Patent Application
20130302347
The instant application contains a Sequence listing submitted via EFS-Web and hereby incorporated by reference in its entirety. Said ACSII copy, created on May 21, 2013, is named P4549R1C1_SeqListing_as_filed.txt, and is 20,808 bytes in size.
The present invention relates generally to methods and compositions for modulating the interaction between a ligand and its receptor. Specifically, the invention relates to various methods, uses, reagents, and kits based on modulation of the interaction between IGFLs and TMEM149.
The TNF receptor family is composed primarily of type I integral membrane glycoproteins which exhibit sequence homology, particularly with respect to cysteine-rich repeats in their extracellular domains. The TNF receptor family includes over 29 members (reviewed in Bodmer et al. TRENDS in Biochem. Sci. 27:19-26, 2002). TMEM149 is a putative TNF receptor (TNFR). See, for example, WO 2002/020762 describing MK61 and the isotypes. SEQ ID NO:2 of WO 2002/020762 is identical to TMEM149 (SEQ ID NO:1) as described herein. However, no ligand was described for MK61 (i.e., TMEM149) in WO 2002/020762.
Recently, a novel IGF-like (IGFL) gene family was identified consisting of four transcribed genes in humans (IGFL1-4; SEQ ID NOs: 6-9, respectively) and one in mice (mIGFL; SEQ ID NO:10) (Emtage et al., 2006. Genomics. 88:513-20). IGFL proteins contain eleven regularly spaced cysteine residues, six of which are conserved within the IGF family, and a signal peptide, but no transmembrane domain, indicating that they are likely secreted. The IGFL sequences are not well conserved between human and mice, the identity of mIGFL and the human IGFLs varies from between 21% for IGFL1 to 38% for IGFL3. The IGFL genes were found to be expressed rarely and at low levels in human tissues, and have no described biological function.
Thus, elucidating the receptor for the IGFL family members is a necessary prerequisite for the prevention/treatment of diseases/conditions associated with IGFL dysfunction. The present invention identifies a receptor (i.e., TMEM149) and provides a method and compositions, such as selective binding agents, to modulate the interactions. Specifically, described herein are novel reagents and methods based on the interaction of IGFL1 and/or IGFL3 for the prevention/treatment of diseases/conditions associated with TMEM149 activity.
The invention relates to various methods, uses, reagents, and kits based on modulation of the interaction between an IGFL and TMEM149. In some aspects, the IGFL is IGFL1. In some aspects, the IGFL is IGFL3.
In one aspect thereof, the present invention relates to an agent that may block the interaction between an IGFL and TMEM149. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In another aspect, the present invention relates to a composition that may comprise an agent that may block the interaction between an IGFL and TMEM149 and a pharmaceutically acceptable carrier. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
The present invention further relates to a method for blocking the interaction between an IGFL and TMEM149; the method may comprise the step of administering an effective amount of an agent that may block the interaction between an IGFL and TMEM149. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In an embodiment of the present invention, the interaction may occur at the cell surface and a method for blocking the cell surface interaction between an IGFL and TMEM149 may comprise contacting cells (a cell expressing an IGFL and/or a cell expressing TMEM149) with an effective amount of an agent that may block the interaction between an IGFL and TMEM149.
In a further aspect, the present invention provides a method for inhibiting production of an inflammatory mediator by a cell, the method may comprise blocking the interaction between an IGFL and TMEM149. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In another aspect, the present invention relates to the use of an agent that may block the interaction between an IGFL to TMEM149 and/or for the preparation of a medicament that may block the interaction between an IGFL and TMEM149. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In a further aspect, the present invention relates to the use of an agent for treating an inflammatory disease/condition in a subject and/or for the preparation of a medicament for treating an inflammatory disease in a subject.
In yet a further aspect, the present invention relates to a method for identifying a compound capable of blocking the interaction between an IGFL and TMEM149; the method may comprise measuring the binding of an IGFL to TMEM149 in the presence versus the absence of an agent, wherein a lower binding of an IGFL to TMEM149 in the presence of the agent (in comparison with the absence of the agent) may be indicative that the agent is capable of blocking the interaction between an IGFL and TMEM149. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In another aspect, the present invention relates to a method for identifying a compound capable of blocking the interaction between an IGFL and TMEM149; the method may comprise measuring an IGFL-mediated TMEM149 activity in the presence or absence of the agent, wherein a lower TMEM149 activity in the presence of the agent may be indicative that the agent is blocking the interaction between an IGFL and TMEM149. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In another aspect, the present invention relates to a method for identifying a compound capable of inhibiting and/or decreasing inflammation; the method may comprise measuring the binding of an IGFL to TMEM149 in the presence versus the absence of the agent, wherein a lower binding of an IGFL to TMEM149 in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation. In some embodiments, the IGFL is IGFL1.
In yet another aspect, the present invention provides a method of identifying a compound capable of inhibiting or decreasing inflammation; the method may comprise measuring an IGFL-mediated TMEM149 activity in the presence versus the absence of the agent, wherein a lower TMEM149 activity in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation.
In a further aspect, the present invention provides a method of treating an inflammatory disease or condition in a subject; the method may comprise blocking the interaction between an IGFL and TMEM149 in the subject. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In another aspect, the present invention relates to a method for treating psoriasis in a subject (in need thereof), the method may comprise administering an agent that may block the interaction between IGFL1 and TMEM149 as described herein.
In a further aspect, the present invention related to a use of an agent capable of blocking the interaction between an IGFL and TMEM149 for treating an inflammatory disease or condition in a subject. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In a further aspect, the present invention relates to a use of an agent capable of blocking the interaction between an IGFL and TMEM149 for the preparation of a medicament for treating an inflammatory disease or condition in a subject. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In a further aspect, the present invention relates to a composition for treating an inflammatory disease or condition in a subject comprising an agent capable of blocking the interaction between an IGFL and TMEM149 and a pharmaceutically acceptable carrier. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In a further aspect, the present invention relates to a package comprising:
In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In an embodiment, the use of a package may be for the treatment and/or prevention (e.g., reduction, blockade, inhibition) of inflammatory-related diseases or condition in the subject.
In another aspect thereof, the present invention relates to a soluble form of an IGFL. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
The present invention also relates to the use of soluble form(s) of an IGFL for treatment of an IGFL-related disorder as well as a method for treating an IGFL-related disorder; the method may comprise administering a soluble form of an IGFL. Soluble form(s) of an IGFL may also be used for detecting a TMEM149 receptor by methods well within the province of a person skilled in the art. In some embodiments, the IGFL is IGFL1. In an embodiment, the IGFL is IGFL2. In some embodiments, the IGFL is IGFL3.
In one aspect thereof, the present invention relates to an agent that may stimulate the interaction between an IGFL and TMEM149. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In another aspect, the present invention relates to a composition that may comprise an agent that may stimulate the interaction between an IGFL and TMEM149 and a pharmaceutically acceptable carrier. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
The present invention further relates to a method for stimulating the interaction between an IGFL and TMEM149; the method may comprise the step of administering an effective amount of an agent that may stimulate the interaction between an IGFL and TMEM149. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In an embodiment of the present invention, the interaction may occur at the cell surface and a method for stimulating the cell surface interaction between an IGFL and TMEM149 may comprise contacting cells (a cell expressing an IGFL and/or a cell expressing TMEM149) with an effective amount of an agent that may stimulate the interaction between an IGFL and TMEM149. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In a further aspect, the present invention provides a method for inhibiting production of an inflammatory mediator by a cell, the method may comprise stimulating the interaction between an IGFL and TMEM149. In some embodiments, the IGFL is IGFL3.
In another aspect, the present invention relates to the use of an agent that may stimulate the interaction between an IGFL to TMEM149 and/or for the preparation of a medicament that may stimulate the interaction between an IGFL and TMEM149. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In a further aspect, the present invention relates to the use of an agent for treating an inflammatory disease in a subject and/or for the preparation of a medicament for treating an inflammatory disease in a subject.
In yet a further aspect, the present invention relates to a method for identifying a compound capable of stimulating the interaction between an IGFL and TMEM149; the method may comprise measuring the binding of an IGFL to TMEM149 in the presence versus the absence of an agent, wherein an elevated binding of an IGFL to TMEM149 in the presence of the agent (in comparison with the absence of the agent) may be indicative that the agent is capable of stimulating the interaction between an IGFL and TMEM149. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In another aspect, the present invention relates to a method for identifying a compound capable of stimulating the interaction between an IGFL and TMEM149; the method may comprise measuring an IGFL-mediated TMEM149 activity in the presence or absence of the agent, wherein an elevated TMEM149 activity in the presence of the agent may be indicative that the agent is stimulating the interaction between an IGFL and TMEM149. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In another aspect, the present invention relates to a method for identifying a compound capable of inhibiting and/or decreasing inflammation; the method may comprise measuring the binding of an IGFL to TMEM149 in the presence versus the absence of the agent, wherein an elevated binding of an IGFL to TMEM149 in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation. In some embodiments, the IGFL is IGFL3.
In yet another aspect, the present invention provides a method of identifying a compound capable of inhibiting or decreasing inflammation; the method may comprise measuring an IGFL-mediated TMEM149 activity in the presence versus the absence of the agent, wherein an elevated TMEM149 activity in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation. In some embodiments, the IGFL is IGFL3.
In a further aspect, the present invention provides a method of treating an inflammatory disease or condition in a subject; the method may comprise stimulating the interaction between an IGFL and TMEM149 in the subject. In some embodiments, the IGFL is IGFL3.
In another aspect, the present invention relates to a method for treating psoriasis in a subject (in need thereof), the method may comprise administering an agent that may stimulate the interaction between an IGFL and TMEM149 as described herein. In an embodiment, the IGFL is IGFL3.
