EP1058500A1 - Das helios gen - Google Patents

Das helios gen

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Publication number
EP1058500A1
EP1058500A1 EP99909641A EP99909641A EP1058500A1 EP 1058500 A1 EP1058500 A1 EP 1058500A1 EP 99909641 A EP99909641 A EP 99909641A EP 99909641 A EP99909641 A EP 99909641A EP 1058500 A1 EP1058500 A1 EP 1058500A1
Authority
EP
European Patent Office
Prior art keywords
helios
cell
polypeptide
gene
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP99909641A
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English (en)
French (fr)
Other versions
EP1058500A4 (de
Inventor
Katia Georgopoulos
Bruce A. Morgan
Clair Kelley
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
General Hospital Corp
Original Assignee
General Hospital Corp
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Filing date
Publication date
Application filed by General Hospital Corp filed Critical General Hospital Corp
Publication of EP1058500A1 publication Critical patent/EP1058500A1/de
Publication of EP1058500A4 publication Critical patent/EP1058500A4/de
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
    • C07K14/4705—Regulators; Modulating activity stimulating, promoting or activating activity
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to the Helios gene, Helios polypeptide, Helios homodimers,
  • the Helios polypeptide encodes amino acid residues 1- 526 of SEQ ID NO:2, residues 1-500 of SEQ ID NO:4 or residues 1-526 of SEQ ID NO:6 or a functionally equivalent residue in the Helios sequence of another vertebrate or mammal, e.g., a monkey.
  • the Helios polypeptide differs in amino acid sequence at 1, 2, 3, 5, 10 or more residues, but preferably less than 15, from a sequence in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
  • the differences are such that the Helios polypeptide exhibits at least one biological activity of an Helios polypeptide, e.g., the Helios polypeptide retains a biological activity of a naturally occurring Helios polypeptide.
  • the Helios polypeptide differs at up to 1, 2, 3, 5, 10 amino acid residues from the sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6.
  • the fragment includes one or more of: a N-terminal zinc finger, e.g., N-zinc fmger 1 (ZFl), N-zinc fmger 2 (ZF2), N-zinc fmger 3 (ZF3), N-zinc fmger 4 (ZF4), a transcriptional activation domain, or a C-terminal zinc finger, e.g., C-zinc finger 1 (ZF5), C-zinc finger 2 (ZF6).
  • a N-terminal zinc finger e.g., N-zinc fmger 1 (ZFl)
  • ZF2 N-zinc fmger 2
  • ZF3 N-zinc fmger 3
  • ZF4 N-zinc fmger 4
  • a transcriptional activation domain e.g., C-terminal zinc finger 1 (ZF5), C-zinc finger 2 (ZF6).
  • the nucleic acid will include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the Helios gene sequence, e.g., to render the Helios gene sequence suitable for use as an expression vector.
  • a transcriptional regulatory sequence e.g. at least one of a transcriptional promoter or transcriptional enhancer sequence, operably linked to the Helios gene sequence, e.g., to render the Helios gene sequence suitable for use as an expression vector.
  • the invention features a method of making a fragment or analog of an Helios polypeptide, e.g., an Helios polypeptide having at least one biological activity of a naturally occurring Helios polypeptide.
  • the method includes altering the sequence, e.g., by substitution or deletion of one or more residues, preferably which are non-conserved residues, of an Helios polypeptide, and testing the altered polypeptide for the desired activity.
  • the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for a disorder, e.g., an immune system disorder, e.g., a T or B cell related disorder, e.g., a nude mouse or a SCID mouse, including administering a therapeutically-effective amount of an Helios polypeptide to the animal.
  • a disorder e.g., an immune system disorder, e.g., a T or B cell related disorder, e.g., a nude mouse or a SCID mouse
  • the Helios polypeptide can be monomeric or an Helios-Helios, an Helios-Aiolos dimer, or Helios-Ikaros dimer.
  • the invention features a method for treating an animal, e.g., a human, a mouse, a transgenic animal, or an animal model for an immune system disorder, e.g., a T or B cell related disorder, e.g., a nude mouse or a SCID mouse.
  • the method - 10 - includes administering to the animal a nucleic acid encoding an Helios peptide and expressing the nucleic acid.
