WO1998010066A1 - Modulators of brca1 activity - Google Patents
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- WO1998010066A1 WO1998010066A1 PCT/US1997/013944 US9713944W WO9810066A1 WO 1998010066 A1 WO1998010066 A1 WO 1998010066A1 US 9713944 W US9713944 W US 9713944W WO 9810066 A1 WO9810066 A1 WO 9810066A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
- C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
- C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
- C07K14/4702—Regulators; Modulating activity
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the invention described herein relates generally to the field of human disease, and more specifically to treating and diagnosing disease based on the presence of modulators of BRCA1 activity.
- Breast cancer is one of the leading causes of cancer deaths of women in the United States, and approximately 170,000 women are affected by the disease each year. About 5% of these reported cases are thought to result from a patient's genetic predisposition to the disease.
- Breast cancer is generally considered to be classifiable as early-age onset and late-age onset, the latter being defined as occurring at about age 50. Approximately 25% of patients diagnosed with breast cancer before the age of 40 are thought to be familial, and thus have an underlying genetic component. Late-age onset breast cancer is also often familial although the risks of a family member developing the disease is less compared to early-age onset if relatives have presented with the disease.
- BRCA1 chromosome 17
- BRCA1 chromosome 17
- Additional studies on sporadic cases of breast cancer have also established a genetic link with this disease to BRCA1 which was more precisely localized to the chromosomal region 17q21. See, Hall, ⁇ . ., et al. Science, vol. 250: 1684-1689 (1990).
- the BRCA1 gene has been cloned, and shown to encode a protein having the properties of a tumor suppressor protein. See, Miki, Y., et al Science, vol. 266: 66-71; and WO96/05306. It has been known for some time that a variety of cancers are caused, at least in part, by mutations to certain normal genes, termed "proto- oncogenes.” Proto-oncogenes are involved in regulating normal cell growth in ways that are only now beginning to be appreciated at the molecular level. The mutated proto-oncogenes, or cancer causing genes termed “oncogenes,” disrupt normal cell growth which ultimately causes the death of the organism, if the cancer is not detected and treated in time.
- tumor suppressor proteins include BRCA1, p53, retinoblastoma protein (Rb), adenomatous polyposis coli protein (APC), Wilm's tumor 1 protein (WT1), neurofibromatosis type 1 protein (NF1), and neurofibromatosis type 2 protein (NF2).
- BRCA1 cDNA encodes a 1863 amino acid protein with a predicted molecular weight of approximately 207,000. See, Miki, Y., et al. (1994) Science vol.
- BRCA1 The cloning and characterization of BRCA1 has facilitated establishing it as a tumor suppressor protein. For example, recent work by several investigators have shown that transfection and expression of the BRCA1 gene sequence into MCF-7 tumor cells retards tumor growth in vivo, and extends the survival time of tumor bearing animals. See, Holt, J. T., et al, (1996) Nat. Genet, vol. 12, pages 298-302. Similar results were obtained using a retroviral vector expressing wild-type BRCA1 against an established MCF-7 peritoneal tumor.
- the interactions of certain tumor suppressor proteins with host cell proteins have begun to be elucidated. See, Levin, A., Annu. Rev. Biochem. 1993, vol. 62: pages 623-651.
- the identification of proteins involved in these interactions will facilitate the development of novel diagnostic methods, as well as novel therapeutics for identifying and treating cancer.
- the retinoblastoma tumor suppressor protein is phosphorylated at serine residues adjacent to a proline. The level of phosphorylation is high through S, G2, and M-phase of the cell cycle.
- the kinase that effects this reaction is, in turn, activated by a cyclin that regulates events in the cell cycle.
- a phosphatase removes the phosphate groups from the protein, and returns the retinoblastoma tumor suppressor protein to an unphosphorylated state in Go-Gl.
- the identification of drugs that can effect these interactions can be expected to play a critical role in regulating cell growth and thus be useful in the treatment of cancer.
- a first object of the invention is to describe a family of related isolated nucleic acid sequences that encode proteins, hereinafter referred to as Modulator Proteins, that bind to the tumor suppressor protein BRCA1.
- a second object of the invention is to describe a family of related isolated nucleic acid sequences that encode BRCA1 Modulator Proteins having a range of molecular weights ranging from about 45-97 kdaltons, at least one leucine zipper domain, and optionally a zinc finger domain, that bind BRCA1 at a discreet sequence for Modulator Protein binding that is encompassed in the first six hundred amino acids of BRCA1.
- a third object of the invention is to describe a BRCA1 Modulator Protein having a calculated molecular weight of about 53 kdaltons that has one leucine zipper domain and a zinc finger domain, both domains near the amino terminal region of the molecule, that bind BRCA1 at a consenus sequence for Modulator Protein binding encompassed within the first six hundred amino acids of BRCA1.
- a fourth object of the invention is to describe isolated nucleic acid or protein fragments of BRCA1 Modulator Protein (s), respectively.
- a fifth object of the invention is to describe host cells transformed with isolated nucleic acid sequences that encodes BRCA1 Modulator Protein(s) or fragments thereof.
- a sixth object of the invention is to describe vectors that contain isolated nucleic acid sequences that encode BRCA1 Modulator Protein(s) or fragments thereof.
- a seventh object of the invention is to describe complexes consisting of full length or fragments of BRCA1 and BRCA1 Modulator Proteins.
- An eighth object of the invention is to describe methods of diagnosing disease, preferably those involving unwanted cell growth, including cancer, using isolated nucleic acid sequences, or fragments thereof, that encode a BRCA1 Modulator Protein, or fragments thereof.
- a ninth object of the invention is to describe an assay for identifying compounds that would have therapeutic applications for the treatment of diseases involving unwanted cell growth, including cancer.
- Figure 1 shows the cDNA and amino acid sequence of the BRCA1 Modulator Protein, depicted in Sequence ID No. 1, 091-21 A31.
- Figure 2 shows the cDNA and amino acid sequence of the BRCA1 Modulator Protein, depicted in Sequence ID No. 3, 091-1F84.
- Figure 3 shows the cDNA and amino acid sequence of the BRCA1 Modulator Protein, depicted in Sequence ID No. 5, 091-132Q20.
- Figure 4 shows the format of an assay to identify compounds that increase the intracellular levels of BRCA1.
- Table 1 shows the regions of BRCA1 that interact with the BRCA1 Modulator Proteins 091-1F84, Sequence ID No. 3, 091-21 A31, Sequence ID No. 1 and 091-132Q20, Sequence ID No. 5.
- the experiment was conducted using the two-hybrid assay as described in U. S. Patent No. 5, 283, 173, or Chien et al., 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582.
- the amino- and carboxy-terminal groups although often not specifically shown, will be understood to be in the form they would assume at physiological pH values, unless otherwise specified.
- the N-terminal H 2 + and C-terminal-O at physiological pH are understood to be present though not necessarily specified and shown, either in specific examples or in generic formulas.
- the left-hand end of the molecule is the amino terminal end and the right-hand end is the carboxy-terminal end, in accordance with standard usage and convention.
- the basic and acid addition salts including those which are formed at nonphysiological pH values are also included in the compounds of the invention.
- amino acid residues described herein are preferably in the "L" isomeric form.
- Stereoisomers e.g., D-amino acids
- unnatural amino acids such as a,a-distributed amino acids, N-alkyl amino acids, lactic acid, and other unconventional amino acids may also be suitable components for polypeptides of the present invention, as long as the desired functional property is retained by the polypeptide.
- each encoded residue where appropriate is represented by a three letter designation, corresponding to the trivial name of the conventional amino acid, in keeping with standard polypeptide nomenclature (described in T. Biol. Chem., 243:3552-59 (1969) and adopted at 37 CFR ⁇ 1.822(b)(2)).
- Free functional groups including those at the carboxy- or amino-terminus, referred to as noninterfering substituents, can also be modified by amidation, acylation or other substitution, which can, for example, change the solubility of the compounds without affecting their activity. This may be particularly useful in those instances where BRCA1 Modulator Proteins are known to have certain regions that bind to BRCA1, and it is desirable to make soluble peptides from these regions.
- BRCA1 Modulator Proteins are known to have certain regions that bind to BRCA1, and it is desirable to make soluble peptides from these regions.
- isolated protein means a protein of cDNA, recombinant RNA, or synthetic origin or some combination thereof, which by virtue of its origin the "isolated protein” (1) is not substantially associated with proteins found in nature, (2) is substantially free of other proteins from the same source, e.g. free of human proteins, (3) may be expressed by a cell from a different species, or (4) does not occur in nature.
