Patent Application
20080009444
The present invention relates generally to a biologically active complex comprising lest two heterologous molecules. More particularly, the biologically active complex of the present invention comprises at least two polypeptides or parts, fragments, truncates or protease-activated forms of one or more of the polypeptides wherein the complex alone or in association with a receptor, ligand or other molecule facilitates proliferation, differentiation and/or survival of a cell. The identification of the biologically active complex of the present invention permits the assay for agonists and antagonists of the formation of the biologically active complex as well as therapeutic and diagnostic reagents based on the biologically active complex or interaction between the biologically active complex and a receptor, ligand or other molecule.
Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.
Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.
The rapidly increasing sophistication of recombinant DNA techniques is greatly facilitating research into the medical and allied health fields. Cytokine research is of particular importance, especially as these molecules regulate the proliferation, differentiation and function of a wide variety of cells. Administration of recombinant cytokines or regulating cytokine function and/or synthesis is becoming increasingly the focus of medical research into the treatment of a range of disease conditions.
Despite the discovery of a range of cytokines and other secreted regulators of cell function, comparatively few cytokines are directly used or targeted in therapeutic regimens. One reason for this is the pleiotropic nature of many cytokines. For example, interleukin (IL)-11 is a functionally pleiotropic molecule (1,2), initially characterized by its ability to stimulate proliferation of the IL-6-dependent plasmacytoma cell line, T11 65 (3). Other biological actions of IL-11 include induction of multipotential haemopoietin progenitor cell proliferation (4,5,6), enhancement of megakaryocyte and platelet formation (7,8,9,10), stimulation of acute phase protein synthesis (11) and inhibition of adipocyte lipoprotein lipase activity (12,13).
Other important cytokines in the IL-11 group include IL-6, leukaemia inhibitory factor (LIF), oncostatin M (OSM), ciliary neurotrophic factor (CNTF) and cardiotrophin-1 (CT-1). All these cytokines exhibit pleiotropic properties with significant activities in proliferation, differentiation and survival of cells. Members of the haemopoietin receptor family are defined by the presence of a conserved amino acid domain in their extracellular region. However, despite the low level of amino acid sequence conservation between other haemopoietin receptor domains of different receptors, they are all predicted to assume a similar tertiary structure, centred around two fibronectin-type III repeats (18,19).
Recently a molecule has been identified which has cardiotrophin-like properties (26). This molecule has been referred to as cardiotrophin-like cytokine (CLC) and novel neurotrophic factor 1 (NNT-1) [U.S. Pat. No. 5,741,772].
Cytokines signal through cell-associated receptors. These receptors are classified into families based on sequence and structural similarities.
The size of the haemopoietin receptor family has now become extensive and includes the cell surface receptors for may cytokines including interleukin-2 (IL-2), IL-3, IL-4, IL-5, IL-6, IL-7, IL-9, IL-11, IL-12, IL-13, IL-15, granulocyte colony stimulating factor (G-CSF), granulocyte-macrophage-CSF (GM-CSF), erythropoietin, thrombopoietin, leptin, LIF, OSM, CNTF, CT-1, growth hormone and prolactin. Although most of the members of the haemopoietin receptor family act as classic cell surface receptors, binding their cognate ligand at the cell surface and initiating intracellular signal transduction, some receptors are also produced in naturally occurring soluble forms. These soluble receptors can either act as cytokine antagonists, by binding to cytokines arid inhibiting productive interactions with cell surface receptors (e.g. LIF binding protein; (20) or as agonists, binding to cytokine and potentiating interaction with cell surface receptor components (e.g. soluble interleukin-6 receptor a-chain; (2.1)). Still other members of the family appear to be produced only as secreted proteins, with no evidence of a cell surface form. In this regard, the IL-12 p40 subunit is a useful example. The cytokine IL-12 is secreted as a heterodimer composed of a p35 subunit which shows similarity to cytokines such as IL-6 (22) and a p40 subunit which shares similarity with the IL-6 receptor a-chain (23). In this case the soluble receptor acts as part of the cytokine itself and essential to formation of an active protein. In addition to acting as cytokines (e.g. IL-12p40), cytokine agonists (e.g. IL-6 receptor a-chain) or cytokine antagonists (LIF binding protein), members of the haemopoietin receptor have been useful in the discovery of small molecule cytokine mimetics. For example, the discovery of peptide mimetics of two commercially valuable cytokines, erythropoietin and thrombopoietin, centred on the selection of peptides capable of binding to soluble versions of the erythropoietin and thrombopoietin receptors (24,25).
Due to the importance and multifactorial nature of these cytokines, there is a need to further investigate and elucidate the molecular interactions not only between cytokines and their receptors but also between cytokines themselves.
Nucleotide and amino acid sequences are referred to by a sequence identifier, i.e. <400>1, <400>2, etc. A sequence listing is provided before the Examples.
Throughout this specification, unless the context requires otherwise, the word “comprise”, or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.
One aspect of the present invention is directed to a biologically active complex comprising at least two heterologous molecules which complex alone or in association with a receptor, ligand or other molecule facilitates proliferation, differentiation and/or survival of a cell.
Another aspect of the present invention provides a biologically active complex comprising at least two polypeptides or parts, fragments, truncates or protease-activated forms of one or more of the polypeptides which complex alone or in association with a receptor, ligand or other molecule facilitates proliferation, differentiation and/or survival of a cell.
Still another aspect of the present invention provides a biologically active complex comprising at least two polypeptides or parts, fragments, truncates or protease-activated forms of one or more of the polypeptides wherein at least one of said polypeptides is NR6 or a part, fragment, truncate or protease-activated form thereof and wherein said complex alone or in association with a receptor, ligand or other molecule facilitates proliferation, differentiation and/or survival of a cell.
