WO2013052168A2 - Detection aib1-delta 4 protein - Google Patents

Detection aib1-delta 4 protein Download PDF

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Publication number
WO2013052168A2
WO2013052168A2 PCT/US2012/039494 US2012039494W WO2013052168A2 WO 2013052168 A2 WO2013052168 A2 WO 2013052168A2 US 2012039494 W US2012039494 W US 2012039494W WO 2013052168 A2 WO2013052168 A2 WO 2013052168A2
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peptide
levels
cells
αιβ1
antibody
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PCT/US2012/039494
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French (fr)
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WO2013052168A3 (en
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Alexander KIRILYUK
Christopher D. CHIEN
Anton Wellstein
Anna T. Riegel
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Georgetown University
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/82—Translation products from oncogenes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • a computer readable text file entitled "036681-5012-WO-SequenceListing.txt" created on or about May 24, 2012 with a file size of a bout 15 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.
  • the invention relates to an isolated peptide comprising the amino acid sequence of SEQ ID NO: 1 ( ⁇ 1- ⁇ 4) and methods of use thereof.
  • Gene transcription in eukaryotes is a complex and highly regulated process.
  • One of the major controls of gene transcription is exerted by the coregulator family of proteins. These include both corepressors, which dampen transcription, and coactivators, which potentiate transcription.
  • a su bgroup of coactivators has been shown to be critical for the malignant progression of cancer and is known as the pl60 steroid receptor coactivators (1).
  • One member in particular was identified to be amplified in breast cancer.
  • AIBl Amplified in Breast Cancer 1
  • SRC-3 NCOA3, ACTR, TRAM-1, pCIP, RAC3
  • a splice variant of AIBl, where exon 3 was spliced from the mature mRNA and was named AIB1- ⁇ 3 has been identified previously (17). More recently, an additional 5' exon 81, 164 bases upstream of the known 5'UTR was also identified. Thus the deleted exon is now known to be exon 4 and is thus referred to as splice variant ⁇ 1- ⁇ 4. It was previously reported that ⁇ 1- ⁇ 4 mRNA results in an N- terminally truncated isoform of the AIBl protein that was found to be a more potent coactivator of steroid dependent transcription on a per mole basis when compared with the full-length AIBl protein.
  • ⁇ 1- ⁇ 4 mRNA expression was elevated in breast tumor tissue relative to normal breast tissue (17). It was also shown to increase the efficacy of estrogenic compounds and the agonist effects of the selective estrogen receptor modulator tamoxifen in breast and endometrial tumor cells (18). Overexpression of ⁇ 1- ⁇ 4 in mice leads to ductal ectasia in the mammary gland with an increased expression of proliferative markers such as PCNA, phospho-histone H3, and Cyclin Dl (19).
  • ⁇ 1- ⁇ 4 was shown to act as a molecular bridge between Epidermal Growth Factor Receptor (EGFR) and Focal Adhesion Kinase (FAK) in the cytoplasm and its overexpression increased the invasiveness of the MDA- MB-231 metastatic breast cancer cell line (20).
  • EGFR Epidermal Growth Factor Receptor
  • FAK Focal Adhesion Kinase
  • the invention relates to an isolated peptide with the amino acid sequence of SEQ ID NO: 1 ( ⁇ 1- ⁇ 4).
  • the invention also relates to methods of stratifying a population of cells based on levels of the ⁇ 1- ⁇ 4 peptide in abnormal cells.
  • the invention also relates to methods of assessing the prognosis of a subject based on levels of the ⁇ 1- ⁇ 4 peptide in abnormal tissue.
  • the invention also relates to methods of assessing sensitivity or resistance to drug treatment of an individual on levels of the ⁇ 1- ⁇ 4 peptide in the diseased tissues.
  • FIGURE 1 depicts the identification of the translation start site of ⁇ 1- ⁇ 4.
  • the MQCFALSQPR (SEQ ID NO: 22) peptide identified through further mass spectrometry analysis containing the translation start site is shown in bold, b, The annotated fragmentation spectra of the MQCFALSQPR peptide are shown. This tryptic peptide is bearing acetylation of the initial methionine as a result of cotranslational modification.
  • the top spectrum shows collision induced dissociation (CID) fragmentation of the double charged peptide with a non-oxidized methionine and m/z ratio of 640.3.
  • the lower spectrum depicts fragmentation of the double charged peptide with m/z ratio of 648.3 due to a mass shift caused by oxidation of the initial methionine.
  • FIGURE 2 depicts that ⁇ 1- ⁇ 4 is found predominantly in the cytoplasm but can be detected in the nucleus
  • a Chinese Hamster Ovary (22) cells were grown in DMEM F12+10%FBS and transfected with FLAG AIBl or FLAG ⁇ 1- ⁇ 4. Cells were fixed and stained for FLAG peptide and DAPI and visualized by direct immunofluorescence. Typical nuclear, nuclear/cytoplasmic, and cytoplasmic FLAG staining is shown in the top panels (green) and DAPI staining DNA in the nucleus is shown in the bottom panels (blue). The percentage of nuclear, nuclear/cytoplasmic, and cytoplasmic staining cells is graphed.
  • Nuclear, nuclear/cytoplasmic, and cytoplasmic stained cells are represented by the black, gray, and white bars respectively, b, CHO cells were treated as in panel a. 24 hours after transfection, either the carrier EtOH or 50 nM leptomycin B (LMB in EtOH) was added to the culture media for 4 hours before fixing and staining cells. The percentage of nuclear, nuclear/cytoplasmic, and cytoplasmic staining cells is shown in the absence and presence of LMB. Cells were quantified as in panel a. Nuclear, nuclear/cytoplasmic, and cytoplasmic stained cells are represented by the black, gray, and white bars respectively.
  • FIGURE 3 depicts that the overexpression of AIBl localizes ⁇ 1- ⁇ 4 to the nucleus
  • a, HEK293T cells grown in DM EM+10%FBS were transfected with either FLAG AIBl alone, ⁇ 1- ⁇ 4 alone, or FLAG AIBl and ⁇ 1- ⁇ 4 together and lysates were immunoprecipitated using FLAG antibody to pulldown FLAG AIBl and any interacting proteins
  • b, AIBl knockout murine embryonic fibroblasts (KO M EFs) were transfected by electroporation with 4 ⁇ g FLAG ⁇ 1- ⁇ 4 alone or with 2, 4, or 6 ⁇ g of untagged AIBl and plated on glass cover slips in DMEM+10% FBS.
  • FIGURE 4 depicts that the overexpression of p300 localizes ⁇ 1- ⁇ 4 to the nucleus
  • a, HEK293T cells were transfected with either p300-HA, FLAG AIBl, or FLAG ⁇ 1- ⁇ 4.
  • Equal amounts of FLAG AIBl or FLAG ⁇ 1- ⁇ 4 protein were incubated with equal amounts of p300-HA.
  • a FLAG Western blot was performed to determine how much AIBl and ⁇ 1- ⁇ 4 immunoprecipitated with p300.
  • AIBl KO MEFs were transfected by electroporation with 4 ⁇ g ⁇ 1- ⁇ 4 alone or with 2, 4, or 6 ⁇ g of p300-HA and plated on glass cover slips in DMEM+10% FBS. Cells were fixed and permeabolized 24 hours after plating and stained for DAPI, FLAG, and HA containing proteins. Cells were then analyzed by confocal microscopy. The number of nuclear, nuclear/cytoplasmic, and cytoplasmic staining cells was quantified as in Fig 2a.
  • Typical nuclear, nuclear/cytoplasmic, and cytoplasmic FLAG staining (green), p300 (red, nuclear) and DAPI staining DNA in the nucleus (blue) is shown with overlay of the images in the right panels.
  • the percentage of nuclear, nuclear/cytoplasmic, and cytoplasmic stained cells is shown in the black, gray, and white bars respectively for three experiments is shown.
  • FIGURE 5 depicts that that ⁇ 1- ⁇ 4, like AIBl, is recruited to ERE in the nucleus, a, HEK293 cells grown in phenol red free IMEM + 10% charcoal stripped serum were transfected with either FLAG AIBl or FLAG ⁇ 1- ⁇ 4 and ERa. 24 hours later cells were stimulated with estrogen and harvested at 0, 15, 30, 45, and 60 minutes after estrogen stimulation. These lysates were subjected to a quantitative ChIP analysis using a FLAG antibody. The percentage of the input recovered after immunoprecipitation for each ERE in pS2, hC3, or HER2 was determined. Data is representative of three independent experiments.
  • FIGURE 6 depicts the N-terminus of AIBl contains an inhibitory domain that is lost in ⁇ 1- ⁇ 4.
  • COS-7 cells were transfected with human progesterone receptor B (25 ng), MMTV luciferase (100 ng), and either pcDNA3 or FLAG AIBl (500 ng) with increasing amounts of FLAG AIBl N term (125, 500, 750 ng). 24 hours later cells were treated with 10 nM R5020 for 24 hours before reporter activity was determined. Luciferase values were normalized to TK Renilla (10 ng) reporter activity. The assay was plated in triplicate and a representative graph is shown from three separate experiments.
  • FIGURE 7 depicts that ⁇ 1- ⁇ 4 expression is increased in metastatic cancer cell lines, a
  • a scorpion primer was designed to specifically recognize the unique splice junction of exon 3 and exon 5.
  • the scorpion primer consists of primer (black half arrow), blocker (blue jagged line), quencher (purple octagon), probe (red - exon3, light blue - exon 5), stem (black attached lines), and reporter (green ball).
  • the stem region will only be dissociated when a target sequence is created during the process of PCR. In the case of AIB1, no appropriate target is generated so the preferred conformation is to remain in the stem loop with the reporter quenched.
  • ⁇ 1- ⁇ 4 transcript an appropriate target is generated and the probe can then bind its target sequence allowing the reporter to fluoresce, b, Scorpion primers were used to quantitate the amount of ⁇ 1- ⁇ 4 transcript from the RNA of human mammary epithelial cells (HMEC), parental MDA-MB-231 breast cancer cells and three tissue specific metastatic variants of MDA-MB-231 cells. The Ct values were normalized to actin expression as a control. Data were analyzed by one way ANOVA with Bonferroni post test.
  • FIGU RE 8 depicts that the AI B1-4 is recruited to the PRE in the M MTV promoter.
  • T-47D (Al-2) cells which have a stable integration of the MMTV luciferase reporter in their genome, were transfected with either pCMV-Flag or FLAG AI B1- 4 for 24 hr and then treated with 10 nM R5020 for 1 hr.
  • the binding of AIB1- 4 to the MMTV promoter was analyzed by ChIP assay with a FLAG antibody.
  • FIGU RE 9 depicts that the AI B1- 4 Scorpion primer is specific for AI B1- 4 transcript.
  • Scorpion primers specific for AIB1- 4 tested on plasmid cDNA constructs of AI B1 and AIB1- 4. Plasmids for AI B1 or AI B1- 4 were subjected to real time PCR analysis with scorpion primers specific for AIB1- 4.
  • FIGURE 10 depicts that the AIB1- 4 is resistant to density induced degradation.
  • HEK293T cells were transfected with FLAG AI B1 or FLAG AI B1- 4. Cells were plated at either low or high density to obtain low or high confluence at the time of lysate harvest. Whole cell lysates were harvested 24 hours after plating at different densities a nd the levels of AIBl and AI Bl- 4 were determined by Western blot for FLAG.
  • the invention relates to a peptide comprising the amino acid sequence of SEQ ID NO: 1 (AIB1- ⁇ 4) and methods of its use.
  • a portion of human chromosome 20q that is frequently amplified in breast cancer contains the gene for the nuclear coactivator AIBl (Amplified in breast cancer 1).
  • the AIBl gene is amplified in five to ten percent of breast cancers and the abundance of the corresponding mRNA and protein is increased in up to 60% of breast tumors and also breast cancer cell lines.
  • AIBl binds directly to estrogen receptor (ER) and this binding can be rate-limiting for estrogen-induced growth of MCF-7 cells.
  • ER estrogen receptor
  • the overall role of AIBl for breast tumorigenesis is not clear, however, since AIBl potentiates not only the action of estrogen and progesterone receptors, but also that of various other nuclear receptors and transcription factors.
  • the ⁇ 1- ⁇ 4 peptide (SEQ ID NO: 1) is a splice variant of AIBl in which the 4th exon has been spliced out and, unexpectedly, translation begins at the methionine at position 224 of the full length AIBl.
  • peptide As used herein, an "isolated peptide” is intended to mean a peptide that has been completely or partially removed from its native environment. For example, peptides that have been removed or purified from cells are considered isolated. In addition, recombinantly produced peptide molecules contained in host cells are considered isolated for the purposes of the present invention. Moreover, a peptide that is found in a cell, tissue or matrix in which it is not normally expressed or found is also considered as “isolated” for the purposes of the present invention. Similarly, polypeptides that have been synthesized are considered to be isolated polypeptides.
  • peptides are substantially free of cellular material, cellular components, chemical precursors or other chemicals beyond, perhaps, buffer or solvent. “Substantially free” is not intended to mean that other components beyond the novel peptides are undetectable.
  • the peptides of the present invention may be isolated or purified.
  • peptides or polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or su bstitution of amino acid residues.
  • the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic and inorganic moieties.
  • Such derivatives may be prepared to increase circulating half-life of a polypeptide, or may be designed to improve the targeting capacity of the polypeptide for desired cells, tissues or organs.
  • the invention further embraces ⁇ 1- ⁇ 4 peptides that have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol or polypropylene glycol.
  • the invention provides fusion proteins comprising at least a first and a second fusion peptide.
  • the fusion partners are, generally speaking, covalently bonded to one another via a typical amine bond between the fusion peptides, thus creating one contiguous amino acid chain.
  • the first peptide of the fusion protein consists of a peptide with an amino acid sequence at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1.
  • Types of fusion proteins provided by the present invention include but are not limited to, fusions with secretion signals and other heterologous functional regions.
  • a region of additional amino acids, particularly charged amino acids may be added to the N-terminus or C-terminus of the protein to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
  • a region may be added to facilitate purification.
  • "histidine tags” (“his tags”) or “lysine tags” (the second fusion peptide) may be appended to the first fusion peptide.
  • histidine tags include, but are not limited to hexaH, heptaH and hexaHN. Additional examples of purification tags are disclosed in Waugh, D.S., Trends in Biotechnology, 23(6):316-320 (June 2005), and Gaberc-Porekar V. and Menart, V., J. Biochem. Biophys. Methods. 49:335-360 (2001), which are incorporated by reference.
  • lysine tags include, but are not limited to pentaL, heptaL and FLAG. Additional examples of solubility tags are also disclosed in Waugh, D.S., Trends in Biotechnology, 23(6) 316-320 (June 2005). Such regions may be removed prior to final preparation of the protein.
  • Other examples of a second fusion peptide include, but are not limited to, glutathione S-transferase (GST) and alkaline phosphatase (AP).
  • the fusion proteins of the current invention can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose
  • Fusion proteins of the present invention include, but are not limited to, products of chemical synthetic procedures and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells.
  • the fusion proteins of the present invention may be glycosylated or may be non-glycosylated.
  • fusion proteins of the invention may also include an initial modified methionine residue, in some cases as a result of host- mediated processes.
  • the invention also relates to isolated nucleic acids and to constructs comprising these nucleic acids.
  • the nucleic acids of the invention can be DNA or RNA, for example, mRNA.
  • the nucleic acid molecules can be dou ble-stranded or single-stranded; single stranded RNA or DNA can be the coding, or sense, strand or the non-coding, or antisense, strand.
  • the nucleic acids may encode any polypeptide of the invention, including, but not limited to, the fusion proteins of the present invention.
  • the nucleic acids of the invention include polynucleotide sequences that encode glutathione-S-transferase (GST) fusion protein, poly-histid ine (e.g., His 6 ), poly-HN, poly-lysine, hemagglutinin, HSV-Tag and at least a portion of H IV-Tat.
  • GST glutathione-S-transferase
  • poly-histid ine e.g., His 6
  • poly-HN poly-lysine
  • hemagglutinin HSV-Tag
  • H IV-Tat H IV-Tat
  • the nucleotide sequence of the isolated nucleic acid can include additional non-coding sequences such as non-coding 3' and 5' sequences (including regulatory sequences, for example).
  • nucleic acid molecules of the invention can be "isolated.”
  • a n "isolated" nucleic acid molecule or nucleotide sequence is intended to mea n a nucleic acid molecule or nucleotide sequence that is not flanked by nucleotide sequences normally flanking the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially removed from its native environment (e.g., a cell, tissue).
  • nucleic acid molecules that have been removed or purified from cells are considered isolated.
  • the isolated material will form part of a composition (for example, a crude extract containing other su bstances), buffer system or reagent mix.
  • an isolated nucleic acid molecule or nucleotide sequence can includes a nucleic acid molecule or nucleotide sequence which is synthesized chemically, using recombinant DNA technology or using any other suita ble method.
  • a nucleic acid contained in a vector wou ld be included in the definition of "isolated" as used herein.
  • isolated nucleotide sequences include recom binant nucleic acid molecules (e.g., DNA, RNA) in heterologous organisms, as well as partially or su bstantially purified nucleic acids in solution.
  • nucleic acid molecules of the present invention may be isolated or purified. Both in vivo and in vitro NA transcripts of a DNA molecule of the present invention are also encompassed by "isolated" nucleotide sequences.
  • the invention also encompasses variations of the nucleotide sequences of the invention, such as those encoding functional fragments or variants of the polypeptides as described above.
  • variants can be naturally-occurring, or non-naturally-occurring, such as those induced by various mutagens and mutagenic processes.
  • Intended variations include, but are not limited to, addition, deletion and substitution of one or more nucleotides which can result in conservative or non- conservative amino acid changes, including additions and deletions.
  • fragments of the isolated nucleic acid molecules described herein also relates to fragments of the isolated nucleic acid molecules described herein.
  • fragment is intended to encompass a portion of a nucleotide sequence described herein which is from at least about 20 contiguous nucleotides to at least about 50 contiguous nucleotides or longer in length.
  • Such fragments may be useful as probes and primers.
  • primers and probes may selectively hybridize to the nucleic acid molecule encoding the polypeptides described herein.
  • fragments which encode polypeptides that retain activity, as described below, are particularly useful.
  • the invention also provides nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to the nucleotide sequences described herein (e.g., nucleic acid molecules which specifically hybridize to a nucleotide sequence encoding polypeptides described herein).
  • Hybridization probes include synthetic oligonucleotides which bind in a base-specific manner to a complementary strand of nucleic acid. Suitable probes include polypeptide nucleic acids, as described in Nielsen et al., Science, 254:1497-1500 (1991).
  • Such nucleic acid molecules can be detected and/or isolated by specific hybridization e.g., under high stringency conditions.
  • "Stringency conditions" for hybridization is a term of art that refers to the incubation and wash conditions, e.g., conditions of temperature and buffer concentration, which permit hybridization of a particular nucleic acid to a second nucleic acid; the first nucleic acid may be perfectly complementary, i.e., 100%, to the second, or the first and second may share some degree of complementarity, which is less than perfect, e.g., 60%, 75%, 85%, 95% or more.
  • certain high stringency conditions can be used which distinguish perfectly complementary nucleic acids from those of less complementarity.
  • 0.2 X SSC, 0.1 X SSC of the wash buffers temperature, e.g., room temperature, 42°C, 68°C, etc., and the concentration of destabilizing agents such as formamide or denaturing agents such as SDS, but also on factors such as the length of the nucleic acid sequence, base composition, percent mismatch between hybridizing sequences and the frequency of occurrence of subsets of that sequence within other non- identical sequences.
  • concentration of destabilizing agents such as formamide or denaturing agents such as SDS
  • Washing is the step in which conditions are usually set so as to determine a minimum level of complementarity of the hybrids.
  • each degree (°C) by which the final wash temperature is reduced allows an increase by 1% in the maximum extent of mismatching among the sequences that hybridize.
  • doubling the concentration of SSC results in an increase in Tm.
  • the washing temperature can be determined empirically for high, moderate or low stringency, depending on the level of mismatch sought.
  • Exemplary high stringency conditions include, but are not limited to, hybridization in 50% formamide, 1 M NaCI, 1% SDS at 37°C, and a wash in 0.1 X SSC at 60°C.
  • Example of progressively higher stringency conditions include, after hybridization, washing with 0.2 X SSC and 0.1% SDS at about room temperature (low stringency conditions); washing with 0.2 X SSC, and 0.1% SDS at about 42°C (moderate stringency conditions); and washing with 0.1 X SSC at about 68°C (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, washing may encompass two or more of the stringency conditions in order of increasing stringency. Optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.
  • Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleic acid molecule and the primer or probe used.
  • Hybridizable nucleotide sequences are useful as probes and primers for identification of organisms comprising a nucleic acid of the invention and/or to isolate a nucleic acid of the invention, for example.
  • the term "primer” is used herein as it is in the art and refers to a single-stranded oligonucleotide which acts as a point of initiation of template- directed DNA synthesis under appropriate conditions in an appropriate buffer and at a suitable temperature.
  • primer site refers to the area of the target DNA to which a primer hybridizes.
  • primer pair refers to a set of primers including a 5' (upstream) primer that hybridizes with the 5' end of the DNA sequence to be amplified and a 3' (downstream) primer that hybridizes with the complement of the 3' end of the sequence to be amplified.
  • nucleic acids described herein can be amplified by methods known in the art. For example, amplification can be accomplished by the polymerase chain reaction (PCR). See PCR
  • the present invention also relates to vectors that include nucleic acid molecules of the present invention, host cells that are genetically engineered with vectors of the invention and the production of proteins of the invention by recombinant techniques.
  • the vector may be, for example, a plasmid vector, a single-or double-stranded phage vector, or a single-or double-stranded NA or DNA viral vector.
  • Such vectors may be introduced into cells as polynucleotides, for example DNA, by well-known techniques for introducing DNA and RNA into cells.
  • Viral vectors may be replication competent or replication defective. In the latter, case viral propagation generally will occur only in complementing host cells.
  • the vectors to be used are those for expression of polynucleotides and proteins of the present invention.
  • such vectors comprise c/ ' s-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed.
  • Appropriate transacting factors are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
  • vectors can be used to express the proteins of the invention.
  • Such vectors include chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as adeno-associated virus, lentivirus, baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. All may be used for expression in accordance with this aspect of the present invention.
  • any vector suitable to maintain, propagate or express polynucleotides or proteins in a host may be used for expression in this regard.
  • the DNA sequence in the expression vector is operatively linked to appropriate expression control sequence(s) including, for instance, a promoter to direct mRNA transcription.
  • promoters include, but are not limited to, the phage lambda PL promoter, the E. coli lac, trp and tac promoters, HIV promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name just a few of the well-known promoters.
  • expression constructs will contain sites for transcription, initiation and termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • constructs may contain control regions that regulate, as well as engender expression. Generally, such regions will operate by controlling transcription, such as repressor binding sites and enhancers, among others.
  • Vectors for propagation and expression generally will include selecta ble markers. Such markers also may be suitable for amplification or the vectors may contain additional markers for this purpose.
  • the expression vectors may contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells.
  • Preferred markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing E. coli and other bacteria.
  • the vector containing the appropriate DNA sequence, as well as an appropriate promoter, and other appropriate control sequences may be introduced into an appropriate host using a variety of well-known techniques suitable to expression therein of a desired polypeptide.
  • appropriate hosts include bacterial cells, such as, but not limited to, E. coli, Streptomyces, Bacillus, and Salmonella cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Hosts for of a great variety of expression constructs are well known, and those of skill in the art will be enabled by the present disclosure to select an appropriate host for expressing one of the proteins of the present invention.
  • vectors that may be useful for fusion proteins include, but are not limited to, pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67, 31 40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione-S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
  • GST glutathione-S-transferase
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Such enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Examples of vectors for expression in yeast 5. cerevisiae include pYepSecl (Baldari, et al, (1987) EM BO J.
  • the ⁇ ⁇ 1- ⁇ 4 peptides can be expressed in insect cells using baculovirus expression vectors.
  • Baculovirus vectors availa ble for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al, (1983) Mol. Cell. Biol. 3, 2156 2165) and the pVL series (Lucklow and Summers (1989) Virology 170, 31 39).
  • a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed (1987) Nature 329, 840) and pMT2PC (Kaufman et al. (1987) EM BO J. 6, 187 195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and Simian Virus 40.
  • the recom binant ma mmalian expression vector is ca pable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art.
  • Non-limiting examples of suita ble tissue-specific promoters include liver-specific promoters (e.g., al bumin promoter), lymphoid-specific promoters such as, but not limited to, T cell receptors and immunoglobulins, neuron-specific promoters (e.g., neurofilament promoter), pancreas- specific promoters, mammary gland-specific promoters (e.g., milk whey promoter), bone-specific promoters (e.g., osteocalcin, osteopontin or bone sialoprotein, promoter regions), cartilage specific promoters (e.g., WARP) a nd muscle specific promoters (Desmin, myglobin, etc) just to name a few.
  • liver-specific promoters e.g., al bumin promoter
  • lymphoid-specific promoters such as, but not limited to, T cell receptors and immunoglobulins
  • neuron-specific promoters e.g., neurofilament
  • the present invention also relates to host cells containing the above-described constructs.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • the host cell can be stably or transiently transfected with the construct.
