WO2007075933A2 - Translocation et kinase csf1r mutante dans la leucemie humaine - Google Patents

Translocation et kinase csf1r mutante dans la leucemie humaine Download PDF

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WO2007075933A2
WO2007075933A2 PCT/US2006/048867 US2006048867W WO2007075933A2 WO 2007075933 A2 WO2007075933 A2 WO 2007075933A2 US 2006048867 W US2006048867 W US 2006048867W WO 2007075933 A2 WO2007075933 A2 WO 2007075933A2
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csf1
csf1r
polypeptide
rbm6
kinase
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PCT/US2006/048867
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WO2007075933A3 (fr
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Ting-Lei Gu
Valerie Goss
Roberto Polakiewicz
Brian Druker
Denise Walters
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Cell Signaling Technology, Inc.
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Publication of WO2007075933A3 publication Critical patent/WO2007075933A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/85—Fusion polypeptide containing an RNA binding domain

Definitions

  • the invention relates generally to proteins and genes involved in cancer, and to the detection, diagnosis and treatment of cancer.
  • AML acute megakaryoblastic leukemia
  • AML-M7 acute megakaryoblastic leukemia
  • BCR- ABL oncoprotein a tyrosine kinase fusion protein
  • CML chronic myelogenous leukemia
  • the BCR-ABL oncoprotein which is found in at least 90-95% of CML cases, is generated by the translocation of gene sequences from the c-ABL protein tyrosine kinase on chromosome 9 into BCR sequences on chromosome 22, producing the so-called Philadelphia chromosome. See, e.g. Kurzock et ai., N. Engl. J. Med. 319: 990-998 (1988).
  • the translocation is also observed in acute lymphocytic leukemia and AML cases.
  • translocations leading to mutant or fusion proteins implicated in a variety of other hematological cancers have been described. See, e.g., review by Falini et ai, Blood 99(2): 409-426 (2002).
  • translocations are the NPM-ALK fusion (involved in anaplastic large cell lymphoma), the E2A-PBX/HLF fusion (involved in B- cell acute lymphoblastic leukemia), and the NPM-MLF1 fusion (involved in myelodysplastic syndrome/AML). See id.
  • CSF1 R expression and/or activation have been found in acute myeloid leukemia and myelodysplastic syndrome (MDS). See, e.g. Casas etai, Leuk. Lymphoma 44: 1935-1941 (2003); Li etal., Leukemia Res. 26: 377-382 (2002). Elevated coexpression CSF1R and its ligand, CSF1 , have been correlated with invasiveness and poor prognosis of epithelial tumors including breast, ovarian and endometrial cancer. See Kacinski BM, Ann. Med. 27: 79-85 (1995).
  • Identifying translocations and mutations in human cancers is highly desirable because it can lead to the development of new therapeutics that target such mutant or fusion proteins, and to new diagnostics for identifying patients that have such gene translocations.
  • BCR-ABL has become a target for the development of therapeutics to treat leukemia.
  • Gleevec® Imatinib mesylate, STI-571
  • STI-571 a small molecule inhibitor of the ABL kinase
  • This drug is the first of a new class of anti-proliferative agents designed to interfere with the signaling pathways that drive the growth of tumor cells.
  • This drug represents a significant advance over the conventional therapies for CML and ALL, chemotherapy and radiation, which are plagued by well known side-effects and are often of limited effect since they fail to specifically target the underlying causes of the malignancies.
  • reagents and methods for specifically detecting BCR-ABL fusion protein in patients, in order to identify patients most likely to respond to targeted inhibitors like Gleevec® have been described.
  • a novel gene translocation, (3p21, 5q33), in human myelogenous leukemia (AML) that results in a fusion protein combining part of RNA Binding Protein-6 (RBM6) with Macrophage Colony Stimulating Factor-1 Receptor (CSF1R) kinase has now been identified.
  • the invention therefore provides, in part, isolated polynucleotides and vectors encoding the disclosed mutant CSF1R polypeptides, probes and assays for detecting them, isolated mutant CSF1R polypeptides, recombinant mutant polypeptides, and reagents for detecting the mutant CSF1R polynucleotides and polypeptides.
  • Fig. 1 - shows the location of the RBM-6 gene and CSF1R gene on chromosomes 3 and 5 respectively (panel A), and the domain locations of full length RBM-6 and CSF1 R proteins as well as those of RBM6-CSF1R fusion protein (panel B); the fusion junction occurs at residue 574 in the juxtamembrane domain of CSF1R.
  • Fig. 2 - is the amino acid sequence (1 letter code) of human RBM6-CSF1 R fusion protein (SEQ ID NO: 1) (top panel) with coding DNA sequence also indicated (SEQ ID NO: 2) (bottom panel); the residues of the RBM-6 moiety are in italics, while the residues of the split kinase domain of CSF1R are in bold.
  • Fig. 3A-3B - is the amino acid sequence (1 letter code) of human RBM-6 protein (SEQ ID NO: 3) (SwissProt Accession No. P78332) with coding DNA sequence also indicated (SEQ ID NO: 4) (GeneBank Accession No. NM_005777); the residues involved in the translocation are underlined.
  • Fig. 4A-4B - is the amino acid sequence (1 letter code) of human CSF1 R kinase (SEQ ID NO: 5) (SwissProt Accession No. P07333) with coding DNA sequence also indicated (SEQ ID NO: 6) (GeneBank Accession No. NM_005211); the residues involved in the translocation are underlined, while the residues of the split kinase domain are in bold.
  • Fig. 5 is a Western blot analysis of extracts from a human AML cell line (MKPL-1) showing expression of a truncated/fusion form of CSF1 R.
  • Fig. 6 - is a Western blot analysis of extracts from a human AML cell line (MKPL-1) showing inhibition of CSF1R kinase activity, as well as its downstream target STAT5 and ERK by Gleevec® (panel A), and a graph depicting the inhibition of cell growth in various cell types by lmatinib (Gleevec®) in a 48 hour MTT assay demonstrating that growth of MKPL-1 is specifically inhibited by Imati ⁇ ib (Gleevec®) (panel B). It is accompanied by an increase in apoptosis (panel C).
  • Fig. 7 - is a Western blot analysis of extracts from a human AML cell line (MKPL-1) showing inhibition of CSF1R kinase activity, as well as its downstream target STAT5 and ERK by siRNA silencing (panel A), and a graph depicting the inhibition of cell growth in various cell types by siRNA silencing in a 72 hour MTT assay demonstrating that growth of MKPL-1 is specifically inhibited by siRNA against CSF1R protein (panel B). It is accompanied by an increase in apoptosis (panel C).
  • Fig. 8 - is a gel depicting detection of CSF1 R via the 5' RACE product with CSF 1 R primers after 2 rounds of PCR; UAP stands for Universal Amplification Primer, GSP for Gene Specific Primer.
  • Fig. 9 - are gels depicting the detection of the fusion gene formed by the RBM-6 and CSF1 R translocation by RT-PCR; the DNA (and protein) sequence of the exon 2/exon 12 fusion junction is shown below (SEQ ID NO: 7 and SEQ ID NO: 8).
  • Fig. 10 presents (top) diagrams showing the location of exon 2 in the RBM-6 gene and exon 12 in the CSF1R gene that are involved in the translocation resulting in the fusion protein; also shown (bottom) are primer locations used for PCR amplification of the fusion protein, and a gel depicting the expected size of PCR product.
  • Fig. 11 - are gels showing the cloning and expression of the RBM6-CSF1 R fusion protein in Baf3 cells (panels A and B), and a graph depicting the IL3-independent growth of Baf3 cells expressing the RBM6- CSF1 R fusion protein, as compared to parental BaF3 cells or BaF3 cells transfected with an empty vector (panel C).
  • AML-M7 human acute megakaryoblastic leukemia
  • AML acute myelogenous leukemia
  • the translocation which occurs between chromosome (3p21) and chromosome (5q33), produces a fusion protein that combines the N- terminus of RNA Binding Protein 6 (RBM-6), an 1123 amino acid RNA binding protein (also known as DEF-3), with the juxtamembrane and split kinase domains of Macrophage Colony Stimulating Factor-I Receptor (CSF1R), a 972 amino acid receptor tyrosine kinase.
  • RBM-6 RNA Binding Protein 6
  • DEF-3 1123 amino acid RNA binding protein
  • CSF1R Macrophage Colony Stimulating Factor-I Receptor
  • the resulting RBM6-CSF1 R fusion protein which is 435 amino acids and retains CSF1R kinase activity independent of the RBM6 moiety, was confirmed to drive the proliferation and survival of a human leukemia cell line, MKPL-1.
  • MKPL-1 human leukemia cell line
  • RBM-6 is an RNA binding protein that is expressed in most human tissues, and which specifically binds poly(G) RNA homopolymers. Defects in RBM-6 expression and/or activity have been found in small cell and non-small cell lung carcinomas. See Drabkin et al., supra.
  • CSF1R the product of the oncogene c-fms, is a transmembrane receptor tyrosine kinase that is the cell surface receptor for macrophage Colony Stimulating Factor-1 (CSF-1).
  • CSF1R is expressed, in humans, in bone marrow and differentiated blood mononuclear cells, and it plays an important role in regulating the normal proliferation and differentiation of macrophages and trophoblasts.
  • CSF 1 R expression and/or activation have been found in acute myeloid leukemia and myelodysplastic syndrome (MDS). See Casas et al., supra.; Li et ai, supra.
  • elevated coexpression CSF 1 R and its ligand, CSF1 has been correlated with invasiveness and poor prognosis of epithelial tumors including breast, ovarian and endometrial cancer. See Kacinski supra.
  • Activating point mutations at codons L301 and Y969 of CSF1R have been detected in AML and CMML. See Ridge et al., supra.; Tobal et al., supra.
  • the RBM6-CSF1 R translocation and the expressed fusion protein have presently been isolated and sequenced, and cDNAs for expressing the mutant kinase protein (both as a fusion and as a truncated active kinase) produced.
  • the invention provides, in part, isolated polynucleotides that encode RBM6- CSF1 R fusion polypeptides or truncated active CSF1R polypeptides, nucleic acid probes that hybridize to such polynucleotides, and methods, vectors, and host cells for utilizing such polynucleotides to produce recombinant mutant CSF1 R polypeptides.
  • the invention also provides, in part, isolated polypeptides comprising amino acid sequences encoding RBM6-CSF1 R fusion polypeptides or truncated active CSF1R polypeptides, recombinant mutant polypeptides, and isolated reagents that specifically bind to and/or detect RBM6-CSF1 R fusion polypeptides, or truncated active CSF1R polypeptides, but do not bind to or detect either wild type RBM-6 or wild type CSF1 R.
  • AML the most common type of leukemia, is an aggressive disease, with a 5% year survival rate of less than 20%. See American Cancer Society, supra.
  • the acute megakaryoblastic (AML-7) subtype of AML is a rare form of pediatric AML that is most prevalent in young children with Down's Syndrome. See A. Verschurr, Ophanet Encyclopedia (May 2004) (orpha.net/data/patho/GB/uk-AMLM7.pdf). Treatment of AML remains difficult, and the first-line therapy of aggressive multi-drug chemotherapy regimes is hard on patients and associated with significant mortality. Alternatively, total body irradiation coupled with chemotherapy and bone marrow transplant may be employed, which are similarly traumatic for patients.
  • the discovery of the RBM6-CSF1 R fusion protein resulting from gene translocation which is presently shown to drive proliferation and survival of a subtype of AML (with truncated kinase activity independent of the RBM moiety), enables important new methods for accurately identifying mammalian leukemias (such as AML), as well as other cancers, in which the RBM6-CSF1 R fusion protein or truncated active CSF1R kinase is expressed. These tumors are most likely to respond to inhibitors of the kinase activity of the CSF1 R mutant protein, such as lmatinib (STI-571 ; Gleevec®).
  • the invention provides, in part, methods for detecting the presence of a RBM6-CSF1 R translocation (t(3; 5)(p21, q33)) and/or fusion polypeptide, or a truncated CSF 1 R polynucleotide or truncated active CSF 1 R polypeptide, in a cancer using fusion-specific and mutant- specific reagents of the invention.
  • Such methods may be practiced, for example, to identify a cancer, such as leukemia, that is likely to respond to an inhibitor of the CSF1R kinase activity of the mutant protein.
  • the invention also provides, in part, methods for determining whether a compound inhibits the progression of a cancer characterized by a RBM6- CSF1 R fusion polypeptide or truncated active CSF1 R polypeptide. Further provided by the invention is a method for inhibiting the progression of a cancer that expresses a RBM6-CSF1 R fusion polypeptide or truncated active CSF1 R polypeptide by inhibiting the expression and/or activity of the mutant polypeptide. Such methods are described in further detail below.
  • Antibody refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE, including F a b or antigen-recognition fragments thereof, including chimeric, polyclonal, and monoclonal antibodies.
  • humanized antibody refers to antibody molecules in which amino acids have been replaced in the non- antigen binding regions in order to more closely resemble a human antibody, while still retaining the original binding ability.
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active refers to the capability of the natural, recombinant, or synthetic RBM6-CSF1 R fusion polypeptide or truncated active CSF1R polypeptide, or any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • biological sample is used in its broadest sense, and means any biological sample suspected of containing RBM6-CSF1R fusion or truncated CSF1 R polynucleotides or polypeptides or fragments thereof, and may comprise a cell, chromosomes isolated from a cell (e.g., a spread of metaphase chromosomes), genomic DNA (in solution or bound to a solid support such as for Southern analysis), RNA (in solution or bound to a solid support such as for northern analysis), cDNA (in solution or bound to a solid support), an extract from cells, blood, urine, marrow, or a tissue, and the like.
  • chromosomes isolated from a cell
  • genomic DNA in solution or bound to a solid support such as for Southern analysis
  • RNA in solution or bound to a solid support such as for northern analysis
  • cDNA in solution or bound to a solid support
  • an extract from cells blood, urine, marrow, or a tissue, and the like.
  • CSF 1 R polynucleotide and polypeptide is meant a cancer in which the RBM6- CSF 1 R gene translocation and/or expressed fusion polypeptide are present, or in which a truncated CSF1R polynucleotide and/or truncated active polypeptide are present, as compared to a cancer in which such translocation and/or fusion polypeptide are not present.
  • the presence of mutant polypeptide may drive, in whole or in part, the growth and survival of such cancer.
  • Consensus refers to a nucleic acid sequence which has been re- sequenced to resolve uncalled bases, or which has been extended using XL-PCRTM (Perkin Elmer, Norwalk, Conn.) in the 5* and/or the 3 1 direction and re-sequenced, or which has been assembled from the overlapping sequences of more than one lncyte clone using the GELVIEWTM Fragment Assembly system (GCG, Madison, Wis.), or which has been both extended and assembled.
  • XL-PCRTM Perkin Elmer, Norwalk, Conn.
  • CSF1R kinase-inhibiting therapeutic means any composition comprising one or more compounds, chemical or biological, which inhibits, either directly or indirectly, the expression and/or activity of wild type or truncated active CSF1 R kinase, either alone and/or as part of the RBM6-CSF1 R fusion protein.
  • Derivative refers to the chemical modification of a nucleic acid sequence encoding RBM6-CSF1 R fusion polypeptide or truncated active CSF1R polypeptide, or the encoded polypeptide itself. Illustrative of such modifications would be replacement of hydrogen by an alkyl, acyl, or amino group. A nucleic acid derivative would encode a polypeptide that retains essential biological characteristics of the natural molecule.
  • Detectable label with respect to a polypeptide, polynucleotide, or reagent disclosed herein means a chemical, biological, or other modification, including but not limited to fluorescence, mass, residue, dye, radioisotope, label, or tag modifications, etc., by which the presence of the molecule of interest may be detected.
  • “Expression” or “expressed” with respect to RBM6-CSF1 R fusion polypeptide or truncated active CSF1 R polypeptide in a biological sample means significantly expressed as compared to control sample in which this fusion polypeptide is not significantly expressed.
  • Heavy-isotope labeled peptide (used interchangeably with AQUA peptide) means a peptide comprising at least one heavy-isotope label, which is suitable for absolute quantification or detection of a protein as described in WO/03016861, "Absolute Quantification of Proteins and Modified Forms Thereof by Multistage Mass Spectrometry” (Gygi et al.), further discussed below.
  • the term “specifically detects” with respect to such an AQUA peptide means the peptide will only detect and quantify polypeptides and proteins that contain the AQUA peptide sequence and will not substantially detect polypeptides and proteins that do not contain the AQUA peptide sequence.
  • isolated refers to nucleic or amino acid sequences that are removed from their natural environment, isolated or separated. They preferably are at least 60% free, more preferably 75% free, and most preferably 90% or more free from other components with * which they are naturally associated.
  • “Mimetic” refers to a molecule, the structure of which is developed from knowledge of the structure of RBM6-CSF1 R fusion polypeptide, or truncated active CSF1 R polypeptide, or portions thereof and, as such, is able to effect some or all of the actions of translocation associated protein-like molecules.
  • “Mutant CSF1R” polynucleotide or polypeptide means a RBM6- CSF1 R fusion polynucleotide or polypeptide, or a truncated CSF1 R polynucleotide or truncated active CSF1R polypeptide, as described herein.
  • Polynucleotide refers to an oligonucleotide, nucleotide, or polynucleotide, and fragments or portions thereof, and to DNA or RNA of genomic or synthetic origin, which may be single- or double-stranded, and represent the sense or anti-sense strand.
  • Polypeptide refers to an oligopeptide, peptide, polypeptide, or protein sequence, and fragments or portions thereof, and to naturally occurring or synthetic molecules. Where "amino acid sequence” is recited herein to refer to an amino acid sequence of a naturally occurring protein molecule, “amino acid sequence” and like terms, such as “polypeptide” or “protein”, are not meant to limit the amino acid sequence to the complete, native amino acid sequence associated with the recited protein molecule.
  • RBM6-CSF1 R fusion polynucleotide refers to the nucleic acid sequence of a substantially purified RBM6-CSF1 R translocation gene product or fusion polynucleotide as described herein, obtained from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic, or recombinant.
  • RBM6-CSF1 R fusion polypeptide refers to the amino acid sequence of a substantially purified RBM6-CSF1 R fusion polypeptide described herein, obtained from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic, or recombinant.
  • the term “does not bind” with respect to an antibody's binding to sequences or antigenic determinants other than that for which it is specific means does not substantially react with as compared to the antibody's binding to antigenic determinant or sequence for which the antibody is specific.
  • stringent conditions with respect to sequence or probe hybridization conditions is the “stringency” that occurs within a range from about Tm minus 5 0 C (5 0 C below the melting temperature (T m ) of the probe or sequence) to about 20 0 C to 25 0 C below T m .
  • Typical stringent conditions are: overnight incubation at 42 0 C in a solution comprising: 50% formamide, 5 X.SSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 X SSC at about 65 0 C.
  • the stringency of hybridization may be altered in order to identify or detect identical or related polynucleotide sequences.
  • Truncated CSF1 R [kinase] polynucleotide refers to the nucleic acid sequence of a substantially purified truncated CSF1 R polynucleotide that encodes an active truncated active CSF1R kinase polypeptide as described herein, obtained from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic, or recombinant.
