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TECHNICAL FIELD
The present invention relates generally to immunotherapy using a combination of polypeptide inhibitors of gene expression and other immunosuppressants. More particularly, the invention relates to polypeptide inhibitors of nuclear protein
translocation which have gene expression modulating activity, immunosuppressive activity, antiviral activity, and antitumor activity used in combination with immunosuppressants such as cyclosporin A.
BACKGROUND OF THE INVENTION
Nuclear transport is essential to a number of biological processes including gene expression and cell division, as well as to viral replication, tumorigenesis and tumor cell proliferation. The mechanism of nuclear transport has only recently
been characterized in detail and has been shown to involve a number of discrete steps. Proteins that are destined to be transported into the nucleus contain within their amino acid sequence a short stretch of amino acids termed a nuclear localization
sequence ("NLS"). These sequences are generally basic in nature, however, there has been no consensus sequence identified. Thus, there is a wide variety of these sequences that appear to be specific for particular proteins.
Within the cell, these NLSs may be either masked or unmasked by accessory proteins or by conformational changes within the NLS-containing protein. An NLS may be masked because it is buried in the core of the protein and not exposed on the
surface of the protein. Unmasking of NLSs, and nuclear translocation of cytoplasmic proteins may be triggered by phosphorylation, dephosphorylation, proteolytic digestion, subunit association or dissociation of an inhibitory subunit, or the like.
Accordingly, the masking and unmasking of NLSs provides a mechanism by which the transport of these cytoplasmic proteins into the nucleus may be regulated.
Nuclear translocation of transcription factors requires the presence of an unmasked or activated NLS in the nucleus-targeted protein. The binding of certain ligands to cell surface receptors activates the nuclear translocation of cytoplasmic
transcription factors. Once in the nucleus, these transcription factors exert gene expression modulatory activity.
NF-.kappa.B is a ubiquitous transcription factor found in various levels and states of activation in different cell types. NF-.kappa.B is composed of several different subunits including p65, p50, c-rel p52 and p105. Recent studies suggest that
distinct NF-.kappa.B complexes contribute to the regulatory control of gene transcription. The function and regulation of NF-.kappa.B has been most well-characterized in lymphocytic cells. In these cells, there is a wide variety of target genes, e.g.,
immunoregulatory genes, that are regulated by NF-.kappa.B including .kappa. Ig light chains. Such genes include those that encode the interleukin-2" ("IL-2.alpha.") receptor, interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), tumor necrosis factor-.alpha. ("TNF-.alpha."), and the like.
In unstimulated cells, a major form of NF-.kappa.B is a heterodimer of p50 and p65 (RelA) subunits. Nonactive NF-.kappa.B is retained in the cytoplasm as an inactive complex by inhibitory proteins such as I.kappa.B.alpha., .beta. and .gamma..
When cells are appropriately stimulated, e.g., by a proinflammatory stimulus such as a cytokine, the I.kappa.Bs are degraded, thereby releasing free NF-.kappa.B dimers, which translocate to the nucleus and activate target genes, e.g., lymphokine genes
and other immunoregulatory genes. This response is transient and is terminated through delayed NF-.kappa.B-mediated I.kappa.B.alpha. induction.
Recently it has been demonstrated that glucocorticoids exert their immunosuppressive activity by inhibiting NF-.kappa.B nuclear translocation. Scheinman et al. (1995) Science 270:283-286 and Auphan et al. (1995) Science 270:286-290 independently
demonstrated that the inhibition is mediated by an increase in the induction by glucocorticoids of I.kappa.B inhibitory proteins. These investigators proposed that inhibitors of NF-.kappa.B may be useful immunosuppressive and anti-inflammatory agents.
Such an NF-.kappa.B nuclear translocation inhibitor, comprising an NLS from the p50 subunit of NF-.kappa.B attached to a membrane-permeable polypeptide motif, was described in Lin et al. (1995) J Biol. Chem. 270:14255-14258.
