WO1998051792A1 - Fac molecules and uses thereof - Google Patents

Fac molecules and uses thereof Download PDF

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Publication number
WO1998051792A1
WO1998051792A1 PCT/US1998/009975 US9809975W WO9851792A1 WO 1998051792 A1 WO1998051792 A1 WO 1998051792A1 US 9809975 W US9809975 W US 9809975W WO 9851792 A1 WO9851792 A1 WO 9851792A1
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Prior art keywords
fac
hematopoietic progenitor
nucleic acid
molecule
leu
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PCT/US1998/009975
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French (fr)
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WO1998051792A8 (en
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Hagop Youssoufian
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Brigham & Women's Hospital, Inc.
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Priority to AU74901/98A priority Critical patent/AU7490198A/en
Publication of WO1998051792A1 publication Critical patent/WO1998051792A1/en
Publication of WO1998051792A8 publication Critical patent/WO1998051792A8/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5403—IL-3
    • 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
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • This invention relates to Fanconi anemia complementation group C (FAC) nucleic acids and encoded polypeptides which modulate apoptosis in hematopoietic progenitor cells.
  • the invention also relates to conjugates, including fusion polypeptides, comprising the FAC molecules and polypeptides which selectively bind hematopoietic progenitor cells.
  • the invention further relates to methods of using such FAC molecules and conjugates in the treatment and/or diagnosis of disease.
  • Fanconi anemia is an autosomal recessive disorder with an incidence of about 1 in
  • Fanconi anemia patients have an actuarial risk of 67 percent of developing acute myelogenous leukemia (AML) and a risk of 80 percent of death from hematologic causes (Butturini, et al., Blood 84:1650-1655, 1994). Earlier estimates also showed a markedly increased (about 15,000-fold) incidence of AML (Auerbach, Cancer Genet. Cytogenet. 51 :1-12, 1991). Although pancytopenia often heralds the development of leukemia, AML can also be the initial hematologic manifestation (Auerbach, et al., Am. J. Hematol. 12:289-300, 1982).
  • CFU-GM colony forming unit-granulocyte-macrophage
  • BFU-E burst-forming unit-erythroid erythroid
  • CFU-GEMM colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte
  • Fanconi anemia is the quintessential model of an inherited "pre-leukemic" disorder that leads to marrow failure and leukemia.
  • pancytopenia and AML are responsible for a large share of the difficulties suffered by FA patients.
  • the most unique aspect of FA as a disease model is the opportunity to prevent or reduce the development of leukemia.
  • bone marrow transplantation provides a respite from leukemia, but the challenges presented by cytoreductive therapy, tissue typing, expense and access to specialized centers are daunting. Thus, there is a need for effective and safer forms of therapy for Fanconi anemia.
  • the invention involves the discovery that the Fanconi anemia complementation group C (FAC) protein may be utilized to inhibit the effects of Fanconi anemia in hematopoietic progenitor cells in vivo and in vitro. It has also been realized that the FAC protein can be effective for ameliorating the effects of chemotherapy given to subject having a non-myeloid malignancy. It further has been discovered that FAC molecules can be delivered to hematopoietic progenitor cells by preparing a conjugate of a FAC molecule and a targeting molecule which binds to a cell surface protein of the cells and is internalized. Such targeting molecules include IL3 and antibodies which recognize CD33.
  • FAC Fanconi anemia complementation group C
  • an isolated conjugate includes a FAC molecule and a polypeptide which selectively binds a hematopoietic progenitor cell.
  • the FAC molecule is a polypeptide or a nucleic acid.
  • the FAC molecule is a polypeptide, preferably it is a protein which has the amino acid sequence of SEQ ID NO:2, or a functional fragment thereof.
  • the FAC molecule is a nucleic acid molecule which hybridizes under stringent conditions to a molecule having the nucleotide sequence of SEQ ID NO: 1, and which codes for a FAC protein, nucleic acids which differ from the foregoing due to the degeneracy of the genetic code, or complementary molecules.
  • Preferred FAC nucleic acids are those which include the nucleotide sequence of SEQ ID NO:l, and those which encode a functional fragment of a FAC protein.
  • the FAC nucleic acid is operably linked to a promoter, preferably one which is active in a hematopoietic progenitor cell, and optionally one which is not active in mature hematopoietic cells.
  • the FAC conjugate includes a targeting molecule in the form of a polypeptide which selectively binds hematopoietic progenitor cells.
  • the polypeptide is an antibody or a ligand which selectively binds to a cell surface protein, such as an antigen or a ligand, of the hematopoietic progenitor cells.
  • the targeting molecule is an antibody
  • the antibody is an anti-CD33 antibody or an antigen binding fragment thereof.
  • the targeting antibody can be conjugated to the FAC molecule in the form of a fusion protein. In such cases, the antibody preferably is a single chain antibody.
  • the conjugate is a fusion protein.
  • the ligand is IL3 or a receptor-binding portion thereof.
  • the IL3-FAC fusion protein can be constructed with either the IL3 portion or the FAC portion at the amino terminus, and can include a linker between the IL3 and FAC portions.
  • Other ligands are also embraced by the invention.
  • an isolated nucleic acid which codes for a fusion polypeptide, including a FAC protein and a polypeptide which selectively binds a hematopoietic progenitor cell, as described above, is provided.
  • the use of the foregoing conjugates and nucleic acids for delivery of a FAC molecule to a hematopoietic progenitor cell is provided.
  • the invention also encompasses methods for treating cells which have reduced FAC expression or which are at an increased risk of apoptosis due to exposure to high-dose chemotherapy.
  • methods for inhibiting apoptosis in a hematopoietic progenitor cell include contacting the hematopoietic progenitor cell with an amount of a composition which includes a FAC molecule effective to inhibit apoptosis.
  • the methods preferably include contacting the hematopoietic progenitor cell with the foregoing conjugates of FAC proteins and nucleic acids.
  • the FAC molecule is conjugated to a polypeptide which selectively binds to the hematopoietic progenitor cell.
  • conjugated can mean that the molecules are linked together noncovalently or covalently.
  • FAC molecules can be contained within a delivery vehicle (e.g., a liposome or a virus) which is targeted to the hematopoietic progenitor cell by a targeting moiety contained on the surface of the delivery vehicle, such as a polypeptide which selectively binds to the hematopoietic progenitor cell.
  • methods for treating a condition characterized by apoptosis of hematopoietic progenitor cells in a subject exposed to high-dose chemotherapy for non-myeloid malignancies comprise administering to the subject an amount of the foregoing compositions which include a FAC molecule effective to inhibit apoptosis of the hematopoietic progenitor cells in the subject.
  • the use of such compositions in the preparation of a medicament is also provided.
  • Fig. 1 is a schematic illustration of His-ILFAC fusion proteins.
  • Fig. 2A depicts the detection of FAC fusion proteins in cell lysates and the purification of such proteins.
  • Fig. 2B depicts an immunoblot of the His-ILFAC fusion protein.
  • Fig. 3 shows that complementation of FA group cells with FAC molecules increases resistance of the cells to treatment with MMC.
  • Fig. 4 shows the intracellular turnover of His-ILFAC fusion protein.
  • SEQ ID NO:l is the nucleotide sequence of the Fanconi anemia complementation group C nucleic acid.
  • SEQ ID NO:2 is the amino acid sequence of the Fanconi anemia complementation group C protein.
  • SEQ ID NO:3 is the nucleotide sequence of the gibbon IL3 nucleic acid.
  • SEQ ID NO:4 is the amino acid sequence of the gibbon IL3 protein.
  • SEQ ID NO: 5 is the nucleotide sequence of the anti CD33 antibody heavy chain nucleic acid.
  • SEQ ID NO: 6 is the amino acid sequence of the anti CD33 heavy chain antibody protein.
  • SEQ ID NO: 7 is the nucleotide sequence of the anti CD33 antibody light chain nucleic acid.
  • SEQ ID NO: 8 is the amino acid sequence of the anti CD33 light chain antibody protein.
  • SEQ ID NO: 9 is the nucleotide sequence of the forward primer for PCR amplification of gibbon IL3 minus the signal sequence.
  • SEQ ID NO: 10 is the nucleotide sequence of the reverse primer for PCR amplification of gibbon IL3.
  • SEQ ID NO: 11 is the nucleotide sequence of the forward primer for PCR amplification of gibbon IL3 including the signal sequence.
  • the present invention in one aspect involves the discovery that the Fanconi anemia complementation group C (FAC) protein is useful in modulating apoptosis in hematopoetic progenitor cells.
  • the sequence of the human FAC cDNA is presented as SEQ ID NO:l, and the predicted amino acid sequence of this gene's protein product is presented as SEQ ID NO:2.
  • the present invention in another aspect involves conjugates comprising a FAC molecule and a molecule which selectively binds to a hematopoetic progenitor cell for delivery of the FAC molecule to the hematopoetic progenitor cell.
  • the invention thus involves in one aspect FAC polypeptides, nucleic acids encoding those polypeptides, functional modifications and variants of the foregoing, useful fragments of the foregoing, as well as therapeutics relating thereto.
  • the invention is directed to conjugates of a FAC molecule and a molecule which targets the FAC molecule to a hematopoietic progenitor cell and related methods for forming such conjugates.
  • conjugates means two entities bound to one another by any physiochemical means, including hydrophobic interaction between an antigen and the non-specific hydrophobic portions of an antibody molecule, antibody-antigen specific binding and covalent coupling.
  • the nature of the preferred bonding will depend, among other things, upon the mode of administration and the pharmaceutical carriers used to deliver the conjugate to the target cell. For example, some bonds are not as well suited as others to withstand certain environments such as the stomach, but can be protected therefrom by delivery systems which bypass the stomach.
  • the conjugate preferably is formed as a fusion protein.
  • a molecule which targets the FAC molecule to a hematopoietic progenitor cell means any entity that can selectively bind or be selectively bound by a cell surface molecule on a hematopoietic progenitor cell.
  • Targeting moieties include those which recognize or are recognized by cell surface epitopes, cell surface receptors and/or channels.
  • Targeting molecules which recognize cell surface epitopes include immunoglobulin and effective fragments thereof. Effective fragments of immunoglobulin include Fab, F(ab)'2 and Fv fragments thereof.
  • Single chain antibodies, humanized antibodies, bifunctional antibodies, chimeric antibodies and other such entities also may be used according to the invention.
  • ligands of cell surface molecules are useful according to the invention.
  • the targeting moiety may be isolated or attached to the surface of a delivery vehicle, such as an encapsulating particle that has the ability to target the delivery of the contents of the particle to a desired location and, simultaneously, encapsulate a FAC molecule for delivery to the target.
  • a delivery vehicle such as an encapsulating particle that has the ability to target the delivery of the contents of the particle to a desired location and, simultaneously, encapsulate a FAC molecule for delivery to the target.
  • a virus particle may be formed around a FAC molecule by incubation of the FAC molecule with sufficient viral coat proteins.
  • the viral coat will self-assemble around the viral genome; this process may be utilized in preparation of the compounds of the invention.
  • liposomes spontaneously form around the constituents of the solution with which the precursor lipids are combined.
  • the binding of the targeting molecule and the cell surface molecule results in active transport of the FAC-targeting molecule complex into the hematopoietic progenitor cell.
  • Preferred examples include IL-3, c-kit, anti-CD33 antibodies and other molecules which selectively bind to the receptors for the foregoing proteins on the surface of a hematopoietic progenitor cell and are transported into the cell by endocytosis.
  • hematopoietic progenitor cells can be isolated by preparing antibodies to such cells.
  • the hematopoietic progenitor cells of interest can be isolated by immunoaffinity procedures using e.g. anti-CD33 antibodies and then used (as whole cells or as cell membrane fragments on which membrane proteins are exposed) to immunize animals for preparation of antibodies by procedures well known to those of skill in the art.
  • Antibodies, cell surface receptor ligands or portions thereof specific for hematopoietic progenitor cells and capable of being transported into such cells once bound can be identified and isolated using well established techniques.
  • random generated molecularly diverse libraries can be screened and molecules that are bound to and transported by hematopoietic progenitor cells can be isolated using conventional techniques.
  • targeting molecules can be prepared by selecting a peptide ligand from a peptide library. It is not intended that the invention be limited by the selection of any particular targeting molecule.
  • the invention thus involves agents such as polypeptides which bind to hematopoietic progenitor cells for targeting of FAC molecules.
  • the agents can be antibodies or fragments of antibodies having the ability to selectively bind to hematopoietic progenitor cells.
  • Antibodies include polyclonal and monoclonal antibodies, prepared according to conventional methodology. Where antibodies which bind a cell surface molecule of an hematopoietic progenitor cell are desired, a specific cell surface molecule can be selected and used as the immunogen for preparation of the antibody. Alternatively, the hematopoietic progenitor cell itself is used as the immunogen for preparation of the antibody.
  • an antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region designated an F(ab') 2 fragment
  • an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region designated an Fab fragment
  • Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd.
  • the Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope- binding ability in isolation.
  • CDRs complementarity determining regions
  • FRs framework regions
  • CDR1 through CDR3 complementarity determining regions
  • the present invention also provides for F(ab') 2 , Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab') 2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non- human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences.
  • the present invention also includes so-called single chain antibodies.
  • polypeptides of numerous size and type that bind specifically to hematopoietic progenitor cells which can be conjugated to FAC molecules for the delivery of FAC molecules to hematopoietic progenitor cells.
  • polypeptide binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form, as bacterial flagella peptide display libraries or as phage display libraries.
  • Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptides and non-peptide synthetic moieties.
  • Phage display can be particularly effective in identifying binding peptides useful according to the invention. Briefly, one prepares a phage library (using e.g. ml3, fd, or lambda phage), displaying inserts from 4 to about 80 amino acid residues using conventional procedures.
  • the inserts may represent, for example, a completely degenerate or biased array.
  • phages that exhibit the best binding characteristics are further characterized by nucleic acid analysis to identify the particular amino acid sequences of the peptides expressed on the phage surface and the optimum length of the expressed peptide to achieve optimum binding to the target mammalian cell.
  • peptide ligands can be selected from combinatorial libraries of peptides containing one or more amino acids. Such libraries can further be synthesized which contain non-peptide synthetic moieties which are less subject to enzymatic degradation compared to their naturally-occurring counterparts. The minimal linear portion of the sequence that binds to the hematopoietic progenitor cells or cell surface molecule can be determined.
  • Yeast two-hybrid screening methods also may be used to identify polypeptides that bind to cell surface molecules of an hematopoietic progenitor cell. These novel ligands can be conjugated to the FAC molecule to deliver the molecule to a hematopoietic progenitor cell.
  • the conjugates of the invention include one or more FAC molecules.
  • the invention also provides isolated polypeptides, which include fusion proteins which include the polypeptide of SEQ ID NO:2 and fragments thereof. Such polypeptides are useful for inhibiting apoptosis, for example, alone, conjugated, or as fusion proteins for FAC replacement therapy, for treatment of high dose chemotherapy, or to generate antibodies, as components of an immunoassay.
  • the invention embraces variants of the FAC polypeptides described above.
  • a "variant" of a FAC polypeptide is a polypeptide which contains one or more modifications to the primary amino acid sequence of a FAC polypeptide.
  • Modifications which create a FAC variant can be made to a FAC polypeptide 1) to reduce or eliminate an activity of a FAC polypeptide, such as protease susceptibility; 2) to enhance a property of a FAC polypeptide, such as protein stability in an expression system or the stability of protein-protein binding; or 3) to provide a novel activity or property to a FAC polypeptide, such as addition of a detectable moiety.
  • Modifications to a FAC polypeptide are typically made to the nucleic acid which encodes the FAC polypeptide, and can include deletions, point mutations, truncations, amino acid substitutions and additions of amino acids or non-amino acid moieties.
  • modifications can be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, addition of a fatty acid, and the like. Modifications also embrace fusion proteins comprising all or part of the FAC amino acid sequence, provided that the part of the FAC amino acid sequence is capable of inhibiting apoptosis.
  • Variants can include FAC polypeptides which are modified specifically to alter a feature of the polypeptide unrelated to its physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages. Similarly, certain amino acids can be changed to enhance expression of a FAC polypeptide by eliminating proteo lysis by proteases in an expression system (e.g., dibasic amino acid residues in yeast expression systems in which KEX2 protease activity is present).
  • Mutations of a nucleic acid which encode a FAC polypeptide preferably preserve the amino acid reading frame of the coding sequence, and preferably do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such a hairpins or loops, which can be deleterious to expression of the variant polypeptide.
  • Mutations can be made by selecting an amino acid substitution, or by random mutagenesis of a selected site in a nucleic acid which encodes the polypeptide. Variant polypeptides are then expressed and tested for one or more activities to determine which mutation provides a variant polypeptide with the desired properties. Further mutations can be made to variants (or to non- variant FAC polypeptides) which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli, are well known to those of ordinary skill in the art. Still other mutations can be made to the noncoding sequences of a FAC gene or cDNA clone to enhance expression of the polypeptide.
  • “conservative amino acid substitution” refers to an amino acid substitution which does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made.
  • Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York.
  • Exemplary functionally equivalent variants of the FAC polypeptides include conservative amino acid substitutions of SEQ ID NO:2.
  • Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.
  • functionally equivalent variants and fragments of FAC polypeptides i.e., variants or fragments of FAC polypeptides which retain the function of the natural FAC polypeptides, are contemplated by the invention.
  • the invention does not contemplate using the natural FAC mutant, R548X, which has a small deletion and the C terminus has no complementing activity.
  • Conservative amino-acid substitutions in the amino acid sequence of FAC polypeptides to produce functionally equivalent variants of FAC polypeptides typically are made by alteration of a nucleic acid encoding FAC polypeptides (SEQ ID NO:l). Such substitutions can be made by a variety of methods known to one of ordinary skill in the art. For example, amino acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis according to the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A.
  • Functionally equivalent fragments of FAC also referred to herein as functional fragments, can be prepared by methods known to one of ordinary skill in the art, such as proteolytic cleavage and preparation of FAC nucleic acid fragments which encode fragments of FAC protein.
  • the activity of functionally equivalent variants or fragments of FAC polypeptides can be tested by cloning the gene encoding the altered FAC polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the altered FAC polypeptide, and testing for a functional capability of the FAC polypeptides as disclosed herein.
  • the FAC molecule component of the conjugates of the invention can also be a FAC nucleic acid.
  • the invention is not limited with respect to homologs and alleles of the FAC nucleic acids, which can be identified by conventional techniques.
  • an aspect of the invention is those nucleic acid sequences which code for FAC polypeptides and which hybridize to a nucleic acid molecule consisting of the coding region of SEQ ID NO:l, under stringent conditions.
  • stringent conditions refers to parameters with which the art is familiar. Nucleic acid hybridization parameters may be found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J.
  • stringent conditions refers, for example, to hybridization at 65°C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% Bovine Serum Albumin, 2.5mM NaH 2 PO 4 (pH7), 0.5% SDS, 2mM EDTA).
  • SSC 0.15M sodium chloride/0.15M sodium citrate, ⁇ H7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetraacetic acid.
  • homologs and alleles typically will share at least 40% nucleotide identity and/or at least 50% amino acid identity to SEQ ID NO:l and SEQ ID NO:2, respectively, in some instances will share at least 50% nucleotide identity and/or at least 65% amino acid identity and in still other instances will share at least 60% nucleotide identity and/or at least 75% amino acid identity.
  • Watson-Crick complements of the foregoing nucleic acids also are embraced by the invention.
  • a Southern blot may be performed using the foregoing conditions, together with a radioactive probe. After washing the membrane to which the DNA is finally transferred, the membrane can be placed against X-ray film or a phosphoimager plate to detect the radioactive signal.
  • the invention also includes degenerate nucleic acids which include alternative codons to those present in the native materials. For example, serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons is equivalent for the purposes of encoding a serine residue.
  • any of the serine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a serine residue into an elongating FAC polypeptide.
  • nucleotide sequence triplets which encode other amino acid residues include, but are not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons).
  • Other amino acid residues may be encoded similarly by multiple nucleotide sequences.
  • the invention embraces degenerate nucleic acids that differ from the biologically isolated nucleic acids in codon sequence due to the degeneracy of the genetic code.
  • the nucleic acids of the invention may be composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5' end of one native nucleotide and the 3' end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleoside linkage.
  • These nucleic acids may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors.
  • the nucleic acids of the invention also may include "modified" nucleic acids. That is, the oligonucleotides may be modified in a number of ways which do not prevent their transcription or translation but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness.
  • modified nucleic acids describes a nucleic acid in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the nucleic acid.
  • a synthetic internucleoside linkage i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide
  • Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.
  • modified nucleic acid also encompasses nucleic acids, including oligonucleotides, with a covalently modified base and/or sugar.
  • modified nucleic acids include nucleic acids having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position.
  • modified nucleic acids may include a 2'-O-alkylated ribose group.
  • modified nucleic acids may include sugars such as arabinose instead of ribose.
  • the present invention contemplates pharmaceutical preparations containing modified nucleic acids together with pharmaceutically acceptable carriers.
  • a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell.
  • Vectors are typically composed of DNA although RNA vectors are also available.
  • Vectors include, but are not limited to, plasmids, phagemids and virus genomes.
  • a cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.
  • An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., ⁇ -galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein).
  • Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
  • a coding sequence and regulatory sequences are said to be "operably” joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences.
  • two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
  • a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
  • regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene.
  • Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired.
  • the vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
  • RNA heterologous DNA
  • FAC polypeptide FAC polypeptide or fragment or variant thereof.
  • RNA heterologous DNA
  • Preferred systems for mRNA expression in mammalian cells are those such as pRc/CMV (available from Invitrogen, Carlsbad, CA) that contain a selectable marker such as a gene that confers G418 resistance (which facilitates the selection of stably transfected cell lines) and the human cytomegalovirus (CMV) enhancer-promoter sequences.
  • pRc/CMV available from Invitrogen, Carlsbad, CA
  • CMV human cytomegalovirus
  • suitable for expression in primate or canine cell lines is the pCEP4 vector (Invitrogen), which contains an Epstein Barr virus (EBV) origin of replication, facilitating the maintenance of plasmid as a multicopy extrachromosomal element.
  • EBV Epstein Barr virus
  • Another expression vector is the pEF-BOS plasmid containing the promoter of polypeptide Elongation Factor l ⁇ , which stimulates efficiently transcription in vitro.
  • the plasmid is described by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), and its use in transfection experiments is disclosed by, for example, Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996).
  • Still another preferred expression vector is an adeno virus, described by Stratford-Perricaudet, which is defective for El and E3 proteins (J. Clin. Invest. 90:626-630, 1992).