  • a "host cell” is a cell that normally does not contain any of the nucleotides of the present invention and contains at least one copy of the nucleotides of the present invention.
  • a host cell as used herein can be a cell in a culture setting or the host cell can be in an organism setting where the host cell is part of an organism, organ or tissue.
  • Suitable host cells for expression of the polypeptides of the invention include, but are not limited to, prokaryotes, yeast, and eukaryotes. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences.
  • Suitable prokaryotic cells include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces and Staphylococcus.
  • eukaryotic cells are cells of higher eukaryotes. Suitable eukaryotic cells include, but are not limited to, non-human mammalian tissue culture cells and human tissue culture cells. Other host cells include, but are not limited to, insect cells, HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), human 293 cells, and murine 3T3 fibroblasts. Propagation of such cells in cell culture has become a routine procedure (see, Tissue Culture, Academic Press, Kruse and Patterson, Eds. (1973), which is incorporated herein by reference in its entirety).
  • a yeast cell may be employed as a host cell.
  • Yeast cells include, but are not limited to, the genera Saccharomyces, Pichia and Kluveromyces.
  • the yeast hosts are 5. cerevisiae or P. pastoris.
  • Yeast vectors may contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replication sequence (A S), a promoter region, sequences for
  • insect cells may be used as host cells.
  • the polypeptides of the invention are expressed using a baculovirus expression system (see, Luckow et al., Bio/Technology, 1988, 6, 47; BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MAN UAL, O'Rielly et al. (Eds.), W.H. Freeman and Company, New York, 1992; and U.S. Pat. No. 4,879,236, each of which is incorporated herein by reference).
  • commercially availa ble complete baculovirus expression systems can, for example, be used for production in insect cells.
  • the recom binant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Introduction of a construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation,
  • Vector DNA can be introd uced into prokaryotic or eukaryotic cells via conventional
  • transfection are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
  • foreign nucleic acid e.g., DNA
  • a host cell including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation.
  • Suita ble methods for transforming or transfecting host cells can be found in Sam brook, et al (MOLECU LAR CLONI NG: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor La boratory, Cold Spring Harbor La boratory Press, Cold Spring Harbor, N.Y., 1989), and other la boratory manuals.
  • a gene that encodes a selecta ble marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selecta ble markers include those that confer resistance to d rugs, such as G418, hygromycin, dihydrofolate reductase (DHFR) and methotrexate.
  • Nucleic acid encoding a selecta ble marker can be introduced into a host cell on the same vector as that encode the ⁇ ⁇ 1- ⁇ 4 peptide or can be introduced on a separate vector. Cells sta bly transfected with the introduced nucleic acid ca n be identified by drug selection (e.g., cells that have incorporated the selecta ble marker gene will survive, while the other cells die).
  • One em bodiment of the invention provides antibodies or antibody fragments that specifically bind to a peptide with an a mino acid sequence of SEQ ID NO: 1. Another aspect of the invention relates to detecting the presence of a peptide with an amino acid sequence of SEQ ID NO: 1.
  • the antibodies or antibody fragments of the present invention specifically bind to a peptide, wherein the polypeptide consists of an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ I D NO: 1.
  • a polypeptide having an amino acid sequence at least, for example, a bout 95% "identical" to a reference an amino acid sequence, e.g., SEQ ID NO: 1, is understood to mean that the amino acid sequence of the polypeptide is identical to the reference sequence except that the amino acid sequence may include up to a bout five modifications per each 100 amino acids of the reference amino acid sequence.
  • a polypeptide having an amino acid sequence at least a bout 95% identical to a reference amino acid sequence up to a bout 5% of the amino acid residues of the reference sequence may be deleted or su bstituted with another amino acid or a num ber of amino acids up to a bout 5% of the total amino acids in the reference sequence may be inserted into the reference sequence.
  • These modifications of the reference sequence may occur at the N- terminus or C-terminus positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
  • identity is a measure of the identity of nucleotide sequences or amino acid sequences compared to a reference nucleotide or amino acid sequence. In general, the sequences are aligned so that the highest order match is obtained. "Identity” per se has an art-recognized meaning and can be calculated using pu blished techniques. (See, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics And Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • identity is well known to skilled artisans (Carillo, H. & Lipton, D., Siam J Applied Math 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J.
  • Computer programs may also contain methods and algorithms that calculate identity and similarity. Examples of computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(i):387 (1984)), BLASTP, ExPASy, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)) and FASTDB. Examples of methods to determine identity and similarity are discussed in Michaels, G. and Garian, R., Current Protocols in Protein Science, Vol 1, John Wiley & Sons, Inc. (2000), which is incorporated by reference.
  • the algorithm used to determine identity between two or more polypeptides is BLASTP.
  • the algorithm used to determine identity between two or more polypeptides is FASTDB, which is based upon the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990), incorporated by reference).
  • FASTDB sequence alignment the query and reference sequences are amino sequences. The result of sequence alignment is in percent identity.
  • the reference sequence is shorter or longer than the query sequence because of N-terminus or C-terminus additions or deletions, but not because of internal additions or deletions, a manual correction can be made, because the FASTDB program does not account for N-terminus and C-terminus truncations or additions of the reference sequence when calculating percent identity.
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N-and C- terminus to the reference sequence that are not matched/aligned, as a percent of the total bases of the query sequence.
  • the results of the FASTDB sequence alignment determine matching/alignment.
  • the alignment percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
  • This corrected score can be used for the purposes of determining how alignments "correspond" to each other, as well as percentage identity. Residues of the reference sequence that extend past the N- or C-termini of the query sequence may be considered for the purposes of manually adjusting the percent identity score. That is, residues that are not matched/aligned with the N- or C-termini of the comparison sequence may be counted when manually adjusting the percent identity score or alignment numbering.
  • a 90 amino acid residue query sequence is aligned with a 100 residue reference sequence to determine percent identity.
  • the deletion occurs at the N-terminus of the query sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 residues at the N- terminus.
  • the 10 unpaired residues represent 10% of the reference sequence (number of residues at the N- and C-termini not matched/total number of residues in the reference sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched (100% alignment) the final percent identity would be 90% (100% alignment - 10% unmatched overhang).
  • a 90 residue query sequence is compared with a 100 reference sequence, except that the deletions are internal deletions.
  • the percent identity calculated by FASTDB is not manually corrected, since there are no residues at the N- or C- termini of the subject sequence that are not matched/aligned with the query.
  • a 110 amino acid query sequence is aligned with a 100 residue reference sequence to determine percent identity. The addition in the query occurs at the N-terminus of the query sequence and therefore, the FASTDB alignment may not show a match/alignment of the first 10 residues at the N-terminus. If the remaining 100 amino acid residues of the query sequence have 95% identity to the entire length of the reference sequence, the N-terminal addition of the query would be ignored and the percent identity of the query to the reference sequence would be 95%.
  • the ⁇ 1- ⁇ 4 peptides or fragments thereof or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies or antibody fragments thereof.
  • Any of the antibodies can be, for example, polyclonal, monoclonal, bi-specific, multispecific, human or chimeric antibodies.
  • the antibody molecules of the invention can be of any type, e.g., IgG, IgE, IgM, IgD, IgA and IgY, class, e.g., IgGl, lgG2, lgG3, lgG4, IgAl and lgA2 or subclass of immunoglobulin molecule.
  • an antibody of the invention comprises, or alternatively consists of, a polypeptide having an amino acid sequence of a VH domain, at least one VH CDR, a VL domain, or at least one VL CDR.
  • the antibodies or antibody fragments of the present invention may be monovalent, bivalent, trivalent or multivalent.
  • monovalent scFvs can be multimerized either chemically or by association with another protein or substance.
  • An scFv that is fused to a hexahistidine tag or a Flag tag can be multimerized using Ni-NTA agarose (Qiagen) or using anti-Flag antibodies (Stratagene, Inc.).
  • the antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity.
  • Multispecific antibodies may be specific for different epitopes of an ⁇ 1- ⁇ 4 peptide, or a domain thereof, or may be specific for both an ⁇ 1- ⁇ 4 peptide, or a doman thereof, and a heterologous epitope, such as a heterologous polypeptide or solid support material.
  • a heterologous epitope such as a heterologous polypeptide or solid support material.
  • an antibody fragment is a fragment of an antibody capable of specifically binding the same epitope that the intact antibody would bind.
  • antibody fragments include but are not limited to Fab and F(ab') 2 fragments, Fd fragments, disulfide-linked Fvs (sd Fvs), antiidiotypic (anti-Id) antibodies, including but not limited to anti-Id antibodies to antibodies of the invention, and epitope- binding fragments of any of the above.
  • Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody and generally clear more rapidly from the circulation, and may have less non-specific tissue binding than that of an intact antibody (Wahl et al., J. Nucl. Med.
  • antibody fragments include but are not limited to single chain Fv fragments (scFv) that are well-known in the art. Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention.
  • the antibodies or fragments of the present invention may be prepared by any of a variety of methods.
  • cells expressing ⁇ 1- ⁇ 4 peptide or an antigenic fragment thereof can be administered to an animal to induce the production of sera containing polyclonal antibodies.
  • a preparation of ⁇ 1- ⁇ 4 peptide is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
  • one aspect of the invention provides a method for making ⁇ 1- ⁇ 4 peptide-specific antibodies.
  • Polyclonal antibodies of the invention may be produced according to standard techniques by immunizing a suitable animal, e.g., rabbit, goat, etc., with an antigen comprising an antigenic portion of the ⁇ 1- ⁇ 4 peptide. Collecting immune serum from the animal and separating the polyclonal antibodies from the immune serum can be carried out in accordance with known procedures, and screening and isolating a polyclonal antibody specific for ⁇ 1- ⁇ 4 peptide can be carried out with well- known procedures and as described below. Methods for immunizing non-human animals such as mice, rats, sheep, goats, pigs, cattle and horses are well known in the art. See, e.g., Harlow and Lane,
  • the immunogen may be the full length protein or a peptide comprising an ⁇ 1- ⁇ 4 peptide portion of interest.
  • the immunogen is a peptide of from 7 to 20 amino acids in length, in particular from about 8 to 17 amino acids in length.
  • the peptide antigen desirably will comprise about 3 to 8 amino acids .
  • Peptide antigens suitable for producing antibodies of the invention may be designed, constructed and employed in accordance with well-known techniques. See, e.g., Antibodies: A Laboratory Manual, Chapter 5, p. 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czemik, Methods in Enzymology, 201: 264-283 (1991); Merrifield, J. Am. Chem. Soc. 85: 21-49 (1962), which are incorporated by reference.
  • the immunogen is administered with an adjuvant.
  • adjuvants will be well known to those of skill in the art.
  • exemplary adjuvants include complete or incomplete Freund's adjuvant, IBI (muramyl dipeptides) or ISCOM (immunostimulating complexes).
  • the polyclonal antibodies which secreted into the bloodstream can be recovered using known techniques. Purified forms of these antibodies can, of course, be readily prepared by standard purification techniques, such as for example, affinity chromatography with Protein A, antiimmunoglobulin, or the antigen itself. In any case, to monitor the success of immunization, the antibody levels with respect to the antigen in serum can be monitored using standard techniques such as ELISA, RIA and the like.
  • the antibodies or fragments thereof of the present invention are monoclonal antibodies.
  • Such monoclonal antibodies can be prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., (1981) pp. 563-681).
  • such procedures involve immunizing an animal (for example a mouse) with an ⁇ 1- ⁇ 4 peptide antigen or with a ⁇ 1- ⁇ 4 peptide-expressing cell.
  • Suitable cells can be recognized by their capacity to bind anti-AIBl-A4 peptide antibody.
  • Such cells may be cultured in any suitable tissue culture medium; however, it is desirable to culture cells in Earl's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56°C), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ⁇ g/ml of streptomycin.
  • the splenocytes of such mice are extracted and fused with a suitable myeloma cell line.
  • a suitable myeloma cell line may be employed in accordance with the present invention; however, it is may be desirable to employ the parent myeloma cell line (SP 2 0), available from the American Type Culture Collection, ockville, Md.
  • SP 2 0 myeloma cell line
  • the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)).
  • the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding ⁇ 1- ⁇ 4 peptide antigen.
  • the secreted antibody may be recovered from tissue culture supernatant by conventional methods such as precipitation, ion exchange or affinity chromatography, or the like.
  • Other methods of generating monclonal antibodies include but are not limited to the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • additional antibodies capable of binding to ⁇ 1- ⁇ 4 peptide antigen may be produced in a two-step procedure through the use of anti-idiotypic antibodies.
  • Such a method makes use of the fact that antibodies are themselves antigens, thus it is possible to obtain an antibody which binds to a second antibody.
  • ⁇ 1- ⁇ 4 peptide specific antibodies are used to immunize an animal, for example a mouse.
  • the splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to ⁇ 1- ⁇ 4 peptide-specific antibody can be blocked by an ⁇ 1- ⁇ 4 peptide antigen, respectively.
  • Such antibodies comprise anti-idiotypic antibodies to ⁇ 1- ⁇ 4 peptide-specific antibody and can be used to immunize an animal to induce formation of ⁇ 1- ⁇ 4 peptide- specific antibodies.
  • the invention also encompasses antibody-producing cells and cell lines, such as hybridomas, as described above.
  • Polyclonal or monoclonal antibodies may also be obtained through in vitro immunization.
  • phage display techniques can be used to provide libraries containing a repertoire of antibodies with varying affinities for a particular antigen. Techniques for the identification of high affinity human antibodies from such libraries are described by Griffiths et al., EMBO J., 13:3245-3260 (1994); which is incorporated by reference.
  • the antibodies may be produced recombinantly using methods well known in the art for example, according to the methods disclosed in U.S. Pat. No. 4,349,893 or U.S. Pat. No. 4,816,567, which are incorporated by reference.
  • the antibodies may also be chemically constructed by specific antibodies made according to the method disclosed in U.S. Pat. No. 4,676,980, which is incorporated by reference..
  • polynucleotides encoding the antibody may be cloned and isolated from antibody-producing cells using means that are well known in the art.
  • the antigen combining site of the monoclonal antibody can be cloned by PCR and single-chain antibodies produced as phage-displayed recombinant antibodies or soluble antibodies in E. coli. See, e.g., Antibody Engineering Protocols, Humana Press, Sudhir Paul, Ed. (1995), which is incorporated by reference.
  • the invention provides such nucleic acids encoding the heavy chain, the light chain, a variable region, a framework region or a CDR of an antibody of the invention.
  • the nucleic acids are operably linked to expression control sequences.
  • the invention thus, also provides vectors and expression control sequences useful for the recombinant expression of an antibody or antigen-binding portion thereof of the invention. Those of skill in the art will be able to choose vectors and expression systems that are suitable for the host cell in which the antibody or antigen-binding portion is to be expressed.
  • a nucleic acid molecule of the invention encodes an antibody comprising, or alternatively consisting of, a VH domain having an amino acid sequence of any one of the VH domains of the antibodies described herein.
  • a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VH CDR1 having an amino acid sequence of any of the antibodies described herein.
  • a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VH CD 2 having an amino acid sequence of any one of the VH CDR2 of any of the antibodies described herein.
  • a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VH CDR3 having an amino acid sequence of any of the antibodies described herein.
  • Nucleic acid molecules encoding antibodies that immunospecifically bind ⁇ 1- ⁇ 4 peptide and comprise, or alternatively consist of, fragments or variants of the VH domains and/or VH CDRs are also encompassed by the invention.
  • a nucleic acid molecule of the invention encodes an antibody, including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof, comprising, or alternatively consisting of, a VL domain having an amino acid sequence of any one of the VL, domains of any of the antibodies described herein.
  • a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VL CDR1 having amino acid sequence of any one of the any of the antibodies described herein.
  • a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VL CDR2 having an amino acid sequence of any one of the VL CDR2 of any of the antibodies described herein.
  • a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VL CDR3 having an amino acid sequence of any, one of the VL CDR3 of any of the antibodies described herein.
  • Nucleic acid encoding antibodies that immunospecifically bind ⁇ 1- ⁇ 4 peptide and comprise, or alternatively consist of, fragments or variants of the VL domains and/or VLCDR(s) are also encompassed by the invention.
  • a nucleic acid molecule of the invention encodes an antibody comprising, or alternatively consisting of, a VH domain having an amino acid sequence of any one of the VH domains of any of the antibodies described herein, and a VL domain having an amino acid sequence of any one of the VL domains of any of the antibodies described herein.
  • a nucleic acid molecule of the invention encodes an antibody comprising, or alternatively consisting of, a VH CDR1, a VL CDR1, a VH CDR2, a VL CDR2, a VH CDR3, a VL CDR3, or any combination thereof having an amino acid sequence of any of the antibodies described herein.
  • Nucleic acid encoding antibodies that immunospecifically bind ⁇ 1- ⁇ 4 peptide and comprise, or alternatively consist of, fragments or variants of the VL and/or domains and/or VHCDR(s) and/or VLCDR(s) are also encompassed by the invention.
  • the present invention also provides antibodies that comprise, or alternatively consist of, variants, including derivatives, of the VH domains, VH CD s, VL domains, and VL CDRs described herein, which antibodies immunospecifically bind to ⁇ 1- ⁇ 4 peptide.
  • Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions.
  • the variants including derivatives, encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference VH domain, VHCDR1, VHCDR2, VHCDR3, VL domain, VLCDR1, VLCDR2, or VLCDR3.
  • the variants encode substitutions of VHCDR3.
  • the variants have conservative amino acid substitutions at one or more predicted non-essential amino acid residues.
  • a “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Conservative substitutions are shown in the Tables below.
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity, e.g., the ability to bind ⁇ 1- ⁇ 4 peptide.
  • the encoded protein may routinely be expressed and the functional and/or biological activity of the encoded protein, e.g., ability to immunospecifically bind AIB1- ⁇ 4 peptide, can be determined using techniques described herein or by routinely modifying techniques known in the art.
  • Trp Tyr Tyr (Y) Trp, Phe, Thr, Ser
  • Val (V) lie, Leu, Met, Phe, Ala
  • the antibodies of the invention include derivatives or variants that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not affect the ability of the antibody to immunospecifically bind ⁇ 1- ⁇ 4 peptide.
  • derivatives of the invention include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known
  • the derivative may contain one or more non-classical amino acids.
  • an antibody or antibody fragment of the invention including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof, that immunospecifically binds ⁇ 1- ⁇ 4 peptide, comprises, or alternatively consists of, an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleotide sequence that is complementary to that encoding one of the VH or VL domains of any of the antibodies described herein under stringent conditions, e.g., hybridization to filter-bound DNA in 6X sodium chloride/sodium citrate (SSQ at about 45°C.
  • SSQ 6X sodium chloride/sodium citrate
  • immunospecifically binds to ⁇ 1- ⁇ 4 peptide comprises, or alternatively consists of, an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleotide sequence that is complementary to that encoding one of the VH CD s or VL CDRs of any of the antibodies described herein under stringent conditions, e.g., hybridization under conditions as described above, or under other stringent hybridization conditions which are known to those of skill in the art.
  • an antibody of antibody fragment that immunospecifically binds to ⁇ 1- ⁇ 4 peptide or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VH domains of any of the antibodies described herein.
  • the invention provides an antibody or antibody fragment of the invention that immunospecifically binds to ⁇ 1- ⁇ 4 peptide comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VH CD s of any of the antibodies described herein. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
  • immunospecifically binds to ⁇ 1- ⁇ 4 peptide comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VL domains of any of the antibodies described herein.
  • the invention provides an antibody of the invention that immunospecifically binds to ⁇ 1- ⁇ 4 peptide comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VL CDRs of any of the antibodies described herein.
  • the invention provides antibodies or antibody fragments that immunospecifically binds to ⁇ 1- ⁇ 4 peptide comprising, or alternatively consisting of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to at least one, two or three of the VH CDRs of any of the antibodies described herein and that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to at least one, two or three of the VL CDRs of any of the antibodies described herein.
  • the invention also provides antibodies or antibody fragments that immunospecifically binds to ⁇ 1- ⁇ 4 peptide comprising, or alternatively consisting of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to at least two or three of the VH CDRs of any of the antibodies described herein and that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to at least two or three of the VL CDRs of any of the antibodies described herein.
  • the invention also provides antibodies or antibody fragments that immunospecifically binds to ⁇ 1- ⁇ 4 peptide comprising, or alternatively consisting of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to three of the VH CDRs of any of the antibodies described herein and that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to three of the VL CDRs of any of the antibodies described herein. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
  • Antibodies or fragments of the present invention may also be described or specified in terms of their binding affinity for ⁇ 1- ⁇ 4 peptide or domains or variants of ⁇ 1- ⁇ 4 peptide.
  • antibodies or fragments of the invention bind ⁇ 1- ⁇ 4 peptide or domains or variants thereof, with a dissociation constant or K d of less than or equal to 5xlO "2 M, 10 "2 M, 5xlO ⁇ 3 M, 10 ⁇ 3 M, 5xlO "4 M, 10 "4 M, 5xlO "5 M, 10 s M, 5xlO "6 M, 10 "6 M, 5xlO "7 M, 10 "7 M, 5xlO "8 M, 10 s M, 5xlO "9 M, 10 "9 M, 5x10 10 M, 10 10 M, 5x10 "iVI, 10 11 M, 5x10 12 M, 10 12 M, 5x10 13 M, 10 13 M, 5x10 14 M, 10 " 14 M, 5x10 15 M or 10 15 M.
  • Monoclonal Fab fragments may also be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246: 1275-81 (1989); Mullinax et al., Proc. Nat'l. Acad. Sci. 87: 8095 (1990).
  • monoclonal antibodies of a single desired isotype are preferred for a particular application, particular isotypes can be prepared directly, by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class-switch variants (Steplewski, et al., Proc. Nat'l. Acad. Sci., 82: 8653 (1985); Spira et al., J. Immunol. Methods, 74: 307 (1984)).
  • the isotype of a monoclonal antibody with desira ble propertied can be changed using antibody engineering techniques that are well- known in the art.
  • ⁇ 1- ⁇ 4 peptide-specific antibodies of the invention may be screened for epitope specificity according to standard techniques. See, e.g., Czernik et al., Methods in Enzymology, 201: 264-283 (1991).
  • the antibodies may be screened against ⁇ ⁇ 1- ⁇ 4 peptide library by ELISA to ensure specificity for both the desired antigen.
  • the a ntibodies may also be tested by Western blotting against cell preparations containing the parent signaling protein, e.g., cell lines over-expressing the parent protein, to confirm reactivity with the desired epitope/target.
  • Antibodies may be further characterized via immunohistochemical (IHC) staining using normal and diseased tissues to examine levels of ⁇ 1- ⁇ 4 peptide in diseased tissue.
  • I HC immunohistochemical
  • I HC may be carried out according to well-known techniques. See, e.g., Antibodies: A La boratory Manual, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor La boratory (1988).
  • paraffin-em bedded tissue e.g., tumor tissue
  • paraffin-em bedded tissue is prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; u nmasking antigen by heating slide in sodium citrate buffer; incu bating sections in hydrogen peroxide; blocking in blocking solution; incu bating slide in primary antibody and secondary antibody; a nd finally detecting using ABC avidin/biotin method according to manufacturer's instructions.
  • Antibodies may be further characterized by flow cytometry carried out according to standard methods. See Chow et al., Cytometry, Communications in Clinical Cytometry 46: 72-78 (2001). Briefly and by way of example, the following protocol for cytometric analysis may be employed : samples may be centrifuged on Ficoll gradients to remove lysed erythrocytes and cell debris. Adhering cells may be scraped off plates and washed with PBS. Cells may then be fixed with 2% paraformaldehyde for 10 minutes at 37°C, followed by permea bilization in 90% methanol for 30 minutes on ice.
  • Cells may then be stained with the primary ⁇ 1- ⁇ 4 peptide-specific antibody of the invention, which can detect an ⁇ ⁇ 1- ⁇ 4 peptide, washed and la beled with a fluorescent-la beled secondary antibody.
  • Additional fluorochrome-conjugated marker antibodies e.g., CD45, CD34, may also be added at this time to aid in the su bsequent identification of specific hematopoietic cell types.
  • the cells would then be analyzed on a flow cytometer, e.g., a Beckman Coulter FC500, according to the specific protocols of the instrument used.
  • Antibodies of the invention may also be conjugated to fluorescent dyes, e.g., Alexa488, PE, etc., for use in multi-parametric analyses along with other signal transduction, e.g., phospho-CrkL, phospho- Erk 1/2, and/or cell marker, e.g., CD34 antibodies.
  • fluorescent dyes e.g., Alexa488, PE, etc.
  • other signal transduction e.g., phospho-CrkL, phospho- Erk 1/2
  • cell marker e.g., CD34 antibodies.
  • bispecific antibodies are within the purview of those skilled in the a rt.
  • the recom binant prod uction of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)).
  • Antibody varia ble domains with the desired binding specificities (antibody-antigen com bining sites) can be fused to immunoglobulin constant domain sequences.
  • the fusion is with an immunoglobulin heavy-chain constant domain, includ ing at least part of the hinge, CH2, a nd CH3 regions.
  • DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suita ble host organism.
