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  • C12N9/10—Transferases (2.)
  • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
  • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
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  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
  • C07K14/723—G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor
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  • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
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  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
  • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
  • C12N9/6424—Serine endopeptidases (3.4.21)
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  • C12Y304/21—Serine endopeptidases (3.4.21)
  • C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin
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  • C07K2319/00—Fusion polypeptide
  • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
  • C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
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  • C07K2319/74—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor
  • C07K2319/75—Fusion polypeptide containing domain for protein-protein interaction containing a fusion for binding to a cell surface receptor containing a fusion for activation of a cell surface receptor, e.g. thrombopoeitin, NPY and other peptide hormones

Definitions

• Polypeptide therapeutic agents despite their promise in a number of disease treatments, are readily decomposed by gastric juices and by intestinal proteinases such as pepsin and trypsin. As a result, when these polypeptides are orally administered, they are barely absorbed and produce no effective pharmacological action. In order to obtain the desired biological activity, the polypeptides are at present usually dispensed in injectable dosage forms. However, the injectable route is inconvenient and painful to the patient, particularly when administration must occur on a regular and frequent basis. Consequently, efforts have focused recently on alternative methods for administration of such polypeptides.
• Such agents usually exhibit a short half-life in the circulation, being rapidly excreted through the kidneys or taken up by the reticuloendothelial system (RES) and other tissues.
• RES reticuloendothelial system
• larger doses are required so that sufficient amounts of drug can concentrate in areas in need of treatment.
• Sustained- release formulations (Putney, S.D. et al. Nature Biotechnology 1998, 16, 153-157) generally reduce the necessary dosage, but still depend on injection or more objectionable forms of delivery.
• a therapeutic protein with a longer half-life in the body would maintain a more stable blood level in much the same way as a sustained-release formulation, but would not entail the difficulties of preparing a sustained-release formulation and would require an even lower dosage because it is destroyed less quickly.
• cytokines such as interferon (IFN-gamma) and interleukin-2 (IL-2) would be more effective, less toxic and could be used in smaller quantities, if their presence in the circulation could be extended.
• One aspect of the present invention provides a chimeric polypeptide comprising a biologically active heterologous peptide fragment inserted into a serum albumin protein or a homolog thereof.
• the heterologous peptide fragment may optionally replace a portion of the serum albumin protein sequence.
• a peptide fragment which replaces a portion of the serum albumin protein sequence need not be of the same length as the fragment it replaces.
• a chimeric polypeptide according to this aspect may include more than one heterologous peptide fragment which replaces a portion of the serum albumin protein sequence.
• the included fragments may be identical, may be distinct sequences from a protein unrelated to serum albumin protein, or may be distinct sequences of unrelated origin.
• a chimeric polypeptide of this aspect may comprise the structure A-B- C, wherein A represents a first fragment of a serum albumin protein or homolog thereof, B represents a biologically active heterologous peptide sequence, and C represents another fragment of a serum albumin protein or a homolog thereof.
• a chimeric polypeptide may comprise the structure A-B-C-D-E, wherein A, C, and E represent fragments of a serum albumin protein and B and D represent identical biologically active heterologous peptide sequences, two different biologically active sequences of a protein unrelated to serum albumin protein, or two different biologically active sequences of two different proteins unrelated to serum albumin protein.
• a chimeric polypeptide may comprise the structure A-B-C-D-E-F-G, wherein A, C, E, and G represent fragments of a serum albumin protein and B, D, and F represent identical biologically active heterologous peptide sequences, at least two different biologically active sequences of a protein unrelated to serum albumin protein, or at least two different biologically active sequences of two different proteins unrelated to serum albumin protein.
• a peptide fragment of serum albumin or a heterologous peptide sequence includes at least 6 amino acids, at least 12 amino acids, or at least 18 amino acids.
• a chimeric polypeptide may comprise the structure (A-B-C) n , e.g., -HN-(A-B-C) n - CO- or H 2 N-(A-B-C) n -CO 2 H, wherein A, independently for each occurrence, represents a fragment of serum albumin (S A), B, independently for each occurrence, represents a biologically active heterologous peptide sequence, C, independently for each occurrence, represents a second biologically active heterologous peptide sequence or a fragment of serum albumin (SA), and n is an integer greater than 0.
• a peptide fragment of serum albumin or a heterologous peptide sequence includes at least 6 amino acids, at least 12 amino acids, or at least 18 amino acids.
• such a chimeric polypeptide may comprise an N-terminal fragment of a serum albumin protein or a homolog thereof, a biologically active heterologous peptide sequence, and a C-terminal fragment of a serum albumin protein or a homolog thereof.
• the heterologous peptide sequence may be between about 3 and about 500 or between about 4 and about 400 residues in length, preferably between about 4 and about 200 residues, more preferably between about 4 and 100 residues, and most preferably between about 4 and about 20 residues.
• the chimeric polypeptide has a half-life in the blood no less than 10 days, preferably no less than about 14 days, and most preferably no less than 50% of the half-life of the native serum albumin protein or homolog thereof.
• the heterologous peptide sequence is capable of binding to a cell surface receptor protein.
• a receptor protein include a G protein-coupled receptor, a tyrosine kinase receptor, a cytokine receptor, an MIRR receptor, and an orphan receptor.
• the chimeric polypeptide is capable of binding to an extracellular receptor or ion channel.
• the chimeric polypeptide may be an agonist or an antagonist of an extracellular receptor or ion channel.
• the chimeric polypeptide of this embodiment may, for example, induce apoptosis, modulate cell proliferation, or modulate differentiation of cell types.
• the invention also comprises a nucleic acid sequence which encodes a chimeric polypeptide as described above.
• the invention further comprises a delivery vector, such as a viral or retroviral vector comprising a nucleic acid sequence encoding the chimeric polypeptide.
• a delivery vector such as a viral or retroviral vector comprising a nucleic acid sequence encoding the chimeric polypeptide.
• Suitable vectors may include, for example, an adenovirus, an adeno-associated virus, a herpes simplex virus, a human immunodeficiency viruses, or a vaccinia virus.
• the invention also comprises a pharmaceutical composition comprising a chimeric polypeptide as described above, and methods for treating a disease in an organism by administering an effective dose of such a pharmaceutical composition to the organism.
• a chimeric polypeptide according to the invention comprises a fragment of an angiogenesis-inhibiting protein, such as angiostatin or endostatin, as the heterologous peptide sequence and is capable of inhibiting angiogenesis.
• angiogenesis-inhibiting protein such as angiostatin or endostatin
• a peptide fragment that inhibits angiogenesis and which may be incorporated into a subject polypeptide is RGD (Arg-Gly-Asp), or a sequence which includes the sequence RGD (e.g., VRGDF).
• Analogous methods may be used to modulate conditions such as cell proliferation, cell differentiation, and cell death.
• the present invention provides a method of treating a disease in an organism by introducing into cells of the organism genetic material encoding a chimeric polypeptide protein comprising serum albumin protein or segments thereof and one or more therapeutic proteins or polypeptides or fragments thereof, such that the introduced genetic material is expressed by the transfected cells of the organism.
• Analogous methods may be used to modulate conditions such as cell proliferation, cell differentiation, and cell death.
• the present invention provides a method for treating a disease in an organism by introducing genetic material encoding a chimeric polypeptide comprising serum albumin protein or segments thereof and one or more therapeutic proteins or polypeptides or fragments thereof into target cells ex vivo under conditions sufficient to cause the genetic material to be incorporated into the cell, thereby causing the cell to express the genetic material encoding said proteins or polypeptides.
• the target cells are then introduced into the host organism such that the introduced genetic material encoding said proteins or polypeptides is expressed by the target cells in the organism.
• the target cells may be selected from the group consisting of blood cells, skeletal muscle cells, smooth muscle cells, stem cells, skin cells, liver cells, secretory gland cells, hematopoietic cells, and marrow cells.
• transfected cells comprising target cells which have been exposed to a delivery vector comprising a nucleic acid encoding the chimeric protein or polypeptide of this invention.
• These cells are preferably selected from the group consisting of blood cells, skeletal muscle cells, smooth muscle cells, stem cells, skin cells, liver cells, secretory gland cells, hematopoietic cells, and marrow cells.