  • Conjugation between a FAC molecule and a targeting moiety according to the invention need not be direct attachment.
  • the functional components of the conjugate i.e., the FAC molecule and a targeting moiety
  • the functional components of the conjugate of the present invention may be synthesized in a single process, whereby the components could be regarded as one and the same entity.
  • a protein targeting moiety specific for an hematopoietic progenitor cell could be synthesized together with a FAC polypeptide as a fusion protein.
  • Covalent linking of a FAC polypeptide to an hematopoietic progenitor cell targeting molecule is intended to include linkage by peptide bonds in a single polypeptide chain to create a fusion protein.
  • Established methods e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY 1989
  • This DNA can be placed in an expression vector and introduced into bacterial or eukaryotic cells by established methods.
  • the fusion protein can then be purified from the cells or from the culture medium by established methods.
  • a fusion polypeptide may be useful for purification of the peptides of the invention.
  • the fusion domain may, for example, include a poly-His tail which allows for purification on Ni + columns or the maltose binding protein of the commercially available vector pMAL (New England BioLabs, Beverly, MA).
  • Specific examples of covalent bonds include those wherein bifunctional cross-linker molecules are used.
  • the cross-linker molecules may be homobifunctional or heterobifunctional, depending upon the nature of the molecules to be conjugated. Homobifunctional cross-linkers have two identical reactive groups. Heterobifunctional cross-linkers are defined as having two different reactive groups that allow for sequential conjugation reaction.
  • cross-linkers are reactive with one or more of the following groups; primary amines, secondary amines, sulfhydryls, carboxyls, carbonyls and carbohydrates.
  • amine-specific cross-linkers are bis(sulfosuccinimidyl) suberate, bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, disuccinimidyl suberate, disuccinimidyl tartarate, dimethyl adipimate»2 HCl, dimethyl pimelimidate » 2 HCl, dimethyl suberimidate «2 HCl, and ethylene glycolbis-[succinimidyl-[succinate]j.
  • Cross-linkers reactive with sulfhydryl groups include bismaleimidohexane, l,4-di-[3'-(2'-pyridyldithio)-propionamido)]butane, 1 -[p-azidosalicylamido]-4-[iodoacetamido]butane, and N-[4-(p-azidosalicylamido)butyl]- 3'-[2'-pyridyldithio]propionamide.
  • Cross-linkers preferentially reactive with carbohydrates include azidobenzoyl hydrazide.
  • Cross-linkers preferentially reactive with carboxyl groups include 4-[p-azidosalicylamido]butylamine.
  • Heterobifunctional cross-linkers that react with amines and sulfhydryls include N-succinimidyl-3-[2-pyridyldithio]propionate, succinimidyl [4- iodoacetyljaminobenzoate, succinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate, m-maleimidobenzoyl-N-hydroxysuccinimide ester, sulfosuccinimidyl
  • Heterobifunctional cross-linkers that react with carboxyl and amine groups include l-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride.
  • Heterobifunctional cross-linkers that react with carbohydrates and sulfhydryls include 4-[N-maleimidomethyl]-cyclohexane- 1 -carboxylhydrazide»HCl, 4-(4-N-maleimidophenyl)-butyric acid hydrazide » HCl, and 3-[2-pyridyldithio]propionyl hydrazide.
  • the cross-linkers may also be nonselective. Examples of nonselective cross-linkers are bis-[ ⁇ -(4-azidosalicylamido)ethyl]disulfide and glutaraldehyde.
  • Amine or thiol groups may be added at any nucleotide of a synthetic nucleic acid so as to provide a point of attachment for a bifunctional cross-linker molecule.
  • the nucleic acid may be synthesized incorporating conjugation-competent reagents such as Uni-Link AminoModifier, 3'-DMT-C6-Amine-ON CPG, AminoModifier II, N-TFA-C6-AminoModifier, C6-ThiolModifier, C6-Disulfide Phosphoramidite and C6-Disulfide CPG (Clontech, Palo Alto, CA).
  • Covalent linkages may be noncleavable in physiological environments or cleavable in physiological environments, such as linkers containing disulfide bonds. Such molecules may resist degradation and/or may be subject to different intracellular transport mechanisms.
  • One of ordinary skill in the art will be able to ascertain without undue experimentation the preferred bond for linking the targeting moiety and FAC molecule, based on the chemical properties of the molecules being linked and the preferred characteristics of the bond.
  • Noncovalent methods of conjugation may also be used to join the targeting moiety and the FAC molecule.
  • Noncovalent conjugation includes hydrophobic interactions, ionic interactions of positively and negatively charged molecules, biotin-avidin and biotin-streptavidin complexation, intercalation of a FAC nucleic acid, interaction with specific nucleotide sequences and other affinity interactions.
  • a FAC polypeptide can be bound by an antibody which also recognizes a hematopoietic progenitor cell.
  • biotinylated FAC nucleic acids can be complexed with avidin or streptavidin antibody conjugates.
  • a FAC nucleic acid can ionically complex a polylysine-antibody or a histone-antibody conjugate.
  • the conjugates of the invention are contacted with a hematopoietic progenitor target cell under conditions to permit selective binding of the conjugate to the receptor on the surface of the hematopoietic progenitor cell and to allow the conjugate to enter the cell.
  • conditions which permit the binding of the ligand to its receptor are the physiological conditions (e.g., the particular pH, ionic strength, viscosity) at which the ligands and receptors are known to bind to one another in vivo and the conditions at which the ligands and receptors are known to bind to one another in vitro, such as in receptor assays for determining the presence of a ligand in, for example, a biological fluid.
  • physiological conditions e.g., the particular pH, ionic strength, viscosity
  • the conditions at which the ligands and receptors are known to bind to one another in vitro such as in receptor assays for determining the presence of a ligand in, for example, a biological fluid.
  • receptor-mediated processes such as receptor-based binding assays and receptor-mediated delivery of therapeutic agents to preselected tissues in situ.
  • the conditions which permit binding to the receptor on the hematopoietic progenitor cell can be determined by conducting in vitro binding assays of the conjugate and the hematopoietic progenitor cell in accordance with standard procedures. Such in vitro assays are known to be predictive of the in vivo conditions for binding.
  • contacting in reference to the FAC conjugate and the target cell, refers to bringing the FAC conjugate into sufficiently close proximity to the target cell to permit the "receptor" on the target cell to selectively bind to the targeting moiety of the FAC conjugate.
  • the targeting moiety can bind with the cell by means of, inter alia, antibody/antigen binding and receptor/ligand binding.
  • Such conditions are well known to those of ordinary skill in the art. See also, e.g., U.S. patent No. 5,108,921, issued to Low et al. which reports the conditions for receptor-mediated delivery of "exogenous molecules” such as peptides, proteins and nucleic acids to animal cells and U.S. patent No.
  • the methods of the invention do not require contacting a FAC conjugate.
  • a non- conjugated FAC molecule can be contacted with a target cell, although it may not enter the cell by a receptor-meditaed endocytosis mechanism.
  • contacting a FAC molecule alone with a target cell can involve methods such as microinjection, electroporation, transfection, and the like.
  • RNA or protein analysis for detecting specific transcription or translation products can be performed in accordance with standard practice.
  • Exemplary detection techniques to measure gene expression include one or more of the following techniques, alone or in combination: northern or western blotting, in situ hybridization, reverse transcription, PCR amplification, immunostaining, RIA and ELISA.
  • a hematopoietic progenitor cell is contacted with a FAC molecule to inhibit apoptosis of the cell.
  • FAC is delivered to the cell for the purpose of reducing apoptosis of the cell.
  • Such inhibition of apoptosis is useful for ameliorating the effects of the Fanconi anemia disease, and for reducing the detrimental effects of high dose chemotherapy on myelopoiesis.
  • the FAC molecule When contacted with the cell, the FAC molecule optionally is part of a conjugate.
  • the targeting moiety is conjugated to the FAC molecule, by covalent or noncovalent linkage, in a manner that does not interfere with the binding of the targeting moiety to the cell.
  • the targeting moiety can be associated with the FAC molecule so as to encompass the FAC molecule. Examples of such associated targeting moieties include liposomes and viruses.
  • the FAC molecule need not be enclosed within the associated targeting moiety, but may merely be associated with the targeting moiety by ionic or hydrogen bonding and found in nucleic acid hybridization.
  • the FAC conjugate can be contacted with a hematopoietic progenitor cell in vitro, for example, for ex vivo FAC replacement therapy or in vitro production of a recombinant protein in cell culture, or in vivo for in vivo FAC replacement therapy or in vivo production of a nucleic acid transcription or translation product.
  • ex vivo immunotherapy involves the introduction in vitro of a functional copy of a gene into a cell(s) of a subject which contains a defective copy of the gene, and returning the genetically engineered cell(s) to the subject to alleviated the condition occasioned by the defective gene.
  • the functional copy of the FAC gene is under the operable control of regulatory elements which permit expression of the gene in the genetically engineered hematopoietic progenitor cell(s).
  • the target cells are not removed from the patient.
  • the nucleic acid molecule encoding e.g. a FAC polypeptide
  • the methods disclosed herein can be practiced by using appropriately targeted FAC conjugates in the procedures currently used for administering gene therapy vectors (or cells containing these vectors) to the subjects ex vivo or in vivo. Such procedures are known to those of skill in the art of human gene therapy.
  • hematopoietic progenitor cells which are to be treated are removed from a subject and the cells are contacted with a FAC conjugate in vitro.
  • the treated cells are expanded in culture before being reimplanted into the mammalian recipient.
  • the target cells subsequently can be introduced into the mammal (e.g., into the portal vein or into the spleen) if desired.
  • the FAC conjugate includes a FAC nucleic acid
  • expression of the nucleic acid molecule encoding a FAC polypeptide is accomplished by allowing the cell to live or propagate in vitro, in vivo, or in vitro and in vivo, sequentially.
  • the FAC conjugates as described herein have a number of uses, some of which are described elsewhere herein.
  • the invention includes the use of the conjugates described herein for the delivery of a FAC polypeptide or nucleic acid molecule to a hematopoietic progenitor cell.
  • the invention includes methods for inhibiting apoptosis in a hematopoietic progenitor cell by contacting a hematopoietic progenitor cell with a FAC molecule.
  • the FAC molecule can, but need not, be part of a conjugate with a targeting molecule such as a polypeptide which selectively binds to the hematopoietic progenitor cell.
  • the invention includes methods for treating a condition characterized by apoptosis of hematopoietic progenitor cells in a subject exposed to high dose chemotherapy for treatment of a non-myeloid malignancy, by administering an amount of a FAC molecule to the subject effective to inhibit the apoptosis.
  • FAC protein when introduced into hematopoietic progenitor cells, can inhibit the apoptosis such cells. Although the exact mechanism of the inhibition of apoptosis is unclear at this time, what is clear is that the addition of functional FAC (e.g. not FAC mutated as in Fanconi anemia) to the cells prevents the toxicity of mitomycin C, which kills the cells by apoptosis.
  • functional FAC e.g. not FAC mutated as in Fanconi anemia
  • This property also permits the treatment of hematopoietic progenitor cells of a subject exposed to chemotherapy.
  • Administration of FAC prior to chemotherapy increases the amount of surviving hematopoietic progenitor cells.
  • FAC hematopoietic progenitor cells
  • the invention embraces the use of the FAC conjugates in vitro to increase the level of FAC in host cells and cell lines, for instance, for determining optimal amounts of FAC molecules to administer to inhibit or even prevent apoptosis or to generally ameliorate the defect in Fanconi anemia cells.
  • mammalian cells such as mouse, hamster, pig, goat, primate, etc. They may be of a wide variety of tissue types, and include primary cells and cell lines. Specific examples include hematopoietic progenitor cells, bone marrow stem cells and embryonic stem cells.
  • the expression vectors require that the pertinent sequence, i.e., those nucleic acids described supra, be operably linked to a promoter.
  • compositions of the present invention are administered in pharmaceutically acceptable preparations.
  • Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary agents such as cytokines and optionally other therapeutic agents.
  • compositions should be sterile and contain a therapeutically effective amount of the FAC molecule in a unit of weight or volume suitable for administration to a patient.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients.
  • physiologically acceptable refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
  • the therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time.
  • the administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.
  • compositions may be conveniently presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the conjugate into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the conjugate into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
  • carrier as used herein, and described more fully below, means one or more solid or liquid filler, dilutants or encapsulating substances which are suitable for administration to a human or other mammal.
  • the “carrier” may be an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate administration.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • the preparations of the invention are administered in effective amounts.
  • An effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, produces the desired response.
  • the desired response is a reduction in the rate or amount of hematopoitic progenitor cell apoptosis.
  • the desired response is increasing the amount of active FAC protein present in a hematopoietic progenitor cell such that the survival of the hematopoietic progenitor cell is increased. This may involve only boosting the survival of hematopoietic progenitor cells temporarily, although more preferably, it involves enhancing survival of hematopoietic progenitor cells permanently.
  • doses ranging from 1 nanogram/kilogram to 100 milligrams/kilogram, depending upon the mode of administration, will be effective.
  • the preferred range is believed to be between about 500 nanograms and 500 micrograms/kilogram, and most preferably between 1 microgram and 100 micrograms/kilogram.
  • the absolute amount will depend upon a variety of factors, including the conjugate selected, the modulation desired, whether the administration is in a single or multiple doses, and individual patient parameters including age, physical condition, size and weight. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.
  • the conjugates of the invention may be administered per se (neat) or in the form of a pharmaceutically acceptable salt.
  • the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulfonic, tartaric, citric, methane sulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzene sulfonic.
  • pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
  • Suitable buffering agents include: acetic acid and a salt (1-2% W/V); citric acid and a salt (1-3% W/N); boric acid and a salt (0.5-2.5% W/V); sodium bicarbonate (0.5-1.0% W/V); and phosphoric acid and a salt (0.8-2% W/V).
  • Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V) and thimerosal (0.004-0.02% W/V).
  • compositions are capable of being commingled with the conjugates of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
  • supplementary agents such as molecules involved in signal transduction which enhance survival of hematopoietic progenitor cells
  • the molecules contemplated are those that may enhance the beneficial effects that result from administering the FAC molecule according to the invention.
  • cytokines Upon binding of molecules involved in signal transduction such as cytokines to their cognate receptors on the cell surface, specific polypeptides, called JAKs and STATs, are involved in transmission of the signal to the nucleus.
  • the preferred signal transduction molecules are STATs (especially STAT 1, 2 and 3) and JAKs that promote myeloid cell growth.
  • the selection of the particular signalling molecule will depend upon the particular modulation of the hematopoietic system that is desired.
  • the activity of molecules involved in signal transduction, e.g. cytokines, on particular cell types is known to those of ordinary skill in the art.
  • the precise amounts of the foregoing signalling molecules used in the invention will depend upon a variety of factors, including the conjugate selected, the dose and dose-timing selected, the mode of administration and the characteristics of the subject. The precise amounts selected can be determined without undue experimentation, particularly since a threshold amount will be any amount which will enhance the desired immune response. Thus, it is believed that nanogram to milligram amounts are useful, depending upon the mode of delivery, but that nanogram to microgram amounts are likely to be sufficient because physiological levels of these molecules are correspondingly low.
  • Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the conjugates of the invention, increasing convenience to the subject and the physician.
  • Many types of release/delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as polylactic and polyglycolic acid, polyanhidrides and polycaprolactone; wax coatings, compressed tablets using conventional binders and excipients, and the like.
  • Bioadhesive polymer systems to enhance delivery of a material to the intestinal epithelium are known and described in published PCT application WO 93/21906. Capsules for delivering agents to the intestinal epithelium also are described in published PCT application WO 93/19660.
  • Example 1 Generation. Targeting and Activity of Interleukin-3-FAC.
  • IL3 Mature human or gibbon interleukin-3 (IL3) is a 133 amino acid glycoprotein that is produced primarily by activated T cells (Lindemann, et al., Cancer Invest. 609-623, 1993). Recombinant IL3 expressed in E. coli maintains its bioactivity despite the lack of carbohydrate residues.
  • the high-affinity human IL3 receptor is a heterodimer of ⁇ and ⁇ subunits present at about 50-1,000 copies per cell and is essential for the survival of hematopoietic progenitor cells. Both murine and human receptors undergo internalization of the receptor-ligand complex, followed by digestion of IL3 within lysosomes and recycling of the receptors back to the cell surface.
  • His-ILFAC Construction and Expression of His-ILFAC.
  • a tripartite protein was constructed consisting of an epitope of six histidine residues (His-TAG) at the amino terminus, gibbon IL3 (minus the signal peptide) at the center, and full-length human FAC (minus the initiation methiodine) at the carboxy terminus (schematically shown in Fig. 1). Based on the predicted tertiary structure of IL3, it was reasoned that placement of FAC fusion partner at the "free" carboxy-terminal end of the IL3 would not result in steric hinderance.
  • the corresponding cDNA cloned in the vector pQE9 (Qiagen, Inc., Santa Clarita, CA) was used to transform E. coli strain Ml 5.
  • a 75 kDa fusion protein was detected by a His epitope reactive antibody (mouse anti-RGS(H) 4 antibody, Qiagen) in cell lysates after induction of the transformed cells with 2 mM IPTG for 5 hours (Fig. 2A).
  • Immunoblotting confirmed that the 75 kDa protein is immunoreactive with polyclonal antibodies directed against FAC (Fig. 2B). His-tagged FAC (without interposed IL3) was also generated (Fig. 1) and expressed (Fig. 2A) using similar procedures.
  • a third fusion protein (secreted ILFAC, Fig. 1) was created with the gibbon IL3 molecule (including a signal peptide) at the amino terminus and full-length human FAC (minus the initiation methiodine) at the carboxy terminus. The specific protocols follow.
  • the polylinker region of the vector pQE9 (Qiagen) was modified to generate a vector of greater versatility and allow the in-frame cloning of additional restriction fragments.
  • pQE9 was cut with BamHI and Hindlll, and the -30 bp BamHI/Hindlll fragment from pGEM3 (Promega) was inserted.
  • the resultant vector is called pQE9Xl .
  • pQE9-FAC without the IL3 ligand at the N-terminus
  • pQE9Xl was cut with BamHI/Xbal.
  • the 1.65 kb insert encoding human FAC was purified by gel electrophoresis from another bacterial expression vector (pGEX2TK-FAC) generated by me and published previously ( Youssoufian et al. J. Clin. Invest. 97:957-962, 1996).
  • the linearized pQE9Xl and the FAC insert were ligated to generate pQE9Xl-FAC.
  • the mature coding region of gibbon IL3 was obtained by PCR: BamHI Forward primer 5'-CGGGATCC GCT CCC ATG ACC CAG ACA A-3'
  • PCR Thirty cycles of PCR were performed using pMX-IL3 as template (vector obtained from Todd Golub, Dana-Farber Cancer Institute, Boston, MA who obtained it from the Genetics Institute).
  • the PCR product was cut with BamHI and gel-purified according to standard procedures. 4.
  • the plasmid pQE9Xl-FAC was cut with BamHI and the ends were phosphatased.
  • the BamHI-digested 0.4 kb IL3 PCR fragment from step 3 was inserted into the BamHI site of vector to generate the final desired product, pQE9Xl-ILFAC.
  • This construct encodes a tripartite protein consisting of an epitope of six histidine (His) residues at the N-terminus, mature gibbon IL3 in the middle, and full-length human FAC (except the initiation codon) at the carboxy- terminus.
  • His histidine
  • His-ILFAC histidine residues
  • gibbon IL3 without the signal peptide
  • full-length human FAC minus the initiation methiodine
  • the mammalian expression construct was prepared by a three-way ligation of a vector backbone, the gibbon IL3 and the human FAC sequence described above.
  • the vector was pED6 (obtained from Genetics Institute), which was disgested with Sail and Xbal.
  • the FAC portion was prepared as above.
  • the IL3 portion was prepared by PCR as above, but with a different forward primer that preserves the natural signal peptide sequence of IL3
  • the PCR product was digested with
  • the ligated plasmid was used to transform bacteria and clones were selected according to standard protocols.
  • the mammalian ILFAC plasmid was used to transfect subconfluent COS cells by a standard DEAE transfection protocol, using about 1 ⁇ g plasmid per milliliter transfection medium (e.g., 5 ⁇ g plasmid per 10mm dish COS cells. After 5-7 days, the conditioned medium was harvested and clarified of debris. The clarified conditioned medium was used directly in bioassays at various concentrations.
  • FAC conjugates include those which include an anti-CD33 antibody (such as M195, see Co et al., J. Immunol.148(4), 1149-1154, 1992), or individual chains thereof, attached to a FAC molecule.
  • an anti-CD33-FAC molecule is prepared according to the method for preparing a mammalian fusion protein above.
  • the nucleic acid encoding the heavy chain of the Ml 95 anti-CD33 antibody (SEQ ID NO: 5) is ligated with the pED6 vector and the human FAC cDNA (SEQ ID NO:3, except the initiation codon).
  • the nucleic acid encoding the light chain of the M195 anti-CD33 antibody (SEQ ID NO:7) can also be used in place of the heavy chain.
  • the fusion protein is expressed and used as above.
  • a chemically crosslinked conjugate of the Ml 95 anti-CD33 antibody and FAC can also be prepared.
  • the nucleic acids encoding the heavy and light chains of Ml 95 are inserted in a bicistronic vector which is transfected into an appropriate cell line to obtain approximately stoichiometric amounts of heavy and light chains which assemble as an antibody molecule.
  • the antibody molecule is purified by e.g. Protein A or Protein G affinity chromatography and conjugated to the FAC protein (SEQ ID NO:2) by a bifunctional crosslinker as identified supra.
  • conjugation also may be performed by derivatizing both FAC protein and the anti- CD33 antibody (or fragments thereof) with sulfosuccinimidyl 6-[3-(2- pyridyldithio)propionamide] hexanoate (sulfo-LC-SPDP, Pierce) for 18 hours at room temperature.
  • Conjugates also may be prepared by derivatizing FAC protein and the anti-CD33 antibody with different crosslinking reagents that will subsequently form a covalent linkage.
  • crosslinking reagents comprising aldehyde, imide, cyano, halogen, carboxyl, activated carboxyl, anhydride and maleimide functional groups are known to persons of ordinary skill in the art and also may be used for conjugation of FAC protein and the anti-CD33 antibody.
  • crosslinking reactions it is important to purify the derivatized compounds free of crosslinking reagent. It is important also to purify the final conjugate substantially free of unconjugated reactants. Purification may be achieved by affinity, gel filtration or ion exchange chromatography based on the properties of either FAC protein or the anti-CD33 antibody.
  • a particularly preferred method is an initial affinity purification step using immobilized CD33 to retain Ml 95 antibody and M195-FAC conjugates, followed by gel filtration or ion exchange chromatography based on the mass, size or charge of the conjugate.
