CN101568262A - Compositions and methods for treating Gram positive bacterial infection in a mammalian subject - Google Patents

Abstract

The present invention provides compositions and methods for treating Gram-positive bacterial infections in mammalian subjects. The invention further provides compositions and methods for treating Gram-positive bacterial skin infections in a mammalian subject. The compositions and methods provided herein comprise administering to the subject an effective amount of a compound that activates the expression of the Scd1 gene or activates the product of the Scd1 gene.

Description

Compositions and methods for treating gram-positive bacterial infections in mammalian subjects

Statement of Government Funding

This invention was made with support from the National Institutes of Health under grant number U54-AI54523. The government has certain rights in this invention.

Cross References to Related Applications

This application claims U.S. Provisional Application 60/701,216, filed July 20, 2005, and EMS EV 670672061 US, filed July 19, 2006, entitled "Compositions for the Treatment of Gram-Positive Bacterial Infections in a Mammalian Subject" and Methods", the entire disclosure of which is hereby incorporated by reference.

technical field

The present invention generally relates to compositions and methods for treating Gram-positive bacterial infections in mammalian subjects. The invention further relates to compositions and methods for treating Gram-positive bacterial skin infections in mammalian subjects. The compositions and methods further comprise administering to the mammalian subject an effective amount of a compound that activates expression of the Scd1 gene or activates the product of the Scd1 gene.

Background technique

Surface epithelial cells constitute the first line of defense against pathogens. This line of defense relies on both barrier function and specific bactericidal effector molecules. For example, mammalian skin provides physical protection in part because it is composed of tightly bound cells covered with a layer of highly cross-linked keratin and is generally impermeable to bacteria. In humans, a number of hereditary diseases affecting skin epithelial constructs at different levels such as mucoepithelial dysplasia or epidermolysis bullosa are associated with a substantially increased susceptibility to infection. Vidal et al., Nat Genet 10:229-34, 2995; Witkop et al., Am J Hum Genet 31:414-27, 1979. But skin can still exhibit bactericidal activity even when its physical integrity is compromised. It contains a large number of bioactive molecules, among which antimicrobial peptides (AMPs) such as defensins and cecropins are essential for host defense against microbial invasion (for review, see Zasloff, Nature 415:389-95, 2002; Zasloff, N Engl J Med 347 : 1199-200, 2002).

Although AMPs are the most studied skin defense molecules, other protective systems exist. Monounsaturated fatty acids (MUFAs) produced by the sebaceous glands are involved in this context, some of which are known to have bactericidal properties. Miller et al., Arch Dermatol 124:209-15, 1988; Wille and Kydonieus, Skin Pharmacol Appl Skin Physiol 16:176-87, 2003. However, their role for antimicrobial defense has not been established in vivo, and their biosynthesis is not regulated by microbial stimuli. There is a need in the art to develop optimized compositions and methods capable of eliciting innate immune responses to microbial infections in mammalian subjects. A further need exists to develop optimized compositions and methods capable of treating Gram-positive bacterial infections and Gram-positive bacterial infections in mammalian subjects.

Contents of the invention

The present invention generally relates to compositions and methods for treating Gram-positive bacterial infections in mammalian subjects. The invention further provides compositions and methods for treating Gram-positive bacterial skin infections in a mammalian subject. The compositions and methods provided by the present invention comprise administering to the mammalian subject an effective amount of a drug capable of activating stearoyl-CoA desaturase 1 (Scd1) gene expression or activating the Scd1 gene product-stearoyl-CoA desaturase compound.

The congenital immunodeficiency phenotype in mice has been attributed to a mutation that affects the structure of a key enzyme for monounsaturated fatty acid (MUFA) synthesis. ENU-induced germline mutations in C57BL/6 mice were used to isolate and identify Flake(flk), a recessive C57BL/6 mouse germline mutation, flk mutant mice are resistant to Streptococcus pyogenes infection and Staphylococcus aureus ( Clearance of skin infections by Gram-positive pathogens, which can trigger an innate immune response through activation of Toll-like receptor 2, is impaired. Positional cloning and sequencing revealed that flk is a novel allele of the stearoyl-CoA desaturase 1 gene (Scdl).

The present invention provides a method of treating a Gram-positive bacterial infection in a mammalian subject comprising administering to the subject an effective amount of a compound capable of activating expression of the Scd1 gene. In one aspect, the compound is an agonist of toll-like receptor 2. In another aspect, the compound is a small chemical molecule, antibody, antisense nucleic acid, short hairpin RNA, or short interfering RNA. Examples of Gram-positive bacterial infections may be Streptococcus pyogenes infection or Staphylococcus aureus infection. In another aspect, the method comprises treating a subject having a loss-of-function or a decrease-of-function mutation of the Scd1 gene.

The present invention provides a method of treating a Gram-positive bacterial infection in a mammalian subject comprising administering to the subject an effective amount of a compound that activates the Scd1 gene product. In one aspect, the compound is an agonist of toll-like receptor 2. In another aspect, the compound is a small chemical molecule, antibody, antisense nucleic acid, short hairpin RNA, or short interfering RNA. Gram-positive bacterial infection can be Streptococcus pyogenes infection or Staphylococcus aureus infection etc. In another aspect, the method comprises treating a subject having a loss-of-function or a decrease-of-function mutation of the Scd1 gene.

The present invention provides a method of treating a Gram-positive bacterial infection in a mammalian subject comprising administering to the subject an effective amount of a monounsaturated fatty acid. The monounsaturated fatty acid may be palm oleate or oleate or the like. Gram-positive bacterial infection can be Streptococcus pyogenes infection or Staphylococcus aureus infection etc. In one aspect, an effective amount of monounsaturated fatty acids can be administered topically or intradermally. In another aspect, an effective amount of monounsaturated fatty acid can be administered intramuscularly, subcutaneously, intraperitoneally or intravenously.

The present invention provides a method of treating a Gram-positive bacterial infection in a mammalian subject comprising administering to the subject an effective amount of a compound that is a product of the Scd1 biosynthetic pathway. In one aspect, the compound is a monounsaturated fatty acid. The monounsaturated fatty acid may be palm oleate or oleate or the like. Gram-positive bacterial infection can be Streptococcus pyogenes infection or Staphylococcus aureus infection etc. In one aspect, an effective amount of monounsaturated fatty acids can be administered topically or intradermally. In another aspect, an effective amount of monounsaturated fatty acid can be administered intramuscularly, subcutaneously, intraperitoneally or intravenously.

The present invention provides a method of identifying a compound that modulates intracellular Gram-positive bactericidal activity, comprising: contacting a test compound with a cell-based assay system comprising cell-expressed toll-like receptor 2, to The assay system provides an amount of ligand selected to be effective in activating toll-like receptor 2 signaling, which can signal a response to the ligand and modulate Scd1 gene expression, and then detects the pair of test compounds against Role of toll-like receptor 2 signaling and regulation of Scd1 gene expression, effectiveness of test compounds in assays as indicators of Gram-positive bactericidal activity. In one aspect, the ligand is an endogenous ligand or an exogenous ligand. In one aspect, the exogenous ligand can specifically be lipopolysaccharide, lipid A, diacyl fatty acyl peptide, triacyl fatty acyl peptide, S-MALP-2, R-MALP-2, bacterial fatty acyl peptide, Pam2CSK4, lipid Teichoic acid or zymosan A. In another more specific aspect, the exogenous ligand is MALP-2. In another more specific aspect, the exogenous ligand is rough lipopolysaccharide, smooth lipopolysaccharide or lipid A from Salmonella Minnesota. In a specific aspect, the exogenous ligand is a component of a Gram-positive bacterium rather than a Gram-negative bacterium. In another more specific aspect, the endogenous ligand is specifically a lipid. The compound may be an agonist of signal transduction of toll-like receptor 2 pathway and the like.

In one embodiment, the method comprises the detecting step further comprising detecting Scd1 gene expression or activation of Scd1 gene product in the cell, wherein Scd1 gene expression or Scd1 gene product is activated in response to contact of the cell with an exogenous ligand activation.

In a further embodiment, the detecting step of the provided method further comprises detecting the compound-enhanced ligand binding to toll-like receptor 2. The testing step of the provided methods further comprises detecting an elevated Scd1 gene product in the cell assay. The testing step of the provided methods further comprises detecting increased Scd1 gene product activity in a cellular assay. The testing step in the provided methods further comprises detecting increased monounsaturated fatty acid synthesis in cell assays. In another aspect, the detecting step further comprises detecting binding of the labeled ligand to toll-like receptor 2. The labeled ligand may be radiolabeled or fluorescently labeled.

In another aspect, the cellular analysis can include macrophages or cells from the sebaceous glands. The sebaceous gland-derived cells may be sebocytes.

In one embodiment, the method further comprises providing toll-like receptor 2 to an assay system, and detecting the effect of the test compound on signal transduction of toll-like receptor 2 in the assay system, the effectiveness of the test compound in the assay being determined by as an indication of adjustment.

In one embodiment, the detecting step further comprises reducing the binding of the ligand to toll-like receptor 2 by the compound. In another embodiment, the detecting step further comprises enhancing the binding of the ligand to toll-like receptor 2 by the compound. In a further embodiment, the testing step further comprises detecting an increase in stearoyl-CoA desaturase 1 activity in a cellular assay. In a further embodiment, the testing step further comprises detecting increased monounsaturated fatty acid synthesis in a cell assay. In a further embodiment, the testing step further comprises detecting an increase in Gram-positive bactericidal activity in a cell assay.

The present invention provides a method of diagnosing risk factors for Gram-positive bacterial infection in a mammalian subject, comprising removing cells or tissues from the subject, combining the cells or tissues with endogenous toll-like receptor 2 Ligand or exogenous ligand exposure, detection of Scd1 gene product production in cells or tissues contacted with the ligand, detection of decreased function or loss of function of the Scd1 gene product in a mammalian subject. The cells or tissues may be from macrophages, sebocytes or sebaceous glands.

In one aspect, the loss of function or deletion of the Scd1 gene product by this approach increases the risk of Gram-positive bacterial infection. In another aspect, the decreased function or deletion of the Scd1 gene product described therein reduces the synthesis of monounsaturated fatty acids in cells. On the other hand, this decreased function or deletion of the Scd1 gene product reduced the inflammatory response to Gram-positive bacterial infection. In a specific aspect, the decreased function or deletion of the Scd1 gene product reduces the inflammatory response at the site of injury in the patient. On the other hand, deletion of the Scd1 gene product increases the risk in cases where inflammation is the intended defense mechanism. The ligand can be an exogenous ligand, lipoteichoic acid (LTA), diacyl fatty acyl peptide, triacyl fatty acyl peptide, S-MALP-2, bacterial fatty acyl peptide, peptidoglycan, mannan Sugars, unmethylated CpG DNA, flagellin or single-stranded RNA. The ligand can be an endogenous ligand, lipid, fat, sterol, lipoprotein, fatty acid, oxidized LDL, thrombospondin or beta amyloid.

The present invention provides a method for diagnosing disorders caused by the loss of function of the Scd1 gene or thus causing genetic predisposition in a mammalian subject, which comprises detecting mutated Scd1 in a cell sample, protein sample or nucleic acid sample obtained from a mammalian subject The presence of a protein or nucleic acid encoding a mutant Scd1 protein, wherein the presence of the protein or nucleic acid is indicative of a disorder caused by a loss of function of the Scd1 gene or a genetic predisposition thereby. In one aspect, the disorder caused by loss of function of the Scd1 gene is increased susceptibility to Gram-positive bacterial infection.

In one embodiment, the method further comprises contacting the protein sample or the cell sample with an anti-Scd1 antibody, and detecting the presence of wild-type or mutant Scd1 protein. In another aspect of the method, the detecting step further comprises performing fluorescence activated cell sorting (FACS) analysis on the mononuclear macrophages or macrophages from the mammal. In another aspect, the method further comprises contacting the nucleic acid sample with a labeled DNA or RNA molecule encoding the mutant Scd1 gene under hybridization conditions, and detecting the labeled DNA or RNA molecule after hybridization, wherein the detection of the labeled DNA or RNA indicates that the sample The presence of a nucleic acid molecule encoding the mutant Scd1 gene in the In another aspect, the method comprises contacting a nucleic acid sample with a restriction enzyme whose recognition sequence is affected by the mutation of the mutant Scd1 gene, and detecting the presence or absence of nucleic acid fragments or the presence of variant fragments following contact with the restriction enzyme , wherein the absence of nucleic acid fragments or the presence of variant fragments after contact with a restriction enzyme indicates the presence of a nucleic acid molecule encoding a mutant Scd1 gene in the sample.

The present invention provides a transgenic non-human animal comprising an exogenous nucleic acid comprising a loss-of-function allele of the Scd1 gene, and the animal exhibits a wild-type Phenotypes of susceptibility to infection with Lambert-positive bacteria. The phenotype of the transgenic non-human animal Scd1 mutant animals can be characterized by atrophied sebaceous glands or loss of ability to synthesize monounsaturated fatty acids. The transgenic non-human animal may have a loss-of-function allele in the Scd1 gene, such as an amino acid substitution at T227K. The transgenic non-human animal can be a mouse or a rat. In one aspect, a cell or cell line can be derived from the transgenic non-human animal.

The present invention provides an in vitro method for screening modulators of Toll-like receptor 2 signal transduction activity, which comprises: contacting cells or cell lines derived from transgenic non-human animals with a test compound, and detecting single Increase or decrease in unsaturated fatty acid synthesis, susceptibility to Gram-positive bacterial infection, or Toll-like receptor 2-induced macrophage activation, thereby identifying the test compound as a Toll-like receptor 2-induced macrophage Modulator of activation activity. The present invention provides an in vivo method for screening for modulators of Toll-like receptor 2 signaling activity, comprising: contacting cells or cell lines derivable from transgenic non-human animals with a test compound, and detecting single Increase or decrease in unsaturated fatty acid synthesis, susceptibility to Gram-positive bacterial infection, or Toll-like receptor 2-induced macrophage activation, thereby identifying the test compound as a Toll-like receptor 2-induced macrophage Modulator of activation activity.

Description of drawings

Figures 1A, 1B, 1C and 1D show the visible phenotypes observed in flake mutant mice.

Figures 2A, 2B and 2C show that flake mutant mice developed persistent skin infections when exposed to Gram-positive bacteria.

Figures 3A, 3B and 3C show maps of flake mutations.

Figures 4A and 4B show the molecular characterization of flake mutations.

Figures 5A and 5B show thin-layer chromatographic analysis of lipid content in wild-type and flake mutant mice.

Figures 6A, 6B, 6C, 6D, 6E and 6F show palmitoleic acid with antibacterial activity in vivo.

Figures 7A, 7B, 7C and 7D show infection and TLR2-dependent induction of Scd1 gene expression in mice.

Figures 8A, 8B, 8C and 8D show the inflammatory response and upregulation of SCD1 and FADS2 genes in human sebocytes stimulated with MALP-2.

Figure 9 shows unsaturated fatty acid biosynthesis via the SCD1 biosynthetic pathway.

Detailed ways

The present invention generally relates to compositions and methods for treating Gram-positive bacterial infections in mammalian subjects. The invention further provides compositions and methods for treating Gram-positive bacterial skin infections in a mammalian subject. The compositions and methods provided herein comprise administering to the subject an effective amount of a compound capable of activating expression of the Stearoyl-CoA desaturase 1 (Scd1) gene or activating the product of the Scd1 gene, Stearoyl-CoA desaturase 1 . The present invention provides a method of treating a Gram-positive bacterial infection comprising administering to the subject an effective amount of a monounsaturated fatty acid.

Flake (flk), an ENU-induced recessive germline mutation in C57BL/6 mice, impairs innate immunity to S. pyogenes infection and S. Clearance of skin infections caused by responsive Gram-positive pathogens). Positional cloning and sequencing revealed that flk is a novel allele of the stearoyl-CoA desaturase 1 gene (Scd1). Flake homozygotes are unable to synthesize the monounsaturated fatty acids (MUFAs) palmitoleate (C16:1) and oleate (C18:1), which kill Gram-positive (but not Gram-negative) bacteria . Intradermal MUFA administration to S. aureus-infected mice enhanced bacterial clearance. In normal mice, the transcription of Scd1, a gene with multiple NF-κB elements in the promoter, is strongly and specifically induced by TLR2 signaling. Similarly, the SCD1 gene is also induced in human sebocytes by TLR2 signaling. These findings reveal a regulated lipid-based pathway for antimicrobial effects in mammals and suggest a new approach to treat or prevent Gram-positive bacterial infections.

"Patient", "subject", "vertebrate" or "mammal" are used interchangeably and refer to mammals, such as human patients or non-human primates, as well as rabbits, rats, mice and other laboratory animals . Animals include all vertebrates such as mammals and non-mammals such as sheep, dogs, cows, chickens, amphibians and reptiles.

"Treatment" includes administration of the antibody composition, compound or reagent of the present invention to prevent or delay the onset of symptoms, complications or biochemical indicators of a disease, alleviate symptoms or prevent or inhibit a disease, disease or disorder (such as cancer or the further development of metastatic cancer). Treatment can be prophylactic (preventing or delaying the onset of disease, or preventing the development of associated clinical or subclinical symptoms) or therapeutic suppression or alleviation of symptoms after disease has manifested.

As used herein, "inhibitors", "activators" and "modulators" of Toll-like receptors in cells refer to inhibitory, activating or modulatory molecules, respectively, which can be identified by in vitro and in vivo assays of Toll-like receptor binding or signal transduction, Such as ligands, agonists, antagonists and their homologues and analogs.

"Modulator" includes both inhibitors and activators. Inhibitor refers to an agent, such as an antagonist, that binds, partially or completely prevents excitation, reduces, prevents, delays activation, inactivates, desensitizes or down-regulates Toll-like receptor activity. Activator refers to an agent, such as an agonist, that binds, stimulates, increases, opens, activates, facilitates, potentiates activation, sensitizes or upregulates the activity of Toll-like receptors. Modulators may include agents capable of altering the interaction of Toll-like receptors with: proteins, receptors, including proteins, peptides, lipids, sugars, polysaccharides, or combinations thereof, such as lipids, capable of binding to activators or inhibitors Proteins, glycoproteins and the like. Modulators include products of naturally occurring Toll-like receptor ligands that have been genetically modified (eg, altered in activity), as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. "Cell-based assays"for inhibitors and activators include, for example, applying putative modulator compounds to cells expressing Toll-like receptors and then testing for functional effects on Toll-like receptor signaling as described herein. "Cell-based assays" include, but are not limited to, in vivo tissue or cell samples from mammalian subjects or containing Toll-like receptors treated with potential activators, inhibitors or modulators versus those without inhibitors, activators or modulators. In vitro cell-based assays of modulator-treated control samples to detect the extent of inhibition. Control samples (no inhibitor treatment) can be assigned a value of 100% relative to Toll-like receptor activity. Toll-like receptor inhibition is achieved when the Toll-like receptor activity value is about 80%, optionally 50% or 25-0% of the control. Toll-like receptor activation is achieved when the Toll-like receptor activity value is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher than the control.

The ability of a molecule to bind Toll-like receptors can be determined, for example, by the ability of a putative ligand to bind Toll-like receptors immunoadherently coated on an assay plate. The specificity of binding can be tested by comparing binding to non-Toll-like receptors.

"Test compound" refers to any compound that is tested as a modulator of Scd1 or toll-like receptor 2. A test compound can be any small organic molecule, or biological entity, such as a protein (such as an antibody or peptide), a sugar, a nucleic acid (such as an antisense oligonucleotide, RNAi), or a ribozyme or a lipid. In addition, the test compound may be a product of gene modification of Scd1 protein or toll-like receptor 2 protein. Typically, test compounds are small organic molecules, peptides, lipids or lipid analogs.

In one example, antibody binding to Toll-like receptors can be assayed by immobilizing the ligand or receptor. For example, the assay may involve immobilization of Toll-like receptors fused to a His-tag to Ni-activated NTA resin beads. Antibodies are added in an appropriate buffer and the beads are incubated at a given temperature for a period of time. After washing to remove unbound material, bound proteins can be released and analyzed by SDS, high pH buffers, and the like.

"Signal transduction response" refers to signal transduction by toll-like receptors, such as toll-like receptor 2. The signal transduction response may refer to an LPS response dependent on a signal-transmitting transmembrane complex formed by Toll-like receptor 2 (TLR2) and Scd1, among others. TLR2 signaling is directly or indirectly induced by MALP2 and increases Scd1 expression. TLR2 signaling can occur in cells such as macrophages or sebocytes. Signal-generating compounds for detection in cell-based assays can be generated by conjugation with enzymes or fluorophores, among others. The target enzymes used as labels are mainly hydrolases, especially phosphatases, esterases and glycosidases, or oxidases, especially peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin and 2,3-dihydrophthalazinediones such as luminol.

"Detecting the effect of a test compound on toll-like receptor 2 signal transduction" may refer to a therapeutic or prophylactic effect on a mammalian subject, such as attenuation, elimination or prevention of a disease, a symptom of a disease, or a side effect of a disease in the subject . "Detecting the effect of a test compound on toll-like receptor 2 signaling" may refer to one having an effect as measured by MALP2 stimulation of TLR2 signaling and upregulation of Scd1 gene expression in a cell-based assay such as a diagnostic assay compound. Loss-of-function mutations of the Scd1 gene, such as the Flake mutation, impair the clearance of S. pyogenes infections and skin infections caused by S. aureus, a Gram-positive pathogen that can elicit an innate immune response by activating Toll-like receptor 2. Flake homozygotes are unable to synthesize the monounsaturated fatty acids (MUFAs) palmitoleate (C16:1) and oleate (C18:1), which kill Gram-positive (but not Gram-negative) bacteria . Intradermal MUFA administration to S. aureus-infected mice enhanced bacterial clearance.

It is to be understood that this invention is not limited to particular methods, reagents, compounds compositions or biological systems, which may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. As used in this specification and claims, the singular forms "a", "an" and "the" include plural referents unless expressly stated otherwise. Thus, reference to "a cell" includes combinations or the like of two or more cells.

As used herein, the term "about" means that a measurement such as quantity, time period, etc. may contain a variation of ± 20% or ± 10% relative to the specified value, the variation is preferably ± 5%, more preferably ± 1%, more preferably ±0.1%, as such variations are suitable for practicing the disclosed methods.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although other methods and materials similar or equivalent to those described herein can also be used in the testing of the present invention, the preferred materials and methods are described herein. The following terms may be used to describe and claim the present invention.

Antibodies that act as modulators of SCD1 gene expression or SCD1 gene product or TOLL-like receptor 2

The antibodies and antigen-binding fragments thereof described herein specifically bind to and/or activate toll-like receptor 2 (TLR2) or specifically bind to and/or activate Scd1 gene expression or Scd1 gene product, and may regulate or activate the expression of the Scd1 gene in mammals Innate immune response to gram-positive bacterial infection in subjects.

Antibodies capable of binding TLR2 or Scd1 gene products are useful as compounds that modulate signal transduction through the toll-like receptor 2 pathway in cells. See, eg, Takeda and Akira, Cell Microbiol 5:143-153, 2003.

In some embodiments, the antibody or antigen-binding fragment thereof can selectively bind (e.g., competitively bind or bind to the same epitope, such as a conformational or linear gauge). Thus, the epitope may be spatially close or functionally related to a known epitope to which the antibody binds, such as an epitope that overlaps or is adjacent in linear sequence or conformational space. Potential epitopes can be identified in silico using a peptide threading program and verified using methods known in the art, for example, by binding of antibodies to mutants or fragments of the toll-like receptor 2 or Scd1 gene product (e.g., toll-like receptor 2 Mutants or fragments of one domain of receptor 2 or Scd1 gene product) were analyzed.

Methods for detecting antibody sequences described herein are well known in the art; for example, the sequence of an antibody can be determined by isolating and identifying cDNA encoding the antibody from a hybridoma cell line using known techniques. Methods for sequencing cDNA are well known in the art.

Antibodies described herein typically have at least one or two heavy chain variable regions ( _VH ), and at least one or two light chain variable regions ( _VL ). The _VH and _VL domains can be further subdivided into hypervariable domains, Complementary Determinant Regions (CDRs), in which are distributed highly conserved framework regions (FRs). These domains have been clearly described (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242, 1991 and Chothia et al., J. MoI. Biol. 196: 901-917, 1987). Antibodies or antibody fragments comprising one or more framework regions are also useful in the present invention. The fragment can specifically bind to toll-like receptor 2 and regulate or activate Scd1 gene product activity in lipopolysaccharide-induced cells, or can regulate or activate innate immune response to Gram-positive bacteria.

The antibodies described herein may include a heavy and/or light chain constant region (constant regions typically mediate the binding of the antibody to host tissues or factors, the latter including effector cells of the immune system as well as components of the classical complement system. first component (Clq)), and thus form immunoglobulin light chains and immunoglobulin heavy chains, respectively. For example, the antibody may be a tetramer (two heavy immunoglobulin chains and two light immunoglobulin chains linked by disulfide bonds or the like). The antibody may comprise only a portion of the heavy chain constant region (e.g., one of the three heavy chain regions designated _CH1 , _CH2 , and _CH3 ), or a portion of the light chain constant region (e.g., CL part of the domain).

The invention also encompasses antigen-binding fragments. The fragment may be: (i) a _Fab fragment (i.e. a monovalent fragment consisting of VL, _VH , _CL and _CHl domains); ₍ ii) a F( _ab ') ₂ fragment (i.e. a bivalent fragment comprising two F _ab fragments linked by a disulfide bond at the hinge region); (iii) an F _d fragment composed of _VH and _CHl domains; (iv) A F _V fragment consisting of the _VL and _VH domains in a single arm of an antibody, (v) a dAb fragment consisting of the _VH domain (Ward et al., Nature 341:544-546, 1989) and/or (vi) an isolated complementary determinant region (CDR).