• FIG 1 shows the tertiary structure of human serum albumin (HSA).
• FIG. 2 illustrates the transfection of cells with mouse serum albumin (MSA)-Myc fusion constructs and successful expression of the fusion protein, as well as binding of MSA and Myc antibodies to MSA-Myc fusion proteins depending on the location of the heterologous sequence in the MSA protein.
• Figure 3 depicts inhibition of FGF-induced proliferation of bovine capillary endothelial cells by RGD peptide and by MSA-myc-RGD fusion proteins.
• SA serum albumin
• SA biologically active heterologous peptide sequences.
• SA is the major protein constituent of the circulatory system, has a half-life in the blood of about three weeks (Rothschild, M.A. et al. Hepatology 1988, 8, 385-401), and is present in quantity (40 g/L in the serum). It is also known that the normal adult human liver produces approximately 15 grams of human serum albumin (HSA) per day, or about 200 mg per kilogram of body weight.
• HSA human serum albumin
• Serum albumin has no immunological activity or enzymatic function, and is a natural carrier protein used to transport many natural and therapeutic molecules.
• Fusion proteins wherein a therapeutic polypeptide has been covalently linked to serum albumin have been shown to have serum half-lives many times longer than the half-life of the therapeutic peptide itself (Syed, S. et al. Blood 1997, 89, 3243-3252; Yeh, P. et al. Proc. Natl. Acad. Sci. USA 1992, 89, 1904-1908).
• the half-life of the fusion protein was more than 140 times greater than that of the therapeutic polypeptide itself, and approached the half-life of unfused serum albumin.
• the amino- terminal portion of serum albumin has been found to favor particularly efficient translocation and export of the fusion proteins in eukaryotic cells (PCT publication WO 90/13653). Generally, this means that such proteins are more efficiently secreted by a cell manufacturing such proteins than are the free therapeutic polypeptides themselves.
• chimeric polypeptides of serum albumin proteins offer substantial promise because serum albumins are found in tissues and secretions throughout the body. It is known, for example, that serum albumin is responsible for the transport of compounds across organ-circulatory interfaces into such organs as the liver, intestine, kidney, and brain. Chimeric proteins of serum albumin may thus manifest their biological activity anywhere in the body, crossing even the daunting blood-brain barrier.
• a chimeric polypeptide of the present invention may include a biologically active heterologous peptide sequence inserted into the peptide sequence of a serum albumin protein.
• the inserted sequence may optionally replace a portion of the serum albumin sequence, whether that portion is of similar or dissimilar length. In some cases, more than one insertion may be required to obtain the desired biological activity.
• a biologically active heterologous peptide sequence may be placed between two fragments of a serum albumin sequence to create such a chimeric polypeptide.
• one or more additional biologically active peptide sequences may be placed between fragments of serum albumin protein.
• Chimeric polypeptides of the present invention may also be described as a biologically active heterologous peptide sequence flanked on one side by an N-terminal fragment of serum albumin protein and on the other side by a C- terminal fragment of serum albumin protein.
• chimeric polypeptides The advantage of such chimeric polypeptides is that the similarity to serum albumin protein in structure may camouflage these polypeptides to biological mechanisms which degrade foreign peptides even more effectively than known fusion proteins, because the foreign polypeptide fragments are carried on a protein that is substantially similar to a protein that is pervasive within the organism.
• Such proteins may retain the beneficial characteristics of serum albumin (non-immunogenicity, high level of expression, efficient secretion, and long half-life), while supporting the additional desired biological function.
• chimeric polypeptides include inclusion of a peptide fragment which inhibits cell proliferation might serve as a treatment for cancer and other diseases characterized by cell proliferation known to those in the art.
• inclusion of a peptide fragment which modulates the differentiation of immature cells into particular cell types may create a chimeric polypeptide which may be effective in the treatment of neurological conditions, e.g., nerve damage and neurodegenerative diseases, hyperplastic and neoplastic disorders of pancreatic tissue, and other conditions characterized by undesirable proliferation and differentiation of tissue.
• Inclusion of a peptide fragment which induces apoptosis may provide a polypeptide effective in treating diseases marked by unwanted cell proliferation, such as cancer, and other conditions known to those in the art as amenable to apoptotic therapy.
• Inclusion of an anti- angiogenic peptide fragment e.g., a fragment of angiostatin or endostatin, may yield a chimeric polypeptide useful in the treatment of cancer and other conditions resulting from or enabled by angiogenesis.
• 'peptide' refers to an oligomer in which the monomers are amino acids (usually alpha-amino acids) joined together through amide bonds. Peptides are two or more amino acid monomers long, but more often are between 5 to 10 amino acid monomers long and can be even longer, i.e., up to 20 amino acids or more, although peptides longer than 20 amino acids are more likely to be called 'polypeptides'.
• the term 'protein' is well known in the art and usually refers to a very large polypeptide, or set of associated homologous or heterologous polypeptides, that has some biological function. For purposes of the present invention the terms 'peptide', 'polypeptide', and 'protein' are largely interchangeable as all three types are collectively referred to as peptides.
• 'fusion' and 'chimeric' relate to polypeptides or proteins wherein two individual polypeptides or portions thereof are fused to form a single amino acid chain. Such fusion may arise from the expression of a single continuous coding sequence formed by recombinant DNA techniques.
• 'fusion' polypeptides and 'chimeric' polypeptides include contiguous polypeptides comprising a first polypeptide covalently linked via an amide bond to one or more amino acid sequences which define polypeptide domains that are foreign to and not substantially homologous with any domain of the first polypeptide.
• Gene constructs encoding fusion proteins are likewise referred to a 'chimeric genes' or 'fusion genes'.
• ⁇ omology' and 'identity' each refer to sequence similarity between two polypeptide sequences, with identity being a more strict comparison.
• Homology and identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When a position in the compared sequence is occupied by the same amino acid residue, then the polypeptides can be referred to as identical at that position; when the equivalent site is occupied by the same amino acid (e.g., identical) or a similar amino acid (e.g., similar in steric and/or electronic nature), then the molecules can be referred to as homologous at that position.
• a percentage of homology or identity between sequences is a function of the number of matching or homologous positions shared by the sequences.
• a 'heterologous peptide sequence' is a peptide sequence substantially dissimilar to a sequence to which it is compared.
• SA serum albumin'
• HSA human serum albumin
• the term 'serum albumin' is intended to include (but not necessarily to be restricted to) serum albumin proteins of living organisms, preferably mammalian serum albumins, even more preferably known or yet-to-be-discovered polymorphic forms of human serum albumin (HSA), and variants thereof.
• HSA human serum albumin
• the human serum albumin Naskapi has Lys-372 in place of Glu-372
• albumin Victoria has an altered pro-sequence.
• the term 'variants' is intended to include (but not necessarily be restricted to) homologs of SA proteins with minor artificial variations in sequence (such as molecules lacking one or a few residues, having conservative substitutions or minor insertions of residues, or having minor variations of amino acid structure).
• polypeptides which have 80%, 85%, 90%, or 99% homology with a native SA are deemed to be 'variants'. It is also preferred for such variants to share at least one pharmacological utility with a native SA. Any putative variant which is to be used pharmacologically should be non-immunogenic in the animal (especially human) being treated. Sequences of a number of contemplated serum albumin proteins can be obtained from GenBank (National Center for Biotechnology Information), including human, bovine, mouse, pig, horse, sheep, and chick serum albumins.
• 'native' is used to describe a protein which occurs naturally in a living organism. Wild-type proteins are thus native proteins. Proteins which are non-native are those which have been generated by artificial mutation, recombinant design, or other laboratory modification and are not known in natural populations.
• substitutions' are those where one or more amino acids are substituted for others having similar properties such that one skilled in the art of polypeptide chemistry would expect at least the secondary structure, and preferably the tertiary structure, of the polypeptide to be substantially unchanged.
• typical such substitutions include asparagine for glutamine, serine for asparagine, and arginine for lysine.
• the term 'physiologically functional equivalents' also encompasses larger molecules comprising the native sequence plus a further sequence at the N-terminus (for example, pro-HSA, pre-pre-HSA, and met-HSA).