  • the initial step of this purification scheme ensures that the conjugate will bind to CD33.
  • immobilized anti-FAC antibodies can be used in the affinity purification step, followed by gel filtration or ion exchange chromatography based on the mass, size or charge of the conjugate.
  • the purification protocol consisted of two successive column chromatography steps that yielded protein of greater than 80 percent homogeneity. Briefly, cells were transferred and induced as described above, and then were sonicated in a buffer containing 50 mM sodium phosphate (pH 8.0) and 300 mM NaCl. The crude protein extract was subjected to nickel- agarose column chromatography, which retains proteins having the His-TAG epitope. After elution with 50 mM imidazole and dialysis against PBS (Fig. 2A), the partially purified protein was subjected to size exclusion chromatography over Superdex-75.
  • Fraction 1 from the void volume contained greater than 80 percent of His-ILFAC as assessed by immunoreactivity with anti-FAC antibody (Fig. 2B).
  • the final yield of partially purified protein was approximately 1 milligram from 4 liters of induced bacterial culture. The specific protocol follows:
  • E. coli strain Ml 5 (Invitrogen) was transformed with pQE9Xl-ILFAC. 2. Transformants were selected for in LB containing Ampicillin (100 ⁇ g/ml) and
  • Kanamycin (25 g/ml).
  • the cells were pelleted by centrifugation. 6. The pellet was washed with cold PBS.
  • the pellet was lysed as follows. The pellet was resuspended in SB-I/2.5% Sarkosyl/0.2 mM PMSF/10 mM ⁇ -ME, at 5 ml resuspension volume per gram wet weight of pellet. The resuspension was sonicated using the following conditions: Duty cycle 50%, Output control 5; four sonications of 30 sec. each (total 2 min), keeping the suspension on ice in between sonications. The sonicated suspension was centrifuged at 12,000 rpm for 15 minutes and the supernatant was recovered. A 1/10th volume of SB-I/20% TRITON X-10 was added.
  • Binding was performed as follows. 0.5 ml Ni-NTA agarose beads (Qiagen) were washed in SB I buffer ( SB-I: 50 mM Na-phosphate pH 7.8, 300 mM NaCl). The supernatant of step 7 was added to the washed beads and rotated at 4°C for 60 min.
  • the beads were washed using the following protocol: Spin beads at 1500 rpm x 2 min
  • SB-II buffer is: 50 mM Na-phosphate pH 6.0, 300 mM NaCl
  • the fusion protein was batch eluted from the washed beads using 1 ml of the following solutions of imidazole in PBS:
  • the protein eluted by 25 mM imidazole was dialyzed against PBS and used in bioassays.
  • the fusion protein was further purified by size exclusion chromatography using Superdex-75 according to standard procedures.
  • Example 2 Receptor-mediated Targeting in Human Lymphoblastoid Cells.
  • the GM4510 FA-C lymphoblastoid cells homozygous for a splicing mutation in FAC (this mutation is predicted to yield a truncated protein product) were incubated with either 1 ⁇ g/ml His-ILFAC or His-FAC for 0-30 minutes at 37 °C, washed in cold PBS, lysed and analyzed by immunoblotting with anti-FAC antibody. Immunoreactive His-ILFAC was detected after two minutes of incubation and reached a maximum after 10 minutes. By contrast, very little if any cell-associated His-FAC was detected under the same conditions.
  • ligand internalization occurred much more readily at 37 °C than 4°C (5-6 fold more).
  • ligand internalization was inhibited (to -20% of control) by addition of recombinant human IL3 (10 ⁇ g/ml) to the medium and inhibited further (to -5% of control) by pre-treatment of intact cells with trypsin prior to incubation with His-ILFAC for 10 minutes.
  • the lack of complete inhibition may relate to inadequate receptor occupancy by IL3, inefficient stripping of surface IL3 receptors with trypsin (as recognized previously: Stevens, et al., J Biol. Chem. 266:4151-4158, 1991), or additional factors.
  • HSC536 cells which are compound heterozygous for the L 554 P mutation and an unknown mutation.
  • Approximately 1 x 10 4 viable cells were incubated with different concentrations of MMC for 5 days in the presence or absence of His-ILFAC. Viable cells were quantitated by trypan blue exclusion. Cells were supplemented as follows (Fig.
  • FAC mutant cells stably transfected with wild-type FAC
  • ILFAC(a) His-ILFAC (1 ⁇ g/ml) added to the culture medium of mutant cell daily for 5 days
  • ILFAC(b) COS-1 conditioned medium containing secreted ILFAC added to the culture medium as a 10% (v/v) supplement
  • IL3 recombinant human IL3 (1 g/ml) added to the culture medium of mutant cells daily for 5 days.
  • cells supplemented with FAC or ILFAC molecules were significantly more resistant to MMC than control cells (untreated or IL3 treated).
  • Example 4 Expression of Other His-ILFAC Molecules.
  • the IL3 cassette encodes the Gibbon protein, it is comparable to the human IL3 in its interaction with the human receptor and in signal transduction (Miyajima, et al., Blood 82:1960-1974, 1994).
  • a His-TAG fusion protein is generated that includes the mature portion of murine IL3 (minus the signal peptide) fused at the downstream end to human FAC as described above in Example 2.
  • additional His-ILFAC proteins comprising various combinations of the components are prepared to determine if the order of the components influences bioactivity.
  • the His-TAG has been exploited to obtain partially pure protein from bacterial lysates by affinity chromatography, and it has been used in conjunction with other methods, especially size exclusion chromatography.
  • the protein was not purified to homogeneity, it retained partial biological activity for both the IL3 domain, as demonstrated by successful receptor targeting, and the FAC domain, as demonstrated by correction of MMC hypersensitivity.
  • the presence of lower molecular size forms may be due to either degradation products or premature termination of the fusion protein at cryptic stop codons.
  • the immunoaffinity column is generated using purified anti-FAC antibodies directed against the carboxy-terminus of FAC.
  • polyclonal anti-FAC antibodies directed against a nearly full-length FAC protein were generated (Youssoufian, et al., J. Biol. Chem. 270:9876-9882, 1995; Youssoufian, Proc. Natl. Acad. Sci. U.S.A. 91 :7975-7979, 1994; Youssoufian, J. Clin. Invest. 97:2003-2010, 1996a).
  • anti-peptide antibodies from the most carboxy- terminal region of FAC are generated in order to select the desired full-length bacterial products that contain intact carboxy-termini.
  • short peptides are synthesized from the carboxyterminal region of FAC, and these peptides are coupled covalently to appropriately antigenic carriers (e.g., keyhole limpet hemocyanin), and rabbits are immunized with such conjugated peptides according to standard procedures.
  • the antigenic peptide is coupled to epoxide-activated silica beads (Waters, Inc., Bedford, MA) as an affinity reagent with which affinity-purified IgG is isolated from the crude rabbit antiserum.
  • affinity-purified anti-FAC antibodies Specific elution of the anti- FAC antibodies with either acidic glycine or imidazole yields a population of affinity-purified anti-FAC antibodies. These antibodies are used to generate a second affinity column for the purification of His-ILFAC. Similar to the affinity column mentioned above, affinity purified anti-FAC antibodies are immobilized onto epoxy-activated silica beads according to the manufacturer's instructions, and the fusion protein obtained from nickel agarose chromatography is subjected to a second round of affinity purification over anti-FAC affinity column. The double affinity purification procedure thus is effective for isolating molecules which retain both the IL3 and FAC portions of the fusion protein.
  • His-ILFAC fusion protein To determine the intracellular turnover of the His-ILFAC fusion protein, purified His- ILFAC or His-FAC were added to HSC536 cells ar 4 °C for 10 minutes in DME containing 10% FBS. Cells were then centrifuged, fresh DME-10%> FBS medium was added in the absence of His-tagged proteins, and cells were rapidly warmed to 37 °C. Internalization of the fusion proteins and the intracellular turnover of the proteins was assessed by washing cells with cold PBS and lysing the cells in IX Laemmli buffer at the indicated times (Fig.
  • the post-nuclear supernatant is immunoprecipitated sequentially with anti-FAC antibody for 16 hours at 4°C and protein A-agarose beads for 2 hours (Bio-Rad, Richmond, CA), and the unbound supernatant is subjected to a second round of immunoprecipitation to maximize antigen recovery.
  • protease inhibitors 0.2 mM PMSF, 2 mM EDTA and 1 ⁇ g/ml aprotinin
  • the combined immuno-complexes are washed three times with NET-gel buffer (Kruyt, et al., Blood 87:938-948, 1996), boiled in 1 x Laemmli buffer in the presence of 200 mM dithiothreitol, and analyzed by SDS-PAGE and autoradiography.
  • the intracellular t. 2 is tested by immunoblotting as described above. In this manner, regions of the protein that are responsible for promoting degradation are identified. After the identification of such a domain, the degradation-promoting region is localized with greater precision and conventional site- directed mutagenesis (Lu, et al., Exp. Hematol. 20:418-424, 1992) is used to alter specific residues.
  • mutations that suppress intracellular degradation are subcloned in pQE9 and expressed as IL3 fusion proteins in E. coli.
  • receptor-mediated uptake assays on IL3 receptor-positive lymphoblastoid B cells are performed as described above. The half life of these proteins is determined by pulse-chase analysis in cells supplemented exogenously with either wild type or mutant His-ILFAC. After treatment for 10 minutes at 37 °C, cells are washed and chased in complete media and the intracellular levels of His-ILFAC in cell lysates from various time points are analyzed by immunoblotting. FAC fusion proteins having longer t, ⁇ will be useful for treatments. The intracellular activities of the purified His-ILFAC fusion proteins having prolonged intracellular half lives are determined. Correction of the MMC hypersensitivity of FA-C cells by exogenous administration to the FAC mutant fusion proteins IL3 receptor-positive cells is assessed as described above.
  • Example 7 Activity of His-ILFAC Proteins in FA-C Lymphoblasts.
  • DAA-CD expression vector that encodes the T cell-specific surface antigen CD4 as well as the full-length FAC cDNA (Youssoufian, 1995; Youssoufian, 1996a, supra).
  • DAA-CD expression vector that encodes the T cell-specific surface antigen CD4 as well as the full-length FAC cDNA (Youssoufian, 1995; Youssoufian, 1996a, supra).
  • cells are subjected to one or more rounds of selection with immunomagnetic beads coated with an anti-CD4 monoclonal antibody, and cell viability assays are performed shortly thereafter.
  • the rapidity of this procedure makes it possible to analyze the functional consequences of a large number of mutations relatively quickly.
  • this assay is useful to identify mutants of FAC, such as are termed previously functional equivalents.
  • the assay is useful to identify FAC proteins which have longer half lives and are effective in reducing apoptosis.
  • mutant FAC cDNAs that result in longer half lives are cloned into the DRA-CD expression vector, electroporated into FA-C cells (Youssoufian, 1995; Youssoufian, 1996a, supra) and the ability of the mutant proteins to confer resistance to crosslinker-treated cells is assessed as described above.
  • Second, intracellular activity of FAC will be expressed in E. coli as His-IL3 fusion proteins.
  • the ability to correct MMC-induced hypersensitivity is assessed directly by targeted delivery of His-ILFAC protein into FA-C lymphoblastoid cells.
  • This dual strategy of assessing the activity of the fusion protein both after transfection and after exogenous administration of the fusion protein permits the detection of subtle quantitative differences between the activities of different mutant proteins.
  • mutations that either partially inactivate the protein or, conversely, result in superactive proteins are identified by this approach.
  • Example 8 Activity of His-ILFAC in Primary Hematopoietic Progenitor Cells of FA-C Patients.
  • bone marrow cells of FA-C patients and normal control subjects are assessed for their capacity to form distinct hematopoietic lineages in the presence or absence of the FAC fusion protein.
  • Hematopoietic cells are obtained from bone marrow donors during harvests prior to transplantation.
  • CD34 +++ cells are obtained after sorting non-adherent low-density T lymphocyte-depleted cells on a flow cytometer (Epics 753; Coulter Corp., Hialeah, FL). This fraction is richest in stem/immature progenitor cells.
  • CD34 +++ population is sorted further to yield the following subsets: CD34 ++ CD33 " (enriched for BFU-E and CFU-E), and CD34 ++ CD33 + (enriched for CFU-GM).
  • CD34 ++ CD33 enriched for BFU-E and CFU-E
  • CD34 ++ CD33 + enriched for CFU-GM.
  • These cells are placed into single wells containing 0.1 ml semisolid culture medium containing Iscove's modified Dulbecco's medium, 1 % methyl cellulose, 30% fetal calf serum and 0.1 mM hemin, as well as maximally active concentrations of recombinant human steel factor, IL3, GM-CSF, and erythropoietin.
  • the criteria of greater than 1 mm diameter with a dense center and composed of greater than 10 3 cells/colony is used.
  • Example 9 Physiological Properties of IL3-FAC in Murine Models. Although the molecular events involved in post-receptor IL3 signaling appear to be highly conserved between human and mouse, the primary sequence homology between human and mouse IL3 is limited, and the interaction of these ligands with their receptors is highly species specific. Thus, a His-tagged murine IL3-human FAC protein (His-mlLFAC) is generated, as indicated above, for the studies described below.
  • His-mlLFAC His-tagged murine IL3-human FAC protein
  • Example 10 Tissue Uptake and Toxicity.
  • mice are injected through their tail veins with increasing doses of His-mlLFAC. After one hour, mice are sacrificed and tissues analyzed by immunoblotting with anti-FAC antibody. Targeting of the protein to IL3 receptor-positive cells is confirmed by finding the immunoreactive protein in the bone marrow, spleen, and other cells that constitute the reticuloendothelial system.
  • the fusion protein is distinguished from endogenous FAC protein by its greater size. The interval between protein injection and tissue processing can be varied to establish the optimal timing of this analysis.
  • this set of experiments establishes the maximum tolerated dose of the fusion protein; the animals will be observed for clinically recognized features of cardiorespiratory distress and analysis of complete blood counts.
  • Example 11 In vivo Bioactivity of IL3-FAC.
  • FA A mice are treated with a dose of the fusion protein that causes little toxicity and results in hematopoietic cell targeting in normal mice.
  • Karyotype analysis is performed on splenic lymphocytes in the presence or absence of the fusion protein. Mice are injected 1-2 hours prior to sacrifice to "load" lymphocytes with exogenous protein. Lymphocytes are then stimulated in vitro for 24-48 hours with concanavalin A prior to metaphase arrest and standard karyotype analysis. The mean number of aberrations per cell with and without treatment with exogenous His-mlLFAC provides evidence for the activity of the fusion protein. Finally, the His-mlLFAC fusion protein is used to rescue male and female FA " ' " mice from cross-linker- induced toxicity. Different schedules of administration (i.e., frequency of administration, pre- administration relative to MMC) are used to identify the conditions that result in hematopoietic rescue.
  • ADDRESSEE WOLF, GREENFIELD & SACKS, P.C.
  • B STREET: 600 ATLANTIC AVENUE
  • AAAAACTCAA AAAGCCAATA CGAGGCAAAG CCAAATTTTC AAGCCACAGA TCCCGGGCGG 120
  • GAG GCT GTA AAC GAG GCC ATT TTG CTG AAG AAG ATT TCT CTC CCC ATG 963 Glu Ala Val Asn Glu Ala He Leu Leu Lys Lys He Ser Leu Pro Met 225 230 235
  • GCA CTC CTG GAA ACC GAT GGG GCC CTG GAA ATC ATA GCC ACT ATT CAG 1203 Ala Leu Leu Glu Thr Asp Gly Ala Leu Glu He He Ala Thr He Gin 305 310 315
  • Trp He Gin Gly Val Leu Ser His He Leu Ser Ala Leu Arg Phe Asp 115 120 125
  • GCT CAA CAG ATG ACT TTG AGC CTT GAG ATC TCT TGAGTCCAAC GTCCAGCTCT 485 Ala Gin Gin Met Thr Leu Ser Leu Glu He Ser 145 150 155
  • CTCTGGGTCG TCTCTCACAC AGTCCAGGAC CAGAAGCATT TCACC-TITIC CTGCGGCATC 605

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Abstract

The invention provides conjugates comprising a FAC molecule and a targeting molecule which selectively binds hematopoietic progenitor cells. The invention further relates to methods of using such FAC molecules and conjugates in the treatment and/or diagnosis of disease.

Description

FAC MOLECULES AND USES THEREOF
Related Applications
This application claims priority under 35 U.S.C. §119 from US provisional application serial number 60/046,546, filed May 15, 1997.
Grant Support
This work was funded in part by the Leukemia Society of America under Translational Research Award No. 6299-97.
Field of the Invention This invention relates to Fanconi anemia complementation group C (FAC) nucleic acids and encoded polypeptides which modulate apoptosis in hematopoietic progenitor cells. The invention also relates to conjugates, including fusion polypeptides, comprising the FAC molecules and polypeptides which selectively bind hematopoietic progenitor cells. The invention further relates to methods of using such FAC molecules and conjugates in the treatment and/or diagnosis of disease.
Background of the Invention Fanconi anemia (FA) is an autosomal recessive disorder with an incidence of about 1 in
350,000 and manifested clinically by birth defects and an exorable progression to pancytopenia and leukemia (Fanconi Semin. Hematol. 4:233-240, 1967; Glanz, et al. J Med. Genet. 19:412- 416, 1982; Liu, et al. Blood 84:3995-4007, 1994). Over the past few years, great strides have been made in the genetic classification of FA into five distinct complementation groups (Strathdee, et al., Nature Genet.1 :196-198, 1992; Joenje, et al., Blood 86:2156-2160, 1995) and in cloning of the gene defective in one group (Strathdee Nature 356:763-767, 1992). This gene (called FAC) is that of the complementation group C (FA-C) which was isolated by virtue of its ability to suppress the toxicity of cross-linkers on Fanconi anemia cells.
Fanconi anemia patients have an actuarial risk of 67 percent of developing acute myelogenous leukemia (AML) and a risk of 80 percent of death from hematologic causes (Butturini, et al., Blood 84:1650-1655, 1994). Earlier estimates also showed a markedly increased (about 15,000-fold) incidence of AML (Auerbach, Cancer Genet. Cytogenet. 51 :1-12, 1991). Although pancytopenia often heralds the development of leukemia, AML can also be the initial hematologic manifestation (Auerbach, et al., Am. J. Hematol. 12:289-300, 1982). The basis of enhanced susceptibility of the hematopoietic system to the FA defect is not well understood. Hematopoietic colony growth is severely deficient in FA patients regardless of their hematologic status. Deficiencies in progenitor cells of granulocyte-macrophage (CFU-GM, colony forming unit-granulocyte-macrophage), erythroid (BFU-E burst-forming unit-erythroid), and multi potential (CFU-GEMM, colony-forming unit-granulocyte, erythroid, macrophage, megakaryocyte) lineages can be demonstrated in pre-symptomatic patients (Daneshbod-Skibba -βr. J Haematol. 44:33-38, 1938; Alter, et al., Blood 78:602-608, 1991; Stark, et al., -Br. J. Haematol. 83:554-559, 1993) as well as during the pancytopenic and leukemic stages of Fanconi anemia (Lui, et al., J. Pediatr. 91 :952-954, 1977; Saunders, et al., Br. J. Haematol. 40:277-287, 1978; Chu, Proc. Soc. Exp. Biol. Med. 161 :609-612, 1979; Prindull, et al, Scand. J. Haematol. 23:59-63, 1979). Long term bone marrow culture assays have shown 15-fold reduction in CFU- GM clonogenicity compared to normal controls (Stark, et al., supra), and repression of FAC gene expression in non-FA cells with antisense oligonucleotides has led to a concomitant suppression in clonal growth of hematopoietic progenitor cells (Segal, et al., J. Clin. Invest. 94:846-852, 1994).
Although a number of therapies for FA have been described (e.g., androgens), the most promising strategy has been provided by HLA-matched sibling bone marrow or umbilical cord blood transplantation for its potential for reconstitution of the hematopoietic system (Sullivan, Leukemia 7:1098-1099, 1993; Glockman, et al., Blood 86:2856-2862, 1995). Frequently, however, no suitable donor can be identified, leaving the patient with the prospect of undergoing a partially-matched or mismatched transplant. The mortality of a mismatched transplant can approach 100 percent. Another disturbing feature of transplantation is its inability to prevent the appearance of other forms of cancer (Witherspoon, et al., Blood 72:413a, 1988). It is possible that cytoreductive therapy and radiation used as part of the transplant conditioning regime contribute to this risk.
Fanconi anemia is the quintessential model of an inherited "pre-leukemic" disorder that leads to marrow failure and leukemia. Although the molecular defect is not restricted to the hematopoietic system, pancytopenia and AML are responsible for a large share of the difficulties suffered by FA patients. Perhaps the most unique aspect of FA as a disease model is the opportunity to prevent or reduce the development of leukemia. Clearly, bone marrow transplantation provides a respite from leukemia, but the challenges presented by cytoreductive therapy, tissue typing, expense and access to specialized centers are daunting. Thus, there is a need for effective and safer forms of therapy for Fanconi anemia.
Summary of the Invention
The invention involves the discovery that the Fanconi anemia complementation group C (FAC) protein may be utilized to inhibit the effects of Fanconi anemia in hematopoietic progenitor cells in vivo and in vitro. It has also been realized that the FAC protein can be effective for ameliorating the effects of chemotherapy given to subject having a non-myeloid malignancy. It further has been discovered that FAC molecules can be delivered to hematopoietic progenitor cells by preparing a conjugate of a FAC molecule and a targeting molecule which binds to a cell surface protein of the cells and is internalized. Such targeting molecules include IL3 and antibodies which recognize CD33.
It is an object of the invention to deliver a FAC molecule to Fanconi anemia cells to complement the genetic defect. It is a further object of the invention to deliver a FAC molecule to hematopoietic progenitor cells to ameliorate the apoptotic effects of high dose chemotherapy on such cells. It is yet another object of the invention to provide compositions which specifically target hematopoietic progenitor cells for the delivery of FAC molecules to such cells.
According to one aspect of the invention, an isolated conjugate is provided. The conjugate includes a FAC molecule and a polypeptide which selectively binds a hematopoietic progenitor cell. In certain embodiments, the FAC molecule is a polypeptide or a nucleic acid. Where the FAC molecule is a polypeptide, preferably it is a protein which has the amino acid sequence of SEQ ID NO:2, or a functional fragment thereof. In other embodiments, the FAC molecule is a nucleic acid molecule which hybridizes under stringent conditions to a molecule having the nucleotide sequence of SEQ ID NO: 1, and which codes for a FAC protein, nucleic acids which differ from the foregoing due to the degeneracy of the genetic code, or complementary molecules. Preferred FAC nucleic acids are those which include the nucleotide sequence of SEQ ID NO:l, and those which encode a functional fragment of a FAC protein. In certain embodiments, the FAC nucleic acid is operably linked to a promoter, preferably one which is active in a hematopoietic progenitor cell, and optionally one which is not active in mature hematopoietic cells.