Nuclear translocation of proteins other than endogenous transcription factors and other cytoplasmic proteins also depends on the presence of an activated or unmasked NLS. For example, nuclear translocation of the retroviral preintegration
complex is a crucial step in human immunodeficiency virus type-1 ("HIV-1 ") replication in nondividing cells such as monocytes and growth-arrested T cells. Such translocation is dependent on the presence of an NLS in the N-terminal portion of HIV matrix
antigen ("MA") p 17. Indeed, the HIV-1 enhancer contains tandem binding sites for NF-.kappa.B that can be essential for virus replication (Ross et al. (I 991) J Virol. 65:4350-4358; Parrott et al. (1991) J Virol 65:1414-1419). Nuclear translocation of
the HIV-1 preintegration complex can be partially inhibited by an excess of the SV40 large T antigen NLS (Gulizia et al. (1994) J Virol. 68:2021-2025). Furthermore, Dubrovsky et al. (1995) Molecular Med. 2:217-230 reported that a series of compounds
capable of binding to and reacting with the HIV-1 MA p17 NLS inhibit HIV-1 replication in human monocytes.
In addition, tumorigenesis and tumor cell proliferation are regulated by the expression of oncoproteins, many of which are cytoplasmic transcription factors that are translocated into the nucleus by virtue of the presence of an NLS. Miller et
al. (1996) J Cell Biochemistry 60:560.
Prior to Applicants invention, those in the art failed to demonstrate the synergism between peptide inhibitors of nuclear translocation and other imunosuppressants, such as cyclosporin. Buelow et al. (1995) Transplantation 59:455 demonstrated
that therapy with a small synthetic peptide derived from the .alpha.1 helix of an HLA class I molecule (called the ALLOTRAP peptide), combined with a subtherapeutic dose of cyclosporin, led to the prolonged survival of allografts. The ALLOTRAP peptide,
however, does not inhibit nuclear translocation of protein, an essential element of Applicants invention.
Many immunosuppressants are known in the art to be useful in treating autoimmune disease and in preventing transplant rejection. Examples of known immunosuppressants useful in compositions of the present invention are cyclosporin A,
mycophenolate mofetil, rapamycin, FK506, and steroids. Compositions of the present invention comprising at least one peptide inhibitor of nuclear translocation of a protein also comprise at least one immunosuppressant. Together, the peptide inhibitor
and immunosuppressant work synergistically to provide better immune suppression than either treatment alone.
Accordingly, inhibitors of nuclear translocation of cytoplasmic proteins would be useful as gene expression modulating agents, immunoregulatory agents, antiviral agents, antitumor agents, and the like. Such inhibitors, in combination with other
immunosuppressant compounds such as cyclosporin A, would provide useful compositions to regulate immune responses (e.g., prevent transplant rejection).
SUMMARY OF THE INVENTION
The present invention provides for compositions comprising at least one immunosuppressant and a polypeptide that can be introduced into an intact cell for the purpose of inhibiting the nuclear translocation of a cytoplasmic protein. The
polypeptide contains at least one, more preferably two, NLSs and an amino acid sequence that can deliver the polypeptide through the cytoplasmic membrane into the cell. The inventors herein have found that such a composition exhibits surprisingly
superior immunosuppressive characteristics compared to using an immunosuppressant or polypeptide alone.
Accordingly, in one embodiment, the composition of the present invention comprises a polypeptide comprising a signal sequence peptide and at least one NLS covalently attached thereto.
In another embodiment, the invention is directed to a method of suppressing an immune response of a subject comprising administering to the subject an immunosuppressive amount of a composition comprising a polypeptide comprising a signal sequence
peptide and at least one NLS and an immunosuppressant. In a preferred embodiment, the immunosuppressant is cyclosporin A.
In a further embodiment, the invention is directed to a method of treating or preventing a viral infection in an individual comprising administering to the individual an effective antiviral amount of a composition comprising a polypeptide
inhibitor of nuclear translocation of a cellular protein, said inhibitor comprising a signal sequence peptide and at least one NLS, and an immunosuppressant.
In yet a further embodiment, the invention is directed to a method of preventing transplant rejection in a subject comprising administering to the subject a composition comprising an immunosuppressant and a polypeptide inhibitor of nuclear
translocation, wherein the polypeptide comprises a signal sequence peptide and at least one NLS. In a preferred embodiment the immunosuppressant is cyclosporin A.
