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WO2000011162A1 - Modulation de l'apoptose a l'aide de bid - Google Patents

Modulation de l'apoptose a l'aide de bid Download PDF

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Publication number
WO2000011162A1
WO2000011162A1 PCT/US1999/016966 US9916966W WO0011162A1 WO 2000011162 A1 WO2000011162 A1 WO 2000011162A1 US 9916966 W US9916966 W US 9916966W WO 0011162 A1 WO0011162 A1 WO 0011162A1
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Prior art keywords
bid
polypeptide
cell
target cell
mutant
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PCT/US1999/016966
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English (en)
Inventor
Atan Gross
Stanley J. Korsmeyer
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Washington University
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Priority claimed from US09/136,879 external-priority patent/US6326354B1/en
Application filed by Washington University filed Critical Washington University
Priority to AU52337/99A priority Critical patent/AU5233799A/en
Priority to EP99937522A priority patent/EP1104460A1/fr
Publication of WO2000011162A1 publication Critical patent/WO2000011162A1/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
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • C12N9/6475—Interleukin 1-beta convertase-like enzymes (3.4.22.10; 3.4.22.36; 3.4.22.63)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • This invention relates generally to the regulation of cell death and to compounds which regulate cell death, and more particularly, to a novel BH3- containing fragment of BID with cell death agonist activity.
  • the BCL-2 family possesses pro-apoptotic (BAX, BAK, BOK) as well as anti-apoptotic (BCL-2, BCL-X L , BCL-W, MCL-1, Al) molecules (Farrow and Brown, supra).
  • the ratio of anti- to pro-apoptotic molecules such as BCL-2/BAX determines the response to a death signal (Oltvai et al., Cell 74:609-619, 1993).
  • a striking characteristic of many BCL-2 family members is their propensity to form homo- and heterodimers (Sedlak et al., Proc. Natl. Acad. Sci. USA 92:7834-7838, 1995; Zha et al., J. Biol.
  • BH3 only members include the mammalian proteins BID, BAD, BIK, RIM, BLK and HRK, as well as the EGL- 1 protein of C. elegans. Of note, all of these molecules are pro-apoptotic, lending credence to the thesis that BH3 represents a minimal death domain (Wang et al.,
  • BID and BAD lack the typical hydrophobic C-terminal sequence that is found in most BCL-2 family members, which for BCL-2 has been shown to function as a signal anchor segment required for its targeting mitochondria (Nguyen et al., J. Biol. Chem. 268:25265-25268, 1993). Consistent with their lack of a putative C-terminal anchor segment, BID and BAD have been observed in cytosolic as well as membrane based localizations (Wang et al., 1996, supra; Zha et al., Cell 87:619-628, 1996).
  • BID and BAD may represent death ligands, sensors that receive death signals in the cytosol and translocate to membranes where they interact with membrane bound, classic BCL-2 members which serve as "receptors" (Wang et al., 1996, supra).
  • a physiologic death stimulus results in the translocation of monomeric BAX from the cytosol to the mitochondria where it is present as a homodimerized, integral membrane protein (Gross et al., supra).
  • the importance of the BH3 domain and caspase activation in apoptosis is also suggested by the recent demonstration that BAX and BID fragments containing the
  • B3 domain i.e., BAX 53-104 and BID 74-128, had death agonist activity when expressed in cells and that death of these cells was significantly inhibited in the presence of the general caspase inhibitor z-VAD-fmk (copending application, U.S.
  • the best characterized signal transduction pathways that mediate apoptosis are the cell surface receptors of the TNF family, including CD95 (Fas/Apo-1) and
  • CD120a p55 TNF receptor
  • DISC Death-Inducing Signaling Complex
  • This complex comprises Fas/TNF receptor, FADD (MORTI), and pro-caspase-8 (MACH/FLICE/Mch5).
  • caspase-8 Once caspase-8 is recruited, it is processed and released from the complex in active form to activate downstream "effector” caspases (Medema et al., supra; Srinivasula et al., Proc. Natl. Acad. Sci. USA 93:14486-14491, 1996; Muzio et al., J. Biol. Chem. 272:2952-2956, 1997).
  • caspase family has been divided into three groups based upon sequence homology and substrate specificity using a positional scanning substrate combinatorial library (Thomberry et al., J. Biol. Chem. 272:17907-17911, 1997).
  • the optimal cleavage sequence for caspases 6, 8, and 9 of (I/L/V)EXD resembles activation sites in the effector caspase proenzymes, suggesting that caspases 6, 8, and 9 represent "initiator" caspases.
  • cytochrome c, Apaf-1 and caspase-9 form a complex that initiates a downstream apoptotic caspase cascade (Li et al., Cell 91:479-489, 1997).
  • cytochrome c was released, leading to the activation of caspases and nuclear apoptosis (Kluck et al., EMBO J. 16:4639-4649, 1997).
  • cytochrome c redistribution from mitochondria to cytosol was also reported to occur in intact cells during apoptosis (Bossy- Wetzel et al., EMBO J.: 17:37-49, 1998).
  • the molecular mechanism responsible for the release of cytochrome c from mitochondria to the cytosol during apoptosis was not known.
  • Some disease conditions are believed to be related to the development of a defective down-regulation of apoptosis in the affected cells. For example, neoplasias may result, at least in part, from a apoptosis-resistant state in which cell proliferation signals inappropriately exceed cell death signal.
  • DNA viruses such as Epstein-Barr virus, African swine fever virus and adenovirus, parasitze the host cellular machinery to drive their own replication and at the same time block apoptosis to repress cell death and allow the target cell to reproduce the virus.
  • certain disease conditions such as lymphoproliferative conditions, cancer including drug resistant cancer, arthritis, inflammation, autoimmune diseases and the like may result from a down regulation of cell death regulation. In such disease conditions it would be desirable to promote apoptotic mechanisms and one advantageous approach might involve treatment with a cell death agonist having a BH3 domain which has been identified as being critical for killing.