As noted previously, the FAC conjugate includes a targeting molecule in the form of a polypeptide which selectively binds hematopoietic progenitor cells. Preferably the polypeptide is an antibody or a ligand which selectively binds to a cell surface protein, such as an antigen or a ligand, of the hematopoietic progenitor cells. In certain of the embodiments in which the targeting molecule is an antibody, the antibody is an anti-CD33 antibody or an antigen binding fragment thereof. The targeting antibody can be conjugated to the FAC molecule in the form of a fusion protein. In such cases, the antibody preferably is a single chain antibody. In certain of the embodiments in which the targeting molecule is a ligand, the conjugate is a fusion protein. Preferably the ligand is IL3 or a receptor-binding portion thereof. The IL3-FAC fusion protein can be constructed with either the IL3 portion or the FAC portion at the amino terminus, and can include a linker between the IL3 and FAC portions. Other ligands are also embraced by the invention.
According to another aspect of the invention, an isolated nucleic acid which codes for a fusion polypeptide, including a FAC protein and a polypeptide which selectively binds a hematopoietic progenitor cell, as described above, is provided. According to a further aspect of the invention, the use of the foregoing conjugates and nucleic acids for delivery of a FAC molecule to a hematopoietic progenitor cell is provided.
The invention also encompasses methods for treating cells which have reduced FAC expression or which are at an increased risk of apoptosis due to exposure to high-dose chemotherapy. Thus, according to still another aspect of the invention, methods for inhibiting apoptosis in a hematopoietic progenitor cell are provided. The methods include contacting the hematopoietic progenitor cell with an amount of a composition which includes a FAC molecule effective to inhibit apoptosis. The methods preferably include contacting the hematopoietic progenitor cell with the foregoing conjugates of FAC proteins and nucleic acids. In preferred embodiments, the FAC molecule is conjugated to a polypeptide which selectively binds to the hematopoietic progenitor cell. As used herein, "conjugated" can mean that the molecules are linked together noncovalently or covalently. Thus, FAC molecules can be contained within a delivery vehicle (e.g., a liposome or a virus) which is targeted to the hematopoietic progenitor cell by a targeting moiety contained on the surface of the delivery vehicle, such as a polypeptide which selectively binds to the hematopoietic progenitor cell. According to yet another aspect of the invention, methods for treating a condition characterized by apoptosis of hematopoietic progenitor cells in a subject exposed to high-dose chemotherapy for non-myeloid malignancies is provided. The methods comprise administering to the subject an amount of the foregoing compositions which include a FAC molecule effective to inhibit apoptosis of the hematopoietic progenitor cells in the subject. The use of such compositions in the preparation of a medicament is also provided.
Brief Description of the Figures
Fig. 1 is a schematic illustration of His-ILFAC fusion proteins.
Fig. 2A depicts the detection of FAC fusion proteins in cell lysates and the purification of such proteins. Fig. 2B depicts an immunoblot of the His-ILFAC fusion protein.
Fig. 3 shows that complementation of FA group cells with FAC molecules increases resistance of the cells to treatment with MMC.
Fig. 4 shows the intracellular turnover of His-ILFAC fusion protein.
Brief Description of the Sequences
SEQ ID NO:l is the nucleotide sequence of the Fanconi anemia complementation group C nucleic acid.
SEQ ID NO:2 is the amino acid sequence of the Fanconi anemia complementation group C protein.
SEQ ID NO:3 is the nucleotide sequence of the gibbon IL3 nucleic acid. SEQ ID NO:4 is the amino acid sequence of the gibbon IL3 protein. SEQ ID NO: 5 is the nucleotide sequence of the anti CD33 antibody heavy chain nucleic acid.
SEQ ID NO: 6 is the amino acid sequence of the anti CD33 heavy chain antibody protein. SEQ ID NO: 7 is the nucleotide sequence of the anti CD33 antibody light chain nucleic acid. SEQ ID NO: 8 is the amino acid sequence of the anti CD33 light chain antibody protein.
SEQ ID NO: 9 is the nucleotide sequence of the forward primer for PCR amplification of gibbon IL3 minus the signal sequence.
SEQ ID NO: 10 is the nucleotide sequence of the reverse primer for PCR amplification of gibbon IL3. SEQ ID NO: 11 is the nucleotide sequence of the forward primer for PCR amplification of gibbon IL3 including the signal sequence. Detailed Description of the Invention
The present invention in one aspect involves the discovery that the Fanconi anemia complementation group C (FAC) protein is useful in modulating apoptosis in hematopoetic progenitor cells. The sequence of the human FAC cDNA is presented as SEQ ID NO:l, and the predicted amino acid sequence of this gene's protein product is presented as SEQ ID NO:2. The present invention in another aspect involves conjugates comprising a FAC molecule and a molecule which selectively binds to a hematopoetic progenitor cell for delivery of the FAC molecule to the hematopoetic progenitor cell.
The invention thus involves in one aspect FAC polypeptides, nucleic acids encoding those polypeptides, functional modifications and variants of the foregoing, useful fragments of the foregoing, as well as therapeutics relating thereto.
According to one aspect of the invention, the invention is directed to conjugates of a FAC molecule and a molecule which targets the FAC molecule to a hematopoietic progenitor cell and related methods for forming such conjugates. As used herein, "conjugate" means two entities bound to one another by any physiochemical means, including hydrophobic interaction between an antigen and the non-specific hydrophobic portions of an antibody molecule, antibody-antigen specific binding and covalent coupling. The nature of the preferred bonding will depend, among other things, upon the mode of administration and the pharmaceutical carriers used to deliver the conjugate to the target cell. For example, some bonds are not as well suited as others to withstand certain environments such as the stomach, but can be protected therefrom by delivery systems which bypass the stomach. It, of course, is important that the bond between the FAC molecule and a molecule which targets the FAC molecule to a hematopoietic progenitor cell be of such a nature that it does not destroy the ability of the molecule which targets the FAC molecule to a hematopoietic progenitor cell to bind to the hematopoietic progenitor cell. Such bonds are well known to those of ordinary skill in the art; examples are provided in greater detail below. The conjugate preferably is formed as a fusion protein.
A molecule which targets the FAC molecule to a hematopoietic progenitor cell (i.e. a targeting moiety or targeting molecule) means any entity that can selectively bind or be selectively bound by a cell surface molecule on a hematopoietic progenitor cell. Targeting moieties include those which recognize or are recognized by cell surface epitopes, cell surface receptors and/or channels. Targeting molecules which recognize cell surface epitopes include immunoglobulin and effective fragments thereof. Effective fragments of immunoglobulin include Fab, F(ab)'2 and Fv fragments thereof. Single chain antibodies, humanized antibodies, bifunctional antibodies, chimeric antibodies and other such entities also may be used according to the invention. Cloned receptors that recognize cell surface molecules also may be used. Likewise, ligands of cell surface molecules are useful according to the invention. The targeting moiety may be isolated or attached to the surface of a delivery vehicle, such as an encapsulating particle that has the ability to target the delivery of the contents of the particle to a desired location and, simultaneously, encapsulate a FAC molecule for delivery to the target. Such "particles"include viruses, bacteria, liposomes and the like. Methods for the encapsulation of compounds in such particles are well known in the art. For example, a virus particle may be formed around a FAC molecule by incubation of the FAC molecule with sufficient viral coat proteins. It is well established that the viral coat will self-assemble around the viral genome; this process may be utilized in preparation of the compounds of the invention. Similarly, liposomes spontaneously form around the constituents of the solution with which the precursor lipids are combined. Preferably the binding of the targeting molecule and the cell surface molecule results in active transport of the FAC-targeting molecule complex into the hematopoietic progenitor cell. Preferred examples include IL-3, c-kit, anti-CD33 antibodies and other molecules which selectively bind to the receptors for the foregoing proteins on the surface of a hematopoietic progenitor cell and are transported into the cell by endocytosis. As will be recognized by those of ordinary skill in the art, other molecules which bind to hematopoietic progenitor cells and are transported therein can be isolated by preparing antibodies to such cells. The hematopoietic progenitor cells of interest can be isolated by immunoaffinity procedures using e.g. anti-CD33 antibodies and then used (as whole cells or as cell membrane fragments on which membrane proteins are exposed) to immunize animals for preparation of antibodies by procedures well known to those of skill in the art.
Antibodies, cell surface receptor ligands or portions thereof specific for hematopoietic progenitor cells and capable of being transported into such cells once bound can be identified and isolated using well established techniques. Likewise, random generated molecularly diverse libraries can be screened and molecules that are bound to and transported by hematopoietic progenitor cells can be isolated using conventional techniques. For example, targeting molecules can be prepared by selecting a peptide ligand from a peptide library. It is not intended that the invention be limited by the selection of any particular targeting molecule. The invention thus involves agents such as polypeptides which bind to hematopoietic progenitor cells for targeting of FAC molecules. In particular, the agents can be antibodies or fragments of antibodies having the ability to selectively bind to hematopoietic progenitor cells. Antibodies include polyclonal and monoclonal antibodies, prepared according to conventional methodology. Where antibodies which bind a cell surface molecule of an hematopoietic progenitor cell are desired, a specific cell surface molecule can be selected and used as the immunogen for preparation of the antibody. Alternatively, the hematopoietic progenitor cell itself is used as the immunogen for preparation of the antibody.
Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W.R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology. 7th Ed., Blackwell Scientific Publications, Oxford). The pFc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope- binding ability in isolation.
Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDR3). The CDRs, and in particular the CDR3 regions, and more particularly the heavy chain CDR3, are largely responsible for antibody specificity.
It is now well-established in the art that the non-CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. Thus, for example, PCT International Publication Number WO 92/04381 teaches the production and use of humanized murine RSV antibodies in which at least a portion of the murine FR regions have been replaced by FR regions of human origin. Such antibodies, including fragments of intact antibodies with antigen-binding ability, are often referred to as "chimeric" antibodies.
Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab')2, Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non- human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences. The present invention also includes so-called single chain antibodies.
The invention involves polypeptides of numerous size and type that bind specifically to hematopoietic progenitor cells, which can be conjugated to FAC molecules for the delivery of FAC molecules to hematopoietic progenitor cells. These polypeptides may be derived also from sources other than antibody technology. For example, such polypeptide binding agents can be provided by degenerate peptide libraries which can be readily prepared in solution, in immobilized form, as bacterial flagella peptide display libraries or as phage display libraries. Combinatorial libraries also can be synthesized of peptides containing one or more amino acids. Libraries further can be synthesized of peptides and non-peptide synthetic moieties. Phage display can be particularly effective in identifying binding peptides useful according to the invention. Briefly, one prepares a phage library (using e.g. ml3, fd, or lambda phage), displaying inserts from 4 to about 80 amino acid residues using conventional procedures. The inserts may represent, for example, a completely degenerate or biased array. One then can select phage-bearing inserts which bind to the hematopoietic progenitor cells or a cell surface molecule thereof. These phages then are subjected to several cycles of reselection to identify the peptide ligand-expressing phages that have the most useful binding characteristics. Typically, phages that exhibit the best binding characteristics (e.g., highest affinity) are further characterized by nucleic acid analysis to identify the particular amino acid sequences of the peptides expressed on the phage surface and the optimum length of the expressed peptide to achieve optimum binding to the target mammalian cell. Alternatively, such peptide ligands can be selected from combinatorial libraries of peptides containing one or more amino acids. Such libraries can further be synthesized which contain non-peptide synthetic moieties which are less subject to enzymatic degradation compared to their naturally-occurring counterparts. The minimal linear portion of the sequence that binds to the hematopoietic progenitor cells or cell surface molecule can be determined. One can repeat the procedure using a biased library containing inserts containing part or all of the minimal linear portion plus one or more additional degenerate residues upstream or downstream thereof. Yeast two-hybrid screening methods also may be used to identify polypeptides that bind to cell surface molecules of an hematopoietic progenitor cell. These novel ligands can be conjugated to the FAC molecule to deliver the molecule to a hematopoietic progenitor cell.
The conjugates of the invention include one or more FAC molecules. Thus, the invention also provides isolated polypeptides, which include fusion proteins which include the polypeptide of SEQ ID NO:2 and fragments thereof. Such polypeptides are useful for inhibiting apoptosis, for example, alone, conjugated, or as fusion proteins for FAC replacement therapy, for treatment of high dose chemotherapy, or to generate antibodies, as components of an immunoassay. The invention embraces variants of the FAC polypeptides described above. As used herein, a "variant" of a FAC polypeptide is a polypeptide which contains one or more modifications to the primary amino acid sequence of a FAC polypeptide. Modifications which create a FAC variant can be made to a FAC polypeptide 1) to reduce or eliminate an activity of a FAC polypeptide, such as protease susceptibility; 2) to enhance a property of a FAC polypeptide, such as protein stability in an expression system or the stability of protein-protein binding; or 3) to provide a novel activity or property to a FAC polypeptide, such as addition of a detectable moiety. Modifications to a FAC polypeptide are typically made to the nucleic acid which encodes the FAC polypeptide, and can include deletions, point mutations, truncations, amino acid substitutions and additions of amino acids or non-amino acid moieties. Alternatively, modifications can be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as biotin, addition of a fatty acid, and the like. Modifications also embrace fusion proteins comprising all or part of the FAC amino acid sequence, provided that the part of the FAC amino acid sequence is capable of inhibiting apoptosis.
Variants can include FAC polypeptides which are modified specifically to alter a feature of the polypeptide unrelated to its physiological activity. For example, cysteine residues can be substituted or deleted to prevent unwanted disulfide linkages. Similarly, certain amino acids can be changed to enhance expression of a FAC polypeptide by eliminating proteo lysis by proteases in an expression system (e.g., dibasic amino acid residues in yeast expression systems in which KEX2 protease activity is present). Mutations of a nucleic acid which encode a FAC polypeptide preferably preserve the amino acid reading frame of the coding sequence, and preferably do not create regions in the nucleic acid which are likely to hybridize to form secondary structures, such a hairpins or loops, which can be deleterious to expression of the variant polypeptide.
Mutations can be made by selecting an amino acid substitution, or by random mutagenesis of a selected site in a nucleic acid which encodes the polypeptide. Variant polypeptides are then expressed and tested for one or more activities to determine which mutation provides a variant polypeptide with the desired properties. Further mutations can be made to variants (or to non- variant FAC polypeptides) which are silent as to the amino acid sequence of the polypeptide, but which provide preferred codons for translation in a particular host. The preferred codons for translation of a nucleic acid in, e.g., E. coli, are well known to those of ordinary skill in the art. Still other mutations can be made to the noncoding sequences of a FAC gene or cDNA clone to enhance expression of the polypeptide.
The skilled artisan will realize that conservative amino acid substitutions may be made in FAC polypeptides to provide functionally equivalent variants of the foregoing polypeptides, i.e, the variants retain the functional capabilities of the FAC polypeptides. As used herein, a
"conservative amino acid substitution" refers to an amino acid substitution which does not alter the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequence known to one of ordinary skill in the art such as are found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. Exemplary functionally equivalent variants of the FAC polypeptides include conservative amino acid substitutions of SEQ ID NO:2. Conservative substitutions of amino acids include substitutions made amongst amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D. Thus functionally equivalent variants and fragments of FAC polypeptides, i.e., variants or fragments of FAC polypeptides which retain the function of the natural FAC polypeptides, are contemplated by the invention. Thus, for example, the invention does not contemplate using the natural FAC mutant, R548X, which has a small deletion and the C terminus has no complementing activity. Conservative amino-acid substitutions in the amino acid sequence of FAC polypeptides to produce functionally equivalent variants of FAC polypeptides typically are made by alteration of a nucleic acid encoding FAC polypeptides (SEQ ID NO:l). Such substitutions can be made by a variety of methods known to one of ordinary skill in the art. For example, amino acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis according to the method of Kunkel (Kunkel, Proc. Nat. Acad. Sci. U.S.A. 82: 488- 492, 1985), or by chemical synthesis of a gene encoding a FAC polypeptide. Functionally equivalent fragments of FAC, also referred to herein as functional fragments, can be prepared by methods known to one of ordinary skill in the art, such as proteolytic cleavage and preparation of FAC nucleic acid fragments which encode fragments of FAC protein. The activity of functionally equivalent variants or fragments of FAC polypeptides can be tested by cloning the gene encoding the altered FAC polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the altered FAC polypeptide, and testing for a functional capability of the FAC polypeptides as disclosed herein.
The FAC molecule component of the conjugates of the invention can also be a FAC nucleic acid. The invention is not limited with respect to homologs and alleles of the FAC nucleic acids, which can be identified by conventional techniques. Thus, an aspect of the invention is those nucleic acid sequences which code for FAC polypeptides and which hybridize to a nucleic acid molecule consisting of the coding region of SEQ ID NO:l, under stringent conditions. The term "stringent conditions" as used herein refers to parameters with which the art is familiar. Nucleic acid hybridization parameters may be found in references which compile such methods, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley & Sons, Inc., New York. More specifically, stringent conditions, as used herein, refers, for example, to hybridization at 65°C in hybridization buffer (3.5 x SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% Bovine Serum Albumin, 2.5mM NaH2PO4(pH7), 0.5% SDS, 2mM EDTA). SSC is 0.15M sodium chloride/0.15M sodium citrate, ρH7; SDS is sodium dodecyl sulphate; and EDTA is ethylenediaminetetraacetic acid. After hybridization, the membrane upon which the DNA is transferred is washed at 2 x SSC at room temperature and then at 0.1 x SSC/0.1 x SDS at temperatures up to 65°C.
There are other conditions, reagents, and so forth which can used, which result in a similar degree of stringency. The skilled artisan will be familiar with such conditions, and thus they are not given here. It will be understood, however, that the skilled artisan will be able to manipulate the conditions in a manner to permit the clear identification of homologs and alleles of FAC nucleic acids of the invention. The skilled artisan also is familiar with the methodology for screening cells and libraries for expression of such molecules which then are routinely isolated, followed by isolation of the pertinent nucleic acid molecule and sequencing. In general homologs and alleles typically will share at least 40% nucleotide identity and/or at least 50% amino acid identity to SEQ ID NO:l and SEQ ID NO:2, respectively, in some instances will share at least 50% nucleotide identity and/or at least 65% amino acid identity and in still other instances will share at least 60% nucleotide identity and/or at least 75% amino acid identity. Watson-Crick complements of the foregoing nucleic acids also are embraced by the invention.
In screening for FAC nucleic acid homologs, a Southern blot may be performed using the foregoing conditions, together with a radioactive probe. After washing the membrane to which the DNA is finally transferred, the membrane can be placed against X-ray film or a phosphoimager plate to detect the radioactive signal. The invention also includes degenerate nucleic acids which include alternative codons to those present in the native materials. For example, serine residues are encoded by the codons TCA, AGT, TCC, TCG, TCT and AGC. Each of the six codons is equivalent for the purposes of encoding a serine residue. Thus, it will be apparent to one of ordinary skill in the art that any of the serine-encoding nucleotide triplets may be employed to direct the protein synthesis apparatus, in vitro or in vivo, to incorporate a serine residue into an elongating FAC polypeptide. Similarly, nucleotide sequence triplets which encode other amino acid residues include, but are not limited to: CCA, CCC, CCG and CCT (proline codons); CGA, CGC, CGG, CGT, AGA and AGG (arginine codons); ACA, ACC, ACG and ACT (threonine codons); AAC and AAT (asparagine codons); and ATA, ATC and ATT (isoleucine codons). Other amino acid residues may be encoded similarly by multiple nucleotide sequences. Thus, the invention embraces degenerate nucleic acids that differ from the biologically isolated nucleic acids in codon sequence due to the degeneracy of the genetic code.
In one set of embodiments, the nucleic acids of the invention may be composed of "natural" deoxyribonucleotides, ribonucleotides, or any combination thereof. That is, the 5' end of one native nucleotide and the 3' end of another native nucleotide may be covalently linked, as in natural systems, via a phosphodiester internucleoside linkage. These nucleic acids may be prepared by art recognized methods which may be carried out manually or by an automated synthesizer. They also may be produced recombinantly by vectors.
In other embodiments, however, the nucleic acids of the invention also may include "modified" nucleic acids. That is, the oligonucleotides may be modified in a number of ways which do not prevent their transcription or translation but which enhance their stability or targeting or which otherwise enhance their therapeutic effectiveness.
The term "modified nucleic acids" as used herein describes a nucleic acid in which (1) at least two of its nucleotides are covalently linked via a synthetic internucleoside linkage (i.e., a linkage other than a phosphodiester linkage between the 5' end of one nucleotide and the 3' end of another nucleotide) and/or (2) a chemical group not normally associated with nucleic acids has been covalently attached to the nucleic acid. Preferred synthetic internucleoside linkages are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.
The term "modified nucleic acid" also encompasses nucleic acids, including oligonucleotides, with a covalently modified base and/or sugar. For example, modified nucleic acids include nucleic acids having backbone sugars which are covalently attached to low molecular weight organic groups other than a hydroxyl group at the 3' position and other than a phosphate group at the 5' position. Thus modified nucleic acids may include a 2'-O-alkylated ribose group. In addition, modified nucleic acids may include sugars such as arabinose instead of ribose. The present invention, thus, contemplates pharmaceutical preparations containing modified nucleic acids together with pharmaceutically acceptable carriers.
As used herein, a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell. Vectors are typically composed of DNA although RNA vectors are also available. Vectors include, but are not limited to, plasmids, phagemids and virus genomes. A cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell. In the case of plasmids, replication of the desired sequence may occur many times as the plasmid increases in copy number within the host bacterium or just a single time per host before the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase. An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector. Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., β-galactosidase or alkaline phosphatase), and genes which visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques (e.g., green fluorescent protein). Preferred vectors are those capable of autonomous replication and expression of the structural gene products present in the DNA segments to which they are operably joined.
As used herein, a coding sequence and regulatory sequences are said to be "operably" joined when they are covalently linked in such a way as to place the expression or transcription of the coding sequence under the influence or control of the regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
The precise nature of the regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribed and 5' non-translated sequences involved with the initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like. Especially, such 5' non- transcribed regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control of the operably joined gene. Regulatory sequences may also include enhancer sequences or upstream activator sequences as desired. The vectors of the invention may optionally include 5' leader or signal sequences. The choice and design of an appropriate vector is within the ability and discretion of one of ordinary skill in the art.