In a further aspect, the present invention relates to a use of an agent capable of stimulating the interaction between an IGFL and TMEM149 for treating an inflammatory disease or condition in a subject. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In a further aspect, the present invention relates to a use of an agent capable of stimulating the interaction between IGFL and TMEM149 for the preparation of a medicament for treating an inflammatory disease or condition in a subject. In some embodiments, the IGFL is IGFL1. In some embodiments, the IGFL is IGFL3.
In a further aspect, the present invention relates to a composition for treating an inflammatory disease or condition in a subject comprising an agent capable of stimulating the interaction between IGFL-1 and TMEM149 and a pharmaceutically acceptable carrier.
In a further aspect, the present invention relates to a package comprising:
In an embodiment, the use of a package may be for the treatment and/or prevention (e.g., reduction, blockade, inhibition) of inflammatory-related diseases or condition in the subject.
Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the scope and spirit of the invention will become apparent to one skilled in the art from this detailed description.
The invention will now be described in detail by way of reference only using the following definitions and examples. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.
Unless defined otherwise herein, all 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. Singleton, et al., D
Numeric ranges are inclusive of the numbers defining the range.
Unless otherwise indicated, nucleic acids are written left to right in 5′ to 3′ orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.
The headings provided herein are not limitations of the various aspects or embodiments of the invention which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.
In the Examples below, we analyzed the gene expression profile of mIGFL and discovered that it is primarily expressed in normal skin in mice. Interestingly, mIGFL expression is further upregulated during inflammatory responses in the skin and during skin wounding. Of the four human IGFL family members, only IGFL1 is upregulated in human psoriatic skin samples. In vitro, IGFL1 is produced by keratinocytes and TNFα enhances its expression. Finally, we found that both mIGFL and IGFL1 specifically interact with high-affinity to a putative tumor necrosis factor receptor (TNFR) family member, designated herein as TMEM149. Murine TMEM149 is expressed most abundantly on mouse T cells, suggesting mIGFL and IGFL1 produced in the skin may potentially exert regulatory functions on T cell responses.
As described herein, Applicants have demonstrated that TMEM149 is a functional receptor for IGFL family members. In addition, Applicants have demonstrated that certain IGFL family members affect T-cell proliferation.
In order to provide a clear and consistent understanding of the terms used in the present disclosure, a number of definitions are provided below. Moreover, unless defined otherwise, all technical and scientific terms as used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention pertains.
The term “antibody” herein is used in the broadest sense and refers to any immunoglobulin (Ig) molecule comprising two heavy chains and two light chains, and any fragment, mutant, variant or derivation thereof which so long as they exhibit the desired biological activity (e.g., epitope binding activity). Examples of antibodies include monoclonal antibodies, polyclonal antibodies, multispecific antibodies and antibody fragments.
In the studies described herein, Applicants have demonstrated that TMEM149 is a functional receptor for IGFL family members. In addition, Applicants have demonstrated that certain IGFL family members affect T-cell proliferation.
As used herein, TMEM149 refers to a widely distributed cell surface receptor. The human TMEM149 (SEQ ID NO: 1) and mouse TMEM149 (SEQ ID NO: 3) are shown in
The terms “TMEM149 gene” or “TMEM149 nucleic acid molecule” or “polynucleotide” refers to a nucleic acid molecule comprising or consisting of a nucleotide sequence as set forth in US7153669 for MK61 gene, nucleic acid molecule or polynucleotide.
The term “TMEM149 polypeptide” refers to a polypeptide comprising the amino acid sequence as defined in US7153669 for the MK61 polypeptide. Related polypeptides include: TMEM149 polypeptide allelic variants, TMEM149 polypeptide orthologs, TMEM149 polypeptide splice variants, TMEM149 polypeptide variants and TMEM149 polypeptide derivatives. TMEM149 polypeptides may be mature polypeptides, as defined herein, and may or may not have an amino terminal methionine residue, depending on the method by which they are prepared.
The term “TMEM149 polypeptide allelic variant” refers to the polypeptide encoded by one of several possible naturally occurring alternate forms of a gene occupying a given locus on a chromosome of an organism or a population of organisms.
The term “TMEM149 polypeptide derivatives” refers to a TMEM149, as defined above, that have been chemically modified.
The term “TMEM149 polypeptide fragment” refers to a polypeptide that comprises a truncation at the amino terminus (with or without a leader sequence) and/or a truncation at the carboxy terminus of the polypeptide whose sequence is as defined in US7153669. TMEM149 polypeptide fragments may result from alternative RNA splicing or from in vivo protease activity. For transmembrane or membrane-bound forms of the TMEM149 polypeptides, preferred fragments include soluble forms such as those lacking a transmembrane or membrane-binding domain.
In preferred embodiments, truncations comprise about 10 amino acids, or about 20 amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or more than about 100 amino acids. The polypeptide fragments so produced will comprise about 25 contiguous amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or about 150 amino acids, or about 200 amino acids. Such TMEM149 polypeptide fragments may optionally comprise an amino terminal methionine residue. It will be appreciated that such fragments can be used, for example, to generate antibodies to TMEM149 polypeptides.
The term “TMEM149 fusion polypeptide” refers to a fusion of one or more amino acids (such as a heterologous peptide or polypeptide) at the amino or carboxy terminus of the TMEM149 polypeptide.
The term “TMEM149 polypeptide ortholog” refers to a polypeptide from another species that corresponds to an TMEM149 polypeptide as defined above. For example, mouse and human TMEM149 polypeptides are considered orthologs of each other. For ease of reference, the human and mouse TMEM149 polypeptide sequences are aligned and shown in
The term “TMEM149 polypeptide splice variant” refers to a nucleic acid molecule, usually RNA, which is generated by alternative processing of intron sequences in an RNA primary transcript containing the non-contiguous coding region of the TMEM149 polypeptide as defined above.
The term “TMEM149 polypeptide variants” refers to TMEM149 polypeptides comprising amino acid sequences having one or more amino acid sequence substitutions, deletions (such as internal deletions and/or TMEM149 polypeptide fragments), and/or additions (such as internal additions and/or TMEM149 fusion polypeptides) as compared to the TMEM149 polypeptide as defined above. Variants may be naturally occurring (e.g., TMEM149 polypeptide allelic variants, TMEM149 polypeptide orthologs and TMEM149 polypeptide splice variants) or may be artificially constructed. Such TMEM149 polypeptide variants may be prepared from the corresponding nucleic acid molecules having a DNA sequence that varies accordingly from the DNA sequence as defined above for the TMEM149 gene. In preferred embodiments, the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 50, or from 1 to 75, or from 1 to 100, or more than 100 amino acid substitutions, insertions, additions and/or deletions, wherein the substitutions may be conservative, or non-conservative, or any combination thereof.
The term “antigen” refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of each antigen. An antigen may have one or more epitopes.
The term “biologically active TMEM149 polypeptides” refers to TMEM149 polypeptides having at least one activity characteristic of the TMEM149 polypeptide as defined above.
IGFL as used herein generally refers to any member of the family of proteins depicted in
The four members of the human IGFL family that are described herein are located within a 220 Kb stretch of chromosome 19 and are referred to herein as IGFL1, IGFL2, IGFL3, and IGFL4. Only a single mouse IGFL has been described; it has the protein sequence shown in
The most striking sequence conservation among the proteins is the two CC motifs within 25 amino acids of each other. Another feature of this family is the conservation of residues immediately following the conserved cysteines. For example, there are two CG (C=cysteine, G=guanine) motifs that are repeated twice among all human IGFL proteins. In addition, there are CT and CFE motifs conserved among all members (wherein T=thymine, F=phenylalanine, E=glutamic acid). Except for these features, the overall level of similarity among the IGFL family members is relatively low. The highest sequence divergence occurs, as expected, within the signal peptide region. Within the mature protein, IGFL-I is 41% identical to IGFL-2, 39% identical to IGFL-3, and only 29% identical to IGFL-4. The highest homology occurs between IGFL-2 and IGFL-3 (at 63.4% identity) and the lowest homology occurs between IGFL-I and IGFL4 (at 20% identity).
Interestingly, the mouse IGFL member (mIGFL) is very divergent from all the human members. Although the 11 cysteines are conserved, there are variations in other conserved residues. In the human proteins, the first cysteine is followed by a glutamine (Q) and in the mouse, it is replaced with an asparagine (N). There are five non-cysteine residues between the first two cysteines in the human proteins, whereas in the mouse, there are only three residues. In all human IGFL proteins, the second cysteine is followed by a glycine (G), whereas in the mouse protein, there is an aspartic acid (D) inserted between the C and the G. The mouse sequence is also substantially longer (141 total amino acids and 118 in the mature protein) than the human genes which range from 111 to 131 total amino acids and to 106 amino acids in the mature proteins. The overall sequence homology between human IGFLs and the mouse IGFL is within 26% to 40% in the mature protein.
The IGFL1 polypeptide (SEQ ID NO: 6) is an approximately 110 amino acid protein with a predicted molecular mass of approximately 12.1 kDa unglycosylated. A signal peptide of 23 residues is predicted from residue 1 to residue 23, inclusive, of SEQ ID NO: 6. The extracellular portion, or mature protein, is useful on its own. The mature protein (i.e., without the signal peptide) is SEQ ID NO:25. One of skill in the art will recognize that the actual cleavage site may be different than that predicted by the computer program.
The IGFL2 polypeptide (SEQ ID NO: 7) is an approximately 123 amino acid protein with a predicted molecular mass of 13.5 kDa, unglycosylated. A signal peptide of 29 residues is predicted from residue 1 to residue 29 of SEQ ID NO: 26. The extracellular portion, or mature protein, is useful on its own. The mature protein (i.e., without the signal peptide) is SEQ ID NO:27. One of skill in the art will recognize that the actual cleavage site may be different than that predicted by the computer program.
