invention also includes (a) contacting a test compound with a cell that expresses a heat shock protein and a CD36; and (b) measuring the level of CD36 activity or expression in the cell, wherein (b) If the level of activity or expression measured is different from the level of CD36 activity in the absence of the test compound, then a compound that modulates the HSP-CD36 mediated process is identified as an HSP-CD36 mediated process. Methods for identifying compounds that modulate are also provided. In another embodiment, the CD36 activity measured is the ability to interact with a heat shock protein.
[0023]
The present invention also provides (a) contacting a heat shock protein with CD36, or a fragment, analog, derivative or mimetic thereof, in the presence of a test compound; and (b) CD36, or a fragment, analog thereof. Determining the amount of heat shock protein bound to the body, derivative or mimetic, wherein the amount of bound heat shock protein measured in (b) is less than the amount of heat shock protein bound in the absence of the test compound. In a different case, a method of identifying a compound that modulates the binding of heat shock protein to CD36 is also included, provided that a compound that modulates the binding of HSP to CD36 has been identified. In another embodiment, the CD36 contacted in step (a) is on the cell surface. In another embodiment, CD36 is immobilized on a solid surface. In another embodiment, the solid surface is a microtiter dish. In another embodiment, the amount of the bound heat shock protein is measured by contacting the cell with a heat shock protein specific antibody. In yet another embodiment, the amount of the bound heat shock protein is determined by labeling the heat shock protein and detecting the label. In another embodiment, the heat shock protein is labeled with a fluorescent label.
[0024]
The invention further provides (a) adding a test compound to a mixture of CD36 expressing cells and one consisting essentially of a heat shock protein associated with an antigen molecule, under conditions that promote CD36 mediated signal transducer production. (B) determining the level of a signal transducer by the CD36-expressing cells, wherein the level measured in (b) is different from the stimulation in the absence of the test compound; Provided is a method for identifying a compound that modulates heat shock protein-mediated cell signaling by CD36-expressing cells, provided that the compound that modulates protein-mediated signal transduction substance stimulation has been identified. In one embodiment of the method, the step of measuring the level of signal transducer stimulation (b) comprises: (i) transforming the CD36-expressing cells formed in step (a) into T cells under conditions that promote signal transducer stimulation. And (ii) comparing the level of signal transducer activation of the T cell with the level of T cell activation by CD36-expressing cells formed in the absence of the test compound. An increase or decrease in the level of cell activation indicates that a compound that modulates heat shock protein-mediated signaling by CD36-expressing cells has been identified.
[0025]
In various embodiments, the heat shock protein used in the methods of the invention is gp96.
[0026]
In another embodiment, the invention comprises measuring the level of an HSP-CD36 mediated process in a patient sample, wherein the measured level is different from the level found in clinically normal individuals. Provided is a method for detecting a heat shock protein-CD36-related disorder in a mammal, wherein a shock-CD36-related disorder is detected.
[0027]
The present invention also includes kits containing the compositions of the present invention. In one embodiment, (a) a purified heat shock protein, a nucleic acid encoding a heat shock protein, or a cell expressing a heat shock protein; and (b) a CD36 polypeptide, a nucleic acid encoding a CD36 polypeptide, or a CD36 polypeptide. Provides a kit, wherein the cells expressing the are packaged in one or more containers. In one embodiment, the CD36 polypeptide, the nucleic acid encoding the CD36 polypeptide, or the cell expressing the CD36 polypeptide has been purified. In another embodiment, the kit further comprises instructions for use in treating an autoimmune disorder, an infectious disease, or a proliferative disorder.
[0028]
The present invention also provides a method of modulating an immune response, comprising administering to a mammal a purified compound that modulates the interaction of a heat shock protein with CD36. In one embodiment, the compound is an agonist that enhances the interaction of heat shock protein with CD36. In another embodiment of the method, the compound is an antagonist that interferes with the interaction of heat shock protein with CD36.
[0029]
The present invention further provides a method of treating an autoimmune disorder, comprising administering to a mammal in need of treatment a purified compound that interferes with the interaction of heat shock protein with CD36. In one embodiment of the method, the compound is an antagonist that interferes with the interaction of heat shock protein with CD36. In another embodiment, the antagonist is an antibody specific for CD36. In another embodiment, the antagonist is an antibody specific for a heat shock protein. In another embodiment, the antagonist is a small molecule. In another embodiment, the antagonist is a peptide. In another embodiment, the peptide comprises at least 5 contiguous amino acids of CD36. In another embodiment, the peptide comprises at least five consecutive amino acids of a heat shock protein sequence.
[0030]
The present invention is further directed to a method of enhancing the immunity of a cancer cell or infected cell, comprising the steps of: A method is provided, comprising transforming.
[0031]
The present invention further provides a method of enhancing the immunity of a cancer cell or infected cell, the method comprising: (a) operably linking to a (i) promoter to obtain a high immune response; Transforming the cells with a nucleic acid comprising the nucleotide sequence of the present invention; and (b) administering the cells to an individual in need of treatment.
[0032]
The invention also provides a recombinant cancer cell, which is (i) operably linked to a promoter, and (ii) transformed with a nucleic acid comprising a nucleotide sequence encoding a CD36 polypeptide. In one embodiment, the recombinant cells are human cells.
[0033]
In yet another embodiment, the invention provides a recombinantly infected cell that is (i) operably linked to a promoter and (ii) transformed with a nucleic acid comprising a nucleotide sequence encoding a CD36 polypeptide. In one embodiment, the recombinant cells are human cells.
[0034]
In another embodiment, the invention provides a method wherein (a) contacting CD36 with one or more test molecules under conditions which promote binding; and (b) any of the test molecules specifically binds to CD36. There is provided a method for screening for a molecule that specifically binds to CD36, comprising a step of determining whether or not the molecule binds to CD36. In one embodiment of the method, the test molecule is a potential immunotherapeutic.
[0035]
The present invention also relates to (a) contacting CD36 with a CD36 ligand or a CD36 binding fragment, analog, derivative or mimetic thereof in the presence of one or more test compounds; and (b) bound to CD36 Determining the amount of CD36 ligand or a fragment, analog, derivative or mimetic thereof, wherein the amount of bound CD36 ligand measured in (b) is less than the amount of bound CD36 measured in the absence of the test compound. If different, a method of identifying a compound that modulates the binding of CD36 ligand to CD36 is also provided, provided that a compound that modulates the binding of CD36 ligand to CD36 has been identified.
[0036]
In another embodiment, the method comprises: (a) contacting CD36 with one or more test compounds and a CD36 ligand; and (b) measuring the level of activity or expression, wherein the activity or If the level of expression differs from the level of CD36 activity in the absence of the one or more test compounds, then a compound that modulates the interaction of CD36 with the CD36 ligand has been identified, and the interaction of CD36 with the CD36 ligand has been identified. Methods for identifying compounds that modulate an interaction are provided.
[0037]
In another embodiment, the present invention modulates an immune response comprising administering to a mammal a purified compound that binds CD36 in an amount effective to modulate an immune response in the mammal. Provide a method.
[0038]
In yet another embodiment, treating or preventing a disease or disorder comprising administering to a mammal a purified compound that binds CD36 in an amount effective to treat or prevent the disease or disorder in the mammal. Provide a way to In one embodiment, the disease or disorder is a cancer or an infectious disease.
[0039]
In a further embodiment, a method of treating an autoimmune disorder comprising administering to a mammal in need thereof a purified compound that binds to CD36 in an amount effective to treat the autoimmune disorder in the mammal. I will provide a.
[0040]
The term "HSP-CD36 mediated process" as used herein refers to a process that directly or indirectly depends and / or responds to the interaction of HSP with CD36. Such processes include binding of various CD36 ligands, including but not limited to lipoprotein complexes, thrombospondin 1, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), LDL and phospholipids. Processes resulting from the expression, synthesis and / or activity of abnormal levels of CD36, such as relevant signaling stimulatory activities. Such processes include, but are not limited to, phagocytosis of apoptotic cells and metabolism of long chain fatty acids.
[0041]
The terms "HSP-CD36-related disorder" and "HSP-C36-related condition" as used herein refer to disorders and conditions, respectively, that involve HSP-CD36 interaction. Such disorders and conditions may include, for example, levels found in normal, unaffected and unimpaired individuals, levels found in clinically normal individuals, and / or baseline, mean HSP and And / or an abnormal level of expression, synthesis and / or activity of HSP and / or CD36 relative to levels found in the population of levels representing CD36 levels. It can be caused by abnormal abilities. Such disorders include autoimmune disorders, diseases and disorders involving cell signaling or growth disruption, diseases and disorders involving cytokine clearance and / or inflammation, proliferative disorders, viral disorders and other infectious diseases, hypercholesterolemia. Disease, Alzheimer's disease, diabetes, and osteoporosis.
[0042]
As used herein, the term “CD36 ligand” refers to a molecule that can bind to CD36. Such CD36 ligands include, but are not limited to, lipoprotein complexes, thrombospondin 1, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), and phospholipids, along with known ligands. In addition, CD36 ligands include molecules that are readily identified as CD36 ligands using standard binding assays well known in the art. Such CD36 ligand is typically taken up by cells by endocytosis upon binding to CD36.
[0043]
4.BRIEF DESCRIPTION OF THE FIGURES
A brief description of the drawings will be described later.
[0044]
5.DETAILED DESCRIPTION OF THE INVENTION
The present invention relates to compositions and methods using CD36 as a heat shock protein ("HSP") receptor. In particular, the present invention relates to isolated CD36-ligand complexes (eg, CD36-HSP complexes) (isolated and / or recombinant cells, and antibodies that modulate the interaction of CD36 with a CD36 ligand, such as HSP, (Including molecules and compounds). The invention further encompasses methods of using CD36 as a heat shock protein receptor, screening assays to identify compounds that modulate the interaction of CD36 with HSP or other CD36 ligands, as well as those molecules and Methods include using the conjugates for the diagnosis and treatment of immune disorders, proliferative disorders and infectious diseases.
[0045]
As used herein, the term “CD36 ligand” refers to a molecule that can bind to CD36. Such CD36 ligands include, but are not limited to, lipoprotein complexes, thrombospondin, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), LDL, and phospholipids, along with known ligands. In addition, CD36 ligands also include molecules that are easily identified as CD36 ligands using standard binding assays well known in the art.
[0046]
HSPs useful in the practice of the present invention may be selected from any cellular protein that meets the following criteria: that the intracellular concentration of HSP increases when the cell is exposed to a stressful stimulus; Be capable of binding to a protein or peptide and releasing the bound protein or peptide in the presence of adenosine triphosphate (ATP) or at a low pH; or that the HSP is at least 35% of any cellular protein having any of the above properties. % Homology. The HSP used in the compositions and methods of the present invention is preferably (but not limited to) HSP90, gp96, BiP, Hsp70, DnaK, Hsc70, PhoE calreticulin, PDI, or sHsp alone or in combination. Is not.)
[0047]
In a preferred embodiment, the HSP is of a mammal (eg, a domestic animal such as a mouse, rat, primate, dog, cat, cow, horse, etc.), and is most preferably a human.
[0048]
Hsps useful in the practice of the present invention include HSP60 family, HSP70 family, HSP90 family, HSP100 family, members of the sHSP family, calreticulin, PDI, and other proteins in the endoplasmic reticulum containing a thioredoxin-like domain ( ERp72 and ERp61, but are not limited thereto).
[0049]
HSP analogs, muteins, derivatives and fragments can also be used in place of the HSPs of the present invention. An HSP peptide-binding "fragment" as used in the present invention refers to a polypeptide comprising an HSP peptide-binding domain that can non-covalently associate with a peptide to form a complex capable of inducing an immune response. In one embodiment, the HSP peptide binding fragment is a polypeptide comprising an HSP peptide binding domain of about 100-200 amino acids.
[0050]
Databases can be searched using programs such as FASTA and BLAST, which rank similar sequences by alignment score and statistics, to identify sequences with varying degrees of similarity to the query sequence. Such nucleotide sequences for non-limiting examples of HSPs that can be used for the preparation of HSPs for use in the method of the invention include the following: human Hsp70, Genbank accession number NM_005345, Sargent et al., 1989, Proc. Natl. Acad. Sci. U. S. A. , 86: 1968-1972; human Hsp90, Genbank accession number X15183, Yamazaki et al., Nucl. Acids Res. 17: 7108; human gp96: Genbank accession number X15187, Maki et al., 1990, Proc. Natl. Acad Sci. , 87: 5658-5562; human BiP: Genbank accession number M19645; Ting et al., 1988, DNA 7: 275-286; human Hsp27, Genbank accession number M24743; Hickey et al., 1986, Nucleic Acids Res. 14: 4127-45; mouse Hsp70: Genbank accession number M35021, Hunt et al., 1990, Gene, 87: 199-204; mouse gp96: Genbank accession number M16370, Srivastava et al., 1987, Proc. Natl. Acad. Sci. 85: 3807-3811; and mouse BiP: Genbank accession number U16277, Haas et al., 1988, Proc. Natl. Acad. Sci. U. S. A. , 85: 2250-2254. Due to the degeneracy of the genetic code, the term “HSP sequence” as used herein encompasses not only natural amino acid and nucleotide sequences, but also all other degenerate sequences that encode HSP.
[0051]
The HSP family also includes proteins that are sequence related to HSPs (eg, have more than 35% amino acid identity) but whose expression levels are not altered by stress. Accordingly, the definition of heat shock protein or stress protein as used herein is defined as at least 35% to 55%, preferably 55% to 55% It is intended to encompass other proteins, variants, analogs, and variants thereof having 75%, and most preferably, 75% to 85% amino acid identity. Determining the percent identity between two sequences can also be accomplished using a mathematical algorithm. Preferred, non-limiting examples of mathematical algorithms utilized to compare two sequences include those described in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. ScL USA 90: 5873-5877, modified by Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. USA 87: 2264-2268. Such an algorithm is described in Altschul et al., 1990, J. Am. Mol. Biol. 215: 403-410, incorporated into the NBLAST and XBLAST programs. BLAST nucleotide searches can be performed with the NBLAST program, score = 100, character length = 12 to obtain nucleotide sequences homologous to the nucleic acid molecules of the invention. BLAST protein searches are performed with the XBLAST program, score = 50, character length = 3, to obtain amino acid sequences homologous to the protein molecules of the present invention. To obtain a gapped alignment for comparison purposes, gapped BLAST was performed as described in Altschul et al., 1997, Nucleic Acids Res. 25: 3389-3402. Alternatively, PSI-Blast can be used to perform an iterated search that detects distant relationships between molecules (Altschul et al., 1997, supra). When utilizing BLAST, Gapped BLAST, and PSI-Blast programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used (see http://www.ncbi.nlm.nih.gov). Another preferred, non-limiting example of a mathematical algorithm utilized for comparing sequences is the algorithm of Myers and Miller, 1988, CABIOS 4: 11-17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 can be used.
[0052]
An immunogenic HSP-peptide conjugate of the invention can include any conjugate comprising HSP and a peptide capable of inducing an immune response in a mammal. Preferably, the peptide associates non-covalently with the HSP. Preferred complexes include gp96-peptide complex, HSP90-peptide complex, HSP70-peptide complex, HSP60-peptide complex, HSP100-peptide complex, calreticulin-peptide complex, and sHSP-peptide complex. Body, but is not limited thereto. For example, HSP gp96, which is present in the endoplasmic reticulum of eukaryotic cells and is associated with cytoplasmic HSP90, can be used to produce an effective vaccine comprising a gp96-peptide complex.
[0053]
The HSP, CD36 and / or antigen molecule used in the present invention can be purified, chemically synthesized, or recombinantly produced from a natural source. The HSP may be allogeneic to the patient, but in a preferred embodiment, the HSP is autologous to the patient to whom it is administered.
[0054]
5.1.Composition of the invention
The present invention provides compositions that modulate the interaction of CD36 with a CD36 ligand (eg, HSP, etc.). Such compositions can be used in a method for inducing or modulating an immune response. Such compositions also include the HSP-CD36 complex, isolated cells expressing the HSP-CD36 complex, and antibodies that specifically recognize the isolated and recombinant cells containing the recombinant CD36 and HSP sequences. In addition, in various methods of the invention, sequences encoding CD36, HSP, and CD36 are used for immunotherapy. Such compositions can be used, for example, for immunotherapy against proliferative disorders, infectious diseases and other HSP-CD36 related disorders. Methods for synthesizing and making such compositions are described herein.
[0055]
5.1.1.Recombinant expression
In various embodiments of the invention, sequences encoding CD36, HSP or other CD36 ligand are inserted into an expression vector and propagated and expressed in recombinant cells. Thus, in one embodiment, the CD36, HSP, or other CD36 ligand coding region is linked to a non-naturally occurring promoter for expression in a recombinant cell.
[0056]
Based on Applicants' discovery, the extracellular portion of CD36 is likely responsible for recognizing and binding HSPs and HSP-antigen peptide complexes. The interaction between CD36 and HSP triggers a signaling pathway that stimulates the immune response. According to the present invention, compositions comprising agonists and antagonists of CD36 and HSP interactions can be used to modulate an immune response. Accordingly, recombinant CD36 polypeptides, complexes of CD36 and HSP or HSP-antigen peptide complexes, and recombinant cells expressing CD36 are used in the methods for immunotherapy and diagnostic methods described herein. it can.
[0057]
In various embodiments of the invention, sequences encoding CD36 and / or heat shock proteins, or fragments thereof, are inserted into expression vectors and propagated and expressed in recombinant cells. An expression construct, as used herein, refers to a specific gene product, such as CD36 or HSP, operably associated with one or more regulatory regions that enables expression of the encoded gene product in a suitable host cell. Refers to the nucleotide sequence "Operably associated" refers to an association in which the nucleotide sequences encoding the regulatory regions and the expressed gene product are ligated and located so that they can be transcribed and ultimately translated.
[0058]
DNA can be obtained from tissues, cell cultures, or cloned DNA (eg, DNA "libraries") by DNA amplification or molecular cloning, based on known sequences obtained from sequence databases by standard procedures known in the art. You may get it directly. Any eukaryotic cell can serve as a source of nucleic acid to obtain the coding region for the hsp gene. Nucleic acid sequences encoding HSPs can be isolated from vertebrate, mammalian, and primate sources, including humans. Clones derived from genomic DNA may contain regulatory and intronic DNA regions in addition to the coding regions; clones derived from cDNA contain only exon sequences. Regardless of the source, the hsp gene should be cloned into a suitable vector for propagation of the gene.
[0059]
E. coli-based vectors are the most common and versatile system for high level expression of foreign proteins (Makrides, 1996, Microbiol Rev, 60: 512-538). Non-limiting examples of regulatory regions that can be used for expression in E. coli include lac, trp, lpp, phoA, recA, tac, λP_LAnd the phage T3 and T7 promoters (Makrides, 1996, Microbiol Rev, 60: 512-538). Non-limiting examples of prokaryotic expression vectors include λgt vector series such as λgt11 (“DNA Cloning Technologies”, Vol. I: A Practical Approach (D. Glover), pp. 49-78, IRL Press, Oxford. Huynh et al., 1984), and the pET vector series (Studier et al., 1990, Methods Enzymol., 185: 60-89). However, a potential disadvantage of prokaryotic host vector systems is that many post-translational processing events in mammalian cells cannot be performed. Thus, eukaryotic host vector systems are preferred, mammalian host vector systems are more preferred, and human host vector systems are most preferred.
[0060]
For example, the expression vector can include regulatory regions required for transcription of the CD36 sequence. A translation initiation codon (ATG) may also be provided for expressing a nucleotide sequence encoding CD36 lacking the initiation codon. In compatible host construct systems, the cellular proteins required for transcription, such as RNA polymerase and transcription factors, bind to regulatory regions on the expression construct, resulting in transcription of the CD36 sequence in the host organism. The exact nature of the regulatory regions required for gene expression may vary from host cell to host cell. Generally, a promoter is required that can bind to RNA polymerase and initiate transcription of an operably associated nucleic acid sequence. Such regulatory regions may include 5 'non-coding sequences involved in the initiation of transcription and translation (TATA box, cap site, CAAT box, etc.). The non-coding region 3 'to the coding sequence can include transcription termination regulatory sequences, such as terminators and polyadenylation sites.