- naturally-occurring refers to the fact that an object can be found in nature.
- a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature and which has not been intentionally modified by man in the laboratory is naturally-occurring.
- polynucleotide as referred to herein means a polymeric form of nucleotides of at least 10 bases in length, either ribonucleotides or deoxynucleotides or a modified form of either type of nucleotide.
- the term includes single and double stranded forms of DNA.
- oligonucleotide includes naturally occurring, and modified nucleotides linked together by naturally occurring, and non-naturally occurring oligonucleotide linkages.
- Oligonucleotides are a polynucleotide subset with 200 bases or fewer in length. Preferably oligonucleotides are 10 to 60 bases in length and most preferably 12, 13, 14, 15, 16, 17, 18, 19, or 20 to 40 bases in length. Oligonucleotides are usually single stranded, e.g. for probes; although oligonucleotides may be double stranded, e.g. for use in the construction of a gene mutant.
- Oligonucleotides of the invention can be either sense or antisense oligonucleotides.
- naturally occurring nucleotides referred to herein includes deoxyribonucleotides and ribonucleotides.
- modified nucleotides referred to herein includes nucleotides with modified or substituted sugar groups and the like.
- oligonucleotide linkages includes oligonucleotides linkages such as phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoroanilothioate, phoshoroaniladate, phosphoroamidate, and the like.
- An oligonucleotide can include a label for detection, if desired.
- sequence homology referred to herein describes the proportion of base matches between two nucleic acid sequences or the proportion amino acid matches between two amino acid sequences.
- sequence homology When sequence homology is expressed as a percentage, e.g., 50%, the percentage denotes the proportion of matches over the length of sequence from BRCA 1 that is compared to some other sequence. Gaps (in either of the two sequences) are permitted to maximize matching; gap lengths of 15 bases or less are usually used, 6 bases or less are preferred with 2 bases or less more preferred.
- sequence homology between the target nucleic acid and the oligonucleotide sequence is generally not less than 17 target base matches out of 20 possible oligonucleotide base pair matches (85%); preferably not less than 9 matches out of 10 possible base pair matches (90%), and most preferably not less than 19 matches out of 20 possible base pair matches (95%).
- Two amino acid sequences are homologous if there is a partial or complete identity between their sequences. For example, 85% homology means that 85% of the amino acids are identical when the two sequences are aligned for maximum matching. Gaps (in either of the two sequences being matched) are allowed in maximizing matching; gap lengths of 5 or less are preferred with 2 or less being more preferred. Alternatively and preferably, two protein sequences (or polypeptide sequences derived from them of at least 30 amino acids in length) are homologous, as this term is used herein, if they have an alignment score of at more than 5 (in standard deviation units) using the program ALIGN with the mutation data matrix and a gap penalty of 6 or greater.
- BRCA1 Modulator Protein is the presence of a leucine zipper domain.
- the latter is defined as a stretch of amino acids rich in leucine residues, generally every seventh residue, which provide a means whereby a protein may dimerize to form either homodimers or heterodimers.
- proteins with leucine zippers include Jun and Fos.
- An optional property of a BRCA1 Modulator Protein is the presence of a zinc finger domain, preferrably of the type C,H 2 C 3 , C 3 HC 4 , or CX 2 CX U 27 CXHX 2 H or CX 2 CX 6 17 CX 2 C; where C, X, and H denote cysteine, an amino acid, and histidine, respectively.
- the domain binds zinc ions, and is often associated with proteins that bind DNA. Such domains are readily identified using an appropriate data base known to a skilled practitioner of this art, particularly the Prosite Protein Database.
- substantially pure means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other macromolecular individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80 percent of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.
- Polypeptide refer to proteins or peptides that affect the activity of the BRCA1 gene or the protein encoded by the gene. Each of these definitions is meant to encompass one or more such entities.
- DNA regions are operably linked when they are functionally related to each other.
- a promoter is operably linked to a coding sequence if it controls the transcription of the sequence
- a ribosome binding site is operably linked to a coding sequence if it is positioned so as to permit translation.
- operably linked means contiguous and, in the case of leader sequences, contiguous and in reading frame.
- Suitable host cells include prokaryotes, yeast cells, or higher eukaryotic cells.
- Prokaryotes include gram negative or gram positive organisms, for example Escherichia coli (E. col ⁇ ) or Bacilli.
- Higher eukaryotic cells include established cell lines of mammalian origin as described below.
- Exemplary host cells are DH5a , £. coli W3110
- Autographa californica nuclear polyhidrosis virus ( AcNPV) may be used as a vector to express foreign genes. (E.g., see Smith et al, 1983, J. Virol.
- Sf9 insect cells are infected with a baculovirus vector expressing a glu-glu epitope tagged BRCA1 Modulator construct.
- a baculovirus vector expressing a glu-glu epitope tagged BRCA1 Modulator construct.
- Other epitope tags may be employed that are known in the art including a 6x histidine tag , myc, or an EE-tag (i.e. Glu-Glu-tag).
- E refers to the amino acid glutamine.
- microbial vectors will contain an origin of replication recognized by the intended host, a promoter which will function in the host and a phenotypic selection gene such as a gene encoding proteins conferring antibiotic resistance or supplying an autotrophic requirement. Similar constructs will be manufactured for other hosts. £. coli is typically transformed using pBR322. See Bolivar et al, Gene 2, 95 (1977). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides easy means for identifying transformed cells. Expression vectors should contain a promoter which is recognized by the host organism. This generally means a promoter obtained from the intended host.
- Promoters most commonly used in recombinant microbial expression vectors include the beta-lactamase (penicillinase) and lactose promoter systems (Chang et al, Nature 275, 615 (1978); and Goeddel et al, Nucleic Acids Res. 8, 4057 (1980) and EPO Application Publication Number 36,776) and the tac promoter (H. De Boer et al, Proc. Natl. Acad. Sci. USA 80, 21 (1983)). While these are commonly used, other microbial promoters are suitable.
- the promoter and Shine-Dalgarno (SD) sequence are operably linked to the DNA encoding BRCA 1, i.e. they are positioned so as to promote transcription of the BRCA 1 messenger RNA from the DNA.
- SD sequence is thought to promote binding of mRNA to the ribosome by the pairing of bases between the SD sequence and the 3' end of E. coli 16S rRNA (Steitz et al. (1979).
- a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription.
- a naturally occurring promoter of non-bacterial origin can also be coupled with a compatible RNA polymerase to produce high levels of expression of some genes in prokaryotes.
- the bacteriophage T7 RNA polymerase/promoter system is an example of a coupled promoter system (Studier et al. (1986) /. Mol. Biol. 189:113; Tabor et al. (1985) Proc. Natl. Acad. Sci. 82:1074).
- a hybrid promoter can also be composed of a bacteriophage promoter and an E. coli operator region (EPO Pub. No. 267,851).
- BRCA1 Modulators can be expressed intracellularly.
- a promoter sequence can be directly linked with a BRCA1 Modulator gene or a fragment thereof, in which case the first amino acid at the N-terminus will always be a methionine, which is encoded by the ATG start codon.
- methionine at the N-terminus can be cleaved from the protein by in vitro incubation with cyanogen bromide or by either in vivo on in vitro incubation with a bacterial methionine N-terminal peptidase (EPO Pub. No. 219,237).
- Eukaryotic microbes such as yeast cultures may be transformed with suitable BRCA1 Modulator vectors. See, e.g. U.S.
- Saccharomyces cerevisiae is the most commonly used among lower eukaryotic host microorganisms, although a number of other strains are commonly available.
- Yeast vectors may contain an origin of replication from the 2 micron yeast plasmid or an autonomously replicating sequence (ARS), a promoter, DNA encoding BRCA1 Modulator, sequences for polyadenylation and transcription termination, and a selection gene.
- ARS autonomously replicating sequence
- Suitable promoting sequences in yeast vectors include the promoters for metallothionein, 3-phosphogly cerate kinase (Hitzeman et al, J. Biol. Chem. 255, 2073 (1980) or other glycolytic enzymes (Hess et al, J. Adv. Enzyme Reg.
- enolase such as enolase, glyceraldehyde-3- phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.
- Suitable vectors and promotes for use in yeast expression are further described in R. Hitzman et al, EPO Publication Number 73,657.
- transcriptional and translational control sequences in expression vectors to be used in transforming vertebrate cells are often provided by viral sources.
- promoters are derived from CMV, polyoma, Adenovirus 2, and Simian Virus 40 (SV40). See, e.g., U.S. Patent Number 4,599,308.