  • Truncated active CSF1R [kinase] polypeptide refers to the amino acid sequence of a substantially purified, truncated CSF1R kinase polypeptide that retains kinase activity (and comprises the split kinase domain) but does not comprise the extracellular or transmembrane domains of wild type CSF1R kinase, as described herein, obtained from any species, particularly mammalian, including bovine, ovine, porcine, murine, equine, and preferably human, from any source whether natural, synthetic, semi-synthetic, or recombinant.
  • a “variant" of a mutant CSF1 R polypeptide refers to an amino acid sequence that is altered by one or more amino acids.
  • the variant may have "conservative" changes, wherein a substituted amino acid has similar structural or chemical properties, e.g., replacement of leucine with isoleucine. More rarely, a variant may have "nonconservative" changes, e.g., replacement of a glycine with a tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both.
  • Guidance in determining which amino acid residues may be substituted, inserted, or deleted without abolishing biological or immunological activity may be found using computer programs well known in the art, for example, DNASTAR software.
  • the novel human gene translocation disclosed herein which occurs between chromosome (3p21) and chromosome (5q33) in human leukemia and results in expression of a fusion protein that combines the N-terminus of RBM-6 with the juxtamembrane and split kinase domains of CS.F1R, was surprisingly identified during examination of global phosphorylated peptide profiles in extracts from a cell line (MKPL-1) of human acute megakaryoblastic leukemia (AML-M7), a subtype of acute myelogenous leukemia (AML).
  • MKPL-1 human acute megakaryoblastic leukemia
  • AML-M7 a subtype of acute myelogenous leukemia
  • the phosphorylation profile of this cell line was elucidated using a recently described technique for the isolation and mass spectrometric characterization of modified peptides from complex mixtures (see U.S. Patent Publication No. 20030044848, Rush et a/., "Immunoaffinity Isolation of Modified Peptides from Complex Mixtures” (the "IAP” technique), as further described in Example 1 below.
  • IAP immunoaffinity Isolation of Modified Peptides from Complex Mixtures
  • Application of the IAP technique using a phosphotyrosine-specific antibody (CELL SIGNALING TECHNOLOGY, INC., Beverly, MA, 2003/04 Cat. #9411), identified that the MKPL-1 cell line expresses CSF1R kinase, but that the kinase was apparently truncated (see Figure 5).
  • the screen identified many other activated kinases in the cell line including CSF1R. Analysis of the sequence 5' to CSF 1 R by 5' RACE then identified that the kinase was fused to the N-terminus of RBM-6 (see Fig. 8).
  • the RBM6-CSF1R fusion gene was amplified by PCR, isolated, and sequenced (see Example 4). As shown in panel B of Figure 1 , the RBM6-CSF1 R translocation combines the N-terminus of RBM-6 (amino acids 1-36) with the juxtamembrane and split kinase domains of CSF1R (amino acids 574-972) (see also SEQ ID NOs: 3 and 5). The RBM6- CSF1 R fusion polypeptide retains the N-terminal 36 amino acid of RBM-6, which includes part of the predicted POZ (poxvirus and zinc finger) domain (POZ domains are known to be involved in protein-protein interactions).
  • POZ poxvirus and zinc finger
  • the resulting RBM6-CSF1 R fusion protein which comprises 435 amino acids (see panel B of Figure 1 and Figure 2 (SEQ ID NO: 1)), retains kinase activity of CSF1R.
  • the exons involved and the fusion junction are shown in Figure 9 (bottom panel).
  • cDNA encoding the fusion protein was prepared and transfected into a murine hematopoietic progenitor cell line (BaF3) to confirm that expression of the mutant CSF1R kinase transforms the cells and drives proliferation and growth (see Example 5; Figure 11 (panel C)).
  • PCR probes were used to detect the presence of the fusion protein in this AML cell line.
  • the present Invention provides, in part, isolated polynucleotides that encode RBM6-CSF1 R fusion polypeptides and truncated active CSF1R polypeptides, nucleotide probes that hybridize to such polynucleotides, and methods, vectors, and host cells for utilizing such polynucleotides to produce recombinant fusion polypeptides.
  • nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Science).
  • Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule.
  • the actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • nucleotide sequence of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides, and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G, C and U), where each thymidine deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U).
  • RNA molecule having the sequence of SEQ ID NO: 2 set forth using deoxyribonucleotide abbreviations is intended to indicate an RNA molecule having a sequence in which each deoxyribonucleotide A, G or C of SEQ ID NO: 2 has been replaced by the corresponding ribonucleotide A, G or C, and each deoxyribonucleotide T has been replaced by a ribonucleotide U.
  • the invention provides an isolated polynucleotide comprising a nucleotide sequence at least 95% identical to a sequence selected from the group consisting of:
  • RNA Binding Protein-6- Macrophage Colony Stimulating Factor-1 Receptor (RBM6-CSF1R) fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 1;
  • nucleotide sequence encoding a RBM6-CSF1 R fusion polypeptide, said nucleotide sequence comprising the nucleotide sequence of SEQ ID NO: 2;
  • nucleotide sequence comprising the N-terminal nucleotide sequence of RBM-6 (residues 1-108 of SEQ ID NO: 4) and the split kinase domain nucleotide sequence of CSF 1 R (residues 1746-2730 of SEQ ID NO: 6);
  • a nucleotide sequence comprising at least six contiguous nucleotides encompassing the fusion junction (residues 106-111 of SEQ ID NO: 2) of a RBM6-CSF1 R fusion polynucleotide;
  • nucleotide sequence encoding a polypeptide comprising at least six contiguous amino acids encompassing the fusion junction (residues 36-37 of SEQ ID NO: 1) of a RBM6-CSF1 R fusion polypeptide;
  • nucleotide sequence comprising up to thirty contiguous nucleotides encompassing the truncation point (residue 1722 of SEQ ID NO: 6) of wild type CSF1R kinase polynucleotide;
  • (k) a nucleotide sequence complementary to any of the nucleotide sequences of (a)-(j).
  • a nucleic acid molecule of the present invention encoding a mutant CSF1 R polypeptide of the invention may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material.
  • the RBM6-CSF1R fusion polynucleotide described in Figure 2 was isolated from genomic DNA from a human AML cell line (as further described in Example 4 below).
  • the fusion gene can also be identified in genomic DNA or cDNA libraries in other leukemias or cancers in which the RBM6-CSF1 R translocation (3p21 , 5q33) occurs, or in which a deletion or alternative translocation results in expression of a truncated active CSF1R kinase lacking the extracellular and transmembrane domains of the wild type kinase.
  • the determined nucleotide sequence of the RBM6-CSF1R translocation gene (SEQ ID NO: 2) encodes a kinase fusion protein of 435 amino acid residues (see Figure 2 (SEQ ID NO: 1) and Figure 1).
  • the RBM6-CSF1 R fusion polynucleotide comprises the portion of the nucleotide sequence of wild type RBM6 (see Figure 3 (SEQ ID NO: 4)) that encodes the N-terminus (amino acids 1-36) of that protein with the portion of the nucleotide sequence of wild type CSF1R (see Figure 4 (SEQ ID NO: 6)) that encodes the juxtamembrane and split kinase domains of that protein. See Figure 1.
  • the split kinase domain comprises residues 45-373 in the fusion protein (encoded by nucleotides 135-1119 of the fusion polynucleotide).
  • the present invention provides, in part, the mature form of the RBM6-CSF1 R fusion protein.
  • proteins secreted by mammalian cells have a signal or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
  • Most mammalian cells and even insect cells cleave secreted proteins with the same specificity.
  • cleavage of a secreted protein is not entirely uniform, which results in two or more mature species on the protein.
  • the present invention provides, in part, nucleotide sequences encoding a mature RBM6-CSF1 R fusion polypeptide having the amino acid sequence encoded by the cDNA clone identified as ATCC Deposit No. PTA-7309, which was deposited with the American Type Culture Collection (Manassas, Virginia, U.S.A.) on December 29, 2005 in accordance with the provisions of the Budapest Treaty.
  • RBM6-CSF1 R polypeptide having the amino acid sequence encoded by the deposited cDNA clone is meant the mature form of this fusion protein produced by expression in a mammalian cell (e.g., COS cells, as described below) of the complete open reading frame encoded by the human DNA sequence of the deposited clone.
  • a mammalian cell e.g., COS cells, as described below
  • polynucleotides of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
  • the DNA may be double-stranded or single-stranded.
  • Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
  • Isolated polynucleotides of the invention are nucleic acid molecules, DNA or RNA, which have been removed from their native environment.
  • recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
  • Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
  • Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention.
  • Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
  • Isolated polynucleotides of the invention include the DNA molecule shown in Figure 2 (SEQ ID NO: 2), DNA molecules comprising the coding sequence for the mature RBM6-CSF1 R fusion protein shown in Figure 1 (SEQ ID NO: 1), and DNA molecules that comprise a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode a CSF1R mutant polypeptide of the invention.
  • the genetic code is well known in the art, thus, it would be routine for one skilled in the art to generate such degenerate variants.
  • the invention provides an isolated polynucleotide encoding the RBM6-CSF1 R fusion polypeptide comprising the RBM6-CSF1R translocation nucleotide sequence contained in the above-described deposited cDNA clone.
  • such nucleic acid molecule will encode the mature fusion polypeptide encoded by the deposited cDNA clone.
  • the invention provides an isolated nucleotide sequence encoding a RBM6-CSF1 R fusion polypeptide comprising the N-terminal amino acid sequence of RBM-6 (residues 1-36 of SEQ ID NO: 3) and the split kinase domain of CSF1R (residues 582-910 of SEQ ID NO: 5).
  • the polypeptide comprising the split kinase domain of CSF1R comprises residues 574-972 of SEQ ID NO: 5 (see Figure 1, panel B).
  • the aforementioned N-terminal amino acid sequence of RBM-6 and split kinase domain of CSF1R are encoded by nucleotide sequences comprising nucleotides 1-108 of SEQ ID NO: 4 and nucleotides 1746-2730 of SEQ ID NO: 6, respectively.
  • the present invention also provides in part a truncated active CSF1R kinase comprising the split kinase domains of the wild type protein but lacking the extracellular and transmembrane domains of the wild type protein (see Figure 1 (panel B) - the truncated kinase comprises residues 574-972 of the wild type CSF1R kinase).
  • an isolated polynucleotide comprising a nucleic acid sequence encoding the truncated active CSF1R kinase (e.g. nucleotides 1722-2916 of SEQ ID NO: 6) but not encoding the extracellular and transmembrane domains of the wild type protein.
  • nucleotide sequences encoding a truncated active CSF1R kinase polypeptide comprising the split kinase domain of CSF1 R (residues 582- 910 of SEQ ID NO: 5) but not comprising the extracellular or transmembrane domains of wild type CSF1R.
  • the present invention also provides, in part, nucleotide sequences encoding a truncated active CSF1 R polypeptide having the amino acid sequence (residues 574-972 of SEQ ID NO: 1) encoded by the deposited cDNA clone described above (ATCC Deposit No. PTA-7309).
  • the invention further provides isolated polynucleotides comprising nucleotide sequences having a sequence complementary to one of the mutant CSF1R polynucleotides of the invention.
  • isolated molecules particularly DNA molecules, are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the RBM6-CSF1 R fusion protein or truncated active CSF1R kinase in human tissue, for instance, by Northern blot analysis, as further described in Section F below.
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated RBM6-CSF1 R polynucleotide or truncated CSF1R polynucleotide of the invention is intended fragments at least about 15 nucleotides, and more preferably at least about 20 nucleotides, still more preferably at least about 30 nucleotides, and even more preferably, at least about 40 nucleotides in length, which are useful as diagnostic probes and primers as discussed herein.
  • fragments of about 50-1500 nucleotides in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the mutant CSF1R nucleotide sequences of the deposited cDNAs or as shown in Figure 2 (SEQ ID NO: 2).
  • a fragment at least 20 nucleotides in length is intended fragments which include 20 or more contiguous bases from the respective nucleotide sequences from which the fragments are derived.
  • DNA fragments are routine to the skilled artisan, and may be accomplished, by way of example, by restriction endonuclease cleavage or shearing by sonication of DNA obtainable from the deposited cDNA clone or synthesized according to the sequence disclosed herein. Alternatively, such fragments can be directly generated synthetically.
  • nucleic acid -fragments or probes of the present invention include nucleic acid molecules encoding the fusion junction of the RBM6- CSF1 R translocation gene product (see Figure 1 , panel B, and Figure 9, bottom panel).
  • an isolated polynucleotide of the invention comprises a nucleotide sequence/fragment comprising at least six contiguous nucleotides encompassing the fusion junction (nucleotides 106-111 of SEQ ID NO: 2) of a RBM6-CSF1 R fusion polynucleotide (see Figure 9, bottom panel (SEQ ID NO: 8)).
  • an isolated polynucleotide of the invention comprises a nucleotide sequence/ fragment that encodes a polypeptide comprising at least six contiguous amino acids encompassing the fusion junction (residues 36-37 of SEQ ID NO: 1 ) of a RBM6-CSF1 R fusion polypeptide (see Figure 9, bottom panel (SEQ ID NO: 7)).
  • an isolated polynucleotide of the invention comprises a nucleotide sequence/ fragment comprising up to thirty contiguous nucleotides encompassing the truncation point (residue 1722 of SEQ ID NO: 6) in the wild type CSF1R kinase gene/polynucleotide.
  • the invention provides an isolated polynucleotide that hybridizes under stringent hybridization conditions to a portion of a mutant CSF1 R polynucleotide of the invention as describe herein.
  • stringent hybridization conditions is intended overnight incubation at 42 0 C in a solution comprising: 50% formamide, 5 X.SSC (750 mM NaCI, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 micrograms/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at about 65 0 C.
  • a polynucleotide that hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. These are useful as diagnostic probes and primers as discussed above and in more detail below.
  • polynucleotides hybridizing to a larger portion of the reference polynucleotide e.g. the mature RBM6-CSF1R fusion polynucleotide described in Figure 2 (SEQ ID NO: 2)
  • a portion 50-750 nt in length, or even to the entire length of the reference polynucleotide are also useful as probes according to the present invention, as are polynucleotides corresponding to most, if not all, of the nucleotide sequences of the deposited cDNAs or the nucleotide sequences shown in Figure 2 (SEQ ID NO: 2), residues 1722-2916 of SEQ ID NO: 2, or Figure 9 panel B, bottom) (SEQ ID NO: 7).
  • a portion of a polynucleotide of "at least 20 nucleotides in length,” for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide. As indicated, such portions are useful diagnostically either as a probe according to conventional DNA hybridization techniques or as primers for amplification of a target sequence by the polymerase chain reaction (PCR), as described, for instance, in MOLECULAR CLONING, A LABORATORY MANUAL, 2nd. edition, Sambrook, J., Fritsch, E. F. and Maniatis, T., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y.
  • PCR polymerase chain reaction
  • a polynucleotide which hybridizes only to a poly A sequence such as the 3 1 terminal poly(A) tract of the RBM6-CSF1R sequence shown in Figure 2 (SEQ ID NO: 2)) or to a complementary stretch of T (or U) resides, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double- stranded cDNA clone).
  • nucleic acid molecules of the present invention which encode a mutant CSF1 R polypeptide of the invention, may include but are not limited to those encoding the amino acid sequence of the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding the leader or secretory sequence, such as a pre-, or pro- or pre-pro-protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non- translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example-ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa- histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37: 767 (1984).
  • other such fusion proteins include the RBM6-CSF1 R fusion polypeptide or truncated active CSF 1 R polypeptide itself fused to Fc at the N- or C- terminus.
  • the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of a RBM6-CSF1 R fusion polypeptide or truncated active CSF1 R polypeptide disclosed herein.
  • Variants may occur naturally, such as a natural allelic variant.
  • allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. See, e.g. GENES II, Lewin, B., ed., John Wiley & Sons, New York (1985).
  • Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
  • variants include those produced by nucleotide substitutions, deletions or additions.
  • the substitutions, deletions or additions may involve one or more nucleotides.
  • the variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities (e.g. kinase activity) of the mutant CSF1R polypeptides disclosed herein. Also especially preferred in this regard are conservative substitutions.
  • inventions include isolated polynucleotides comprising a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to a mutant CSF1 R polynucleotide of the invention (for example, a nucleotide sequence encoding the RBM6-CSF1 R fusion polypeptide having the complete amino acid sequence shown in Figure 2 (SEQ ID NO: 1; or a nucleotide sequence encoding the N-terminal of RBM-6 and the split kinase domains of CSF1R (see Figure 1, panel B; and Figures 3 and 4); or a nucleotide complementary to such exemplary sequences).
  • a mutant CSF1 R polynucleotide of the invention for example, a nucleotide sequence encoding the RBM6-CSF1 R fusion polypeptide having the complete amino acid sequence shown in Figure 2 (SEQ ID NO: 1; or a nucleotide sequence
  • polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding a mutant CSF1R polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence encoding the mutant CSF1 R polypeptide.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
  • These mutations of the reference sequence may occur at the 5' or 3 1 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • nucleotide sequence shown in Figure 2 SEQ ID NO: 2
  • nucleotide sequence of the deposited cDNA clones described above can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of homology between two sequences.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • the present invention includes in its scope nucleic acid molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in Figure 2 (SEQ ID NO: 2), or to nucleotides 1722-2916 of SEQ ID NO: 2, or to the nucleic acid sequences of the deposited cDNAs, irrespective of whether they encode a polypeptide having CSF1R kinase activity. This is because even where a particular nucleic acid molecule does not encode a fusion polypeptide having CSF1R kinase activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
  • PCR polymerase chain reaction
  • nucleic acid molecules of the present invention that do not encode a polypeptide having kinase include, inter alia, (1) isolating the RBM6-CSF1 R translocation gene, or truncated CSF1 R gene, or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the RBM6-CSF1 R translocation gene or truncated CSF1 R gene, as described in Verma ef a/., HUMAN CHROMOSOMES: A MANUAL OF BASIC TECHNIQUES, Pergamon Press, New York (1988); and Northern Blot analysis for detecting RBM6-CSF1 R fusion protein or truncated CSF1 R kinase mRNA expression in specific tissues.
  • FISH in situ hybridization
  • nucleic acid molecules having sequences at least 95% identical to a mutant CSF1R polypeptide of the invention or to the nucleic acid sequence of the deposited cDNAs which do, in fact, encode a fusion polypeptide having CSF1R kinase activity.
  • Such activity may be similar, but not necessarily identical, to the activity of the RBM6- CSF1 R fusion protein and truncated active CSF1R kinase disclosed herein (either the full-length protein, the mature protein, or a protein fragment that retains kinase activity), as measured in a particular biological assay.
  • the kinase activity of CSF1R can be examined by determining its ability to phosphorylate one or more tyrosine containing peptide substrates, for example, "Src-related peptide" (RRLIEDAEYAARG), which is a substrate for many receptor and nonreceptor tyrosine kinases.
  • tyrosine containing peptide substrates for example, "Src-related peptide” (RRLIEDAEYAARG), which is a substrate for many receptor and nonreceptor tyrosine kinases.
  • SEQ ID NO: 2 nucleic acid sequence shown in Figure 2
  • degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay.
  • nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide that retains CSF1 R kinase activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid).
  • Bowie et al. "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions," Science 247: 1306-1310 (1990), which describes two main approaches for studying the tolerance of an amino acid sequence to change.
  • the first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection.