• 'Tertiary structure' refers to the three-dimensional structure of a protein. Proteins which have similar tertiary structures will have similar shapes and surfaces, even if the amino acid sequences (the 'secondary structure') is not identical. Tertiary structure is a consequence of the folding and twisting of an amino acid chain upon itself and can be disrupted by chemical means, e.g., strong acid or base, or by physical means, e.g., heating.
• biologically active refers to an entity which interacts in some way with a living organism on a molecular level. Entities which are biologically active may activate a receptor, provoke an immune reaction, interact with a membrane or ion channel, or otherwise induce a change in a biological function of an organism or any part of an organism.
• 'ligand' refers to a molecule that is recognized by a particular protein, e.g., a receptor. Any agent bound by or reacting with a protein is called a 'ligand', so the term encompasses the substrate of an enzyme and the reactants of a catalyzed reaction.
• the term 'ligand' does not imply any particular molecular size or other structural or compositional feature other than that the substance in question is capable of binding or otherwise interacting with a protein.
• a 'ligand' may serve either as the natural ligand to which the protein binds or as a functional analogue that may act as an agonist or antagonist.
• vectors refers to a DNA molecule, capable of replication in a host cell, into which a gene can be inserted to construct a recombinant DNA molecule.
• vectors include plasmids and infective microorganisms such as viruses, or non-viral vectors such as ligand-DNA conjugates, liposomes, or lipid-DNA complexes.
• 'cell surface receptor' refers to molecules that occur on the surface of cells, interact with the extracellular environment, and (directly or indirectly) transmit or transduce the information regarding the environment intracellularly in a manner that may modulate intracellular second messenger activities or transcription of specific promoters, resulting in transcription of specific genes.
• 'extracellular signals' include a molecule or other change in the extracellular environment that is transduced intracellularly via cell surface proteins that interact, directly or indirectly, with the signal.
• An extracellular signal or effector molecule includes any compound or substance that in some manner alters the activity of a cell surface protein. Examples of such signals include, but are not limited to, molecules such as acetylcholine, growth factors and hormones, lipids, sugars and nucleotides that bind to cell surface and/or intracellular receptors and ion channels and modulate the activity of such receptors and channels.
• extracellular signals' also include as yet unidentified substances that modulate the activity of a cellular receptor, and thereby influence intracellular functions.
• extracellular signals are potential pharmacological agents that may be used to treat specific diseases by modulating the activity of specific cell surface receptors.
• Orphan receptors' is a designation given to receptors for which no specific natural ligand has been described and/or for which no function has been determined.
• Target cells as used herein means cells, either in vivo or ex vivo, into which it is desired to introduce exogenous genetic material.
• Target cells may be any type of cell, including blood cells, skeletal muscle cells, stem cells, skin cells, liver cells, secretory gland cells, hematopoietic cells, and marrow cells.
• An 'effective amount' of a fusion polypeptide refers to an amount of the polypeptide in a preparation which, when applied as part of a desired dosage regimen, provides inhibition of angiogenesis so as to reduce or cure a disorder according to clinically acceptable standards.
• 'Serum half-life' refers to the time required for half of a quantity of a peptide in the bloodstream to be degraded.
• the chimeric polypeptide of the present invention can be constructed as a chimeric polypeptide containing a sequence homologous to at least a portion of a serum albumin and at least a portion of one or more heterologous proteins, expressed as one contiguous polypeptide chain.
• a fusion gene is constructed comprising DNA encoding at least one sequence each of a serum albumin, a heterologous protein, and, optionally, a peptide linker sequence to span the fragments.
• heterologous sequences are included in the chimeric polypeptide, they may be identical, related, or unrelated sequences. Identical sequences may be included to increase the effective concentration of the sequence.
• chimeric polypeptide might include a sequence that has antiangiogenic activity and a sequence which induces apoptosis of tumor cells.
• an entire protein can be cloned and expressed as part of the protein, or alternatively, a suitable fragment thereof containing a biologically active moiety can be used.
• a suitable fragment thereof containing a biologically active moiety can be used.
• Both the coding sequence of a gene and its regulatory regions can be redesigned to change the functional properties of the protein product, the amount of protein made, or the cell type in which the protein is produced.
• the coding sequence of a gene can be extensively altered, for example, by fusing part of it to the coding sequence of a different gene to produce a novel hybrid gene that encodes a fusion protein.
• fusion genes are well known. Essentially, the joining of various DNA fragments coding for different polypeptide sequences is performed in accordance with conventional techniques, employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
• the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
• PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, Eds. Ausubel et al. John Wiley & Sons: 1992).
• This invention also provides expression vectors comprising a nucleotide sequence encoding a subject chimeric polypeptide operably linked to at least one regulatory sequence.
• 'Operably linked' is intended to mean that the nucleotide sequence is linked to a regulatory sequence in a manner which allows expression of the nucleotide sequence.
• Regulatory sequences are art-recognized and are selected to direct expression of the encoded polypeptide. Accordingly, the term regulatory sequence includes promoters, enhancers and other expression control elements. Exemplary regulatory sequences are described in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
• any of a wide variety of expression control sequences-sequences that control the expression of a DNA sequence when operatively linked to it may be used in these vectors to express DNA sequences encoding the chimeric polypeptides of this invention.
• useful expression control sequences include, for example, the early and late promoters of SV40, adenovirus or cytomegalovirus immediate early promoter, the lac system, the trp system, the TAC or TRC system, T7 promoter whose expression is directed by T7 RNA polymerase, the major operator and promoter regions of phage lambda, the control regions for fd coat protein, the promoter for 3-phosphoglycerate kinase or other glycolytic enzymes, the promoters of acid phosphatase, e.g.,
• Pho5 the promoters of the yeast ⁇ -mating factors, the polyhedron promoter of the baculovirus system and other sequences known to control the expression of genes of prokaryotic or eukaryotic cells or their viruses, and various combinations thereof. It should be understood that the design of the expression vector may depend on such factors as the choice of the host cell to be transformed and or the type of protein desired to be expressed. Moreover, the vector's copy number, the ability to control that copy number and the expression of any other proteins encoded by the vector, such as antibiotic markers, should also be considered.
• the subject gene constructs can be used to cause expression of the subject chimeric polypeptides in cells propagated in culture, e.g., to produce chimeric polypeptides, for purification. This represents a method for preparing substantial quantities of the polypeptide, e.g., for research, clinical, and pharmaceutical uses.
• the ex v vo-derived chimeric polypeptides are utilized in a manner appropriate for therapy in general.
• the polypeptides of the invention can be formulated for a variety of modes of administration, including systemic and topical or localized administration.
• the polypeptide may by combined with a pharmaceutically acceptable excipient, e.g., a non-pyrogenic excipient.
• a pharmaceutically acceptable excipient e.g., a non-pyrogenic excipient.
• the polypeptides of the invention can be formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
• the polypeptides may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
• Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally.
• penetrants appropriate to the barrier to be permeated are used in the formulation.
• penetrants are generally known in the art, and include, for example, for transmucosal administration bile salts and fusidic acid derivatives.
• detergents may be used to facilitate permeation.
• Transmucosal administration may be through nasal sprays or using suppositories.
• the peptides are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
• the oligomers of the invention are formulated into ointments, salves, gels, or creams as generally known in the art.
• Genetic material of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces the desired chimeric polypeptide.
• the genetic material is provided by use of an "expression" construct, which can be transcribed in a cell to produce the chimeric polypeptide.
• expression constructs may be administered in any biologically effective carrier, e.g., any formulation or composition capable of effectively transfecting cells either ex vivo or in vivo with genetic material encoding a chimeric polypeptide.
• Approaches include insertion of the antisense nucleic acid in viral vectors including recombinant retroviruses, adenoviruses, adeno-associated viruses, human immunodeficiency viruses, and herpes simplex viruses-1, or recombinant bacterial or eukaryotic plasmids.
• Viral vectors can be used to transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (lipofectin) or derivatized (e.g., antibody conjugated), polylysine conjugates, gramacidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or calcium phosphate precipitation carried out in vivo.