Antibody fragments (including the aforementioned antigen-binding fragments) can be synthesized by methods known in the art such as an automatic peptide synthesizer, or expressed by full-length genes or gene fragments, for example, Scd1 gene product F( _ab ') ₂ fragment, the F _ab fragment can be obtained by reducing the disulfide bond of the F( _ab ') ₂ fragment. In addition, _Fab expression libraries can be constructed (Huse et al., Science 246: 1275-81, 1989) for relatively rapid identification of monoclonal _Fab fragments with the desired specificity.

Methods for making other antibodies and antibody fragments are known in the art. For example, although the two domains _VL and _VH of the _Fv fragment are encoded by two separate genes, they can be paired with the VL _{and VH} domains by recombinant methods or synthetic linkers to form a single protein chain of a monovalent molecule (known as Known as single-chain _Fv ( _scFv ); see Bird et al., Science 242:423-426, 1988; Huston et al., Proc.Natl.Acad.Sci.USA 85:5879-5883, 1988; Colcher et al., Ann. NY Acad. Sci. 880:263-80, 1999; and Reiter, Clin. Cancer Res. 2:245-52, 1996).

Techniques for preparing single-chain antibodies are described in U.S. Pat. Nos. 4,946,778 and 4,704,692. Such single chain antibodies are encompassed within the term "antigen-binding fragment" of an antibody. These antibody fragments can be obtained by conventional techniques known to those of ordinary skill in the art, and the fragments can be screened for use by screening intact antibodies. In addition, a single chain antibody can form complexes or multimers, thereby becoming multivalent antibodies with specificity for different epitopes of the same target protein.

The antibodies and portions thereof described herein may be monoclonal antibodies, may be produced from monoclonal antibodies, or may be obtained synthetically by methods known in the art. Antibodies can be produced recombinantly (e.g., by phage display or combinatorial methods, see U.S. Pat. No. 5,223,409; WO 92/18619; WO 91/17271; WO 92/20791; WO 92/15679; WO 93/01288 WO 92/01047; WO 92/09690; WO 90/02809; Fuchs et al., Bio/Technology 9:1370-1372, 1991; Hay et al., Human Antibody Hybridomas 3:81-85, 1992; Huse et al, Science 246:1275-1281, 1989; Griffiths et al., EMBO J.12:725-734, 1993; Hawkins et al., J.MoI.Biol.226:889-896, 1992; Clackson et al., Nature 352:624- 628, 1991; Gram et al., Proc. Natl. Acad. Sci. USA 89:3576-3580, 1992; Garrad et al., Bio/Technology 9:1373-1377, 1991; -4137, 1991; and Barbas et al., Proc. Natl. Acad. Sci. USA 88:7978-7982, 1991).

For example, an antibody to toll-like receptor 2 or an antibody to a Scd1 gene product can be obtained by using a TLR2 polypeptide or a Scd1 polypeptide, or a fragment derived from TLR24 or a corresponding Scd1 gene product (i.e., having a sequence of a portion thereof) to obtain an antigenic peptide Fragments, or cells expressing TLR2 antigen or Scd1 antigen or corresponding antigen fragments are prepared after immunization of animals. In some embodiments, an antibody or antigen-binding fragment thereof described herein binds to purified TLR2 or Scd1 gene product. In some embodiments, antibodies or antigen-binding fragments thereof can bind to TLR2 or Scdl gene products in tissue sections, whole cells (live, lysed, or fractionated cells), or membrane fractions. The antibody can be tested in an in vitro system, for example, the modulation, activation or inhibition of Scd1 gene expression or Scd1 protein activity can be detected by MALP-2 activation of macrophages.

When using an antigenic peptide derived from a TLR2 or Scd1 gene product, it generally includes at least 8 (eg, 10, 15, 20, 30, 50, 100 or more) contiguous amino acid residues of a region of the TLR2 or Scd1 gene product . In some embodiments, the antigenic peptide will comprise all regions of the TLR2 or Scd1 gene product. The antibody produced will specifically bind to one of the proteins in its native form (thus, antibodies with linear or conformational epitopes are encompassed by the invention), in a denatured or other non-native form, or in both forms. Potentially antigenic peptides can be identified by methods known in the art, such as computer-based antigenicity prediction algorithms. Sometimes conformational epitopes can be identified by identifying antibodies that bind to proteins in their native, rather than non-denatured, form.

A host animal (such as a rabbit, mouse, guinea pig or rat) can be immunized with an antigen, optionally linked to a carrier (i.e., a substance that stabilizes or otherwise enhances the immunogenicity of the linked molecule), and Adjuvants may optionally be administered (see, for example, Ausubel et al., supra) Exemplary carriers include keyhole limpet hemocyanin (KLH), Exemplary adjuvants (usually selected based on the species of host animal) include Freud Adjuvants (complete or incomplete), auxiliary mineral gels (such as aluminum hydroxide), surface active substances such as lysolecithin, polyols, polyanions, peptides, oil emulsions, dinitrophenol, BCG (BCG) and Corynebacterium brevis. KLH is also sometimes referred to as an adjuvant. Antibodies produced in the host can be purified by methods such as affinity chromatography, in which polypeptide antigens or fragments thereof are immobilized on resins.

Epitopes contained in antigenic peptides are usually located on the surface of a protein (eg, a hydrophilic region), or in a region that is highly antigenic (eg, a region that is first selected for containing multiple charged residues). An Emini surface probabilistic analysis of human protein sequences can be used to indicate domains with a high probability of being localized on the protein surface.

Antibodies can be fully human antibodies (e.g., antibodies derived from human immunoglobulin sequences derived from human immunoglobulin genes (κ, λ, α (IgA ₁ and IgA ₂ ), γ (IgG ₁ , IgG ₂ , IgG ₃ , IgG ₄ ), δ, ξ, and μ constant region genes or various immunoglobulin variable region genes). In addition, antibodies also Can be a non-human antibody (eg, a rodent (eg, mouse or rat), goat, rabbit, or non-human primate (eg, monkey) antibody).

Human monoclonal antibodies are produced by transgenic mice carrying human rather than mouse immunoglobulin genes. Splenocytes obtained from these mice (after immunization with an antigen of interest) can be used to generate hybridomas capable of secreting human mAbs with specific affinity for epitopes of human proteins (see, e.g., WO 91/00906, WO 91/00906, WO 91/10741; WO92/03918; WO 92/03917; Lonberg et al., Nature 368:856-859, 1994; Green et al., Nature Genet. 7:13-21, 1994; Morrison et al., Proc.Natl.Acad .Sci.USA 81:6851-6855,1994; Bruggeman et al., Immunol.7:33-40,1993; Tuaillon et al., Proc.Natl.Acad.Sci.USA 90:3720-3724,1993; and Bruggeman et al. People, Eur. J. Immunol. 21:1323-1326, 1991).

The anti-TLR2 antibody or anti-Scd1 antibody can be an antibody whose variable regions or portions thereof (eg, CDRs) are produced by a non-human organism (eg, rat or mouse). Thus, the invention encompasses chimeric, CDR-grafted, humanized antibodies as well as antibodies produced by non-human organisms and subsequently modified (eg, in variable framework or constant regions) to reduce antigenicity in humans. Chimeric antibodies (ie, antibodies in which different portions are derived from different animal species (eg, variable regions of a murine mAb and constant regions of a human immunoglobulin)) can be prepared by recombinant techniques known in the art. For example, the gene encoding the mouse Fc constant region can be removed by restriction endonuclease digestion of the gene encoding _{the Fc} constant region of a murine (or other species) monoclonal antibody molecule, and then the equivalent of the gene encoding the human _Fc constant region Partially substituted (see, for example, European Patent Application Application Nos. 125,023; 184,187; 171,496 and 173,494; see also WO 86/01533; USPat. No. 4,816,567; Better et al., Science 240:1041-1043, 1988; Liu USA 84: 3439-3443, 1987; Liu et al., J. Immunol. 139: 3521-3526, 1987; Sun et al., Proc. Natl. : 214-218, 1987; Nishimura et al., Cancer Res.47: 999-1005, 1987; Wood et al., Nature 314: 446-449, 1985; Shaw et al., J. Natl. Cancer Inst. 80: 1553- 1559, 1988; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851, 1984; Neuberger et al., Nature 312:604, 1984; and Takeda et al., Nature 314:452, 1984).

In a humanized or CDR-grafted antibody, at least one or two, and usually all three, acceptor CDRs (of the immunoglobulin heavy or light chain) will be replaced by donor CDRs (see, for example, U.S. Pat. No. 5, 225, 539; Jones et al., Nature 321:552-525, 1986; Verhoeyan et al., Science 239:1534, 1988; and Beidler et al., J. Immunol. 141:4053-4060, 1988). One need only replace the number of CDRs required to bind the humanized antibody to toll-like receptor 2, the Scd1 gene or the Scd1 gene product. The donor can be a rodent antibody and the acceptor can be a human framework or human consensus framework. Usually the immunoglobulin that provides the CDRs is called the "donor" (and is often of rodent origin), while the immunoglobulin that provides the framework is called the "acceptor". The acceptor framework can be a naturally occurring (eg, human) framework, a consensus framework or sequence, or a sequence at least 85% (eg, 90%, 95%, 99%) identical thereto. A "consensus sequence" is a sequence consisting of the most frequently occurring amino acids (or nucleotides) in a family of related sequences (see, for example, Winnaker, From Genes to Clones, Verlagsgesellschaft, Weinheim, Germany, 1987). Each position in the consensus sequence is occupied by the most frequently occurring amino acid residue at the same position in the family (when two amino acid residues occur with the same frequency, either can be included). "Consensus framework" refers to the framework regions in the consensus immunoglobulin sequences. Humanized antibodies against toll-like receptor 2, the Scd1 gene or the Scd1 gene product can be prepared with specific amino acid residues substituted, removed or added (eg, in the framework regions to increase antigen binding). For example, a humanized antibody will have framework residues that are identical to those of the donor or amino acids of the acceptor but not the acceptor framework residues. To generate such antibodies, a small number of acceptor framework residues of selected humanized immunoglobulin chains are replaced by the corresponding donor amino acids. This substitution can occur near a CDR or in a region that interacts with a CDR (U.S. Pat. No. 5,585,089, see especially lines 12-16). Other techniques for humanizing antibodies are described in EP 519596A1.

Antibodies to the toll-like receptor 2 or Scd1 gene product can be humanized as described above or other methods known in the art. For example, _Fv variable regions not directly involved in antigen binding can be replaced with equivalent sequences of human _Fv variable regions to generate humanized antibodies. Conventional methods for generating humanized antibodies are described in Morrison, Science 229:1202-1207, 1985; Oi et al., BioTechniques 4:214, 1986, and Queen et al. (US Pat. Nos. 5,585,089; 5,693,761 and 5,693,762). The desired nucleic acid sequence can be obtained from a hybridoma that can produce TLR2 or Scd1 or TLR2 or Scd1 with desired properties (such as the ability to detect Scd1 gene expression or the regulation, activation or inhibition of Scd1 protein activity by MALP-2-activated macrophages). Antibodies to its fragments. Recombinant DNA encoding humanized antibodies or fragments thereof can then be cloned into suitable expression vectors.

In certain embodiments, the antibody has effector function and is capable of fixing complement, while in other embodiments it neither recruits effector cells nor fixes complement. The antibody may also bind weakly to _Fc receptors or not bind at all. For example, it may be an isotype or subtype that does not bind the _Fc receptor, or a fragment or other mutant (eg, the antibody may have a mutation (eg, deletion) in the _Fc receptor binding region). Antibodies lacking the _Fc region are generally unable to fix complement and are therefore less likely to cause cell death to which they bind.

In other embodiments, the antibody can be conjugated to an exogenous substance, such as a therapeutic agent (eg, antibiotic), or a detectable label. Detectable labels can include enzymes (such as horseradish peroxidase, alkaline phosphatase, beta-galactosidase or acetylcholinesterase), prosthetic groups (such as streptavidin-biotin and avidin-biotin fluorescein), or fluorescent, luminescent, bioluminescent or radioactive materials (such as umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazine sesamine fluorescein, dandelion Sulfonyl chloride or phycoerythrin (non-fluorescent), luminol (luminescent), luciferase, luciferin and aequorin (bioluminescent), and ⁹⁹ mTc, ¹⁸⁸ Re, ¹¹¹ In, ¹²⁵ I , ¹³¹ I, ³⁵ S or ³ H (radioactive)).

Antibodies (e.g., monoclonal antibodies) described herein can also be used to isolate toll-like receptor 2 or Scd1 proteins or fragments thereof, as in relation to activation of macrophages by MALP-2 (e.g., by affinity chromatography or immunoprecipitation ) regulates, activates or inhibits Scd1 gene expression or Scd1 protein activity fragments, or can be present in cell lysates or supernatants (by Western blot, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays and other methods) or tissue detect it in slices. These methods support the determination of the abundance and pattern of expression of specific proteins. This information will be useful when making a diagnosis or when evaluating the efficacy of a clinical trial or treatment.

The present invention also includes nucleic acid encoding the above-mentioned antibody, as well as vectors and cells (for example, mammalian cells such as CHO cells or lymphocytes) comprising the nucleic acid (for example, to encode a protein that can specifically bind to toll-like receptor 2 or Scd1 protein) Antibody nucleic acid transformed cells). Likewise, the invention includes cell lines (eg, hybridomas) capable of producing antibodies of the invention and methods of producing these cell lines.

Immunological detection of SCD1 polypeptide or TOLL-like receptor 2 polypeptide and its modulators

In addition to detecting Scd1 gene or toll-like receptor 2 gene and gene expression by nucleic acid hybridization technology, people can also use immunoassay to detect Scd1 or toll-like receptor 2 protein. This assay is very useful for screening modulators of Scd1 or toll-like receptor 2, as well as for therapeutic and diagnostic applications. Immunoassays can be used for qualitative or quantitative analysis of Scd1 protein or toll-like receptor 2 protein. A review of suitable techniques can be found in Harlow & Lane, Antibodies: A Laboratory Manual, 1988.

A. Antibody Preparation

Preparation methods of polyclonal and monoclonal antibodies capable of specifically reacting with Scd1 protein or toll-like receptor 2 protein are known to those skilled in the art (for example, see Coligan, Current Protocols in Immunology, 1991; Harlow & Lane, supra; Goding, Monoclonal Antibodies: Principles and Practice, 2d ed.1986; and Kohler et al., Nature 256:495-497, 1975. This type of technology includes selecting antibodies from recombinant antibody libraries of phage or similar vectors for antibody preparation, and by Polyclonal and monoclonal antibodies are produced by immunizing rabbits or mice (see, eg, Huse et al., Science 246:1275-1281, 1989; Ward et al., Nature 341:544-546, 1989).

A series of immunogens comprising portions of Scd1 protein or toll-like receptor 2 protein can be used to prepare antibodies specifically reactive with Scd1 protein or toll-like receptor 2 protein. For example, recombinant Scd1 protein or toll-like receptor 2 protein or antigenic fragments thereof can be isolated as described herein. Recombinant proteins can be expressed in eukaryotic or prokaryotic cells as described above, and purified as generally described above. Recombinant proteins are the preferred immunogens for monoclonal or polyclonal antibody production. In addition, a synthetic peptide derived from the sequences disclosed herein conjugated to a carrier protein can be used as an immunogen. Native proteins, either in purified or impure form, may also be used. The product is then injected into animals capable of producing the antibody. Monoclonal or polyclonal antibodies are produced for use in immunoassays that detect proteins.

Methods for preparing polyclonal antibodies are well known to those skilled in the art. The protein is used to immunize inbred mice (such as BALB/C mice) or rabbits with standard adjuvants (such as Freund's adjuvant) and standard immunization schemes. The animal's immune response to the immunogen preparation can be monitored by taking a blood sample and determining the titer of the response to the beta subunit. When suitably high titers of antibodies to the immunogen are obtained, the animal's blood is collected and antiserum is prepared. Antisera can be further isolated to enrich for antibodies reactive with the protein if desired (see Harlow & Lane, supra).

Monoclonal antibodies can be obtained by various techniques well known to those skilled in the art. Briefly, spleen cells obtained from animals immunized with the desired antigen are generally immortalized by fusion with myeloma cells (see Kohler et al., Eur. J. Immunol. 6:511-519, 1976). Other methods of immortalization include transformation with Epstein Barr virus, oncogene or retroviruses, or other methods well known in the art. Clones selected from individual immortalized cells can produce antibodies with the desired specificity and affinity for the antigen, and the yield of monoclonal antibodies produced by the cells can be enhanced by a variety of techniques, including injection into vertebrate hosts of the abdominal cavity. Alternatively, one can screen DNA libraries of human B cells to isolate DNA sequences encoding monoclonal antibodies or binding fragments thereof by following the general protocol outlined in Huse et al., Science 246:1275-1281, 1989.

Monoclonal antibodies and polyclonal sera are collected and titrated against the immunogenic protein in an immunoassay (eg, a solid phase immunoassay with the immunogen immobilized on a solid support). Polyclonal antisera with a titer of ¹⁰⁴ or higher are usually selected and tested for cross-reactivity to non-Scd1 or toll-like receptor 2 proteins using a competitive binding immunoassay. Specific polyclonal antisera and monoclonal antibodies will generally bind with a Kd of at least about 0.1 mM, more usually at least about 1 μM, more preferably at least about 0.1 μM or more, most preferably 0.01 μM or more. Antibodies specific only for a particular Scd1 ortholog or toll-like receptor 2 ortholog (e.g., human Scd1 protein or human toll-like receptor 2) can also be obtained by subtracting from one species, such as a non-human mammal. Prepare from other cross-reactive orthologs. Antibodies that bind only Scd1 or toll-like receptor 2 can be obtained by this method.

Once specific antibodies against Scd1 protein or toll-like receptor 2 protein are obtained, the protein can be detected by various immunoassay methods. In addition, the antibody may be therapeutically useful as a modulator of the Scd1 gene product or toll-like receptor 2. See Basic and Clinical Immunology (Stites & Terreds., 7 ^th ed. 1991) for immunology and immunoassay procedures. Furthermore, the immunoassays of the present invention can be performed in any of a variety of configurations, which have been thoroughly reviewed, see Enzyme Immunoassay (Maggio, ed., 1980) and Harlow & Lane, supra.

B. Immunological Binding Assays

Scd1 protein or toll-like receptor 2 protein can be detected and/or quantified by any one of a series of widely accepted immunological binding assays (eg, see U.S. Patents 4,366,241; 4,376,110; 4,517,288 and 4,837,168). For a review of conventional immunoassays, see also Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993); Basic and Clinical Immunology (Stites & Terr, eds., 7th ed. 1991). Immunological binding assays (or immunoassays) generally employ an antibody that can specifically bind to a target protein or antigen (in the present invention, Scd 1 protein or toll-like receptor 2 protein or an antigenic subsequence thereof). The antibody (such as anti-Scd1 gene product or anti-toll-like receptor 2) can be prepared by any one of a series of methods well known to those skilled in the art as described above.

In immunoassays, a labeling reagent is often used to specifically bind and label the complex formed by the antibody and the antigen. The labeling reagent may itself comprise one of the parts of the antibody/antigen complex. Thus, the labeling reagent can be to label the Scd1 gene product or to label toll-like receptor 2. In addition, the labeling reagent can also be a third moiety, such as a second antibody capable of specifically binding the antibody/Scd1 gene product or antibody/toll-like receptor 2 complex (the second antibody is usually specific for the first antibody from which the first antibody can be derived). type of antibody). Other proteins capable of specifically binding to the constant regions of immunoglobulins, such as protein A or protein G, can also be used as labeling reagents. These proteins exhibit strong non-immunogenic reactivity to immunoglobulin constant regions from various classes (see, e.g., Kronval et al., J. Immunol. 111:1401-1406, 1973; Akerstrom et al., J. Immunol. 135:2589-2542, 1985). Labeling reagents can be modified with a detectable moiety, such as biotin, that can be specifically bound by molecules such as streptavidin. Various detectable moieties are well known to those skilled in the art.

Incubation and/or washing steps may be required after each reagent pool throughout the assay. The incubation step can last anywhere from 5 seconds to several hours, optionally from about 5 minutes to about 24 hours. However, the time of incubation will depend on the assay format, antigen, solution volume, concentration and other factors. Although the assay can be performed at a range of temperatures (eg, 10°C to 40°C), it is usually performed at room temperature.

Noncompetitive assay formats: Immunoassays that detect the Scd1 gene product or toll-like receptor 2 in a sample can be competitive or noncompetitive. A noncompetitive immunoassay is an assay that directly detects the amount of antigen. For example, if one chooses a "sandwich" assay, an anti-Scd1 gene product or an anti-toll-like receptor 2 antibody can be directly bound to a solid substrate for immobilization. These immobilized antibodies can capture the Scd1 gene product or toll-like receptor 2 in the test sample. The Scd1 protein or toll-like receptor 2 protein is thus immobilized and bound by a labeling reagent (eg, a secondary antibody to the Scd1 gene product carrying the label or an antibody to toll-like receptor 2). Alternatively, the second antibody may lack a label, but may be bound by a labeled third antibody specific for an antibody of the species from which the second antibody was derived. The second or third antibody will typically be modified with a detectable moiety, such as biotin, that can be specifically bound by a molecule such as streptavidin to provide the detectable moiety.

Competitive assay format: In a competitive assay, the amount of Scd1 protein or toll-like receptor 2 protein in the sample can be detected by detecting the unknown Scd1 protein or toll-like receptor protein in the sample by anti-Scd1 protein or anti-toll-like receptor 2 antibody. Known amount of added (exogenous) Scd1 protein or toll-like receptor 2 protein replaced (competed away) by body 2 protein. In a competition assay, a known amount of Scd1 protein or toll-like receptor 2 protein is added to a sample, which is subsequently bound to an antibody that specifically binds Scd1 protein or toll-like receptor 2 protein. The amount of exogenous Scd1 protein or toll-like receptor 2 protein bound to the antibody is inversely proportional to the concentration of Scd1 protein or toll-like receptor 2 protein in the sample. In a particularly preferred embodiment, the antibody is immobilized on a solid substrate. The amount of Scd1 protein or toll-like receptor 2 protein bound to the antibody can be obtained by measuring the amount of Scd1 gene product or toll-like receptor 2 in the Scd1 protein/antibody complex or toll-like receptor 2 protein/antibody complex, or It is obtained by detecting the amount of residual uncomplexed protein. The amount of Scd1 protein or toll-like receptor 2 protein can be determined by providing labeled Scd1 protein molecule or toll-like receptor 2 molecule.

Hapten inhibition assays are another preferred competitive assay. In this assay it is known that Scd1 protein or toll-like receptor 2 protein is immobilized on a solid substrate. A known amount of anti-Scd1 antibody or anti-toll-like receptor 2 antibody is added to the sample, and the sample is then contacted with immobilized Scd1 gene product or toll-like receptor 2. The amount of anti-Scd1 antibody or anti-toll-like receptor 2 antibody bound to a known immobilized Scd1 gene product or toll-like receptor 2 is inversely proportional to the amount of Scd1 protein or toll-like receptor 2 protein in the sample. In addition, the amount of immobilized antibody can be detected by detecting the immobilized portion of the antibody or the portion remaining in solution. This detection can be performed directly when the antibody is labeled, or indirectly by the subsequent addition of a labeling moiety that specifically binds to the antibody, as described above.

Cross-reactivity assays: Competitive binding format immunoassays can also be used for cross-reactivity assays. For example, Scd1 protein or toll-like receptor 2 protein can be immobilized on a solid support. Proteins capable of competing with the immobilized antigen for binding to the antiserum (eg, Scd1 gene product or toll-like receptor 2 and homologues) are added to the assay. The ability of the added protein to compete with the immobilized protein for antiserum binding was compared to the ability of the Scd1 protein or the toll-like receptor 2 protein to compete with itself. The percent cross-reactivity for the above proteins was calculated using standard algorithms. Antisera with less than 10% cross-reactivity to each of the added proteins listed above were selected and pooled. Cross-reactive antibodies are selectively removed from pooled antisera by immunoadsorption with added proteins of interest (eg, distant homologues).

The immunoadsorbed and pooled antisera were used in a competitive binding immunoassay as described above for a second protein thought to be likely an allelic or polymorphic variant of the Scd1 protein or the toll-like receptor 2 protein Compare with immunogenic protein. For this comparison, a wide concentration range was used for both proteins in the assay and the concentration required for each protein to inhibit 50% of the binding of the antiserum to the immobilized protein was determined. If the amount of the second protein required to inhibit 50% binding is less than 10 times the amount of Scd1 protein or toll-like receptor 2 protein required to inhibit 50% binding, the second protein is considered to be able to bind to the Scd1 gene product or Polyclonal antibodies to the toll-like receptor 2 immunogen specifically bind.

Other assay formats: Western blot (immunoblot) analysis can be used to detect and quantify the presence of Scd1 protein or toll-like receptor 2 protein in a sample. This technique usually involves separating sample proteins by gel electrophoresis according to molecular weight, transferring the separated proteins to a suitable solid support (such as nitrocellulose filter, nylon filter or derivatized nylon filter), and then mixing the sample with a protein that can specifically bind to Scd1 Protein or toll-like receptor 2 protein antibodies were incubated. The anti-Scd1 protein antibody or the anti-toll-like receptor 2 antibody is specifically bound to the Scd1 gene product or the toll-like receptor 2 on the solid support. These antibodies can be directly labeled, or a labeled antibody capable of specifically binding to an anti-Scd1 protein antibody or an anti-toll-like receptor 2 antibody (such as a labeled goat anti-mouse antibody) can be used for subsequent detection.