  • the second approach uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality. These studies have revealed that proteins are surprisingly tolerant of amino acid substitutions. Skilled artisans familiar with such techniques also appreciate which amino acid changes are likely to be permissive at a certain position of the protein. For example, most buried amino acid residues require nonpolar side chains, whereas few features of surface side chains are generally conserved. Other such phenotypically silent substitutions are described in Bowie et a!., supra., and the references cited therein.
  • Methods for DNA sequencing may be used to practice any polynucleotide embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE® (US Biochemical Corp, Cleveland, Ohio), Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, III.), or combinations of recombinant polymerases and proofreading exonucleases such as the ELONGASE Amplification System marketed by Gibco BRL (Gaithersburg, Md.).
  • the process is automated with machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI 377 DNA sequencers (Perkin Elmer).
  • machines such as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), Peltier Thermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI 377 DNA sequencers (Perkin Elmer).
  • Polynucleotide sequences encoding a mutant CSF1R polypeptide of the invention may be extended utilizing a partial nucleotide sequence and employing various methods known in the art to detect upstream sequences such as promoters and regulatory elements.
  • one method that may be employed "restriction-site" PCR, uses universal primers to retrieve unknown sequence adjacent to a known locus (Sarkar, G., PCR Methods Applic. 2: 318-322 (1993)).
  • genomic DNA is first amplified in the presence of primer to linker sequence and a primer specific to the known region.
  • Exemplary primers are those described in Example 4 herein (see also Figure 10, bottom panel).
  • amplified sequences are then subjected to a second round of PCR with the same linker primer and another specific primer internal to the first one.
  • Products of each round of PCR are transcribed with an appropriate RNA polymerase and sequenced using reverse transcriptase.
  • Inverse PCR may also be used to amplify or extend sequences using divergent primers based on a known region (Triglia etal., Nucleic Acids Res. 16: 8186 (1988)).
  • the primers may be designed using OLIGO 4.06 Primer Analysis software (National Biosciences Inc., Madison, Wis.
  • Minn. or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68-72 0 C.
  • the method uses several restriction enzymes to generate a suitable fragment in the known region of a gene. The fragment is then circularized by intramolecular ligation and used as a PCR template.
  • Another method which may be used is capture PCR which involves PCR amplification of DNA fragments adjacent to a known sequence in human and yeast artificial chromosome DNA (Lagerstrom et al., PCR Methods Applic. 1: 111-119 (1991)). In this method, multiple restriction enzyme digestions and ligations may also be used to place an engineered double-stranded sequence into an unknown portion of the DNA molecule before performing PCR. Another method which may be used to retrieve unknown sequences is that described in Parker et al., Nucleic Acids Res. 19: 3055-3060 (1991)). Additionally, one may use PCR, nested primers, and PROMOTERFINDER® libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). This process avoids the need to screen libraries and is useful in finding intron/exon junctions.
  • libraries that have been size-selected to include larger cDNAs.
  • random-primed libraries are preferable, in that they will contain more sequences that contain the 5' regions of genes. Use of a randomly primed library may be especially preferable for situations in which an oligo d(T) library does not yield a full-length cDNA.
  • Genomic libraries may be useful for extension of sequence into the 5* and 3' non-transcribed regulatory regions.
  • Capillary electrophoresis systems which are commercially available, may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capillary sequencing may employ flowable polymers for electrop.horetic separation, four different fluorescent dyes (one for each nucleotide) which are laser activated, and detection of the emitted wavelengths by a charge coupled device camera.
  • Output/light intensity may be converted to electrical signal using appropriate software (e.g. GENOTYPERTM and SEQUENCE NAVIGATORTM, Perkin Elmer) and the entire process from loading of samples to computer analysis and electronic data display may be computer controlled.
  • Capillary electrophoresis is especially preferable for the sequencing of small pieces of DNA that might be present in limited amounts in a particular sample.
  • the present invention also provides recombinant vectors that comprise an isolated polynucleotide of the present invention, host cells which are genetically engineered with the recombinant vectors, and the production of recombinant RBM6-CSF1R polypeptides, truncated active CSF1 R polypeptides, or fragments thereof by recombinant techniques.
  • Recombinant constructs may be introduced into host cells using well-known techniques such infection, transduction, transfection, transvection, electroporation and transformation.
  • the vector may be, for example, a phage, plasmid, viral or retroviral vector.
  • Retroviral vectors may be replication competent or replication defective. In the latter case, viral propagation generally will occur only in complementing host cells.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
  • vectors comprising cis-acting control regions to the polynucleotide of interest.
  • Appropriate trans-acting factors may be supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
  • the vectors provide for specific expression, which may be inducible and/or cell type- specific. Particularly preferred among such vectors are those inducible by environmental factors that are easy to manipulate, such as temperature and nutrient additives.
  • Expression vectors useful in the present invention include chromosomal-, episomal- and virus-derived vectors, e.g., vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as cosmids and phagemids.
  • vectors derived from bacterial plasmids, bacteriophage, yeast episomes, yeast chromosomal elements, viruses such as baculoviruses, papova viruses, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses and vectors derived from combinations thereof, such as cosmids and phagemids.
  • the DNA insert comprising a RBM6-CSF1 R polynucleotide or truncated CSF1R polynucleotide of the invention should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few. Other suitable promoters are known to the skilled artisan.
  • the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium 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. Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • bacterial promoters suitable for use in the present invention include the E. coli lacl and lacZ promoters, the T3 and T7 promoters, the gpt promoter, the lambda PR and PL promoters and the trp promoter.
  • Suitable eukaryotic promoters include the CMV immediate early promoter, the HSV thymidine kinase promoter, the early and late SV40 promoters, the promoters of retroviral LTRs, such as those of the Rous sarcoma virus (RSV), and metallothionein promoters, such as the mouse metallothionein-l promoter.
  • yeast Saccharomyces cerevisiae
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH.
  • Introduction of the 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 laboratory manuals, such as Davis et al., BASIC METHODS IN MOLECULAR BIOLOGY (1986). Transcription of DNA encoding a RBM6-CSF1R fusion polypeptide of the present invention by higher eukaryotes may be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting elements of DNA 1 usually about from 10 to 300 bp that act to increase transcriptional activity of a promoter in a given host cell-type.
  • enhancers examples include the SV40 enhancer, which is located on the late side of the replication origin at basepairs 100 to 270, the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • secretion signals may be incorporated into the expressed polypeptide.
  • the signals may be endogenous to the polypeptide or they may be heterologous signals.
  • the polypeptide may be expressed in a modified form, such as a fusion protein (e.g. a GST-fusion), and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage.
  • peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide.
  • a preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins.
  • RBM6-CSF1 R polypeptides or truncated active CSF1R polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • Polypeptides of the present invention include naturally purified products, 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 upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
  • the invention provides a method for producing a recombinant RBM6-CSF1 R fusion polypeptide or truncated active CSF 1 R polypeptide by culturing a recombinant host cell (as described above) under conditions suitable for the expression of the fusion polypeptide and recovering the polypeptide.
  • Culture conditions suitable for the growth of host cells and the expression of recombinant polypeptides from such cells are well known to those of skill in the art. See, e.g., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel FM et a/., eds., Volume 2, Chapter 16, Wiley Interscience.
  • the invention also provides, in part, isolated mutant CSF1R kinase polypeptides and fragments thereof.
  • the invention provides an isolated polypeptide comprising an amino acid sequence at least 95% identical to a sequence selected from the group consisting of: (a) an amino acid sequence encoding a RBM6-CSF1R fusion polypeptide comprising the amino acid sequence of SEQ ID NO: 1 ;
  • the invention provides an isolated RBM6-CSF1 R fusion polypeptide having the amino acid sequence encoded by the first deposited cDNA described above (ATCC Deposit No. PTA-7309).
  • the invention provides an isolated truncated active CSF1R kinase polypeptide having the amino acid sequence (residues 574-972 of SEQ ID NO: 1) encoded the second deposited cDNA described above.
  • recombinant mutant CSF1R polypeptides of the invention are provided, which may be produced using a recombinant vector or recombinant host cell as described above.
  • the invention further includes variations of a RBM6-CSF1R fusion polypeptide or truncated active CSF1 R kinase polypeptide that retain substantial CSF 1 R kinase activity or that include other regions of RBM6 or CSF1R proteins, such as the protein portions discussed below.
  • Such mutants include deletions, insertions, inversions, repeats, and type substitutions (for example, substituting one hydrophilic residue for another, but not strongly hydrophilic for strongly hydrophobic as a rule). Small changes or such "neutral" amino acid substitutions will generally have little effect on activity.
  • conservative substitutions are the replacements, one for another, among the aliphatic amino acids Ala, VaI, Leu and lie; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and GIu 1 substitution between the amide residues Asn and GIn, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.
  • Aromatic phenylalanine tryptophan tyrosine
  • Hydrophobic leucine isoleucine valine
  • Polar glutamine asparagines
  • Basic arginine lysine histidine
  • Acidic aspartic acid glutamic acid
  • Small alanine serine threonine methionine glycine.
  • polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified.
  • a recombinantly produced version of a RBM6-CSF1 R fusion polypeptide or truncated active CSF1R kinase polypeptide of the invention can be substantially purified by the one-step method described in Smith and Johnson, Gene 67: 31-40 (1988).
  • polypeptides of the present invention include the RBM6- CSF1 R fusion polypeptide of Figure 2 (SEQ ID NO: 1) (whether or not including a leader sequence), the fusion polypeptide encoded by the deposited cDNA clone (ATCC No.
  • RBM6-CSF1 R fusion polypeptide comprising the N-terminal amino acid sequence of RBM-6 (residues 1-36 of SEQ ID NO: 3) and the split kinase domain of CSF1R (residues 582-910 of SEQ ID NO: 5), and an amino acid sequence encoding a polypeptide comprising at least six contiguous amino acids encompassing the fusion junction (residues 36-37 of SEQ ID NO: 1) of a RBM6-CSF1 R fusion polypeptide (see Figure 9, bottom panel), as well as polypeptides that have at least 90% similarity, preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98% or 99% similarity to those described above.
  • polypeptides of the present invention also include an amino acid sequence encoding a truncated active CSF1R kinase polypeptide comprising the amino acid sequence of residues 574-972 of SEQ ID NO: 5, but not comprising the extracellular or transmembrane domains of wild type CSF1R; an amino acid sequence encoding a truncated active
  • CSF1R kinase polypeptide comprising the split kinase domain of CSF1R (residues 582-910 of SEQ ID NO: 5), but not comprising the extracellular or transmembrane domains of wild type CSF1R; and the truncated active CSF1R polypeptide encoded by nucleotides 109-1305 of coding sequence of the deposited cDNA clone (ATCC No. PTA-7309), as well as polypeptides that have at least 90% similarity, more preferably at least 95% similarity, and still more preferably at least 96%, 97%, 98% or 99% similarity to those described above.
  • % similarity for two polypeptides is intended a similarity score produced by comparing the amino acid sequences of the two polypeptides using the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, Wis. 53711) and the default settings for determining similarity. Bestfit uses the local homology algorithm of Smith and Waterman (Advances in Applied Mathematics 2: 482-489 (1981)) to find the best segment of similarity between two sequences.
  • polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a RBM6- CSF1 R fusion polypeptide or truncated active CSF 1 R kinase polypeptide of the invention is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid sequence of the mutant CSF1 R polypeptide.
  • a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
  • a RBM6-CSF1 R fusion polypeptide or truncated active CSF 1 R polypeptide of the present invention may be used as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns, for example, using methods well known to those of skill in the art.
  • polypeptides of the present invention can also be used to generate fusion polypeptide specific reagents, such as polyclonal and monoclonal antibodies, or truncated polypeptide specific reagents, which are useful in assays for detecting mutant CSF1R polypeptide expression as described below, or as agonists and antagonists capable of enhancing or inhibiting the function/activity of the mutant CSF1R protein.
  • fusion polypeptide specific reagents such as polyclonal and monoclonal antibodies, or truncated polypeptide specific reagents, which are useful in assays for detecting mutant CSF1R polypeptide expression as described below, or as agonists and antagonists capable of enhancing or inhibiting the function/activity of the mutant CSF1R protein.
  • polypeptides can be used in the yeast two-hybrid system to "capture" RBM6-CSF1 R fusion polypeptide or truncated active CSF1R kinase polypeptide binding proteins,
  • the invention provides a peptide or polypeptide comprising an epitope-bearing portion of a polypeptide of the invention, for example, an epitope comprising the fusion junction of a RBM6-CSF1R fusion polypeptide (see Figure 9, bottom panel).
  • the epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide of the invention.
  • An "immunogenic epitope" is defined as a part of a protein that elicits an antibody response when the whole protein is the immunogen. These immunogenic epitopes are believed to be confined to a few loci on the molecule.
  • an antigenic epitope a region of a protein molecule to which an antibody can bind is defined as an "antigenic epitope.”
  • the number of immunogenic epitopes of a protein generally is less than the number of antigenic epitopes. See, for instance, Geysen et ai, Proc. Natl. Acad. Sci. USA 87:3998-4002 (1983).
  • the production of fusion polypeptide-specific antibodies of the invention is described in further detail below.
  • the antibodies raised by antigenic epitope-bearing peptides or polypeptides are useful to detect a mimicked protein, and antibodies to different peptides may be used for tracking the fate of various regions of a protein precursor which undergoes post-translational processing.
  • the peptides and anti-peptide antibodies may be used in a variety of qualitative or quantitative assays for the mimicked protein, for instance in competition assays since it has been shown that even short peptides (e.g., about 9 amino acids) can bind and displace the larger peptides in immunoprecipitation assays. See, for instance, Wilson et a/., Cell 37: 767- 778 (1984) at 777.
  • the anti-peptide antibodies of the invention also are useful for purification of the mimicked protein, for instance, by adsorption chromatography using methods well known in the art. Immunological assay formats are described in further detail below.
  • Recombinant mutant CSF1R polypeptides are also within the scope of the present invention, and may be producing using polynucleotides of the invention, as described in Section B above.
  • the invention provides, in part, a method for producing a recombinant RBM6-CSF1 R fusion polypeptide or truncated active CSF1R kinase polypeptide by culturing a recombinant host cell (as described above) under conditions suitable for the expression of the fusion polypeptide and recovering the polypeptide.
  • Culture conditions suitable for the growth of host cells and the expression of recombinant polypeptides from such cells are well known to those of skill in the art.
  • Mutant CSF1R polypeptide-specific reagents useful in the practice of the disclosed methods include, among others, fusion polypeptide specific antibodies and AQUA peptides (heavy-isotope labeled peptides) corresponding to, and suitable for detection and quantification of, RBM6- CSF1 R fusion polypeptide expression in a biological sample. Also useful are truncation-specific reagents, such as antibodies or AQUA peptides, suitable for detecting the presence or absence of a truncated active CSF1R kinase polypeptide of the invention.
  • a fusion polypeptide-specific reagent is any reagent, biological or chemical, capable of specifically binding to, detecting and/or quantifying the presence/level of expressed RBM6-CSF1 R fusion polypeptide in a biological sample.
  • the term includes, but is not limited to, the preferred antibody and AQUA peptide reagents discussed below, and equivalent reagents are within the scope of the present invention.
  • Reagents suitable for use in practice of the methods of the invention include a RBM6-CSF1 R fusion polypeptide-specific antibody.
  • a fusion-specific antibody of the invention is an isolated antibody or antibodies that specifically bind(s) a RBM6-CSF1 R fusion polypeptide of the invention (e.g. SEQ ID NO: 1) but does not substantially bind either wild type RBM6 or wild type CSF1R.
  • Suitable reagents include epitope-specific antibodies that specifically bind to an epitope in the extracelluar domain of wild type CSF 1 R protein sequence (which domain is not present in the truncated, active CSF1R kinase disclosed herein), and are therefore capable of detecting the presence (or absence) of wild type CSF1R in a sample.
  • Human RBM6-CSF1 R fusion polypeptide-specific antibodies may also bind to highly homologous and equivalent epitopic peptide sequences in other mammalian species, for example murine or rabbit, and vice versa.
  • Antibodies useful in practicing the methods of the invention include (a) monoclonal antibodies, (b) purified polyclonal antibodies that specifically bind to the target polypeptide (e.g.
  • antibody refers to all types of immunoglobulins, including IgG, IgM, IgA, IgD, and IgE.
  • the antibodies may be monoclonal or polyclonal and may be of any species of origin, including (for example) mouse, rat, rabbit, horse, or human, or may be chimeric antibodies. See, e.g., M. Walker et a/., Molec. Immunol. 26: 403-11 (1989); Morrision et al., Proc. Nat'l. Acad. ScL 81: 6851 (1984); Neuberger etal., Nature 312: 604 (1984)).
  • the antibodies may be recombinant monoclonal antibodies produced according to the methods disclosed in U.S. Pat. No. 4,474,893 (Reading) or U.S. Pat. No.4,816,567 (Cabilly et a/.)
  • the antibodies may also be chemically constructed specific antibodies made according to the method disclosed in U.S. Pat. No.
  • the preferred epitopic site of a RBM6-CSF1 R fusion polypeptide specific antibody of the invention is a peptide fragment consisting essentially of about 11 to 17 amino acids of the human RBM6-CSF1R fusion polypeptide sequence (SEQ ID NO: 1) which fragment encompasses the fusion junction (which occurs at residues 36-37 in the fusion protein (see Figure 1 (bottom panel) and Figure 9 (bottom panel)). It will be appreciated that antibodies that specifically binding shorter or longer peptides/epitopes encompassing the fusion junction of the RBM6- CSF1 R fusion polypeptide are within the scope of the present invention.
  • the invention is not limited to use of antibodies, but includes equivalent molecules, such as protein binding domains or nucleic acid aptamers, which bind, in a fusion-protein or truncated-protein specific manner, to essentially the same epitope to which a RBM6-CSF1 R fusion polypeptide-specific antibody or CSF1 R truncation point epitope-specific antibody useful in the methods of the invention binds. See, e.g., Neuberger et ai, Nature 312: 604 (1984). Such equivalent non-antibody reagents may be suitably employed in the methods of the invention further described below.
  • Polyclonal antibodies useful in practicing the methods of the invention may be produced according to standard techniques by immunizing a suitable animal (e.g., rabbit, goat, etc.) with an antigen encompassing a desired fusion-protein specific epitope (e.g. the fusion junction (see Figure 9, bottom panel), collecting immune serum from the animal, and separating the polyclonal antibodies from the immune serum, and purifying polyclonal antibodies having the desired specificity, in accordance with known procedures.
  • the antigen may be a synthetic peptide antigen comprising the desired epitopic sequence, selected and constructed in accordance with well-known techniques. See, e.g., ANTIBODIES: A LABORATORY MANUAL, Chapter 5, p.
  • Monoclonal antibodies may also be beneficially employed in the methods of the invention, and may be produced in hybridoma cell lines according to the well-known technique of Kohler and Milstein. Nature 265: 495-97 (1975); Kohler and Milstein, Eur. J. Immunol. 6: 511 (1976); see also, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Ausubel et a/. Eds. (1989). Monoclonal antibodies so produced are highly specific, and improve the selectivity and specificity of assay methods provided by the invention. For example, a solution containing the appropriate antigen (e.g.
  • a synthetic peptide comprising the fusion junction of RBM6-CSF1R fusion polypeptide may be injected into a mouse and, after a sufficient time (in keeping with conventional techniques), the mouse sacrificed and spleen cells obtained.
