Patent Application
20100041596
1. Field of the Invention
This invention provides a novel function for a conserved domain in the extracellular region of the members of the TNF receptor (TNFR) superfamily in mediating specific ligand-independent assembly of receptor oligomers.
2. Background Art
The members of the TNFR superfamily typically contain one to six cysteine rich domains (CRDs) in their extracellular regions, a single transmembrane domain and variably sized intracytoplasmic domains. The members of this receptor family typically bind to ligands of the TNF cytokine family that are defined by structural, functional and sequence similarities. These receptors form trimers in their active liganded state and several members contain a cytoplasmic domain referred to as a death domain.
According to the present invention, the extracellular region of these receptors is further characterized by a novel self-association or homotypic association function that is mediated via a pre-ligand receptor assembly domain (PLAD) that contains at least one cysteine rich domain. More specifically, members of the TNFR superfamily, including TRAIL receptor 1, CD40, 60 kDa TNFR and 80 kDa TNFR show this homotypic association. Other members of the TNFR superfamily, including Fas, LTβR, CD40, CD30, CD27, HVEM, RANK, OX40 and DR4 contain this PLAD. The PLAD is necessary for ligand binding and receptor function. Thus, members of the TNFR superfamily appear to signal through distinct pre-formed complexes rather than through ligand-induced cross-linking of individual receptor subunits. Therefore, PLAD can be targeted by pharmaceutical agents in order to block the formation of these preformed complexes and thus block receptor function.
It is well established that many microbes have evolved effective strategies for countering the immune response directed against them (79). More specifically, several viruses and bacteria express homologs of cellular proteins designed to modulate or directly block the action of immune effector molecules, including cytokines and chemokines (80). Viral homologs of TNF receptor-like receptors (vTNFRs) have been identified for several large DNA viruses, including several poxviruses and human cytomegalovirus. The existence of or any role for PLAD-mediated self-association of vTNFRs or heterologous association with TNF family receptors has not been elucidated.
Two major arthritides are rheumatoid arthritis (RA) and septic arthritis (SA). RA is a common human autoimmune disease with chronic joint inflammation and progressive bone destruction (48). Although the etiology and pathogenesis of RA are not yet fully understood, cytokines such as TNF-α, IL-1, IL-6 and receptor activator of NF-κB ligand (RANKL), are involved in disease progression (56-60). Nuclear factor-kappa B (NF-κB) is a critical regulator of these cytokines (56-60). TNF-α plays a key role in the pathogenesis of RA and its antagonists such as etanercept (also known as Enbrel), a TNFR II immunoglobulin Fc fusion protein, and infliximab (also known as Remicade), an anti-TNF-α monoclonal antibody, can improve the clinical course of RA (48). SA is a rapidly progressive and highly destructive joint disease induced by bacterial infection in which TNF-α also plays an important role (49). Experimental mouse models of arthritis induced by TNF-α (59), lipopolysaccharide (LPS), CpG-DNA (50, 61), and collagen (62) have been useful for testing new treatments. These agents induce synovitis, pannus formation, and bone and cartilage destruction as well as other features observed in human RA and SA.
According to the invention, in vitro and in vivo data indicate that TNFR PLAD proteins can potently inhibit TNF-α and its consequences in experimental inflammatory arthritis.
The present invention provides a polypeptide comprising the isolated amino acid sequence of a pre-ligand assembly domain (PLAD) of a TNF receptor-like receptor.
Also provided by this invention is a polypeptide comprising the isolated amino acid sequence of a pre-ligand assembly domain (PLAD), wherein the PLAD is selected from the group consisting of: the PLAD of TNF-R, the PLAD of p60, the PLAD of p80, the PLAD of Fas (CD95/APO-1), the PLAD of TRAIL receptors, the PLAD of LTβR, the PLAD of CD40, the PLAD of CD30, the PLAD of CD27, the PLAD of HVEM, the PLAD of OX40, the PLAD of DR4, the PLAD of NGFR, the PLAD of Troy, the PLAD of EDAR, the PLAD of XEDAR, the PLAD of DcR3, the PLAD of AITR, the PLAD of 4-1BB, the PLAD of DR3, the PLAD of RANK, the PLAD of TACI, the PLAD of BCMA, the PLAD of DR6, the PLAD of OPG, the PLAD of DRS, the PLAD of DcR1, and the PLAD of DcR2. TNF-R, p60 TNFR, p80 TNFR, Fas, TRAIL receptors, LTβR, CD40, CD30, CD27, HVEM, OX40, DR4, NGFR, Troy, EDAR, XEDAR, DcR3, AITR, 4-1BB, DR3, RANK, TACI, BCMA, DR6, OPG, DRS, DcR1, and DcR2 are all members of the TNF receptor superfamily also referred to herein as the TNF receptor-like receptor family. The invention also provides the PLAD for other members of the TNF receptor superfamily and how it can be identified by one of skill in the art.
The polypeptides of the present invention can be utilized to inhibit PLAD self-association as well as oligomerization of members of the TNF receptor superfamily. These polypeptides can also be utilized to inhibit ligand binding to members of the TNF receptor superfamily.
The present invention also provides a composition comprising an inhibitor of TNF receptor oligomerization. Further provided by this invention are members of the TNF receptor superfamily that are lacking a PLAD.
The present invention may be understood more readily by reference to the following detailed description of the preferred embodiments of the invention and the Examples included therein and to the Figures and their previous and following description.
As used in the specification and in the claims, “a” can mean one or more, depending upon the context in which it is used. Thus, for example, reference to “a nucleic acid” means that at least one nucleic acid is utilized.
The present invention provides a polypeptide comprising the isolated amino acid sequence of a pre-ligand assembly domain (PLAD). The present invention also provides a polypeptide consisting of the amino acid sequence of a pre-ligand assembly domain. The PLAD of the present invention can be the PLAD of a TNF-R, the PLAD of p60, the PLAD of p80, the PLAD of Fas (CD95/APO-1), the PLAD of TRAIL, the PLAD of LTβR, the PLAD of CD40, the PLAD of CD30, the PLAD of CD27, the PLAD of HVEM, the PLAD of OX40, the PLAD of DR4 or any other PLAD domain from a member of the TNFR superfamily. Since the PLAD domain is highly conserved among members of the TNFR superfamily, one skilled in the art could identify the PLAD domain of any TNF receptor by searching available databases for the conserved motif that characterizes the PLAD domain. Identification of these regions in TNF receptor-like receptors is made routine by the provision of exemplary PLAD sequences herein and their comparison to published sequences of other members of the family (see
The PLADs provided herein can comprise as few as 38 amino acids of the N-terminus of a mature TNF receptor-like receptor. A mature TNF receptor-like receptor is a TNF receptor-like receptor that does not include a signal sequence. Examples of PLADs are disclosed in the sequence listing, which includes amino acid sequences of examples of TNF receptor-like receptors including their signal sequences. The residues of the signal sequences of the respective receptors can be found by reference to the GenBank accession numbers for these TNF receptor-like receptors listed in Table 1. Thus, the sequences of the mature TNF receptor-like receptors and their corresponding PLADs are disclosed in the provided sequences. Table 3 provides additional information about the TNF receptor-like receptors and receptor ligands disclosed herein. It also provides information regarding the uses for the isolated PLADs and polypeptides containing the isolated PLADs disclosed herein. The PLADs can be used to study the implications of interfering with a signal transduction pathway mediated by a receptor of the TNFR superfamily. For example, if signaling via a receptor of the TNFR superfamily is known or shown to be associated with a disease pathway, the inhibition of receptor pre-ligand assembly by the present polypeptides, can treat or prevent the disease. For example, diseases that can be treated include cancer, heart disease and inflammatory diseases. Modifications of PLAD can also change the affinity of ligand/receptor interactions, which can be used in in vitro studies such as measuring ligand and receptor binding, receptor signals etc. Fluorescence-tagged PLAD proteins may also be utilized as reagents for determining relative expression of specific TNFRs on the surface of cells via flow cytometry or fluorescence microscopy.
The present invention also provides a polypeptide of 38 to 125 amino acids comprising an isolated PLAD. For example, the polypeptide can be from 50 to 125 amino acids comprising an isolated PLAD. In a further example, the polypeptide can comprise the subsequence R1-TNF receptor-like receptor PLAD-R2, wherein R1 and R2 are optional and when present can be H, acyl, NH2, an amino acid or a peptide. When present, R1 and/or R2 can be any amino acid. When R1 and/or R2 is a peptide, this peptide can vary in length. For example, R1 and/or R2 can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more amino acids in length as long as the entire polypeptide comprising the isolated TNF-like PLAD is no more than 125 amino acid residues, and can be 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124 or 125 amino acids in length. R1 and R2 can also be sequences of the TNF receptor-like receptor that normally flank the TNF-like PLAD in a naturally occurring TNF receptor-like receptor, wherein the polypeptide comprising the TNF-like receptor PLAD is not the entire extracellular domain of a TNF receptor-like receptor.
Further provided by this invention is a polypeptide of any size, comprising the isolated amino acid sequence of a pre-ligand assembly domain (PLAD) of a TNF receptor-like receptor, wherein the polypeptide is R1-TNF receptor-like receptor PLAD-R2, wherein R1 or R2 comprise an amino acid sequence that does not flank the TNF receptor-like receptor PLAD in a naturally occurring TNF receptor-like receptor. R1 or R2, but not both can be full or partial sequences of the TNF receptor-like receptor that normally flank the TNF-like PLAD in a naturally occurring TNF receptor-like receptor. For example, the PLAD can be from a TNF receptor-like receptor and R1 or R2, can be amino acid sequences that are not present in the TNF receptor-like receptor from which the TNF-like PLAD of the polypeptide was derived or any other TNF receptor-like receptor. R1 or R2 can be any amino acid sequence as long as R1-TNF-like PLAD-R2 is not a naturally occurring full-length TNF receptor-like receptor. In another example, the PLAD can be from one TNF receptor-like receptor and R1 or R2 or both, if present, can be peptide sequences from another TNF receptor-like receptor. Therefore, one skilled in the art can combine the PLAD of one TNF receptor-like receptor with R1 or R2 sequences from a different TNF receptor-like receptor to obtain this polypeptide. Since the sequences of known TNF receptor-like receptors are publicly available, the structure of R1 and R2 of the present polypeptide are numerous but well known and contemplated herein. Alternatively, R1 or R2 can be peptide sequences that are not related to any of the TNF receptor-like receptor sequences. In one embodiment the polypeptide comprising an isolated PLAD is not the 124 amino acid sequence of the mature (lacking the signal sequence) TNF1 receptor disclosed in U.S. Pat. No. 5,633,145 (Feldman et al.) and shown in SEQ ID NO:40.
Examples of polypeptides comprising the above-mentioned subsequence include: R1-amino acids 1-38, 1-39, 1-40, 141, 142, 143, 1-44, 1-45, 1-46, 1-47, 1-48, 1-49, 1-50, 1-51, 1-52, 1-53, or 1-54 of mature p60-R2 (e.g., SEQ ID NO: 1); R1-amino acids 10-48, 10-49, 10-50, 10-51, 10-52, 10-53, or 10-54 of mature p80-R2 (SEQ ID NO: 2); R1-amino acids 1-38, 1-39, 1-40, 1-41, 142, or 1-43 of mature Fas-R2 (SEQ ID NO: 3); R1-amino acids 1-38, 1-39, 1-40, 1-41, 1-42, 1-43, 1-44, 145, 1-46, 1-47, 1-48, 1-49, 1-50, 1-51, 1-52, 1-53, 1-54, 1-55, 1-56, 1-57, 1-58, 1-59, 1-60, 1-61, 1-62, 1-63, 1-64, 1-65, or 1-66 of mature Fas-R2 (SEQ ID NO: 4); R1-amino acids 13-50 of mature LtβR-R2 (SEQ ID NO: 5); R1-amino acids 6-39 of mature CD40-R2 (SEQ ID NO: 6); R1-amino acids 11-49, 11-50, or 11-51 of mature CD30-R2 (SEQ ID NO: 7); R1-amino acids 7-42 of mature CD27-R2 (SEQ ID NO: 8), R1-amino acids 6-37 of mature HVEM-R2 (SEQ ID NO: 9); R1-amino acids 3-36 of mature OX40-R2 (SEQ ID NO: 10), and R1-amino acids 109-138 of mature DR4-R2 (SEQ ID NO: 11).