The IGFL3 (SEQ ID NO: 8) is an approximately 125 amino acid protein with a predicted molecular mass of approximately 13.7 kDa unglycosylated. A signal peptide of 36 residues is predicted (SEQ ID NO: 28). The extracellular portion is useful on its own. The mature protein (i.e., without the signal peptide) is SEQ ID NO:29. One of skill in the art will recognize that the actual cleavage site may be different than that predicted by the computer program.
The IGFL4 (SEQ ID NO: 9) is an approximately 124 amino acid protein with a predicted molecular weight of approximately 13.6 kD unglycosylated. A signal peptide of 19 residues is predicted (SEQ ID NO: 30). The extracellular portion, or mature protein, is useful on its own. The mature protein (i.e., without the signal peptide) is SEQ ID NO:31. One of skill in the art will recognize that the actual cleavage site may be different than that predicted by the computer program.
In one aspect thereof, the present invention relates to an agent that may block the interaction between an IGFL and TMEM149. In another aspect, the present invention relates to an agent that may modulate the interaction between an IGFL and TMEM149.
In an embodiment of the present invention, an “agent” that may block the interaction between an IGFL and TMEM149 may be a protein. For example, such protein may be an (isolated) antibody, or antigen-binding fragment (portion) thereof, that may specifically bind to an IGFL and/or TMEM149. The antibody may be, for example, a monoclonal antibody and/or a polyclonal antibody. Monoclonal antibodies (MAbs) may be made by one of several procedures available to one of skill in the art, for example, by fusing antibody producing cells with immortalized cells and thereby making a hybridoma. The general methodology for fusion of antibody producing B cells to an immortal cell line is well within the province of one skilled in the art. Another example is the generation of MAbs from mRNA extracted from bone marrow and spleen cells of immunized animals using combinatorial antibody library technology. One drawback of MAbs derived from animals or from derived cell lines is that although they may be administered to a patient for diagnostic or therapeutic purposes, they are often recognized as foreign antigens by the immune system and are unsuitable for continued use. Antibodies that are not recognized as foreign antigens by the human immune system have greater potential for both diagnosis and treatment. Methods for generating human and humanized antibodies are now well known in the art.
Polyclonal antibodies may be obtained by immunizing a selected animal with a protein or polypeptide (for example without limitation an IGFL and TMEM149). Serum from the animal may be collected and treated according to known procedures. Polyclonal antibodies to the protein or polypeptide of interest may then be purified by affinity chromatography. Techniques for producing polyclonal antisera are well known in the art.
Antibodies may originate for example, from a mouse, rat or any other mammal. The antibody may also be a human antibody which may be obtained, for example, from a transgenic non-human mammal capable of expressing human immunoglobulin genes. The antibody may also be a humanized antibody which may comprise, for example, one or more complementarity determining regions of non-human origin. It may also comprise a surface residue of a human antibody and/or framework regions of a human antibody. The antibody may also be a chimeric antibody which may comprise, for example, variable domains of a non-human antibody and constant domains of a human antibody. Suitable antibodies may also include, for example, an antigen-binding fragment, a Fab fragment; a F(ab′)2 fragment, and Fv fragment; or a single-chain antibody comprising an antigen-binding fragment (e.g., a single chain Fv). An antibody encompassed in the present invention may be an antibody binding specifically to TMEM149. In an embodiment, an antibody encompassed in the present invention may be an antibody binding specifically to an IGFL.
Anti-TMEM149 agents (e.g. antibodies) may be experimentally tested and validated using in vivo and in vitro assays. Suitable assays include, but are not limited to, activity assays and binding assays. For example, assays for testing an IGFL activity for TMEM149 includes T-cell proliferation assays.
The activity of an IGFL interaction with TMEM149 may, among other means, be measured by the following methods:
Suitable assays for thymocyte or splenocyte cytotoxicity include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3. 1-3.19; Chapter 7, Immunologic studies in Humans); Herrmann, et al., Proc. Natl. Acad. Sci. USA 78:2488-2492 (1981); Herrmann, et al., J. Immunol. 128:1968-1974 (1982); Handa, et al., J. Immunol. 135:1564-1572 (1985); Takai, et al., I. Immunol. 137:3494-3500 (1986); Takai, et al., J. Immunol. 140:508-512 (1988); Bowman, et al., J. Virology 61:1992-1998; Bertagnolli, et al., Cellular Immunology 133:327-341 (1991); Brown, et al., J. Immunol. 153:3079-3092 (1994).
Assays for T-cell-dependent immunoglobulin responses and isotype switching (which will identify, among others, proteins that modulate T-cell dependent antibody responses and that affect Th1/Th2 profiles) include, without limitation, those described in: Maliszewski, J. Immunol. 144:3028-3033 (1990); and Assays for B cell function: In vitro antibody production, Mond, J. J. and Brunswick, M. In Current Protocols in Immunology. J. E. e.a. Coligan eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto. 1994.
Mixed lymphocyte reaction (MLR) assays (which will identify, among others, proteins that generate predominantly Th1 and CTL responses) include, without limitation, those described in: Current Protocols in Immunology, Ed by J. E. Coligan, A. M. Kruisbeek, D. H. Margulies, E. M. Shevach, W. Strober, Pub. Greene Publishing Associates and Wiley-Interscience (Chapter 3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai, et al., J. Immunol. 137:3494-3500 (1986); Takai, et al., J. Immunol. 140:508-512 (1988); Bertagnolli, et al., J. Immunol. 149:3778-3783 (1992).
Dendritic cell-dependent assays (which will identify, among others, proteins expressed by dendritic cells that activate naive T-cells) include, without limitation, those described in: Guery et al., J. Immunol. 134:536-544 (1995); Inaba et al., J. Exp. Med. 173:549-559 (1991); Macatonia, et al., J. Immunol. 154:5071-5079 (1995); Porgador, et al., J. Exp. Med. 182:255-260 (1995); Nair, et al., J. Virology 67:4062-4069 (1993); Huang, et al., Science 264:961-965 (1994); Macatonia, et al., J. Exp. Med. 169:1255-1264 (1989); Bhardwaj, et al., J. Clin. Invest. 94:797-807 (1994); and Inaba, et al., J. Exp. Med. 172:631-640 (1990).
Assays for lymphocyte survival/apoptosis (which will identify, among others, proteins that prevent apoptosis after superantigen induction and proteins that regulate lymphocyte homeostasis) include, without limitation, those described in: Darzynkiewicz et al., Cytometry 13:795-808 (1992); Gorczyca, et al., Leukemia 7:659-670 (1993); Gorczyca, et al., Cancer Res. 53:1945-1951 (1993); Itoh, et al., Cell 66:233-243 (1991); Zacharchuk, J. Immunol. 145:4037-4045 (1990); Zamai, et al., Cytometry 14:891-897 (1993); Gorczyca, et al., Int. J. Oncol. 1:639-648 (1992).
Assays for proteins that influence early steps of T-cell commitment and development include, without limitation, those described in: Antica, et al., Blood 84:111-117 (1994); Fine, et al., Cell. Immunol. 155:111-122, (1994); Galy, et al., Blood 85:2770-2778 (1995); Toki, et al., Proc. Nat. Acad. Sci. USA 88:7548-7551 (1991).
According to the present invention, a (protein) agent may also be a “soluble protein”. Soluble proteins (purified) of the invention may be obtained from any techniques well known in the art. For example, a soluble protein may be obtained by transfecting a recombinant DNA molecule expressing solely the extracellular region of a molecule and/or portion thereof followed by purification. In another example, a protein and/or a portion of a protein (for example an extracellular region exempt of its transmembrane and cytoplasmic domains) may be fused to a constant domain (Fc portion) of an immunoglobulin. A (purified) soluble protein of the present invention may be soluble an IGFL and/or soluble TMEM149 and/or portion thereof. By “portion” (of soluble protein for example) it is meant a portion that exhibits similar (biological) activity yet is smaller in size. An agent of the present invention may be soluble TMEM149. An agent of the present invention may be portions of soluble TMEM149. An agent of the present invention may be soluble an IGFL. An agent of the present invention may be portions of an IGFL. Human TMEM149 (SEQ ID NO: 1) is a 355 amino acid type Ia protein. Its extracellular domain is approximately 138 amino acids in length (SEQ ID NO: 23). A (purified) soluble human TMEM149 of the invention may have a sequence that may consist from about residue 23 to residue 160 of SEQ ID NO:1. The present invention relates to and explicitly incorporates herein each and every specific member and combination of sub-ranges therein whatsoever. Thus, any specified range or group is to be understood as a shorthand way of referring to each and every member of a range or group individually as well as each and every possible sub-ranges or sub-groups encompassed therein; and similarly with respect to any sub-ranges or sub-groups therein.
As used herein, the term “block” or “inhibit” refers to a decrease in one or more given measurable activity by at least 10% relative to a reference and/or control. Where inhibition is desired, such inhibition is preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, up to and including 100%, i.e., complete inhibition or absence of the given activity. As used herein, the term “substantially inhibits/blocks” refers to a decrease in a given measurable activity by at least 50% relative to a reference. For example, “substantially inhibits” refers to a decrease in a given measurable activity of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% and up to and including 100% relative to a reference. As used herein, “blocks/prevents/inhibits/impairs/lowers the interaction”, with reference to the binding of an IGFL that binds to TMEM149 refers to a decrease in binding by at least 10% relative to a reference. An agent may block the binding of an IGFL to TMEM149 expressing cells. “Inhibits the interaction” and/or “block the binding” preferably refers to a decrease in binding of at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, up to and including 100%.
In another aspect, the present invention relates to a composition including an agent that blocks the interaction between an IGFL and TMEM149 and a pharmaceutically acceptable carrier.