[0061]
Both constitutive and inducible regulatory regions can be used for expression of CD36, HSP or other CD36 ligand. Where optimal conditions for the growth of recombinant cells and conditions for high level expression of the gene product are different, it may be desirable to use an inducible promoter. Examples of useful regulatory regions are described below in the following section.
[0062]
For expression of CD36, HSP or other CD36 ligand gene products in mammalian host cells, for example, the SV40 early and late promoters, the cytomegalovirus (CMV) immediate early promoter, and the Rous sarcoma virus long terminal repeat Various regulatory regions such as the (RSV-LTR) promoter can be used. Inducible promoters that may be useful in mammalian cells include those with the metallothionein II gene, the mouse mammary adenocarcinoma virus glucocorticoid-responsive long terminal repeat (MMTV-LTR), the β-interferon gene, and the Hsp70 gene. (Williams et al., 1989, Cancer Res. 49: 2735-42; Taylor et al., 1990, Mol. Cell Biol., 10: 165-75). It may be advantageous to use a heat shock promoter or a stress promoter to drive expression of CD36 in the recombinant host cell.
[0063]
The following animal regulatory regions that show tissue specificity and have been utilized in transgenic animals can also be used in tumor cells of certain tissue types: elastase I gene regulatory regions active in pancreatic acinar cells (Swift et al., 1984, Cell 38: 639-646; Ornitz et al., 1986, Cold Spring Harbor Symp. Quant. Biol. 50: 399-409; MacDonald, 1987, Hepatology 7: 425-515); Regions (Hanahan, 1985, Nature 315: 115-122); immunoglobulin gene regulatory regions active in lymphocytes (Grosschedl et al., 1984, Cell 38: 647-658). Adames et al., 1985, Nature 318: 533-538; Alexander et al., 1987, Mol. Cell. Biol. 7: 1464-1444); a mouse mammary adenocarcinoma virus control region active in testis, breast, lymphocytes and mast cells ( Leder et al., 1986, Cel 45: 485-495); an albumin gene regulatory region active in the liver (Pinkert et al., 1987, Genes and Dvel. 1: 268-276); an alpha-fetoprotein gene regulatory region active in the liver (Krumlauf et al.). 5: 1639-1648; Hammer et al., 1987, Science 235: 53-58); α1-antitrypsin gene regulatory region active in liver (Kelsey et al., 1987, Genes and Level. 1: 161-171); β-globin gene regulatory region active in bone marrow cells (Mogram et al., 1985, Nature 315: 338-340; Kollias et al., 1986, Cell 46: 89-). 94); myelin basic protein gene regulatory region active in oligodendrocytes in the brain (Readhead et al., 1987, Cell 48: 703-712); myosin light chain 2 gene regulatory region active in skeletal muscle (Sani, 1985) , Nature 314: 283-286); and a gonadotropic releasing hormone gene regulatory region active in the hypothalamus (Mason et al., 1986, Scie). ce 234: 1372-1378).
[0064]
The efficiency of CD36 expression in host cells is determined by including appropriate transcription enhancer elements (such as those found in SV40 virus, hepatitis B virus, cytomegalovirus, immunoglobulin genes, metallothionein, β-actin) in the expression vector. (See Bittner et al., 1987, Methods in Enzymol. 153: 516-544; Gorman, 1990, Curr. Op. In Biotechnol. 1: 36-47).
[0065]
Expression vectors may also contain sequences that allow for the maintenance and replication of the vector in more than one type of host cell, or for integration of the vector into the host chromosome. Such sequences include, but are not limited to, an origin of replication, an autonomously replicating sequence (ARS), centromeric DNA and telomeric DNA. It may also be advantageous to use a shuttle vector that can replicate and maintain in at least two types of host cells.
[0066]
Further, the expression vector may include a selectable or screenable marker gene to initially isolate or identify a host cell containing DNA encoding CD36. For long-term, high-yield production of CD36, stable expression in mammalian cells is preferred. Numerous selection systems are available for mammalian cells, including herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11: 223), hypoxanthine guanine phosphoribosyltransferase (Szybalski and Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48: 2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22: 817) genes can be employed in, but not limited to, tk, hgprf or aprt cells, respectively. Also, dihydrofolate reductase (dhfr) that confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77: 3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78: USA). Gpt (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78: 2072); neomycin phosphotransferase (neo) (Colberre-) that confers resistance to aminoglycoside G-418. Garapin et al., 1981, J. Mol. Biol. 150: 1); and hygromycin phosphotrans conferring resistance to hygromycin Antimetabolite resistance can be used as a criterion for the selection of ferase (hyg) (Santere et al., 1984, Gene 30: 147). Histidinol and Zeocin^TMOther selectable markers, such as, but not limited to, can also be used.
[0067]
In order to insert a DNA sequence encoding CD36, HSP, or another CD36 ligand into a cloning site of a vector, a DNA sequence having a regulatory function such as a promoter encodes a CD36, HSP, or other CD36 ligand, respectively. Must attach to the DNA sequence. To this end, a linker or adapter providing an appropriate compatible restriction site may be ligated to the end of the cDNA encoding CD36 or synthetic DNA by techniques well known in the art (Wu et al., 1987, Methods in Enzymol). 152: 343-349). Modifications may be made after restriction enzyme digestion to digest or fill in the single-stranded DNA ends before ligation to create blunt ends. Alternatively, the desired restriction enzyme site can be introduced into the DNA fragment by amplifying the DNA using PCR with primers containing the desired restriction enzyme site.
[0068]
In one embodiment, an expression construct comprising a CD36 sequence operably associated with a regulatory region can be introduced directly into a suitable host cell to express and produce CD36 without further cloning (eg, US Pat. , 580, 859). The expression construct may also include a DNA sequence that facilitates integration of the CD36 sequence (eg, via homologous recombination) into the genome of the host cell. In this case, in order to propagate and express CD36 in the host cell, it is not necessary to employ an expression vector containing an origin of replication suitable for a suitable host cell.
[0069]
Expression constructs containing cloned nucleotide sequences encoding CD36, HSP, or other CD36 ligands can be introduced into host cells by various techniques known in the art, and for prokaryotic cells, bacterial transformation ( Hanahan, 1985, DNA Cloning, A Practical Approach, 1: 109-136), and for eukaryotic cells, calcium phosphate mediated transfection (Wigler et al., 1977, Cell 11: 223-232), liposome mediated transfection. (Schaefer-Ridder et al., 1982, Science 215: 166-168), electroporation (Wolff et al., 1987, Proc. Natl Acad S.). i 84: 3344) and microinjection (Cappechi, 1980, Cell 22: 479-488) but the like without limitation.
[0070]
For long-term, high-yield production of correctly processed CD36, HSP or other CD36 ligand, stable expression in mammalian cells is preferred. Cell lines that stably express CD36, HSP or other CD36 ligands or CD36-peptide complexes may be engineered with vectors containing selectable markers. As a non-limiting example, after introduction of the expression construct, the engineered cells may be allowed to grow for 1-2 days in an enriched media before being replaced with a selective media. The selectable marker in the expression construct preferably confers resistance to selection and optimally allows cells to stably integrate the expression construct into the chromosome, grow in culture, and expand into cell lines. Such cells can be cultured for long periods, during which the desired gene product is continuously expressed.
[0071]
Recombinant cells can be cultured at standard conditions of temperature, incubation time, optical density and media composition. Alternatively, the recombinant antigen cells may be cultured under conditions that mimic the nutritional and physiological requirements of the cancer cells or infected cells. However, the growth conditions of the recombinant cells can be different from those for expression of CD36, HSP or other CD36 ligand, or antigenic peptides.
[0072]
5.1.2Peptide synthesis
An alternative to recombinantly producing peptides and polypeptides containing HSP, CD36, or other CD36 sequence ligands is peptide synthesis. For example, a peptide corresponding to a portion of the HSP or CD36 peptide containing a receptor binding domain that can be used as an antagonist in the therapeutic methods described herein can be synthesized using a peptide synthesizer. Synthetic peptides corresponding to the CD36 sequence useful in the therapeutic methods described herein can also be produced synthetically. Conventional peptide synthesis, or other synthetic protocols well known in the art, may be used.
[0073]
For example, amino acid sequences of CD36, HSP or other CD36 ligands, or peptides having analogs, muteins, fragments or derivatives thereof, are disclosed in Merrifield, 1963, J. Am. Am. Chem. Soc. , 85: 2149, by solid phase peptide synthesis using procedures similar to those described in US Pat. During synthesis, N-α-protected amino acids with protected side chains are added stepwise to the growing polypeptide chain (linked to the C-terminus), and to the insoluble polymer support (ie, polystyrene beads). The peptide is synthesized by linking the amino group of the N-α-deprotected amino acid to the α-carboxyl group of the N-α-protected amino acid activated by reaction with a reagent such as dicyclohexylcarbodiimide. Attachment of a free amino group to the activated carboxyl results in peptide bond formation. The most commonly used N-α-protecting groups include Boc, which is acid labile, and Fmoc, which is base labile. Details of suitable chemicals, resins, protecting groups, protected amino acids and reagents are well known in the art and will not be discussed in detail here (Atherton et al., 1989, Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, and Bodanszky, 1993, Peptide Chemistry, A Practical Textbook, 2nd edition, Springer-Verlag).
[0074]
Purification of the resulting CD36, HSP or other CD36 ligand peptide is accomplished using conventional procedures such as preparative HPLC using gel permeation, partitioning and / or ion exchange chromatography. The selection of appropriate matrices and buffers is well known in the art and will not be described in detail herein.
[0075]
In addition, analogs and derivatives of CD36, HSP or other CD36 ligand proteins can be chemically synthesized. In addition, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into CD36, HSP or other CD36 ligand sequences. Non-classical amino acids include D-nuclear isomers of common amino acids, α-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, 2-aminobutyric acid Aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoro- These include, but are not limited to, amino acids, designer amino acids (β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids), and common amino acid analogs.
[0076]
5.1.3CD36-HSP Complex-specific antibodies
Described herein are methods for producing antibodies capable of specifically recognizing a CD36 epitope, an HSP-CD36 complex epitope, or an epitope of a conserved variant or peptide fragment of a receptor or receptor complex. Such antibodies are useful in the therapeutic and diagnostic methods of the present invention.
[0077]
Such antibodies include polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F (ab ')₂Fragments, fragments produced by a Fab expression library, anti-idiotype (anti-Id) antibodies, and any of the above epitope-binding fragments. Such antibodies may be used, for example, in detecting CD36 or HSP-CD36 complex in a biological sample. Such antibodies can also be used in conjunction with compound screening schemes to assess the effect of a test compound on the interaction of HSP with CD36, for example, as described in Section 5.2 below.
[0078]
Anti-CD36-HSP complex antibodies can further be used as a method of inhibiting aberrant receptor product activity. Accordingly, such antibodies can be utilized as part of a method of treating an HSP-CD36-related disorder (eg, an autoimmune disorder).
[0079]
To produce antibodies against CD36 or the receptor complex, various host animals can be immunized by injection with CD36 or HSP-CD36, or a portion thereof. The antigen portion of CD36 or the HSP-CD36 complex can be easily predicted by algorithms known in the art.
[0080]
Host animals include, but are not limited to, rabbits, mice, and rats. Various adjuvants can be used depending on the host species to enhance the immunological response, including Freund (complete and incomplete), mineral gels (such as aluminum hydroxide), surfactants (such as lysolecithin), pluronic polyols (Pururonic polyol), polyanions, peptides, oily emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjuvants (BCG (Bacille Calmette-Guerin) and Corynebacterium parvum) (Corynebacterium parvum)).
[0081]
Polyclonal antibodies are a heterogeneous population of antibody molecules derived from the sera of animals immunized with an antigen, such as CD36 or the HSP-CD36 complex, or an antigen-functional derivative thereof. To produce polyclonal antibodies, a host animal as described above can be immunized by injecting a CD36 or HSP-CD36 complex, also supplemented with an adjuvant as described above, or a portion thereof.
[0082]
Monoclonal antibodies, a homogenous population of antibodies to a particular antigen, can be obtained by any technique that produces antibody molecules by continuous cell lines in culture. These include the hybridoma technology of Kohler and Milstein (1975, Nature 256, 495-497; and U.S. Pat. No. 4,376,110), the human B cell hybridoma technology (Kosbor et al., 1983, Immunology Today 4:72; Cole). Natl. Acad. Sci. USA 80, 2026-2030), and EBV-hybridoma technology (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan In. ), But is not limited thereto. These antibodies can be of any immunoglobulin class, including IgG, IgM, IgE, IgA, IgD and any subclasses thereof. Hybridomas producing the mAbs of the invention can be cultured in vitro or in vivo. This is the presently preferred method of production because high titers of mAbs are produced in vivo.
[0083]
In addition, techniques developed for the production of “chimeric antibodies” by splicing genes from mouse antibody molecules of appropriate antigen specificity with genes from human antibody molecules of appropriate biological activity (Morrison Natl. Acad. Sci., 81: 6851-6855; Neuberger et al., 1984, Nature 312: 604-608; Takeda et al., 1985, Nature 314: 452-454) can be used. Chimeric antibodies are molecules in which different portions are derived from different animal species, including those having a variable region derived from a mouse mAb and a human immunoglobulin constant region (eg, Cabilly et al., US Pat. No. 4,816). No., 567; and Boss et al., US Pat. No. 4,816,397, which is hereby incorporated by reference in its entirety).
[0084]
In a further embodiment of the invention, monoclonal antibodies can be produced in sterile animals (see PCT International Publication No. WO 89/12690 published December 12, 1989). According to the present invention, human antibodies may be used, which can be performed using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. USA 80: 2026-2030). Or by transforming human B cells in vitro with the EBV virus (Monoclonal Antibodies an d Cancer Therapy, Alan R .; Liss, pp. Cole et al., 1985), pp. 77-96. Techniques developed for the production of "chimeric antibodies" by splicing genes from mouse antibody molecules specific for the CD36-HSP complex with genes from human antibody molecules of appropriate biological activity ( Morrison et al., 1984, Proc. Natl. Acad. Sci. USA 81: 6851-6855; Neuberger et al., 1984, Nature 312: 604-608; Takeda et al., 1985, Nature 314: 452-4). Such antibodies can be used; such antibodies are within the scope of the present invention.
[0085]
Humanized antibodies are also provided (see Winter US Pat. No. 5,225,539). An immunoglobulin light or heavy chain variable region is comprised of a "framework" region that is interrupted by three hypervariable regions called complementarity determining regions (CDRs). The extent and CDRs of the framework regions are well defined (see "Sequences of Proteins of Immunological Interest", Kabat, E. et al., US Department of Health and Human Services (1983). Briefly, a humanized antibody is an antibody molecule from a non-human species that has one or more CDRs from a non-human species and framework regions from a human immunoglobulin molecule. Such CDR grafting antibodies have been conveniently constructed against various antigens, see, for example, Queen et al., 1989, Proc. Natl. Acad. Sci. USA 86: 10029; an antibody to the IL-2 receptor described in Riechmann et al., 1988, Nature 332: 323; an antibody to the cell surface receptor CAMPATH; Co et al., 1991, Proc. Natl. Acad. Sci. USA 88: 2869; antibodies against respiratory syncytial virus antigens described in Tempest et al., 1991, Bio-Technology 9: 267. For therapeutic use in humans, humanized antibodies are most preferred.
[0086]
Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778; Bird, 1988, Science 242: 423-426; Huston et al., 1988, Proc. Natl. Acad. Sci. USA. 85: 5879-5883; and Ward et al., 1989, Nature 334: 544-546) can be applied to produce single-chain antibodies to CD36 or HSP-CD36 complexes, or portions thereof. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge to obtain a single chain polypeptide.
[0087]
An antibody fragment that recognizes a specific epitope may be prepared by a known technique. For example, such fragments include F (ab '), which can be generated by pepsin digestion of an antibody molecule.₂Fragment, and F (ab ')₂Examples include, but are not limited to, Fab fragments that can be generated by reducing the disulfide bridges of the fragment. Alternatively, a Fab expression library may be constructed (Huse et al., 1989, Science, 246: 1275-1281) to allow fast and simple identification of monoclonal Fab fragments with the desired specificity.
[0088]
Alternatively, antibodies to CD36 can be utilized to generate anti-idiotypic antibodies that "mimic" CD36 by techniques well known to those of skill in the art (eg, Greenspan and Bona, 1993, FASEB J 7 (5): 437-). 444; and Nissinoff, 1991, J. Immunol. 147 (8): 2429-2438). For example, antibodies that bind to the CD36 ECD and competitively inhibit HSP binding to CD36 can be used to create anti-idiotypes that "mimic" the ECD and thus bind and neutralize HSP. Such neutralizing anti-idiotypes, or Fab fragments of such anti-idiotypes, can be used in therapeutic contexts to neutralize native ligands and produce HSP-CD36 related disorders (immunological disorders, proliferative disorders, And infectious diseases).
[0089]
Alternatively, antibodies to CD36 can be made that can act as agonists of CD36 activity. Such antibodies bind to CD36 and activate the signaling activity of the receptor. In addition, antibodies that act as antagonists of CD36 activity (ie, inhibit CD36 activation) are particularly useful for treating autoimmune disorders, proliferative disorders (such as cancer), and infectious diseases. Methods for assaying such agonists and antagonists are described in detail in Section 5.2 below.
[0090]
5.2CD36 Assays to identify compounds that interact with
The present invention is based on the discovery that CD36 recognizes the HSP-antigen peptide complex and induces a signaling pathway that elicits an immune response. Thus, included in the invention are methods for identifying compounds that interact with the receptor or enhance or block the function of the receptor. The invention includes the following subsections, which can be used to identify compounds or compositions that interact with CD36 or modulate the activity of CD36 and the interaction of CD36 with HSP or HSP-peptide complexes. The described in vitro and in vivo assay systems are provided.
[0091]
The present invention provides screening methodology useful for identifying small molecules, proteins, and other compounds that interact with CD36 or that modulate the interaction of HSP with CD36. Such compounds may bind to the CD36 gene or gene product with different affinities and serve as in vivo regulators of receptor activity with therapeutic applications useful for modulating an immune response. For example, certain compounds that inhibit receptor function can be used in patients to down-regulate the destructive immune response resulting from cellular release of HSPs.
[0092]
Methods for screening potential agents for the ability to interact with CD36 or modulate CD36 expression and activity are described in the present book on receptors and their role in HSP or HSP-peptide complex binding and recognition. It can be designed based on the findings of the inventors. CD36 proteins, nucleic acids, and derivatives are used in screening assays to identify molecules that specifically bind to HSP proteins, derivatives, or nucleic acids, and thus have potential uses to modulate the immune response as agonists or antagonists of CD36. Can be detected. In a preferred embodiment, such assays are molecules with potential utility as anti-autoimmune disease agents, anti-cancer and anti-infectives (such as anti-virals and antibiotics), or lead compounds for drug development. Performed to screen for For example, recombinant cells that express CD36 nucleic acid can be used to recombinantly produce CD36 in these assays and screen for molecules that interfere with the binding of HSP to CD36. Similar methods can be used to screen for molecules that bind to CD36 derivatives or nucleic acids. Methods that can be used to perform the above are generally known in the art.
