WO1999032609A1

WO1999032609A1 - Molecules associated with the human fused gene

Info

Publication number: WO1999032609A1
Application number: PCT/SE1998/002384
Authority: WO
Inventors: Rune TOFTGÅRD; Peter G. Zaphiropoulos
Original assignee: Karolinska Innovations Ab
Priority date: 1997-12-19
Filing date: 1998-12-18
Publication date: 1999-07-01
Also published as: AU1991899A

Abstract

The present invention relates to proteins, polypeptides and nucleotides related to the human homologue of the Drosophila fused gene, which is involved in the transduction of signals in the hedgehog-patched (HH-PTC) pathway. The invention also relates to antibodies raised against the polypeptides according to the invention. The molecules according to the invention are useful in diagnostic and therapeutic methods relating to conditions associated with defects in said pathway, especially cancer and certain malformations. Other fields of application of the molecules according to the invention are e.g. studies of embryonic development, gene transcription and tissue repair.

Description

MOLECULES ASSOCIATED WITH THE HUMAN FUSED GENE

Technical field

The present invention relates to novel molecules, such as proteins, polypeptides and nucleotides, involved in the transduction of signals in the hedgehog-patched (HH- PTC) pathway, which takes place during the development of the cells of a human body. The invention also relates to certain advantageous uses of the molecules according to the invention in diagnosis and therapy.

Background

In the study of the development of cells, fruit flies have extensively been used as a model, as they are less complex than mammalian cells.

Pattern formation takes place through a series of logical steps, reiterated many times during the development of an organism. Viewed from a broader evolutionary perspective, across species, the same sort of reiterative pattern formations are seen. The central dogma of pattern formation has been described (Lawrence and Struhl, 1996). Three interlocking and overlapping steps are defined. Firstly, positional information in the form of morphogen gradients allocate cells into non-overlapping sets, each set founding a compartment. Secondly, each of these compartments acquire a genetic address, as a result of the function of active "selector" genes, that specify cell fate within a compartment and also instruct cells and their descendents how to communicate with cells in neighboring compartments. The third step involves interactions between cells in adjacent compartments, initiating new morphogen gradients, which directly organize the pattern.

Taking these steps in greater detail, one finds the first step in patterning to be the definition of sets of cells in each primordium. Cells are allocated according to their positions with respect to both dorsoventral and anterior/posterior axes by morphogen gradients. Allocation of cells in the dorsoventral axis constitutes the germ layers, such as mesoderm or neurectoderm. In segmentation, the second step (the specification of cell fate in each compartment) is carried out by the gene engrailed and elements of the bithorax complex. Engrailed defines anterior and posterior compartments both in segmentation and in limb specification.

The third step in pattern formation, secretion of morphogens, functions to differentiate patterns within compartments (and thereby establish segment polarity). Initially, all cells within a compartment are equipotent, but they become diversified to fom pattern. Pattern formation depends on gradients of morphogens, gradients initiated along compartment boundaries. Such gradients are established by a short-range signal induced in all the cells of the compartment in which the above mentioned selector gene engrailed is active. For segment polarity, this signal is Hedgehog. In the adjacent compartment the selector gene is inactive, ensuring that the cells are sensitive to the signal. The Hedgehog signal range is probably only a few rows of cells wide; responding cells become a linear source of a long-range morphogen, that diffuses outward in all directions. There are three known Hedgehogs, Sonic (SHH), Indian (IHH) and Desert (DHH). The proteins they encode can substitute each for each other, but in wildtype animals, their distinct distributions result in unique activities. SHH controls the polarity of limb growth, directs the development of neurons in the ventral neural tube and patterns somities. IHH controls endochondral bone development and DHH is necessary for spermiogenesis. Vertebrate hedgehog genes are expressed in many other tissues, including the peripheral nervous system, brain, lung, liver, kidney, tooth primordia, genitalia and hindgut and foregut endoderm.

Thus, segment polarity genes have been identified in flies as mutations, which change the pattern of structures of the body segments. Mutations in these genes cause animals to develop the changed patterns on the surfaces of body segments, the changes affecting the pattern along the head to tail axis. For example, mutations in the gene patched cause each body segment to develop without the normal structures in the center of each segment. Instead there is a mirror image of the pattern normally found in the anterior segment. Thus, cells in the center of the segment make the wrong structures, and point them in the wrong direction with reference to the over all head-to-tail polarity of the animal.

About sixteen genes in the class are known. The encoded proteins include kinases, transcription factors, a cell junction protein, two secreted proteins called wingless (WG) and the above mentioned Hedgehog (HH), a single transmembrane protein called patched (PTC) and some novel proteins not related to any known protein. All of these proteins are beleived to work together in signaling pathways that inform cells about their neighbors in order to set cell fates and polarities.

PTC has been proposed as a receptor for HH protein based on genetic experiments in flies. A model for the relationship is that PTC acts through a largely unknown pathway to inactivate both its own transcription and the transcription of the wingless segment polarity gene. This model proposes that HH protein, secreted from adjacent cells, binds to the PTC receptor, inactivates it and thereby prevents PTC from turning off its own transcription or that of wingless. A number of experiments have shown coordinate events between PTC and HH.

WO 96/11260 discloses the isolation of patched genes and the use of the PTC protein to identify ligands, other than the established ligand Hedgehog, that bind thereto. However, even though it is briefly suggested that drugs may be identified which can prevent the transduction of signals by the PTC protein, there are no teachings as regards how such signals are transduced.

In order to elucidate how the Hedgehog elecits signal transduction, a large complex containing the kinesin-related protein costal 2 has been proposed (Robbins et al; Cell: July 25, 1997, 90(2), p. 225-34). Said complex includes the products of at least three genes -.fused (a protein-serine/threonine kinase), cubitus interruptus (a tran- scription factor) and costal2 (a kinesin-like protein). It is concluded that in Droso- phila, the complex may facilitate signaling from HH by governing access of the cu- bitus interruptus protein to the nucleus.

Therond et al have also studied signaling from Hedgehog in Drosophila (Proc. Natl. Acad. Sci. USA, April 30, 1996, 93(9), p. 4224-8). The Drosophila gene fused (fu) encodes a serine/threonine-protein kinase, that genetic experiments have implicated in signaling initiated by hedgehog. It is proposed that the fused protein is phospho- rylated during the course of Drosophila embryogenesis, as a result of hedgehog activity. The conclusion of this reference is, that a reconstruction of signaling from hed- gehog in cell culture should provide further access to the mechanisms by which the hedgehogs acts.

Thus, even though the gene fused (fu) has been identified in Drosophila, it has not been isolated from human beings in the prior art. The prior art teachings about the interactions between hedgehog and patched in mammals are not at all sufficient for practical application in regard of human beings. Firstly, there is a need of a better understanding of how signals are actually elicited and transduced between the hedgehog ligand and the patched receptor. Secondly, the human homologues of the genes and proteins implicated in this pathway must also be fully sequenced, and the cDNA thereof identified, in order to enable recombinant reproduction thereof.

However, due to substantial genetic differences between fruit flies and human beings and the complex nature of the human genome, the isolation of the human homologues to genes identified in fruit flie is in no way a straightforward task.

Summary of the invention

The present invention fulfill the above defined desire by providing novel human homologues to molecules associated with the Drosophila røec. gene and implicated in the transduction of the signals that are elicited by the interaction between the pat- ched receptor (sometimes denoted the NBCCS gene) and any one of the hedgehog ligands. Thus, the present gene has according to the invention been localised to human chromosome 15q22-24. The molecules according to the invention are nucleic acids as well as polypeptides and proteins encoded thereof, which are useful within several fields, including the study of different conditions, such as cancer and deve- lopment of cancer therapies, the regulation of gene transcription, studies of embryonic development, tissue repair etc. Two specific syndromes have been mapped to the 15q22-24 genomic region, namely "Bardet-Biedl Syndrome" and "Cerebral Gigan- tism/Sotus Syndrome" (15q22 or 5q35). The latter is a cancer predisposition syndrome also involving craniofacial defects and growth disturbances making the FUH an interesting candidate gene. The great importance of the present pathway also makes the FUH a potential "oncogene" or "tumor suppressor gene".

The present invention provides an important basic understanding of a signaling pathway that is central to normal development and often disrupted in disease. The new knowledge will also be of great value when considering new therapeutic strategies involving modification of SHH-PTCH signaling. Potential areas include tissue repair/wound healing (brain, bone, cartilage, skin), neurodegenerative disease, testi- cular function and cancer.

Detailed description of the invention

Accordingly, in a first aspect, the present invention relates to a protein molecule capable of being involved in eliciting intracellular signaling in the human HH-PTC pathway, preferably the SHH-PTC pathway. The protein according to the invention exhibits a substantial similarity with the sequence disclosed in SEQ ID NO 2 of the Sequence Listing shown below. In a particular embodiment, the protein according to the invention is comprised of about 80% of said sequence and in a specific embodiment, it is comprised of substantially all of said amino acid sequence. However, it should probably include at least about 60% of said sequence in order to exhibit the advantageous properties as disclosed in the present application. As someone of skill in this area easily realises, the percentages given herein will depend on possible further added fragments or sequence parts, as a protein exhibiting 80% of the sequence of SEQ ID NO 2, to which a further fragment has been added, naturally in itself will include a smaller percentage of the present sequence. Thus, it is to be understood that any sequence including the herein given percentages of SEQ ID NO 2 as disclosed is within the scope of the invention. Analogues of, and functional equivalents to, the proteins according to the invention are also encompassed by the present invention. Thus, the protein according to the invention is an isolated human fused protein, (FUH).

According to the present invention, it has been found that the human homologue of Drosophila/wsec. is an intracellular transducer of the signal elicited at the cell membrane by interaction of ligands, such as sonic hedgehog (SHH), indian hedgehog (IHH) and desert hedgehog (DHH), with the receptor human homologue of Drosophila patched (PTC). This interaction leads to initiation of a signaling pathway by allowing PTC or its coreceptor, human homologue of Drosophila smoothened

(SMOH), to activate intracellular signal transducers. As mentioned above, in Drosophila cells, fused interacts with the microtubule associated protein costal-2 (cos-2) and the transcription factor cubitus interruptus (ci). The latter has human homologues denoted GLI 1-3.

Thus, in a second aspect, the present invention also relates to polypeptides comprised of suitable fragments or parts of the amino acid sequence of the above described protein. Thus, such a polypeptide is also associated with the HH-PTC pathway, such as SHH-PTC, and is comprised of a subsequence of up to about 20, such as 8-15, preferably 9-12 and most preferred 10 contiguous amino acids of the sequence disclosed in SEQ ID NO 2 or conservative substitutions of said sequence. The polypeptide according to the invention may be encoded by a nucleic acid amplified from genomic DNA or RNA using appropriate primers which the skilled man in this field easily chooses. In a specific embodiment, the polypeptide according to the invention is presented as an antigen, which elicits the production of an antibody which speci- fically binds to a polypeptide encoded by a nucleic acid according to SEQ ID NO 1 , and said polypeptide does not bind to antisera raised against a polypeptide encoded by a nucleic acid sequence according to SEQ ID NO 1 , which has been fully immu- nosorbed with a polypeptide encoded by a sequence of SEQ ID NO 2.

A third aspect of the present invention is a nucleic acid encoding any one of the above defined proteins or polypeptides. In a preferred embodiment, the nucleic acid according to the invention is such a DNA sequence as disclosed in SEQ ID NO 1. Thus, according to the present invention, for the first time, a human homologue of the

has been isolated, the cDNA thereof has been established, sequenced and cloned. The complete cDNA sequence thereof is disclosed in SEQ ID NO 1. The present invention also encompasses nucleotides that hybridises to part or all. of the sequence of SEQ ID NO 1 as well as to nucleotides hybridising to the DNA encoding the proteins and polypeptides according to the invention. Such nuc- leotides may e.g. be RNA sequences.

The human homologue according to the invention exhibits an amino acid similarity with Drosophilay sefi. of 54%. Within the kinase domain, the similarity increases to 59,5%. Further, the present human homologue of fused exhibits an amino acid iden- tity with Drosophila w^ec. of 33,5%). Within the kinase domain, the identity increases to 37%).

In a fourth aspect, the present invention also relates to a vector comprising a nucleic acid according to the invention, such as part of, or all of, the DNA sequence disclo- sed in SEQ ID NO 1. The vector preferably include the above described nucleic acid operably linked, i.e. under the control of, a promoter; either constitutive or inducib- le. The vector can also include suitable initiation and termination codons. In addition, the invention also relates to an expression cassette comprising any one of the nucleic acids according to the invention. Accordingly, yet another aspect of the invention is a cell comprising an expression cassette according to the invention. In one further aspect, the invention relates to an antibody which specifically binds to such a polypeptide as defined above, which preferably comprises at least about 10, more preferably at least 20, 40 or 50 and most preferably at least 100 or 200, or even 400, amino acids. Alternatively, the antibody binds to a protein encoded by essentially all of the sequence as disclosed in SEQ ID NO 1. The antibody is polyclonal or monoclonal, preferably, it is a monoclonal antibody. It can be humanized or human.

One specific embodiment of the present invention is a kinase-activating substance, which is capable of activating a protein according to the invention. Alternatively, the invention relates to a kinase-inhibiting substance. Such kinase-regulating substances may be used as drugs. Pharmaceutical compositions to this end comprise a kinase-activator or kinase-inhibitor capable of influencing the protein according to the invention together with a pharmaceutically acceptable carrier.