Yet another aspect of the present invention is directed to a biologically-active complex comprising the structure:
[X1]n (a) [X2]n1 (b) [X3]n2 . . . [Xd]n3
wherein
Even yet another aspect of the present invention provides a biologically active complex comprising the structure:
[X1]a3 [NR6]a [CLC]a1 [NR6]a2 [X1]a4
wherein:
Another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides substantially as set forth in <400>1 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>1 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
Still another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides substantially as set forth in <400>3 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>3 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
Yet another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides substantially as set forth in <400>5 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>5 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
Even yet another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides substantially as set forth in <400>7 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>7 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
Another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides substantially as set forth in <400>9 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>9 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
Still another aspect of the present invention is directed to a genetic construct substantially as set forth in <400>11 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>11 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
Yet another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides substantially set forth in <400>12 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>12 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
Even yet another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides substantially set forth in <400>13 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>13 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
Another aspect of the present invention provides an expression vector comprising a nucleic acid molecule encoding NR6 and CLC or modified forms thereof said expression vector capable of expression in a selected host cell.
Still another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides encoding NR6 or a derivative thereof having an amino acid sequence as set forth in <400>2 or having at least about 50% similarity to all or part thereof, said genetic construct further comprising a sequence of nucleotides encoding CLC or a modified form thereof.
Yet another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides encoding NR6 or a derivative thereof having an amino acid sequence as set forth in <400>4 or having at least about 50% similarity to all or part thereof, said genetic construct further comprising a sequence of nucleotides encoding CLC or a modified form thereof.
Even still another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides encoding NR6 or a derivative thereof having an amino acid sequence as set forth in <400>6 or having at least about 50% similarity to all or part thereof, said genetic construct further comprising a sequence of nucleotides encoding CLC or a modified form thereof.
Another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides encoding NR6 or a derivative thereof having an amino acid sequence as set forth in <400>8 or having at least about 50% similarity to all or part thereof, said genetic construct further comprising a sequence of nucleotides encoding CLC or a modified form thereof.
Still another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides encoding NR6 or a derivative thereof having an amino acid sequence as set forth in <400>14 or having at least about 50% similarity to all or part thereof, said genetic construct further comprising a sequence of nucleotides encoding CLC or a modified form thereof.
Yet another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides encoding NR6 or a derivative thereof having an amino acid sequence as set forth in one or more of <400>15 or having at least about 50% similarity to all or part thereof, said genetic construct further comprising a sequence of nucleotides encoding CLC or a modified form thereof.
Another aspect of the present invention contemplates a method of modulating activity of the complex as hereinbefore described, said method comprising administering to a subject a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease the biological activity of the complex.
Still another aspect of the present invention is directed to antibodies to the complex and its derivatives.
Yet another aspect of the present invention contemplates a method for detecting the complex as hereinbefore described in a biological sample from a subject, said method comprising contacting said biological sample with an antibody specific for the complex (or a component thereof) or its derivatives or homologues for a time and under conditions sufficient for an antibody complex to form, and then detecting said antibody.
Even yet another further aspect of the present invention contemplates the use of the biologically active complex or its functional derivatives in the manufacture of a medicament for the treatment of conditions resulting from aberrations in the complex or in reduced or excessive amounts of the complex.
Another aspect of the present invention contemplates a ligand or receptor for the complex such as in isolated or recombinant form, or a derivative of said ligand or receptor.
Still another aspect of the present invention further contemplates knockout animals such as mice or other murine species for components of the complex gene including homozygous and heterozygous knockout animals.
Even still another aspect of the present invention contemplates a method of identifying an agent capable of modulating the effects of a biologically active complex as herein defined, said method comprising screening for agents which are capable of interacting with the complex or interfering or otherwise antagonizing or promoting or otherwise agonizing interaction between the heterologous molecules of said complex.
The complex and its components and in particular CLC, NR6 and/or a CLC-NR6 complex are particularly useful in inducing neurotrophic activity.
Conditioned medium was collected from CHO cells following transfection with either FLAG-NR6 or HA-CLC, or FLAG-NR6 and HA-CLC together. The conditioned media was immunoprecipitated with either anti-haemagglutinin (HA) antibody-sepharose (panel A and B), or anti-FLAG (M2) antibody-agarose (panel C and D). All samples were electrophoresed under reducing conditions except for samples in lanes 1 and 2 of panel C and D (*). Following electrophoretic transfer to PVDF, membranes were probed with anti-FLAG antibody (panel A) and anti-HA antibody (panel C). These membranes were subsequently stripped (incompletely) and reprobed with the antibody used for immunoprecipitation (panel B and D).
SDS-PAGE analysis of proteins purified from conditioned media by anti-FLAG affinity chromatography (panel A). The 27-29 kD band was transferred to PVDF and excised for NH2-terminal amino acid sequencing. The determined sequence of the 27-29 kD band is shown in panel B. This sequence is identical to mature human CLC.
Table 1 is a list of single and three letter abbreviations used throughout the specification.
Table 2 is a summary of amino acid and nucleotide sequence identifiers.
1The polyadenylation signal AATAAATAAA is at nucleotide position 1451 to 1460; NR6.1 (<400>1) and NR6.2 (<400>3) are identical to nucleotide 1223 encoding Q407, the represents the end of an exon. NR6.1 splices out an exon present only in NR6.2 and uses a different reading frame for the final exon which is
2The polyadenylation signal AATAAA is at nucleotide positions 1494 to 1503. The WSXWS motif is at amino acid residues 330 to 334. NR6.1 and NR6.2 are identical to nucleotide 1223 encoding Q407 which represents the end of an exon. NR6.2 splices in an exon beginning at amino acid residue D408, nucleotide 1224 and ends
3The polyadenylation signal AATAAA is at nucleotide positions 1494 to 1503. The WSXWS motif is at amino acid residues 330 to 334. NR6.1 and NR6.2 are identical to nucleotide 1223 encoding Q407 which represents the end of an exon. NR6.2 splices in an exon beginning at amino acid residue D408, nucleotide 1224 and ends
4The nucleotide sequence is identical to NR6.1, NR6.2 and NR6.3 from nucleotide C151, the first nucleotide for Pro51. The numbering from this nucleotide is the same as for <400>3 and <400>5. The 5 of this point is unique to the products generated by 5′ RACE not being found in NR6.1, Nr6.2 and NR6.3 and is represented in <400>16 and <400>17.