• lipofectin cationic liposomes
• derivatized e.g., antibody conjugated
• polylysine conjugates e.g., gramacidin S
• artificial viral envelopes e.g., artificial viral envelopes or other such intracellular carriers
• a preferred approach for in vivo introduction of genetic material encoding one of the subject proteins into a cell is by use of a viral vector containing said genetic material.
• Infection of cells with a viral vector has the advantage that a large proportion of the targeted cells can receive the nucleic acid.
• chimeric polypeptides encoded by genetic material in the viral vector e.g., by a nucleic acid contained in the viral vector, are expressed efficiently in cells which have taken up viral vector nucleic acid.
• Such a strategy may be particularly effective when skeletal muscle cells are the targets of the vector (Fisher, K.J. et al. Nature Medicine 1997, 3, 306-312).
• Retrovirus vectors and adeno-associated virus vectors are generally understood to be the recombinant gene delivery system of choice for the transfer of exogenous genes in vivo, particularly into humans. These vectors provide efficient delivery of genes into cells, and the transferred nucleic acids are stably integrated into the chromosomal DNA of the host.
• a major prerequisite for the use of retroviruses is to ensure the safety of their use, particularly with regard to the possibility of the spread of wild-type virus in the cell population.
• the development of specialized cell lines (termed "packaging cells") which produce only replication-defective retroviruses has increased the utility of retroviruses for gene therapy, and defective retroviruses are well characterized for use in gene transfer for gene therapy purposes (for a review see Miller, A.D.
• recombinant retrovirus can be constructed in which part of the retroviral coding sequence (gag, pol, env) has been replaced by nucleic acid encoding one of the antisense E6AP constructs, rendering the retrovirus replication defective.
• the replication defective retrovirus is then packaged into virions which can be used to infect a target cell through the use of a helper virus by standard techniques. Protocols for producing recombinant retroviruses and for infecting cells in vitro or in vivo with such viruses can be found in Current Protocols in Molecular Biology, Ausubel, F.M. et al.
• retroviruses include pLJ, pZIP, pWE and pEM which are well known to those skilled in the art.
• suitable packaging virus lines for preparing both ecotropic and amphotropic retroviral systems include ⁇ Crip, ⁇ Cre, ⁇ 2 and ⁇ Am. Retroviruses have been used to introduce a variety of genes into many different cell types, including neural cells, epithelial cells, endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrow cells, in vitro and/or in vivo (see for example Eglitis, et al.
• retroviral vectors as a gene delivery system for genetic material encoding the subject chimeric polypeptides, it is important to note that a prerequisite for the successful infection of target cells by most retroviruses, and therefore of stable introduction of the genetic material, is that the target cells must be dividing. In general, this requirement will not be a hindrance to use of retroviral vectors. In fact, such limitation on infection can be beneficial in circumstances wherein the tissue (e.g., nontransformed cells) surrounding the target cells does not undergo extensive cell division and is therefore refractory to infection with retroviral vectors.
• tissue e.g., nontransformed cells
• retroviral-based vectors by modifying the viral packaging proteins on the surface of the viral particle.
• strategies for the modification of the infection spectrum of retroviral vectors include: coupling antibodies specific for cell surface antigens to the viral env protein (Roux et al. (1989) PNAS 86:9079-9083; Julan et al. (1992) J. Gen Virol 73:3251-3255; and Goud et al.
• Coupling can be in the form of the chemical cross-linking with a protein or other variety (e.g., lactose to convert the env protein to an asialoglycoprotein), as well as by generating chimeric proteins (e.g., single- chain antibody/e «v chimeric proteins).
• This technique while useful to limit or otherwise direct the infection to certain tissue types, and can also be used to convert an ecotropic vector in to an amphotropic vector.
• retroviral gene delivery can be further enhanced by the use of tissue- or cell-specific transcriptional regulatory sequences which control expression of the genetic material of the retroviral vector.
• Another viral gene delivery system useful in the present invention utilizes adenovirus- derived vectors.
• the genome of an adeno virus can be manipulated such that it encodes a gene product of interest, but is inactive in terms of its ability to replicate in a normal lytic viral life cycle (see, for example, Berkner et al. (1988) BioTechniques 6:616; Rosenfeld et al. (1991) Science 252:431-434; and Rosenfeld et al. (1992) Cell 68:143-155).
• adenoviral vectors derived from the adenovirus strain Ad type 5 dl324 or other strains of adenovirus are well known to those skilled in the art.
• Recombinant adenoviruses can be advantageous in certain circumstances in that they are capable of infecting non-dividing cells and can be used to infect a wide variety of cell types, including airway epithelium (Rosenfeld et al. (1992) cited supra), endothelial cells (Lemarchand et al. (1992) Proc. Natl. Acad. Sci. USA 89:6482-6486), hepatocytes (Herz and Gerard (1993) Proc.
• virus particle is relatively stable and amenable to purification and concentration, and, as above, can be modified so as to affect the spectrum of infectivity.
• introduced adenoviral DNA and foreign DNA contained therein is not integrated into the genome of a host cell but remains episomal, thereby avoiding potential problems that can occur as a result of insertional mutagenesis in situations where introduced DNA becomes integrated into the host genome (e.g., retroviral DNA).
• the carrying capacity of the adenoviral genome for foreign DNA is large (up to 8 kilobases) relative to other gene delivery vectors (Berkner et al., supra; Haj-Ahmand and Graham (1986) J. Virol. 57:267).
• Most replication-defective adenoviral vectors currently in use and therefore favored by the present invention are deleted for all or parts of the viral El and E3 genes but retain as much as 80% of the adenoviral genetic material (see, for example, Jones et al. (1979) Cell 16:683; Berkner et al., supra; and Graham et al. in Methods in Molecular Biology. E.J. Murray, Ed.
• Expression of the inserted genetic material can be under control of, for example, the El A promoter, the major late promoter (MLP) and associated leader sequences, the E3 promoter, or exogenously added promoter sequences.
• MLP major late promoter
• Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
• Adeno-associated virus is a naturally occurring defective virus that requires another virus, such as an adenovirus or a herpes virus, as a helper virus for efficient replication and a productive life cycle.
• Adeno-associated virus is also one of the few viruses that may integrate its DNA into non-dividing cells, and exhibits a high frequency of stable integration (see for example Flotte et al. (1992) Am. J. Respir. Cell. Mol. Biol.
• Vectors containing as little as 300 base pairs of AAV can be packaged and can integrate. Space for exogenous DNA is limited to about 4.5 kb.
• An AAV vector such as that described in Tratschin et al. (1985) Mol. Cell. Biol. 5:3251-3260 can be used to introduce DNA into cells.
• a variety of nucleic acids have been introduced into different cell types using AAV vectors (see for example Hermonat et al. (1984) Proc. Natl. Acad. Sci.
• viral vector systems that may have application in gene therapy have been derived from herpes virus, vaccinia virus, and several RNA viruses.
• non- viral methods can also be employed to cause expression of genetic material encoding the subject chimeric polypeptides in the tissue of an animal.
• Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules.
• non-viral gene delivery systems of the present invention rely on endocytic pathways for the uptake of genetic material by the targeted cell.
• Exemplary gene delivery systems of this type include liposomal derived systems, polylysine conjugates, and artificial viral envelopes.
• genetic material can be entrapped in liposomes bearing positive charges on their surface (e.g., hpofectins) and, optionally, which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075).
• lipofection of papilloma-infected cells can be carried out using liposomes tagged with monoclonal antibodies against PV-associated antigen (see Viac et al. (1978) J Invest Dermatol 70:263-266; see also Mizuno et al. (1992) Neurol. Med. Chir. 32:873-876).
• the gene delivery system comprises an antibody or cell surface ligand which is cross-linked with a gene binding agent such as polylysine (see, for example, PCT publications WO93/04701, WO92/22635, WO92/20316, WO92/19749, and WO92/06180).
• a gene binding agent such as polylysine
• genetic material encoding the subject chimeric polypeptides can be used to transfect hepatocytic cells in vivo using a soluble polynucleotide carrier comprising an asialoglycoprotein conjugated to a polycation, e.g., polylysine (see U.S. Patent 5,166,320).