These and other embodiments of the subject invention will readily occur to those of ordinary skill in the art in view of the disclosure herein.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1A is a graphical representation of the effect of PKKKRKVAAVALLPAVLLALLAPKKKRKVC (SEQ ID NO:1) (the "SV40MEM" polypeptide) on lipopolysaccharide ("LPS")-stimulated surface antigen expression in 70Z/3 murine leukemia pre-B cells (solid bar:
.kappa. Ig light chain; stippled bar: IL-2.alpha. receptor; striped bar: CD45).
FIG. 1B is a graphical representation of the effect of three inhibitory polypeptides on LPS-stimulated .kappa. Ig light chain production in 70Z/3 murine leukemia pre-B cells (diamonds: the SV40MEM polypeptide; squares: KKKYKAAVALLPAVLLALLAKKKYKC
(SEQ ID NO:2) (the "HIV-1 MEM" polypeptide); triangles: AKRVKLAAVALLPAVLLALLAKRVKLC (SEQ ID NO:3) (the "C-MYCMEM" polypeptide)).
FIG. 1C is a graphical representation of the effect of increasing LPS concentrations on SV40MEM-inhibited .kappa. Ig light chain production in 70Z/3 murine leukemia pre-B cells.
FIG. 2 is a graphical representation of the effect of the SV40MEM polypeptide on LPS-stimulated cytokine production in 70Z/3 murine leukemia pre-B cells (squares: untreated; diamonds: the SV40MEM polypeptide (10 .mu.M); circles: the SV40MEM
polypeptide (5 .mu.M); triangles: the SV40 NLS (10 .mu.M)).
FIG. 3 is a graphical representation of dose-response relationships of three inhibitory polypeptides on LPS-stimulated .kappa. Ig light chain production in 70Z/3 murine leukemia pre-B cells (triangles: a peptide containing only the fibroblast
growth factor ("FGF") signal sequence AAVALLPAVLLALLAP (SEQ ID NO:4) (the "MEM" peptide); squares: a polypeptide having the FGF signal sequence and the NLS of NF-.kappa.B p50 on the carboxy terminus of the signal sequence, namely,
AAVALLPAVLLALLAPVQRKRQKLMP (SEQ ID NO:5) polypeptide (the "NF-.kappa.BMEM" polypeptide); diamonds: the SV40MEM polypeptide comprised of L-amino acids; circles: the SV40MEM polypeptide comprised of D-amino acids.).
FIG. 4A is a graphical representation of the effect of inhibitory polypeptides on LPS-stimulated TNF-.alpha. production.
FIG. 4B is a graphical representation of the effect of inhibitory polypeptides on LPS-stimulated interleukin-8 ("IL-8") production. In both FIG. 4A and FIG. 4B the following symbols are used: squares--media control; crossed squares--the MEM
peptide (5 .mu.M); diamonds--the NF-.kappa.BMEM polypeptide (5 .mu.M); circles--the SV40MEM polypeptide comprised of L-amino acids (5 1.mu.M); and triangles--the SV40MEM polypeptide comprised of D-amino acids (5 .mu.M).
FIG. 5 is a graphical representation of the effect of the SV40MEM polypeptide on LPS-induced CD40 expression in 70Z/3 murine leukemia pre-B cells.
FIG. 6A is a graphical representation of the effect of the HIV-1MEM polypeptide on .sup.3 H-deoxyribothymidine uptake into peripheral blood mononuclear cells ("PBMCs").
FIG. 6B is a graphical representation of the effect of HIV-1MEM on viral p24 production in anti-CD3 stimulated PBMCs infected with HIV-1 primary isolate M1.
FIG. 6C is a photograph of a gel depicting the effect of HIV-1MEM polypeptide on the expression of proviral gag sequences in anti-CD3 activated PBMCs infected with HIV-1 primary isolate M1.
FIG. 7 is a photograph of a gel depicting the results of a polymerase chain reaction analysis of the effect of HIV-1MEM polypeptide on the expression of proviral gag sequences in H9 human lymphoma T-cells or Jurkat human leukemia T-cells infected
with HIV-1 primary isolate M1.
FIG. 8A is a photograph of gels depicting the results of polymerase chain analyses of the effect of HIV-1MEM and the NF-.kappa.BMEM polypeptide on the expression of proviral gag sequences in Jurkat T-cells infected with HIV.sub.LAI.