In work leading up to the present invention, the inventors identified an interaction between a haemopoietin receptor and a cytokine or cytokine-like molecule resulting in a biologically active complex.
The identification of this biologically active complex permits the rational design of agonist and antagonists of the interaction as well as interaction between the biologically active complex and other receptors, cytokines or cytokine-like molecules or other molecules. The biologically active complex itself may also be used in the development of therapeutics and diagnostics.
In one embodiment, the complex and/or its components have neurotrophic activity.
One aspect of the present invention is directed to a biologically active complex comprising at least two heterologous molecules which complex alone or in association with a receptor, ligand or other molecule facilitates proliferation, differentiation and/or survival of a cell.
Reference herein to “heterologous” means inter alia that two molecules differ in physical structure, biological activity or origin. For example, when the molecules are polypeptides, they are either encoded by different genes or represent, for example, splice variants of mRNA from the one gene. Preferably, the heterologous molecules differ at the amino acid level, after optimal alignment, by at least 5%.
Reference to a “complex” and in particular a “biologically active complex” means an association or other form of interaction between at least two molecules. The molecules may form a complex by a variety of mechanisms including covalent bonds, disulphide bridges, van der waals forces, ionic latticing, hydrogen bonds, amide bonds or physically being held together as a consequence of mutual folding.
In a particularly preferred embodiment, the at least two molecules in the complex are peptides, polypeptides or proteins.
Accordingly, another aspect of the present invention provides a biologically active complex comprising at least two polypeptides or parts, fragments, truncates or protease-activated forms of one or more of the polypeptides which complex alone or in association with a receptor, ligand or other molecule facilitates proliferation, differentiation and/or survival of a cell.
Preferably, the at least two polypeptides are heterologous with respect to each other meaning, as stated above, that both polypeptides are encoded by different genes or are, for example, splice variants from a single gene.
Parts and fragments of a polypeptide include peptides. A “protease-activated polypeptide” is a polypeptide where some portion has been enzymatically cleaved or processed. For example, an N-terminal or C-terminal amino acid sequence may be cleaved off by the protease. Reference herein to a “protease” means a protease in its most general sense and includes any enzyme which cleaves an amino acid chain at a defined location or at a defined sequence and includes a peptidase and a proteinase.
In a particularly preferred embodiment, at least one polypeptide in the complex is a soluble receptor and more particularly a soluble haemopoietin receptor. In a most preferred embodiment, the receptor is referred to as “NR6” which is described in International Patent Publication Number WO 98/11225.
Accordingly, another aspect of the present invention provides a biologically active complex comprising at least two polypeptides or parts, fragments, truncates or protease-activated forms of one or more of the polypeptides wherein at least one of said polypeptides is NR6 or a part, fragment, truncate or protease-activated form thereof and wherein said complex alone or in association with a receptor, ligand or other molecule facilitates proliferation, differentiation and/or survival of a cell.
Another polypeptide in the complex is preferably a cytokine or cytokine-like molecule.
According to this embodiment, the present invention contemplates a biologically active complex comprising at least two polypeptides or parts, fragments, truncates or protease-activated forms of the polypeptides wherein at least one polypeptide is NR6 and at least one other polypeptide is a cytokine or cytokine-like molecule or a part, fragment, truncate or protease-activated form of NR6 and/or the cytokine or cytokine-like molecule and wherein said complex alone or in association with a receptor, ligand or other molecule facilitates proliferation, differentiation and/or survival of a cell.
In a particularly preferred embodiment, at least one of the polypeptides is cardiotrophin-like cytokine (CLC). Accordingly, a particularly preferred biologically active complex comprises at least NR6 and CLC or parts, fragments, truncates or protease-activated forms is of NR6 and/or CLC.
The biologically active complex identified in accordance with the present invention may comprise two molecules and in particular two peptides or may comprise more than two molecules. Additional members of the complex include receptors, ligands, cytokines and cytokine-like molecules. For example, the complex may include gp130 or a cytokine and gp130.
Accordingly, another aspect of the present invention is directed to a biologically-active complex comprising the structure:
[X1]n (a) [X2]n1 (b) [X3]n2 . . . [Xd]n3
wherein
According to this embodiment, at least the NR6 and/or CLC may be in multiple form. Furthermore, X1, X2, X3 . . . Xd may be in any order. Preferred values for n and n1 are from about 1 to about 5.
In a particularly preferred embodiment, X3 is CNTFR, gp130, LIFR or other receptor molecule or cytokine-like molecule.
Accordingly, another aspect of the present invention provides a biologically active complex comprising the structure:
[X1]a3 [NR6]a [CLC]a1 [NR6]a2 [X1]a4
wherein
X1 is optionally present and is a cytokine or cytokine-like molecule;
Reference herein to NR6 and CLC means a molecule from any animal or avian species such as from humans, primates, laboratory test animals (e.g. mice, rats, rabbits, guinea pigs), companion animals (e.g. cats, dogs), captive wild animals, poultry birds, game birds, caged birds, reptiles or fish.
Preferably, the NR6 and/or CLC is of human, primate or murine origin.
Nucleotide sequences encoding NR6 are disclosed in WO 98/11225. The nucleotide sequence encoding CLC is represented in Genbank AR002595, AC005849 and AF172854 (see also U.S. Pat. No. 5,741,772 and International Patent Publication No. WO 99/00415).