• a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g., by intravenous injection, and specific transduction of the target cells occurs predominantly from specificity of transfection provided by the gene delivery vehicle, cell-type or tissue-type expression due to the transcriptional regulatory sequences controlling expression of the gene, or a combination thereof.
• initial delivery of the recombinant gene is more limited with introduction into the animal being quite localized.
• the gene delivery vehicle can be introduced by catheter (see U.S. Patent 5,328,470) or by stereotactic injection (e.g., Chen et al. (1994) PNAS 91: 3054-3057).
• the pharmaceutical preparation can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
• the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.
• methods of introducing the viral packaging cells may be provided by, for example, rechargeable or biodegradable devices.
• Various slow release polymeric devices have been developed and tested in vivo in recent years for the controlled delivery of drugs, including proteinaceous biopharmaceuticals, and can be adapted for release of viral particles through the manipulation of the polymer composition and form.
• biocompatible polymers including hydrogels
• biodegradable and non-degradable polymers can be used to form an implant for the sustained release of an the viral particles by cells implanted at a particular target site.
• Such embodiments of the present invention can be used for the delivery of an exogenously purified virus, which has been incorporated in the polymeric device, or for the delivery of viral particles produced by a cell encapsulated in the polymeric device.
• the amount of water, porosity and consequent permeability characteristics can be controlled.
• the selection of the shape, size, polymer, and method for implantation can be determined on an individual basis according to the disorder to be treated and the individual patient response. The generation of such implants is generally known in the art. See, for example, Concise Encyclopedia of Medical & Dental Materials, ed. by David Williams (MIT Press: Cambridge, MA, 1990); and the Sabel et al. U.S. Patent No. 4,883,666.
• a source of cells producing a the recombinant virus is encapsulated in implantable hollow fibers.
• Such fibers can be pre- spun and subsequently loaded with the viral source (Aebischer et al. U.S. Patent No. 4,892,538; Aebischer et al. U.S. Patent No. 5,106,627; Hoffman et al. (1990) Expt. Neurobiol. 110:39-44; Jaeger et al. (1990) Prog. Brain Res. 82:41-46; and Aebischer et al. (1991) J. Biomech. Eng. 113:178-183), or can be co-extruded with a polymer which acts to form a polymeric coat about the viral packaging cells (Lim U.S. Patent No. 4,391,909; Sefton U.S. Patent No.
• Chimeric polypeptides of the present invention can be designed by using molecular modeling.
• a computer model of serum albumin may be altered to include a selected heterologous sequence and the resulting structure may be submitted to calculations designed to determine how the resulting peptide will change in shape, how much strain the alteration introduces into the polypeptide, how the heterologous sequence is displayed in three dimensions, and other data relevant to the resulting structure of the chimeric polypeptide.
• the nature of the sequence to be included might be determined by the calculation, based on knowledge of a receptor or binding pocket.
• the calculations might best determine how to insert a desired sequence to maintain the tertiary structure of the serum albumin backbone, or to display the insertion in the proper orientation.
• Other calculational strategies will be known to those skilled in the art. Calculations such as these can be useful for directing the synthesis of chimeric polypeptides of the present invention in a time- and material-efficient manner, before actual synthesis and screening techniques begin.
• peptide libraries are one way of screening large numbers of polypeptides at once.
• the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind a target molecule, such as a receptor protein via this gene product is detected in a "panning assay".
• the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting chimeric polypeptide detected by panning (Ladner et al., WO 88/06630; Fuchs et al. (1991) Bio /Technology 9:1370-1371; and Goward et al. (1992) TIBS 18:136-140).
• the peptide library is expressed as chimeric polypeptides on the surface of a viral particle.
• foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits.
• coli filamentous phages Ml 3, fd, and fl are most often used in phage display libraries, as either of the phage gill or gVIII coat proteins can be used to generate chimeric polypeptides without disrupting the ultimate packaging of the viral particle (Ladner et al. PCT publication WO 90/02809; Garrard et al, PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007-16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461).
• U.S. patent application 09/174,943, filed October 19, 1998 discloses a method for isolating biologically active peptides. Using the techniques disclosed therein, a chimeric polypeptide of the present invention may be developed which interacts with a chosen receptor.
• this method is utilized to identify polypeptides which have antiproliferative activity with respect to one or more types of cells.
• the chimeric polypeptide library can be panned with the target cells for which an antiproliferative is desired in order to enrich for polypeptides which bind to that cell.
• the polypeptide library can also be panned against one or more control cell lines in order to remove polypeptides which bind the control cells.
• the polypeptide library which is then tested in the secretion mode can be enriched for polypeptides which selectively bind target cells (relative to the control cells).
• the display mode can produce a polypeptide library enriched for polypeptides which preferentially bind tumor cells relative to normal cells, which preferentially bind p53- cells relative to p53+ cells, which preferentially bind hair follicle cells relative to other epithelial cells, or any other differential binding characteristic.
• the polypeptides are tested for antiproliferative activity against the target cell using any of a number of techniques known in the art. For instance, BrdU or other nucleotide uptake can be measured as an indicator of proliferation.
• the secretion mode can include negative controls in order to select for polypeptides with specific antiproliferative activity.
• polypeptides can be isolated from the library based on their ability to induce apoptosis or cell lysis, for example, in a cell-selective manner.
• this method can be used to identify polypeptides with angiogenic or antiangiogenic activity.
• the polypeptide library can be enriched for polypeptides that bind to endothelial cells but which do not bind to fibroblasts.
• the resulting sub-library can be screened for polypeptides which inhibit capillary endothelial cell proliferation and/or endothelial cell migration. Polypeptides scoring positive for one or both of these activities can also be tested for activity against other cell types, such as smooth muscle cells or fibroblasts, in order to select polypeptides active only against endothelial cells.
• this method can be used to identify anti-infective polypeptides, for example, which are active as anti-fungal or antibacterial agents.
• this assay can be used for identifying effectors of a receptor protein or complex thereof.
• the assay is characterized by the use of a test cell which includes a target receptor or ion channel protein whose signal transduction activity can be modulated by interaction with an extracellular signal, the transduction activity being able to generate a detectable signal.
• such assays are characterized by the use of a mixture of cells expressing a target receptor protein or ion channel capable of transducing a detectable signal in the reagent cell.
• the receptor/channel protein can be either endogenous or heterologous.
• a culture of the instant reagent cells will provide means for detecting agonists or antagonists of receptor function.
• the ability of particular polypeptides to modulate a signal transduction activity of the target receptor or channel can be scored for by detecting up or down-regulation of the detection signal.
• second messenger generation e.g., GTPase activity, phospholipid hydrolysis, or protein phosphorylation patterns as examples
• an indicator gene can provide a convenient readout.
• a detection means consists of an indicator gene.
• polypeptides which induce a signal pathway from a particular receptor or channel can be identified. If a test polypeptide does not appear to induce the activity of the receptor/channel protein, the assay may be repeated as described above, and modified by the introduction of a step in which the reagent cell is first contacted with a known activator of the target receptor/channel to induce signal transduction, and the test peptide can be assayed for its ability to inhibit the activated receptor/channel, for example, to identify antagonists. In yet other embodiments, peptides can be screened for those which potentiate the response to a known activator of the receptor.
• the assays can be used to test functional ligand-receptor or ligand-ion channel interactions for cell surface- localized receptors and channels.
• the subject assay can be used to identify effectors of, for example, G protein- coupled receptors, receptor tyrosine kinases, cytokine receptors, and ion channels.
• the method described herein is used for identifying ligands for "orphan receptors" for which no ligand is known.
• the receptor is a cell surface receptor, such as: a receptor tyrosine kinase, for example, an EPH receptor; an ion channel; a cytokine receptor; an multisubunit immune recognition receptor, a chemokine receptor; a growth factor receptor, or a G-protein coupled receptor, such as a chemoattracttractant peptide receptor, a neuropeptide receptor, a light receptor, a neurotransmitter receptor, or a polypeptide hormone receptor.