A “composition” of the invention including an agent may be manufactured in a conventional manner. In particular, it is formulated with a pharmaceutically acceptable diluent or carrier, e.g., water or a saline solution such as phosphate buffer saline. In general, a diluent or carrier is selected on the basis of the mode and route of administration, as well as standard pharmaceutical practice. Therapeutic compositions typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. In many cases, it may be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of compositions may be brought about by including in the composition an agent which delays absorption, for example, monostearate salts and gelatin. Moreover, an agent of the invention may be administered in a time release formulation, for example in a composition which includes a slow release polymer. The active agents may be prepared with carriers that will protect the agent against rapid release, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable, biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid and polylactic, polyglycolic copolymers (PLG). Many methods for the preparation of such formulations are patented or generally known to those skilled in the relevant art. The present invention relates to compositions that may comprise an agent capable of modulating IGFL activity and a pharmacologically acceptable carrier. In one embodiment, such compositions include an agent that may block the interaction between an IGFL and TMEM149 to treat an IGFL-related disease (for example an immune-related disease and/or inflammatory disease).
As used herein “pharmaceutically acceptable carrier” or excipient includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In one embodiment, the carrier is suitable for parenteral administration. Alternatively, the carrier may be suitable for intravenous, intraperitoneal, intramuscular, sublingual or oral administration. Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media is incompatible with the active agent, use thereof in the pharmaceutical compositions of the invention is contemplated. Supplementary active compounds may also be incorporated into the compositions.
The present invention further relates to a method for blocking the interaction between an IGFL and TMEM149; the method may comprise the step of administering (to a subject) an effective amount of an agent that blocks the interaction between an IGFL and TMEM149. In an embodiment of the present invention, the interaction may occur at the cell surface and a method for blocking the cell surface interaction between an IGFL and TMEM149 may comprise contacting the cell with an effective amount of an agent.
“Administration” of a composition may be performed by any suitable routes. Such routes may include parenteral, pulmonary, nasal and/or oral routes. In one embodiment, the pharmaceutical composition may be intra-muscular (IM), subcutaneous (SC), intra-dermal (ID), intra-venous (IV) and/or intra-peritoneal (IP) routes using any suitable means.
The term “effective amount” is intended to mean an amount of an agent sufficient to substantially block the interaction between an IGFL and TMEM149. An effective amount may also encompass either “therapeutically effective amount” and/or “prophylactically effective amount”. A “therapeutically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result, such as a reduction in disease progression and/or alleviation of the symptoms associated with a disease. A therapeutically effective amount of modulators of an IGFL activity may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the agent to elicit a desired response in the individual. Dosage regimens may be adjusted to provide the optimum therapeutic response. A therapeutically effective amount is also one in which any toxic or detrimental effects of the agent are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result, such as preventing and/or inhibiting (reducing) the rate of disease onset or progression. A prophylactically effective amount may be determined as described above for the therapeutically effective amount. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the person administering of the compositions.
In a further aspect, the present invention provides a method of inhibiting production of an inflammatory mediator (by a cell), the method comprising blocking the interaction between an IGFL and TMEM149.
During inflammation, various molecules may be secreted by cells. Such molecules may be referred to as “inflammatory mediators”. As will be appreciated by one skilled in the art, these inflammatory mediators may be, for example and without limitation, amines, eicosanoids, growth factors, reactive oxygen species, enzymes (for example a proteinase), chemokines, cytokines, etc.
In yet another aspect, the present invention relates to the use of an agent that may block the interaction between an IGFL to TMEM149 and/or its use for the preparation of a medicament that may block the interaction between an IGFL and TMEM149. In one aspect, the IGFL may be IGFL1. In another aspect, the IGFL may be IGFL3.
In a further aspect, the present invention relates to the use of an agent for treating an inflammatory disease in a subject and/or for the preparation of a medicament for treating an inflammatory disease in a subject.
In an embodiment, the subject is a mammal, in a further embodiment, a human.
Given the IGFL1-mediated T cell proliferation, agents capable of inhibiting the IGFL-mediated activation of T cell proliferation may be used for the prevention and treatment of inflammation-related disorders.
In view of the IGFL3-mediated inhibition of T cell proliferation, agents capable of enhancing the IGFL3-mediated inhibition of T cell proliferation may be used for the prevention and treatment of inflammation-related disorders.
Alternatively, in view of the IGFL3-mediated inhibition of T cell proliferation, agents capable of inhibiting the IGFL3-mediated inhibition of T cell proliferation may be used for enhancing an immune response.
The present invention also further relates to screening methods for the identification and characterization of compounds capable of blocking the interaction between an IGFL and TMEM149 and/or the IGFL1-mediated activation of T-cell proliferation and/or IGFL3-mediated inhibition of T-cell proliferation.
The above-mentioned compounds/agents may be used for prevention and/or treatment of inflammation-related diseases or conditions, or may be used as lead compounds for the development and testing of additional compounds having improved specificity, efficacy and/or pharmacological (e.g. pharmacokinetic) properties. In an embodiment the compound may be a prodrug which is altered into its active form at the appropriate site of action. In certain embodiments, one or a plurality of the steps of the screening/testing methods of the invention may be automated.
A method of identifying a compound capable of blocking the interaction between an IGFL and TMEM149 may comprise measuring the binding of an IGFL to TMEM149 in the presence versus the absence of an agent, wherein a lower binding of an IGFL to TMEM149 in the presence of the agent may be indicative that the agent is capable of blocking the interaction between an IGFL and TMEM149.
The methods for identifying a compound (screening method) mentioned herein may be employed either with a single test compound or a plurality or library (e.g. a combinatorial library) of test compounds. In the latter case, synergistic effects provided by combinations of compounds may also be identified and characterized.
Measuring the binding of an IGFL to TMEM149 may be performed using (without limitation) such suitable assays as quantitative comparisons comparing kinetic and equilibrium binding constants. The kinetic association rate (kon) and dissociation rate (koff), and the equilibrium binding constants (Kd) may be determined using surface plasmon resonance on a BIAcore™ instrument following the standard procedure in the literature. Binding properties of these interactions may also be assessed by flow cytometry and/or by solid phase binding assay.
The present invention also relates to a method of identifying a compound capable of blocking the interaction between an IGFL and TMEM149; the method may comprise measuring an IGFL-mediated TMEM149 activity in the presence or absence of the agent, wherein a lower TMEM149 activity in the presence of the agent may be indicative that the agent is blocking the interaction between an IGFL and TMEM149.
As used herein, “an activity mediated by an IGFL” or “an IGFL-mediated TMEM149 activity” is an activity involving or resulting from the binding of an IGFL to TMEM149, and includes, but is not limited to, binding to TMEM149, the induction of T cells to produce and secrete cytokines (for example IL-2, IL-10, IFN-γ and TNF-α), the synthesis of inflammatory molecules (inflammatory mediators) such as IL-6, IL-8 and metalloproteinases and T-cell proliferation (or inhibition thereof), etc. It will be understood that the IGFL-mediated activity may depend on the specific IGFL, e.g., IGFL1 or IGFL3, being evaluated.
In an aspect, the present invention relates to a method for identifying a compound capable of inhibiting or decreasing inflammation; the method may comprise measuring the binding of IGFL1 to TMEM149 in the presence versus the absence of the agent. A lower binding of an IGFL1 to TMEM149 in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation.
In an aspect, the present invention relates to a method for identifying a compound capable of inhibiting or decreasing inflammation; the method may comprise measuring the binding of IGFL3 to TMEM149 in the presence versus the absence of the agent. An enhanced binding of an IGFL3 to TMEM149 in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation.
In a further aspect, the present invention provides a method of identifying a compound capable of inhibiting or decreasing inflammation; the method may comprise measuring an IGFL-mediated TMEM149 activity in the presence versus the absence of the agent, wherein a lower TMEM149 activity in the presence of the agent may be indicative that the agent is capable of inhibiting or decreasing inflammation.
In a further aspect, the present invention provides a method of treating an inflammatory disease or condition in a subject; the method may comprise blocking the interaction between an IGFL and TMEM149 in the subject.
In various embodiments, agents blocking the interaction between an IGFL and TMEM149 may be used therapeutically in formulations or medicaments to prevent or treat IGFL-related disorders. IGFL-related disorders generally relate to various immune-mediated and/or inflammatory-related diseases/conditions. The modulators of an IGFL activity may find use in disease conditions for which antagonism of immune cell activation, and more particularly an IGFL1-mediated immune activation, is desirable, including a variety of inflammatory and autoimmune diseases. Such diseases include, but are not limited to: systemic lupus erythematosus (SLE), arthritis, psoriasis, multiple sclerosis, allergic encephalitis, Crohn's disease, diabetes, Hodgkin's and non-Hodgkin's Lymphomas (NHL), chronic renal failure, nephrotic syndrome, Hashimoto's thyroiditis, sickle cell anemia, inflammatory bowel disease, Hodgkin's disease, rheumatoid vasculitis, chronic lymphocytic leukemia, myasthenia gravis, preeclampsia and cardiovascular conditions including atherosclerosis, thrombocytopenia (Purpura) and thrombosis. The blocking agents may find particular use in psoriasis.
In a further aspect, the present invention provides a use of an agent capable of blocking the interaction between an IGFL and TMEM149 for treating an inflammatory disease or condition in a subject.
In a further aspect, the present invention provides a use of an agent capable of blocking the interaction between an IGFL and TMEM149 for the preparation of a medicament for treating an inflammatory disease or condition in a subject.
In various embodiments, agents modulating the interaction between an IGFL and TMEM149 may be used therapeutically in formulations or medicaments to prevent or treat IGFL-related disorders. The modulators of an IGFL activity (agents) have potential utility for treatment of other IGFL-related disorders such as non-autoimmune conditions wherein immunomodulation is desirable, e.g., infection. In an aspect, the IGFL is IGFL3. In one aspect, the agent is an IGFL3 agonist.
In a further aspect, the present invention provides a composition for treating an inflammatory disease or condition in a subject comprising an agent capable of blocking the interaction between an IGFL and TMEM149 and a pharmaceutically acceptable carrier.