- An origin of replication may be provided either by construction of the vector to include an exogenous origin, such as may be derived from SV40 or other viral source (e.g. Polyoma, Adenovirus, VSV, or BPV), or may be provided by the host cell chromosomal replication mechanism. If the vector is integrated into the host cell chromosome, the latter may be sufficient.
- BRC Al Modulators can be identified using several different techniques for detecting protein-protein interactions. Among the traditional methods which may be employed are co-immunoprecipitation, crosslinking and co-purification through gradients or chromatographic columns of cell lysates, or proteins obtained from cell lysates using BRCA1 to identify proteins in the lysate that interact with BRCA1. Such assays may employ full length BRCA1 or a BRCA1 peptide. Once isolated, such an intracellular protein can be identified and can, in turn, be used, in conjunction with standard techniques, to identify proteins with which it interacts.
- amino acid sequence of an intracellular protein which interacts with BRCA1 can be ascertained using techniques well known to those of skill in the art, such as the Edman degradation technique. (See, e ⁇ g., Creighton, 1983, "Proteins: Structures and Molecular Principles", W.H. Freeman & Co., N.Y., pp.34-49).
- the amino acid sequence obtained may be used as a guide for the generation of oligonucleotide mixtures that can be used to screen for gene sequences encoding such intracellular proteins. Screening may be accomplished, for example, by standard hybridization or PCR techniques. Techniques for the generation of oligonucleotide mixtures and the screening are well-known.
- methods may be employed which result in the simultaneous identification of genes which encode the intracellular proteins interacting with BRCAl.
- methods include, for example, probing expression libraries, in a manner similar to the well known technique of antibody probing of ⁇ gtll libraries, using labeled BRCAl protein, or fusion protein, e.g., BRCAl fused to a marker (e.g., an enzyme, fluor, luminescent protein, or dye), or an Ig-Fc domain.
- a marker e.g., an enzyme, fluor, luminescent protein, or dye
- plasmids are constructed that encode two hybrid proteins: one plasmid consists of nucleotides encoding the DNA-binding domain of a transcription activator protein fused to a BRCAl nucleotide sequence encoding BRCAl, or BRCAl peptide or fusion protein, and the other plasmid consists of nucleotides encoding the transcription activator protein's activation domain fused to a cDNA encoding an unknown protein which has been recombined into this plasmid as a part of the cDNA library.
- the DNA-binding domain fusion plasmid and the cDNA library are transformed into a strain of the yeast Saccharomyces cerevisiae that contains a reporter gene (e.g., HIS3 or lacZ) whose regulatory region contain the transcription activator's binding site.
- a reporter gene e.g., HIS3 or lacZ
- Either hybrid protein alone cannot activate transcription of the reporter gene; the DNA-binding domain hybrid cannot because it does not provide activation function, and the activation domain hybrid cannot because it cannot localize to the activator's binding sites. Interaction of the two hybrid proteins reconstitutes the functional activator protein and results in transcriptional activation of the reporter gene, which is detected by an assay for the reporter gene product.
- the two-hybrid system or related methodology may be used to screen activation domain libraries for proteins that interact with the "bait" gene product.
- BRCAl peptides, or fusion proteins are used as the bait gene product.
- Full length BRCAl alone can act as a transcriptional activator protein and thus cannot serve as "bait.”
- Total genomic or cDNA sequences are fused to the DNA encoding an activation domain.
- This library and a plasmid encoding a hybrid of a bait BRCAl gene product fused to the DNA-binding domain are cotransformed into a yeast reporter strain, and the resulting tranformants are screened for those that have transcriptionally activated reporter gene.
- a bait BRCAl gene sequence such as the open reading frame of BRCAl (or a domain of BRCAl) can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein.
- These colonies are purified and the library plasmids responsible for reporter gene transcription are isolated.
- DNA sequencing is then used to determine the nucleotide sequence of the clones which, in turn, reveals the identity of the protein sequences encoded by the library plasmids.
- a cDNA library of the cell line from which proteins that interact with bait can be cloned into a vector such that it is translationally fused to the DNA encoding the DNA-binding domain of the GAL4 protein.
- the cDNA fragments can be inserted into a vector such that they are translationally fused to the transcriptional activation domain of GAL4.
- This library can be co-transformed along with the bait BRCAl gene-GAL4 fusion plasmid into a yeast strain which contains a lacZ gene driven by a promoter which contains GAL4 activation sequence.
- a cDNA encoded protein, fused to GAL4 transcriptional activation domain, that interacts with bait BRCAl gene product will reconstitute an active GAL4 protein and thereby drive expression of the HIS3 gene.
- Colonies which express HIS3 can be detected by their growth on petri dishes containing semi-solid agar based media lacking histidine. The cDNA can then be purified from these strains, and used to produce and isolate the bait BRCAl gene-interacting protein using techniques routinely practiced in the art.
- BRCAl Modulator cDNA The cDNA, and deduced amino acid sequences, of three representative BRCAl Modulator Proteins are shown in Figures 1-3.
- the cDNAs or the proteins that they encode are hereinafter referred to as 091-21 A31, Sequence ID No. 1, 091-1F84, Sequence ID No. 3, and 091-132Q20, Sequence ID No. 5.
- the cDNAs encode proteins that have calculated molecular weights in the range of about 45-97kd. Particularly noteworthy is the presence of at least one leucine zipper motif, and optionally a zinc finger domain.
- the BRCAl Modulator Protein nucleotide sequences of the invention include: (a)the DNA sequences shown in Figures 1-3 or contained in the cDNA clones as deposited with the American Type Culture Collection on August 14, 1996 (ATCC) under accession numbers 98141 (091-1F84, Sequence ID No. 3), 98142 (091-21 A31, Sequence ID No. 1), and 98143 (091-132Q20, Sequence ID No.
- Functional equivalents include naturally occurring BRCAl Modulator Protein genes present in other species, and mutant BRCAl Modulator Protein genes whether naturally occurring or engineered which retain at least some of the functional activities of a BRCAl Modulator Protein (i.e., binding to BRCAl).
- the invention also includes degenerate variants of sequences (a) through (c).
- the invention also includes nucleic acid molecules, preferably DNA molecules, that hybridize to, and are therefore the complements of, the nucleotide sequences (a)- through (c), in the preceding paragraph.
- Such hybridization conditions may be highly stringent or less highly stringent, as described above.
- highly stringent conditions may refer, e ⁇ , to washing in 6xSSC/0.05% sodium pyrophosphate at 37°C (for 14-base oligos), 48°C (for 17-base oligos), 55°C (for 20-base oligos), and 60°C (for 23-base oligos).
- nucleic acid molecules may encode or act as BRCAl Modulator gene antisense molecules, useful, for example, in gene regulation (for and/or as antisense primers in amplification reactions of BRCAl Modulator gene nucleic acid sequences).
- Such sequences may be used as part of ribozyme and /or triple helix sequences, also useful for BRCAl Modulator gene regulation.
- such molecules may be used as components of diagnostic methods whereby, for example, the presence of a particular BRCAl Modulator allele associated with uncontrolled cell growth (i.e. cancer) may be detected.
- a BRCAl Modulator gene homolog may be isolated from nucleic acid of an organism of interest by performing PCR using two degenerate oligonucleotide primer pools designed on the basis of amino acid sequences within the BRCAl Modulator gene product disclosed herein.
- the template for the reaction may be cDNA obtained by reverse transcription of mRNA prepared from, for example, human or non-human cell lines or cell types, such as breast or ovarian cells, known or suspected to express a BRCAl Modulator gene allele.
- the PCR product may be subcloned and sequenced to ensure that the amplified sequences represent the sequences of a BRCAl Modulator gene.
- the PCR fragment may then be used to isolate a full length cDNA clone by a variety of methods.
- the amplified fragment may be labeled and used to screen a cDNA library, such as a bacteriophage cDNA library.
- the labeled fragment may be used to isolate genomic clones via the screening of a genomic library.
- RNA may be isolated, following standard procedures, from an appropriate cellular source (i.e.. one known, or suspected, to express a BRCAl Modulator gene, such as, for example, from breast or ovarian cells).
- a reverse transcription reaction may be performed on the RNA using an oligonucleotide primer specific for the most 5' end of the amplified fragment for the priming of first strand synthesis.
- the resulting RNA/DNA hybrid may then be "tailed" with guanines using a standard terminal transferase reaction, the hybrid may be digested with RNAase H, and second strand synthesis may then be primed with a poly-C primer.