FIG. 8B is a photograph of gels depicting the results of polymerase chain analyses of the effect of HIV-1MEM and the NF-.kappa.BMEM polypeptide on the expression of 2-long terminal repeat ("LTR") circles in Jurkat T-cells infected with
HIV.sub.LAI.
FIG. 9 is a graphical representation of the effect of SV40MEM prepared from D-amino acids on proliferation of 70Z/3 murine leukemia pre-B cells.
FIG. 10 is a graphical representation of the effect of SV40MEM prepared from D-amino acids on proliferation of RAJI human B-cell leukemia cell line.
FIG. 11A is a graphical representation of the effect of an intravenous administration of the SV40MEM polypeptide on the in vivo response of mice to sheep red blood cells.
FIG. 11B is a graphical representation of the effect of an oral administration of the SV40MEM polypeptide on the in vivo response of mice to sheep red blood cells.
FIG. 12 shows the effect of BMS-205820 and C-MYCMEM (BMS-214572) on the anti-hemocyanin (KLH) response in mice.
FIG. 13A, shows the effect of the BMS-205820 polypeptide on the production of TNF-.alpha. in vivo.
FIG. 13B shows the effect of the BMS-205820 polypeptide on the production of IL-6 in vivo.
FIG. 13C shows the effect of the BMS-205820 polypeptide on the production of IL-10 in vivo.
FIG. 14 depicts the effect of BMS-205820, C-MYCMEM (BMS-214572), an SV40 NLS polypeptide containing a single NLS, a c-myc NLS alone, without a translocation sequence (AKRVKL (SEQ ID NO:6)) and a control non-NLS polypeptide, 377G, on
lipopolysaccharide (LPS) binding to CD14.
FIG. 15A is a time line depicting the administration scheme for the intraperitoneal injection of ovalbumin (OVA), nebulized OVA, BMS-205820 and C-MYCMEM.
FIG. 15B shows the % eosinophils in lung following the treatment outlined in FIG. 15A.
FIG. 16 shows the effect of BMS-214572 on splenic T-cell proliferation.
FIG. 17 demonstrates that BMS-205820 acts synergistically with cyclosporin A to inhibit human peripheral blood T-cell proliferation in response to activation with CD3.times.CD28.
DETAILED DESCRIPTION
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of protein chemistry and biochemistry, molecular biology, microbiology and recombinant DNA technology, which are within the skill of the art.
Such techniques are explained fully in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning. A Laboratory Manual, Second Edition (1989); DNA Cloning, Vols. I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J. Gait
ed. 1984); Nucleic Acid Hybridization (B. D. Hames & S. J. Higgins eds. 1984); Animal Cell Culture (R. K. Freshney ed. 1986); Immobilized Cells and Enzymes (IRL press, 1986); Perbal, B., A Practical Guide to Molecular Cloning (1984); the series,
Methods In Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).
All patents, patent applications and publications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.
As used in this specification and the appended claims, the singular forms "a," "an" and "the" include plural references unless the content clearly dictates otherwise.
A. Definitions
In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.
As used herein, the term "signal sequence" or "signal sequence peptide" is used to indicate a peptide that is capable of directing the movement of the polypeptide of which it is a part through a cell membrane. In particular, the term is used to
indicate a peptide that directs the movement of a polypeptide across the cytoplasmic membrane into the cell. The term "signal sequence" is intended to encompass not only the signal sequence of a particular polypeptide, but also fragments or derivatives
thereof that are capable of delivering a polypeptide through a cell membrane. A "signal sequence" may be composed of L- or D-amino acids, preferably D-amino acids.
The terms "nuclear localization sequence" and "NLS" are used interchangeably to indicate a peptide that directs the transport of a protein with which it is associated from the cytoplasm of a cell across the nuclear envelope barrier. The term
"NLS" is intended to encompass not only the nuclear localization sequence of a particular peptide, but also derivatives thereof that are capable of directing translocation of a cytoplasmic polypeptide across the nuclear envelope barrier. NLSs are
capable of directing nuclear translocation of a polypeptide when attached to the N-terminus, the C-terminus, or both the N- and C- termini of the polypeptide. In addition, a polypeptide having an NLS coupled by its N- or C-terminus to amino acid side
chains located randomly along the amino acid sequence of the polypeptide will be translocated. Adam et al. (1990) J Cell. Biol. 111:807-818. "Nuclear localization sequences" may be composed of D- or L-amino acids.