  • illustrative currently known methods for generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986); WO 96127011; Brennan et al., Science 229:81 (1985); Shala by et ai, J. Exp. Med. 175:217-225 (1992); Kostelny et al. . Immunol.
  • Bispecific antibodies also include cross-linked or heteroconjugate antibodies.
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suita ble cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • bispecific antibodies have been produced using leucine zippers.
  • the leucine zipper peptides from the Fos and Jun proteins may be linked to the Fa b' portions of two different antibodies by gene fusion.
  • the antibody homodimers may be reduced at the hinge region to form monomers a nd then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers.
  • the antibodies ca n be "linear antibodies” as described in Zapata et al. Protein Eng. 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (V H -C H i-V H - C H i) which form a pair of a ntigen binding regions. Linear antibodies can be bispecific or monospecific. To produce the chimeric antibodies, the portions derived from two different species, e.g., human constant region and murine varia ble or binding region can be joined together chemically by
  • Fully human antibodies may be produced by a variety of techniques.
  • One example is trioma methodology.
  • the basic approach and an exemplary cell fusion partner, SPAZ-4, for use in this approach have been described by Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and U.S. Pat. No. 4,634,666, which are incorporated by reference.
  • Hu man antibodies can also be produced from non-human transgenic animals having transgenes encoding at least a segment of the hu man immunoglobulin locus. The production and properties of animals having these properties are described in detail by, see, e.g., W093/12227; U.S. Pat. No.
  • Various recom binant antibody library technologies may also be utilized to produce fully human antibodies.
  • one approach is to screen a DNA library from human B cells according to the general protocol outlined by Huse et al., Science 246:1275-1281 (1989). The protocol described by Huse is rendered more efficient in combination with phage-display technology. See, WO 91/17271, WO 92/01047 and U.S. Pat. No. 5,969, 108, which are incorporated by reference.
  • Eukaryotic ribosome can also be used as means to display a library of antibodies and isolate the binding human antibodies by screening against the target antigen, as described in Coia G, et al., J.
  • the yeast system is also suita ble for screening mammalian cell-surface or secreted proteins, such as antibodies.
  • Antibody libraries may be displayed on the surface of yeast cells for the purpose of obtaining the human antibodies against a target antigen. This approach is described by Yeung, et al., Biotechnol. Prog. 18(2):212-20 (2002); Boeder, E. T., et al., Nat. Biotechnol. 15(6):553-7 (1997), which are incorporated by reference.
  • human antibody libraries may be expressed intracellularly and screened via the yeast two-hybrid system. See WO0200729A2, which is incorporated by reference.
  • Recom binant DNA techniques can be used to produce the recom binant ⁇ 1- ⁇ 4 peptide-specific antibodies described herein, as well as the chimeric or huma nized ⁇ ⁇ 1- ⁇ 4 peptide-specific antibodies, or any other genetically-altered antibod ies and the fragments or conjugate thereof in any expression systems including both prokaryotic and eukaryotic expression systems, such as bacteria, yeast, insect cells, plant cells, mammalian cells (for example, NSO cells).
  • prokaryotic and eukaryotic expression systems such as bacteria, yeast, insect cells, plant cells, mammalian cells (for example, NSO cells).
  • the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present application can be purified according to standard procedures of the art, including a mmonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Once purified, partially or to the desired levels of homogeneity, the polypeptides may then be used for performing assay procedures, immu nofluorescent staining, and the like.
  • Fa b and F(a b') 2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein.
  • Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fa b fragments) or pepsin (to produce F(a b') 2 fragments).
  • enzymes such as papain (to produce Fa b fragments) or pepsin (to produce F(a b') 2 fragments).
  • ⁇ ⁇ 1- ⁇ 4 peptide-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.
  • “Humanized” chimeric antibodies can also be used. Such antibodies can be produced using genetic constructs derived from hybridoma cells prod ucing the monoclonal antibodies described a bove. Methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Ca billy et al., U.S. Pat. No.
  • the antibodies and methods described herein can be used to measure levels ⁇ ⁇ 1- ⁇ 4 peptide
  • the measurement of the levels of peptide may be qualitative or quantitative.
  • the levels peptide may be quantified is some numerical expression, such as a ratio or a percentage.
  • this determination can then be compared to normal levels or baseline levels of the peptide. "Normal levels" of the peptide may be assessed by measuring levels of the peptide in a known healthy su bject, including the same su bject that is later screened or being diagnosed.
  • Normal levels may also be assessed over a population sample, where a population sample is intended to mea n either multiple samples from a single su bject or at least one sample from a multitude of su bjects.
  • the samples used to generate the population can be ta ken from previously harvested tissues that, for exa mple, may be stored in paraffin or cryogenically stored.
  • the population of samples can continually grow as additional samples are added to the population to gain statistical confidence in the data.
  • Normal levels of the peptide in terms of a population of samples, may or may not be categorized according to characteristics of the population including, but not limited to, sex, age, weight, ethnicity, geographic location, fasting state, state of pregnancy or post- pregnancy, menstrual cycle, general health of the su bject, alcohol or drug consumption, caffeine or nicotine intake and circadian rhythms.
  • baseline levels are determined by measuring levels of the peptide in a su bject where the condition is minimally aggressive or is considered to be benign.
  • baseline levels of the peptide can be esta blished from a population of su bjects that have, for example, cancer or tumors, but the cancer has been graded as not aggressive and/or well differentiated.
  • the American Joint Commission on Cancer has developed the following guidelines for grading tumors and are believed to be current as of the filing date of the application:
  • baseline levels of the peptide may be esta blished in GX, Gl, G2, G3 and/or G4 graded tumors.
  • GX Gl
  • G2 G3
  • G4 G4 graded tumors.
  • other means of stratifying tu mors may also be used, for example the Bloom-Richardson system in breast cancer, and these other means for stratifying tumors can be used to esta blish baseline levels of the ⁇ ⁇ 1- ⁇ 4 peptide.
  • a difference between normal/baseline levels and the measured levels of the peptide may indicate that the su bject has a disease or a bnormal condition or has a higher (or lower) proba bility of developing a disease or a bnormal condition than do normal su bjects or su bjects within the range of baseline levels of the peptide.
  • differences between normal/baseline levels and the measured levels of the ⁇ ⁇ 1- ⁇ 4 peptide may indicate that the su bject has a more or less severe or aggressive form of the disease or a bnorma l condition compared to others.
  • the magnitude of difference between measured levels and normal/baseline levels of the peptide may also indicate the severity of disease or a bnormal condition or the level of proba bility of developing a disease or a bnormal condition, compared to normal su bjects or su bjects within the range of baseline levels of the peptide, such as the likelihood of metastasis.
  • the difference between measured levels of the peptide and normal/baseline levels may be a relative or a bsolute qua ntity.
  • Levels of peptide is used to mean any measure of the quantity of the peptide such as, but not limited to, mass, concentration and biological activity.
  • Example of biological activities that may be used to quantify biomarkers include, but are not limited to, chemotactic, cytotoxic, enzymatic or other biological activities, such as quantifia ble activities that are used, for example, by the National Institute for Biological Standards and Control (N IBSC) in the United Kingdom for the quantification of interferon, cytokine and growth-factor activity.
  • the difference in levels of peptide may be equal to zero, indicating that the su bject is or may be normal or within the range of baseline levels of the peptide, or that there has been no change in levels of peptide since the previous assay.
  • the difference may simply be, for exa mple, a measured fluorescent value, radiometric value, densitometric value, DNA quantification, mass value etc., without any additional measurements or manipulations.
  • the levels or d ifferences of the ⁇ 1- ⁇ 4 peptide may be expressed as a percentage or ratio of the measured value of the peptide to a measured value of another compound including, but not limited to, a standard or internal standard.
  • the differences or levels may also be expressed as a percentage or ratio of ⁇ ⁇ 1- ⁇ 4: ⁇ ⁇ 1 (or perhaps the reciprocal).
  • the differences may be negative, indicating a decrease in the amount of measured peptide over normal/baseline values or from a previous measurement, or the difference may be positive, indicating an increase in the amount of measured peptide over normal/baseline values or from a previous measurement.
  • the difference may also be expressed as a difference or ratio of the peptide to itself, measured at a d ifferent point in time. The d ifference may also be determined using in an algorithm, wherein the raw data is manipulated.
  • levels of peptide which are higher than normal/baseline levels of peptide may confirm that the su bject has the a bnormal condition or that the su bject may have a higher proba bly than normal of developing the a bnormal condition, or that the su bject has a condition that is considered more less severe or aggressive.
  • a ratio of ⁇ ⁇ 1- ⁇ 4: ⁇ ⁇ 1 that is higher in a test patient compared to normal/baseline levels would indicate that the patient may have an increased likelihood that the condition, e.g. a tumor, is more aggressive and, perhaps, more likely to metastasize.
  • levels of peptide that are equal to or lower than normal/baseline levels of peptide may confirm that the su bject either does not have the a bnormal condition or that the su bject may have a lower proba bility of developing the a bnormal condition or that the condition is less severe or aggressive.
  • the present invention also relates to methods of monitoring the progression of a bnormal conditions in su bjects, with the methods comprising determining the levels of ⁇ ⁇ 1- ⁇ 4 peptide in a sample from the su bject at first and second, third, fourth, etc. time points. The levels of ⁇ 1- ⁇ 4 peptide at each time point are then compared to determine differences of the ⁇ 1- ⁇ 4 peptide over time. Any differences between the levels of ⁇ ⁇ 1- ⁇ 4 peptide over time are indicative of the progression, regression or stasis of the a bnormal condition in the su bject. In one em bodiment, an increase in the levels over time indicates that the a bnormal condition is worsening in the su bject.
  • a decrease in the levels over time indicates that the a bnormal condition is improving in the su bject.
  • the improvement or worsening of the su bject based on the levels of ⁇ ⁇ 1- ⁇ 4 peptide over time can then possibly dictate or suggest a change in therapy.
  • the phrase "monitor the progression" is used to indicate that the a bnormal cond ition in the su bject is being periodically checked to determine if the a bnormal condition is progressing (worsening), regressing (improving) or remaining static (no detecta ble change) in the individual by assaying the levels of ⁇ 1- ⁇ 4 peptide in the su bject using the methods of the present invention.
  • the methods of monitoring may be used in conjunction with other monitoring methods or treatment regimens for the a bnormal condition a nd to monitor the efficacy of these treatments.
  • the methods of the present invention may be used to monitor a su bject after mastectomy.
  • the methods may be used to monitor patients that have had a successful mastectomy, such that the methods can be used to monitor the patient for followup mammogram.
  • the methods can be used to monitor patients that have received treatment, such as a tumor removal, but may need followup or concurrent treatment for that cancer.
  • the present invention provides methods of individualizing a therapeutic regimen, comprising assessing levels of the ⁇ 1- ⁇ 4 peptide and correlating these levels with a likely response to a variety of therapies. For example, patient populations may be stratified according to their response to therapy as their responsiveness correlates to levels of ⁇ 1- ⁇ 4 peptide.
  • Monitoring may include assessing the levels of ⁇ 1- ⁇ 4 peptide at two time points from which a sample is taken, or it may include more time points, where any of the levels of ⁇ 1- ⁇ 4 peptide at one particular time point from a given subject may be compared with the levels of biomarker in the same subject, respectively, at one or more other time points.
  • the expression ratio of ⁇ 1- ⁇ 4: ⁇ 1 may also be used to predict or assess a drug regimen to which a given patient is more or less sensitive. Knowledge of the ratio at the outset of therapy can guide a clinician in choosing a particular therapy. For example, comparing a subject's initial levels of the ⁇ 1- ⁇ 4: ⁇ 1 ratio to levels that characteristically present a good response to a particular therapeutic regimen can guide a clinician in choosing a similar, if not identical, regimen. Conversely, comparing a subject's initial levels of the ⁇ 1- ⁇ 4: ⁇ 1 ratio to levels that characteristically present a negative response or no response to a particular therapeutic regimen can guide a clinician in choosing a different regimen.
  • the levels to which the subject's ratio is compared can be generated from a population of samples. Methods of determining levels in a population of samples has been discussed herein and these same principles can be used to correlate the ⁇ 1- ⁇ 4: ⁇ 1 ratio to a given response in a population of samples which can, in turn, be used for comparison purposes.
  • increase in levels of the peptide compared to normal levels indicates that the tumor has a higher probability of being sensitive or resistant to a therapy, i.e., that the tumor in the subject will not respond well to the therapy.
  • increase in levels of the peptide compared to normal levels indicates that the tumor has a lower probability of being sensitive or resistant to a therapy, i.e., that the tumor in the subject will respond well to the therapy.
  • the therapy that is being considered may or may not be preselected by the clinician.
  • the subject's calculated ratio may guide the physician to a therapy that was not previously considered, or the subject's calculated ratio may confirm as a good choice the therapy that the clinician had pre-determined.
  • the subject's calculated ratio may indicate to the clinician that the pre-determined therapy should be re-evaluated for a different choice.
  • the therapeutic regimen may be a single compound or it may be a com bination of compounds.
  • the levels of ⁇ ⁇ 1- ⁇ 4 will guide the clinician towards or away from a specific combination of compounds of the therapeutic regimen.
  • Plasmids- p300-HA and ERa constructs were provided by Dr. Maria L. Avantaggiati (Georgetown University) and from Dr. Pierre Cham bon (INSERM, Strassbourg) respectively.
  • AI Bl, ⁇ ⁇ 1- ⁇ 4, FLAG AI Bl, and FLAG ⁇ 1- ⁇ 4 were described previously (17,21).
  • a C-terminal FLAG was added to the ⁇ ⁇ 1- ⁇ 4 cDNA by deletion of the stop codon in ⁇ 1- ⁇ 4 and addition of FLAG peptide sequence by site-directed mutagenesis (Stratagene).
  • the AI Bl N term construct was created by PCR amplification of the
  • ACTR/AI Bl cDNA (184 bp to 777bp) to add a new 5' NotI site and 3' Bgl ll site.
  • the PCR product was then cloned into p3XFLAG-CMV-10 (Sigma).
  • H EK293, H EK293T, COS-7, and M DA-M B-231 were obtained from the Tissue Culture Shared Resource at Georgetown University.
  • the Human mammary epithelial cells (H M EC) were purchased and cultured in commercially supplied medium (BulletKit, Lonza).
  • H EK293T, COS-7, COLO 357, COLO SL, and COLO PL, M DA-M B-231, M DA231-BrM2 (brain), 4175-TR (lung), SCP2-TR (bone), and AIBl KO M EFs were grown in Dul becco modified Eagle maxim m (DM EM, Invitrogen) with 10% FBS.
  • DM EM Dul becco modified Eagle maxim m
  • CHO cells were grown in DM EM F12 (Invitrogen) with 10% FBS.
  • HEK293 cells were grown in Iscove's modified Eagle medium (I M EM, Invitrogen) with 10% Charcoal stripped serum (CCS).
  • I M EM Iscove's modified Eagle medium
  • CCS Charcoal stripped serum
  • T47D Al-2 were grown in I M EM+5%CCS.
  • HEK293, HEK293T, CHO, COS-7, and T47D Al-2 cells were transiently transfected with FuGEN E 6 (Roche).
  • Extracted tryptic peptides were analyzed using the MIDAS-MS based algorithm on an LC-ESI-MS 4000QT AP instrument (AB SCIEX, Framingham, MA).
  • MIDAS-MS based algorithm on an LC-ESI-MS 4000QT AP instrument
  • silico predicted peptides and corresponding collision energy settings were generated using recommended settings in MRMpilot software (AB SCIEX, Framingham, MA).
  • the list of predicted precursors includes the potential variable modification of methionine oxidation and the fixed modification of cysteine alkylation.
  • a final MS method was created for detection of the tryptic peptides produced from the full-length AIB1 protein.
  • Nuclear staining was defined as protein specific signal that overlaid with the DAPI signal only.
  • Nuclear/cytoplasmic was defined as protein specific staining that overlaid with the DAPI signal but also showed staining in the cytoplasmic compartment. Cytoplasmic staining was defined as protein specific staining that did not overlay with the DAPI staining of the nucleus, (ii) 2x10 s AIB1 MEFs were transiently transfected with 4 ⁇ g FLAG ⁇ 1- ⁇ 4 and either 2, 4, or 6 ⁇ g of p300-HA or AIB1 using the MEF 2 Nucleofector kit (Amaxa, Lonza) and plated on glass coverslips after transfection. 24 hours later cells were fixed, permeabolized, stained, mounted, and quantified as in (i). p300 (1:500, Abeam) and anti- rabbit AlexaFluor568 (1:1000, Invitrogen) were used to stain for p300-HA.
  • Chromatin immunoprecipitation assays (ChIP)- HEK293 cells in a 10 cm dish were transfected with 5 ⁇ g E a and either 6 ⁇ g FLAG AIB1 or 3 ⁇ g FLAG ⁇ 1- ⁇ 4. 24 hours later cells were treated with Estrogen (E2) for 0, 15, 30, 45, or 60 minutes. Cells were fixed with formaldehyde fixation solution (3.7% formaldehyde, 100 mM NaCI, 50 mM Tris/HCI pH 8.0, 1 mM EDTA, 0.5 mM EGTA) for 10 minutes at 37°C and stopped with 0.125 M Glycine in lx PBS for 5 minutes at 25°C.
  • formaldehyde fixation solution 3.7% formaldehyde, 100 mM NaCI, 50 mM Tris/HCI pH 8.0, 1 mM EDTA, 0.5 mM EGTA
  • Cells were washed three times IxPBS and resuspended in SDS lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 1% SDS). Cells were sonicated and resuspended in ChIP dilution buffer (20 mM Tris pH8.0, 2 mM EDTA pH 8.0, 150mM NaCI, 1% Triton-X-100) and pre-cleared with 30 ⁇ of protein G agarose/salmon sperm DNA (Millipore) for 1 hour.
  • SDS lysis buffer 50 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 1% SDS. Cells were sonicated and resuspended in ChIP dilution buffer (20 mM Tris pH8.0, 2 mM EDTA pH 8.0, 150mM NaCI, 1% Triton-X-100) and pre-cleared with 30 ⁇ of protein G agarose/sal
  • Cycling conditions for real time PCR using iCycler were 95°C 3 minutes followed by 40 cycles of 95°C 20 sec, 57°C 30 sec, 72°C 40 sec for hC3 ERE and pS2 ERE.
  • HER2 ERE cycling conditions were 95°C 3 minutes followed by 40 cycles of 95°C 20 sec, 65°C 30 sec, 72°C 40 sec. The percentage of the input for each time point is plotted on the graphs and normalized time 0 for each transfection.
  • T47D Al-2 cells were transfected with either FLAG ⁇ 1- ⁇ 4 or FLAG empty vector. 24 hours later cells were stimulated with ⁇ R5020 for 1 hour and lysates were processed as described above.
  • endpoint PCR was used with the following conditions 96°C 4 minutes followed by 30 cycles of 94°C 1 min, 60°C 1 min, 72°C 1 min.
  • Primer sequences in the MMTV promoter were MMTV s: 5'-CGGTTCCCAGGGCTTAAGTAAGTT-3' (SEQ ID NO: 8) and M MTV as: 5'-GGATGGCGAACAGACACAAACACA-3' (SEQ ID NO: 9). All primers were synthesized by Integrated DNA Technologies (IDT).
  • Primer sequences used were: pS2 s: 5'-CCCCGTGAAAGACAGAATTGT-3' (SEQ ID NO: 10), pS2 as: 5'-GGTGTCGTCGAAACAGCAG-3' (SEQ ID NO: 11), hC3 s: 5'-CTGTCCACGACTTCCCAGG-3' (SEQ ID NO: 12), hC3 as: 5'-CCCTTTTCTGACTTGAACTCCC-3' (SEQ ID NO: 13), HER2 s: 5'- AAAGGCCCAAGACTCTCTCC-3' (SEQ ID NO: 14), HER2 as: 5'-CAAGTACTCGGGGTTCTCCA-3' (SEQ ID NO: 15), human actin s: 5'-CCTGGCACCCAGCACAAT-3' (SEQ ID NO: 16), human actin as: 5'- GCCGATCCACACGGAGTACT-3' (SEQ ID NO: 17).
  • Cycling conditions for the AIBl-A4-scorpion primer consist of an initial denaturing step at 94°C (2 min), and 50 cycles (20 seconds at 94°C, 15 seconds at 55.5°C and 20 seconds at 72°C). Unlike SYBR green real time PCR analysis where data is collected during the extension step, data for the Scorpion primer reactions were collected during the 55.5°C annealing step (iCycler; Bio-Rad). Cycling conditions for the human actin primers include a denaturing step at 94°C (2 min), and 45 cycles (20 seconds at 94°C, 30 seconds at 58°C and 40 seconds at 72°C). The ⁇ 1- ⁇ 4 scorpion primer was custom designed and purchased from Sigma-Aldrich.
  • ⁇ 1- ⁇ 4 Scorpion reaction 5'FAMCCCGCGCTTGGAAATAGTTTTTCCCTTGTCCGCGCGGGBHQlHEGCGCAAATTGCCATGTGATAC-3' (SEQ ID NO: 18).
  • ⁇ 1- ⁇ 4 reverse primer 5'-CCATCCAATGCCTGAAGTAA-3' (SEQ ID NO: 19).
  • the expression level of ⁇ 1- ⁇ 4 is normalized to actin expression levels and multiplied by either 10,000 or 100,000 to obtain whole number values.
  • Luciferase assay- 25,000 COS-7 cells per well in a 24 well dish were transfected in DMEM without serum with 100 ng M MTV luciferase, 25 ng Progesterone receptor (PR), 5 ng Thymidine Kinase (TK) Renilla luciferase, and either 500 ng pcDNA3, 500 ng FLAG AIB1, 500 ng FLAG N term, or 125, 500, and 750 ng of FLAG N term with 500 ng FLAG AIB1. 24 hours later cells were treated with 10 nM R5020 or an equivalent volume of ethanol.
  • luciferase values were measured using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase values were normalized to Renilla luciferase values and averaged for each transfection condition plated in triplicate.
  • ⁇ 1- ⁇ 4 is a splice variant of the nuclear receptor coactivator AIB1 (17) which results in the translation of an N-terminally truncated isoform of the full-length AIB1 protein with a molecular weight of approximately 130 kDa.
  • the translation start site of ⁇ 1- ⁇ 4 was predicted to be at the methionine at position 199 in the full-length AIB1 protein since this was the next in frame methionine residue, however the translation start site was not identified experimentally.
  • AM235M236EEGEDLQSCM246ICVAR (SEQ ID NO: 21) (underlined in Fig. la). This ruled out the M235, M236, M246, and M289 methionines as possible translation start sites and limited the possibilities to the M217 and M224 methionine residues. As a next step these methionines were tested using in silico predictions for tryptic peptide with an initial methionine as the first amino acid residue. Also included were predicted precursors that could appear as a result of cotranslational modifications such as N- terminal acetylation. In eukaryotes, 80% of all proteins have been described with an acetyl moiety added to the N-terminus (23,24).
  • Fig. lc The full-length and alternatively spliced AIBl mRNAs and proteins derived from these data are depicted schematically in Fig. lc.
  • the full-length human AIBl transcript has 23 exons coding for a 1424 amino acid protein of 155 kDa. Translation of AIBl is initiated in exon 3 and continues until the stop codon in exon 23.
  • the ⁇ 1- ⁇ 4 transcript lacks exon 4 due to alternative splicing. Translation of the ⁇ 1- ⁇ 4 isoform is initiated in exon 7 and the resultant protein lacks the N-terminal 223 amino acids of the full-length protein.
  • ⁇ 1- ⁇ 4 is now defined as a 130 kDa protein of 1201 amino acids.
  • ⁇ 1- ⁇ 4 is a predominantly cytoplasmic protein that enters the nucleus.
  • CHO cells transfected with AIBl showed nuclear and nuclear/cytoplasmic staining in 75% and 25% of the cells respectively.
  • Cells transfected with ⁇ 1- ⁇ 4 showed nuclear, nuclear/cytoplasmic, and cytoplasmic staining in 1.5%, 41.3%, and 57.2% of cells respectively.
  • Typical staining for nuclear, nuclear/cytoplasmic, and cytoplasmic stained cells are shown. At least some nuclear and nuclear/cytoplasmic staining was detected for ⁇ 1- ⁇ 4.
  • the nuclear export inhibitor leptomycin B which inhibits C M 1 dependent nuclear export of proteins that contain a nuclear export sequence (NES) (16). Both AIBl and ⁇ 1- ⁇ 4 contain the NES that is in the C-terminal half of the proteins (Fig. lc) (26).
  • CHO cells were transfected with FLAG AIBl or FLAG ⁇ 1- ⁇ 4 in media with 10% FBS and were either treated with vehicle or leptomycin B. Cells were fixed, stained, and the localization was quantified as before (Fig. 2b). It was observed the same distribution of staining in the transfected CHO cells treated with vehicle, however there was an increase in the number of cells showing nuclear staining in both AIBl and ⁇ 1- ⁇ 4 transfected cells.
  • CHO cells transfected with FLAG AIBl and treated with leptomycin B showed 88%, 12%, and 0% of cells staining in the nuclear, nuclear/cytoplasmic, and the cytoplasmic compartments respectively.