  • the spleen cells are then immortalized by fusing them with myeloma cells, typically in the presence of polyethylene glycol, to produce hybridoma cells.
  • Rabbit fusion hybridomas for example, may be produced as described in U.S Patent No. 5,675,063, K. Knight, Issued October 7, 1997.
  • the hybridoma cells are then grown in a suitable selection media, such as hypoxanthine-aminopterin-thymidine (HAT), and the supernatant screened for monoclonal antibodies having the desired specificity, as described below.
  • a suitable selection media such as hypoxanthine-aminopterin-thymidine (HAT)
  • HAT hypoxanthine-aminopterin-thymidine
  • the secreted antibody may be recovered from tissue culture supernatant by conventional methods such as precipitation, ion exchange or affinity chromatography, or the like.
  • 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. NaVI Acad. Sci. 87: 8095 (1990). If monoclonal antibodies of one 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, er a/., Proc. Nat'l. Acad.
  • 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, 1995, Humana Press, Sudhir Paul editor.)
  • U.S. Pat. No. 5,194,392 Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent of the epitope (i.e., a "mimotope") which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, this method involves detecting or determining a sequence of monomers which is a topographical equivalent of a ligand which is complementary to the ligand binding site of a particular receptor of interest.
  • U.S. Pat. No. 5,480,971 Houghten etal.
  • Antibodies useful in the methods of the invention may be screened for epitope and fusion protein specificity according to standard techniques. See, e.g. Czernik et a/., Methods in Enzymology, 201: 264-283 (1991).
  • the antibodies may be screened against a peptide library by ELISA to ensure specificity for both the desired antigen and, if desired, for reactivity only with a RBM6-CSF1 R fusion polypeptide of the invention and not with wild type RBM6 or wild type CSF 1 R.
  • the antibodies may also be tested by Western blotting against cell preparations containing target protein to confirm reactivity with the only the desired target and to ensure no appreciable binding to other fusion proteins involving CSF1R.
  • the production, screening, and use of fusion protein-specific antibodies is known to those of skill in the art, and has been described. See, e.g., U.S. Patent Publication No. 20050214301, Wetzel et al., September 29, 2005.
  • Fusion polypeptide-specific antibodies useful in the methods of the invention may exhibit some limited cross-reactivity with similar fusion epitopes in other fusion proteins or with the epitopes in wild type RBM6 and wild type CSF1 R that form the fusion junction. This is not unexpected as most antibodies exhibit some degree of cross-reactivity, and anti-peptide antibodies will often cross-react with epitopes having high homology or identity to the immunizing peptide. See, e.g., Czernik, supra. Cross-reactivity with other fusion proteins is readily characterized by Western blotting alongside markers of known molecular weight.
  • Amino acid sequences of cross-reacting proteins may be examined to identify sites highly homologous or identical to the RBM6-CSF1 R fusion polypeptide sequence to which the antibody binds.
  • Undesirable cross- reactivity can be removed by negative selection using antibody purification on peptide columns (e.g. selecting out antibodies that bind either wild type RBM6 and/or wild type CSF1R).
  • RBM6-CSF1 R fusion polypeptide-specific antibodies of the invention that are useful in practicing the methods disclosed herein are ideally specific for human fusion polypeptide, but are not limited only to binding the human species, perse.
  • the invention includes the production and use of antibodies that also bind conserved and highly homologous or identical epitopes in other mammalian species (e.g. mouse, rat, monkey). Highly homologous or identical sequences in other species can readily be identified by standard sequence comparisons, such as using BLAST, with the human RBM6-CSF1 R fusion polypeptide sequence disclosed herein (SEQ ID NO: 1).
  • Antibodies employed in the methods of the invention may be further characterized by, and validated for, use in a particular assay format, for example FC, IHC, and/or ICC.
  • a particular assay format for example FC, IHC, and/or ICC.
  • the use of RBM6-CSF1 R fusion polypeptide-specific antibodies in such methods is further described in Section F below.
  • Antibodies may also be advantageously conjugated to fluorescent dyes (e.g. Alexa488, PE), or labels such as quantum dots, for use in multi-parametric analyses along with other signal transduction (phospho-AKT, phospho-Erk 1/2) and/or cell marker (cytokeratin) antibodies, as further described in Section F below.
  • the expression and/or activity of wild type RBM6 and/or wild type CSF1R in a given biological sample may also be advantageously examined using antibodies (either phospho-specific or total) for these wild type proteins.
  • CSF receptor phosphorylation-site specific antibodies are commercially available (see CELL SIGNALING TECHNOLOGY, INC., Beverly MA, 2005/06 Catalogue, #'s 3151, 3155, and 3154; and Upstate Biotechnology, 2006 Catalogue, #06-457).
  • Such antibodies may also be produced according to standard methods, as described above.
  • the amino acid sequences of both human RBM-6 and CSF1R are published (see Figures 3 and 4, and referenced SwissProt Accession Nos.), as are the sequences of these proteins from other species.
  • Detection of wild type RBM-6 and wild type CSF1 R expression and/or activation, along with RBM6-CSF1 R fusion polypeptide expression, in a biological sample can provide information on whether the fusion protein alone is driving the tumor, or whether wild type CSF1 R is also activated and driving the tumor. Such information is clinically useful in assessing whether targeting the fusion protein or the wild type protein(s), or both, or is likely to be most beneficial in inhibiting progression of the tumor, and in selecting an appropriate therapeutic or combination thereof.
  • Antibodies specific for the wild type CSF1 R kinase extracellular domain, which is not present in the truncated active CSF1R kinase disclosed herein, may be particularly useful for determining the presence/absence of the mutant CSF1 R kinase.
  • RBM6-CSF1 R fusion polypeptide-specific antibodies together with one or more antibodies specific for another kinase, receptor, or kinase substrate that is suspected of being, or potentially is, activated in a cancer in which RBM6-CSF1 R fusion polypeptide is expressed may be simultaneously employed to detect the activity of such other signaling molecules in a biological sample comprising cells from such cancer.
  • RBM6-CSF1R fusion polypeptides of the present invention and the fusion junction epitope- bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • These fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature 331: 84-86 (1988)).
  • Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric RBM6-CSF1 R fusion polypeptide alone (Fountoulakis et aL, J Biochem 270: 3958-3964 (1995)).
  • RBM6-CSF1R fusion polypeptide-specific reagents useful in the practice of the disclosed methods may also comprise heavy-isotope labeled peptides suitable for the absolute quantification of expressed RBM6-CSF1 R fusion polypeptide or truncated active CSF1 R kinase polypeptide in a biological sample.
  • AQUA peptides for the absolute quantification of proteins (AQUA) in complex mixtures has been described. See WO/03016861, "Absolute
  • the AQUA methodology employs the introduction of a known quantity of at least one heavy-isotope labeled peptide standard (which has a unique signature detectable by LC-SRM chromatography) into a digested biological sample in order to determine, by comparison to the peptide standard, the absolute quantity of a peptide with the same sequence and protein modification in the biological sample.
  • the AQUA methodology has two stages: peptide internal standard selection and validation and method development; and implementation using validated peptide internal standards to detect and quantify a target protein in sample.
  • the method is a powerful technique for detecting and quantifying a given peptide/protein within a complex biological mixture, such as a cell lysate, and may be employed, e.g., to quantify change in protein phosphorylation as a result of drug treatment, or to quantify differences in the level of a protein in different biological states.
  • a particular peptide (or modified peptide) within a target protein sequence is chosen based on its amino acid sequence and the particular protease to be used to digest.
  • the peptide is then generated by solid-phase peptide synthesis such that one residue is replaced with that same residue containing stable isotopes ( 13 C, 15 N).
  • the result is a peptide that is chemically identical to its native counterpart formed by proteolysis, but is easily distinguishable by MS via a 7-Da mass shift.
  • the newly synthesized AQUA internal standard peptide is then evaluated by LC-MS/MS. This process provides qualitative information about peptide retention by reverse-phase chromatography, ionization efficiency, and fragmentation via collision- induced dissociation. Informative and abundant fragment ions for sets of native and internal standard peptides are chosen and then specifically monitored in rapid succession as a function of chromatographic retention to form a selected reaction monitoring (LC-SRM) method based on the unique profile of the peptide standard.
  • LC-SRM reaction monitoring
  • the second stage of the AQUA strategy is its implementation to measure the amount of a protein or modified protein from complex mixtures.
  • Whole cell lysates are typically fractionated by SDS-PAGE gel electrophoresis, and regions of the gel consistent with protein migration are excised. This process is followed by in-gel proteolysis in the presence of the AQUA peptides and LC-SRM analysis.
  • AQUA peptides are spiked in to the complex peptide mixture obtained by digestion of the whole cell lysate with a proteolytic enzyme and subjected to immunoaffinity purification as described above.
  • the retention time and fragmentation pattern of the native peptide formed by digestion e.g.
  • trypsin ization is identical to that of the AQUA internal standard peptide determined previously; thus, LC-MS/MS analysis using an SRM experiment results in the highly specific and sensitive measurement of both internal standard and analyte directly from extremely complex peptide mixtures.
  • the ratio of the areas under the curve can be used to determine the precise expression levels of a protein or phosphorylated form of a protein in the original cell lysate.
  • the internal standard is present during in-gel digestion as native peptides are formed, such that peptide extraction efficiency from gel pieces, absolute losses during sample handling (including vacuum centrifugation), and variability during introduction into the LC-MS system do not affect the determined ratio of native and AQUA peptide abundances.
  • An AQUA peptide standard is developed for a known sequence previously identified by the IAP-LC-MS/MS method within in a target protein.
  • one AQUA peptide incorporating the modified form of the particular residue within the site may be developed, and a second AQUA peptide incorporating the unmodified form of the residue developed.
  • the two standards may be used to detect and quantify both the modified an unmodified forms of the site in a biological sample.
  • Peptide internal standards may also be generated by examining the primary amino acid sequence of a protein and determining the boundaries of peptides produced by protease cleavage.
  • a protein may actually be digested with a protease and a particular peptide fragment produced can then sequenced.
  • Suitable proteases include, but are not limited to, serine proteases (e.g. trypsin, hepsin), metallo proteases (e.g. PUMP1), chymotrypsin, cathepsin, pepsin, thermolysin, carboxypeptidases, etc.
  • a peptide sequence within a target protein is selected according to one or more criteria to optimize the use of the peptide as an internal standard.
  • the size of the peptide is selected to minimize the chances that the peptide sequence will be repeated elsewhere in other non-target proteins.
  • a peptide is preferably at least about 6 amino acids.
  • the size of the peptide is also optimized to maximize ionization frequency.
  • peptides longer than about 20 amino acids are not preferred.
  • the preferred ranged is about 7 to 15 amino acids.
  • a peptide sequence is also selected that is not likely to be chemically reactive during mass spectrometry, thus sequences comprising cysteine, tryptophan, or methionine are avoided.
  • a peptide sequence that does not include a modified region of the target region may be selected so that the peptide internal standard can be used to determine the quantity of all forms of the protein.
  • a peptide internal standard encompassing a modified amino acid may be desirable to detect and quantify only the modified form of the target protein.
  • Peptide standards for both modified and unmodified regions can be used together, to determine the extent of a modification in a particular sample (i.e. to determine what fraction of the total amount of protein is represented by the modified form).
  • peptide standards for both the phosphorylated and unphosphorylated form of a protein known to be phosphorylated at a particular site can be used to quantify the amount of phosphorylated form in a sample.
  • the peptide is labeled using one or more labeled amino acids (i.e. the label is an actual part of the peptide) or less preferably, labels may be attached after synthesis according to standard methods.
  • the label is a mass-altering label selected based on the following considerations: The mass should be unique to shift fragments masses produced by MS analysis to regions of the spectrum with low background; the ion mass signature component is the portion of the labeling moiety that preferably exhibits a unique ion mass signature in MS analysis; the sum of the masses of the constituent atoms of the label is preferably uniquely different than the fragments of all the possible amino acids.
  • the labeled amino acids and peptides are readily distinguished from unlabeled ones by the ion/mass pattern in the resulting mass spectrum.
  • the ion mass signature component imparts a mass to a protein fragment that does not match the residue mass for any of the 20 natural amino acids.
  • the label should be robust under the fragmentation conditions of MS and not undergo unfavorable fragmentation. Labeling chemistry should be efficient under a range of conditions, particularly denaturing conditions, and the labeled tag preferably remains soluble in the MS buffer system of choice.
  • the label preferably does not suppress the ionization efficiency of the protein and is not chemically reactive.
  • the label may contain a mixture of two or more isotopically distinct species to generate a unique mass spectrometric pattern at each labeled fragment position. Stable isotopes, such as 2 H, 13 C, 15 N, 17 0, 18 O, or 34 S, are among preferred labels. Pairs of peptide internal standards that incorporate a different isotope label may also be prepared. Preferred amino acid residues into which a heavy isotope label may be incorporated include leucine, proline, valine, and phenylalanine.
  • Peptide internal standards are characterized according to their mass-to-charge (m/z) ratio, and preferably, also according to their retention time on a chromatographic column (e.g. an HPLC column). Internal standards that co-elute with unlabeled peptides of identical sequence are selected as optimal internal standards.
  • the internal standard is then analyzed by fragmenting the peptide by any suitable means, for example by collision-induced dissociation (CID) using, e.g., argon or helium as a collision gas.
  • CID collision-induced dissociation
  • the fragments are then analyzed, for example by multi-stage mass spectrometry (MS”) to obtain a fragment ion spectrum, to obtain a peptide fragmentation signature.
  • MS multi-stage mass spectrometry
  • peptide fragments have significant differences in m/z ratios to enable peaks corresponding to each fragment to be well separated, and a signature is that is unique for the target peptide is obtained. If a suitable fragment signature is not obtained at the first stage, additional stages of MS are performed until a unique signature is obtained.
  • Fragment ions in the MS/MS and MS 3 spectra are typically highly specific for the peptide of interest, and, in conjunction with LC methods, allow a highly selective means of detecting and quantifying a target peptide/protein in a complex protein mixture, such as a cell lysate, containing many thousands or tens of thousands of proteins.
  • a complex protein mixture such as a cell lysate, containing many thousands or tens of thousands of proteins.
  • Any biological sample potentially containing a target protein/peptide of interest may be assayed. Crude or partially purified cell extracts are preferably employed.
  • the sample has at least 0.01 mg of protein, typically a concentration of 0.1-10 mg/mL, and may be adjusted to a desired buffer concentration and pH.
  • a known amount of a labeled peptide internal standard, preferably about 10 femtomoles, corresponding to a target protein to be detected/quantified is then added to a biological sample, such as a cell lysate.
  • the spiked sample is then digested with one or more protease(s) for a suitable time period to allow digestion.
  • a separation is then performed (e.g. by HPLC, reverse-phase HPLC, capillary electrophoresis, ion exchange chromatography, etc.) to isolate the labeled internal standard and its corresponding target peptide from other peptides in the sample.
  • Microcapillary LC is a preferred method.
  • Each isolated peptide is then examined by monitoring of a selected reaction in the MS. This involves using the prior knowledge gained by the characterization of the peptide internal standard and then requiring the MS to continuously monitor a specific ion in the MS/MS or MS" spectrum for both the peptide of interest and the internal standard. After elution, the area under the curve (AUC) for both peptide standard and target peptide peaks are calculated. The ratio of the two areas provides the absolute quantification that can be normalized for the number of cells used in the analysis and the protein's molecular weight, to provide the precise number of copies of the protein per cell. Further details of the AQUA methodology are described in Gygi et a/., and Gerber et a/, supra.
  • AQUA internal peptide standards may desirably be produced, as described above, to detect any quantify any unique site (e.g. the fusion junction within RBM6-CSF1R fusion polypeptide) within a mutant CSF1 R polypeptide of the invention.
  • an AQUA phosphopeptide may be prepared that corresponds to the fusion junction sequence of RBM6-CSF1 R fusion polypeptide (see Figure 9 (bottom panel)).
  • Peptide standards for may be produced for the RBM6-CSF1 R fusion junction and such standards employed in the AQUA methodology to detect and quantify the fusion junction (i.e. the presence of RBM6-CSF1 R fusion polypeptide) in a biological sample.
  • an exemplary AQUA peptide of the invention comprises the amino acid sequence PLKKWE (see Figure 9, bottom panel), which corresponds to the three amino acids immediately flanking each side of the fusion junction in RBM6-CSF1 R fusion polypeptide (see SEQ ID NO: 7).
  • PLKKWE amino acid sequence PLKKWE
  • SEQ ID NO: 7 amino acid sequence PLKKWE
  • larger AQUA peptides comprising the fusion junction sequence and additional residues downstream or upstream of it
  • a smaller AQUA peptide comprising less than all of the residues of such sequence (but still comprising the point of fusion junction itself) may alternatively be constructed.
  • AQUA peptides are within the scope of the present invention, and the selection and production of preferred AQUA peptides may be carried out as described above (see Gygi et al., Gerber et a/., supra.). Nucleic Acid Probes.
  • Fusion-specific reagents provided by the invention also include nucleic acid probes and primers suitable for detection of a RBM6-CSF1R polynucleotide or truncated CSF1 R kinase polynucleotide, as described in detail in Section B above.
  • the specific use of such probes in assays such as fluorescence in-situ hybridization (FISH) or polymerase chain reaction (PCR) amplification is described in Section F below.
  • the methods of the invention may be carried out in a variety of different assay formats known to those of skill in the art.
  • Immunoassays useful in the practice of the methods of the invention may be homogenous immunoassays or heterogeneous immunoassays.
  • the immunological reaction usually involves a mutant CSF1 R kinase polypeptide-specific reagent (e.g. a RBM6-CSF1 R fusion polypeptide-specific antibody), a labeled analyte, and the biological sample of interest.
  • the signal arising from the label is modified, directly or indirectly, upon the binding of the antibody to the labeled analyte.
  • Both the immunological reaction and detection of the extent thereof are carried out in a homogeneous solution.
  • Immunochemical labels that may be employed include free radicals, radio-isotopes, fluorescent dyes, enzymes, bacteriophages, coenzymes, and so forth.
  • Semi-conductor nanocrystal labels, or “quantum dots”, may also be advantageously employed, and their preparation and use has been well described. See generally, K. Barovsky, Nanotech. Law & Bus. 1(2): Article 14 (2004) and patents cited therein.
  • the reagents are usually the biological sample, a mutant CSF1R kinase polypeptide-specific reagent (e.g., a RBM6-CSF1 R fusion-specific antibody), and suitable means for producing a detectable signal.
  • Biological samples as further described below may be used.
  • the antibody is generally immobilized on a support, such as a bead, plate or slide, and contacted with the sample suspected of containing the antigen in a liquid phase. The support is then separated from the liquid phase and either the support phase or the liquid phase is examined for a detectable signal employing means for producing such signal.
  • the signal is related to the presence of the anaiyte in the biological sample.
  • Means for producing a detectable signal include the use of radioactive labels, fluorescent labels, enzyme labels, quantum dots, and so forth.
  • an antibody which binds to that site can be conjugated to a detectable group and added to the liquid phase reaction solution before the separation step.
  • the presence of the detectable group on the solid support indicates the presence of the antigen in the test sample.
  • suitable immunoassays are the radioimmunoassay, immunofluorescence methods, enzyme-linked immunoassays, and the like.