  • the transgenic animal e.g., a transgenic mouse
  • is homozygous for null mutations e.g., it is homozygous for a deletion of the C terminal end of the protein, at the Helios locus and includes a mutation at Ikaros or Aiolos, e.g., a dominant negative mutation at Ikaros or Aiolos.
  • the Ikaros mutation is heterozygous.
  • the transgenic animal or cell is heterozygous for an Helios transgene; homozygous for an Helios transgene; includes a first Helios transgene and a second Helios transgene; includes an Helios transgene and a second transgene which is other than an Helios transgene, e.g., an Ikaros or Aiolos transgene.
  • the invention also includes: a preparation of cells, e.g., cultured cells or a stem cells, including a cell a containing purified Ikaros- or Aiolos-protein-encoding-DNA and a cell encoding purified Helios-protein-encoding -DNA.
  • a preparation of cells e.g., cultured cells or a stem cells, including a cell a containing purified Ikaros- or Aiolos-protein-encoding-DNA and a cell encoding purified Helios-protein-encoding -DNA.
  • the invention also includes substantially pure preparation of an antibody, preferably a monoclonal antibody directed against an Ikaros-Helios dimer or an Aiolos-Helios dimer (which preferably does not bind to an Ikaros-Ikaros, Aiolos- Aiolos or Helios-Helios dimer); a therapeutic composition including an Ikaros-Helios dimer or an Aiolos-Helios dimer and a - 14 - pharmaceutically acceptable carrier; a therapeutic composition which includes a purified
  • DNA e.g., hematopoietic stem cells transformed with Ikaros or Aiolos and or Heliospeptide-encoding DNA.
  • the Ikaros Aiolos and Helios DNA can be present in the same or in different cells.
  • the invention features a method for determining if a subject, e.g., a human, is at risk for a disorder related to mis-expression of the Ikaros gene, e.g., a leukemic disorder or other disorder of the immune system, e.g., an immunodeficiency, or a T or B cell related disorder, e.g., a disorder characterized by a shortage of T or B cells, including examining the subject for the expression of the Ikaros-Helios or Aiolos-Helios dimers, non- wild type expression or mis-expression being indicative of risk.
  • a disorder related to mis-expression of the Ikaros gene e.g., a leukemic disorder or other disorder of the immune system, e.g., an immunodeficiency, or a T or B cell related disorder, e.g., a disorder characterized by a shortage of T or B cells
  • the method further includes: allowing the cell to proliferate in the mammal.
  • the mammal is a non-human mammal, e.g., a swine, a nonhuman primate, e.g., a monkey, a goat, or a rodent, e.g., a rat or a mouse.
  • a non-human mammal e.g., a swine
  • a nonhuman primate e.g., a monkey, a goat
  • a rodent e.g., a rat or a mouse.
  • the Helios-misexpressing cell is a lymphocyte and is: a cell which secretes one or more anti-inflammatory cytokines; a cell which is antigen or idiotype specific.
  • the donor of the Helios-misexpressing cell is heterozygous or homozygous for an Helios transgene; the donor of theHelios-misexpressing cell is heterozygous at the Helios locus; the donor of the Helios-misexpressing cell carries a point mutation in or a deletion for all or part of the Helios gene, e.g., mutation in the DNA binding region, e.g., a point mutation in, or a deletion for all or part of one or more of the four N-terminal zinc finger regions which mediate Helios binding to DNA or in one or both of the C-terminal zinc finger regions which mediates Helios dimerization; the donor of the Helios-misexpressing cell is human or a non-human mammal, e.g., a swine, a monkey, a goat, or a rodent, e.g., a rat or a mouse. In preferred embodiments, e.g.
  • the cell is a lymphocyte and is: a T cell; a cell which secretes one or more anti-inflammatory cytokines; a T cell which is antigen or idiotype specific.
  • the donor mammal can be, e.g., a human or a nonhuman mammal, e.g., a swine, a monkey, a goat, or a rodent, e.g., a rat or a mouse. If the donor mammal is human, the manipulation that gives rise to Helios misexpression e.g., an the introduction of an Helios lesion, can be made in vitro.
  • the donor mammal and the recipient mammal can be different individuals or the same individual.