[0093]
Compounds that can specifically bind to CD36 may be useful for immunotherapy. In one embodiment, an assay for identifying compounds that specifically bind to CD36, comprising: (a) contacting CD36 with one or more test compounds under conditions that effect binding; and (b) A) identifying one or more test compounds that specifically bind to CD36, wherein the compounds capable of specifically binding to CD36 are identified as compounds useful for immunotherapy;
[0094]
Another method of identifying compounds useful in immunotherapy and encompassed by the present invention involves identifying compounds that modulate the binding of CD36 ligand to CD36. As used herein, the term "CD36 ligand" refers to a CD36 molecule capable of binding CD36. Such CD36 ligands include, but are not limited to, lipoprotein complex, thrombospondin 1, Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), LDL, and phospholipids. The method comprises the steps of (a) contacting CD36 with a CD36 ligand, or a fragment, analog, derivative or mimetic thereof, in the presence of one or more test compounds; and (b) CD36 ligand bound to CD36 Or the amount of a fragment, analog, derivative or mimetic thereof, wherein the amount of bound CD36 ligand measured in (b) is different from the amount of bound CD36 measured in the absence of the test compound. Suppose that a compound useful for immunotherapy modulating the binding between CD36 ligand and CD36 has been identified.
[0095]
In another embodiment, the invention provides a method of identifying a compound useful in immunotherapy that modulates the interaction between CD36 and a CD36 ligand. The method comprises: (a) contacting CD36 with one or more test compounds; and (b) measuring the level of CD36 activity or expression, wherein the level of activity or expression measured in (b) is: If the level of CD36 activity in the absence of the one or more test compounds is different, then a compound that modulates the interaction of CD36 with a CD36 ligand is identified.
[0096]
In another embodiment, disclosed is an assay for identifying compounds that modulate an HSP-CD36 mediated process. The assay comprises: (a) contacting the test compound with HSP and CD36; and (b) measuring the level of CD36 activity or expression, wherein the level of activity or expression measured in (b) is A compound that modulates an HSP-CD36 mediated process is identified as different from the level of CD36 activity in the absence of the test compound. In another embodiment, where the identified compound is an antagonist that interferes with the interaction of HSP with CD36, the method comprises determining whether the level interferes with the interaction of HSP with CD36. In addition.
[0097]
In another embodiment, a cell-based method for identifying a compound that modulates an HSP-CD36 mediated process is described. The method comprises: (a) contacting the test compound with a heat shock protein and a CD36 expressing cell; and (b) measuring the level of CD36 activity or expression in the cell, wherein the activity measured in (b) Or, if the level of expression differs from the level of CD36 activity in the absence of the test compound, then a compound that modulates an HSP-CD36 mediated process is identified.
[0098]
In another embodiment, a receptor-ligand binding assay for identifying compounds that interact with CD36 or modulate the binding of HSP to CD36 is described. One such method includes: (a) contacting HSP with CD36, or a fragment, analog, derivative or mimetic thereof, in the presence of a test compound; and (b) CD36, or a fragment thereof, Measuring the amount of heat shock protein bound to the analog, derivative or mimetic, wherein the amount of bound heat shock protein measured in (b) is less than the amount of bound heat shock protein measured in the absence of the test compound. If the amount is different, it is assumed that a compound that modulates the binding between HSP and CD36 is identified.
[0099]
In another embodiment, the invention provides a method of identifying a compound that modulates signaling by a CD36-expressing cell. In one embodiment, such a method comprises: (a) administering one or more test compounds to a mixture of CD36-expressing cells and a complex comprising a CD36 ligand and an antigenic molecule to stimulate CD36-mediated signaling stimulation; (B) measuring the level of signaling activity by CD36-expressing cells, wherein the level measured in (b) is the level of said stimulus in the absence of said one or more test compounds. If not, it is assumed that a compound that modulates heat shock protein-mediated signal transduction stimulation by CD36-expressing cells has been identified. In another embodiment, a test compound is added to a mixture of CD36-expressing cells and a complex consisting essentially of HSPs non-covalently associated with an antigen molecule under conditions that promote CD36-mediated signaling stimulation. A compound that modulates the stimulation of HSP-mediated signaling by CD36-expressing cells, wherein the level of signal-stimulating stimulation by CD36-expressing cells is measured and if the measured level is different from the level of the stimulation in the absence of the test compound; Is identified.
[0100]
The assays of the invention may be initially optimized on a small scale (ie, in vitro) and then scaled up for high-throughput assays. In various embodiments, the in vitro screening assays of the invention can be performed using purified components or cell lysates. In another embodiment, screening assays can be performed on cultured intact cells and animal models. According to the present invention, test compounds that have been shown in vitro to modulate the activity of CD36 as described herein can be further assayed in vivo (including cultured cells and animal models) to test It was determined whether the compound had a similar effect in vivo, as well as cytokine release, intracellular Ca⁺⁺The effect of the test compound on release, T cytotoxicity, tumor development, nitric oxide release, chemokine release, accumulation or degradation of positive and negative regulators, cell proliferation, etc. may be determined.
[0101]
5.2.1CD36- Ligand binding assay
The screening assays described herein can be used to identify compounds and compositions, such as peptides and organic non-protein molecules, that interact with CD36 or modulate the interaction of HSP with CD36. Recombinant, synthetic, or exogenous compounds may have binding ability and thus may be candidates for pharmaceutical agents. Alternatively, the proteins and compounds include endogenous cellular components that interact with the identified genes and proteins in vivo. Such endogenous components may provide new targets for pharmaceutical and therapeutic intervention.
[0102]
Thus, in a preferred embodiment, both natural and / or synthetic compounds (eg, libraries of small molecules or peptides) may be screened for interaction with CD36 and / or modulation of CD36 activity. In another series of embodiments, cell lysates or tissue homogenates may be screened for proteins or other compounds that bind to one of the normal or mutant genes and polypeptides.
[0103]
The screening assays described herein identify small molecules, peptides or proteins, or derivatives, analogs and fragments thereof, that interact with CD36 and / or modulate the interaction of HSP with CD36 activity. Can be used to Such compounds can be used as agonists or antagonists for the binding of CD36 ligands, such as HSPs and HSP complexes, to cell surface receptors. For example, compounds that modulate the CD36-ligand interaction include compounds that bind to CD36 and thereby inhibit (antagonist) or enhance (agonist) the binding of ligands such as HSPs and HSP complexes to receptors. And, but not limited to, compounds that bind to a ligand, such as HSP, thereby preventing or enhancing the binding of the ligand to the receptor. Compounds that affect gene activity (by affecting gene expression), such as molecules capable of affecting transcription or interfering with splicing events to modulate the expression of full-length or blunt-ended forms of CD36 (eg, protein or organic) ) Can be identified by the screening of the present invention. It is further noted that the assays described can also identify CD36 ligands (eg, HSPs), compounds that modulate signaling by CD36 (eg, compounds that affect downstream signaling in the CD36 signaling pathway). The identification and use of such compounds that affect signaling events downstream of CD36 and thus modulate the effect of the receptor on the immune response are within the scope of the invention.
[0104]
Compounds that affect CD36 gene activity (by affecting CD36 gene expression), such as molecules capable of affecting transcription or interfering with splicing events to modulate the expression of full-length or blunt-ended forms of CD36 (eg, protein Or small organic molecules) can also be identified by the screening of the present invention. However, it is noted that the assays described can also identify compounds that modulate CD36 signaling (eg, compounds that act on downstream signaling events that are activated by ligands bound to CD36). The identification and use of such compounds that affect signaling events downstream of CD36 and thus modulate the effect of the receptor on allergic response are within the scope of the invention.
[0105]
The screening assays described herein are designed to detect compounds that modulate (ie, block or enhance) ligand-receptor interactions (such as HSP-CD36 interactions). As described in detail below, such assays are functional assays (such as binding assays) applicable to high-throughput screening methodologies.
[0106]
Binding assays can be used to identify compounds that modulate the interaction of a ligand (eg, HSP) with CD36. In one aspect of the invention, screens may be designed to identify compounds that disrupt the interaction of CD36 with a ligand, such as HSP, or a peptide derived from HSP or another CD36 ligand. Such compounds are useful as lead compounds for antagonists of HSP-CD36 related disorders and conditions such as immune disorders, proliferative disorders and infectious diseases.
[0107]
Binding assays can be performed by either direct or competitive binding assays. In a direct binding assay, test compounds are tested for binding to either CD36 or a CD36 ligand (such as HSP). Then, in a second step, the test compound is tested for its ability to modulate the ligand-CD36 interaction. On the other hand, competitive binding assays assess the ability of a test compound to compete with a ligand (ie, HSP) for binding to CD36.
[0108]
In a direct binding assay, either the ligand and / or CD36 is contacted with a test compound under conditions that allow the test compound to bind to the ligand or receptor. Binding can occur in solution or on a solid surface. Preferably, the test compound is pre-labeled for detection. Any detectable compound can be used for labeling, including, but not limited to, luminescent, fluorescent or radioisotopes, or groups comprising them, or non-isotopic labels (such as enzymes or dyes). . After an incubation period sufficient for binding to occur, the reaction is subjected to conditions and manipulations to remove excess or non-specifically bound test compound. This typically involves washing with a suitable buffer. Finally, the presence of the ligand-test compound (eg, HSP-test compound) or CD36 test compound complex is detected.
[0109]
In competitive binding assays, test compounds are assayed for the ability to disrupt or enhance the binding of a ligand (eg, HSP) to CD36. A labeled ligand (eg, HSP) may be mixed with CD36 or a fragment or derivative thereof and placed under conditions where their interaction normally occurs with or without the addition of test compounds. The amount of labeled ligand (eg, HSP) that binds to CD36 may be compared to the amount that binds in the presence or absence of a test compound.
[0110]
In a preferred embodiment, a binding assay is performed with one or more components immobilized on a solid surface to facilitate complex formation and detection. In various embodiments, the solid support can be, but is not limited to, polycarbonate, polystyrene, polypropylene, polyethylene, glass, nitrocellulose, dextran, nylon, polyacrylamide, and agarose. Support structures include beads, membranes, microparticles, the inner surface of a reaction vessel (such as a microtiter plate), test tubes, or other reaction vessels. Immobilization of CD36 or other components can be achieved by covalent or non-covalent attachment. In one embodiment, the attachment may be indirect (ie, via an attached antibody). In another embodiment, CD36 and the negative control are tagged with an epitope (such as glutathione S-transferase (GST)) and adhere to a solid surface by a commercially available antibody (such as anti-GST (Santa Cruz Biotechnology)). Be able to be interposed.
[0111]
For example, such an affinity binding assay can be performed using CD36 immobilized on a solid support. Typically, the non-immobilized component of the binding reaction (in this case, either the ligand (eg, HSP) or the test compound) is labeled and made detectable. Various labeling methods are available and can be used, such as luminescence, chromophores, fluorescent or radioisotopes, or groups comprising them, and non-isotopic labels (such as enzymes or dyes). In a preferred embodiment, the test compound is labeled with a fluorophore such as fluorescein isothiocyanate (FITC, available from Sigma Chemicals, St. Louis).
[0112]
The labeled test compound or ligand (eg, HSP) and the test compound are then contacted with the solid support under conditions that cause specific binding. After the binding reaction has taken place, unbound and non-specifically bound test compounds are separated by washing the surface. Attachment of the solid phase to the binding partner can be achieved by various methods known to those skilled in the art, including chemical crosslinking, non-specific adhesion to plastic surfaces, interaction with the antibody attached to the solid phase, and attachment to the binding partner. Examples include, but are not limited to, the interaction between a ligand (such as biotin) and a ligand-binding protein (such as avidin or streptavidin) attached to a solid phase.
[0113]
Finally, the label remaining on the solid surface can be detected by any detection method known in the art. For example, if the test compound is labeled with a fluorophore, the complex may be detected using a fluorometer.
[0114]
Preferably, CD36 is added to the binding assay in the form of intact cells expressing CD36 or an isolated membrane containing CD36. Accordingly, the ability of a test compound to directly bind to CD36 or modulate a ligand-CD36 complex (eg, an HSP-CD36 complex) in the presence or absence of a test compound, in cultured intact cells or animal models. May be. A labeled ligand (eg, HSP) may be mixed with cells expressing CD36, or a crude extract obtained from such cells, and a test compound added. Isolated membranes can be used to identify compounds that interact with CD36. For example, in a typical experiment using an isolated membrane, cells are engineered to express CD36. The membrane is recovered by standard techniques and can be used in an in vitro binding assay. Labeled ligands (eg,¹²⁵I-labeled HSP) is bound to a membrane and assayed for specific activity; specific binding is compared to a binding assay performed in the presence of excess unlabeled (cold) ligand. decide. Alternatively, soluble CD36 may be recombinantly expressed and used in non-cell based assays to identify compounds that bind to CD36. Recombinantly expressed CD36 polypeptides or fusion proteins comprising the extracellular domain of CD36 (ECD), or one or more subdomains thereof, can be used in non-cell based screening assays. Alternatively, a peptide corresponding to one or more CDs of CD36, or a fusion protein comprising one or more CDs of CD36, is used in a non-cell based assay system to identify compounds that bind to the cytoplasmic portion of CD36. Yes; such compounds may be useful for modulating the CD36 signaling pathway. In non-cell based assays, recombinantly expressed CD36 is attached to a solid substrate such as a test tube, microtiter well, or column by means well known to those of skill in the art (see Ausubel et al., Supra). Thereafter, test compounds are assayed for their ability to bind to CD36.
[0115]
Alternatively, the binding reaction may be performed in solution. In this assay, a labeled component interacts with its binding partner in solution. If separation is possible due to the size difference between the labeling component and its binding partner, the binding reaction product is passed through (the unbound labeling component is passed, but the labeling component bound to its binding partner or partner is passed through. Separation can be achieved by passing through an ultrafiltration device (with no holes). Separation can also be achieved by using any reagent that can capture the binding partner of the labeling component from solution, such as an antibody to the binding partner, a ligand binding protein that can interact with a ligand previously attached to the binding partner, and the like.
[0116]
In one embodiment, for example, by passing phage from a continuous phage display library through a column containing purified CD36 or a derivative, analog, fragment or domain thereof linked to a solid phase (such as plastic beads), Phage libraries can be screened. By changing the stringency of the wash buffer, it is possible to enrich for phage expressing peptides with high affinity for CD36. Phage isolated from the column can be cloned to directly measure the affinity of short peptides. Combinations of more than one oligonucleotide sequence can be tested for even higher affinity binding to CD36. Knowing which amino acid sequences contribute the strongest binding to CD36, computer models can be used to identify molecular contacts between CD36 and test compounds. This allows the design of non-protein compounds that mimic this contact. Such compounds have the advantage that they have the same activity as the peptides, can be used therapeutically, and can be made efficiently and inexpensively.
[0117]
In another particular embodiment of this aspect of the invention, the solid support is a membrane comprising CD36 attached to a microtiter dish. For example, a test compound, such as a cell that expresses a library member, is cultured under conditions that allow expression of the library member in a microtiter dish. The library members that bind to the protein (or nucleic acid or derivative) are recovered. Such methods are described, for example, in Parmley and Smith, 1988, Gene 73: 305-318; Fowlkes et al., 1992, BioTechniques 13: 422-427; PCT Publication No. WO 94/18318; and the references cited above. Have been.
[0118]
In another embodiment of the invention, the interaction of CD36 or a ligand (eg, HSP) with a test compound may be assayed in vitro. Known or unknown molecules are assayed for specific binding to CD36 nucleic acid, protein or derivative under conditions that promote binding to identify molecules that specifically bind to CD36. The two components can be measured by various techniques. One approach involves labeling one of the components with a readily detectable label, placing it together with the test component under conditions that cause binding, and performing a separation step to separate bound labeled component from unbound labeled component; Then, the amount of the binding component is measured. In one embodiment, CD36 is labeled and added to the test substance using conditions that cause binding. Test substance binding can be determined by using polyacrylamide gel analysis to compare complexes formed in the presence or absence of the test substance.
[0119]
In yet another embodiment, binding of a ligand (eg, HSP) to CD36 may be assayed in intact cells in an animal model. A labeled ligand (eg, HSP) may be administered directly to an animal with or without a test compound. Signal transduction stimulation of a ligand (eg, HSP) may be measured in the presence and absence of a test compound. For these assays, the host cells to which the test compound is added are genetically engineered to express CD36 and / or a ligand (eg, HSP) (either transient, inducible, constitutive, or stable). It is possible). For the purposes of the screening methods of the present invention, various types of host cells can be used, including but not limited to tissue culture cells, mammalian cells, yeast cells and bacteria. Mammalian cells such as macrophages or other cells that express CD36 (ie, cells of the monocyte cell lineage, liver parenchymal cells, fibroblasts, keratinocytes, neurons and placental syncytiotrophoblasts) of the present invention It may be a preferred cell type for performing the assay in. Bacteria and yeast are relatively easy to culture, but treat proteins differently than mammalian cells.
[0120]
5.2.2Activity assay
After identifying a test compound that modulates an interaction with CD36, or a ligand (eg, HSP) with CD36, the test compound is further characterized to determine its effect on CD36 activity and ligand-CD36 cell signaling pathway. Can be measured. For example, test compounds can be characterized by testing their effect on ligand (eg, HSP) CD36 cell activity in vivo. Such assays include downstream signaling assays, assays for antigen-specific activation of cytotoxic T cells, nitric oxide assays, chemokine assays, and the like.
[0121]
In various embodiments, candidate compounds identified in the primary assay may be tested for an effect on intrinsic CD36 signaling transduction activity. For example, downstream signaling effects of activation that can be assayed include enhanced locomotion and chemotaxis of macrophages (Forrester et al., 1983, Immunology 50: 251-259), down-regulation of proteinase synthesis, and intracellular Increasing calcium, inositol phosphate and cyclic AMP (Misra et al., 1993, Biochem. J., 290: 885-891). Other essential immune responses that can be tested are the release of cytokines (ie, IL-12, IL1β, GMCSF and TNFα), the release of nitric oxide, and the release of chemokines. Thus, as a secondary assay, any candidate compounds identified can be tested for such changes in activity in the presence and absence.
[0122]
For example, in one embodiment, a chemotaxis assay can be used to further characterize the candidates identified by the first screening assay. A number of techniques are available for testing chemotaxis migration in vitro (eg, Current Protocols in Immunology, 6.12.1-6.12.28, edited by Colligan et al., John Wiley and Sons, Inc. See Leonard et al., 1995, "Measurement of α and β Chemokines", published in 1995). For example, in one embodiment, chemokine gradients can be used in a multiwell Boyden chemotactic chamber to test candidate compounds for the ability to modulate the ability of CD36 to induce migration of cells expressing the receptor. . In an embodiment of this method, a serial dilution of a ligand (eg, HSP) / CD36 antagonist or agonist test compound identified in the primary screen is placed in the bottom well of a Boyden chemotaxis chamber. A fixed amount of ligand is also added to the dilution series. As a control, at least one aliquot contains only the ligand (eg, HSP). Chemotaxis of CD36 by comparing the number of migrating cells on the bottom of the membrane filter of an aliquot containing only the ligand (eg, HSP) with the number of cells in the aliquot containing the test compound and the ligand (eg, HSP) The contribution of the antagonist or agonist compound to sexual activity is measured. Addition of a test compound to a ligand (eg, HSP) solution reduces the number of cells detected on the membrane compared to the number of cells detected using a solution containing only the ligand (eg, HSP). In this case, a ligand (eg, HSP) -induced antagonist of chemotactic activity of CD36-expressing cells has been identified.
[0123]
In another embodiment, a modulator of ligand-CD36 interaction can be further characterized using a calcium flux (flux) assay as a secondary screen. Intracellular calcium ion concentration can be measured in cells expressing CD36 in the presence of a ligand, in the presence and absence of a test compound. For example, calcium migration can be detected and measured by flow cytometry by labeling with a fluorescent dye trapped in cells. Fluorescent dyes such as Indo-1 show a change in the emission spectrum when bound to calcium, and use the ratio of the fluorescence generated by the calcium-bound dye to the fluorescence generated by the unbound dye. Alternatively, the intracellular calcium concentration may be estimated. In a specific embodiment, cells are incubated and excited at 37 ° C. in India 1 containing media in cuvettes and fluorescence is measured using a fluorimeter (Photon Technology Corporation, International). Ligands are added at specific time points in the presence and absence of test compound, EGTA is added to the cuvette to release and chelate total calcium, and the response is measured. Upon binding of the ligand, Ca in the cells expressing CD36²⁺The concentration increases. The agonist is intracellular Ca²⁺Resulting in a relative increase in concentration, where the antagonist²⁺This causes a relative decrease in concentration.