In a further aspect, the present invention provides a package comprising:
In an embodiment the use may be for the treatment or prevention of inflammatory-related diseases or condition in the subject.
Although various embodiments of the invention are disclosed herein, many adaptations and modifications may be made within the scope of the invention in accordance with the common general knowledge of those skilled in the relevant art. Such modifications include the substitution of known equivalents for any aspect of the invention in order to achieve the same result in substantially the same way. Furthermore, numeric ranges are inclusive of the numbers defining the range. In the claims, the word “comprising” is used as an open-ended term, substantially equivalent to the phrase “including, but not limited to”. The following examples are illustrative of various aspects of the invention, and do not limit the broad aspects of the invention as disclosed herein.
In the experimental disclosure which follows, the following abbreviations apply: eq (equivalents); M (Molar); μM (micromolar); N (Normal); mol (moles); mmol (millimoles); μmol (micromoles); nmol (nanomoles); g (grams); mg (milligrams); kg (kilograms); μg (micrograms); L (liters); ml (milliliters); μl (microliters); cm (centimeters); mm (millimeters); μm (micrometers); nm (nanometers); ° C. (degrees Centigrade); h (hours); min (minutes); sec (seconds); msec (milliseconds); SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis); CRD (cysteine rich domain); ECD (extracellular domain); HVEM (herpes virus entry mediator); IGF (insulin growth factor); IGFL (IGF-like); NGFR (nerve growth factor receptor); nHEK (normal human epidermal keratinocytes); SPR (Surface plasmon resonance); TNFR (TNF receptor); RPL19 (Ribosomal Protein L19);
The present invention is described in further detail in the following examples which are not in any way intended to limit the scope of the invention as claimed. The attached Figures are meant to be considered as integral parts of the specification and description of the invention. All references cited are herein specifically incorporated by reference for all that is described therein. The following examples are offered to illustrate, but not to limit the claimed invention.
Cells and Reagents used in the Examples were as follows: Neonatal normal human epidermal keratinocytes were purchased from Lonza (Walkersville, Md.), Keratinocyte-SFM with supplements were from Invitrogen (Carlsbad, Calif.) and collagen IV was purchased form Sigma-Aldrich (St. Louis, Mo.). All transfections were performed with Fugene 6 (Roche) according to the manufacturers protocol. Mouse total RNA adult tissue panel was purchased from Zyagen (San Diego, Calif.). Recombinant human cytokines were from Peprotech (Rocky Hill, N.J.). The following anti-mouse antibodies were used for flow cytometry: anti-B220, anti-CD3, anti-CD11b, anti-CD11c, anti-DX5, anti-GR1, anti-NKG2D, and anti-F4/80 all from BD Biosciences (San Jose, Calif.), anti-FLAG (M2) monoclonal antibody was purchased from Sigma-Aldrich and labeled with Alexa Flour-647 monoclonal antibody labeling kit from Invitrogen, and anti-mouse IgG1-PE was from Jackson Immunoresearch (West Grove, Pa.).
This example illustrates preparation of potentially glycosylated forms of the desired IGFL or TMEM149 proteins (either of which is referred to in this example as a desired protein) by recombinant expression in mammalian cells.
The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), was employed as the expression vector in all instances. Optionally, DNA encoding the desired protein was ligated into pRK5 with selected restriction enzymes to allow insertion of such DNA using ligation methods such as described in Sambrook et al., supra. Epitope-tagged variants of the desired protein may also be expressed in cells. The DNA encoding the desired protein was ligated into pRK5 containing the desired epitope tag (poly-His, FLAG, human IgG1 Fc) in frame with the desired epitope tag.
The predicted extracellular domains of human and mouse TMEM149 were cloned without the transmembrane domain into the pRK5 vector containing a C-terminal human IgG1-Fc or 8×-His tag, or with the transmembrane domain into the pRK5 vector containing a C-terminal GFP tag.
Soluble forms of these proteins were produced in a CHO cell transient transfection and purified by affinity chromatography using anti-FLAG (M2) agarose affinity gel (Sigma-Aldrich) for FLAG-tagged proteins, Ni-NTA agarose (Qiagen) for 8×-His-tagged proteins, or protein-A Sepharose (Amersham Pharmacia) for IgG1-Fc fusion proteins. Proteins were further separated from aggregates and contaminants with a Superdex 200 gel-filtration column and/or MonoQ/S ion exchange columns (Amersham). Protein purity was assessed by SDS-PAGE followed by SimplyBlue Safe Stain (Invitrogen) and purified proteins were aliqouted and frozen at −80° C. until needed.
In one embodiment, the selected host cells may be HEK293T cells. Human 293 cells (ATCC CCL 1573) were grown to 50-80% confluence in tissue culture plates in medium such as DMEM supplemented with fetal calf serum and optionally, nutrient components and/or antibiotics. 1-10 μg of DNA encoding the desired protein ligated into pRK5 was introduced into HEK293T cells using commercially available transfection reagents Superfect® (Qiagen), Lipofectamine® (Invitrogen) or Fugene® (Roche) according to manufacturer's instructions. 18-24 hours after the transfections, the culture medium was removed and tested in selected bioassays or cells were harvested using 10 mM EDTA in 20 mM Na phosphate buffer, pH7.4, and tested in selected bioassays.
Stable expression of TMEM149 was achieved in HEK293T cells by cloning DNA encoding the desired protein into pRK5 vector with a selection marker that confers resistance to the antibiotic Geneticin®. For stable expression of desired proteins, cells were transfected as described and allowed to grow in DMEM with a concentration of Geneticin® that would permit growth of cells in which the desired vector had integrated into the genome (1-0.5 μg/ml).
In another embodiment, the epitope tagged versions of the desired protein can be expressed in host CHO cells. Twelve micrograms of the desired plasmid DNA was introduced into approximately 10 million CHO cells using commercially available transfection reagents Superfect® (Qiagen), Dosper®, Lipofectamine® (Invitrogen) or Fugene® (Boehringer Mannheim) according to manufacturer's instructions. The cells are grown as described in Lucas et al. (Nucl. Acids Res. (1996) 24:9 1774-1779). Approximately 3×10−7 cells are frozen in an ampule for further growth and production as described below.
The ampules containing the plasmid DNA are thawed by placement into a water bath and mixed by vortexing. The contents are pipetted into a centrifuge tube containing 10 mLs of media and centrifuged at 1000 rpm for 5 minutes. The supernatant was aspirated and the cells were resuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5% 0.2 μm diafiltered fetal bovine serum). The cells are then aliquoted into a 100 mL spinner containing 90 mL of selective media. After 1-2 days, the cells are transferred into a 250 mL spinner filled with 150 mL selective growth medium and incubated at 37° C. After another 2-3 days, 250 mL, 500 mL and 2000 mL spinners are seeded with 3×105 cells/mL. The cell media was exchanged with fresh media by centrifugation and resuspension in production medium. Although any suitable CHO media may be employed, a production medium described in U.S. Pat. No. 5,122,469, issued Jun. 16, 1992 may actually be used. A 3 L production spinner was seeded at 1.2×106 cells/mL. On day 0, the cell number and pH was determined. On day 1, the spinner was sampled and sparging with filtered air was commenced. On day 2, the spinner was sampled, the temperature shifted to 33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g., 35% polydimethylsiloxane emulsion, Dow Corning 365 Medical Grade Emulsion) taken. Throughout the production, the pH was adjusted as necessary to keep it at around 7.2. After 10 days, or until the viability dropped below 70%, the cell culture was harvested by centrifugation and filtering through a 0.22 μm filter. The filtrate was either stored at 4° C. or immediately loaded onto columns for purification.
For the poly-His tagged constructs, the proteins are purified using a Ni-NTA column (Qiagen). Before purification, imidazole was added to the conditioned media to a concentration of 5 mM. The conditioned media was pumped onto a 6 ml Ni-NTA column equilibrated at 4° C., in 20 mM Hepes, pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rate of 4-5 ml/min. After loading, the column was washed with additional equilibration buffer and the protein eluted with equilibration buffer containing 0.25 M imidazole. The purified protein was then run over a Superdex S200 gel filtration column and/or a MonoQ/S ion exchange column (Applied Biosystems) to remove aggregated or proteolysed protein or any contaminants and subsequently concentrated and dialyzed into PBS. The homogeneity was assessed by SDS polyacrylamide gels and by N-terminal amino acid sequencing by Edman degradation. Proteins were stored at −80° C. until used in bioassays.
For the FLAG-epitope tagged constructs, the proteins are purified using an anti-FLAG (M2) agarose column (Sigma). The conditioned media was pumped onto a 6 ml anti-FLAG column equilibrated at 4° C. with 20 mM Na phosphate buffer, pH 7.4. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 μL of 1 M Tris buffer, pH 9. The highly purified protein was subsequently run over size exclusion chromatography, dialyzed, analyzed, and stored as above for the poly-His tagged proteins.
Immunoadhesin (Fc-containing) constructs are purified from the conditioned media as follows. The conditioned medium was pumped onto a 5 ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Na phosphate buffer, pH 7.4. After loading, the column was washed extensively with equilibration buffer before elution with 100 mM citric acid, pH 3.5. The eluted protein was immediately neutralized by collecting 1 ml fractions into tubes containing 275 μL of 1 M Tris buffer, pH 9. The highly purified protein was subsequently run over size exclusion chromatography, dialyzed, analyzed, and stored as above for the poly-His tagged proteins.
This example describes the generation of monoclonal antibodies to TMEM149.