  • Helios gene e.g., a point mutation or a deletion, which, inactivates one or both of - 21 - transcriptional activation or dimerization, which decreases the half life of the protein, or which inactivates one or both of the C terminal Zinc finger domains.
  • the method is performed in vivo, and the recipient mammal or the donor mammal or both are immunized with an antigen.
  • the antigen can be: an alloantigen; a xenoantigen or an autoantigen; a protein; or an antigen which gives rise to an anti-idiotypic lymphocyte.
  • the invention features, a method of evaluating the interaction of an Helios misexpressing cell, e.g., a hematopoietic cell, a T lymphocyte, with an immune system component.
  • the method includes: supplying an animal, e.g., a swine, a nonhuman primate, e.g., a monkey, a goat, or a rodent, e.g., a rat or a mouse; introducing the cell and the immune component into the animal; and evaluating the interaction between the Helios misexpressing cell and the immune system component.
  • the cell or cells include a gene which misexpress an endogenous Helios gene, e.g., a gene the expression of which is disrupted, e.g., a knockout.
  • a gene which misexpress an endogenous Helios gene e.g., a gene the expression of which is disrupted, e.g., a knockout.
  • Such cells can serve as a model for studying disorders which are related to mutated or mis-expressed Helios alleles or for use in drug screening.
  • the method further includes: allowing the Helios- misexpressing cell to divide and give rise to a proliferation-deregulated or antibody producing cell, e.g., a lymphocyte.
  • the mammal e.g., a mouse
  • is homozygous for null mutations e.g., it is homozygous for a deletion of the C terminal end of the protein, at the Helios locus.
  • the mammal carries homozygous mutations at the Helios gene, e.g., a point mutation or a deletion, which, inactivates one or both of transcriptional activation or dimerization. which decreases the half life of the protein, or which inactivates one or both of the C terminal Zinc finger domains.
  • the cell is a lymphocyte and is: a cell which secretes one or more anti-inflammatory cytokines; a cell which is antigen or idiotype specific; a cell which produces, or over produces, antibodies, e.g., IgG, IgA, or IgE antibodies.
  • the Helios-misexpressing cell e.g., a lymphocyte
  • the mammal e.g., to a homozygous wild-type Helios mammal or a mammal carrying a mutation at the Helios gene, e.g., a point mutation or a deletion for all or part of the Helios gene.
  • the cell can be a human or a nonhuman, e.g., a swine, nonhuman primate, e.g., a monkey, a goat, or a rodent, e.g., a rat or a mouse, lymphocyte.
  • the exogenously supplied cell can be homozygous for null mutations, e.g., homozygous for a deletion of the C terminal end of the protein, at the Helios locus.
  • the method further comprises isolating one or more cells, e.g., lymphocytes, from the mammal, and allowing the cell or cells to proliferate into a clonal population of cells, e.g., lymphocytes.
  • cells e.g., lymphocytes
  • the method further comprises isolating one or more cells, e.g., lymphocytes, from the mammal, and allowing the cell or cells to proliferate into a clonal population of cells, e.g., lymphocytes, and isolating the antibody therefrom.
  • cells e.g., lymphocytes
  • a cell from the animal is fused with a second cell to provide a hybridoma and the antibody is isolated from the hybridoma.
  • An Helios-deregulated cell is a cell which has a mutant or misexpressed Helios gene, e.g., an inactiviated Helios gene.
  • a hematopoietic cell can be, e.g., stem cell, e.g., a totipotent or a pluripotent stem cell, or a descendent of a stem cell, e.g., a lymphocyte, e.g. a B lymphocyte or a T lymphocyte.
  • stem cell e.g., a totipotent or a pluripotent stem cell
  • a descendent of a stem cell e.g., a lymphocyte, e.g. a B lymphocyte or a T lymphocyte.
  • a mutation at the Helios locus includes any mutation which alters the expression, structure, or activity of the Helios gene or its gene product. These include point mutations in and in particular deletions of all or part of the Helios coding region or its control region.
  • An exogenously supplied cell, tissue, or cell product e.g., a cytokine, as used herein, is a cell, tissue, or a cell product which is derived from an animal other than the one to which is supplied or administered. It can be from the same species or from different species than the animal to which it is supplied.