The mature p60 (TNFR1) polypeptide starts at position 30 of the full-length p60 coding sequence set forth as SEQ ID NO: 12. Therefore, the present invention provides a polypeptide comprising amino acids 1-54 of the mature p60 protein (amino acids 30-83 of SEQ ID NO: 12), a polypeptide comprising amino acids 1-53 of the mature p60 protein (amino acids 30-82 of SEQ ID NO: 0.12), a polypeptide comprising amino acids 1-52 of the mature p60 protein (amino acids 30-81 of SEQ ID NO: 12), a polypeptide comprising amino acids 1-51 of the mature p60 protein (amino acids 30-80 of SEQ ID NO: 12), a polypeptide comprising amino acids 1-50 of the mature p60 protein (amino acids 30-79 of SEQ ID NO: 12), a polypeptide comprising amino acids 1-49 of the mature p60 protein (amino acids 30-78 of SEQ ID NO: 12), a polypeptide comprising amino acids 1-48 of the mature p60 protein (amino acids 30-77 of SEQ ID NO: 12), a polypeptide comprising amino acids 1-47 of the mature p60 protein (amino acids 30-76 of SEQ ID NO: 12), a polypeptide comprising amino acids 1-46 of the mature p60 protein (amino acids 30-75 of SEQ ID NO: 12), a polypeptide comprising amino acids 1-45 of the mature p60 protein (amino acids 30-74 of SEQ ID NO: 12), a polypeptide comprising amino acids 1-44 of the mature p60 protein (amino acids 30-73 of SEQ ID NO: 12), a polypeptide comprising amino acids 1-43 of the mature p60 protein (amino acids 30-72 of SEQ ID NO: 12), a polypeptide comprising amino acids 1-42 of the mature p60 protein (amino acids 30-71 of SEQ ID NO: 12), a polypeptide comprising amino acids 1-41 of the mature p60 protein (amino acids 30-70 of SEQ ID NO: 12), a polypeptide comprising amino acids 1-40 of the mature p60 protein (amino acids 30-69 of SEQ ID NO: 12), and a polypeptide comprising amino acids 1-39 of the mature p60 protein (amino acids 30-68 of SEQ ID NO: 12) as well as other polypeptides comprising fragments of amino acids 1-54 of the mature p60 protein that retain PLAD activity.
The mature p80 (TNFR2) polypeptide starts at position 23 of the full-length p80 coding sequence set forth as SEQ ID NO: 13. Therefore the present invention provides a polypeptide comprising amino acids 10-54 of the mature p80 protein (amino acids 32-76 of SEQ ID NO: 13), a polypeptide comprising amino acids 10-53 of the mature p80 protein (amino acids 32-75 of SEQ ID NO: 13), a polypeptide comprising amino acids 10-52 of the mature p80 protein (amino acids 32-74 of SEQ ID NO: 13), a polypeptide comprising amino acids 10-51 of the mature p80 protein (amino acids 32-73 of SEQ ID NO: 13), a polypeptide comprising amino acids 10-50 of the mature p80 protein (amino acids 32-72 of SEQ ID NO: 13), a well as other polypeptides comprising fragments of amino acids 10-54 of the mature p80 protein that retain PLAD activity.
The mature Fas receptor polypeptide starts at position 17 of the full-length Fas coding sequence set forth as SEQ ID NO: 14. Therefore the present invention provides a polypeptide comprising amino acids 1-43 of the mature Fas protein (amino acids 17-59 of SEQ ID NO: 14), a polypeptide comprising amino acids 1-42 of the mature Fas protein (amino acids 17-58 of SEQ ID NO: 14), a polypeptide comprising amino acids 1-41 of the mature Fas protein (amino acids 17-57 of SEQ ID NO: 14), a polypeptide comprising amino acids 1-40 of the mature Fas protein (amino acids 17-56 of SEQ ID NO: 14), a polypeptide comprising amino acids 1-39 of the mature Fas protein (amino acids 17-55 of SEQ ID NO: 14), a well as other polypeptides comprising fragments of amino acids 1-43 of the mature Fas protein that retain PLAD activity.
The present invention also provides a polypeptide comprising amino acids 1-66 of the mature Fas protein (amino acids 17-82 of SEQ ID NO: 14), a polypeptide comprising amino acids 1-65 of the mature Fas protein (amino acids 17-81 of SEQ ID NO: 14), a polypeptide comprising amino acids 1-64 of the mature Fas protein (amino acids 17-80 of SEQ ID NO: 14), a polypeptide comprising amino acids 1-63 of the mature Fas protein (amino acids 17-79 of SEQ ID NO: 14), a polypeptide comprising amino acids 1-62 of the mature Fas protein (amino acids 17-78 of SEQ ID NO: 14), as well as other polypeptides comprising fragments of amino acids 1-66 of the mature Fas protein that retain PLAD activity.
The present invention also provides a polypeptide comprising amino acids 43-80 of the full-length LtβR protein set forth as SEQ ID NO: 15 as well as other polypeptides comprising fragments of amino acids 43-80 of SEQ ID NO: 15 that retain PLAD activity.
The present invention also provides a polypeptide comprising amino acids 26-59 of the full-length CD40 protein set forth as SEQ ID NO: 16 as well as other polypeptides comprising fragments of amino acids 26-59 of SEQ ID NO: 16 that retain PLAD activity.
The mature CD30 polypeptide starts at position 19 of the full-length CD30 coding sequence set forth as SEQ ID NO: 17. Therefore the present invention provides a polypeptide comprising amino acids 11-51 of the mature CD30 protein (amino acids 29-69 of SEQ ID NO: 17), a polypeptide comprising amino acids 11-50 of the mature CD30 protein (ammo acids 29-68 of SEQ ID NO: 17), a polypeptide comprising amino acids 11-49 of the mature CD30 protein (amino acids 29-67 of SEQ ID NO: 17), a polypeptide comprising amino acids 11-48 of the mature CD30 protein (amino acids 29-66 of SEQ ID NO: 17), a polypeptide comprising amino acids 11-47 of the mature CD30 protein (amino acids 29-65 of SEQ ID NO: 17), a well as other polypeptides comprising fragments of amino acids 11-51 of the mature CD30 protein that retain PLAD activity.
The present invention also provides a polypeptide comprising amino acids 27-62 of the full-length CD27 protein set forth as SEQ ID NO: 18 as well as other polypeptides comprising fragments of amino acids 27-62 of SEQ ID NO: 18 that retain PLAD activity
The present invention also provides a polypeptide comprising amino acids 42-75 of the full-length HVEM protein set forth as SEQ ID NO: 19 as well as other polypeptides comprising fragments of the polypeptide comprising amino acids 42-75 of SEQ ID NO: 19 that retain PLAD activity.
The mature OX40 polypeptide starts at position 29 of the full-length OX40 coding sequence set forth as SEQ ID NO: 20. Therefore the present invention provides a polypeptide comprising amino acids 3-36 of the mature OX40 protein (amino acids 31-64 of SEQ ID NO: 20), a polypeptide comprising amino acids 3-35 of the mature OX40 protein (amino acids 31-63 of SEQ ID NO: 20), a polypeptide comprising amino acids 3-34 of the mature OX40 protein (amino acids 31-62 of SEQ ID NO: 20), a polypeptide comprising amino acids 3-33 of the mature OX40 protein (amino acids 31-61 of SEQ ID NO: 20), a polypeptide comprising amino acids 3-32 of the mature CD30 protein (amino acids 31-60 of SEQ ID NO: 20), a well as other polypeptides comprising fragments of the polypeptide comprising amino acids 3-36 of the mature OX40 protein that retain PLAD activity.
The present invention also provides a polypeptide comprising amino acids 132-170 of the full-length DR4 protein set forth as SEQ ID NO: 21 as well as other polypeptides comprising fragments of the polypeptide comprising amino acids 132-170 of SEQ ID NO: 21 that retain PLAD activity.
Table 1 sets forth examples of TNF receptor-like receptors comprising a PLAD of the present invention. The nucleotide and polypeptide sequences for these receptors can be found under the GenBank Accession Nos. set forth in Table 1. The nucleotide sequences, the polypeptide sequences and any information (e.g., signal sequence and mature protein residue numbers) set forth under the GenBank Accession Nos. set forth in Table 1 are hereby incorporated in their entireties by this reference. For example, the nucleotide sequence, the polypeptide sequence and additional information (e.g., signal sequence and mature protein residue numbers) for p60 can be found under GenBank Accession No. M75866. These p60 sequences and additional information set forth under GenBank Accession No. M75866 are hereby incorporated in their entireties by this reference. Similarly, the nucleotide sequence, the polypeptide sequence and additional information (e.g., signal sequence and mature protein residue numbers) set forth for p80 can be found under GenBank Accession No. M32315. These p80 sequences and additional information set forth under GenBank Accession No. M32315 are hereby incorporated in their entireties by this reference. By accessing the GenBank Accession Nos. set forth in Table 1, one of skill in the art can access additional GenBank Accession Nos. listed therein to obtain additional information concerning signal sequences and mature protein sequences. For example, upon accessing GenBank Accession No. M75866, one of skill in the art can access GenBank Accession No. AAA61201 which sets forth the signal sequence and mature protein sequences information for p60. This information can also be found by directly accessing GenBank Accession Nos. AAA51201 (p60), GenBank Accession No. AAA59929 (p80), GenBank Accession No. AAA63174 (Fas), GenBank Accession No. AAA36757 (LTBR), GenBank Accession No. CAA43045 (CD40), GenBank Accession No. AAA51947 (CD30), GenBank Accession No. AAA58411 (CD27), GenBank Accession No. AAB58354, GenBank Accession No. CAA53576 (OX40), GenBank Accession No. AAC51226 (DR4), and is incorporated herein by this reference.
Table 1 also provides Locus Link Accession Nos. for the TNF-like receptors. Locus Link Accession Nos. are now equivalent to Entrez Gene Identification Numbers (Gene ID numbers) that can be accessed at the National Center for Biotechnology Information at the U.S. National Library of Medicine. For example, one of skill in the art can obtain additional information, regarding p60, including nucleotide and protein sequences, by accessing Locus Link number 7132 (now Gene D 7132 in Entrez Gene) in the Entrez Gene database. Similarly one of skill in the art can obtain additional information regarding p80, including nucleotide and protein sequences, by accessing Locus Link number 7133 (now Gene ID 7133 in Entrez Gene) in the Entrez Gene database. Thus, one of skill in the art can readily obtain information regarding any of the TNF-like receptors listed in Table 1 by accessing their respective Locus Link (Gene ID) numbers in Entrez Gene. All of the information provided under the Locus Link (Gene ID) numbers set forth in Table 1 is hereby incorporated by reference in its entirety.
Provided are polypeptides comprising the isolated amino acid sequences for PLAD domains of vTNFR proteins (SEQ ID NOS: 28-39). Other vTNFR PLADs can be identified using protein-protein BLAST database searches of homologs for TNFR1 and TNFR2 PLAD sequences. Also included are full length amino acid sequences for each vTNFR and its modified protein (SEQ. ID NOS: 44-55). Also provided is methodology for identification, production, and functional testing of and additional vTNFR PLAD polypeptides by one of skill in the art.
The vTNFR PLAD domains can disrupt self-association of host TNFRs and/or subsequent ligand binding to dampen anti-viral immunity and/or protect infected cells from TNF-mediated cell death. The M-T2 protein, a TNF-receptor like protein encoded by myxoma virus, can protect myxoma-infected T cells from TNF-induced death independently of its extracellular TNF binding capacity (82). vTNFR PLAD sequences can serve as more potent inhibitors of TNF-induced effects than P60 or P80 PLADs themselves, as a consequence of evolutionary selection for higher affinity binding to host TNFR PLAD domains. In this regard, isolated viral PLAD proteins represent improved agents for clinical use in blocking TNF-associated pathogenesis associated with rheumatoid arthritis and other autoimmune diseases.
It is understood that techniques such as those described herein can be employed to identify additional microbial proteins with homology to PLAD domains found in other TNF receptor-like receptors (e.g. Fas) described herein. For example, three examples of microbial polypeptides containing sequences homologous to the Fas PLAD domain (SEQ ID NOS: 41-43, 56-58) are disclosed.
As used herein an “isolated amino acid sequence of a PLAD” means a sequence which is substantially free from the naturally occurring materials with which the amino acid sequence is normally associated in nature. The polypeptides of this invention can comprise the entire amino acid sequence of a PLAD domain or fragments thereof that have PLAD activity. The polypeptides or fragments thereof of the present invention can be obtained by isolation and purification of the polypeptides from cells where they are produced naturally or by expression of exogenous nucleic acid encoding a PLAD. Fragments of a PLAD can be obtained by chemical synthesis of peptides, by proteolytic cleavage of the PLAD or the polypeptide comprising a PLAD and by synthesis from nucleic acid encoding the portion of interest. The PLAD can include conservative substitutions where a naturally occurring amino acid is replaced by one having similar properties. Such conservative substitutions do not alter the function of the polypeptide. Mutations that enhance binding and effectiveness can be found by creating various amino acid substitutions and testing them in binding assays described within the specification using techniques available to those of ordinary skill in the art.