[0124]
Increased nitric oxide is also an indicator of CD36 activation. Thus, in another embodiment, a nitric oxide assay can be used for screening to characterize modulators of ligand-CD36 interaction. Nitric oxide concentration can be measured in cells expressing CD36 in the presence of a ligand, or in the presence and absence of a test compound. For example, after 20 hours of incubation, the supernatant can be collected and reacted with a Greiss reagent (Nims, RW, et al., 1996. Methods in Enzymology 268, 93). Such a reaction can be quantified by spectrophotometry.
[0125]
An increase in MCP-1 chemokines is also an indicator of CD36 activation. Thus, in another embodiment, the modulator of ligand-CD36 interaction can be characterized using the MCP-1 chemokine assay for screening. MCP-1 chemokine concentrations can be measured in cells expressing CD36 in the presence of a ligand, or in the presence and absence of a test compound. For example, after 20 hours of incubation, the supernatant can be collected and analyzed by ELISA (enzyme-linked immunosorbent assay) using an antibody specific for MCP-1 chemokine.
[0126]
5.2.3Compounds that can be screened by the present invention
Using the screening assays described herein, small molecules, peptides or proteins, or derivatives, analogs and the like, that interact with CD36 or modulate the interaction of a ligand (eg, HSP) with CD36. Fragments can be identified. Compounds that can be screened according to the present invention include those that bind to the ECD of CD36 and either block the activity caused by the natural ligand (ie, antagonists) or mimic the activity caused by the natural ligand (ie, agonists) Molecules, peptides, antibodies and fragments thereof, and other organic compounds (eg, peptidomimetics) include, but are not limited to, small molecules, peptides, antibodies or fragments thereof, and other organic compounds. In one embodiment, such compounds comprise a sequence of CD36, such as the ECD of CD36 (or a portion thereof) that can bind or "neutralize" a native ligand such as HSP, LDL. In another embodiment, such compounds include a ligand sequence, such as an HSP sequence, that can bind to the active site of CD36 and block its activity.
[0127]
Compounds that can be used for screening include, for example, peptides such as soluble peptides, including but not limited to members of a random peptide library (eg, Lam et al., 1991, Nature 354: 82-84; Houghten et al., 1991). , Nature 354: 84-86), members of a combinatorial chemically-derived molecular library of D- and / or L-structured amino acids, phosphopeptides (random or partially degenerate targeted phosphodiesters). Examples include, but are not limited to, members of a peptide library; see, eg, Songyang et al., 1993, Cell 72: 767-778), antibodies (polyclonal, monoclonal). , Humanized, anti-idiotypic, chimeric or single chain antibodies, FAb, F (ab ')₂And FAb expression library fragments, and their epitope-binding fragments, including, but not limited to), and small organic or inorganic molecules.
[0128]
In one embodiment of the invention, a peptide library can be used as a source of test compounds that can be used to screen for modulators of CD36 interaction (such as HSP-CD36). Diversity libraries, such as random or combinatorial peptide or non-peptide libraries, can be screened for molecules that specifically bind to CD36. Many available libraries are known in the art. (Eg, chemical synthesis libraries, recombination (eg, phage display libraries), and in vitro translation-based libraries).
[0129]
Examples of chemically synthesized libraries are described in Fodor et al., 1991, Science 251: 767-773; Houghten et al., 1991, Nature 354: 84-86; Lam et al., 1991, Nature 354: 82-84; Medynski, 1994, Bio /. Technology 12: 709-710; Gallop et al., 1994, J. Am. Medicinal Chemistry 37 (9): 1233-1251; Ohlmeyer et al., 1993, Proc. Natl. Acad. Sci. ScL USA 90: 10922-10926; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91: 11422-11426; Houghten et al., 1992, Biotechniques 13: 412; Jaywickickeme et al., 1994, Proc. Natl. Acad. Sci. USA 91: 1614-1618; Salmon et al., 1993, Proc. Natl. Acad. Sci. USA 90: 11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner, 1992, Proc. Natl. Acad. Sci. USA 89: 5381-5383.
[0130]
Examples of phage display libraries are described in Scott and Smith, 1990, Science 249: 386-390; Devlin et al., 1990, Science, 249: 404-406; Christian et al., 1992, J. Am. Mol. Biol. 227: 711-718; Lenstra, 1992, J. Am. Immunol. Meth. 152: 149-157; Kay et al., 1993, Gene 128: 59-65; and PCT Publication No. WO 94/18318, Aug. 18, 1994.
[0131]
As a non-limiting example of a non-peptide library, a benzodiazepine library (see, for example, Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91: 4708-4712) can be used. A peptoid library (Simon et al., 1992, Proc. Natl. Acad. Sci. USA 89: 9397-9371) can also be used. Another example of a library that can be used to completely methylate the amide functionality in a peptide to create a chemically transformed combinatorial library is described in Ostresh et al. (1994, Proc. Natl. Acad. Sci. USA. 91: 11138-11142).
[0132]
Screening the library may be accomplished by any of a variety of well-known methods. See, for example, the following references disclosing screening of peptide libraries: Palmley and Smith, 1989, Adv. Exp. Med. Biol. 251: 215-218; Scott and Smith, 1990, Science 249: 386-390; Fowlkes et al., 1992; BioTechniques 13: 422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89: 5393-5397; Yu et al., 1994, Cell 76: 933-945; Staudt et al., 1988, Science 241: 577-580; Bock et al., 1992, Nature 355: 564-566; Turk et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6988-6992; Ellington et al., 1992, Nature 355: 850-852; U.S. Pat. No. 5,096,815, U.S. Pat. No. 5,223,409 and U.S. Pat. 198,346; Rebar and Pabo, 1993, Science 263: 671-673; and PCT Publication No. WO 94/18318.
[0133]
In another embodiment of the invention, a labeled ligand (eg, HSP) is added to an in vitro translation system, such as a rabbit reticulocyte lysate (RRL) system, followed by a subsequent in vitro priming immunoreaction. Screening may be performed. In vitro translation-based libraries include PCT Publication No. WO 91/05058, dated April 18, 1991; and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91: 9022-9026, but is not limited thereto.
[0134]
Compounds that can be tested and identified according to the methods described herein include Aldrich (Milwaukee, WI 53233), Sigma Chemical (St. Louis, MO), Fluka Chemie AG (Buchs, Swisserland), Fluk. (Ronkonkoma, NY), Eastman Chemical Company, Fine Chemicals (Kingsport, TN), Boehringer Mannheim GmbH (Mannheim, Germany), Takasago (Rockleigh, NJ), SST Corporation (Clifton, NJ), Ferro (Zachary, LA 70791), Riedel-deHaen Aktiengesellschaft (Seelze, Germany), PPG Industries Inc. , Fine Chemicals (Pittsburgh, PA 15272), including, but not limited to, compounds obtained from any commercial source. Any further type of natural product, including bacterial, fungal, plant or animal extracts, can be screened using the methods of the present invention.
[0135]
In addition, a diverse library of test compounds, including small molecule test compounds, may be utilized. For example, the library is Specs and BioSpecs B.R. V. (Rijswijk, The Netherlands), Chembridge Corporation (San Diego, CA), Contract Services Company (Dolgoprudny, Moscow Region, USA). (Princeton, NJ), Maybridge Chemicals Ltd. (Cornwall PL34 OHW, United Kingdom) and Asinex (Moscow, Russia).
[0136]
In addition, combinatorial library methods known in the art, including, but not limited to, the following: biological libraries; spatially addressable parallel solid phase or solution phase Libraries; synthetic library methods requiring deconvolution; "one bead one compound" library method; and synthetic library methods using affinity chromatography selection. Biological library approaches are limited to peptide libraries, and the other four approaches are applicable to peptide, non-peptide oligomer, or small molecule compound libraries (Lam, 1997, Anticancer Drug Des. 12). 145). Combinatorial libraries of test compounds, including small molecule test compounds, are available and are described, for example, in Eichler and Houghten, 1995, Mol. Med. Today 1: 174-180; Dolle, 1997, Mol. Divers. 2: 223-236; and Lam, 1997, Anticancer Drug Des. 12: 145-167.
[0137]
Examples of methods for synthesizing molecular libraries are described, for example, in DeWitt et al., 1993, Proc. Natl. Acad. Sci. ScL USA 90: 6909; Erb et al., 1994, Proc. Natl. Acad. Sci. USA 91: 11422; Zuckermann et al., 1994, J. Am. Med. Chem. 37: 2678; Cho et al., 1993, Science 261: 1303; Carrell et al., 1994, Angew. Chem. Int. Ed. Engl. 33: 2059; Carell et al., 1994, Angew. Chem. Int. Ed. Engl. 33: 2061; and Gallop et al., 1994, J. Am. Med. Chem. 37: 1233 etc. can be found in the art.
[0138]
Compound libraries may be in solution (eg, Houghten, 1992, BioTechniques 13: 412-421), or beads (Lam, 1991, Nature 354: 82-84), chips (Fodor, 1993, Nature 364: 555-556). , Bacteria (US Pat. No. 5,223,409), pores (US Pat. Nos. 5,571,698; 5,403,484; and 5,223,409), plasmids (Cull et al.). Natl. Acad. Sci. USA 89: 1865-1869) or phage (Scott and Smith, 1990, Science 249: 386-390; Devlin, 1990, Science 249: 404-406; Cwi. Rla et al., 1990, Proc. Natl. Acad. Sci. USA 87: 6378-6382; and Felici, 1991, J. Mol. Biol. 222: 301-310).
[0139]
Screening of the library can be accomplished by various commonly known methods. See, for example, the following references disclosing screening of peptide libraries: Palmley and Smith, 1989, Adv. Exp. Med. Biol. 251: 215-218; Scott and Smith, 1990, Science 249: 386-390; Fowlkes et al., 1992; BioTechniques 13: 422-427; Oldenburg et al., 1992, Proc. Natl. Acad. Sci. USA 89: 5393-5397; Yu et al., 1994, Cell 76: 933-945; Staudt et al., 1988, Science 241: 577-580; Bock et al., 1992, Nature 355: 564-566; Turk et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6988-6992; Ellington et al., 1992, Nature 355: 850-852; U.S. Patent Nos. 5,096,815, 5,223,409 and 5,198, issued to Ladner et al. 346; Rebar and Pabo, 1993, Science 263: 671-673; and PCT Publication No. WO 94/18318.
[0140]
5.3Useful for immunotherapy, CD36 and / Or CD36 Ligand (HSP etc ) Identification of fragments of
The invention also encompasses methods of identifying ligand binding CD36 fragments (such as "HSP binding domains") and analogs, muteins or derivatives thereof capable of binding to a CD36 ligand peptide (such as an HSP peptide complex). Such ligand binding CD36 fragments (eg, HSP binding domains) can then be tested for activity in vivo and in vitro using the CD36 / ligand binding assay described in Section 5.2.1 above. In one embodiment, such a method of identifying a CD36 fragment capable of binding to a heat shock protein comprises: (a) contacting the heat shock protein with one or more CD36 fragments; and (b) specific for the heat shock protein. Identifying a CD36 polypeptide fragment that binds to.
[0141]
A ligand binding domain (eg, an HSP binding domain) of CD36 capable of binding to a ligand-antigen peptide complex (such as an HSP-antigen peptide complex) can be determined by an in vivo binding assay as described in Section 5.2.2 above. Activity can be further tested using any of the CTL assays. For example, one such method of identifying a CD36 fragment capable of inducing an HSP-CD36 mediated process includes: (a) contacting a heat shock protein with a cell that expresses the CD36 fragment; and (b) the cell. Measuring the level of CD36 activity in the HSP-CD36 mediated process or activity measured in (b), if the level of CD36 activity is greater than the level of CD36 activity in the absence of the CD36 fragment. Assume that a CD36 fragment that can induce a CD36-mediated process has been identified. Depending on the effect in such assays, such molecules can be administered or expressed in vivo to either enhance or also block receptor function. For example, these assays can be used to identify CD36-HSP binding domains that can bind to the HSP-antigen complex and negatively interfere with signaling activity. These antagonists can be used to down-modulate the immune response generated by the cellular release of HSP. Alternatively, certain CD36 HSP binding domains may be used to enhance HSP-antigen complex uptake and signaling. Such agonists can be administered or expressed in a subject to elicit an immune response against the antigen of interest.
[0142]
In another embodiment, the invention encompasses methods of identifying ligand fragments (such as HSP fragments) capable of binding CD36 ("CD36 binding domain"), and analogs, muteins or derivatives thereof. As described for the assays for CD36-related polypeptides above, such CD36 binding domains can then be tested for activity in vivo and in vitro using the binding assays described in Section 5.2.1 above. . For example, one such method of identifying heat shock protein fragments capable of binding to CD36 includes: (a) contacting CD36 with one or more heat shock protein fragments; and (b) specifically binding to CD36. Identifying a heat shock protein fragment that does.
[0143]
A ligand fragment of interest (such as an HSP fragment) may be further tested in cells using an in vivo binding assay, as described in section 5.2.2, or a CTL assay. For example, in one embodiment, such a method of identifying a heat shock protein fragment capable of inducing an HSP-CD36 mediated process includes: (a) contacting the fragment with a cell that expresses a heat shock protein; and (b) C.) Measuring the level of CD36 activity in said cells, wherein the level of HSP-CD36 mediated process or activity measured in (b) exceeds the level of CD36 activity in the absence of said heat shock protein fragment. In this case, it is assumed that an HSP fragment capable of inducing an HSP-CD36-mediated process has been identified. Alternatively, the CD36 binding domain may be used to block HSP uptake by CD36. In one embodiment, such an HSP fragment containing a CD36 binding domain sequence may be used to construct a recombinant fusion protein consisting of a heat shock protein CD36 binding domain and an antigenic peptide sequence. Such recombinant fusion proteins may be used to elicit an immune response and to treat or prevent immune diseases and disorders (Suzue et al., 1997, Proc. Natl. Acad. Sci. USA). .94: 13146-51).
[0144]
The CD36 fragments, analogs, muteins and derivatives, and / or ligand (eg, HSP) fragments, analogs, muteins and derivatives of the present invention may be obtained using recombinant DNA technology, synthetic methods, or native CD36 and / or derivatives. Alternatively, it can be produced by enzymatic or chemical cleavage of a ligand (eg, HSP).
[0145]
Any eukaryotic cell can serve as a source of nucleic acid to obtain the coding region for the CD36 ligand (eg, HSP) gene. Nucleic acid sequences encoding a ligand (eg, HSP) and / or CD36 can be isolated from vertebrate, mammalian, and primate sources, including humans. Amino acid and nucleotide sequences for natural ligands (eg, HSP) and CD36 are generally available from sequence databases such as Genbank.
[0146]
DNA is obtained from tissue, cell culture, or cloned DNA (eg, a DNA “library”) by DNA amplification or molecular cloning, using standard procedures known in the art. Clones derived from genomic DNA may contain regulatory and intronic DNA regions, in addition to coding regions; clones derived from cDNA contain only exon sequences. In a preferred embodiment, polymerase chain reaction (PCR) amplification allows amplification of DNA from genomic or cDNA using primers designed from known sequences of ligands (eg, HSP or other CD36 ligand). Polymerase chain reaction (PCR) is commonly used to obtain a gene or gene fragment of interest. For example, a nucleotide sequence encoding a fragment of any desired length can be generated using PCR primers flanking the nucleotide sequence encoding the peptide binding domain. Alternatively, the CD36 ligand (eg, HSP) or other CD36 ligand receptor gene sequence is cleaved with restriction endonucleases at appropriate sites, if any, to release a DNA fragment encoding the peptide binding domain. I do. If there are no convenient restriction sites, such sites may be created at appropriate locations by site-directed mutagenesis and / or DNA amplification methods known in the art (eg, Shankarappa et al., 1992, PCR Methods Appl. 1: 277-278). The DNA fragment encoding the ligand (eg, HSP) or a fragment of the CD36 gene is then isolated and ligated to a suitable expression vector, taking care to ensure that the proper translational reading frame is maintained. Alternatives to isolating genomic DNA include, but are not limited to, chemically synthesizing the gene sequence itself from a known sequence, or turning cDNA into mRNA encoding a ligand (eg, HSP) and / or CD36. Not done.
[0147]
Any mutagenesis techniques known in the art may be used to make amino acid substitutions in the peptide sequence to be expressed or to create / delete restriction sites to facilitate further manipulation in the DNA sequence. Individual nucleotides can be modified. Such techniques include chemical mutagenesis, in vitro site-directed mutagenesis (Hutchinson, C. et al., 1978, J. Biol. Chem 253: 6551), oligonucleotide-directed mutagenesis (Smith, 1985). 19: 423-463; Hill et al., 1987, Methods Enzymol. 155: 558-568), PCR-based overlap extension (Ho et al., 1989, Gene 77: 51-59), PCR-based type. Megaprimer mutagenesis (Sarkar et al., 1990, Biotechniques, 8: 404-407) and the like, but are not limited thereto. Modifications can be confirmed by double-stranded dideoxy DNA sequencing.
[0148]
An alternative to producing CD36 and / or ligand (eg, HSP) fragments by recombinant techniques is peptide synthesis. For example, a peptide that includes a substrate binding domain or that corresponds to a portion of CD36 and / or a ligand (eg, HSP) that binds the peptide in vitro can be synthesized using a peptide synthesizer. Conventional peptide synthesis or other synthetic protocols well known in the art can be used.
[0149]
In addition, analogs or derivatives of CD36 and / or ligand (eg, HSP) can be chemically synthesized. In addition, if desired, nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the CD36 and / or ligand (eg, HSP) sequence. Non-classical amino acids include D-isomers of general amino acids, α-isobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, γ-Abu, ε-Ahx, 6-aminohexanoic acid, Aib, -Aminoisobutyric acid, 3-aminopropionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, fluoroamino acid, designer Examples include, but are not limited to, amino acids (β-methyl amino acids, Cα-methyl amino acids, Nα-methyl amino acids, and the like), as well as common amino acid analogs.
[0150]
CD36 and / or ligand (eg, HSP) peptides, or variants or derivatives thereof, are described in Merrifield, 1963, J. Am. Am. Chem. Soc. , 85: 2149, by solid phase peptide synthesis. During synthesis, N-α-protected amino acids with protected side chains are added stepwise to the growing polypeptide chain linked at the C-terminus and to the insoluble polymer support (ie, polystyrene beads). The peptide is synthesized by linking the amino group of the N-α-deprotected amino acid to the α-carboxyl group of the N-α-protected amino acid activated by reacting with an agent such as dicyclohexylcarbodiimide. Attachment of a free amino group to the activated carboxyl results in peptide bond formation. The most commonly used N-α-protecting groups include Boc, which is acid labile, and Fmoc, which is base labile. Suitable chemicals, resins, protecting groups, protected amino acids and reagents are well known in the art and will not be discussed in detail here (Atherton et al., 1989, Solid Phase Peptide Synthesis: A Practical Approach, IRL Press, And Bodanszky, 1993, Peptide Chemistry, A Practical Textbook, 2nd edition, Springer-Verlag).
[0151]
Purification of the resulting fragments is performed using conventional procedures, such as preparative HPLC using gel permeation, partition and / or ion exchange chromatography. The selection of suitable matrices and buffers is well known in the art and will not be described in detail herein.