B6 mice were immunized by repeated footpad injections of recombinant human TMEM149-Fc or mouse TMEM149-His resuspended in monophosphoryl lipid A/trehalose icorynomycolate adjuvant (Corixa Corp., Seattle, Wash.). Three days after the final boost, lymph nodes and spleens harvested were fused with SP2/0 myeloma cells (ATCC, Manassas, Va.) using the Cyto Pulse CEEF-50 apparatus (Cyto Pulse Sciences, Glen Burnie, Md.). After 7-10 days single hybridoma clones were picked by ClonePix (Genetix, United Kingdom) and 2-3 days later culture supernatants were collected and screened by ELISA for specific binding to recombinant human or murine TMEM149. The selected hybridomas were then cloned by limiting dilution and monoclonal antibodies were purified from hybridoma culture supernatants by affinity chromatography.
This example illustrates that recombinant mIGFL is a disulphide-linked homodimer.
Recombinant FLAG-mIGFL was express as described above and purified from CHO supernatants. Expressed mIGFL-FLAG was run on an SDS-PAGE gel under both reducing and non-reducing conditions (
This example illustrates that mIGFL is likely produced as a secreted soluble dimeric protein.
To determine if mIGFL can be produced as a soluble protein in vivo, a hydrodynamic tail vein injection-based transfection system was used to express an N-terminal FLAG epitope tagged mIGFL transgene. The technique for expressing exogenous genes in mice by rapidly injecting DNA dissolved in a saline solution equal to 8-12% of the body weight via the tail vein of the animal was adapted from Liu et al. (Gene Ther. (1999) 6:1258-66) For hydrodynamic tail vein (HTV) injection-induced expression of mIGFL, 8-10 wk old Balb/c mice were placed under a heat lamp for 5 minutes prior to the injection to dilate the tail veins. Mice were then restrained in an acrylic chamber to allow access to their tails and 50 μg of empty pRK5 or pRK5 with N-terminal FLAG-tagged mIGFL in a volume of sterile Ringers solution equal to 10% of the mouse's body weight was injected into the tail vein over 5-8 seconds. Mice were bled 6 hours after injections and euthanized via CO2 inhalation at 24 hours after injections and blood was collected via ventricular puncture.
mIGFL was detected in the serum via sandwich ELISA. To capture mIGFL, 384 well plates were coated with mTMEM149-Fc overnight at 4° C. Following 3 washes, plates were blocked for non-specific binding with 0.5% BSA in PBS for 1 hr. Plates were again washed and mouse serum diluted with 50% assay buffer (PBS with 0.5% PBS and 0.05% Tween 20) or a dilution series of purified FLAG-mIGFL standards in assay buffer with 50% mouse serum were added to plates, incubated for 2 hr at RT, and washed 6 times. To detect FLAG-mIGFL, plates were incubated with HRP conjugated anti-FLAG-HRP, washed and incubated with Moss substrate solution to for development. The reaction was stopped with 1M H3PO4 after and plates were read at 450/650 nM.
FLAG-mIGFL was detectible by ELISA in serum from mice injected with the mIGFL transgene, but not control vector, as early as 6 hours post injection and persisted in the serum for at least 24 hours (
This example illustrates that mIGFL is most highly expressed in skin and its expression is enhanced during inflammation.
We determined the tissue distribution of mIGFL expression by RT-PCR on a panel of normal mouse tissues and found that mIGFL mRNA had high relative expression in skin (
Imiquimod is a TLR7/8 agonist that can be topically applied to induce skin lesions that share many of the phenotypic and histological features of psoriasis, including epidermal alterations such as acanthosis, an inflammatory infiltration that includes T cells, neutrophils and dendritic cells, and neoangiogenesis (van der Fits et al. (2009) J Immunol. 182:5836-45).
The Imiquimod-induced psoriasis like model was carried out in 8-12 wk old C57B/6 mice (Charles River). Three days before treatment, mice were anesthetized with isoflurane and hair on their back hindquarters was removed with depilatory cream. Mice were anesthetized with isoflurane and 62.5 mg of Imiquimod cream was administered to the shaved back and right ear daily. Ear thickness was monitored and mice were scored for clinical signs of inflammation every two days according to the following scale: 0=no disease; 1=very mild erythema with very mild thickening and scaling involving a small area; 2=mild erythema with mild thickening and scaling (irregular and patchy) involving a small area; 3=moderate erythema with moderate thickening and scaling (irregular and patchy) involving a moderate area; 4=severe erythema with marked thickening and scaling (irregular and patchy) involving a large area. One day after the last Imiquimod treatment, mice were euthanized via CO2 inhalation and the skin covering the treated area was harvested for RNA purification.
In B6 mice, inflammatory alterations peaked after five days of daily application of Imiquimod (
The following example demonstrates that mIGFL is most highly expressed in skin and its expression is enhanced during injury.
Skin wounding assays were carried out in 8-10 wk old B6 mice. Briefly, mice were put under mild anesthesia and, using sterile conditions, the dorsal region of mice were shaved, excess hair removed with hair removal lotion, and the region was prepped with betadine followed by alcohol. Then, a 6 mm diameter full thickness skin punch was removed at the midline between the scapula and a 0.5 mm silicone frame with 10-12 mm diameter was placed around each wound, which was then dressed. Dressings were changed every other day. Mice were euthanized 7 days after wounding and skin from the area of wounding was collected for RNA purification.
RNA levels were determined using mIGFL primers and probes purchased from Applied Biosystems, Inc, member of Life Sciences, Carlsbad, Calif.
mIGFL mRNA levels were augmented in skin samples taken from healing full-thickness dorsal skin punches (
This example illustrates the tissue distribution of mTMEM149 using RT-PCR on isolated RNA from normal mice.
Total RNA was purified using Qiagen (Valencia, Calif.) RNEasy (cells) according to the manufacturers protocol with DNAse digest. One-step RT-PCR was performed on 25 or 50 ng of total RNA using TaqMan Gold with Buffer A kit on a Strategene (La Jolla, Calif.) Mx3000P system. Copy numbers of mTMEM149 in the mouse tissue panel were determined using a dilution series of mTMEM149 cDNA and then normalized to the average expression level of all tissues examined. For RT-PCR performed on leukocytes, results were normalized to the housekeeping gene RPL19 using comparative Ct method. mTMEM 149 primers and probes were:
RNA for mTMEM149 was ubiquitously expressed throughout the tissue panel, with the highest expression levels observed in the lymph node (
We also found that expression levels of mouse TMEM149 was not altered in psoriasis models (
To more closely analyze the expression pattern of mTMEM149 within the immune system, we analyzed RNA from sorted leukocyte populations of mouse spleens and found mTMEM149 was most highly expressed in T cells and monocytes, though it was expressed in all cell types analyzed (
We could also compete anti-mTMEM149 T cell surface staining with soluble recombinant mTMEM149-His. These results indicate that mouse T cells could be the target cell population for mIGFL produced in the skin.
This example illustrates the expression profile of human TMEM149.
Total RNA from normal human tissues were obtained from Clonetech (Mt. View, Calif.). Total RNA from human leukocyte populations was obtained using Qiagen (Valencia, Calif.) RNEasy kit (cells) according to the manufacturer's protocol with DNAse digest. All RNA was diluted to 10 ng/μl in RNAse free water. One-step RT-PCR was performed on 25 to 50 ng of total RNA using TaqMan Gold with Buffer A kit on a Strategene (La Jolla, Calif.) Mx3000P system. Relative expression levels of human TMEM149 were determined by normalizing to the housekeeping gene RPL19 using comparative Ct method. TMEM 149 primers and probes were:
RNA for TMEM149 was ubiquitously expressed throughout the tissue panel, with the highest expression levels observed in liver, fetal liver and lymphoid tissues (
We also found that the expression level human TMEM149 was not altered in samples from psoriasis patients when compared to normal controls (
To more closely analyze the expression pattern of human TMEM149 within the immune system, we analyzed RNA from leukocytes sorted form human peripheral blood and found that human TMEM149 was most highly expressed in monocyte derived dendritic cells (MDDC), macrophages, and myeloid dendritic cells (mDC), though RNA for human TMEM149 was detectible in all cell types analyzed (
For flow cytometry, cells were washed and stained in PBS containing 2% BSA and 0.1 mM NaN3, and maintained at 4° C. throughout the procedure. HEK293T cells stably transfected with GFP-tagged human TMEM149 were used as a positive control. Primary human peripheral blood leukocytes were purified by centrifuging over a ficoll (GE Lifesciences, Piscataway, N.J.) gradient. MDDC were matured from CD14+ peripheral blood leukocytes purified by magnetic separation according to the manufacturers protocol (Miltenyi, Auburn, Calif.) for one week in 100 ng/ml GM-CSF and 6.7 ng/ml IL-4 (Peprotech, Rock Hill, N.J.). Non-specific antibody binding was blocked with an anti-Fc receptor antibody. Cells were then incubated with anti-human TMEM149 in the presence or absence or recombinant His-tagged human TMEM149, washed, and then incubated with an anti-mouse IgG-PE or -APC labeled secondary antibody (Jackson Immunoresearch, West Grove, Pa.). Cells were then labeled with fluorescently labeled, lineage specific antibodies: CD3, CD4, and CD8 for T cell populations, B220 for B cells, DX5 for NK cells, CD11c for mDC, and CD14 for monocytes (BD Biosciences, San Jose, Calif.), and analyzed by FACS.
Antibody staining of leukocytes indicated that human TMEM149 was expressed on the surface of all cell types analyzed (
These results indicate that all human leukocyte populations could be target cell populations for IGFL1.
This example characterizes the various IGFL family members.
Human IGFL1 was expressed in CHO cells and purified as described above. Expressed IGFL1-FLAG was run on an SDS-PAGE gel under both reducing and non-reducing conditions (
Human IGFL2, IGFL3 and IGFL4 were each expressed as homodimers as well when expressed in CHO cells. (
In addition, IGFL1 appeared to be larger than mIGFL. This could be due to glycosylation. In order to confirm that the IGFL members are glycosylated the polypeptides were treated with a glycosidase (PNGase F; New England Biolabs, Ipswich, Mass.) and run on an SDS-PAGE gel. Results are shown in
This example illustrates the binding of IGFL1 to TMEM149.