The present invention also relates to a cell, such as a recombinant cell, e.g. hybri- domas or triomas, expressing such an antibody as defined above. Also encompassed by the invention is the use of such an antibody as a medicament or in the manufacture of a specific drug or therapy.

Thus, another aspect of the invention is a pharmaceutical composition which comprises a molecule selected from the group consisting of a vector encoding a polypep- tide according to the invention or a subsequence thereof, a polypeptide according to the invention or a subsequence thereof and an antibody raised against the present polypeptides together with a pharmaceutically acceptable carrier.

Yet another aspect of the invention is a kit for detection of a human fused gene, which kit comprises a nucleic acid sequence capable of hybridising to an essential part of the sequence disclosed in SEQ ID NO 1, preferably under stringent conditions. The invention also relates to a kit for detection of a protein encoded by a human fused gene sequenced according to the present invention, comprising a container containing an antibody as defined above.

In a last aspect, the invention also relates to methods for detecting any deviations from a normal human fused gene. The method includes the steps of a) providing a biological sample of the organism and b) detecting a human fused gene or gene product in the sample. The provision of a biological sample and detection methods will be described below.

The invention also relates to diagnostic and prognostic methods, wherein mutations in pathway components are detected, as well as to kits for performing such methods.

Finally, the present invention also relates to methods of treatment, wherein the above defined molecules are used. Conditions that may be contemplated include cancers, such as basal cell cancer of the skin, medulloblastomas, trichoepitheliomas and breast cancer, conditions caused by defects in brain development, lung development, tooth and hair development and sperm formation, and tissues repair. The present molecules are also useful in gene therapy. The methods according to the invention can involve transfecting cells of a mammal with a vector expressing a polypeptide or antibody according to the invention. The transfection can be in vivo or ex vivo. Ex vivo transfection is suitably followed by re-infusing the cells into the organism. Other methods involve administering to the mammal, e.g. a human, of a therapeuti- cally effective dose of a composition comprising a polypeptide according to the invention and a pharmacological excipient and/or carrier.

Definitions

The term "antibody" refers to a polypeptide substantially encoded by an immunog- lobulin gene or immunoglobulin genes, or fragments thereof which specifically bind and recognize an analyte (antigen). The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Light chains are classified as either kappa or lambda. Heavy chains are classified as gamma, mu alpha, del- ta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.

An exemplary immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light" (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition. The terms variable light chain (V ) and variable haeavy chain (V_H) refer to these light and heavy chains, respectively.

Antibodies exist e.g., as intact immunoglobulins or as a number of well characteri- zed fragments produced by digestion with various peptidases. Thus, for example, pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'₂, a dimer of Fab which itself is a light chain joined to V_H-C_H1 by a disulfide bond. The F(ab)'₂ may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'₂ dimer into an Fab' mo- nomer. The Fab' monomer is essentially a Fab with part of the hinge region (see, Fundamental Immunology, Third Edition, W.E. Paul, ed., Raven Press, N.Y. 1993). While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv).

A "chimeric antibody" is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site (variable region) is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.

The term "immunoassay" is an assay that utilizes an antibody to specifically bind an analyte. The immunoassay is characterized by the use of specific binding properties of a particular antibody to isolate, target, and/or quantify the analyte.

The terms "isolated" "purified" or "biologically pure" refer to material which is substantially or essentially free from components which normally accompany it as found in its native state.

The term "nucleic acid" refers to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, encompasses known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.

A "label" is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include ³²P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in a ELISA), biotin, dioxigenin, or haptens and proteins for which antisera or monoclonal antibo- dies are available (e.g., the peptide of SEQ ID NO 1 can be made detectable, e.g., by incorporating a radio-label into the peptide, and used to detect antibodies specifically reactive with the peptide).

As used herein a "nucleic acid probe" is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation. As used herein, a probe may include natural (i.e. A, G, C, U or T) or modified bases (7-deazaguanosine, inosine, etc.) In addition, the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it do- es not interfere with hybridization. Thus, for example, probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather that phosphodiester linkages. It will be understood by one of skill in the art that probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions. The probes are preferably directly labeled as with isotopes, chromophores, lumiphore, chromo- gens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.

A "labeled nucleic acid probe" is a nucleic acid probe that is bound, either cova- lently, through a linker, or through ionic, van der Waals or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.

The term "target nucleic acid" refers to a nucleic acid (often derived from a biological sample), to which a nucelic acid probe is designed to specifically hybridize. It is either the presence or absence of the target nucleic acid that is to be detected, or the amount of the target nucleic acid that is to be quantified. The target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding probe directed to the target. The term target nucleic acid may refer to the specific subsequence of a larger nucleic acid to which the probe is directed or to the overall sequence (e.g., gene or mRNA) whose expression level it is desired to detect. The difference in usage will be apparent from the context.

"Subsequence" refers to a sequence of nucleic acids or amino acids that comprise a part of a longer sequence of nucleic acids or amino acids (e.g., polypeptide), respectively.

The term "recombinant" when used with reference to a cell, or nucleic acid, or vector, indicates that the cell, or nucleic acid, or vector, has been modified by the intro- duction of a heterologous nucleic acid or the alteration of a native nucleic acid, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.

The term "identical" in the context of two nucleic acids or polypeptide sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl. Math. 2: 482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci. USA 85: 2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wl) or by inspection.

An additional algorithm that is suitable for determining sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/). This algorithm involves first identifying high scoring sequence pair (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al, supra.) These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are extended in both directions along each sequence for as fas as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achie- ved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T and X determine the sensitivity and speed of the alignment. The BLAST program uses as defaults a word length (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Hen ikoff (1992) Proc. Natl. Acad. Sci. USA 89 : 10915 - 10919) alignments (B) of 50, expectation (E) of 10, M=5, N=4, and a comparison of both strands.

The BLAST algorithm performs a statistical analysis of the similarity between two sequences; see e.g., Karlin and Altschul (1993) Proc. Nat 'I Acad. Sci. USA 90: 5873-5787. One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance. For example, a nucleic acid is considered similar to a fused gene or cDNA if the smallest sum probability in a comparison of the test nucleic acid to a fused nuc- leic acid is less than about 1, preferably less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.

The term "substantial identity" or "substantial similarity" in the context of a polypeptide indicates that a polypeptides comprises a sequence with at least 70% sequence identity to a reference sequence, or preferably 80%, or more preferably 85% sequence identity to the reference sequence, or most preferably 90%) identity over a comparison window of about 10-20 amino acid residues. An indication that two polypeptide sequences are substantially identical is that one peptide is immu- nologically reactive with antibodies raised against the second peptide. Thus, a poly- peptide is substantially identical to a second polypeptide, for example, where the two peptides differ only by a conservative substitution.

An indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.

Another indication that two nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions.

"Bind(s) substantially" refers to complementary hybridization between a probe nucleic acid and a target nucleic acid and embraces minor mismatches that can be accomodated by reducing the stringency of the hybridization media to achieve the desired detection of the target polynucleotide sequence.

The phrase "hybridizing specifically to", refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA. The term "stringent conditions" refers to conditions under which a probe will hybridize to its target subsequence, but to no other sequences. Stringent condi- tions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C lower than the thermal melting point Tm for the specific sequence at a defined ionic strength and pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at Tm, 50%> of the probes are occupies at equilibrium). Typically, stringent conditions will be those in which the salt concentration is less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. The phrases "specifically binds to a protein" or "specifically immunoreactive with", when referring to an antibody refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind preferentially to a particular protein and do not bind in a significant amount to other proteins present in the sample. Specific binding to a protein under such conditions requires an antibody that is selected for its specificity for a particu- lar protein. A variety of immunoassay formats may be used to select antibodies specifically immunoreactive with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select monoclonal antibodies specifically immunoreactive with a protein. See Harlow and Lane (1988) Antibodies, A Laboratory Manual, Cold Spring Harbour Publications, New York, for a description of immu- noassay formats and conditions that can be used to determine specific immunoreac- tivity.

A "conservative substitution", when describing a protein, refers to a change in the amino acid composition of the protein that does not substantially alter the protein's activity. Thus, "conservatively modified variations" of a particular amino acid sequence refers to amino acid substitutions of those amino acids that are not critical for protein activity or substitution of amino acids with other amino acids having similar properties (e.g., acidic, basic, positively or negatively charged, polar or non- polar, etc.) such that the substitutions of even critical amino acids do not substanti- ally alter activity. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are conservative substitutions for one another:

1) Alanine (A), Serine (S), Threonine (T);

2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), glutamine (Q);

4) Arginine (R), Lysine (K);

5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

See also, Creighton (1984) Protein, W.H. Freeman and Company. In addition, indi- vidual substitutions, deletions or additions which alter, add or delete a single amino acid or a small percentage of amino acids in an encoded sequence are also "conservatively modified variations".

A "gene product", as used herein, refers to a nucleic acid whose presence, absence, quantity, or nucleic acid sequence is indicative of a presence, absence, quantity, or nucleic acid composition of the gene. Gene products thus include, but are not limited to, an mRNA transcript a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA or subsequences of any of these nucleic acids. Polypeptides expressed by the gene or subsequences thereof are also gene products. The particular type of gene product will be evident from the context of the usage of the term.

EXPERIMENTAL General methods L Uses ofthe /j/MK/ cDNA

Among other applications, detection of defective fused gene expression is of clinical value. The presence of a fused gene, cDNA, mRNA, protein or subsequences of the gene, cDNA or protein in a biological sample is useful, e.g. as a marker to asses in vivo and/or in situ RNA transcription and/or translation, in cancer diagnostics, in prophylaxis etc. Full-length fused cDNA, individual exons, or subsequences thereof are also useful as probes (particularly when labeled) for the detection of the presence or absence and/or quantification of normal or abnormal (e.g. truncated or mutated) fused DNA or RNA in a biological sample. The labeled probes can also be useful as in karyotyping analysis as markers of the fused gene. The proteins and polypeptides according to the invention are also useful as lead compounds in the design of organic molecules resembling the native protein, fully or in part. The designed molecules preferably exhibits the activity of the native one. Methods for protein mimicking are today used widely and the skilled man can perform such a method using the present molecules as lead compounds with reference to the literature and well known techniques. In addition, the molecules according to the invention are also useful in screening for various substances.

Further, vectors according to the invention may be used for expression of those proteins to provide immunogens for antibody production. Vectors encoding the pre- sent proteins and /or polypeptides are also useful for transforming cells in vitro or in vivo to express the proteins and/or polypeptides.

Cells and/or tissues expressing the fused gene may be used to monitor expression levels of fused polypeptides in a wide variety of contexts.

II. The fused gene

A) The human fused gene (FUH)

SEQ ID NO 1 provides the cDNA sequence for the largest nucleic acid according to the invention, while SEQ ID NO 2 provides the corresponding protein sequence.

B) Isolation of cDNA and/or probes.

The nucleic acids of the present invention are cloned, or amplified by in vitro methods, such as the polymerase chain reaction (PCR), the ligase chain reaction (LCR), the transcription-based amplification system (TAS), the self-sustained sequence replication system (SSR). A wide variety of cloning and in vitro amplification methodologies are well-known to persons of skill. Examples of these techniques and instructions sufficient to direct persons of skill through many cloning exercises are found in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology 152 Academic Press, Inc., san Diego, CA (Berger); Sambrook et al. (1989) Molecular Cloning - A Laboratory Manual (2nd ed.) Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring harbor Press, NY, (Sambrook et al); Current Protocols in Molecular Biology, F.M. Ausubel et al, Current Protocols, a joint venture between Greene Publishing Associates, Inc. And John Wiley & Sons, Inc., (1994 Supplement) (Ausubel); Cashion et al., U.S. patent number 5,017,478; and Carr, European Patent No. 0,246,864. Examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, Sambrook, and Ausubel, as well as Mullis et al, (1987) U.S. Patent No. 4,683,202; PCR Protocols A Guide to Methods and Applications (Innis et al. Eds) Academic Press Inc. San Die- go, CA (1990) (Innis); Arnheim & Levinson (October 1, 1990)Cά£N 36-47; The Journal Of NIH Research (1991) 3: 81-94 (Kwoh et al. (1989) Proc. Natl Acad. Sci. USA 86: 1173; Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87, 1874; Lo- mell et al (1989) J. Clin. Chem., 35: 1826; Landegren et α/., (1988) Science, 241 : 1077-1080; Van Brunt (1990) Biotechnology, 8: 291-294; Wu and Wallace, (1989) Gene, 4: 560; and Barringer et al. (1990) Gene, 89: 117.

In one preferred embodiment, the human fused cDΝA can be isolated by routine cloning methods. The cDΝA sequence provided in SEQ ID NO: 1 can be used to provide probes that specifically hybridize to the fused gene, in a genomic DNA sample, or to the fused mRNA, in a total RNA sample (e.g., in a Northern blot). Once the target fused nucleic acid is identified (e.g., in a Southern blot), it can be isolated according to standard methods known to those of skill in the art (see, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd Ed. Vols. 1- 3, Cold Spring Harbor Laboratory; Berger and Kimmel (1987) Methods in Enzy- mology, Vol. 152: Guide to Molecular Cloning Techniques, San Diego: Academic press, Inc.; or Ausubel et al. (1987) Current Protocols in Molecular Biology, Greene Publishing and Wiley-Interscience, New York).

In another preferred embodiment, the human fused cDNA is isolated by amplifica- tion methods, such as polymerase chain reaction (PCR).

C Labeling of nucleic acid probes.