  • the gene can be used to prepare antisense constructs capable of inhibiting expression of a mutant or wild type Helios gene encoding a polypeptide having an undesirable function.
  • an Helios polypeptide can be used to raise antibodies capable of detecting proteins or protein levels associated with abnormal cell proliferation or lymphocyte differentiation, e.g., T cell maturation.
  • Helios peptides, antibodies or nucleic acids can be used to identify the stage of lymphocyte differentiation, e.g., the stage of T cell differntiation.
  • a gene encoding an Helios polypeptide can be entrapped in liposomes bearing positive charges on their surface (e.g., lipofectins) and (optionally) which are tagged with antibodies against cell surface antigens of the target tissue (Mizuno et al. (1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309; Japanese patent application 1047381; and European patent publication EP-A-43075).
  • the gene delivery systems for the therapeutic Helios gene can be introduced into a patient by any of a number of methods, each of which is familiar in the art.
  • a pharmaceutical preparation of the gene delivery system can be introduced systemically, e.g.
  • the pharmaceutical preparation of the gene therapy construct can consist essentially of the gene delivery system in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can comprise one or more cells which produce the gene delivery system.
  • antisense therapy refers to administration or in situ generation of oligonucleotides or their derivatives which specifically hybridize (e.g. bind) under cellular conditions, with the cellular mRNA and/or genomic DNA encoding an Helios polypeptide, or mutant thereof, so as to inhibit expression of the encoded protein, e.g. by inhibiting transcription and/or translation.
  • the binding may be by conventional base pair complementarity, or, for example, in the case of binding to DNA duplexes, through specific interactions in the major groove of the double helix.
  • antisense refers to the range of techniques generally employed in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences.
  • the antisense construct binds to a naturally-occurring sequence of an Helios gene which, for example, is involved in expression of the gene.
  • sequences include, for example, start codons, stop codons, and RNA primer binding sites.
  • an antisense construct of the present invention can be delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a Helios polypeptide.
  • the antisense construct is an oligonucleotide probe which is generated ex vivo and which, when introduced into the cell causes inhibition of expression by hybridizing with the mRNA and/or genomic sequences of an Helios gene.
  • Such oligonucleotide probes are preferably modified oligonucleotide which are resistant to endogenous nucleases, e.g. exonucleases and/or endonucleases, and is therefore stable in vivo.
  • target sequence refers to a nucleotide sequence that is genetically recombined by a recombinase.
  • the target sequence is flanked by recombinase recognition sequences and is - 43 - generally either excised or inverted in cells expressing recombinase activity.
  • Recombinase catalyzed recombination events can be designed such that recombination of the target sequence results in either the activation or repression of expression of the subject Helios gene.
  • excision of a target sequence which interferes with the expression of a recombinant Helios gene can be designed to activate expression of that gene.
  • This interference with expression of the protein can result from a variety of mechanisms, such as spatial separation of the Helios gene from the promoter element or an internal stop codon.
  • the inventor has provided the primary amino acid structure of an Helios polypeptide. Once an example of this core structure has been provided, one skilled in the art can alter the disclosed structure by producing fragments or analogs, and testing the newly produced structures for activity. Examples of prior art methods which allow the production and testing of fragments and analogs are discussed below. These, or analogous methods can be used to make and screen fragments and analogs of an Helios polypeptide having at least one biological activity e.g., which react with an antibody (e.g., a monoclonal antibody) specific for an Helios polypeptide.
  • an antibody e.g., a monoclonal antibody
  • Fragments of a protein can be produced in several ways, e.g., recombinantly, by proteolytic digestion, or by chemical synthesis. Internal or terminal fragments of a polypeptide can be generated by removing one or more nucleotides from one end (for a terminal fragment) or both ends (for an internal fragment) of a nucleic acid which encodes the polypeptide. Expression of the mutagenized DNA produces polypeptide fragments. Digestion with "end-nibbling" endonucleases can thus generate DNA's which encode an array of fragments. DNA's which encode fragments of a protein can also be generated by random shearing, restriction digestion or a combination of the above-discussed methods.
  • Fragments can also be chemically synthesized using techniques known in the art such as conventional Merrifield solid phase f-Moc or t-Boc chemistry.