Other test formats include liposome immunoassays (LIAs), which employ liposomes designed to bind to specific molecules, such as antibodies, and release encapsulated reagents or labels. The released chemicals are then measured according to standard techniques (see Monroe et al., Amer. CHn. Prod. Rev. 5:34-41, 1986).

Reduction of Non-Specific Binding: Those skilled in the art will appreciate that in immunoassays there is often a need to minimize non-specific binding. Especially when assays involve antigens or antibodies immobilized on solid substrates, there is a need to minimize the amount of non-specific binding to the substrate. Methods of reducing this non-specific binding are well known to those skilled in the art. Typically the technique involves coating the substrate with a proteinaceous composition. Protein compositions such as bovine serum albumin (BSA), skim milk powder and gelatin are particularly widely used, with milk powder being particularly preferred.

Labels: The specific labels or detectable groups used in the assay do not form an essential aspect of the invention as long as they do not significantly interfere by specific binding to the antibodies used in the assay. A detectable group can be any material having detectable physical or chemical properties. Such detectable labels are well developed in the field of immunoassays, and in general most labels that can be used in this method are useful in the present invention. Thus, a label refers to any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electronic, optical or chemical means. Labels that can be used in the present invention include magnetic beads (such as DYNABEADS ^TM ), fluorescent dyes (such as fluorescein isothiocyanate, Texas red, rhodamine, etc.), radioactive labels (such as ³ H, ¹²⁵ I, ³⁵ S, ¹⁴ C or ³² P), enzymes (such as horseradish peroxidase, alkaline phosphatase, and other enzymes commonly used in ELISA), chemiluminescent labels, and colorimetric labels such as colloidal gold or colored glass or plastic beads (such as polystyrene , polypropylene, latex, etc.).

The label can be directly or indirectly coupled to the desired components in the assay according to methods well known in the art. As indicated above, a wide variety of labels are available, and the choice of label depends on the desired sensitivity, ease of conjugation to the compound, desired stability, available instrumentation, and handling regulations.

Non-radioactive labels are often attached by indirect methods. Usually the ligand molecule (such as biotin) is covalently bound to the molecule. The ligand is then bound to another molecule (such as a streptavidin molecule) that is either naturally detectable or covalently bound to a signaling system such as a detectable enzyme, fluorescent compound or Chemiluminescent compounds. The ligand and its target can be used in appropriate combination with an antibody that can recognize Scd1 protein or toll-like receptor 2 protein, or a second antibody that can recognize anti-Scd1 protein antibody or anti-toll-like receptor 2 antibody.

The molecule can also be directly conjugated to a signal generating compound, such as an enzyme or a fluorophore. The target enzymes used as labels are mainly hydrolases, especially phosphatases, esterases and glycosidases, or oxidases, especially peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin and 2,3-dihydrophthalazinediones such as luminol. For a review of various available label or signal generating systems see U.S. Patent No. 4,391,904.

Methods for detecting labels are well known to those skilled in the art. Thus, for example when the label is a radioactive label, methods of detection include scintillation counters or autoradiographic film. When the label is a fluorescent label, it can be detected by exciting the fluorochrome with light of the appropriate wavelength and measuring the resulting fluorescence. This fluorescence can be detected visually using electronic detectors such as charge-coupled devices (CCDs) or similar instruments such as photomultipliers. Similarly, enzyme labeling can be detected by providing an appropriate substrate for the enzyme and then detecting the resulting reaction product. Finally simple colorimetric markers can be detected simply by observing the color associated with the marker. Therefore, in various test paper assays, conjugated gold often appears pink, while various conjugated beads show the color of the beads.

Some assay formats do not require the use of labeled components. For example, agglutination assays can be used to detect the presence of antibodies of interest. In this assay, antigen-coated particles are agglutinated by a sample containing the antibody of interest. In this format, all components are label-free, and the presence of the antibody of interest can be detected by simple visual inspection.

High-throughput assay for modulators of the SCD1 gene product or TOLL-like receptor 2

Compounds to be tested as modulators of the Scd1 gene product or toll-like receptor 2 can be any small organic molecule, or biological entity such as a protein (e.g. antibody or peptide), sugar, nucleic acid (e.g. antisense oligonucleotide, RNAi ) or ribozymes, or lipids. In addition, the modulator may be the product of gene modification of Scd1 protein or toll-like receptor 2 protein. Typically, test compounds are small organic molecules, peptides, lipids or lipid analogs.

Essentially any compound can be used as a potential modulator or ligand in the assays of the invention, although compounds that are soluble in aqueous or organic (particularly DMSO-based) solutions will most likely be used. The assay can screen large chemical libraries by automating the assay step and assaying compounds from any source conveniently, often in parallel (eg, by microtiter format on microtiter plates in robotic assays). It should be appreciated that there are numerous suppliers of compounds including Sigma (St.Louis, MO), Aldrich (St.Louis, MO), Sigma-Aldrich (St.Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) etc.

In a preferred embodiment, the high throughput screening method involves providing a combinatorial small organic molecule or peptide library comprising a large number of potential therapeutic compounds (potential modulator or ligand compounds). This "combinatorial chemical library" or "ligand library" is then screened in one or more assays as described herein to identify library members (particular chemical species or subclasses) that exhibit the desired characteristic activity. The compounds thus identified may be used as traditional "lead compounds" or may themselves be used as potential or actual therapeutic agents.

A combinatorial chemical library is a collection of various compounds (whether obtained by chemical synthesis or biosynthesis) obtained by combining a certain amount of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library can be formed by combining a batch of compound building blocks (amino acids) at a given compound length (ie, the number of amino acids in a polypeptide compound) in every possible way. Thousands of compounds can be synthesized through the combination and mixing of such chemical building blocks.

Preparation and screening of combinatorial chemical libraries is well known to those skilled in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, for example, U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493, 1991 and Houghton et al., Nature 354:84-88, 1991 ). Other chemistries capable of generating chemically diverse libraries can be used. Such chemicals include, but are not limited to: peptoids (e.g. PCT Publication No. WO 91/19735), encoded peptides (e.g. PCT Publication No. WO 93/20242), random biooligomers (e.g. PCT Publication No. WO 92/00091 ), benzo(a)diazepines (such as U.S.Pat.No. 5,288,514), hydantoins, benzo(a)diazepines and dipeptides (diversomers) (Hobbs et al., Proc.Nat.Acad. Sci.USA 90:6909-6913,1993), vinyllogous polypeptides (Hagihara et al., J.Amer.Chem.Soc.114:6568,1992), non-peptidic peptide analogs with glucose scaffolds (Hirschmann et al., J.Amer.Chem.Soc.114:9217-9218,1992), organic synthetic analogs of small compound libraries (Chen et al., J.Amer.Chem.Soc.116:2661,1994), oligocarbamic acid ester (Cho et al., Science 261:1303, 1993), and/or peptidyl phosphate (Campbell et al., J.Org.Chem.59:658, 1994), nucleic acid library (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid library (see, for example, U.S. Patent 5,539,083), antibody library (see, for example, Vaughn et al., Nature Biotechnology, 14:309-314, 1996 and PCT/US96/10287), sugar library (see, for example, Liang et al., Science 274:1520-1522, 1996 and U.S. Patent 5,593,853), libraries of small organic molecules (see, for example, benzodiazepines Baum C&EN, Jan 18, page 33 (1993); isopentenes, U.S. Patent 5,569,588; thiazolinones and m-buprofezinones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholine compounds, U.S. Patent 5,506,337; benzodiazepines 5,288,514, etc.).

Instruments for preparing combinatorial libraries are commercially available (see, e.g., 357MPS, 390MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050Plus, Millipore, Bedford , MA). In addition, many combinatorial libraries themselves have entered the market (see, for example, ComGenex, Princeton, N.J., Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.)

Candidate compounds may be used as part of a strategy to identify drugs capable of treating disorders involving the induction of macrophages by MALP-2 through the toll-like receptor 2/Scd1 interaction pathway. Test compounds that bind to TLR2 or Scd1 are considered candidate compounds.

Also included in the invention are screening assays to identify candidate or test compounds that bind to TLR2 or Scdl, or that modulate the activity of a TLR2 or Scdl protein or polypeptide, or a biologically active portion thereof. The test compound can be acquired by any one of various combinatorial library methods known in the art, including but not limited to: biological library; space-addressable parallel solid-phase or liquid-phase library; synthetic library method requiring overlapping legality; " one-bead-one-compound” libraries; and synthetic libraries using affinity chromatography selection. The biobank method can be used for peptide libraries etc., while the other four methods are suitable for small chemical molecule libraries of peptides, non-peptide oligomers or compounds (Lam, Anticancer Drug Des. 12:145, 1997). Examples of small molecule library synthesis methods exist in the art, for example: De Witt et al., Proc. Natl. Acad. Sci. U.S.A. 90:6909, 1993; 11422, 1994; Zuckermann et al., J.Med.Chem.37:2678, 1994; Cho et al., Science 261:1303, 1993; Carrell et al., Angew.Chem.Int.Ed.Engl.33:2059, 1994; Carrell et al., Angew. Chem. Int. Ed. Engl. 33:2061, 1994; and Gallop et al., J. Med. Chem. In some embodiments, the test compound is an activating variant of TLR2 or Scd1.

Compound libraries can exist in solution (e.g. Houghten, Bio/Techniques 13:412-421, 1992), or on beads (Lam, Nature 354:82-84, 1991), on a chip (Fodor, Nature 364:555 -556, 1993), in bacteria (U.S.Pat.No.5,223,409), in spores (U.S.Pat.Nos.5,571,698, 5,403,484, and 5,223,409), in plasmids (Cull et al., Proc.Natl.Acad.Sci.USA 89 : 1865-1869, 1992) or on phage (Scott et al., Science 249: 386-390, 1990; Devlin, Science 249: 404-406, 1990; Cwirla et al., Proc.NatlAcad.Sci.USA.S7: 6378-6382, 1990; and Felici, J. Mol Biol. 222:301-310, 1991).

The ability of a test compound to modulate TLR2 or Scd1 or a biologically active portion thereof can be determined, for example, by monitoring the ability to form a TLR2/Scd1 complex in the presence of the test compound. Modulation of the activity of TLR2 or Scd1 or biologically active parts thereof can be determined by measuring the induction of macrophages by MALP-2 through a pathway involving toll-like receptor 2/Scd1 interaction. The ability of a test compound to modulate the activity of toll-like receptor 2 or Scd1 or a biologically active portion thereof can also be detected by monitoring the ability of toll-like receptor 2 to bind Scd1. The binding assay can be cell-based or performed in a cell-free state.

The ability of a toll-like receptor 2 protein to bind or interact with Scdl can be determined by one of the methods for measuring direct binding described herein or known in the art. In one embodiment, the ability of the toll-like receptor 2 protein to bind or interact with Scd1 can be detected by monitoring the induction of MALP-2 on macrophages. Detection of macrophage induction by MALP-2 may include detection of expression of recombinant Scd1 that simultaneously encodes a FLAG sequence or a detectable marker such as luciferase. This assay can be added to the direct binding assay. In general, the assay is used to detect the ability of a test compound to affect toll-like receptor 2 protein binding to Scdl or toll-like receptor 2 to activate Scdl protein or gene expression.

Overall, the ability of test compounds to bind Scd1, interfere with signal transduction through toll-like receptor 2, or affect MALP-2 induction of macrophages was compared to controls in which binding or MALP-2 Induction of macrophages was all measured in the absence of test compounds. In some cases predetermined reference values are used. The reference value can be determined relative to a control, wherein a test sample different from the reference will be responsive to the compound binding to the target molecule (e.g., toll-like receptor 2) or modulating expression (e.g., modulating, activating or inhibiting the induction of MALP-2 in cells). macrophages, or regulation, activation or inhibition of macrophages in response to Gram-positive bacterial infection) are indicated. When looking at a criterion (e.g., antibody affinity for toll-like receptor 2, or modulation of MALP-2-induced Scd 1 expression), reference values can also reflect binding or MALP-2-induced macrophage activity. quantity. In this case, a test compound that is similar to (eg, equal to or less than) the reference would indicate that the compound is a candidate compound (eg, binds toll-like receptor 2 to an equal or greater extent than the reference antibody).

The invention further relates to novel agents identified by the screening assays described above and their use in the treatments described herein.

In one embodiment the invention provides a solubility assay using native or recombinant Scd1 gene product or toll-like receptor 2 protein, or cells or tissues expressing Scd1 gene product or toll-like receptor 2 protein. In another embodiment, the present invention provides a high-throughput format of a solid phase-based in vitro assay wherein the Scd1 gene product or toll-like receptor 2 protein or its ligand is attached to the solid phase either covalently or non-covalently. substrate. Any of the assays described herein can be used for high throughput screening.

In the high throughput assays of the present invention, whether soluble or solid phase assays, it is possible to screen thousands of different modulators or ligands in one day. The method can be used for the Scd1 gene product or toll-like receptor 2 protein in vitro, or for cell-based or membrane-based assays comprising the Scd1 gene product or toll-like receptor 2 protein. In particular, each well of a microtiter plate can be used for an independent assay of selected potential modulators, or every 5-10 wells can be tested for an individual modulator when observing the effect of concentration or incubation time . Thus, about 100 (eg 96) modulators can be assayed in a single standard microtiter plate. If a 1536-well plate is used, about 100 to about 1500 different compounds can easily be assayed on a single plate. Multiple plate assays can be performed in one day; approximately 6,000, 20,000, 50,000 or more than 100,000 assay screens per day of different compounds can be performed using the integrated system of the present invention.

In a solid phase reaction, the target protein or its fragment (e.g. extracellular region), or cells or membranes containing the target protein or its fragment as part of the fusion protein can be directly or indirectly linked covalently or non-covalently (e.g. , via a label) bound to the solid-phase component. The tag can be any kind of component. Generally speaking, a molecule that can be combined with a tag (tag-binding agent) is first immobilized on a solid-phase support, and then a tagged target molecule is attached to the solid-phase support through the interaction between the tag and the tag-binding agent.

A variety of tags and tag-binding agents are available, based on detailed descriptions of known intermolecular reactions in the literature. For example, when a tag has a natural binding agent (such as biotin, protein A or protein G), it can be used with the appropriate tag binding agent (avidin, streptavidin, neutralizing affinity Proteins, Fc regions of immunoglobulins, etc.) for binding. Antibodies to molecules with natural binders such as biotin also exist widely and are suitable tag binders (see SIGMA Immunochemicals 1998 catalog SIGMA, St. Louis MO).

Similarly, any hapten or antigenic compound can be used for conjugation with an appropriate antibody to form a tag/tag-binding agent pair. There are thousands of specific antibodies already on the market and many others described in the literature. For example, in one conventional configuration, the tag is a primary antibody and the tag-binding agent is a secondary antibody that recognizes the primary antibody. In addition to antibody-antigen responses, receptor-ligand responses are also available as appropriate tag and tag-binding agent pairs. For example, agonists and antagonists of cell membrane receptors (e.g., cell receptor-ligand responses such as toll-like receptors, transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors body, interleukin receptor, immunoglobulin receptor and antibody, cadherin family, integrin family, selectin family, etc.; see, for example, Pigott & Power, The Adhesion Molecule Facts Book I, 1993. Similarly, toxins and venoms , viral epitopes, hormones (such as opioids, steroids, etc.), intracellular receptors (such as receptors that mediate the action of various small ligands, including steroids, thyroid hormones, retinoic acid, and vitamin D; peptides), Drugs, lectins, sugars, nucleic acids (linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids, and antibodies all interact with a variety of cellular receptors.)

Synthetic polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides and polyacetates are capable of forming suitable labels and Label binder. As will be appreciated by those skilled in the art upon reading this disclosure, many other tag/tag-binding agent pairs can also be used in the assay systems described herein.

Common linkers such as peptides, polyethers, etc. can also be used as tags, which also include polypeptide sequences, such as polyglycine sequences between about 5 and 200 amino acids. Such flexible joints are known to those skilled in the art. For example, polyethylene glycol linkers are available from Shearwater Polymers, Inc. Huntsville, Alabama. These linkers can optionally have amide linkages, sulfhydryl linkages and heterofunctional linkages.

Tag-binding agents can be immobilized to a solid substrate by any of a variety of known methods. Solid phase substrates can typically be derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent that immobilizes to the surface a chemical group capable of reacting with a portion of the tag-binding agent. For example, groups suitable for attachment of longer chain moieties include amine, hydroxyl, thiol and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid-phase biopolymer arrays has been described in detail in the literature. See, for example, Merrifield, J.Am.Chem.Soc.85:2149-2154, 1963 (describing solid-phase synthesis of peptides, etc.); Geysen et al., J.Immun.Meth.102:259-274, 1987 (described in Solid-phase component synthesis on cellulose plates); Frank & Doring, Tetrahedron 44:6031-6040, 1988 (describing the synthesis of various peptide sequences on cellulose plates); Fodor et al., Science 251:767-777, 1991; Sheldon et al., Clinical Chemistry 39:718-719, 1993; and Kozal et al., Nature Medicine 2:753-759, 1996 (both describe arrays of biopolymers immobilized to solid substrates). Non-chemical methods of immobilizing tag-binding agents to substrates include other commonly used methods such as heating, cross-linking by UV radiation, and the like.

Bispecific compounds as modulators of SCD1 and TOLL-like receptor 2

In one aspect, the invention provides a method of identifying a candidate or testing a bispecific compound by which the serum and/or circulating concentration of a substance in a non-human animal can be reduced. Compounds selected or optimized using this rapid method can be used to treat subjects, such as human subjects, who would benefit from administration of such compounds.

Candidate compounds that can be tested in embodiments of this method of the invention are bispecific compounds. As used herein, the term "bispecific compound" includes compounds that have two different binding specificities. Exemplary bispecific compounds include bispecific antibodies, heteropolymers, and antigen-based heteropolymers.

Bispecific molecules that can be tested in embodiments of the invention preferably comprise a binding moiety specific for Scd1, preferably human Scd1, cross-linked to a target substance (e.g. a unique antibody or antigen) the second binding part. Examples of binding moieties specific for toll-like receptor 2 include, but are not limited to, toll-like receptor 2 ligands, such as MALP-2 or in more preferred embodiments, antibodies against toll-like receptor 2.

In another example, novel toll-like receptor 2 binding molecules can be identified based on their ability to bind toll-like receptor 2. For example, libraries of compounds or small chemical molecules can be tested by cell-free binding assays. Any number of test compounds, such as peptide analogs, small chemical molecules or other drugs, can be tested and can be obtained by any of a number of combinatorial library methods known in the art, including: biobanking; spatial Addressable parallel solid-phase or liquid-phase libraries; synthetic libraries requiring overlap legality; "one-bead-one-compound" libraries; and synthetic libraries using affinity chromatography selection. Biolibrary methods are limited to peptide libraries, while the other four methods are applicable to small chemical molecule libraries of peptides, non-peptide oligomers, or compounds (Lam, Anticancer Drug Des. 12:145, 1997).

High-throughput assays are required in many drug screening programs testing libraries of modulators and natural extracts to maximize the number of modulators examined within a given time frame. Assays performed in cell-free systems (e.g., by purified or semi-purified proteins) are often chosen as "primary" screens that allow rapid development and relatively easy detection of modification of target molecules mediated by test modulators . In addition, the effects of test modulators on cytotoxicity and/or bioavailability in in vitro systems can often be ignored, allowing assays to focus primarily on the effect of the drug on the molecular target, such as upstream or downstream elements on binding affinity. Remodel as shown.

In another example, phage display techniques known in the art can be used to identify novel TLR2 or Scd1 binding molecules.

In one embodiment, the invention provides assays for screening candidate or test compounds capable of binding TLR2 or Scd1 or biologically active portions thereof.

Cell-based assays that can identify molecules capable of binding TLR2 or Scdl can be used to identify other agents useful in the bispecific compounds of the invention. For example, cells expressing TLR2 or Scd1 can be employed in screening assays. For example, compounds capable of producing a statistically significant difference in binding of TLR2 or Scd1 can be identified.

In one embodiment, the assay is a cell-free assay wherein the toll-like receptor 2 binding molecule is identified based on its ability to bind TLR2 or Scd1 protein in vitro. The TLR2 or Scd1 protein binding molecule can be provided, and the ability of the protein to bind to the TLR2 or Scd1 protein can be tested using art-recognized methods for assaying direct binding. The detection of the binding ability of the protein to the target molecule can be realized by techniques such as real-time biomolecular interaction analysis (BIA). Sjolander et al., Anal. Chem. 63: 2338-2345, 1991, and Szabo et al., Curr. Opin. Struct. Biol. 5: 699-705, 1995. "BIA" as described here is a technique (such as BIAcore) for studying biologically specific interactions in real time without labeling any interactants. Changes in optical phenomena in surface plasmon resonance (SPR) can be used as indicators of real-time reactions between biomolecules.

X-100，

X-114，

，异十三烷基聚乙二醇醚，3-[3-(胆酰氨丙基)二甲铵基]丙磺酸内盐(CHAPS)，3-[3-(胆酰氨丙基)二甲铵基]-2羟基-丙磺酸内盐(CHAPSO)或N-十二烷基＝N，N-二甲基-3-铵基-丙磺酸内盐。Cell-free assays in the present invention may be adapted to use soluble and/or membrane-bound proteins. In the case of cell-free assays using membrane-bound proteins, co-solvents are required to keep the membrane-bound proteins in solution. Examples of such co-solvents include non-ionic detergents such as n-octyl glucoside, n-dodecyl glucoside, octanoyl-N-methylglucamine, capryl-N-methylglucosamine,

X-100,

X-114,

, Isotridecyl polyethylene glycol ether, 3-[3-(cholamidopropyl)dimethylammonio]propanesulfonic acid inner salt (CHAPS), 3-[3-(cholamidopropyl) Dimethylammonio]-2hydroxy-propanesulfonic acid inner salt (CHAPSO) or N-dodecyl=N,N-dimethyl-3-ammonio-propanesulfonic acid inner salt.

Suitable assays that detect protein-protein interactions are known in the art (eg, immunoprecipitation, two-hybrid assays, etc.). This test, performed in the presence or absence of a test compound, can be used to identify compounds capable of modulating (eg, inhibiting or enhancing) the interaction of a protein of the invention with a molecule of interest.

The detection of the ability of the protein to bind or interact with the target molecule can be achieved by methods such as direct binding. In direct binding assays, the protein is coupled to a radioactive isotope or enzymatic label so that binding of the protein to the target molecule can be detected by detecting the labeled protein in the complex. For example, proteins can be directly or indirectly labeled with ¹²⁵ 1, ³⁵ S, ¹⁴ C or ³ H, and the radioisotope can be detected by direct radiation counting or scintillation counting. Alternatively, the molecule can be labeled with an enzymatic label, eg, horseradish peroxidase, alkaline phosphatase, or luciferase, which can then be detected by measuring the conversion of the appropriate substrate to product.

Often it is desirable to immobilize the protein of the invention or its binding protein to facilitate the separation of the complex from the uncomplexed form of one or both proteins and to accommodate automated assays. Binding to upstream or downstream binding elements, in the presence or absence of a candidate reagent, can be accomplished in any vessel suitable for containing the reactants. Examples include microtiter plates, test tubes, and microcentrifuge tubes. In one embodiment a fusion protein is provided that is supplemented with a region that allows the protein to bind to a matrix. For example, glutathione s-transferase/TLR2 (GST/TLR2) fusion proteins can be adsorbed to glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione microtiter plates, One of the latter is then combined with cell lysates (e.g. ³⁵ S markers) and test modulators, and the mixture is incubated under conditions favorable for complex formation (e.g. at physiological salinity and pH, harsher conditions may also be used). conditions of). After incubation, the beads are washed to remove unbound label, the matrix is immobilized and the radioactive label is detected directly (eg, by placing the beads in scintillant), or in the supernatant after subsequent dissociation of the complexes. Alternatively, the complexes can be dissociated from the matrix and separated by SDS-PAGE, and the levels of TLR2-bound protein in the bead fraction determined from the gel using standard electrophoretic techniques.

Other techniques for immobilizing proteins on substrates can also be used for this subject assay. For example, biotinylated molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, Hi.), and then immobilized on chains. Wells of mycoavidin-coated 96-well plates (Pierce Chemical).

It is also within the scope of the present invention to determine the ability of a compound to modulate the interaction between TLR2 and Scd1 without labeling either interactor. For example, a microphysiometer can be used to detect the interaction between the protein of the present invention or the target molecule without labeling the two. McConnell et al., Science 257:1906-1912, 1992. As described therein, a "microphysiometer" (e.g., a cell sensor) is a system that uses light-addressable potentiometric sensors (LAPS) to detect the rate at which a cell acidifies its environment. analytical instruments. Changes in the acidification rate can be an indicator of the interaction between the compound and the receptor.