  • Immunoassay formats and variations thereof, which may be useful for carrying out the methods disclosed herein, are well known in the art. See generally E. Maggio, Enzyme-lmmunoassay, (1980) (CRC Press, Inc., Boca Raton, FIa.); see also, e.g., U.S. Pat. No. 4,727,022 (Skold et al., “Methods for Modulating Ligand-Receptor Interactions and their Application”); U.S. Pat. No. 4,659,678 (Forrest et al., "Immunoassay of Antigens"); U.S. Pat. No.
  • the concentration of detectable reagent should be sufficient such that the binding of RBM6-CSF1R fusion polypeptide is detectable compared to background.
  • Antibodies useful in the practice of the methods disclosed herein may be conjugated to a solid support suitable for a diagnostic assay (e.g., beads, plates, slides or wells formed from materials such as latex or polystyrene) in accordance with known techniques, such as precipitation.
  • Antibodies or other RBM6-CSF1 R fusion polypeptide- or truncated active CSF1R kinase polypeptide- binding reagents may likewise be conjugated to detectable groups such as radiolabels (e.g., 35 S 1 125 I, 131 I), enzyme labels (e.g., horseradish peroxidase, alkaline phosphatase), and fluorescent labels (e.g., fluorescein) in accordance with known techniques.
  • radiolabels e.g., 35 S 1 125 I, 131 I
  • enzyme labels e.g., horseradish peroxidase, alkaline phosphatase
  • fluorescent labels e.g., fluorescein
  • Cell-based assays such flow cytometry (FC), immuno- histochemistry (IHC), or immunofluorescence (IF) are particularly desirable in practicing the methods of the invention, since such assay formats are clinically-suitable, allow the detection of mutant CSF1R kinase polypeptide expression in vivo, and avoid the risk of artifact changes in activity resulting from manipulating cells obtained from, e.g. a tumor sample in order to obtain extracts.
  • the methods of the invention are implemented in a flow- cytometry (FC) 1 immuno-histochemistry (IHC), or immunofluorescence (IF) assay format.
  • Flow cytometry may be employed to determine the expression of mutant CSF1R kinase polypeptide in a mammalian tumor before, during, and after treatment with a drug targeted at inhibiting CSF1R kinase activity.
  • tumor cells from a bone marrow sample may be analyzed by flow cytometry for RBM6-CSF1R fusion polypeptide expression and/or activation, as well as for markers identifying cancer cell types, etc., if so desired.
  • Flow cytometry may be carried out according to standard methods. See, e.g. Chow et a/., Cytometry (Communications in Clinical Cytometry) 46: 72-78 (2001).
  • the following protocol for cytometric analysis may be employed: fixation of the cells with 2% paraformaldehyde for 10 minutes at 37 0 C followed by permeabilization in 90% methanol for 30 minutes on ice. Cells may then be stained with the primary RBM6-CSF1 R fusion polypeptide-specific antibody, washed and labeled with a fluorescent-labeled secondary antibody. 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. Such an analysis would identify the level of expressed RBM6-CSF1 R fusion polypeptide in the tumor.
  • a flow cytometer e.g. a Beckman Coulter FC500
  • IHC immunohistochemical staining may be also employed to determine the expression and/or activation status of mutant CSF1R kinase polypeptide in a mammalian cancer (e.g. AML) before, during, and after treatment with a drug targeted at inhibiting CSF1 R kinase activity. IHC may be carried out according to well-known techniques.
  • paraffin-embedded tissue e.g. tumor tissue from a biopsy
  • paraffin-embedded tissue e.g. tumor tissue from a biopsy
  • paraffinizing tissue sections with xylene followed by ethanol hydrating in water then PBS
  • unmasking antigen by heating slide in sodium citrate buffer
  • incubating sections ⁇ n hydrogen peroxide blocking in blocking solution
  • incubating slide in primary anti-RBM6-CSF1 R fusion polypeptide antibody and secondary antibody and finally detecting using ABC avidin/biotin method according to manufacturer's instructions.
  • Immunofluorescence assays may be also employed to determine the expression and/or activation status of mutant CSF1R kinase polypeptide in a mammalian cancer before, during, and after treatment with a drug targeted at inhibiting CSF1 R kinase activity.
  • IF may be carried out according to well-known techniques. See, e.g., J. M. polak and S. Van Noorden (1997) INTRODUCTION TO JMMUNOCYTOCHEMISTRY, 2nd Ed.; ROYAL MICROSCOPY SOCIETY MICROSCOPY HANDBOOK 37, BioScientific/Springer-Verlag.
  • patient samples may be fixed in paraformaldehyde followed by methanol, blocked with a blocking solution such as horse serum, incubated with the primary antibody against RBM6-CSF1 R fusion polypeptide followed by a secondary antibody labeled with a fluorescent dye such as Alexa 488 and analyzed with an epifluorescent microscope.
  • a blocking solution such as horse serum
  • Antibodies employed in the above-described assays may be advantageously conjugated to fluorescent dyes (e.g. Alexa488, PE), or other labels, such as quantum dots, for use in multi-parametric analyses along with other signal transduction (EGFR, phospho-AKT, phospho-Erk 1/2) and/or cell marker (cytokeratin) antibodies.
  • fluorescent dyes e.g. Alexa488, PE
  • other labels such as quantum dots
  • RBM6-CSF1 R fusion polypeptide expression is established by combining body fluids or cell extracts taken from normal mammalian subjects, preferably human, with antibody to RBM6-CSF1 R fusion polypeptide under conditions suitable for complex formation. The amount of standard complex formation may be quantified by various methods, but preferably by photometric means. Quantities of RBM6-CSF1 R fusion polypeptide expressed in subject, control, and disease samples from biopsied tissues are compared with the standard values. Deviation between standard and subject values establishes the parameters for diagnosing disease.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent-activated cell sorting
  • the RBM6- CSF1 R fusion polypeptide-specific reagent comprises a heavy isotope labeled phosphopeptide (AQUA peptide) corresponding to a peptide sequence comprising the fusion junction of RBM6-CSF1 R fusion polypeptide, as described above in Section E.
  • Mutant CSF1R polypeptide-specific reagents useful in practicing the methods of the invention may also be mRNA, oligonucleotide or DNA probes that can directly hybridize to, and detect, fusion or truncated polypeptide expression transcripts in a biological sample. Such probes are discussed in detail in Section B above. Briefly, and by way of example, formalin-fixed, paraffin-embedded patient samples may be probed with a fluorescein-labeled RNA probe followed by washes with formamide, SSC and PBS and analysis with a fluorescent microscope.
  • Polynucleotides encoding mutant CSF1R kinase polypeptide may also be used for diagnostic purposes.
  • the polynucleotides which may be used include oligonucleotide sequences, antisense RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantitate gene expression in biopsied tissues in which expression of RBM6-CSF1 R fusion polypeptide or truncated active CSF1R kinase polypeptide may be correlated with disease.
  • the diagnostic assay may be used to distinguish between absence, presence, and excess expression of RBM6-CSF1R fusion polypeptide, and to monitor regulation of RBM6-CSF1 R fusion polypeptide levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding RBM6-CSF1 R fusion polypeptide or truncated active CSF1 R kinase polypeptide, or closely related molecules, may be used to identify nucleic acid sequences which encode mutant CSF1R polypeptide.
  • PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding RBM6-CSF1 R fusion polypeptide or truncated active CSF1 R kinase polypeptide, or closely related molecules.
  • the specificity of the probe whether it is made from a highly specific region, e.g., 10 unique nucleotides in the fusion junction, or a less specific region, e.g., the 3' coding region, and the stringency of the hybridization or amplification (maximal, high, intermediate, or low) will determine whether the probe identifies only naturally occurring sequences encoding mutant CSF1 R polypeptide, alleles, or related sequences.
  • Probes may also be used for the detection of related sequences, and should preferably contain at least 50% of the nucleotides from any of the mutant CSF1 R polypeptide encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and derived from the nucleotide sequence of SEQ ID NO: 2, most preferably encompassing the fusion junction (see Figure 7, bottom panel), or from genomic sequence including promoter, enhancer elements, and introns of the naturally occurring RBM6 and CSF1R polypeptides, as further described in Section B above.
  • a RBM6-CSF1 R fusion polynucleotide or truncated CSF1R polynucleotide of the invention may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; or in dip stick, pin, ELISA or chip assays utilizing fluids or tissues from patient biopsies to detect altered CSF1R polypeptide expression. Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding a mutant CSF1 R polypeptide of the invention may be useful in assays that detect activation or induction of various cancers, including leukemias.
  • Mutant CSF1 R polynucleotides may be labeled by standard methods, and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantitated and compared with a standard value. If the amount of signal in the biopsied or extracted sample is significantly altered from that of a comparable control sample, the nucleotide sequences have hybridized with nucleotide sequences in the sample, and the presence of altered levels of nucleotide sequences encoding RBM6-CSF1 R fusion polypeptide or truncated active CSF1R kinase polypeptide in the sample indicates the presence of the associated disease. Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or in monitoring the treatment of an individual patient.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, which encodes RBM6-CSF1 R fusion polypeptide or truncated active CSF1R kinase polypeptide, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with those from an experiment where a known amount of a substantially purified polynucleotide is used. Standard values obtained from normal samples may be compared with values obtained from samples from patients who are symptomatic for disease. Deviation between standard and subject values is used to establish the presence of disease.
  • hybridization assays may be repeated on a regular basis to evaluate whether the level of expression in the patient begins to approximate that which is observed in the normal patient.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • PCR polymerase chain reaction
  • MOLECULAR CLONING A LABORATORY MANUAL, 2nd. edition, Sambrook, J., Fritsch, E. F. and Maniatis, T., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989).
  • PCR oligomers may be chemically synthesized, generated enzymatically, or produced from a recombinant source.
  • Oligomers will preferably consist of two nucleotide sequences, one with sense orientation (5 1 to 3 1 ) and another with antisense (3 1 to 5'), employed under optimized conditions for identification of a specific gene or condition.
  • the same two oligomers, nested sets of oligomers, or even a degenerate pool of oligomers may be employed under less stringent conditions for detection and/or quantitation of closely related DNA or RNA sequences.
  • Methods which may also be used to quantitate the expression of RBM6-CSF1 R fusion polypeptide or truncated active CSF1R kinase polypeptide include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and standard curves onto which the experimental results are interpolated (Melby etal., J. Immunol. Methods, 159: 235-244 (1993); Duplaa et al. Anal. Biochem. 229-236 (1993)).
  • the speed of quantitation of multiple samples may be accelerated by running the assay in an ELISA format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • the mutant CSF1R polynucleotides of the invention may be used to generate hybridization probes that are useful for mapping the naturally occurring genomic sequence.
  • the sequences may be mapped to a particular chromosome or to a specific region of the chromosome using well known techniques.
  • Such techniques include fluorescence in-situ hybridization (FISH), FACS 1 or artificial chromosome constructions, such as yeast artificial chromosomes, bacterial artificial chromosomes, bacterial P1 constructions or single chromosome cDNA libraries, as reviewed in Price, C. M., Blood Rev. 7: 127-134 (1993), and Trask, B. J., Trends Genet. 7: 149-154 (1991).
  • FISH fluorescence in-situ hybridization
  • the nucleotide sequences of the subject invention may be used to detect differences in gene sequences between normal, carrier, or affected individuals.
  • In situ hybridization of chromosomal preparations and physical mapping techniques such as linkage analysis using established chromosomal markers may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms, or parts thereof, by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • nucleic acid detection such as minor groove-binding conjugated oligonuclotide probes (see, e.g. U.S. Patent No. 6,951,930, "Hybridization-Triggered Fluorescent Detection of Nucleic Acids") are known to those of skill in the art.
  • Biological samples useful in the practice of the methods of the invention may be obtained from any mammal in which a cancer characterized by the expression of a RBM6-CSF1 R fusion polypeptide or truncated active CSF1 R kinase polypeptide is present or developing.
  • the mammal is a human, and the human may be a candidate for a CSF1R-inhibiting therapeutic for the treatment of a leukemia, e.g. AML.
  • the human candidate may be a patient currently being treated with, or considered for treatment with, a CSF1R kinase inhibitor, such as Gleevec®.
  • the mammal is large animal, such as a horse or cow, while in other embodiments, the mammal is a small animal, such as a dog or cat, all of which are known to develop cancers, including leukemias.
  • any biological sample comprising cells (or extracts of cells) from a mammalian cancer is suitable for use in the methods of the invention.
  • Serum and bone marrow samples may be particularly preferred for patients with leukemia, and may be obtained by standard methods. Circulating tumor cells may also be obtained from serum using tumor markers, cytokeratin protein markers or other methods of negative selection as described (see Ma et al., Anticancer Res. 23(1A): 49-62 (2003)).
  • the biological sample may comprise cells obtained from a tumor biopsy, which maybe be obtained according to standard clinical techniques. For example, aberrant expression of CSF1 R has been observed in a spectrum of cancers including breast and ovarian cancer, and its expression stimulates tumor invasion.
  • a biological sample may comprise cells (or cell extracts) from a cancer in which RBM6-CSF1R fusion polypeptide or truncated active CSF1R kinase polypeptide is expressed and/or activated but wild type CSF1R kinase is not.
  • the sample may comprise cells from a cancer in which both the mutant CSF1R polypeptide and wild type CSF1R kinase are expressed and/or activated, or in which wild type CSF1 R kinase and/or RBM-6 are expressed and/or active, but mutant CSF1R polypeptide is not.
  • Cellular extracts of the foregoing biological samples may be prepared, either crude or partially (or entirely) purified, in accordance with standard techniques, and used in the methods of the invention.
  • biological samples comprising whole cells may be utilized in preferred assay formats such as immunohistochemistry (IHC), flow cytometry (FC) 1 and immunofluorescence (IF), as further described above.
  • IHC immunohistochemistry
  • FC flow cytometry
  • IF immunofluorescence
  • whole-cell assays are advantageous in that they minimize manipulation of the tumor cell sample and thus reduce the risks of altering the in vivo signaling/activation state of the cells and/or introducing artifact signals.
  • Whole cell assays are also advantageous because they characterize expression and signaling only in tumor cells, rather than a mixture of tumor and normal cells.
  • biological samples comprising cells from mammalian bone marrow transplant models or xenografts may also be advantageously employed.
  • Preferred xenografts are small mammals, such as mice, harboring human tumors (or leukemias) that express a mutant CSF1R kinase polypeptide.
  • Xenografts harboring human tumors are well known in the art (see KaI, Cancer Treat Res. 72: 155-69 (1995)) and the production of mammalian xenografts harboring human tumors is well described (see Winograd et al., In Vivo. 1(1): 1-13 (1987)). Similarly the generation and use of bone marrow transplant models is well described (see, e.g., Schwaller, et al., EMBO J. 17: 5321- 333 (1998); Kelly et al., Blood 99: 310-318 (2002)).
  • cancer characterized by a RBM6-CSF1 R translocation and/or fusion polypeptide, or a truncated CSF1R kinase polynucleotide and/or or truncated active CSF1R kinase polypeptide, is meant a cancer in which such mutant CSF1R gene and/or expressed polypeptide are present, as compared to a cancer in which such translocation, truncated gene, and/or fusion polypeptide are not present.
  • a control sample representing a cell in which such translocation and/or fusion protein do not occur may desirably be employed for comparative purposes.
  • the control sample comprises cells from a subset of the particular cancer (e.g. leukemia) that is representative of the subset in which the mutation (e.g. RBM6-CSF1R translocation) does not occur and/or the fusion polypeptide is not expressed. Comparing the level in the control sample versus the test biological sample thus identifies whether the mutant CSF 1 R polynucleotide and/or polypeptide is/are present.
  • any tissue that similarly does not express such mutant CSF1 R polypeptide (or harbor the mutant polynucleotide) may be employed as a control.
  • biological samples may be obtained from a subject that has not been previously diagnosed as having a cancer characterized by a RBM6- CSF1 R translocation and/or fusion polypeptide, nor has yet undergone treatment for such cancer, and the method is employed to diagnostically identify a tumor in such subject as belonging to a subset of tumors (e.g. leukemias) in which RBM6-CSF1 R fusion polynucleotide and/or polypeptide is present and/or expressed.
  • a subset of tumors e.g. leukemias
  • the methods of the invention may also be employed to monitor the progression or inhibition of a mutant CSF1R kinase polypeptide-expressing cancer following treatment of a subject with a composition comprising a CSF 1 R kinase-inhibiting therapeutic or combination of therapeutics.
  • Such diagnostic assay may be carried out subsequent to or prior to preliminary evaluation or surgical surveillance procedures.
  • the identification method of the invention may be advantageously employed as a diagnostic to identify patients having cancer, such as AML, driven by the RBM6-CSF1 R fusion protein or by truncated active CSF1R kinase, which patients would be most likely to respond to therapeutics targeted at inhibiting CSF1R kinase activity, such as Gleevec® or its analogues.
  • the ability to select such patients would also be useful in the clinical evaluation of efficacy of future CSF1 R-targeted therapeutics as well as in the future prescription of such drugs to patients.
  • the ability to selectively identify cancers in which a RBM6-CSF1 R translocation and/or fusion polypeptide, or a truncated CSF1R polynucleotide or truncated active CSF1R polypeptide, is/are present enables important new methods for accurately identifying such tumors for diagnostic purposes, as well as obtaining information useful in determining whether such a tumor is likely to respond to a CSF1R- inhibiting therapeutic composition, or likely to be partially or wholly non- responsive to an inhibitor targeting a different kinase when administered as a single agent for the treatment of the caner.
  • the invention provides a method for detecting the presence of a mutant CSF 1 R polynucleotide and/or polypeptide in a cancer, the method comprising the steps of:
  • the mutant CSF1R polynucleotide comprises a translocation polynucleotide, and in a preferred embodiment, the translocation polynucleotide comprises a RBM6-CSF1 R fusion polynucleotide.
  • the cancer is a leukemia, such as acute myelogenous leukemia (AML).
  • AML acute myelogenous leukemia
  • the presence of a mutant CSF1R polypeptide identifies a cancer that is likely to respond to a composition comprising at least one CSF1R kinase-inhibiting therapeutic.
  • Exemplary CSF1R- inhibiting therapeutics include, but are not limited to, lmatinib mesylate (STI-571 ; Gleevec®) or its analogues, such as SU11248 and GW2580.
  • the diagnostic methods of the invention are implemented in a flow-cytometry (FC), immuno- histochemistry (IHC), or immuno-fluorescence (IF) assay format, as described above.
  • FC flow-cytometry
  • IHC immuno- histochemistry
  • IF immuno-fluorescence
  • the activity of the RBM6-CSF1 R fusion polypeptide and/or truncated active CSF1R kinase polypeptide is detected.
  • the diagnostic methods of the invention are implemented in a fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR) assay format, as described above.
  • FISH fluorescence in situ hybridization
  • PCR polymerase chain reaction
  • the invention further provides a method for determining whether a compound inhibits the progression of a cancer characterized by a RBM6- CSF1R fusion polynucleotide, a truncated CSF1R polynucleotide, a RBM6-CSF1 R fusion polypeptide, and/or or a truncated active CSF 1 R polypeptide, said method comprising the step of determining whether said compound inhibits the expression and/or activity of said RBM6-CSF1 R fusion polypeptide or a truncated active CSF1R polypeptide in said cancer.