Expression vectors containing all the necessary elements for expression are commercially available and known to those skilled in the art. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, 1989. Cells are genetically engineered by the introduction into the cells of heterologous DNA (RNA) encoding FAC polypeptide or fragment or variant thereof. That heterologous DNA (RNA) is placed under operable control of transcriptional elements to permit the expression of the heterologous DNA in the host cell.
Preferred systems for mRNA expression in mammalian cells are those such as pRc/CMV (available from Invitrogen, Carlsbad, CA) that contain a selectable marker such as a gene that confers G418 resistance (which facilitates the selection of stably transfected cell lines) and the human cytomegalovirus (CMV) enhancer-promoter sequences. Additionally, suitable for expression in primate or canine cell lines is the pCEP4 vector (Invitrogen), which contains an Epstein Barr virus (EBV) origin of replication, facilitating the maintenance of plasmid as a multicopy extrachromosomal element. Another expression vector is the pEF-BOS plasmid containing the promoter of polypeptide Elongation Factor lα, which stimulates efficiently transcription in vitro. The plasmid is described by Mishizuma and Nagata (Nuc. Acids Res. 18:5322, 1990), and its use in transfection experiments is disclosed by, for example, Demoulin (Mol. Cell. Biol. 16:4710-4716, 1996). Still another preferred expression vector is an adeno virus, described by Stratford-Perricaudet, which is defective for El and E3 proteins (J. Clin. Invest. 90:626-630, 1992).
Conjugation between a FAC molecule and a targeting moiety according to the invention need not be direct attachment. The functional components of the conjugate (i.e., the FAC molecule and a targeting moiety) may be provided with functionalized groups to facilitate their attachment and/or linker groups may be interposed between the functional components of the conjugate to facilitate their attachment. In addition, the functional components of the conjugate of the present invention may be synthesized in a single process, whereby the components could be regarded as one and the same entity. For example, a protein targeting moiety specific for an hematopoietic progenitor cell could be synthesized together with a FAC polypeptide as a fusion protein. These and other modifications are intended to be embraced by the present invention.
Covalent linking of a FAC polypeptide to an hematopoietic progenitor cell targeting molecule is intended to include linkage by peptide bonds in a single polypeptide chain to create a fusion protein. Established methods (e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY 1989) can be used to engineer a DNA encoding a fusion protein comprised of the FAC polypeptide to an hematopoietic progenitor cell targeting molecule. This DNA can be placed in an expression vector and introduced into bacterial or eukaryotic cells by established methods. The fusion protein can then be purified from the cells or from the culture medium by established methods. A fusion polypeptide may be useful for purification of the peptides of the invention. The fusion domain may, for example, include a poly-His tail which allows for purification on Ni+ columns or the maltose binding protein of the commercially available vector pMAL (New England BioLabs, Beverly, MA). Specific examples of covalent bonds include those wherein bifunctional cross-linker molecules are used. The cross-linker molecules may be homobifunctional or heterobifunctional, depending upon the nature of the molecules to be conjugated. Homobifunctional cross-linkers have two identical reactive groups. Heterobifunctional cross-linkers are defined as having two different reactive groups that allow for sequential conjugation reaction. Various types of commercially available cross-linkers are reactive with one or more of the following groups; primary amines, secondary amines, sulfhydryls, carboxyls, carbonyls and carbohydrates. Examples of amine-specific cross-linkers are bis(sulfosuccinimidyl) suberate, bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, disuccinimidyl suberate, disuccinimidyl tartarate, dimethyl adipimate»2 HCl, dimethyl pimelimidate»2 HCl, dimethyl suberimidate«2 HCl, and ethylene glycolbis-[succinimidyl-[succinate]j. Cross-linkers reactive with sulfhydryl groups include bismaleimidohexane, l,4-di-[3'-(2'-pyridyldithio)-propionamido)]butane, 1 -[p-azidosalicylamido]-4-[iodoacetamido]butane, and N-[4-(p-azidosalicylamido)butyl]- 3'-[2'-pyridyldithio]propionamide. Cross-linkers preferentially reactive with carbohydrates include azidobenzoyl hydrazide. Cross-linkers preferentially reactive with carboxyl groups include 4-[p-azidosalicylamido]butylamine. Heterobifunctional cross-linkers that react with amines and sulfhydryls include N-succinimidyl-3-[2-pyridyldithio]propionate, succinimidyl [4- iodoacetyljaminobenzoate, succinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate, m-maleimidobenzoyl-N-hydroxysuccinimide ester, sulfosuccinimidyl
6-[3-[2-pyridyldithio]propionamido]hexanoate, and sulfosuccinimidyl 4-[N-maleimidomethyl] cyclohexane-1-carboxylate. Heterobifunctional cross-linkers that react with carboxyl and amine groups include l-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride. Heterobifunctional cross-linkers that react with carbohydrates and sulfhydryls include 4-[N-maleimidomethyl]-cyclohexane- 1 -carboxylhydrazide»HCl, 4-(4-N-maleimidophenyl)-butyric acid hydrazide»HCl, and 3-[2-pyridyldithio]propionyl hydrazide. The cross-linkers may also be nonselective. Examples of nonselective cross-linkers are bis-[β-(4-azidosalicylamido)ethyl]disulfide and glutaraldehyde. Amine or thiol groups may be added at any nucleotide of a synthetic nucleic acid so as to provide a point of attachment for a bifunctional cross-linker molecule. The nucleic acid may be synthesized incorporating conjugation-competent reagents such as Uni-Link AminoModifier, 3'-DMT-C6-Amine-ON CPG, AminoModifier II, N-TFA-C6-AminoModifier, C6-ThiolModifier, C6-Disulfide Phosphoramidite and C6-Disulfide CPG (Clontech, Palo Alto, CA). Covalent linkages may be noncleavable in physiological environments or cleavable in physiological environments, such as linkers containing disulfide bonds. Such molecules may resist degradation and/or may be subject to different intracellular transport mechanisms. One of ordinary skill in the art will be able to ascertain without undue experimentation the preferred bond for linking the targeting moiety and FAC molecule, based on the chemical properties of the molecules being linked and the preferred characteristics of the bond.
Noncovalent methods of conjugation may also be used to join the targeting moiety and the FAC molecule. Noncovalent conjugation includes hydrophobic interactions, ionic interactions of positively and negatively charged molecules, biotin-avidin and biotin-streptavidin complexation, intercalation of a FAC nucleic acid, interaction with specific nucleotide sequences and other affinity interactions. For example, a FAC polypeptide can be bound by an antibody which also recognizes a hematopoietic progenitor cell. Similarly, biotinylated FAC nucleic acids can be complexed with avidin or streptavidin antibody conjugates. As another example, a FAC nucleic acid can ionically complex a polylysine-antibody or a histone-antibody conjugate.
In accordance with certain methods of the invention, the conjugates of the invention are contacted with a hematopoietic progenitor target cell under conditions to permit selective binding of the conjugate to the receptor on the surface of the hematopoietic progenitor cell and to allow the conjugate to enter the cell. In cases in which the targeting molecule of the conjugate is a ligand for a receptor on the surface of the cell, conditions which permit the binding of the ligand to its receptor are the physiological conditions (e.g., the particular pH, ionic strength, viscosity) at which the ligands and receptors are known to bind to one another in vivo and the conditions at which the ligands and receptors are known to bind to one another in vitro, such as in receptor assays for determining the presence of a ligand in, for example, a biological fluid. Such conditions are known to those of ordinary skill in the art of receptor-mediated processes, such as receptor-based binding assays and receptor-mediated delivery of therapeutic agents to preselected tissues in situ. In cases in which a non-natural ligand is used as a targeting moiety for the FAC molecule conjugate, the conditions which permit binding to the receptor on the hematopoietic progenitor cell can be determined by conducting in vitro binding assays of the conjugate and the hematopoietic progenitor cell in accordance with standard procedures. Such in vitro assays are known to be predictive of the in vivo conditions for binding.
As used herein, "contacting", in reference to the FAC conjugate and the target cell, refers to bringing the FAC conjugate into sufficiently close proximity to the target cell to permit the "receptor" on the target cell to selectively bind to the targeting moiety of the FAC conjugate. The targeting moiety can bind with the cell by means of, inter alia, antibody/antigen binding and receptor/ligand binding. Such conditions are well known to those of ordinary skill in the art. See also, e.g., U.S. patent No. 5,108,921, issued to Low et al. which reports the conditions for receptor-mediated delivery of "exogenous molecules" such as peptides, proteins and nucleic acids to animal cells and U.S. patent No. 5,166,320, issued to Wu et al., which reports the conditions for the receptor mediated delivery of a ligand-gene conjugate to a mammalian cell. For a further discussion of the conditions and mechanisms by which receptor mediated delivery can be used to deliver an exogenous molecule into a target cell, and in particular, into a mammalian cell, see, e.g., S. Michael, et al., J. Biol. Chem. 268(10):6866 (1993), "Binding-incompetent Adenovirus Facilitates Molecular Conjugate-mediated Gene Transfer by the Receptor-mediated Endocytosis Pathway"; M. Barry, et al., Nature Medicine 2(3):299 (1996), "Toward cell-targeting gene therapy vectors: Selection of cell-binding peptides from 20random peptide-presenting phage libraries"; S. I. Michael, Gene Ther. 2:660 (1995), "Addition of a short peptide ligand to the adenovirus fiber protein".
The methods of the invention do not require contacting a FAC conjugate. A non- conjugated FAC molecule can be contacted with a target cell, although it may not enter the cell by a receptor-meditaed endocytosis mechanism. Thus, contacting a FAC molecule alone with a target cell can involve methods such as microinjection, electroporation, transfection, and the like.
In general, internalization of a FAC molecule in a mammalian cell is detected by measuring the functional or immunological activity of the expressed gene in the targeted cell. Alternatively, direct RNA or protein analysis for detecting specific transcription or translation products can be performed in accordance with standard practice. Exemplary detection techniques to measure gene expression include one or more of the following techniques, alone or in combination: northern or western blotting, in situ hybridization, reverse transcription, PCR amplification, immunostaining, RIA and ELISA.
In accordance with certain methods, a hematopoietic progenitor cell is contacted with a FAC molecule to inhibit apoptosis of the cell. By contacting the cell in this manner, FAC is delivered to the cell for the purpose of reducing apoptosis of the cell. Such inhibition of apoptosis is useful for ameliorating the effects of the Fanconi anemia disease, and for reducing the detrimental effects of high dose chemotherapy on myelopoiesis.
When contacted with the cell, the FAC molecule optionally is part of a conjugate. Where the FAC molecule is part of a conjugate, the targeting moiety is conjugated to the FAC molecule, by covalent or noncovalent linkage, in a manner that does not interfere with the binding of the targeting moiety to the cell. For example, the targeting moiety can be associated with the FAC molecule so as to encompass the FAC molecule. Examples of such associated targeting moieties include liposomes and viruses. The FAC molecule need not be enclosed within the associated targeting moiety, but may merely be associated with the targeting moiety by ionic or hydrogen bonding and found in nucleic acid hybridization.
The FAC conjugate can be contacted with a hematopoietic progenitor cell in vitro, for example, for ex vivo FAC replacement therapy or in vitro production of a recombinant protein in cell culture, or in vivo for in vivo FAC replacement therapy or in vivo production of a nucleic acid transcription or translation product.
A procedure for performing ex vivo gene therapy is outlined in U.S. Patent 5,399,346 and in exhibits submitted in the file history of that patent, all of which are publicly available documents. In general, ex vivo immunotherapy involves the introduction in vitro of a functional copy of a gene into a cell(s) of a subject which contains a defective copy of the gene, and returning the genetically engineered cell(s) to the subject to alleviated the condition occasioned by the defective gene. Thus, the functional copy of the FAC gene is under the operable control of regulatory elements which permit expression of the gene in the genetically engineered hematopoietic progenitor cell(s). In in vivo immunotherapy, the target cells are not removed from the patient. Rather, the nucleic acid molecule encoding, e.g. a FAC polypeptide, is introduced into the cells of the mammalian recipient in situ, i.e., within the recipient. In general, the methods disclosed herein can be practiced by using appropriately targeted FAC conjugates in the procedures currently used for administering gene therapy vectors (or cells containing these vectors) to the subjects ex vivo or in vivo. Such procedures are known to those of skill in the art of human gene therapy.
In ex vivo gene therapy, hematopoietic progenitor cells which are to be treated are removed from a subject and the cells are contacted with a FAC conjugate in vitro. Optionally, the treated cells are expanded in culture before being reimplanted into the mammalian recipient. The target cells subsequently can be introduced into the mammal (e.g., into the portal vein or into the spleen) if desired. In cases in which the FAC conjugate includes a FAC nucleic acid, expression of the nucleic acid molecule encoding a FAC polypeptide is accomplished by allowing the cell to live or propagate in vitro, in vivo, or in vitro and in vivo, sequentially. The FAC conjugates as described herein have a number of uses, some of which are described elsewhere herein. First, the invention includes the use of the conjugates described herein for the delivery of a FAC polypeptide or nucleic acid molecule to a hematopoietic progenitor cell. Second, the invention includes methods for inhibiting apoptosis in a hematopoietic progenitor cell by contacting a hematopoietic progenitor cell with a FAC molecule. The FAC molecule can, but need not, be part of a conjugate with a targeting molecule such as a polypeptide which selectively binds to the hematopoietic progenitor cell. Third, the invention includes methods for treating a condition characterized by apoptosis of hematopoietic progenitor cells in a subject exposed to high dose chemotherapy for treatment of a non-myeloid malignancy, by administering an amount of a FAC molecule to the subject effective to inhibit the apoptosis.
As is shown in the Examples which follow, FAC protein, when introduced into hematopoietic progenitor cells, can inhibit the apoptosis such cells. Although the exact mechanism of the inhibition of apoptosis is unclear at this time, what is clear is that the addition of functional FAC (e.g. not FAC mutated as in Fanconi anemia) to the cells prevents the toxicity of mitomycin C, which kills the cells by apoptosis.
This property also permits the treatment of hematopoietic progenitor cells of a subject exposed to chemotherapy. Administration of FAC prior to chemotherapy increases the amount of surviving hematopoietic progenitor cells. By supplementing the subject's hematopoietic progenitor cells with FAC, the subject does not require extensive bone marrow transplants to restore hematopoiesis following chemotherapy.
It will also be recognized that the invention embraces the use of the FAC conjugates in vitro to increase the level of FAC in host cells and cell lines, for instance, for determining optimal amounts of FAC molecules to administer to inhibit or even prevent apoptosis or to generally ameliorate the defect in Fanconi anemia cells. Especially useful are mammalian cells such as mouse, hamster, pig, goat, primate, etc. They may be of a wide variety of tissue types, and include primary cells and cell lines. Specific examples include hematopoietic progenitor cells, bone marrow stem cells and embryonic stem cells. The expression vectors require that the pertinent sequence, i.e., those nucleic acids described supra, be operably linked to a promoter.
When administered, the therapeutic compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, supplementary agents such as cytokines and optionally other therapeutic agents.
The pharmaceutical compositions should be sterile and contain a therapeutically effective amount of the FAC molecule in a unit of weight or volume suitable for administration to a patient. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. The term "physiologically acceptable" refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics of the carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
The therapeutics of the invention can be administered by any conventional route, including injection or by gradual infusion over time. The administration may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, subcutaneous, or transdermal.
Compositions may be conveniently presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the conjugate into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the conjugate into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. The term "carrier" as used herein, and described more fully below, means one or more solid or liquid filler, dilutants or encapsulating substances which are suitable for administration to a human or other mammal. The "carrier" may be an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate administration.
Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
The preparations of the invention are administered in effective amounts. An effective amount is that amount of a pharmaceutical preparation that alone, or together with further doses, produces the desired response. In the case of inhibiting apoptosis, the desired response is a reduction in the rate or amount of hematopoitic progenitor cell apoptosis. In the case of treating FAC deficiency, the desired response is increasing the amount of active FAC protein present in a hematopoietic progenitor cell such that the survival of the hematopoietic progenitor cell is increased. This may involve only boosting the survival of hematopoietic progenitor cells temporarily, although more preferably, it involves enhancing survival of hematopoietic progenitor cells permanently. This can be monitored by routine methods. It is believed that doses ranging from 1 nanogram/kilogram to 100 milligrams/kilogram, depending upon the mode of administration, will be effective. The preferred range is believed to be between about 500 nanograms and 500 micrograms/kilogram, and most preferably between 1 microgram and 100 micrograms/kilogram. The absolute amount will depend upon a variety of factors, including the conjugate selected, the modulation desired, whether the administration is in a single or multiple doses, and individual patient parameters including age, physical condition, size and weight. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation.
The conjugates of the invention may be administered per se (neat) or in the form of a pharmaceutically acceptable salt. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulfonic, tartaric, citric, methane sulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzene sulfonic. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts of the carboxylic acid group.
Suitable buffering agents include: acetic acid and a salt (1-2% W/V); citric acid and a salt (1-3% W/N); boric acid and a salt (0.5-2.5% W/V); sodium bicarbonate (0.5-1.0% W/V); and phosphoric acid and a salt (0.8-2% W/V). Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V) and thimerosal (0.004-0.02% W/V).
The components of the pharmaceutical compositions are capable of being commingled with the conjugates of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficiency.
Other supplementary agents, such as molecules involved in signal transduction which enhance survival of hematopoietic progenitor cells, may be delivered in conjunction with the conjugates of the invention. The molecules contemplated are those that may enhance the beneficial effects that result from administering the FAC molecule according to the invention. Upon binding of molecules involved in signal transduction such as cytokines to their cognate receptors on the cell surface, specific polypeptides, called JAKs and STATs, are involved in transmission of the signal to the nucleus. The preferred signal transduction molecules are STATs (especially STAT 1, 2 and 3) and JAKs that promote myeloid cell growth. The selection of the particular signalling molecule will depend upon the particular modulation of the hematopoietic system that is desired. The activity of molecules involved in signal transduction, e.g. cytokines, on particular cell types is known to those of ordinary skill in the art.
The precise amounts of the foregoing signalling molecules used in the invention will depend upon a variety of factors, including the conjugate selected, the dose and dose-timing selected, the mode of administration and the characteristics of the subject. The precise amounts selected can be determined without undue experimentation, particularly since a threshold amount will be any amount which will enhance the desired immune response. Thus, it is believed that nanogram to milligram amounts are useful, depending upon the mode of delivery, but that nanogram to microgram amounts are likely to be sufficient because physiological levels of these molecules are correspondingly low.
Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the conjugates of the invention, increasing convenience to the subject and the physician. Many types of release/delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as polylactic and polyglycolic acid, polyanhidrides and polycaprolactone; wax coatings, compressed tablets using conventional binders and excipients, and the like. Bioadhesive polymer systems to enhance delivery of a material to the intestinal epithelium are known and described in published PCT application WO 93/21906. Capsules for delivering agents to the intestinal epithelium also are described in published PCT application WO 93/19660.
Examples
Example 1 : Generation. Targeting and Activity of Interleukin-3-FAC.
Mature human or gibbon interleukin-3 (IL3) is a 133 amino acid glycoprotein that is produced primarily by activated T cells (Lindemann, et al., Cancer Invest. 609-623, 1993). Recombinant IL3 expressed in E. coli maintains its bioactivity despite the lack of carbohydrate residues. The high-affinity human IL3 receptor is a heterodimer of α and β subunits present at about 50-1,000 copies per cell and is essential for the survival of hematopoietic progenitor cells. Both murine and human receptors undergo internalization of the receptor-ligand complex, followed by digestion of IL3 within lysosomes and recycling of the receptors back to the cell surface. In this example I describe the construction, expression and partial purification of a chimeric protein including IL3 and FAC (His-ILFAC). Also demonstrated is receptor-specific uptake of the chimeric protein into B-lymphoblastoid cells and correction of the cross-linker hypersensitivity of FA-C cells.
A: Construction and Expression of His-ILFAC. To express His-ILFAC in bacteria, a tripartite protein was constructed consisting of an epitope of six histidine residues (His-TAG) at the amino terminus, gibbon IL3 (minus the signal peptide) at the center, and full-length human FAC (minus the initiation methiodine) at the carboxy terminus (schematically shown in Fig. 1). Based on the predicted tertiary structure of IL3, it was reasoned that placement of FAC fusion partner at the "free" carboxy-terminal end of the IL3 would not result in steric hinderance. The corresponding cDNA cloned in the vector pQE9 (Qiagen, Inc., Santa Clarita, CA) was used to transform E. coli strain Ml 5. As expected, a 75 kDa fusion protein was detected by a His epitope reactive antibody (mouse anti-RGS(H)4 antibody, Qiagen) in cell lysates after induction of the transformed cells with 2 mM IPTG for 5 hours (Fig. 2A). Immunoblotting confirmed that the 75 kDa protein is immunoreactive with polyclonal antibodies directed against FAC (Fig. 2B). His-tagged FAC (without interposed IL3) was also generated (Fig. 1) and expressed (Fig. 2A) using similar procedures. A third fusion protein (secreted ILFAC, Fig. 1) was created with the gibbon IL3 molecule (including a signal peptide) at the amino terminus and full-length human FAC (minus the initiation methiodine) at the carboxy terminus. The specific protocols follow.
Construction of the prokaryotic expression plasmid encoding His-ILFAC:
1. The polylinker region of the vector pQE9 (Qiagen) was modified to generate a vector of greater versatility and allow the in-frame cloning of additional restriction fragments. pQE9 was cut with BamHI and Hindlll, and the -30 bp BamHI/Hindlll fragment from pGEM3 (Promega) was inserted. The resultant vector is called pQE9Xl .
2. To construct pQE9-FAC without the IL3 ligand at the N-terminus, pQE9Xl was cut with BamHI/Xbal. The 1.65 kb insert encoding human FAC was purified by gel electrophoresis from another bacterial expression vector (pGEX2TK-FAC) generated by me and published previously ( Youssoufian et al. J. Clin. Invest. 97:957-962, 1996). The linearized pQE9Xl and the FAC insert were ligated to generate pQE9Xl-FAC.