Where the fused cDNA or its subsequences are to be used as nucleic acid probes, it is often desirable to label the sequences with detectable labels. The labels may be incorporated by any of a number of means well known to those of skill in the art. However, in a preferred embodiment, the label is simultaneously incorporated during the amplification step in the preparation of the sample nucleic acids. Thus, for example, polymerase chain reaction (PCR) with labeled primers or labeled nucleotides will provide a labeled amplification product. In another preferred embodiment, transcription amplification using a labeled nucleotide (e.g. fluorescein-labeled UTP and/or CTP) incorporates a label into the transcribed nucleic acids.

Alternatively, a label may be added directly to an original nucleic acid sample (e.g., mRNA, polyA mRNA, cDNA, etc.) or to the amplification product after the ampli- fication is completed. Means of attaching labels to nucleic acids are well known to those of skill in the art and include, for example nick translation or end labeling (e.g. with a labeled RNA) by kinasing of the nucleic acid and subsequent attachment (ligation) of a nucleic acid linker joining the sample nucleid acid to a label (e.g., a fluorophore).

Detectable labels suitable for use in the present invention include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include biotin for staining with labeled streptavidin conjugate, magnetic beads (e.g., Dynabe- ads™), fluorescent dyes (e.g., fluorescein, texas red, thodamine, green fluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as collodial gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads. Patents teaching the use of such labels incluse U.S. Patent Nos. 3,871,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241.

Means of detecting such labels are well known to those of skill in the art. Thus, for example, radiolabels may be detected using photographic film or scintillation counters, fluorescent markers may be detected using a photodetector to detect emitted light. Enzymatic labels are typically detected by providing the enzyme with a substrate and detecting the reaction product produced by the action of the enzyme on the substrate, and colorimetric labels are detected by simply visualizing the colored label.

III. Antibodies to the fused polypeptide(s).

Antibodies are raised to the fused polypeptides of the present invention, including individual, ellelic, strain, or species variants, and fragments thereof, both in their naturally occurring (full-length) forms and in recombinant forms. Additionally, antibodies are raised to these polypeptides in either their native configurations or in non-native configurations. Anti-idiotypic antibodies can also be generated. Many methods of making antibodies are known to persons of skill. The following discussion is presented as a general overview of the techniques available; however, one of skill will recognize that many variations upon the following methods are known.

A) Antibody Production

A number of immunogens are used to produce antibodies specifically reactive with polypeptides according to the invention. Recombinant or synthetic polypeptides of 10 amino acids in length, or greater, selected from amino acid sub-sequences of SEQ ID NO 2 are the preferred polypeptide immunogen (antigen) for the production of monoclonal or polyclonal antibodies. In one class of preferred embodiments, and immunogenic peptide conjugate is also included as an immunogen. Naturally oc- curing polyptides are also used either in pure or impure form.

Recombinant polypeptides are expressed in eukaryotic or prokaryotic cells (as described below) and purified using standard techniques. The polypeptide, or a synthetic version thereof, is then injected into an animal capable of producing antibodies. Either monoclonal or polyclonal antibodies can be generated for subsequent use in immunoassays to measure the presence and quantity of the polypetide.

Methods of producing polyclonal antibodies are known to those of skill in the art. In brief, an immunogen (antigen), preferably a purified polypeptide, a polypeptide coupled to an appropriate carrier (e.g., GST, keyhole limpet hemanocyanin, etc.), or a polypeptide incorporated into an immunization vector such as a recombinant vaccinia virus (see, U.S. Patent No. 4,722,848) is mixed with an adjuvant and animals are immunized with the mixture. The animal's immune response to the immunogen preparation is monitored by taking test bleeds and determining the titer of reactivity to the polypeptide of interest. When appropriately high titers of antibody to the immunogen are obtained, blood is collected from the animal and antisera are prepared. Further fractionation of the antisera to enrich for antibodies reactive to the polypep- tide is performed where desired (see, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY; and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold Spring Harbor Press, NY).

Antibodies, including binding fragments and single chain recombinant versions the- reof, against predetermined fragments of the present polypeptides are raised by immunizing animals, e.g., with conjugates of the fragments with carrier proteins as described above. Typically, the immunogen of interest is a peptide of at least about 5 amino acids, more typically the peptide is 10 amino acids in length, preferably, the fragment is 15 amino acids in length and more preferably the fragment is 20 amino acids in length or greater. The peptides are typically coupled to a carrier protein (e.g., as a fusion protein), or are recombinantly expressed in a immunization vector. Antigenic determinants on peptides to which antibodies bind are typically 3 to 10 amino acids in length.

Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies are screened for binding to normal or modified polypeptides, or screened for agonistic or antagonistic activity, e.g., activity mediated through a fused protein. Specific monoclonal and polyclonal antibodies will usually bind with a K_D of at least about 0.1 mM, more usually at least about 50 μM, and most preferably at least about 1 μM or better.

In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies are found in, e.g. Stites et αl. (eds) Basic and Clinical Immunology (4th ed.) Lange Medical Publications, Los Altos, CA, and references cited therin; Harlow and Lane, supra; Goding (1986) Monoclonal Antiboides: Principles and Practice (2d ed.) Academic Press, New York, NY; and Kohler and Milstein (1975) Nature 256: 495-497. Summarized briefly, this method proceeds by injecting an animal with an immunogen. The animal is then sa- crificed and cells taken from its spleen, which are fused with myeloma cells. The result is a hybrid cell or "hybridoma" that is capable of reproducing in vitro. The population of hybridomas is then screened to isolate individual clones, each of which secrete a single antibody species to the immunogen. In this manner, the individual antibody species obtained are the products of immortalized and cloned single B cells from the immune animal generated in response to a specific site recognized on the immunogenic substance.

Alternative methods of immortalization include transformation with Epstein Ban- Virus, oncogenes, or retroviruses, or other methods known in the art. Colonies arising from single immortalized cells are screened for production of antibodies of the desired specificity and affinity for the antigen, and yield of the monoclonal antibodies produced by such cells in enhanced by various techniques, including injection into the peritoneal cavity of a vertebrate (preferably mammalian) host. The po- lypetides and antibodies of the present invention are used with or without modifica- tion, and include chimeric antibodies such as humanized murine antibodies.

Other suitable techniques involve selection of libraries of recombinant antibodies in phage or similar vectors (see, e.g., Huse et al. (1989) Science 246: 1275-1281 ; and Ward, et al. (1989) Nature 341 : 544-546; and Vaughan et al. (1996) Nature Bio- technology, 14: 309-314).

Frequently, the polypetides and antibodies will be labeled by joining, either cova- lently or non-covalently, a substance which provides for a detectable signal. A wide variety of labels, and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionucleotides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents teaching the use of such labels include U.S. Patent Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and 4,366,241. Also recombinant immunoglobulins may be produced. See, Cabalitty, U.S. Patent No. 4,816,567; and Queen et al. (1989) Proc. Nat'l Acad. Sci. USA 86: 10029-10033.

The antibodies of this invention are also used for affinity chromatography in isola- ting fused polypeptides. Columns are prepared, e.g., with the antibodies linked to a solid support, e.g., particles, such as agarose, Sephadex, or the like, where a cell ly- sate is passed through the column, washed, and treated with increasing concentrations of a mild denaturant, whereby purified/t/sec. polypeptides are released.

The antibodies can be used to screen expression libraries for particular expression products such as normal or abnormal human fused protein. Usually the antibodies in such a procedure are labeled with a moiety allowing easy detection of presence of antigen by antibody binding.

Antibodies raised against fused polypeptides can also be used to raise antiidiotypic antibodies. These are useful for detecting or diagnosing various pathological conditions related to the presence of the respective antigens.

B) Human or humanized (chimeric) antibody production.

The antibodies of this invention can also be administered to an organism (e.g., a human patient) for therapeutic purposes. Antibodies administered to an organism other than the species in which they are raised are often immunogenic. Thus, for example, murine antibodies administered to a human often induce an immunologic response against the antibody (e.g., the human anti-mouse antibody (HAMA) response) on multiple administrations. The immunogenic properties of the antibody are reduced by altering portions, or all, of the antibody into characteristically human sequences thereby producing chimeric or human antibodies, respectively.

i) Humanized (chimeric) antibodies.

Humanized (chimeric) antibodies are immunoglobulin molecules comprising a hu- man and non-human portion. More specifically, the antigen combining region (or variable region) of a humanized chimeric antibody is derived from a non-human source (e.g., murine) and the constant region of the chimeric antibody (which confers biological effector function to the immunoglobulin) is derived from a human source. The humanized chimeric antibody should have the antigen binding specifi- city of the non-human antibody molecule and the effector function conferred by the human antibody molecule. A large numer of methods of generating chimeric antibodies are well known to those of skill in the art (see, e.g., U.S. Patent Nos: 5,502,167, 5,500,362, 4,491,088, 5,482,856, 5,472,693, 5,354,847, 5,292,867, 5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235, 5,075,431, and 4,975,369). In general, the procedures used to produce these chimeric antibodies consist of the following steps (the order of some steps may be interchanged): (a) identifying and cloning the correct gene segment encoding the antigen binding portion of the antibody molecule; this gene segment (known as the DVJ, variable, diversity and joi- ning regions for heavy chains or VJ, variable, joining regions, for light chains (or simply as the V or Variable region) may be in either the cDNA or genomic form; (b) cloning the gene segments encoding the constant region or desired part thereof; (c) ligating the variable region to the constant region so that the complete chimeric antibody is encoded in a transcribable and translatable form; (d) ligating this construct into a vector containing a selectable marker and gene control regions such as promoters, enhancers and poly(A) addition signals; (e) amplifying this construct in a host cell (e.g., bacteria; (f) introducing the DNA into eukaryotic cells (transfections) most often mammalian lymphocytes; and culturing the host cell under conditions suitable for expression of the chimeric antibody.

Antibodies of several distinct antigen bindning specificities have been manipulated by these protocols to produce chimeric proteins (e.g., anti-TNP: Boulianne et al. (1984) Nature, 312: 643; and anti-tumor antigens: Sahagan et al. (1986) J. Immunol, 137: 1066). Likewise several different effector functions have been achieved by linking new sequences to those encoding the antigen binding region. Some of these inlcude enzymes (Neuberger et al. (1984) Nature 312:604), immunoglobulin constant regions from another species and constant regions of another immunoglobulin chain (Sharon et al. (1984) Nature 309: 364; Tan et al, (1985) J. Immunol. 135: 3565-3567).

In one preferred embodiment, a recombinant DNA vector is used to transfect a cell line that produces an

antibody. The novel recombinant DNA vector contains a "replacement gene" to replace all or a portion of the gene encoding the immunoglobulin constant region in the cell line (e.g., a replacement gene may encode all or a portion of a constant region of a human immunoglobulin, a specific immu- noglobulin class, or an enzyme, a toxin, a biologically active peptide, a growth factor, inhibitor, or a linker peptide to facilitate conjugation to a drug, toxin, or other molecule, etc.), and a "target sequence" which allows for targeted homologous recombination with immunoglobulin sequences within the antibody producing cell.

In another embodiment, a recombinant DNA vector is used to transfect a cell line that produces an antibody having a desired effector function, (e.g., a constant region of a human immunoglobulin) in which case, the replacement gene contained in the recombinant vector may encode all or a portion of a region of an anti-fused antibody and the target sequence contained in the recombinant vector allows for homologous recombination and targeted gene modification within the antibody producing cell. In either embodiment, when only a portion of the variable or constant region is replaced, the resulting chimeric antibody may define the same antigen and/or have the same effector function yet be altered or improved so that the chimeric antibody may demonstrate a greater antigen specificity, greater affinity binding constant, increased effector function, or increased secretion and production by the transfected antibody producing cell line, etc.

Regardless of the embodiment practiced, the processes of selection for integrated DNA (via a selectable marker), screening for chimeric antibody production, and cell cloning, can be used to obtain a clone of cells producing the chimeric antibody.

Thus, a piece of DNA which encodes a modification for a monoclonal antibody can be targeted directly to the site of the expressed immunoglobulin gene within a B-cell or hybridoma cell line. DNA constructs for any particular modification may be used to alter the protein product of any monoclonal cell line or hybridoma. Such a procedure circumvents the costly and time consuming task of cloning both heavy and light chain variable region genes from each B-cell close expressing a useful antigen specificity. In addition to circumventing the process of cloning variable region ge- nes, the level of expression of chimeric antibody should be higher when the gene is at its natural chromosomal location rather than at a random position. Detailed methods for preparation of chimeric (humanized) antibodies can be found in U.S. Patent 5,482,856.

ii) Human antibodies.

In another embodiment, this invention provides for fully human anti-fused antibodies. Human antibodies consist entirely of characteristically human polypeptide sequences. The human antibodies of this invention can be produced in using a wide variety of methods (see, e.g., Larrick et αl, U.S. Pat. No 5,001,065, for review). In one preferred embodiment, the human antibodies of the present invention are usually produced initially in trioma cells. Genes encoding the antibodies are then cloned and expressed in other cells, particularly, nonhuman mammalian cells.

The general approach for producing human antibodies by trioma technology has be- en described by Ostberg et αl. (1983) Hybridoma 2: 361-367, Ostberg, U.S. Pat. No. 4,634,664, and Engelman et al. U.S. Pat. No. 4,634,666. The antibody-producing cell lines obtained by this method are called triomas because they are descended from three cells; two human and one mouse. Triomas have been found to produce antibody more stably than ordinary hybridomas made from human cells.