Antigen-based heteropolymers that can be tested in the present invention preferably comprise a binding moiety specific for TLR2 or Scd1, preferably human TLR2 or Scd1, cross-linked to an antigen recognized by autoantibodies. Examples of antigens that can be recognized by autoantibodies include, but are not limited to, any of the following: Factor VIII (antibodies related to the treatment of hemophilia by replacing recombinant Factor VIII); muscle acetylcholine receptors (associated with the disease myasthenia gravis Antibodies against Sjogren's syndrome); Diphosphatidylglycerol (associated with the disease lupus); platelet-associated protein (associated with the disease idiopathic thrombocytopenic purpura); complex antigens associated with Sjögren's syndrome; antigens involved in the autoimmune response to tissue transplantation; myocardial antigens found in the disease autoimmune myocarditis; antigens associated with immune complex-mediated nephropathy; dsDNA and ssDNA antigens (associated with lupus nephritis); herpes and pemphigoid); or any other well-characterized antigen associated with disease pathogenesis.

Exemplary heteropolymers tested in the present invention as well as antigen-based heteropolymers and methods for their preparation are known in the art. For example, exemplary heteropolymers can be referred to WO0300797Ial; U.S.20020103343A1; U.S.Pat.No.5,879,679; U.S.Pat.No.5,487,890; U.S.Pat.No.5,470,570; 0180883A1, WO/0145669A1, WO 9205801A1, Lindorfer et al., J.Immunol.Methods.248:125, 2001; Hahn et al., J.Immnol.166:1057, 2001; Nardin et al., J.Immunol.Methods.211 : 21, 1998; Kuhn et al., J. Immunol. 160: 5088, 1998; Taylor et al., Cancer Immunol Immunother. 45: 152, 1997; Taylor et al., J. Immunol. 159: 4035, 1997; and Taylor et al. People, J. Immunol. 148:2462, 1992. In addition, variants of these heteropolymers can also be prepared. For example, bispecific molecular formats prepared with different linker chemistries may be used in one embodiment. Exemplary reagents that can be used to cross-link bispecific molecular components include polyethylene glycol, SATA, SMCC, and others known in the art and available from Pierce Biotechnology, among others. Exemplary formats of bispecific molecules that can be tested can be found in U.S. Ser. No. 60/411,731, filed September 16, 2006, the contents of which are incorporated herein by reference.

In another example, different multimeric forms of bispecific molecules can be prepared (eg, dimers, trimers, tetramers, pentamers, or higher multimeric forms). In another example, purified forms of bispecific molecules can be tested, as seen, for example, in U.S. Ser. No. 60/380,211, filed May 13, 2002, the contents of which are incorporated herein by reference.

In another example, when one of the binding moieties of the heteropolymer is an antibody, the various isotypes of the antibody (e.g., IgA, IgD, IgE, IgGl, _IgG2 (e.g., _IgG2a ), _IgG3 , IgG ₄ , or IgM). In another example, a portion of an antibody molecule (eg, a Fab fragment) can be used as one of the binding moieties. In a preferred embodiment at least one binding moiety is an antibody comprising an Fc domain. In one embodiment, the antibody is a mouse antibody.

In another example, the effect of antibody modification can be tested, for example, the antibody can be tested for deimmunization (eg, U.S. Ser. No. 60/458,869 filed March 28, 2003).

In the methods provided by the present invention, substances (such as pathogens) in the serum, circulation and/or tissues of non-human animals can be reduced by at least about 20%, about 30%, about 40%, about 50%, about 60% , about 70%, about 80%, about 90%, or about 100%.

In another embodiment, the concentration of the substance in the serum, circulation and/or tissues of the subject can be detected indirectly. For example, pathology due to the presence of the agent in serum and/or circulation can be tested, eg, on a tissue sample from an animal. Another indirect measure of the concentration of the substance in the serum, circulation and/or tissue of the non-human animal is the ability of the agent to cause an infection in the non-human animal. For example, the effect of the bispecific compound on clinical manifestations and symptoms of infection can be tested. The effect of the bispecific compound on inhibiting the spread of infection (eg, from one organ system to another, or from one individual to another) can also be tested.

In another example, bispecific compounds are tested for their ability to bind TLR2 or Scd1 bearing cells in non-human animals. For example, in one embodiment, detection of the binding ability of the bispecific compound to TLR2 or Scd1 target molecules can be achieved by techniques such as real-time biomolecular interaction analysis (BIA) (Sjolander et al., Anal. Chem. 63: 2338 -2345, 1991 and Szabo et al., Curr. Opin. Struct. Biol. 5:699-705, 1995). "BIA" as described here is a technique (such as BIAcore) for studying biologically specific interactions in real time without labeling any interactants. Changes in optical phenomena in surface plasmon resonance (SPR) can be used as indicators of real-time reactions between biomolecules.

In another example, destruction of the formulation by cells in a non-human animal (eg, inactivation by macrophages) is tested.

Compounds can be selected for the ability to reduce the serum, and/or circulating concentration of the agent in the non-human animal as compared to the concentration observed in the non-human animal not receiving the bispecific compound.

The compounds tested in the subject assays can be selected from a wide variety of compounds tested. In another example, a bispecific compound tested in a rapid assay may have already been identified as capable of binding TLR2 or Scd1 (eg, in an in vitro assay) and may be further evaluated or optimized by the rapid assay. At this point, the ability of a bispecific compound to reduce the concentration of a substance in serum and/or circulation can be compared to another bispecific compound or a non-optimized version of the same compound to determine its effect in serum and/or circulation The ability to reduce the concentration of the formulation.

In a preferred embodiment, the bispecific compound of the invention may be administered in the range of about 1 μg compound/kg body weight to about 100 μg compound/kg body weight. As defined herein, the therapeutically effective amount (i.e. effective dose) of the bispecific compound is in the range of about 0.01 to 5000 μg/kg body weight, preferably about 0.1 to 500 μg/kg body weight, more preferably about 2 to 80 μg/kg body weight, more preferably about 5 to 70 μg/kg body weight, 10 to 60 μg/kg body weight, 20 to 50 μg/kg body weight, 24 to 41 μg/kg body weight, 25 to 40 μg/kg body weight, 26 to 39 μg/kg body weight, 27 to 38 μg/kg body weight, 28 to 37 μg/kg body weight, 29 to 36 μg/kg body weight, 30 to 35 μg/kg body weight, 31 to 34 μg/kg body weight, or 32 to 33 μg/kg body weight. The skilled artisan will appreciate that certain factors can affect dosages required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the subject's general health and/or age and the presence of other diseases. In addition, the treatment of a subject with a therapeutically effective amount of a protein, polypeptide or antibody may include a single treatment, or preferably multiple treatments.

In a preferred embodiment, the animal is treated with the bispecific compound in the range of about 1 to 500 [mu]g/kg body weight following intravenous (iv) injection of a substance. It is understood that the effective dose of the bispecific compound used in therapy may be raised or lowered following a particular treatment. Modifications in dosage may be evident as a result of diagnostic assays as described herein.

The route of administration of test compounds and/or substances can be chosen from intravenous (iv) injection into the animal's circulation. Other routes of administration include, but are not limited to, topical, parenteral, subcutaneous or inhalation administration. The term "parenteral" includes injection by subcutaneous, intravenous or intramuscular routes, as well as topical administration, eg, at the site of disease or injury. Methods for sustained release of compounds from implants are also known in the art. Those skilled in the relevant art will recognize that appropriate dosages will vary depending on factors such as the nature of the disorder being treated, the weight, age and general condition of the patient and the route of administration. The preliminary dose can be determined with reference to animal experiments, and the adjustment of the human dose can be performed with reference to practices accepted in the art.

Candidate compounds and substances can be administered to animals at a range of dosages. When the formulation is also administered to the animal, the administration of the candidate compound can be before, concurrently with or after administration of the formulation.

Transgenic animals (such as mice) expressing TLR2 or Scd1 in the present invention can be used for screening or evaluating the ability to deal with human disorders or diseases related to the appearance of harmful substances (such as autoantibodies, infectious pathogens or toxins) in the subject's serum and/or circulation candidate compounds.

Exemplary target substances capable of being bound by the bispecific compounds of the invention include blood-derived substances including, but not limited to, any of the following: viruses, viral particles, toxins, bacteria, polynucleotides, antibodies (such as with autoantibodies associated with autoimmune disorders). In one embodiment, exemplary target viral agents include, but are not limited to, any of the following: cytomegalovirus, influenza, Newcastle disease virus, vesicular stomatitis virus, rabies virus, herpes simplex virus, hepatitis, adenovirus-2, Bovine viral diarrhea virus, human immunodeficiency virus (HIV), dengue virus, Marburg virus, Epstein-Barr virus.

Exemplary Gram-positive bacterial targets include S. pyogenes infection, S. aureus, M. tuberculosis, S. pneumoniae or Bacillus subtilis. Any of the above methods and compositions can be used for the treatment of skin infection, community acquired pneumonia, upper and lower respiratory tract infection, skin and soft tissue infection, hospital acquired lung infection, bone and joint infection, respiratory tract infection, acute bacterial otitis media , bacterial pneumonia, urinary tract infection, concurrent infection, uncomplicated infection, pyelonephritis, intra-abdominal infection, deep abscess, bacterial sepsis, central nervous system infection, bacteremia, wound infection, peritonitis, meningitis, burns Posterior infections, genitourinary tract infections, gastrointestinal infections, pelvic inflammatory disease, endocarditis, and other intravascular infections. These pending infections may be caused by Gram-positive bacteria. These infection-causing bacteria include, but are not limited to, Staphylococcus aureus, Staphylococcus epidermidis, Enterococcus faecalis, Enterococcus faecium, Clostridium, Clostridium difficile, Streptococcus pyogenes, Streptococcus pneumoniae, other Streptococcus species, and other Clostridia. More specifically, the infection may be caused by a gram-positive coccus, or by a drug-resistant gram-positive coccus. Exemplary Gram-positive cocci include, but are not limited to, Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, Streptococcus pyogenes, Moraxella catarrhalis, Clostridium difficile, Helicobacter pylori, Chlamydia, and Enterococcus .

Bacteremia can be caused by Gram-negative or Gram-positive bacteria. Gram-negative bacteria have a thin-walled cell membrane consisting of a peptidoglycan monolayer and an outer layer of lipopolysaccharides, lipoproteins, and phospholipids. Exemplary Gram-negative bacteria include, but are not limited to, those consisting of Escherichia, Shigella, Edwardsiella, Salmonella, Citrobacter, Klebsiella, Enterobacter, Harbin Funiella, Serratia, Proteus, Morganella, Providencia, Yersinia, Erwinia, Buttlauxella, Sietia, Love The Enterobacteriaceae family consists of the genera Venella, Kluverella, Tatumella, and Lahnella. Other exemplary Gram-negative bacteria that do not belong to the Enterobacteriaceae family include, but are not limited to, Pseudomonas aeruginosa, Stenotrophomonas maltophilia, Burkholderia cepacia, Gardnerella vaginalis, and Acinetobacter. Gram-positive bacteria have a thick cell membrane composed of multiple layers of peptidoglycan and a teichoic (acid) outer layer. Exemplary Gram-positive bacteria include, but are not limited to, Staphylococcus aureus, coagulase-negative Staphylococcus, Streptococcus, Enterococcus, Corynebacterium, and Bacillus.

In one embodiment, the substance of interest is resistant to conventional therapy, eg resistant to antibiotics.

In one embodiment, the formulation may be administered to the transgenic animal prior to, at the time of, or after administration of the bispecific compound during the practice of an assay of the invention.

Bispecific compounds of the invention, or any portion thereof, may be modified to increase their half-life. Peptide analogs are often used in the pharmaceutical industry as non-peptide drugs with similar properties to the template peptide. These non-peptidic compounds are called "peptide analogs" (Fauchere, Adv. Drug Res. 15:29, 1986; Veber et al., TINS p.392, 1985; and Evans et al., J. Med. Chem 30: 1229 , 1987, both incorporated herein by reference) and are often assisted in their development by computer-aided molecular modeling. Peptide analogs that are structurally similar to therapeutic peptides are capable of producing comparable therapeutic or prophylactic effects. In general, a peptide analog is structurally similar to an exemplary polypeptide (i.e., one having biological or pharmacological activity), such as an antigenic polypeptide, but wherein one or more peptide bonds may optionally be selected from: _-CH2NH -, -CH ₂ S-, -CH ₂ -CH ₂ -, -CH=CH- (cis and trans), -COCH ₂ -, -CH(OH)CH ₂ -, and -CH ₂ SO- Bond substitution by methods known in the art and further described in the following references: Spatola, AFinChemistry and Biochemistry of Amino Acids, Peptides, and Proteins Weinstein, B., ed., Marcel Dekker, New York, p .267, 1983; Spatola, AF, Vega Data, Vol.1, Issue 3, "Peptide Backbone Modifications,"1983; Morley, Trends.Pharm.Sci.pp.463-468, 1980; Hudson et al., Int.J .Pept.Prot.Res.14:177-185, 1979 (-CH ₂ NH-, CH ₂ CH ₂ -); Spatola et al., Life.Sci.38:1243-1249, 1986 (-CH ₂ -S) ; Hann, J.Chem.Soc.Perkin.Trans.1:307-314, 1982 (--CH-CH-, cis and trans); Almquist et al., J.Med.Chem.23:1392-1398 , 1980 (-COCH ₂ -); People such as Jennings-White, Tetrahedron Lett.23: 2533, 1982 (-COCH ₂ -); People such as Szelke, European Patent Application No.EP 45665CA: 97: 39405, 1982 (-CH ( OH)CH ₂ -); Holladay et al., Tetrahedron. Lett. 24:4401-4404, 1983 (-C(OH)CH ₂ -); and Hruby, Life Sci. 31:189-199, 1982 (-CH ₂ -S-); each of which is hereby incorporated by reference. A particularly preferred non-peptide bond is _-CH2NH- . Such peptide analogs have significant advantages over polypeptide examples, including, for example: more economical preparation, higher chemical stability, stronger pharmacological properties (half-life, absorption, potency, efficacy, etc.), changes in specificity (such as a broad spectrum of biological activity), antigenicity reduction, and others. Labeling of peptide analogs generally involves coordinating one or more labels, directly or via spacers (e.g., amino groups), with non-interfering sites of the peptide analog predicted by quantitative structure-activity data and/or molecular modeling. price combination. This non-interfering site is usually a site that does not directly contact the macromolecule that produces the therapeutic effect by binding to the peptide analog. Derivatization (eg, labeling) of a peptide analog should not substantially affect the intended biological or pharmacological activity of the peptide analog.

Systematic substitution of one or more amino acids in an amino acid sequence with a D amino acid of the same type (eg D lysine for L lysine) can be used to obtain more stable peptides. In addition, constrained peptides can be produced by methods well known in the art (Rizo et al., Annu. Rev. Biochem. 61:387, 1992, incorporated herein by reference); for example, by adding The internal cysteine residue of the bond cyclizes the peptide.

Such modified polypeptides can be produced by prokaryotic or eukaryotic host cells. In addition, such peptides can also be synthesized by chemical methods. Methods for expression of exogenous polypeptides in recombinant hosts, chemical synthesis of polypeptides, and in vitro translation are well known in the art and are further described in: Maniatis et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor , N.Y., 1989; Berger et al., Methods in Enzymology, Volume 152, Guide to Molecular Cloning Techniques, 1987, Academic Press, Inc., San Diego, Calif.; Merrifield, J.Am.Chem.Soc.91:501, 1969 ; Chaiken, CRC Crit. Rev. Biochem.11: 255, 1981; Kaiser et al., Science 243: 187, 1989; Merrifield, Science 232: 342, 1986; Kent, Annu. Rev. Biochem. 57: 957, 1988; and Offord, Semisynthetic Proteins, Wiley Publishing, 1980, all of which are cited herein by reference).

Polypeptides can often be prepared by direct chemical synthesis and used as binding moieties of heteropolymers. Peptides can be prepared as modified peptides in which non-peptide moieties are covalently linked to the N-terminus and/or C-terminus. In some preferred embodiments one or both of the carboxy terminus or amino terminus are chemically modified. The most common modifications to terminal amino and carboxyl groups are acetylation and amidation, respectively. Amino-terminal modifications such as acylation (e.g. acetylation) or alkylation (e.g. methylation) and carboxy-terminal modifications such as amidation, as well as other terminal modifications, including cyclization, can be used for various test compound examples . Certain amino-terminal and/or carboxy-terminal modifications and/or peptide extensions of the core sequence can provide advantages in physical, chemical, biochemical and pharmacological properties such as: increased stability, enhanced potency and/or efficacy, effects on serum proteases drug resistance, desired pharmacokinetic properties, and more.

Construction of transgenic animals

In one aspect, the present invention provides an animal whose genome comprises a polynucleotide encoding TLR2 or Scd1 operably linked to a promoter, whereby the non-human or human TLR2 gene or Scd1 gene is expressed in the macrophage of the animal. Functionally expressed in phagocytes, or the non-human or human TLR2 gene or Scd1 gene becomes accumulation of functional mutations in macrophages of animals. The present invention further provides a method for constructing transgenic non-human animals expressing non-human or human TLR2 or Scd1 in macrophages.

The transgenic animals used in the methods of the present invention can be: mammals, birds, reptiles or amphibians. Suitable mammals as described herein include: rodents; ruminants; ungulates; domesticated mammals and dairy animals. Preferred animals include: rodents, goats, sheep, camels, cows, pigs, horses, bulls, llamas, chickens, geese and turkeys. In a preferred embodiment, the non-human animal is a mouse.

Various methods of constructing transgenic animals are known in the art (see, for example, Watson et al., "The Introduction of Foreign Genes Into Mice," in Recombinant DNA, 2d Ed., W.H. Freeman & Co., New York, pp. 255-272 , 1992; Gordon, Intl. Rev. Cytol. 115:171-229, 1989; Jaenisch, Science 240:1468-1474, 1989; Rossant, Neuron 2:323-334, 1990). For exemplary protocols for constructing transgenic pigs see White and Yannoutsos, Current Topics in Complement Research: 64th Forum in Immunology, pp.88-94; U.S. Pat. No. 5,523,226; U.S. Pat. No. 5,573,933; PCTApplication WO93/25071; and PCT application WO95/04744. For an exemplary protocol for constructing transgenic rats see Bader et al., Clinical and Experimental Pharmacology and Physiology, Supp. 3:S81-S87, 1996. An exemplary protocol for constructing a transgenic cow can be found in Transgenic Animal Technology, A Handbook, 1994, ed., Carl A. Pinkert, Academic Press, Inc. An exemplary protocol for the construction of transgenic sheep can be found in Transgenic Animal Technology, A Handbook, 1994, ed., Carl A. Pinkert, Academic Press, Inc. Some more specific methods are provided below.

A. Injection into pronuclei

Transgenic animals can be constructed by introducing the nucleic acid construct of the present invention into egg cells. The resulting egg cells are implanted into the uterus of female animals for normal fetal development, and then the developed transgene-carrying animals are backcrossed to establish transgene hybrids. Embryonic target cells of different developmental stages were used to introduce the transgenes of the present invention. Different methods can be used according to the different developmental stages of embryonic target cells. Exemplary methods of introducing a transgene include, but are not limited to, microinjection of fertilized eggs or zygotes (Brinster et al., Proc. Natl. Acad. Sci. USA 82:4438-4442, 1985), and viral integration ( Jaenisch, Proc.Natl.Acad.Sci.USA 73:1260-1264, 1976; Jahner et al., Proc.Natl.Acad.Sci.USA 82:6927-6931, 1985; Van der Putten et al., Proc.Natl. Acad. Sci. USA 82:6148-6152, 1985). The procedure of nuclear microinjection for embryo manipulation can be found in Manipulating the Mouse Embryo (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY., 1986, the contents of which are incorporated herein by reference). Similar methods can be used for the construction of other transgenic animals.

In an exemplary embodiment, the following steps are used for the construction of transgenic mice. Male and female mice from the genetic background of the indicated inbred lines were mated. Mated female mice were previously treated with pregnant horse serum (PMS) to induce follicle growth and human chorionic gonadotropin (hCG) to induce ovulation. After mating, the female is sacrificed and fertilized eggs are removed from her uterine tube. At this point, the pronuclei have not yet fused, so they can be visualized using light microscopy. In another approach embryos can be harvested at different developmental stages, for example embryo sacs can be harvested. Embryos can be reconstituted with Duchenne's Modified Phosphate Buffered Saline (DPBS) and maintained with Duchenne's Modified Essential Medium (DMEM) supplemented with 10% fetal calf serum.

Exogenous DNA or recombinant constructs (such as TLR or Scd1 expression constructs) are then microinjected (100-1000 molecules/egg cell) into pronuclei. Microinjection of expression constructs can be performed by standard micromanipulators attached to microscopes. For example, embryos are maintained in 100 microliter drops of DPBS under oil while microinjecting. DNA solutions were microinjected into male pronuclei. The success of the injection can be detected by the expansion of the pronuclei. Shortly thereafter the pronuclei (female and male) will fuse and, in some cases, foreign DNA will insert into (usually) one chromosome of the fertilized egg or zygote. Recombinant ES cells prepared as described below can be injected into embryo sacs by similar techniques.

B. Embryonic stem cells

In another method of constructing transgenic mice, recombinant DNA molecules of the invention can be introduced into mouse embryonic stem (ES) cells. The resulting recombinant ES cells were then microinjected into mouse embryo sacs using a technique similar to the previous subsection.

ES cells were obtained from preimplantation embryos and cultured in vitro (Evans et al., Nature 292:154-156, 1981; Bradley et al., Nature 309:255-258, 1984; Gossler et al., Proc.Natl.Acad.Sci .USA 83:9065-9069, 1986; Robertson et al., Nature 322:445-448, 1986). Any ES cell capable of integrating into and becoming part of the germline of a developing embryo, thereby establishing germline transmission of the construct of interest is suitable for use herein. For example, the 129J strain is a mouse strain that can be used to produce ES cells. The murine cell line D3 is a preferred ES cell line (American TypeCulture Collection catalog no. CRL 1934). Methods of culturing ES cells and preparing them for DNA insertion are well known in the art and can be found in Robertson, Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E.J. Robertson, ed. IRL Press, Washington, D.C., 1987; Bradley et al., Current Topics in Devel. Biol. 20:357-371, 1986 and Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1986, the contents of which are incorporated herein by reference.

Expression constructs can be introduced into ES cells by methods known in the art, for example, such methods can be found in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed., ed., Cold Spring Harbor Laboratory Press: 1989, the contents of which are here Cited as reference. Suitable methods include, but are not limited to, electroporation, microinjection and calcium phosphate treatment. The expression construct (eg TLR2 or Scd1) to be introduced into ES cells is preferably linear. Linearization can be achieved by digestion with appropriate restriction enzyme lines for selective excision in vector sequences other than genes (eg TLR2 or Scd1 genes).

After introduction of the expression construct, ES cells harboring the construct are screened. Cells can be screened in a variety of ways. When a marker gene is used in the construct, animal cells can be tested for the presence of the marker gene. For example, when the marker gene is an antibiotic resistance gene, the cells can be grown in the presence of an otherwise lethal concentration of the antibiotic (eg, G418 for Neo selection). Those cells that survived were presumed to have integrated the transgene construct. If the marker gene is a gene encoding an enzyme with measurable activity (eg, beta-galactosidase), a substrate for the enzyme can be added to cells under appropriate conditions, and the enzyme activity can be analyzed. Alternatively, or in addition, ES cell genomic DNA can be detected directly. For example, DNA can be extracted from ES cells using standard methods and then probed on Southern blots using probes designed to hybridize specifically to the transgene. Genomic DNA can also be amplified by PCR using probes specially designed to amplify DNA fragments of a specific size and sequence within the transgene, allowing only those cells that contain the construct of interest to produce DNA fragments of the correct size.

C. Implantation

Fertilized eggs carrying recombinant nucleic acid molecules of the present invention (eg, TLR2 or Scd1) are implanted into pseudopregnant female mice that were previously mated with vasectomized males. In the conventional protocol, recipient females are anesthetized, a paralumbar incision is made to expose the fallopian tubes, and the embryos are transferred into the ampullary region of the fallopian tubes. The body wall was sutured and the skin closed with wound clips. After the embryos have developed through a full gestation period, potential transgenic mice are delivered by surrogate mothers. Finally, newborn mice are tested for the presence of exogenous or recombinant DNA. Of all the eggs injected, an average of 10 percent developed normally and formed mice. Of all mice born, an average of one quarter (25%) were transgenic, resulting in an overall efficacy rate of 2.5%. Once these mice were bred, they passed on the foreign genes linked to the mouse chromosomes in the conventional (Mendelian) way.

D. Screening for the presence of transgenic constructs

Transgenic animals can be identified postnatally using standard protocols. DNA from tail tissue can be screened for the presence of the transgene construct, for example by southern blot and/or PCR. If they are believed to carry the transgene, offspring that appear to be chimeras can be crossed with each other to produce homozygous animals. If it is not known whether the offspring have germline transmission, they can be crossed to the mother or other lines and their offspring screened for heterozygosity. Heterozygotes can be identified by southern blot and/or PCR amplification of DNA. The hybrids can be crossed with each other to produce homozygous transgenic offspring. Identification of homozygotes can be performed by Southern blots of equal amounts of genomic DNA from mice obtained from the cross, mice known to be heterozygous, and wild-type mice. Probes for screening southern blots can be designed based on human or non-human TLR2 or Scd1 gene sequences, or marker genes, or both.

Other methods of identifying and characterizing transgenic progeny are known in the art. For example, by probing a western blot with antibodies against the protein encoded by the introduced gene (such as human or non-human TLR2 or Scd1 protein), or against a marker gene product, the western blot can be used to assess the various The expression level of the introduced gene in the tissue expressing the gene.