  • apoptosis such as in the treatment of immunodeficiency diseases, including AIDS, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, wound-healing, and the like.
  • immunodeficiency diseases including AIDS, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, wound-healing, and the like.
  • apoptosis such as in the treatment of immunodeficiency diseases, including AIDS, senescence, neurodegenerative disease, ischemic and reperfusion cell death, infertility, wound-healing, and the like.
  • cytosolic BID activation of the TNF and FAS death signal pathways induces a caspase-mediated cleavage of cytosolic BID to produce a BID polypeptide fragment of approximately 15 kDa containing the BH3 domain.
  • This polypeptide fragment referred to herein as pl5 BID, translocates from the cell cytosol to the mitochondria where it resides as an integral membrane protein and is required for the release of cytochrome c.
  • N-terminal sequence analysis of pi 5 produced by incubation of full-length BID, also referred to herein as p22 BID, with Caspase-8 or Caspase-3 demonstrated that BID is cleaved between Asp and Gly residues corresponding to positions 60 and 61 of human BID (SEQ ID NO:l) and 59 and 60 of murine BID (SEQ ID NO:2) (see Fig. 1). Mutation of Asp59 to Ala in murine BID prevents caspase-mediated cleavage at this site. Therefore, in one aspect the invention provides an isolated and purified polypeptide comprising a pl5 BID polypeptide which has cell death agonist activity.
  • Preferred pl5 BID polypeptides identified herein include human and murine pi 5 BID as shown in FIGS. 2A and 2C (SEQ ID NO:3 and SEQ ID NO:5, respectively).
  • the invention provides isolated and purified polynucleotides encoding a pi 5 BID polypeptide having cell death agonist activity. These polynucleotides may be used to transfect a target cell in which expression of the encoded pi 5 BID polypeptide promotes death of the target cell.
  • a recombinant cell stably transformed with a polynucleotide encoding for expression a pl5 BID polypeptide is also provided by the invention.
  • the recombinant cell may be used in a method for producing the pi 5 BID polypeptide.
  • the present invention provides a composition comprising a pi 5 BID polypeptide which has cell death agonist activity and a carrier which facilitates delivery of the pi 5 polypeptide into a cell.
  • the invention also provides a method for promoting death of a target cell which comprises treating the cell with an effective amount of a pi 5 BID polypeptide having cell death agonist activity.
  • the cell can be treated in vitro or in vivo in a patient
  • the cell is treated in vivo by administering to the patient a polynucleotide encoding the pl5 BID polypeptide, through which the pi 5 polypeptide is expressed in the target cell.
  • the treating step comprises administering the pi 5 BID polypeptide to the patient, preferably with a carrier that facilitates delivery of the pi 5 BID polypeptide into the target cell.
  • the invention is directed to inhibiting cell death using compositions and methods which inhibit the in vivo generation of pi 5 BID and/or translocation of p 15 BID to the mitochondria.
  • the invention provides a composition comprising an agent that specifically inhibits cleavage of p22 BID at the pi 5 cleavage site and a pharmaceutically acceptable carrier.
  • an agent that specifically inhibits cleavage of p22 BID at the pi 5 cleavage site and a pharmaceutically acceptable carrier.
  • One such agent is a peptide comprising LQTD which is believed can act as a competitive inhibitor for the active site of Caspase-8 and would thus inhibit Caspase-8 cleavage of p22 BID.
  • the agent may also be a peptide mimetic of LQTD.
  • the invention also provides a method for inhibiting death of a target cell comprising treating the cell with a polynucleotide encoding a mutant p22 BID polypeptide comprising a mutation in the pi 5 cleavage site which blocks caspase cleavage at the pi 5 site.
  • the mutation is designed to not significantly reduce binding of the mutant p22 BID to caspases and therefore can act as a competitive inhibitor for Caspase-8 binding to wild-type p22 BID.
  • the invention provides a method for inhibiting death of a target cell comprising treating the cell with a mutant pi 5 BID polypeptide having an inactivating mutation in the BH3 domain.
  • the mutant pi 5 BID lacks cell death agonist activity but is capable of serving as a competitive inhibitor in the cellular mechanism which translocates pl5 BID to the mitochondria.
  • the target cell is treated with a mutant p22 BID polypeptide which is cleaved by Caspase-8 to produce mutant pi 5 BID.
  • the mutant pi 5 BID and or mutant p22 BID may be delivered to the target cell with a carrier which facilitates entry of the mutant polypeptide into the cell or alternatively may be expressed in the cell by a recombinant polynucleotide introduced into the cell.
  • a new pi 5 BID polypeptide having cell death agonist activity may be noted the provision of a new pi 5 BID polypeptide having cell death agonist activity; the provision of polynucleotides encoding pl5 BID; the provision of pi 5 BID or mutant pi 5 BID compositions which can be readily delivered intracellularly to produce a death agonist or death antagonist activity, respectively; the provision of methods for modulating cell death using these compositions; and the provision of methods for inhibiting cell death by inhibiting in vivo production of pi 5 BID.
  • Figure 1 illustrates the aligned human and murine BID polypeptides (SEQ ID NO: 1
  • FIG. 2 illustrates the amino acid sequences of (FIG. 2 A) human pi 5 BID (SEQ ID NO:3), (FIG. 2B) a human variant pl5 BID (SEQ ID NO:4) containing an additional five amino acids indicated by the underline, and (FIG. 2C) murine pi 5 BID (SEQ ID NO:5);
  • Figure 3 A is a graph showing cell viability, mitochondrial membrane potential and ROS production for cells treated with TNF ⁇ /cycloheximide in the presence or absence (w/o) of the general caspase inhibitor zVAD-ftnk.