To identify a binding partner for IGFL1, we screened a protein microarray containing ˜700 unique mostly human proteins that are secreted or have transmembrane domains (Clark, et al. 2003. Genome Res. 13:2265-70; Ramani et al., Identifying Extracellular Protein Interactions using a Secreted Protein Microarray Platform.) and identified the top hit as TMEM149 (
Proteins from the Genentech SPDI library (Clark, et al. 2003, supra) were printed and immobilized in duplicate on epoxy derivatized slides (Schott, Elmsford, N.Y.), using a NanoPrint microarrayer (Arrayit). Slides were blocked in 5% milk PBST and stained with Cy-5 labeled IGFL-1 or TMEM149-Fc for 20 min. (20 μg/ml) followed by 3×5 min. washes with PBST, using an A-Hyb hybridization station (Miltenyi, Auburn, Calif.). Fluorescent intensity at 635 nm minus local background was measured for each arrayed protein and the scores normalized by the average intensity over the entire slide.
In a reverse screen, TMEM149 uniquely bound to 5 individual protein preparations of IGFL1 in the library (
To further characterize the interaction between TMEM149 and IGFL1, we analyzed the binding of recombinant IGFL1 to HEK293T cells expressing TMEM149 or other TNFR family members. An antibody generated against the extracellular domain of TMEM149 was used to demonstrate surface expression of TMEM149 on transiently transfected cells (
This example demonstrates that TMEM149 binds to certain members of the IGFL family.
To determine the affinity of IGFL with TMEM149, ligands were iodinated with 125I using the lactoperoxidase method and free 125I-Na was removed from the labeled protein by gel filtration using a NAP-5 column. 50 μL competition reaction mixtures containing a fixed concentration of iodinated ligand and decreasing concentrations of serially diluted, unlabeled ligand were placed into 96-well plates in triplicate. To each well, HEK293T cells stably expressing human or mouse TMEM149 were added at a density of 100,000 cells in 0.2 mL of binding buffer (Dulbecco's Modified Eagle Medium with 1% bovine serum albumin, 50 mM HEPES pH 7.2 and 2 mM sodium azide). The final concentration of the iodinated ligand with cells in each competition reaction was 100 pM (100,000 cpms per 0.25 mL). The final concentration of the unlabeled ligand with cells in the competition reaction varied, starting at 500 nM and then decreasing by 1- to 2-fold dilution for ten concentrations, and included a zero-added, buffer-only sample. Competition reactions were incubated for 2 hours at room temperature, then transferred to a Millipore Multiscreen filter plate (Billerica, Mass.) and washed 4 times with binding buffer to separate the free from bound iodinated antibody. The filters were counted on a Wallac Wizard 1470 gamma counter (PerkinElmer Life and Analytical Sciences Inc.; Wellesley, Mass.). The binding data was evaluated using NewLigand software (Genentech), which uses the fitting algorithm of Munson and Robard to determine the binding affinity of the antibody (Munson, P. J., and D. Rodbard. 1980. Anal Biochem. 107:220-39).
A radioligand binding assay performed using IGFL1 and TMEM149 expressing cells revealed a high-affinity interaction with an equilibrium dissociation constant (KD) of 0.31 nM (
Surface plasmon resonance (SPR) was run on a BiaCore 3000 (GE healthcare, Piscataway, N.J.) at 25° C. using anti-human IgG F(ab′)2 (Jackson Immunoresearch, West Grove, Pa.) amine-coupled to BiaCore CM5 sensor chips. For kinetic analysis of protein interactions, Fc-fusion proteins at 25 μg/ml were first captured onto sensor chips by injecting them for 3 min at 5 μl/min. Then, test proteins diluted to their indicated concentrations in HBS-P buffer (0.01 M Hepes, pH 7.4 0.15 M NaCl 0.005% Surfactant P20) were injected for 3 min at 30 μl/min. Dissociation in HBS-P buffer alone was then measured at a flow rate of 30 ml/min. Sensor chips surfaces were regenerated by injecting 10 mM glycine pH 1.5 for 30 s after each cycle. Kinetic parameters were determined by fitting the data to a 1:1 Langmuir model using the Biacore 3000 evaluation software (GE Healthcare). For TNF screen, EDA-A1, OX40L, FasL, 4-1 BBL, CD30L were purchased from R&D and GITRL, ApoL, RANKL, TNFα, APRIL, LTα, LTβ, TL1A, TNFSF14, TRAIL, C40L, BAFF, and CD27L were produced in house.
SPR resulted in a KD of 1.2 nM (
To determine if this interaction was conserved between species, we analyzed the ability of mIGFL to interact with mouse TMEM149 (mTMEM149). Using an antibody directed against mTMEM149, we could demonstrate surface expression of mTMEM149 on transfected cells (
A sequence analysis of the ectodomain (ECD) of human TMEM149 demonstrated protein homology to the TNFR family (Zhang, 2004). The ECD of TNFR are comprised of cysteine-rich domains (CRDs) that can be further dissected into modular subdomains with conserved cysteine registers, disulphide bonding, and overall structures (Naismith and Sprang, 1998). Alignment of the ECD of TMEM149 with these modules revealed that the first potential CRD was similar to A1-B2 CRD modules that are commonly observed in other TNFR family members (
Since TMEM149 is an unrecognized TNFR family member, we screened TNF-like ligands for binding to TMEM149 via SPR. There was no significant binding between 18 TNF family members screened and TMEM149 (not shown). Thus, we have positively identified a specific and high-affinity protein interaction between mIGFL/IGFL1 and TMEM149 with a putative cytoplasmic DD. This interaction has not been described previously.
This example illustrates the expression of IGFL1 on keratinocytes.
As sequence identity between mIGFL and the human IGFL genes is low, there is no clear human ortholog to mIGFL. We therefore sought to determine if the expression of any human IGFL genes were altered during skin inflammation.
RNA microarrays were performed on two sets of normal control and psoriasis patient samples. Samples from normal skin, psoriatic skin or adjacent nonlesional skin were run on HGU133A and HGU133B Genechips (Affymetrix, Santa Clara, Calif.) or WHG 44×44 k microarray chips (Agilent Technologies, Foster City, Calif.). The following probes were used: 239430_at for IGFL1, 231148_at for IGFL2, and P_A51261_at for IGFL3 on HGU33 or HuGenen1 Affymetrix microarray chips (n=5 normal and n=11 psoriasis/PSNE, ±SE), and A—23_P119407 for IGFL4 on an Agilent WHG 4×44 k microarray (n=7 normal and n=15 psoriasis/PSNE, ±SE). Expression values were measured by signal intensity from Affymetrix Microarray Analysis Suite version 5 and samples were further normalized by using Robust Multi-chip Average quantile normalization.
First, microarray analysis of skin RNA isolated from effected or non-effected skin from psoriasis patients or normal controls revealed that IGFL1 was upregulated in effected skin (
To confirm microarray data, we performed RT-PCR with primers specific for each of the human IGFL genes on skin RNA samples from psoriasis patients. Total RNA was purified using Qiagen (Valencia, Calif.) RNeasy Fibrous Tissue according to the manufacturer's protocol with DNAse digest. The primer and probe sets for IGFL2 and IGFL4 were purchased from ABI (Foster City, Calif.) and primer and probes for IGFL1 and 3 were synthesized in house. One-step RT-PCR was performed on 25 or 50 ng of total RNA using TaqMan Gold with Buffer A kit on a Strategene (La Jolla, Calif.) Mx3000P system. For RT-PCR performed on skin results were normalized to the housekeeping gene RPL19 using comparative Ct method.
Once again, IGFL1 was highly upregulated and IGFL2 was down-regulated (
This example illustrates that TNFα produced in the psoriatic lesion could contribute to the IGFL1 expression levels observed in patient skin.
Psoriatic skin is characterized as epidermal keratinocyte hyperplasia and leukocytes infiltration and activation. Both the infiltrating leukocyte populations and the keratinocytes are crucial sources for producing factors that enhance this tissue inflammation (Tonel and Conrad (2009) Int J Biochem Cell Biol. 41:963-8).
To further identify the source of IGFL1, we analyzed mRNA expression levels by RT-PCR and found that IGFL1 was expressed in primary keratinocytes but not in peripheral blood mononuclear cells (PBMC; not shown). Furthermore, activation of human PBMCs or mouse splenocytes by ConA, phytohaemagglutanin, or lipopolysaccharide did not induce IGFL1 or mIGFL expression (not shown).
To gain insights to the regulation of IGFL1 in skin we used cultured primary human keratinocytes to determine if any of the cytokines known to play a major role in psoriasis altered IGFL1 expression. Primary human keratinocytes were cultured in supplemented Keratinocyte-SFM in tissue culture flasks coated with 0.67 μg/cm2 Collagen IV in a 37° C. incubator with 5% CO2. Medium was replaced every 2-3 days until cells reached 70-80% confluency at which time cells were subcultured or used for experiments. For stimulations, 3-5×105 cells were added to Collagen IV coated 12-well tissue culture plates in 1 ml Keratinocyte-SFM and allowed to adhere overnight. The following day cytokines were added to a final concentration of 50 ng/ml TNFα, 50 ng/ml IL-16, 50 ng/ml IFNγ, 40 ng/ml IL-17A or 50 ng/ml IL-22. After 6 hr and 24 hr cells were harvested, snap frozen on dry ice, and stored at −80° C. for RNA purification. Treatment of cultured keratinocytes with TNFα for 6 h resulted in a five-fold upregulation of mIGFL that remained elevated after 24 h of stimulation (
This example illustrates that binding of hIGFL1 to TMEM149 can be blocked by antibodies directed against TMEM149.