The present invention preferably provides the biologically active complex in isolated form such that it has undergone at least one purification or co-precipitation step from culture medium or biological fluid. Reference herein to “biologically active” means that the complex has a direct effect on a cell or biochemical pathway or physiological process or has this effect after processing or interaction with receptor, ligand or other molecule.
The present invention further comprises genetic constructs comprising a first nucleotide sequence encoding one or other of NR6 or CLC or modified forms thereof, and a second nucleotide sequence encoding the other of NR6 or CLC. The genetic construct may also comprise other nucleotide sequences encoding further members of the complex. Preferably, each nucleotide sequence encoding NR6 and CLC is operably linked to the same or a separate promoter sequence. The genetic construct according to this aspect of the present invention is conveniently used to co-express nucleotide sequences encoding NR6 and CLC. Alternatively, separate genetic constructs each encoding one or other of NR6 and CLC are used to transfect a cell for co-expression.
Reference herein to “NR6” and “CLC” or other modified forms includes reference to parts, fragments and truncates thereof These terms also include various splice forms of NR6 and CLC.
The present invention further provides a genetic construct comprising a sequence of nucleotides substantially as set forth in <400>1 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>1 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
In a related embodiment, the present invention provides a genetic construct comprising a sequence of nucleotides substantially as set forth in <400>3 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>3 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
In another related embodiment, the present invention provides a genetic construct comprising a sequence of nucleotides substantially as set forth in <400>5 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>5 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
In a further related embodiment, the present invention provides a genetic construct comprising a sequence of nucleotides substantially as set forth in <400>7 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>7 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
In yet a further related embodiment, the present invention provides a genetic construct comprising a sequence of nucleotides substantially as set forth in <400>9 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>9 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
Still yet a further embodiment of the present invention is directed to a genetic construct substantially as set forth in <400>11 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>11 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
In still yet another embodiment, the present invention provides a genetic construct comprising a sequence of nucleotides substantially set forth in <400>12 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>12 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
Another embodiment of the present invention provides a genetic construct comprising a sequence of nucleotides substantially set forth in <400>13 or a nucleotide sequence having at least 60% similarity to the nucleotide sequence set forth in <400>13 or a nucleotide sequence capable of hybridizing thereto under low stringency conditions at 42° C. and a sequence of nucleotides encoding CLC or a modified form thereof.
Reference to CLC or a modified form includes a molecule having an amino acid sequence set forth in <400>20 or having an amino acid sequence of at least 60% similarity thereto or encoded by a nucleotide sequence substantially as set forth in <400>19 or having at least 60% similarity thereto or a sequence capable of hybridizing thereto under low stringency conditions at 42° C.
The term “receptor” is used in its broadest sense and includes any molecule capable of binding, associating or otherwise interacting with a ligand. Generally, the interaction will have a signaling effect although the present invention is not necessarily so limited. For example, the “receptor” may be in soluble form, often referred to as a cytokine binding protein. A receptor may be deemed a receptor notwithstanding that its ligand or ligands has or have not been identified.
Different forms of NR6 are referred to as, for example, NR6.1, NR6.2 and NR6.3. The nucleotide and corresponding amino acid sequences for these molecules are represented in <400>1, <400>3 and <400>5, respectively.
Preferred human and murine nucleic acid sequences for NR6 or its derivatives include sequences from brain, liver, kidney, neonatal, embryonic, cancer or tumour-derived tissues.
Reference herein to a low stringency at 42° C. includes and encompasses from at least about 0% v/v to at least about 15% v/v forrnamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions.
The term “similarity” as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, “similarity” includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. Where there is non-identity at the amino acid level, “similarity” includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particularly preferred embodiment, nucleotide and sequence comparisons are made at the level of identity rather than similarity.
Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include “reference sequence”, “comparison window”, “sequence similarity”, “sequence identity”, “percentage of sequence similarity”, “percentage of sequence identity”, “substantially similar” and “substantial identity”. A “reference sequence” is at least 12 but frequently 15 to 18 and often at least 25 or above, such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides, may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of the two polynucleotides over a “comparison window” to identify and compare local regions of sequence similarity. A “comparison window” refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerised implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Drive Madison, Wis., USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as for example disclosed by Altschul et al. (1997) (30). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al. (1998) (31).
The terms “sequence similarity” and “sequence identity” as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a “percentage of sequence identity”, for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, “sequence identity” will be understood to mean the “match percentage” calculated by the DNASIS computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, Calif., USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.
Reference herein to a low stringency includes and encompasses from at least about 0 to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is at from about 25-30° C. to about 42° C. The temperature may be altered and higher temperatures used to replace formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% vlv formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out Tm=69.3+0.41 (G+C)% (Marmur and Doty, 1962 (32). However, the Tm of a duplex DNA decreases by 1° C. with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, 1974 (33)). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows: low stringency is 6×SSC buffer, 0.1% w/v SDS at 25-42° C.; a moderate stringency is 2×SSC buffer, 0.1% w/v SDS at a temperature in the range 20° C. to 65° C.; high stringency is 0.1×SSC buffer, 0.1% w/v SDS at a temperature of at least 65° C.
The nucleic acid molecules contemplated by the present invention are generally in isolated form and are preferably cDNA or genomic DNA molecules. In a particularly preferred embodiment, the nucleic acid molecules are in vectors and most preferably expression vectors to enable expression in a suitable host cell. Particularly useful host cells include prokaryotic cells, mammalian cells, yeast cells and insect cells. The cells may also be in the form of a cell line.
Accordingly, another aspect of the present invention provides an expression vector comprising a nucleic acid molecule encoding NR6 and CLC or modified forms thereof said expression vector capable of expression in a selected host cell.
Preferred percentage similarities to the reference nucleotide sequences include at least about 70%, more preferably at least about 80%, still more preferably at least about 90% and even more preferably at least about 95% or above.
Another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides encoding NR6 or a derivative thereof having an amino acid sequence as set forth in <400>2 or having at least about 50% similarity to all or part thereof, said genetic construct further comprising a sequence of nucleotides encoding CLC or a modified form thereof.