  • Figure 3B is a digitized image of proteins from whole cell lysates of FL5.12 cells or 2B4 cells treated with TNF ⁇ /Chx in the presence or absence of zVAD-fink which were fractionated by SDS-PAGE and analyzed by Western blot with anti-BID Ab;
  • Figure 4A is a digitized image of a Western blot of murine BID incubated with the cytosol of FL5.12 cells treated with TNF ⁇ /Chx (S 1 OO(TNF)) in the presence or absence of zVAD-fmk;
  • Figure 4B is a digitized image of a Western blot of the products of incubating wt BID or a mutant BID having a D59A with the cytosol of FL5.12 cells treated with TNF ⁇ /Chx (SIOO(TNF)) in the presence or absence of zVAD-fmk;
  • Figure 4C shows aligned partial sequences of murine and human BID showing the caspase cleavage sites in murine BID;
  • Figure 5A is a digitized image of a Western blot of BID, cytochrome c (Cyt c), and anticytochrome c oxidase subunit IV (Cyt oxi) detected in various subcellular fractions using appropriate antibodies;
  • Figure 5B is a digitized image of a Western blot showing that BID cleavage products in mitochondria are resistant to alkali and salt extraction.
  • Figure 5C is a digitized image of a Western blot showing that cytosolic pi 5 BID targets mouse liver mitochondria but full-length p22 BID does not.
  • Figures 6A-6C are digitized images of Western blots showing that targeting of cytosolic pi 5 BID to mouse liver mitochondria is required to release cytochrome c.
  • Figures 7A-7C are digitized images of Western blots showing that anti-Fas Ab injection of mice results in accumulation of pi 5 BID in the cytosol of hepatocytes and is subsequently translocated to mitochondria.
  • Figure 8 is a model of BID cleavage and translocation following TNF receptor/Fas engagement.
  • the present invention is based on the surprising discovery that cell death mediated by the TNF and FAS signaling pathways includes the generation of a pi 5 BID polypeptide which is translocated to the mitochondria where it exerts cell death agonist activity, probably by inducing the release of cytochrome c.
  • the cell death induced by pi 5 BID is intended to include death by apoptosis and or death by necrosis.
  • sequence of biochemical and molecular events in apoptosis vary, at least to some degree, depending on the cell type and death-inducing stimulus, all apoptotic cell death appears to undergo common terminal events including distinct morphological features of cytoplasmic shrinking, plasma membrane blebbing, condensation of nuclear chromatin, and fragmentation of genomic DNA.
  • apoptotic cells are eventually engulfed by neighboring cells or phagocytes, thereby avoiding an inflammatory response.
  • Necrosis is a pathological type of cell death observed following physical or chemical injury, exposure to toxins, or ischemia and characterized by swelling, rupture of the plasma membrane and cellular organelles, and in vivo inflammation due to release of the cellular content into the surrounding tissue. Hengartner, M., in Molecular Biolosv and Biotechnology. Robert A. Myers, ed., VCH Publishers, Inc., 1995, p. 158.
  • pi 5 BID is produced by caspase cleavage of BID after the internal aspartic acid residue which corresponds to Asp ⁇ O and Asp59 of human and murine BID.
  • BID or p22 BID refers to the full-length BID polypeptide synthesized by a cell.
  • Human and murine BID are 195 amino acids in length and it is believed that BID synthesized by other mammalian species is of similar length and can be identified by alignment with the human and murine BID amino acid sequences shown in Fig. 1.
  • one embodiment of the invention provides an isolated and purified polypeptide comprising a pi 5 BID polypeptide which has cell death agonist activity.
  • a pi 5 BID polypeptide contains the BH3 domain but lacks the N-terminal region of BID. Based on the conservation of the putative LQTD caspase recognition site in human and murine BID (see Fig. 4C), it is believed that BID proteins from other species, particularly mammalian species, are cleaved by a caspase after an aspartic acid corresponding to Asp 60 and Asp59 of human and murine BID to produce pi 5 BID polypeptides having cell death agonist activity.
  • pi 5 BID polypeptides embraced by the invention include any naturally- occurring pi 5 BID molecules as well as engineered pi 5 BID polypeptides comprising an amino acid sequence substantially identical to a naturally-occurring amino acid sequence.
  • substantially identical is meant that two amino acid sequences of the same length share at least 85% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity or more.
  • amino acid positions which are not identical differ by conservative amino acid substitutions.
  • Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains.
  • Conservatively substituted amino acids can be grouped according to the chemical properties of their side chains.
  • one grouping of amino acids includes those amino acids that have neutral and hydrophobic side chains (A, V, L, I, P, W, F, and M); another grouping is those amino acids having neutral and polar side chains (G, S, T, Y, C, N, and Q); another grouping is those amino acids having basic side chains (K, R, and H); another grouping is those amino acids having acidic side chains (D and E); another grouping is those amino acids having aliphatic side chains (G, A, V, L, and I); another grouping is those amino acids having aliphatic-hydroxyl side chains (S and T); another grouping is those amino acids having amine-containing side chains (N, Q, K, R, and H); another grouping is those amino acids having aromatic side chains (F, Y, and W); and another grouping is those amino acids having sulfur-containing side chains (C and M).
  • Preferred conservative amino acid substitutions groups are: R-K; E-D, Y-F, L-M; V-I, and Q-H
  • a pi 5 BID polypeptide can comprise a modified amino acid or unusual amino acid at one or more positions in its amino acid sequence, and can also comprise amino acids that are glycosylated or phosphorylated so long as the pi 5 BID polypeptide has cell death agonist activity.
  • Preferred embodiments of the pl5 BID polypeptide comprise SEQ ID NO:3 or SEQ ID NO: 5 or conservatively substituted variants thereof. Even more preferably, the pl5 BID polypeptide consists of SEQ ID NO:3.
  • the pi 5 BID polypeptides of the present invention can be made by recombinant DNA technology by expressing a nucleotide sequence encoding the desired amino acid sequence in a suitable transformed host cell. Using methods well known in the art, a polynucleotide encoding a pl5 BID polypeptide may be operably linked to an expression vector, transformed into a host cell and culture conditions established that are suitable for expression of the pl5 BID polypeptide by the transformed cell.