In situ analysis (e.g. fixing cells and labeling with antibodies) of various cells from progeny (e.g. erythrocytes), and/or FACS (fluorescence activated cell sorting) analysis to check for transgene product by using appropriate antibodies present or absent. For example, to verify the expression of TLR2 or Scd1 in macrophages, it can be directly conjugated or used in combination with a secondary antibody (conjugated with a fluorophore and recognizing TLR2 or Scd1 antibody) specific for human TLR2 or Scd1. Antibodies for flow cytometry. In this assay, human erythrocytes can be used as a positive control for the presence of TLR2 or Scd1, while normal mouse erythrocytes can be used as a negative control.

E. Mice with multivalent transgenes or additional mutations

Transgenic mice expressing TLR2 or Scd1 described herein can be crossed with mice that a) carry additional transgenes, or b) contain mutations in other genes. Mice heterozygous or homozygous for the various mutations can be generated and maintained using standard crossbreeding procedures. Mice that can be bred with TLR2 or Scd1 transgenic mice include, but are not limited to, mouse strains that are more susceptible to autoimmune diseases, such as mouse strains that serve as lupus models, such as the mouse strain NZB/W, MRL+ or SJL.

The invention further relates to cells from transgenic animals. The progeny may not actually be identical to the parent cell due to certain modifications that may result from mutations or environmental influences, but it is still encompassed within the scope of the term as used herein.

recombinant nucleic acid technology

Nucleic acids used in the practice of the present invention, whether RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses, or hybrids thereof, can be isolated from various sources and subjected to genetic engineering, amplification, and/or or recombinant expression/production. Recombinant polypeptides produced from these nucleic acids can be isolated or cloned individually and tested for the activity of interest. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.

In addition, these nucleic acids can be synthesized in vitro using known chemical synthesis techniques, as described in documents such as Adams, J.Am.Chem.Soc.105:661, 1983; Belousov, Nucleic Acids Res.25:3440-3444 , 1997; Frenkel, Free Radic.Biol.Med.19:373-380, 1995; Blommers, Biochemistry 33:7886-7896, 1994; Narang, Meth.Enzymol.68:90, 1979; Brown Meth.Enzymol.68: 109, 1979; Beaucage, Tetra. Lett. 22:1859, 1981; U.S. Pat. No. 4,458,066.

The invention provides oligonucleotides comprising sequences of the invention (eg, subsequences of exemplary sequences of the invention). An oligonucleotide may comprise, for example, a single stranded polydeoxyribonucleic acid or two complementary polydeoxyribonucleic acid strands, which may be chemically synthesized.

Nucleic acid manipulation techniques such as subcloning, labeling probes (e.g., random primer labeling using Klenow large fragment polymerase, nick translation, amplification), sequencing, hybridization, etc. are well described in the scientific and patent literature, see for example Sambrook , ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), VoIs.1-3, Cold Spring Harbor Laboratory, 1989; CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley&Sons, Inc., New York, 1997; LABORATORY TECHNIQUES INBIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y., 1993.

Nucleic acids, vectors, capsids, polypeptides, etc. can be analyzed and quantified by any conventional method known to those skilled in the art. It includes, for example, analytical biochemical methods (such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and superdiffusion chromatography), various immunological Methods (e.g. fluid or gel precipitation reactions, immunodiffusion, immunoelectrophoresis, radioimmunoassays (RIAs), enzyme-linked immunosorbent assays (ELISAs), immunofluorescence assays), Southern analysis, Northern analysis, dot blot analysis, gel electrophoresis (eg SDS-PAGE), nucleic acid or target or signal amplification methods, radioactive labeling, scintillation counting and affinity chromatography.

Nucleic acids used to practice the methods of the invention can be obtained and manipulated by cloning from genomic samples and, if desired, screening and recloning of inserts isolated or amplified from genomic or cDNA clones. Sources of nucleic acids used in the methods of the invention include genomic or cDNA libraries, e.g., mammalian artificial chromosomes (MACs), e.g. see U.S. Pat. Nos. 5,721,118; 6,025,155; human artificial chromosomes, e.g. see Rosenfeld, Nat. Genet. 15:333-335, 1997; Yeast artificial chromosome (YAC); Bacterial artificial chromosome (BAC); P1 artificial chromosome, see e.g. Woon, Genomics 50:306-316, 1998; Vectors (PACs) derived from P1, see e.g. Kern, Biotechniques 23:120-124, 1997; cosmid, recombinant virus, phage or plasmid.

The invention provides fusion proteins and nucleic acids encoding them. The Scd1 gene product or toll-like receptor 2 polypeptide can be fused to an exogenous peptide or polypeptide, such as an N-terminal identification peptide that confers desired properties such as increased stability or ease of purification. Peptides and polypeptides of the invention can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto to generate, for example, more immunogenic peptides, allowing for easier isolation of recombinant synthetic peptides for Identification and isolation of antibodies and antibody-expressing B cells, and more. Regions that facilitate detection and purification include, for example, metal-chelating peptides (such as polyhistidine tracts and histidine-tryptophan modules that can be purified on immobilized metals, proteins that can be purified on immobilized immunoglobulins A domain, and a domain that can be used in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.). Contains a cleavable linker sequence such as factor Xa or enterokinase ( Invitrogen, San DiegoCalif.) to aid in purification. For example, expression vectors may contain nucleic acid sequences that encode epitopes linked to six histidine residues, followed by thioredoxin and enterokinase cleavage sites (see, for example, Williams, Biochemistry 34:1787-1797, 1995; Dobeli, ProteinExpr.Purif 12:404-414, 1998). Histidine residues can help detection and purification, while the enterokinase cleavage site provides a residue from the fusion protein Method for purifying epitopes. On the one hand, the nucleic acid encoding the polypeptide of the present invention is assembled into a guide sequence for direct secretion of the translated polypeptide or fragments thereof with a suitable phase. Relating to the vector encoding the fusion protein and the application of the fusion protein in science and technology and patent literature It has been described in detail, see eg Kroll, DNA Cell. Biol. 12:441-53,1993.

A. Transcriptional Control Elements

A nucleic acid of the invention can be operably linked to a promoter. A promoter may be a motif or array of nucleic acid control sequences capable of directing the transcription of a nucleic acid. A promoter may contain an essential nucleic acid sequence near the site of initiation of transcription, such as a TATA element in a polymerase II type promoter. A promoter may also optionally include distal enhancer or repressor elements thousands of base pairs from the transcription start site. A "constitutive" promoter refers to a promoter that is capable of functioning under most environmental and developmental conditions. An "inducible" promoter refers to a promoter that is regulated by the environment or development. A "tissue-specific" promoter refers to a promoter that acts in a particular tissue type of an organism, but not in other tissue types of that organism. The term "operably linked" refers to a functional linkage between a nucleic acid expression control sequence (e.g., a promoter, or an array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the corresponding nucleic acid of the second sequence .

B. Expression Vectors and Cloning Vectors

The invention provides expression vectors and cloning vectors comprising a nucleic acid of the invention (eg, a sequence encoding a protein of the invention). Expression vectors and cloning vectors of the present invention may include viral particles, baculoviruses, phages, plasmids, cosmids, Fossmids, bacterial artificial chromosomes, viral DNA (such as vaccinia, adenovirus, stinkpox virus, pseudorabies and SV40) derivatives), P1-based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for a particular target host (eg, Bacillus, Aspergillus, and yeast). Vectors of the present invention may include chromosomal, non-chromosomal and synthetic DNA sequences. A large number of suitable vectors are known to those skilled in the art and are commercially available.

The nucleic acid of the present invention can be cloned into any vector by conventional molecular biology methods if necessary; methods for cloning nucleic acids amplified in vitro can be found in U.S. Pat. No. 5,426,039 and the like. To facilitate cloning of amplified sequences, restriction sites can be "built into" the PCT primer pairs.

The invention provides libraries of expression vectors encoding the polypeptides and peptides of the invention. These nucleic acids can be introduced into the genome or the cytoplasm and nucleus of the cell and expressed by a variety of conventional techniques well described in the scientific and patent literature. See, for example, Roberts, Nature 328:731, 1987; Schneider, Protein Expr. Purif. 6435:10, 1995; Sambrook, Tijssen or Ausubel. These vectors can be isolated from natural sources, obtained from sources such as ATCC or gene banks, or obtained through Prepared synthetically or recombinantly. For example, nucleic acids of the invention can be expressed in expression cassettes, vectors or viruses (eg, episomal expression systems) capable of stable or transient expression in cells. Selectable markers can be included in expression cassettes and vectors to confer a selectable phenotype on transformed cells and sequences. For example, a selectable marker can encode episomal maintenance and replication so that integration into the host genome is not required.

In one aspect, a nucleic acid of the invention is introduced in vivo to obtain in situ expression of a peptide or polypeptide of the invention. The nucleic acid can be introduced as "naked DNA" (see, eg, U.S. Pat. No. 5,580,859) or in the form of an expression vector such as a recombinant virus. As described below, the nucleic acid can be introduced by any route including peritumoral or intratumoral. Vectors for in vivo administration may be derived from viral genomes, including recombinantly modified enveloped or non-enveloped DNA and RNA viruses, preferably from baculoviridiae, parvoviridiae, picornoviridiae, herpesveridiae, poxyiridae, adenoviridiae or picornaviridiae. Chimeric vectors can also be used that take advantage of the attributes of each parent vector (see, eg, Feng, Nature Biotechnology 15:866-870, 1997). The viral gene can be modified by recombinant DNA technology to contain the nucleic acid of the present invention; and further engineered to become replication-deficient, conditionally replicable or replicable. In another aspect, the vector may be derived from an adenovirus (eg, a non-replicable vector from a human adenovirus genome, eg, see U.S. Pat. Nos. 6,096,718; 6,110,458; 6,113,913; 5,631,236); adeno-associated virus and retrovirus genomes. Retroviral vectors can be based on viruses including murine leukemia virus (MuLV), gibbon leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof; see, e.g., U.S. Pat. Nos. 6,117,681; 6,107,478; 5,658,775; 5,449,614; Buchscher, J. Virol. 66:2731-2739, 1992; Johann, J. Virol. 66:1635-1640, 1992). Adeno-associated virus (AAV)-based vectors can be used to irradiate cells with nucleic acids of interest, e.g., for the production of nucleic acids and peptides in vitro, and during gene therapy in vivo and ex vivo; see, e.g., U.S. Pat. Nos. 6,110,456; 5,474,935; Okada , Gene Ther. 3:957-964, 1996.

"Expression cassette" as used herein refers to a nucleotide sequence capable of affecting the expression of a structural gene (ie, a sequence encoding a protein such as a polypeptide of the present invention) in a host compatible with the sequence. The expression cassette includes at least one promoter operably linked to the sequence encoding the polypeptide; and optionally other sequences, such as transcription termination signals. Other additional factors necessary or helpful to affect expression, such as enhancers, may also be used.

A nucleic acid is "operably linked" when it enters into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence when it affects the transcription of the sequence. For transcriptional regulatory sequences, operably linked means that the DNA sequences being linked are adjacent to each other and, when necessary, join two adjacent protein coding regions in reading frame. With respect to a switch sequence, operably linked means that the sequence is capable of effecting switch recombination. Thus, expression cassettes may also include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like.

"Vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid," which refers to a circular double-stranded DNA loop capable of ligation of additional DNA segments. Another type of vector is a viral vector, in which additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication within the host cell into which they are introduced (eg, bacterial vectors with an origin of replication and episomal mammalian vectors). Other vectors (eg, non-episomal mammalian vectors) can be integrated into the genome of the host cell when introduced into the host cell and replicate along with the host genome. In addition, some vectors are capable of directing the expression of genes to which they are operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). In general, expression vectors employed in recombinant DNA techniques are usually in the form of plasmids. In this specification, "plasmid" and "vector" are used interchangeably, since plasmids are the most commonly used form of vectors. However, the invention is intended to encompass other forms of expression vectors, such as viral vectors (eg, replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

C. Host Cells and Transformed Cells

The present invention also provides a transformed cell comprising the nucleic acid sequence of the present invention (eg, the sequence encoding the polypeptide of the present invention) or the vector of the present invention. The host cell may be any host cell familiar to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells or plant cells. Exemplary bacterial cells include Escherichia coli, Streptomyces, Bacillus subtilis, Salmonella typhimurium and species of Pseudomonas, Streptomyces and Staphylococci. Exemplary insect cells include Drosophila S2 and Spodoptera Sf9. Exemplary animal cells include CHO, COS or Bowes melanoma or any mouse or human cell line. Selection of an appropriate host is within the ability of those skilled in the art.

The vector can be introduced into host cells by any of a variety of available techniques, including transformation, transfection, transduction, viral infection, biolistic or Ti-mediated gene transfer. Specific methods include calcium phosphate transfection, DEAE-dextran mediated transfection, lipofection or electroporation.

The engineered host cells can be cultured in modified conventional nutrient media suitable for activation of promoters, selection of transformants, or amplification of the genes of the invention. After transforming a suitable host strain and allowing the host strain to grow to an appropriate cell density, the selected promoter can be induced by appropriate methods (e.g., temperature shift or chemical induction), and the cells are then cultured for a period of time to produce the desired polypeptides or fragments thereof.

Cells can be harvested by centrifugation, disrupted physically or chemically, and the resulting crude extract retained for further purification. Microbial cells used to express proteins can be disrupted by any conventional method, including freeze-thaw cycles, sonication, mechanical disruption or use of cell lysis reagents. These methods are well known to those skilled in the art. Expressed polypeptides or fragments can be recovered and purified from recombinant cells by a variety of methods, including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity Chromatography, hydroxyapatite chromatography and lectin chromatography. Protein refolding steps can be employed, if desired, in completing the conformation of the polypeptide. High performance liquid chromatography (HPLC) can be used as a final purification step if desired.

Various mammalian culture systems can also be used to express recombinant proteins. Examples of mammalian expression systems include the COS-7 line of monkey kidney fibroblasts and other cell lines capable of expressing proteins from compatible vectors such as C127, 3T3, CHO, HeLa and BHK cell lines.

Such a construct in a host cell can be used to produce the gene product encoded by the recombinant sequence in a conventional manner. Depending on the host used in the recombinant production process, the polypeptide produced by the host cell carrying the vector may be a glycosylated polypeptide or a non-glycosylated polypeptide. Polypeptides of the invention may or may not have the initial methionine amino acid residue.

Cell-free translation systems can also be used to produce the polypeptides of the invention. Cell-free translation systems may employ mRNAs transcribed from a DNA construct comprising a promoter operably linked to a nucleic acid encoding a polypeptide or a fragment thereof. In some cases, the DNA construct can be linearized prior to the in vitro transcription reaction. The transcribed mRNA is then cultured in an appropriate cell-free translation extract (eg, rabbit reticulocyte extract) to produce the desired polypeptide and fragments thereof.

Expression vectors may contain one or more selectable marker genes to provide phenotypic traits required for selection of transformed host cells, such as dihydrofolate reductase or neomycin resistance in eukaryotic cell culture, or e.g. Tetracycline or ampicillin resistance.

D. Amplification of nucleic acids

During the implementation of the present invention, the nucleic acid encoding the polypeptide of the present invention or the modified nucleic acid can be replicated by means of amplification or the like. The present invention provides a pair of amplification primers for amplifying nucleic acid encoding a polypeptide of the present invention, for example, a pair of primers capable of amplifying nucleic acid comprising Scd1 protein or toll-like receptor 2 sequence or subsequence thereof.

Amplification methods include, such as polymerase chain reaction, PCR (PCR PROTOCOLS, A GUIDE TO METHODS AND APPLICATIONS, ed. Innis, Academic Press, N.Y., 1990 and PCR STRATEGIES, 1995, ed. Innis, Academic Press, Inc., N.Y.), Ligase chain reaction (LCR) (see, for example, Wu, Genomics 4:560, 1989; Landegren, Science 241:1077, 1988; Barringer, Gene 89:117, 1990); transcriptional amplification (see, for example, Kwoh, Proc. USA 86:1173,1989); and self-sustaining sequence amplification (see, for example, Guatelli, Proc.Natl.Acad.Sci.USA 87:1874,1990); Q Beta replicase amplification ( See, e.g., Smith, J. Clin. Microbiol. 35:1477-1491, 1997), automated Q-beta replicase amplification assays (e.g., see Burg, MoI. Cell. Probes 10: 257-271, 1996) and others RNA polymerase-mediated techniques (e.g. NASBA, Cangene, Mississauga, Ontario); see also Berger, Methods Enzymol. 152:307-316, 1987; Sambrook; Ausubel; U.S. Pat. Nos. 4,683,195 and 4,683,202; Sooknanan, Biotechnology 13: 563-564, 1995.

E. Nucleic acid hybridization

The invention provides isolated or recombinant nucleic acids that hybridize under stringent conditions to an exemplary sequence of the invention (eg, the Scd1 sequence or the toll-like receptor 2 sequence, or the complement of either, or a nucleic acid encoding a polypeptide of the invention). In another aspect, the stringent conditions are conditions of high stringency, conditions of moderate stringency or conditions of low stringency, as known in the art and as described herein. These methods can be used to isolate nucleic acids of the invention.

In another aspect, nucleic acids of the invention (as defined by their ability to hybridize under stringent conditions) can be from about 5 residues to the full length of the nucleic acids of the invention; for example, they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 or more residues, or the full length of a gene or coding sequence, eg cDNA. Nucleic acids shorter than full length are also included. These nucleic acids can be used in a variety of ways, such as hybridization probes, labeling probes, PCR oligonucleotide probes, iRNA, antisense or sequences encoding antibody binding polypeptides (epitopes), motifs, active sites wait.

"Selective (or specific) hybridization" refers to the binding of molecules to, or hybridization to, a specific nucleotide sequence present in a complex mixture (such as total cell or library DNA or RNA) under stringent hybridization conditions, where the specific The detection value of the nucleotide sequence should be at least 10 times the background. In one example, a nucleic acid can be identified by its ability to hybridize under stringent conditions to a nucleic acid otherwise determined to be within the scope of the invention, such as the exemplary sequences described herein. within the scope of the present invention.

"Stringent hybridization conditions" mean that a probe hybridizes to its target subsequence (usually in a complex mixture of nucleic acids) by about 10 times to the background of other sequences (positive signal (e.g., identification of nucleic acids of the invention) in significant numbers) ) conditions for hybridization. Stringent conditions are sequence dependent and vary with circumstances. Longer sequences hybridize specifically at higher temperatures. Extensive guidance on nucleic acid hybridization can be found, for example, in Sambrook, ed., MOLECULAR CLONING: A LABORATORY MANUAL (2ND ED.), VoIs. 1-3, Cold Spring Harbor Laboratory, 1989; CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel, ed. John Wiley & Sons , Inc., New York, 1997; LABORATORY TECHNIQUES INBIOCHEMISTRY AND MOLECULAR BIOLOGY: HYBRIDIZATION WITH NUCLEIC ACID PROBES, Part I. Theory and Nucleic Acid Preparation, Tijssen, ed. Elsevier, N.Y., 1993.

Generally, stringent conditions can be selected to be about 5-10°C lower than the thermal melting point I at a particular ionic strength pH. is (at a given ionic strength, pH and nucleic acid concentration) the temperature at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (since the target sequence is present in excess, at Tm, 50% of the probe is occupied). Stringent conditions can be a salt concentration of less than about 1.0 M sodium ion at pH 7.0 to 8.3, typically about 0.01 to 1.0 M sodium ion concentration (or other salts), and for short probes (e.g., 10 to 50 nucleotides) ) is at least 30°C and for long probes (eg, greater than 50 nucleotides), the temperature is at least 60°C. Stringent conditions can also be achieved by adding a destabilizing agent such as formamide with reference to Sambrook (cited below). For high stringency hybridizations, a positive signal is at least two times background, preferably 10 times background hybridization. Very high stringency or stringent hybridization conditions include: 50% formamide, 5x SSC and 1% SDS at 42°C, or 5x SSC and 1% SDS at 65°C, and 0.2x SSC at 65°C and Wash in 1% SDS. For selective or specific hybridization, a positive signal (eg, identification of a nucleic acid of the invention) is about 10 times background hybridization. Stringent hybridization conditions for identifying nucleic acids within the scope of the invention include: hybridization at 42°C in a buffer containing 50% formamide, 5x SSC and 1% SDS, or at 65°C in a buffer containing 5x SSC and 1% SDS. Hybridization was performed in buffer with % SDS, and both were washed with 0.2x SSC and 1% SDS at 65°C. In the present invention, genomic DNA or cDNA comprising a nucleic acid of the invention can be identified by standard Southern blotting under stringent conditions using the nucleic acids disclosed herein. Additional stringent conditions for this hybridization (to identify nucleic acids within the scope of the invention) are conditions that include hybridization at 37°C in a buffered solution of 40% formamide, 1M NaCl, 1% SDS.

However, the choice of hybridization format is not critical, and it is the stringency of the wash conditions that determine the conditions for determining whether a nucleic acid is within the scope of the invention. Washing conditions for identifying whether a nucleic acid is within the scope of the invention include, for example, a salt concentration of about 0.02 molar at pH 7 and a temperature of at least about 50°C or about 55°C to about 60°C; or, for about 15 minutes, at A salt concentration of about 0.15M NaCl at 72°C; or, for about 15 to 20 minutes, a salt concentration of about 0.2X SSC at a temperature of at least about 50°C or about 55°C to about 60°C; A solution of approximately 2X SSC containing 0.1% SDS was washed twice at room temperature for 15 minutes, followed by a wash in 0.1X SSC containing 0.1% SDS at 68°C for 15 minutes and repeated twice; or equivalent conditions. See Sambrook, Tijssen and Ausubel for a description of SSC buffer and equivalent conditions.

F. Oligonucleotide probes and methods of use thereof

The present invention also provides nucleic acid probes for identifying nucleic acids encoding polypeptides that are modulators of TLR2 or Scd1 signaling activity. In one aspect, the probe comprises at least 10 contiguous bases of the nucleic acid of the invention. In addition, the probes of the present invention can be at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, or about 10 to 50, about 20 to 60, about 30 to 70 consecutive bases in the nucleic acid sequence of the present invention. The probes identify nucleic acids by binding and/or hybridization. Such probes can be used in arrays of the invention, see discussion below. The probes of the present invention can also be used for the isolation of other nucleic acids or polypeptides.

G. Determination of sequence identity

The invention provides polynucleotides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , nucleic acids with 99% or greater sequence identity. The invention provides a protein having 90%, at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to Scd1 protein or toll-like receptor 2 protein Sexual polynucleotides. Sequence identity can be determined by analysis of sequence comparison algorithms or visual inspection. Sequence identity (homology) of proteins and/or nucleic acids can be assessed using any number of sequence comparison algorithms and programs known in the art.

In sequence comparison, typically one sequence acts as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, and, if necessary, sequence algorithm program parameters are designated. Default program parameters can be used, or additional parameters can be specified. The sequence comparison algorithm will then calculate the percent sequence similarity for the test sequences relative to the reference sequence, based on the program parameters. For nucleic acid and protein sequence comparisons, BLAST and BLAST 2.2.2 or FASTA version 3.0t78 can be used with the default parameters discussed below.

As used herein, a "comparison window" refers to a range that includes fragments for any number of adjacent positions selected from the group consisting of 20 to 600, usually about 50 to about 200, more typically about 100 to About 150, the range in which a sequence can be compared to a reference sequence with the same number of adjacent positions when the two are optimally aligned. Methods for comparison by aligning sequences are well known in the art. Comparison by optimal alignment of sequences can be achieved, for example, by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482, 1981, by Needleman & Wunsch, J. MoI. Biol. 48: 443, the homology comparison algorithm of 1970, by the similarity search method of Pearson&Lipman, Proc. Biomedical Information Center), GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Package, Genetics Computer Group, 575 ScienceDr., Madison, WI), or by manual alignment and visual inspection (see, for example, Ausubel et al., (1999Suppl.), Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y., 1987).

A preferred example of an algorithm suitable for detecting percent sequence identity and sequence similarity is the FASTA algorithm, described in Pearson & Lipman, Proc. See also Pearson, Methods Enzymol. 266:227-258,1996. The preferred parameters for FASTA alignment of DNA sequences used to calculate percent identity have been optimized, BL50Matrix 15: -5, k-tuple=2; joining penalty=40, optimization=28; gap penalty-12, gap length penalty=- 2; and width=16.

Another preferred example of an algorithm suitable for detecting percent sequence identity and sequence similarity is the BLAST and BLAST2.0 algorithms described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul, respectively. et al., J. MoI. Biol. 215:403-410, 1990. BLAST and BLAST 2.0 can detect percent sequence identity for nucleic acids and proteins of the invention using the parameters described herein. Software for performing BLAST analyzes is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). The algorithm consists of first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the test sequence that either match or satisfy some positive numerical cutoff score T when aligned with a word of the same length in a database sequence. T refers to the neighborhood word score bound (Altschul et al., supra). These initial neighboring word hits serve as seeds to initiate searches for longer HSPs containing them. The term is stretched in both directions for each sequence until the cumulative alignment score is increased. Cumulative scores for nucleotide sequences are calculated using the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix can be used to calculate the cumulative score. Stretching of a term in each direction is stopped when: the cumulative alignment score is an amount X less than its maximum achieved value; the cumulative score falls to 0 or below due to the accumulation of one or more negative-scoring residue alignments; or end of any sequence. The parameters W, T and X of the BLAST algorithm measure the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) defaults to a word length (w) of 11, an expectation (E) of 5, M=5, N=-4, and compares two strands simultaneously. For amino acid sequences, the default word length (w) of the BLASTP program is 3, the expected value (E) is 10, and the alignment of the score BLOSUM62 matrix (see Henikoff&Henikoff, Proc.Natl.Acad.Sci.U.S.A.89:10915,1989) ( B) is 50, the expected value (E) is 10, M=5, N=-4, and the two chains are compared at the same time.