Still yet another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides encoding NR6 or a derivative thereof having an amino acid sequence as set forth in <400>4 or having at least about 50% similarity to all or part thereof, said genetic construct further comprising a sequence of nucleotides encoding CLC or a modified form thereof.
Even yet another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides encoding NR6 or a derivative thereof having an amino acid sequence as set forth in <400>6 or having at least about 50% similarity to all or part thereof, said genetic construct further comprising a sequence of nucleotides encoding CLC or a modified form thereof.
A further aspect of the present invention provides a genetic construct comprising a sequence of nucleotides encoding NR6 or a derivative thereof having an amino acid sequence as set forth in <400>8 or having at least about 50% similarity to all or part thereof, said genetic construct further comprising a sequence of nucleotides encoding CLC or a modified form thereof.
Even yet a another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides encoding NR6 or a derivative thereof having an amino acid sequence as set forth in <400>14 or having at least about 50% similarity to all or part thereof, said genetic construct further comprising a sequence of nucleotides encoding CLC or a modified form thereof.
Another aspect of the present invention provides a genetic construct comprising a sequence of nucleotides encoding NR6 or a derivative thereof having an amino acid sequence as set forth in one or more of <400>15 or having at least about 50% similarity to all or part thereof, said genetic construct further comprising a sequence of nucleotides encoding CLC or a modified form thereof.
Preferably, the percentage amino acid similarity is at least about 60%, more preferably at least about 70%, even more preferably at least about 80-85% and still even more preferably at least about 90-95% or greater.
The biologically active complex of the present invention may be in soluble form or may be expressed on a cell surface or conjugated or fused to a solid support or another molecule.
As stated above, the present invention further contemplates a range of derivatives of members of the complex. Derivatives include fragments, parts, portions, mutants, homologues and analogues of the polypeptides and corresponding genetic sequences. Derivatives also include single or multiple amino acid substitutions, deletions and/or additions to polypeptides or single or multiple nucleotide substitutions, deletions and/or additions to the genetic sequence encoding the polypeptides. “Additions” to amino acid sequences or nucleotide sequences include fusions with other peptides, polypeptides or proteins or fusions to nucleotide sequences. Reference herein to “NR6” or “CLC” or other polypeptides includes reference to all derivatives thereof including functional derivatives or immunologically interactive derivatives.
Analogues of the polypeptides contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecule or their analogues.
Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH4.
The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.
The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.
Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.
Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-nitrotyrosine derivative.
Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.
Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy-5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid, contemplated herein is shown in Table 3.
These types of modifications may be important to stabilize the complex if administered to an individual or for use as a diagnostic reagent.
Crosslinkers can be used, for example, to stabilize 3D conformations, using homo-bifunctional crosslinkers such as the bifunctional imido esters having (CH2)n spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of Cα and Nα-methylamino acids, introduction of double bonds between Cα and Cβ atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.
The present invention further contemplates chemical analogues of the polypeptides in the complex capable of acting as antagonists or agonists of the biologically active complex or of polypeptides interacting with or within the complex or which can act as functional analogues of the complex. Chemical analogues may not necessarily be derived from polypeptides in the complex but may share certain conformational similarities. Alternatively, chemical analogues may be specifically designed to mimic certain physiochemical properties of one or more polypeptides in the complex. Chemical analogues may be chemically synthesized or may be detected following, for example, natural product screening.
Other derivatives contemplated by the present invention include a range of glycosylation variants from a completely unglycosylated molecule to a modified glycosylated molecule. Altered glycosylation patterns may result from expression of recombinant molecules in different host cells.
The identification of the complex permits the generation of a range of therapeutic molecules capable of modulating expression of polypeptides in the complex or modulating the activity of the complex. Modulators contemplated by the present invention includes agonists and antagonists of polypeptide expression. Antagonists of polypeptide expression include antisense molecules, ribozymes and co-suppression molecules. Agonists include molecules which increase promoter ability or interfere with negative regulatory mechanisms. Agonists of polypeptide include molecules which overcome any negative regulatory mechanism. Antagonists of the polypeptide include antibodies and inhibitor peptide fragments.
Another aspect of the present invention contemplates a method of modulating activity of the complex as hereinbefore described, said method comprising administering to a subject a modulating effective amount of a molecule for a time and under conditions sufficient to increase or decrease the biological activity of the complex. The molecule may be a proteinaceous molecule or a chemical entity and may also be a derivative of a polypeptide of the complex or its ligand or a chemical analogue or truncation mutant of a polypeptide of the complex or its ligand. The complex and its components such as but not limited to CLC, NR6 or a CLC-NR6 complex, is proposed, in one embodiment, to possess neurotrophic activity.
The present invention, therefore, contemplates a pharmaceutical composition comprising the complex or a modulator of complex activity and one or more pharmaceutically acceptable carriers and/or diluents. These components are referred to as the “active ingredients”.
The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dilution medium comprising, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of superfactants. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.
When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 1% by weight of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 ug and 2000 mg of active compound. Alternative dosage amounts include from about 1 μg to about 1000 mg and from about 10 μg to about 500 mg.
The present invention also extends to forms suitable for topical application such as creams, lotions and gels as well as a range of “paints” which are applied to skin and through which the active ingredients are absorbed. In addition, the complex or components thereof may be associated with penetration or the TAT protein of HIV.
Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art and except insofar as any conventional media or agent is incompatible with the active ingredient, their use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired as herein disclosed in detail.
The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.5 μg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.5 μg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.
Dosages may also be expressed per body weight of the recipient. For example, from about 10 ng to about 1000 mg/kg body weight, from about 100 ng to about 500 mg/kg body weight and for about 1 μg to above 250 mg/kg body weight may be administered.
The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating levels of polypeptides involved the complex. The vector may, for example, be a viral vector.