Thus, it is understood that, where desired, modifications and changes can be made in the nucleic acid encoding the polypeptides of this invention and/or amino acid sequence of the polypeptides of the present invention and still obtain a polypeptide having like or otherwise desirable characteristics. Such changes can occur in natural isolates or can be synthetically introduced using site-specific mutagenesis, the procedures for which, such as mis-match polymerase chain reaction (PCR), are well known in the art.
For example, certain amino acids can be substituted for other amino acids in a polypeptide without appreciable loss of functional activity. It is thus contemplated that various changes can be made in the amino acid sequence of the PLAD (or underlying nucleic acid sequence) without appreciable loss of biological utility or activity and possibly with an increase in such utility or activity. For example, the Q24A mutation, the D49R mutation and the K19E mutation in the natural sequence of p60 TNFR do not impair PLAD self-association.
These polypeptides can also be obtained in any of a number of procedures well known in the art. One method of producing a polypeptide is to link two peptides or polypeptides together by protein chemistry techniques. For example, peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, Calif.). One skilled in the art can readily appreciate that a peptide or polypeptide corresponding to a particular protein can be synthesized by standard chemical reactions. For example, a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a hybrid peptide can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment. By peptide condensation reactions, these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form a larger polypeptide. (Grant, Asynthetic Peptides: A User Guide, W.H. Freeman and Co., N.Y. (1992) and Bodansky and Trost, Ed., Principles of Peptide Synthesis, Springer-Verlag Inc., N.Y. (1993)). Alternatively, the peptide or polypeptide can be independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides can be linked to form a larger protein via similar peptide condensation reactions.
For example, enzymatic ligation of cloned or synthetic peptide segments can allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen et al. Biochemistry, 30:4151 (1991)). Alternatively, native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. A Synthesis of Proteins by Native Chemical Ligation, Science, 266:776-779 (1994)). The first step is the chemoselective reaction of an unprotected synthetic peptide-%-thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site. Application of this native chemical ligation method to the total synthesis of a protein molecule is illustrated by the preparation of human interleukin 8 (IL-8) (Clark-Lewis et al. FEBS Lett., 307:97 (1987), Clark-Lewis et al., J. Biol. Chem., 269:16075 (1994), Clark-Lewis et al. Biochemistry, 30:3128 (1991), and Rajarathnam et al. Biochemistry, 29:1689 (1994)).
Alternatively, unprotected peptide segments can be chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer et al. Science, 256:221 (1992)). This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton et al. ATechniques in Protein Chemistry IV, Academic Press, New York, pp. 257-267 (1992)).
The present invention also provides peptide mimetics for the disclosed polypeptides. A “peptide mimetic” is defined to include a chemical compound, or an organic molecule, or any other peptide mimetic, the structure of which is based on or derived from a binding region of a protein. For example, one can model predicted chemical structures to mimic the structure of a binding region, such as a PLAD. Such modeling can be performed using standard methods. Alternatively, peptide mimetics can also be selected from combinatorial chemical libraries in much the same way that peptides are. (Ostresh, J. M. et al., Proc Natl Acad Sci USA 1994 Nov. 8; 91(23):11138-42; Dorner, B. et al., Bioorg Med Chem 1996 May; 4(5):709-15; Eichler, J. et al., Med Res Rev 1995 November; 15(6):481-96; Blondelle, S. E. et al. Biochem J 1996 Jan. 1; 313 (Pt 1):141-7; Perez-Paya, E. et al., J Biol Chem 1996 Feb. 23; 271(8):4120-6). Functional assays can also be utilized to select peptide mimetics.
The polypeptides of this invention can be linked to another moiety such as a nucleic acid, a protein, a peptide, a ligand, a carbohydrate moiety, viral proteins, a monoclonal antibody, a polyclonal antibody or a liposome. Furthermore, two or more PLAD containing polypeptides can also be linked to each other. For example, a bifunctional or multifunctional polypeptide containing two or more different PLADs can be made such that the polypeptide is capable of modulating the activity of more than one TNF receptor-like receptor. The polypeptide can also contain two or more PLADs from the same TNF receptor-like receptor in order to increase the avidity of this polypeptide for a particular TNF receptor-like receptor.
Disclosed herein are fusion proteins containing PLAD and the nucleic acids encoding them. The fusion protein can comprise the PLAD of a TNF receptor-like receptor disclosed herein linked to a fusion tag. The functional molecule can be an antibody or targeting portion thereof or other fusion tag. Since PLAD is on the cell surface, the PLAD containing fusion protein can include a component that targets the PLAD to the cell surface of PLAD-expressing cells. For example, marker-binding portions of ligands for non-TNF receptor-like markers on the surfaces of intended target cells can be fused to PLAD. The PLAD can be fused to various carrier proteins such as immunoglobulin or other serum, soluble, and/or stable proteins. The fusion tag can be GST or other molecule that facilitates purification of the fusion protein. The PLAD-containing fusion protein can include a signal sequence to facilitate secretion of a recombinantly expressed PLAD.
The nucleic acids encoding a polypeptide comprising or consisting of a PLAD can also be functionally linked to other nucleic acids to encode an immunoadhesin. For the purposes of the invention, the term “immunoadhesin” is defined as including any polypeptide encoded by a nucleic acid where at least a portion of a nucleic acid encoding a non-immunoglobulin molecule such as a PLAD is coupled to at least a portion of a nucleic acid encoding an immunoglobulin heavy chain polypeptide, IgG for example. The Fc regions of IgG2, IgG3, IgM, IgA, IgE can also be utilized to construct an immunoadhesin. In a particular example, the fusion protein comprises PLAD fused to an Ig Fc portion, especially that of Ig Gamma 4. The coupling can be achieved in a manner which provides for a functional transcribing and translating of the nucleic acid segment and message derived therefrom, respectively.
The PLAD polypeptide fusion protein can be expressed by transient or stable transfection in a variety of mammalian host cells as well as in baculovirus-infected cells. The expressed fusion protein can be purified according to standard methods. Similar, to antibodies, IgG immunoadhesins can be purified from the culture medium into which they are secreted by single-step protein A or protein G affinity chromatography.
Provided are PLAD-encoding transgenes. By a “transgene” is meant a nucleic acid sequence that is inserted by artifice into a cell and becomes a part of the genome of that cell and its progeny. Such a transgene can be (but is not necessarily) partly or entirely heterologous (e.g., derived from a different species) to the cell. The term “transgene” broadly refers to any nucleic acid that is introduced into an animal's genome, including but not limited to genes or DNA having sequences which are perhaps not normally present in the genome, genes which are present, but not normally transcribed and translated (“expressed”) in a given genome, or any other gene or DNA which one desires to introduce into the genome. This can include genes which may normally be present in the nontransgenic genome but which one desires to have altered in: expression, or which one desires to introduce in an altered or variant form. A transgene can include one or more transcriptional regulatory sequences and any other nucleic acid, such as introns, that may be necessary for optimal expression of a selected nucleic acid. A transgene can be as few as a couple of nucleotides long, but is preferably at least about 50, 100, 150, 200, 250, 300, 350, 400, or 500 nucleotides long or even longer. A transgene can be coding or non-coding sequences, or a combination thereof. A transgene usually comprises a regulatory element that is capable of driving the expression of one or more transgenes under appropriate conditions.
Also provided by the present invention are antibodies that specifically bind to a PLAD of a TNF receptor-like receptor. For example, the antibodies of the present invention can be antibodies that specifically bind to a PLAD of a TNF receptor, antibodies that specifically bind to a PLAD of FAS or antibodies that specifically bind a PLAD of DR4, to name a few. The antibody (either polyclonal or monoclonal) can be raised to any of the polypeptides provided and contemplated herein, both naturally occurring and recombinant polypeptides, and immunogenic fragments, thereof. The antibody can be used in techniques or procedures such as diagnostics, treatment, or vaccination. Anti-idiotypic antibodies and affinity matured antibodies are also considered.
Antibodies can be made by many well-known methods (See, e.g. Harlow and Lane, “Antibodies; A Laboratory Manual” Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., (1988)). Briefly, purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells can then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen nucleic acid clone libraries for cells secreting the antigen. Those positive clones can then be sequenced. (See, for example, Kelly et al. Bio/Technology, 10:163-167 (1992); Bebbington et al. Bio/Technology, 10:169-175 (1992)). Humanized and chimeric antibodies are also comtemplated in this invention. Heterologous antibodies can be made by well known methods (See, for example, U.S. Pat. Nos. 5,545,806, 5,569,825, 5,625,126, 5,633,425, 5,661,016, 5,770,429, 5,789,650, and 5,814,318)
The phrase “specifically binds” with the polypeptide refers to a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biologics. Thus, under designated immunoassay conditions, the specified antibodies bound to a particular protein do not bind in a significant amount to other proteins present in the sample. Selective binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein. A variety of immunoassay formats can be used to select antibodies that selectively bind with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a protein. See Harlow and Lane “Antibodies, A Laboratory Manual” Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding.
The present invention also provides nucleic acids that encode polypeptides of up to 125 amino acids comprising a PLAD of a TNF receptor-like receptor as well as nucleic acids that encode polypeptides consisting of a TNF receptor-like receptor PLAD.
The present invention also provides nucleic acids that encode a polypeptide of up to 125 amino acids comprising an isolated PLAD, wherein the polypeptide comprises the subsequence R1-PLAD-R2, wherein R1 and R2 are optional and when present can be H, acyl, NH2, an amino acid or a peptide.
The invention further provides a nucleic acid that encodes a polypeptide comprising the isolated amino acid sequence of a pre-ligand assembly domain (PLAD) of a TNF receptor-like receptor, wherein the polypeptide is R1-TNF receptor-like receptor PLAD-R2, wherein R1 or R2 comprise an amino acid sequence that does not flank the TNF receptor-like receptor PLAD in a naturally occurring TNF receptor-like receptor.
As used herein, the term “nucleic acid” refers to single- or multiple stranded molecules which may be DNA or RNA, or any combination thereof, including modifications to those nucleic acids. The nucleic acid may represent a coding strand or its complement, or any combination thereof. Nucleic acids may be identical in sequence to the sequences which are naturally occurring for any of the novel genes discussed herein or can include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence. These nucleic acids can also be modified from their typical structure. Such modifications include, but are not limited to, methylated nucleic acids, the substitution of a non-bridging oxygen on the phosphate residue with either a sulfur (yielding phosphorothioate deoxynucleotides), selenium (yielding phosphorselenoate deoxynucleotides), or methyl groups (yielding methylphosphonate deoxynucleotides).
A nucleic acid molecule encoding a PLAD can be isolated from the organism in which it is normally found. For example, a genomic DNA or cDNA library can be constructed and screened for the presence of the nucleic acid of interest. Methods of constructing and screening such libraries are well known in the art and kits for performing the construction and screening steps are commercially available (for example, Stratagene Cloning Systems, La Jolla, Calif.). Once isolated, the nucleic acid can be directly cloned into an appropriate vector, or if necessary, be modified to facilitate the subsequent cloning steps. Such modification steps are routine, an example of which is the addition of oligonucleotide linkers which contain restriction sites to the termini of the nucleic acid. General methods are set forth in Sambrook et al., “Molecular Cloning, a Laboratory Manual,” Cold Spring Harbor Laboratory Press (1989). Also contemplated by the present invention are nucleic acids encoding a PLAD that do not contain a ligand binding site.
Once the nucleic acid sequence of the desired PLAD is obtained, the sequence encoding specific amino acids can be modified or changed at any particular amino acid position by techniques well known in the art. For example, PCR primers can be designed which span the amino acid position or positions and which can substitute any amino acid for another amino acid. Then a nucleic acid can be amplified and inserted into the wild-type PLAD coding sequence in order to obtain any of a number of possible combinations of amino acids at any position of the PLAD. Alternatively, one skilled in the art can introduce specific mutations at any point in a particular nucleic acid sequence through techniques for point mutagenesis. General methods are set forth in Smith, M. “In vitro mutagenesis” Ann. Rev. Gen., 19:423-462 (1985) and Zoller, M. J. “New molecular biology methods for protein engineering” Curr. Opin. Struct. Biol., 1:605-610 (1991). Techniques such as these can be used to alter the coding sequence without altering the amino acid sequence that is encoded.