  • inhibition of expression and/or activity of the RBM6-CSF1 R fusion polypeptide or said truncated active CSF1R polypeptide is determined using at least one reagent that detects an RBM6-CSF1 R fusion polynucleotide or polypeptide of the invention.
  • Compounds suitable for inhibition of CSF1R kinase activity are discussed in more detail in Section G below.
  • Mutant CSF1 R polynucleotide probes and polypeptide-specific reagents useful in the practice of the methods of the invention are described in further detail in sections B and D above.
  • the RBM6-CSF1 R fusion polypeptide-specific reagent comprises a fusion polypeptide-specific antibody.
  • the fusion polypeptide-specific reagent comprises a heavy- isotope labeled phosphopeptide (AQUA peptide) corresponding to the fusion junction of RBM6-CSF1 R fusion polypeptide (see Figure 9 (bottom panel)).
  • AQUA peptide heavy- isotope labeled phosphopeptide
  • the methods of the invention described above may also optionally comprise the step of determining the level of expression or activation of other kinases, such as wild type CSF1R and EGFR, or other downstream signaling molecules in said biological sample.
  • Profiling both RBM6- CSF1R fusion polypeptide, or truncated active CSF1R kinase polypeptide, expression/ activation and expression/activation of other kinases and pathways in a given biological sample can provide valuable information on which kinase(s) and pathway(s) is/are driving the disease, and which therapeutic regime is therefore likely to be of most benefit.
  • the invention also provides, in part, a method for determining whether a compound inhibits the progression of a cancer characterized by a RBM6-CSF1R fusion polynucleotide, a truncated CSF1R polynucleotide, a RBM6-CSF1 R fusion polypeptide, and/or a truncated active CSF1R kinase polypeptide, said method comprising the step of determining whether said compound inhibits the expression and/or activity of said RBM6-CSF1 R fusion polypeptide or said truncated active CSF1 R kinase polypeptide in said cancer.
  • inhibition of expression and/or activity of the RBM6-CSF1 R fusion polypeptide or truncated active CSF1R kinase polypeptide is determined using at least one reagent that detects a mutant CSF1R polynucleotide and/or mutant CSF1R polypeptide of the invention.
  • Preferred reagents of the invention have been described above.
  • Compounds suitable for the inhibition of CSF1R kinase activity are described in more detail in Section G below.
  • the compound may, for example, be a kinase inhibitor, such as a small molecule or antibody inhibitor. It may be a pan-kinase inhibitor with activity against several different kinases, or a kinase-specific inhibitor.
  • CSF1R kinase-inhibiting compounds are discussed in further detail in Section G below.
  • Patient biological samples may be taken before and after treatment with the inhibitor and then analyzed, using methods described above, for the biological effect of the inhibitor on CSF1 R kinase activity, including the phosphorylation of downstream substrate protein.
  • Such a pharmacodynamic assay may be useful in determining the biologically active dose of the drug that may be preferable to a maximal tolerable dose. Such information would also be useful in submissions for drug approval by demonstrating the mechanism of drug action.
  • CSF1R activity in cancers characterized by expression of a mutant CSF1 R polypeptide may be inhibited by contacting the cancer (e.g. a tumor) with a CSF1R kinase-inhibiting therapeutic, such as a small-molecule kinase inhibitor like lmatinib mesylate (STI-571; Gleevec®).
  • a CSF1R kinase-inhibiting therapeutic such as a small-molecule kinase inhibitor like lmatinib mesylate (STI-571; Gleevec®).
  • growth inhibition of RBM6-CSF1 R fusion protein-expressing leukemia tumors can be accomplished by inhibiting this fusion kinase using an exemplary CSF1R-inhibiting therapeutic, Gleevec®, or by exemplary slRNA silencing.
  • the invention provides, in part, a method for inhibiting the progression of a cancer that expresses RBM6-CSF1 R fusion polypeptide and/or a truncated active CSF1R kinase polypeptide by inhibiting the expression and/or activity of the mutant CSF1 R kinase(s) in the cancer.
  • a CSF1 R kinase-inhibiting therapeutic may be any composition comprising at least one compound, biological or chemical, which inhibits, directly or indirectly, the expression and/or activity of CSF1R kinase in vivo, including the exemplary classes of compounds described below.
  • Such compounds include therapeutics that act directly on CSF1R kinase itself, or on proteins or molecules that modify the activity of CSF1R, or that act indirectly by inhibiting the expression of CSF1 R.
  • Such compositions also include compositions comprising only a single CSF1 R kinase inhibiting compound, as well as compositions comprising multiple therapeutics (including those against other RTKs), which may also include a non-specific therapeutic agent like a chemotherapeutic agent or general transcription inhibitor.
  • a CSF 1 R-inhibiting therapeutic useful in the practice of the methods of the invention is a targeted, small molecule inhibitor, such as Gleevec® (STI-571), and its analogues.
  • Gleevec® STI-571
  • administration of Gleevec® to a transgenic leukemia cell line expressing the RBM6-CSF1R fusion protein, or the truncated active CSF1 R kinase (lacking the RBM6 moiety) selectively inhibited the progression of the disease in those cells, but not in control cells that do not express the mutant CSF1R proteins.
  • Gleevec® which specifically binds to and blocks the ATP-binding site of CSF1 R kinase (as well as other kinases) thereby preventing phosphorylation and activation of this enzyme, is commercially available and its properties are well known.
  • lmatinib is an exemplary small molecule inhibitor that should be considered for patients with a cancer characterized by the RBM6-CSF1 R fusion or CSF1R mutant kinase.
  • Small molecule targeted inhibitors are a class of molecules that typically inhibit the activity of their target enzyme by specifically, and often irreversibly, binding to the catalytic site of the enzyme, and/or binding to an ATP-binding cleft or other binding site within the enzyme that prevents the enzyme from adopting a conformation necessary for its activity.
  • Small molecule inhibitors may be rationally designed using X-ray crystallographic or computer modeling of CSF1R kinase three- dimensional structure, or may found by high throughput screening of compound libraries for inhibition of CSF1 R. Such methods are well known in the art, and have been described.
  • CSF1R inhibition may be confirmed, for example, by examining the ability of such compound to inhibit CSF1 R activity, but not other kinase activity, in a panel of kinases, and/or by examining the inhibition of CSF 1 R activity in a biological sample comprising leukemia tumor cells, as described above. Such screening methods are further described below.
  • CSF 1 R kinase-inhibiting therapeutics useful in the methods of the invention may also be targeted antibodies that specifically bind to critical catalytic or binding sites or domains required for CSF1 R activity, and inhibit the kinase by blocking access of ligands (e.g. CSF) 1 substrates or secondary molecules to ⁇ and/or preventing the enzyme from adopting a conformation necessary for its activity.
  • ligands e.g. CSF
  • the production, screening, and therapeutic use of humanized target-specific antibodies has been well- described. See Merluzzi et al., Adv CHn Path. 4(2): 77-85 (2000).
  • Commercial technologies and systems, such as Morphosys, Inc.'s Human Combinatorial Antibody Library (HuCAL®), for the high-throughput generation and screening of humanized target-specific inhibiting antibodies are available.
  • a library of antibody fragments displayed on the surface of bacteriophages may be produced (see, e.g. U. S. Patent 6,300,064, October 9, 2001, Knappik et al.) and screened for binding to a soluble dimeric form of a receptor protein tyrosine kinase (like CSF1R).
  • An antibody fragment that binds to the soluble dimeric form of the RTK used for screening is identified as a candidate molecule for blocking constitutive activation of the target RTK in a cell. See European Patent No. EP1423428, Borges etal., supra.
  • CSF1R binding targeted antibodies identified in screening of antibody libraries as describe above may then be further screened for their ability to block the activity of CSF 1 R, both in vitro kinase assay and in vivo in cell lines and/or tumors.
  • CSF1 R inhibition may be confirmed, for example, by examining the ability of such antibody therapeutic to inhibit CSF1R kinase activity, but not other kinase activity, in a panel of kinases, and/or by examining the inhibition of CSF1R activity in a biological sample comprising cancer cells, as described above. Methods for screening such compounds for CSF1R kinase inhibition are further described above.
  • CSF1R-inhibiting compounds useful in the practice of the disclosed methods may also be compounds that indirectly inhibit CSF1 R activity by inhibiting the activity of proteins or molecules other than CSF1 R. kinase itself.
  • Such inhibiting therapeutics may be targeted inhibitors that modulate the activity of key regulatory kinases that phosphorylate or de- phosphorylate (and hence activate or deactivate) CSF 1 R itself, or interfere with binding of ligands, such as CSF.
  • CSF1R regulates downstream signaling through a network of adaptor proteins and downstream kinases. As a result, induction of cell growth and survival by CSF1R activity may be inhibited by targeting these interacting or downstream proteins.
  • Drugs currently in development that could be used in this manner include AKT inhibitors
  • RX-0201 rapamycin and its analogs such as CC 1- 779, Rapamune and RAD001.
  • CSF1R kinase activity may also be indirectly inhibited by using a compound that inhibits the binding of an activating molecule, such as the Macrophage Colony Stimulating Factor (CSF) 1 or 2, necessary for CSF1 R to adopt its active conformation.
  • an activating molecule such as the Macrophage Colony Stimulating Factor (CSF) 1 or 2, necessary for CSF1 R to adopt its active conformation.
  • CSF Macrophage Colony Stimulating Factor
  • Indirect inhibitors of CSF1R activity may be rationally designed using X-ray crystallographic or computer modeling of CSF1R three dimensional structure, or may found by high throughput screening of compound libraries for inhibition of key upstream regulatory enzymes and/or necessary binding molecules, which results in inhibition of CSF1 R kinase activity. Such approaches are well known in the art, and have been described. CSF1R inhibition by such therapeutics may be confirmed, for example, by examining the ability of the compound to inhibit CSF1R activity, but not other kinase activity, in a panel of kinases, and/or by examining the inhibition of CSF1R activity in a biological sample comprising cancer cells, e.g. AML cells, as described above.
  • cancer cells e.g. AML cells
  • CSF1R inhibiting therapeutics may also comprise anti-sense and/or transcription inhibiting compounds that inhibit CSF1R kinase activity by blocking transcription of the gene encoding CSF1 R and/or the RBM6-CSF1 R fusion gene or truncated CSF1R gene.
  • antisense therapeutics for the treatment of cancer has been described. See, e.g., U.S. Patent Nos. 6,734,017; 6,710,174, 6,617,162; 6,340,674; 5,783,683; 5,610,288.
  • Antisense oligonucleotides may be designed, constructed, and employed as therapeutic agents against target genes in accordance with known techniques. See, e.g. Cohen, J., Trends in Pharmacol. ScL 10(11): 435-437 (1989); Marcus-Sekura, Anal. Biochem. 172: 289-295 (1988); Weintraub, H., ScL AM. pp. 40-46 (1990); Van Der Krol etal., BioTechniques 6(10): 958-976 (1988); Skorski etal., Proc. Natl. Acad. ScL USA (1994) 97: 4504-4508. Inhibition of human carcinoma growth in vivo using an antisense RNA inhibitor of EGFR has recently been described.
  • a CSF1R-inhibiting therapeutic comprising at least one antisense oligonucleotide against a mammalian CSF 1 R gene (see Figure 4 (SEQ ID NO: 6)) or RBM6-CSF1 R fusion polynucleotide or truncated CSF1R polynucleotide (see Figure 2 (SEQ ID NO: 2)) may be prepared according to methods described above.
  • Pharmaceutical compositions comprising CSF1 R-inhibiting antisense compounds may be prepared and administered as further described below.
  • Small Interfering RNA Small interfering RNA molecule (siRNA) compositions, which inhibit translation, and hence activity, of CSF1 R through the process of RNA interference, may also be desirably employed in the methods of the invention.
  • RNA interference and the selective silencing of target protein expression by introduction of exogenous small double-stranded RNA molecules comprising sequence complimentary to mRNA encoding the target protein, has been well described. See, e.g. U.S. Patent Publication No. 20040038921 , "Composition and Method for Inhibiting Expression of a Target Gene," February 26, 2004, Kreutzer etal.; U.S. Patent Publication No. 20020086356, "RNA Sequence-Specific Mediators of RNA Interference," June 12, 2003, Tuschl et al.; U.S. Patent
  • siRNA- mediated silencing of expression of the RBM6-CSF1 R fusion protein in a human leukemia cell line expressing the fusion protein selectively inhibited the progression of the disease in those cells, but not in control cells that do not express the mutant CSF1R protein.
  • RNA interference Double-stranded RNA molecules (dsRNA) have been shown to block gene expression in a highly conserved regulatory mechanism known as RNA interference (RNAi). Briefly, the RNAse III Dicer processes dsRNA into small interfering RNAs (siRNA) of approximately 22 nucleotides, which serve as guide sequences to induce target-specific mRNA cleavage by an RNA-induced silencing complex RISC (see Hammond et al., Nature (2000) 404: 293-296). RNAi involves a catalytic- type reaction whereby new siRNAs are generated through successive cleavage of longer dsRNA. Thus, unlike antisense, RNAi degrades target RNA in a non-stoichiometric manner. When administered to a cell or organism, exogenous dsRNA has been shown to direct the sequence- specific degradation of endogenous messenger RNA (mRNA) through RNAi.
  • mRNA messenger RNA
  • RNAi RNAi Technical Reference & Application Guide
  • RNAi RNAi: A Guide to Gene Silencing
  • CSF1 R-inhibiting siRNA products are also commercially available, and may be suitably employed in the method of the invention. See, e.g. Dharmacon, Inc., Lafayette, CO (Cat Nos. M-003162-03, MU-003162-03, D-003162-07 thru -10 (siGENOMETM SMARTselection and SMARTpool® siRNAs).
  • small dsRNA less than 49 nucleotides in length, and preferably 19-25 nucleotides, comprising at least one sequence that is substantially identical to part of a target mRNA sequence, and which dsRNA optimally has at least one overhang of 1-4 nucleotides at an end, are most effective in mediating RNAi in mammals.
  • 21-23 nt RNAs can be produced and tested for their ability to mediate RNAi in a mammalian cell, such as a human or other primate cell. Those 21-23 nt RNA molecules shown to mediate RNAi can be tested, if desired, in an appropriate animal model to further assess their in vivo effectiveness.
  • Target sites that are known, for example target sites determined to be effective target sites based on studies with other nucleic acid molecules, for example ribozymes or antisense, or those targets known to be associated with a disease or condition such as those sites containing mutations or deletions, can be used to design siRNA molecules targeting those sites as well.
  • the sequences of effective dsRNA can be rationally designed/predicted screening the target mRNA of interest for target sites, for example by using a computer folding algorithm.
  • the target sequence can be parsed in silico into a list of all fragments or subsequences of a particular length, for example 23 nucleotide fragments, using a custom Perl script or commercial sequence analysis programs such as Oligo, MacVector, or the GCG Wisconsin Package.
  • RNA sequence can be determined which sites are the most suitable target sites within the target RNA sequence. These parameters include but are not limited to secondary or tertiary RNA structure, the nucleotide base composition of the target sequence, the degree of homology between various regions of the target sequence, or the relative position of the target sequence within the RNA transcript. Based on these determinations, any number of target sites within the RNA transcript can be chosen to screen siRNA molecules for efficacy, for example by using in vitro RNA cleavage assays, cell culture, or animal models. See, e.g., U.S. Patent Publication No. 20030170891, September 11 , 2003, McSwiggen J. An algorithm for identifying and selecting RNAi target sites has also recently been described. See U.S.
  • Patent Publication No. 20040236517 "Selection of Target Sites for Antisense Attack of RNA," November 25, 2004, Drlica et at.
  • Commonly used gene transfer techniques include calcium phosphate, DEAE-dextran, electroporation and microinjection and viral methods (Graham et al. (1973) Virol. 52: 456; McCutchan etal., (1968), J. Natl. Cancer Inst. 41: 351 ; Chu et al. (1987), Nucl. Acids Res. 15: 1311; Fraley etal. (1980), J. Biol. Chem. 255: 10431; Capecchi (1980), Cell 22: 479).
  • DNA may also be introduced into cells using cationic liposomes (Feigner et al. (1987), Proc. Natl. Acad. Sci USA 84: 7413).
  • cationic liposomes include Tfx 50 (Promega) or Lipofectamin 200 (Life Technologies).
  • viral vectors may be employed to deliver dsRNA to a cell and mediate RNAi. See U.S Patent Publication No. 20040023390, "siRNA-mediated Gene Silencing with Viral Vectors," Feb. 4, 2004, Davidson et al.
  • RNAi in mammalian cells are commercially available and have been well desribed. See, e.g. Dharmacon, Inc., DharmaFECTTM system; Promega, Inc., siSTRIKETM U6 Hairpin system; see also Gou et al. (2003) FEBS. 548, 113-118; Sui, G. et al. A DNA vector-based RNAi technology to suppress gene expression in mammalian cells (2002) Proc. Natl. Acad. Sci. 99, 5515-5520; Yu etal. (2002) Proc. Natl. Acad. Sci. 99, 6047- 6052; Paul, C. et al.
  • siRNA interference in a mammal using prepared dsRNA molecules may then be effected by administering a pharmaceutical preparation comprising the dsRNA to the mammal.
  • the pharmaceutical composition is administered in a dosage sufficient to inhibit expression of the target gene.
  • dsRNA can typically be administered at a dosage of less than 5 mg dsRNA per kilogram body weight per day, and is sufficient to inhibit or completely suppress expression of the target gene.
  • a suitable dose of dsRNA will be in the range of 0.01 to 2.5 milligrams per kilogram body weight of the recipient per day, preferably in the range of 0.1 to 200 micrograms per kilogram body weight per day, more preferably in the range of 0.1 to 100 micrograms per kilogram body weight per day, even more preferably in the range of 1.0 to 50 micrograms per kilogram body weight per day, and most preferably in the range of 1.0 to 25 micrograms per kilogram body weight per day.
  • a pharmaceutical composition comprising the dsRNA is administered once daily, or in multiple sub- doses, for example, using sustained release formulations well known in the art. The preparation and administration of such pharmaceutical compositions may be carried out accordingly to standard techniques, as further described below.
  • Such dsRNA may then be used to inhibit CSF1 R expression and activity in a cancer, by preparing a pharmaceutical preparation comprising a therapeutically-effective amount of such dsRNA, as described above, and administering the preparation to a human subject having a cancer expressing RBM6-CSF1 R fusion protein or truncated active CSF1 R kinase, for example, via direct injection to the tumor.
  • a pharmaceutical preparation comprising a therapeutically-effective amount of such dsRNA, as described above, and administering the preparation to a human subject having a cancer expressing RBM6-CSF1 R fusion protein or truncated active CSF1 R kinase, for example, via direct injection to the tumor.
  • siRNA inhibitors has recently been described. See U.S. Patent Publication No. 20040209832, October 21 , 2004, McSwiggen et a/.; U.S. Patent Publication No. 20030170891 , September 11 , 2003, McSwiggen;
  • CSF 1 R kinase-inhibiting therapeutic compositions useful in the practice of the methods of the invention may be administered to a mammal by any means known in the art including, but not limited to oral or peritoneal routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal and topical (including buccal and sublingual) administration.
  • a CSF1R-inhibiting therapeutic will generally be provided in the form of tablets or capsules, as a powder or granules, or as an aqueous solution or suspension.