  • Any suitable expression vector may be employed to produce recombinant pi 5 BID polypeptides such as, for example, the mammalian expression vector pCB6 (Brewer, Meth Cell Biol 4.3:233-245, 1994) or the E. coli pET expression vectors, specifically, pET-30a (Studier et al., Methods Enzymol 185:60-89, 1990).
  • suitable expression vectors for expression in mammalian and bacterial cells are known in the art as are expression vectors for use in yeast or insect cells. Baculovirus expression systems can also be employed.
  • Mammalian host cells include, but are not limited to, monkey COS cells, Chinese Hamster Ovary (CHO) cells, human kidney 293 cells, human epidermal A431 cells, human Colo 205 cells, 3T3 cells, CV-1 cells, other transformed primate cell lines, normal diploid cells, cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK and Jurkat cells.
  • monkey COS cells Chinese Hamster Ovary (CHO) cells
  • human kidney 293 cells human epidermal A431 cells
  • human Colo 205 cells human Colo 205 cells
  • CV-1 cells other transformed primate cell lines
  • normal diploid cells cell strains derived from in vitro culture of primary tissue, primary explants, HeLa cells, mouse L cells, BHK, HL-60, U937, HaK and Jurkat cells.
  • Yeast strains that may act as suitable host cells include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces strains, Candida, and any other yeast strain capable of expressing heterologous proteins.
  • Host bacterial strains include Escherichia coli, Bacillus subtilis, Salmonella typhimurium and any other yeast strain capable of expressing heterologous proteins. If the pi 5 BID polypeptide is made in yeast or bacteria, it may be necessary to modify the polypeptide, for example, by phosphorylation or glycosylation of the appropriate sites using known chemical or enzymatic methods, to obtain a biologically active pi 5 BID polypeptide.
  • the expressed pl5 BID polypeptide can be isolated and purified using known purification procedures, such as gel filtration and ion exchange chromatography.
  • isolated and purified means that a designated polypeptide constitutes at least about 50 percent of a composition on a molar basis compared to total proteins or other macromolecular species present in the composition.
  • the polypeptide of the invention will constitute at least about 75 to about 80 mole percent of the total protein or other macromolecular species present. More preferably, an isolated and purified polypeptide will constitute about 85 to about 90 mole percent of a composition and still more preferably, at least about 95 mole percent or greater.
  • Purification may also include affinity chromatography using an agent that will specifically bind the pi 5 BID polypeptide, such as a polyclonal or monoclonal antibody raised against BID or a C-terminal fragment thereof.
  • affinity resins typically used in protein purification may also be used such as concanavalin A- agarose, heparin-toyopearl ® or Cibacrom blue 3GA Sepharose ® .
  • Purification of pBID polypeptides can also include one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether.
  • a pi 5 BID polypeptide may be expressed as a fusion protein to facilitate purification.
  • fusion proteins include a pi 5 BID polypeptide fused to a histidine tag such as when expressed in the pET bacterial expression system as well as a pi 5 BID polypeptide fused to maltose binding protein (MBP), glutathione-S-transferase (GST) or thioredoxin (TRX).
  • MBP maltose binding protein
  • GST glutathione-S-transferase
  • TRX thioredoxin
  • the polypeptide of the invention can be tagged with a heterologous epitope and subsequently purified by immunoaffinity chromatography using an antibody that specifically binds such epitope. Kits for expression and purification of such fusion proteins and tagged proteins are commercially available.
  • pi 5 BID polypeptides of the invention may be produced by incubating BID, which has been isolated from cells or produced by recombinant DNA technology, with Caspase-8 or Caspase-3.
  • pi 5 BID may be chemically synthesized using methods known to those skilled in the art.
  • the pi 5 BID polypeptide can be tested for cell death agonist activity using the assays described below or any known model of apoptotic and/or necrotic cell death.
  • the present invention provides an isolated and purified polynucleotide comprising a nucleotide sequence that encodes a pi 5 BID polypeptide.
  • a polynucleotide includes DNA and/or RNA and thus the nucleotide sequences recited in the Sequence Listing as DNA sequences also include the identical RNA sequences with uracil substituted for thymine residues.
  • Nucleotide sequences included in the invention are those encoding the pl5 BID polypeptides set forth in SEQ ID NOS:3-5. It is understood by the skilled artisan that degenerate nucleotide sequences can encode the pi 5 BID polypeptides described herein and these are also intended to be included within the present invention.
  • nucleotide sequences include modifications of naturally-occurring sequences in which at least one codon is substituted with a corresponding redundant codon preferred by a given host cell, such as E. coli or insect cells, so as to improve expression of recombinant pi 5 BID therein.
  • Preferred polynucleotides of the invention encode human and mouse pi 5 BID as set forth in SEQ ID NO:3 and 5 and their nucleotide sequences can be determined by inspection of the known cDNA sequences for BID, see, e.g., copending Application Serial No. 08/706,741.
  • the present invention also encompasses vectors comprising an expression regulatory element operably linked to any of the pl5 BID-encoding nucleotide sequences included within the scope of the invention.
  • This invention also includes host cells, of any variety, that have been transformed with such vectors.
  • compositions comprising a pi 5 BID polypeptide which cell death agonist activity and a method for promoting death of a target cell which comprises treating the cell with an effective amount of the pi 5 BID polypeptide.
  • BID BH3 domain destroy its cell death agonist activity but not necessarily its ability to interact with other BCL-2 family members.
  • death of a target cell can be inhibited by treatment with a mutant pi 5 BID polypeptide lacking cell death agonist activity but retaining the ability to be translocated to the mitochondria or retaining the ability to competitively block translocation of pl5 BID.
  • other embodiments of the invention include therapeutic or pharmaceutical compositions comprising a mutant pl5 BID polypeptide lacking cell death agonist activity and a method for inhibiting death of a target cell which comprises treating the cell with an effective amount of the mutant pi 5 BID polypeptide.