Still another aspect of the present invention is directed to antibodies to the complex and its derivatives. Such antibodies may be monoclonal or polyclonal and may be selected from naturally occurring antibodies to the complex or may be specifically raised to the complex or derivatives thereof. In the case of the latter, the complex or its derivatives may first need to be associated with a carrier molecule. The antibodies and/or recombinant complex or its derivatives of the present invention are particularly useful as therapeutic or diagnostic agents. For example, complex antibodies or antibodies to its ligand may act as antagonists.
For example, the complex and its derivatives can be used to screen for naturally occurring antibodies to the complex. These may occur, for example in some autoimmune diseases. Alternatively, specific antibodies can be used to screen for the complex. Techniques for such assays are well known in the art and include, for example, sandwich assays and ELISA. Knowledge of complex levels may be important for diagnosis of certain disease conditions or a predisposition for a disease condition to occur or for monitoring certain therapeutic protocols.
Fragments of antibodies may be used such as Fab fragments. Furthermore, the present invention extends to recombinant and synthetic antibodies and to antibody hybrids. A “synthetic antibody” is considered herein to include fragments and hybrids of antibodies. The antibodies of this aspect of the present invention are particularly useful for immunotherapy and may also be used as a diagnostic tool for assessing apoptosis or monitoring the program of a therapeutic regimen.
For example, specific antibodies can be used to screen for the complex. The latter would be important, for example, as a means for screening for levels of the complex in a cell extract or other biological fluid or purifying the complex made by recombinant means from culture supernatant fluid. Techniques for the assays contemplated herein are known in the art and include, for example, sandwich assays and ELISA.
It is within the scope of this invention to include any second antibodies (monoclonal, polyclonal or fragments of antibodies or synthetic antibodies) directed to the first mentioned antibodies discussed above. Both the first and second antibodies may be used in detection assays or a first antibody may be used with a commercially available anti-immunoglobulin antibody. An antibody as contemplated herein includes any antibody specific to any region of the complex.
Both polyclonal and monoclonal antibodies are obtainable by immunization with the enzyme or protein and either type is utilizable for immunoassays. The methods of obtaining both types of sera are well known in the art. Polyclonal sera are less preferred but are relatively easily prepared by injection of a suitable laboratory animal with an effective amount of NR6, or antigenic parts thereof, collecting serum from the animal, and isolating specific sera by any of the known immunoadsorbent techniques. Although antibodies produced by this method are utilizable in virtually any type of immunoassay, they are generally less favoured because of the potential heterogeneity of the product.
The use of monoclonal antibodies in an immunoassay is particularly preferred because of the ability to produce them in large quantities and the homogeneity of the product. The preparation of hybridoma cell lines for monoclonal antibody production derived by fusing an immortal cell line and lymphocytes sensitized against the immunogenic preparation can be done by techniques which are well known to those who are skilled in the art.
Another aspect of the present invention contemplates a method for detecting the complex as hereinbefore described in a biological sample from a subject said method comprising contacting said biological sample with an antibody specific for the complex (or a component thereof) or its derivatives or homologues for a time and under conditions sufficient for an antibody complex to form, and then detecting said antibody.
The presence of the complex may be accomplished in a number of ways such as by Western blotting and ELISA procedures. A wide range of immunoassay techniques are available as can be seen by reference to U.S. Pat. Nos. 4,016,043, 4,424,279 and 4,018,653. These, of course, includes both single-site and two-site or “sandwich” assays of the non-competitive types, as well as in the traditional competitive binding assays. These assays also include direct binding of a labelled antibody to a target.
Sandwich assays are among the most useful and commonly used assays and are favoured for use in the present invention. A number of variations of the sandwich assay technique exist, and all are intended to be encompassed by the present invention. Briefly, in a typical forward assay, an unlabelled antibody is immobilized on a solid substrate and the sample to be tested brought into contact with the bound molecule. After a suitable period of incubation, for a period of time sufficient to allow formation of an antibody-antigen complex, a second antibody specific to the antigen, labelled with a reporter molecule capable of producing a detectable signal is then added and incubated, allowing time sufficient for the formation of another complex of antibody-antigen-labelled antibody. Any unreacted material is washed away, and the presence of the antigen is determined by observation of a signal produced by the reporter molecule. The results may either be qualitative, by simple observation of the visible signal, or may be quantitated by comparing with a control sample containing known amounts of hapten. Variations on the forward assay include a simultaneous assay, in which both sample and labelled antibody are added simultaneously to the bound antibody. These techniques are well known to those skilled in the art, including any minor variations as will be readily apparent. In accordance with the present invention, the sample is one which might contain the complex including cell extract, tissue biopsy or possibly serum, saliva, mucosal secretions, lymph, tissue fluid and respiratory fluid. The sample is, therefore, generally a biological sample comprising biological fluid but also extends to fermentation fluid and supernatant fluid such as from a cell culture.
In the typical forward sandwich assay, a first antibody having specificity for the NR6 or antigenic parts thereof, is either covalently or passively bound to a solid surface. The solid surface is typically glass or a polymer, the most commonly used polymers being cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene. The solid supports may be in the form of tubes, beads, discs of microplates, or any other surface suitable for conducting an immunoassay. The binding processes are well-known in the art and generally consist of cross-linking covalently binding or physically adsorbing, the polymer-antibody complex is washed in preparation for the test sample. An aliquot of the sample to be tested is then added to the solid phase complex and incubated for a period of time sufficient (e.g. 2-40 minutes or overnight if more convenient) and under suitable conditions (e.g. from about room temperature to about 37° C.) to allow binding of any subunit present in the antibody. Following the incubation period, the antibody subunit solid phase is washed and dried and incubated with a second antibody specific for a portion of the hapten. The second antibody is linked to a reporter molecule which is used to indicate the binding of the second antibody to the hapten.