• Preferred G protein-coupled receptors include ⁇ l A-adrenergic receptor, ⁇ lB-adrenergic receptor, ⁇ 2-adrenergic receptor, ⁇ 2B-adrenergic receptor, 1-adrenergic receptor, ⁇ 2-adrenergic receptor, ⁇ 3-adrenergic receptor, ml acetylcholine receptor (AChR), m2 AChR, m3 AChR, m4 AChR, m5 AChR, Dl dopamine receptor, D2 dopamine receptor, D3 dopamine receptor, D4 dopamine receptor, D5 dopamine receptor, Al adenosine receptor, A2b adenosine receptor, 5- HTla receptor, 5-HTlb receptor, 5HTl-like receptor, 5-HTld receptor, 5HTld-like receptor, 5HTld beta receptor, substance K (neurokinin A) receptor, fMLP receptor, fMLP-like receptor, angiotens
• EPH receptors inlcude eph, elk, eck, sek, mek4, hek, hek2, eek, erk, tyrol, tyro4, tyro5, tyro ⁇ , tyrol 1, cek4, cek5, cek ⁇ , cek7, cek8, cek9, ceklO, bsk, rtkl, rtk2, rtk3, mykl, myk2, ehkl, ehk2,pagliaccio, htk, erk and n P receptors.
• the target receptor is a cytokine receptor.
• Cytokines are a family of soluble mediators of cell-to-cell communication that includes interleukins, interferons, and colony-stimulating factors. The characteristic features of cytokines lie in their functional redundancy and pleiotropy. Most of the cytokine receptors that constitute distinct superfamilies do not possess intrinsic protein tyrosine kinase domains, yet receptor stimulation usually invokes rapid tyrosine phosphorylation of intracellular proteins, including the receptors themselves. Many members of the cytokine receptor superfamily activate the Jak protein tyrosine kinase family, with resultant phosphorylation of the STAT transcriptional activator factors.
• IL-2, IL-7, IL-2 and Interferon ⁇ have all been shown to activate Jak kinases (Frank et al (1995) Proc Natl Acad Sci USA 92:7779-7783); Scharfe et al. (1995) Blood 86:2077-2085); (Bacon et al. (1995) Proc Natl Acad Sci USA 92:7307-7311); and (Sakatsume et al (1995) J. Biol Chem 270:17528- 17534). Events downstream of Jak phosphorylation have also been elucidated.
• STAT signal transducers and activators of transcription
• STATl ⁇ signal transducers and activators of transcription
• STAT2 ⁇ signal transducers and activators of transcription
• STAT3 two STAT -related proteins, p94 and p95.
• the STAT proteins were found to translocate to the nucleus and to bind to a specific DNA sequence, thus suggesting a mechanism by which IL-2 may activate specific genes involved in immune cell function (Frank et al. supra).
• Jak3 is associated with the gamma chain of the IL-2, IL-4, and IL-7 cytokine receptors (Fujii et al.
• Jak kinases have also been shown to be activated by numerous ligands that signal via cytokine receptors such as, growth hormone and erythropoietin and IL-6 (Kishimoto (1994) Stem cells Suppl 12:37-44).
• Detection means which may be scored for in the present assay, in addition to direct detection of second messengers, such as by changes in phosphorylation, includes reporter constructs or indicator genes which include transcriptional regulatory elements responsive to the STAT proteins. Described infra.
• MIRR Multisubunit Immune Recognition Receptor
• the receptor is a multisubunit receptor.
• Receptors can be comprised of multiple proteins referred to as subunits, one category of which is referred to as a multisubunit receptor is a multisubunit immune recognition receptor (MIRR).
• MIRRs include receptors having multiple noncovalently associated subunits and are capable of interacting with src-family tyrosine kinases.
• MIRRs can include, but are not limited to, B cell antigen receptors, T cell antigen receptors, Fc receptors and CD22.
• An MIRR is an antigen receptor on the surface of a B cell.
• the MIRR on the surface of a B cell comprises membrane-bound immunoglobulin (mlg) associated with the subunits Ig- ⁇ and Ig- or Ig- ⁇ , which forms a complex capable of regulating B cell function when bound by antigen.
• An antigen receptor can be functionally linked to an amplifier molecule in a manner such that the amplifier molecule is capable of regulating gene transcription.
• Src-family tyrosine kinases are enzymes capable of phosphorylating tyrosine residues of a target molecule.
• a src-family tyrosine kinase contains one or more binding domains and a kinase domain.
• a binding domain of a src-family tyrosine kinase is capable of binding to a target molecule and a kinase domain is capable of phosphorylating a target molecule bound to the kinase.
• Members of the src family of tyrosine kinases are characterized by an N-terminal unique region followed by three regions that contain different degrees of homology among all the members of the family.
• src homology region 1 SHI
• SH2 src homology region 2
• SH3 src homology region 3
• Both the SH2 and SH3 domains are believed to have protein association functions important for the formation of signal transduction complexes.
• the amino acid sequence of an N-terminal unique region varies between each src-family tyrosine kinase.
• An N-terminal unique region can be at least about the first 40 amino acid residues of the N-terminal of a src-family tyrosine kinase.
• Syk-family kinases are enzymes capable of phosphorylating tyrosine residues of a target molecule.
• a syk-family kinase contains one or more binding domains and a kinase domain.
• a binding domain of a syk-family tyrosine kinase is capable of binding to a target molecule and a kinase domain is capable of phosphorylating a target molecule bound to the kinase.
• Members of the syk family of tyrosine kinases are characterized by two SH2 domains for protein association function and a tyrosine kinase domain.
• a primary target molecule is capable of further extending a signal transduction pathway by modifying a second messenger molecule.
• Primary target molecules can include, but are not limited to, phosphatidylinositol 3-kinase (PI-3K), P21 ras GAPase-activating protein and associated PI 90 and P62 protein, phospholipases such as PLC ⁇ l and PLC 2, MAP kinase, She and VAV.
• PI-3K phosphatidylinositol 3-kinase
• P21 ras GAPase-activating protein and associated PI 90 and P62 protein phospholipases such as PLC ⁇ l and PLC 2, MAP kinase, She and VAV.
• a primary target molecule is capable of producing second messenger molecule which is capable of further amplifying a transduced signal.
• Second messenger molecules include, but are not limited to diacylglycerol and inositol 1,4,5-triphosphate (
• Second messenger molecules are capable of initiating physiological events which can lead to alterations in gene transcription. For example, production of IP3 can result in release of intracellular calcium, which can then lead to activation of calmodulin kinase II, which can then lead to serine phosphorylation of a DNA binding protein referred to as ets-1 proto-onco-protein.
• Diacylglycerol is capable of activating the signal transduction protein, protein kinase C which affects the activity of the API DNA binding protein complex.
• Signal transduction pathways can lead to transcriptional activation of genes such as c-fos, egr-1, and c-myc.
• An adaptor molecule comprises a protein that enables two other proteins to form a complex (e.g., a three molecule complex). She protein enables a complex to form which includes Grb2 and SOS. She comprises an SH2 domain that is capable of associating with the SH2 domain of Grb2.
• Molecules of a signal transduction pathway can associate with one another using recognition sequences.
• Recognition sequences enable specific binding between two molecules. Recognition sequences can vary depending upon the structure of the molecules that are associating with one another. A molecule can have one or more recognition sequences, and as such can associate with one or more different molecules.
• MIRR-induced signal transduction pathways can regulate the biological functions of specific types of cells involved in particular responses by an animal, such as immune responses, inflammatory responses and allergic responses.
• Cells involved in an immune response can include, for example, B cells, T cells, macrophages, dendritic cells, natural killer cells and plasma cells.
• Cells involved in inflammatory responses can include, for example, basophils, mast cells, eosinophils, neutrophils and macrophages.
• Cells involved in allergic responses can include, for example mast cells, basophils, B cells, T cells and macrophages.
• the detection signal is a second messenger, such as a phosphorylated src-like protein, including reporter constructs or indicator genes which include transcriptional regulatory elements such as serum response element (SRE), 12-O-tetradecanoyl-phorbol-13- acetate response element, cyclic AMP response element, c- fos promoter, or a CREB-responsive element.
• SRE serum response element
• cyclic AMP response element c- fos promoter
• CREB-responsive element a second messenger
• the target receptor is a receptor tyrosine kinase.