  • the mutant pi 5 BID polypeptide is intended to be identical to a pi 5 BID polypeptide other than having an inactivating mutation in the BH3 domain that destroys its cell death agonist activity. It is also contemplated that the target cell can be treated with the mutant pi 5 BID polypeptide by delivering into the cell a mutant p22 BID polypeptide which is cleaved in the target cell by a caspase by a mechanism similar to or identical to that which produces pl5 BID.
  • death of a target cell may be inhibited by treating the cell with a mutant p22 BID polypeptide that has a mutation in the pi 5 cleavage site which blocks cleavage of the mutant p22 BID at the cleavage site.
  • pi 5 cleavage site is meant a sequence of four contiguous amino acids in a BID protein which corresponds to the putative Caspase-8 LQTD recognition site in human and murine BID.
  • a mutant BID containing an alanine substituted for the aspartic acid residue in this sequence is not cleaved at pi 5 when incubated in vitro with Caspase 8 (data not shown).
  • mutant pi 5 BID polypeptide and both types of mutant p22 BID polypeptides are intended to be included within the terms mutant BID or mutant BID polypeptide.
  • cell death is promoted or inhibited by treating the target cell with a polynucleotide encoding pi 5 BID or mutant BID operably linked to a promoter that produces expression of pi 5 BID or mutant BID in the target cell.
  • the polynucleotide can comprise an expression plasmid, a retrovirus vector, an adenovirus vector, an adenovirus associated vector (AAV) or other viral or nonviral vector used in the art to deliver genes into cells.
  • the polynucleotide can be administered to the target cell by microinjection.
  • the polynucleotide encoding pi 5 BID or mutant BID is administered to the patient.
  • the target cell may be treated with pi 5 BID or mutant BID by co infection with a replication-defective adenovirus expressing pl5 BID or mutant BID and another replication competent adenovirus that complements the replication defective virus to increase expression of the recombinant polypeptide in infected cells.
  • the polynucleotide is selectively delivered to target cells within the patient so as not to affect viability of other tissues.
  • Targeted delivery of the polynucleotide can be done for example by using delivery vehicles such as polycations, liposomes or viral vectors containing a targeting moiety that recognizes and binds to a specific marker on the target cell.
  • delivery vehicles such as polycations, liposomes or viral vectors containing a targeting moiety that recognizes and binds to a specific marker on the target cell.
  • delivery vehicles such as polycations, liposomes or viral vectors containing a targeting moiety that recognizes and binds to a specific marker on the target cell.
  • delivery vehicles such as polycations, liposomes or viral vectors containing a targeting moiety that recognizes and binds to a specific marker on the target cell.
  • viral vectors that can only replicate in specific cell types which is accomplished by placing the viral genes necessary for replication under the transcriptional control of a response element for a
  • the target cell is treated with a composition comprising a pl5 BID polypeptide or a mutant BID polypeptide.
  • a composition comprising a pl5 BID polypeptide or a mutant BID polypeptide.
  • pi 5 BID or mutant BID is administered with a carrier that facilitates delivery of the polypeptide into the cell, such as liposomes.
  • the liposomes can have targeting moieties exposed on the surface such as antibodies, ligands or receptors to specific cell surface molecules to limit delivery of p 15 BID or mutant BID to targeted cells. Liposome drug delivery is known in the art (see, e.g., Amselem et al., Chem. Phys. Lipid 64:219-237, 1993).
  • pl5 BID or mutant BID can be modified to include a specific transit peptide that is capable of delivering the polypeptide into the cytoplasm of the target cell or the polypeptide can be delivered directly into a target cell by microinjection.
  • compositions comprising a pl5 BID polypeptide or a mutant BID polypeptide can be administered by any suitable route known in the art including, for example, intravenous, subcutaneous, intramuscular, transdermal, intrathecal or intracerebral or administration to cells in ex vivo treatment protocols. Administration can be either rapid as by injection or over a period of time as by slow infusion or administration of slow release formulation. For treating tissues in the central nervous system, administration can be by injection or infusion into the cerebrospinal fluid (CSF).
  • CSF cerebrospinal fluid
  • the pi 5 BID polypeptide or mutant BID polypeptide can also be administered with one or more agents capable of promoting penetration of the polypeptide across the blood- brain barrier.
  • the pi 5 BID and mutant BID polyeptides can also be linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties, including for example, substances known in the art to promote penetration or transport across the blood-brain barrier such as an antibody to the transferrin receptor (Friden et al., Science 259:373-377, 1993), or a polymer such as polyethylene glycol to obtain desirable properties of solubility, stability, half- life and other pharmaceutically advantageous properties (Davis et al. Enzyme Eng 4:169-73, 1978; Burnham, Am JHosp Pharm 57:210-218, 1994).
  • agents that provide desirable pharmaceutical or pharmacodynamic properties including for example, substances known in the art to promote penetration or transport across the blood-brain barrier such as an antibody to the transferrin receptor (Friden et al., Science 259:373-377, 1993), or a polymer such as polyethylene glycol to obtain desirable properties of solubility, stability, half- life and other pharmaceutically advantageous properties (Davis
  • the compositions can also include absorption enhancers which increase the pore size of the mucosal membrane.
  • absorption enhancers include sodium deoxycholate, sodium glycocholate, dimethyl- ⁇ - cyclodextrin, lauroyl-1-lysophosphatidylcholine and other substances having structural similarities to the phospholipid domains of the mucosal membrane.
  • the compositions are usually employed in the form of pharmaceutical preparations. Such preparations are made in a manner well known in the pharmaceutical art. One preferred preparation utilizes a vehicle of physiological saline solution, but it is contemplated that other pharmaceutically acceptable carriers such as physiological concentrations of other non-toxic salts, five percent aqueous glucose solution, sterile water or the like may also be used.
  • a suitable buffer be present in the composition.