  • Tablets for oral use may include the active ingredients mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrating agents, binding agents, lubricating agents, sweetening agents, flavoring agents, coloring agents and preservatives.
  • suitable inert diluents include sodium and calcium carbonate, sodium and calcium phosphate, and lactose, while corn starch and alginic acid are suitable disintegrating agents.
  • Binding agents may include starch and gelatin, while the lubricating agent, if present, will generally be magnesium stearate, stearic acid or talc.
  • the tablets may be coated with a material such as glyceryl monostearate or glyceryl distearate, to delay absorption in the gastrointestinal tract.
  • Capsules for oral use include hard gelatin capsules in which the active ingredient is mixed with a solid diluent, and soft gelatin capsules wherein the active ingredients is mixed with water or an oil such as peanut oil, liquid paraffin or olive oil.
  • the pharmaceutical compositions of the invention will generally be provided in sterile aqueous solutions or suspensions, buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride.
  • the carrier may consists exclusively of an aqueous buffer ("exclusively" means no auxiliary agents or encapsulating substances are present which might affect or mediate uptake of the CSF 1 R-inhibiting therapeutic).
  • aqueous suspensions may include suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone and gum tragacanth, and a wetting agent such as lecithin.
  • Suitable preservatives for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.
  • CSF1R kinase-inhibiting therapeutic compositions may also include encapsulated formulations to protect the therapeutic (e.g. a dsRNA compound) against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • encapsulated formulations to protect the therapeutic (e.g. a dsRNA compound) against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions (including liposomes targeted to infected cells with monoclonal antibodies to viral antigens) can also be used as pharmaceutically acceptable carriers.
  • An encapsulated formulation may comprise a viral coat protein.
  • the viral coat protein may be derived from or associated with a virus, such as a polyoma virus, or it may be partially or entirely artificial.
  • the coat protein may be a Virus Protein 1 and/or Virus Protein 2 of the polyoma virus, or a derivative thereof.
  • CSF1R-inhibiting compositions can also comprise a delivery vehicle, including liposomes, for administration to a subject, carriers and diluents and their salts, and/or can be present in pharmaceutically acceptable formulations.
  • a delivery vehicle including liposomes
  • methods for the delivery of nucleic acid molecules are described in Akhtar et a/., 1992, Trends Cell Bio., 2, 139; DELIVERY STRATEGIES FOR ANTISENSE OLIGONUCLEOTIDE THERAPEUTICS, ed. Akbtar, 1995, Maurer et a/., 1999, MoI. Membr. Biol., 16, 129-140; Hofland and Huang, 1999, Handb. Exp.
  • CSF1R-inhibitlng therapeutics can be administered to a mammalian tumor by a variety of methods known to those of skill in the art, including, but not restricted to, encapsulation in liposomes, by iontophoresis, or by incorporation into other vehicles, such as hydrogels, cyclodextrins, biodegradable nanocapsules, and bioadhesive microspheres, or by proteinaceous vectors (O'Hare and Normand, International PCT Publication No. WO 00/53722).
  • the therapeutic/vehicle combination is locally delivered by direct injection or by use of an infusion pump.
  • compositions whether subcutaneous, intramuscular, or intradermal, can take place using standard needle and syringe methodologies, or by needle-free technologies such as those described in Conry et al., 1999, Clin. Cancer Res., 5, 2330-2337 and Barry etal., International PCT Publication No. WO 99/31262.
  • Pharmaceutically acceptable formulations of CSF1 R kinase- inhibitory therapeutics include salts of the above described compounds, e.g., acid addition salts, for example, salts of hydrochloric, hydrobromic, acetic acid, and benzene sulfonic acid.
  • a pharmacological composition or formulation refers to a composition or formulation in a form suitable for administration, e.g., systemic administration, into a cell or patient, including for example a human. Suitable forms, in part, depend upon the use or the route of entry, for example oral, transdermal, or by injection. Such forms should not prevent the composition or formulation from reaching a target cell. For example, pharmacological compositions injected into the blood stream should be soluble. Other factors are known in the art, and include considerations such as toxicity and forms that prevent the composition or formulation from exerting its effect.
  • Administration routes that lead to systemic absorption are desirable and include, without limitation: intravenous, subcutaneous, intraperitoneal, inhalation, oral, intrapulmonary and intramuscular.
  • Each of these administration routes exposes the CSF1R-inhibiting therapeutic to an accessible diseased tissue or tumor.
  • the rate of entry of a drug into the circulation has been shown to be a function of molecular weight or size.
  • the use of a liposome or other drug carrier comprising the compounds of the instant invention can potentially localize the drug, for example, in certain tissue types, such as the tissues of the reticular endothelial system (RES).
  • RES reticular endothelial system
  • a liposome formulation that can facilitate the association of drug with the surface of cells, such as, lymphocytes and macrophages is also useful. This approach can provide enhanced delivery of the drug to target cells by taking advantage of the specificity of macrophage and lymphocyte immune recognition of abnormal cells, such as cancer cells.
  • compositions or formulations that allows for the effective distribution of the nucleic acid molecules of the instant invention in the physical location most suitable for their desired activity.
  • agents suitable for formulation with the nucleic acid molecules of the instant invention include: P-glycoprotein inhibitors (such as Pluronic P85), which can enhance entry of drugs into the CNS (Jolliet-Riant and Tillement, 1999, Fundam. Clin. Pharmacol., 13, 16-26); biodegradable polymers, such as poly (DL-lactide-coglycolide) microspheres for sustained release delivery after intracerebral implantation (Emerich et al, 1999, Cell Transplant, 8, 47-58) (Alkermes, Inc.
  • CSF1R- inhibiting compounds useful in the method of the invention include material described in Boado et al., 1998, J. Pharm. Sci., 87, 1308-1315; Tyler et at., 1999, FEBS Lett., 421, 280-284; Pardridge et al., 1995, PNAS USA, 92, 5592-5596; Boado, 1995, Adv.
  • compositions comprising surface-modified liposomes containing poly (ethylene glycol) lipids (PEG-modified, or long-circulating liposomes or stealth liposomes) may also be suitably employed in the methods of the invention. These formulations offer a method for increasing the accumulation of drugs in target tissues.
  • This class of drug carriers resists opsonization and elimination by the mononuclear phagocytic system (MPS or RES), thereby enabling longer blood circulation times and enhanced tissue exposure for the encapsulated drug (Lasic et al. Chem. Rev. 1995, 95, 2601-2627; Ishiwata et al., Chem. Pharm. Bull. 1995, 43, 1005-1011).
  • Such liposomes have been shown to accumulate selectively in tumors, presumably by extravasation and capture in the neovascularized target tissues (Lasic etal., Science 1995, 267, 1275-1276; Oku et al.,1995, Biochim. Biophys. Acta, 1238, 86-90).
  • the long-circulating liposomes enhance the pharmacokinetics and pharmacodynamics of DNA and RNA, particularly compared to conventional cationic liposomes which are known to accumulate in tissues of the MPS (Liu et al., J. Biol. Chem. 1995, 42, 24864-24870; Choi etal., International PCT Publication No. WO 96/10391; Ansell et al., International PCT Publication No. WO 96/10390; Holland et al., International PCT Publication No. WO 96/10392). Long-circulating liposomes are also likely to protect drugs from nuclease degradation to a greater extent compared to cationic liposomes, based on their ability to avoid accumulation in metabolically aggressive MPS tissues such as the liver and spleen.
  • compositions may include a pharmaceutically effective amount of the desired compounds in a pharmaceutically acceptable carrier or diluent.
  • Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in REMINGTON'S PHARMACEUTICAL SCIENCES, Mack Publishing Co. (A. R. Gennaro edit. 1985).
  • preservatives, stabilizers, dyes and flavoring agents can be provided. These include sodium benzoate, sorbic acid and esters of p-hydroxybenzoic acid.
  • antioxidants and suspending agents can be used.
  • a pharmaceutically effective dose is that dose required to prevent, inhibit the occurrence, or treat (alleviate a symptom to some extent, preferably all of the symptoms) of a disease state.
  • the pharmaceutically effective dose depends on the type of disease, the composition used, the route of administration, the type of mammal being treated, the physical characteristics of the specific mammal under consideration, concurrent medication, and other factors that those skilled in the medical arts will recognize. Generally, an amount between 0.1 mg/kg and 100 mg/kg body weight/day of active ingredients is administered dependent upon potency of the negatively charged polymer.
  • Dosage levels of the order of from about 0.1 mg to about 140 mg per kilogram of body weight per day are useful in the treatment of the above-indicated conditions (about 0.5 mg to about 7 g per patient per day).
  • the amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the host treated and the particular mode of administration.
  • Dosage unit forms generally contain between from about 1 mg to about 500 mg of an active ingredient. It is understood that the specific dose level for any particular patient depends upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy.
  • the composition can also be added to the animal feed or drinking water. It can be convenient to formulate the animal feed and drinking water compositions so that the animal takes in a therapeutically appropriate quantity of the composition along with its diet. It can also be convenient to present the composition as a premix for addition to the feed or drinking water.
  • a CSF1R-inhibiting therapeutic useful in the practice of the invention may comprise a single compound as described above, or a combination of multiple compounds, whether in the same class of inhibitor (i.e. antibody inhibitor), or in different classes (i.e antibody inhibitors and small-molecule inhibitors). Such combination of compounds may increase the overall therapeutic effect in inhibiting the progression of a fusion protein-expressing cancer.
  • the therapeutic composition may a small molecule inhibitor, such as STI-571 (Gleevec®) alone, or in combination with other Gleevec® analogues (e.g. SU 11248 or GW2580) targeting CSF1R activity and/or other small molecule inhibitors.
  • the therapeutic composition may also comprise one or more non-specific chemotherapeutic agent in addition to one or more targeted inhibitors.
  • Such combinations have recently been shown to provide a synergistic tumor killing effect in many cancers. The effectiveness of such combinations in inhibiting CSF1R activity and tumor growth in vivo can be assessed as described below. Identification of Mutant CSF1 R Kinase-inhibitin ⁇ Compounds.
  • the invention also provides, in part, a method for determining whether a compound inhibits the progression of a cancer characterized by a RBM6-CSF1 R translocation and/or fusion polypeptide, or a truncated CSF1 R polynucleotide and/or truncated active CSF1 R kinase polypeptide, by determining whether the compound inhibits the activity of RBM6-CSF1 R kinase fusion polypeptide or truncated active CSF 1 R kinase polypeptide in the cancer.
  • inhibition of activity of CSF1R is determined by examining a biological sample comprising cells from bone marrow, blood, or a tumor.
  • inhibition of activity of CSF1R is determined using at least one mutant CSF1R polynucleotide or polypeptide-specific reagent of the invention.
  • the tested compound may be any type of therapeutic or composition as described above. Methods for assessing the efficacy of a compound, both in vitro and in vivo, are well established and known in the art. For example, a composition may be tested for ability to inhibit CSF1R in vitro using a cell or cell extract in which CSF1R is activated. A panel of compounds may be employed to test the specificity of the compound for CSF1 R (as opposed to other targets, such as EGFR or PDGFR).
  • mutant CSF1 R polypeptides large numbers of different small test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the test compounds are reacted with mutant CSF1 R polypeptide, or fragments thereof, and washed.
  • Bound mutant polypeptide e.g. RBM6-CSF1R fusion polypeptide or truncated active CSF 1 R kinase polypeptide
  • Purified mutant CSF1R polypeptide can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non- neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • a compound found to be an effective inhibitor of CSF1 R activity in vitro may then be examined for its ability to inhibit the progression of a cancer expressing RBM6-CSF1 R fusion polypeptide and/or truncated active CSF1R kinase polypeptide, in vivo, using, for example, mammalian bone marrow transplants (e.g. mice) harboring human leukemias that are driven by the mutant CSF1R protein(s).
  • mammalian bone marrow transplants e.g. mice harboring human leukemias that are driven by the mutant CSF1R protein(s).
  • bone marrow cells known to be driven by mutant CSF1R kinase are transplanted in the mouse.
  • the growth of the cancerous cells may be monitored.
  • the mouse may then be treated with the drug, and the effect of the drug treatment on cancer phenotype or progression be externally observed.
  • mammalian xenografts may be prepared, by standard methods, to examine drug response in solid tumors expressing a mutant CSF1R kinase. In this way, the effects of the drug may be observed in a biological setting most closely resembling a patient.
  • the drug's ability to alter signaling in the cancerous cells or surrounding stromal cells may be determined by analysis with phosphorylation-specific antibodies.
  • the drug's effectiveness in inducing cell death or inhibition of cell proliferation may also be observed by analysis with apoptosis specific markers such as cleaved caspase 3 and cleaved PARP.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit high therapeutic indices are preferred.
  • the IAP technique was performed using a phosphotyrosine-specific antibody (CELL SIGNALING TECHNOLOGY, INC., Beverly, MA 1 2003/04 Cat. #9411) to isolate, and subsequently characterize, phosphotyrosine-containing peptides from extracts of the AML cell lines.
  • STAT5 is a member of the STAT family of transcription factors.
  • the activated tyrosine kinases typically phosphorylate one or more signal transducer and activator (STAT) of transcription factors, which translocate to the cell nucleus and regulate the expression of genes associated with survival and proliferation.
  • STAT signal transducer and activator
  • the phosphorylation and activation of STAT family members has been previously been described in a wide range of human leukemias.
  • animal models have demonstrated the important role of STAT in leukemogenesis.
  • STAT5 has been found to be constitutively tyrosine phosphorylated in about 70% of patients with AML.
  • Activating mutations of FLT3 or KIT can account for up to 35% of patients with STAT5 phosphorylation. However, a significant percentage of patients lacking these mutations maintain phosphorylation of STAT5. Hence, it was hypothesized that the upstream activator of STAT5 in some of these patients is an activated tyrosine kinase, and activation of these kinases was examined.
  • K562 cells were obtained from American Type Culture Collection (ATCC). MKPL-1, GDM-1, NKM-1, CMK, BaF3, and BaF3/BCR-ABL cell lines were generously provided by Dr. Brian Druker (OHSU). BaF3/FLT3- ITD cells were a kind gift from Dr. Donald Small. BaF3 cells were maintained in RPMM 640 medium (Invitrogen) with 10% fetal bovine serum (FBS) (Sigma) and 1.0 ng/ml IL-3 (R&D Systems). Other cell lines were grown in RPMI-1640 with 10% FBS. 293T cells were grown in DMEM with 10% fetal calf serum.
  • ATCC American Type Culture Collection
  • MKPL-1, GDM-1, NKM-1, CMK, BaF3, and BaF3/BCR-ABL cell lines were generously provided by Dr. Brian Druker (OHSU).
  • BaF3/FLT3- ITD cells were a kind gift from Dr. Donald Small.
  • a total of 2 x 10 8 cells were lysed in urea lysis buffer (2OmM HEPES pH 8.0, 9M urea, 1 mM sodium vanadate, 2.5 noM sodium pyrophosphate, 1mM beta-glycerophosphate) at 1.25 x 10 8 cells/ml and sonicated. Sonicated lysates were cleared by centrifugation at 20,000 x g, and proteins were reduced and alkylated as described previously (see Rush et al, Nat. Biotechnol. 23(1): 94-101 (2005)). Samples were diluted with 20 mM HEPES pH 8.0 to a final urea concentration of 2M. Trypsin (1mg/ml in 0.001 M HCI) was added to the clarified lysate at 1:100 v/v. Samples were digested overnight at room temperature.
  • urea lysis buffer 2OmM HEPES pH 8.0, 9M urea, 1 mM sodium vanadate, 2.5
  • lysates were acidified to a final concentration of 1% TFA.
  • Peptide purification was carried out using Sep-Pak Ci 8 columns as described previously (see Rush et al., supra.). Following purification, all elutions (8%, 12%, 15%, 18%, 22%, 25%, 30%, 35% and 40% acetonitrile in 0.1% TFA) were combined and lyophilized. Dried peptides were resuspended in 1.4 ml MOPS buffer (50 mM MOPS/NaOH pH 7.2, 10 mM Na 2 HPO-I, 50 mM NaCI) and insoluble material removed by centrifugation at 12,000 x g for 10 minutes.
  • MOPS buffer 50 mM MOPS/NaOH pH 7.2, 10 mM Na 2 HPO-I, 50 mM NaCI
  • the phosphotyrosine monoclonal antibody P-TyMOO (Cell Signaling Technology) from ascites fluid was coupled non-covalently to protein G agarose beads (Roche) at 4 mg/ml beads overnight at 4 0 C. After coupling, antibody-resin was washed twice with PBS and three times with MOPS buffer. Immobilized antibody (40 ⁇ l, 160 ⁇ g) was added as a 1:1 slurry in MOPS IP buffer to the solubilized peptide fraction, and the mixture was incubated overnight at 4 0 C. The immobilized antibody beads were washed three times with MOPS buffer and twice with ddHaO. Peptides were eluted twice from beads by incubation with 40 ⁇ l of 0.1% TFA for 20 minutes each, and the fractions were combined.
  • Peptides in the IP eluate (40 ⁇ l) were concentrated and separated from eluted antibody using Stop and Go extraction tips (StageTips) (see Rappsilber et al., Anal. Chem., 75(3): 663-70 (2003)). Peptides were eluted from the microcolumns with 1 ⁇ l of 60% MeCN, 0.1% TFA into 7.6 ⁇ l of 0.4% acetic acid/0.005% heptafluorobutyric acid (HFBA).
  • StageTips Stop and Go extraction tips
  • the sample was loaded onto a 10 cm x 75 ⁇ m PicoFrit capillary column (New Objective) packed with Magic C18 AQ reversed-phase resin (Michrom Bioresources) using a Famos autosampler with an inert sample injection valve (Dionex).
  • the column was developed with a 45-min linear gradient of acetonitrile in 0.4% acetic acid, 0.005% HFBA delivered at 280 nl/min (Ultimate, Dionex).
  • Tandem mass spectra were collected in a data-dependent manner with an LCQ Deca XP Plus ion trap mass spectrometer
  • ThermoFinnigan using a top-four method, a dynamic exclusion repeat count of 1, and a repeat duration of 0.5 min.
  • MS/MS spectra were evaluated using TurboSequest (ThermoFinnigan) (in the Sequest Browser package (v. 27, rev. 12) supplied as part of BioWorks 3.0). Individual MS/MS spectra were extracted from the raw data file using the Sequest Browser program CreateDta, with the following settings: bottom MW, 700; top MW, 4,500; minimum number of ions, 20; minimum TIC, 4 x 10 5 ; and precursor charge state, unspecified. Spectra were extracted from the beginning of the raw data file before sample injection to the end of the eluting gradient. The IonQuest and VuDta programs were not used to further select MS/MS spectra for Sequest analysis.
  • TurboSequest ThermoFinnigan
  • MS/MS spectra were evaluated with the following TurboSequest parameters: peptide mass tolerance, 2.5; fragment ion tolerance, 0.0; maximum number of differential amino acids per modification, 4; mass type parent, average; mass type fragment, average; maximum number of internal cleavage sites, 10; neutral losses of water and ammonia from b and y ions were considered in the correlation analysis.
  • Proteolytic enzyme was specified except for spectra collected from elastase digests.