3. The mature coding region of gibbon IL3 was obtained by PCR: BamHI Forward primer 5'-CGGGATCC GCT CCC ATG ACC CAG ACA A-3'
(SEQ ID NO.9) A P M T Q T
BamHI S I E L S L T Reverse primer: 5*-CGGGATCC AGA GAT CTC AAG GCT CAA AGT-3'
(SEQ ID NO: 10) Mutated Stop codon
Thirty cycles of PCR were performed using pMX-IL3 as template (vector obtained from Todd Golub, Dana-Farber Cancer Institute, Boston, MA who obtained it from the Genetics Institute). The PCR product was cut with BamHI and gel-purified according to standard procedures. 4. To clone the coding region of gibbon IL3 at the 5' end of FAC, the following steps were performed. The plasmid pQE9Xl-FAC was cut with BamHI and the ends were phosphatased. The BamHI-digested 0.4 kb IL3 PCR fragment from step 3 was inserted into the BamHI site of vector to generate the final desired product, pQE9Xl-ILFAC. This construct encodes a tripartite protein consisting of an epitope of six histidine (His) residues at the N-terminus, mature gibbon IL3 in the middle, and full-length human FAC (except the initiation codon) at the carboxy- terminus.
To express His-ILFAC in bacteria, a fusion protein was constructed consisting of an epitope of six histidine residues (His-TAG) at the amino terminus, gibbon IL3 (without the signal peptide), and full-length human FAC (minus the initiation methiodine) at the carboxy terminus.
The mammalian expression construct was prepared by a three-way ligation of a vector backbone, the gibbon IL3 and the human FAC sequence described above. The vector was pED6 (obtained from Genetics Institute), which was disgested with Sail and Xbal. The FAC portion was prepared as above. The IL3 portion was prepared by PCR as above, but with a different forward primer that preserves the natural signal peptide sequence of IL3
(SEQ ID NO:l 1): 5*-CGGTCGACCACCATGGGCTGCCTGCCCGTCCTG-3'.
Sail Initiation codon
The same reverse primer, SEQ ID NO: 10, was used. The PCR product was digested with
BamHI and Sail prior to ligation. The ligated plasmid was used to transform bacteria and clones were selected according to standard protocols. The mammalian ILFAC plasmid was used to transfect subconfluent COS cells by a standard DEAE transfection protocol, using about 1 μg plasmid per milliliter transfection medium (e.g., 5 μg plasmid per 10mm dish COS cells. After 5-7 days, the conditioned medium was harvested and clarified of debris. The clarified conditioned medium was used directly in bioassays at various concentrations.
Other FAC conjugates include those which include an anti-CD33 antibody (such as M195, see Co et al., J. Immunol.148(4), 1149-1154, 1992), or individual chains thereof, attached to a FAC molecule. One example of an anti-CD33-FAC molecule is prepared according to the method for preparing a mammalian fusion protein above. The nucleic acid encoding the heavy chain of the Ml 95 anti-CD33 antibody (SEQ ID NO: 5) is ligated with the pED6 vector and the human FAC cDNA (SEQ ID NO:3, except the initiation codon). The nucleic acid encoding the light chain of the M195 anti-CD33 antibody (SEQ ID NO:7) can also be used in place of the heavy chain. The fusion protein is expressed and used as above.
A chemically crosslinked conjugate of the Ml 95 anti-CD33 antibody and FAC can also be prepared. The nucleic acids encoding the heavy and light chains of Ml 95 are inserted in a bicistronic vector which is transfected into an appropriate cell line to obtain approximately stoichiometric amounts of heavy and light chains which assemble as an antibody molecule. The antibody molecule is purified by e.g. Protein A or Protein G affinity chromatography and conjugated to the FAC protein (SEQ ID NO:2) by a bifunctional crosslinker as identified supra. For example, conjugation also may be performed by derivatizing both FAC protein and the anti- CD33 antibody (or fragments thereof) with sulfosuccinimidyl 6-[3-(2- pyridyldithio)propionamide] hexanoate (sulfo-LC-SPDP, Pierce) for 18 hours at room temperature. Conjugates also may be prepared by derivatizing FAC protein and the anti-CD33 antibody with different crosslinking reagents that will subsequently form a covalent linkage. An example of this reaction is derivatization of the antibody with sulfosuccinimidyl 4-(N- maleimidomethyl) cyclo-hexane-1-carboxylate (Sulfo-SMCC, Pierce) and thiolation of the FAC protein with N-succinimidyl S-acetylthioacetate (SAT A). The derivatized components are purified free of crosslinker and combined at room temperature for one hour to allow crosslinking. Other crosslinking reagents comprising aldehyde, imide, cyano, halogen, carboxyl, activated carboxyl, anhydride and maleimide functional groups are known to persons of ordinary skill in the art and also may be used for conjugation of FAC protein and the anti-CD33 antibody. In the foregoing crosslinking reactions it is important to purify the derivatized compounds free of crosslinking reagent. It is important also to purify the final conjugate substantially free of unconjugated reactants. Purification may be achieved by affinity, gel filtration or ion exchange chromatography based on the properties of either FAC protein or the anti-CD33 antibody. A particularly preferred method is an initial affinity purification step using immobilized CD33 to retain Ml 95 antibody and M195-FAC conjugates, followed by gel filtration or ion exchange chromatography based on the mass, size or charge of the conjugate. The initial step of this purification scheme ensures that the conjugate will bind to CD33. Alternatively, immobilized anti-FAC antibodies can be used in the affinity purification step, followed by gel filtration or ion exchange chromatography based on the mass, size or charge of the conjugate.
B: Purification of Soluble His-ILFAC.
The purification protocol consisted of two successive column chromatography steps that yielded protein of greater than 80 percent homogeneity. Briefly, cells were transferred and induced as described above, and then were sonicated in a buffer containing 50 mM sodium phosphate (pH 8.0) and 300 mM NaCl. The crude protein extract was subjected to nickel- agarose column chromatography, which retains proteins having the His-TAG epitope. After elution with 50 mM imidazole and dialysis against PBS (Fig. 2A), the partially purified protein was subjected to size exclusion chromatography over Superdex-75. Fraction 1 from the void volume contained greater than 80 percent of His-ILFAC as assessed by immunoreactivity with anti-FAC antibody (Fig. 2B). The final yield of partially purified protein was approximately 1 milligram from 4 liters of induced bacterial culture. The specific protocol follows:
Expression and purification of His-ILFAC:
1. E. coli strain Ml 5 (Invitrogen) was transformed with pQE9Xl-ILFAC. 2. Transformants were selected for in LB containing Ampicillin (100 μg/ml) and
Kanamycin (25 g/ml).
3. Transformants were grown from a single colony in liquid culture to OD600=0.5.
4. Production of the fusion protein was induced with 1 mM IPTG for 3 hrs at 37 °C.
5. The cells were pelleted by centrifugation. 6. The pellet was washed with cold PBS.
7. The pellet was lysed as follows. The pellet was resuspended in SB-I/2.5% Sarkosyl/0.2 mM PMSF/10 mM β-ME, at 5 ml resuspension volume per gram wet weight of pellet. The resuspension was sonicated using the following conditions: Duty cycle 50%, Output control 5; four sonications of 30 sec. each (total 2 min), keeping the suspension on ice in between sonications. The sonicated suspension was centrifuged at 12,000 rpm for 15 minutes and the supernatant was recovered. A 1/10th volume of SB-I/20% TRITON X-10 was added.
8. Binding was performed as follows. 0.5 ml Ni-NTA agarose beads (Qiagen) were washed in SB I buffer ( SB-I: 50 mM Na-phosphate pH 7.8, 300 mM NaCl). The supernatant of step 7 was added to the washed beads and rotated at 4°C for 60 min.
9. The beads were washed using the following protocol: Spin beads at 1500 rpm x 2 min
SB I (25ml)* X 15 min
SB I (25 ml)/NaC 1 to 0.6M X 15 min
SBII (25 ml)** X 15 min
SB II (25 Ml)/20% glycerol X 15 min
PBS (25 ml) quick rinse
SB-II buffer is: 50 mM Na-phosphate pH 6.0, 300 mM NaCl
10. The fusion protein was batch eluted from the washed beads using 1 ml of the following solutions of imidazole in PBS:
Imidazole Final Cone. Outcome 10 mM No protein in eluate 25 mM*** >75% of eluted protein is full-length
His-ILFAC
50 mM <10% of eluted protein is full-length
His-ILFAC; remainder is low molecular weight species
(incompletely characterized)
100 mM No further elution of His-ILFAC
The protein eluted by 25 mM imidazole was dialyzed against PBS and used in bioassays. In some experiments, the fusion protein was further purified by size exclusion chromatography using Superdex-75 according to standard procedures. Example 2: Receptor-mediated Targeting in Human Lymphoblastoid Cells. To determine whether cells expressing IL3 receptors are capable of binding and internalizing His-ILFAC, the GM4510 FA-C lymphoblastoid cells homozygous for a splicing mutation in FAC (this mutation is predicted to yield a truncated protein product) were incubated with either 1 μg/ml His-ILFAC or His-FAC for 0-30 minutes at 37 °C, washed in cold PBS, lysed and analyzed by immunoblotting with anti-FAC antibody. Immunoreactive His-ILFAC was detected after two minutes of incubation and reached a maximum after 10 minutes. By contrast, very little if any cell-associated His-FAC was detected under the same conditions. At the 10 minute time point, ligand internalization occurred much more readily at 37 °C than 4°C (5-6 fold more). In addition, ligand internalization was inhibited (to -20% of control) by addition of recombinant human IL3 (10 μg/ml) to the medium and inhibited further (to -5% of control) by pre-treatment of intact cells with trypsin prior to incubation with His-ILFAC for 10 minutes. The lack of complete inhibition may relate to inadequate receptor occupancy by IL3, inefficient stripping of surface IL3 receptors with trypsin (as recognized previously: Stevens, et al., J Biol. Chem. 266:4151-4158, 1991), or additional factors. Cells were also cooled to 4°C, preincubated with recombinant IL3 for 30 minutes to saturate surface receptors, and subsequently incubated with His-ILFAC for 10 minutes at 37 °C. Under those conditions, no cell-associated His-ILFAC was detected. Taken together, these data strongly support the notion that His-ILFAC is internalized by lymphoblastoid cells in a temperature dependent manner via IL3 receptors.
Finally, in view of the ability of IL3 receptor to recycle, the delivery of ligand back to the cell surface and its release into the medium could create a cycle and lower the effective intracellular concentrations of His-ILFAC. To evaluate this possibility, 2 X 106 cells were incubated with His-ILFAC for 10 minutes at 37 °C, washed with PBS to remove unbound His- ILFAC, and subsequently incubated in RPMI with 10% fetal bovine serum for up to one hour. The medium was then clarified of cell debris and analyzed by immunoblotting. There was no detectable His-ILFAC in the medium. The lack of His-ILFAC in the medium suggests that once endocytosed, His-ILFAC remains confined within the intracellular compartment.
Example 3: Correction of MMC Hypersensitivity in FA-C cells.
The effect of His-ILFAC on cell survival in the presence of MMC was evaluated in HSC536 cells, which are compound heterozygous for the L554P mutation and an unknown mutation. Approximately 1 x 104 viable cells were incubated with different concentrations of MMC for 5 days in the presence or absence of His-ILFAC. Viable cells were quantitated by trypan blue exclusion. Cells were supplemented as follows (Fig. 3): FAC, mutant cells stably transfected with wild-type FAC; ILFAC(a), His-ILFAC (1 μg/ml) added to the culture medium of mutant cell daily for 5 days; ILFAC(b), COS-1 conditioned medium containing secreted ILFAC added to the culture medium as a 10% (v/v) supplement; IL3, recombinant human IL3 (1 g/ml) added to the culture medium of mutant cells daily for 5 days. As shown in Fig. 3, cells supplemented with FAC or ILFAC molecules were significantly more resistant to MMC than control cells (untreated or IL3 treated). When cells were treated with His-ILFAC at 200 μg/ml twice daily for 2 days prior to and during the assay period, their resistance to MMC appeared to be greater than those treated with a single dose of His-ILFAC. Thus, administration of His- ILFAC rescued FA-C cells from the toxicity of MMC.
Example 4: Expression of Other His-ILFAC Molecules. Although the IL3 cassette encodes the Gibbon protein, it is comparable to the human IL3 in its interaction with the human receptor and in signal transduction (Miyajima, et al., Blood 82:1960-1974, 1994). However, for experiments involving murine models, a His-TAG fusion protein is generated that includes the mature portion of murine IL3 (minus the signal peptide) fused at the downstream end to human FAC as described above in Example 2. Further, additional His-ILFAC proteins comprising various combinations of the components are prepared to determine if the order of the components influences bioactivity.
Example 5: FAC affinity purification of His-ILFAC.
The His-TAG has been exploited to obtain partially pure protein from bacterial lysates by affinity chromatography, and it has been used in conjunction with other methods, especially size exclusion chromatography. Importantly, although the protein was not purified to homogeneity, it retained partial biological activity for both the IL3 domain, as demonstrated by successful receptor targeting, and the FAC domain, as demonstrated by correction of MMC hypersensitivity. The presence of lower molecular size forms may be due to either degradation products or premature termination of the fusion protein at cryptic stop codons. Theoretically, such a heterogeneous population would not only compromise the specific activity of the fusion protein, but carboxy-terminal truncated forms could also become receptor antagonists by competition with full length His-ILFAC for binding to IL3 receptors. To overcome these problems, a dual affinity purification strategy is used that takes advantage of specific epitopes at both ends of the fusion protein: the amino terminal His-TAG, and the most carboxy terminal portion of FAC. For the former, nickel-agarose beads available commercially are used to immobilize the fusion protein as described above. After extensive washing, the protein is eluted with 50-100 mM imidazole and then dialyzed against PBS. For the second step, the immunoaffinity column is generated using purified anti-FAC antibodies directed against the carboxy-terminus of FAC. Previously, polyclonal anti-FAC antibodies directed against a nearly full-length FAC protein were generated (Youssoufian, et al., J. Biol. Chem. 270:9876-9882, 1995; Youssoufian, Proc. Natl. Acad. Sci. U.S.A. 91 :7975-7979, 1994; Youssoufian, J. Clin. Invest. 97:2003-2010, 1996a). In this case, anti-peptide antibodies from the most carboxy- terminal region of FAC are generated in order to select the desired full-length bacterial products that contain intact carboxy-termini. First, short peptides are synthesized from the carboxyterminal region of FAC, and these peptides are coupled covalently to appropriately antigenic carriers (e.g., keyhole limpet hemocyanin), and rabbits are immunized with such conjugated peptides according to standard procedures. The antigenic peptide is coupled to epoxide-activated silica beads (Waters, Inc., Bedford, MA) as an affinity reagent with which affinity-purified IgG is isolated from the crude rabbit antiserum. Specific elution of the anti- FAC antibodies with either acidic glycine or imidazole yields a population of affinity-purified anti-FAC antibodies. These antibodies are used to generate a second affinity column for the purification of His-ILFAC. Similar to the affinity column mentioned above, affinity purified anti-FAC antibodies are immobilized onto epoxy-activated silica beads according to the manufacturer's instructions, and the fusion protein obtained from nickel agarose chromatography is subjected to a second round of affinity purification over anti-FAC affinity column. The double affinity purification procedure thus is effective for isolating molecules which retain both the IL3 and FAC portions of the fusion protein.
Example 6: Modification of Amino Acids Responsible for Protein Degradation.
To determine the intracellular turnover of the His-ILFAC fusion protein, purified His- ILFAC or His-FAC were added to HSC536 cells ar 4 °C for 10 minutes in DME containing 10% FBS. Cells were then centrifuged, fresh DME-10%> FBS medium was added in the absence of His-tagged proteins, and cells were rapidly warmed to 37 °C. Internalization of the fusion proteins and the intracellular turnover of the proteins was assessed by washing cells with cold PBS and lysing the cells in IX Laemmli buffer at the indicated times (Fig. 4): no protein added (lane 2), 5 min post-incubation (lane 3), 15 min post-incubation (lane 4), 30 min post-incubation (lane 5), 60 min post-incubation (lane 6), 120 min post-incubation (lane 7), and 240 min post- incubation (lane 8). Visual inspection of the immunoblot indicates that the intracellular t,/2 of His-ILFAC is about 60 minutes. This correlates well with the intracellular t,A of transfected FAC (see below). It also can be seen that in the absence of the IL3 moiety, the FAC molecule is not internalized (His-FAC arrow).
In a previous study, the intracellular t,Λ of FAC within transiently transfected COS cells was established to be approximately 40-45 minutes (Youssoufian, et al., J. Clin. Invest. 97:957- 962, 1996b). Thus, the protein is short lived, although normally the endogenous rate of FAC transcription and translation are sufficient to maintain homeostasis by generating physiologically adequate levels of protein. In the context of protein replacement therapy in genetically deficient cells unable to manufacture wild type protein, it is desirable to engineer modifications that result in longer acting forms of exogenous protein.
To identify amino acid residues responsible for the rapid degradation of FAC, a panel of six deletion mutants that lack progressively larger portions from the carboxyterminus of FAC were generated. The intracellular A of this panel of truncated proteins is tested by pulse-chase analysis. Briefly, COS-1 cells are transfected with the truncated constructs subcloned into the pED6 expression vector and metabolically labeled with Expre35S35S label (0.2 mCi/ml; DuPont, Wilmington, DE) for 30 minutes in cysteine and methionine-deficient medium. The labeled cells are washed rapidly with serum-free medium and the radiolabel is chased by incubation for 15- 120 minutes. After washing with ice cold PBS and lysing these cells in non-ionic detergents in the presence of protease inhibitors (0.2 mM PMSF, 2 mM EDTA and 1 μg/ml aprotinin), the post-nuclear supernatant is immunoprecipitated sequentially with anti-FAC antibody for 16 hours at 4°C and protein A-agarose beads for 2 hours (Bio-Rad, Richmond, CA), and the unbound supernatant is subjected to a second round of immunoprecipitation to maximize antigen recovery. The combined immuno-complexes are washed three times with NET-gel buffer (Kruyt, et al., Blood 87:938-948, 1996), boiled in 1 x Laemmli buffer in the presence of 200 mM dithiothreitol, and analyzed by SDS-PAGE and autoradiography. Alternatively, the intracellular t.2 is tested by immunoblotting as described above. In this manner, regions of the protein that are responsible for promoting degradation are identified. After the identification of such a domain, the degradation-promoting region is localized with greater precision and conventional site- directed mutagenesis (Lu, et al., Exp. Hematol. 20:418-424, 1992) is used to alter specific residues. Finally, mutations that suppress intracellular degradation are subcloned in pQE9 and expressed as IL3 fusion proteins in E. coli. After affinity purification of the IPTG-induced protein, receptor-mediated uptake assays on IL3 receptor-positive lymphoblastoid B cells are performed as described above. The half life of these proteins is determined by pulse-chase analysis in cells supplemented exogenously with either wild type or mutant His-ILFAC. After treatment for 10 minutes at 37 °C, cells are washed and chased in complete media and the intracellular levels of His-ILFAC in cell lysates from various time points are analyzed by immunoblotting. FAC fusion proteins having longer t,Λ will be useful for treatments. The intracellular activities of the purified His-ILFAC fusion proteins having prolonged intracellular half lives are determined. Correction of the MMC hypersensitivity of FA-C cells by exogenous administration to the FAC mutant fusion proteins IL3 receptor-positive cells is assessed as described above.
Example 7: Activity of His-ILFAC Proteins in FA-C Lymphoblasts. A rapid scheme has been devised for the selection of transfected B-lymphoblastoid cells using an expression vector (DRA-CD) that encodes the T cell-specific surface antigen CD4 as well as the full-length FAC cDNA (Youssoufian, 1995; Youssoufian, 1996a, supra). After electroporation, cells are subjected to one or more rounds of selection with immunomagnetic beads coated with an anti-CD4 monoclonal antibody, and cell viability assays are performed shortly thereafter. The rapidity of this procedure makes it possible to analyze the functional consequences of a large number of mutations relatively quickly. Thus this assay is useful to identify mutants of FAC, such as are termed previously functional equivalents. As one example, the assay is useful to identify FAC proteins which have longer half lives and are effective in reducing apoptosis. First, mutant FAC cDNAs that result in longer half lives are cloned into the DRA-CD expression vector, electroporated into FA-C cells (Youssoufian, 1995; Youssoufian, 1996a, supra) and the ability of the mutant proteins to confer resistance to crosslinker-treated cells is assessed as described above. Second, intracellular activity of FAC will be expressed in E. coli as His-IL3 fusion proteins. As described above, the ability to correct MMC-induced hypersensitivity is assessed directly by targeted delivery of His-ILFAC protein into FA-C lymphoblastoid cells. This dual strategy of assessing the activity of the fusion protein both after transfection and after exogenous administration of the fusion protein permits the detection of subtle quantitative differences between the activities of different mutant proteins. Thus, mutations that either partially inactivate the protein or, conversely, result in superactive proteins are identified by this approach.
Example 8: Activity of His-ILFAC in Primary Hematopoietic Progenitor Cells of FA-C Patients.
To evaluate the possibility that exogenous His-ILFAC could influence the proliferation or differentiation of hematopoietic cells, bone marrow cells of FA-C patients and normal control subjects are assessed for their capacity to form distinct hematopoietic lineages in the presence or absence of the FAC fusion protein. Hematopoietic cells are obtained from bone marrow donors during harvests prior to transplantation. CD34+++ cells are obtained after sorting non-adherent low-density T lymphocyte-depleted cells on a flow cytometer (Epics 753; Coulter Corp., Hialeah, FL). This fraction is richest in stem/immature progenitor cells. To evaluate the effect of ILFAC on progenitors destined to particular lineages, the CD34+++ population is sorted further to yield the following subsets: CD34++ CD33" (enriched for BFU-E and CFU-E), and CD34++ CD33+ (enriched for CFU-GM). These cells are placed into single wells containing 0.1 ml semisolid culture medium containing Iscove's modified Dulbecco's medium, 1 % methyl cellulose, 30% fetal calf serum and 0.1 mM hemin, as well as maximally active concentrations of recombinant human steel factor, IL3, GM-CSF, and erythropoietin. For quantitation of colonies derived from high proliferative-potential cells, the criteria of greater than 1 mm diameter with a dense center and composed of greater than 103 cells/colony is used.
Example 9: Physiological Properties of IL3-FAC in Murine Models. Although the molecular events involved in post-receptor IL3 signaling appear to be highly conserved between human and mouse, the primary sequence homology between human and mouse IL3 is limited, and the interaction of these ligands with their receptors is highly species specific. Thus, a His-tagged murine IL3-human FAC protein (His-mlLFAC) is generated, as indicated above, for the studies described below.
Example 10: Tissue Uptake and Toxicity.
To establish correct targeting to hematopoietic cells in vivo, mice are injected through their tail veins with increasing doses of His-mlLFAC. After one hour, mice are sacrificed and tissues analyzed by immunoblotting with anti-FAC antibody. Targeting of the protein to IL3 receptor-positive cells is confirmed by finding the immunoreactive protein in the bone marrow, spleen, and other cells that constitute the reticuloendothelial system. The fusion protein is distinguished from endogenous FAC protein by its greater size. The interval between protein injection and tissue processing can be varied to establish the optimal timing of this analysis. In addition, this set of experiments establishes the maximum tolerated dose of the fusion protein; the animals will be observed for clinically recognized features of cardiorespiratory distress and analysis of complete blood counts.