- cDNA sequences upstream of the amplified fragment may easily be isolated.
- a cDNA of a mutant BRCAl Modulator gene may also be isolated, for example, by using PCR.
- the first cDNA strand may be synthesized by hybridizing an oligo-dT oligonucleotide to mRNA isolated from cells known or suspected to be expressed in an individual putatively carrying the mutant BRCAl Modulator allele, and by extending the new strand with reverse transcriptase.
- the second strand of the cDNA is then synthesized using an oligonucleotide that hybridizes specifically to the 5' end of the normal gene. Using these two primers, the product is then amplified via PCR, cloned into a suitable vector, and subjected to DNA sequence analysis through methods well known to those of skill in the art. By comparing the DNA sequence of the mutant BRCAl Modulator allele to that of the normal BRCAl Modulator allele, the mutation(s) responsible for the loss or alteration of function of the mutant BRCAl Modulator gene product can be ascertained.
- a genomic library can be constructed using DNA obtained from an individual suspected of or known to carry the mutant BRCAl Modulator allele, or a cDNA library can be constructed using RNA from a cell type known, or suspected, to express the mutant BRCAl Modulator allele.
- the normal BRCAl Modulator gene or any suitable fragment thereof may then be labeled and used as a probe to identify the corresponding mutant BRCAl Modulator allele in such libraries.
- Clones containing the mutant BRCAl Modulator gene sequences may then be purified and subjected to sequence analysis according to methods well known to those of skill in the art.
- an expression library can be constructed utilizing cDNA synthesized from, for example, RNA isolated from a cell type known, or suspected, to express a mutant BRCAl Modulator allele in an individual suspected of or known to carry such a mutant allele.
- gene products made by the putatively mutant cell type may be expressed and screened using standard antibody screening techniques in conjunction with antibodies raised against the normal BRCAl Modulator gene product, as described, below.
- screening techniques see, for example, Harlow, E. and Lane, eds., 1988, "Antibodies: A Laboratory Manual", Cold Spring Harbor Press, Cold Spring Harbor.
- screening can be accomplished by screening with labeled fusion proteins.
- a polyclonal set of antibodies to a BRCAl Modulator are likely to cross-react with the BRCAl Modulator mutant.
- Such BRCAl Modulator mutants detected via their reaction with labeled antibodies can be purified and subjected to sequence analysis according to methods well known to those of skill in the art.
- the invention also encompasses nucleotide sequences that encode peptide fragments of a BRCAl Modulator, truncated BRCAl Modulators, and fusion proteins of a BRCAl Modulator.
- Nucleotides encoding fusion proteins may include but are not limited to full length BRCAl Modulators, truncated BRCAl Modulators or peptide fragments to an unrelated protein or peptide, such as for example, an epitope tag which aids in purification or detection of the resulting fusion protein; or an enzyme, fluorescent protein, luminescent protein which can be used as a marker.
- the preferred epitope tag is glu-glu as described by Rubinfeld, B., et al., J. Biol. Chem. vol. 270, no. 10, pp 5549-5555 (1995), and Grussenmyer, T., et al., Proc. Natl. Acad. Sci. U. S. A. vol. 82, pp. 7952-7954 (1985).
- the invention also encompasses (a) DNA vectors that contain any of the foregoing BRCAl Modulator coding sequences and /or their complements (i.e., antisense); (b) DNA expression vectors that contain any of the foregoing BRCAl Modulator coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences; and (c) genetically engineered host cells that contain any of the foregoing BRCAl Modulator coding sequences operatively associated with a regulatory element that directs the expression of the coding sequences in the host cell.
- regulatory elements include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression.
- Such regulatory elements include but are not limited to the baculovirus promoter, cytomegalovirus hCMV immediate early gene, the early or late promoters of SV40 adenovirus, the lac system, the trp_ system, the TAC system, the TRC system, the major operator and promoter regions of phage A, the control regions of fd coat protein, the promoter for 3- phosphoglycerate kinase, the promoters of acid phosphatase, and the promoters of the yeast-mating factors.
- BRCAl Modulator Proteins include but are not limited to inducible and non-inducible promoters, enhancers, operators and other elements known to those skilled in the art that drive and regulate expression.
- Such regulatory elements include but are not limited to the baculovirus promoter, cytomegal
- Figures 1-3 shows the cDNA, and deduced amino acid sequences, of three representative BRCAl Modulator Proteins; 091-21A31, Sequence ID No. 1, 091-1F84, Sequence ID No. 3, and 091-132Q20, Sequence ID No. 5.
- 091-132Q20, Sequence ID No. 5 is not a full length sequence.
- the proteins have calculated molecular weights in the range of about 45-97kd. Particularly noteworthy is the presence of at least one leucine zipper motif, and optionally a zinc finger domain.
- 091-1F84, Sequence ID No. 3 has two leucine zippers
- 091-132Q20, Sequence ID No. 5 has a single leucine zipper
- 091-21A31, Sequence ID No. 1 has a single leucine zipper and a zinc finger domain.
- Such domains are readily identified using the Prosite Protein Database.
- BRCAl Modular Proteins, peptide fragments, mutated, truncated or deleted forms of and fusion proteins of these can be prepared for a variety of uses, including but not limited to the generation of antibodies, as reagents in diagnostic assays, the identification and /or the interaction with other cellular gene products involved in cell growth, as reagents in assays for screening for compounds that can be used in the treatment of unwanted cell growth disorders, including but not limited to cancer, and as pharmaceutical reagents useful in the treatment of such diseases.
- the 091-21 A31, Sequence ID No. 1 BRCAl Modulator Protein sequence begins with a methionine in a DNA sequence context consistent with a translation initiation site.
- the predicted molecular mass of this BRCAl Modulator Protein is 53.3 kD.
- the BRCAl Modulator Protein amino acid sequences of the invention include the amino acid sequence shown in FIG. 1, or the amino acid sequence encoded by the cDNA clone, as deposited with the ATCC, as described above. Further, BRCAl Modulator Proteins of other species are encompassed by the invention. In fact, any BRCAl Modulator Protein protein encoded by the cDNAs described above, are within the scope of the invention.
- the invention also encompasses proteins that are functionally equivalent to the BRCAl Modulator Protein encoded by the nucleotide sequences described above, as judged by any of a number of criteria, including but not limited to the ability to bind BRCAl, the binding affinity for BRCAl , a change in cellular metabolism or change in phenotype when the BRCAl Modulator Protein equivalent is present in an appropriate cell type (such as ovarian or breast cells).
- Such functionally equivalent BRCAl Modulator Protein proteins include but are not limited to additions or substitutions of amino acid residues within the amino acid sequence encoded by the BRCAl Modulator nucleotide sequences described, above, but which result in a silent change, thus producing a functionally equivalent gene product.
- Nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine
- polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine
- positively charged (basic) amino acids include arginine, lysine, and histidine
- negatively charged (acidic) amino acids include aspartic acid and glutamic acid.
- mutant BRCAl Modulator Proteins can be engineered so that regions of interspecies identity are maintained, whereas the variable residues are altered, e.g., by deletion or insertion of an amino acid residue(s) or by substitution of one or more different amino acid residues.
- Conservative alterations at the variable positions can be engineered in order to produce a mutant BRCAl Modulator Protein that retains function.
- Non-conservative changes can be engineered at these variable positions to alter function.
- deletion or non-conservative alterations of the conserved regions can be engineered.
- One of skill in the art may easily test such mutant or deleted BRCAl Modulator Proteins for these alterations in function using the teachings presented herein.
- BRCAl Modulator coding sequence can be made to generate BRCAl Modulator Proteins that are better suited for expression, scale up, etc. in the host cells chosen.
- the triplet code for each amino acid can be modified to conform more closely to the preferential codon usage of the host cell's translational machinery.
- fusion proteins include but are not limited to fusions to an epitope tag (such as is exemplified herein); or fusions to an enzyme, fluorescent protein, or luminescent protein which provide a marker function.
- BRCAl Modulator Proteins and peptides can be chemically synthesized (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y.), large polypeptides derived from the BRCAl Modulator Protein and the full length BRCAl Modulator Protein itself may advantageously be produced by recombinant DNA technology using techniques well known in the art for expressing nucleic acid containing BRCAl Modulator gene sequences and/or coding sequences. Such methods can be used to construct expression vectors containing the BRCAl Modulator nucleotide sequences described above and appropriate transcriptional and translational control signals.