By "interchangeably flanked at its amino- and carboxy-termini by a first and a second NLS" is intended to mean that the first or second NLS may be located at either the amino- or carboxy-terminus of the signal sequence polypeptide.
An "inhibitor of nuclear translocation" is a polypeptide composed of a signal sequence peptide and at least two NLSs which inhibits, e.g., either decreases or halts, nuclear localization of a cytoplasmic protein. Preferably, the polypeptide
comprises a signal sequence peptide interchangeably flanked at its amino- and carboxy-termini by a first and a second NLS. The NLSs at the N- and C-termini may be the same or different. In one preferred embodiment, the signal sequence peptide and the
NLSs are each composed of L-amino acids. In another preferred embodiment, the signal sequence peptide and the NLSs are each composed of D-amino acids.
A "derivative" of a polypeptide is intended to include homologous polypeptides in which conservative amino acid substitutions have been made, as well as to include other amino acid substitutions that result in a polypeptide that retains its
function, e.g., as a signal sequence peptide, an NLS, or an inhibitor of nuclear localization. A "derivative" of a peptide may be a peptide mimetic.
"Peptide mimetics" are structures which serve as substitutes for peptides in interactions with acceptor molecules (see Morgan et al. (1989) Ann. Reports Med. Chem. 24:243-252 for a review of peptide mimetics). Peptide mimetics, as used herein,
include synthetic structures which may or may not contain amino acids and/or peptide bonds, but retain the structural and functional features of a peptide ligand. The term, "peptide mimetics" also includes peptoids and oligopeptoids, which are peptides
or oligomers of N-substituted amino acids (Simon et al. (1972) Proc. Natl. Acad. Sci. USA 89:9367-9371). Further included as peptide mimetics are peptide libraries, which are collections of peptides designed to be of a given amino acid length and
representing all conceivable sequences of amino acids corresponding thereto. Methods for the production of peptide mimetics are described more fully below.
Two polypeptide sequences are "substantially homologous" when at least about 85% (preferably at least about 85% to 90%, and most preferably at least about 95%) of the nucleotides or amino acids match over a defined length of the molecule. As
used herein, substantially homologous also refers to sequences showing identity to the specified polypeptide sequence.
The terms "polypeptide", "peptide" and "protein" are used interchangeably and refer to any polymer of amino acids (dipeptide or greater) linked through peptide bonds. Thus, the terms "polypeptide", "peptide" and "protein" include oligopeptides,
protein fragments, analogues, muteins, fusion proteins and the like.
The following single-letter amino acid abbreviations are used throughout the text:
______________________________________ Alanine A Arginine R Asparagine N Aspartic acid D Cysteine C Glutamine Q Glutamic acid E Glycine G Histamine H Isoleucine I Leucine L Lysine K Methionine M Phenylalanine F Proline P Serine S
Threonine T Tryptophan W Tyrosine Y Valine V ______________________________________
By an "isolated polypeptide" is meant a polypeptide which is devoid of, in whole or part, tissue or cellular components with which the protein is normally associated in nature. Thus, a polypeptide contained in a tissue extract would constitute
an "isolated" polypeptide, as would a polypeptide synthetically or recombinantly produced.
By "mammalian subject" is meant any member of the class Mammalia, including, without limitation, humans and non-human primates, such as chimpanzees and other apes and monkey species; farm animals such as cattle, sheep, pigs, goats and horses;
domestic mammals such as dogs and cats; and laboratory animals including rodents such as mice, rats and guinea pigs. The term does not denote a particular age. Thus, adult, newborn and fetal mammals are intended to be covered.
The term "treatment" as used herein refers to either (i) the prevention of infection or reinfection (prophylaxis), or (ii) the reduction or elimination of symptoms of the disease of interest (therapy).