  • CHO cells transfected with AIB1- ⁇ 4 and treated with leptomycin B showed staining in the nuclear, nuclear/cytoplasmic, and the cytoplasmic compartments in 18%, 60%, and 22% of cells respectively.
  • M EFs mouse embryonic fibroblasts derived from AI Bl knockout mice were used to analyze the localization of ⁇ ⁇ 1- ⁇ 4.
  • the AIBl KO M EFs were transfected with FLAG ⁇ 1- ⁇ 4 in the presence of increasing amounts of AI Bl (Fig. 3 b). It was observed increased nuclear staining of ⁇ 1- ⁇ 4 after transfection with increasing amounts of AIBl.
  • the pattern of staining of ⁇ ⁇ 1- ⁇ 4 that was cytoplasmic, nuclear/cytoplasmic, or nuclear was 86 ⁇ 1%, 14 ⁇ 1%, and 0% respectively and shifted to 70.3 ⁇ 3.5%, 29.3 ⁇ 4%, and 0.3 ⁇ 0.6% with full-length AIBl (Fig. 3c).
  • ⁇ ⁇ 1- ⁇ 4 is recruited to active transcriptional complexes and coactivates estrogen responsive genes.
  • HEK293 cells were transfected with ER and either FLAG AI Bl or FLAG ⁇ 1- ⁇ 4 and Chromatin Immunoprecipitation (ChI P) analysis (Fig. 5a) was performed.
  • Cells were treated with estrogen for 15, 30, 45, and 60 minutes to observe the recruitment of AIBl or ⁇ 1- ⁇ 4 over time.
  • Both AIBl and ⁇ 1- ⁇ 4 were recruited to the three different estrogen response elements in pS2, hC3, and HER2 (22,36,37).
  • HEK293 cells were transfected with ER and either FLAG AI Bl or FLAG ⁇ 1- ⁇ 4 and stimulated with estrogen for 4, 8, and 24 hours.
  • RNA was harvested from the cells and the gene expression examined by real time PCR. Maximal gene expression in ⁇ 1- ⁇ 4 transfected cells occurred at 8 hours for H ER2 a nd at 24 hours for pS2 and hC3. Significant differences in gene expression were only observed in the AI B1- ⁇ 4 transfected cells suggesting that ⁇ ⁇ 1- ⁇ 4 is better a ble to stimulate expression of estrogen responsive genes than full-length AI Bl.
  • the region deleted from ⁇ 1- ⁇ 4 contains an inhibitory domain.
  • ⁇ ⁇ 1- ⁇ 4 also stimulates expression of endogenous estrogen dependent genes. This potent coactivator function cannot be explained merely based on the cellular localization of ⁇ 1- ⁇ 4 because it is mostly cytoplasmic.
  • a FLAG tagged N-terminal AI Bl construct (AI Bl N term) was created which contains the bH LH and PAS A domains not present in the ⁇ ⁇ 1- ⁇ 4 protein (Fig. lc). This construct should have no coactivator function since it lacks the C-terminal activation domain responsible for binding to nuclear receptors and molecules that affect transcription such as p300/CBP and CARM 1.
  • AI Bl was a ble to coactivate transcription of the luciferase reporter relative to empty vector transfected cells (AIBl 0 vs. 500).
  • ⁇ 1- ⁇ 4 expression is correlated with metastatic potential.
  • a quantitative assay to measure ⁇ 1- ⁇ 4 m RNA levels was designed. Through the use of Scorpion primer technology (39,40) it was shown that the expression of ⁇ 1- ⁇ 4 is increased in breast cancer cell lines relative to normal breast cell lines.
  • a Scorpion primer with a sequence complimentary to a sequence in exon 3 and a probe sequence complimentary to the junction of exon 3 and exon 5 were designed. This scorpion primer specifically recognizes the ⁇ 1- ⁇ 4 transcript and not the AI Bl transcript due to the fact that after amplification of AI Bl transcript, the probe sequence is not complimentary to any sequence generated in the full-length AI Bl transcript.
  • Plasmids containing either AIBl or ⁇ 1- ⁇ 4 cDNA were su bjected to an analysis by real time PC with the ⁇ 1- ⁇ 4 scorpion primer.
  • the ⁇ 1- ⁇ 4 scorpion primer specifically identified ⁇ 1- ⁇ 4 cDNA and not full-length AIBl cDNA.
  • total RNA was harvested from human mammary epithelial cells (HM EC), parental M DA-M B-231, and three tissue specific metastatic variants of M DA-M B-231 cells. The three tissue specific variants home either to the brain, bone, or lung after intravenous or intracardiac injection (41-43). After reverse transcription, cDNA generated from these RNAs was su bjected to real time PCR analysis with Scorpion primers for ⁇ 1- ⁇ 4 (Fig. 7b).
  • ⁇ ⁇ 1- ⁇ 4 m RNA was higher in the parental and three tissue specific metastatic variants of M DA-M B-231 cells relative to H M ECs confirming increased expression of ⁇ 1- ⁇ 4 in breast cancer cell lines relative to normal breast cells.
  • Neoplasia 1 50- 62

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Abstract

The invention relates to an isolated peptide comprising the amino acid sequence of SEQ ID NO: 1 and methods of use thereof.

Description

DETECTION OF AIB1-DELTA 4 PROTEIN
Cross-Reference to Related Applications
[0001] This application claims the benefit of U.S. provisional patent application number 61/490,565, filed May 26, 2011, which is incorporated herein by reference.
Statement Regarding Federally Sponsored Research or Development
[0002] Part of the work performed during development of this invention utilized U.S. Government funds under National Institutes of Health Grant Number 01 CA113477, United States Department of Defense Grant Number W81XWH-06-10590. The U.S. Government has certain rights in this invention.
Sequence Listing Submission via EFS-Web
[0003] A computer readable text file, entitled "036681-5012-WO-SequenceListing.txt" created on or about May 24, 2012 with a file size of a bout 15 kb contains the sequence listing for this application and is hereby incorporated by reference in its entirety.
Background of the Invention
Field of the Invention
[0004] The invention relates to an isolated peptide comprising the amino acid sequence of SEQ ID NO: 1 (ΑΙΒ1-Δ4) and methods of use thereof.
Background of the Invention
[0005] Gene transcription in eukaryotes is a complex and highly regulated process. One of the major controls of gene transcription is exerted by the coregulator family of proteins. These include both corepressors, which dampen transcription, and coactivators, which potentiate transcription. A su bgroup of coactivators has been shown to be critical for the malignant progression of cancer and is known as the pl60 steroid receptor coactivators (1). One member in particular was identified to be amplified in breast cancer. Amplified in Breast Cancer 1 (AIBl, SRC-3, NCOA3, ACTR, TRAM-1, pCIP, RAC3) has been shown to be gene amplified in breast cancer (2) and is also overexpressed at the mRNA and protein level in various cancers (1,3,4) Its role in tumorigenesis is attributed to its ability to coactivate both steroid hormone and growth factor dependent transcription (3,5-7). In several oncogene driven mouse models (8-11) reduction of AIBl levels lead to a decrease in tumorigenesis and overexpression of AIBl lead to the formation of various tumors (12). Clinically, AIBl expression in breast cancer cases is correlated with high HER2 levels, larger tumor size, higher tumor grade and shorter disease free survival (13-15). Also, high levels of AIBl in conjunction with high HER2 levels coincide with reduced disease free survival in patients treated with tamoxifen suggesting a role for AIBl in tamoxifen resistance (16).
[0006] A splice variant of AIBl, where exon 3 was spliced from the mature mRNA and was named AIB1- Δ3 has been identified previously (17). More recently, an additional 5' exon 81, 164 bases upstream of the known 5'UTR was also identified. Thus the deleted exon is now known to be exon 4 and is thus referred to as splice variant ΑΙΒ1-Δ4. It was previously reported that ΑΙΒ1-Δ4 mRNA results in an N- terminally truncated isoform of the AIBl protein that was found to be a more potent coactivator of steroid dependent transcription on a per mole basis when compared with the full-length AIBl protein. ΑΙΒ1-Δ4 mRNA expression was elevated in breast tumor tissue relative to normal breast tissue (17). It was also shown to increase the efficacy of estrogenic compounds and the agonist effects of the selective estrogen receptor modulator tamoxifen in breast and endometrial tumor cells (18). Overexpression of ΑΙΒ1-Δ4 in mice leads to ductal ectasia in the mammary gland with an increased expression of proliferative markers such as PCNA, phospho-histone H3, and Cyclin Dl (19). More recently, ΑΙΒ1-Δ4 was shown to act as a molecular bridge between Epidermal Growth Factor Receptor (EGFR) and Focal Adhesion Kinase (FAK) in the cytoplasm and its overexpression increased the invasiveness of the MDA- MB-231 metastatic breast cancer cell line (20).
[0007] To date the potent coactivator function of ΑΙΒ1-Δ4 has not been attributed solely to either a cytoplasmic or nuclear function of the protein. It has been discovered that ΑΙΒ1-Δ4 enters the nucleus by a non-canonical nuclear import mechanism and that ΑΙΒ1-Δ4 is recruited to estrogen response elements of endogenous estrogen-regulated genes and increases their expression. It has also been discovered that the N-terminal region that is absent from the ΑΙΒ1-Δ4 protein contains an inhibitory domain. Through the use of Scorpion primer technology, the first quantitative assay for the ΑΙΒ1-Δ4 transcript was created and it was found that there was a correlation between ΑΙΒ1-Δ4 expression and the metastatic phenotype of human cancer cell lines.
Summary of the Invention
[0008] The invention relates to an isolated peptide with the amino acid sequence of SEQ ID NO: 1 (ΑΙΒ1-Δ4).
[0009] The invention also relates to methods of stratifying a population of cells based on levels of the ΑΙΒ1-Δ4 peptide in abnormal cells.
[0010] The invention also relates to methods of assessing the prognosis of a subject based on levels of the ΑΙΒ1-Δ4 peptide in abnormal tissue.
[0011] The invention also relates to methods of assessing sensitivity or resistance to drug treatment of an individual on levels of the ΑΙΒ1-Δ4 peptide in the diseased tissues.
Brief Description of the Drawings
[0012] FIGURE 1 depicts the identification of the translation start site of ΑΙΒ1-Δ4. a, After
overexpression of a C-terminal FLAG ΑΙΒ1-Δ4 construct in HEK293T cells, mass spectrometric analysis determined that the ΑΙΒ1-Δ4 transcript initiates translation at the methionine at position 224 in the full- length AIBl protein. The TPHDILEDINASPEMR (SEQ ID NO: 20) peptide was not identified in ΑΙΒ1-Δ4 (boxed) and the AMMEEGEDLQSCMICVAR (SEQ ID NO: 21) peptide was identified (underlined) by mass spectrometric analysis. The MQCFALSQPR (SEQ ID NO: 22) peptide identified through further mass spectrometry analysis containing the translation start site is shown in bold, b, The annotated fragmentation spectra of the MQCFALSQPR peptide are shown. This tryptic peptide is bearing acetylation of the initial methionine as a result of cotranslational modification. The top spectrum shows collision induced dissociation (CID) fragmentation of the double charged peptide with a non-oxidized methionine and m/z ratio of 640.3. The lower spectrum depicts fragmentation of the double charged peptide with m/z ratio of 648.3 due to a mass shift caused by oxidation of the initial methionine. Stars indicate the position of parental ions in the MS/MS spectra. Based on the analysis of the CID fragmentation acetylation was assigned to the initial methionine residue, c, The full-length human AIBl transcript consists of 23 exons. This leads to the creation of a 155 kDa protein consisting of 1424 amino acids. The ΑΙΒ1-Δ4 transcript lacks exon 4 due to alternative splicing and the resultant protein lacks the N-terminal 223 amino acids of the full-length protein. This results in a 130 kDa protein consisting of 1201 amino acids.
[0013] FIGURE 2 depicts that ΑΙΒ1-Δ4 is found predominantly in the cytoplasm but can be detected in the nucleus, a, Chinese Hamster Ovary (22) cells were grown in DMEM F12+10%FBS and transfected with FLAG AIBl or FLAG ΑΙΒ1-Δ4. Cells were fixed and stained for FLAG peptide and DAPI and visualized by direct immunofluorescence. Typical nuclear, nuclear/cytoplasmic, and cytoplasmic FLAG staining is shown in the top panels (green) and DAPI staining DNA in the nucleus is shown in the bottom panels (blue). The percentage of nuclear, nuclear/cytoplasmic, and cytoplasmic staining cells is graphed. 200 cells were counted per transfection and cell compartment staining was quantified for three separate experiments. Nuclear, nuclear/cytoplasmic, and cytoplasmic stained cells are represented by the black, gray, and white bars respectively, b, CHO cells were treated as in panel a. 24 hours after transfection, either the carrier EtOH or 50 nM leptomycin B (LMB in EtOH) was added to the culture media for 4 hours before fixing and staining cells. The percentage of nuclear, nuclear/cytoplasmic, and cytoplasmic staining cells is shown in the absence and presence of LMB. Cells were quantified as in panel a. Nuclear, nuclear/cytoplasmic, and cytoplasmic stained cells are represented by the black, gray, and white bars respectively.
[0014] FIGURE 3 depicts that the overexpression of AIBl localizes ΑΙΒ1-Δ4 to the nucleus, a, HEK293T cells grown in DM EM+10%FBS were transfected with either FLAG AIBl alone, ΑΙΒ1-Δ4 alone, or FLAG AIBl and ΑΙΒ1-Δ4 together and lysates were immunoprecipitated using FLAG antibody to pulldown FLAG AIBl and any interacting proteins, b, AIBl knockout murine embryonic fibroblasts (KO M EFs) were transfected by electroporation with 4 μg FLAG ΑΙΒ1-Δ4 alone or with 2, 4, or 6 μg of untagged AIBl and plated on glass cover slips in DMEM+10% FBS. Cells were fixed and permeabolized 24 hours after plating and stained for FLAG containing proteins and nuclei stained with DAPI. Cells were then analyzed by confocal microscopy, c, The number of nuclear, nuclear/cytoplasmic, and cytoplasmic staining cells was quantified for three experiments as in Fig 2a. Data were analyzed by one way ANOVA with Tukey's multiple comparison post test. *=p<0.05, ***=P<0.001 when compared to ΑΙΒ1-Δ4 transfection alone.
[0015] FIGURE 4 depicts that the overexpression of p300 localizes ΑΙΒ1-Δ4 to the nucleus, a, HEK293T cells were transfected with either p300-HA, FLAG AIBl, or FLAG ΑΙΒ1-Δ4. Equal amounts of FLAG AIBl or FLAG ΑΙΒ1-Δ4 protein were incubated with equal amounts of p300-HA. After immunoprecipitation with HA antibody to pull down p300 and associated proteins, a FLAG Western blot was performed to determine how much AIBl and ΑΙΒ1-Δ4 immunoprecipitated with p300. b, AIBl KO MEFs were transfected by electroporation with 4 μg ΑΙΒ1-Δ4 alone or with 2, 4, or 6 μg of p300-HA and plated on glass cover slips in DMEM+10% FBS. Cells were fixed and permeabolized 24 hours after plating and stained for DAPI, FLAG, and HA containing proteins. Cells were then analyzed by confocal microscopy. The number of nuclear, nuclear/cytoplasmic, and cytoplasmic staining cells was quantified as in Fig 2a. Typical nuclear, nuclear/cytoplasmic, and cytoplasmic FLAG staining (green), p300 (red, nuclear) and DAPI staining DNA in the nucleus (blue) is shown with overlay of the images in the right panels. The percentage of nuclear, nuclear/cytoplasmic, and cytoplasmic stained cells is shown in the black, gray, and white bars respectively for three experiments is shown. Data were analyzed by one way ANOVA with Tukey's multiple comparison post test. *=p<0.05, ***=p<0.001 when compared to ΑΙΒ1-Δ4 transfection alone.
[0016] FIGURE 5 depicts that that ΑΙΒ1-Δ4, like AIBl, is recruited to ERE in the nucleus, a, HEK293 cells grown in phenol red free IMEM + 10% charcoal stripped serum were transfected with either FLAG AIBl or FLAG ΑΙΒ1-Δ4 and ERa. 24 hours later cells were stimulated with estrogen and harvested at 0, 15, 30, 45, and 60 minutes after estrogen stimulation. These lysates were subjected to a quantitative ChIP analysis using a FLAG antibody. The percentage of the input recovered after immunoprecipitation for each ERE in pS2, hC3, or HER2 was determined. Data is representative of three independent experiments. The relative amounts of transfected proteins and actin after transfection for each time point is shown by Western blot, b, HEK293 cells were grown as in panel a and total RNA was harvested from cells at 0, 4, 8, and 24 hours after estrogen treatment to determine the relative gene expression for pS2, hC3, and HER2. Data were analyzed by two way ANOVA with Bonferroni post test where *=p<0.05 **=p<0.01 ***=p<0.001 relative to time 0. The relative amounts of protein after transfection for each time point is shown by the Western blot.
[0017] FIGURE 6 depicts the N-terminus of AIBl contains an inhibitory domain that is lost in ΑΙΒ1-Δ4. a, COS-7 cells were transfected with human progesterone receptor B (25 ng), MMTV luciferase (100 ng), and either pcDNA3 or FLAG AIBl (500 ng) with increasing amounts of FLAG AIBl N term (125, 500, 750 ng). 24 hours later cells were treated with 10 nM R5020 for 24 hours before reporter activity was determined. Luciferase values were normalized to TK Renilla (10 ng) reporter activity. The assay was plated in triplicate and a representative graph is shown from three separate experiments. Data were analyzed by one way ANOVA with Tukey's multiple comparison post test. **=p<0.01, ***=p<0.001 when compared to FLAG AIB1 transfection alone, b, The relative amount of FLAG proteins in the COS-7 cells is shown by Western blot.
[0018] FIGURE 7 depicts that ΑΙΒ1-Δ4 expression is increased in metastatic cancer cell lines, a, A scorpion primer was designed to specifically recognize the unique splice junction of exon 3 and exon 5. The scorpion primer consists of primer (black half arrow), blocker (blue jagged line), quencher (purple octagon), probe (red - exon3, light blue - exon 5), stem (black attached lines), and reporter (green ball). The stem region will only be dissociated when a target sequence is created during the process of PCR. In the case of AIB1, no appropriate target is generated so the preferred conformation is to remain in the stem loop with the reporter quenched. For the ΑΙΒ1-Δ4 transcript an appropriate target is generated and the probe can then bind its target sequence allowing the reporter to fluoresce, b, Scorpion primers were used to quantitate the amount of ΑΙΒ1-Δ4 transcript from the RNA of human mammary epithelial cells (HMEC), parental MDA-MB-231 breast cancer cells and three tissue specific metastatic variants of MDA-MB-231 cells. The Ct values were normalized to actin expression as a control. Data were analyzed by one way ANOVA with Bonferroni post test. **=p<0.01, ***=p<0.001 when compared to HMEC AIB1- Δ4 expression levels, c, Scorpion primers were used as in panel b to analyze RNA from parental COLO 357 pancreatic cancer cells and two metastatic variants which metastasized from pancreas to liver (COLO PL) or from spleen to liver (COLO SL). Ct values were normalized to actin expression as a control. Data were analyzed by t test. *=p<0.05 when compared to parental COLO 357 cells.
[0019] FIGU RE 8 depicts that the AI B1-4 is recruited to the PRE in the M MTV promoter. T-47D (Al-2) cells, which have a stable integration of the MMTV luciferase reporter in their genome, were transfected with either pCMV-Flag or FLAG AI B1- 4 for 24 hr and then treated with 10 nM R5020 for 1 hr. The binding of AIB1- 4 to the MMTV promoter was analyzed by ChIP assay with a FLAG antibody.
[0020] FIGU RE 9 depicts that the AI B1- 4 Scorpion primer is specific for AI B1- 4 transcript. Scorpion primers specific for AIB1- 4 tested on plasmid cDNA constructs of AI B1 and AIB1- 4. Plasmids for AI B1 or AI B1- 4 were subjected to real time PCR analysis with scorpion primers specific for AIB1- 4.
[0021] FIGURE 10 depicts that the AIB1- 4 is resistant to density induced degradation. HEK293T cells were transfected with FLAG AI B1 or FLAG AI B1- 4. Cells were plated at either low or high density to obtain low or high confluence at the time of lysate harvest. Whole cell lysates were harvested 24 hours after plating at different densities a nd the levels of AIBl and AI Bl- 4 were determined by Western blot for FLAG.
Detailed Description of the Invention
[0022] The invention relates to a peptide comprising the amino acid sequence of SEQ ID NO: 1 (AIB1- Δ4) and methods of its use.
MQCFALS QPRAMMEEGE DLQSCMICVA RRITTGERTF PSNPESFITR HDLSGKWNI 57 DTNSLRSSMR PGFEDI IRRC IQRFFSLNDG QSWSQKRHYQ EAYLNGHAET PVYRFSLADG 117 TIVTAQTKSK LFRNPVTNDR HGFVSTHFLQ REQNGYRPNP NPVGQGIRPP MAGCNSSVGG 177 MSMSPNQGLQ MPSSRAYGLA DPSTTGQMSG ARYGGSSNIA SLTPGPGMQS PSSYQNNNYG 237 LNMSSPPHGS PGLAPNQQNI MISPRNRGSP KIASHQFSPV AGVHSPMASS GNTGNHSFSS 297 SSLSALQAIS EGVGTSLLST LSSPGPKLDN SPNMNITQPS KVSNQDSKSP LGFYCDQNPV 357 ESSMCQSNSR DHLSDKESKE SSVEGAENQR GPLESKGHKK LLQLLTCSSD DRGHSSLTNS 417 PLDSSCKESS VSVTSPSGVS SSTSGGVSST SNMHGSLLQE KHRILHKLLQ NGNSPAEVAK 477 ITAEATGKDT SSITSCGDGN WKQEQLSPK KKENNALLRY LLDRDDPSDA LSKELQPQVE 537 GVDNKMSQCT SSTIPSSSQE KDPKIKTETS EEGSGDLDNL DAILGDLTSS DFYNNSISSN 597 GSHLGTKQQV FQGTNSLGLK SSQSVQSIRP PYNRAVSLDS PVSVGSSPPV KNI SAFPMLP 657 KQPMLGGNPR MMDSQENYGS SMGGPNRNVT VTQTPSSGDW GLPNSKAGRM EPMNSNSMGR 717 PGGDYNTSLP RPALGGSIPT LPLRSNSIPG ARPVLQQQQQ MLQMRPGEIP MGMGANPYGQ 777 AAASNQLGSW PDGMLSMEQV SHGTQNRPLL RNSLDDLVGP PSNLEGQSDE RALLDQLHTL 837 LSNTDATGLE EIDRALGIPE LVNQGQALEP KQDAFQGQEA AVMMDQKAGL YGQTYPAQGP 897 PMQGGFHLQG QSPSFNSMMN QMNQQGNFPL QGMHPRANIM RPRTNTPKQL RMQLQQRLQG 957 QQFLNQSRQA LELKMENPTA GGAAVMRPMM QPQVSSQQGF LNAQMVAQRS RELLSHHFRQ 1017 QRVAMMMQQQ QQQQQQQQQQ QQQQQQQQQQ QQQQQQTQAF SPPPNVTASP SMDGLLAGPT 1077 MPQAPPQQFP YQPNYGMGQQ PDPAFGRVSS PPNAMMSSRM GPSQNPMMQH PQAASIYQSS 1137 EMKGWPSGNL ARNSSFSQQQ FAHQGNPAVY SMVHMNGSSG HMGQMNMNPM PMSGMPMGPD 1197 QKYC 1201 (SEQ ID NO: 1)
[0023] A portion of human chromosome 20q that is frequently amplified in breast cancer contains the gene for the nuclear coactivator AIBl (Amplified in breast cancer 1). The AIBl gene is amplified in five to ten percent of breast cancers and the abundance of the corresponding mRNA and protein is increased in up to 60% of breast tumors and also breast cancer cell lines. AIBl binds directly to estrogen receptor (ER) and this binding can be rate-limiting for estrogen-induced growth of MCF-7 cells. The overall role of AIBl for breast tumorigenesis is not clear, however, since AIBl potentiates not only the action of estrogen and progesterone receptors, but also that of various other nuclear receptors and transcription factors.
[0024] The ΑΙΒ1-Δ4 peptide (SEQ ID NO: 1) is a splice variant of AIBl in which the 4th exon has been spliced out and, unexpectedly, translation begins at the methionine at position 224 of the full length AIBl.
[0025] The terms "peptide," "polypeptide" and "protein" are used interchangeably herein. As used herein, an "isolated peptide" is intended to mean a peptide that has been completely or partially removed from its native environment. For example, peptides that have been removed or purified from cells are considered isolated. In addition, recombinantly produced peptide molecules contained in host cells are considered isolated for the purposes of the present invention. Moreover, a peptide that is found in a cell, tissue or matrix in which it is not normally expressed or found is also considered as "isolated" for the purposes of the present invention. Similarly, polypeptides that have been synthesized are considered to be isolated polypeptides. "Purified," on the other hand, is well understood in the art and generally means that the peptides are substantially free of cellular material, cellular components, chemical precursors or other chemicals beyond, perhaps, buffer or solvent. "Substantially free" is not intended to mean that other components beyond the novel peptides are undetectable. The peptides of the present invention may be isolated or purified.