- RNA capable of encoding BRCAl Modulator nucleotide sequences may be chemically synthesized using, for example, synthesizers. See, for example, the techniques described in "Oligonucleotide Synthesis", 1984, Gait, M.J. ed., IRL Press, Oxford, which is incorporated by reference herein in its entirety.
- a variety of host-expression vector systems may be utilized to express the BRCAl Modulator nucleotide sequences of the invention. Where a BRCAl Modulator Protein peptide or polypeptide is a soluble secreted derivative the peptide or polypeptide can be recovered from the culture medium. If the BRCAl Modulator Protein peptide or polypeptide is not secreted, it may be isolated from the host cells. However, such engineered host cells themselves may be used in situations where it is important not only to retain the structural and functional characteristics of a BRCAl Modulator Protein, but to assess biological activity, e.g., in drug screening assays.
- the expression systems that may be used for purposes of the invention include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing BRCAl Modulator nucleotide sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing the BRCAl Modulator nucleotide sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing the BRCAl Modulator sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing BRCAl Modulator nucleotide sequences; or mammalian
- a number of expression vectors may be advantageously selected depending upon the use intended for the BRCAl Modulator gene product being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of BRCAl Modulator Protein or for raising antibodies to the BRCAl Modulator Protein, for example, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
- vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., 1983, EMBO J.
- pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
- the inserted sequence encodes a relatively small polypeptide (less than 25 kD)
- such fusion proteins are generally soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
- the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
- the inclusion bodies may be purified and the recombinant protein solubilized using techniques well known to one of skill in the art.
- Autographa californica nuclear polyhidrosis virus may be used as a vector to express foreign genes.
- AcNPV Autographa californica nuclear polyhidrosis virus
- Sf9 insect cells are infected with a baculovirus vectors expressing either a 6 x HIS- tagged construct, or an (EE)-tagged BRCAl Modulator construct.
- a number of viral-based expression systems may be utilized. Specific embodiments described more fully below express tagged BRCAl Modulator cDNA sequences using a CMV promoter to transiently express recombinant protein in U937 cells or in Cos-7 cells.
- retroviral vector systems well known in the art may be used to insert the recombinant expression construct into host cells. For example, retroviral vector systems for transducing hematopoietic cells are described in published PCT applications WO 96/09400 and WO 94/29438. In cases where an adenovirus is used as an expression vector, the BRCAl
- Modulator nucleotide sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g.. the late promoter and tripartite leader sequence.
- This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the BRCAl Modulator gene product in infected hosts.
- a non-essential region of the viral genome e.g., region El or E3
- Specific initiation signals may also be required for efficient translation of inserted BRCAl Modulator nucleotide sequences. These signals include the ATG initiation codon and adjacent sequences. In cases where an entire BRCAl Modulator gene or cDNA, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only a portion of the BRCAl Modulator coding sequence is inserted, exogenous translational control signals, including, perhaps, the ATG initiation codon, must be provided. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert.
- exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic.
- the efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (See Bittner et al., 1987, Methods in Enzymol. 153:516-544).
- a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
- Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript may be used.
- Such mammalian host cells include but are not limited to CHO, VERO, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and U937 cells.
- stable expression is preferred.
- cell lines which stably express the BRCAl Modulator sequences described above may be engineered.
- host cells can be transformed with DNA controlled by appropriate expression control elements (e.g.. promoter, enhancer sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
- engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
- the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form colonies which in turn can be cloned and expanded into cell lines.
- This method may advantageously be used to engineer cell lines which express the BRCAl Modulator gene product.
- Such engineered cell lines may be particularly useful in screening and evaluation of compounds that affect the endogenous activity of the BRCAl Modulator gene product.
- a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., 1977, Cell 11:223), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc. Natl. Acad. Sci. USA 48:2026), and adenine phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes can be employed in tk “ , hgprt " or aprt " cells, respectively.
- antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567; O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin et al., 1981, J. Mol. Biol.
- the BRCAl Modulator gene products can also be expressed in transgenic animals. Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate BRCAl Modulator transgenic animals.
- Any technique known in the art may be used to introduce the BRCAl Modulator transgene into animals to produce the founder lines of transgenic animals.
- Such techniques include, but are not limited to pronuclear microinjection (Hoppe, P.C. and Wagner, T.E., 1989, U.S. Pat. No. 4,873,191); retrovirus mediated gene transfer into germ lines (Van der Putten et al, 1985, Proc. Natl. Acad. Sci., USA 82:6148-6152); gene targeting in embryonic stem cells (Thompson et al., 1989, Cell 56:313-321); electroporation of embryos (Lo, 1983, Mol Cell. Biol.
- the present invention provides for transgenic animals that carry the BRCAl Modulator transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, Li , mosaic animals.
- the transgene may be integrated as a single transgene or in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
- the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Lasko, M. et al., 1992, Proc. Natl. Acad. Sci. USA 89: 6232-6236).
- the regulatory sequences required for such a cell- type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
- gene targeting is preferred.
- vectors containing some nucleotide sequences homologous to the endogenous BRCAl Modulator gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous BRCAl Modulator gene.
- the expression of the endogenous BRCAl Modulator gene may also be eliminated by inserting non-functional sequences into the endogenous gene.
- the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous BRCAl Modulator gene in only that cell type, by following, for example, the teaching of Gu et al. (Gu et al., 1994, Science 265: 103-106).
- the regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
- BRCAl Modulator gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to assay whether integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include but are not limited to Northern blot analysis of cell type samples obtained from the animal, in situ hybridization analysis, and RT-PCR. Samples of BRCAl Modulator gene-expressing tissue, may also be evaluated immunocytochemically using antibodies specific for the BRCAl Modulator transgene product, as described below. Antibodies to BRCAl Modulator Proteins
- Antibodies that specifically recognize one or more epitopes of a BRCAl Modulator Protein, or epitopes of conserved variants, or peptide fragments are also encompassed by the invention.
- Such antibodies include but are not limited to polyclonal antibodies, monoclonal antibodies (mAbs), humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab')2 fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies, and epitope- binding fragments of any of the above.
- the antibodies of the invention may be used, for example, in the detection of the BRCAl Modulator Protein in a biological sample and may, therefore, be utilized as part of a diagnostic or prognostic technique whereby patients may be tested for abnormal amounts of these proteins. Such antibodies may also be utilized in conjunction with, for example, compound screening schemes, as described herein for the evaluation of the effect of test compounds on expression and/or activity of the BRCAl Modulator Protein. Additionally, such antibodies can be used in conjunction with the gene therapy techniques described herein, to, for example, evaluate the normal and /or engineered BRCAl Modulator Protein expressing cells prior to their introduction into the patient. Such antibodies may additionally be used as a method for the inhibition of abnormal BRCAl Modulator Protein activity.
- various host animals may be immunized by injection with the BRCAl Modulator Protein, a BRCAl Modulator Protein peptide, truncated BRCAl Modulator Protein polypeptides, functional equivalents of the BRCAl Modulator Protein or mutants of the BRCAl Modulator Protein.
- Such host animals may include but are not limited to rabbits, mice, and rats, to name but a few.
- adjutants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjutants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum.
- BCG Bacille Calmette-Guerin
- Monoclonal antibodies which are homogeneous populations of antibodies to a particular antigen, may be obtained by any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique of Kohler and Milstein, (1975, Nature 256:495-497; and U.S. Patent No. 4,376,110), the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R.
- Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof.
- the hybridoma producing the mAb of this invention may be cultivated in vitro or in vivo. Production of high titers of mAbs in vivo makes this the presently preferred method of production.
- chimeric antibodies In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, Proc. Natl. Acad. Sci. USA, 81:6851-6855; Neuberger et al., 1984, Nature, 312:604-608; Takeda et al., 1985, Nature, 314:452-454) by splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity can be used.
- a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a mAb and a human immunoglobulin constant region.
- single chain antibodies can be adapted to produce single chain antibodies against BRCAl Modulator Protein gene products.
- Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
- Antibody fragments which recognize specific epitopes may be generated by known techniques.
- such fragments include but are not limited to: the F(ab')2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragments.
- Fab expression libraries may be constructed (Huse et al., 1989, Science, 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
- Antibodies to the BRCAl Modulator Protein can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" the BRCAl Modulator Protein using techniques well known to those skilled in the art.
- BRCAl gene encodes a protein that has been shown to have tumor suppressor activity. See, Holt, J. T., et al, (1996) Nat. Genet, vol. 12, pages 298-302. Such studies have shown that certain cancer cells have low levels of BRCAl, and that increasing the levels causes a reversion to the normal cell phenotype. Thus, compounds that increase BRCAl levels will have significant therapeutic use for the treatment of cancer.