Example 11 : In vivo Bioactivity of IL3-FAC.
Mice homozygous for an inactivated Fac allele (F A"'" mice) have a number of biological features similar to those observed in the human Fanconi anemia disorder (Chen, et al., Nature Genet. 12:448-451, 1996). Splenic lymphocytes from FA~Λ mice have elevated chromosomal anomalies that are induced by the crosslinkers MMC and DEB. Although these mice do not develop leukemia or cancer at elevated rates, intravenous administration of MMC causes a rapidly fatal pancytopenia and death. Thus, the hematopoietic system of these FA"Λ mice is indeed exquisitely sensitive to cross-linker toxicity, and is thus a valuable in vivo model for these studies. FAA mice are treated with a dose of the fusion protein that causes little toxicity and results in hematopoietic cell targeting in normal mice. Karyotype analysis is performed on splenic lymphocytes in the presence or absence of the fusion protein. Mice are injected 1-2 hours prior to sacrifice to "load" lymphocytes with exogenous protein. Lymphocytes are then stimulated in vitro for 24-48 hours with concanavalin A prior to metaphase arrest and standard karyotype analysis. The mean number of aberrations per cell with and without treatment with exogenous His-mlLFAC provides evidence for the activity of the fusion protein. Finally, the His-mlLFAC fusion protein is used to rescue male and female FA"'" mice from cross-linker- induced toxicity. Different schedules of administration (i.e., frequency of administration, pre- administration relative to MMC) are used to identify the conditions that result in hematopoietic rescue.
Those skilled in the art will be able to ascertain with no more than routine experimentation numerous equivalents to the specific processes and products described herein. Such equivalents are considered to be within the scope of the invention and are intended to be embraced by the following claims.
All documents referred to in this application are incorporated in their entirety by reference. A Sequence Listing is presented followed by what is claimed:
SEQUENCE LISTING
(1) GENERAL INFORMATION:
(I) APPLICANT:
(A) NAME: BRIGHAM AND WOMEN'S HOSPITAL, INC.
(B) STREET: 75 FRANCIS STREET
(C) CITY: BOSTON (D) STATE: MASSACHUSETTS
(E) COUNTRY: UNITED STATES OF AMERICA
(F) POSTAL CODE: 02115
(ii) TITLE OF INVENTION: FAC MOLECULES AND USES THEREOF
(iii) NUMBER OF SEQUENCES: 11
(iv) CORRESPONDENCE ADDRESS:
(A) ADDRESSEE: WOLF, GREENFIELD & SACKS, P.C. (B) STREET: 600 ATLANTIC AVENUE
(C) CITY: BOSTON
(D) STATE: MASSACHUSETTS
(E) COUNTRY: UNITED STATES OF AMERICA
(F) POSTAL CODE: 02210
(v) COMPUTER READABLE FORM:
(A) MEDIUM TYPE: Floppy disk
(B) COMPUTER: IBM PC compatible
(C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25
(vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUMBER:
(B) FILING DATE: (C) CLASSIFICATION:
(vii) PRIOR APPLICATION DATA:
(A) APPLICATION NUMBER: US 60/046,546
(B) FILING DATE: 15-MAY-1997
(viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: Plumer, Elizabeth R.
(B) REGISTRATION NUMBER: 36,637
(C) REFERENCE/DOCKET NUMBER: B0801/7086WO
(ix) TELECOMMUNICATION INFORMATION:
(A) TELEPHONE: 617-720-3500
(B) TELEFAX: 617-720-2441 (2) INFORMATION FOR SEQ ID NO:l:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 4567 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 256..1929
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
ACTGCTGACA CGTGTGCGCG CGCGCGGCTC C-ACTGCCGGG CGACCGCGGG AAAATTCCAA 60
AAAAACTCAA AAAGCCAATA CGAGGCAAAG CCAAATTTTC AAGCCACAGA TCCCGGGCGG 120
TGGCTTCCTT TCCGCCACTG CCCAAACTGC TGAAGCAGCT CCCGCGAGGA CCACCCGATT 180
TAATGTGTGC CGACCATTTC CTTCAGTGCT GGACAGGCTG CTGTGAAGGG ACATCACCTT 240
TTCGClTiTr CCAAG ATG GCT CAA GAT TCA GTA GAT CTT TCT TGT GAT TAT 291
Met Ala Gin Asp Ser Val Asp Leu Ser Cys Asp Tyr 1 5 10
CAG TTT TGG ATG CAG AAG CTT TCT GTA TGG GAT CAG GCT TCC ACT TTG 339 Gin Phe Trp Met Gin Lys Leu Ser Val Trp Asp Gin Ala Ser Thr Leu 15 20 25
GAA ACC CAG CAA GAC ACC TGT CTT CAC GTG GCT CAG TTC CAG GAG TTC 387 Glu Thr Gin Gin Asp Thr Cys Leu His Val Ala Gin Phe Gin Glu Phe 30 35 40
CTA AGG AAG ATG TAT GAA GCC TTG AAA GAG ATG GAT TCT AAT ACA GTC 435 Leu Arg Lys Met Tyr Glu Ala Leu Lys Glu Met Asp Ser Asn Thr Val 45 50 55 60
ATT GAA AGA TTC CCC ACA ATT GGT CAA CTG TTG GCA AAA GCT TGT TGG 483 He Glu Arg Phe Pro Thr He Gly Gin Leu Leu Ala Lys Ala Cys Trp
65 70 75
AAT CCT TTT ATT TTA GCA TAT GAT GAA AGC CAA AAA ATT CTA ATA TGG 531 Asn Pro Phe He Leu Ala Tyr Asp Glu Ser Gin Lys He Leu He Trp 80 85 90
TGC TTA TGT TGT CTA ATT AAC AAA GAA CCA CAG AAT TCT GGA CAA TCA 579 Cys Leu Cys Cys Leu He Asn Lys Glu Pro Gin Asn Ser Gly Gin Ser 95 100 105
AAA CTT AAC TCC TGG ATA CAG GGT GTA TTA TCT CAT ATA CTT TCA GCA 627 Lys Leu Asn Ser Trp He Gin Gly Val Leu Ser His He Leu Ser Ala 110 115 120
CTC AGA TTT GAT AAA GAA GTT GCT CTT TTC ACT CAA GGT CTT GGG TAT 675 Leu Arg Phe Asp Lys Glu Val Ala Leu Phe Thr Gin Gly Leu Gly Tyr 125 130 135 140
GCA CCT ATA GAT TAC TAT CCT GGT TTG CTT AAA AAT ATG GTT TTA TCA 723 Ala Pro He Asp Tyr Tyr Pro Gly Leu Leu Lys Asn Met Val Leu Ser 145 150 155
TTA GCG TCT GAA CTC AGA GAG AAT CAT CTT AAT GGA TTT AAC ACT CAA 771 Leu Ala Ser Glu Leu Arg Glu Asn His Leu Asn Gly Phe Asn Thr Gin 160 165 170
AGG CGA ATG GCT CCC GAG GGA GTG GCG TCC CTG TCA CGA GTT TGT GTC 819 Arg Arg Met Ala Pro Glu Arg Val Ala Ser Leu Ser Arg Val Cys Val 175 180 185
CCA CTT ATT ACC CTG ACA GAT GTT GAC CCC CTG GTG GAG GCT CTC CTC 867 Pro Leu He Thr Leu Thr Asp Val Asp Pro Leu Val Glu Ala Leu Leu 190 195 200
ATC TGT CAT GGA CGT GAA CCT CAG GAA ATC CTC CAG CCA GAG TTC TTT 915 He Cys His Gly Arg Glu Pro Gin Glu He Leu Gin Pro Glu Phe Phe 205 210 215 220
GAG GCT GTA AAC GAG GCC ATT TTG CTG AAG AAG ATT TCT CTC CCC ATG 963 Glu Ala Val Asn Glu Ala He Leu Leu Lys Lys He Ser Leu Pro Met 225 230 235
TCA GCT GTA GTC TGC CTC TGG CTT CGG CAC CTT CCC AGC CTT GAA AAA 1011 Ser Ala Val Val Cys Leu Trp Leu Arg His Leu Pro Ser Leu Glu Lys 240 245 250
GCA ATG CTG CAT CTT TTT GAA AAG CTA ATC TCC AGT GAG AGA AAT TGT 1059 Ala Met Leu His Leu Phe Glu Lys Leu He Ser Ser Glu Arg Asn Cys 255 260 265
CTG AGA AGG ATC GAA TGC TTT ATA AAA GAT TCA TOG CTG CCT CAA GCA 1107 Leu Arg Arg He Glu Cys Phe He Lys Asp Ser Ser Leu Pro Gin Ala 270 275 280
GCC TGC CAC CCT GCC ATA TTC CGG GTT GTT GAT GAG ATG TTC AGG TGT 1155 Ala Cys His Pro Ala He Phe Arg Val Val Asp Glu Met Phe Arg Cys 285 290 295 300
GCA CTC CTG GAA ACC GAT GGG GCC CTG GAA ATC ATA GCC ACT ATT CAG 1203 Ala Leu Leu Glu Thr Asp Gly Ala Leu Glu He He Ala Thr He Gin 305 310 315
GTG TTT ACG CAG TGC TTT GTA GAA GCT CTG GAG AAA GCA AGC AAG CAG 1251 Val Phe Thr Gin Cys Phe Val Glu Ala Leu Glu Lys Ala Ser Lys Gin 320 325 330
CTG CGG TTT GCA CTC AAG ACC TAC TTT CCT TAC ACT TCT CCA TCT CTT 1299 Leu Arg Phe Ala Leu Lys Thr Tyr Phe Pro Tyr Thr Ser Pro Ser Leu 335 340 345
GCC ATG GTG CTG CTG CAA GAC CCT CAA GAT ATC CCT CGG GGA CAC TGG 1347 Ala Met Val Leu Leu Gin Asp Pro Gin Asp He Pro Arg Gly His Trp 350 355 360
CTC CAG ACA CTG AAG CAT ATT TCT GAA CTG CTC AGA GAA GCA GTT GAA 1395 Leu Gin Thr Leu Lys His He Ser Glu Leu Leu Arg Glu Ala Val Glu 365 370 375 380
GAC CAG ACT CAT GGG TCC TGC GGA GGT CCC TTT GAG AGC TGG TTC CTG 1443 Asp Gin Thr His Gly Ser Cys Gly Gly Pro Phe Glu Ser Trp Phe Leu 385 390 395
TTC ATT CAC TTC GGA GGA TGG GCT GAG ATG GTG GCA GAG CAA TTA CTG 1491 Phe He His Phe Gly Gly Trp Ala Glu Met Val Ala Glu Gin Leu Leu 400 405 410
ATG TOG GCA GCC GAA CCC CCC ACG GCC CTG CTG TGG CTC TTG GCC TTC 1539 Met Ser Ala Ala Glu Pro Pro Thr Ala Leu Leu Trp Leu Leu Ala Phe 415 420 425
TAC TAC GGC CCC CGT GAT GGG AGG CAG AGA GCA CAG ACT ATG GTC CAG 1587 Tyr Tyr Gly Pro Arg Asp Gly Arg Gin Arg Ala Gin Thr Met Val Gin 430 435 440
GTG AAG GCC GTG CTG GGC CAC CTC CTG GCA ATG TCC AGA AGC AGC AGC 1635 Val Lys Ala Val Leu Gly His Leu Leu Ala Met Ser Arg Ser Ser Ser 445 450 455 460
CTC TCA GCC CAG GAC CTG CAG ACG GTA GCA GGA CAG GGC ACA GAC ACA 1683 Leu Ser Ala Gin Asp Leu Gin Thr Val Ala Gly Gin Gly Thr Asp Thr 465 470 475
GAC CTC AGA GCT CCT GCA CAA CAG CTG ATC AGG CAC CTT CTC CTC AAC 1731 Asp Leu Arg Ala Pro Ala Gin Gin Leu He Arg His Leu Leu Leu Asn 480 485 490
TTC CTG CTC TGG GCT CCT GGA GGC CAC ACG ATC GCC TGG GAT GTC ATC 1779 Phe Leu Leu Trp Ala Pro Gly Gly His Thr He Ala Trp Asp Val He 495 500 505
ACC CTG ATG GCT CAC ACT GCT GAG ATA ACT CAC GAG ATC ATT GGC TTT 1827 Thr Leu Met Ala His Thr Ala Glu He Thr His Glu He He Gly Phe 510 515 520
CTT GAC CAG ACC TTG TAC AGA TGG AAT CGT CTT GGC ATT GAA AGC CCT 1875 Leu Asp Gin Thr Leu Tyr Arg Trp Asn Arg Leu Gly He Glu Ser Pro 525 530 535 540
AGA TCA GAA AAA CTG GCC CGA GAG CTC CTT AAA GAG CTG CGA ACT CAA 1923 Arg Ser Glu Lys Leu Ala Arg Glu Leu Leu Lys Glu Leu Arg Thr Gin 545 550 555
GTC TAGAAGGCAC GCAGGCCGTG TGGGTGCCCG GCGTGAGGGA TCAGGCTCGC 1976 Val
CAGGGCCACA GGACAGGTGA TGACCTGTGG CCACGCATTT GTGGAGTAAG TGCCCTCGCT 2036
GGGCTGTGAG AATGAGCTGT ACACATCTTG GGACAATCTG CTAGTATCTA TTTTACAAAA 2096
TGCAGAGCCA GGTCCCTCAG CCCAGACTCA GTCAGACATG TTCACTAATG ACTCAAGTGA 2156
GCTTCGGTAC TCCTGGTGCC CGCCCGGCCA GACCGTCAGC TTGATAATTA CTAAAGCAAA 2216
GGCCTGGGTG GGAGAACAGG TTTCTAGTTT TTACCCAAGT CAAGCTGCAC ATCTATTATT 2276
TAAAAATTCA AAGTCTTAGA ACCAAGAATT TGGTCATGAA CCATTAAAGA ATTTAGAGAG 2336
AACTTAGCTC TTTTTAGACT CTTTTTAGGA GTCAGGGATC TGGGATAAAG CCACACTGTC 2396
TTGCTGTATG GAGAAATTCT TCAAGGGGAG TCAGGGTCCC TCAGGCTTCC CTTGTGTCTC 2456
CCTGGACCTG CCTGACAGGC CACAGGAGCA GACAGCACAC CCAAGCCCGG GCCTCCGGCA 2516
CACTCTTTCC ACTCTGTATT TGCTAAATGA TGCTAACTGC TACCAAAAGG CCCTTGGGAC 2576
ATCAGAGGAG CCGGCAGCGA AGGTAGAGGA TGTGTTCCAG AAACATTAGA AGGCAGGATT 2636 AATTCAGTTA GTTAGTCTCT TGTTAAATGG AAATGGGAAT TGGAAATTCC TGATAAAGAA 2696
Tπ-K^CTGGC TGGGTGCAGT GGCTCACACC TGTGATCCCA GCACπ-TGGG AGGCCAAGGC 2756
AGGGGGATTA CTTCAGCCCA GGAGTTCCAG ACTGCCTGGC TAACATGGCA ATACCCTATC 2816
TCTACTAAAA ATACAAAAAT TATCGGGGTG CAATGGCATG CATCTGTAAC CCAGCTATTC 2876
AAGAGGCTGA GGCATGAGGA TCTCTTGAAC CCGGGAGGTG GGAGTTGTAG TGAGCCGAGA 2936
TCATGACACT GCACTCCAGC CTGGGCAACA GAGCGAGACC ATCTCTTAAA AAAAGGCATT 2996
GTTAGTGTAA CTCAAGGTTA ACATTTATTT CATGTCAGTA CAGGGTGCTT TTTCCTTTCA 3056
GGGACATTCT GGAATTGTAT TGGTTGTACA TTCTTTTGTG TCTATTCTGT TTGTCAAGTG 3116
AGTCAAGACT TGCTTTTGTC CATTTTGATT TGTGTGTATT AGTCTGAGTC TTGGCTCCGT 3176
TTTGAGGTAT GAGCAAAGTT TTGCTGGATA GAGTTAACCT TTAGGGAAAT TCCTTATTTT 3236
GGTATGTGGC AATGCTAATA GATCCACTGA AGATCTGGAA AATTCCAGGA ACTTTTTCAC 3296
CTGAGCCTTT CTTCTGAGAA ATGCTGCAGT CAGAAGGGTG TGCTGGTAAA GTATTTTGGT 3356
GGCAGCTGCC ATCATGGTCA TTGCCTTCAT ATAACATGCT TCGTGCTCAT GGTCATTGCC 3416
TTCATATAAC ATGCTTCGTG CCATCATGAT CCTTGCCTTC ATATAACAAA CATGCTTCGT 3476
CAGAGGTGTT GGGGTTGAAA AAGGAGCTGC ATGCTTCACT GGAGTTGAGG GCCTCTCCTG 3536
TCTGACTTTA AGCCAGAACT TGTGGCTGGG CCATGGAAGC TGTGACTCCT CTGTGGACAT 3596
GGTGGCAGCA GGGAACCCCT AGAGAGAGGG GCCACTGGGA CCAGGCCTCC TGTTGTGGAG 3656
GGACTCCTGG GACAGTCCTC CACCCTGTCC TGTGGTCCTG TGTACAGGGT TGGCCTCTTC 3716
CTCCTCCCCT GCCAGGCCTC TGCCCATGCC CCTTCCTTCC TTCTCCTGGG ACTGGTGAAG 3776
CTAGGCATCT GGAAGACTTC TTCCTAGCCT GGAAGCCCTG ACCTCGGCCC ATCTGCAGAA 3836
TCTCCCAGTT CCTTCACAGC TGCCGAGTCC TCTCACGGGT GCGGTGGAGG CGGCCTTGCG 3896
GTGGTGCTTT CTGGGCAGCC AGGGGTTCCT GGGTGGGAGG ACTGTCCCTC TGGGGACGTG 3956
GCACTGAAGT GCCTGCTGGC TTCATGTGGC CCTTTGCCCT TTCCCAGCCT GAG-AGATGCT 4016
CAAAGGTGGG GAGCTGGGGG AGCCACCCCT CGGCCATTCC CTCCACCTCC AAGACAGGTG 4076 GCGGCCGGGC AGGCACTCTT AAGCCCACCT CCCCCTCTTG TTGCCTTCGA TITCGGCAAA 4136
GCCTGGGCAG GTGCCACCGG GAAGGAATGG CATCGAGATG CTO--X3CGGGG A∞CGGCGTG 4196
GCGAGGGGGC TTGACGGCGT TGGCGGGGCT GGGCACAGGG GCAGCCGCAG GGAGGCAGGG 4256
ATGGCAAGGC GTGAAGCCAC CCTGGAAGGA ACTGGACCAA GGTCTTCAGA GGTGCGACAG 4316
GGTCTGGAAT CTGACCTTAC TCTAGCAGGA GTTTTTGTAG ACTCTCCCTG ATAGTTTAGT 4376
TTTTGATAAA GCATGCTGGT AAAACCACTA CCCTCAGAGA GAGCCAAAAA TACAGAAGAG 4436
GCGGAGAGCG CCCCTCCAAC CAGGCTGTTA TTCCCCTGGA CTCCGTGACA TCTGTGGAAT 4496
TTTTTAGCTC TTTAAAATCT GTAATTTGTT GTCTATTTTT TCATTCTAAA TAAAACTTCA 4556
GTTTGCACCT A 4567
(2) INFORMATION FOR SEQ ID NO: 2:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 557 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2:
Met Ala Gin Asp Ser Val Asp Leu Ser Cys Asp Tyr Gin Phe Trp Met 1 5 10 15
Gin Lys Leu Ser Val Trp Asp Gin Ala Ser Thr Leu Glu Thr Gin Gin 20 25 30
Asp Thr Cys Leu His Val Ala Gin Phe Gin Glu Phe Leu Arg Lys Met 35 40 45
Tyr Glu Ala Leu Lys Glu Met Asp Ser Asn Thr Val He Glu Arg Phe 50 55 60
Pro Thr He Gly Gin Leu Leu Ala Lys Ala Cys Trp Asn Pro Phe He 65 70 75 80
Leu Ala Tyr Asp Glu Ser Gin Lys He Leu He Trp Cys Leu Cys Cys
85 90 95 Leu He Asn Lys Glu Pro Gin Asn Ser Gly Gin Ser Lys Leu Asn Ser 100 105 110
Trp He Gin Gly Val Leu Ser His He Leu Ser Ala Leu Arg Phe Asp 115 120 125
Lys Glu Val Ala Leu Phe Thr Gin Gly Leu Gly Tyr Ala Pro He Asp 130 135 140
Tyr Tyr Pro Gly Leu Leu Lys Asn Met Val Leu Ser Leu Ala Ser Glu 145 150 155 160
Leu Arg Glu Asn His Leu Asn Gly Phe Asn Thr Gin Arg Arg Met Ala 165 170 175
Pro Glu Arg Val Ala Ser Leu Ser Arg Val Cys Val Pro Leu He Thr 180 185 190
Leu Thr Asp Val Asp Pro Leu Val Glu Ala Leu Leu He Cys His Gly 195 200 205
Arg Glu Pro Gin Glu He Leu Gin Pro Glu Phe Phe Glu Ala Val Asn 210 215 220
Glu Ala He Leu Leu Lys Lys He Ser Leu Pro Met Ser Ala Val Val 225 230 235 240
Cys Leu Trp Leu Arg His Leu Pro Ser Leu Glu Lys Ala Met Leu His 245 250 255
Leu Phe Glu Lys Leu He Ser Ser Glu Arg Asn Cys Leu Arg Arg He 260 265 270
Glu Cys Phe He Lys Asp Ser Ser Leu Pro Gin Ala Ala Cys His Pro 275 280 285
Ala He Phe Arg Val Val Asp Glu Met Phe Arg Cys Ala Leu Leu Glu 290 295 300
Thr Asp Gly Ala Leu Glu He He Ala Thr He Gin Val Phe Thr Gin 305 310 315 320
Cys Phe Val Glu Ala Leu Glu Lys Ala Ser Lys Gin Leu Arg Phe Ala 325 330 335
Leu Lys Thr Tyr Phe Pro Tyr Thr Ser Pro Ser Leu Ala Met Val Leu 340 345 350 Leu Gin Asp Pro Gin Asp He Pro Arg Gly His Trp Leu Gin Thr Leu 355 360 365
Lys His He Ser Glu Leu Leu Arg Glu Ala Val Glu Asp Gin Thr His 370 375 380
Gly Ser Cys Gly Gly Pro Phe Glu Ser Trp Phe Leu Phe He His Phe 385 390 395 400
Gly Gly Trp Ala Glu Met Val Ala Glu Gin Leu Leu Met Ser Ala Ala
405 410 415
Glu Pro Pro Thr Ala Leu Leu Trp Leu Leu Ala Phe Tyr Tyr Gly Pro 420 425 430
Arg Asp Gly Arg Gin Arg Ala Gin Thr Met Val Gin Val Lys Ala Val 435 440 445
Leu Gly His Leu Leu Ala Met Ser Arg Ser Ser Ser Leu Ser Ala Gin 450 455 460
Asp Leu Gin Thr Val Ala Gly Gin Gly Thr Asp Thr Asp Leu Arg Ala 465 470 475 480
Pro Ala Gin Gin Leu He Arg His Leu Leu Leu Asn Phe Leu Leu Trp
485 490 495
Ala Pro Gly Gly His Thr He Ala Trp Asp Val He Thr Leu Met Ala 500 505 510
His Thr Ala Glu He Thr His Glu He He Gly Phe Leu Asp Gin Thr 515 520 525
Leu Tyr Arg Trp Asn Arg Leu Gly He Glu Ser Pro Arg Ser Glu Lys 530 535 540
Leu Ala Arg Glu Leu Leu Lys Glu Leu Arg Thr Gin Val 545 550 555
(2) INFORMATION FOR SEQ ID NO: 3:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 675 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(vi) ORIGINAL SOURCE:
(A) ORGANISM: Hylobates lar
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..468
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3:
AAT CCA AAC ATG AGC TGC CTG CCC GTC CTG CTC CTG CTC CAA CTC CTG 48 Asn Pro Asn Met Ser Cys Leu Pro Val Leu Leu Leu Leu Gin Leu Leu 1 5 10 15
GTC AGC CCC GGA CTC CAA GCT CCC ATG ACC CAG ACA ACG TCC TTG AAG 96 Val Ser Pro Gly Leu Gin Ala Pro Met Thr Gin Thr Thr Ser Leu Lys 20 25 30
ACA AGC TGG GTT AAC TGT TCT AAC ATG ATC GAT GAA ATT ATA ACA CAC 144 Thr Ser Trp Val Asn Cys Ser Asn Met He Asp Glu He He Thr His 35 40 45
TTA AAG CAG CCA CCT TTG CCC TTG CTG GAC TTC AAC AAC CTC AAT GGG 192 Leu Lys Gin Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly 50 55 60
GAA GAC CAA GAC ATT CTG ATG GAA AAT AAC CTT CGA AGG CCA AAC CTG 240 Glu Asp Gin Asp He Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu 65 70 75 80
GAG GCA TTC AAC AAG GCT GTC AAG AGT TTA CAG AAT GCA TCA GCA ATC 288 Glu Ala Phe Asn Lys Ala Val Lys Ser Leu Gin Asn Ala Ser Ala He
85 90 95
GAG AGC ATT CTT AAG AAT CTC CCC CCA TGC CTG CCC ATG GCC ACA GCC 336 Glu Ser He Leu Lys Asn Leu Pro Pro Cys Leu Pro Met Ala Thr Ala 100 105 110
GCA CCC ACG CGA CAT CCA ATC CGT ATC AAG GAC GGT GAC TGG AAT GAA 384 Ala Pro Thr Arg His Pro He Arg He Lys Asp Gly Asp Trp Asn Glu 115 120 125
TTC CGG AGG AAA CTG AAG TTC TAT CTG AAA ACC CTT GAG AAT GAG CAA 432 Phe Arg Arg Lys Leu Lys Phe Tyr Leu Lys Thr Leu Glu Asn Glu Gin 130 135 140
GCT CAA CAG ATG ACT TTG AGC CTT GAG ATC TCT TGAGTCCAAC GTCCAGCTCT 485 Ala Gin Gin Met Thr Leu Ser Leu Glu He Ser 145 150 155
CTCTCTGGGC CGTCTCACCG CAGAGCCTCA GGACATCAAA AACAGCAGAA CTTCTGAAAC 545