  • the spectrum contained at least one major peak (at least 10% as intense as the most intense ion in the spectrum) that could not be mapped to the assigned sequence as an a, b, or y ion, as an ion arising from neutral-loss of water or ammonia from a b or y ion, or as a multiply protonated ion;
  • the spectrum did not contain a series of b or y ions equivalent to at least six uninterrupted residues; or
  • the sequence was not observed at least five times in all the studies we have conducted (except for overlapping sequences due to incomplete proteolysis or use of proteases other than trypsin).
  • the foregoing IAP analysis identified 512 non-redundant phosphotyrosine-containing peptides, 437 phosphotyrosine sites, and 300 tyrosine phosphorylated proteins, the majority of which are novel, from MKPL-1 cells (data not shown).
  • tyrosine phosphorylated kinases several of those detected are not normally detected by MS analysis in other leukemia cell lines (unpublished data), including CSF1R, C-KIT, Jak2, and Jak3.
  • MKPL-1 cells were lysed in 1 x cell lysis buffer (Cell Signaling Technology) supplemented with Protease ArrestTM (G Biosciences) and separated by electrophoresis. All antibodies and reagents for immunoblotting were from Cell Signaling Technology, Inc. (Beverly, MA). Western blotting was carried out as described in "Western Immunoblotting Protocol” (Cell Signaling Technology, Inc., 2005-2006 catalogue).
  • Figure 5 shows the western blot results. While wild type CSF1 R was detected in two of the AML cell lines (GDM-1 and NKM-1), a truncated form of CSF1R was detected in the MKPL-1 cell line (see panel A, left column). In addition, phosphorylation of CSF1 R kinase's downstream targets, STAT5 and ERK 1 was also detected in the MKPL-1 cell line (as well as cell lines CMK and K562), validating the presence of activated (but truncated) CSF1 R kinase in this AML cell line (see Fig. 5, panel B). Beta-actin expression was used as a control.
  • lmatrnib (STI-571 ; Gleevec® (Novartis, Basel, Switzerland)) is a potent tyrosine kinase inhibitor for ABL, ARG, PDGFR a and b, and C- KIT 1 and also inhibits CSF1 R.
  • CSF1 R drives the proliferation of MKPL-1 cells
  • the effect of lmatinib on the growth of MKPL-1 cells was examined. For Western blotting, cells were treated for two hours with 10 ⁇ M lmatinib before lysis.
  • a standard MTT cell proliferation assay (see Mosmann, J. Immunol Methods. 65(1-2): 55-63 (1983)) was performed on the MKPL-1 cell line (as well as AML cell line GDM-1) using a range of Gleevec® concentrations.
  • the K562 cell line which is known to be driven by the BCR/ABL translocation and inhibited by Gleevec®, was employed as a positive control.
  • CMK was employed as a negative control.
  • the results of the assay are presented in Figure 6 (panel B). The results confirm that the MKPL-1 cell line (as well as the GDM-1 cell line, which expresses wild type CSF1R) is inhibited by Gleevec®, while the control cells are not.
  • MKPL-1 cells were inhibited by Imatinib, though at much higher concentrations than required in K562 cells expressing BCR-ABL.
  • concentration of Imatinib required to inhibit MKPL-1 cells by 50 percent (IC 50 ) was 1.26 mM, where as the ICsofor BCR-ABL in K562 cells was 0.31 mM.
  • treatment of MKPL-1 cells with lmatinib resulted in induction of apoptosis (% cleaved caspase-3), which was not observed in the control cell line CMK 1 a known AML-M7 cell line (see Figure 6, panel C).
  • Wild type CSF1 R has been reported as a target of lmatinib, with an IC 50 of 1.42 mM, which is very similar to what we observed in MKPL-1 cells expressing the truncated form of the kinase disclosed herein. These data indicate that truncated CSF1 R is the target of lmatinib in MKPL-1 cells.
  • CSF1 R contributes to the growth and viability of the AML (MKPL-1) cells
  • siRNA For Western blotting, cells were treated for two hours with 10 ⁇ M lmatinib before lysis.
  • dose response curves cells were incubated for 48 hours in the presence of lmatinib, and the number of viable cells was determined with the CellTiter 96 AQueous One solution cell proliferation assay (Promega). IC 5 O was calculated with the use of OriginPro 6.1 software (OriginLab). The percentage of apoptotic cells at 48 hours was determined by flow cytometric analysis of Cleaved- Caspase-3 (Cell Signaling Technology).
  • CSF1 R SMARTpoo] siRNA duplexes (proprietary target sequences - data not shown) were purchased from Dharmacon Research, Inc. (Lafayette, CO). A non-specific SMARTpoo] siRNA was used as a control.
  • Cells were transfected with the siRNA via electroporation. Briefly, 2 x 10 7 cells were pulsed once (MKPL-1 20ms; 275V, K562 20ms; 285V) using a square-wave electroporator (BTX Genetronics, San Diego, CA), incubated at room temperature for 30 minutes and transferred to T150 flasks with 30 ml RPMM 640/10% FBS.
  • RNeasy Mini Kit (Qiagen) was used to extract RNA from human leukemia cell lines. DNA was extracted with the use of DNeasy Tissue Kit (Qiagen). Rapid amplification of cDNA ends was performed with the use of 5 * RACE system (Invitrogen) with primers CSF1 R-P1 for cDNA synthesis and CSF1 R-P2 and CSF1 R-P3 for a nested PCR reaction.
  • first-strand cDNA was synthesized from 2.5 mg of total RNA with the use of SuperscriptTM III first-strand synthesis system (Invitrogen) with oligo (dT)2o. Then, the RBM6-CSF1 R fusion gene was amplified with the use of primer pairs RBM6-F1 and CSF1R-P3. The reciprocal fusion was detected with the use of primer pairs CSF1R-F and RBM6-R. Wild type RBM6 and CSF 1 R were amplified with the use of primer pairs RBM6-F1 and RBM6-R, CSF1R-F and CSF1R-P3, respectively.
  • amplification of the fusion gene was performed with the use of Platinum Taq DNA polymerase high fidelity (Invitrogen) with primer pairs gRBM6-F1 and gCSF1R-R1 , or gRBM6-F1 and gCSF1R-R2.
  • RBM6-CSF1 R fusion gene The open reading frame of the RBM6-CSF1 R fusion gene was amplified by PCR from cDNA of MKPL-1 cells with the use of Platinum Taq DNA polymerase high fidelity (Invitrogen) and primer pairs RBM6-Fc1 and CSFIR-Rc. This PCR product was cloned in the retroviral vector MSCV-Neo or MSCV-GFP. Construct with deletion mutation was obtained by PCR from RBM6-CSF1 R clone with primer pairs RBM6-Fc2 and CSFIR-Rc. The following primers were used:
  • RBM6-F1 5'GACCTGCTAACAGAACTGGACCTT (SEQ ID NO: 9)
  • RBM6-R 5'CTCTGAAGTCAACAGCGTGAGCAT (SEQ ID NO: 10)
  • CSF1R-F 5'ATCGAGAGCTATGAGGGCAACAGT
  • CSF1 R-P1 5'CCTTCCTTCGCAGAAAGTTGAGCA
  • CSF1 R-P2 5'AAAGTTGAGCAGGTCGCCATAGCA
  • CSF1R-P3 5'AGCAACAGTACTCCGTGATGACCA
  • gRBM6-F1 5TGTAAAAGGAGGGCCAGUCCTCGTGTTCTGAAATGGGAGCA
  • GAPDH-F 5TGGAAATCCCATCACCATCT (SEQ ID NO: 18)
  • GAPDH-R 5'GTCTTCTGGGTGGCAGTGAT (SEQ ID NO: 19)
  • RBM6-Fc1 ⁇ 'CGGAATTCGATAAAAAGAGATGTGGGGGGAT
  • RBM6-Fc2 5'CGGAATTCACCATGAAGTGGGAGTTCCCCCGGA
  • FIG 8 shows the detection of the PCR amplification product after 2 rounds. Sequence analysis of the resultant product revealed that the split kinase domain of CSF1R was fused to RNA Binding Motif 6 (RBM6) gene N-terminus (see Figure 1, panel B).
  • RBM6-CSF1 R fusion gene was in-frame and fused the first 36 amino acids of RBM6 to the last 399 amino acids of CSF1 R (see Figure 1 , panel B).
  • RBM6 was located on chromosome 3p21.3, whereas CSF1R was on chromosome 5q33.
  • the fusion gene was created by t(3;5)(p21;q33).
  • RBM6 and CSF 1 R were confirmed by reverse- transcriptase-PCR on RNA and PCR on DNA from MKPL-1 cells (see Figures 9 and 10).
  • the reciprocal CSF1R-RBM6 fusion gene was not detected in RNA.
  • the MKPL-1 cell line did not express wild- type CSF1R, but did express wild-type RBM6 (see Figure 9), strongly suggesting that RBM6-CSF1 R fusion is the major genetic abnormality responsible for proliferation and survival the MKPL-1 AML cell line.
  • BaF3 cells were transformed with the cDNA construct described above.
  • BaF3 cells were a kind gift from Dr. Donald Small. Cells were maintained in RPMI-1640 medium (Invitrogen) with 10% fetal bovine serum (FBS) (Sigma) and 1.0 ng/ml IL-3 (R&D Systems). Production of retroviral supernatant and transduction was carried out as previously described. See Schwaller etal., Embo J. 17(18): 5321- 33 (1998).
  • BaF3 cells were transduced with retroviral supernatant containing either the MSCV-Neo/RBM6-CSF1R or MSCV-Neo/CSF1R (truncation) vectors, respectively, and selected for G418 (1 mg/ml).
  • IL-3 independent growth was accessed by plating transduced BaF3 cells in IL- 3 free medium, after the cells were washed three times in PBS. For Western blotting, cells were treated for two hours with 10 ⁇ M lmatinib before lysis.
  • the in vivo transforming ability of activated CSF1R may be further shown using murine bone marrow transplantation experiments, as previously described. See, e.g., Stover ef al., Blood 106(9): 3206-3213 (2005). Briefly, MSCV-GFP retroviral supernatants were titered by transducing Ba/F3 cells with supernatant (plus polybrene, 10 ⁇ g/mL) and analyzing for the percentage of GFP+ cells by flow cytometry at 48 hours after transduction. Balb/C donor mice (Taconic, Germantown, NY) were treated for 5 to 6 days with 5-fluorouracil (150 mg/kg, Intraperitoneal injection).
  • Bone marrow cells from donor mice were harvested, treated with red blood cell lysis buffer, and cultured 24 hours in transplantation medium (RPMI + 10% FBS + 6 ng/mL IL-3, 10 ng/mL IL-6, and 10 ng/mL stem-cell factor). Cells were treated by spin infection with retroviral supernatants (1 ml_ supernatant per 4 x 10 6 cells, plus polybrene) and centrifuged at 180Og for 90 minutes.
  • FISH fluorescence in situ hybridization
  • bone marrow samples may be obtained from a patient having AML using standard techniques.
  • FISH probes against truncated CSF1R kinase or RBM6-CSF1 R fusion protein are constructed, and FISH analysis is performed as described below. See, e.g., Dierlamm etal., Genes, Chromosomes and Cancer 6: 261-4 (1996).
  • Probes Preparation At room temperature mix 7 A ⁇ L of LSI Hybridization Buffer, 1 A ⁇ L LSI DNA probe, and 2 A ⁇ L purified H2O. Centrifuge for 1-3 seconds, vortex and then re-centrifuge.
  • Slide Preparation Mark hybridization areas with a diamond tipped scribe. Immerse the slide in the 73+/-1 0 C denaturant bath (70% formamide/2X SSC) for 5 minutes. If metaphase chromosome morphology is problematic, a denaturant temperature of 70-73 0 C may provide for better results. Dehydrate slide 1 minute in 70% EtOH 1 1 minute in 85% EtOH, and 1 minute in 100% EtOH. Dry slide and place on a 45-50 0 C slide warmer for 2 minutes.
  • Hybridization Apply 10 A ⁇ L of probe mix to slide. Apply coverslip immediately upon placing probe on slide, and seal coverslip with diluted rubber cement. Place slide in a pre-warmed humidified box and allow for hybridization to proceed overnight for 12-16 hrs in a 37 0 C incubator. Prepare one wash tank with 0.4X SSC/0.3%NP-40 and place into the 73+/-1 0 C water bath for at least 30 minutes. Discard after 1 day of use. Prepare a second tank of 2X SSC/0.1 % NP-40 at room temperature.
  • Such an analysis will identify a patient having a cancer characterized by expression of the truncated CSF 1 R kinase (and/or RBM6-CSF1 R fusion protein), which patient is a candidate for treatment using a CSF1 R-inhibiting therapeutic, such as Gleevec®.
  • truncated CSF1R kinase and/or RBM6-CSF1R fusion protein in a human cancer sample may be detected using either genomic or reverse transcriptase (RT) polymerase chain reaction (PCR) 1 previously described. See, e.g., Cools etal., N. Engl. J. Med. 348: 1201- 1214 (2003).
  • RT reverse transcriptase
  • bone marrow samples may be obtained from a patient having AML using standard techniques. PCR probes against truncated CSF1R kinase or RBM6-CSF1 R fusion protein are constructed. RNeasy Mini Kit (Qiagen) may be used to extract RNA from human bone marrow samples. DNA may be extracted with the use of DNeasy Tissue Kit (Qiagen).
  • first-strand cDNA is synthesized from, e.g., 2.5 ⁇ g of total RNA with the use, for example, of SuperscriptTM III first-strand synthesis system (Invitrogen) with oligo (dT)2o- Then, the RBM6-CSF1R fusion gene is amplified with the use of primer pairs, e.g. RBM6-F1 and CSF1R-P3 (see Example 4 above).
  • amplification of the fusion gene may be performed with the use of Platinum Taq DNA polymerase high fidelity (Invitrogen) with primer pairs, e.g. gRBM6-F1 and gCSF1R-R1, or gRBM6-F1 and gCSF1R-R2 (see Example 4, above).
  • Such an analysis will identify a patient having a cancer characterized by expression of the truncated CSF1 R kinase (and/or RBM6-CSF1 R fusion protein), which patient is a candidate for treatment using a CSF1R-inhibiting therapeutic, such as Gleevec®.

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Abstract

Selon la présente invention, une nouvelle translocation de gène, (3p21, 5q33), dans la leucémie myélogène humaine (AML) qui produit une protéine hybride combinant une partie de la protéine 6 de liaison à l'ARN (RBM6) avec la kinase du récepteur du facteur 1 de stimulation colonies de macrophages (CSF1 R) a maintenant été identifiée. La protéine hybride RBM6-CSF1 R et la kinase CSF1R tronquée elle-même, qui retiennent toutes les deux l'activité de la tyrosine kinase CSF1 R, ont été confirmées comme entraînant la prolifération et la survie de la leucémie mégacaryoblaste aiguë (AML-M7). Par conséquent, l'invention concerne, en partie, des polynucléotides isolés et des vecteurs codant pour des polypeptides de la kinase CSFR1 mutante de l'invention, des sondes pour sa détection, des polypeptides mutants isolés, des polypeptides recombinants, et des réactifs pour la détection de la fusion et des polypeptides tronqués. L'identification de l'invention de cette nouvelle protéine hybride permet de nouveaux procédés pour la détermination de la présence de ces polypeptides de la kinase CSFR1 mutants dans un échantillon biologique, des procédés pour le criblage pour des composés inhibiteurs des protéines, et des procédés pour l'inhibition de la progression d'un cancer caractérisé par les polynucléotides ou polypeptides mutants de l'invention.
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US10982001B2 (en) 2012-05-11 2021-04-20 Five Prime Therapeutics, Inc. Methods of treating conditions with antibodies that bind colony stimulating factor 1 receptor (CSF1R)
US11421034B2 (en) 2017-09-13 2022-08-23 Five Prime Therapeutics, Inc. Combination anti-CSF1R and anti-PD-1 antibody combination therapy for pancreatic cancer

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US8183207B2 (en) 2008-11-26 2012-05-22 Five Prime Therapeutics, Inc. Treatment of osteolytic disorders and cancer using CSF1R extracellular domain fusion molecules
US9957327B2 (en) 2010-05-04 2018-05-01 Five Prime Therapeutics, Inc. Antibodies that bind colony stimulating factor 1 receptor (CSF1R)
US8747845B2 (en) 2010-05-04 2014-06-10 Five Prime Therapeutics, Inc. Methods of treatment by administering antibodies that bind colony stimulating factor 1 receptor (CSF1R)
US9200075B2 (en) 2010-05-04 2015-12-01 Five Prime Therapeutics, Inc. Nucleic acids encoding antibodies that bind colony stimulating factor 1 receptor (CSF1R)
US9695243B2 (en) 2010-05-04 2017-07-04 Five Prime Therapeutics, Inc. Antibodies that bind colony stimulating factor 1 receptor (CSF1R)
US11186646B2 (en) 2010-05-04 2021-11-30 Five Prime Therapeutics, Inc. Antibodies that bind colony stimulating factor 1 receptor (CSF1R)
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US10982001B2 (en) 2012-05-11 2021-04-20 Five Prime Therapeutics, Inc. Methods of treating conditions with antibodies that bind colony stimulating factor 1 receptor (CSF1R)
US10221243B2 (en) 2012-08-31 2019-03-05 Five Prime Therapeutics, Inc. Methods of treating conditions with antibodies that bind colony stimulating factor 1 receptor (CSF1R)
US10822421B2 (en) 2012-08-31 2020-11-03 Five Prime Therapeutics, Inc. Methods of treating conditions with antibodies that bind colony stimulating factor 1 receptor (CSF1R)
CN103664800A (zh) * 2012-09-25 2014-03-26 杨子娇 一类治疗房角狭窄的化合物及其用途
US10975153B2 (en) 2014-06-23 2021-04-13 Five Prime Therapeutics, Inc. Methods of treating conditions with antibodies that bind colony stimulating factor 1 receptor (CSF1R)
US11566076B2 (en) 2014-10-29 2023-01-31 Five Prime Therapeutics, Inc. Anti-CSF1R antibody and anti-PD-1 antibody combination therapy for selected cancers
US10618967B2 (en) 2014-10-29 2020-04-14 Five Prime Therapeutics, Inc. Anti-CSF1R antibody and anti PD-1 antibody combination therapy for cancer
US10221244B2 (en) 2014-10-29 2019-03-05 Five Prime Therapeutics, Inc. Anti-CSF1R antibody and anti PD-1 antibody combination therapy for cancer
US9765147B2 (en) 2014-10-29 2017-09-19 Five Prime Therapeutics, Inc. Anti-CSFR1 antibody and anti PD-1 antibody combination therapy for cancer
US10040858B2 (en) 2014-12-22 2018-08-07 Five Prime Therapeutics, Inc. Anti-CSF1R antibodies for treating PVNS
US10730949B2 (en) 2014-12-22 2020-08-04 Five Prime Therapeutics, Inc. Method of treating PVNS with anti-CSF1R antibodies
US11559583B2 (en) 2015-04-13 2023-01-24 Five Prime Therapeutics, Inc. Anti-CSF1R antibody and agonistic anti-CD40 antibody combination therapy for cancer
CN107709365A (zh) * 2015-04-13 2018-02-16 戊瑞治疗有限公司 癌症组合疗法
US11421034B2 (en) 2017-09-13 2022-08-23 Five Prime Therapeutics, Inc. Combination anti-CSF1R and anti-PD-1 antibody combination therapy for pancreatic cancer

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