  • peptides of the present invention may be arbitrarily divided into fragments of desired length with no overlap of the fragments, or divided into overlapping fragments of a desired length.
  • Amino acid sequence variants of a protein can be prepared by random mutagenesis of DNA which encodes a protein or a particular domain or region of a protein. Useful methods include PCR mutagenesis and saturation mutagenesis. A library of random amino - 45 - acid sequence variants can also be generated by the synthesis of a set of degenerate oligonucleotide sequences. (Methods for screening proteins in a library of variants are elsewhere herein.)
  • a library of homologs can also be generated from a set of degenerate oligonucleotide sequences. Chemical synthesis of a degenerate sequences can be carried out in an automatic DNA synthesizer, and the synthetic genes then ligated into an appropriate expression vector. The synthesis of degenerate oligonucleotides is known in the art (see for example, Narang, SA (1983) Tetrahedron 39:3; Itakura et al. (1981) - 46 -
  • Non-random or directed, mutagenesis techniques can be used to provide specific sequences or mutations in specific regions. These techniques can be used to create variants which include, e.g., deletions, insertions, or substitutions, of residues of the known amino acid sequence of a protein.
  • the sites for mutation can be modified individually or in series, e.g., by (1) substituting first with conserved amino acids and then with more radical choices depending upon results achieved, (2) deleting the target residue, or (3) inserting residues of the same or a different class adjacent to the located site, or combinations of options 1-3.
  • Alanine scanning mutagenesis is a useful method for identification of certain residues or regions of the desired protein that are preferred locations or domains for mutagenesis, Cunningham and Wells (Science 244:1081-1085, 1989).
  • a residue or group of target residues are identified (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) and replaced by a neutral or negatively charged amino acid (most preferably alanine or polyalanine).
  • Replacement of an amino acid can affect the interaction of the amino acids with the surrounding aqueous environment in or outside the cell.
  • Those domains demonstrating functional sensitivity to the substitutions are then refined by introducing further or other variants at or for the sites of substitution.
  • the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation per se need not be predetermined.
  • alanine scanning or random mutagenesis may be conducted at the - 47 - target codon or region and the expressed desired protein subunit variants are screened for the optimal combination of desired activity.
  • a DNA polymerase is used to synthesize an entire second complementary strand of the template that will thus inco ⁇ orate the oligonucleotide primer, and will code for the selected alteration in the desired protein DNA.
  • oligonucleotides of at least 25 nucleotides in length are used.
  • An optimal oligonucleotide will have 12 to 15 nucleotides that are completely complementary to the template on either side of the nucleotide(s) coding for the mutation. This ensures that the oligonucleotide will hybridize properly to the single-stranded DNA template molecule.
  • the oligonucleotides are readily synthesized using techniques known in the art such as that described by Crea et al. (Proc. Natl. Acad. Sci. USA, 75: 5765[1978]).
  • the starting material is a plasmid (or other vector) which includes the protein subunit DNA to be mutated.
  • the codon(s) in the protein subunit DNA to be mutated are identified.
  • a double-stranded oligonucleotide encoding the sequence of the DNA between the restriction sites but containing the desired mutation(s) is synthesized using standard procedures. The two strands are synthesized separately and then - 48 - hybridized together using standard techniques.
  • This double-stranded oligonucleotide is referred to as the cassette.
  • This cassette is designed to have 3' and 5' ends that are comparable with the ends of the linearized plasmid, such that it can be directly ligated to the plasmid.
  • This plasmid now contains the mutated desired protein subunit DNA sequence.
  • Combinatorial mutagenesis can also be used to generate mutants, e.g., a library of variants which is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library.
  • mutants e.g., a library of variants which is generated by combinatorial mutagenesis at the nucleic acid level, and is encoded by a variegated gene library.
  • a mixture of synthetic oligonucleotides can be enzymatically ligated into gene sequences such that the degenerate set of potential sequences are expressible as individual peptides, or alternatively, as a set of larger fusion proteins containing the set of degenerate sequences.
  • Techniques for screening large gene libraries often include cloning the gene library into replicable expression vectors, transforming appropriate cells with the resulting library of vectors, and expressing the genes under conditions in which detection of a desired activity, e.g., in this case, binding to an antibody specific for a Helios polypeptide.