[0152]
In alternative embodiments, fragments of CD36 and / or ligand (eg, HSP) can be obtained by chemical or enzymatic cleavage of native or recombinant CD36 and / or ligand (eg, HSP) molecules. . Specific chemical cleavages include cyanogen bromide, NaBH₄, Acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, and the like. Endoproteases that cleave specific sites can also be used. Such proteases are known in the art and include, but are not limited to, trypsin, α-chymotrypsin, V8 protease, papain, and proteinase K (“Current Protocols in Molecular Biology”, Green Publishing Associates, Wisconsin, Wis.) (See Ausubel et al. (Eds.) In York, 17.4.6-17.4.8). The CD36 and / or ligand (eg, HSP) amino acid sequence of interest can be tested for recognition sites for these proteases. The enzyme is selected so as to release a peptide binding domain or a peptide binding fragment. The CD36 and / or ligand (eg, HSP) molecule is then incubated with the protease under conditions that cause digestion by the protease and release of the specifically designated peptide binding fragment. Alternatively, such protease digestion is performed blindly (ie, which digestion products bind the peptide binding domain) using a protease (proteinase K or pronase) with specific or general specificity. (Without knowing whether to include).
[0153]
After preparation of the fragments, the digestion products may be purified as described above and then tested for their ability to bind to the peptide or for immunogenicity. Methods for determining the immunogenicity of ligand (eg, HSP) conjugates by cytotoxicity assays are described in Section 5.2.2.
[0154]
5.4Drug design
In identifying compounds that interact with CD36 or modulate the interaction of CD36 with a CD36 ligand (such as HSP), such compounds can be further investigated to test for their ability to alter an immune response. . In particular, for example, compounds identified by the methods of the invention can be used in vivo in HSP-CD36 mediated processes and in permissive animal models of HSP-CD36 related disorders (eg, immune disorders, proliferative disorders and infectious diseases). Can test.
[0155]
Computer modeling and search techniques allow the identification of compounds that can modulate the interaction of CD36 with its ligand (eg, HSP), or the improvement of previously identified compounds. Such compounds or compositions are identified to identify active sites or regions. Such an active site is typically a ligand binding site. The active site is identified using methods known in the art, such as, for example, from the amino acid sequence of a peptide, from the nucleotide sequence of a nucleic acid, or from a survey of the complex of a related compound or composition and its natural ligand. it can. In the latter case, the active site can be sought by using chemical or X-ray crystallographic methods to find where the complexed ligand is found on the factor.
[0156]
Next, the three-dimensional geometric structure of the active site is determined. This can be performed by a known method such as an X-ray crystal method that can determine a complete molecular structure. Alternatively, solid or liquid phase NMR can be used to determine a particular intra-molecular distance. Any other experimental method of structure determination can be used to obtain a partial or complete geometry. The geometry can be measured using natural or artificial conjugated ligands that can increase the accuracy of the determined active site structure.
[0157]
If an imperfect or inaccurate structure is determined, computer-aided numerical modeling methods can be used to complete the structure or improve its accuracy. A parameterized model specific to a specific biological macromolecule such as a protein or a nucleic acid, a molecular mechanics model based on computing molecular motion, a statistical mechanics model based on thermal ensembles , Or any recognized modeling method, including combinatorial models. For most types of models, a standard molecular force field representing the forces between the constituent atoms and groups is required and can be selected from force fields known in the field of physical chemistry. Incomplete or inaccurate experimental structures are a constraint on the complete and more accurate structures computed by these modeling methods.
[0158]
Finally, candidate modulating compounds can be identified by experimentally determining the structure of the active site, either by modeling or by combination, and then searching a database containing the compounds along with information about their molecular structure. Such a search finds compounds that have a structure that matches the determined active site structure and that interacts with the groups that define the active site. Such searches may be performed manually, but are preferably computer assisted. These compounds found in this search are potential CD36 modulating compounds.
[0159]
Alternatively, improved modulating compounds can be identified from known modulating compounds or ligands using the methods described above. The structural effects of the modifications can be determined by modifying the composition of the known compound and applying the experimental and computer modeling methods described above applied to the novel compositions. The altered structure is then compared to the active site structure of the compound to determine whether an improved fit or interaction occurs. In this way, a systematic change in the composition, such as a change in side group, can be quickly evaluated to obtain a modified modulating compound or ligand with improved specificity or activity. be able to.
[0160]
Additional experimental and computer modeling methods useful for identifying modulating compounds based on the identification of the active site of either CD36 or HSP, as well as other ligands and analogs thereof, will be apparent to those skilled in the art.
[0161]
Examples of molecular modeling systems include CHARMm and the QUANTA program (Polygen Corporation, Waltham, Mass.). CHARMm performs energy minimization and molecular mechanics functions. QUANTA performs the construction, image modeling and analysis of molecular structures. QUANTA allows the interactive construction, modification, visualization and analysis of molecular interactions.
[0162]
Rotivinen et al. (1988, Acta Pharmaceutical Fennica 97: 159-166); Ripka (1988 New Scientist 54-57); McKinally and Rossmann (1989, Annu. Rev. Pharma. OSAR: Quantitative Structure-Activity Relationships in Drug Design pp. 189-193 Alan R.A. Liss, Inc. Numerous articles such as Lewis and Dean (1989, Proc. R. Soc. London. 236: 125-140 and 141-162) outline computer modeling of drugs that interact with specific proteins. . Askew et al. (1989, J. Am. Chem. Soc. 111: 1082-1090) outline model receptors for nucleic acid components. Other computer programs that screen and image chemicals are available from BioDesign, Inc. (Pasadena, CA), Allelix, Inc. (Mississauga, Ontario, Canada) and Hypercube, Inc. (Cambridge, Ontario). These are primarily designed for application to drugs specific to a particular protein, but can be applied to the design of drugs specific to a DNA or RNA region once the region is identified.
[0163]
5.5Diagnostic applications
CD36 was initially identified as a heat shock protein receptor by interacting with gp96, which is exclusively located intracellularly and is released as a result of necrosis rather than apoptotic cell death. Thus, gp96 bound to CD36 can act as a sensor for necrotic cell death. Thus, CD36-ligand complexes may be used to detect and diagnose proliferative disorders (such as cancer), autoimmune disorders and infectious diseases. Thus, CD36 proteins, analogs, derivatives and subsequences thereof, CD36 nucleic acids (and their complementary sequences), and anti-CD36 antibodies are useful for detecting and diagnosing such disorders.
[0164]
CD36 and CD36 nucleic acids can be used in assays to detect, prognose, or diagnose immune system disorders that can result in tumorigenesis, cancer, adenomas, and the like and viral diseases.
[0165]
The molecules of the invention can be used in assays, such as immunoassays, to detect, prognose, diagnose or monitor, or monitor the treatment of, various conditions, diseases and disorders that affect expression. In particular, such immunoassays comprise contacting a sample obtained from a patient with an HSP-CD36-specific antibody under conditions that allow immunospecific binding to occur, as well as detecting the amount of any immunospecific binding by the antibody. Or measuring. In certain embodiments, such antibody binding in tissue sections is used to detect abnormal localization or abnormal (eg, low or absent) CD36 levels. In certain embodiments, antibodies to CD36 can be used to assay patient tissue or serum samples for the presence of CD36, where abnormal levels of CD36 are indicative of disease symptoms. By “abnormal level” is meant a level that is higher or lower than the level in a similar sample obtained from an intact body part or subject (or a representative level representative thereof).
[0166]
Available immunoassays include Western blot, immunohistochemistry radioimmunoassay, ELISA, "sandwich" immunoassay, immunoprecipitation assay, sedimentation reaction, gel diffusion sedimentation reaction, immunodiffusion assay, agglutination assay, complement fixation assay, immunoradiometric Competitive and non-competitive assay systems using techniques such as assays, fluorescent immunoassays, protein A immunoassays, include but are not limited to.
[0167]
The CD36 gene and related nucleic acid sequences and subsequences, including complementary sequences, can also be used in hybridization assays. The CD36 nucleic acid sequence, or a subsequence thereof comprising at least about 8 nucleotides, can be used as a hybridization probe. Hybridization assays can be used to detect, prognose, diagnose or monitor conditions, disorders or disease states associated with abnormal changes in expression and / or activity as described above. In particular, such hybridization assays involve contacting a sample containing nucleic acids with a nucleic acid probe capable of hybridizing to CD36 DNA or RNA under conditions that allow hybridization to occur, as well as any resulting hybridization. Detection or measurement is performed.
[0168]
In certain embodiments, a CD36 RNA that detects reduced levels of CD36 protein, CD36 RNA or CD36 functional activity (eg, binding to HSP, antibody binding activity, etc.), or that reduces CD36 expression or activity, By detecting mutations in the DNA or CD36 protein (eg, transposition in the CD36 nucleic acid, blunting in the CD36 gene or protein, alteration in nucleotide or amino acid sequence as compared to wild-type CD36), Diseases and disorders with poor immunoresponsiveness can be diagnosed, screened for their presence, or the predisposition to suffer such disorders can be detected. Such diseases and disorders include, but are not limited to, those described in Sections 5.7, 5.8, and 5.9. By way of example, the level of CD36 protein can be detected by an immunoassay, the level of CD36 RNA can be detected by a hybridization assay (eg, Northern blot, in situ hybridization), and the CD36 activity can be measured in vivo or in vitro. Can be assayed. Transpositions, deletions and point mutagenesis in CD36 nucleic acids were obtained from Southern blots, FISH, RFLP analysis, SSCP, PCR using primers (preferably, primers that generate fragments that span at least almost the entire CD36 gene), from patients. CD36 genomic DNA or cDNA can be detected.
[0169]
In a preferred embodiment, the level of CD36 mRNA or protein in the patient sample is detected or measured relative to the level in a similar sample obtained from a subject not suffering from a malignancy or hyperproliferative disorder. A decrease in the level indicates that the subject has, or is predisposed to, a viral infection, malignancy or hyperproliferative disorder.
[0170]
In another specific embodiment, a CD36 RNA, DNA or protein that detects elevated levels of CD36 protein, RNA or CD36 functional activity (eg, HSP binding or antibodies, etc.) or that increases CD36 expression or activity. Detection of mutations (eg, transposition in a CD36 nucleic acid, truncation in a gene or protein, alterations in nucleotide or amino acid sequence relative to wild type), resulting in loss of immune responsiveness resulting in cell proliferation, or Diseases and disorders for which proliferation is desirable for treatment can be diagnosed, screened for their possible presence, or a predisposition to suffer such disorders can be detected. Such diseases and disorders include, but are not limited to, those described in Sections 5.7, 5.8, and 5.9. By way of example, levels of CD36 protein, CD36 RNA, CD36 binding activity, and the presence of transpositions or point mutations can be determined as described above.
[0171]
In certain embodiments, the level of CD36 mRNA or protein in a patient sample is detected or measured relative to the level in a similar sample from a non-disordered patient (where the elevated level indicates that the subject has Suffering from or predisposed to suffer from an autoimmune disorder).
[0172]
Also provided is a kit for diagnostic use, wherein the anti-CD36 antibody, and optionally, a labeled binding partner for the antibody, is contained in one or more containers. Alternatively, the anti-CD36 antibody can be labeled (with a detectable marker, such as a chemiluminescent, enzymatic, fluorescent, or radioactive moiety). Kits are also provided that contain a nucleic acid probe capable of hybridizing to CD36 RNA in one or more containers. In certain embodiments, the kits are prepared under appropriate reaction conditions for at least a portion of the nucleic acid (eg, polymerase chain reaction (eg, Innis et al., 1990, PCR Protocols, Academic Press, Inc., San Diego, CA). Ligase chain reaction (see EP 320,308), use of Qβ replicase, circular probe reaction, or other methods known in the art) to initiate a pair of primers (eg, 6-6 each). (30 nucleotides in size) in one or more containers. The kit may optionally further include a predetermined amount of a purified CD36 protein or nucleic acid (eg, as a standard or control) in a container.
[0173]
5.6Therapeutic uses
The invention further includes a method of modulating an immune response. For the purpose of stimulating the immune system and eliciting an immune response, CD36 recognizes HSP proteins such as gp96 and stimulates chemokine and nitric oxide release (eg, HSP-antigen peptide complex). Thus, the compositions and methods of the present invention may be used in the treatment of HSP-CD36 related disorders and conditions such as autoimmune diseases, cancer and infectious diseases. In particular, as described in detail below, HSP-antigen peptide complexes, antibodies, and other compounds that interact with CD36 or modulate the interaction of CD36 with its ligand (eg, HSP); And recombinant cells containing a CD36 complex, such as other compounds that modulate HSP-CD36 mediated processes, to elicit or block an immune response to treat such HSP-CD36 related disorders and conditions May be used.
[0174]
5.6.1Therapeutic uses of identified agonists and antagonists
Compounds that interact with CD36 or modulate the interaction of CD36 with its ligand (eg, HSP), such as those identified by the screening methods provided herein, are useful as therapeutics. obtain. Such compounds include, but are not limited to, agonists, antagonists (such as antibodies), antisense RNAs and ribozymes. Compounds that interfere with the ligand (eg, HSP) CD36 interaction can be used to block the immune response and can be used to treat autoimmune responses and conditions. Other antibodies, agonists, antagonists, antisense RNAs and ribozymes can up-regulate ligand (eg, HSP) -CD36 interaction, activity, or expression, and uptake of antigen complexes (eg, HSP-antigen complexes). It may be potentiated and thus be useful for stimulating the host's immune system prior to or simultaneously with the administration of the vaccine. Described below are methods and compositions for using such compounds in the treatment of HSP-CD36 related disorders such as immune disorders, proliferative disorders and infectious diseases.
[0175]
In one embodiment, an antagonist of a CD36-ligand (eg, HSP-CD36) interaction is used to block an immune response. Such antagonists include compounds that compete for binding to CD36, a ligand or a ligand-CD36 complex, thereby preventing the ligand (eg, HSP) from binding to the receptor.
[0176]
In one embodiment, the antagonist is an antibody specific for CD36, or a fragment thereof that includes an HSP ligand binding site. In another embodiment, the antagonist is an antibody specific to HSP that prevents HSP from binding to the receptor.
[0177]
In another embodiment, the antagonist is a peptide comprising at least 10 contiguous amino acids of the HSP sequence. Such peptides can bind to the ligand binding site of CD36 and block the interaction of HSP or HSP complex. In another embodiment, the antagonist is a peptide comprising at least 10 contiguous amino acids of the CD36 sequence, which, like HSP, can bind to CD36 and prevent binding and signaling activity. In yet another embodiment, the antagonist is a peptide comprising at least 10 contiguous amino acids of the CD36 sequence, particularly a CD36 (or a portion thereof) capable of binding and "neutralizing" natural ligands (HSP, LDL, etc.). ECD.
[0178]
Such peptides may be produced synthetically or by using standard molecular biology techniques. Amino acid sequences and nucleotide sequences of native CD36 ligands (such as HSP) are generally available from sequence databases such as GenBank. Using a computer program such as Entrez, a database can be browsed, and any amino acid sequence and desired gene sequence data can be searched from the accession number. Methods for the recombinant and synthetic production of such peptides are described in sections 5.1.1 and 5.1.2.
[0179]
In addition, compounds capable of modulating CD36 gene product activity, identified via techniques such as those described in Section 5.2 above, can be administered using standard techniques well known to those skilled in the art.
[0180]
5.6.1.1CD36- Competitive antagonist of ligand interaction
In one embodiment, an antagonist of the CD36-ligand (eg, HSP-CD36) interaction is used to block an immune response to the antigen complex (eg, to treat an autoimmune disorder). Such antagonists include molecules that prevent binding by binding to CD36, thereby preventing ligand (eg, HSP) from binding to the receptor. An example of this type of competitive inhibitor is an antibody against CD36, or a fragment of CD36 that contains an HSP ligand binding site.
[0181]
A CD36-ligand (eg, HSP) competition inhibitor can be any type of molecule, including but not limited to proteins, nucleic acids or drugs. In a preferred embodiment, the HSP-CD36 competition inhibitor is a CD36 binding or HSP binding peptide. Examples of such peptides are provided below.
[0182]
5.6.1.1.1HSP Binding peptide
CD36 peptide
In one embodiment of the invention, the HSP-CD36 competitive antagonist is a CD36 peptide, preferably a soluble peptide, that binds to HSP and competitively inhibits HSP from binding to the native receptor.
[0183]
Preferably, functional expression of the HSP binding portion of CD36 is performed. Briefly, to maintain proper folding, the protein is expressed as a GST fusion, recombinantly expressed, the GST portion is cleaved, the uncleaved protein is removed on GSH-Sepharose, and the cleaved protein is refolded. Let it. Since the complement repeat binds to calcium, proper folding is assayed by measuring the binding of the refolded protein to calcium.
[0184]
In a particular mode of this embodiment, the HSP binding portion of CD36 consists of or comprises at least one complement repeat. In another particular mode of this embodiment, the HSP binding portion of CD36 comprises a cluster of complement repeats, most preferably C1-II. In another mode of this embodiment, the HSP binding moiety comprises at least 10, more preferably at least 20, even more preferably at least 30, even more preferably at least 40 and most preferably at least 80 (consecutive) amino acids. In certain modes of this embodiment, such fragments are no more than 40-45 amino acids. In another particular mode of this embodiment, such fragments are no more than 80-90 amino acids.
[0185]
Derivatives or analogs of the HSP binding portion of CD36 are also contemplated as competitive antagonists of the HSP-CD36 complex. Such derivatives or analogs include substantially homologous to the extracellular domain of CD36 or a fragment thereof (eg, in various embodiments, amino acid sequences of the same size, or computer homology known in the art). Molecules comprising at least 60%, 70%, 80%, 90% or 95% identity compared to alignment sequences aligned by the program), or stringent, moderately stringent or non-stringent conditions Below, those in which the encoding nucleic acid is capable of hybridizing to the sequence encoding the CD36 HSP binding sequence include, but are not limited to. In certain embodiments, the CD36 derivative preferably comprises at least one complement repeat of C1-II linked at the amino or carboxy terminus to the amino acid sequence of a different protein via a peptide bond, wherein the HSP binding of CD36 A chimeric or fusion protein comprising a moiety. Such chimeric proteins can be produced recombinantly, as described above, by omitting the cleavage repurification step.
[0186]
Other HSP-binding CD36 derivatives can be made by altering the CD36 coding sequence by substitutions, additions or deletions that provide a functionally equivalent molecule. Due to the degeneracy of the nucleotide coding sequence, other DNA sequences encoding substantially the same amino acid sequence as the CD36 gene or gene fragment may be used in the practice of the present invention. Selection of the appropriate modification and production of HSP-bound CD36 derivatives applies the same principles as described above for CD36 derivatives and uses the general methods described in sections 5.1.1 and 5.1.2. Can be implemented.
[0187]
HSP peptide
In another mode of this embodiment, the antagonist is a peptide comprising at least 10 contiguous amino acids of the HSP sequence. Such peptides can bind to the ligand binding site of CD36 and block the interaction of HSP or HSP complex.
[0188]
Such peptides can be produced synthetically or using standard molecular biology techniques. Amino acid and nucleotide sequences of native HSP are commonly available from sequence databases such as GenBank. Using a computer program such as Entrez, a database can be browsed, and any amino acid sequence and desired gene sequence data can be searched from the accession number. Methods for the recombinant and synthetic production of such peptides are described in sections 5.1.1 and 5.1.2.
[0189]
In addition, compounds identified via techniques as described in Section 5.2 above that are capable of modulating gene product activity can be administered using standard techniques well known to those skilled in the art.
[0190]
5.6.2For cancer and infectious diseases CD36 Therapeutic use of
In another embodiment, the symptoms of certain CD36 gene disorders (autoimmune disorders, or proliferative or differentiating disorders) that result in tumorigenesis or cancer modulate the level of CD36 gene expression and / or CD36 gene product activity. This can be alleviated. In one embodiment, for example, reducing CD36 gene expression may be useful for reducing CD36 activity and alleviating the symptoms of an autoimmune disorder. In this case, the level of CD36 gene expression can be reduced by using the CD36 gene sequence with the well-known antisense gene "knockout" ribozyme and / or triple helix method. In another embodiment, increased CD36 gene expression may be desirable to compensate for a mutant or damaged gene in the HSP-CD36 mediated pathway and to alleviate the symptoms of an HSP-CD36 related disorder.