- An aspect of the instant invention is the description of an assay using BRCAl and BRCAl Modulators that facilitates the identification of compounds that increase intracellular levels of BRCAl.
- One format of the assay is shown in schematic form in Figure 4. Briefly, the assay makes use of two events: firstly, BRCAl is known to be a general transcriptional activator, and secondly, BRCAl Modulators bind to BRCAl.
- the assay makes use of certain features of the two-hybrid assay described above. Two plasmids are constructed and transfected into a suitable cell line, preferrably a breast or ovarian cell line. A preferred breast cell line would be MCF-7.
- One plasmid contains the nucleotide sequence recognized by GAL4 operably linked to an activator sequence, and a reporter gene downstream of this sequence.
- An example of a preferred reporter gene is the gene that encodes luciferase.
- the second plasmid encodes and expresses the GAL4 DNA binding domain fused to a BRCAl Modulator.
- the preferred Modulator is 091-21 A31, Sequence ID No. 1.
- the GAL4 DNA binding domain-BRCAl Modulator fusion protein binds to the GAL4 DNA binding domain on the first plasmid which, in turn, recruits any BRCAl present to form a complex consisting of GAL4 DNA binding domain-BRCAl Modulator fusion and BRCAl.
- BRCAl is in proximity to the activator sequence which in turn initiates transcription of the reporter gene.
- compounds can be tested for their capacity to stimulate the production of BRCAl. Those that do will cause an increase in the reporter gene product.
- the above assay is schematically presented in Figure 4.
- BRCAl is a known tumor suppressor. See, Holt, J. T., et al,
- the basic principle of the assay systems used to identify such compounds that affect BRCAl interactions with BRCAl Modulator Proteins involves preparing a reaction mixture containing BRCAl protein, polypeptide, peptide or fusion protein as described above, and a BRCAl Modulator Protein under conditions and for a time sufficient to allow the two to interact and bind, thus forming a complex.
- the reaction mixture is prepared in the presence and absence of the test compound.
- the test compound may be initially included in the reaction mixture, or may be added at a time subsequent to the addition of the BRCAl moiety and its BRCAl Modulator Protein. Control reaction mixtures are incubated without the test compound or with a placebo.
- any complexes between the BRCAl moiety and the BRCAl Modulator Protein is then detected.
- the formation of a complex in the control reaction, but not in the reaction mixture containing the test compound, indicates that the compound interferes with the interaction of the BRCAl and the interactive BRCAl Modulator Protein.
- complex formation within reaction mixtures containing the test compound and normal BRCAl protein may also be compared to complex formation within reaction mixtures containing the test compound and a mutant BRCAl. This comparison may be important in those cases wherein it is desirable to identify compounds that disrupt interactions of mutant but not normal BRCAl.
- the assay for compounds that interfere with the interaction of the BRCAl and BRCAl Modulator Proteins can be conducted in a heterogeneous or homogeneous format.
- Heterogeneous assays involve anchoring either the BRCAl moiety or the
- test compounds that interfere with the interaction by competition can be identified by conducting the reaction in the presence of the test substance; e_., by adding the test substance to the reaction mixture prior to or simultaneously with the BRCAl moiety and interactive BRCAl Modulator Protein.
- test compounds that disrupt preformed complexes e.g. compounds with higher binding constants that displace one of the components from the complex, can be tested by adding the test compound to the reaction mixture after complexes have been formed. Representative formats are described briefly below.
- either the BRCAl moiety or the interactive BRCAl Modulator Protein is anchored onto a solid surface, while the non-anchored species is labeled, either directly or indirectly.
- the anchored species may be immobilized by non-covalent or covalent attachments. Non-covalent attachment may be accomplished simply by coating the solid surface with a solution of BRCAl or BRCAl Modulator Protein and drying. Alternatively, an immobilized antibody specific for the species to be anchored may be used to anchor the species to the solid surface. The surfaces may be prepared in advance and stored.
- the partner of the immobilized species is exposed to the coated surface with or without the test compound. After the reaction is complete, unreacted components are removed (e.g.. by washing) and any complexes formed will remain immobilized on the solid surface.
- the detection of complexes anchored on the solid surface can be accomplished in a number of ways. Where the non-immobilized species is pre-labeled, the detection of label immobilized on the surface indicates that complexes were formed.
- an indirect label can be used to detect complexes anchored on the surface; e.g., using a labeled antibody specific for the initially non-immobilized species (the antibody, in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody).
- the antibody in turn, may be directly labeled or indirectly labeled with a labeled anti-Ig antibody.
- test compounds which inhibit complex formation or which disrupt preformed complexes can be detected.
- the reaction can be conducted in a liquid phase in the presence or absence of the test compound, the reaction products separated from unreacted components, and complexes detected; e.g., using an immobilized antibody specific for one of the binding components to anchor any complexes formed in solution, and a labeled antibody specific for the other partner to detect anchored complexes.
- test compounds which inhibit complex or which disrupt preformed complexes can be identified.
- a homogeneous assay can be used.
- a preformed complex of the BRCAl moiety and the interactive BRCAl Modulator Protein is prepared in which either the BRCAl or its BRCAl Modulator Proteins is labeled, but the signal generated by the label is quenched due to formation of the complex (see, e.g., U.S. Patent No.4,109,496 by Rubenstein which utilizes this approach for immunoassays).
- the addition of a test substance that competes with and displaces one of the species from the preformed complex will result in the generation of a signal above background. In this way, test substances which disrupt BRCAl /intracellular BRCAl Modulator Protein interaction can be identified.
- a BRCAl fusion protein can be prepared for immobilization.
- BRCAl or a peptide fragment can be fused to a glutathione-S-transferase (GST) gene using a fusion vector, such as pGEX-5X-l, in such a manner that its binding activity is maintained in the resulting fusion protein.
- GST glutathione-S-transferase
- the interactive BRCAl Modulator Protein can be purified and used to raise a monoclonal antibody, using methods routinely practiced in the art and described above. This antibody can be labeled with the radioactive isotope l- ⁇ I, for example, by methods routinely practiced in the art.
- the GST-BRCA1 fusion protein can be anchored to glutathione-agarose beads.
- the interactive BRCAl Modulator Protein can then be added in the presence or absence of the test compound in a manner that allows interaction and binding to occur.
- unbound material can be washed away, and the labeled monoclonal antibody can be added to the system and allowed to bind to the complexed components.
- the interaction between the BRCAl protein and the interactive BRCAl Modulator Protein can be detected by measuring the amount of radioactivity that remains associated with the glutathione-agarose beads. A successful inhibition of the interaction by the test compound will result in a decrease in measured radioactivity.
- the GST-BRCA1 fusion protein and the interactive BRCAl Modulator Protein can be mixed together in liquid in the absence of the solid glutathione-agarose beads.
- the test compound can be added either during or after the species are allowed to interact. This mixture can then be added to the glutathione- agarose beads and unbound material is washed away. Again the extent of inhibition of the BRCAl /BRCAl Modulator Protein interaction can be detected by adding the labeled antibody and measuring the radioactivity associated with the beads.
- these same techniques can be employed using peptide fragments that correspond to the binding domains of the BRCAl and /or the interactive or BRCAl Modulator in place of one or both of the full length proteins.
- Any number of methods routinely practiced in the art can be used to identify and isolate the binding domains. Such domains are discussed more fully in the examples, below. These methods include, but are not limited to, mutagenesis of the gene encoding one of the proteins and screening for disruption of binding in a co-immunoprecipitation assay. Compensating mutations in the gene encoding the second species in the complex can then be selected. Sequence analysis of the genes encoding the respective proteins will reveal the mutations that correspond to the region of the protein involved in interactive binding.
- the two hybrid assay may also be used, as discussed more fully in the examples below.
- short gene segments can be engineered to express peptide fragments of the protein, which can then be tested for binding activity and purified or synthesized.
- Toxicity and therapeutic efficacy of compounds identified above that affect the interaction of BRCAl with BRCAl Modulator Proteins, and thus affect cell growth can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD5 Q (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
- LD5 Q the dose lethal to 50% of the population
- ED50 the dose therapeutically effective in 50% of the population.
- Numerous model systems are known to the skilled practitioner of the art that can be employed to test the cell growth properties of the instant compounds including growth of cells in soft agar, and effect on tumors in vivo. Such experiments can be conducted on cells co- transfected with BRCAl and BRCAl Modulators
- the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5 Q /ED5 Q .
- Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
- the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
- the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
- the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
- the therapeutically effective dose can be estimated initially from cell culture assays.
- a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC5 Q (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
- IC5 Q i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
- levels in plasma may be measured, for example, by high performance liquid chromatography.
- BRCAl modulators were identified initially using the yeast two hybrid assay system described in U. S. Patent No. 5, 283, 173, or Chien et al, 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582.
- the assay components are also commercially available from Clontech (Palo Alto, CA).
- the cDNA encoding human BRCAl (See, Miki, Y., et al Science, vol. 266: 66-71; and PCT /US95/ 10202) was digested with Mvnl-Nhel and the fragment representing BRCAl amino acids 8-1293 was fused to the GAL4 binding domain in the Smal-Nhel sites of pGBT8 plasmid, which is the pMA424 plasmid of Chien et al. as described in Proc. Natl. Acad. Sci. vol. 88: pages 9578-9582 (1991), modified by the insertion of the sequence 5'-
- the YGH1 strain carrying the plasmid GAL4-BRCA1 (8-1293) was subsequently transformed with a HeLa cell cDNA library fused to the GAL4 activation domain in the pGAD plasmid (Chien et al., Proc. Natl. Acad. Sci. vol. 88: pages 9578-9582 (1991).
- a cDNA encodes a protein that interacts with the BRCAl protein (amino acids 8- 1293)
- the YGH1 strain is expected to grow in the absence of histidine supplemented with 7.5mM 3AT and produce ⁇ -galactosidase.
- One of the plasmids contained the novel cDNA encoding for the BRCAl Modulator Protein hereinafter termed, 091-21 A31, Sequence ID No. 1.
- the nucleotide and protein sequence are shown in Figure 1.
- the calculated molecular weight is about 53kd, and it has an estimated pi of 9.05. Particularly noteworthy is the presence of a zinc finger domain and a leucine zipper motif.
- the nucleotide sequence of the second cDNA and amino acid sequence that it encodes hereinafter termed, 091-1F84, Sequence ID No. 3, is shown in Figure 2. Note that this clone displays two leucine zipper domains.
- the protein has a calculated molecular weight of 96, 443. 3 and an estimated pi of 4.95.
- the construct containing the first 300 amino acids of BRCAl was generated by subcloning the Ncol-EcoRl blunted BRCAl fragment into the blunted EcoRI site of pGBT8.
- the BRCAl containing amino acids 8-1293 was generated as described above.
- the BRCAl construct containing amino acids 1-600 was generated by subcloning the Ncol-Spel BRCAl fragment into the Ncol-Nhel site of pGBT8.
- Table 1 shows those regions of BRCAl that interact with the proteins encoded by 091-1F84, Sequence ID No. 3, 091-21A31, Sequence ID No. 1, and 091-132Q20, Sequence ID No. 5.
- the "+” sign is a subjective measure of the amount of b-galactosidase activity. One “+” being the lowest, and three “+++” being the highest activity. It is apparent from Table 1 that the first 300 amino acids of BRCAl do not bind to any of the three BRCAl Modulators, but that all three BRCAl Modulators bind to the BRCAl construct containing the first 600 amino acids of BRCA.
- Modulator 091-21 A31 Sequence ID No. 1 that interact with BRCAl.
- the assay was run essentially as described in Example 1. Transformation and growth of yeast cultures were performed essentially as described in U. S. Patent No. 5, 283, 173; Chien et al, 1991, Proc. Natl. Acad. Sci. USA, 88:9578-9582; or Spaargaren, M., et al., (1994) Biochem. J. 300, 303-307.
- the YGH1 yeast strain was co-transformed with cDNA encoding 091- 21A31, Sequence ID No. 1, or cDNA encoding 091-21A31, Sequence ID No. 1 fragments containing amino acids 78-469, 1-300, or 300-469 fused to the GAL4 activation domain.
- bcl-2 cDNA See, U. S. Patent 5, 539, 085
- cDNAs encoding BRCAl fragments having amino acids 1-300, 1- 600, or 8-1293 were fused to the GAL4 binding domain as described in Example 2.
- the 091-21A31, Sequence ID No. 1 constructs were generated using the plasmids pGADGH or pGAD424; both are available from Clontech.
- the 091-21A31, Sequence ID No. 1 construct containing amino acids 75-469 was generated by subcloning the EcoR -Xhol 091-21A31, Sequence ID No. 1 fragment into the EcoRl-Sall site of ⁇ GAD424.
- the 091-21A31, Sequence ID No. 1 construct containing amino acids 1-300 was generated by subcloning the BamHl-Sall 091-21A31, Sequence ID No. 1 fragment into the BamHl-Sall site of pGADGH.
- the 091-21A31, Sequence ID No. 1 construct containing amino acids 300-469 was generated by subcloning the BamHl blunted-Sall 091-21 A31, Sequence ID No. 1 fragment into the Sail blunted-Xhol site of pGADGH.
- Table 2 shows the results of the co-transformation studies. It is apparent that the first 300 amino acids of BRCAl do not to bind to any of three 091-21 A31, Sequence ID No. 1 fragment constructs, nor to 091-21 A31, Sequence ID No. 1.
- the BRCAl construct containing amino acids 1-600 does bind to 091-21 A31, Sequence ID No. 1, and to the construct containing 091-21A31, Sequence ID No. 1 amino acids 78-469, but not to the amino acid 091-21A31, Sequence ID No. 1 constructs 1-300 and 300-469.
- the BRCAl construct having amino acids 8-1293 also binds 091-21A31, Sequence ID No. 1, the 78-469 and 1-300 amino acid constructs, but not to the 091-21 A31, Sequence ID No. 1 construct having amino acids 300-469.
- BRCAl Modulators were expressed in and purified from baculovirus SF9 infected cells. Methods for producing baculovirus, as well as growing SF9 cells are well known in the art, and detailed procedures can be found in M. Summers and G. Smith in "A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures," Texas Agricultural Experiment Station, Bulletin No. 1555 (May, 1987 or in EPS 127,839 to G. E. Smith and M. D. Summers.
- the following constructs were generated using pAcC13 (See, Rubinfeld, B., et al. Cell 65, 1033-1042 (1991)) or pAcOG, a derivative of pAcCl3 in which the polylinker was replaced with a synthetic linker engineered to encode an initiating methionine, the Glu-Glu (See, Grussenmyer, T., et al. Proc. Natl. Acad. Sci. U.S.A. 82, 7952 (1985)) epitope tag, and a multiple cloning site containing several stop codons (See, Rubinfeld, B., et al. J. Biol. Chem, 270, 5549-5555 (1995)).
- the construct containing 091-21A31, Sequence ID No. 1 was generated by subcloning the Kpnl-Xbal 091-21A31, Sequence ID No. 1 fragment into pAcC13 at the Kpnl-Xbal site.
- the construct containing 091-1F84, Sequence ID No. 3 was generated by subcloning the Ncol - Xbal 091-1F84, Sequence ID No. 3 fragment into pAcOGl at the Ncol-Xbal sites.
- the construct containing 091-132Q20, Sequence ID No. 5 was generated by subcloning the Kpnl-Xbal fragment of 091-132Q20, Sequence ID No. 5 into pAcC13 at the Kpnl-Xbal site.
- Baculovirus containing the appropriate BRCAl Modulator was produced by transfecting the above described plasmids into SF9 cells, and isolating the corresponding baculovirus using essentially the methods described in Pharmingen's cat. no. 21100D, BaculoGoldtm /Baculovirus DNA. Virus was isolated from individual plaques, and used to infect Sf9 cells. The cells were grown for 4 days, isolated by centrifugation, and cell extracts made by solubilizing the cell pellet.
- recombinant Sf9 cells were pelleted, lysed in 5 volumes of [20mM Tris (pH8.0), ImM EDTA, lO ⁇ g/ml each of leupeptin, pepstatin, pefabloc, ImM aprotinin and ImM DTT] and incubated on ice for 10 minutes. NaCl was then added to a final concentration of 150mM, incubated at room temperature for 10 minutes and centrifuged. The resulting supernatant was loaded onto a 1-ml affinity column containing a mouse Glu-Glu monoclonal antibody covalently cross-linked to protein G-Sepharose. See,
- the clarified supernatent was then diluted 1:10 in 1 X TG buffer [20mM Tris (pH8.0), 137mM NaCl, ImM EGTA, 1.5mM MgCl,, 1% Triton X100, 10% glycerol, lO ⁇ g/ml each of leupeptin, pepstatin, pefabloc, ImM aprotinin and ImM DTT], filtered through a 3uM Gelman Versapore filter and loaded onto a 1-ml anti-Glu- Glu affinity column. See, Rubinfeld, B., et al., Mol. Cell. Bio. 12, 4634-4642 (1992).