The term "immunosuppressant" is used to refer to any compound that is known or found to suppress or prevent an undesired immune response, e.g., prevent the immune system's rejection of a transplanted organ. Examples of "immunosuppressants"
include, but are not limited to, cyclosporin A, mycophenolate mofetil, rapamycin, FK506, steroids, and any other known immunosuppressant compound. One or more immunosuppressants may be used in a composition of the present invention.
The term "composition" when used herein refers to a composition comprising a polypeptide inhibitor of nuclear translocation and an immunosuppressant.
By "immunosuppressive amount" is meant an amount of a composition of the present invention sufficient to stop or suppress an undesired immune response (e.g., stop or slow progression of an autoimmune disease) or prevent an immune response from
occurring (e.g., prevent immune rejection of a transplanted tissue or organ). The exact amount of a composition of the present invention that is immunosuppressive can be determined by one skilled in the art, and depends upon such factors as target
indication, a subject's age, weight and health, and mode of delivery.
B. General Methods
Central to the present invention is the discovery that compositions comprising at least one immunosuppressant in combination with at least one polypeptide molecule that inhibits nuclear localization of cytoplasmic proteins are more effective at
modulating an immune response than either the immunosuppressant or polypeptide alone. The polypeptide molecules comprise a signal sequence peptide and at least on e NLS. The polypeptide inhibitors, and derivatives thereof, provide useful tools for
introducing an exogenous polypeptide comprising an NLS into an intact cell to inhibit nuclear translocation of a cellular protein, for studying the role of nuclear translocation in the regulation of cellular processes.
Since the nuclear translocation of certain cellular peptides is required for the host organism to mount an immune response, the polypeptide inhibitors in combination with other immunosuppressants are useful as immunosuppression compositions.
Immune responses are typically manifested by the expression of antibodies, the production of a number of cytokines, and/or the expression of cell surface receptors. Thus, inhibition of immune responses by the compositions of the present invention can
take the form of: inhibition of antibody production, including the production of antibody component peptides such as a .kappa. light chain polypeptide; inhibition of cytokine production, including such cytokines as interleukin-1, interleukin-2,
interleukin-4, interleukin-6, interleukin-10, tumor necrosis factor, or granulocyte-macrophage colony-stimulating factor; and/or the inhibition of the expression of cell-surface receptors such as an interleukin-2 receptor, gp39, CD40, CD45, CD80, CD86,
ICAM, ELAM, major histocompatibility complex ("MHC") class II, or VCAM. Clark et al. (1994) Nature 367:425.
By virtue of their superior immunosuppressive activities, the compositions of the present invention, comprising at least one polypeptide inhibitor and at least one additional immunosuppressive agent, are useful in the treatment of a wide variety
of immune disorders, including but not limited to, the treatment of autoimmune diseases such as rheumatoid arthritis, multiple sclerosis, juvenile-onset diabetes, systemic lupus erythematosus (SLE), autoimmune uveoretinitis, autoimmune vasculitis,
bullous pemphigus, myasthenia gravis, autoimmune thyroiditis or Hashimoto's disease, Sjogren's syndrome, granulomatous orchitis, autoimmune oophoritis, Crohn's disease, sarcoidosis, rheumatic carditis, ankylosing spondylitis, Grave's disease, and
autoimmune thrombocytopenic purpura (see e.g., Paul, W. E. (1993) Fundamental Immunology, Third Edition, Raven Press, New York, Chapter 30, pp. 1033-1097; and Cohen et al. (1994) Autoimmune Disease Models, A Guidebook, Academic Press, 1994), as well as
to prevent the rejection of transplanted tissue or organs.
Similarly, the compositions of the present invention are useful for treating physical symptoms manifested by responses to allergens which can initiate a state of hypersensitivity, or which can provoke a hypersensitivity reaction in a subject
already sensitized with the allergen. Such physical symptoms include asthma, joint swelling, urticaria, and the like. Additionally, due to the superior immunosuppressive properties, the compositions of the present invention are useful in the treatment
of sepsis and in the prevention of septic shock, a potentially lethal condition caused by the uncontrolled production of certain cytokines due to the presence of endotoxins, such as lipopolysaccharide (LPS), from extracellular bacteria.