CTCTGGGTCG TCTCTCACAC AGTCCAGGAC CAGAAGCATT TCACC-TITIC CTGCGGCATC 605
AGATGAATTG TTAATTATCT AATTTCTGAA ATGTGCAGCT CCCATTTGGC CTTGTGTGGT 665
TGTGTTCTCA 675
(2) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 155 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:
Asn Pro Asn Met Ser Cys Leu Pro Val Leu Leu Leu Leu Gin Leu Leu 1 5 10 15
Val Ser Pro Gly Leu Gin Ala Pro Met Thr Gin Thr Thr Ser Leu Lys 20 25 30
Thr Ser Trp Val Asn Cys Ser Asn Met He Asp Glu He He Thr His 35 40 45
Leu Lys Gin Pro Pro Leu Pro Leu Leu Asp Phe Asn Asn Leu Asn Gly 50 55 60
Glu Asp Gin Asp He Leu Met Glu Asn Asn Leu Arg Arg Pro Asn Leu 65 70 75 80
Glu Ala Phe Asn Lys Ala Val Lys Ser Leu Gin Asn Ala Ser Ala He
85 90 95
Glu Ser He Leu Lys Asn Leu Pro Pro Cys Leu Pro Met Ala Thr Ala 100 105 110 Ala Pro Thr Arg His Pro He Arg He Lys Asp Gly Asp Trp Asn Glu 115 120 125
Phe Arg Arg Lys Leu Lys Phe Tyr Leu Lys Thr Leu Glu Asn Glu Gin 130 135 140
Ala Gin Gin Met Thr Leu Ser Leu Glu He Ser 145 150 155
(2) INFORMATION FOR SEQ ID NO: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 405 base pairs (B) TYPE: nucleic acid
(C) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE:
(A) NAME/KEY: CDS
(B) LOCATION: 1..405
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5:
ATG GGA TGG AGC TGG ATC TTT CTC TTC CTC CTG TCA GGA ACT GCA GGC 48 Met Gly Trp Ser Trp He Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15
GTC CAC TCT GAG GTC CAG CTT CAG CAG TCA GGA CCT GAG CTG GTG AAA 96 Val His Ser Glu Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys 20 25 30
CCT GGG GCC TCA GTG AAG ATA TCC TGC AAG GCT TCT GGA TAC ACA TTC 144 Pro Gly Ala Ser Val Lys He Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45
ACT GAC TAC AAC ATG CAC TGG GTG AAG CAG AGC CAT GGA AAG AGC CTT 192 Thr Asp Tyr Asn Met His Trp Val Lys Gin Ser His Gly Lys Ser Leu 50 55 60
GAG TGG ATT GGA TAT ATT TAT CCT TAC AAT GGT GGT ACT GGC TAC AAC 240 Glu Trp He Gly Tyr He Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn 65 70 75 80
CAG AAG TTC AAG AGC AAG GCC ACA TTG ACT GTA GAC AAT TCC TCC AGC 288 Gin Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Asn Ser Ser Ser 85 90 95
ACA GCC TAC ATG GAC GTC CGC AGC CTG ACA TCT GAG GAC TCT GCA GTC 336 Thr Ala Tyr Met Asp Val Arg Ser Leu Thr Ser Glu Asp Ser Ala Val 100 105 110
TAT TAC TGT GCA AGA GGG CGC CCC GCT ATG GAC TAC TGG GGT CAA GGA 384 Tyr Tyr Cys Ala Arg Gly Arg Pro Ala Met Asp Tyr Trp Gly Gin Gly 115 120 125
ACC TCA GTC ACC GTC TCC TCA 405
Thr Ser Val Thr Val Ser Ser 130 135
(2) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 135 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6:
Met Gly Trp Ser Trp He Phe Leu Phe Leu Leu Ser Gly Thr Ala Gly 1 5 10 15
Val His Ser Glu Val Gin Leu Gin Gin Ser Gly Pro Glu Leu Val Lys 20 25 30
Pro Gly Ala Ser Val Lys He Ser Cys Lys Ala Ser Gly Tyr Thr Phe 35 40 45
Thr Asp Tyr Asn Met His Trp Val Lys Gin Ser His Gly Lys Ser Leu 50 55 60
Glu Trp He Gly Tyr He Tyr Pro Tyr Asn Gly Gly Thr Gly Tyr Asn 65 70 75 80
Gin Lys Phe Lys Ser Lys Ala Thr Leu Thr Val Asp Asn Ser Ser Ser
85 90 95 Thr Ma Tyr Met Asp Val Arg Ser Leu Thr Ser Glu Asp Ser Ma Val 100 105 110
Tyr Tyr Cys Ma Arg Gly Arg Pro Ma Met Asp Tyr Trp Gly Gin Gly 115 120 125
Thr Ser Val Thr Val Ser Ser 130 135
(2) INFORMATION FOR SEQ ID NO: 7:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 393 base pairs
(B) TYPE: nucleic acid (C) STRANDEDNESS : double
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(ix) FEATURE: (A) NAME/KEY: CDS
(B) LOCATION: 1..393
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
ATG GAG AAA GAC ACA CTC CTG CTA TGG GTC CTG CTT CTC TGG GTT CCA 48 Met Glu Lys Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15
GGT TCC ACA GGT GAC ATT GTG CTG ACC CAA TCT CCA GCT TCT TTG GCT 96 Gly Ser Thr Gly Asp He Val Leu Thr Gin Ser Pro Ma Ser Leu Ma 20 25 30
GTG TCT CTA GGG CAG AGG GCC ACC ATC TCC TGC AGA GCC AGC GAA AGT 144 Val Ser Leu Gly Gin Arg Ma Thr He Ser Cys Arg Ma Ser Glu Ser 35 40 45
GTT GAT AAT TAT GGC ATT AGT TTT ATG AAC TGG TTC CAA CAG AAA CCA 192 Val Asp Asn Tyr Gly He Ser Phe Met Asn Trp Phe Gin Gin Lys Pro 50 55 60
GGA CAG CCA CCC AAA CTC CTC ATC TAT GCT GCA TCC AAC CAA GGA TCC 240
Gly Gin Pro Pro Lys Leu Leu He Tyr Ma Ma Ser Asn Gin Gly Ser
65 70 75 80 GGG GTC CCT GCC AGG TTT AGT GGC AGT GGG TCT GGG ACA GAC TTC AGC 288 Gly Val Pro Ma Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser
85 90 95
CTC AAC ATC CAT CCT ATG GAG GAG GAT GAT ACT GCA ATG TAT TTC TGT 336 Leu Asn He His Pro Met Glu Glu Asp Asp Thr Ma Met Tyr Phe Cys 100 105 110
CAG CAA AGT AAG GAG GTT CCG TGG ACG TTC GGT GGA GGC ACC AAG CTG 384 Gin Gin Ser Lys Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125
GAA ATC AAA 393
Glu He Lys 130
(2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 131 amino acids
(B) TYPE: amino acid (D) TOPOLOGY: linear
(ii) MOLECULE TYPE: protein
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:
Met Glu Lys Asp Thr Leu Leu Leu Trp Val Leu Leu Leu Trp Val Pro 1 5 10 15
Gly Ser Thr Gly Asp He Val Leu Thr Gin Ser Pro Ma Ser Leu Ma 20 25 30
Val Ser Leu Gly Gin Arg Ma Thr He Ser Cys Arg Ma Ser Glu Ser 35 40 45
Val Asp Asn Tyr Gly He Ser Phe Met Asn Trp Phe Gin Gin Lys Pro 50 55 60
Gly Gin Pro Pro Lys Leu Leu He Tyr Ma Ma Ser Asn Gin Gly Ser 65 70 75 80
Gly Val Pro Ma Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Ser 85 90 95
Leu Asn He His Pro Met Glu Glu Asp Asp Thr Ma Met Tyr Phe Cys 100 105 110 Gln Gin Ser Lys Glu Val Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu 115 120 125
Glu He Lys 130
(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 27 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS : single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:
CGGGATCCGC TCCCATGACC CAGACAA 27
(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS : (A) LENGTH: 29 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: YES
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:
CGGGATCCAG AGATCTCAAG GCTCAAAGT 29
(2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 33 base pairs
(B) TYPE: nucleic acid
(C) STRANDEDNESS: single
(D) TOPOLOGY: linear
(ii) MOLECULE TYPE: cDNA
(iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE : NO
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:
CGGTCGACCA CCATGGGCTG CCTGCCCGTC CTG 33

Claims

1. An isolated conjugate comprising a Fanconi anemia complementation group C (FAC) molecule and a polypeptide which selectively binds a hematopoietic progenitor cell.
2. The isolated conjugate of claim 1 wherein the FAC molecule is a FAC protein.
3. The isolated conjugate of claim 2 wherein the FAC protein consists essentially of the amino acid sequence of SEQ ID NO:2.
4. The isolated conjugate of claim 2 wherein the FAC protein is a functional fragment of SEQ ID NO:2.
5. The isolated conjugate of claim 1 wherein the FAC molecule is an isolated nucleic acid molecule selected from the group consisting of:
(a) nucleic acid molecules which hybridize under stringent conditions to a molecule having the nucleic acid sequence of SEQ ID NO: 1 and which code for a FAC protein,
(b) nucleic acid molecules which differ from the nucleic acid molecules of (a) in codon sequence due to degeneracy of the genetic code, and (c) complements of (a) and (b).
6. The isolated conjugate of claim 5 wherein the isolated nucleic acid molecule comprises the nucleotide sequence of SEQ ID NO: 1.
7. The isolated conjugate of claim 5 wherein the isolated nucleic acid molecule encodes a functional fragment of a FAC protein.
8. The isolated conjugate of claim 5 wherein the isolated nucleic acid molecule is operably linked to a promoter.
9. The isolated conjugate of claim 8, wherein the promoter is active in a hematopoietic progenitor cell.
10. The isolated conjugate of claim 1 wherein the polypeptide which selectively binds the hematopoietic progenitor cell is an antibody which selectively binds to an antigen on the surface of the hematopoietic progenitor cell.
11. The isolated conjugate of claim 10, wherein the antibody is an antigen-binding fragment.
12. The isolated conjugate of claim 10, wherein the antibody is an anti-CD33 antibody.
13. The isolated conjugate of claim 10, wherein the conjugate is a fusion polypeptide.
14. The isolated conjugate of claim 13, wherein the antibody is a single chain antibody.
15. The isolated conjugate of claim 1 , wherein the polypeptide which selectively binds the hematopoietic progenitor cell is a ligand which binds to a receptor of the hematopoietic progenitor cell.
16. The isolated conjugate of claim 15, wherein the conjugate is a fusion polypeptide.
17. The isolated conjugate of claim 16, wherein the ligand is IL3 or a receptor-binding portion thereof.
18. The isolated conjugate of claim 17, wherein IL3 is the amino terminal portion of the fusion polypeptide.
19. The isolated conjugate of claim 17, wherein the FAC protein is the amino terminal portion of the fusion polypeptide.
20. The isolated conjugate of any of claims 17-19, wherein IL3 and FAC are separated by a linker polypeptide.
21. An isolated nucleic acid which codes for a fusion polypeptide comprising a FAC protein and a polypeptide which selectively binds to a hematopoietic progenitor cell.
22. The isolated nucleic acid of claim 21 wherein the polypeptide which selectively binds hematopoietic progenitor cells is a ligand which binds to a receptor of the hematopoietic progenitor cells.
23. The isolated nucleic acid of claim 22 wherein the ligand which binds to a receptor of the hematopoietic progenitor cells is IL3.
24. The isolated nucleic acid of claim 21 wherein the polypeptide which selectively binds the hematopoietic progenitor cell is an antibody which selectively binds to an antigen on the surface of the hematopoietic progenitor cell .
25. The isolated nucleic acid of claim 24, wherein the antibody is an antigen-binding fragment.
26. The isolated nucleic acid of claim 24, wherein the antibody is an anti-CD33 antibody.
27. The use of the compositions of any of claims 1-20 for delivery of a FAC molecule to a hematopoietic progenitor cell.
28. A method for inhibiting apoptosis in a hematopoietic progenitor cell comprising contacting the hematopoietic progenitor cell with an amount of a composition comprising a FAC molecule effective to inhibit apoptosis in the hematopoietic progenitor cell.
29. The method of claim 28, wherein the FAC molecule is a FAC protein.
30. The method of claim 29, wherein the composition further comprises a polypeptide which selectively binds to the hematopoietic progenitor cell, wherein the polypeptide is conjugated to the FAC protein.
31. The method of claim 30, wherein the FAC protein is contained within a liposome and wherein the liposome contains on its surface the polypeptide which selectively binds to the hematopoietic progenitor cell.
32. The method of claim 28,wherein the FAC molecule is an isolated nucleic acid:
(a) which hybridizes under stringent conditions to a molecule consisting of the nucleic acid sequence of SEQ ID NO: 1 and which codes for a FAC protein,
(b) nucleic acid molecules which differ from the nucleic acid molecules of (a) in codon sequence due to degeneracy of the genetic code, and
(c) complements of (a) and (b).
33. The method of claim 32, wherein the isolated nucleic acid is operably linked to a promoter.
34. The method of claim 33, wherein the promoter is active in the hematopoietic progenitor cell.
35. The method of claim 33, wherein the isolated nucleic acid is contained in a virus which is contains on its surface the polypeptide which selectively binds to the hematopoietic progenitor cell.
36. A method for treating a condition characterized by apoptosis of hematopoietic progenitor cells in a subject exposed to high-dose chemotherapy for non-myeloid malignancies, comprising administering to the subject an amount of a FAC molecule effective to inhibit apoptosis of the hematopoietic progenitor cells in the subject.
37. The method of claim 36, wherein the FAC molecule is a FAC protein.
38. The method of claim 37, wherein the composition further comprises a polypeptide which selectively binds to the hematopoietic progenitor cells of the subject.
39. The method of claim 38, wherein the FAC protein is conjugated to the polypeptide which selectively binds the hematopoietic progenitor cells of the subject.
40. The method of claim 38, wherein the FAC protein is contained within a liposome and wherein the liposome is targeted to the hematopoietic progenitor cells of the subject by the polypeptide which selectively binds to the hematopoietic progenitor cells of the subject.
41. The method of claim 36,wherein the FAC molecule is an isolated nucleic acid:
(a) which hybridizes under stringent conditions to a molecule consisting of the nucleic acid sequence of SEQ ID NO: 1 and which codes for a FAC protein,
(b) nucleic acid molecules which differ from the nucleic acid molecules of (a) in codon sequence due to degeneracy of the genetic code, and
(c) complements of (a) and (b).
42. The method of claim 41 , wherein the isolated nucleic acid is operably linked to a promoter.
43. The method of claim 42, wherein the promoter is active in the hematopoietic progenitor cells of the subject.
44. The method of claim 42, wherein the isolated nucleic acid is contained in a virus which targeted to the hematopoietic progenitor cells of the subject by a polypeptide which selectively binds to the hematopoietic progenitor cells of the subject.
PCT/US1998/009975 1997-05-15 1998-05-15 Fac molecules and uses thereof WO1998051792A1 (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2853910A1 (en) * 2003-04-16 2004-10-22 Galderma Res & Dev Using polynucleotide probes based on the FANCC gene (Fanconi anemia, complementation group C), for diagnosis of psoriasis, or predisposition, and in screening for antipsoriatic agents

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5681942A (en) * 1992-04-29 1997-10-28 Hsc Research & Development Limited Partnership Fanconi Anemia Type C gene

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5681942A (en) * 1992-04-29 1997-10-28 Hsc Research & Development Limited Partnership Fanconi Anemia Type C gene

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
LIEBETRAU W., ET AL.: "P53 ACTIVATES FANCONI ANEMIA GROUP C GENE EXPRESSION.", HUMAN MOLECULAR GENETICS, OXFORD UNIVERSITY PRESS, GB, vol. 06., no. 02., 1 January 1997 (1997-01-01), gb, pages 277 - 283., XP002910692, ISSN: 0964-6906, DOI: 10.1093/hmg/6.2.277 *
LIU J. M., YOUNG N. S.: "RETROVIRAL MEDIATED GENE TRANSFER OF THE FANCONI ANEMIA COMPLEMENTATION GROUP C GENE TO HEMATOPOIETIC PROGENITORS OF GROUP C PATIENTS.", HUMAN GENE THERAPY, MARY ANN LIEBERT, INC. PUBLISHERS, US, vol. 08., 20 September 1997 (1997-09-20), US, pages 1715 - 1730., XP002910694, ISSN: 1043-0342 *
MARATHI U. K., ET AL.: "THE FANCONI ANEMIA COMPLEMENTATION GROUP C PROTEIN CORRECTS DNA INTERSTRAND CROSS-LINK-SPECIFIC APOPTOSIS IN HSC536N CELLS.", BLOOD, AMERICAN SOCIETY OF HEMATOLOGY, US, vol. 88., no. 06., 15 September 1996 (1996-09-15), US, pages 2298 - 2305., XP002910696, ISSN: 0006-4971 *
WHITNEY M. A., ET AL.: "THE ASHKENAZI JEWISH FANCONI ANEMIA MUTATION: INCIDENCE AMONG PATIENTS AND CARRIER FREQUENCY IN THE AT-RISK POPULATION.", HUMAN MUTATION, JOHN WILEY & SONS, INC., US, vol. 03., 1 January 1994 (1994-01-01), US, pages 339 - 341., XP002910693, ISSN: 1059-7794, DOI: 10.1002/humu.1380030402 *
YOUSSOUFIAN H.: "CYTOPLASMIC LOCALIZATION OF FAC IS ESSENTIAL FOR THE CORRECTION OF A PREREPAIR DEFECT IN FANCONI ANEMIA GROUP C CELLS.", JOURNAL OF CLINICAL INVESTIGATION, AMERICAN SOCIETY FOR CLINICAL INVESTIGATION, US, vol. 97., no. 09., 1 January 1996 (1996-01-01), US, pages 2003 - 2010., XP002910695, ISSN: 0021-9738, DOI: 10.1172/JCI118635 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2853910A1 (en) * 2003-04-16 2004-10-22 Galderma Res & Dev Using polynucleotide probes based on the FANCC gene (Fanconi anemia, complementation group C), for diagnosis of psoriasis, or predisposition, and in screening for antipsoriatic agents
WO2004094661A2 (en) * 2003-04-16 2004-11-04 Galderma Research & Development, S.N.C. Fancc, dad1, grim19 and hadhii marker genes of psoriasis
WO2004094661A3 (en) * 2003-04-16 2005-10-13 Galderma Res & Dev Fancc, dad1, grim19 and hadhii marker genes of psoriasis

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