Another example of a method of obtaining a DNA molecule encoding a PLAD is to synthesize a recombinant DNA molecule which encodes the PLAD. For example, oligonucleotide synthesis procedures are routine in the art and oligonucleotides coding for a particular protein region are readily obtainable through automated DNA synthesis. A nucleic acid for one strand of a double-stranded molecule can be synthesized and hybridized to its complementary strand. One can design these oligonucleotides such that the resulting double-stranded molecule has either internal restriction sites or appropriate 5′ or 3′ overhangs at the termini for cloning into an appropriate vector. Double-stranded molecules coding for relatively large proteins can readily be synthesized by first constructing several different double-stranded molecules that code for particular regions of the protein, followed by ligating these DNA molecules together. For example, Cunningham, et al., “Receptor and Antibody Epitopes in Human Growth Hormone Identified by Homolog-Scanning Mutagenesis,” Science, 243:1330-1336 (1989), have constructed a synthetic gene encoding the human growth hormone gene by first constructing overlapping and complementary synthetic oligonucleotides and ligating these fragments together. See also, Ferretti, et al., Proc. Nat. Acad. Sci. 82:599-603 (1986), wherein synthesis of a 1057 base pair synthetic bovine rhodopsin gene from synthetic oligonucleotides is disclosed. By constructing a PLAD in this manner, one skilled in the art can readily obtain any particular PLAD with desired amino acids at any particular position or positions within the PLAD. See also, U.S. Pat. No. 5,503,995 which describes an enzyme template reaction method of making synthetic genes. Techniques such as this are routine in the art and are well documented. These nucleic acids or fragments of a nucleic acid encoding a PLAD can then be expressed in vivo or in vitro as discussed below.
The invention also provides for the isolated nucleic acids encoding a PLAD in a vector suitable for expressing the nucleic acid. Once a nucleic acid encoding a particular PLAD of interest, or a region of that nucleic acid, is constructed, modified, or isolated, that nucleic acid can then be cloned into an appropriate vector, which can direct the in vivo or in vitro synthesis of that wild-type and/or modified PLAD. The vector is contemplated to have the necessary functional elements that direct and regulate transcription of the inserted gene, or nucleic acid. These functional elements include, but are not limited to, a promoter, regions upstream or downstream of the promoter, such as enhancers that may regulate the transcriptional activity of the promoter, an origin of replication, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic resistance genes or other markers which can serve to select for cells containing the vector or the vector containing the insert, RNA splice junctions, a transcription termination region, or any other region which may serve to facilitate the expression of the inserted gene or hybrid gene. (See generally, Sambrook et al.).
There are numerous E. coli (Escherichia coli) expression vectors known to one of ordinary skill in the art which are useful for the expression of the nucleic acid insert. Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species. In these prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Trp) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda. The promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences for example, for initiating and completing transcription and translation. If necessary, an amino terminal methionine can be provided by insertion of a Met codon 5′ and in-frame with the downstream nucleic acid insert. Also, the carboxy-terminal extension of the nucleic acid insert can be removed using standard oligonucleotide mutagenesis procedures.
Additionally, yeast expression can be used. There are several advantages to yeast expression systems. First, evidence exists that proteins produced in a yeast secretion systems exhibit correct disulfide pairing. Second, post-translational glycosylation is efficiently carried out by yeast secretory systems. The Saccharomyces cerevisiae pre-pro-alpha-factor leader region (encoded by the MF′-1 gene) is routinely used to direct protein secretion from yeast. (Brake, et al., Alpha-Factor-Directed Synthesis and Secretion of Mature Foreign Proteins in Saccharomyces cerevisiae. Proc. Nat. Acad. Sci., 81:4642-4646 (1984)). The leader region of pre-pro-alpha-factor contains a signal peptide and a pro-segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene: this enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage signal sequence. The nucleic acid coding sequence can be fused in-frame to the pre-pro-alpha-factor leader region. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter. The nucleic acid coding sequence is followed by a translation termination codon which is followed by transcription termination signals. Alternatively, the nucleic acid coding sequences can be fused to a second protein coding sequence, such as Sj26 or β-galactosidase, used to facilitate purification of the fusion protein by affinity chromatography. The insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast. Efficient post translational glycosylation and expression of recombinant proteins can also be achieved in Baculovirus systems.
Mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active protein. Vectors useful for the expression of active proteins in mammalian cells are characterized by insertion of the protein coding sequence between a strong viral promoter and a polyadenylation signal. The vectors can contain genes conferring hygromycin resistance, genticin or G418 resistance, or other genes or phenotypes suitable for use as selectable markers, or methotrexate resistance for gene amplification. The chimeric protein coding sequence can be introduced into a Chinese hamster ovary (CHO) cell line using a methotrexate resistance-encoding vector, or other cell lines using suitable selection markers. Presence of the vector DNA in transformed cells can be confirmed by Southern blot analysis. Production of RNA corresponding to the insert coding sequence can be confirmed by Northern blot analysis. A number of other suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc. Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc. The vectors containing the nucleic acid segments of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transformation is commonly utilized for prokaryotic cells, whereas calcium phosphate, DEAE dextran, or lipofectin mediated transfection or electroporation can be used for other eukaryotic cellular hosts.
Alternative vectors for the expression of genes or nucleic acids in mammalian cells, those similar to those developed for the expression of human gamma-interferon, tissue plasminogen activator, clotting Factor VIII, hepatitis B virus surface antigen, protease Nexin1, and eosinophil major basic protein, can be employed. Further, the vector can include CMV promoter sequences and a polyadenylation signal available for expression of inserted nucleic acids in mammalian cells (such as COS-7).
Insect cells also permit the expression of mammalian proteins. Recombinant proteins produced in insect cells with baculovirus vectors undergo post-translational modifications similar to that of wild-type proteins. Briefly, baculovirus vectors useful for the expression of active proteins in insect cells are characterized by insertion of the protein coding sequence downstream of the Autographica californica nuclear polyhedrosis virus (AcNPV) promoter for the gene encoding polyhedrin, the major occlusion protein. Cultured insect cells such as Spodoptera frugiperda cell lines are transfected with a mixture of viral and plasmid DNAs and the viral progeny are plated. Deletion or insertional inactivation of the polyhedrin gene results in the production of occlusion negative viruses which form plaques that are distinctively different from those of wild-type occlusion positive viruses. These distinctive plaque morphologies allow visual screening for recombinant viruses in which the AcNPV gene has been replaced with a hybrid gene of choice.
The invention also provides for the vectors containing the contemplated nucleic acids in a host suitable for expressing the nucleic acids. The vectors containing the nucleic acid segments of interest can be transferred into host cells by well-known methods, which vary depending on the type of cellular host; For example, calcium chloride transformation, transduction, and electroporation are commonly utilized for prokaryotic cells, whereas calcium phosphate, DEAE dextran, or lipofection mediated transfection or electroporation can be used for other cellular hosts.
Alternatively, the nucleic acids of the present invention can be operatively linked to one or more of the functional elements that direct and regulate transcription of the inserted nucleic acid and the nucleic acid can be expressed. For example, a nucleic acid can be operatively linked to a bacterial or phage promoter and used to direct the transcription of the nucleic acid in vitro. A further example includes using a nucleic acid provided herein in a coupled transcription-translation system where the nucleic acid directs transcription and the RNA thereby produced is used as a template for translation to produce a polypeptide. One skilled in the art will appreciate that the products of these reactions can be used in many applications such as using labeled RNAs as probes and using polypeptides to generate antibodies or in a procedure where the polypeptides are being administered to a cell or a subject.
Expression of the nucleic acid, in combination with a vector, can be by either in vivo or in vitro. In vivo synthesis comprises transforming prokaryotic or eukaryotic cells that can serve as host cells for the vector. Alternatively, expression of the nucleic acid can occur in an in vitro expression system. For example, in vitro transcription systems are commercially available which are routinely used to synthesize relatively large amounts of mRNA. In such in vitro transcription systems, the nucleic acid encoding a PLAD would be cloned into an expression vector adjacent to a transcription promoter. For example, the Bluescript II cloning and expression vectors contain multiple cloning sites which are flanked by strong prokaryotic transcription promoters. (Stratagene Cloning Systems, La Jolla, Calif.). Kits are available which contain all the necessary reagents for in vitro synthesis of an RNA from a DNA template such as the Bluescript vectors. (Stratagene Cloning Systems, La Jolla, Calif.). RNA produced in vitro by a system such as this can then be translated in vitro to produce the desired PLAD polypeptide. (Stratagene Cloning Systems, La Jolla, Calif.).
Using gene therapy methods, a nucleic acid encoding a polypeptide comprising or consisting of a PLAD can be administered. The nucleic acid encoding the polypeptide of this invention can be placed into a vector and delivered to the cells of a subject either in vivo or ex vivo by standard methods.
The nucleic acid encoding the polypeptide of this invention can be functionally attached to a specific leader peptide which can specify for secretion of the polypeptide. For example the polypeptide can have a signal sequence, such as the murine Ig-kappa signal sequence (Blezinger et al. Nat. Biotechnol. 17: 343-8, 1999), rat insulin leader sequence (Fakhral et al. J. Immunother. 20: 437-8, 1997), FGF-4 signal sequence (Ueno et al. Aterioscler. Thromb. Vasc. Biol., 17: 2453-2460, 1997), human growth hormone signal peptide (Rade et al. Gene Ther. 6: 385-92, 1999), beta lactamase signal sequence (Hughes et al. Hum. Gene Ther. 5: 1445-55, 1994), bovine prolactin signal sequence (Gorman et al. Bran Res. Mol. Brain. Res. 44:143-146, 1997) and other similar signal sequences.
For in vivo administration, the cells can be in a subject and the nucleic acid can be administered in a pharmaceutically acceptable carrier. The subject can be any animal in which it is desirable to selectively express a nucleic acid in a cell. In a preferred embodiment, the animal of the present invention is a human. In addition, non-human animals which can be treated by the method of this invention can include, but are not limited to, cats, dogs, birds, horses, cows, goats, sheep, guinea pigs, hamsters, gerbils and rabbits, as well as any other animal in which selective expression of a nucleic acid in a cell can be carried out according to the methods described herein.
In the method described above which includes the introduction of exogenous DNA into the cells of a subject (i.e., gene transduction or transfection), the nucleic acids of the present invention can be in the form of naked DNA or the nucleic acids can be in a vector for delivering the nucleic acids to the cells for expression of the nucleic acid inside the cell. The vector can be a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as Lipofectin®, Lipofectamine® (GIBCO-BRL, Inc., Gaithersburg, Md.), Superfect® (Qiagen, Inc. Hilden, Germany) and Transfectam® (Promega Biotec, Inc., Madison, Wis.), as well as other liposomes developed according to procedures standard in the art. In addition, the nucleic acid or vector of this invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, Calif.) as well as by means of a Sonoporation machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).
As one example, vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome. The recombinant retrovirus can then be used to infect and thereby deliver nucleic acid to the infected cells. The exact method of introducing the nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors, adeno-associated viral (AAV) vectors, lentiviral vectors, pseudotyped retroviral vectors, and pox virus vectors, such as vaccinia virus vectors. Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanism. This invention can be used in conjunction with any of these or other commonly used gene transfer methods.
The nucleic acid and the nucleic acid delivery vehicles of this invention, (e.g., viruses; liposomes, plasmids, vectors) can be in a pharmaceutically acceptable carrier for in vivo administration to a subject. By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to a subject, along with the nucleic acid or vehicle, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
The nucleic acid or vehicle can be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like. The exact amount of the nucleic acid or vector required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity or mechanism of any disorder being treated, the particular nucleic acid or vehicle used, its mode of administration and the like.
The present invention further provides a composition comprising an inhibitor of PLAD self-association or TNF-like receptor oligomerization. An “inhibitor” is defined as a compound that binds a PLAD or a compound, including antibodies, that binds the target for a PLAD and prevents an activity of a PLAD. Upon binding to a PLAD, the inhibitor can disrupt or prevent PLAD self-association, thus inhibiting TNF receptor-like receptor oligomerization. The inhibitor of TNF-like receptor oligomerization can be an antibody, either polyclonal or monoclonal, that specifically binds to a PLAD, a ligand that binds to a PLAD, a polypeptide that binds to a PLAD, a compound that binds to a PLAD or a peptide mimetic based on a PLAD. For example, a polypeptide comprising or consisting of a PLAD can associate with the PLAD of a naturally occurring TNF receptor-like receptor, thus preventing or inhibiting the TNF receptor-like receptor from self-associating with other naturally occurring TNF receptor-like receptors. The polypeptide comprising or consisting of a PLAD can be a soluble PLAD. Anti-idiotypic antibodies and affinity matured antibodies are also considered. Other inhibitors include, but are not limited to molecules or compounds designed to block PLAD self-association. The inhibitor can be a whole protein or a fragment of a protein that inhibits PLAD self-association, thus preventing TNF receptor-like receptor oligomerization. The inhibitor can be an organic molecule identified according to the methods described herein. Crystal structures of the TNF receptors and their oligomeric complexes can be utilized to design molecules that can disrupt PLAD self-association. The crystal structures can also be analyzed to design molecules that mimic PLAD and disrupt PLAD self-association.