Preparation of trioma cells requires an initial fusion of a mouse myeloma cell line with unimmunized human peripheral B lymphocytes. This fusion generates a xenogenic hybrid cell containing both human and mouse chromosomes (see, Engelman, supra.). Xenogenic cells that have lost the capacity to secrete antibodies are selec- ted. Preferably, a xenogenic cell is selected that is resistant to 8-azaguanine. Cells possessing resistance to 8-azaguanine are unable to propagate on hypoxanthine- aminopterin-thymidine (HAT) or azaserine-hypoxanthine (AH) media.

The capacity to secrete antibodies is conferred by a further fusion between the xe- nogenic cell and B-lymphocytes immunized against a polypeptide according to the invention or an epitope thereof. The B-lymphocytes are obtained from the spleen, blood or lymph nodes of human donor. If antibodies against a specific antigen or epitope are desired, it is preferable to use that antigen or epitope thereof as the immunogen rather than fused polypeptide. Alternatively, B-lymphocytes are obtained from an unimmunized individual and stimulated with the present polypeptide, or an epitope thereof, in vitro. In a further variation, B-lymphocytes are obtained from an infected, or otherwise immunized individual, and then hyperimmunized by exposure to a fused polypeptide for about seven to fourteen days, in vitro. The immunized B-lymphocytes prepared by one of the above procedures are fused with a xenogenic hybrid cell by well known methods. For example, the cells are treated with 40-50% polyethylene glycol of MW 1000-4000, at about 37°C for about 5-10 min. Cells are separated from the fusion mixture and propagated in media selective for the desired hybrids. When the xenogenic hybrid cell is resistant to 8- azaguanine, immortalized trioma cells are conveniently selected by successive pas- sage of cells on HAT or AH medium. Other selective procedures are, of course, possible depending on the nature of the cells used in fusion. Clones secreting antibodies having the required binding specificity are identified by assaying the trioma culture medium for the ability to bind to the present polypeptide or an epitope thereof. Triomas producing human antibodies having the desired specificity are subcloned by the limiting dilution technique and grown in vitro in culture medium, or are injected into selected host animals and grown in vitro.

The trioma cell lines obtained are then tested for the ability to bind a polypeptide or an epitope thereof. Antibodies are separated from the resulting culture medium or body fluids by conventional antibody-fractionation procedures, such as ammonium sulfate precipitation, DEAE cellulose chromatography and affinity chromatography.

Although triomas are genetically stable they do not produce antibodies at very high levels. Expression levels can be increased by cloning antibodies genes from the tri- oma into one or more expression vectors, and transforming the vector into a cell line, such as the cell lines typically used for expression of recombinant or humanized immunoglobulins. As well as increasing yield of antibody, this strategy offers the additional advantage that immunoglobulins are obtained from a cell line that does not have a human component, and does not therefore need to be subjected to the especially extensive viral screening required for human cell lines.

The genes encoding the heavy and light chains of immunoglobulins secreted by trioma cell lines are cloned according to methods, including the polymerase chain reaction, known in the art (see, e.g., Sambrook et al, Molecular Cloning: A Laborato- ry Manual, 2nd ed., Cold Spring Harbor, N.Y., 1989; Berger & Kimmel, Methods in Enzymology, Vol. 152: Guide to Molecular Cloning Techniques, Academic Press, Inc., San Diego, Calif, 1987; Co et al. (1992) J. Immunol, 148: 1149). For example, genes encoding heavy and light chains are cloned from a trioma' s genomic DNA or cDNA produced by reverse transcription of the trioma' s RNA. Cloning is ac- complished by conventional techniques including the use of PCR primers that hybridize to the sequences flanking or overlapping the genes, or segments of genes, to be cloned.

Typically, recombinant constructs comprise DNA segments encoding a complete human immunoglobulin heavy chain and/or a complete human immunoglobulin light chain of an immunoglobulin expressed by a trioma cell line. Alternatively, DNA segments encoding only a portion of the primary antibody genes are produced, which portions possess binding and/or effector activities. Other recombinant constructs contain segments of trioma cell line immunoglobulin genes fused to seg- ments of other immunoglobulin genes, particularly segments of other human constant region requences (heavy and/or light chain). Human constant region sequences can be selected from various reference sources, including but not limited to those listed in Kabat et al. (1987) Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services. In addition to the DNA segments encoding an -fused immunoglobulins or fragments therof, other substantially homologous modified immunoglobulins can be readily designed and manufactured utilizing various recombinant DNA techniques known to those skilled in the art such as site-directed mutagenesis (see Gillman & Smith (1979) Gene, 8: 81-97; Roberts et αl (1987) Nature 328: 731-734). Such modified segments will usually retain antigen binding capacity and/or effector function. Moreover, the modified segments are usually not so far changed from the original trioma genomic sequences to prevent hybridization to these sequences under stringent conditions. Because, like many genes, immunoglobulin genes contain separate functional regions, each having one or more distinct biological activites, the genes may be fused to functional regions from other genes to produce fusion proteins (e.g., immunotoxins) having novel properties or novel combinations of properties.

The recombinant polynucleotide constructs will typically include an expression control sequence operably linked to the coding sequences, including naturally associated or heterologous promoter regions. Preferably, the expression control sequences will be eukaryotic promoter systems in vectors capable of transforming or transfecting eukaryotic host cells. Once the vector has been incorporated into the appropriate host, the host is maintained under conditions suitable for high level ex- pression of the nucleotide sequences, and the collection and purification of the human anti-fused immunoglobulins.

These expression vectors are typically replicable in the host organisms either as epi- somes or as an integral part of the host chromosomal DNA.. Commonly, expression vectors will contain selection markers, e.g. ampicillin-resistance or hygromycin- resistance, to permit detection of those cells transformed with the desired DNA sequences.

In general, prokaryotes can be used for cloning the DNA sequences encoding a hu- man anti-fused immunoglobulin chain. E. coli is one prokaryotic host particularly useful for cloning the DNA sequences of the present invention. Microbes, such as yeast are also useful for expression. Saccharomyces is a preferred yeast host, with suitable vectors having expression control sequences, an origin of replication, termination sequences and the like as desired. Typical promoters include 3- phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include, among others, promoters from alcohol dehydrogenase 2, isocytochrome C, and enzymes responsible for maltose and galactose utilization.

Mammalian cells are a particularly preferred host for expressing nucleotide seg- ments encoding immunoglobulins or fragments thereof (see, e.g. Winnacker, From Genes to Clones, VCH Publishers, N.Y., 1987). A number of suitable host cell lines capable of secreting intact heterologous proteins have been developed in the art, and include CHO cell lines, various COS cell lines, HeLa cells, L cells and myeloma cell lines. Preferably, the cells are nonhuman. Expression vectors for these cells can include expression control sequences, such as an origin of replication , a promoter, an enhancer (Queen et al. (1986) Immunol. Rev. 89:49), and necessary processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences. Preferred expression control sequences are promoters derived from endogenous genes, cytomegalovirus, SV40, adeno- virus, bovine pappillomavirus, and the like (see, e.g., Co et al. (1992) J. Immunol. 1458: 1149).

The vectors containing the DNA segments of interest can be transferred into the host cell by well-known methods, depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, whereas calcium phosphate treatment, electroporation, lipofection, biolistics or viral-based transfection may be used for other cellular hosts. Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection (see, generally, Sambrook et al, supra). Once expressed, human anti-fused immunoglobulins of the invention can be purified according to standard procedures of the art, including HPLC purification, fraction column chromatography, gel electrophoresis and the like (see, generally, Scopes, Protein Purification, Springer- Verlag, NY, 1982). Detailed protocols for the pro- duction of human antibodies can be found in U.S. Patent 5,506,132.

Other approaches in vitro immunization of human blood. In this approach, human blood lymphocytes capable of producing human antibodies are produced. Human peripheral blood is collected from the patient and is treated to recover mononuclear cells. The suppressor T-cells then are removed and remaining cells are suspended in a tissue culture medium to which is added the antigen and autologous serum and, preferably, a nonspecific lymphocyte activator. The cells then are incubated for a period of time so that they produce the specific antibody desired. The cells then can be fused to human myeloma cells to immortalize the cell line, thereby to permit continuous production of antibody (see U.S. Patent 4,716,111).

In another approach, mouse-human hybridomas which produces human anti-fused are prepared (see, e.g. 5,506,132). Other approaches include immunization of muri- nes transformed to express human immunoglobulin genes, and phage display scre- ening (Vaughan et αl. Supra).

IV. Expression of fused polypeptides.

A) De novo chemical synthesis.

The present proteins or subsequences thereof may be synthesized using standard chemical peptide synthesis techniques. Where the desired subsequnces are relatively short (e.g., when a particular antigenic determinant is desired) the molecule may be synthesized as a single contiguous polypeptide. Where larger molecules are desired, subsequences can be synthesized separately (in one or more units) and then fused by condensation of the amino terminus of one molecule with the carboxyl terminus of the other molecule thereby forming a peptide bond. Solid phase synthesis in wich the C-terminal amino acid of the sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is the preferred method for the chemical synthesis of the polypepti- des of this invention. Techniques for solid phase synthesis are described by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. w: Special Methods in peptide Synthesis, Part A., Merrifi- led, et al. J. Am. Chem. Soc, 85: 2149-2156 (1963), and Stewart et al, Solid Phase Peptide Synthesis, 2nd ed. Pierce Chem. Co., Rockford, 111. (1984). B. Recombinant expression.

In a preferred embodiment, the present proteins or subsequences thereof are synthesized using recombinant DNA methodology. Generally this involves creating a DNA sequence that encodes the fusion protein, placing the DNA in an expression casette under the control of a particular promoter, expressing the protein in a host, isolating the expressed protein and, if required, renaturing the protein.

DNA encoding the present proteins or subsequences of this invention may be prepared by any suitable method as described above, including, for example, cloning and restriction of appropriate sequences or direct chemical synthesis by methods such as the phosphotriester method of Narang et al. Meth. Enzymol. 68: 90-99 (1979); the phosphodiester method of Brown et al, Meth Enzymol. 68: 109-151 (1979); the di- ethylphosphoramidite method of Beaucage et al, Tetra . Lett., 22: 1859-1862 (1981); and the solid support method of U.S. Patent No. 4,458,066.

Chemical synthesis produces a single stranded oligonucleotide. This may be converted into double stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template. One of skill would recognize that while chemical synthesis of DNA is limited to sequences of about 100 bases, longer sequences may be obtained by the ligation of shorter sequences. Alternatively, subsequences may be cloned and the appropriate subsequences cleaved using appropriate restriction enzymes. The fragments may then be ligated to produce the desired DNA sequence.

In one embodiment, proteins of this invention may be cloned using DNA amplification methods such as polymerase chain reaction (PCR). Thus, for example, the nucleic acid sequence or subsequence is PCR amplified, using a sense primer containing one restriction site (e.g., Ndel) and an antisense primer containing another re- striction site (e.g., Hindlll). This will produce a nucleic acid encoding the desired sequence or subsequence and having terminal restriction sites. This nucleic acid can then be easily ligated into a vector containing a nucleic acid encoding the second molecule and having the appropriate corresponding restriction sites. Suitable PCR primers can be determined by one of skill in the art using the Sequence information provided in SEQ ID No: 1. Appropriate restriction sites can also be added to the nucleic acid encoding the protein or protein subsequence by site-directed mutagene- sis. The plasmid containing the sequence or subsequence is cleaved with the appropriate restriction endonuclease and then ligated into the vector encoding the second molecule according to standard methods.

The nucleic acid sequences encoding tse proteins or protein subsequences may be expressed in a variety of host cells, including E. coli, other bacterial hosts, yeast, and various higher eukaryotic cells such as the COS, CHO and HeLa cells lines and myeloma cell lines. As the present proteins are typically found in eukaryotes, a eu- karyote host is preferred. The recombinant protein gene will be operably linked to appropriate expression control sequences for each host. For E. coli this includes a promoter such as the T7, trp, or lambda promoters, a ribosome binding site and preferably a transcription termination signal. For eukaryotic cells, the control sequences will include a promoter and preferably and enhancer derived from immu- noglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.

The plasmids of the invention can be transferred into the chosen host cell by well- known methods such as calcium chloride transformation for E. coli and calcium phosphate treatment or electroporation for mammalian cells. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo and hyg genes.

Once expressed, the recombinant proteins according to the invention can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, generally, R. Scopes, Protein Purification, springer-Verlag, N.Y. (1982), Deutscher, Methods in Enzymology Vol. 183: Guide to Protein Purification., Aca- demic Press, Inc. N.Y. (1990)). Substantially pure compositions of at least about 90 to 95%o homogeneity are preferred, and 98 to 99% or more homogeneity are most preferred. Once purified, partially or to homogeneity as desired, the polypeptides may then be used (e.g., immunogens for antibody production).

One of skill in the art would recognize that after chemical synthesis, biological expression, or purification, the present protein(s) may possess a conformation substantially different than the native conformations of the constituent polypeptides. In this case, it may be necessary to denature and reduce the polypeptide and then to cause the polypeptide to re-fold into the preferred conformation. Methods or reducing and denaturing proteins and inducing re-folding are well known to those of skill in the art (See, Debinski et al. (1993) J. Biol. Chem., 268: 14065-14070; Kreitman and pastan (1993) Bioconjug. Chem., 4: 581-585; and Buchner, et al, (1992) Anal. Biochem., 205: 263-270). Debinski et al, for example, describe the denaturation and reduction of inclusion body proteins in guanidine-DTE. The protein is then refolded in a redox buffer containing oxidized glutathione and L-arginine. One of skill would recognize that modifications can be made to the fused proteins without diminishing their biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion protein. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.