  • detection of a desired activity e.g., in this case, binding to an antibody specific for a Helios polypeptide.
  • Each of the techniques described below is amenable to high through-put analysis for screening large numbers of sequences created, e.g., by random mutagenesis techniques.
  • the candidate peptides are displayed on the surface of a cell or viral particle, and the ability of particular cells or viral particles to bind an appropriate receptor protein via the displayed product is detected in a "panning assay".
  • the gene library can be cloned into the gene for a surface membrane protein of a bacterial cell, and the resulting fusion protein detected by panning (Ladner et al., WO 88/06630; Fuchs et al. ( 1991 ) Bio/Technology 9: 1370- 1371 ; and Goward et al. ( 1992) 7755 18: 136-140).
  • a gene library can be expressed as a fusion protein on the surface of a viral particle.
  • foreign peptide sequences can be expressed on the surface of infectious phage, thereby conferring two significant benefits.
  • the physical link between the peptide and its encoding DNA occurs by the containment of the DNA within a particle (cell or phage) that carries the peptide on its surface. Capturing the peptide captures the particle and the DNA within.
  • An alternative scheme uses the DNA- binding protein Lad to form a link between peptide and DNA (Cull et al. (1992) PNAS USA 89: 1865-1869). This system uses a plasmid containing the Lad gene with an oligonucleotide cloning site at its 3'-end. Under the controlled induction by arabinose, a Lacl-peptide fusion protein is produced.
  • This fusion retains the natural ability of Lad to bind to a short DNA sequence known as LacO operator (LacO).
  • LacO operator By installing two copies of LacO on the expression plasmid, the Lacl-peptide fusion binds tightly to the plasmid that encoded it. Because the plasmids in each cell contain only a single oligonucleotide sequence and each cell expresses only a single peptide sequence, the peptides become specifically and stably associated with the DNA sequence that directed its synthesis. The cells of the library are gently lysed and the peptide-DNA complexes are exposed to a matrix of immobilized receptor to recover the complexes containing active peptides.
  • peptides-on-plasmids differs in two important ways from the phage display methods.
  • the peptides are attached to the C- - 51 - terminus of the fusion protein, resulting in the display of the library members as peptides having free carboxy termini.
  • Both of the filamentous phage coat proteins, pill and pVIII are anchored to the phage through their C-termini, and the guest peptides are placed into the outward-extending N-terminal domains.
  • the phage-displayed peptides are presented right at the amino terminus of the fusion protein. (Cwirla, et al. (1990) Proc.
  • Useful analogs of an Helios polypeptide can be agonists or antagonists.
  • Antagonists of an Helios polypeptide can be molecules which form the Helios-Ikaros dimers but which lack some additional biological activity such as transpriptional activation of genes that control lymphocyte development.
  • Helios antagonists and agonists are derivatives which can modulate, e.g., inhibit or promote, lymphocyte maturation and function.
  • the invention also includes antibodies specifically reactive with a subject Helios polypeptide or Helios-Ikarod dimers.
  • Anti-protein/anti-peptide antisera or monoclonal antibodies can be made by standard protocols (See, for example, Antibodies: A Laboratory Manual ed. by Harlow and Lane (Cold Spring Harbor Press: 1988)).
  • a mammal such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide.
  • Techniques for conferring immunogenicity on a protein or peptide include conjugation to carriers or other techniques well known in the art.
  • An immunogenic portion of the subject Helios polypeptide can be administered in the presence of adjuvant.
  • Antibodies which specifically bind Helios-Ikaros dimers or Helios polypeptide epitopes can also be used in immunohistochemical staining of tissue samples in order to evaluate the abundance and pattern of expression of Helios-Ikaros dimer or Helios polypeptide.
  • Anti-Helios polypeptide antibodies can be used diagnostically in immuno- precipitation and immuno-blotting to detect and evaluate wild type or mutant Helios polypeptide levels in tissue or bodily fluid as part of a clinical testing procedure.
  • the ability to monitor Helios-Ikaros dimer or Helios polypeptide levels in an individual can allow determination of the efficacy of a given treatment regimen for an individual afflicted with disorders associated with modulation of lymphocyte differentiation and/or proliferation.