Thus, a method of making a small molecule inhibitor is provided, as is the inhibitor produced by this method. Provided are small molecules (SM) and PLAD-peptide derivatives that interfere with TNFR assembly and TNF binding. Based on the crystal structure of dimerized TNFR1 protein, a search for the binding surfaces of PLAD association was performed. This in combination with a conservative homology search and data on PLAD mutagenesis described herein, allows the identification of some potential inter-chain association sites. For example, as illustrated in the dimerized PLAD structure (
Disclosed herein are compounds that can interfere with TNFR assembly and TNF binding. Generally, suitable inhibitors are those compounds that can enter the inter-chain pocket of the PLAD dimerized structure and interfere with the interaction between the two histidine rings at position 34. In this sense, inhibitors containing bulky substituents that hinder entry into the inter-chain pocket or prevent the disruption of the interaction between the two histidine residues at position 34 are not preferred. Conversely, inhibitors that contain substituents that allow easy entry into the inter-chain pocket and facilitate insertion and consequent disruption of the interaction between the two histidine residues at position 34 are preferred. Further, inhibitors containing substituents that have increased affinity for other residues in the inter-chain pocket, thus facilitating and/or enhancing binding of the inhibitor in the PLAD dimerized structure are even more preferred. Analyzing putative inhibitors to evaluate whether certain substituents enhance or hinder binding to the PLAD dimerized structure can be performed in silico by those of skill in the art.
As used herein, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.
“A1,” “A2,” “A3,” and “A4” are used herein as generic symbols to represent various specific substituents. These symbols can be any substituent, not limited to those disclosed herein, and when they are defined to be certain substituents in one instance, they can, in another instance, be defined as some other substituents.
The term “alkyl” as used herein is a branched or unbranched saturated hydrocarbon group of 1 to 24 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, eicosyl, tetracosyl, and the like. The alkyl group can also be substituted or unsubstituted. The alkyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.
Throughout the specification “alkyl” is generally used to refer to both unsubstituted alkyl groups and substituted alkyl groups; however, substituted alkyl groups are also specifically referred to herein by identifying the specific substituent(s) on the alkyl group. For example, the term “halogenated alkyl” specifically refers to an alkyl group that is substituted with one or more halide, e.g., fluorine, chlorine, bromine, or iodine. The term “alkoxyalkyl” specifically refers to an alkyl group that is substituted with one or more alkoxy groups, as described below. The term “alkylamino” specifically refers to an alkyl group that is substituted with one or more amino groups, as described below, and the like. When “alkyl” is used in one instance and a specific term such as “alkylalcohol” is used in another, it is not meant to imply that the term “alkyl” does not also refer to specific terms such as “alkylalcohol” and the like.
This practice is also used for other groups described herein. That is, while a term such as “cycloalkyl” refers to both unsubstituted and substituted cycloalkyl moieties, the substituted moieties can, in addition, be specifically identified herein; for example, a particular substituted cycloalkyl can be referred to as, e.g., an “alkylcycloalkyl.” Similarly, a substituted alkoxy can be specifically referred to as, e.g., a “halogenated alkoxy,” a particular substituted alkenyl can be, e.g., an “alkenylalcohol,” and the like. Again, the practice of using a general term, such as “cycloalkyl,” and a specific term, such as “alkylcycloalkyl,” is not meant to imply that the general term does not also include the specific term.
The term “alkoxy” as used herein is an alkyl group bound through a single, terminal ether linkage; that is, an “alkoxy” group can be defined as —OA1 where A1 is alkyl as defined above.
The term alkoxylalkyl as used herein is an alkyl group that contains an alkoxy substituent and can be defined as -A1-O-A2, where A1 and A2 are all groups.
The term “alkenyl” as used herein is a hydrocarbon group of from 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon double bond. Asymmetric structures such as (A1A2)C═C(A3A4) are intended to include both the E and Z isomers. This may be presumed in structural formulae herein wherein an asymmetric alkene is present, or it may be explicitly indicated by the bond symbol C═C. The alkenyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.
The term “alkynyl” as used herein is a hydrocarbon group of 2 to 24 carbon atoms with a structural formula containing at least one carbon-carbon triple bond. The alkynyl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol, as described below.
The term “aryl” as used herein is a group that contains any carbon-based aromatic group including, but not limited to, benzene, naphthalene, phenyl, biphenyl, phenoxybenzene, and the like. The term “aryl” also includes “heteroaryl,” which is defined as a group that contains an aromatic group that has at least one heteroatom incorporated within the ring of the aromatic group. Examples of heteroatoms include, but are not limited to, nitrogen, oxygen, sulfur, and phosphorus. Likewise, the term “non-heteroaryl,” which is also included in the term “aryl,” defines a group that contains an aromatic group that does not contain a heteroatom. The aryl group can be substituted or unsubstituted. The aryl group can be substituted with one or more groups including, but not limited to, alkyl, halogenated alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein. The term “biaryl” is a specific type of aryl group and is included in the definition of aryl. Biaryl refers to two aryl groups that are bound together via a fused ring structure, as in naphthalene, or are attached via one or more carbon-carbon bonds, as in biphenyl.
The term “cycloalkyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms. Examples of cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. The term “heterocycloalkyl” is a cycloalkyl group as defined above where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkyl group and heterocycloalkyl group can be substituted or unsubstituted. The cycloalkyl group and heterocycloalkyl group can be substituted with one or more groups including, but not limited to, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.
The term “cycloalkenyl” as used herein is a non-aromatic carbon-based ring composed of at least three carbon atoms and containing at least one double bound, i.e., C═C. Examples of cycloalkenyl groups include, but are not limited to, cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, and the like. The term “heterocycloalkenyl” is a type of cycloalkenyl group as defined above, and is included within the meaning of the term “cycloalkenyl,” where at least one of the carbon atoms of the ring is substituted with a heteroatom such as, but not limited to, nitrogen, oxygen, sulfur, or phosphorus. The cycloalkenyl group and heterocycloalkenyl group can be substituted or unsubstituted. The cycloalkenyl group and heterocycloalkenyl group can be substituted with one or more groups including, but not limited to, alkyl alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol as described herein.
The term “cyclic group” is used herein to refer to either aryl groups, non-aryl groups (i.e., cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl groups), or both. Cyclic groups have one or more ring systems that can be substituted or unsubstituted. A cyclic group can contain one or more aryl groups, one or more non-aryl groups, or one or more aryl groups and one or more non-aryl groups.
The term “aldehyde” as used herein is represented by the formula —C(O)H. Throughout this specification “C(O)” is a short hand notation for C═O.
The terms “amine” or “amino” as used herein are represented by the formula NA1A2A3, where A1, A2, and A3 can be, independently, hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
The term “carboxylic acid” as used herein is represented by the formula —C(O)OH. A “carboxylate” as used herein is represented by the formula —C(O)O−.
The term “ester” as used herein is represented by the formula —OC(O)A1 or —C(O)OA1, where A1 can be an alkyl, halogenated alkyl alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
The term “ether” as used herein is represented by the formula A1OA2, where A1 and A2 can be, independently, an alkyl halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
The term “ketone” as used herein is represented by the formula A1C(O)A2, where A1 and A2 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
The term “halide” as used herein refers to the halogens fluorine, chlorine, bromine, and iodine.
The term “hydroxyl” as used herein is represented by the formula —OH.
The term “nitro” as used herein is represented by the formula —NO2.
The term “silyl” as used herein is represented by the formula —SiA1A2A3, where A1, A2, and A3 can be, independently, hydrogen, alkyl, halogenated alkyl, alkoxy, alkenyl alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
The term “sulfo-oxo” as used herein is represented by the formulas —S(O)A1, —S(O)2A1, —OS(O)2A1, or —OS(O)2OA1, where A1 can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkyl, aryl, heteroaryl, cycloalkyl cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above. Throughout this specification “S(O)” is a short hand notation for S═O
The term “sulfonyl” is used herein to refer to the sulfo-oxo group represented by the formula —S(O)2A1, where A1 can be hydrogen, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
The term “sulfonylamino” or “sulfonamide” as used herein is represented by the formula —S(O)2NH—.
The term “sulfone” as used herein is represented by the formula A1S(O)2A2, where A1 and A2 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
The term “sulfoxide” as used herein is represented by the formula A1S(O)A2, where A1 and A2 can be, independently, an alkyl, halogenated alkyl, alkenyl, alkynyl, aryl, heteroaryl, cycloalkyl, cycloalkenyl, heterocycloalkyl, or heterocycloalkenyl group described above.
The term “thiol” as used herein is represented by the formula —SH.
“R1,” “R2,” “R3,” “Rn,” where n is an integer, as used herein can, independently, possess one or more of the groups listed above. For example, if R1 is a straight chain alkyl group, one of the hydrogen atoms of the alkyl group can optionally be substituted with a hydroxyl group, an alkoxy group, an alkyl group, a halide, and the like. Depending upon the groups that are selected, a first group can be incorporated within second group or, alternatively, the first group can be pendant (i.e., attached) to the second group. For example, with the phrase “an alkyl group comprising an amino group,” the amino group can be incorporated within the backbone of the alkyl group. Alternatively, the amino group can be attached to the backbone of the alkyl group. The nature of the group(s) that is (are) selected will determine if the first group is embedded or attached to the second group.
Unless stated to the contrary, a formula with chemical bonds shown only as solid lines and not as wedges or dashed lines contemplates each possible isomer, e.g., each enantiomer and diastereomer, and a mixture of isomers, such as a racemic or scalemic mixture.
Provided is a method of inhibiting ligand binding to a TNF receptor-like receptor by administering an effective amount of an imidazole or imidazole derivative.
Examples of inhibitors that are contemplated herein are generally 5-membered nitrogen containing heterocycles functionalized with various substituents. Examples of such inhibitors are shown below and generically identified by the name of the basic unsubstituted heterocylic structure (i.e., where Rn is H).
In many examples of inhibitors having the structures shown above, R1, R2, R3, R4, and R5, when present, are independently H, alkyl, alkoxy, alkenyl, alkynyl, aryl, heteroaryl, aldehyde, amino, carboxylic acid, ester, ether, halide, hydroxy, ketone, nitro, silyl, sulfo-oxo, sulfonyl, sulfone, sulfoxide, or thiol. In specific examples, R1-5 are independently a C1-C6 alkyl, a C1-C6 alkoxyalkyl group, or a C1-C6 alkoxy group. Exemplary C1-C6 alkyl groups and C1-C4 alkyl groups include methyl, ethyl, propyl, iso-propyl, butyl, sec-butyl, iso-butyl, pentyl, iso-pentyl, hexyl, 2-ethylbutyl, 2-methylpentyl, and the like. Corresponding C1-C6 alkoxy groups contain the above C1-C6 alkyl group bonded to an oxygen atom that is also bonded to the cation ring. An alkoxyalkyl group contains an ether group bonded to an alkyl group, and here contains a total of up to six carbon atoms. It is to be noted that there are two isomeric 1,2,3-triazoles.
The invention also contemplates targeting other regions of TNF receptor-like receptors such that upon binding that region, the conformation of a PLAD in the receptor is disrupted thus preventing it from associating with another PLAD. For example, one skilled in the art could target the CRD3 of p60 TNFR, such that upon binding the CRD3 region of p60 TNFR, the conformation of the receptor is changed, thus preventing the PLAD of the p60 TNFR from associating with another PLAD.
Thus, further provided by the present invention is a method of inhibiting TNF receptor-like receptor oligomerization in a cell by administering an effective amount of an inhibitor of TNF receptor-like receptor oligomerization.
The invention also contemplates enhancing PLAD self-association in order to enhance the effects of a TNF receptor-like receptor. For example, there are circumstances in which it would be desirous to enhance TNFR signaling. In such instances, agonists of PLAD self association, such as certain antibodies or molecules that bind to a PLAD and have the specific property of enhancing PLAD self association, can be utilized to convert cells that are resistant to TNFR effects due to weak PLAD interactions, into cells that are responsive to TNFR effects. Such enhanced PLAD self association can increase ligand binding as well as signaling. Examples of disease states where such enhanced PLAD interactions would be desirable include, but are not limited to, autoimmune lymphoproliferative syndrome (ALPS) and hyper IgM syndrome.
The invention also provides for utilizing a PLAD as a targeting moiety to deliver biological agents to cells. For example, a PLAD linked to a toxin can be delivered to cells, such that upon binding to a naturally occurring PLAD on a TNF-R, oligomerization is inhibited and upon internalization of the naturally occurring TNF-R, the PLAD linked to the toxin is internalized as well, thus delivering the toxin to the cell.