[0026] It should be understood that the definition of peptides or polypeptides of the invention is intended to include polypeptides bearing modifications other than insertion, deletion, or su bstitution of amino acid residues. By way of example, the modifications may be covalent in nature, and include for example, chemical bonding with polymers, lipids, other organic and inorganic moieties. Such derivatives may be prepared to increase circulating half-life of a polypeptide, or may be designed to improve the targeting capacity of the polypeptide for desired cells, tissues or organs. Similarly, the invention further embraces ΑΙΒ1-Δ4 peptides that have been covalently modified to include one or more water-soluble polymer attachments such as polyethylene glycol, polyoxyethylene glycol or polypropylene glycol.
[0027] In one embodiment, the invention provides fusion proteins comprising at least a first and a second fusion peptide. The fusion partners are, generally speaking, covalently bonded to one another via a typical amine bond between the fusion peptides, thus creating one contiguous amino acid chain. In one specific embodiment, the first peptide of the fusion protein consists of a peptide with an amino acid sequence at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO: 1. Types of fusion proteins provided by the present invention include but are not limited to, fusions with secretion signals and other heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus or C-terminus of the protein to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
[0028] For example, in one embodiment of the fusion proteins of the present invention, a region may be added to facilitate purification. For example, "histidine tags" ("his tags") or "lysine tags" (the second fusion peptide) may be appended to the first fusion peptide. Examples of histidine tags include, but are not limited to hexaH, heptaH and hexaHN. Additional examples of purification tags are disclosed in Waugh, D.S., Trends in Biotechnology, 23(6):316-320 (June 2005), and Gaberc-Porekar V. and Menart, V., J. Biochem. Biophys. Methods. 49:335-360 (2001), which are incorporated by reference. Examples of lysine tags include, but are not limited to pentaL, heptaL and FLAG. Additional examples of solubility tags are also disclosed in Waugh, D.S., Trends in Biotechnology, 23(6) 316-320 (June 2005). Such regions may be removed prior to final preparation of the protein. Other examples of a second fusion peptide include, but are not limited to, glutathione S-transferase (GST) and alkaline phosphatase (AP).
[0029] The fusion proteins of the current invention can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose
chromatography, hydrophobic interaction chromatography, affinity chromatography, e.g., immobilized metal affinity chromatography (IMAC), hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography ("HPLC") may also be employed for purification. Well-known techniques for refolding protein may be employed to regenerate active conformation when the fusion protein is denatured during isolation and/or purification. [0030] Fusion proteins of the present invention include, but are not limited to, products of chemical synthetic procedures and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending u pon the host employed in a recombinant production proced ure, the fusion proteins of the present invention may be glycosylated or may be non-glycosylated. In addition, fusion proteins of the invention may also include an initial modified methionine residue, in some cases as a result of host- mediated processes.
[0031] The invention also relates to isolated nucleic acids and to constructs comprising these nucleic acids. The nucleic acids of the invention can be DNA or RNA, for example, mRNA. The nucleic acid molecules can be dou ble-stranded or single-stranded; single stranded RNA or DNA can be the coding, or sense, strand or the non-coding, or antisense, strand. In particular, the nucleic acids may encode any polypeptide of the invention, including, but not limited to, the fusion proteins of the present invention. For example, the nucleic acids of the invention include polynucleotide sequences that encode glutathione-S-transferase (GST) fusion protein, poly-histid ine (e.g., His6), poly-HN, poly-lysine, hemagglutinin, HSV-Tag and at least a portion of H IV-Tat. If desired, the nucleotide sequence of the isolated nucleic acid can include additional non-coding sequences such as non-coding 3' and 5' sequences (including regulatory sequences, for example).
[0032] The nucleic acid molecules of the invention can be "isolated." As used herein, a n "isolated" nucleic acid molecule or nucleotide sequence is intended to mea n a nucleic acid molecule or nucleotide sequence that is not flanked by nucleotide sequences normally flanking the gene or nucleotide sequence (as in genomic sequences) and/or has been completely or partially removed from its native environment (e.g., a cell, tissue). For example, nucleic acid molecules that have been removed or purified from cells are considered isolated. In some instances, the isolated material will form part of a composition (for example, a crude extract containing other su bstances), buffer system or reagent mix. In other circumstances, the material may be purified to near homogeneity, for example as determined by PAGE or column chromatography such as H PLC. Thus, an isolated nucleic acid molecule or nucleotide sequence can includes a nucleic acid molecule or nucleotide sequence which is synthesized chemically, using recombinant DNA technology or using any other suita ble method. To be clear, a nucleic acid contained in a vector wou ld be included in the definition of "isolated" as used herein. Also, isolated nucleotide sequences include recom binant nucleic acid molecules (e.g., DNA, RNA) in heterologous organisms, as well as partially or su bstantially purified nucleic acids in solution. "Purified," on the other hand is well understood in the art and generally means that the nucleic acid molecules are substantially free of cellular material, cellular components, chemical precursors or other chemicals beyond, perhaps, buffer or solvent. "Substantially free" is not intended to mean that other components beyond the novel nucleic acid molecules are undetecta ble. The nucleic acid molecules of the present invention may be isolated or purified. Both in vivo and in vitro NA transcripts of a DNA molecule of the present invention are also encompassed by "isolated" nucleotide sequences.
[0033] The invention also encompasses variations of the nucleotide sequences of the invention, such as those encoding functional fragments or variants of the polypeptides as described above. Such variants can be naturally-occurring, or non-naturally-occurring, such as those induced by various mutagens and mutagenic processes. Intended variations include, but are not limited to, addition, deletion and substitution of one or more nucleotides which can result in conservative or non- conservative amino acid changes, including additions and deletions.
[0034] The invention described herein also relates to fragments of the isolated nucleic acid molecules described herein. The term "fragment" is intended to encompass a portion of a nucleotide sequence described herein which is from at least about 20 contiguous nucleotides to at least about 50 contiguous nucleotides or longer in length. Such fragments may be useful as probes and primers. In particular, primers and probes may selectively hybridize to the nucleic acid molecule encoding the polypeptides described herein. For example, fragments which encode polypeptides that retain activity, as described below, are particularly useful.
[0035] The invention also provides nucleic acid molecules that hybridize under high stringency hybridization conditions, such as for selective hybridization, to the nucleotide sequences described herein (e.g., nucleic acid molecules which specifically hybridize to a nucleotide sequence encoding polypeptides described herein). Hybridization probes include synthetic oligonucleotides which bind in a base-specific manner to a complementary strand of nucleic acid. Suitable probes include polypeptide nucleic acids, as described in Nielsen et al., Science, 254:1497-1500 (1991).
[0036] Such nucleic acid molecules can be detected and/or isolated by specific hybridization e.g., under high stringency conditions. "Stringency conditions" for hybridization is a term of art that refers to the incubation and wash conditions, e.g., conditions of temperature and buffer concentration, which permit hybridization of a particular nucleic acid to a second nucleic acid; the first nucleic acid may be perfectly complementary, i.e., 100%, to the second, or the first and second may share some degree of complementarity, which is less than perfect, e.g., 60%, 75%, 85%, 95% or more. For example, certain high stringency conditions can be used which distinguish perfectly complementary nucleic acids from those of less complementarity.
[0037] "High stringency conditions", "moderate stringency conditions" and "low stringency conditions" for nucleic acid hybridizations are explained in Current Protocols in Molecular Biology, John Wiley & Sons, (1998)), which is incorporated by reference. The exact conditions which determine the stringency of hybridization depend not only on ionic strength, e.g.,
0.2 X SSC, 0.1 X SSC of the wash buffers, temperature, e.g., room temperature, 42°C, 68°C, etc., and the concentration of destabilizing agents such as formamide or denaturing agents such as SDS, but also on factors such as the length of the nucleic acid sequence, base composition, percent mismatch between hybridizing sequences and the frequency of occurrence of subsets of that sequence within other non- identical sequences. Thus, high, moderate or low stringency conditions may be determined empirically.
[0038] By varying hybridization conditions from a level of stringency at which no hybridization occurs to a level at which hybridization is first observed, conditions which will allow a given sequence to hybridize with the most similar sequences in the sample can be determined.
[0039] Exemplary conditions are described in Krause, M. H. and S. A. Aaronson, Methods in
Enzymology, 200:546-556 (1991), which is incorporated by reference. Washing is the step in which conditions are usually set so as to determine a minimum level of complementarity of the hybrids.
Generally, starting from the lowest temperature at which only homologous hybridization occurs, each degree (°C) by which the final wash temperature is reduced, while holding SSC concentration constant, allows an increase by 1% in the maximum extent of mismatching among the sequences that hybridize. Generally, doubling the concentration of SSC results in an increase in Tm. Using these guidelines, the washing temperature can be determined empirically for high, moderate or low stringency, depending on the level of mismatch sought. Exemplary high stringency conditions include, but are not limited to, hybridization in 50% formamide, 1 M NaCI, 1% SDS at 37°C, and a wash in 0.1 X SSC at 60°C. Example of progressively higher stringency conditions include, after hybridization, washing with 0.2 X SSC and 0.1% SDS at about room temperature (low stringency conditions); washing with 0.2 X SSC, and 0.1% SDS at about 42°C (moderate stringency conditions); and washing with 0.1 X SSC at about 68°C (high stringency conditions). Washing can be carried out using only one of these conditions, e.g., high stringency conditions, washing may encompass two or more of the stringency conditions in order of increasing stringency. Optimal conditions will vary, depending on the particular hybridization reaction involved, and can be determined empirically.
[0040] Equivalent conditions can be determined by varying one or more of the parameters given as an example, as known in the art, while maintaining a similar degree of identity or similarity between the target nucleic acid molecule and the primer or probe used. Hybridizable nucleotide sequences are useful as probes and primers for identification of organisms comprising a nucleic acid of the invention and/or to isolate a nucleic acid of the invention, for example. The term "primer" is used herein as it is in the art and refers to a single-stranded oligonucleotide which acts as a point of initiation of template- directed DNA synthesis under appropriate conditions in an appropriate buffer and at a suitable temperature. The appropriate length of a primer depends on the intended use of the primer, but typically ranges from about 15 to about 30 nucleotides. Short primer molecules generally require cooler temperatures to form sufficiently stable hybrid complexes with the template. A primer need not reflect the exact sequence of the template, but must be sufficiently complementary to hybridize with a template. The term "primer site" refers to the area of the target DNA to which a primer hybridizes. The term "primer pair" refers to a set of primers including a 5' (upstream) primer that hybridizes with the 5' end of the DNA sequence to be amplified and a 3' (downstream) primer that hybridizes with the complement of the 3' end of the sequence to be amplified.
[0041] The nucleic acids described herein can be amplified by methods known in the art. For example, amplification can be accomplished by the polymerase chain reaction (PCR). See PCR
Technology: Principles and Applications for DNA Amplification (ed. H. A. Erlich, Freeman Press, NY, N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (eds. Innis, et al., Academic Press, San Diego, Calif., 1990); Eckert et al., PCR Methods and Applications 1:17 (1991); PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202, all of which are incorporated by reference. Other suitable amplification methods include the ligase chain reaction (LCR) (see Wu and Wallace, Genomics, 4:560
(1989) , Landegren et al., Science, 241:1077 (1988), both of which are incorporated by reference), transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA, 86:1173 (1989), incorporated by reference), and self-sustained sequence replication (Guatelli et al., Proc. Nat. Acad. Sci. USA, 87:1874
(1990) incorporated by reference) and nucleic acid based sequence amplification (NASBA). [0042] The present invention also relates to vectors that include nucleic acid molecules of the present invention, host cells that are genetically engineered with vectors of the invention and the production of proteins of the invention by recombinant techniques.
[0043] In accordance with this aspect of the invention, the vector may be, for example, a plasmid vector, a single-or double-stranded phage vector, or a single-or double-stranded NA or DNA viral vector. Such vectors may be introduced into cells as polynucleotides, for example DNA, by well-known techniques for introducing DNA and RNA into cells. Viral vectors may be replication competent or replication defective. In the latter, case viral propagation generally will occur only in complementing host cells.
[0044] In certain respects, the vectors to be used are those for expression of polynucleotides and proteins of the present invention. Generally, such vectors comprise c/'s-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed. Appropriate transacting factors are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
[0045] A great variety of expression vectors can be used to express the proteins of the invention. Such vectors include chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as adeno-associated virus, lentivirus, baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. All may be used for expression in accordance with this aspect of the present invention. Generally, any vector suitable to maintain, propagate or express polynucleotides or proteins in a host may be used for expression in this regard.
[0046] The DNA sequence in the expression vector is operatively linked to appropriate expression control sequence(s) including, for instance, a promoter to direct mRNA transcription. Representatives of such promoters include, but are not limited to, the phage lambda PL promoter, the E. coli lac, trp and tac promoters, HIV promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name just a few of the well-known promoters. In general, expression constructs will contain sites for transcription, initiation and termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
[0047] In addition, the constructs may contain control regions that regulate, as well as engender expression. Generally, such regions will operate by controlling transcription, such as repressor binding sites and enhancers, among others.
[0048] Vectors for propagation and expression generally will include selecta ble markers. Such markers also may be suitable for amplification or the vectors may contain additional markers for this purpose. In this regard, the expression vectors may contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Preferred markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing E. coli and other bacteria.
[0049] The vector containing the appropriate DNA sequence, as well as an appropriate promoter, and other appropriate control sequences, may be introduced into an appropriate host using a variety of well-known techniques suitable to expression therein of a desired polypeptide. Representative examples of appropriate hosts include bacterial cells, such as, but not limited to, E. coli, Streptomyces, Bacillus, and Salmonella cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Hosts for of a great variety of expression constructs are well known, and those of skill in the art will be enabled by the present disclosure to select an appropriate host for expressing one of the proteins of the present invention.
[0050] Examples of vectors that may be useful for fusion proteins include, but are not limited to, pGEX (Pharmacia Biotech Inc; Smith and Johnson (1988) Gene 67, 31 40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione-S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. [0051] Examples of vectors for expression in yeast 5. cerevisiae include pYepSecl (Baldari, et al, (1987) EM BO J. 6, 229 234), pM Fa (Kurjan and Herskowitz (1982) Cell 30, 933 943), pJ Y88 (Schultz et al., (1987) Gene 54, 113 123), pYES2 (Invitrogen Corporation, San Diego, Calif., and picZ (InVitrogen Corp, San Diego, Calif.).
[0052] Alternatively, the ΑΙ Β1-Δ4 peptides can be expressed in insect cells using baculovirus expression vectors. Baculovirus vectors availa ble for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith et al, (1983) Mol. Cell. Biol. 3, 2156 2165) and the pVL series (Lucklow and Summers (1989) Virology 170, 31 39).
[0053] In yet another em bodiment, a nucleic acid of the invention is expressed in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed (1987) Nature 329, 840) and pMT2PC (Kaufman et al. (1987) EM BO J. 6, 187 195). When used in mammalian cells, the expression vector's control functions are often provided by viral regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus and Simian Virus 40. For other suita ble expression systems for both prokaryotic and eukaryotic cells, see, e.g., Chapters 16 and 17 of Sam brook et al., (Eds.) Molecular Cloning: A La boratory Manual. 2nd Ed., Cold Spring Harbor La boratory, Cold Spring Harbor La boratory Press, Cold Spring Harbor, N.Y., 1989 and later editions.
[0054] In another em bodiment, the recom binant ma mmalian expression vector is ca pable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suita ble tissue-specific promoters include liver-specific promoters (e.g., al bumin promoter), lymphoid-specific promoters such as, but not limited to, T cell receptors and immunoglobulins, neuron-specific promoters (e.g., neurofilament promoter), pancreas- specific promoters, mammary gland-specific promoters (e.g., milk whey promoter), bone-specific promoters (e.g., osteocalcin, osteopontin or bone sialoprotein, promoter regions), cartilage specific promoters (e.g., WARP) a nd muscle specific promoters (Desmin, myglobin, etc) just to name a few. Developmentally-regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss (1990) Science 249, 374 379) and the a-fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3, 537 546). [0055] The present invention also relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. The host cell can be stably or transiently transfected with the construct. The polynucleotides may be introduced alone or with other polynucleotides. Such other polynucleotides may be introduced independently, co- introduced or introduced joined to the polynucleotides of the invention. As used herein, a "host cell" is a cell that normally does not contain any of the nucleotides of the present invention and contains at least one copy of the nucleotides of the present invention. Thus, a host cell as used herein can be a cell in a culture setting or the host cell can be in an organism setting where the host cell is part of an organism, organ or tissue.
[0056] Suitable host cells for expression of the polypeptides of the invention include, but are not limited to, prokaryotes, yeast, and eukaryotes. If a prokaryotic expression vector is employed, then the appropriate host cell would be any prokaryotic cell capable of expressing the cloned sequences.
Suitable prokaryotic cells include, but are not limited to, bacteria of the genera Escherichia, Bacillus, Salmonella, Pseudomonas, Streptomyces and Staphylococcus.
[0057] If a eukaryotic expression vector is employed, then the appropriate host cell would be any eukaryotic cell capable of expressing the cloned sequence. In one embodiment, eukaryotic cells are cells of higher eukaryotes. Suitable eukaryotic cells include, but are not limited to, non-human mammalian tissue culture cells and human tissue culture cells. Other host cells include, but are not limited to, insect cells, HeLa cells, Chinese hamster ovary cells (CHO cells), African green monkey kidney cells (COS cells), human 293 cells, and murine 3T3 fibroblasts. Propagation of such cells in cell culture has become a routine procedure (see, Tissue Culture, Academic Press, Kruse and Patterson, Eds. (1973), which is incorporated herein by reference in its entirety).
[0058] In addition, a yeast cell may be employed as a host cell. Yeast cells include, but are not limited to, the genera Saccharomyces, Pichia and Kluveromyces. In one embodiment, the yeast hosts are 5. cerevisiae or P. pastoris. Yeast vectors may contain an origin of replication sequence from a 2T yeast plasmid, an autonomously replication sequence (A S), a promoter region, sequences for
polyadenylation, sequences for transcription termination and a selectable marker gene. Shuttle vectors for replication in both yeast and E. coli are also included herein. [0059] Alternatively, insect cells may be used as host cells. In one em bodiment, the polypeptides of the invention are expressed using a baculovirus expression system (see, Luckow et al., Bio/Technology, 1988, 6, 47; BACULOVIRUS EXPRESSION VECTORS: A LABORATORY MAN UAL, O'Rielly et al. (Eds.), W.H. Freeman and Company, New York, 1992; and U.S. Pat. No. 4,879,236, each of which is incorporated herein by reference). In addition, commercially availa ble complete baculovirus expression systems can, for example, be used for production in insect cells.
[0060] Suita ble host cells are d iscussed further in Goeddel, GENE EXPRESSION TECH NOLOGY:
M ETHODS I N ENZYMOLOGY 185, Academic Press, Sa n Diego, Calif. (1990). Alternatively, the recom binant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
[0061] Introduction of a construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation,
transduction, infection or other methods. Such methods are described in many standard la boratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986), incorporated by reference.
[0062] Vector DNA can be introd uced into prokaryotic or eukaryotic cells via conventional
transformation or transfection techniques. As used herein, the terms "transformation" and
"transfection" are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suita ble methods for transforming or transfecting host cells can be found in Sam brook, et al (MOLECU LAR CLONI NG: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor La boratory, Cold Spring Harbor La boratory Press, Cold Spring Harbor, N.Y., 1989), and other la boratory manuals.
[0063] For sta ble transfection of mammalian cells, it is known that, depending upon the expression vector and transfection technique used, only a small fraction of cells may integrate the foreign DNA into their genome. To identify and select these integrants, a gene that encodes a selecta ble marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest. Various selecta ble markers include those that confer resistance to d rugs, such as G418, hygromycin, dihydrofolate reductase (DHFR) and methotrexate. Nucleic acid encoding a selecta ble marker can be introduced into a host cell on the same vector as that encode the ΑΙ Β1-Δ4 peptide or can be introduced on a separate vector. Cells sta bly transfected with the introduced nucleic acid ca n be identified by drug selection (e.g., cells that have incorporated the selecta ble marker gene will survive, while the other cells die).
[0064] One em bodiment of the invention provides antibodies or antibody fragments that specifically bind to a peptide with an a mino acid sequence of SEQ ID NO: 1. Another aspect of the invention relates to detecting the presence of a peptide with an amino acid sequence of SEQ ID NO: 1.
[0065] In an additional em bodiment, the antibodies or antibody fragments of the present invention specifically bind to a peptide, wherein the polypeptide consists of an amino acid sequence at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to SEQ I D NO: 1.
[0066] A polypeptide having an amino acid sequence at least, for example, a bout 95% "identical" to a reference an amino acid sequence, e.g., SEQ ID NO: 1, is understood to mean that the amino acid sequence of the polypeptide is identical to the reference sequence except that the amino acid sequence may include up to a bout five modifications per each 100 amino acids of the reference amino acid sequence. In other words, to obtain a polypeptide having an amino acid sequence at least a bout 95% identical to a reference amino acid sequence, up to a bout 5% of the amino acid residues of the reference sequence may be deleted or su bstituted with another amino acid or a num ber of amino acids up to a bout 5% of the total amino acids in the reference sequence may be inserted into the reference sequence. These modifications of the reference sequence may occur at the N- terminus or C-terminus positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.
[0067] As used herein, "identity" is a measure of the identity of nucleotide sequences or amino acid sequences compared to a reference nucleotide or amino acid sequence. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using pu blished techniques. (See, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics And Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); von Heinje, G., Sequence Analysis In Molecular Biology, Academic Press (1987); and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York (1991)). While there are several methods to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known to skilled artisans (Carillo, H. & Lipton, D., Siam J Applied Math 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego (1994) and Carillo, H. & Lipton, D., Siam J Applied Math 48:1073 (1988). Computer programs may also contain methods and algorithms that calculate identity and similarity. Examples of computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCG program package (Devereux, J., et al., Nucleic Acids Research 12(i):387 (1984)), BLASTP, ExPASy, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)) and FASTDB. Examples of methods to determine identity and similarity are discussed in Michaels, G. and Garian, R., Current Protocols in Protein Science, Vol 1, John Wiley & Sons, Inc. (2000), which is incorporated by reference.
[0068] In one embodiment of the present invention, the algorithm used to determine identity between two or more polypeptides is BLASTP. In another embodiment of the present invention, the algorithm used to determine identity between two or more polypeptides is FASTDB, which is based upon the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990), incorporated by reference). In a FASTDB sequence alignment, the query and reference sequences are amino sequences. The result of sequence alignment is in percent identity. In one embodiment, parameters that may be used in a FASTDB alignment of amino acid sequences to calculate percent identity include, but are not limited to: Matrix=PAM, k-tuple=2, Mismatch Penalty=l, Joining Penalty=20, Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty 0.05, Window Size=500 or the length of the subject amino sequence, whichever is shorter.
[0069] If the reference sequence is shorter or longer than the query sequence because of N-terminus or C-terminus additions or deletions, but not because of internal additions or deletions, a manual correction can be made, because the FASTDB program does not account for N-terminus and C-terminus truncations or additions of the reference sequence when calculating percent identity. For query sequences truncated at the N- or C- termini, relative to the reference sequence, the percent identity is corrected by calculating the number of residues of the query sequence that are N-and C- terminus to the reference sequence that are not matched/aligned, as a percent of the total bases of the query sequence. The results of the FASTDB sequence alignment determine matching/alignment. The alignment percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score can be used for the purposes of determining how alignments "correspond" to each other, as well as percentage identity. Residues of the reference sequence that extend past the N- or C-termini of the query sequence may be considered for the purposes of manually adjusting the percent identity score. That is, residues that are not matched/aligned with the N- or C-termini of the comparison sequence may be counted when manually adjusting the percent identity score or alignment numbering.
[0070] For example, a 90 amino acid residue query sequence is aligned with a 100 residue reference sequence to determine percent identity. The deletion occurs at the N-terminus of the query sequence and therefore, the FASTDB alignment does not show a match/alignment of the first 10 residues at the N- terminus. The 10 unpaired residues represent 10% of the reference sequence (number of residues at the N- and C-termini not matched/total number of residues in the reference sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched (100% alignment) the final percent identity would be 90% (100% alignment - 10% unmatched overhang). In another example, a 90 residue query sequence is compared with a 100 reference sequence, except that the deletions are internal deletions. In this case the percent identity calculated by FASTDB is not manually corrected, since there are no residues at the N- or C- termini of the subject sequence that are not matched/aligned with the query. In still another example, a 110 amino acid query sequence is aligned with a 100 residue reference sequence to determine percent identity. The addition in the query occurs at the N-terminus of the query sequence and therefore, the FASTDB alignment may not show a match/alignment of the first 10 residues at the N-terminus. If the remaining 100 amino acid residues of the query sequence have 95% identity to the entire length of the reference sequence, the N-terminal addition of the query would be ignored and the percent identity of the query to the reference sequence would be 95%.