• the receptor tyrosine kinases can be divided into five subgroups on the basis of structural similarities in their extracellular domains and the organization of the tyrosine kinase catalytic region in their cytoplasmic domains. Sub-groups I (epidermal growth factor (EGF) receptor-like), II (insulin receptor-like) and the eph/eck family contain cysteine-rich sequences (Hirai et al., (1987) Science 238:1717-1720 and Lindberg and Hunter, (1990) Mo/. Cell. Biol. 10:6316-6324).
• EGF epidermal growth factor
• the functional domains of the kinase region of these three classes of receptor tyrosine kinases are encoded as a contiguous sequence ( Hanks et al. (1988) Science 241 :42-52).
• Subgroups III platelet-derived growth factor (PDGF) receptor-like) and IV (the fibro-blast growth factor (FGF) receptors) are characterized as having immunoglobulin (Ig)-like folds in their extracellular domains, as well as having their kinase domains divided in two parts by a variable stretch of unrelated amino acids (Yanden and Ullrich (1988) supra and Hanks et al. (1988) supra).
• the family with by far the largest number of known members is the EPH family. Since the description of the prototype, the EPH receptor (Hirai et al. (1987) Science 238:1717-1720), sequences have been reported for at least ten members of this family, not counting apparently orthologous receptors found in more than one species. Additional partial sequences, and the rate at which new members are still being reported, suggest the family is even larger (Maisonpierre et al. (1993) Oncogene 8:3277-3288; Andres et al. (1994) Oncogene 9:1461-1467; Henkemeyer et al. (1994) Oncogene 9:1001-1014; Ruiz et al.
• the expression patterns determined for some of the EPH family receptors have implied important roles for these molecules in early vertebrate development.
• the timing and pattern of expression of sek, mek4 and some of the other receptors during the phase of gastrulation and early organogenesis has suggested functions for these receptors in the important cellular interactions involved in patterning the embryo at this stage (Gilardi-Hebenrison et al. (1992) Oncogene 7:2499-2506; Nieto et al. (1992) Development 116:1137-1150; Henkemeyer et al., supra; Ruiz et al., supra; and Xu et al., supra).
• Sek shows a notable early expression in the two areas of the mouse embryo that show obvious segmentation, namely the somites in the mesoderm and the rhombomeres of the hindbrain; hence the name sek, for segmentally expressed kinase (Gilardi-Hebenrison et al., supra; Nieto et al., supra).
• these segmental structures of the mammalian embryo are implicated as important elements in establishing the body plan.
• the observation that Sek expression precedes the appearance of morphological segmentation suggests a role for sek in forming these segmental structures, or in determining segment-specific cell properties such as lineage compartmentation (Nieto et al, supra).
• EPH receptors have been implicated, by their pattern of expression, in the development and maintenance of nearly every tissue in the embryonic and adult body. For instance, EPH receptors have been detected throughout the nervous system, the testes, the cartilaginous model of the skeleton, tooth primordia, the infundibular component of the pituitary, various epithelial tissues, lung, pancreas, liver and kidney tissues. Observations such as this have been indicative of important and unique roles for EPH family kinases in development and physiology, but further progress in understanding their action has been severely limited by the lack of information on their ligands.
• EPH receptor or "EPH-type receptor” refer to a class of receptor tyrosine kinases, comprising at least eleven paralogous genes, though many more orthologs exist within this class, e.g., homologs from different species.
• EPH receptors in general, are a discrete group of receptors related by homology and easily recognizable, for example, they are typically characterized by an extracellular domain containing a characteristic spacing of cysteine residues near the N-terminus and two fibronectin type III repeats (Hirai et al. (1987) Science 238:1717-1720; Lindberg et al. (1990) Mol Cell Biol 10:6316-6324; Chan et al.
• EPH receptors include the eph, elk, eck, sek, mek4, hek, hek2, eek, erk, tyrol, tyro4, tyro5, tyro6, tyrol 1, cek4, cek5, cek ⁇ , cek7, cek8, cek9, ceklO, bsk, rtkl, rtk2, rtk3, mykl, myk2, ehkl, ehk2, pagliaccio, htk, erk and nuk receptors.
• the term "EPH receptor” refers to the membrane form of the receptor protein, as well as soluble extracellular fragments which retain the ability to bind the ligand of the present invention.
• the detection signal is provided by detecting phosphorylation of intracellular proteins, e.g., MEKKs, MEKs, or Map kinases, or by the use of reporter constructs or indicator genes which include transcriptional regulatory elements responsive to c-fos and/or c-jun. Described infra.
• G proteins One family of signal transduction cascades found in eukaryotic cells utilizes heterotrimeric "G proteins." Many different G proteins are known to interact with receptors. G protein signaling systems include three components: the receptor itself, a GTP-binding protein (G protein), and an intracellular target protein.
• G protein GTP-binding protein
• the cell membrane acts as a switchboard. Messages arriving through different receptors can produce a single effect if the receptors act on the same type of G protein. On the other hand, signals activating a single receptor can produce more than one effect if the receptor acts on different kinds of G proteins, or if the G proteins can act on different effectors.
• the G proteins which consist of alpha ( ⁇ ), beta ( ⁇ ) and gamma ( ⁇ ) subunits, are complexed with the nucleotide guanosine diphosphate (GDP) and are in contact with receptors.
• GDP nucleotide guanosine diphosphate
• the receptor changes conformation and this alters its interaction with the G protein. This spurs the ⁇ subunit to release GDP, and the more abundant nucleotide guanosine triphosphate (GTP), replaces it, activating the G protein.
• GTP nucleotide guanosine triphosphate
• the effector (which is often an enzyme) in turn converts an inactive precursor molecule into an active "second messenger,” which may diffuse through the cytoplasm, triggering a metabolic cascade. After a few seconds, the G ⁇ converts the GTP to
• the inactivated G ⁇ may then reassociate with the G ⁇ complex.
• G protein-coupled receptors are comprised of a single protein chain that is threaded through the plasma membrane seven times. Such receptors are often referred to as seven- transmembrane receptors (STRs). More than a hundred different STRs have been found, including many distinct receptors that bind the same ligand, and there are likely many more STRs awaiting discovery.
• STRs seven- transmembrane receptors
• STRs have been identified for which the natural ligands are unknown; these receptors are termed "orphan" G protein-coupled receptors, as described above. Examples include receptors cloned by Neote et al. (1993) CelI 72, 4l5; Kouba et a ⁇ . FEBSLett. (1993) 321, 173; Birkenbach et al.(1993) J. Virol. 67, 2209.
• the 'exogenous receptors' of this example may be any G protein-coupled receptor which is exogenous to the cell which is to be genetically engineered for the purpose of the present invention.
• This receptor may be a plant or animal cell receptor. Screening for binding to plant cell receptors may be useful in the development of, for example, herbicides.
• an animal receptor it may be of invertebrate or vertebrate origin. If an invertebrate receptor, an insect receptor is preferred, and would facilitate development of insecticides.
• the receptor may also be a vertebrate, more preferably a mammalian, still more preferably a human, receptor.
• the exogenous receptor is also preferably a seven transmembrane segment receptor.
• ligands for G protein coupled receptors include: purines and nucleo tides, such as adenosine, cAMP, ATP, UTP, ADP, melatonin and the like; biogenic amines (and related natural ligands), such as 5-hydroxytryptamine, acetylcholine, dopamine, adrenaline, adrenaline, adrenaline., histamine, noradrenaline, noradrenaline, noradrenaline., tyramine/octopamine and other related compounds; peptides such as adrenocorticotrophic hormone (acth), melanocyte stimulating hormone (msh), melanocortins, neurotensin (nt), bombesin and related peptides, endofhelins, cholecystokinin, gastrin, neurokinin b (nk3), invertebrate tachykinin-like peptides, substance k (nk2), substance p (
• G-protein coupled receptors include, but are not limited to, dopaminergic, muscarinic cholinergic, a-adrenergic, b-adrenergic, opioid (including delta and mu), cannabinoid, serotoninergic, and GABAergic receptors.
• Preferred receptors include the 5HT family of receptors, dopamine receptors, C5a receptor and FPRL-1 receptor, cyclo-histidyl- proline-diketoplperazine receptors, melanocyte stimulating hormone release inhibiting factor receptor, and receptors for neurotensin, thyrotropin releasing hormone, calcitonin, cholecytokinin-A, neurokinin-2, histamine-3, cannabinoid, melanocortin, or adrenomodulin, neuropeptide- Yl or galanin.