  • Such solutions can, if desired, be lyophilized and stored in a sterile ampoule ready for reconstitution by the addition of sterile water for ready injection.
  • the primary solvent can be aqueous or alternatively non-aqueous.
  • the carrier can also contain other pharmaceutically-acceptable excipients for modifying or maintaining the pH, osmolarity, viscosity, clarity, color, sterility, stability, rate of dissolution, or odor of the formulation.
  • the carrier may contain still other pharmaceutically-acceptable excipients for modifying or maintaining release or absorption or penetration across the blood-brain barrier.
  • excipients are those substances usually and customarily employed to formulate dosages for parenteral administration in either unit dosage or multi-dose form or for direct infusion by continuous or periodic infusion.
  • formulations comprising a pl5 BID polypeptide or a mutant BID polypeptide are to be administered orally.
  • Such formulations are preferably encapsulated and formulated with suitable carriers in solid dosage forms.
  • suitable carriers, excipients, and diluents include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, gelatin, syrup, methyl cellulose, methyl- and propylhydroxybenzoates, talc, magnesium, stearate, water, mineral oil, and the like.
  • the formulations can additionally include lubricating agents, wetting agents, emulsifying and suspending agents, preserving agents, sweetening agents or flavoring agents.
  • the compositions may be formulated so as to provide rapid, sustained, or delayed release of the active ingredients after administration to the patient by employing procedures well known in the art.
  • the formulations can also contain substances that diminish proteolytic degradation and/or substances which promote absorption such as, for example, surface active agents.
  • the pi 5 BID polypeptide or mutant BID is administered to patients in an amount effective to promote death of target cells within the patient.
  • the specific dose is calculated according to the approximate body weight or body surface area of the patient or the volume of body space to be occupied.
  • the dose will also be calculated dependent upon the particular route of administration selected. Further refinement of the calculations necessary to determine the appropriate dosage for treatment is routinely made by those of ordinary skill in the art. Such calculations can be made without undue experimentation by one skilled in the art in light of the activity disclosed herein in cell death assays. Exact dosages are determined in conjunction with standard dose-response studies.
  • the amount of the composition actually administered will be determined by a practitioner, in the light of the relevant circumstances including the condition or conditions to be treated, the choice of composition to be administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the chosen route of administration. Dose administration can be repeated depending upon the pharmacokinetic parameters of the dosage formulation and the route of administration used.
  • death of a target cell can be inhibited by treating the cell with a peptide which serves as a competitive inhibitor of p22 BID binding to Caspase-8.
  • a peptide which serves as a competitive inhibitor of p22 BID binding to Caspase-8.
  • One such peptide comprises LQTD.
  • Peptide mimetics of LQTD are also contemplated and may be advantageous to improve stability, binding affinity and other characteristics.
  • the design and synthesis of peptide mimetics are well known in the art.
  • Administration and dosage of caspase-cleavage blocking peptides and peptide mimetics can be accomplished using the detailed descriptions above.
  • Preferred embodiments of the invention have been described above. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification and the examples below or by practice of the invention as disclosed herein. It is intended that the specification, together with the Examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims which
  • Example 1 This Example illustrates that cell death induced by TNF ⁇ /cycloheximide treatment includes mitochondrial dysfunction and caspase-mediated cleavage of BID.
  • a protein synthesis inhibitor e.g. cycloheximide
  • FL15.2 cells were treated with recombinant mouse TNF- ⁇ (1 ng/ml; Sigma) and cycloheximide (Chx; 1 ⁇ g/ml; Sigma) in the presence or absence of the general caspase inhibitior 50 ⁇ M zVAD-fmk.
  • cell viability was determined by propidium iodide (PI) dye exclusion.
  • PI propidium iodide
  • ROS reactive oxygen species
  • FL5.12 cells or 2B4 T cells treated as described above were lysed at 5 hr or 2.5 hr., respectively, in 50 mM Tris (pH 7.5), 150 mM NaCl, and 1% Triton X-100 supplemented with a protease inhibitor cocktail (Sigma; added at a 1 : 100 dilution).
  • the lysates were separated by SDS-PAGE, and transferred to a polyvinylidine difluoride (PVDF; Bio-Rad) membrane. The membrane was first blocked with 5% milk for 1 hr., then incubated for 1 hr.
  • PVDF polyvinylidine difluoride
  • Fig. 3B The results, which are shown in Fig. 3B, revealed that in cells treated with TNF/Chx the intracellular pro-apoptotic molecule p22 BID was cleaved to yield a major pi 5 and minor pl3 and pi 1 fragments (Fig. 3B).
  • the 2B4 T cell hybridoma which is also killed by TNF and displays mitochondrial dysfunction (data not shown) also demonstrated the p 15 fragment (Fig. 3B).
  • Pre-treatment of cells with 5 0 ⁇ M zVAD-fmk markedly inhibited BID cleavage in FL5.12 cells and to a large extent in 2B4 cells (Fig. 3B).
  • Example 2 This example illustrates that BID is cleaved at three internal aspartic acid residues.
  • a recombinant murine BID (rBID) was incubated with the cytosol of TNF ⁇ /Chx treated FL5.12 cells.
  • Murine rBID was prepared by cloning the murine BID cDNA into pGEX-KG. Expression of GST-BID fusion protein was induced in BL21DE3 by I mM IPTG.
  • the bacterial pellet was resuspended in lysis buffer (1% Triton X- 100; 1 mM EDTA, I mM DTT in PBS) supplemented with a protease inhibitor cocktail (Sigma; added at a 1:100 dilution), and sonicated. After centrifugation at 10,000 g for 20 min., the supernatant was applied to glutathione-agarose beads (Sigma). The beads were washed with buffer and treated with 10 units of thrombin per original liter. Cleaved BID was eluted from beads and the cleavage reaction was terminated by adding 50 ⁇ g/ml of N ⁇ -p-tosyl-L-lysine chloromethyl ketone (TLCK). To remove the GST protein and incompletely cleaved fusion proteins, the preparation was further purified on a monoQ column and the proteins were eluted with a NaCl gradient.