An alternative method involves immobilizing the target molecules in the biological sample and then exposing the immobilized target to specific antibody which may or may not be labelled with a reporter molecule. Depending on the amount of target and the strength of the reporter molecule signal, a bound target may be detectable by direct labelling with the antibody. Alternatively, a second labelled antibody, specific to the first antibody is exposed to the target-first antibody complex to form a target-first antibody-second antibody tertiary complex. The complex is detected by the signal emitted by the reporter molecule.
In another alternative method, a ligand or receptor of the complex is immobilized to a solid support and a biological sample containing the complex brought into contact with its immobilised ligand. Binding between the complex and its ligand or receptor can then be determined using an antibody to the complex or a component thereof which itself may be labelled with a reporter molecule or a further anti-immunoglobulin antibody labelled with a reporter molecule could be used to detect antibody bound to the complex.
By “reporter molecule” as used in the present specification, is meant a molecule which, by its chemical nature, provides an analytically identifiable signal which allows the detection of antigen-bound antibody. Detection may be either qualitative or quantitative. The most commonly used reporter molecules in this type of assay are either enzymes, fluorophores or radionuclide containing molecules (i.e. radioisotopes) and chemiluminescent molecules.
In the case of an enzyme immunoassay, an enzyme is conjugated to the second antibody, generally by means of glutaraldehyde or periodate. As will be readily recognized, however, a wide variety of different conjugation techniques exist, which are readily available to the skilled artisan. Commonly used enzymes include horseradish peroxidase, glucose oxidase, beta-galactosidase and alkaline phosphatase, amongst others. The substrates to be used with the specific enzymes are generally chosen for the production, upon hydrolysis by the corresponding enzyme, of a detectable colour change. Examples of suitable enzymes include alkaline phosphatase and peroxidase. It is also possible to employ fluorogenic substrates, which yield a fluorescent product rather than the chromogenic substrates noted above. In all cases, the enzyme-labelled antibody is added to the first antibody hapten complex, allowed to bind, and then the excess reagent is washed away. A solution containing the appropriate substrate is then added to the complex of antibody-antigen-antibody. The substrate will react with the enzyme linked to the second antibody, giving a qualitative visual signal, which may be further quantitated, usually spectrophotometrically, to give an indication of the amount of hapten which was present in the sample. “Reporter molecule” also extends to use of cell agglutination or inhibition of agglutination such as red blood cells on latex beads, and the like.
Alternately, fluorescent compounds, such as fluorescein and rhodamine, may be chemically coupled to antibodies without altering their binding capacity. When activated by illumination with light of a particular wavelength, the fluorochrome-labeled antibody adsorbs the light energy, inducing a state to excitability in the molecule, followed by emission of the light at a characteristic colour visually detectable with a light microscope. As in the EIA, the fluorescent labelled antibody is allowed to bind to the first antibody-hapten complex. After washing off the unbound reagent, the remaining tertiary complex is then exposed to the light of the appropriate wavelength the fluorescence observed indicates the presence of the hapten of interest. Immunofluorescene and EIA techniques are both very well established in the art and are particularly preferred for the present method. However, other reporter molecules, such as radioisotope, chemiluminescent or bioluminescent molecules, may also be employed.
The present invention further contemplates a method for identifying agonists and antagonists of the biologically active complex as herein defined for use in therapy.
Accordingly, another aspect of the invention contemplates a method of identifying an agent capable of modulating the effects of a biologically active complex as herein defined, said method comprising screening for agents which are capable of interacting with the complex or interfering or otherwise antagonizing or promoting or otherwise agonizing interaction between the heterologous molecules of said complex.
The agent capable of agonizing or antagonizing interaction of the biologically active complex or between the heterologous molecules within the biologically active complex may be a proteinaceous or non-proteinaceous molecule. A proteinaceous molecule includes a peptide, polypeptide or protein or a complex thereof with, for example, a lipid, phospholipid or carbohydrate. A non-proteinaceous molecule includes a range of chemical entities including aromatic and/or pentanoid containing structures. Conveniently, the agent is identified following natural product screening of members of the biosphere such as but not limited to coral, plants and plant parts including bark, roots, flowers, leaves and stems, river beds, sea beds, micro-organisms, insects, soil and rock deposits as well as arctic, antarctic and even extraterrestrial (e.g. lunar) environments. The agent may also be identified following a screening of chemical libraries or using combinatorial chemical approaches.
Any number of screening procedures may be adopted to identify the agonists and antagonist. In one example, one or both heterologous molecules within the complex is/are linked to a reporter molecule such that upon interaction wither another molecule or a receptor or ligand, the reporter molecule provides an identifiable signal. An “identifiable signal” may be presence of a signal or absence of a signal. The amount or extent of signaling is then measured, quantitatively or qualitatively in the presence of potential agonists and/or antagonists. Any number of variations may be adopted to screen for agonists and antagonists. Variations of two hybrid screening and phage labelling may also be employed.
Once identified, the agonists and antagonists may be incorporated into a composition such as a pharmaceutical composition which comprises the agonist/antagonist and one or more pharmaceutically acceptable carriers and/or diluents. Alternatively, the agonist/antagonist may be provided genetically such as in the form of a DNA or RNA composition or the agonist/antagonist may comprise antisense, sense or riboyzme molecules.
A further aspect of the present invention contemplates the use of the biologically active complex or its functional derivatives in the manufacture of a medicament for the treatment of conditions resulting from aberrations in the complex or in reduced or excessive amounts of the complex.
Still a further aspect of the present invention contemplates a ligand or receptor for the complex such as in isolated or recombinant form, or a derivative of said ligand or receptor.
The present invention further contemplates knockout animals such as mice or other murine species for components of the complex gene including homozygous and heterozygous knockout animals. Such animals provide a particularly useful live in vivo model for studying the effects of the complex as well as screening for agents capable of acting as agonists or antagonists of the complex.