[0071] The ΑΙΒ1-Δ4 peptides or fragments thereof or other derivatives, or analogs thereof, or cells expressing them can be used as an immunogen to produce antibodies or antibody fragments thereof. Any of the antibodies can be, for example, polyclonal, monoclonal, bi-specific, multispecific, human or chimeric antibodies. The antibody molecules of the invention can be of any type, e.g., IgG, IgE, IgM, IgD, IgA and IgY, class, e.g., IgGl, lgG2, lgG3, lgG4, IgAl and lgA2 or subclass of immunoglobulin molecule. In one embodiment, an antibody of the invention comprises, or alternatively consists of, a polypeptide having an amino acid sequence of a VH domain, at least one VH CDR, a VL domain, or at least one VL CDR. [0072] The antibodies or antibody fragments of the present invention may be monovalent, bivalent, trivalent or multivalent. For example, monovalent scFvs can be multimerized either chemically or by association with another protein or substance. An scFv that is fused to a hexahistidine tag or a Flag tag can be multimerized using Ni-NTA agarose (Qiagen) or using anti-Flag antibodies (Stratagene, Inc.).
[0073] The antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of an ΑΙΒ1-Δ4 peptide, or a domain thereof, or may be specific for both an ΑΙΒ1-Δ4 peptide, or a doman thereof, and a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al, J. Immunol. 147:60 69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al, J.
Immunol. 148:1547-1553 (1992), which are incorporated by reference.
[0074] As used herein, an antibody fragment is a fragment of an antibody capable of specifically binding the same epitope that the intact antibody would bind. Examples of antibody fragments include but are not limited to Fab and F(ab')2 fragments, Fd fragments, disulfide-linked Fvs (sd Fvs), antiidiotypic (anti-Id) antibodies, including but not limited to anti-Id antibodies to antibodies of the invention, and epitope- binding fragments of any of the above. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody and generally clear more rapidly from the circulation, and may have less non-specific tissue binding than that of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Other types of antibody fragments include but are not limited to single chain Fv fragments (scFv) that are well-known in the art. Techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) can be adapted to produce single chain antibodies to immunogenic polypeptide products of this invention.
[0075] The antibodies or fragments of the present invention may be prepared by any of a variety of methods. For example, cells expressing ΑΙΒ1-Δ4 peptide or an antigenic fragment thereof can be administered to an animal to induce the production of sera containing polyclonal antibodies. In one method, a preparation of ΑΙΒ1-Δ4 peptide is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
[0076] Accordingly, one aspect of the invention provides a method for making ΑΙΒ1-Δ4 peptide-specific antibodies. [0077] Polyclonal antibodies of the invention may be produced according to standard techniques by immunizing a suitable animal, e.g., rabbit, goat, etc., with an antigen comprising an antigenic portion of the ΑΙΒ1-Δ4 peptide. Collecting immune serum from the animal and separating the polyclonal antibodies from the immune serum can be carried out in accordance with known procedures, and screening and isolating a polyclonal antibody specific for ΑΙΒ1-Δ4 peptide can be carried out with well- known procedures and as described below. Methods for immunizing non-human animals such as mice, rats, sheep, goats, pigs, cattle and horses are well known in the art. See, e.g., Harlow and Lane,
Antibodies: A Laboratory Manual, New York: Cold Spring Harbor Press, 1990, which is incorporated by reference.
[0078] The immunogen may be the full length protein or a peptide comprising an ΑΙΒ1-Δ4 peptide portion of interest. In some embodiments the immunogen is a peptide of from 7 to 20 amino acids in length, in particular from about 8 to 17 amino acids in length. In some embodiments, the peptide antigen desirably will comprise about 3 to 8 amino acids . Peptide antigens suitable for producing antibodies of the invention may be designed, constructed and employed in accordance with well-known techniques. See, e.g., Antibodies: A Laboratory Manual, Chapter 5, p. 75-76, Harlow & Lane Eds., Cold Spring Harbor Laboratory (1988); Czemik, Methods in Enzymology, 201: 264-283 (1991); Merrifield, J. Am. Chem. Soc. 85: 21-49 (1962), which are incorporated by reference.
[0079] In some embodiments the immunogen is administered with an adjuvant. Suitable adjuvants will be well known to those of skill in the art. Exemplary adjuvants include complete or incomplete Freund's adjuvant, IBI (muramyl dipeptides) or ISCOM (immunostimulating complexes).
[0080] When the above-described methods are used for producing polyclonal antibodies, following immunization, the polyclonal antibodies which secreted into the bloodstream can be recovered using known techniques. Purified forms of these antibodies can, of course, be readily prepared by standard purification techniques, such as for example, affinity chromatography with Protein A, antiimmunoglobulin, or the antigen itself. In any case, to monitor the success of immunization, the antibody levels with respect to the antigen in serum can be monitored using standard techniques such as ELISA, RIA and the like.
[0081] In one aspect of the present invention, the antibodies or fragments thereof of the present invention are monoclonal antibodies. Such monoclonal antibodies can be prepared using hybridoma technology (Kohler et al., Nature 256:495 (1975); Kohler et al., Eur. J. Immunol. 6:511 (1976); Kohler et al., Eur. J. Immunol. 6:292 (1976); Hammerling et al., In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., (1981) pp. 563-681). In general, such procedures involve immunizing an animal (for example a mouse) with an ΑΙΒ1-Δ4 peptide antigen or with a ΑΙΒ1-Δ4 peptide-expressing cell. Suitable cells can be recognized by their capacity to bind anti-AIBl-A4 peptide antibody. Such cells may be cultured in any suitable tissue culture medium; however, it is desirable to culture cells in Earl's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56°C), and supplemented with about 10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 μg/ml of streptomycin. The splenocytes of such mice are extracted and fused with a suitable myeloma cell line. Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is may be desirable to employ the parent myeloma cell line (SP20), available from the American Type Culture Collection, ockville, Md. After fusion, the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding ΑΙΒ1-Δ4 peptide antigen. The secreted antibody may be recovered from tissue culture supernatant by conventional methods such as precipitation, ion exchange or affinity chromatography, or the like. Other methods of generating monclonal antibodies include but are not limited to the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today 4:72), and the EBV-hybridoma technique to produce human monoclonal antibodies (Cole, et al., 1985, in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
[0082] Alternatively, additional antibodies capable of binding to ΑΙΒ1-Δ4 peptide antigen may be produced in a two-step procedure through the use of anti-idiotypic antibodies. Such a method makes use of the fact that antibodies are themselves antigens, thus it is possible to obtain an antibody which binds to a second antibody. In accordance with this method, ΑΙΒ1-Δ4 peptide specific antibodies are used to immunize an animal, for example a mouse. The splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to ΑΙΒ1-Δ4 peptide-specific antibody can be blocked by an ΑΙΒ1-Δ4 peptide antigen, respectively. Such antibodies comprise anti-idiotypic antibodies to ΑΙΒ1-Δ4 peptide- specific antibody and can be used to immunize an animal to induce formation of ΑΙΒ1-Δ4 peptide- specific antibodies. [0083] The invention also encompasses antibody-producing cells and cell lines, such as hybridomas, as described above.
[0084] Polyclonal or monoclonal antibodies may also be obtained through in vitro immunization. For example, phage display techniques can be used to provide libraries containing a repertoire of antibodies with varying affinities for a particular antigen. Techniques for the identification of high affinity human antibodies from such libraries are described by Griffiths et al., EMBO J., 13:3245-3260 (1994); which is incorporated by reference.
[0085] The antibodies may be produced recombinantly using methods well known in the art for example, according to the methods disclosed in U.S. Pat. No. 4,349,893 or U.S. Pat. No. 4,816,567, which are incorporated by reference. The antibodies may also be chemically constructed by specific antibodies made according to the method disclosed in U.S. Pat. No. 4,676,980, which is incorporated by reference..
[0086] Once a desired ΑΙΒ1-Δ4 peptide antibody is identified, polynucleotides encoding the antibody, such as heavy, light chains or both (or single chains in the case of a single chain antibody) or portions thereof such as those encoding the variable region, may be cloned and isolated from antibody- producing cells using means that are well known in the art. For example, the antigen combining site of the monoclonal antibody can be cloned by PCR and single-chain antibodies produced as phage-displayed recombinant antibodies or soluble antibodies in E. coli. See, e.g., Antibody Engineering Protocols, Humana Press, Sudhir Paul, Ed. (1995), which is incorporated by reference.
[0087] Accordingly, in a further aspect, the invention provides such nucleic acids encoding the heavy chain, the light chain, a variable region, a framework region or a CDR of an antibody of the invention. In some embodiments, the nucleic acids are operably linked to expression control sequences. The invention, thus, also provides vectors and expression control sequences useful for the recombinant expression of an antibody or antigen-binding portion thereof of the invention. Those of skill in the art will be able to choose vectors and expression systems that are suitable for the host cell in which the antibody or antigen-binding portion is to be expressed.
[0088] In one embodiment, a nucleic acid molecule of the invention encodes an antibody comprising, or alternatively consisting of, a VH domain having an amino acid sequence of any one of the VH domains of the antibodies described herein. In another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VH CDR1 having an amino acid sequence of any of the antibodies described herein. In another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VH CD 2 having an amino acid sequence of any one of the VH CDR2 of any of the antibodies described herein. In yet another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VH CDR3 having an amino acid sequence of any of the antibodies described herein. Nucleic acid molecules encoding antibodies that immunospecifically bind ΑΙΒ1-Δ4 peptide and comprise, or alternatively consist of, fragments or variants of the VH domains and/or VH CDRs are also encompassed by the invention.
[0089] In another embodiment, a nucleic acid molecule of the invention encodes an antibody, including molecules comprising, or alternatively consisting of, antibody fragments or variants thereof, comprising, or alternatively consisting of, a VL domain having an amino acid sequence of any one of the VL, domains of any of the antibodies described herein. In another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VL CDR1 having amino acid sequence of any one of the any of the antibodies described herein. In another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VL CDR2 having an amino acid sequence of any one of the VL CDR2 of any of the antibodies described herein. In yet another embodiment, a nucleic acid molecule of the present invention encodes an antibody comprising, or alternatively consisting of, a VL CDR3 having an amino acid sequence of any, one of the VL CDR3 of any of the antibodies described herein. Nucleic acid encoding antibodies that immunospecifically bind ΑΙΒ1-Δ4 peptide and comprise, or alternatively consist of, fragments or variants of the VL domains and/or VLCDR(s) are also encompassed by the invention.
[0090] In another embodiment, a nucleic acid molecule of the invention encodes an antibody comprising, or alternatively consisting of, a VH domain having an amino acid sequence of any one of the VH domains of any of the antibodies described herein, and a VL domain having an amino acid sequence of any one of the VL domains of any of the antibodies described herein. In another embodiment, a nucleic acid molecule of the invention encodes an antibody comprising, or alternatively consisting of, a VH CDR1, a VL CDR1, a VH CDR2, a VL CDR2, a VH CDR3, a VL CDR3, or any combination thereof having an amino acid sequence of any of the antibodies described herein. Nucleic acid encoding antibodies that immunospecifically bind ΑΙΒ1-Δ4 peptide and comprise, or alternatively consist of, fragments or variants of the VL and/or domains and/or VHCDR(s) and/or VLCDR(s) are also encompassed by the invention. [0091] The present invention also provides antibodies that comprise, or alternatively consist of, variants, including derivatives, of the VH domains, VH CD s, VL domains, and VL CDRs described herein, which antibodies immunospecifically bind to ΑΙΒ1-Δ4 peptide. Standard techniques known to those of skill in the art can be used to introduce mutations in the nucleotide sequence encoding a molecule of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis which result in amino acid substitutions. In select embodiment, the variants, including derivatives, encode less than 50 amino acid substitutions, less than 40 amino acid substitutions, less than 30 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions relative to the reference VH domain, VHCDR1, VHCDR2, VHCDR3, VL domain, VLCDR1, VLCDR2, or VLCDR3. In specific embodiments, the variants encode substitutions of VHCDR3. In a preferred embodiment, the variants have conservative amino acid substitutions at one or more predicted non-essential amino acid residues. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a side chain with a similar charge. Conservative substitutions are shown in the Tables below. Alternatively, mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity, e.g., the ability to bind ΑΙΒ1-Δ4 peptide. Following mutagenesis, the encoded protein may routinely be expressed and the functional and/or biological activity of the encoded protein, e.g., ability to immunospecifically bind AIB1- Δ4 peptide, can be determined using techniques described herein or by routinely modifying techniques known in the art.
Table I - Conservative Substitutions
SIDE CHAIN CHARACTERISTIC AMINO ACID
Aliphatic
Non-polar G A P
1 L V
Polar - uncharged C S T M
N Q
Polar - charged D E
K R
Aromatic H F w Y
Other N Q D E
[0092] Alternatively, conservative amino acids can be grouped as described in Lehninger,
[Biochemsitry, Second Edition; Worth Publishers, Inc. NY, N.Y. (1975), pp. 71 77] as set out below.
Table II - Conservative Substitutions
SIDE CHAIN CHARACTERISTIC AMINO ACID
Non-polar (hydrophobic)
A. Aliphatic: V
B. Aromatic: F W
C. Sulfur-containing: M
D. Borderline: G Uncharged-polar
A. Hydroxyl:
B. Amides: N
C. Sylfhydryl: C
D. Borderline: G Positively Charged (Basic):
Negativelv Charged (Acidic)
[0093] And still other alternative, exemplary conservative substitutions are set out below. Table III - Conservative Substitutions
Original Residue Exemplary Substitution
Ala (A) Val, Leu, lie
Arg (R) Lys, Gin, Asn
Asn (N) Gin, His, Lys, Arg
Asp (D) Glu
Cys (C) Ser
Gin (Q) Asn
Glu (E) Asp
His (H) Asn, Gin, Lys, Arg
He (1) Leu, Val, Met, Ala, Phe
Leu (L) lie, Val, Met, Ala, Phe
Lys (K) Arg, Gin, Asn
Met (M) Leu, Phe, lie
Phe (F) Leu, Val, lie, Ala
Pro (P) Gly
Ser (S) Thr
Thr (T) Ser
Trp (W) Tyr Tyr (Y) Trp, Phe, Thr, Ser
Val (V) lie, Leu, Met, Phe, Ala
[0094] The antibodies of the invention include derivatives or variants that are modified, e.g., by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not affect the ability of the antibody to immunospecifically bind ΑΙΒ1-Δ4 peptide. For example, but not by way of limitation, derivatives of the invention include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known
protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not, limited to, specific chemical cleavage, acetylation, formulation, metabolic synthesis of tunicamycin, etc.
Additionally, the derivative may contain one or more non-classical amino acids.
[0095] In a specific embodiment, an antibody or antibody fragment of the invention, including a molecule comprising, or alternatively consisting of, an antibody fragment or variant thereof, that immunospecifically binds ΑΙΒ1-Δ4 peptide, comprises, or alternatively consists of, an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleotide sequence that is complementary to that encoding one of the VH or VL domains of any of the antibodies described herein under stringent conditions, e.g., hybridization to filter-bound DNA in 6X sodium chloride/sodium citrate (SSQ at about 45°C. followed by one or more washes in 0.2XSSC/0.1% SDS at about 50°-65° C, under highly stringent conditions, e.g., hybridization to filter-bound nucleic acid in 6XSSC at about 45°C, followed by one or more washes in O.lXSSC/0.2% SDS at a bout 68°C, or under other stringent hybridization conditions which are known to those of skill in the art.
[0096] In another embodiment, an antibody or antibody fragment of the invention that
immunospecifically binds to ΑΙΒ1-Δ4 peptide, comprises, or alternatively consists of, an amino acid sequence encoded by a nucleotide sequence that hybridizes to a nucleotide sequence that is complementary to that encoding one of the VH CD s or VL CDRs of any of the antibodies described herein under stringent conditions, e.g., hybridization under conditions as described above, or under other stringent hybridization conditions which are known to those of skill in the art. [0097] In another embodiment, an antibody of antibody fragment that immunospecifically binds to ΑΙΒ1-Δ4 peptide, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VH domains of any of the antibodies described herein. In another embodiment, the invention provides an antibody or antibody fragment of the invention that immunospecifically binds to ΑΙΒ1-Δ4 peptide comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VH CD s of any of the antibodies described herein. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
[0098] In another embodiment, an antibody or antibody fragment of the invention that
immunospecifically binds to ΑΙΒ1-Δ4 peptide comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VL domains of any of the antibodies described herein. In another embodiment, the invention provides an antibody of the invention that immunospecifically binds to ΑΙΒ1-Δ4 peptide comprises, or alternatively consists of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to any one of the VL CDRs of any of the antibodies described herein.
[0099] In yet another embodiment, the invention provides antibodies or antibody fragments that immunospecifically binds to ΑΙΒ1-Δ4 peptide comprising, or alternatively consisting of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to at least one, two or three of the VH CDRs of any of the antibodies described herein and that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to at least one, two or three of the VL CDRs of any of the antibodies described herein. Of course, the invention also provides antibodies or antibody fragments that immunospecifically binds to ΑΙΒ1-Δ4 peptide comprising, or alternatively consisting of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to at least two or three of the VH CDRs of any of the antibodies described herein and that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to at least two or three of the VL CDRs of any of the antibodies described herein. The invention also provides antibodies or antibody fragments that immunospecifically binds to ΑΙΒ1-Δ4 peptide comprising, or alternatively consisting of, a polypeptide having an amino acid sequence that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical, to three of the VH CDRs of any of the antibodies described herein and that is at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identical to three of the VL CDRs of any of the antibodies described herein. Nucleic acid molecules encoding these antibodies are also encompassed by the invention.
[00100] Antibodies or fragments of the present invention may also be described or specified in terms of their binding affinity for ΑΙΒ1-Δ4 peptide or domains or variants of ΑΙΒ1-Δ4 peptide.
[00101] In specific embodiments, antibodies or fragments of the invention bind ΑΙΒ1-Δ4 peptide or domains or variants thereof, with a dissociation constant or Kd of less than or equal to 5xlO"2M, 10"2 M, 5xlO~3M, 10~3 M, 5xlO"4M, 10"4 M, 5xlO"5M, 10 s M, 5xlO"6M, 10"6 M, 5xlO"7M, 10"7 M, 5xlO"8M, 10 s M, 5xlO"9M, 10"9 M, 5x10 10M, 10 10 M, 5x10 "iVI, 10 11 M, 5x10 12M, 10 12 M, 5x10 13M, 10 13 M, 5x10 14M, 10" 14 M, 5x10 15M or 10 15 M.
[00102] Monoclonal Fab fragments may also be produced in Escherichia coli by recombinant techniques known to those skilled in the art. See, e.g., W. Huse, Science 246: 1275-81 (1989); Mullinax et al., Proc. Nat'l. Acad. Sci. 87: 8095 (1990).
[00103] If monoclonal antibodies of a single desired isotype are preferred for a particular application, particular isotypes can be prepared directly, by selecting from the initial fusion, or prepared secondarily, from a parental hybridoma secreting a monoclonal antibody of different isotype by using the sib selection technique to isolate class-switch variants (Steplewski, et al., Proc. Nat'l. Acad. Sci., 82: 8653 (1985); Spira et al., J. Immunol. Methods, 74: 307 (1984)). Alternatively, the isotype of a monoclonal antibody with desira ble propertied can be changed using antibody engineering techniques that are well- known in the art.
[00104] ΑΙΒ1-Δ4 peptide-specific antibodies of the invention, whether polyclonal or monoclonal, may be screened for epitope specificity according to standard techniques. See, e.g., Czernik et al., Methods in Enzymology, 201: 264-283 (1991). For example, the antibodies may be screened against ΑΙ Β1-Δ4 peptide library by ELISA to ensure specificity for both the desired antigen. The a ntibodies may also be tested by Western blotting against cell preparations containing the parent signaling protein, e.g., cell lines over-expressing the parent protein, to confirm reactivity with the desired epitope/target.
[00105] Antibodies may be further characterized via immunohistochemical (IHC) staining using normal and diseased tissues to examine levels of ΑΙΒ1-Δ4 peptide in diseased tissue. I HC may be carried out according to well-known techniques. See, e.g., Antibodies: A La boratory Manual, Chapter 10, Harlow & Lane Eds., Cold Spring Harbor La boratory (1988). Briefly, paraffin-em bedded tissue, e.g., tumor tissue, is prepared for immunohistochemical staining by deparaffinizing tissue sections with xylene followed by ethanol; hydrating in water then PBS; u nmasking antigen by heating slide in sodium citrate buffer; incu bating sections in hydrogen peroxide; blocking in blocking solution; incu bating slide in primary antibody and secondary antibody; a nd finally detecting using ABC avidin/biotin method according to manufacturer's instructions.
[00106] Antibodies may be further characterized by flow cytometry carried out according to standard methods. See Chow et al., Cytometry, Communications in Clinical Cytometry 46: 72-78 (2001). Briefly and by way of example, the following protocol for cytometric analysis may be employed : samples may be centrifuged on Ficoll gradients to remove lysed erythrocytes and cell debris. Adhering cells may be scraped off plates and washed with PBS. Cells may then be fixed with 2% paraformaldehyde for 10 minutes at 37°C, followed by permea bilization in 90% methanol for 30 minutes on ice. Cells may then be stained with the primary ΑΙΒ1-Δ4 peptide-specific antibody of the invention, which can detect an ΑΙ Β1-Δ4 peptide, washed and la beled with a fluorescent-la beled secondary antibody. Additional fluorochrome-conjugated marker antibodies, e.g., CD45, CD34, may also be added at this time to aid in the su bsequent identification of specific hematopoietic cell types. The cells would then be analyzed on a flow cytometer, e.g., a Beckman Coulter FC500, according to the specific protocols of the instrument used. [00107] Antibodies of the invention may also be conjugated to fluorescent dyes, e.g., Alexa488, PE, etc., for use in multi-parametric analyses along with other signal transduction, e.g., phospho-CrkL, phospho- Erk 1/2, and/or cell marker, e.g., CD34 antibodies.
[00108] Methods for making bispecific antibodies are within the purview of those skilled in the a rt. Traditionally, the recom binant prod uction of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983)). Antibody varia ble domains with the desired binding specificities (antibody-antigen com bining sites) can be fused to immunoglobulin constant domain sequences. In certain em bodiments, the fusion is with an immunoglobulin heavy-chain constant domain, includ ing at least part of the hinge, CH2, a nd CH3 regions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suita ble host organism. For fu rther details of illustrative currently known methods for generating bispecific antibodies see, for example, Suresh et al., Methods in Enzymology, 121:210 (1986); WO 96127011; Brennan et al., Science 229:81 (1985); Shala by et ai, J. Exp. Med. 175:217-225 (1992); Kostelny et al. . Immunol. 148(5):1547-1553 (1992); Hollinger ei al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Gru ber et al., J. Immunol. 152:5368 (1994); and Tutt et al., J. Immunol. 147:60 (1991). Bispecific antibodies also include cross-linked or heteroconjugate antibodies. Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suita ble cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
[00109] Various techniques for making a nd isolating bispecific antibody fragments directly from recom binant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins may be linked to the Fa b' portions of two different antibodies by gene fusion. The antibody homodimers may be reduced at the hinge region to form monomers a nd then re-oxidized to form the antibody heterodimers. This method can also be utilized for the production of antibody homodimers. A strategy for making bispecific antibody fragments by the use of single-chain Fv (scFv) dimers has also been reported. See Gru ber at al., J. Immunol., 152:5368 (1994). Alternatively, the antibodies ca n be "linear antibodies" as described in Zapata et al. Protein Eng. 8(10): 1057-1062 (1995). Briefly, these antibodies comprise a pair of tandem Fd segments (VH-CHi-VH- CHi) which form a pair of a ntigen binding regions. Linear antibodies can be bispecific or monospecific. To produce the chimeric antibodies, the portions derived from two different species, e.g., human constant region and murine varia ble or binding region can be joined together chemically by
conventional techniques or can be prepared as single contiguous proteins using genetic engineering techniques. The DNA molecules encoding the proteins of both the light chain and heavy chain portions of the chimeric antibody ca n be expressed as contiguous proteins. The method of making chimeric antibodies is disclosed in U.S. Pat. No. 5,677,427; U.S. Pat. No. 6, 120,767; and U.S. Pat. No. 6,329,508 which are incorporated by reference.
[00110] Fully human antibodies may be produced by a variety of techniques. One example is trioma methodology. The basic approach and an exemplary cell fusion partner, SPAZ-4, for use in this approach have been described by Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; and U.S. Pat. No. 4,634,666, which are incorporated by reference.
[00111] Hu man antibodies can also be produced from non-human transgenic animals having transgenes encoding at least a segment of the hu man immunoglobulin locus. The production and properties of animals having these properties are described in detail by, see, e.g., W093/12227; U.S. Pat. No.
5,545,806, WO91/10741 and U.S. Pat. No. 6, 150,584, which are herein incorporated by reference.
[00112] Various recom binant antibody library technologies may also be utilized to produce fully human antibodies. For example, one approach is to screen a DNA library from human B cells according to the general protocol outlined by Huse et al., Science 246:1275-1281 (1989). The protocol described by Huse is rendered more efficient in combination with phage-display technology. See, WO 91/17271, WO 92/01047 and U.S. Pat. No. 5,969, 108, which are incorporated by reference.
[00113] Eukaryotic ribosome can also be used as means to display a library of antibodies and isolate the binding human antibodies by screening against the target antigen, as described in Coia G, et al., J.