• Other suitable receptors are listed in the art.
• the term 'receptor,' as used herein, encompasses both naturally occurring and mutant receptors.
• G protein-coupled receptors like the yeast a- and ⁇ -factor receptors, contain seven hydrophobic amino acid-rich regions which are assumed to lie within the plasma membrane.
• Specific human G protein-coupled STRs for which genes have been isolated and for which expression vectors could be constructed include those listed herein and others known in the art.
• the gene would be operably linked to a promoter functional in the cell to be engineered and to a signal sequence that also functions in the cell.
• suitable promoters include Sl ⁇ 2, Sle3_ and g ⁇ LLQ.
• Suitable signal sequences include those of Sle2, Ste3 and of other genes which encode proteins secreted by yeast cells.
• the codons of the gene would be optimized for expression in yeast. See Hoekema et al.,(1987) Mol. Cell. Biol, 7:2914-24; Sharp, et al., (1986)14:5125-43.
• STRs The homology of STRs is discussed in Dohlman et al., Ann. Rev. Biochem., (1991) 60:653-88. When STRs are compared, a distinct spatial pattern of homology is discernible. The transmembrane domains are often the most similar, whereas the N- and C-termmal regions, and the cytoplasmic loop connecting transmembrane segments V and VI are more divergent
• a foreign receptor will fail to functionally integrate into the yeast membrane, and there interact with the endogenous yeast G protein More likely, either the receptor will need to be modified (e g , by replacing its V-VI loop with that of the yeast STE2 or STE3 receptor), or a compatible G protein should be provided
• the wild-type exogenous G protem-coupled receptor cannot be made functional m yeast, it may be mutated for this purpose
• a compa ⁇ son would be made of the amino acid sequences of the exogenous receptor and of the yeast receptors, and regions of high and low homology identified T ⁇ al mutations would then be made to distinguish regions involved m ligand or G protein binding, from those necessary for functional integration m the membrane
• the exogenous receptor would then be mutated in the latter region to more closely resemble the yeast receptor, until functional integration was achieved If this were insufficient to achieve functionality, mutations would next be made in the regions involved m G protein binding Mutations would be made in regions involved in ligand binding only as a last resort, and then an effort would be made to preserve ligand binding by making conservative substitutions whenever possible
• the yeast genome is modified so that it is unable to produce the yeast receptors which are homologous to the exogenous receptors in functional form Otherwise, a positive assay score might reflect the ability of a peptide to activate the endogenous G protem- coupled receptor, and not the receptor of interest
• N-formyl peptide receptor is a classic example of a calcium mobilizing G protein- coupled receptor expressed by neutrophils and other phagocytic cells of the mammalian immune system (Snyderman et al (1988) In Inflammation Basic Principles and Clinical Correlates, pp 309-323)
• N-Formyl peptides of bacte ⁇ al origin bind to the receptor and engage a complex activation program that results m directed cell movement, release of inflammatory granule contents, and activation of a latent NADPH oxidase which is important for the production of metabolites of molecular oxygen.
• This pathway initiated by receptor-ligand interaction is critical in host protection from pyogenic infections. Similar signal transduction occurs in response to the inflammatory peptides C5a and IL-8.
• FPRL formyl peptide receptor like
• FPRLl was found to be 69% identical in amino acid sequence to NFPR, it did not bind prototype N-formyl peptides ligands when expressed in heterologous cell types. This lead to the hypothesis of the existence of an as yet unidentified ligand for the FPRLl orphan receptor (Murphy et al. supra).
• the yeast cell In the case of an exogenous Gprotein-coupled receptor, the yeast cell must be able to produce a G protein which is activated by the exogenous receptor, and which can in turn activate the yeast effector(s).
• the endogenous yeast G ⁇ subunit e.g., GPA
• GPA endogenous yeast G ⁇ subunit
• the G ⁇ subunit of the yeast G protein may be replaced by the G ⁇ subunit natively associated with the exogenous receptor.
• the G ⁇ subunit may be modified to improve coupling. These modifications often will take the form of mutations which increase the resemblance of the G ⁇ subunit to the yeast G ⁇ while decreasing its resemblance to the receptor- associated G ⁇ . For example, a residue may be changed so as to become identical to the corresponding yeast G ⁇ residue, or to at least belong to the same exchange group of that residue. After modification, the modified G ⁇ subunit might or might not be "substantially homologous" to the foreign and or the yeast G ⁇ subunit.
• the modifications are preferably concentrated in regions of the G ⁇ which are likely to be involved in G ⁇ binding.
• the modifications will take the form of replacing one or more segments of the receptor-associated G ⁇ with the corresponding yeast G ⁇ segment(s), thereby forming a chimeric G ⁇ subunit.
• segment refers to three or more consecutive amino acids.
• point mutations may be sufficient.
• This chimeric G ⁇ subunit will interact with the exogenous receptor and the yeast G ⁇ complex, thereby permitting signal transduction. While use of the endogenous yeast G ⁇ is preferred, if a foreign or chimeric G ⁇ is capable of transducing the signal to the yeast effector, it may be used instead.
• Serum albumin loop regions A space-filling model of human serum albumin (HSA) is shown in Figure 1.
• HSA human serum albumin
• the tertiary structure of HSA reveals the presence of ten approximate helical regions or loops, each constrained by disulfide bonded cysteine pairs.
• the space-filling model was used to predict loop regions that are exposed on the surface of the protein.
• Two amino acid segments were chosen to represent surface exposed regions (loop 53-62 and loop 360-369) and a third to represent a region assumed to be buried within the protein (loop 450-463).
• Myc epitome display in MSA loop regions In order to determine whether the predicted loops were indeed exposed on the surface of the albumin molecule, mouse serum albumin (MSA) was modified to include the myc epitope, EQKLISEEDL. The myc epitope was inserted in the middle of each of three amino acid segments: between amino acids 57-58 for loop 53-62, amino acids 364-365 for loop 360-369 and amino acids 467-468 for loop 450-467. Cos7 cells were transfected with either wild type MSA or the various myc containing MSA constructs. The presence of the proteins in the medium was first determined by Western blot analysis using antibodies specific for MSA and the myc epitope.
• the conditioned media was either mixed directly with the antibody (N, native) or first denatured in the presence of 0.1%) SDS, 1 mM ⁇ -mercapthoethanol and heat (100 °C for 10 min) and then antibody added (D, denatured). Following immunoprecipitation the presence of the proteins that could be precipitated by the myc antibody were revealed by Western blot analysis using the MSA specific antibody.
• the right panel of Figure 2 shows that, as predicted, the albumin proteins with myc inserted in loops 53-62 and 360-369 were bound by the myc antibody regardless of whether the protein was in its native or denatured form.
• loop 450-463 the protein only bound the antibody when it was first denatured. This experiment clearly demonstrates that loops 53-62 and 360-369 are exposed on the surface of the MSA protein and therefore good for display. Additionally, the 450-463 loop is buried.
• Cos7 cells were grown in the lower chamber of a Transwell® tissue culture plate with BCE cells in the upper chamber.
• FGF was added to the lower chamber or not in the case of no FGF control and the cells allowed to grow for 72 hours.
• those with pAM7-stuffer 6.25 ⁇ M c-RGD peptide was also added.
• Cell growth was determined by a Calcein-binding fluorescence assay.
• the left panel of Figure 3 is a graph of the optical density (OD) for each. The data reveals the addition of FGF results in a 2-fold stimulation of growth of the BCE cells.
• This growth was inhibited by the c-RGD peptide and also by the secreted MSA-myc-RGD protein.
• the right panel is a different way of looking at the same data. In this instance the degree of inhibition of growth is graphed for each.
• the data shows that the MSA-Myc-RGD protein inhibited the growth of the BCE cell by 53% and the degree of inhibition was equivalent to that of the added RGD peptide.
• the RGD peptide displayed on the surface of the MSA molecule inhibited BCE cell growth as efficiently as the endogenously added free RGD peptide demonstrating that the peptide retains its activity in the looped orientation.

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