The BLAST algorithm can also perform a statistical analysis of the similarity between two sequences (see, eg, Karlin & Altschul, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5787, 1993). One test of similarity provided by the BLAST algorithm is the overall probability (P(N)), which provides an indication of the likelihood by which a match between two nucleotide or amino acid sequences would result by chance. For example, a nucleic acid is considered to be similar to a reference nucleic acid when the smallest overall probability obtained from the comparison between the test nucleic acid and the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, more preferably less than about 0.001.

Another example of a useful algorithm is PILEUP. PILEUP uses progressive, pairwise alignments to build multiple sequence alignments from groups of related sequences, showing relatedness and percent sequence identity. It also draws a dendrogram that shows the clustering relationships used to build the alignment. PILEUP adopts the simplified progressive comparison method of Feng & Doolittle, J.MoI.Evol.35:351-360,1987. The method employed is similar to that described by Higgins & Sharp, CABIOS 5:151-153, 1989. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids. This multiple alignment procedure begins with a pairwise alignment of the two most similar sequences, resulting in a cluster of the two aligned sequences. This cluster is then aligned to the next closest sequence or cluster of aligned sequences. Two sequence clusters can be aligned by a simple extension of the pairwise alignment of two separate sequences. The final alignment can be achieved through a series of incremental, pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates as regions for sequence comparison and designating program parameters. Using PILEUP will determine the percent sequence identity relationship by comparing a reference sequence to other test sequences using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. PILEUP is available from GCG Sequence Analysis Software Version 7.0 and others (Devereaux et al., Nuc. Acids Res. 12:387-395, 1984.

Another example of a preferred algorithm suitable for multiple alignments of DNA and amino acid sequences is the CLUSTALW program (Thompson et al., Nucl. Acids. Res. 22:4673-4680, 1994). ClustalW can perform multiple pairwise comparisons between groups of sequences and load them into homology-based multiple alignments. Interval opening and interval extension penalties are 10 and 0.05, respectively. For amino acid alignments, the BLOSUM algorithm can be used as a protein weight matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. U.S.A. 89:10915-10919, 1992).

"Sequence identity" refers to a measure of similarity between amino acid or nucleotide sequences and can be measured using methods well known in the art, such as described below:

"Identity" or percent "identity" in the context of two or more nucleic acid or nucleotide sequences means that two or more sequences or subsequences are identical to each other or have a certain percentage of identical amino acid residues or nucleotides (i.e. for a specified region or comparison window, when comparing and aligning the maximum correspondence, one of the following sequence comparison algorithms or by manual alignment and visual inspection detects 60% concordance, preferably 65%, 70%, 75% , 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or higher concordance).

"Substantial identity" of two nucleic acids or nucleotide sequences in this context means that two or more have at least 60%, usually at least 70%, preferably at least 80%, most preferably at least 80% when compared and aligned for maximum correspondence. A sequence or subsequence that is 90% or at least 95% nucleotide or amino acid residue identical, as determined by one of the following sequence comparison algorithms or by visual inspection. Such sufficient identity is preferably present over a sequence region of at least 50 bases or residues in length, more preferably at least 100 bases or residues, and most preferably at least 150 bases or residues in length. In a most preferred embodiment, the sequences are substantially identical over the entire length of the coding region.

"Homology" and "identity" in the context of two or more nucleic acids or nucleotide sequences means (when compared and aligned for maximum correspondence over a comparison window or specified region) that two or more sequences or Subsequences are identical to each other or have a certain percentage of identical amino acid residues or nucleotides, as determined by one of the following sequence comparison algorithms or by manual alignment and visual inspection. For sequence comparison, one sequence can be used as a reference sequence (exemplary sequences for Scd1 gene product or toll-like receptor 2 polynucleotide or polypeptide) to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, and, if necessary, sequence algorithm program parameters are designated. Default program parameters can be used, or additional parameters can be specified. The sequence comparison algorithm will then calculate the percent sequence similarity for the test sequences relative to the reference sequence, based on the program parameters.

As used herein, "comparison window" refers to the range of fragments that includes any number of contiguous residues. For example, in another aspect of the invention, adjacent residues at any position ranging from 20 to the full length of an exemplary polypeptide or nucleic acid sequence of the invention (e.g., Scd1 or toll-like receptor 2 polynucleotides) are used Comparisons are made to a reference sequence having the same number of adjacent positions after the two sequences are optimally aligned. If the reference sequence has the necessary sequence identity to an exemplary polypeptide or nucleic acid sequence of the present invention, for example at least 90%, 91%, 92%, 93%, 94% to a Scd1 or toll-like receptor 2 polynucleotide or polypeptide %, 95%, 96%, 97%, 98%, 99% or higher sequence identity, then the sequence is within the scope of the present invention.

Motifs that can be detected by the above procedures include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta folds, encoding signals capable of directing the secretion of encoded proteins Peptide signal sequence, sequences involved in transcription regulation such as homeobox, acidic stretch, enzyme active site, substrate binding site and enzyme cleavage site.

H. COMPUTER SYSTEMS AND COMPUTER PROGRAM PRODUCTS

In order to detect and identify sequence identity, structural homology, motifs, etc. in silico, the sequences of the present invention can be stored, recorded and manipulated on any medium that can be read and accessed by a computer. Accordingly, the present invention provides computers, computer systems, computer readable media, computer program products, etc. for recording or storing the nucleic acid and polypeptide sequences of the present invention. The words "recording" and "storage" as used herein refer to the process of storing information on computer media. Those skilled in the art can quickly use various known methods to record information on computer-readable media, thereby producing products comprising one or more nucleic acid and/or polypeptide sequences of the present invention.

Another aspect of the present invention is a computer readable medium on which at least one nucleic acid and/or polypeptide sequence of the present invention is recorded. Computer readable media includes magnetically readable media, optically readable media, electronically readable media, and magnetic/optical media. For example, the computer readable medium can be a hard disk, floppy disk, magnetic tape, CD-ROM, digital versatile disk (DVD), random access memory (RAM) or read only memory (ROM) and other memory devices known to those skilled in the art. media.

As used herein, the terms "computer", "computer program" and "processor" are used in the broadest context and include all such devices.

Modulation or inhibition of peptides and transcripts

The invention further provides nucleic acids (eg antisense sequences) that are complementary to the nucleic acid sequences of the invention. Antisense sequences are capable of regulating or inhibiting the translocation, splicing or transcription of a protein-encoding gene (eg, nucleic acid encoding TLR2 or Scd1). This modulation or inhibition can be effected by targeting genomic DNA or messenger RNA. The transcription or function of the targeted nucleic acid can be inhibited by methods such as hybridization and/or cleavage. A particularly useful class of inhibitors provided by the invention includes oligonucleotides that bind to a gene or message while preventing or inhibiting the production and action of the protein. This association can be achieved by sequence-specific hybridization. Another class of useful inhibitors includes oligonucleotides that cause inactivation or cleavage of protein messengers. The oligonucleotide may have enzymatic activity, such as a ribozyme, that causes such cleavage. The oligonucleotide can be chemically modified or linked to an enzyme or composition capable of cleaving a complementary nucleic acid. One can screen many different collections of such oligonucleotides for the desired activity.

Conventional gene therapy methods using antisense, ribozyme technology and RNAi technology to control gene expression, or to express foreign genes in this way are well known in the art. These methods all employ systems, such as vectors, that encode antisense or ribozyme transcripts of the phosphatase polypeptides of the invention. The term "RNAi" stands for RNA interference. The term is understood in the art to encompass techniques using RNA molecules capable of silencing genes. See, eg, McManus et al. Nature Reviews Genetics 3:737, 2002. In this application, the term "RNAi" includes short interfering RNA (siRNA), microRNAs (mRNA), small sequential RNA (stRNA) and other molecules. In general, RNA interference results from the interaction of double-stranded RNA and genes.

A. Antisense oligonucleotides

The present invention provides antisense oligonucleotides capable of binding to TLR2 or Scd1 messenger RNA (which can inhibit the activity of the polypeptide by targeting mRNA). Strategies for designing antisense oligonucleotides are well described in the scientific and patent literature, and the skilled artisan can design such oligonucleotides using the reagents of the present invention. For example, gene walking/RNA localization schemes for screening effective antisense oligonucleotides are well known in the art, see, for example, Ho, Methods Enzymol. 314:168-183, 2000, which describes molecular-based technology provides simple and reliable RNA localization analysis for the selection of potential antisense sequences. See also Smith, Eur. J. Pharm. Sci. 11:191-198, 2000.

Naturally occurring nucleic acids can be used as antisense oligonucleotides. The antisense oligonucleotides can be of any length; eg, in another aspect, the antisense oligonucleotides are between about 5-100, about 10-80, about 15-60, about 18-40. The optimal length can be determined by routine screening. The antisense oligonucleotide can be present in any concentration. Optimal concentrations can be determined by routine screening. A variety of synthetic, non-naturally occurring nucleotide and nucleic acid analogs are known that can address this potential problem. For example, peptide nucleic acids (PNAs) comprising nonionic backbones such as N-(2-aminoethyl)glycine units can be used. Antisense oligonucleotides with phosphorothioate linkages can also be used as described in WO 97/03211; WO 96/39154; Mata, Toxicol Appl Pharmacol. 144:189-197, 1997; Antisense Therapeutics, ed. Agrawal , Humana Press, Totowa, N.J., 1996. As mentioned above, antisense oligonucleotides with synthetic DNA backbone analogs provided by the present invention may also include phosphorodithioate, methylphosphonate, phosphoramide, alkylphosphotriester, sulfamic acid salt, 3'-thioacetal, methylene (methimino), 3'-N-carbamate, and morpholine carbamate nucleic acids.

Combinatorial chemistry methods can be used to construct a wide variety of oligonucleotides that can be rapidly screened for specific oligonucleotides with appropriate binding affinity and specificity for any target (such as the sense and antisense polypeptide sequences of the invention) (See, eg, Gold, J. of Biol. Chem. 270:13581-13584, 1995).

B. siRNA

"Small interfering RNA" (siRNA) refers to double-stranded RNA molecules 10 to 30 nucleotides in length and named for their ability to specifically interfere with protein expression by RNA interference (RNAi). The siRNA is preferably 12-28 nucleotides in length, more preferably 15-25 nucleotides in length, more preferably 19-23 nucleotides in length, and most preferably 21-23 nucleotides in length. Thus, preferred siRNA molecules are 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 2728 or 29 nucleotides in length.

RNAi is a two-step mechanism. Elbashir et al., Genes Dev., 15:188-200, 2001. First, long dsRNAs are cleaved with Dicer enzyme at 21-23 ribonucleotide (nt) fragments called small interfering RNAs (siRNAs). The siRNA is then linked to a ribonuclease complex (called RISC, for RNA-induced silencing complex) that targets complementary mRNAs. Target mRNAs against complementary siRNAs are then cleaved by RISC, which can sensitize mRNAs to other RNA degradation pathways.

The siRNA designed in the present invention can interact with the target ribonucleotide sequence, that is, it is sufficiently complementary to a target sequence so as to be able to combine with the target sequence. The invention also includes siRNA molecules that have been chemically modified to increase stability against nuclease degradation but retain the ability to bind target nucleic acids where present.

C. Inhibition of ribozymes

The present invention provides ribozymes capable of binding a messenger that can inhibit the activity of a polypeptide (eg, inhibit a polypeptide having TLR2 activity such as TLR2 signal transduction activity or Scd1 activity) by targeting mRNA. Strategies for designing ribozymes and selectin-specific antisense sequences have been described in detail in the scientific and patent literature, and skilled artisans can use the reagents of the present invention to design such ribozymes.

A ribozyme acts by binding to a target RNA through its target RNA binding site, which is very close to the site of the enzyme that cleaves the RNA of the target RNA. In this way, the nucleic acid recognizes and binds to the target RNA through complementary base pairing, and once bound to the correct site, enzymatically cleaves and inactivates the target RNA. If this cleavage occurs within the coding sequence, cleavage of the target RNA by this means will destroy its ability to directly synthesize the encoded protein. After a ribozyme binds and cleaves its RNA target, it is usually released from that RNA, allowing it to repeatedly bind and cleave new targets.

In some cases, the enzymatic properties of ribozymes are superior to other technologies, such as antisense technology (in which a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation, or binding to other molecules), because of the The effective concentration of the ribozyme can be lower than the corresponding concentration of the antisense oligonucleotide. This potential advantage reflects the enzymatic capabilities of ribozymes. Thus, a single ribozyme molecule is capable of cleaving multiple target RNA molecules. In addition, ribozymes are generally highly specific inhibitors, and the specificity of their inhibition depends not only on the base-pairing mechanism of binding, but also on the mechanism by which the molecule is able to inhibit the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target, so specificity can be defined as the ratio of the cleavage rate of the target RNA to the cleavage rate of the non-target RNA. The cleavage mechanism also depends on additional factors regarding base pairing. Thus, the specificity of ribozyme action may be greater than that of an antisense oligonucleotide binding to the same RNA site.

The enzymatic ribozyme RNA molecule can be formed in a hammerhead motif, or in a hairpin, hepatitis delta virus, type I intron, or RNaseP RNA-like (associated RNA guide sequence) motif. Examples of such hammerhead motifs are described in Rossi, Aids Research and Human Retroviruses 8: 183, 1992; hairpin motifs are described in Hampel, Biochemistry 28: 4929, 1989, and Hampel, Nuc. Acids Res. 18: 299, 1990; See Perrotta, Biochemistry 31:16, 1992 for hepatitis delta virus motifs; Guerrier-Takada, Cell 35:849, 1983 for RnaseP motifs; and Cech U.S. Pat. No. 4,987,071 for type I intron motifs. References to these specific motifs are not intended to be limiting; those skilled in the art will appreciate that the enzyme-action RNA molecules of the present invention have specific substrate binding sites complementary to one or more target gene RNA domains, and upon substrate binding Within or around the site is a nucleotide sequence that confers RNA cleavage activity to the molecule.

treatment method

Also described herein is a prophylactic or therapeutic approach to treating a subject at risk (or predisposition) for a disorder or having a disorder associated with deleterious toll-like receptor 2 expression or activity, or Scd1 gene expression activity, or Scd1 gene product activity method.

prevention method

The present invention relates to a method for preventing a subject from harmful amounts of toll-like receptor 2 expression or activity, Scd1 gene expression or activity by administering to the subject a substance that modulates signal transduction through toll-like receptor 2, Scd1 gene expression activity, or Scd1 gene product activity Methods of preventing diseases or conditions associated with the expression or activity of the Scd1 gene product. Subjects at risk of having disordered or deleterious symptoms caused or induced by toll-like receptor 2 or Scd1-mediated signal transduction can be evaluated by any combination of diagnostic or pre-screening assays described herein or known in the art. identification. Generally, such disturbances involve deleterious activation of the innate immune system, for example, by Gram-positive bacterial infection. Administration of the formulation as a prophylactic agent may occur prior to the manifestation of symptoms such that the symptoms are prevented, delayed or reduced as compared to the symptoms in the absence of the formulation. In some embodiments, the formulation reduces ligand binding to toll-like receptor 2 and Scd1. In some embodiments, the agent reduces the binding of a ligand that binds toll-like receptor 2 and Scd1. Such suitable substances can be identified by the screening assays described herein. Generally, the substance specifically binds toll-like receptor 2 and/or Scd1 gene product.

treatment method

Another aspect of the invention relates to methods of modulating or activating TLR2 activity or Scd1 gene expression or Scd1 gene product activity for therapeutic purposes. The method may comprise contacting the cell with a substance that modulates the activity of one or more toll-like receptor 2 and/or Scd1 associated with the cell, for example, specifically binds to TLR2 or Scd1 or activates signaling through toll-like receptor 2 divert. The agent can specifically bind to toll-like receptor 2, Scd1 gene or Scd1 gene product and selectively activate TLR2 activity in lipopolysaccharide-induced cells, or can activate macrophage responses to Gram-positive bacteria . The agent may be an antibody or protein, a naturally occurring cognate ligand of the toll-like receptor 2 protein, a peptide, a peptide analog of the toll-like receptor 2 or Scd1 protein, a small non-nucleic acid organic molecule or a small inorganic molecule. These methods of modulation can be performed in vitro (eg, by culturing cells in the presence of the formulation) or, alternatively, in vivo (eg, by administering the formulation to a subject).

The invention provides a gram-positive bacterial infection or a gram-positive bacterial skin infection characterized by a disease or disorder (e.g., a lack of expression or activity of toll-like receptor 2 protein activity, Scd1 gene expression, or Scd1 gene product activity) ) treatment of affected individuals. In one embodiment, the method includes administering a therapeutic agent, such as a monounsaturated fatty acid or the like, eg, palmitoleate (palmitoleic acid) or oleate (oleic acid), or the like.

The present invention provides methods of treating individuals affected by diseases or disorders characterized by loss of expression or activity of toll-like receptor 2 protein activity, Scd1 gene expression, or Scd1 gene product activity. In one embodiment, the method comprises administering a substance (eg, a substance identified by a screening assay described herein) or combination of substances to increase signal transduction through toll-like receptor 2, or to increase Scd1 gene expression or Scd1 gene product activity. In cases where a subject is being treated for a Gram-positive bacterial infection, the formulation may also be used for treatment.

Other disorders treatable by the novel methods and compositions include fungal infections, sepsis, cytomegalovirus infection, tuberculosis, leprosy, bone resorption (e.g. in periodontal disease), arthritis (e.g. with Lyme arthritis) and viral hepatitis. Compounds capable of activating signal transduction through toll-like receptor 2 (eg, by activating Scd1 gene expression or Scd1 gene product activity) are also useful in the treatment of Gram-positive bacterial infections.

Disorders associated with Gram-positive bacterial infection can be successfully treated by techniques capable of activating binding to toll-like receptor 2, Scd1 gene expression or the Scd1 gene product. For example, compounds demonstrated to exhibit negative regulatory activity (e.g., substances identified by the assays described herein, such as antibodies) may be used to prevent and/or ameliorate symptoms of disorders caused by deleterious Scd1 gene product or toll-like receptor 2 activity . Such molecules may include, but are not limited to, peptides, phosphopeptides, small organic or inorganic molecules or antibodies (including, for example, polyclonal, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and F _ab , F ( _ab ') ₂ and _Fab expression library fragments, scFv molecules and their epitope-binding fragments). In particular, it can specifically bind to toll-like receptor 2 in lipopolysaccharide-induced cells and can regulate or activate Scd1 activity (Scd1 gene expression or Scd1 gene product), or regulate or activate macrophages in response to Gram-positive bacterial infection Antibodies and derivatives thereof (eg, antigen-binding fragments thereof) to which the cells respond.

Reagent test kit

The invention provides kits comprising compositions (eg, nucleic acids, expression cassettes, vectors, cells, polypeptides (eg, Scd1 polypeptides or toll-like receptor 2 signal-activating polypeptides)) and/or antibodies of the invention. The kit may also contain introductory material describing the methods and uses of the invention as described herein.

therapeutic application

Compounds and modulators identified by the methods of the invention are useful in a variety of therapeutic approaches. Accordingly, the present invention provides compositions and methods for treating infectious diseases, Gram-positive bacterial infections, toll-like receptor 2 signal transduction defects, Scd1 gene mutations or gene expression defects or Scd1 gene product defects.

Exemplary include, but are not limited to, Gram-positive bacterial skin infections, such as Streptococcus pyogenes infection or Staphylococcus aureus. Infection with the gram-positive coccus Streptococcus pyogenes or Staphylococcus aureus is the main bacterium responsible for human pustule, cellulite and wound infections.

Exemplary include, but are not limited to, viral or bacterial diseases. The polypeptides or polynucleotides of the invention are useful in the treatment or detection of infectious agents. For example, infectious diseases can be treated by increasing the immune response, particularly by increasing the proliferation and differentiation of B and/or T cells. Improving immune responses can be achieved either by enhancing existing immune responses or by initiating new immune responses. In addition, the polypeptides or polynucleotides of the invention can also be used to directly inhibit infectious agents without eliciting an immune response.

Similarly, bacteria or fungi capable of causing disease or symptoms that can be treated or detected by the polypeptides or polynucleotides of the present invention include, but are not limited to, the following Gram-negative and Gram-positive bacterial families and fungi: Filaments (eg, Corynebacterium, Mycobacterium, Nocardia), Aspergillus, Bacillus (eg, Anthrax, Clostridium), Bacteroides, Blastomycetes, Bordetella, Borrelia, Candida bacteria, Campylobacter, Coccidioides, Addictococcus, dermatophytes, Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionella, Leptospirosis , Listeria, Mycoplasma, Neisseria (e.g., Acinetobacter, gonorrhea, meningitis), Pasteur infection (e.g., Actinobacillus, Haemophilus, Pasteurella), Pseudomonas, Cretschia, Chlamydia, Syphilis and Staphylococcus. These families of bacteria or fungi can cause diseases or symptoms including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections ( For example, AIDS-related infections), paronychia, repair-related infections, Reiter's disease, respiratory infections such as whooping cough or empyema, sepsis, Lyme arthritis, cat scratch disease, dysentery, paratyphoid fever, food poisoning , typhoid fever, pneumonia, gonorrhea, meningitis, chlamydia, syphilis, diphtheria, leprosy, paratuberculosis, tuberculosis, lupus, botulism, gangrene, tetanus, impetigo, rheumatic fever, scarlet fever, sexually transmitted diseases , skin disorders (eg, cellulitis, dermatophytitis), hemorrhages, urinary tract infections, wound infections. The polypeptides or polynucleotides of the present invention can be used to treat or detect any of these symptoms and diseases.

In addition, parasites capable of causing disease or symptoms that can be treated or detected by the polypeptides or polynucleotides of the present invention include, but are not limited to, the following families: Amoeba, Babesia, Coccidia, Cryptosporidium, Dikaryotic amoebae, equine trypanosoma, ectoparasites, giardia, intestinal worms, leishmania, piroma therella, toxoplasma, trypanosoma and trichomonas. These parasites can cause a variety of diseases or conditions including, but not limited to: scabies, chiggers, eye infections, intestinal diseases (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections ( Examples include AIDS-related), malaria, pregnancy complications, and toxoplasmosis. The polypeptides or polynucleotides of the present invention can be used to treat or detect any of these symptoms and diseases.

Preferably, the treatment with the polypeptide or polynucleotide of the present invention can be performed by administering an effective amount of the polypeptide to the patient, or by taking the cells from the patient, providing the cells with the polynucleotide of the present invention, and Introducing the engineered cells back into the patient (ex vivo therapy). In addition, the polypeptides or polynucleotides of the present invention can be used as antigens in vaccines to elicit immune responses to infectious diseases.

Pharmaceutical composition formulation and administration

The invention provides pharmaceutical compositions comprising the nucleic acids, peptides and polypeptides (including Abs) of the invention. As discussed above, the nucleic acids, peptides and polypeptides of the invention can be used to activate the expression of an endogenous Scd1 gene or Scd1 polypeptide. Such activation in cells or non-human animals may form a screening feature for compounds capable of treating or ameliorating infectious diseases or Gram-positive bacterial infections. Administration of a pharmaceutical composition of the invention to a subject can be used to create a tolerogenic immune environment in the subject. This can be used to tolerize the subject to the antigen.

The nucleic acids, peptides and polypeptides of the present invention can be combined with pharmaceutically acceptable carriers (excipients) to form pharmaceutical compositions. The pharmaceutically acceptable carrier may contain physiologically acceptable compounds that have the functions of making the pharmaceutical composition of the present invention more stable, or increasing or decreasing its absorption or clearance rate. Physiologically acceptable compounds may include, for example, carbohydrates (such as glucose, sucrose or dextran), antioxidants (such as vitamin C or glutathione), chelating agents, low molecular weight proteins, that reduce the clearance of the peptide or polypeptide or Hydrolyzed compositions, or excipients or other stabilizers and/or buffers. Detergents, including lipid carriers, can also be used to stabilize the pharmaceutical composition or to increase or decrease its absorption. Pharmaceutically acceptable carriers and formulations for peptides and polypeptides are well known to those skilled in the art and are described in detail in the scientific and patent literature, see, for example, the latest edition of Remington's Pharmaceutical Science, Mack Publishing Company, Easton, Pa. ("Remington's").

Other physiologically acceptable compounds include wetting agents, emulsifying agents, dispersing agents or preservatives which are particularly effective against the growth or action of microorganisms. Various preservatives are widely known including, for example, phenol and vitamin C. Those skilled in the art can understand that the selection of pharmaceutically acceptable carriers including physiologically acceptable compounds depends on the route of administration of the peptide or polypeptide of the present invention and its specific physiological-chemical properties.

In one aspect, the nucleic acid, peptide or polypeptide of the invention can be dissolved in a pharmaceutically acceptable carrier, for example an aqueous carrier when the composition is water soluble. Examples of aqueous solutions of formulations that can be used for enteral, parenteral or transmucosal drug delivery include, water, saline, phosphate buffered saline, Hank's solution, Ringer's solution, dextrose/saline, glucose solution, and the like. The formulation may contain pharmaceutically acceptable auxiliary substances required to approximate physiological conditions, such as buffers, tonicity regulators, wetting agents, detergents and the like. Additives may also include additional active ingredients such as bactericides, or stabilizers. For example, the solution may contain sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitol monolaurate or triethanolamine oleate. These compositions may be sterilized by conventional, well-known sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as such or lyophilized, the lyophilized product being mixed with a sterile aqueous solution prior to administration. The concentration of peptide in these formulations can vary widely, the concentration being selected primarily based on fluid volumes, viscosities, body weight, etc. consistent with the particular mode of administration chosen and the needs of the patient.