Immunol. Methods 1: 254 (l-2):191-7 (2001); Hanes J. et al., Nat. Biotechnol. 18(12):1287-92 (2000); Proc. Natl. Acad. Sci. U.S.A. 95(24):14130-5 (1998); Proc. Natl. Acad. Sci. U.S.A. 94(10):4937-42 (1997), which are incorporated by reference.
[00114] The yeast system is also suita ble for screening mammalian cell-surface or secreted proteins, such as antibodies. Antibody libraries may be displayed on the surface of yeast cells for the purpose of obtaining the human antibodies against a target antigen. This approach is described by Yeung, et al., Biotechnol. Prog. 18(2):212-20 (2002); Boeder, E. T., et al., Nat. Biotechnol. 15(6):553-7 (1997), which are incorporated by reference. Alternatively, human antibody libraries may be expressed intracellularly and screened via the yeast two-hybrid system. See WO0200729A2, which is incorporated by reference.
[00115] Recom binant DNA techniques can be used to produce the recom binant ΑΙΒ1-Δ4 peptide-specific antibodies described herein, as well as the chimeric or huma nized ΑΙ Β1-Δ4 peptide-specific antibodies, or any other genetically-altered antibod ies and the fragments or conjugate thereof in any expression systems including both prokaryotic and eukaryotic expression systems, such as bacteria, yeast, insect cells, plant cells, mammalian cells (for example, NSO cells).
[00116] Once produced, the whole antibodies, their dimers, individual light and heavy chains, or other immunoglobulin forms of the present application can be purified according to standard procedures of the art, including a mmonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like. Once purified, partially or to the desired levels of homogeneity, the polypeptides may then be used for performing assay procedures, immu nofluorescent staining, and the like.
[00117] It will be appreciated that Fa b and F(a b')2 and other fragments of the antibodies of the present invention may be used according to the methods disclosed herein. Such fragments are typically produced by proteolytic cleavage, using enzymes such as papain (to produce Fa b fragments) or pepsin (to produce F(a b')2 fragments). Alternatively, ΑΙ Β1-Δ4 peptide-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.
[00118] "Humanized" chimeric antibodies can also be used. Such antibodies can be produced using genetic constructs derived from hybridoma cells prod ucing the monoclonal antibodies described a bove. Methods for producing chimeric antibodies are known in the art. See, for review, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Ca billy et al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neu berger et al., WO 8601533; Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984); Neu berger et al., Nature 314:268 (1985).
[00119] The antibodies and methods described herein can be used to measure levels ΑΙ Β1-Δ4 peptide The measurement of the levels of peptide may be qualitative or quantitative. For example, the levels peptide may be quantified is some numerical expression, such as a ratio or a percentage. [00120] Once the levels of peptide have been determined, this determination can then be compared to normal levels or baseline levels of the peptide. "Normal levels" of the peptide may be assessed by measuring levels of the peptide in a known healthy su bject, including the same su bject that is later screened or being diagnosed. Normal levels may also be assessed over a population sample, where a population sample is intended to mea n either multiple samples from a single su bject or at least one sample from a multitude of su bjects. The samples used to generate the population can be ta ken from previously harvested tissues that, for exa mple, may be stored in paraffin or cryogenically stored. The population of samples can continually grow as additional samples are added to the population to gain statistical confidence in the data. Normal levels of the peptide, in terms of a population of samples, may or may not be categorized according to characteristics of the population including, but not limited to, sex, age, weight, ethnicity, geographic location, fasting state, state of pregnancy or post- pregnancy, menstrual cycle, general health of the su bject, alcohol or drug consumption, caffeine or nicotine intake and circadian rhythms.
[00121] Similarly, "baseline levels" are determined by measuring levels of the peptide in a su bject where the condition is minimally aggressive or is considered to be benign. For exa mple, baseline levels of the peptide can be esta blished from a population of su bjects that have, for example, cancer or tumors, but the cancer has been graded as not aggressive and/or well differentiated. For example, the American Joint Commission on Cancer has developed the following guidelines for grading tumors and are believed to be current as of the filing date of the application:
Grade
GX Grade cannot be assessed (Undetermined grade)
Gl Well-differentiated (Low grade)
G2 Moderately differentiated (Intermediate grade)
G3 Poorly differentiated (High grade)
G4 Undifferentiated (High grade)
[00122] For the purposes of the present invention, baseline levels of the peptide may be esta blished in GX, Gl, G2, G3 and/or G4 graded tumors. Of course, other means of stratifying tu mors may also be used, for example the Bloom-Richardson system in breast cancer, and these other means for stratifying tumors can be used to esta blish baseline levels of the ΑΙ Β1-Δ4 peptide. [00123] A difference between normal/baseline levels and the measured levels of the peptide may indicate that the su bject has a disease or a bnormal condition or has a higher (or lower) proba bility of developing a disease or a bnormal condition than do normal su bjects or su bjects within the range of baseline levels of the peptide. Alternatively, or in addition to, differences between normal/baseline levels and the measured levels of the ΑΙ Β1-Δ4 peptide may indicate that the su bject has a more or less severe or aggressive form of the disease or a bnorma l condition compared to others. In addition, the magnitude of difference between measured levels and normal/baseline levels of the peptide may also indicate the severity of disease or a bnormal condition or the level of proba bility of developing a disease or a bnormal condition, compared to normal su bjects or su bjects within the range of baseline levels of the peptide, such as the likelihood of metastasis.
[00124] The difference between measured levels of the peptide and normal/baseline levels may be a relative or a bsolute qua ntity. "Levels of peptide" is used to mean any measure of the quantity of the peptide such as, but not limited to, mass, concentration and biological activity. Example of biological activities that may be used to quantify biomarkers include, but are not limited to, chemotactic, cytotoxic, enzymatic or other biological activities, such as quantifia ble activities that are used, for example, by the National Institute for Biological Standards and Control (N IBSC) in the United Kingdom for the quantification of interferon, cytokine and growth-factor activity. The difference in levels of peptide may be equal to zero, indicating that the su bject is or may be normal or within the range of baseline levels of the peptide, or that there has been no change in levels of peptide since the previous assay.
[00125] The difference may simply be, for exa mple, a measured fluorescent value, radiometric value, densitometric value, DNA quantification, mass value etc., without any additional measurements or manipulations. Alternatively, the levels or d ifferences of the ΑΙΒ1-Δ4 peptide may be expressed as a percentage or ratio of the measured value of the peptide to a measured value of another compound including, but not limited to, a standard or internal standard. The differences or levels may also be expressed as a percentage or ratio of ΑΙ Β1-Δ4:ΑΙ Β1 (or perhaps the reciprocal). The differences may be negative, indicating a decrease in the amount of measured peptide over normal/baseline values or from a previous measurement, or the difference may be positive, indicating an increase in the amount of measured peptide over normal/baseline values or from a previous measurement. The difference may also be expressed as a difference or ratio of the peptide to itself, measured at a d ifferent point in time. The d ifference may also be determined using in an algorithm, wherein the raw data is manipulated. [00126] In general, levels of peptide which are higher than normal/baseline levels of peptide may confirm that the su bject has the a bnormal condition or that the su bject may have a higher proba bly than normal of developing the a bnormal condition, or that the su bject has a condition that is considered more less severe or aggressive. For example, a ratio of ΑΙ Β1-Δ4:ΑΙ Β1 that is higher in a test patient compared to normal/baseline levels would indicate that the patient may have an increased likelihood that the condition, e.g. a tumor, is more aggressive and, perhaps, more likely to metastasize.
Conversely, levels of peptide that are equal to or lower than normal/baseline levels of peptide may confirm that the su bject either does not have the a bnormal condition or that the su bject may have a lower proba bility of developing the a bnormal condition or that the condition is less severe or aggressive.
[00127] The present invention also relates to methods of monitoring the progression of a bnormal conditions in su bjects, with the methods comprising determining the levels of ΑΙ Β1-Δ4 peptide in a sample from the su bject at first and second, third, fourth, etc. time points. The levels of ΑΙΒ1-Δ4 peptide at each time point are then compared to determine differences of the ΑΙΒ1-Δ4 peptide over time. Any differences between the levels of ΑΙ Β1-Δ4 peptide over time are indicative of the progression, regression or stasis of the a bnormal condition in the su bject. In one em bodiment, an increase in the levels over time indicates that the a bnormal condition is worsening in the su bject. In another embodiment, a decrease in the levels over time indicates that the a bnormal condition is improving in the su bject. The improvement or worsening of the su bject based on the levels of ΑΙ Β1-Δ4 peptide over time can then possibly dictate or suggest a change in therapy.
[00128] As used herein, the phrase "monitor the progression" is used to indicate that the a bnormal cond ition in the su bject is being periodically checked to determine if the a bnormal condition is progressing (worsening), regressing (improving) or remaining static (no detecta ble change) in the individual by assaying the levels of ΑΙΒ1-Δ4 peptide in the su bject using the methods of the present invention. The methods of monitoring may be used in conjunction with other monitoring methods or treatment regimens for the a bnormal condition a nd to monitor the efficacy of these treatments. Thus, "monitor the progression" is also intended to indicate assessing the efficacy of a treatment regimen by periodically assessing the levels of ΑΙΒ1-Δ4 peptide and correlating any differences in the levels of AIB1- Δ4 peptide in the su bject over time with the progression, regression or stasis of the a bnormal condition. Thus, for example, the methods of the present invention may be used to monitor a su bject after mastectomy. In particular, the methods may be used to monitor patients that have had a successful mastectomy, such that the methods can be used to monitor the patient for followup mammogram. In another particular embodiment, the methods can be used to monitor patients that have received treatment, such as a tumor removal, but may need followup or concurrent treatment for that cancer. The methods can thus be used to determine a suitable follow up therapeutic regimen, after an initial treatment. Thus, in one embodiment, the present invention provides methods of individualizing a therapeutic regimen, comprising assessing levels of the ΑΙΒ1-Δ4 peptide and correlating these levels with a likely response to a variety of therapies. For example, patient populations may be stratified according to their response to therapy as their responsiveness correlates to levels of ΑΙΒ1-Δ4 peptide. Monitoring may include assessing the levels of ΑΙΒ1-Δ4 peptide at two time points from which a sample is taken, or it may include more time points, where any of the levels of ΑΙΒ1-Δ4 peptide at one particular time point from a given subject may be compared with the levels of biomarker in the same subject, respectively, at one or more other time points.
[00129]The expression ratio of ΑΙΒ1-Δ4:ΑΙΒ1 may also be used to predict or assess a drug regimen to which a given patient is more or less sensitive. Knowledge of the ratio at the outset of therapy can guide a clinician in choosing a particular therapy. For example, comparing a subject's initial levels of the ΑΙΒ1-Δ4:ΑΙΒ1 ratio to levels that characteristically present a good response to a particular therapeutic regimen can guide a clinician in choosing a similar, if not identical, regimen. Conversely, comparing a subject's initial levels of the ΑΙΒ1-Δ4:ΑΙΒ1 ratio to levels that characteristically present a negative response or no response to a particular therapeutic regimen can guide a clinician in choosing a different regimen. The levels to which the subject's ratio is compared can be generated from a population of samples. Methods of determining levels in a population of samples has been discussed herein and these same principles can be used to correlate the ΑΙΒ1-Δ4:ΑΙΒ1 ratio to a given response in a population of samples which can, in turn, be used for comparison purposes. In one embodiment, increase in levels of the peptide compared to normal levels indicates that the tumor has a higher probability of being sensitive or resistant to a therapy, i.e., that the tumor in the subject will not respond well to the therapy. In another embodiment, increase in levels of the peptide compared to normal levels indicates that the tumor has a lower probability of being sensitive or resistant to a therapy, i.e., that the tumor in the subject will respond well to the therapy. The therapy that is being considered may or may not be preselected by the clinician. In other words, the subject's calculated ratio may guide the physician to a therapy that was not previously considered, or the subject's calculated ratio may confirm as a good choice the therapy that the clinician had pre-determined. Likewise, the subject's calculated ratio may indicate to the clinician that the pre-determined therapy should be re-evaluated for a different choice. The therapeutic regimen may be a single compound or it may be a com bination of compounds. In one embodiment, the levels of ΑΙ Β1-Δ4 will guide the clinician towards or away from a specific combination of compounds of the therapeutic regimen.
[00130] The examples herein are illustrative in nature and are not intended to limit the scope of the claimed invention.
Examples
[00131] Experimental Methods
[00132] Plasmids- p300-HA and ERa constructs were provided by Dr. Maria L. Avantaggiati (Georgetown University) and from Dr. Pierre Cham bon (INSERM, Strassbourg) respectively. AI Bl, ΑΙ Β1-Δ4, FLAG AI Bl, and FLAG ΑΙΒ1-Δ4 were described previously (17,21). A C-terminal FLAG was added to the ΑΙ Β1-Δ4 cDNA by deletion of the stop codon in ΑΙΒ1-Δ4 and addition of FLAG peptide sequence by site-directed mutagenesis (Stratagene). The AI Bl N term construct was created by PCR amplification of the
ACTR/AI Bl cDNA (184 bp to 777bp) to add a new 5' NotI site and 3' Bgl ll site. The PCR product was then cloned into p3XFLAG-CMV-10 (Sigma).
[00133] Cell lines a nd transient transfection- M DA231-BrM2 were kindly provided by Dr. Joan Massague (Sloan Kettering Institute), 4175-TR and SCP2-TR cells by Yibin Kang (Princeton University), AI Bl KO/SRC3-/- mouse em bryonic fibroblasts (M EFs) by Dr. Jianming Xu (Baylor College of Medicine), T47D Al-2 cells by Dr. Steve Nordeen (University of Colorado), and COLO SL and COLO PL cells by Dr. John M. Jessup (Georgetown University). Chinese Hamster Ovary (22), COLO 357 were purchased from ATCC. H EK293, H EK293T, COS-7, and M DA-M B-231 were obtained from the Tissue Culture Shared Resource at Georgetown University. The Human mammary epithelial cells (H M EC) were purchased and cultured in commercially supplied medium (BulletKit, Lonza). H EK293T, COS-7, COLO 357, COLO SL, and COLO PL, M DA-M B-231, M DA231-BrM2 (brain), 4175-TR (lung), SCP2-TR (bone), and AIBl KO M EFs were grown in Dul becco modified Eagle mediu m (DM EM, Invitrogen) with 10% FBS. CHO cells were grown in DM EM F12 (Invitrogen) with 10% FBS. HEK293 cells were grown in Iscove's modified Eagle medium (I M EM, Invitrogen) with 10% Charcoal stripped serum (CCS). T47D Al-2 were grown in I M EM+5%CCS. HEK293, HEK293T, CHO, COS-7, and T47D Al-2 cells were transiently transfected with FuGEN E 6 (Roche).
[00134] Identification of N-terminus of ΑΙΒ1-Δ4- HEK293T cells grown to 80% confluence were transiently transfected with 18 μg C-terminal FLAG ΑΙΒ1-Δ4 cDNA. 24 hours later whole cell lysates were prepared and subjected to immunoprecipitation using Anti-FLAG M2 affinity gel (Sigma). After washing ΑΙΒ1-Δ4 protein was recovered by heating the affinity gel to 95°C and the sample was subjected to SDS-PAGE. A band corresponding to ΑΙΒ1-Δ4 protein was isolated and trypsinized using a conventional in-gel digestion protocol where cysteines were reduced with DTT and alkylated by iodoacetamide. Extracted tryptic peptides were analyzed using the MIDAS-MS based algorithm on an LC-ESI-MS 4000QT AP instrument (AB SCIEX, Framingham, MA). In silico predicted peptides and corresponding collision energy settings were generated using recommended settings in MRMpilot software (AB SCIEX, Framingham, MA). The list of predicted precursors includes the potential variable modification of methionine oxidation and the fixed modification of cysteine alkylation. A final MS method was created for detection of the tryptic peptides produced from the full-length AIB1 protein. This approach allows detecting only tryptic peptides that overlaps with the spliced version of AIB1 and was tested on endogenous AIB1 protein isolated by immunoprecipitation followed by SDS-PAGE. In silico predictions for tryptic peptides with methionine as an initial amino acid residue were applied to data for identification of the N-terminus. More detailed methods are provided in the supplemental material.
[00135] Western blot (WB) analysis and Immunoprecipitation (IP) - Western blotting was done with the following antibodies: AIB1 (5E11, Cell Signaling), FLAG M2 (Sigma), HA (Cell Signaling), ERa (Ab-10, Neomarkers), ERa (G-20, Santa Cruz), human Actin (Millipore). (i) Interaction of AIB1 with ΑΙΒ1-Δ4. HEK293T cells were transfected with 6μg of either FLAG AIB1, ΑΙΒ1-Δ4, or FLAG AIB1 and ΑΙΒ1-Δ4 together. After washing with cold IX PBS whole cell lysates were prepared by adding 1% NP-40 lysis buffer containing ImM Na03V04 and lx Complete protease inhibitor tablet (Roche). IP was performed with Anti-FLAG M2 affinity gel as described previously (6) and samples were subjected to SDS-PAGE. (ii) Interaction of AIB1 and ΑΙΒ1-Δ4 with p300-HA. HEK293T cells were transfected with either FLAG AIB1, FLAG ΑΙΒ1-Δ4, or p300-HA. Whole cell lysates were prepared as in section (i). Equal amounts of FLAG AIB1 and FLAG ΑΙΒ1-Δ4 were added to equal amounts of p300-HA lysate. After immunoprecipitation using HA antibody (Cell Signaling) the amounts of FLAG AIB1 or FLAG ΑΙΒ1-Δ4 were detected with FLAG M2 antibody (Sigma). Densitometry was performed by using Adobe Photoshop 7.0 normalizing ΑΙΒ1-Δ4 bands to AIB1 bands for both input and IP.
[00136] Immunofluorescence and leptomycin B treatment- (i) CHO cells were plated on glass coverslips and transfected with 500ng of either FLAG AIB1 or FLAG ΑΙΒ1-Δ4. 24 hours later cells were fixed with 3.7% formaldehyde for 15 minutes at 37°C. Cells were then washed three times with lx PBS and permeabolized with lx PBS containing 0.2% Triton X-100 for 5 minutes 25°C. Cells were then washed with two times with IxPBS and incubated with FLAG M2 antibody (1:500, Sigma) for 20 minutes. After three 5 minute washes with IxPBS, cells were incubated with anti-mouse IgG AlexaFluor488 (1:1000, Invitrogen) for 20 minutes. Coverslips were then washed three times with lx PBS and mounted with ProLong Gold antifade reagent with DAPI (Invitrogen) on to glass slides. 50 nM leptomycin B was added into the culture medium 4 hours before fixation. 200 cells were counted and the percentage of nuclear, nuclear/cytoplasmic, and cytoplasmic stained cells was quantified from 3 different experiments.
Nuclear staining was defined as protein specific signal that overlaid with the DAPI signal only.
Nuclear/cytoplasmic was defined as protein specific staining that overlaid with the DAPI signal but also showed staining in the cytoplasmic compartment. Cytoplasmic staining was defined as protein specific staining that did not overlay with the DAPI staining of the nucleus, (ii) 2x10s AIB1 MEFs were transiently transfected with 4 μg FLAG ΑΙΒ1-Δ4 and either 2, 4, or 6 μg of p300-HA or AIB1 using the MEF 2 Nucleofector kit (Amaxa, Lonza) and plated on glass coverslips after transfection. 24 hours later cells were fixed, permeabolized, stained, mounted, and quantified as in (i). p300 (1:500, Abeam) and anti- rabbit AlexaFluor568 (1:1000, Invitrogen) were used to stain for p300-HA.
[00137] Chromatin immunoprecipitation assays (ChIP)- HEK293 cells in a 10 cm dish were transfected with 5μg E a and either 6μg FLAG AIB1 or 3 μg FLAG ΑΙΒ1-Δ4. 24 hours later cells were treated with Estrogen (E2) for 0, 15, 30, 45, or 60 minutes. Cells were fixed with formaldehyde fixation solution (3.7% formaldehyde, 100 mM NaCI, 50 mM Tris/HCI pH 8.0, 1 mM EDTA, 0.5 mM EGTA) for 10 minutes at 37°C and stopped with 0.125 M Glycine in lx PBS for 5 minutes at 25°C. Cells were washed three times IxPBS and resuspended in SDS lysis buffer (50 mM Tris pH 8.0, 10 mM EDTA pH 8.0, 1% SDS). Cells were sonicated and resuspended in ChIP dilution buffer (20 mM Tris pH8.0, 2 mM EDTA pH 8.0, 150mM NaCI, 1% Triton-X-100) and pre-cleared with 30 μΙ of protein G agarose/salmon sperm DNA (Millipore) for 1 hour. 500 μg of total protein was immunoprecipitated with 2 μΙ FLAG M2 antibody (Sigma) 16 hours and immunoprecipitated with 30 μΙ protein G agarose/salmon sperm DNA for 2 hours. Agarose was washed with once with low salt buffer (20 mM Tris pH 8.0, 2 mM EDTA pH 8.0, NaCI 150 mM, 0.1% SDS, 1% Triton-X-100), twice with high salt buffer (20 mM Tris pH 8.0, 2 mM EDTA pH 8.0, 500 mM NaCI, 0.1% SDS, 1% Triton-X-100), once with LiCI salt buffer (10 mM Tris pH 8.0, 1 mM EDTA pH 8.0, 250 mM LiCI, 1% Na deoxycholate, 1% NP-40), and twice with TE buffer (10 mM Tris pH 8.0, ImM EDTA, pH 8.0). Samples were eluted with elution buffer (1%SDS, 0.1M NaHC03) for 15 minutes on rotator and 10 minutes on vortexer. Crosslinks were removed with 200mM NaCI for 6 hours at 65°C and proteins digested with 1 μg proteinase K for 1 hour at 45°C. DNA was purified using GENECLEAN Turbo kit (Q- Biogene). Samples were analyzed by real time PCR to examine the ERE recruitment of FLAG AIBl or FLAG ΑΙΒ1-Δ4 with the following primers: pS2 ERE s: 5 ' G G CC ATCTCTC ACT ATG A ATC ACTTC-3 ' (SEQ ID NO: 2), pS2 ERE as: 5 ' - G G C AG G CTCTGTTTG CTT A A AG AG CG - 3 ' (SEQ ID NO: 3), hC3 ERE s: 5'- GAGAAAGGTCTGTGTTCACCAGG-3' (SEQ ID NO: 4), hC3 ERE as: 5'-TGCAGGGTCAGAGGGACAGA-3' (SEQ ID NO: 5), HER2 ERE s: 5'-GTTCCTCCCTCCTGTTCC TC-3' (SEQ ID NO: 6), HER2 ERE as: 5'- CC AC AA ACTG GTG GTCTCCT-3 ' (SEQ ID NO: 7). Cycling conditions for real time PCR using iCycler were 95°C 3 minutes followed by 40 cycles of 95°C 20 sec, 57°C 30 sec, 72°C 40 sec for hC3 ERE and pS2 ERE. For HER2 ERE cycling conditions were 95°C 3 minutes followed by 40 cycles of 95°C 20 sec, 65°C 30 sec, 72°C 40 sec. The percentage of the input for each time point is plotted on the graphs and normalized time 0 for each transfection.
[00138]T47D Al-2 cells were transfected with either FLAG ΑΙΒ1-Δ4 or FLAG empty vector. 24 hours later cells were stimulated with ΙΟηΜ R5020 for 1 hour and lysates were processed as described above. To examine the binding of ΑΙΒ1-Δ4 to the M MTV promoter endpoint PCR was used with the following conditions 96°C 4 minutes followed by 30 cycles of 94°C 1 min, 60°C 1 min, 72°C 1 min. Primer sequences in the MMTV promoter were MMTV s: 5'-CGGTTCCCAGGGCTTAAGTAAGTT-3' (SEQ ID NO: 8) and M MTV as: 5'-GGATGGCGAACAGACACAAACACA-3' (SEQ ID NO: 9). All primers were synthesized by Integrated DNA Technologies (IDT).