Furthermore, since many viruses, e.g., herpes virus, cytomegalovirus, retroviruses, and the like, make use of the host cell's nuclear translocation machinery, the compositions of the present invention comprising inhibitory polypeptides are useful
as antiviral agents. In addition, since tumorigenesis and tumor cell proliferation appear to be mediated by the expression of oncogenes to make oncoproteins, many of which are transcription factors that are translocated into the nucleus, Miller et al.
(1996), supra, the compositions of the present invention comprising polypeptide inhibitors, or derivatives thereof, can be used to suppress tumor growth.
Polypeptide inhibitors useful in the compositions of the present invention include sequences of amino acids that comprise signal sequences from such polypeptides as the antennapedia homeodomain, FGF, HIV Tat, or Hsc70, and derivatives or mimetics
thereof capable of delivering the inhibitor through the cytoplasmic membrane into the cell. Preferred signal sequences include RQIKIWFQNRRMKWKK (SEQ ID NO:7), AAVALLPAVLLALLA (SEQ ID NO:8), AAVALLPAVLLALLAP (SEQ ID NO:4), CFITKALGISYGRKKRRQRRRPPQGSQTH
(SEQ ID NO:9), and the like, or derivatives or mimetics thereof capable of delivering the inhibitor through the cytoplasmic membrane into the cell.
Candidate signal sequences can be tested for their ability to direct the translocation of proteins across cell membranes, for example, by monitoring the localization of exogenous detectably labeled proteins into the cell cytoplasm. Lin et al.
(1995), supra, describe the use of radiolabeled proteins. In vitro nuclear peptide import can be measured using NLS peptides coupled to a fluorescent protein by methods described in Adam et al. (1990), supra.
Polypeptide inhibitor useful in compositions of the present invention further comprise at least one NLS, more preferably at least two NLSs. The NLSs can be covalently bonded to the N-terminus, to the C-terminus, to both the N- and C-termini of
the signal sequence polypeptide, to amino acid side chains located along the amino acid sequence of the signal sequence polypeptide, or any combination thereof. Preferably, the signal sequence polypeptide is interchangeably flanked at its aminoand
carboxy-termini by a first and a second NLS. If at least two NLSs are present, first and second NLSs may be the same or different. A discussion of NLSs and a list of NLSs can be found in Boulikas (1993) Crit. Rev. Eukaryotic Gene Expression 3:193-227,
and references cited therein.
Approaches for identifying NLSs include: (1) gene fusion experiments between a candidate NLS-coding DNA segment and the gene coding for a cytoplasmic protein (see, e.g., Silver et al. (1984) Proc. NatL. Acad. Sci. U.S.A. 81:5951; Moreland et
al. (1987) Mol. Cell. Biol. 7:4048; and Picard et al. (1987) EMBO J 6:3333); (2) nuclear import of nonnuclear proteins conjugated to synthetic NLS peptides (see, e.g., Goldfarb et al. (1986) Nature 322:641; Markland et al. (1987) Mol Cell. Biol
7:4255; and Chelsky et al. (1989) Mol. Cell. Biol. 9:2487); and (3) site-directed mutagenesis of a specific segment of a nuclear protein, resulting in its cytoplasmic retention (see, e.g., Greenspan et al. (1988) J Virol. 62:3020; van Etten et al.
(1989) Cell 58:669; and Boulukos et al. (1989) Mol. Cell. Biol.9:5718).
Preferred NLSs include PKKKRKV (SEQ ID NO: 10) and KKKRKVC (SEQ ID NO:1 1) from the SV40 large T antigen (see, Kalderon et al. (1984) Cell 39:499), GKKRSKA (SEQ ID NO: 12) from yeast histone H2B (see, Moreland et al. (1987) Mol. Cell. Biol.
7:4048), KRPRP (SEQ ID NO:13) from adenovirus EIA (see, Lyons et al. (1987) Mol. Cell. Biol. 7:2451), GNKAKRQRST (SEQ ID NO:14) from the v-rel oncogene of the avian reticuloendotheliosis retrovirus strain T (see, Gilmore et al. (1988) J Virol.