V. Detection of fused

As indicated above, abnormal (e.g., altered or deficient) expression of the human fused gene is beleived to be a causal factor in the development of several different conditions, such as cancers.

Thus, it is desirable to determine the presence or absence, or quantify, the expression of fused polypeptides of the nucleic acids encoding the fused polypeptides. This may be accomplished by assaying the gene product; fused polypeptides themselves, or alternatively, by assaying the nucleic acids (DNA or mRNA) that encode the fu- sed polypeptides. In particular, it is desirable to determine whether fused expression is present, absent, or abnormal (e.g., because of an abnormal gene product or because of abnormal expression levels as, for example, with a hemizygous gene). Particularly, where it is desired to determine a heritable propensity for abnormal fused gene expression, it is preferred to assay the host DNA for abnormal fused genes or gene transcripts (mRNAs).

A) Sample Collection and Processing

The fused gene or gene product (i.e., mRNA or polypeptide) is preferably detected and/or quantified in a biological sample. As used herein, a biological sample is a sample of biological tissue or fluid that, in a healthy and/or pathological state, con- tains a fused nucleic acid or polypeptide. Such samples include, but are not limited to, sputum, amniotic fluid, blood, blood cells (e.g., white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and pleural fluid, or cells therefrom. Biological samples may also include sections of tissues such as frozen sections taken for histological purposes. Although the sample is typically taken from a human patient, the assays can be used to detect fused genes or gene products in samples from any mammal, such as dogs, cats, sheep, cattle, and pigs.

The sample may be pretreated as necessary by dilution in a appropriate buffer solu- tion or concentrated, if desired. Any of a number of standard aqueous buffer solutions, employing one of a variety of buffers, such as phosphate, Tris, or the like, at physiological pH can be used.

B) Nucleic acid assays. In one embodiment, this invention provides for methods of detecting and/or quantifying human fused expression by assaying the underlying fused gene (or a fragment thereof) or by assaying the fused gene transcrip (mRNA). The assay can be for the presence or absence of the normal gene or gene product, for the presence or absence of an abnormal gene or gene product, or quantification of the transcription levels of normal or abnormal fused gene product.

i) Nucleic acid sample.

In a preferred embodiment, nucleic acid assays are performed with a sample of nucleic acid isolated from the organism to be tested. In the simplest embodiment, such a nucleic acid sample is the total mRNA isolated from a biological sample. The nucleic acid (e.g., either genomic DNA or mRNA) may be isolated from the sample according to any of a number of methods well known to those of skill in the art. One of skill will appreciate that where alterations in the copy number of the fused gene are to be detected genomic DNA is preferably isolated. Conversely, where ex- pression levels of a gene or genes are to be detected, preferably RNA (mRNA) is isolated.

Methods of isolating total DNA or mRNA are well known to those of skill in the art. For example, methods of isolation and purificaytion of nucleic acids are described in detail in Chapter 3 of Laboratory Techniques in Biochemistry and Molecular Biology: Hybridization With Nucleic Acid Probes, part I. Theory and Nucleic Acid Preparation, P. Tijssen, ed. elsevier, N.Y. (1993) and Chapter 3 of Laboratory Techniques in Biochemistry and Molecular Biology: Gybridization With Nucleic Acid Probes, Part I. Theory and Nucleic Acid Preparation. P. Tij ssen ed. Elsevier, N.Y. (1993)).

In a preferred embodiment, the total nucleic acid is isolated from a given sample using, for example, an acid guanidinium-phenol-chloroform extraction method and poly A⁺ mRNA is isolated by oligo dT column chromatography or by using (dT)n magnetic beads (see, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual 2nd ed.), Vols.1-3, Cold Spring Harbor Laboratory, (1989), or Current Protocols in Molecular Biology, F. Ausubel et al, ed. Greene Publishing and Wiley- Interscience, New York (1987)).

Frequently, it is desirable to amplify the nucleic acid sample prior to hybridization. One of skill in the art will appreciate that whatever amplification method is used, if a quantitative result is desired, care must be taken to use a method that maintains or controls for the relative frequencies of the amplified nucleic acids.

Methods of "quantitative" amplification are well known to those of skill in the art. For example, quantitative PCR involves simultaneously co-amplifying a known quantity of a control sequence using the same primers. This provides an internal standard that may be used to calibrate the PCR reaction. The high density array may then include probes specific to the internal standard for quantification of the ampli- fied nucleic acid. One preferred internal standard is a synthetic AW 106 cRNA. The AW106 cRNA is combined with RNA isolated from the sample according to standard techniques known to those of skill in the art. The RNA is then reverse transcribed using a rever- se transcriptase to provide copy DNA. The cDNA sequences are then amplified (e.g., by PCR) using labeled primers. The amplification products are separated, typically by electrophoresis, and the amount of radioactivity (proportional to the amount of amplified product) is determined. The amount of mRNA in the sample is then calculated by comparison with the signal produced by the known AW106 RNA standard. Detailed protocols for quantitative PCR are provided in PCR Protocols, A guide to Methods and Applications, Innis et al, Academic Press, Inc. N.Y., (1990). Other suitable amplification methods include, but are not limited to polymerase chain reaction (PCR) (Innis et al, (1990) PCR Protocols. A guide to Methods and Application. Academic Press, Inc. San Diego), ligase chain reaction (LCR) (see Wu and Wallace (1989) Genomics 4: 560, Landegren et al. (1988) Science 241 : 1077, and Barringer et al. (1990) Gene 89: 117, transcription amplification (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86: 1173), and self-sustained sequence replication (Guatelli et al. (1990) Proc. Nat. Acad. Sci. USA 87: 1874).

ii) Hybridization assays.

A variety of methods for specific DNA and RNA measurement using nucleic acid hybridization techniques are known to those of skill in the art (see Sambrook et al. Supra). For example, one method for evaluating the presence, absence, or quantity of DNA encoding/used proteins in a sample involves a Southern transfer. Briefly, the digested genomic DNA is run on agarose slab gels in buffer and transferred to membranes.

Hybridization is carried our using the nucleic acid probes specific for the target fused sequence or subsequence. Nucleic acid probes are designed based on the nucleic acid sequences encoding /wsβc. proteins (see SEQ ID NO: 2). The probes can be full length of the nucleic acid sequence encoding the fused protein. Shorter probes are empirically tested for specificity. Preferably nucleic acid probes are 20 bases or longer in length. (See Sambrook et al. For methods of selecting nucleic acid probe sequences for use in nucleic acid hybridization.) Visualization of the hybridized portions allows the qualitative determination of the presence or absence of DNA en- coding^wsec. proteins.

Similarly, a Northern transfer may be used for the detection of mRNA encoding w- sed proteins. In brief, the mRNA is isolated from a given cell sample using, for ex- ample, and acid guanidinium-phenol-chloroform extraction method. The mRNA is then electrophoresed to separate the mRNA species and the mRNA is transferred from the gel to a nitrocellulose membrane. As with the Southern blots, labeled probes are used to identify the presence or absence of fused proteins.

A variety of nucleic acid hybridization formats are known to those skilled in the art. For example, common formats include sandwich assays and competition or displacement assays. Hybridization techniques are generally described in 'Nucleic Acid Hybridization, A Practical Approach," Ed. Hames, B.D. and Higgins, S.J., IRL Press, (1985); Gall and Pardue Proc. Natl. Acad. Sci. U.S.A. 63: 378-383 (1969); and John et al. Nature 223: 582-587 (1969).

For example, sandwich assays are commercially useful hybridization assays for detecting or isolating nucleic acid sequences. Such assays utilize a "capture" nucleic acid covalently immobilized to a solid support and a labeled "signal" nucleic acid in solution. The clinical sample will provide the target nucleic acid. The "capture" nucleic acid and "signal" nucleic acid probe hybridize with the target nucleic acid to form a "sandwich" hybridization complex. To be effective, the signal nucleic acid cannot hybridize with the capture nucleic acid. Typically, labeled signal nucleic acids are used to detect hybridization. Complementary nucleic acids or signal nucleic acids may be labeled by any one of several methods typically used to detect the presence of hybridized polynucleotides. The most common method of detection is the use of autoradiography with ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P-labelled probes or the like. Other labels include ligands which bind to labeled antibodies, fluorophores, chemiluminescent agents, enzymes, and antibodies which can serve as specific binding pair members for a labeled ligand.

Detection of a hybridization complex may require the binding of a signal generating complex to a duplex of target and probe polynucleotides or nucleic acids. Typically, such binding occurs through ligand and anti-ligand interactions as between a ligand- conjugated probe and an anti-ligand conjugated with a signal. The label may also allow indirect detection of the hybridization complex. For example, where the label is a hapten or antigen, the sample can be detected by using antibodies. In these systems, a signal is generated by attaching fluorescent or enzyme molecules to the antibodies or, in some cases, by attachment to a radioactive label. (Tijssen, P., "Practice and Theory of Enzyme Immunoassays," Laboratory Techniques in Ciochemistry and Molecular Biology, Burdon, R.H., van Knippen- berg, P.H., Eds., Elsevier (1985), pp. 9-20.).

The sensitivity of the hybridization assays may be enhanced through use of a nucleic acid amplification system which multiplies the target nucleic acid being detected. Examples of such systems include the polymerase chain reaction (PCR) system and the ligase chain reaction (LCR) system. Other methods recently described in the art are the nucleic acid sequence based amplification (NASBA™, Cangene, Missis- sauga, Ontario) and Q Beta Replicase systems.

An alternative means for determining the level of expression of a gene encoding a fused protein is in situ hybridization. In situ hybridization assays are well known and are generally described in Angerer, et al, Methods Enzymol, 152: 649-660 (1987). In an in situ hybridization assay, cells or tissue specimens are fixed to a solid support, typically a glass slide. If DNA is to be probed, the cells are denatured with heat or alkali. The cells are then contacted with a hybridization solution at a moderate temperature to permit annealing of labeled probes specific to fused prote- ins. The probes are preferably labeled with radioisotopes of fluorescent reporters.

iii) Amplification based assays.

In another embodiment, the fused gene or gene product can be detected (assayed) using an amplification based assay. In an amplification based assay, all or part of the fused gene or transcript (e.g., mRNA or cDNA) is amplified and the amplification product is then detected. Where there is no underlying gene or gene product to act as a template amplification is non-specific or non-existent and there is no single amplification product. Where the underlying gene or gene product is present, the target sequence is amplified providing an indication of the presence, absence, or quantity of the underlying gene or mRNA.

Amplification-based assays are well known to those of skill in the art (see, e.g., Innis, supra.). The cDNA sequence provided for the fused gene is sufficient to enable one of skill to routinely select primers to amplify any portion of the gene.

Amplification primers can be selected to provide amplification products that span specific deletions, truncations, and insertions, thereby facilitating the detection of specific abnormalities.

iv) Specific detection of abnormalities (e.g., mutations).

Abnormal fused genes or gene products are characterized by premature stop codons, deletions, insertions or change of particular amino acids. Premature stop codons and deletions can be detected by decreased size of the gene or gene product (mRNA transcript or cDNA). Similarly, insertions can be detected by increased size of the gene or gene product. Alternatively, mutations can be determined by sequencing of the gene or gene product according to standard methods.

In addition, amplification assays and hybridization probes can be selected to specifi- cally target particular abnormalities. For example, where the abnormality is a deletion, nucleic acid probes or amplification primers can be selected that specifically hybridize to or amplify, respectively, the nucleic acid sequence that is cdeleted in the abnormal gene. The probe will fail to hybridize, or the amplification reaction will fail to provide specific amplification, to abnormal versions of the fused nucleic acids which have the deletion. Alternatively, the probe or amplification reaction can be designed to span the entire deletion or either end of the deletion (deletion junction). Similarly, probes and amplification primers can be selected that specifically target point mutations or insertions. Methods for detecting specific mutations were described in, for example, US Patent No. 5,512,441. In the case of PCR, amp- lification primers can be designed to hybridize to a portion of the fused gene but the terminal nucleotide at the 3' end of the primer can be used to discriminate between the mutant and wild-type forms of fused gene. If the terminal base matches the point mutation or the wild-type sequence, polymerase dependent extension can proceed and an amplification product is detected. This method for detecting point mutations or polymorphisms was described in detail by Sommer et al, (1989) Mayo Clin. Proc. 64:1361-1372. By using appropriate controls, one can develop a kit having both positive and negative amplification products. The products can be detected using specific probes or by simply detecting their presence or absence. A variation of the PCR method uses LCR where the point of discrimination, i.e., either the point mutation or the wild-type bases fall between the LCR oligonucleotides. The ligation of the oligonucleotides becomes the means for discriminating between the mutant and wild-type forms of the fused gene.