[0191]
Compounds that may exhibit an ability to modulate activity, CD36 expression or synthesis of the CD36 gene, including the ability to alleviate the symptoms of an HSP-related disorder, include antisense, ribozymes and triple helix molecules. Such molecules can be designed to reduce or inhibit either the undamaged target gene activity or, if appropriate, the mutant target gene activity. Techniques for making and using such molecules are well known to those skilled in the art.
[0192]
Antisense RNA and DNA molecules act to block translation of mRNA directly by hybridizing to the targeted mRNA and preventing protein translation. The antisense approach involves the design of an oligonucleotide that is complementary to the target gene mRNA. Antisense oligonucleotides bind to complementary target gene mRNA transcripts and prevent translation. Absolute complementarity is preferred but not required.
[0193]
A sequence "complementary" to a portion of the RNA referred to herein refers to a sequence that has sufficient complementarity to hybridize to the RNA and form a stable double helix; In the case of single-stranded antisense nucleic acids, single strands of double helix DNA may be tested or triple helix formation may be assayed. The ability to hybridize depends on both the degree of complementarity and the length of the antisense nucleic acid. In general, the longer the hybridizing nucleic acid, the more stable the double helix (or triple helix in some cases) is formed with more base mismatches with the RNA. One skilled in the art can ascertain the degree of tolerable mismatch using standard procedures to determine the melting point of the hybridized complex.
[0194]
In one embodiment, oligonucleotides complementary to the non-coding region of the CD36 gene may be used in an antisense approach to inhibit translation of endogenous CD36 mRNA. Preferably, the antisense nucleic acid is an oligonucleotide that is at least 6 nucleotides in length and 6 to about 50 nucleotides in length. In certain aspects, the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides, or at least 50 nucleotides.
[0195]
In one embodiment of the invention, an oligonucleotide complementary to the nucleic acid encoding the HSP receptor ligand binding domain is used.
[0196]
Regardless of the choice of target sequence, it is preferred that an in vitro study be performed first to quantify the ability of the antisense oligonucleotide to inhibit gene expression. In these studies, it is preferable to utilize controls that distinguish antisense gene inhibition from nonspecific biological effects of oligonucleotides. In these studies, it is also preferable to compare the level of the target RNA or protein with the level of an internal control RNA or protein. It is further envisioned that results obtained using the antisense oligonucleotide are compared to results obtained using the control oligonucleotide. It is preferred that the control oligonucleotide be about the same length as the test oligonucleotide and that the nucleotide sequence of the oligonucleotide differ from the antisense sequence by more than prevent specific hybridization with the target sequence.
[0197]
The oligonucleotide may be single-stranded or double-stranded, DNA or RNA, or a chimeric mixture, derivative or modified variant thereof. Oligonucleotides can be modified at the base moiety, sugar moiety, or phosphate backbone to improve, for example, stability of the molecule, hybridization, and the like. Oligonucleotides can be used to bind other accessory groups such as peptides (eg, to target host cell receptors in vivo), cell membranes (eg, Letsinger et al., 1989, Proc. Natl. Acad. Sci. US. A. 86, 6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci. 84, 648-652; see PCT Publication No. WO 88/09810, issued December 15, 1988) or blood. Agents that facilitate transport across the brain barrier (see, eg, PCT Publication No. WO 89/10134, issued April 25, 1988), hybridization-triggered cleavage agents (eg, Kroll et al., 1988, BioTechniques 6, 958-976), Or intercalating agents (see, for example, Zon, 1988, Pharm. Res. 5, 539-549). For this purpose, the oligonucleotide may be conjugated to another molecule (eg, a peptide, hybridization triggered cross-linking agent, transport agent, hybridization triggered cleavage agent, etc.).
[0198]
Antisense oligonucleotides include 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl -2-thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, β-D-galactosylcuosin, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2, -Methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, β-D-man Nosylcuosin, 5'-methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, cuocin, 2-thiocytosine , 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acid methyl ester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3 At least one modified base selected from the group consisting of, but not limited to,-(3-amino-3-N-2-carboxypropyl) uracil, (acp3) w, and 2,6-diaminopurine Parts.
[0199]
The antisense oligonucleotide can also include at least one modified sugar moiety selected from the group consisting of, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
[0200]
In yet another embodiment, the antisense oligonucleotide is a phosphorothioate (S-ODN), phosphorodithioate, phosphoramidothioate, phosphoramidite, phosphorodiamidite, methylphosphonate, alkyl phosphotriester, And at least one modified phosphate backbone selected from the group consisting of formacetals or analogs thereof.
[0201]
In yet another embodiment, the antisense oligonucleotide is an α-anomeric oligonucleotide. Alpha-anomeric oligonucleotides form specific double-stranded hybrids with complementary RNAs, in which the strands run parallel to one another, in contrast to the normal beta unit (Gautier et al., 1987, Nucl. Acids Res. 15, 6625-6641). Oligonucleotides can be 2'-O-methyl ribonucleotides (Inoue et al., 1987, Nucl. Acids Res. 15, 6131-6148), or chimeric RNA-DNA analogs (Inoue et al., 1987, FEBS Lett. 215, 327-). 330).
[0202]
Oligonucleotides of the invention can be synthesized by standard methods known in the art (eg, using an automated DNA synthesizer (commercially available from Biosearch, Applied Biosystems, etc.)). As an example, phosphorothioate oligonucleotides can be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16, 3209), and methylphosphonate oligonucleotides can be used with controlled pore glass polymer supports. (Sarin et al., 1988, Proc. Natl. Acad. Sci. USA 85, 7448-7451).
[0203]
While antisense nucleotides complementary to the target gene coding region sequence can be used, those complementary to the transcribed untranslated region are most preferred.
[0204]
In one embodiment of the invention, a specific target mRNA is hybridized with an antisense phosphorothioate oligonucleotide (S-ODN) followed by digestion with RNase H to perform down-regulation of gene expression. The best strategy is completely empirical, as there are no rules to predict which antisense S-ODN is more convenient, consisting of trying several antisense S-ODNs. Antisense phosphorothioate oligonucleotides (S-ODNs) are designed to target specific regions of the mRNA of interest. The control S-ODN, which consists of a disordered scrambled sequence of the antisense S-ODN, is also designed to ensure that the nucleotide content is identical and that potential differences in nucleic acid content are minimized. You. All S-ODNs are Oligos Etc. (Wilsonville, OR). Cultivate cells to 100-80% confluence on 100 mm tissue culture plates to test the effectiveness of the antisense molecule when applied to cultured cells, such as assays for research purposes or ex vivo gene therapy protocols. Then, rinse with PBS and cover with a lipofection mix (final concentration 200 nM S-ODN) consisting of 8 ml Opti-MEM, 52.8 μl lipofectin. Lipofection is performed using Lipofectin reagent and Opti-MEM (Gibco BRL). The cells are incubated for 5 hours in the presence of the lipofection mix. After incubation, the medium is replaced with complete DMEM. Cells are harvested at different times after lipofection and analyzed for protein levels by Western blot.
[0205]
The antisense molecule should target cells that express the target gene, either directly to the subject in vivo or to cultured cells (such as ex vivo gene therapy protocols). Numerous methods have been developed for delivering antisense DNA or RNA to cells; for example, by injecting the antisense molecule directly into a tissue site, or expressing the desired cell (eg, expressed on the surface of a target cell). A modified antisense molecule designed to target a peptide or antibody that specifically binds to a receptor or an antigen) can be administered systemically.
[0206]
However, it is often difficult to obtain an intracellular concentration of antisense sufficient to suppress translation of endogenous mRNA. Thus, a preferred approach utilizes a recombinant DNA construct in which the antisense oligonucleotide is placed under the control of a strong pol III or pol II promoter. By transfecting target cells in a patient using such a construct, a sufficient amount of one strand will form complementary base pairs with the endogenous target gene transcript and prevent translation of the target gene mRNA. Transcription of the strand RNA occurs. For example, a vector can be introduced, for example, to be taken up by a cell and direct the transcription of an antisense RNA. Such vectors may remain episomal or may be integrated into the chromosome as long as they can be transcribed to produce the desired antisense RNA. Such vectors can be constructed by recombinant DNA techniques standard in the art. Vectors can be plasmids, viral or others known in the art, used for replication and expression in mammalian cells. Expression of the sequence encoding the antisense RNA can be driven by any promoter known in the art to act in mammals, preferably human cells. Such a promoter may be inducible or constitutive. Such promoters include the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290, 304-310), the promoter contained in the 3 'long terminal repeat of Rous sarcoma virus (Yamamoto et al., 1980, Cell 22, 787). -797), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA 78, 1441-1445), the regulatory sequence of the metallothionein gene (Brinster et al., 1982, Nature 296, 39). -42) and the like, but are not limited thereto. Any type of plasmid, cosmid, YAC or viral vector can be used to prepare a recombinant DNA construct that can be introduced directly into a tissue site. Alternatively, a viral vector that selectively infects the desired tissue can be used (in this case, administered by another route (eg, systemically)).
[0207]
Ribozyme molecules designed to catalytically cleave the target gene mRNA transcript can also be used to prevent translation of the target gene mRNA and thus expression of the target gene product. (See, e.g., PCT International Publication No. WO 90/11364, published October 4, 1990; Sarver et al., 1990, Science 247, 1222-1225). In one embodiment of the invention, the oligonucleotide that hybridizes to the HSP receptor gene is designed to be complementary to the nucleic acid encoding the HSP receptor ligand binding domain.
[0208]
Ribozymes are enzymatic RNA molecules that can catalyze the specific cleavage of RNA. (See Rossi, 1994, Current Biology 4, 469-471 for an overview). The mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage event. The composition of the ribozyme molecule must include one or more sequences that are complementary to the target gene mRNA, and must include the well-known catalytic sequence that causes mRNA cleavage. See, for example, US Pat. No. 5,093,246, which is hereby incorporated by reference in its entirety.
[0209]
Although a ribozyme that cleaves mRNA at the site-specific recognition sequence can be used to destroy the target gene mRNA, it is preferred to use a hammerhead ribozyme. Hammerhead ribozymes cleave mRNAs at locations defined by flanking regions that form complementary base pairs with the target mRNA. The only requirement is that the target mRNA has the two base sequence "5'-UG-3 '". The construction and production of hammerhead ribozymes is well known in the art, and is described in Myers, 1995, Molecular Biology and Biotechnology: A Comprehensive Desk Reference, VCH Publishers, and especially in New York, see FIG. , 1988, Nature, 334, 585-591, which is hereby incorporated by reference in its entirety.
[0210]
The ribozyme is preferably engineered such that the cleavage recognition site is located near the 5 'end of the target gene mRNA (i.e., increases efficiency and minimizes accumulation of non-functional mRNA transcripts in cells).
[0211]
The ribozymes of the present invention also include those naturally occurring in tetrahymena thermophila (known as IVS or L-19 IVS RNA), and those described by Thomas Cech and coworkers (Zaug et al., 1984, Science, 224, 574-578; Zaug and Cech, 1986, Science, 231, 470-475; Zaug et al., 1986, Nature 324, 429-433; International Patent Application Publication No. WO 88/043 by University Patents Inc. RNA endoribonucleases (hereinafter "chi"), such as those described extensively by Been and Cech, 1986, Cell, 47, 207-216). Click-type ribozymes "), and the like. Check ribozymes have an eight base pair active site that hybridizes to the target RNA sequence and results in cleavage of the target RNA. The present invention includes a check-type ribozyme that targets an eight base pair active site sequence present in a target gene.
[0212]
In the antisense approach, ribozymes should consist of modified oligonucleotides (eg, for improved stability, targeting, etc.) and be delivered in vivo to cells expressing the target gene. A preferred method of delivery uses a DNA construct that “encodes” the ribozyme under the control of a strong constitutive pol III or pol II promoter, and the transfected cells produce a sufficient amount of the ribozyme to generate the endogenous target gene. Destroy messages and destroy translations. Unlike antisense molecules, ribozymes are catalytic and therefore require lower intracellular concentrations for efficiency.
[0213]
Endogenous target gene expression can also be reduced by inactivating or "knocking out" the target gene or its promoter using targeted homologous recombination (eg, Smithies et al., 1985, Nature 317, 230-234; Thomas and Capecchi, 1987, Cell 51, 503-512; see Thompson et al., 1989, Cell 5, 313-321; each of which is incorporated herein by reference in its entirety). For example, a mutant non-functional target gene (or a completely unrelated DNA sequence) flanked by DNA homologous to the endogenous target gene (either the coding region or the regulatory region of the target gene) can be selected with a selectable marker and / or Alternatively, they can be used with or without a negative selectable marker to transfect cells that express the target gene in vivo. Insertion of the DNA construct via targeted homologous recombination results in inactivation of the target gene. Such an approach is a particularly suitable modification to ES (embryonic stem) cells and can be used to generate animal progeny with an inactive target gene (see, eg, Thomas and Capecchi, 1987, and Thompson, 1989, supra). reference). However, this approach is applicable for use in humans, provided that the recombinant DNA construct is administered or targeted directly to the required site in vivo with an appropriate viral vector.
[0214]
Alternatively, by targeting a deoxyribonucleotide sequence that is complementary to a regulatory region of the target gene (ie, the target gene promoter and / or enhancer), a triple helical structure is formed that prevents transcription of the target gene in target cells of the body. Thus, endogenous target gene expression can be reduced. (Helene, 1991, Anticancer Drug Des., 6 (6), 569-584; Helene et al., 1992, Ann. NY Acad. Sci., 660, 27-36; and Maher, 1992, Bioassay (12). ), 807-815).
[0215]
Nucleic acid molecules used in triple helix formation to inhibit transcription are single-stranded and consist of deoxyribonucleotides. The base composition of these oligonucleotides is generally via Hoogsteen base pairing rules, where a large stretch of either purines or pyrimidines must be present on one strand of the duplex. Must be designed to promote triple helix formation. The nucleotide sequence is pyrimidine-based, which contains TAT and CGC over the three associated strands of the resulting triple helix.⁺This produces triplets. Pyrimidine-enriched molecules provide bases complementary to the purine-enriched region of one strand of the double helix in a direction parallel to the strand. In addition, purine-rich (eg, comprising the sequence of G residues) nucleic acid molecules can be selected. These molecules form a triple helix with a GC-pair rich DNA duplex, where most of the purine residues are located on one strand of the targeted duplex and the triple strand of the triple helix. GGC triplets occur over time.
[0216]
Alternatively, the potential sequences that can be targeted for triple helix formation can be increased by making so-called "switchback" nucleic acid molecules. The switchback molecule is synthesized in an alternating 5'-3 ', 3'-5' fashion to base pair with the first strand of the duplex and then with the other, whether purine or pyrimidine. That large sequence need not be on one strand of the double helix.
[0217]
Where the antisense, ribozyme, and / or triple helix molecules described herein are utilized to inhibit mutant gene expression, the present technique provides for the transcription of mRNA produced by a normal target gene allele (triple helix). ) And / or translation (antisense ribozymes) may be reduced or inhibited too efficiently, creating the possibility that the concentration of normal target gene product present will be lower than that required for a normal phenotype. In such a case, to ensure that substantially normal levels of target gene activity are maintained, nucleic acid molecules encoding and expressing target gene polypeptides exhibiting normal target gene activity are identified in 5.6. Via gene therapy, as described in Section 3, cells may be introduced into cells that do not contain any antisense, ribozyme, or triple helix therapy-sensitive sequences utilized. Alternatively, if the target gene encodes an extracellular protein, it is preferable to co-administer a normal target gene protein to maintain the essential level of target gene activity.
[0218]
The antisense RNA and DNA, ribozymes, and triple helix molecules of the present invention can be prepared by any of the methods for synthesizing DNA and RNA molecules known in the art as discussed above. These include oligodeoxyribonucleotides and techniques for chemically synthesizing oligoribonucleotides well known in the art (eg, solid phase phosphoramidite chemical synthesis and the like). Alternatively, an RNA molecule can be produced by in vitro and in vivo transcription of a DNA sequence encoding the antisense RNA molecule. Such a DNA sequence can be incorporated into various vectors that incorporate an appropriate RNA polymerase promoter, such as a T7 or SP6 polymerase promoter. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly, depending on the promoter used, can be stably introduced into cell lines.
[0219]
5.6.3Gene replacement therapy
For increased levels of normal CD36 gene expression and / or CD36 gene product activity, the CD36 gene nucleic acid sequence can be used to treat an immune disorder that results in a proliferative disorder, such as, for example, cancer. Such treatment can be provided, for example, in the form of gene replacement therapy. Specifically, one or more copies of the normal CD36 gene or a portion of the CD36 gene, which directs the production of a CD36 gene product exhibiting normal CD36 gene function, is transferred to a vector (including adenovirus, adeno-associated virus and retroviral vectors). (But not limited to), may be inserted into appropriate cells of the patient along with other particles (such as liposomes) that introduce the DNA into the cells.
[0220]
Gene replacement therapy techniques must be able to deliver the CD36 gene sequence to cell types that express the HSP receptor in patients. Thus, in one embodiment, using techniques well known to those of skill in the art (see, eg, PCT Publication No. WO 89/10134, issued Apr. 25, 1988), the CD36 gene sequence is transformed into a myeloid lineage (myeloid). (eg, bone marrow). In another specific embodiment, gene replacement can be performed by using macrophages in vitro and delivering them to a patient using adoptive immunotherapy techniques.
[0221]
In another embodiment, the delivery technique involves administering such a gene sequence directly to a cell site where the CD36 gene sequence is expressed (eg, directly at the tumor site).
[0222]
A further method that can be used to increase the overall level of CD35 gene expression and / or CD36 gene product activity is to use appropriate CD36 expressing cells, preferably progenitor cells, to alleviate the symptoms of CD36 disorder. In sufficient number and in sufficient number of patients. Such cells can be either recombinant or non-recombinant cells.
[0223]
Cells that can be administered to increase the overall level of CD36 gene expression in a patient usually include cells that express the CD36 gene.
[0224]
Alternatively, cells (preferably progenitor cells) are engineered to express the CD36 gene sequence and are subsequently suitable for alleviating the symptoms of a CD36 disorder or a proliferative or viral disease (eg, cancer and tumorigenesis). May be introduced to the patient at a different location. Alternatively, cells derived from an individual that expresses the intact CD36 gene and matches the MHC can be used, such as brain cells. Expression of the CD36 gene sequence is controlled by appropriate gene regulatory sequences to allow such expression in the required cell type. Such gene regulatory sequences are well known to those skilled in the art. Such cell-based gene therapy techniques are well known to those of skill in the art (see, eg, Anderson, US Pat. No. 5,399,349).
[0225]
When the cells to be administered are non-progenitor cells, they can be administered using well-known techniques that prevent the generation of a host immune response against the introduced cells. For example, cells may be introduced in an encapsulated form that allows the exchange of components with the immediate extracellular environment while not allowing the host immune system to recognize the introduced cells.
[0226]
5.6.4Delivery of soluble polypeptides
Engineered cells expressing a soluble ECD or fusion protein (eg, a fusion Ig molecule) can be administered in vivo, which can function as a “bioreactor” to deliver a supply of soluble molecules. Such soluble CD36 polypeptides and fusion proteins, when expressed at appropriate concentrations, neutralize or "mop up" HSPs or other natural ligands for CD36, act as inhibitors of activity, Thus, it can be used to treat HSP-CD36 related disorders and diseases such as autoimmune disorders, proliferative disorders and infectious diseases.
[0227]
5.6.5Delivery of dominant negative mutant
In another embodiment of the invention, a dominant negative mutant ("dominant negative") is used for treatment to block the immune response to the HSP-antigen complex (e.g., to treat an autoimmune disorder). May be. Generally, such dominant negatives, when expressed, interact with a ligand (ie, HSP-antigen molecule complex), but have one or more functions of normal CD36 (ie, growth factor production and / or Or signaling function). Such variants disrupt normal CD36 function in the same or different cells (eg, due to the titration of HSP-peptide complexes from wild-type receptors). Such a mutation can be, for example, one or more point mutations, deletions, insertions or other mutations in either the extracellular, transmembrane or intracellular domains.