  • lysis buffer 1% Triton X- 100; 1
  • FL5.12 cells were treated for 5 hr. with TNF ⁇ /Chx, washed once in phosphate-buffered saline (PBS), resuspended in isotonic HIM buffer (200 mM mannitol, 70 mM sucrose, I mM EGTA, 10 mM HEPES, pH 7.5) supplemented with a protease inhibitor cocktail (Sigma; added at a 1:100 dilution), and homogenized using a polytron homogenizer (Brinkmann Instruments) at setting 6.5 for 10 sec. Nuclei and unbroken cells were separated at 120 g for 5 min. as the low speed pellet (PI). This supernatant was centrifuged at 10,000 g for 10 min.
  • PBS phosphate-buffered saline
  • isotonic HIM buffer 200 mM mannitol, 70 mM sucrose, I mM EGTA, 10 mM HEPES, pH 7.5
  • HM heavy membrane pellet
  • LM light membrane pellet
  • SI 00 final soluble fraction
  • the rBID (5 ⁇ g) was incubated for 2 hrs. at 37°C with this SI 00 fraction in the presence or absence of 50 ⁇ M zVAD-fmk.
  • the proteins were denatured, separated by 16% SDS-PAGE and transferred to a polyvinylidine difluoride (PVDF; Bio-Rad) membrane.
  • PVDF polyvinylidine difluoride
  • the S100 fraction from TNF-treated cells caused complete cleavage of p22 rBID and generated major pi 5 and minor pl3 fragments (lane 3). This cleavage was inhibited by the inclusion of 50 ⁇ M zVAD-fmk in the reaction mixture (lane 4). Incubation of rBID with either recombinant active Caspase-8 or Caspase-3 also generated the p 15 fragment (data not shown).
  • Fig. 3B To determine whether the third cleavage site responsible for the less abundant pi 1 fragment observed in TNF-treated FL5.12 cells (Fig. 3B) was the predicted Asp98 residue, 35 S-labeled rBID and a mutant rBID having alanine substituted for aspartic acid at position 98 (BIDD98A) were prepared by in vitro translation and incubated with the SI 00 fraction from TNF-treated FL5.12 cells and analyzed by SDS-PAGE as described above. Cleavage of wild type rBID generated the three cleavage products seen in vivo, whereas cleavage of BID(D98A) generated only the pi 5 and pl3 fragments (not shown).
  • TNF/Chx treatment leads to accumulation of pi 5 BID in mitochondria as an integral membrane protein and release of cytochrome c.
  • p22 BID was consistently in the soluble SI 00 fraction (S) representing the cytosol as well as the mitochondria-enriched heavy membrane (HM) fraction as documented by the mitochondrial markers cytochrome c, which is located in the intermembrane space and cytochrome c oxidase, which is located in the inner membrane (Fig. 5 A, lanes 1-4).
  • the low speed pellet (PI) comprised of residual whole cells, nuclei and some mitochondria, also displays BID.
  • the pi 5 BID fragment was often still present in the cytosol (Fig. 5 A, lane 5) but was predominantly in the mitochondrial HM fraction, (Fig. 5 A, lane 7).
  • pl5 BID was almost exclusively in the mitochondria (data not shown).
  • the pl3 and pi 1 minor fragments were associated exclusively with the mitochondrial fraction (data not shown).
  • most of the cytochrome c was released from the mitochondrial HM fraction and was found either in the SI 00 fraction or presumably as part of membrane fragments in the LM fraction (Fig. 3 A, middle panel).
  • HEPES/hypotonic buffer Hypo
  • alkaline buffer Na 2 CO , pH 11.5
  • high salt 0.5 M NaCl
  • p22 BID was sensitive to treatment with hypotonic buffer, alkaline buffer or high salt (>50% found in the supernatant (Fig. 5B)); whereas, pi 5, pl3 and pll were markedly resistant to these treatments (Fig. 5B), indicative of an integral membrane position for these BID fragments.
  • Example 4 This example illustrates that cytosolic pi 5 BID targets mouse liver mitochondria while p22 BID does not.
  • the cytosol of TNF ⁇ /Chx-treated FL5.12 cells was incubated with mitochondria from mouse liver.
  • the liver from one mouse was minced and washed in ice-cold HIM buffer (supplemented with 2 mg/ml de-lipidated BSA).
  • the minced liver (-2 g wet weight) was gently homogenized in 6 ml HIM buffer in a 15 ml Wheaten Dounce glass homogenizer using two complete up and down cycles of a glass 'B'-type pestle.
  • the homogenate was diluted 6-fold with HIM buffer and centrifuged at 4'C for 10 min. at 600 g in a sorval SS34 rotor.
  • the supernatant was recovered, centrifuged at 7000 g for 15 min. and the pellet resuspended in twice the original homogenate volume in HIM buffer w/o BSA. After centrifuging at 600 g, mitochondria were recovered from the supernatant by centrifuging at 7000 g for 15 min.
  • the mitochondrial pellet was suspended in 0.5 ml MRM buffer (250 mM sucrose, 10 mM HEPES, 1 mM ATP, 5 mM NaSuccinate, 0.08 mM ADP, 2 mM K 2 HP0 4 , pH 7.5) at a concentration of 1 mg of mitochondrial protein per ml, and adjusted to 1 mM DTT just before use (McBride et al., Biochem. Biophys. Ada.. 1237:162-168, 1995). A standard import reaction was then performed. The soluble fraction (60 ⁇ l) prepared from FL5.12 cells after a 5 hr.