As used throughout, “TNF receptor-like receptor” refers to any member of the TNF receptor superfamily that includes, but is not limited to: TNF-R, p60 (also known as p55 and TNFR1), p80 (also known as p75, TNFR2), Fas (CD95/APO-1), TRAIL receptor, LTβR, CD40, CD30, CD27, HVEM, OX40, DR4, TROY, EDAR, XEDAR, DCR3, AITR, 4-1BB, DR3, RANK, TACT BCMA, DR6, DPG, DR5, DCR1 AND DCR2 (See Table 1). The nucleotide sequences, the polypeptide sequences and any information (e.g., signal sequence and mature protein residue numbers) set forth under the provided GenBank Accession Nos. set forth in Table 1 are hereby incorporated in their entireties by this reference.
As previously stated, inhibitors of TNF receptor-like receptor oligomerization include antibodies, ligands, peptide mimetics, compounds and polypeptides that specifically bind to a PLAD. These polypeptides include polypeptides comprising or consisting of an isolated (e.g., soluble) PLAD.
The present invention also provides a method of inhibiting ligand binding to a TNF receptor-like receptor by administering an effective amount of an inhibitor of TNF receptor-like receptor oligomerization. For example, by administering an inhibitor, such as a polypeptide comprising or consisting of a TNFR-PLAD, TNF receptor oligomerization would be inhibited, thus preventing the binding of TNF-α to the TNF receptor and diminishing the deleterious effects of TNF-α Similarly, the administration of a polypeptide comprising a CD40 receptor-PLAD (CD40R-PLAD), would inhibit CD40R oligomerization, thus preventing the binding of CD40 ligand to the CD40R and diminishing the deleterious effects of CD40 in disease states such as allograft rejection, rheumatoid arthritis and systemic lupus erythematosis. Inhibition of ligand binding to a TNF receptor-like receptor results in inhibition of signal transduction via TNF receptor-like receptors, thus providing a method of modulating signaling via TNF receptor-like receptors. Furthermore, the present invention has established that TNF receptor-like receptors bind ligand and signal via homotypic association, i.e. TNFR-PLAD interacts with TNFR-PLAD; Fas-PLAD interacts with Fas-PLAD; CD40-PLAD interacts with CD40-PLAD etc. Therefore, therapy with PLAD self-association disrupting peptides and peptide mimetics would ensure receptor specific therapy because the present invention shows that each receptor associates only with itself through the PLAD. For example, disrupting TNF-R1 function without affecting TNF-R2 has major benefits above current non-selective therapeutics. Similarly, the specific disruption of a particular TNF receptor-like receptor function without affecting other TNF receptor-like receptor functions is highly desirable and provided by the teaching herein.
The present invention also provides a method of treating inflammation in a subject by administering an effective amount of an inhibitor of PLAD self-association. The present invention also provides a method of treating inflammation associated with an autoimmune disease in a subject by administering an effective amount of an inhibitor of PLAD self-association. Such diseases include, but are not limited to, periodic fever syndromes, sepsis syndromes and adult respiratory distress syndrome. Thus, provided is a method wherein the inflammation is associated with septic arthritis and the inhibitor is a soluble PLAD of a TNF receptor-like receptor.
In the present invention, the subject can be any mammal, preferably human, and can include but is not limited to mouse, rat, guinea pig, hamster, rabbit, cat, dog, goat, monkey, horse and chimpanzee.
As used herein, “treating” describes an improvement in the patient's clinical state. The improvement may range from reduction of the inflammatory response to complete amelioration of the inflammatory disease.
As used herein, “autoimmune disease” describes a disease state or syndrome whereby a subject's body produces a dysfunctional immune response against the subject's own body components, with adverse effects. This may include production of B cells which produce antibodies with specificity for all antigens, allergens or major histocompatibility (MHC) antigens, or it may include production of T cells bearing receptors that recognize self-components and produce cytokines that cause inflammation. Examples of autoimmune diseases include, but are not limited to, ulcerative colitis, Crohn's disease, multiple sclerosis, rheumatoid arthritis, septic arthritis, diabetes mellitus, pernicious anemia, autoimmune gastritis, psoriasis, Bechet's disease, Wegener's granulomatosis, Sarcoidois, autoimmune thyroiditis, autoimmune oophoritis, bullous pemphigoid, phemphigus, polyendocrinopathies, Still's disease, Lambert-Eaton myasthenia syndrome, myasthenia gravis, Goodpasture's syndrome, autoimmune orchitis, autoimmune uveitis, systemic lupus erythematosus, Sjogren's Syndrome and ankylosing spondylitis.
Since certain TNFR receptors, such as HVEA, are viral receptors, and these receptors may depend on oligomerization, the present invention also contemplates blocking viral entry by preventing PLAD assembly.
Optimal dosages used will vary according to the individual being treated and the inhibitor being used. The amount of inhibitor will also vary among individuals on the basis of age, size, weight, condition, etc. One skilled in the art will realize that dosages are best optimized by the practicing physician and methods for determining dose amounts and regimens and preparing dosage forms are described, for example, in Remington's Pharmaceutical Sciences. For example, suitable doses and dosage regimens can be determined by comparison to agents presently used in the treatment or prevention of inflammation or autoimmune disorders.
Typically, the inhibitor of this invention can be administered orally or parenterally in a dosage range of 0.1 to 100 mg/kg of body weight depending on the clinical response that is to be obtained. For example, when the inhibitor is a soluble PLAD or PLAD-containing compound, the inhibitor can be administered at an amount of 0.5 to 100 mg/kg, for example 5 mg/kg. In a further example, when the inhibitor is soluble (isolated) p60 PLAD and the disease is arthritis (e.g., septic arthritis), the PLAD can be administered in a dosage or 0.5 to 100 mg per joint. For example, doses of p60 PLAD of 4 mg/kg for intra-articular administration or 16 mg/kg for parenteral administration are effective to treat septic arthritis. Administration of inhibitor can be stopped completely following a prolonged remission or stabilization of disease signs and symptoms and readministered following a worsening of either the signs or symptoms of the disease, or following a significant change in immune status, as determined by routine follow-up immunological studies well known to a clinician in this field.
The efficacy of administration of a particular dose of inhibitor in treating inflammation or an autoimmune disorder as described herein can be determined by evaluating the particular aspects of the medical history, the signs, symptoms and objective laboratory tests that have a documented utility in evaluating pathophysiological activity of the particular disorder being treated. These signs, symptoms and objective laboratory tests will vary depending on the particular disorder being treated, as will be well known to any clinician in this field. For example, if, based on a comparison with an appropriate control group and knowledge of the normal progression of the disorder in the general population or the particular individual, 1) a subject's frequency or severity of recurrences is shown to be improved; 2) the progression of the disease or disorder is shown to be stabilized; or 3) the need for use of other immunosuppressive medications is lessened, then a particular treatment can be considered efficacious.
Once it is established that disease activity is significantly improved or stabilized by a particular inhibitor, specific signs, symptoms and laboratory tests can be evaluated in accordance with a reduced or discontinued treatment schedule. If a disease activity recurs, based on standard methods of evaluation of the particular signs, symptoms and objective laboratory tests as described herein, treatment can be reinitiated.
Additionally, the efficacy of administration of a particular dose of a peptide ligand in preventing an autoimmune disorder in a subject not known to have an autoimmune disorder, but known to be at risk of developing an autoimmune disorder, can be determined by evaluating standard signs, symptoms and objective laboratory tests, known to one of skill in the art, over time. This time interval may be long (i.e., years/decades). The determination of who would be at risk for the development of an autoimmune disorder would be made based on current knowledge of the known risk factors for a particular disorder familiar to clinicians and researchers in this field, such as a particularly strong family history of a disorder or exposure to or acquisition of factors or conditions which are likely to lead to development of an autoimmune disorder.
By “pharmaceutically acceptable” is meant a material that is not biologically or otherwise undesirable, i.e., the material can be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in a undesirable manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier chosen depends on the method of administration and the particular patient. Methods of administration can be oral, sublingual, mucosal, inhaled, absorbed, or by injection. It is also noted that not all methods of administering the inhibitors of TNF receptor-like receptor oligomerization described herein require a pharmaceutically acceptable carrier.
In the present invention, the inhibitors of PLAD self-association or TNF-like oligomerization can be orally or parenterally administered in a carrier pharmaceutically acceptable to human subjects. Suitable carriers for oral or inhaled administration can include one or more of the carriers pharmaceutically acceptable to human subjects. Suitable carriers for oral administration include one or more substances which may also act as flavoring agents, lubricants, suspending agents, or as protectants. Suitable solid carriers include calcium phosphate, calcium carbonate, magnesium stearate, sugars, starch, gelatin, cellulose, carboxypolymethylene, or cyclodextrans. Suitable liquid carriers include water, pyrogen free saline, pharmaceutically accepted oils, or a mixture of any of these. The liquid can also contain other suitable pharmaceutical addition such as buffers, preservatives, flavoring agents, viscosity or osmo-regulators, stabilizers or suspending agents. Examples of suitable liquid carriers include water with or without various additives, including carboxypolymethylene as a ph-regulated gel. The inhibitor can be contained in enteric coated capsules that release the polypeptide into the intestine to avoid gastric breakdown. For parenteral administration of the antagonist, a sterile solution or suspension is prepared in saline that can contain additives, such as ethyl oleate or isopropyl myristate, and can be injected for example, into subcutaneous or intramuscular tissues, as well as intravenously.
A method of screening for an inhibitor of PLAD association comprising: a) transfecting a cell with a plasmid containing a nucleic acid comprising a nucleic acid sequence encoding an isolated PLAD and a plasmid comprising a nucleic acid sequence encoding a second isolated PLAD; b) contacting the cell with a putative inhibitor and; c) measuring PLAD self association, wherein a decrease in PLAD association in the cell of step b) as compared to PLAD association in a cell that was not contacted with the putative inhibitor indicates the presence of an inhibitor of PLAD-association.
One example of this screening method is a method of screening for an inhibitor of PLAD-association comprising: a) transfecting a cell with a plasmid containing a nucleic acid comprising a nucleic acid sequence encoding an isolated PLAD functionally linked to a flourescence donor and a plasmid comprising a nucleic acid sequence encoding an isolated PLAD functionally linked to a flourescence acceptor; b) contacting the cell with the inhibitor; and c) measuring FRET, wherein a decrease in FRET as compared to FRET measurement in a cell that was not contacted with the inhibitor indicates the presence of an inhibitor of PLAD-association.
Also provided by the present invention is a method of screening for an agonist of PLAD association comprising: a) transfecting a cell with a plasmid containing a nucleic acid comprising a nucleic acid sequence encoding an isolated PLAD and a plasmid comprising a nucleic acid sequence encoding a second isolated PLAD; b) contacting the cell with a putative agonist and; c) measuring PLAD self association, wherein an increase in PLAD association in the cell of step b) as compared to PLAD association in a cell that was not contacted with the putative agonist indicates the presence of an agonist of PLAD-association.
The Examples below exemplify the use of FRET to measure PLAD association. Furthermore, in performing the screening methods described above, a single plasmid can be utilized to deliver more than one nucleic acid encoding a PLAD. In methods involving FRET analysis, a single plasmid can be utilized to deliver more than one nucleic acid encoding a PLAD functionally linked to a fluorescence donor or acceptor.
One skilled in the art could also utilize a yeast two hybrid screening method to screen for inhibitors or agonists of PLAD association. Inhibitors or agonists of PLAD association can also be identified by utilizing cellular assays which can include, but are not limited to, apoptosis induction, NF-KB induction, lymphocyte maturation or activation, and necrosis induction (3, 4, 8, 15, 29, 33, 42, 45).
The present invention is more particularly described in the following examples which are intended as illustrative only since numerous modifications and variations therein will be apparent to those skilled in the art.
H9 lymphoma cells were washed and resuspended in PBS. The cells were then incubated with 100 ng/ml of human recombinant TNFα (R&D Systems) for 1 hour at 4° C. with rotation. Cells were then treated with 2 mM of the crosslinker DTSSP (Pierce) for 30 minutes and the reaction was quenched with 20 mM Tris.Cl [pH 7.5] for 15 minutes on ice. The cells were lysed in 150 mM NaCl, 20 M Tris.Cl [pH 7.5], 1 μM EDTA, 30 mM NaF, 2 mM b-glycerophosphate and 1 mM sodium orthovanadate with protease inhibitors added (Boehringer Mannheim). Equal amounts of the lysates were subjected to electrophoresis under non-reducing (without β-mercaptoethanol) or reducing (with 280 mM β-mercaptoethanol) conditions and analyzed for p60 and p80 complexes with specific antibodies (19). Densitometry was performed with a Kodak Image Station 440.