  • the level of an Helios-Ikaros dimer or Helios polypeptide can be measured in tissue, such as produced by biopsy.
  • anti-Helios antibodies of the present invention is in the immunological screening of cDNA libraries constructed in expression vectors such as g t l l , gtl8-23, ZAP, and ORF8.
  • Messenger libraries of this type having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins.
  • gtl 1 will produce fusion proteins whose amino termini consist of ⁇ - galactosidase amino acid sequences and whose carboxy termini consist of a foreign polypeptide.
  • Antigenic epitopes of a subject Helios polypeptide can then be detected with antibodies, as, for example, reacting nitrocellulose filters lifted from infected plates with anti-Helios polypeptide antibodies.
  • Phage, scored by this assay, can then be isolated from the infected plate.
  • the presence of Helios homologs can be detected and cloned from - 56 - other animals, and alternate isoforms (including splicing variants) can be detected and cloned from human sources.
  • Fragments include those expressed in native or endogenous cells, e.g., as a result of post- translational processing, e.g., as the result of the removal of an amino-terminal signal sequence, as well as those made in expression systems, e.g., in CHO cells. Because peptides, such as an Helios polypeptide, often exhibit a range of physiological properties and because such properties may be attributable to different portions of the molecule, a useful Helios polypeptide fragment or Helios polypeptide analog is one which exhibits a biological activity in any biological assay for Helios polypeptide activity.
  • the fragment or analog possesses 10%, preferably 40%, or at least 90% of the activity of an Helios polypeptide (SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6), in any in vivo or in vitro Helios polypeptide activity assay.
  • an Helios polypeptide SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6
  • Glutamic Acid E D-Glu, D-Asp, Asp, Asn, D-Asn, Gin, D-Gln

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EP99909641A 1998-02-27 1999-02-26 Das helios gen Withdrawn EP1058500A4 (de)

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US7632598P 1998-02-27 1998-02-27
US76325P 1998-02-27
PCT/US1999/004224 WO1999043208A1 (en) 1998-02-27 1999-02-26 The helios gene

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EP1058500A1 true EP1058500A1 (de) 2000-12-13
EP1058500A4 EP1058500A4 (de) 2002-05-08

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CA3052473A1 (en) * 2017-02-09 2018-08-16 Indapta Therapeutics, Inc. Engineered natural killer (nk) cells and compositions and methods thereof

Citations (1)

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Publication number Priority date Publication date Assignee Title
WO1996004372A1 (en) * 1994-07-29 1996-02-15 The General Hospital Corporation Ikaros transgenic cells and animals

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1996004372A1 (en) * 1994-07-29 1996-02-15 The General Hospital Corporation Ikaros transgenic cells and animals

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL_OV [Online] EMBL; Acc AF024439, 29 October 1997 (1997-10-29) TURPEN ET AL.: "Xenopus laevis ikaros-related transcription factor" XP002189272 *
DATABASE SWISSPROT [Online] Swissprot; Accession Nr: 042244, 1 January 1998 (1998-01-01) TURPEN J. ET AL.: "Ikaros-related transcription factor (fragment)" XP002189273 *
HANSEN JOHN D ET AL: "Conservation of a master hematopoietic switch gene during vertebrate evolution: Isolation and characterization of Ikaros and teleost and amphibian species." EUROPEAN JOURNAL OF IMMUNOLOGY, vol. 27, no. 11, November 1997 (1997-11), pages 3049-3058, XP001056033 ISSN: 0014-2980 *
MOLNAR ARPAD ET AL: "The Ikaros gene encodes a family of lymphocyte-restricted zinc finger DNA binding proteins, highly conserved in human and mouse." JOURNAL OF IMMUNOLOGY, vol. 156, no. 2, 1996, pages 585-592, XP001055887 ISSN: 0022-1767 *
NIETFELD WILFRIED ET AL: "Cloning and sequencing of hIk-1, a cDNA encoding a human homologue of mouse Ikaros/LyF-1." IMMUNOLOGY LETTERS, vol. 49, no. 1-2, 1996, pages 139-141, XP002189271 ISSN: 0165-2478 *
See also references of WO9943208A1 *

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