Solid formulations are available for enteral (oral) administration. They can be formed into pills, tablets, powders or capsules etc. For solid compositions, conventional nontoxic solid carriers can be used, including pharmaceutical grade mannitol, lactose, starch, magnesium stearate, sodium saccharin, talc, cellulose, glucose, sucrose, magnesium carbonate and the like. For oral administration, pharmaceutically acceptable non-toxic compositions may consist of any commonly used excipients (such as the carriers listed above), and generally account for 10% to 95% of the active ingredient (eg, peptide). Non-solid formulations can also be used for enteral administration. The carrier can be selected from a variety of oils, including those of petroleum, animal, vegetable or synthetic origin, for example, peanut oil, soybean oil, mineral oil, sesame oil and the like. Suitable pharmaceutical excipients include, for example, starch, cellulose, talc, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, magnesium stearate, sodium stearate, glyceryl monostearate Esters, Sodium Chloride, Skim Milk Powder, Glycerin, Propylene Glycol, Water, Ethanol.

When the nucleic acids, peptides of the invention are orally administered, they may be protected from digestion. This can be done either by complexing the nucleic acid, peptide or polypeptide with a composition which makes it resistant to acid and enzymatic hydrolysis, or by packaging the nucleic acid, peptide or polypeptide in a resistant carrier such as liposomes. Methods of protecting compounds from digestion are well known in the art, see, for example, Fix, Pharm Res. 13:1760-1764, 1996; Samanen, J.Pharm.Pharmacol. 48:119-135, 1996; U.S. Pat. No. 5,391,377, which describes lipid compositions for oral delivery of therapeutic agents (liposomal delivery is discussed further below).

Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, appropriate penetrants for the barrier to be permeated may be employed in the formulation. Such penetrants are well known in the art and include, for example, for transmucosal administration, bile salts and fusidic acid derivatives. In addition, detergents can be used to facilitate penetration. Transmucosal administration can be accomplished by nasal spray or using suppositories. See, eg, Sayani, Crit. Rev. Ther. Drug Carrier Syst. 13:85-184, 1996. For topical, transdermal administration, this drug is available in ointments, creams, salves, powders, and gels, among others. Transdermal delivery systems may also include, for example, ointments.

Nucleic acids, peptides or polypeptides of the invention may also be administered using sustained delivery or sustained release mechanisms that deliver the formulation in vivo. For example, biodegradable droplets or capsules or other biodegradable polymeric structures capable of sustained delivery of peptides may be included in the formulations of the invention (see, eg, Putney, Nat. Biotechnol. 16:153-157, 1998).

For inhalation, systems known in the art including dry powder sprays, liquid delivery systems, jet nebulizers, propellant systems and the like can be used to deliver the nucleic acids, peptides or polypeptides of the present invention. See, eg, Patton, Biotechniques 16:141-143, 1998; in Dura Pharmaceuticals (San Diego, Calif.), Aradigrn (Hayward, Calif.), Aerogen (Santa Clara, Calif.), Inhale Therapeutic Systems (San Carlos, Calif. .) and other companies’ peptide macromolecule products and inhalation delivery systems. For example, the pharmaceutical formulation can be administered in aerosol or spray form. For aerosol application, the formulation may be presented in finely divided form with the surfactant and propellant. In another aspect, the device used to deliver the agent into the respiratory tissue is an inhaler in which the agent is vaporized. Another liquid delivery system includes, for example, a jet nebulizer.

In preparing the medicaments of the present invention, various methods of formulation modification can be employed to alter the pharmacokinetics and biodistribution. Numerous methods of altering pharmacokinetics and biodistribution are known to those of ordinary skill in the art. Such methods include, for example, protecting the composition with a carrier composed of proteins, lipids (eg, liposomes, see below), carbohydrates or synthetic polymers (discussed above). A general discussion of pharmacokinetics can be found in Remington's, Chapters 37-39 et al.

Nucleic acids, peptides or polypeptides of the invention may be delivered alone or as pharmaceutical compositions by any method known in the art, e.g., systemically, regionally, locally (e.g., directly into or directly to a tumor); as subcutaneous, tracheal Intraarterial, intrathecal (IT), intravenous (IV) , parenterally, intrapleurally, topically, orally, or topically. Actual methods of preparing compositions for administration are known or will be apparent to those skilled in the art and are described in detail in the scientific and patent literature, see, eg, Remington's. Modes of administration include intraarterial or intrathecal (IT) injection for "regional action", e.g., focusing on specific organs, e.g., focusing on specific organs, such as the brain and CNS (see e.g. Gurun, Anesth Analg. 85:317 -323, 1997). For example, carotid artery injection is preferred when direct delivery of a nucleic acid, peptide or polypeptide of the invention into the brain is desired. Parenteral administration is the preferred route of delivery when high systemic doses are required. Actual methods of preparing compositions for parenteral administration are known or will be apparent to those skilled in the art and are described in detail, for example Remington's, see also Bai, J Neuroimmunol. 80:65-75, 1997 ; Warren, J. Neurol. Sci. 152:31-38, 1997; Tonegawa, J. Exp. Med.

In one aspect, the pharmaceutical formulation comprising a nucleic acid, peptide or polypeptide of the invention is incorporated into a lipid monolayer or bilayer (e.g., liposomes), see, e.g., U.S. Pat. Nos. 6,110,490; 6,096,716; 5,283,185; 5,279,833 . The invention also provides formulations for linking a water-soluble nucleic acid, peptide or polypeptide of the invention to a monolayer or bilayer surface. For example, peptides can be linked to liposomes comprising hydrazide-PEG-(distearoylphosphatidylethanolamine) (see, eg, Zalipsky, Bioconjug. Chem. 6:705-708, 1995). Liposomes or any form of lipid membrane, such as planar lipid membranes or cell membranes of intact cells such as red blood cells, may be used. Lipid formulations can be administered by any means including intravenous, transdermal (eg, see Vutla, J. Phαrm. Sci. 85:5-8, 1996), transmucosal or oral administration. The present invention also provides pharmaceutical formulations in which nucleic acids, peptides and/or polypeptides of the present invention are incorporated into micelles and/or liposomes (for example, see Suntres, J.Pharm.Pharmacol.46:23-28, 1994; Woodle, Pharm. Res. 9:260-265, 1992). Liposomes and lipid formulations can be prepared according to standard methods and are also well known in the art, for example, see Remington's; Akimaru, Cytokines MoI. Ther. 1:197-210, 1995; Alving, Immunol. Rev. 145:5-31, 1995; Szoka, Ann. Rev. Biophys. Bioeng. 9:467, 1980, U.S. Pat. Nos. 4,235,871, 4,501,728 and 4,837,028.

In one embodiment, the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a sustained release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthophosphoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers. They can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Pat. No. 4,522,811.

The preparation of oral or parenteral compositions in dosage unit form will facilitate administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined in cell culture or experimental animals by standard pharmaceutical procedures, eg, determination of the _LD50 (the dose causing 50% death) and the _ED50 (the dose causing 50% therapeutic effect). The ratio between toxicity and efficacy is a therapeutic index and can be expressed as the ratio _LD50 / _ED50 . Compounds with high therapeutic indices are preferred. Although compounds that exhibit toxic side effects may also be used, care should be taken when designing delivery systems to target the compound to the site of action in the tissue to minimize damage to uninfected cells, thereby reducing side effects.

The data obtained from cell culture assays and animal experiments can be used in formulating a range of dosage for use in humans. The dosage of such compounds lies preferably within a range of circulating concentrations that include the _ED50 with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration. For any compound used in the methods of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. Dose can be extrapolated from animal models (eg, of disorders caused by inflammation or deleterious inflammation) to obtain circulating plasma concentration ranges that contain the _IC50 (ie, the concentration of the test compound which achieves a half-maximal inhibition of symptoms) measured in cell culture. Such information can be used to more accurately formulate useful doses in humans. The level in plasma can generally be determined by methods such as high performance liquid chromatography for markers. Animal models useful in assays (e.g. preclinical protocols) are well known in the art, e.g., as (Springer, Sent. Immunopathol. 25:35-45, 2003) and Nikula et al., Inhal. Toxicol. 4(12): 123, 53, 2000), and known in the art, for example, for fungal infection, sepsis, cytomegalovirus infection, tuberculosis, leprosy, viral hepatitis and infection (for example, caused by mycobacterium Infection-induced animal models of inflammatory disorders.

As specified herein, a therapeutically effective amount (i.e., an effective dose) of a protein or polypeptide such as an antibody ranges from about 0.001 to 30 mg/kg body weight, for example, about 0.01 to 25 mg/kg body weight, about 0.1 to 20 mg/kg body weight, or about 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6 mg/kg body weight. The protein or polypeptide can be administered daily or once a week for about 1 to 10 weeks (e.g., between 2 to 8 weeks, will be between about 3 to 7 weeks, or about 4, 5, or 6 weeks) Apply multiple times. In some instances, several months or more are required. The skilled artisan will appreciate that certain factors can affect the dosage and time required to effectively treat a subject, including, but not limited to, the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and the presence of other diseases. Furthermore, treatment of a subject with a therapeutically effective amount of a substance such as a protein or polypeptide (including antibodies) may include a single treatment, or preferably multiple treatments.

For antibodies, the dose is typically 0.1 mg/kg body weight (eg, 10 mg/kg body weight to 20 mg/kg body weight). Partially human antibodies and fully human antibodies generally have a longer half-life in the human body than other antibodies. Accordingly, lower dosages and less frequent administrations will generally be employed. Modifications such as lipidation can be used to increase antibody stability, enhance uptake and tissue penetration (eg, to the brain). A description of a method for lipidating antibodies can be found in Cruikshank et al., J. Acquired Immune Deficiency Syndromes and Human Retrovir'ology, 14:193, 1997).

The present invention encompasses agents or compounds capable of modulating Scd1 gene expression or Scd1 gene product or activity through toll-like receptor 2-modulated signal transduction. The agent can be a small chemical molecule or the like. Such small chemical molecules include, but are not limited to, peptides, peptide analogs (e.g., peptoids), amino acids, amino acid analogs, small non-nucleic acid organic or inorganic compounds (i.e., containing iso organic or organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

Exemplary include milligram or microgram amounts of small chemical molecules per kilogram of subject or sample weight (e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram. kg to about 50 micrograms per kg). It is further understood that the appropriate dosage of the small chemical molecule depends on the potency of the small chemical molecule for the expression or activity to be modulated. When one or more such small chemical molecules are administered to animals (for example, humans) to regulate the expression or activity of the polypeptides or nucleic acids of the present invention, doctors, veterinarians or researchers can first use a relatively low dose and then increase the dose until an appropriate response is obtained. Furthermore, it is understood that the particular dosage level for any particular animal subject will depend upon a variety of factors, including the activity of the particular compound employed, the subject's age, weight, general health, sex and diet, time of administration, route of administration, rate of excretion As well as the degree to which the drug combination and expression or activity need to be modulated.

An antibody or fragment thereof can be linked (eg, covalently and/or via a linker) to another therapeutic moiety (eg, a therapeutic agent or a radioactive metal ion) to form a conjugate. Therapeutic agents include, but are not limited to, antibiotics (eg, dactinomycin (formerly actinomycin), bleomycin, mithramycin, and antramycin (AMC)).

The conjugates described herein can be used to alter a given biological response. For example, the moiety to which an antibody binds can be a protein or polypeptide having the desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin A, pseudomonas exotoxin or diphtheria toxin; tumor necrosis factor, alpha-interferon, beta-interferon, nerve growth factor, platelet-derived growth factor, Tissue plasminogen activators; or, proteins such as biological response modifiers.

In addition, one is capable of binding to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Pat. No. 4,676,980.

The pharmaceutical composition can be contained in a container, pack or dispenser together with instructions for administration.

The compounds described herein are useful in the preparation of medicaments which can be employed in any of the methods of treatment described herein.

The pharmaceutical composition is formulated sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the US Food and Drug Administration.

Treatment options: drug metabolism

Depending on the mode of administration, the pharmaceutical compositions of the present invention can be administered in a variety of unit dosage forms. Dosages for typical nucleic acid, peptide and polypeptide pharmaceutical compositions are well known to those skilled in the art. Such dosage is usually indicative and can be adjusted according to the specific treatment background, patient tolerance and the like. The amount of nucleic acid, peptide or polypeptide sufficient to accomplish this is defined as a "therapeutically effective dose". The dosage schedule and amount effective for this use, the "administration regimen" will depend on various factors, including the stage of the disease or symptoms, the severity of the disease or symptoms, the general state of the patient's health, the patient's physical condition, age, drug formulation and active agent concentrations. The mode of administration should also be considered when calculating an administration regimen to a patient. The administration regimen should also take into account the pharmacokinetics, ie, the rate of absorption, bioavailability, metabolism, clearance, etc. of the pharmaceutical composition. See, eg, the latest edition of Remington's; Egleton, Peptides 18:1431-1439, 1997; Langer, Science 249:1527-1533, 1990.

In therapeutic applications, the composition is administered to a patient suffering from autoimmune disease, infectious disease, antigen presenting cell deficiency or CD4 cell deficiency in an amount sufficient to partially suppress symptoms or disease and/or its complications. For example, in one aspect, the soluble peptide pharmaceutical composition is administered at a dose of about 0.01 mg/hr to about 1.0 mg/hr for intravenous (IV) administration for several hours (usually 1, 3 or 6 hours), which can be Repeat in intermittent cycles for several weeks. Largely increased doses (e.g., adjusted to about 10 mg/ml) may also be used, especially when administered at an isolated site without entering the bloodstream (e.g., into a body cavity or lumen of an organ, e.g., cerebrospinal fluid (CSF )).

The following examples of specific embodiments for carrying out the invention are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way.

Example

Example 1

Flake: a visible phenotypic variant with associated immunodeficiency

ENU-induced germline mutations were used in an effort to identify genes required for general immune function. A total of 20,792F1 and 33,202F3 animals were screened for visible and immune phenotypes. Among them, a recessive mutation known as "flake" (flk) was found to cause progressive hair loss and chronic exfoliative dermatitis. These features appear at weaning age and are more pronounced in older animals (Figure 1). Visible disruption of epidermal integrity and spontaneous skin infections requiring antibiotic treatment prompted us to examine the integrity of innate immune function in these mice.

Figure 1 shows the visible phenotypes observed in flake mutant mice. A. 6 week old mouse B. 8 month old mouse C. Ocular infection in 8 month old mouse D. Magnification of mouse in B to highlight severe dermatitis.

Example 2

Persistent Streptococcus pyogenes and Staphylococcus aureus skin infections in flk/flk mutant mice

mutant mice

The gram-positive cocci Streptococcus pyogenes and Staphylococcus aureus are the main pathogens of impetigo, cellulitis and wound infections in humans. Guay, Expert. Opin. Pharmacother. 4:1259-1275, 2003; Hedrick, Paediatr. Drugs 1:35-46, 2003. Experimental full-thickness skin infection in murine models can be reliably established by direct subcutaneous injection with fine-pitch needles, thereby overcoming the need for trauma and poor infectivity and reproducibility associated with patch vaccination. Bunce et al., Infect. Immun. 60:2636-2640, 1992; Kraft et al., Infect. Immun. 52:707-713, 1986; Nizet et al., Nature 414:454-457, 2001.

Luminescently labeled S. pyogenes and S. aureus and E. coli strains, each constitutively expressing the bacterial luciferase gene operon from Achromobacter, can be used. Kuklin et al., Antimicrob Agents Chemother 47:2740-8, 2003. The course of infection can be observed in anesthetized mice within 16 days by external luminescence detection. As shown in Figure 2A, it took 8 days for normal C57BL/6 mice to completely clear the skin infection initiated by ^5x105 cfu of S. pyogenes inoculation. The flk/flk mutant mice showed similar microbial clearance kinetics within the first 6 days after inoculation, but after that the microbial load in the flk/flk mutants began to differ from control values, rising to a high value and in This value is maintained throughout the entire test thereafter. Luminescence in flk/flk mutants slowly decreased to background levels 4 weeks after inoculation.

S. pyogenes produced a small ulcerated wound that healed almost completely by day 8 in control mice. Ulcers could still be observed in flk/flk mutant mice after 28 days, although there was no detectable luminescence in vivo. Luminescent S. pyogenes can be recovered by culturing ulcers of flk/flk mutants. Thus, flk/flk mutant mice were persistently infected with S. pyogenes even 4 weeks after experimental inoculation.

The results obtained from S. aureus infection (Fig. 2B) were formally consistent with the above results. Bacterial loads of flk/flk mutants closely matched controls at the beginning of the observation period, but a divergence of the two curves was observed at day 7 post-inoculation, where clearance was gradually achieved in control animals (but not in flk/flk mutant realized in vivo), and the control was completely cured within 2 weeks. In contrast, the luminescence of flake mice remained strongly detectable after 3 weeks and reached background levels 4 weeks after inoculation.

On the other hand, the flk/flk mutant was able to clear infection by the Gram-negative bacterium E. coli (Fig. 2C). No difference was observed between flk/flk mutants and normal controls when Gram-positive infection was elicited by other routes (eg, by intravenous inoculation of L. monocytogenes, or intrapulmonary challenge with S. aureus). On the basis of the data obtained from these experiments, it can be seen that: 1. the ability of flk/flk mutant mice to kill Gram-positive skin infections is disrupted; 2. This phenotype does not extend to all biological divisions, and may be limited to the skin; 3. The isolated Gram-negative infection detected was not recognized by the mutant; 4. The fact that flk/flk mice can normally heal skin lesions caused by E. coli suggests that the mutation does not affect Wound healing per se, but with a selective effect on pathogen clearance.

Figure 2 shows the persistent skin infection developed by flake mutant mice upon exposure to Gram-positive bacteria. A. Time course analysis of bacterial growth in control (C57BL/6, n=4) and mutant (flake/flake, n=4) animals infected subcutaneously with S. pyogenes. The upper panel shows the graph after quantification of luminescence (expressed as a percentage of initial inoculation) in 4 animals of each genotype. The lower panel shows the image overlay and light detection of two representative mice of each genotype 1, 6, 8 and 14 days after inoculation. B. Infected with Staphylococcus aureus. Images show infected animals at 1, 6, 9 and 15 days. C. Infection with E. coli.

Example 3

Mapping of the FLK mutation at the stearoyl-CoA desaturase 1 site

The visible phenotype conferred by flk was used for localization, and concordance between the visible and immunophenotype was subsequently established by examining the progeny of crossed F1 mice and other allelic variants at this locus, flk A set of 59 informative markers distributed throughout the mouse genome were initially located on chromosome 19 in 39 gametes in a backcross to C3H/HeN. This phenotype was fully penetrated in mixed backgrounds, and the mutation was placed between markers D19Mit96 and D19MM7 (Fig. 3A). Twelve internal chromosome 19 markers were then used for precise mapping, thereby confining the mutation to a 2.6 Mbp critical region bounded by D19Mitl1 and D19Mit53 on the 283rd gamete. Of the 43 genes shown in this region of the Ensembl database (Fig. 3C), the stearoyl-CoA desaturase 1 (Scdl) gene was considered a possible candidate because of the names asebia-J and asebia Both mutant alleles of -2J have been described as Scd1 and in both cases mutant mice display a skin phenotype called "squamous skin" similar to that observed in flk homozygotes. Sundberg et al., Am J Pathol 156:2067-75, 2000; Zheng et al., Nat Genet 23:268-70, 1999.

Figure 3 shows the location of the flake mutation. A. Transgene log-likelihood ratio (Lod value, Z) analysis shows a single peak of linkage on mouse chromosome 19. A total of 59 informative markers (horizontal axis) were included in the analysis, and 39 gametes (19 wild type and 17 mutant animals) were genotyped for all markers. B. Precise mapping of the terminal region of chromosome 19. Analysis of a total of 283 gametes (3 representatives of which are shown) confined the flake mutation to 2 adjacent markers separated by 2.6 Mb. C. Gene structure of the flake locus according to the ENSEMBL database. The Scd1 gene is highlighted.

The six exons of Scd1 can be amplified from genomic DNA isolated from C57BL/6 control mice and flk/flk mutants Direct sequencing of the amplicon revealed a mutation point (C to A) in exon 5 corresponding to position #938 in the cDNA sequence (accession number BC055453, see Figure 4A). It was predicted that ENU-induced base conversion may cause a missense mutation (T227K) in SCD1. No mutations were detected in Scd2 and Scd3 cDNA.

The microsomal enzyme SCD1 is an ion-binding 41 kDa protein of 355 amino acids with six predicted transmembrane domains. It catalyzes the Δ9 desaturation of long-chain unsaturated fatty acids, leading to the biosynthesis of palmitoleate (C16:1) and oleate (C18:1) as its major products. As shown in Figure 4B, the substitution of a neutral amino acid (T) for a charged residue (K) in the mutein occurs within the predicted transmembrane region and is predicted to disrupt the structural integrity of SCD1.

Figure 4 shows the molecular characterization of the flake mutation. A. Amplified genomic DNA trace files from homozygous flake mutant mice (top panel) and normal animals (bottom panel). B. Schematic representation of the localization of the SCD1 protein and flake mutation. Blue boxes correspond to the transmembrane domains predicted by SMART analysis.

To test this hypothesis, thin layer chromatography (TLC) analysis of the lipid composition of skin sections from control and flk/flk mice was performed. Similar to what was reported for Scdl KO, the latter animal showed a decrease in cholesteryl esters (Fig. 5A), suggesting that the flk phenotype was in fact caused by the observed Scdl allelic variant.

Figure 5 shows TLC analysis of lipid content in wild-type and flake mutant mice. A. TLC of lipids extracted from skin sections of wild-type (B6) or flake (flk) mutant mice. B. TLC of lipids purified from the skin of wild-type mice (B6+) 1 hour or 24 hours after subcutaneous infection with S. aureus. M: marker, Cs: cholesterol, TG: triglyceride, CE: cholesteryl ester.

Example 4

Palm oleate and oleate with intrinsic antimicrobial activity in vitro and in vivo

The loss of C18 and C16 fatty acid desaturase activity in Scd1 ^flk/flk mutant mice prompted us to consider whether loss of oleate and/or palmitoleate could explain the immunodeficiency phenotype in the skin described above. In fact, although there is no evidence that MUFAs exert protective effects in vivo, many reports indicate that MUFAs exhibit antibacterial activity against Gram-positive bacteria. Miller et al., Arch Dermatol 124:209-15, 1988; Wille and Kydonieus, SkinPharmacol Appl Skin Physiol 16:176-87, 2003. To test this working hypothesis, a series of in vitro experiments were first performed in which the effect of various lipids on the growth of S. pyogenes, S. aureus and Escherichia coli was examined.

This result confirms that both palm oleate and oleate have strong bacteriostatic and bactericidal activities against Streptococcus pyogenes and Staphylococcus aureus. The minimum inhibitory concentrations (MICs, see Table 1) for both compounds were in the micromolar range and were comparable to those observed with murine cecropin AMP (CRAMP). Relative to weight, MUFAs are approximately 20 times more potent than cecropins. MUFA is also active against Staphylococcus aureus, while CRAMP is completely inactive against it. On the other hand, no bacteriostatic or bactericidal activity was observed even at millimolar MUFA concentrations, consistent with a specific effect on Gram-positive bacteria.

Table 1. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MGBC) of cecropin (CRAMP), oleic acid and palmitoleic acid against Streptococcus pyogenes and Staphylococcus aureus expressed in μM

Values represent mean of 3 experiments, nd, not tested

To investigate the physiological relevance of this antibacterial activity, wild-type mice were inoculated with Staphylococcus aureus, and infected animals were repeatedly treated with palmitoleate (100 μl of 100 μM in DMSO) or DMSO alone at the site of infection (per Two days) subcutaneous injection for treatment. The results of this experiment are shown in Figures 6A and B. Luminescence was expressed as a percentage of the initial inoculum for two groups of mice (n=6 animals) and was measured 24 hours after infection. Nine days after S. aureus inoculation, palm oleate-treated animals showed a 90% reduction in luminescence compared to vehicle-treated mice. As a result of the increased clearance of S. aureus, the diameter of ulcerated wounds (measured at day 9) in lipid-treated animals was one-fourth that of controls (Fig. 6C). These data, which clearly show the in vitro and in vivo antibacterial activity of MUFAs, also reveal that this lipid-based defense mechanism is not maximally effective in normal mice.

Under similar palm oleate application conditions, the flake mutant showed a marked reduction in bacterial growth between days 1 and 4 (also observed in wild-type mice), but S. Still measurable after 2 weeks. As shown in Figures 6D and E, higher doses of palmitoleic acid (100 μl of a 75 mM solution) moderately improved bacterial clearance and subsequent ulcer healing in flk/flk mutants (Figure 6F). However, this drug therapy did not achieve complete rescue of this phenotype.