A variety of automated solid-phase detection techniques are also appropriate for detecting the presence or absence of mutations in the fused gene. For instance, very large scale immobilized polymer arrays (VLSIPS™), available from Affymetrix, Inc. In Santa Clara, CA are used for the detection of nucleic acids having specific sequences of interest. See, Fodor et al. (1991) Science, 251 : 767-777; Sheldon et al. (1993) Clinical Chemistry 39(4): 718-719, and Kozal et al. (1996) Natural Medicine 2(7): 753-759. For example, oligonucleotides that hybridize to all known/used mutations can be synthesized on a DNA chip (such chips are available from Affymetrix) and the nucleic acids from samples hybridized to the chip for simultaneous analysis of the sample nucleic acid for the presence or absence of any of the known fused mutations. Protocols for detecting mutations are also described in, for examp- le, Tij ssen (1993) Laboratory Techniques in biochemistry and molecular biology — hybridization with nucleic acid probes parts I and II, Elsevier, new York, and Choo (ed) (1994) Methods In Molecular Biology Volume 33-In situ Hybridization Protocols, Humana Press Inc., New Jersey (see also, other books in the Methods in Molecular Biology series).

v. Detection of expression levels.

Where it is desired to quantify the transcription level (and thereby expression) of a normal or mutated/wsec. genes in a sample, the nucleic acid sample is one in which the concentration of the mRNA transcript(s) of the fused gene, or the concentration of the nucleic acids derived from the mRNA transcrip(s), is proportional to the transcription level (and therefore expression level) of that gene. Similarly, it is preferred that the hybridization signal intensity be proportional to the amount of hybridized nucleic acid. While it is preferred that the proportionality be relatively strict (e.g., a doubling in transcription rate results in a doubling in mRNA transcrip in the sample nucleic acid pool and a doubling in hybridization signal), one of skill will appreciate that the proportionality can be more relaxed and even non-linear. Thus, for example, an assay where a 5 fold difference in concentration of the target mRNA results in a 3 to 6 fold difference in hybridization intensity is sufficient for most purposes. Where more precise quantification is required appropriate controls can be run to correct for variations introduced in sample preparation and hybridization as described herein. In addition, serial dilutions of "standard" target mRNAs can be used to prepare calibration curves according to methods well known to those of skill in the art. Of course, where simple detection of the presence or absence of a transcrip is desired, no elaborate control or calibration is required.

C) Polypeptide assays.

The expression of the human fused gene can also be detected and/or quantified by detecting or quantifying the expressed/wsec/ polypeptide. The fused polypeptides can be detected and quantified by any of a number of means well known to those of skill in the art. These may include analytic biochemical methods such as electropho- resis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), hyperdiffusion chromatography, and the like, or various immunological methods such as fluid or gel precipitin reactions, immuno- diffusion (single or double), immunoelectrophoresis, radioimmunoassay(RIA), en- zyme-linked immunosorbent assays (ELISAs), immunofluorescent assays, Western blotting, and the like.

In a particularly preferred ambodiment, the fused polypeptides are detected in an electrophoretic protein separation, more preferably in a two-dimensional electrophoresis, while in a most preferred embodiment, the fused polypeptides are detected using an immunoassay.

As used herein, an immunoassay is an assay that utilizes an antibody to specifically bind to the analyte (fused polypeptide). The immunoassay is thus characterized by detection of specific binding of a fused polypeptide to an anti-fused antibody as op- posed to the use of other physical or chemical properties to isolate, target, and quantify the analyte.

1) Electrophoretic Assays.

As indicated above, the presence or absence of fused polypeptides in a biological sample can be determined using electrophoretic methods. Means of detecting pro- teins using electrophoretic techniques are well known to those of skill in the art (see generally, R. Scopes (1982) Protein Purification, Springer- Verlag, N.Y.; Deutscher, (1990) Methods in Enzymology Vol. 182: Guide to Protein Purification., Academic Press, Inc., N.Y.).

2) Immunological Binding Assays.

In a preferred embodiment, the fused polypeptides are detected and/or quantified using any of a number of well recognized immunological binding assays (see, e.g., U.S. Patent 4,366,241; 4,376,110; 4,517,288; and 4,837,168). For a review of the general immunoassays, see also Methods in Cell Biology Volume 37: antibodies in Cell Biology, Asain, ed. Academic Press, Inc. New York (1993); Basic and Clinical Immunology 7th Edition, Stites & Terr, eds. (1991). Immunological binding assays (or immunoassays) typically utilize a "capture agent" to specifically bind to and often immobilize the analyte (in this case fused polypeptide or subsequence). The capture agent is a moiety that specifically binds to the analyte. In a preferred embodiment, the capture agent is an antibody that specifically binds /tt ed polypeptide(s). The antibody (anti-fused) may be produced by any of a number of means well known to those of skill in the art as described above in Section III(A).

Immunoassays also often utilize a labeling agent to specifically bind to and label the binding complex formed by the capture agent and the analyte. The labeling agent may itself be one of the moieties comprising the antibody/analyte complex. Thus, the labeling agent may be a labeled fused polypeptide or a labeled anti-fused antibody. Alternatively, the labeling agent may be a third moiety, such as another antibo- dy, that specifically binds to the antibody/ wsec. complex.

In a preferred embodiment, the labeling agent is a second human fused antibody bearing a label. Alternatively, the second fused antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is derived. The second can be modified with a de- tectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme labeled streptavidin.

Other proteins capable of specifically binding immunoglobulin constant regions, such as protein A or protein G, may also be used as the label agent. The protein are normal constituents of the cell walls of strepococcal bacteria. They exhibit a strong non-immunogenic reactivity with immunoglobulin constant regions from a variety of species (see, generally Kronval, et al. (1973) J. Immunol, 111: 1401-1406, and Akerstrom, et al. (1985) J Immunol, 135:2589-2542).

Throughout the assays, incubation and/or washing steps may be required after each combination of reagents. Incubation steps can vary from about 5 seconds to several hours, preferably from about 5 minutes to about 24 hours. However, the incubation time will depend upon the assay format, analyte, volume of solution, concentrations, and the like. Usually, the assays will be carried out at ambient temperature, although they can be conducted over a range of temperatures, such as 10°C to 40°C.

a) Non-Competitive Assay Formats.

Immunoassays fot detecting fused polypeptide mays be either competitive or non- competitive. Noncompetitive immunoassays are assays in which the amount of captured analyte (in this case fused) is directly measured. In one preferred "sandwich" assay, for example, the capture agent (anti-fused antibodies) can be bound directly to a solid substrate where the are immobilized. These immobilized antibodies then capture fused present in the test sample. The fused thus immobilized in then bound by a labeling agent, such as a second human fused antibody bearing a label. Alternatively, the second/wse antibody may lack a label, but it may, in turn, be bound by a labeled third antibody specific to antibodies of the species from which the second antibody is dervied. The second can be modified with a detectable moiety, such as biotin, to which a third labeled molecule can specifically bind, such as enzyme-labeled streptavidin. b) Competitive assay formats.

In competitive assays, the amount of analyte (fused) present in the sample is measured indirectly by measuring the amount of an added (exogenous) analyte (fused) displaced (or competed away) from a capture agent (anti-fused antibody) by the analyte present in the sample. In one competitive assay, a known amount of, in this case, fused is added to the sample and the sample is then contacted with a capture agent, in this case an antibody that specifically binds fused. The amount of fused bound to the antibody is inversely proportional to the concentration of fused present in the sample.

In a particularly preferred embodiment, the antibody is immobilized on a solid substrate. The amount of fused bound to the antibody may be determined either by measuring the amount of fused present in a/wseaf/antibody complex, or alternatively by measuring the amount of remaining uncomplexed wsec.. The amount of fused may be detected by providing a labeled wseύ. molecule.

A hapten inhibition assay is another preferred competivite assay. In this assay a known analyte, in this case fused, is immobilized on a solid substrate. A known amount of an -fused antibody is added to the sample, and the sample is then con- tacted with the immobilized/wseύ.. In this case, the amount of anti-fused antibody bound to the immobilized/wsec. is inversely proportional to the amount of fused present in the sample. Again the amount of immobilized antibody may be detected by detecting either the immobilized fraction of antibody or the fraction of the antibody that remains in solution. Detection may be direct where the antibody is labeled or indirect by the subsequent addition of a labeled moiety that specifically binds to the antobdy as described above.

c) Other assay formats.

In a particularly preferred embodiment, Western blot Immunoblot analysis is used to detect and quantify the presence of fused in the sample. The technique generally comprises separating sample proteins by gel electrophoresis on the basis of molecular weight, transferring the separated proteins to a suitable solid support, (such as a nitrocellulose filter, a nylon filter, or derivatized nylon filter), and incubating the sample with the antibodies that specifically bind fused. The anti-fused antibodies specifically bind to fused on the solid support. These antibodies may be directly labeled or alternatively may be subsequently detected using labeled antibodies (e.g., labeled sheep anti-mouse antibodies) that specifically bind to the anti-fuse d.

Other assay formats include liposome immunoassays (LIA), which use liposomes designed to bind specific molecules (e.g., antibodies) and release encapsulated reagents or markers. The released chemicals are then detected according to standard techniques (see, Monroe et αl. (1986) Amer. Clin. Prod. Rev. 5:34-41).

d) Scoring of the assay. The assays of this invention are scored (as positive or negative for fused polypeptide) according to standard methods well known to those of skill in the art. The particular method of scoring will depend on the assay format and choice of label. For example, a Western Blot assay can be scored by visualizing the colored product produced by the enzymatic label. A clearly visible colored band or spot at the correct molecular weight is scored as a positive result, while the absence of a clearly visible spot or band is scored as a negative. In a preferred embodiment, a positive test will show a signal intensity (e.g., fused polypeptide quantity) at least twice that of the background and/or control and more preferably at least 3 times or even at least 5 times greater than the background and/or negative control.

e) Reduction of non-specific binding.

One of skill in the art will appreciate that it is often desirable to reduce non-specific binding in immunoassays. Particularly, where the assay involves an antigen or antibody immobilized on a solid substrate it is desirable to minimize the amount of non- specific bindning to the substrate. Means of reducing such non-specific binding are well known to those of skill in the art. Typically, this involves coating the substrate with a proteinaceous composition. In particular, protein compositions such as bovine serum albumin (BSA), nonfat powdered milk, and gelatin are widely used with powdered milk being most preferred.

f) Labels.

The particular label or detectable group used in the assay is not a critical aspect of the invention, so long as it does not significantly interfere with the specific binding of the antibody used in the assy. The detectable group can be any material having a detectable physical or chemical property. Such detectable labels have been well- developed in the field of immunoassays and, in general, most any label useful in such methods can be applied to the present invention. Thus, a label is any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads (e.g., Dynabeads ), fluorescent dyes (e.g., fluorescein isothiocya- nate, texas red, rhodamine, and the like), radiolabels (e.g., ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horse radish peroxidase, alkaline phosphatase and others commonly used in an ELISA), and colorimetric labels such as colloidal gold or colored glass or plastic (e.g., polystyrene, polypropylene, latex, etc.) beads.

The label may be coupled directly or indirectly to the desired component of the assay according to methods well known in the art. As indicated above, a wide variety of labels may be used, with the choice of label depending on sensitivity required, ease of conjugation with the compound, stability requirements, available instru- mentation, and disposal provisions.

Non-radioactive labels are often attached by indirect means. Generally, a ligand molecule (e.g., biotin) is covalently bound to the molecule. The ligand then binds to an anti-ligand (e.g., streptavidin) molecule which is either inherently detectable or covalently bound to a signal system, such as a detectable enzyme, a fluorescent compound, or a chemiluminescent compound. A number of ligands and anti-ligands can be used. Where a ligand has a natural anti-ligand, for example, biotin, thyroxine, and cortisol, it can be used in conjunction with the labeled, naturally occurring anti- ligands. Alternatively, any haptenic or antigenic compound can be used in combina- tion with an antibody.

The molecules can also be conjugated directly to signal generating compounds, e.g., by conjugation with an enzyme or fluorophore. Enzymes of interest as labels will primarily be hydrolases, particularly phosphatases, esterases and glycosidases, or oxidoreductases, particularly peroxidases. Fluorescent compounds include fluorescein and its derivatives, rhodamine and its derivatives, dansyl, umbelliferone, etc. Chemiluminescent compounds include luciferin, and 2,3-dihydrophtalazinediones, e.g., luminol. For a review of various labeling or signal producing systems which may be used, see, U.S. Patent No. 4,391,904). Means of detecting labels are well known to those of skill in the art. Thus, for example, where the label is a radioactive label, means for detection include a scintillation counter or photographic film as in autoradiography. Where the label is a fluorescent label, it may be detected by exciting the fluorochrome with the appropriate wavelength of light and detecting the resulting fluorescence. The fluorescence may be detected visually, by means of photographic film, by the use of electronic detectors such as charge coupled devices (CCDs) or photomultipliers and the like. Similarly, enzymatic labels may be detected by providing the appropriate substrates for the enzyme and detecting the resulting reaction product. Finally simple colorimetric labels may be detected simply by observing the color associated with the label. Thus, in various dipstick assays, conjugated gold often appears pink, while various conjugated beads appear the color of the bead.