[0228]
Expression of such a dominant negative mutation in a cell can block uptake of the ligand by normal functional receptors in the same or neighboring cells by exhausting the total amount of available ligand (titrating out) . Thus, recombinant antigen presenting cells expressing such dominant negatives can be used to consume HSP-antigen molecule complexes when administered to patients in need of treatment for an autoimmune disorder.
[0229]
5.7Targeted autoimmune disease
Autoimmune diseases that can be treated by the method of the present invention include insulin-dependent diabetes (ie, IDDM, ie, autoimmune diabetes), multiple sclerosis, systemic lupus erythematosus, Sjogren's syndrome, scleroderma, polymyositis, chronic activity Hepatitis, mixed connective tissue disease, primary biliary cirrhosis, pernicious anemia, autoimmune thyroiditis, idiopathic Addison's disease, vitiligo, gluten-sensitive enteropathy, Graves' disease, myasthenia gravis, autoimmune neutropenia, Sudden thrombocytopenic purpura, rheumatoid arthritis, cirrhosis, pemphigus vulgaris, autoimmune infertility, Goodpasture's disease, bullous pemphigoid, discoid lupus, ulcerative colitis, and dense deposit Glomerulonephritis (dense deposit disease). The diseases referred to herein are those exhibited by animal models for such diseases (eg, non-obese diabetic (NOD) mice for IDDM, and experimental autoimmune encephalomyelitis for multiple sclerosis). (EAE) mouse).
[0230]
The methods of the present invention may be used to reduce or eliminate an immune response to a patient's own tissue, or alternatively, to replace an existing tissue or organ transplanted to replace the autologous tissue or organ damaged by the autoimmune response. Such autoimmune diseases can be treated by reducing or eliminating the autoimmune response.
[0231]
5.8Target infectious disease
Infectious diseases that can be treated or prevented using the methods and compositions of the present invention include those caused by intracellular pathogens, such as viruses, bacteria, protozoa, and intracellular parasites. Examples of the virus include hepatitis B virus, parvovirus (such as adeno-associated virus and cytomegalovirus), papovavirus (such as papilloma virus), polyoma virus, SV40, adenovirus, and herpes virus (herpes simplex type I (HSV-I)). , Herpes simplex type II (HSV-II)) and Epstein-Barr virus), poxvirus (pox (smallpox) and vaccinia virus, etc.), RNA virus (human immunodeficiency virus type I (HIV-I), human immunity) Defective virus type II (HIV-II), human T-cell lymphotropic virus type I (HTLV-I) and human T-cell lymphotropic virus type II (HTLV-II). Not limited); influenza Rus, measles virus, rabies virus, Sendai virus, picornavirus (poliovirus, coxsackievirus, rhinovirus, etc.), reovirus, togavirus (rubella virus (german measle) and Semliki Forest fever virus), arbovirus, And viral diseases caused by hepatitis A virus and the like, but are not limited thereto.
[0232]
In another embodiment, Streptococcus pyogenes, Streptococcus pneumoniae, Streptococcus pneumoniae, Neisseria gonorrhoeae, osteomyelitis, diphtheria, botulinum, Clostridium perfringens, tetanus, influenza, Klebsiella pneumoniae, Klebsiella ozenaena, Klebsiella rhinoscleromotis, Staphylococcus aureus, V. cholerae, Escherichia coli, Pseudomonas aeruginosa, Campylobacter (Vibrio) phitus, Campylobacter aphia erojua phiera moss ), Bacillus cereus, Edwardsiella Tarda tarda), Ernesia enterocolitica, plague bacteria, pseudotuberculosis bacteria, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Salmonella typhimurium, Salmonella typhi, Treponema pallidum, Treponema endemic syphilis (Treponema pertenue), Spiroreta (Treponeeta) carateneum), Borrelia vincenti, Borrelia burgdorferi, Jaundice hemorrhagic leptospirosis, Mycobacterium tuberculosis, Toxoplasma, Pneumocystis carini, Haemophilus spp. (Mycoplasma spp.), Typhus rickettsia, tsutsugamushi disease rickettsia, chlamydia (Chlamydia spp.), And Bacterial infections such as failure caused by a pathogen bacteria fine Helicobacter pylori such as (but not limited to) (but not limited to) can be treated or prevented.
[0233]
In another preferred embodiment, dysentery amoeba, Trichomonas buccalis, Trichomonas intestine, Trichomonas vaginalis, Trypanosoma gambia, Trypanosoma rhodesia, Trypanosoma cruzi, Donovan leishmania, Tropical leishmania, Leishmania brazil, Pneumocystis pneumoniae pneumoniae pneumonia The above methods can be used to treat or prevent infections caused by pathogenic protozoa such as, but not limited to, P. falciparum, P. falciparum, and P. vivax.
[0234]
5.9Targeted proliferative cell injury
For certain proliferative and oncogenic diseases associated with HSP-CD36 activity, diseases that can be treated or prevented by the methods of the present invention include human sarcomas and cancers (eg, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, bone). Primordial sarcoma, chordoma, hemangiosarcoma, endotheliosarcoma, lymphatic sarcoma, lymphatic endothelioma, periosteoma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, pancreatic cancer, breast cancer , Ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous carcinoma, papillary carcinoma, papillary adenocarcinoma, cystic adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, liver cancer , Bile duct carcinoma, choriocarcinoma, seminoma, embryonic carcinoma, Wilms tumor, cervical carcinoma, testicular tumor, lung cancer, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glia , Astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblast Tumors, retinoblastoma; leukemias (eg, acute lymphocytic leukemia and acute myeloid leukemia (myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)); chronic leukemia (chronic myeloid (Granulocytic and chronic lymphocytic leukemia); and polycythemia vera, lymphomas (Hodgkin's and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, and heavy chain disease. But not limited to these.
[0235]
Diseases and disorders involving cell growth deficiency or where cell proliferation is desirable for treatment or prevention and which can be treated or prevented by inhibiting CD36 function include degenerative disorders, growth deficiencies, hypoproliferative disorders. disorder), physical trauma, lesions and wounds (eg, promotes wound healing or promotes regeneration in degenerative, lesioned or damaged tissue, etc.).
[0236]
5.10Pharmaceutical dosage form and method of administration
A compound determined to affect CD36 gene expression or gene product activity can be administered to a patient in a therapeutically effective dose to treat or alleviate a cell proliferative disorder. A therapeutically effective dose refers to that amount of the compound that is sufficient to alleviate the symptoms of such a disorder.
[0237]
5.10.1Effective dose
The toxicity and therapeutic efficacy of such compounds can be demonstrated in cell cultures or experimental animals (eg, LD₅₀(Lethal dose for 50% of population) and ED₅₀It can be determined by standard pharmaceutical procedures (to determine the therapeutically effective dose for 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index, and the LD₅₀/ ED₅₀It can be expressed as a ratio. Compounds that exhibit high therapeutic indices are preferred. Compounds that exhibit toxic side effects may be used, but minimize potential damage to uninfected cells when designing a delivery system that targets such compounds to the site of the affected tissue. Care must be taken to reduce side effects.
[0238]
The data obtained from the cell culture assays and animal studies can be used in formulating a variety of dosages for use in humans. The dosage of such compounds should be such that there is little or no toxicity,₅₀Is preferably within the range of the circulating concentration containing Dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. IC determined by cell culture₅₀A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the (ie, the concentration of the test compound at which half-maximal inhibition of symptoms is achieved). Such information can be used to more accurately determine useful doses in humans. Plasma levels can be measured, for example, by high performance liquid chromatography.
[0239]
5.10.2Formulation and use
Pharmaceutical compositions for use according to the invention may be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.
[0240]
Thus, the compounds and their physiologically acceptable salts and solvates are formulated for administration by inhalation or infusion (either via the mouth or nose) or for oral, parenteral or rectal administration. Is also good.
[0241]
For oral administration, the pharmaceutical composition may include, for example, a binder (eg, pregelatinized corn starch, polyvinylpyrrolidone or hydroxypropylmethylcellulose); a filler (eg, lactose, microcrystalline cellulose or calcium hydrogen phosphate); Pharmaceutically acceptable excipients such as bulking agents (eg, magnesium stearate, talc or silica); disintegrating agents (eg, sodium starch or sodium starch glycolate); or humectants (eg, sodium lauryl sulfate). With the form, they can take the form of tablets or capsules prepared by conventional means, which can be coated by methods well known in the art.Liquid preparations for oral administration include, for example, solutions, syrups or suspensions Liquid It may take the form or be provided as a dry product for reconstitution with water or other suitable vehicle before use. Such liquid preparations may be suspension agents such as sorbitol syrup, cellulose derivatives or hydrogenated Edible fats); emulsifiers (eg, lecithin or acacia); water-insoluble vehicles (eg, almond oil, oily esters, ethyl alcohol or fractionated vegetable oil); and preservatives (eg, methyl or propyl-p-hydroxybenzoate or sorbic acid). ) Can be prepared by conventional means with pharmaceutically acceptable excipients, etc. The preparations can also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
[0242]
Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
[0243]
For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.
[0244]
For administration by inhalation, the compounds used in accordance with the invention may be compressed using a suitable propellant (eg, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). It is conveniently delivered in the form of an aerosol spray from a pack or nebulizer. In the case of a compressed aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.
[0245]
The compounds can be formulated for parenteral administration by injection (eg, by bolus injection or continuous infusion). Formulations for injection may be presented in unit dosage form, eg, in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, eg, sterile pyrogen-free water, before use.
[0246]
The compounds can also be formulated in rectal compositions such as suppositories (eg, with conventional suppository substrates such as cocoa butter or other glycerides) or retention enemas.
[0247]
In addition to the formulations described previously, the compounds may also be formulated as a depot form. Such long acting formulations may be administered by implantation (for example, subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compound may be formulated with a suitable polymeric or hydrophobic material (eg, as an emulsion in an acceptable oil), an ion exchange resin, or a sparingly soluble derivative (eg, a sparingly soluble salt).
[0248]
If desired, the compositions can be presented in a pack or dispenser which can contain one or more unit dosage forms containing the active ingredient. The pack may for example comprise metal or plastic foil (such as a blister pack). The pack or dispenser may be accompanied by instructions for administration.
[0249]
6.Example: HSP As a receptor CD36 Identification
6.1preface
The examples presented herein describe the successful identification of the interaction of gp96 with the CD36 receptor in macrophages and dendritic cells. The experiments presented herein underlie the isolation of CD36 receptor polypeptides and the screening, diagnostic and therapeutic methods of the present invention.
[0250]
The applicant of the present invention has noticed that certain observations conflict with the "direct transport" model of HSP chaperoned peptide antigen presentation. First, the immunogenicity of HSP preparations depends on the presence of functional phagocytes and not on B cells or other non-professional antigen presenting cells (Udono and Srivastava, 1993, supra; Suto and Srivastava, 1995, supra), but free peptides can sensitize all cell types. Second, very small amounts of the HSP-peptide conjugate are more effective at eliciting specific immunity (ie, the gp96 chaperone peptide sensitizes macrophages for CTL recognition than free peptide). (Several hundred times more effective), suggesting a possible specific ingestion mechanism. Third, the gp96 chaperone peptide elicited an MHC I response that was not limited by peptide size. Finally, treatment of the gp96-peptide complex in macrophages was found to be sensitive to Brefeldin A (BFA), which blocks transport through the Golgi apparatus, suggesting that treatment occurs via an intracellular mechanism. . These observations have led to the hypothesis that HSP chaperone peptides can be internally processed by MHC class I molecules and redisplayed on the cell surface of macrophages (Suto and Srivastava, 1995, supra). Although there is a hypothesis that the mannose receptor is used in the ingestion of gp96, no mechanism has been proposed for non-glycosylated HSPs such as HSP70 (Ciupitu et al., 1998, J. Exp. Med., 187: 385-). 691). Others have suggested that a novel intracellular transfer pathway may be involved in the transport of peptides from the extracellular medium into the lumen of the ER (Day et al., 1997, Proc. Natl. Acad. Sci. 94: 8065-8069; Nicchitta, 1998, Curr. Opin. In Immunol. 10: 103-109). Further suggestions include (a) an implicit pathway that directs the protein from the phagosome to the cytosol, where it enters the normal class I pathway; (b) digests the ingested material in the lysosome and converts the peptide to Released and placed on the surface for class I molecules (Bevan, 199, J. Exp. Med. 192: 639-41). The discovery of receptors for heat shock proteins will help elucidate the paradox of how extracellular antigen peptides complexed with HSP can stimulate an immune response. In addition, cell surface receptors may have other functions, such as inducing signaling pathways. CD36 disclosed herein has been identified as such a receptor.
[0251]
6.2Materials and methods
Macrophage isolation. Wild type and CD36 null mice were inoculated with 0.2 ml of pristane. Five days later, macrophages were extracted from peritoneal perfusion and plated and cultured for 3-4 hours using standard cell culture techniques.
[0252]
Dendritic cell isolation. Bone marrow cells were extracted from femurs of wild-type and CD36 null mice. Cells were incubated for 6 days in the presence of GM-CSF. After 6 days, cells were replated for another day and selected for non-adherent dendritic cells.
[0253]
MCP-1 Chemokine assay. 1 × 10 for each well of a 96-well plate⁵Wild type and CD36 null dendritic cells or macrophages were prepared and incubated with increasing concentrations of gp96, LPS, HSP70, TNF-α and BSA for 20 hours. The supernatant was collected and analyzed by MCP-1 sandwich ELISA (R and D Systems) according to the manufacturer's instructions, and the optical density was measured at 450 nm.
[0254]
Nitric oxide assay. As described above, wild-type and CD36 null macrophages and dendritic cells were prepared and 1 × 10 6 in 96-well plates.⁵Cells were incubated with increasing concentrations of gp96, LPS, HSP70, TNF-α and BSA. After 20 hours, the supernatant was collected and analyzed by an enzymatic grease assay (Calbiochem) according to the manufacturer's instructions. Optical density was measured at 540 nm.
[0255]
Binding assay. HEK293 cells were transfected with FuGENE 6 (Roche Diagnostics) and a plasmid construct containing CD36 cDNA (CD36 cDNA cloned into pCDNA3.1 expression vector). After expressing the transgene for two days, cells were harvested and incubated with various concentrations of fluorescent protein probes (GP96-FITC, histone FITC, AcLDL-DiI, Ova-FITC and BSA (free of fatty acids) -FITC). . Some experiments were performed with the addition of anti-CD36 monoclonal antibody only, followed by the addition of a second fluorescently labeled anti-mouse antibody to detect CD36 expression. Cells were analyzed by flow cytometry. Low and high CD36 expressing populations were gated for binding of the indicated proteins and analyzed. The average fluorescence intensity of each population was plotted. Competitive inhibition experiments were performed by preincubating cells at 4 ° C. with 50 ug / ml normal / modified LDL or 25 ug / ml anti-CD36 antibody.
[0256]
6.3result
gp96 But CD36 To be joined to. Homogeneous preparations of gp96 were bound to FITC, and HEK293 cells and HEK293 CD36 transfectants were stained with gp96-FITC. CD36 expressing cells bind to gp96-FITC, non-expressing and CD36^lowDid not bind (FIG. 1A). This binding was specific for GP96, as indicated by the lack of any staining of HEK293 cells for histone-FITC, Ova-FITC, BSA (faf) -FITC (FIGS. 1B-D). The positive control, acetylated low-density lipoprotein, stained CD36-expressing cells, but did not^lowCells did not stain (FIG. 1E), transfected HEK293 cells expressed functional CD36 protein, demonstrating that GP96 binding was specific for CD36. To confirm that the transfected HEK293 cells correctly expressed CD36, HEK293 transfected cells and mock transfected cells were incubated with mouse anti-CD36 antibody and then stained with anti-mouse FITC antibody. Mock transfected cells were not stained (FIG. 8A) and transfected cells were stained (FIGS. 8C and 8D). When gp96-FITC was incubated with wild type and CD36 null macrophages, CD36 null cells showed a 52% loss of binding to gp96 compared to wild type cells (FIG. 9). No inhibition occurred when LDL or anti-CD36 was used as a competitive inhibitor of gp96 binding (FIGS. 10A and 10B). This indicates that gp96 does not bind to CD36 at amino acids 155-183 or amino acids 28-93 (the respective binding regions of the anti-CD36 antibody and LDL) under the conditions tested.
[0257]
CD36 To gp96 Binding affects chemokine production in macrophages and dendritic cells Induce. Both wild type and CD36 null mice were injected with 0.2 ml pristane. Macrophages were extracted from these mice. Dendritic cells were extracted from the bone marrow of non-pristane injected mice and cultured. Macrophages and dendritic cells were incubated for 20 hours in the presence of increasing concentrations of various native and denatured proteins (boiled for 30 minutes). The level of chemokine production from mice was measured using a standard ELISA sandwich assay. Native gp96 caused strong chemokine production in both wild-type macrophages and dendritic cells compared to the CD36 null population (FIGS. 2A and 3A). Boiling gp96 did not cause any chemokine production in either wild-type macrophages and dendritic cells or CD36 null macrophages and dendritic cells (FIGS. 2A and 3A). LPS induced chemokine production, but not by CD36. Because levels of chemokine production were higher in CD36 null cells than in wild type cells (FIGS. 2B and 3B). In addition, chemokine levels were the same in both native and boiling samples of LPS (FIGS. 2B and 3B). Both native and boiling HSP70 induced the same amount of chemokines in macrophages and dendritic cells, both wild type and CD36 null cells, demonstrating that this induction was independent of CD36 (FIGS. 2C and 3C). . TNF-α and BSA induced equal amounts of chemokine production in both wild-type and CD36 null cells, demonstrating that they also did not affect chemokine production via CD36 (FIGS. 2D and 3D).
[0258]
CD36 To gp96 Binding induces nitric oxide production in macrophages and dendritic cells. The same experiment was performed to determine the levels of nitric oxide produced by wild-type and CD36 null cells. Macrophages and dendritic cells were incubated for 20 hours in the presence of increasing concentrations of various native and denatured proteins (boiled for 30 minutes) and the levels of nitric oxide production were measured by an enzymatic grease assay. Native gp96 caused strong nitric oxide production in both macrophages and wild type dendritic cells compared to CD36 null cells (FIGS. 4A and 5A). Boiling gp96 did not cause any nitric oxide production in either wild-type macrophages and dendritic cells or CD36 null macrophages and dendritic cells (FIGS. 4A and 5A). LPS induced nitric oxide production, but again not by CD36. Because the level of nitric oxide production was higher in CD36 null cells than in wild type cells (FIGS. 4B and 5B). Both native and boiling HSP70 induced the same amount of nitric oxide in macrophages and dendritic cells, both wild-type and CD36 null cells, demonstrating that this induction was independent of CD36 (Fig. 4C and 5C). TNF-α and BSA induce equal amounts of nitric oxide production in both wild-type and CD36 null cells, also indicating that the induction of nitric oxide in CD36-expressing cells is due to the gp96-CD36 interaction. Demonstrated (FIGS. 2D and 3D).
[0259]
6.4Consideration
Studies reported herein indicate that the heat shock protein gp96 is an additional ligand for the CD36 receptor. The human gp96-encoding gene has already been mapped by the present inventors on chromosome 12 (q24.2 → q24.3) (Maki et al., 1993, Somatic Cell Mol. Gen. 19: 73-81).