According to this embodiment there is provided a transgenic animal comprising a mutation in at least one allele of the gene encoding a component of the complex. Additionally, the present invention provides a transgenic animal comprising a mutation in two alleles of the gene encoding a component of the complex. Preferably, the transgenic animal is a murine animal such as a mouse or rat.
The present invention is further described by the following non-limiting Examples.
Standard methods for DNA cloning and protein expression are set forth in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. [1989]) and in Ausubel et al. Eds. (Current Protocols in Molecular Biology, Wiley, New York, N.Y. [1995]).
Plasmid vectors for the expression of untagged and NH2-terminal haemagglutinin (HA) tagged CLC were prepared using PCR based approaches.
For untagged CLC a human CLC cDNA was cloned by PCR amplification from human fetal lung marathon-ready cDNA library (Clontech #7433-1). Based on the published sequence (GenBank AR002595 and AC005849), a sense primer with an EcoRI site at the 5′ end: (5′-CGAATTCCCCATGGACCTCCGAGCAG-3′) (<400>21) and an anti-sense primer with a BamHI site at the 5′ end: (5′-GGGATCCTTTGAAGGGGGAGCGAAGAG-3′) (<400>22) were synthesized and used in PCR to amplify the cDNA. After digestion with EcoRI and BamHI, the amplified fragment was ligated into the vector pCOS-1 (WO 98/11225). pCOS-1 is a mammalian expression vector with a G418-resistance marker.
Splice-overlap-extension PCR (SOE-PCR) was used to generate a CLC cDNA incorporating two modifications, a HA epitope tag (YPYDVPDYAS [<400>23]) and an IL-3 signal sequence (MVLASSTTSIHTMLLLLLMLFHLGLQASIS [<400>24]) directly NH2-terminal of the coding region of mature CLC (Leu28). Fragment A was amplified using a sense primer (5′-CCATTTCAGGTGTCGTGAGG-3′) (<400>25) and an anti-sense primer (5′-GTAGTCGGGCACGTCATAAGG ATACGAGATTGAAGCTTGGAG TCC-3′) (<400>26) with the plasmid pEFBOS-S-FLAG (29) that contains the murine IL-3 signal sequence as a template. Fragment B was amplified using a sense primer (5′-CCTTATGACGTGCCCGACTACGCCAGTCTCAATCGCACAGGGGACCCA-3′ (<400>27) and an anti-sense primer with human CLC cDNA as a template. After mixing fragments A and B, the SOE-PCR product was amplified using the end primers. Following digestion with EcoRI and BamHI, the amplified fragment was ligated into the expression vectors pCOS-1 and pCHO-1 (WO 98/11225). pCHO-1 is a mammalian expression vector incorporating a dhfr (dihydrofolate reductase) gene, selectable in dhfr-deficient CHO cells.
CHO cell lines expressing CLC alone, NR6 alone or co-expressing CLC and NR6 were established. Using a mammalian cell transfection kit (Stratagene Cat. No. 200285) dhfr-deficient CHO cells were transfected with:
The transfected cells were selected in medium (GIBCO Alpha-MEM medium without nucleic acids, with 10% v/v FCS) in the presence of 0.4 mg/ml G418.
For each immunoprecipitation, 1 ml of media conditioned by the indicated transfected cell line was used. Tagged proteins were precipitated using 0.05 ml of a suspension of either anti-FLAG M2-agarose (Sigma Cat. No. A1205) or anti-HA sepharose (BAbCo; Richmond, Calif., #AFC-101P). Immunoprecipitates were electrophoresed on SDS-PAGE, transferred to PVDF membrane and probed with an anti-HA antibody (Boehringer-Mannheim Cat. No. 1 666 851) or an anti-FLAG BioM2 antibody (Sigma Cat. No. F9291). Bound antibody was detected using an ECL detection system (Amersham-Pharmacia Biotech Cat. No. RPN2209). After stripping the bound antibodies, the membranes were re-probed with the same antibodies as used for the immunoprecipitation.
FLAG-tagged NR6 was co-immunoprecipitated with the HA-tagged CLC (
The pCHO-1 expression vector encoding FLAG-tagged NR6 and the pCOS-1 expression vector encoding CLC with the native signal sequence were transfected simultaneously into dhfr-deficient CHO cells using a mammalian cell transfection kit (Stratagene #200285). The introduced genes were amplified by addition of methotrexate (MTX) and by step-wise increase of the concentration of MTX and G418. The established cell line was cultured in GIBCO IMDM medium with 1% v/v FCS without MTX/G418 and the conditioned medium was collected on day 3. The recombinant secreted protein was purified by anti-FLAG M2-agarose (Sigma Cat. No. A1205) affinity chromatography according to the manufacturer's protocol. SDS-PAGE analysis identified a 27-29 kDa protein that co-purified with FLAG-tagged NR6 (55-60 kDa;
FLAG tagged NR6-CLC expressed in stable transfected CHO cells (see Example 3) was purified using anti-FLAG M2 affinity chromatography. The FLAG-NR6-CLC was displaced from the M2 column using 100 μg/ml FLAG peptide in 1% w/v ammonium bicarbonate. Material purified by M2 affinity was then subjected to size exclusion chromatography using a Pharmacia Superose 12 10/30 column at a flow rate of 1 ml/min on a Pharmacia Smart system. The elution profile (OD 215 nm) and non-reducing SDS-PAGE analysis of fractions collected at 0.5 minute intervals is shown in
The inventors sought to determine whether CLC co-secreted with NR6 from mammalian cells displayed neurotrophic activity. Two pools of material (
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.
This application is a continuation of U.S. application Ser. No. 11/110,172, filed Aug. 26, 2002 as a 371 application based on PCT/AU00/01216 having an international filing date of Oct. 6, 2000.
| Number | Date | Country | |
|---|---|---|---|
| Parent | 11110172 | Apr 2005 | US |
| Child | 11704086 | Feb 2007 | US |