[00139] Real time PCR analysis- HEK293 cells were transfected with FLAG AIBl or FLAG ΑΙΒ1-Δ4 at levels that give equal amounts of transfected protein in IMEM +10% CCS. 16 hours later cells were stimulated with E2 for 0, 4, 8, and 24 hours. Total RNA was harvested using RNeasy mini kit (Qiagen) and reverse transcribed with iScript cDNA synthesis kit (Bio-Rad) using 1 μg of total RNA. Samples were analyzed by real time PCR (iCycler, Bio-Rad) using the following conditions: 95°C 3 min, 40 cycles of 95°C 20 sec 56°C 30 sec 72°C 40 sec. Primer sequences used were: pS2 s: 5'-CCCCGTGAAAGACAGAATTGT-3' (SEQ ID NO: 10), pS2 as: 5'-GGTGTCGTCGAAACAGCAG-3' (SEQ ID NO: 11), hC3 s: 5'-CTGTCCACGACTTCCCAGG-3' (SEQ ID NO: 12), hC3 as: 5'-CCCTTTTCTGACTTGAACTCCC-3' (SEQ ID NO: 13), HER2 s: 5'- AAAGGCCCAAGACTCTCTCC-3' (SEQ ID NO: 14), HER2 as: 5'-CAAGTACTCGGGGTTCTCCA-3' (SEQ ID NO: 15), human actin s: 5'-CCTGGCACCCAGCACAAT-3' (SEQ ID NO: 16), human actin as: 5'- GCCGATCCACACGGAGTACT-3' (SEQ ID NO: 17). All primers were synthesized by IDT. Expression level for each gene is normalized to actin expression and multiplied by either 1000 or 100,000 to obtain whole value numbers. [00140] Quantitation of AIBl-A4mRNA levels using Scorpion primer based quantitative RT-PCR- A total of 2 x 106 cells were plated for each cell line. 24 hours later total RNA was extracted with RNeasy mini kit (Qiagen) and reverse transcribed with iScript cDNA Synthesis Kit (Bio-Rad) using l^g of total RNA. Real-time PCR was performed using IQ SYBR Green Supermix (Bio-Rad) with AIBlA4-scorpion primer and human actin primers. Cycling conditions for the AIBl-A4-scorpion primer consist of an initial denaturing step at 94°C (2 min), and 50 cycles (20 seconds at 94°C, 15 seconds at 55.5°C and 20 seconds at 72°C). Unlike SYBR green real time PCR analysis where data is collected during the extension step, data for the Scorpion primer reactions were collected during the 55.5°C annealing step (iCycler; Bio-Rad). Cycling conditions for the human actin primers include a denaturing step at 94°C (2 min), and 45 cycles (20 seconds at 94°C, 30 seconds at 58°C and 40 seconds at 72°C). The ΑΙΒ1-Δ4 scorpion primer was custom designed and purchased from Sigma-Aldrich. Primer sequences for the ΑΙΒ1-Δ4 Scorpion reaction: 5'FAMCCCGCGCTTGGAAATAGTTTTTCCCTTGTCCGCGCGGGBHQlHEGCGCAAATTGCCATGTGATAC-3' (SEQ ID NO: 18). ΑΙΒ1-Δ4 reverse primer: 5'-CCATCCAATGCCTGAAGTAA-3' (SEQ ID NO: 19). The expression level of ΑΙΒ1-Δ4 is normalized to actin expression levels and multiplied by either 10,000 or 100,000 to obtain whole number values.
[00141] Luciferase assay- 25,000 COS-7 cells per well in a 24 well dish were transfected in DMEM without serum with 100 ng M MTV luciferase, 25 ng Progesterone receptor (PR), 5 ng Thymidine Kinase (TK) Renilla luciferase, and either 500 ng pcDNA3, 500 ng FLAG AIB1, 500 ng FLAG N term, or 125, 500, and 750 ng of FLAG N term with 500 ng FLAG AIB1. 24 hours later cells were treated with 10 nM R5020 or an equivalent volume of ethanol. 24 hours after stimulation cells were lysed and luciferase values were measured using the Dual-Luciferase Reporter Assay System (Promega). Firefly luciferase values were normalized to Renilla luciferase values and averaged for each transfection condition plated in triplicate.
[00142] Results
[00143] Identification of the N-terminus of ΑΙΒ1-Δ4.
[00144] As was previously identified, ΑΙΒ1-Δ4 is a splice variant of the nuclear receptor coactivator AIB1 (17) which results in the translation of an N-terminally truncated isoform of the full-length AIB1 protein with a molecular weight of approximately 130 kDa. The translation start site of ΑΙΒ1-Δ4 was predicted to be at the methionine at position 199 in the full-length AIB1 protein since this was the next in frame methionine residue, however the translation start site was not identified experimentally. There is a cluster of methionines at positions 199, 201, 217, 224, 235, 236, 246, and 289 of full-length AIBl sequence from which initiation of translation could result in an approximately 130 kDa protein (Fig. la). Mass spectrometry was used to determine which of these 8 methionines is the translation start site of the ΑΙΒ1-Δ4 protein. Isolated ΑΙΒ1-Δ4 protein was isolated from HEK293T cells transfected with a C- terminal FLAG ΑΙΒ1-Δ4 cDNA construct. After immunoprecipitation with FLAG antibody, ΑΙΒ1-Δ4 was isolated from a Coomassie stained SDS-PAGE gel and subjected the tryptic peptides to mass
spectrometric analysis. Through an initial protein identification an AIBl related protein was detected based on the initial analysis of the peptides. A search for the peptide TPHDILEDINASPEM217 (SEQ ID NO: 20) (boxed in Fig. la) was employed because it is an easily detectable fragment present in the full- length AIBl protein in previous studies (21). This peptide was not found in the analysis of the tryptic fragments from ΑΙΒ1-Δ4. This eliminated the possibility of the translation start site at M199 and M201. The most N-terminal peptide identified from the ΑΙΒ1-Δ4 protein was
AM235M236EEGEDLQSCM246ICVAR (SEQ ID NO: 21) (underlined in Fig. la). This ruled out the M235, M236, M246, and M289 methionines as possible translation start sites and limited the possibilities to the M217 and M224 methionine residues. As a next step these methionines were tested using in silico predictions for tryptic peptide with an initial methionine as the first amino acid residue. Also included were predicted precursors that could appear as a result of cotranslational modifications such as N- terminal acetylation. In eukaryotes, 80% of all proteins have been described with an acetyl moiety added to the N-terminus (23,24). Interestingly, it was observed that only one predicted peptide M224QCFALSQPR (SEQ ID NO: 22) retained an initiator methionine, which was M224. This peptide harbored N-terminal acetylation and double charge state of this peptide defines an m/z value of 640.3 (Fig. lb). Due to chemical oxidation of methionines, the precursor ions are split between the m/z ratio of 640.3 and an m/z shift of +8 amu to 648.3. Fragmentation analysis of both precursors matched to corresponding peptides with different oxidation states of methionine and with Mascot scores assigned of 45 and 65 respectively where scores above 23 indicate peptide identity. Therefore, the N-terminus of ΑΙΒ1-Δ4 is defined to be at the M224 residue of full-length AIBl.
[00145]The full-length and alternatively spliced AIBl mRNAs and proteins derived from these data are depicted schematically in Fig. lc. The full-length human AIBl transcript has 23 exons coding for a 1424 amino acid protein of 155 kDa. Translation of AIBl is initiated in exon 3 and continues until the stop codon in exon 23. The ΑΙΒ1-Δ4 transcript lacks exon 4 due to alternative splicing. Translation of the ΑΙΒ1-Δ4 isoform is initiated in exon 7 and the resultant protein lacks the N-terminal 223 amino acids of the full-length protein. Thus, ΑΙΒ1-Δ4 is now defined as a 130 kDa protein of 1201 amino acids.
[00146] ΑΙΒ1-Δ4 is a predominantly cytoplasmic protein that enters the nucleus.
[00147] Others have published the presence of a bipartite nuclear localization signal (NLS) in the N- terminus of the AIBl protein at amino acids 16-19 and 35-38 (25-27). Due to the loss of exon 4 in the ΑΙΒ1-Δ4 transcript, the NLS, basic helix loop helix (bHLH), and PAS A domain are lost from the ΑΙΒ1-Δ4 protein. Lack of a NLS in the ΑΙΒ1-Δ4 isoform is intriguing since ΑΙΒ1-Δ4 was found to be a more potent transcriptional coactivator than the full-length AIBl (17,18). It is not clear if the effects of coactivation of transcription are due to a cytoplasmic and/or nuclear function of the ΑΙΒ1-Δ4 protein (17,18,20). A FLAG tagged construct of ΑΙΒ1-Δ4 (21) was used to determine the cellular localization of ΑΙΒ1-Δ4.
Chinese Hamster Ovary cells (22) were transfected with either FLAG AIBl or FLAG ΑΙΒ1-Δ4 in media with 10% FBS and the number of nuclear, nuclear/cytoplasmic, and cytoplasmic stained cells were quantified. We confirmed that the majority of ΑΙΒ1-Δ4 resides in the cytoplasm (Fig. 2a) (20). CHO cells transfected with AIBl showed nuclear and nuclear/cytoplasmic staining in 75% and 25% of the cells respectively. Cells transfected with ΑΙΒ1-Δ4 showed nuclear, nuclear/cytoplasmic, and cytoplasmic staining in 1.5%, 41.3%, and 57.2% of cells respectively. Typical staining for nuclear, nuclear/cytoplasmic, and cytoplasmic stained cells are shown. At least some nuclear and nuclear/cytoplasmic staining was detected for ΑΙΒ1-Δ4.
[00148]The nuclear export inhibitor leptomycin B which inhibits C M 1 dependent nuclear export of proteins that contain a nuclear export sequence (NES) (16). Both AIBl and ΑΙΒ1-Δ4 contain the NES that is in the C-terminal half of the proteins (Fig. lc) (26). CHO cells were transfected with FLAG AIBl or FLAG ΑΙΒ1-Δ4 in media with 10% FBS and were either treated with vehicle or leptomycin B. Cells were fixed, stained, and the localization was quantified as before (Fig. 2b). It was observed the same distribution of staining in the transfected CHO cells treated with vehicle, however there was an increase in the number of cells showing nuclear staining in both AIBl and ΑΙΒ1-Δ4 transfected cells. CHO cells transfected with FLAG AIBl and treated with leptomycin B showed 88%, 12%, and 0% of cells staining in the nuclear, nuclear/cytoplasmic, and the cytoplasmic compartments respectively. CHO cells transfected with AIB1- Δ4 and treated with leptomycin B showed staining in the nuclear, nuclear/cytoplasmic, and the cytoplasmic compartments in 18%, 60%, and 22% of cells respectively. The increase in the number of nuclear stained cells after leptomycin B treatment suggests that ΑΙΒ1-Δ4 is imported into the nucleus however, lack of a canonical N LS and this present data suggests that nuclear import of ΑΙ Β1-Δ4 is an inefficient process, which contributes to the lower steady state levels of nuclear staining of the protein.
[00149] Nuclear import of ΑΙΒ1-Δ4 can be facilitated by other N LS containing proteins.
[00150] Various proteins involved in signaling have been shown to be imported into the nucleus in a n N LS independent manner (28-33). One possible explanation of nuclear import of ΑΙΒ1-Δ4 is by interaction with other N LS containing proteins. To determine if ΑΙ Β1-Δ4 could interact with full-length AIBl, H EK293T cells were transfected with FLAG AI Bl alone, ΑΙ Β1-Δ4 alone, or FLAG AIBl and ΑΙΒ1-Δ4 together and immunoprecipitated with FLAG antibody to pulldown AI Bl. Western blotting with an AI Bl antibody showed that ΑΙΒ1-Δ4 immunoprecipitates with AIBl (Fig. 3a, lane 6, top panel). Interestingly, there is a detecta ble amou nt of endogenous ΑΙΒ1-Δ4 protein in H EK293T cells and immunoprecipitation with FLAG AIBl is a ble to pulldown endogenous ΑΙ Β1-Δ4 protein as well (Fig. 3a, lane 4, bottom panel). When no FLAG AI Bl is present, we do not detect any ΑΙ Β1-Δ4 present after immunoprecipitation (Fig. 3a, lane 5, top and bottom panels).
[00151] To study if the localization of ΑΙΒ1-Δ4 could be altered by other N LS containing proteins such as AI Bl, mouse embryonic fibroblasts (M EFs) derived from AI Bl knockout mice were used to analyze the localization of ΑΙ Β1-Δ4. The AIBl KO M EFs were transfected with FLAG ΑΙΒ1-Δ4 in the presence of increasing amounts of AI Bl (Fig. 3 b). It was observed increased nuclear staining of ΑΙΒ1-Δ4 after transfection with increasing amounts of AIBl. The pattern of staining of ΑΙ Β1-Δ4 that was cytoplasmic, nuclear/cytoplasmic, or nuclear was 86±1%, 14±1%, and 0% respectively and shifted to 70.3±3.5%, 29.3±4%, and 0.3±0.6% with full-length AIBl (Fig. 3c).
[00152] The interaction of AI Bl with other proteins involved in transcription is well characterized (7). One of these proteins is p300/CBP (35) and it is disclosed herein that ΑΙΒ1-Δ4 interacts with p300 by immunoprecipitation (Fig. 4a). H EK293T cells were transfected with either p300-HA, FLAG AIBl, or FLAG ΑΙΒ1-Δ4 and cell lysates were harvested. After normalization for protein content, equal amounts of either FLAG AI Bl or FLAG ΑΙΒ1-Δ4 were added to p300-HA cell lysate. The amount of FLAG AIBl and FLAG ΑΙΒ1-Δ4 proteins were observed after immunoprecipitation with p300-HA. It was found that both AIBl and ΑΙ Β1-Δ4 are associated with p300. Interestingly, with equal amounts of FLAG AI Bl or FLAG ΑΙΒ1-Δ4 in the lysate more ΑΙ Β1-Δ4 was found to be associated with p300, suggesting that ΑΙ Β1-Δ4 may be in active transcriptional complexes since p300 is typically found in active transcriptional complexes. [00153] ΑΙΒ1-Δ4 with p300-HA was also transfected into the AIBl KO M EFs and to determine the shift of ΑΙΒ1-Δ4 to the nucleus (Fig. 3 b). Again, nuclear staining of ΑΙΒ1-Δ4 increased by increased expression of p300-HA. The pattern of staining of ΑΙ Β1-Δ4 that was cytoplasmic, nuclear/cytoplasmic, or nuclear was 86±1%, 14±1%, and 0% respectively and shifted to 23.7±2.5%, 63±4.6%, and 13.3±2.3% (Fig. 4c). These data suggest that ΑΙΒ1-Δ4 may interact with other N LS containing proteins to traffic into the nucleus.
[00154] ΑΙ Β1-Δ4 is recruited to active transcriptional complexes and coactivates estrogen responsive genes.
[00155] To examine the recruitment of ΑΙΒ1-Δ4 to estrogen responsive elements (ERE), HEK293 cells were transfected with ER and either FLAG AI Bl or FLAG ΑΙΒ1-Δ4 and Chromatin Immunoprecipitation (ChI P) analysis (Fig. 5a) was performed. Cells were treated with estrogen for 15, 30, 45, and 60 minutes to observe the recruitment of AIBl or ΑΙΒ1-Δ4 over time. Both AIBl and ΑΙΒ1-Δ4 were recruited to the three different estrogen response elements in pS2, hC3, and HER2 (22,36,37). Maximal recru itment of FLAG AI Bl was at 15 minutes to 8.7%, 4.3%, and 5.0% of the EREs in the pS2, hC3, and H ER2 genes respectively. In contrast, maximal recruitment of FLAG ΑΙ Β1-Δ4 was at 30 minutes to 3.5%, 4.2%, and 3.1% of the EREs in the pS2, hC3, and HER2 genes respectively. Thus, both AI Bl and ΑΙΒ1-Δ4 are recruited to these EREs confirming that ΑΙ Β1-Δ4 is found in the nucleus a nd found at active sites of transcription. Interestingly, the kinetics of ΑΙ Β1-Δ4 recruitment is delayed which is possibly due to inefficient or altered nuclear transport because of lack of the N LS. The corresponding protein levels are shown by Western blot for all the d ifferent times of estrogen stimulation.
[00156] To confirm that recruitment of ΑΙ Β1-Δ4 leads to an increase in these estrogen-regulated genes, HEK293 cells were transfected with ER and either FLAG AI Bl or FLAG ΑΙΒ1-Δ4 and stimulated with estrogen for 4, 8, and 24 hours. RNA was harvested from the cells and the gene expression examined by real time PCR. Maximal gene expression in ΑΙΒ1-Δ4 transfected cells occurred at 8 hours for H ER2 a nd at 24 hours for pS2 and hC3. Significant differences in gene expression were only observed in the AI B1- Δ4 transfected cells suggesting that ΑΙ Β1-Δ4 is better a ble to stimulate expression of estrogen responsive genes than full-length AI Bl.
[00157] It was also observed that recruitment to a progesterone responsive element by taking advantage of the T47D Al-2 cells that have a sta ble integration of the M MTV luciferase construct (38). After transfection with PR and either empty vector or FLAG ΑΙ Β1-Δ4 it was detected that ΑΙ Β1-Δ4 is recruited to the M MTV promoter in T47D Al-2 after 5020 treatment (supplemental Fig. 1) indicating that recruitment of ΑΙ Β1-Δ4 is not exclusive to estrogen-regulated promoters.
[00158] The region deleted from ΑΙΒ1-Δ4 contains an inhibitory domain.
[00159] ΑΙ Β1-Δ4 also stimulates expression of endogenous estrogen dependent genes. This potent coactivator function cannot be explained merely based on the cellular localization of ΑΙΒ1-Δ4 because it is mostly cytoplasmic. A FLAG tagged N-terminal AI Bl construct (AI Bl N term) was created which contains the bH LH and PAS A domains not present in the ΑΙ Β1-Δ4 protein (Fig. lc). This construct should have no coactivator function since it lacks the C-terminal activation domain responsible for binding to nuclear receptors and molecules that affect transcription such as p300/CBP and CARM 1. It was postulated that a repressive function associated with the N-terminal region of AI Bl would be revealed by co-transfection of AIBl N term with AIBl, and this would relieve the repression on the coactivator function of AIBl. COS-7 cells were transfected with progesterone receptor and AIBl with increasing amounts of AI Bl N term and monitored the coactivator function of AI Bl on a progesterone inducible M MTV luciferase reporter construct (Fig. 6a). As expected, AI Bl was a ble to coactivate transcription of the luciferase reporter relative to empty vector transfected cells (AIBl 0 vs. 500). Co-transfection of AIBl with AIBl N term significantly increased the amount of transcription from the luciferase reporter. This effect was increased with the amount of AI Bl N term co-transfected with AIBl (AIBl co- transfection with AI Bl N term 125, 500, 750). A relief of repression on endogenous AIBl coactivator activity due to co-expression of the AI Bl N term construct alone was also apparent (AI Bl N term 0 vs. 500). The levels of AI Bl and AI Bl N term protein are shown by Western blot a nd there is no increase in the AIBl protein with co-transfection of higher levels of AIBl N term (Fig. 6b). These data suggest a suppressor role of the N-terminal region of AIBl in the regulation of the coactivator function of AIBl. This region is the most highly conserved region among steroid receptor coactivator proteins and has been suggested to contain an inhibitory function for AI Bl also in the cytoplasm (20).
[00160] ΑΙΒ1-Δ4 expression is correlated with metastatic potential.
[00161] A quantitative assay to measure ΑΙΒ1-Δ4 m RNA levels was designed. Through the use of Scorpion primer technology (39,40) it was shown that the expression of ΑΙΒ1-Δ4 is increased in breast cancer cell lines relative to normal breast cell lines. A Scorpion primer with a sequence complimentary to a sequence in exon 3 and a probe sequence complimentary to the junction of exon 3 and exon 5 (Fig. 7a) were designed. This scorpion primer specifically recognizes the ΑΙΒ1-Δ4 transcript and not the AI Bl transcript due to the fact that after amplification of AI Bl transcript, the probe sequence is not complimentary to any sequence generated in the full-length AI Bl transcript. Plasmids containing either AIBl or ΑΙΒ1-Δ4 cDNA were su bjected to an analysis by real time PC with the ΑΙΒ1-Δ4 scorpion primer. The ΑΙΒ1-Δ4 scorpion primer specifically identified ΑΙΒ1-Δ4 cDNA and not full-length AIBl cDNA. To confirm the increased expression of ΑΙ Β1-Δ4 in breast cancer cell lines, total RNA was harvested from human mammary epithelial cells (HM EC), parental M DA-M B-231, and three tissue specific metastatic variants of M DA-M B-231 cells. The three tissue specific variants home either to the brain, bone, or lung after intravenous or intracardiac injection (41-43). After reverse transcription, cDNA generated from these RNAs was su bjected to real time PCR analysis with Scorpion primers for ΑΙΒ1-Δ4 (Fig. 7b).
Expression of ΑΙ Β1-Δ4 m RNA was higher in the parental and three tissue specific metastatic variants of M DA-M B-231 cells relative to H M ECs confirming increased expression of ΑΙΒ1-Δ4 in breast cancer cell lines relative to normal breast cells.
[00162] Increased levels of ΑΙΒ1-Δ4 were also found in the more metastatic variants of the COLO 357 pancreatic cancer cells, compared to the parental COLO 357 pancreatic cancer cells, suggesting a correlation between increased metastatic capa bility and ΑΙΒ1-Δ4 expression. (Fig. 7c)
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Claims

What is Claimed is:
1. An isolated peptide consisting of the amino acid sequence of SEQ ID NO: 1.
2. A nucleic acid encoding the isolated peptide of claim 1.
3. A vector comprising the nucleic acid of claim 2.
4. A host cell comprising the vector of claim 3.
5. A method of making a peptide with an amino acid sequence of SEQ ID NO: 1, the method
comprising culturing the host cell of claim 4 under conditions suitable for protein expression and isolating the peptide.
6. An antibody fragment that specifically binds to a peptide, the peptide consisting of the amino acid sequence of SEQ ID NO: 1.
7. The antibody fragment of claim 6, wherein an antibody comprises the antibody fragment.
8. The antibody fragment of claim 6 or 7 wherein the antibody fragment is humanized.
9. The antibody fragment of claim 7, wherein the antibody is monoclonal.
10. A method for determining the likelihood that a tumor in a subject will metastasize, the method comprising determining the levels of a peptide in tumor cell taken from the subject, the peptide comprising the amino acid sequence of SEQ ID NO: 1, wherein an increase in levels of the peptide compared to levels in a normal cell indicates that the tumor has a higher probability of metastasizing.
11. The method of claim 10, wherein determining the levels of the peptide comprises the use of an antibody fragment that specifically binds to a peptide with the amino acid of SEQ ID NO: 1.
12. The method of any of claims 10-11, wherein an antibody comprises the antibody fragment.
13. The method of any of claims 11-12, wherein determining the levels of the peptide comprises the use of an antibody fragment that specifically binds to a peptide with the amino acid of SEQ ID NO: 1.
14. The method of any claims 12-13, wherein the antibody is humanized.
15. The method of any of claims 10-14, wherein the abnormal cell is taken from tissue or body fluid of the subject
16. The method of any of claims 10-15, wherein determining with said antibody or antibody
fragment is an in vitro assay selected from the group consisting of immunohistochemical assay, enzyme linked immunoassay (ELISA), and radioimmunoassay ( IA).
17. The method of any of claims 10-16, wherein determining with said antibody or antibody
fragment is an in vivo imaging assay selected from the group consisting of X-radiography, nuclear magnetic resonance (NM R), and electron spin resonance (ESR).
18. A method of stratifying a population of a group of abnormal cells, the method comprising
determining the levels of a peptide in each of the cells, the peptide comprising the amino acid sequence of SEQ ID NO: 1, wherein the cells are stratified based on the levels of the peptide within the abnormal cells.
19. The method of claim 18, wherein determining the levels of the peptide comprises the use of an antibody fragment that specifically binds to a peptide with the amino acid of SEQ ID NO: 1.
20. The method of any of claims 18-19, wherein an antibody comprises the antibody fragment.
21. The method of any of claims 19-20, wherein determining the levels of the peptide comprises the use of an antibody fragment that specifically binds to a peptide with the amino acid of SEQ ID NO: 1.
22. The method of any claims 20-21, wherein the antibody is humanized.
23. The method of any of claims 18-22, wherein the abnormal cell is taken from tissue or body fluid of a subject
24. The method of any of claims 18-23, wherein determining with said antibody or antibody
fragment is an in vitro assay selected from the group consisting of immunohistochemical assay, enzyme linked immunoassay (ELISA), and radioimmunoassay (RIA).
25. The method of any of claims 18-24, wherein determining with said antibody or antibody fragment is an in vivo imaging assay selected from the group consisting of X-radiography, nuclear magnetic resonance (NM ), and electron spin resonance (ESR).
26. A method for determining the likelihood that a tumor in a subject will respond to a given
therapy, the method comprising determining the levels of a peptide in tumor cell taken from the subject, the peptide comprising the amino acid sequence of SEQ ID NO: 1, wherein an increase in levels of the peptide compared to levels in a normal cell indicates that the tumor has a higher probability of being sensitive or resistant to therapy.
27. A method for determining the likelihood that a tumor in a subject will respond to a given
therapy, the method comprising determining the levels of a peptide in tumor cells from the subject, the peptide comprising the amino acid sequence of SEQ ID NO: 1, wherein an increase in levels of the peptide compared to levels in a normal cell indicates that the tumor has a lower probability of being sensitive or resistant to therapy.
28. The method of claim 26 or 27 wherein the therapy is a combination therapy.
29. A method of monitoring the progression of an abnormal condition in a subject, the method comprising determining the levels of ΑΙΒ1-Δ4 peptide in tumor cells from the subject at a first and second time point, the peptide comprising the amino acid sequence of SEQ ID NO: 1, wherein a difference in the levels of ΑΙΒ1-Δ4 peptide over time are indicative of the progression of the abnormal condition in the subject.
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Citations (2)

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US7163789B2 (en) * 2000-05-26 2007-01-16 Xu Qi Chen Cancer diagnosis method
US20080102491A1 (en) * 2004-03-12 2008-05-01 Unigene Laboratories Inc. Bacterial host cell for the direct expression of peptides

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7163789B2 (en) * 2000-05-26 2007-01-16 Xu Qi Chen Cancer diagnosis method
US20080102491A1 (en) * 2004-03-12 2008-05-01 Unigene Laboratories Inc. Bacterial host cell for the direct expression of peptides

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