Some assay formats do not require the use of labeled components. For instance, agglutination assays can be used to detect the presence of the target antibodies. In this case, antigen-coated particles are agglutinated by samples comprising the target antibodies. In this format, none of the components need be labeled and the presence of the target antibody is detected by simple visual inspection.

g) Substrates. As mentioned above, depending upon the assay, various components, including the antigen, target antibody, or anti-human antibody, may be bound to a solid surface. Many methods for immobilizing biomolecules to a variety of solid surfaces are known in the art. For instance, the solid surface may be a membrane (e.g., nitrocellulose), a mictrotiter dish (e.g., PVC, polypropylene, or polystyrene), a test tube (glass or plastic), a dipstick (e.g., glass, PVC, polypropylene, polystyrene, latex and the like), a microcentrifuge tube, or a glass or plastic bead. The desired component may be covalently bound or noncovalently attached through nonspecific bonding. A wide variety of organic and inorganic polymers, both natural and synthetic, may be employed as the material for the solid surface. Illustrative polymers include poly- ethylene, polypropylene, poly(4-methylbutene), polystyrene, polymethacrylate, polyethylene terephthalate), rayon, nylon, poly(vinyl butyrate), polyvinylidene difluo- ride (PVDF), silicones, polyformaldehyde, cellulose, cellulose acetate, nitrocellulose, and the like. Other materials which may be employed include paper, glasses, ceramics, metals, metalloids, semiconductive materials, cements or the like. In addi- tion, are included substances that form gels, such as proteins (e.g., gelatins) lipopo- lysaccharides, silicates, agarose and polyacrylamides can be used. Polymers which form several aqueous phases, such as dextrans, polyalkylene glycols or surfactants, such as phospholipids, long chain (12-24 carbon atoms) alkyl ammonium salts and the like are also suitable. Where the solid surface is porous, various pore sizes may be employed depending upon the nature of the system.

In preparing the surface, a plurality of different materials may be employed, particularly as laminates, to obtain various properties. For example, protein coatings, such as gelatin, can be used to avoid non-specific binding, simplify covalent conju- gation, enhance signal detection or the like. If covalent bonding between a compound and the surface is desired, the surface will usually be polyfunctional or be capable of being polyfunctionalized. Functional groups which may be present on the surface and used for linking can include carboxylic acids, aldehydes, amino groups, cyano groups, ethylenic groups, hydroxyl groups, mercapto groups and the like. The manner of linking a wide variety of compounds to various surfaces is well known and is amply illustrated in the literature. See, for example, Immobilized Enzymes, Ichiro Chibata, Halsted Press, New York, 1978, and Cuatrecasas (1970) J. Biol. Chem. 245 3059).

In addition to covalent binding, various methods for noncovalently binding an assay component can be used. Noncovalent binding is typically nonspecific absorption of a compound to the surface. Typically, the surface is blocked with a second compound to prevent nonspecific binding of labeled assay components. Alternatively, the surface is designed such that is nonspecifically binds one component but does not significantly bind another. For example, a surface bearing a lectin such as Concana- valin A will bind a carbohydrate containing compound but not a labeled protein that lacks glycosylation. Various solid surfaces for use in noncovalent attachment of assay components are reviewed in U.S. Patent Nos. 4,447,576 and 4,254,082.

D) Evaluation of fused expression levels and/or abnormal expression. One of skill will appreciate that abnormal expression levels or abnormal expression products (e.g., mutated transcripts, truncated or non-sense polypeptides) are identified by comparison to normal expression levels and normal expression products. Normal levels of expression or normal expression products can be determined for any particular population, subpopulation, or group of organisms according to standard methods well known to those of skill in the art. Typically this involves identifying healthy organisms and measuring expression levels of the fused gene (as described herein) or sequencing the gene, mRNA, or reverse transcribed cDNA, to obtain typical (normal) sequence variations. Application of standard statistical met- hods used in molecular genetics permits determination of baseline levels of expression, and normal gene products as well as significant deviations from such baseline levels.

E) Detection kits.

The present invention also provides for kits for the diagnosis of organisms (e.g., patients). The kits preferably include one or more reagents for determining the presence or absence of the fused gene, for quantifying expression of the fused gene, or for detecting an abnormal fused gene or expression products of an abnormal fused gene. Preferred reagents inlcude nucleic acid probes that specifically bind to the normal fused gene, cDNA, or subsequence thereof, probes that specifically bind to abnormal fused gene (e.g., fused containing premature truncations, insertions, or deletions), antibodies that specifically bind to normal fused polypeptides or subsequences thereof, or antibodies that specifically bind to abnormal /wsec. polypepti- des or subsequences thereof. The antibody or hybridization probe may be free or immobilized on a solid support such as a test tube, a microtiter plate, a dipstick and the like. The kit may also contain instructional materials teaching the use of the antibody or hybridization probe in an assay for the detection of a deviation from the normal sequence of a fused gene.

The kits may include alternatively, or in combination with any of the other components described herein, an anti-fused antibody. The antibody can be monoclonal or polyclonal. The antibody can be conjugated to another moiety such as a label and/or it can be immobilized on a solid support (substrate).

The kits(s) may also contain a second antibody for detection of polypeptide/antibody complexes according to the invention or for detection of hybridized nucleic acid probes. The kit may contain appropriate reagents for detection of labels, positive and negative controls, washing solutions, dilution buffers and the like. VI. Modulation of expression of endogenous fused genes.

In still another emobidment, this invention provides methods of regulating the expression of endogenous fused genes. The expression of a fused gene product may be increased. Regulation of fused gene may provide a convenient and controllable mo- del system for the study of the HH-PTC pathway, especially in human beings.

Methods of altering the expression of endogenous genes are well known to those of skill in the art. Typically such methods involve altering or replacing all or a portion of the regulatory sequences controlling expression of the particular gene that is to be regulated. In a preferred embodiment, the regulatory sequences (e.g., the native promoter) upstream of the /wse gene is altered.

This is typically accomplished by the use of homologous recombination to introduce a heterologous nucleic acid into the native regulatory sequences. To downregulate expression the fused gene product, simple mutations that either alter the reading frame or disrupt the promoter are suitable. To upregulate expression of the fused gene product, the native promoter(s) can be substituted with heterologous promoter(s) that induce higher than normal levels of transcription.

In a particularly preferred embodiment, nucleic acid sequences comprising the structural gene in question or upstream sequences are utilized for targeting heterologous recombination constructs. Upstream and downstream sequences can be readily determined using the information provided herein. Such sequences, for example, can be extended using 5'- or 3 '-RACE and homologous recombination constructs created with only routine experimentation.

The use of homologous recombination to alter expression of endogenous genes is described in detail in U.S. Patent 5,272,071, WO 91/09955, WO 93/09222, WO 96/29411, WO 95/31560, and WO 91/12650. VII. Therapeutics A) Pharmaceutical Compositions

The proteins, polypeptides, antibodies and anti-fused antibody-effector (e.g., enzyme toxin, hormone, growth factor, drug, etc.) conjugates or fusion proteins of this invention are useful for parenteral, topical, oral, or local administration, such as by aerosol or transdermally, for prophylactic and/or therapeutic treatment. The pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration. For example, unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and lozenges. It is recog- nized that the polypeptides and related compounds decribed of, when administered orally, must be protected from digestion. This is typically accomplished either by complexing the protein with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the protein in an appropriately resistant carrier such as a liposome. Means of protecting proteins from digestion are well known in the art.

The pharmaceutical compositions of this invention may be used for topical administration, e.g. to treat basal cell carcinomas, or their precursors, solar keratoses. In another embodiment, the compositions are useful for parenteral administration, such as intravenous administration or administration into a body cavity or lumen of an organ. The compositions for administration will commonly comprise a solution of the polypeptide, antibody or antibody chimera/fusion dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter. These compositions may be sterilized by conventio- nal, well known sterilization techniques. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like. The concentration of chimeric molecule in the- se formulations can vary widely, and will be selected primarily based on fluid volu- mes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.

Thus, a typical pharmaceutical composition for intravenous administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg per patient per day may be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ. Substantially higher dosages are possible in topical administration. Actual methods for preparing parenterally administrable compositions will be known or ap- parent to those skilled in the art and are described in more detail in such pulications as Remington 's Pharmaceutical Science, 15^l ., Mack Publishing Company, Easton, Pennsylvania (1980).

The compositions containing the present polypeptides, antibodies or antibody chi- mer/fusion, or a cocktail therof (i.e., with other proteins), can be administered for therapeutic treatments. In therapeutic applications, compositions are administered to a patient suffering from a disease in an amount sufficient to cure or at least partially arrest the disease and its complications. An amount adequate to accomplish this is defined as a "therapeutically effective dose." Amounts effective for this use will de- pend upon the severity of the disease and the general state of the patient's health.

Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the proteins of this in- vention to effectively treat the patient.

B) Cellular Transformation and Gene Therapy.

The present invention provides packageable human fused nucleic acids (cDNAs) for the transformation of cells in vitro and in vivo. These packageable nucleic acids can be inserted into any of a number of well known vectors for the transfection and transformation of target cells and organisms as described below. The nucleic acids are transfected into cells, ex vivo or in vivo, through the interaction of the vector and the target cell. The fused cDNA, under the control of a promoter, then expresses the fused protein thereby mitigating the effects of absent fused genes or partial inactiva- tion of the fused gene or abnormal expression of the fused gene.

Such gene therapy procedures have been used to correct acquired and inherited genetic defects, cancer, and viral infection in a number of contexts. The ability to arti- fical genes in humans facilitates the prevention and or cure of many important hu- man diseases, including many diseases which are not amenable to treatment by other therapies. As an example, in vivo expression of cholesterol-regulating genes, genes which selectively block the replication of HIV, and tumor- suppressing genes in human patients dramatically improves the treatment of heart disease, AIDS, and cancer, respectively. For a review of gene therapy procedures, see Anderson, Science (1992) 256:808-813; Nabel and Feigner (1993) TIBTECH 11 : 211-217; Mitani and Caskey (1993) TIBTECH 11 : 162-166; Mulligan (1993) Science 926-932; Dillon (1993) TIBTECH 11 : 167-175; Miller (1992) Nαtwre 357: 455-460; Van Brunt (1988) Biotechnology 6(10): 1149-1154; Vigne (1995) Restorative Neurology and Neuroscience 8: 35-36; Kremer and Perricaudet (1995) British Medical Bulletin 51(1) 31 -44; Haddada et al. ( 1995) in Current Topics in Microbiology and Immunology Doerfler and Bohm (eds) Springer- Verlag, Heidelberg Germany; and Yu et al, Gene Theraphy(l994) 1 :13-26.

Delivery of the gene or genetic material into the cell is the first critical step in gene therapy treatment of disease. A large number of delivery methods are well known to those of skill in the art. Such methods include, for example liposome-based gene delivery (Debs and Zhu (1993) WO 93/24640; Mannino and Gould-Fogerite (1988) BioTechniques 6(7): 682-691; Rose U.S. Pat No. 5,279,833; Brigham (1991) WO 91/06309; and Feigner et al. (1987) Proc. Natl. Acad. Sci. USA 84: 7413-7414), and replication-defective retroviral vectors harboring a therapeutic polynucleotide sequence as part of the retroviral genome (see, e.g., Miller et al. (1990) Mol. Cell. Biol. 10:4239 (1990; Kolberg (1992) J. NIH Res. 4:43, and Cornetta et al. Hum. Gene Ther. 2:215 (1991)). Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immu- no deficiency virus (STV), human immuno deficiency virus (HIV), and combinations thereof. See, e.g., Buchscher et al. (1992) J. Virol. 66 (5) 2731-2739; Johann et al. (1992) J. Virol. 66 (5): 1635-1640 (1992); Sommerfelt et al, (1990) Virol. 176:58- 59; Wilson et al. (1989) J. Virol. 63:2374-2378; Miller et al, J Virol. 65:2220-2224 (1991); Wong-Staal et al, PCT/US94/05700, and Rosenburg and Fauci (1993) in Fundamental Immunology, Third Edition Paul (ed) Raven Press, Ltd., New York and the references therein, and Yu et al, Gene Therapy (1994) supra).

AAV-based vectors are also used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and in in vivo and ex vivo gene therapy procedures. See, West et al. (1987) Virology 160:38-47; Carter et al. (1989) U.S. Patent No. 4.797,368; Carter et al. WO 93/24641 (1993); Kotin (1994) Human Gene Therapy 5:793-801 ; Myzyczka (1994) J. Clin. Invest. 94: 1351 and Samulski (supra) for an overview of AAV vectors. Construction of recombinant AAV vectors are described in a number of pulbications, including Lebkowski, U.S. Pat. No. 5,173,414; Tratschin et al. (1985) Mol. Cell. Biol. 5(11):3251-3260; Tratschin, et al. (1984) Mol. Cell. Biol. 4:2072-2081 ; Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81 :6466-6470; McLaughlin et al. (1988) and Samulski et al. (1989) J. Virol. 63:03822-3828. Cell lines that can be transformed by rAAV include those described in Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

VIII.

A) Ex vivo transformation of cells.

Ex vivo cell transformation for diagnostics, reserach, or for gene therapy (e.g., via re-infusion of the transformed cells into the host organism) is well known to those of skill in the art. In a preferred embodiment, cells are isolated from the subject orga- nism, transfected with the fused gene or cDNA of this invention, and re-infused back into the subject organism (e.g., patient). Various cell types suitable for ex vivo transformation are well known to those of skill in the art. Paricular preferred cells are progenitor or stem cells (see, e.g., Freshney et al,m Culture of Animal Cells, a Manual of Basic Technique, third edition Wiley-Liss, New York (1994)) and the references cited therein for a discussion of how to isolate and culture cells from patients).

As indicated above, in a preferred embodiment, the packageable nucleic acid enco- des a fused polypeptide under the control of an activated or constitutive promoter.

In one particularly preferred embodiment, stem cells are used in ex-vivo procedures for cell transformation and gene therapy. The advantage to using stem cells is that they can be differentiated into other cell types in vitro, or can be introduced into a mammal (such as the donor of the cells) where they will engraft in the bone marrow. Methods for differentiating CD34⁺ cells in vitro into clinically important immune cell types using cytokines such a GM-CSF, IFN-γ and TNF-α are known (see, Inaba et αl (1992) J. Exp. Med. 176: 1693-1702, and Szabolcs et αl. (1995) 154: 5851- 5861).