  • MRM buffer 250 mM sucrose, 10 mM HEPES, 1 mM ATP, 5 mM NaSuccinate, 0.08 mM ADP, 2 mM K 2 HP0 4 , pH 7.5
  • Fig. 5C As shown in Fig. 5C, at 37°C, greater than 90% of pl5 BID, but less than 10% of p22 BID targeted mitochondria (lanes 3, 4). Moreover, the targeted pl5 but not p22 was resistant to alkali extraction (lanes 9-12), indicating that pl5 BID was now an integral membrane protein. Targeting of pi 5 BID did not occur when the protein import reaction was perfromed at 40°C (Fig. 5C, lanes 5- 6). Moreover, the inclusion of zVAD-fmk in the reaction did not inhibit targeting of pre-existing pi 5 (lanes 7-8), suggesting that this event does not require an additional caspase cleavage at the mitochondria.
  • Example 5 This example illustrates that targeting of cytosolic pl5 BID to mitochondria is required for the release of cytochrome c.
  • pi 5 BID fragment targeted the mitochondria and up to about 50% of cytochrome c was released into the supernatant (lanes 1-2).
  • pi 5 BID was resistant to alkali extraction whereas, as expected, cytochrome c was not (lanes 3-4). Strikingly, depletion of pi 5 BID from the cytosol by an anti-BID Ab not only eliminated pi 5 targeting as expected, but also prevented the release of cytochrome c (lanes 5-8).
  • mice results in accumulation of pi 5 BID in the cytosol of hepatocytes and its subsequent translocation to mitochondria.
  • mice were injected with anti-Fas Ab which results in massive hepatocyte cell death.
  • Six to eight week old (20 g) C57B16 mice were injected intravenously with 5 ⁇ g of purified hamster mAb to mouse Fas (JO2; PharMingen) in 100 ⁇ l of 0.9% w/v saline. Animals were sacrificed 1 hr or 3 hr following injection.
  • hepatocytes from untreated mice of mice treated with anti-Fas Ab were disrupted using isotonic lysis conditions as described above for mouse liver which kept the hepatocyte mitochondria intact with a retained outer membrane.
  • the homogenates were separated by differential centrifugation into soluble (S) and heavy membrane (HM) fractions representing the cytosol and intact mitochondria, respectively.
  • S soluble
  • HM heavy membrane
  • Intracellular p22 BID was cleaved at three internal Asp sites: D59, D75 and D98 ( Figure 4B).
  • D59, D75 and D98 Figure 4B
  • the minor fragments of pi 3 and pl l resulting from cleavage at D75 and D98 respectively are only detected in the mitochondria.
  • Caspase-8 prefers the D59 site
  • other caspases perhaps at the level of mitochondria may be responsible for the pl3 and pl l fragments, which are of less certain importance as they are not prominent in TNF-activated 2B4 cells or Fas-activated hepatocytes.
  • the LQTD recognition motif for the predominant p 15 fragment is an atypical site for "initiator" caspases ((I/LN)EXD).

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Abstract

L'invention porte sur un nouveau polypeptide p15 BID ayant une activité agoniste de la mort cellulaire. Ce polypeptide p15 BID est produit par clivage de BID par médiation des caspases dans les cellules destinées à mourir, la mort étant induite par les récepteurs de TNT ou FAS. Le polypeptide p15 BID est utile dans des procédés de modulation de la mort d'une cellule cible qui consistent à traiter la cellule avec p15 BID. L'invention porte également sur des procédés d'inhibition de la mort cellulaire qui consistent à traiter une cellule cible avec un p15 BID mutant n'ayant pas d'activité agoniste de la mort cellulaire.
PCT/US1999/016966 1998-08-19 1999-07-28 Modulation de l'apoptose a l'aide de bid WO2000011162A1 (fr)

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US6503754B1 (en) 2000-09-07 2003-01-07 Isis Pharmaceuticals, Inc. Antisense modulation of BH3 interacting domain death agonist expression
US10413549B2 (en) 2012-11-21 2019-09-17 Eutropics Pharmaceuticals, Inc. Methods and compositions useful for treating diseases involving Bcl-2 family proteins with isoquinoline and quinoline derivatives
US10640803B2 (en) 2013-10-30 2020-05-05 Eutropics Pharmaceuticals, Inc. Methods for determining chemosensitivity and chemotoxicity
US10732182B2 (en) 2013-08-01 2020-08-04 Eutropics Pharmaceuticals, Inc. Method for predicting cancer sensitivity
US10765673B2 (en) 2012-06-20 2020-09-08 Eutropics Pharmaceuticals, Inc. Methods and compositions useful for treating diseases involving Bcl-2 family proteins with quinoline derivatives
US10793915B2 (en) 2015-01-12 2020-10-06 Eutropics Pharmaceuticals, Inc. Context dependent diagnostics test for guiding cancer treatment

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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6503754B1 (en) 2000-09-07 2003-01-07 Isis Pharmaceuticals, Inc. Antisense modulation of BH3 interacting domain death agonist expression
US10765673B2 (en) 2012-06-20 2020-09-08 Eutropics Pharmaceuticals, Inc. Methods and compositions useful for treating diseases involving Bcl-2 family proteins with quinoline derivatives
US10413549B2 (en) 2012-11-21 2019-09-17 Eutropics Pharmaceuticals, Inc. Methods and compositions useful for treating diseases involving Bcl-2 family proteins with isoquinoline and quinoline derivatives
US10732182B2 (en) 2013-08-01 2020-08-04 Eutropics Pharmaceuticals, Inc. Method for predicting cancer sensitivity
US11656230B2 (en) 2013-08-01 2023-05-23 Eutropics Pharmaceuticals, Inc. Method for predicting cancer sensitivity
US10640803B2 (en) 2013-10-30 2020-05-05 Eutropics Pharmaceuticals, Inc. Methods for determining chemosensitivity and chemotoxicity
US11519015B2 (en) 2013-10-30 2022-12-06 Entropics Pharmaceuticals, Inc. Methods for determining chemosensitivity and chemotoxicity
US10793915B2 (en) 2015-01-12 2020-10-06 Eutropics Pharmaceuticals, Inc. Context dependent diagnostics test for guiding cancer treatment

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