Complexes were found for p80 that exhibited molecular sizes approximately three times the unit size, consistent with glycosylated and non-glycosylated trimers (
The complexes were resolved into monomers by cleaving the crosslinker with β-mercaptoethanol (
To validate the possibility of ligand-independent self-assembly, a domain in the TNFR that would mediate this phenomenon was identified. It is well established that the cytoplasmic death domain of p60 can self-associate and trigger apoptosis when over-expressed (8). However, since the pre-assembled complexes observed were apparently non-signaling, it was hypothesized that the assembly domain resides outside of the cytoplasmic region. It was found that the N-terminal regions of the ECDs of p60 and p80 could specifically self-associate in a yeast two-hybrid interaction assay (9).
The various truncations and mutations of p60, p80, HVEM, DR4 and CD40 were generated by Polymerase chain reaction (PCR) and sequenced. Briefly, the leader sequence and the first ten amino acid residues from p80 was amplified so that the HA epitope tag was included at the 3′ end to create a HA tag at the N-terminus of the receptors. The PCR product was digested with BamHI and EcoRI and cloned into pcDNA3. The PCR fragments containing the receptor fragments were then introduced into this plasmid using the EcoRI and XhoI sites. For the GFP/CFP/YFP chimeras, the fragments were amplified by PCR and introduced in-frame into the XhoI and XbaI sites of p60ΔCD-HA. 293T cells were transfected with Fugene 6 (Boehringer Mannheim) as per manufacturer's protocol. Cells were lysed in 150 mM NaCl, 20 mM Tris.Cl [pH 7.5], 1 mM EDTA, 30 mM NaF, 2 mM β-glycerophosphate, 1 mM sodium orthovanadate, 5 mM iodoacetamide, 2 mM dithiothreitol (DTT), 1% TRITON X-100 and protease inhibitors (Boehringer Mannheim). After pre-clearing with protein G agarose beads (Boehringer Mannheim) and normal mouse IgG, proteins were immunoprecipitated from the lysates with 2 mg anti-GFP and protein G agarose beads. Immune complexes were washed twice with lysis buffer containing 0.5 M NaCl and then three times with regular lysis buffer. Immune-complexes were resolved on Tris/Glycine gels (Novex). Transfection in Jurkat cells showed similar results. In mammalian cells, it was found that a chimeric p60 receptor with the cytoplasmic domain replaced by the green fluorescent protein (GFP) interacted strongly with a tailless p60 (p60ΔCD-HA) but not with the TNFR-like receptor herpesvirus entry mediator (HVEMΔCD-HA) (
GFP alone failed to associate with p60ΔCD-HA (
Moreover, removal of as little as amino acids (a.a)10-54 of p80, overlapping CRD1, completely abrogated ligand-independent association with intact p80 (
The importance of the N-terminal portion of p80 (a.a.10-54) is further illustrated by appending it to the p60 receptor which then interacted with full-length p80 (
The deletion of the PLAD from either p60 or p80 completely abrogated ligand binding (Table 2 and
Unlike monomeric receptor chains, the cytoplasmic portions of the receptor chains within a pre-assembled receptor complex might be expected to be in close proximity to each other. TNFα binding could then cause fighter association of the cytoplasmic domains leading to the recruitment of signaling proteins. To evaluate this hypothesis, a novel flow cytometric approach described in Example II (11) was employed to analyze fluorescence resonance energy transfer (FRET) between two spectral variants of GFP, cyan fluorescent protein (CFP) as the fluorescence donor and yellow fluorescent protein (YFP) as the fluorescence acceptor (12). FRET is a powerful approach to measure molecular interactions in living cells. Since energy transfer is rapidly attenuated as the distance between fluorophores increases, FRET between GFP variants allows the detection of molecular interactions within 100 Å.
Chimeric proteins were generated in which the cytoplasmic regions of p60 and p80 were replaced by either CFP or YFP and tested to determine if energy transfer occurs, between different receptor pairs. FRET was performed with a dual laser FACSvantage machine that excites the YFP protein at 514 nm prior to exciting the CFP protein at 413 nm. Energy transfer from CFP to YFP was then detected as emission at 546 nm. Cells were transfected with a large excess of YFP protein compared with CFP protein. FRET was then analyzed on the CFP positive populations using the program Flowjo (Treestar Inc.).
Energy transfer between p60ΔCD-CFP and p60ΔCD-YFP which increased substantially following the addition of TNFα (
Comparison of the PLAD of p60 and p80 with the first CRD of a number of receptors in the TNFR family reveals conservation beyond the cysteines that form the disulfide bond scaffold of the domain (
Strikingly, the ECDs of TRAIL receptor 1 (DR4) and CD40 both self-associate but do not significantly interact with ECDs from other TNFR-like receptors (
This invention reveals that the p60 and p80 TNFRs pre-assemble into functional complexes in the absence of ligand via a novel N-terminal domain termed PLAD. This reveals how CRD1 plays a crucial role in ligand-binding and receptor signaling for p60 and p80 (10). Until now, the fundamental concept of signaling by members of the TNFR superfamily is that ligand brings monomer receptor chains into apposition in three-fold complexes which leads to recruitment of cytoplasmic signal transduction proteins (1, 3, 5, 6). This model was based largely upon the crystal structure of p60 complexed with ligand, which showed that three receptor chains embrace the trimeric ligand in its intersubunit grooves and remain at least 40 Å apart. The ligand makes contact with the elongated CRD2 and CRD3 domains whereas the CRD1 domains do not interact with ligand or with each other (5). The recent description of the structure of DR5/TRAIL complex reveals similar receptor-ligand interactions (13). However, the liganded structure does not appear to reflect the receptor structure prior to ligand binding. It is now clear that p60 and p80 self-associate on the cell surface and are only found as monomers if the PLAD is deleted. Cross-linking the endogenous p60 and p80 receptors suggests that trimers are a favored conformation, but other oligomeric complexes may also occur.
How ligand interacts with the pre-associated receptor complex is of great interest since it is now evident that pre-association is required for TNFα binding. TNFR signaling could be explained by one of two broad classes of models: 1) chain rotation and rearrangement, and 2) supercluster formation models (
Pre-assembly has been described for other receptor families, notably IL-1 and IL-2, which are comprised of heteromers of different polypeptides (16). Of particular interest is a recent description of pre-association of the erythropoietin receptor dimers that apparently undergo a “scissors-type” movement to accommodate ligand (17). In that case, self-association of the receptor chains occurs via the same amino acid contacts that are critical for ligand binding (17). By contrast, the T superfamily utilizes a dedicated self-association domain distinct from the CRD2/3 ligand contact region. Identification of the PLAD could allow new treatments of diseases caused by TNFα or related ligands through the use of therapeutics that specifically inhibit the pre-ligand assembly of TNFR-like receptors and thereby prevent signaling.
Heterozygous mutations encoding abnormal forms of Fas (CD95/APO-1) dominantly interfere with Fas-induced lymphocyte apoptosis in the human Autoimmune Lymphoproliferative Syndrome (ALPS). This invention demonstrates that, rather than depending on ligand-induced receptor oligomerization, this stems from pre-association of wild-type and mutant Fas receptors through the extracellular domain Pre-associated Fas receptor complexes were found to be essential for signal transduction, and were demonstrated in living cells using a novel application of FRET between variants of the Green Fluorescent Protein (GFP). These results provide a new molecular mechanism for Fas signaling and dominant interference in human disease.
Fas (APO-1/CD95) is a cell surface receptor that transduces apoptotic signals critical for immune homeostasis and tolerance (19-21). Fas is a 317 amino-acid type 1 membrane glycoprotein with three extracellular cysteine-rich domains (CRD) that are characteristic of the tumor necrosis factor receptor (TINE) superfamily.
In humans, lymphocytes from patients with ALPS Type 1A harboring heterozygous Fas mutations have reduced Fas-induced apoptosis, and transfection of the mutant allele causes dominant interference with apoptosis induced through Fas (29-34). This was thought to be due to ligand-mediated cross-linking of wild-type and defective Fas chains into mixed trimer complexes that cannot recruit downstream signaling molecules. However, a dominant-interfering mutation that causes an extracellular domain (ECD) deletion of most of CRD 2 (Pt 2, deletion a.a. 52-96) through altered RNA splicing has been studied. Expression of this mutant on Fas-negative 293T cells shows no binding to agonistic antibodies (
In order to conduct these immunoprecipitation studies, 293T cells were transfected with Fugene 6 (Boehringer Mannheim) according to the manufacturer's instructions. Cells were lysed in 150 mM NaCl, 20 mM Tris.Cl [pH 7.5], 1 mM EDTA, 5 mM iodoacetamide, 2 mM dithiothreitol (DTT), 10% glycerol, 1% TRITON X-100 and protease inhibitors Boehringer Mannheim). After pre-clearing with protein G agarose beads (Boehringer Mannheim) and normal mouse IgG, proteins were immunoprecipitated with 1 mg anti-GFP] (Roche Molecular Biochemicals) and protein G agarose beads. Immune complexes were washed three times with lysis buffer. AU1 was immunoprecipitated with 2 μl of anti-AU1 (Covance) and protein A beads. Proteins were electropheresed on Tris/Glycine gels (Novex), transferred to nitrocellulose membranes, and blotted with the indicated antibodies. Bands were visualized with SuperSignal WestDura (Pierce). Densitometry was performed with 1D image analysis software (Kodak).
In Example I, a conserved N-terminal domain, termed the “pre-ligand assembly domain” (PLAD) is described that mediates specific self-association of other members of the TNFR superfamily. However, the N-terminus of Fas is lacking several key amino-acids conserved in other TNFR-family receptors, raising the issue of whether Fas contains a functional PLAD.
N-terminal Fas mutants truncating or eliminating the first CRD were constructed and tested for ligand binding, Fas-Fas association, and apoptotic function (
These studies indicated that deleting the first 43 amino acids (a.a.) of the mature Fas protein that make up the first CRD subdomain (39) substantially reduced ligand binding but did not prevent binding of the APO-1 agonist antibody. Deleting the first 66 a.a. encoding the entire CRD1 abrogated binding of both FasL and APO-1 (
To quantitate Fas receptor self-association in living cells, flow cytometric and microscopic assays based on fluorescence resonance energy transfer (FRET) between spectrally distinct mutants of GFP, Cyan fluorescent protein (CFP) and Yellow fluorescent protein (YFP) were developed. CFP and YFP have spectral properties favorable for FRET in that the emission maximum for CFP is near the absorption maximum for YFP (40). Since FRET between these proteins rapidly declines at distances larger than 50 Å-100 Å, the presence of FRET between CFP and YFP fusion proteins indicates close proximity of their fluorescent protein domains. When Fas receptors with C-terminal in-frame fusions to CFP and YFP (at position 210 in place of the death domain) were co-transfected into 293T HEK cells, they were appropriately expressed on the cell surface (36).
In-frame CFP and YFP fusions with Fas and other TNF family receptors were generated by standard PCR cloning techniques and correct protein expression was confirmed by western blotting and fluorescence microscopy. 293T cells were transfected with 1 μg of the indicated YFP fusion protein constructs and 2 μg of the indicated CFP constructs. 24-36 hours later cells were harvested in PBS and analyzed on a FACSvantage cytometer with a krypton laser (Spectrophysics) tuned to 413 nm for CFP and an ILT air-cooled laser tuned to 514 nm for YFP. CFP was detected with a 470 nm/20 nm bandpass filter. YFP and FRET were detected with 546 nm/10 nm bandpass filters with signals from the 514 and 413 nm lasers respectively. Cells were sequentially illuminated with the 514 and 413 nm lasers so that all three signals could be detected from each cell. Compensation was applied so that there was no FRET signal visible from cells transfected with CFP or YFP alone. 50,000 events were collected from each sample and the data was analyzed by the FlowJo software package (Treestar) For FRET efficiency measurements, CFP emission intensities from co-transfected cells were measured on a fluorescence microscope before and after bleaching the YFP with 5 min illumination through a 505-545 nm bandpass filter. Controls showed that this much intensity bleached YFP essentially completely, with very little direct bleaching of CFP. Such direct bleaching was corrected for. FRET efficiencies were calculated using the formula
E%=[1−(CFP emission before YFP-bleach/CFP emission after YFP bleach)]*100%.