Stem cells are isolated for transduction and differentiation using known methods. For example, in mice, bone marrow cells are isolated by sacrificing the mouse and cutting the leg bones with a pair of scissors. Stem cells are isolated from bone marrow cells by panning the bone marrow cells with antibodies which bind unwanted cells, such as CD4⁺ and CD8⁺ (T cells), CD45⁺ (panB cells), GR-1 (granulocytes), and Ia^d (defferentiated antigen presenting cells). For an example of this protocol see, Inaba et αl. (1992) J. Exp. Med. 176: 1693-1702.

In humans, bone marrow aspirations from iliac crests are performed e.g., under ge- neral anesthesia in the operating room. The bone marrow aspirations is approxima- tely 1,000 ml in quantity and is collected from the posterior iliac bones and crests. If the total number of cells collected is less than about 2 x 10⁸/kg, a second aspiration using the sternum and anterior iliac crests in addition to posterior crests is performed. During the operation, two units of irradiated packed red cells are administered to replace the volume of marrow taken by the aspiration. Human hematopoietic progenitor and stem cells are characterized by the presence of a CD34 surface membrane antigen. This antigen is used for purification, e.g., on affinity columns which bind CD34. After the bone marrow is harvested, the mononuclear cells are separated from the other components by means of ficoll gradient centrifugation. This is per- formed by a semi-automated method using a cell separator (e.g., a Baxter Fenwal CS3000+ or Terumo machine). The light density cells, composed mostly of mononuclear cells are collected and the cells are incubated in plastic flasks at 37°C for 1.5 hours. The adherent cells (monocytes, macrophages and B-Cells) are discarded. The non-adherent cells are then collected and incubated with a monoclonal anti-CD34 antibody (e.g., the murine antibody 9C5) at 4°C for 30 minutes with gentle rotation. The final concentration for the anti-CD34 antibody is 10 μg/ml. After two washes, paramagnetic microspheres (DynaBeads, supplied by Baxter Immunotherapy Group, Santa Ana, California) coated with sheep antimouse IgG (Fc) antibody are added to the cell suspension at a ratio of 2 cells/bead. After a further incubation period of 30 minuter at 4°C, the rosetted cells with magnetic beads are collected with a magnet. Chymopapain (supplied by Baxter Immunotherapy Group, Santa Ana, California) at a final concentration 200 U/ml is added to release the beads from the CD34+ cells. Alternatively, and preferably, an affinity column isolation procedure can be used which binds to CD34, or to antibodies bound to CD34 (see, the examples below). See, Ho et αl. (1995) Stem Cells 13 Usuppl. 3): 100-105. See also, Brenner (1993) Journal of hematotherapy 2: 7-17 ^'.

In another embodiment, hematopoietic stem cells are isolated from fetal cord blood. Yu et al. (1995) Proc. Natl. Acad. Sci. USA 92: 699-703 describe a preferred met- hod of transducing CD34⁺ cells from human fetal cord blood using retroviral vectors.

B) In vivo transformation Vectors (e.g., retroviruses, adenoviruses, liposomes, etc.) containing therapeutic nucleic acids can be administered directly to the organism for transduction of cells in vivo. Administration is by any of the routes normally used for introducing a molecule into ultimate contact with blood or tissue cells. The packaged nucleic acids are administered in any suitable manner, preferably with pharmaceutically accep- table carriers. Suitable methods of administering such packaged nucleic acids are available and well known to those of skill in the art, and, although more than one route can be used to administer a particular composition, a particular route can often provide a more immediate and more effective reaction than another route.

Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions of the present invention.

Formulations suitable for oral administration can consist of (a) liquid solutions, such as an effective amount of the packagecd nucleic acid suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a predetermined amount of the active ingredient, as liquids, solids, granules or gelatin; (c) suspensions in an appropriate liquid; and (d) suitable emulsions. Tablet forms can include one or more of lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, tragacanth, microcrystalline cellulose, acacia, gelatin, colloidal silicon dioxide, croscarmellose sodium, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffering agents, moistening agents, preservatives, flavoring agents, dyes, disintegrating agents, and pharmaceu- tically compatible carriers. Lozenge forms can comprise the active ingredient in a flavor, usually sucrose and acacia or tragacanth, as well as pastilles comprising the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels, and the like containing, in addition to the active ingredient, carriers known in the art.

The packaged nucleic acids, alone or in combination with other suitable components, can be made into aerosol formulations (i.e., they can be "nebulized") to be administered via inhalation. Aerosol formulations can be placed into pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

Suitable formulations for rectal administration include, for example, suppositiories, which consist of the packaged nucleic acid with a suppository base. Suitable suppository bases include natural or synthetic triglycerides or paraffin hydrocarbons. In addition, it is also possible to use gelatin rectal capsules which consist of a combination of the packaged nucleic acid with a base, including, for example, liquid triglycerides, polyethylene glycols, and paraffin hydrocarbons. Formulations suitable for parenteral administration, such as, for example, by intra- articular (in the joints), intravenous, intramuscular, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous, isotonic sterile injection solutions, which can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that can include suspending agents, solubili- zers, thickening agents, stabilizers, and preservatives. In the practice of this inven- tion, compositions can be administered, for example, by intravenous infusion, orally, topically, intraperitoneally, intravesically or intrathecally. Parenteral administration and intravenous administration are the preferred methods of administration. The formulations of packaged nucleic acid can be presented in unit-dose or multi-dose sealed containers, such as ampules and vials. Injection solutions and suspensions can be prepared from sterile powders, granules, and tablets of the kind previously described. Cells transduced by the packaged nucleic acid as decribed above in the context of ex vivo therapy can also be administered intravenously or parenterally as described above.

The dose administered to a patient, in the context of the present invention should be sufficient to effect a beneficial therapeutic response in the patient over time. The dose will be determined by the efficacy of the particular vector employed and the condition of the patient, as well as the body weight or surface area of the patient to be treated. The size of the dose also will be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a particular vector, or transduced cell type in a particular patient.

For administration, inhibitors and transduced cells of the present invention can be administered at a rate determined by the LD-50 of the inhibitor, vector, or transduced cell type, and the side-effects of the inhibitor, vector or cell type at various concentrations, as applied to the mass and overall health of the patient. Administration can be accomplished via single or divided doses. In a preferred embodiment, prior to infusion, blood samples are obtained and saved o n for analysis. Between 1 X 10 and 1 X 10 transduced cells are infused intravenously over 60-200 minutes. Vital signs and oxygen saturation by pulse oximetry are closely monitored. Blood samples are obtained 5 minutes and 1 hour following infusion and saved for subsequent analysis. Leukopheresis, transduction and reinfu- sion can be repeated are repeated every 2 to 3 months. After the first treatment, infu- sions can be performed on a outpatient basis at the discretion of the clinician. If the reinfusions is given as an outpatient, the participant is monitored for at least 4, and preferably 8 hours following the therapy.

Transduced cells are prepared for reinfusion according to established methods. See, Abrahamsen et al. (1991) J. Clin. Apheresis, 6: 48-53; Carter et al. ( 1988) J. Clin. Apheresis, 4:113-117; Aebersold et al. (1988) J. Immunol. Meth, 1 12: 1-7; Muul et al. (1987) J. Immunol. Methods 101 :171-181 and Carter et al. (1987) Transfusion 27: 362-365. After a period of about 2-4 weeks in culture, the cells should number between 1 X 10⁸ and 1 X 10¹². In this regard, the growth characteristics of cells vary from patient to patient and from cell type to cell type. About 72 hours prior to rein- fusion of the transduced cells, an aliquot is taken for analysis of phenotype, and percentage of cells expressing the therapeutic agent.

EXAMPLES The following examples are offered to illustrate, but not to limit the present invention.

Cloning of a human Fused homolog.

By searching the EST database a partial cDNA clone (GenBank Ace: F07835) was identified having significant sequence identity with the Drosophila Fused. Based on the sequence of this clone. PCR primers were designed in order to use the RACE technology to isolate the missing sequence of the human homolog (Zaphiropoulos and Toftgard, DNA Cell Biology, 15, 1049-1056,1996). The PCR primers used to extend the sequence in the 5' direction from epidermal cDNA were 5' AGA TGC GAC GGC TGG GGT C and 5' GCG TCG ACT GCA GTA GGT CCC GGC AGT CTC (nested primer). The PCR primers used to extend the sequence in the 3' direction from epidermal cDNA were 5' TCT TCG GGC AGC CCC CCT T and 5' GCG TCG ACG TTC TCG GAG CTG GAA GAG AAG AT (nested primer).

The products of the RACE amplification were cloned into the pGEM 5 vector (Promega) and sequenced with dye-didioxy nucleotides using the facilities of Cy- bergene AB.

Two additional splice variants of the human fused homolog were also identified. Variant I had 4 extra nucleotides (GTAG) between positions 124 and 125, and splice variant II lacked the 7 nucelotides (GTGTGCA) at positions 118 to 124. In these variant 5' sequences there are no methionine codons in frame with the remaining fused sequence, but instead termination codons, suggesting that a downstream methionine or non-methionine codons are likely to nbe used for translation initia- tions.

Cloning of a human fused homolog.

By searching the EST database a partial cDNA clone (GenBank Ace: F07835) was identified having significant sequence identity with the drosophila fused. Based on the sequence of this clone, PCR primers were designed in order to use the RACE technology to isolate the missing sequence of the human homolog (Zaphiropoulos and Toftgard, DNA Cell Biology, 15, 1049-1056, 19969). The PCR primers used to extend the sequence in the 5' direction from epidermal cDNA were 5' AGA TGC GAC GGC TGG GGT C and 5' GCG TCG ACT GCA GTA GGT CCC GGC AGT CTC (nested primer=. The PCR primers used to extend the sequence in the 3' direction from epidermal cDNA were 5' TCT TCG GGC AGC CCC CCT T and 5' GCG TCG ACG TTC TCG GAG CTG GAA GAG AAG AT (nested primer). The product of the RACE amplification were cloned into the pGEM 5 vector (Promega) and sequenced with dye-dideoxy necleotides using the facilities of Cy- bergene AB.

Two additional splice variants of the human fused homolog were also identified. Variant I had 4 extra nucleotides (GTAG) between positions 124 and 125, and splice variant II lacked the 7 nucleotides (GTGTGCA) at positions 118 to 124. In these variant 5' sequences there are no methionine codons in frame with the remaining fused sequence, but instead termination codons, suggesting that a downstream methionine or non-methionine codons are likely to be used for translation initiation.

The human fused homolog the the following properties: a. 33,5% amino acid identity with Drosophila fused (within the kinase domain the identity increases to 37,0%>). b. 54,0%) amino acid similarity with Drosophila fused (within the kinase domain the similarity increases to 59,9%>). c. Detected mRNA expresion in epidermis, pancreas, kidney, skeletal muscle, liver, placeta brain, with highest levels in lung.

4. Observed evolutionary conservation in other species including monkey, rat, mouse, dog and cow.

Claims

1. An isolated protein molecule capable of involvement in eliciting an intracellular signal in the human HH-PTC pathway and exhibiting a substantial similarity with the sequence disclosed in SEQ ID NO 2.

2. A protein according to claim 1, which exhibits a sequence similarity of about 80%) with the sequence disclosed in SEQ ID NO 2.

3. A protein according to claim 1 or 2, which is comprised of the amino acid sequence disclosed in SEQ ID NO 2, or an analogue or functional equivalent thereof.

4. An isolated polypeptide capable of involvement in eliciting an intracellular signal in the human HH-PTC pathway and comprised of a subsequence of about 8-15 con- tiguous amino acids of the sequence disclosed in SEQ ID NO 2.

5. A polypeptide according to claim 4 comprised of 10 contiguous amino acids of the sequence disclosed in SEQ ID NO 2.

6. A polypeptide according to claim 4 or 5 for use as a medicament.

7. A substance capable of activating or inhibiting a protein according to any one of claims 1-3 or a polypeptide according to claim 4 or 5, which substance is characterized in the Kinase-regulating property thereof.

8. A substance according to claim 7 for use as a medicament.

9. A DNA sequence encoding a protein according to any one of claims 1-3, or a variant thereof.

10. A DNA sequence encoding a polypeptide according to claim 4 or 5, or a variant thereof.

11. An isolated human DNA sequence capable of involvment in eliciting an intra- cellular signal in the HH-PTCH pathway comprising essentially all of the sequence as disclosed in SEQ ID NO 1.

12. A vector comprising such a DNA sequence as defined in any one of claims 9-11.

13. An expression cassette comprising such a DNA sequence as defined in any one of claims 9-11.

14. A cell comprising such an expression cassette as defined in claim 13 or a vector according to claim 12.

15. An antibody which specifically binds to such a polypeptide as defined in claim 4 or 5.

16. An antibody according to claim 15, which is a monoclonal antibody.

17. A recombinant cell expressing such an antibody as defined in claim 15 or 16.

18. An antibody according to claim 15 or 16 for use as a medicament.

19. A pharmaceutical composition which comprises a polypeptide according to claim 4 or 5 and a pharmaceutically acceptable carrier.

20. A kit for detection of a hu an fused gene, which kit comprises a nucleic acid sequence capable of hybridising specifically with the sequence disclosed in SEQ ID NO 1.

21. A kit for detection of a protein encoded by a human fused gene, comprising a container containing such an antibody as defined in claim 15 or 16.