[0260]
As shown herein, the gp96-CD36 receptor interaction provides a new class of functions for the CD36 receptor (i.e., not only the function of sensors in the extracellular environment with known plasma-based ligands, Also provides the function of a sensor in the intracellular environment). HSPs, such as gp96, are absolute intracellular molecules and are released into the extracellular environment only under conditions of necrotic (but not apoptotic) cell death. Thus, just as the recently identified phosphatidylserine binding protein acts as a sensor for apoptotic cell death and a receptor for apoptotic cells, the CD36 receptor can act as a sensor for necrotic cell death (Savil et al., 1992, J. Clin. Invest. 90: 1513-1522; Fadok et al., 2000, Nature 405: 85-90). Interactions between macrophages and apoptotic cells lead to the down-regulation of inflammatory cytokines such as TNF (Fadok et al., 2000, supra), while the gp96-APC interaction induces a signaling pathway, leading to antigen-specific Stimulation of T cells (Suto and Srivastava, 1995, supra), as well as secretion of proinflammation cytokines such as TNF, GM-CSF and IL-12.
[0261]
Thus, the CD36 receptor allows the APC to (i) regulate the extracellular environment of blood via CD36 and other plasma ligands and (ii) the extracellular environment of tissues via HSPs, especially of the gp96 family. , Can be sampled. The former allows the APC to perform a primordial phagocytic function and the latter allows the APC to perform a unique and adaptive immunological function. From another perspective, recognition of apoptotic cells by APCs via phosphatidylserine leads to anti-inflammatory signals, and interaction of APCs with necrotic cells via CD36 receptor leads to inflammatory immune responses (Srivastava et al.). , 1998, Immunity 8: 657-665).
[0262]
The invention is not limited by the scope of the particular embodiments described, which are intended as a single illustration of each aspect of the invention, and methods and components that are functionally equivalent are within the scope of the invention. . Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.
[0263]
All references, including patent applications, patents, and other references, cited herein are hereby incorporated by reference in their entirety for all purposes.
[Brief description of the drawings]
FIG.
1A-E show the binding of fluorescent protein probes to the CD36 expression gated population of transiently transfected HEK293 cells. Shaded points and solid bars represent CD36 expressing cells. Unshaded points and open bars represent non-expressed and CD36^lowRepresents a cell. The horizontal axis represents the protein concentration in micrograms / microliter, and the vertical axis represents the average fluorescence intensity. A. Gp96-FITC. B. Histone-FITC. C. Acetylated low density lipoprotein-Dil (AcLDL-Dil). D. Ova-FITC. E. FIG. BSA (faf) -FITC.
FIG. 2
Figures 2A-D compare MCP-1 chemokine production by CD36 null and wild type macrophages in response to various stimuli. Filled dots represent wild-type macrophages, open dots represent CD36 null macrophages. The horizontal axis represents the protein concentration in micrograms / microliter for A, C and D and the endotoxin in units / milliliter for B: the horizontal axis represents the protein concentration in picogram / milliliter. A. Macrophages incubated with native gp96 and boiled gp96. B. Macrophages incubated with native LPS and boiled LPS. C. Macrophages incubated with native HSP70 and boiled HSP70. D. Macrophages incubated with TNF-α and BSA.
FIG. 3
3A-D are results of a comparison of MCP-1 chemokine production by CD36 null and wild type dendritic cells in response to various stimuli. Filled dots represent wild-type macrophages, open dots represent CD36 null macrophages. The horizontal axis represents the protein concentration in micrograms / microliter for A, C and D, and the endotoxin in units / milliliter for B. A. Macrophages incubated with native gp96 and boiled gp96. B. Macrophages incubated with native LPS and boiled LPS. C. Macrophages incubated with native HSP70 and boiled HSP70. D. Macrophages incubated with TNF-α and BSA.
FIG. 4
4A-D show the results of a comparison of nitric oxide production by CD36 null and wild type macrophages in response to various stimuli. Filled dots represent wild-type macrophages, open dots represent CD36 null macrophages. The horizontal axis represents the protein concentration in micrograms / microliter for A, C and D, and the endotoxin in units / milliliter for B. A. Macrophages incubated with native gp96 and boiled gp96. B. Macrophages incubated with native LPS and boiled LPS. C. Macrophages incubated with native HSP70 and boiled HSP70. D. Macrophages incubated with TNF-α and BSA.
FIG. 5
5A-E show the results of a comparison of nitric oxide production by CD36 null and wild type dendritic cells in response to various stimuli. Solid dots represent wild-type macrophages and open dots represent CD36 null macrophages. The horizontal axis represents the protein concentration in micrograms / microliter for A, C and D, and the endotoxin in units / milliliter for B. A. Macrophages incubated with native gp96 and boiled gp96. B. Macrophages incubated with native LPS and boiled LPS. C. Macrophages incubated with native HSP70 and boiled HSP70. D. Macrophages incubated with TNF-α and BSA.
FIG. 6
6A-E show the human CD36 cDNA (Genbank accession number L06850), and the putative open reading frame of the CD36 protein (Genbank accession number AAA16068).
FIG. 7
7A-C show increased binding of gp96 to CD36 expressing cells compared to non-expressing controls. Solid dots represent mock transfected cells, and open dots represent CD36 transfected cells. The vertical axis represents the average fluorescence intensity, and the horizontal axis represents the protein concentration in micrograms / microliter. A. Gp96-FITC. B. Transferrin-FITC. C. Histone-FITC.
FIG. 8
8A-E show expression of the CD36 transgene on the cell surface of HEK293 transfectants and control cell lines. The horizontal axis represents the fluorescence intensity, and the vertical axis represents the number of cells. A. HEK293 mock transfected cells. B. U937 cells (positive control). C. CD36 HECK293 transfected cells. D. CD36 HECK293 transfected cells.
FIG. 9
FIG. 9 shows expression of the CD36 transgene on the cell surface of HEK293 transfected cells and control cell lines. The horizontal axis represents the fluorescence intensity, and the vertical axis represents the number of cells. The filled area represents unstained CD36 cells. The first open area, furthest to the left, represents unstained CD36 wild-type cells. The open area in the middle represents CD36 null cells stained with gp96-FITC. The rightmost open area represents CD36 wild-type cells stained with gp96-FITC.
FIG. 10
FIGS. 10A, B, Analysis of blocking of gp-FITC binding by putative competitors. A. Solid squares represent PC93 mock transfected cells. Open squares represent CD36 transfected cells that have not been preincubated. Open triangles represent CD36 transfected cells pre-incubated with acetylated LDL. Open circles represent CD36 transfected cells pre-incubated with oxidized LDL. B. Solid squares represent PC93 mock transfected cells. Open squares represent CD36 transfected cells that have not been preincubated. Open triangles represent CD36 transfected cells pre-incubated with anti-CD36 antibody. Open circles represent CD36 transfected cells pre-incubated with anti-CD36 antibody.

Claims (78)

(A) contacting the test compound with a heat shock protein and CD36; and (b) measuring the level of CD36 activity or expression;
And if the level of activity or expression measured in (b) is different from the level of CD36 activity in the absence of the test compound, then a compound that modulates an HSP-CD36 mediated process is identified. , Methods for identifying compounds that modulate HSP-CD36 mediated processes. 2. The method of claim 1, wherein the identified compound is an antagonist that interferes with the interaction of heat shock protein with CD36.
(C) determining whether said level interferes with the interaction of heat shock protein with CD36;
The method further comprising: 2. The method of claim 1, wherein said test compound is an antibody specific for CD36. 2. The method of claim 1, wherein the test compound is an antibody specific for a heat shock protein. 2. The method of claim 1, wherein said test compound is a small molecule. 2. The method of claim 1, wherein said test compound is a peptide. 7. The method of claim 6, wherein said peptide comprises at least five consecutive amino acids of CD36. 7. The method of claim 6, wherein said peptide comprises at least 10 contiguous amino acids of CD36. 7. The method of claim 6, wherein said peptide comprises at least 5 contiguous amino acids of the sequence of a heat shock protein. 2. The method of claim 1, wherein the compound is an agonist that enhances the interaction between heat shock protein and CD36. The HSP-CD36 mediated process may be associated with an autoimmune disorder, a disease or disorder with disruption of signal transducer activity, a disease or disorder with cytokine clearance or inflammation, a proliferative disorder, a viral disorder or other infectious disease, 2. The method of claim 1, wherein the method affects cholesterol, Alzheimer's disease, diabetes, or osteoporosis. (A) contacting a test compound with a heat shock protein and CD36 expressing cell; and (b) measuring the level of CD36 activity or expression in the cell;
And if the level of activity or expression measured in (b) is different from the level of CD36 activity in the absence of the test compound, then a compound that modulates an HSP-CD36 mediated process is identified. , Methods for identifying compounds that modulate HSP-CD36 mediated processes. 13. The method of claim 1 or 12, wherein the measured CD36 activity is the ability to interact with a heat shock protein. 13. The method of claim 12, wherein the heat shock protein is non-covalently associated with the antigenic peptide and the CD36 activity measured is the ability to induce a signaling activity. 13. The method of claim 12, wherein the heat shock protein is non-covalently associated with the antigenic peptide and the CD36 activity measured is the ability to stimulate a cytotoxic T cell response to the antigenic peptide. (A) contacting a heat shock protein with CD36 or a fragment, analog, derivative or mimetic thereof in the presence of a test compound; and (b) CD36 or a fragment, analog, derivative or mimetic of CD36 or a fragment thereof. Measuring the amount of heat shock protein bound to the body;
Wherein the amount of bound heat shock protein measured in (b) is different from the amount of bound heat shock protein in the absence of the test compound, a compound that modulates the binding of HSP to CD36 is identified. A method for identifying a compound that modulates the binding between heat shock protein and CD36. 17. The method of claim 16, wherein the CD36 contacted in step (a) is on a cell surface. 17. The method of claim 16, wherein said CD36 is immobilized on a solid surface. 73. The method of claims 18 and 72, wherein the solid surface is a microtiter dish. 17. The method of claim 16, wherein the amount of the bound heat shock protein is measured by contacting a cell with a heat shock protein specific antibody. 17. The method of claim 16, wherein the heat shock protein is labeled and the amount of the bound heat shock protein is determined by detecting the label. 22. The method of claim 21, wherein said heat shock protein is labeled with a fluorescent label. (A) adding a test compound to a mixture of CD36-expressing cells and a complex consisting essentially of a heat shock protein non-covalently associated with an antigen molecule under conditions that promote CD36-mediated signaling;
(B) measuring the level of stimulation by CD36 expressing cells;
If the level measured in (b) is different from the stimulation in the absence of the test compound, then a compound that modulates heat shock protein mediated signaling by CD36 expressing cells has been identified; CD36 expression A method for identifying a compound that modulates heat shock protein-mediated signaling by cells. The step (b) of measuring the stimulation of antigen-specific cytotoxic T cells by CD36-expressing cells comprises:
(I) adding the CD36-expressing cells formed in the step (a) to T cells under conditions that promote T cell activation; and (ii) determining the activation level of the cytotoxic T cells. Comparing the level of T cell activation by CD36 expressing cells formed in the absence of the test compound;
24. The method of claim 23, wherein increasing or decreasing the level of T cell activation indicates that a compound that modulates heat shock protein mediated signaling by CD36 expressing cells has been identified. 24. The method of claim 23, wherein said signaling activity is nitric oxide production. 24. The method of claim 23, wherein said signaling activity is the production of MCP-1 chemokines. 24. The method of claim 1, 17, 71 or 23, wherein the heat shock protein is gp96. Determining the level of activity from the HSP-CD36 mediated process in the patient sample, wherein the level of heat shock protein-CD36 related disorder is different if the measured level differs from the level found in clinically normal individuals. A method for detecting a heat shock protein-CD36-related disorder in a mammal, which is detected. 29. The method of claim 28, comprising contacting a sample obtained from the patient with an antibody specific for CD36 under conditions that promote immunospecific binding of the antibody. 29. The method of claim 28, comprising contacting a sample obtained from the patient with an antibody specific for a heat shock protein under conditions that promote immunospecific binding of the antibody. 29. The method of claim 28, comprising contacting a sample obtained from the patient with an antibody specific for the HSP-CD36 complex under conditions that promote immunospecific binding of the antibody. A method of modulating an immune response, comprising administering to a mammal a purified compound that modulates the interaction of heat shock protein with CD36. 33. The method of claim 32, wherein the compound is an agonist that enhances the interaction of heat shock protein with CD36. A method for treating an autoimmune disorder, comprising administering to a mammal in need of treatment a purified compound that interferes with the interaction of heat shock protein with CD36. 35. The method of claim 34, wherein said compound is an antagonist that interferes with the interaction of heat shock protein with CD36. 36. The method of claim 35, wherein said antagonist is an antibody specific for CD36. 36. The method of claim 35, wherein said antagonist is an antibody specific for a heat shock protein. 36. The method of claim 35, wherein said antagonist is a small molecule. 36. The method of claim 35, wherein said antagonist is a peptide. 40. The method of claim 39, wherein the peptide comprises at least 5 contiguous amino acids of CD36. 40. The method of claim 39, wherein the peptide comprises at least five consecutive amino acids of a heat shock protein sequence. 40. The method of claim 39, wherein said peptide comprises at least 10 contiguous amino acids of a heat shock protein sequence. A method for treating an autoimmune disorder, comprising administering to a mammal in need thereof, a recombinant cell expressing CD36, which reduces the signaling activity of functional CD36. A method of increasing the immunity of a cancer cell or an infected cell,
A method comprising: (i) operably linked to a promoter, and (ii) transforming the cell with a nucleic acid comprising a nucleotide sequence encoding a CD36 polypeptide. A method for enhancing the immunity of a cancer cell or an infected cell, comprising: (a) a nucleotide sequence encoding a CD36 polypeptide operably linked to a (i) promoter to obtain a high immune response;
Transforming a cell with a nucleic acid comprising: and (b) administering the cell to an individual in need of treatment;
Including, methods. A recombinant cancer cell operably linked to (i) a promoter and (ii) transformed with a nucleic acid comprising a nucleotide sequence encoding a CD36 polypeptide. Recombinantly infected cells (i) operably linked to a promoter and (ii) transformed with a nucleic acid comprising a nucleotide sequence encoding a CD36 polypeptide. 48. The recombinant cell according to claim 46 or 47, which is a human cell. (A) a nucleic acid probe hybridizable to an anti-CD36 antibody or CD36 nucleic acid; (b) a purified heat shock protein, a nucleic acid encoding a heat shock protein, or a cell expressing a heat shock protein; and (c) a heat shock protein. -A kit comprising, in one or more containers, instructions for use in detecting a CD36-related disorder. 50. The kit of claim 49, wherein said antibody or nucleic acid probe is labeled with a detectable marker. 50. The kit of claim 49, further comprising a labeled CD36 polypeptide. (A) a purified heat shock protein, a nucleic acid encoding a heat shock protein, or a cell expressing the heat shock protein; and (b) a CD36 polypeptide, a nucleic acid encoding the CD36 polypeptide, or a cell expressing the CD36 polypeptide. A kit comprising in one or more containers. 53. The kit of claim 52, wherein the CD36 polypeptide, a nucleic acid encoding the CD36 polypeptide, or a cell expressing the CD36 polypeptide has been purified. 53. The kit of claim 52, further comprising instructions for use in treating an autoimmune disorder, an infectious disease, or a proliferative disorder. (A) contacting a heat shock protein or peptide binding fragment thereof with one or more CD36 fragments; and (b) identifying a CD36 fragment that specifically binds to the heat shock protein or peptide binding fragment thereof;
A method for identifying a CD36 fragment capable of binding to a heat shock protein, comprising: (A) contacting the heat shock protein with a cell that expresses a CD36 fragment; and (b) measuring CD36 activity in the cell;
A CD36 fragment capable of inducing an HSP-CD36 mediated process, wherein the level or level of activity of the HSP-CD36 mediated process measured in (b) is greater than the level of CD36 activity in the absence of the CD36 fragment And a method for identifying a CD36 fragment capable of inducing an HSP-CD36-mediated process. 57. The method of claim 56, wherein the measured CD36 activity is the ability to interact with a heat shock protein. 57. The method of claim 56, wherein the heat shock protein is non-covalently associated with the antigenic peptide, and wherein the measured CD36 activity is the ability to stimulate signaling activity. 59. The method of claim 58, wherein said signaling activity is nitric oxide production. 59. The method of claim 58, wherein said signaling activity is the production of a MCP-I chemokine. 57. The method of claim 56, wherein the heat shock protein is non-covalently associated with the antigenic peptide, and wherein the measured CD36 activity is the ability to stimulate a cytotoxic T cell response to the antigenic peptide. (A) contacting CD36 with one or more heat shock protein fragments; and (b) identifying heat shock protein fragments that specifically bind to CD36;
A method for identifying a heat shock protein fragment capable of binding to CD36, comprising: (A) contacting the CD36 fragment with a cell expressing a heat shock protein; and (b) measuring the level of CD36 activity in the cell;
Wherein the level of HSP-CD36 mediated process or activity measured in (b) is greater than the level of CD36 activity in the absence of the heat shock protein fragment, is capable of inducing an HSP-CD36 mediated process. A method for identifying a heat shock protein fragment capable of inducing an HSP-CD36 mediated process, wherein the heat shock protein fragment has been identified. 64. The method of claim 63, wherein the measured CD36 activity is the ability to interact with a heat shock protein fragment. 64. The method of claim 63, wherein the heat shock protein fragment is non-covalently associated with the antigenic peptide, and wherein the measured CD36 activity is the ability to stimulate signal transducer activity. 64. The method of claim 63, wherein the heat shock protein fragment is non-covalently associated with the antigenic peptide, and wherein the measured CD36 activity is the ability to stimulate a cytotoxic T cell response to the antigenic peptide. (A) contacting CD36 with one or more test molecules under conditions that promote binding; and (b) identifying one or more test molecules that specifically bind to CD36;
A method for identifying a molecule that specifically binds to CD36, comprising: 68. The method of claim 67, wherein said test molecule is a potential immunotherapeutic. (A) contacting CD36 with one or more test molecules under conditions that promote binding; and (b) determining whether any of the test molecules specifically binds CD36;
A method for screening for a molecule that specifically binds to CD36, comprising: (A) contacting CD36 with a CD36 ligand or a CD36 binding fragment, analog, derivative or mimetic thereof in the presence of one or more test compounds; and (b) CD36 ligand or a CD36 ligand bound thereto. Measuring the amount of fragments, analogs, derivatives or mimetics;
If the amount of the bound CD36 ligand measured in (b) is different from the amount of the bound CD36 measured in the absence of the test compound, a compound that modulates the binding between the CD36 ligand and CD36 is identified. A method for identifying a compound that modulates the binding between CD36 ligand and CD36. 71. The method of claim 70, wherein the CD36 contacted in step (a) is on a cell surface. 71. The method of claim 70, wherein said CD 36 is immobilized on a solid surface. (A) contacting CD36 with one or more test compounds; and (b) measuring the level of CD36 activity or expression;
Wherein the level of activity or expression measured in (b) is different from the level of activity in the absence of the one or more test compounds, a compound that modulates the interaction of CD36 with a CD36 ligand is identified. A method for identifying a compound that modulates the interaction between CD36 and a CD36 ligand. (A) adding one or more test compounds to a mixture of CD36-expressing cells and a complex of a CD36 ligand and an antigen molecule under conditions that promote CD36-mediated signaling;
(B) measuring the level of stimulation of antigen-specific cytotoxic T cells by CD36 expressing cells;
If the level measured in (b) is different from the level of the stimulus in the absence of the one or more test compounds, then a compound that modulates signaling by CD36 expressing cells has been identified, CD36. A method for identifying a compound that modulates signal transduction by an expression cell. A method of modulating an immune response, comprising administering to a mammal a purified compound that binds CD36 in an amount effective to modulate an immune response in said mammal. A method for treating or preventing a disease or disorder, comprising administering to a mammal a purified compound that binds to CD36 in an amount effective to treat or prevent the disease or disorder in the mammal. 77. The method of claim 76, wherein said disease or disorder is a cancer or an infectious disease. A method of treating an autoimmune disorder, comprising administering to a mammal in need thereof a purified compound that binds to CD36 in an amount effective to treat the autoimmune disorder in the mammal.

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