This assay was performed on HEK293T cells transfected with human TMEM149-GFP DNA as described above. Cells were lifted from the 6 well plates they were transfected in and washed with FACS buffer (PBS with 2% FBS and 0.1% NaN3). Cells were then incubated with 10 μg/ml anti-TMEM149 antibodies or isotype controls for 15 min at 4° C. Then FLAG-IGFL1 was added to cells at 10 μg/ml, and allowed to incubate for 30 min at 4° C. Antibodies/IGFL1 were then washed off with FACS buffer and fluorescently labeled anti-FLAG was added to the cells to detect IGFL1 binding for 30 min at 4° C. Cells were then washed and analyzed by flow cytometry.
The results indicate that certain antibodies either prevent binding of IGFL3 by direct competition or by inducing a conformational change such that the IGFL3 can no longer bind. See
This example illustrates that binding of hIGFL3 to TMEM149 can be blocked by antibodies directed against TMEM149.
This assay was performed as described in Example 14, above, except that IGFL3 was used instead of IGFL1.
See
This example shows that IGFL1 is active as a stimulator of the proliferation of T-lymphocytes. Compounds which stimulate proliferation of lymphocytes are useful therapeutically where enhancement of an immune response is beneficial. A therapeutic agent may also take the form of antagonists of IGFL1, for example, murine-human chimeric, humanized or human antibodies against the polypeptide, which would be expected to inhibit T-lymphocyte proliferation.
The basic protocol for this assay is described in Current Protocols in Immunology, unit 3.12; edited by J. E. Coligan, A. M. Kruisbeek, D. H. Marglies, E. M. Shevach, W. Strober, National Institutes of Health, Published by John Wiley & Sons, Inc.
More specifically, in one assay variant, peripheral blood mononuclear cells (PBMC) are isolated from mammalian individuals, for example a human volunteer, by leukopheresis (one donor will supply stimulator PBMCs, the other donor will supply responder PBMCs). If desired, the cells are frozen in fetal bovine serum and DMSO after isolation. Frozen cells may be thawed overnight in assay media (37° C., 5% CO2) and then washed and resuspended to 3×106 cells/ml of assay media (RPMI; 10% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential amino acids, 1% pyruvate).
The stimulator PBMCs are prepared by irradiating the cells (about 3000 Rads). The assay is prepared by plating in triplicate wells a mixture of: 100 μl of test sample diluted to 1% or to 0.1%; 50 μl of irradiated stimulator cells and 50 μl of responder PBMC cells. 100 microliters of cell culture media or 100 microliter of CD4-IgG is used as the control. The wells are then incubated at 37° C., 5% CO2 for 4 days. On day 5 and each well is pulsed with tritiated thymidine (1.0 μCi/well; Amersham). After 6 hours the cells are washed 3 times and then the uptake of the label is evaluated.
In another variant of this assay, PBMCs are isolated from the spleens of Balb/c mice and C57B6 mice. The cells are teased from freshly harvested spleens in assay media (RPMI; 10% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential amino acids, 1% pyruvate) and the PBMCs are isolated by overlaying these cells over Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20 minutes, collecting and washing the mononuclear cell layer in assay media and resuspending the cells to 1×107 cells/ml of assay media. The assay is then conducted as described above. IGFL1 at a concentration of 58.32 nM stimulated T-cell proliferation 230.1% compared to controls. Positive increases over control are considered positive with increases of greater than or equal to 180% being preferred. However, any value greater than control indicates a stimulatory effect for the test protein.
This example shows that IGFL3 is active as an inhibitor of the proliferation of stimulated T-lymphocytes. Compounds which inhibit proliferation of lymphocytes are useful therapeutically where suppression of an immune response is beneficial.
The basic protocol for this assay is described in Current Protocols in Immunology, unit 3.12; edited by J. E. Coligan, A. M. Kruisbeek, D. H. Marglies, E. M. Shevach, W. Strober, National Institutes of Health, Published by John Wiley & Sons, Inc.
More specifically, in one assay variant, peripheral blood mononuclear cells (PBMC) are isolated from mammalian individuals, for example a human volunteer, by leukopheresis (one donor will supply stimulator PBMCs, the other donor will supply responder PBMCs). If desired, the cells are frozen in fetal bovine serum and DMSO after isolation. Frozen cells may be thawed overnight in assay media (37° C., 5% CO2) and then washed and resuspended to 3×106 cells/ml of assay media (RPMI; 10% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential amino acids, 1% pyruvate). The stimulator PBMCs are prepared by irradiating the cells (about 3000 Rads).
The assay is prepared by plating in triplicate wells a mixture of:
100:1 of test sample diluted to 1% or to 0.1%,
50:1 of irradiated stimulator cells, and
50:1 of responder PBMC cells.
100 microliters of cell culture media or 100 microliter of CD4-IgG is used as the control. The wells are then incubated at 37° C., 5% CO2 for 4 days. On day 5, each well is pulsed with tritiated thymidine (1.0 μCi/well; Amersham). After 6 hours the cells are washed 3 times and then the uptake of the label is evaluated.
In another variant of this assay, PBMCs are isolated from the spleens of Balb/c mice and C57B6 mice. The cells are teased from freshly harvested spleens in assay media (RPMI; 10% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential amino acids, 1% pyruvate) and the PBMCs are isolated by overlaying these cells over Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20 minutes, collecting and washing the mononuclear cell layer in assay media and resuspending the cells to 1×107 cells/ml of assay media. The assay is then conducted as described above.
Any decreases below control is considered to be a positive result for an inhibitory compound, with decreases of less than or equal to 80% being preferred. However, any value less than control indicates an inhibitory effect for the test protein. IGFL3 at a concentration of 244 nM inhibits T-cell proliferation by 58.9% compared to control.
This example illustrates that human IGFL3 is active as an inhibitor of the stimulation of CD4+ enriched lymphocytes. Compounds which inhibit proliferation of lymphocytes are useful therapeutically where suppression of an inflammatory immune response is beneficial. This assay is a variation of the MLR assay above wherein the IGFL3 was examined for its inhibitory effect upon the co-stimulation of CD4+ enriched lymphocytes with both anti-CD3 and anti-CD28. The inhibition of the stimulatory effect of anti-CD3 and anti-CD28 on PBMCs is proposed to correlate with a general antiproliferative effect similar to the engagement of the TCR with a costimulatory signal.
The basic protocol for the isolation of PBMCs used in this assay is described in Current Protocols in Immunology, unit 3.12; edited by J. E. Coligan, A. M. Kruisbeek, D. H. Marglies, E. M. Shevach, W. Strober, National Institutes of Health, Published by John Wiley & Sons, Inc.
More specifically, in one assay variant, peripheral blood mononuclear cells (PBMC) are isolated from mammalian individuals, for example a human volunteer, by leukopheresis. Cells are isolated and enriched using negative selection. If desired, the enriched cells are frozen in 90% fetal bovine serum and 10% DMSO. Frozen cells may be thawed overnight in assay media (37° C., 5% CO2) and then washed and resuspended to 1×106 cells/ml of assay media (RPMI; 10% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential amino acids, 1% pyruvate).
The assay is prepared by plating in triplicate wells a mixture of:
The wells are then incubated at 37° C., 5% CO2 for about 3 days. On day 4, each well is pulsed with tritiated thymidine (1.0 μCi/well; Amersham). After 6 hours, the plate is harvested and then the uptake of the label is evaluated.
A result which shows an inhibitory effect (i.e., 3[H]-thymidine incorporation) less than 70% of that observed in the control is considered to be a positive result.
In another variant of this assay, CD4+ splenocytes are isolated from the spleens of Balb/c mice. The cells are teased from freshly harvested spleens in assay media (RPMI; 10% fetal bovine serum, 1% penicillin/streptomycin, 1% glutamine, 1% HEPES, 1% non-essential amino acids, 1% pyruvate) and the splenocytes are isolated by overlaying these cells over Lympholyte M (Organon Teknika), centrifuging at 2000 rpm for 20 minutes, collecting and washing the mononuclear cell layer in assay media, negative selection and resuspending the cells to 1×107 cells/ml of assay media. The assay is then conducted as described above.
Human IGFL3 at a concentration of 8.44 nM exhibited an inhibitory effect.
This example illustrates the tissue distribution of human IGFL1, IGFL2 and IGFL3 using RT-PCR.
These assays were performed as described in Example 8 using the same panel of normal human tissue RNA, but with primer/probe sets specific for IGFL1, IGFL2 and IGFL3.
IGFL1 primers and probes were:
IGFL2 probes were purchased from Applied Biosystems, Inc, member of Life Sciences, Carlsbad, Calif.
IGFL3 primers and probes were:
Human IGFL1 appeared to have the highest expression in tonsils, kidneys, testis and thymus. There was also some IGFL1 expression evident in retina, stomach, skin, bladder, and mammary gland, but not other tissues examined. See
Human IGFL2 appeared to be most highly expressed in testis and skin, with some expression evident in thymus, prostate, brain and spinal cord. See
Human IGFL3 appeared to be most highly expressed in testis and brain. Skin, Kidney, spinal cord, retina, mammary gland and thymus also expressed IGFL3. See
These data demonstrate that in normal human tissues, members of the IGFL family are expressed differently. It also demonstrates that expression of these genes is restricted to specific tissues and that all of the IGFL genes examined are expressed in skin.
It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.
MGPSWLLWTVAVAVLLLTRAASMEASSFCGHLEYWNSDKRCCSRCLQRFGPPACPDHE
This application is a continuation of International Application No. PCT/US2011/062812 having an international filing date of Dec. 1, 2011, the entire contents of which are incorporated herein by reference, and which claims benefit under 35 U.S.C. §119 to U.S. Provisional Application Ser. No. 61/419,209 filed 2 Dec. 2010.
| Number | Date | Country | |
|---|---|---|---|
| 61419209 | Dec 2010 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2011/062812 | Dec 2011 | US |
| Child | 13900707 | US |