62:703), GGAAKRVKLD (SEQ ID NO:15) from the human c-myc oncoprotein (see, Chelsky et al. (1989) Mol. Cell. Biol. 9:2487), SALIKKKKKMAP (SEQ ID NO:16) from the murine c-abl (IV) gene product (see, Van Etten et al. (1989) Cell 58:669), RKLKKLGN (SEQ ID
NO:17) from the human or rat androgen receptor (see, Guiochon-Mantel et al. (1989) Cell 57:1147), PQPKKKP (SEQ ID NO:18) from protein p53 (see, Dang et al. (1989) J Biol. Chem. 264:18019)), ASKSRKRKL (SEQ ID NO:19) from viral Jun, a transcription factor
of the AP-1 complex (see, Chida et al. (1992) Proc. Natl. Acad. Sci. USA 89:4290), KKKYK (SEQ ID NO:20) and KKKYKC (SEQ ID NO:21), both of which are from the human immunodeficiency virus matrix protein (see, Bukrinsky et al. (1993) Nature 365:666),
KSKKK (SEQ ID NO:22) from the human immunodeficiency virus matrix 2 protein (see, Bukrinsky et al. (1993), supra), AKRVKL (SEQ ID NO:6) and KRVKLC (SEQ ID NO:23) both of which are from the human c-myc oncoprotein (see, Chelsky et al. (1989), supra), and
derivatives and mimetics thereof that are effective as an NLS.
Polypeptide inhibitors useful in compositions of the present invention may be synthesized by conventional techniques known in the art, for example, by chemical synthesis such as solid phase peptide synthesis. Such methods are known to those
skilled in the art. In general, these methods employ either solid or solution phase synthesis methods, well known in the art. See, e.g., J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd Ed., Pierce Chemical Co., Rockford, Ill. (1984)
and G. Barany and R. B. Merrifield, The Peptide: Analysis Synthesis, Biology, editors E. Gross and J. Meienhofer, Vol. 2, Academic Press, New York, (1980), pp. 3-254, for solid phase peptide synthesis techniques; and M. Bodansky, Principles of Peptide
Synthesis, Springer-Verlag, Berlin (1984) and E. Gross and J. Meienhofer, Eds., The Peptides: Analysis, Synthesis, Biology, supra, Vol. 1, for classical solution synthesis.
In general, these methods comprise the sequential addition of one or more amino acids or suitably protected amino acids to a growing peptide chain. Normally, either the amino or carboxyl group of the first amino acid is protected by a suitable
protecting group. The protected or derivatized amino acid can then be either attached to an inert solid support or utilized in solution by adding the next amino acid in the sequence having the complementary (amino or carboxyl) group suitably protected,
under conditions suitable for forming the amide linkage. The protecting group is then removed from this newly added amino acid residue and the next amino acid (suitably protected) is then added, and so forth. After all the desired amino acids have been
linked in the proper sequence, any remaining protecting groups and any solid support are removed either sequentially or concurrently to afford the final polypeptide. By simple modification of this general procedure, it is possible to add more than one
amino acid at a time to a growing chain, for example, by coupling (under condition that do not racemize chiral centers) a protected tripeptide with a properly protected dipeptide to form, after deprotection, a pentapeptide.
Typical protecting groups include t-butyloxycarbonyl (Boc), 9-fluorenylmethoxycarbonyl (Fmoc), benxyloxycarbonyl (Cbz), p-toluenesulfonyl (Tos); 2,4-dinitrophenyl, benzyl (Bzl), biphenylisopropyloxycarboxycarbonyl, cyclohexyl, isopropyl, acetyl,
o-nitrophenylsulfonyl, and the like. Of these, Boc and Fmoc are preferred.
Typical solid supports are generally cross-linked polymeric materials. These include divinylbenzene cross-linked styrene-based polymers, for example, divinylbenzene-hydroxymethylstyrene copolymers, divinylbenzene-chloromethylstyrene copolymers,
and divinylbenzene-benzhydrylaminopolystyrene copolymers. The divinylbenzene-benzhydrylaminopolystyrene copolymers, as illustrated herein using p-methyl-benzhydrylamine resin, offers the advantage of directly introducing a terminal amide functional
group into the peptide chain, which function is retained by the chain when the chain is cleaved from the support.
In one preferred method, the polypeptides are prepared by conventional solid phase chemical synthesis on, for example, an A | | |