When examined by flow cytometry, CFP excitation of cells co-transfected with the CFP and YFP Fas fusion proteins triggered strong fluorescence emission at the YFP wavelength attributable to FRET (
To test whether native Fas receptors self-associate on the surface of untransfected T lymphocytes, chemical cross-linking studies (
The conserved N-terminal PLAD was required for appropriate Fas receptor function, and could thus play a key role in dominant interference in ALPS. Comparing the structure, dominant interference (DI), and Fas-Fas self-association (SA) of a large number of ALPS patients that have been studied at the National Institutes of Health (29, 33-35) (
Together these findings redefine the mechanism by which Fas mutations in ALPS dominantly interfere with normal Fas function. It is now evident that dominant-interfering Fas mutations preserve the N-terminal PLAD because this domain is responsible for complex formation between wild-type and mutant Fas molecules. The central molecular principle of genetic dominant interference is that mutant proteins must physically interact with wild-type proteins in a specific functional complex (43). Previously, dominant negative receptor mutations associated with human diseases have been shown to interfere with normal receptor signaling by sequestering ligand, blocking intracellular signaling or preventing transport of the WT chain to the cell surface (44). For Fas, the data show that dominant interference stems from a novel mechanism involving PLAD-mediated association between wild type and mutant receptors prior to ligand binding. These findings explain why the abnormal Fas protein in ALPS Pt 2 and other Fas ECD mutants can fail to bind FasL and yet exert dominant interference sufficient to cause disease. PLAD-mediated interactions also account for the dominant-interfering interactions of the large number of ALPS patients that carry mutations affecting the death domain of Fas, since removing the PLAD abrogated the dominant negative function of Fas molecules with deleted or mutated death domains. PLAD interactions are also likely involved in the down-modulation of Fas-induced apoptosis by soluble alternatively spliced forms of Fas that all include this domain (45). Natural receptor mutants that do not encode a functional PLAD would not be expected to be dominant-interfering. PLAD-mediated dominant interference may also play a role in modulation of signaling by decoy receptors (20) and in the pathogenesis of diseases due to heterozygous genetic abnormalities in other members of the TNFR family.
These results also suggest a new model for understanding transmembrane signaling by Fas, involving conversion of pre-associated trimers to a signaling state by ligand, rather than ligand-induced oligomerization of individual receptor chains. The FET studies allow estimation of the distance between CFP and YFP-tagged Fas molecules on the cell surface in the absence of ligand. The Förster radius, R0, for randomly oriented CFP and YFP is 50 Å (23). Assuming that CFP and YFP in the fusions to Fas are equally expressed, randomly oriented with respect to each other, and randomly assorted into equilateral trimers, the observed FRET efficiencies (
Signaling through receptor complex rearrangement may be a widely-used mechanism to ensure rapid and specific cellular responses to ligands. However, this signaling mechanism also confers susceptibility to dominant interference by naturally occurring receptor variants or pathogenic heterozygous mutations in ALPS.
Mice and reagents. BALB/c, C57BL/6, DBA/1J, C3H/HeJ, C3H/HeN, TNFR1 and TNFR2 knockout mice were purchased from the Jackson Laboratory. TNF-transgenic mice were purchased from Taconic. Male mice at 6-8 weeks of age were used in all experiments and were housed in the animal facility of the Laboratory of Immunology, NIAID, NIH. CpG DNA was synthesized by CBER. CpG DNA 1668 sequence is 5′-TCCATGACGTTCCTGATGCT-3′ (SEQ ID NO:61) (50). Mouse monoclonal antibodies (MAbs) against P60 (1H11) or P80 PLAD (3H11) were prepared by using P60 PLAD and P80 PLAD.
Purification of PLAD-GST fusion protein. Cloning and purification of PLAD-GST fusion protein was carried out according to an established procedure (78). Purified PLAD-GST fusion proteins were validated on a 4-20% Tris-Glycine gel and by mass spectroscopy and stored at 80° C. LPS was removed with Detoxi-Gel AffinityPak Columns (Pierce).
In vitro tests of P60 and P80 PLAD proteins. L929 cells were collected after trypsin treatment. Cells were pretreated with different doses of PLAD protein for 30 min, after which TNF-α (2 ng) was added. 42.3 cells pretreated similarly with PLAD protein or GST. Following the 30 minute' pretreatment, TNF-α (3 ng), antibody to Fas (11) (10 ng), and protein A (20 ng) were added and incubated at 37° C. Quantitation of cell death was performed by hemocytometer or flow cytometry at indicated time-points. TNF-α binding was measured according to the manufacturer's protocol (R&D systems).
Immunogenicity and half-life of PLAD proteins. Immunogenicity in vivo and half-life of PLAD proteins by use of ELISA were investigated. Both PLAD proteins could elicit an antibody response at least half of which was directed at the GST portion (Supplementary
Development of arthritis triggered by TNF-αe, CpG DNA with PLAD protein treatment. TNF-α, CpG DNA or LPS were intramuscularly injected with or without 100 μg of PLAD protein into mouse knee joints. The mice were sacrificed and joints were removed for histopathological examination 3 d after injection.
Induction and treatment of CIA with PLAD protein. Chicken collagen type II (Sigma) emulsified with an equal volume of complete Freund's adjuvant (Sigma) was intradermally injected with 0.1 ml of emulsion containing 100 μg of type II collagen at the base of the tail of male DBA/1J mice. Mice were boosted with 100 μg of type II collagen emulsified with an equal volume of incomplete Freund's adjuvant (Sigma) at 21 d. Preventive treatment with PLAD protein or PBS began at 22 d after primary immunization and was administered intraperitoneally three times per week until day 52 in a coded fashion. Therapeutic treatment with P60 PLAD protein or PBS began three d after mice have already developed arthritis. P60 PLAD protein (400 μg administered every other day) was given intraperitoneally for two weeks and then the treatment was switched between groups. In the “masked” experiments, mice were analyzed by two independent examiners unaware of the treatment every third day and assessed for extent of arthritis: paw swelling and clinical score. Joint swelling was determined by measuring the thickness of the paws with a caliper. Clinical arthritis was evaluated using the following scale: grade 0, no swelling; grade 1, slight swelling and erythema; grade 2, pronounced swelling; grade 3, joint rigidity. Each limb was graded as score of 0-3 with a maximum possible score of 12 for each animal.
Histopathological examination of joints. After routine fixation, decalcification and paraffin embedding of the tissue, joint sections were cut and stained with hematoxylin and eosin. All the slides were coded and evaluated blindly by researchers. The extent of synovitis, pannus formation, or bone/cartilage destruction was judged on a scale from grade 0 (no signs of inflammation), grade 1 (mild inflammation with hyperplasia of the synovial lining layer, minimal without cartilage destruction) to grades 2 and 4 (increasing degrees of inflammatory cell infiltrate, or cartilage and bone destruction).
In vitro osteoclastogenesis assay. Bone marrow cells were extracted from the tibia of BALB/c mice and cultured in α-MEM medium in presence of M-CSF. After 3 d, bone marrow macrophages were induced further for 4 d with TNF-α (20 ng/ml) and M-CSF (50 ng/ml) in the presence or absence of PLAD proteins. Cells were then fixed and stained for the osteoclast marker TRAP using the manufacturer's procedure (Sigma).
Immunofluorescence (66). Mononuclear cells isolated from spleen of TNFR1 or TNFR2 knockout mice were treated with TNF-α with or without P60 and P80 PLAD protein in vitro. Fixed and permeabilized cells were stained with Rabbit antibody to NF-κB p65 (C-20, Santa Cruz). Cells were then washed and incubated with a FITC-conjugated donkey anti-rabbit antibody. Nuclear NF-κB was analyzed and scored in masked fashion on a confocal microscope.
Statistical analysis. The difference of incidence and severity in groups was analyzed by use of the Fisher's test and Mann-Whitney U test, respectively. P≦0.05 was considered to be significant.
To obtain soluble proteins containing the PLAD domain of the human P60 or P80 TNFRs, glutathione-S-transferase (GST) fusion proteins were prepared. Affinity purification provided P60 and P80 PLAD-GST fusion proteins whose full-length molecule weights were 34 kD and 33 kD, respectively, the expected sizes based on amino acid sequences (
This experiment was performed to determine whether the purified PLAD proteins inhibit the cytopathic effect of TNF-α on L929 cells (63). The purified P60 PLAD protein clearly inhibited mouse and human TNF-α-induced cell death in a dose-dependent fashion (
Whether PLAD proteins could inhibit TNF-α-induced arthritis in vivo was also tested. Arthritis was induced by intra-articular injection of mouse TNF-α (45 ng) with or without 100 μg PLAD protein added. The P60 PLAD protein powerfully inhibited the features of TNF-α-induced arthritis including synovitis, pannus, and bone erosion (
TNF-α plays an important role in the pathogenesis of SA (49, 50, 61). CpG-containing bacterial DNA and LPS are bacterial components that strongly induce TNF-α release from monocytes and macrophages that can trigger arthritis (49, 61). Co-injection of 100 μg P60 PLAD protein could significantly reduce pannus and bone destruction in CpG DNA-induced arthritis (P<0.05) (
Matrix metalloproteinases (MMP) are important mediators of destruction of cartilage and bones during arthritis, and MMP expression can be induced by TNFα. Therefore, this experiment addressed whether P60-PLAD protein inhibited MW expression in CIA. The results showed that P60 PLAD significantly inhibited MMP expression in CIA rig. 25). Note that the dark (brown in its original color slide) staining in the left panel indicates M expression.
P60 PLAD Inhibits iNOS Expression in Collagen-Induced Arthritis (CIA)
Nitric oxide (NO) is a short-lived free radical gas. Excessive nitric oxide is toxic and causes chronic inflammation. Importantly, it has been shown that TNFα can stimulate macrophages/monocytes to produce nitric oxide by activating inducible nitric oxide synthase (iNOS) and increase NO level, thus causing local joint damage. Therefore, it was determined whether P60-PLAD protein inhibits iNOS expression in CIA. The results showed that P60-PLAD protein markedly inhibited iNOS expression in affected joints of CIA (
The present results demonstrate that the PLAD in TNFR1 is an important therapeutic, target. CD95 PLAD was found to participate in pathogenesis of ALPS by mediating dominant interference (11). The P60 and P80 PLAD proteins block binding of TNF-α to TNFR1 but do not directly bind to TNF-α. Most importantly, the P60 PLAD exhibited a striking ameliorative effect in several mouse models of arthritis through systemic as well as intra-articular administration. Moreover, inflammatory cell infiltration of the joints was markedly reduced by P60 PLAD treatment. In previous examples, it was found that the PLADs for P60 and P80 are selective for homotypic binding and do not cross-bind with each other or the CD95 PLAD (11, 53). Those experiments demonstrated that P60 and P80 PLAD had preferential effects on the corresponding receptors but might not be completely specific (
The P60 PLAD protein potently arrested NF-κB induction by TNF-α. The P60 and
P80 PLAD proteins can inhibit TNF-α-induced NF-κB translocation in a receptor preferential manner. Moreover, NF-κB is required for RANKL-induced osteoclastogenesis (71). Osteoclast-mediated bone and cartilage erosion causes most of the permanent damage in arthritis (67, 68, 69). The present results show osteoclast activation in pannus and sites of bone destruction in TNF-α transgenic mice. Also, there was abundant expression of RANK and RANKL in arthritic joint of the TNF-α transgenic mice and collagen-induced arthritis (69). P60 PLAD treatment dramatically reduced RANK and RANKL in the lining layer and pannus.
Infliximab and etanercept both can directly bind and block the effects of TNF-α and they are now widely used in the treatment of RA and other diseases (48). However, side effects such as lupus-like disease, mycobacterial infections, and an increased incidence of lymphoma have been observed (72-75). Both drugs directly block TNF-o binding to both TNFRs. Thus, they could inhibit potentially beneficial effects mediated by TNF2 while arresting the disease-causing effects of TNFR1. The P60 PLAD is small protein domain that could preferentially target TNFR1. In our models, a dose of P60 PLAD protein of about 4 mg/kg (intra-articularly) or 16 mg/kg (parenterally) had similar effects to doses of infliximab (10 mg/kg) (76) and etanercept (0.4 mg/kg)33 that have been used clinically in the amelioration of arthritis. The P60 PLAD protein offers a new approach to inhibit the pathogenic effects of TNF-α that is useful in RA.
To determine whether the PLAD protein directly binds to TNFR, P80-PLAD protein was mixed with etanercept, a TNFR2 fusion protein that contains complete extracellular domain of TNFR2, was tested by mixing with P80 PLAD protein. The results of immunoprecipitation (IP) and Western Blot (WB) show that P80-PLAD-GST protein but not the GST-only proteins can associate with etanercept, indicating that PLAD protein directly binds to TNFR and therefore inhibit TNFα activity (
, p75, TNFBR,
F12
SL-LR, DDR3, TR3,
IL
disease
indicates data missing or illegible when filed
Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
This application claims priority to U.S. Provisional Application No. 60/694,015, filed on Jun. 24, 2005, and U.S. Provisional Application No. 60/717,589, filed on Sep. 16, 2005, hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US06/24909 | 6/26/2006 | WO | 00 | 3/27/2008 |
| Number | Date | Country | |
|---|---|---|---|
| 60694015 | Jun 2005 | US | |
| 60717589 | Sep 2005 | US |