- BRCAl constructs were generated and tested for BRCAl Modulator binding.
- the BRCAl constructs were Glu-Glu tagged BRCAl 5' (1-1293), and BRCAl 3' (1293-1863).
- the Glu- Glu epitope tag facilitated immunoaffinity purification as described in the above examples.
- a control construct consisted of rapGAP. This construct was made as described by Rubinfeld, B. and Polakis P., "Purification of Baculovirus-Produced Rapl GTPase-activating Proteins". In: Methods and Enzymology, W.E.
- the BRCAl constructs were generated as follows:. pAcO BRCAl 5' (1-1293) was generated by subcloning the Ncol-Nhel BRCAl fragment into pAcO G1S Ncol-Nhel sites. pAcO BRCAl 3' (1293-1863) was generated by subcloning the Nhel blunted-Notl BRCAl fragment into pAcO G2 Stul-Notl sites. Using standard methods, the constructs were transfected into Sf9 cells. The BRCAl constructs were purified using the immunoaffinity purification methods essentially as described in the preceding Examples.
- PCAN myc 091-21A31, Sequence ID No. 1 was generated by subcloning the BamH - Xhol 091-21A31, Sequence ID No. 1 fragment into PCAN myc3 BamHl-Xhol sites.
- the plasmid containing the BRCAl Modulator 091-132Q20, Sequence ID No. 5, PCAN myc 091-132Q20, Sequence ID No. 5, was generated by subcloning the EcoRl- Xhol 091-132Q20, Sequence ID No. 5 fragment into PCAN myc3 EcoRl-Xhol sites.
- the BRCAl Modulator cDNAs (091-1F84, Sequence ID No. 3, 091-21A31, Sequence ID No. 1, 091-132Q20, Sequence ID No. 5) were transcribed and translated in vitro in the presence of [ 5 S]Methionine using the TNT- coupled wheat germ cell lysate system (Promega).
- one-two ⁇ g of purified recombinant BRCAl protein either Glu-Glu tagged BRCAl 5' (1-1293), or BRCAl 3' (1293-1863) was added to 25 ⁇ l of precleared lysate along with lO ⁇ l of anti-Glu Glu coupled protein G-Sepharose beads.
- the beads were washed three times with 1 ml each of ice cold buffer B (20mM tris pH 7.5, 150mM NaCl, 0.5% Nonidet P-40), eluted with 20 ⁇ l of SDS-PAGE sample buffer and subjected to SDS-PAGE and fluorography.
- ice cold buffer B (20mM tris pH 7.5, 150mM NaCl, 0.5% Nonidet P-40
- NaCl was then added to a final concentration of 150mM, incubated at room temperature for 10 minutes and centrifuged. Then resulting supernatant was loaded onto a 1-ml affinity column containing the Glu-Glu antibody covalently cross-linked to protein G-Sepharose. The column was washed with 10-15ml of lysis buffer and eluted with lOO ⁇ g of Glu-Glu peptide (EYMPME) per ml in the same buffer.
- EYMPME lOO ⁇ g of Glu-Glu peptide
- recombinant Sf9 cells were pelleted, lysed in 5 volumes of [20mM Tris (pH8.0), 137mM NaCl, ImM EGTA, 1.5mM MgCl 2 , 2%SDS, lOug/ml each of leupeptin, pepstatin, pefabloc, ImM aprotinin and ImM DTT], incubated at room temperature for 20-30 minutes and ultra centifuged. The upper phase was removed, NaCl was adjusted to 400mM and recentrifuged.
- the clarified supernatent was then diluted 1:10 in 1 X TG buffer [20mM Tris (pH8.0), 137mM NaCl, ImM EGTA, 1.5mM MgCl 2 , 1% Triton X100, 10% glycerol, lOug/ml each of leupeptin, pepstatin, pefabloc, ImM aprotinin and ImM DTT], filtered through a 3um Gelman Versapore filter and loaded onto a 1-ml anti-Glu-Glu affinity column.
- 1 X TG buffer 20mM Tris (pH8.0), 137mM NaCl, ImM EGTA, 1.5mM MgCl 2 , 1% Triton X100, 10% glycerol, lOug/ml each of leupeptin, pepstatin, pefabloc, ImM aprotinin and ImM DTT
- BRCAl Modulators the latter are used to immunize rabbits as follows.
- the immunization protocol generally consisted of two immunizations; the first was a subcutaneous injection of 0.500mg in CFA, followed by a second intramuscular injection of 0.250 mg about four weeks later in ICFA.
- the rabbits were bled, antisera collected and antibody purified as setforth below.
- BRCAl Modulator antibodies are affinity purified using BRCAl Modulator immunogens which have been coupled to a support matrix.
- the BRCAl Modulator 091-21A31, Sequence ID No. 1 is coupled to CNBr activated Sepharose 6MB (Pharmacia) as follows.
- GCC GCC ATC CAC TGC GGC CAC ACC TTC CAC TTG CAG TGC CTA ATT CAG 210 Ala Ala He His Cys Gly His Thr Phe His Leu Gin Cys Leu He Gin
- AAG ACA GTG CCT TCT CTC TTC CAG GCC AAG CTG GAC ACC TTC CTG TGG 1506 Lys Thr Val Pro Ser Leu Phe Gin Ala Lys Leu Asp Thr Phe Leu Trp 455 460 465
- GAG AAC CTA ACG GCT AAA CTG GCC AGC ACC ATA GCA GAT AAC CAG GAG 1542
- GCA AAA GAA GCA GAC ATA GAG AAG CTG AAC CAG GCC TTG TGC TTG CGC 2070 Ala Lys Glu Ala Asp He Glu Lys Leu Asn Gin Ala Leu Cys Leu Arg 665 670 675
Abstract
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EP97935333A EP0929668A1 (en) | 1996-09-04 | 1997-08-06 | Modulators of brca1 activity |
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JP10512659A JP2001502893A (en) | 1996-09-04 | 1997-08-06 | Modulators of BRCA1 activity |
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WO2000050021A2 (en) * | 1999-02-26 | 2000-08-31 | Napro Biotherapeutics, Inc. | Treatment regimen for prostate cancer, initiated after androgen ablative therapy has started |
US6225292B1 (en) * | 1997-06-06 | 2001-05-01 | The Regents Of The University Of California | Inhibitors of DNA immunostimulatory sequence activity |
WO2001070982A2 (en) * | 2000-03-23 | 2001-09-27 | Immusol Incorporated | Brca-1 regulators and methods of use |
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EP0786523A2 (en) * | 1996-01-29 | 1997-07-30 | Eli Lilly And Company | Protein kinase and method of using |
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- 1997-08-06 CA CA002259959A patent/CA2259959A1/en not_active Abandoned
- 1997-08-06 EP EP97935333A patent/EP0929668A1/en not_active Withdrawn
- 1997-08-06 JP JP10512659A patent/JP2001502893A/en active Pending
- 1997-08-06 CN CN97197589A patent/CN1228811A/en active Pending
- 1997-08-06 AU AU38293/97A patent/AU735512B2/en not_active Ceased
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EP0786523A2 (en) * | 1996-01-29 | 1997-07-30 | Eli Lilly And Company | Protein kinase and method of using |
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Cited By (6)
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US6225292B1 (en) * | 1997-06-06 | 2001-05-01 | The Regents Of The University Of California | Inhibitors of DNA immunostimulatory sequence activity |
US8729039B2 (en) | 1997-06-06 | 2014-05-20 | The Regents Of The University Of California | Use of inhibitory oligonucleotides to treat autoimmune disease |
WO2000050021A2 (en) * | 1999-02-26 | 2000-08-31 | Napro Biotherapeutics, Inc. | Treatment regimen for prostate cancer, initiated after androgen ablative therapy has started |
WO2000050021A3 (en) * | 1999-02-26 | 2001-03-08 | Napro Biotherapeutics Inc | Treatment regimen for prostate cancer, initiated after androgen ablative therapy has started |
WO2001070982A2 (en) * | 2000-03-23 | 2001-09-27 | Immusol Incorporated | Brca-1 regulators and methods of use |
WO2001070982A3 (en) * | 2000-03-23 | 2002-08-22 | Immusol Inc | Brca-1 regulators and methods of use |
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