Figure 6 shows that palmitoleic acid has antibacterial activity in vivo. A. Palmitoleic acid injection accelerates bacterial clearance in wild-type mice. Luminescence (indicated as initial inoculum) of control (C57BL/6) mice inoculated with S. aureus (on day 0) and treated with vehicle (DMSO) or palmitoleate injections (arrows) every two days A percentage of ) was measured. B. Pictures of control (C57BL/6) mice treated with DMSO (top) or palmitoleate (bottom) injections 9 days after S. aureus infection. C. Bar graph showing lesion size measured in control (B6) mice treated with DMSO or palmitoleate 9 days post infection. ** means P value < 0.01. D. Palmitoleate treatment of S. aureus-infected flake mice. The protocol was similar to A except that 75 mM palmitoleate was used for 100 μl injections. E. Photographs of infected flake mice treated with DMSO (top) or palmitoleate (bottom) for 12 days. F. Lesion size (measured at 12 days) in infected flk mutants treated with DMSO or palmitoleate. * means P value < 0.05.

Example 5

TLR2-dependent Scd1 transcriptional activation during Gram-positive bacterial infection

The undoubted antibacterial function of MUFA in vivo prompted us to consider whether its synthesis is enhanced in the immune response, similar to the case of other effector molecules such as CRAMP, we analyzed a 5kb fragment of the Scd1 promoter and noticed multiple NF Presence of -κB binding site (Fig. 7A). Semi-quantitative RT-PCR experiments on skin sections from normal or infected mice were performed. Figure 7B shows that Scd1 mRNA accumulation was strongly induced in the skin of control (C57BL/6) mice under S. aureus infection, whereas E. coli inoculation had no effect. Furthermore, the Scd1 gene for mice with a site-mutated Tlr2 gene (Tlr ^−/− ) did not respond to inoculation with Gram-positive bacteria. However, assuming a 24-h lag between infection and RNA isolation, Scd1 transcriptional induction may also be caused by an indirect mechanism.

Figure 7 shows infection and TLR2-dependent induction of Scdl gene expression in mice. A. Signal scanning analysis of the Scd1 promoter. NF-κB and ISRE (Interferon-stimulated regulatory element) are shown. B. In uninfected control (C57BL/6, lane 1) and Tlr2-/- (lane 4) animals or infected with S. aureus (lanes 2 and 5) or E. coli (lane 3) Post-RT-PCR detection of Scd1 and β-actin transcription. PCR products after 30 and 40 cycles are shown. M, standard size. C. RT-PCR detection of Scd1 and β-actin transcription in control (0) and MALP-induced peritoneal macrophages isolated from wild-type mice after 2, 4 and 18 hours. D. Quantification of the Scdl/β-actin ratio.

According to recent reports, macrophages, which represent an ideal system for studying TLR signaling, can also express the Scd1 gene. Uryu et al., Biochem Biophys ResCommun 303:302-5, 2003. To test whether isolated macrophages are able to upregulate Scd1 and to examine the kinetics of this response, they can be stimulated with a known TLR2 agonist, synthetic macrophage-activating fatty acyl peptide (MALP-2, EMC microcollections GmbH, Germany). Peritoneal macrophages isolated from wild-type mice. Takeuchi et al., J Immunol 164:554-7, 2000. Scd1 expression was examined by RT-PCR on isolated RNA samples at 2, 4, 8 and 18 hours after stimulation. As shown in Figure 7C and D, Scd1 expression started to increase 2 hours after MALP induction and reached a 4-fold within 18 hours. This transcriptional induction of Scd1 was associated with increased lipid synthesis in the skin of infected animals (see Figure 5B).

As noted previously, Scd1 is predominantly expressed in the sebaceous glands, and flake, asebia and Scd1 KO mice exhibit atrophy of these structures. To demonstrate the potential association of inducible Scd1 expression in human skin defenses with Gram-positive pathogens, the role of MALP-2 was examined in the immortalized sebocyte cell line SZ95. Zouboulis et al., J. Invest Dermatol. 113:1011-1020, 1999. First, MALP-2, but not LPS treatment, induced a rapid and potent inflammatory response, shown by increased IL-6 and IL-8 production (Fig. 8A and B). Subsequently, upregulation of SCD1 transcription was also observed after 4 hours of MALP-2 stimulation in this human cell line (Fig. 8C and D). These observations were amplified by monitoring the expression of the fatty acid desaturase (FADS2) gene. FADS2 encodes a protein with similar enzymatic properties to SCD1 and was recently shown to be absent in a severe skin disorder manifested by chapped lips, keratotic rashes on the arms and legs, and perineal dermatitis. Williard et al., J. Lipid Res. 42:501-508, 2001. In human sebocytes, FADS2 was slightly but specifically induced by MALP-2 stimulation for 18 hours.

Figure 8 shows the inflammatory response and up-regulation of SCD1 and FADS2 genes in human sebocytes stimulated with MALP-2. A. IL-6 production in SZ95 cells after treatment with MALP-2 (50 ng/ml). LPS (100 ng/ml) stimulation showed minimal effect. B. Quantification of IL-8 under the conditions of A. C. RT-PCR detection of SCD1 and FADS2 expression 4 and 18 hours after LPS and MALP-2 stimulation. GAPDH expression was used as a control. D. SCD1 and FADS2 signals measured in two independent experiments (+/- s.e.m) normalized to GAPDH signal.

Example 6

A Toll-like receptor 2-responsive lipid-effector pathway that equips mammalian defense against Gram-positive bacterial skin infection

SCD1 is an enzyme responsible for the biosynthesis of MUFAs, mainly palmitoleate (C16:1) and oleate (C18:1). Ntambi, Prog Lipid Res 34:139-50, 1995. It catalyzes the Δ9 cis desaturation of carbon chains and uses palmitoyl-CoA and stearoyl-CoA as substrates. The function of this enzyme in lipid metabolism has been intensively studied. Ntambi and Miyazaki, Prog Lipid Res 43:91-104, 2004. Scd1 ^-/- mice were significantly leaner than wild-type animals and protected against diet-induced obesity, an effect mediated by increased expression of genes involved in fatty acid oxidation. Furthermore, compounds homozygous for hypomorphic mutations in the obese (ob) and Scd1 genes showed a significant reduction in the obese phenotype. Ntambi et al., Proc Natl Acad Sci 99:11482-6, 2002. The overexpression of Scd1 in ob mutants suggests that at least part of the leptin metabolism is caused by inhibition of Scd1. Cohen et al., Science 297:240-3, 2002. Two spontaneous mutant alleles of Scd1 have been described and named asebia(ab)-J and -2J. Sundberg et al., Am J Pathol 156:2067-75, 2000; Zheng et al., Nat Genet 23:268-70, 1999. Although phenotypic differences were small, homozygosity for each of these alleles was associated with the phenotypes observed in mice carrying a targeted disruption of this gene, including sebaceous atrophy, alopecia, and scaly skin. Miyazaki et al., J Nutr 131:2260-8, 2001.

Presented in this study is a flake mutation - a visible recessive phenotypic variant with a highly selective innate immunodeficiency phenotype that does not clear Gram-positive (rather than Gram-positive) from the skin. Lang's negative) organisms. Using phenotype-driven therapy, flk mutations were traced to an antisense error (T227K) within the fourth region of the six transmembrane domains of the SCD1 protein. Substitution of neutral amino acids with charged residues in this region may alternatively modify the configuration of the desaturase, which is normally located within the microsomal membrane, or affect the coordination of iron atoms required for enzyme activity. Regardless of the mechanism, a reduction in the level of cholesteryl esters, whose synthesis requires MUFA, was shown in lipids isolated from the skin of flake mutant mice, confirming the atrophic function of the new allele.

Here described are the products of SCD1 and its catalytic activity for epithelial innate immunity against Gram-positive bacteria. It was shown that feeding Scd1-deficient mice on a MUFA-enriched diet did not reduce the mutant phenotype, suggesting that de novo synthesis of MUFA is required for normal skin appearance and function. Therefore, to extend the in vitro observations, the effect of intradermal administration of palmitoleate to S. aureus-infected mice was monitored. These in vivo experiments showed that repeated subcutaneous injections of palmitoleate reduced bacterial expansion and significantly improved recovery in infected mice (as evidenced by a reduction in ulcerated wounds). However, this beneficial effect of palmitoleate remained less pronounced in flake mutants even with repeated injections of higher doses of palmitoleate. The hyperactivated lipid catabolism observed in Scd1 mutants may contribute to the shortened half-life of injected lipids and explain this difference. However, it has been noted that persons dealing with acne problems with retinoids (which induce atrophy of the sebaceous glands) may suffer from a side effect - recurrent Staphylococcus aureus infections. Leyden et al., J Invest Dermatol 86:390-3, 1986. Ocular Gram-positive bacterial infections have also been observed in such patients. Egger et al., Ophthalmologe 92:17-20, 1995. In fact, ocular infection was also observed in flake mutants as shown earlier in Scd1 KO mice (see Figure 1C). Miyazaki et al., J Nutr 131:2260-8, 2001. The data obtained from flk/flk mice emphasize the important role of the sebaceous glands, as well as other lipid-producing organs, possibly including the specialized meibomian glands of the eyelids, in the innate immune response,

The mechanism by which MUFA selectively lyses Gram-positive bacteria remains to be determined. The length of the carbon chain and/or the level of unsaturation may be an important determinant of efficacy. In addition, synergy between lipids and AMPs can also be determined. The Flake/CRAMP double knockout mouse will prove to be a useful tool to study this question. This trial does not rule out the possibility that palm oleate and oleate may indirectly promote tolerance in addition to antimicrobial activity. Regulation of signal conversion through protein modification may be one of the mechanisms. Mass spectrometry can identify palmitoleate from post-translational modifications in the other src homology domain 3 kinase, Fyn, which may affect immune cell activation, as reported recently in insulin signaling in muscle cells. Liang et al., J Biol Chem279:8133-9, 2004; Rahman et al., Proc Natl Acad Sci 100:11110-5, 2003.

SCD1 transcription is strongly upregulated in a TLR-2-dependent manner in mouse and human cells. Human patients with rare skin disorders such as ichthyosis follicularis with atrichosis and photophobia (IFAP syndrome, OMIM 308205) have atrophic sebaceous glands, alopecia with occasional Flake phenotype and recurrent skin infections (Reviewed in Alfadley et al., Pediatr. Dermatol. 20:48-51, 2003). In light of the recent recognition of the expression of TLR2 and 6 in human sebaceous glands (Zouboulis et al., in preparation), this result points to a significant and unambiguous role of the sebaceous gland in the innate immune defense of the skin. Altogether, the data demonstrate the existence of an inducible lipid-based bactericidal effector pathway in the skin and establish a clear link between lipid metabolism and innate immunity.

Example 7

Materials and methods

mice. Germline mutation using N-ethylN-nitrosourea (ENU) has been described in Hoebe et al., J Endotoxin Res 9:250-5, 2003. Animals were housed under pathogen-free conditions in the animal room of the Immunology Division of the Scripps Research Institute. The mice used in this experiment were all 8-12 weeks old. The handling of mice and the conduct of the experimental process were carried out in accordance with the institutional guidelines for animal care and use.

bacteria. S. aureus Xen8.1 (parental strain 8325-4), S. pyogenes Xen20 (serotype M49 derived from strain 591), and E. coli Xen14 (derived from EPECWS2572) were all obtained from Xenogen (Carnbury, NJ).

cell culture. SZ95 sebocytes with/without 50ng/ml MALP-2 or 100ng/ml LPS supplemented with 10% heat-inactivated FCS, 5ng/ml human epidermal growth factor, 1mM CaCl ₂ , 10 ^-5 M palmitic acid, 50μg/ HSG-Med (Sebomed, Berlin, Germany) with ml gentamicin was cultured for 2, 4, 8, and 18 hours, and the supernatant was collected to measure IL6 and IL8 by ELISA. RNA was isolated from 4 and 18 h samples using the RNeasy Midi kit (Qiagen, Hilden, Germany) and purified with RNase-Free DNaseset (Qiagen) for RT-PCR.

reagent. Both palmitoleic acid and oleic acid were purchased from Sigma. S. minesota Re595LPS was purchased from Alexis (Carlsbad, CA) and MALP-2 was purchased from EMC microcollections GmbH (Tubingen, Germany).

skin infection. Bacterial cultures were centrifuged in exponential phase and resuspended in 10 volumes of PBS containing inert Cytodex beads (Sigma) at 10 mg/ml as carrier. Approximately 100 μl of 5×10 ⁵ cfu of luminescent bacteria were injected subcutaneously in the back of anesthetized animals. Chemical hair removal is used to remove hair before vaccination. Daily luminescence detection was performed with a CCD camera (exposing the animals for 5 minutes) and quantification was performed with Xenogen's IVIS program.

TLC. Total lipids extracted from skin sections using chloroform/methanol can be separated by TLC on silica gel. Using hexane/diethyl ether/acetic acid (70:30:1) as a developer, lipids were visualized under UV light after spraying a primrose solution (5 mg, dissolved in 100 ml acetone/water, 80/20).

Semi-quantitative RT-PCR. Wild-type and Tlrr2 ^−/− mutant mice were shaved and injected subcutaneously with S. aureus or E. coli (5×10 ⁵ pfu). After 24 hours, the skin of the infected area was incised and total RNA was extracted by the Trizol (Gibco) method. 1 μg of RNA was used to synthesize cDNA primed with oligo-dT, which was then used as a template for a PCR reaction using a pair of Scd1 (3'-ctctatggatatcgcccctacgacaagaacattc-5' at exon 5 and 3' at exon 6). '-gaagctaggaacaaggagggatgtattcaggagg-5', which allows differentiation of genomic and cDNA amplification) or β-actin gene-specific primers. 4 μl of the PCR reaction was loaded onto an agarose gel. Isolation and stimulation of peritoneal macrophages has been described elsewhere. Hoebe et al., J Endotoxin Res 9:250-5, 2003. The expression of hSCD1 and hFADS2 in SZ95 sebocytes can be detected by semi-quantitative RT-PCR using the following oligonucleotides:

hSCD1f 5′-TTCAGAAACACATGCTGATCCTCATAATTCCC-3′,

hSCD1r 5′-ATTAAGCACCACAGCATATCGCAAGAAAGTGG-3′

hFADS2f5'-ACTTTGGCAATGGCTGGATTCCTACCCTC-3'

hFADS2r5'-ACATCGGGATCCTTGTGGAAGATGTTAGG-3'

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was used as a control.

All publications and patent applications cited in this specification are incorporated by reference in their entirety for all purposes as if each individual publication and patent application were specifically and individually indicated to be incorporated by reference for all purposes.

Although the foregoing invention has been described in detail by illustrations and examples for illustrative purposes, those of ordinary skill in the art should be able to readily understand that certain modifications can be made thereto without departing from the spirit and scope of the appended claims based on the teachings of the present invention. Changes and adjustments.

Claims (68)

CLAIMS 1. A method of treating a Gram-positive bacterial infection in a mammalian subject comprising administering to the subject an effective amount of a compound capable of activating expression of the Scd1 gene. 2. The method of claim 1, wherein the compound is an agonist of toll-like receptor 2. 3. The method of claim 1, wherein said compound is a small chemical molecule, antibody, antisense nucleic acid, short hairpin RNA, or short interfering RNA. 4. The method of claim 1, wherein the Gram-positive bacterial infection is a Streptococcus pyogenes infection or a Staphylococcus aureus infection. 5. The method of claim 2, wherein said subject has a loss-of-function or down-function mutation of the Scd1 gene. 6. A method of treating a Gram-positive bacterial infection in a mammalian subject comprising administering to the subject an effective amount of a compound that activates the Scd1 gene product. 7. The method of claim 6, wherein said compound is an agonist of toll-like receptor 2. 8. The method of claim 6, wherein the compound is a small chemical molecule, an antibody, an antisense nucleic acid, short hairpin RNA, or short interfering RNA. 9. The method of claim 6, wherein the Gram-positive bacterial infection is a Streptococcus pyogenes infection or a Staphylococcus aureus infection. 10. The method of claim 7, wherein said subject has a loss-of-function or down-function mutation of the Scd1 gene. 11. A method of treating a Gram-positive bacterial infection in a mammalian subject comprising administering to the subject an effective amount of a monounsaturated fatty acid. 12. The method of claim 11, wherein the monounsaturated fatty acid is palmitoleate or oleate. 13. The method of claim 11, wherein the Gram-positive bacterial infection is a Streptococcus pyogenes infection or a Staphylococcus aureus infection. 14. The method of claim 11, wherein the administration of the effective amount of monounsaturated fatty acid is topical or intradermal. 15. The method of claim 11, wherein the effective amount of monounsaturated fatty acid is administered intramuscularly, subcutaneously, intraperitoneally or intravenously. 16. A method of treating a Gram-positive bacterial infection in a mammalian subject comprising administering to the subject an effective amount of a compound that is a product of the Scd1 biosynthetic pathway. 17. The method of claim 16, wherein said compound is a monounsaturated fatty acid. 18. The method of claim 17, wherein the monounsaturated fatty acid is palmitoleate or oleate. 19. The method of claim 16, wherein the Gram-positive bacterial infection is a Streptococcus pyogenes infection or a Staphylococcus aureus infection. 20. The method of claim 16, wherein the administration of the effective amount of monounsaturated fatty acid is topical or intradermal. 21. The method of claim 16, wherein the effective amount of monounsaturated fatty acid is administered intramuscularly, subcutaneously, intraperitoneally or intravenously. 22. A method of identifying a compound that modulates intracellular gram-positive bactericidal activity, comprising: Exposure of the test compound to a cell-based assay system comprising cell-expressed toll-like receptor 2, providing the assay system with an amount of the ligand selected to be effective in activating toll-like receptor 2 signaling, wherein toll-like receptor 2 signaling transduces a response to the ligand and modulates Scd1 gene expression, and The effect of the test compound on toll-like receptor 2 signaling and regulation of Scd1 gene expression was detected, and the effectiveness of the test compound in this assay was taken as an indication of Gram-positive bactericidal activity. 23. The method of claim 22, wherein the ligand is an endogenous ligand or an exogenous ligand. 24. The method of claim 23, wherein said exogenous ligand is lipopolysaccharide, lipid A, diacyl fatty acyl peptide, triacyl fatty acyl peptide, S-MALP-2, R-MALP- 2. Bacterial fatty acylpeptides, Pam2CSK4, lipoteichoic acid or zymosan A. 25. The method of claim 24, wherein the exogenous ligand is S-MALP-2 or R-MALP-2. 26. The method of claim 23, wherein the exogenous ligand is rough lipopolysaccharide, smooth lipopolysaccharide, or lipid A from Salmonella Minnesota. 27. The method of claim 23, wherein said testing step further comprises detecting activation of Scd1 gene expression or Scd1 gene product in cells, wherein Scd1 gene expression or Scd1 gene product responds to interaction between cells and exogenous ligands activated by contact. 28. The method of claim 27, wherein the exogenous ligand is a component of a gram-positive bacterium other than a gram-negative bacterium. 29. The method of claim 23, wherein said endogenous ligand is a lipid. 30. The method of claim 22, wherein said compound is an agonist of toll-like receptor 2 pathway signaling. 31. The method of claim 22, wherein said testing step further comprises detecting the binding of the compound-enhanced ligand to toll-like receptor 2. 32. The method of claim 22, wherein said testing step further comprises detecting an elevated Scd1 gene product in a cellular assay. 33. The method of claim 22, wherein said testing step further comprises detecting increased Scd1 gene product activity in a cellular assay. 34. The method of claim 22, wherein said testing step further comprises detecting increased monounsaturated fatty acid synthesis in a cellular assay. 35. The method of claim 22, wherein said analysis of cells further comprises macrophages. 36. The method of claim 22, wherein said cell analysis further comprises cells from sebaceous glands. 37. The method of claim 36, wherein said cell analysis further comprises sebocytes. 38. The method of claim 22, wherein said detecting step further comprises detecting binding of the labeled ligand to toll-like receptor 2. 39. The method of claim 38, wherein the labeled ligand is radiolabeled or fluorescently labeled. 40. The method of claim 22, further comprising providing the toll-like receptor 2 to the analysis system, and detecting the effect of the test compound on the signal transduction of the toll-like receptor 2 in the analysis system, the effect of the test compound in the analysis Effectiveness is taken as an indication of conditioning. 41. The method of claim 22, wherein said detecting step further comprises reducing ligand binding to toll-like receptor 2 by said compound. 42. The method of claim 22, wherein said detecting step further comprises enhancing ligand binding to toll-like receptor 2 by said compound. 43. The method of claim 22, wherein said testing step further comprises detecting an increase in stearoyl-CoA desaturase 1 activity in a cellular assay. 44. The method of claim 43, wherein said testing step further comprises detecting increased monounsaturated fatty acid synthesis in a cellular assay. 45. The method of claim 22, wherein said testing step further comprises detecting an increase in Gram-positive bactericidal activity in a cell assay. 46. A method of diagnosing risk factors for Gram-positive bacterial infection in a mammalian subject, comprising: remove cells or tissue from the subject, contacting the cell or tissue with an endogenous or exogenous ligand for toll-like receptor 2, Detecting production of a Scd1 gene product in a cell or tissue contacted with a ligand, detecting decreased function or loss of function of a Scd1 gene product in a mammalian subject. 47. The method of claim 46, wherein said cells or tissues are from macrophages, sebocytes or sebaceous glands. 48. The method of claim 46, wherein said decreased function or deletion of the Scd1 gene product increases the risk of Gram-positive bacterial infection. 49. The method of claim 46, wherein the decreased function or deletion of the Scd1 gene product reduces intracellular synthesis of monounsaturated fatty acids. 50. The method of claim 46, wherein the reduced function or deletion of the Scd1 gene product reduces the inflammatory response to Gram-positive bacterial infection. 51. The method of claim 50, wherein the reduced function or deletion of the Scd1 gene product reduces the inflammatory response at the site of injury in the patient. 52. The method of claim 46, wherein deletion of the Scd1 gene product increases risk in the event of inflammation as the intended defense mechanism. 53. The method of claim 46, wherein said ligand is an exogenous ligand, lipoteichoic acid (LTA), diacyl fatty acyl peptide, triacyl fatty acyl peptide, S-MALP- 2. Bacterial fatty acyl peptides, peptidoglycan, mannan, unmethylated CpG DNA, flagellin or single-stranded RNA. 54. The method of claim 46, wherein the exogenous ligand is S-MALP-2. 55. The method of claim 46, wherein said ligand is an endogenous ligand, lipid, fat, sterol, lipoprotein, fatty acid, oxidized LDL, thrombospondin or amyloid beta . 56. A method of diagnosing a disorder induced by loss of function of the Scd1 gene or thus a genetic predisposition induced in a mammalian subject, comprising detecting a mutated Scd1 protein or in a cell sample, protein sample or nucleic acid sample obtained from a mammalian subject The presence of a nucleic acid encoding a mutant Scd1 protein, wherein the presence of the protein or nucleic acid is indicative of a loss-of-function-induced disorder of the Scd1 gene or a genetic predisposition thereby. 57. The method of claim 56, wherein the loss-of-function-induced disorder of the Scd1 gene is increased susceptibility to Gram-positive bacterial infection. 58. The method of claim 56, further comprising contacting the protein sample or the cell sample with an anti-Scd1 antibody and detecting the presence of wild-type or mutant Scd1 protein. 59. The method of claim 58, wherein said detecting step further comprises fluorescence activated cell sorting (FACS) sorting of mononuclear macrophages or macrophages from the mammalian subject. 60. The method of claim 56, further comprising contacting the nucleic acid sample with a marker DNA or RNA molecule encoding a mutant Scd1 gene under hybridization conditions, and detecting the marker DNA or RNA molecule after hybridization, wherein the marker DNA or Detection of RNA indicates the presence of a nucleic acid molecule encoding the mutant Scd1 gene in the sample. 61. The method of claim 56, further comprising contacting the nucleic acid sample with a restriction endonuclease whose recognition sequence is affected by the mutation of the mutant Scd1 gene, and detecting the presence or absence of nucleic acid fragments after contacting with the restriction endonuclease Or the presence of a variant fragment, wherein the absence of the nucleic acid fragment after contact with a restriction enzyme or the presence of a variant fragment is indicative of the presence of a nucleic acid molecule encoding a mutant Scd1 gene in the sample. 62. A transgenic non-human animal comprising an exogenous nucleic acid, wherein said nucleic acid comprises a loss-of-function allele of the Scd1 gene, and the animal exhibits a Gram-containing expression relative to a wild-type phenotype Phenotype of susceptibility to positive bacterial infection. 63. The transgenic non-human animal of claim 62, wherein said Scd1 mutant animal is phenotypically characterized by atrophied sebaceous glands or loss of ability to synthesize monounsaturated fatty acids. 64. The transgenic non-human animal of claim 62, wherein the loss-of-function allele of the Scd1 gene is a T227K amino acid substitution. 65. The transgenic non-human animal of claim 62, wherein said animal is a mouse or a rat. 66. A cell or cell line derived from the transgenic non-human animal of claim 62. 67. An in vitro method for screening Toll-like receptor 2 signal transduction activity modulators, the method comprising: contacting the cell or cell line according to claim 66 with a test compound, and detecting the amount of monounsaturated fatty acid synthesis in the cell , susceptibility to Gram-positive bacterial infection, or an increase or decrease in Toll-like receptor 2-induced macrophage activation activity, thereby identifying the test compound as a modulator of Toll-like receptor 2-induced macrophage activation activity . 68. An in vivo method for screening Toll-like receptor 2 signal transduction activity modulators, the method comprising: contacting the transgenic animal according to claim 62 with a test compound, and detecting the amount of monounsaturated fatty acid synthesis in cells, the effect on Susceptibility to Gram-positive bacterial infection or an increase or decrease in Toll-like receptor 2-induced macrophage activation activity, thereby identifying the test compound as a modulator of Toll-like receptor 2-induced macrophage activation activity.

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