WO1994007366A1 - Therapie a base de pp14 - Google Patents

Therapie a base de pp14 Download PDF

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WO1994007366A1
WO1994007366A1 PCT/US1993/009216 US9309216W WO9407366A1 WO 1994007366 A1 WO1994007366 A1 WO 1994007366A1 US 9309216 W US9309216 W US 9309216W WO 9407366 A1 WO9407366 A1 WO 9407366A1
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polypeptide
receptor
cell
patient
cells
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Mark L. Tykocinski
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Priority to JP6509255A priority patent/JPH08502257A/ja
Priority to AU51659/93A priority patent/AU5165993A/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
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4715—Pregnancy proteins, e.g. placenta proteins, alpha-feto-protein, pregnancy specific beta glycoprotein
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/08—Antiallergic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • 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
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/74—Inducing cell proliferation

Definitions

  • PP14 placental protein 14
  • Human PP14 is a 28 kilodalton glycoprotein expressed during the first and second trimesters of pregnancy by the endometrium. During this period, it constitutes up to 5-10% of the total secreted protein from the endometrial decidua (Julkunen et al. , 92 Br. J. Obstet. Gynaecol. 1145, 1985) .
  • PP14 accumulates to significant levels in serum from pregnant women as well.
  • PP14 is found at high concentrations in male seminal fluid (19-515 mg/1) (Pockley et al., 43 Biochem. Soc. Trans. 317, 1989), although the cellular source of PP14 in the male reproduc- tive tract has not been determined.
  • PP14 belongs to a class of proteins called 3-lacto- globulins, which include lactoferrin (Rado et al. , 64 Blood 1103, 1984) and the retinoic acid receptor (Papiz et al., 324 Nature 383, 1986).
  • lactoferrin Roso et al. , 64 Blood 1103, 1984
  • retinoic acid receptor Papiz et al., 324 Nature 383, 1986.
  • a PP14 cDNA has been cloned from endometrium, and analysis of this endometrial PP14 cDNA has shown the PP14-coding region to be approxi ⁇ mately 70% homologous to other S-lactoglobulin family members, and their genomes are similar in size and organ ⁇ ization (Vaisse et al., 9 DNA Cell. Biol 401, 1990).
  • PP14 has been shown to inhibit lymphocyte prolifera ⁇ tion (Bolton et al., in Lancet 593, 1987). Extracts of human decidual tissue were added to mixed lymphocyte cultures (MLCs) , and a linear relationship was observed between the quantity of PP14 present, and the inhibition of lymphocyte proliferation observed. Id. Moreover, an anti-PP14 monoclonal antibody added to the MLCs inhibited the anti-proliferative effect, indicating a functional link between PP14 and anti-proliferative activity. Subse- quent studies have noted an inhibitory effect of PP14 on the synthesis of IL-1, IL-2 and soluble IL-2 receptors by peripheral blood mononuclear cells (Pockley et al.
  • Bolton et al. U.S. Patent 5,039,521. Specific ⁇ ally, Bolton et al. propose use of PP14 to treat arthri- tis, rheumatoid arthritis, asthma, graft-versus-host dis ⁇ ease, organ rejection, osteoarthritis, systemic lupus erythematosus, atopic allergy, multiple sclerosis, aller ⁇ gic dermatitis, inflammatory bowel disease, psoriasis, sarcoidosis, and other inflammatory disorders.
  • lymphoproliferative disorder such as malignant non-Hodgkin's lymphoma, Hodgkin's disease, or malignant histiocystotis.
  • Sugges ⁇ tion is also made to treat inflammatory and autoimmune diseases, and to treat infertility and a neoplastic disorder such as leukemia.
  • monoclonal antibodies to PP14 can be used to treat an immune system disorder, specifically, acquired immuno ⁇ deficiency syndrome (AIDS) .
  • AIDS acquired immuno ⁇ deficiency syndrome
  • PP14 is expressed in cells outside of the female and male reproductive tracts, and specifically, that PP14-encoding mRNA and PP14 protein is expressed in hematopoietic cells.
  • PP14 cDNAs are detectable.
  • This hematopoietically expressed PP14 has two poly- peptide isoforms, encoded by two mRNA species that arise through alternative splicing of the PP14 gene. These two polypeptide isoforms, and their corresponding mRNAs, are designated “PP14.1” and “PP14.2” herein.
  • PP14.1 polypeptide isoforms
  • PP14.2 polypeptide isoforms
  • App ⁇ licant discloses several methods for treatment of patients suffering from untoward effects of platelets, and for blocking immunosuppression associated with PP14 release from platelets in such patients. These methods are based upon blocking PP14 effects. This comprises, but is not limited to, the therapeutic use of anti-PP14 antibodies, peptides which block PP14 activity, solubilized PP14 receptor, and anti-PP14 receptor antibodies, or their equivalent (e.g.. active fragments thereof, which can be identified by standard procedures) . Compositions or therapeutic formulations, including these therapeutic agents are also disclosed.
  • the invention also includes administration, to a patient suffering from platelet-induced immunosuppression, of a reagent that will inhibit PP14 production by hemato- poietic cells of the patient.
  • a reagent that will inhibit PP14 production by hemato- poietic cells of the patient.
  • Antisense PP14 oligonucleo- tides and ribozymes are suitable reagents for interfering with PP14 transcription and/or translation. Methods for identifying additional reagents for blocking PP14 produc- tion at the mRNA or protein levels are straightforward, and readily practiced by those familiar with the art.
  • the invention features methods for treating a patient suffering from leukemia, where the leukemic cells of the patient have megakaryocytic differ- entiative potential and produce a PP14 polypeptide with immunosuppressive activity.
  • the methods for blocking PP14 function and reducing PP14 production cited above can also be applied in this clinical setting.
  • the invention also features production of anti-PP14.1 and PP14.2-specific antibodies and PP14 receptors for therapeutic and diagnostic applications.
  • the discovery of a second PP14 isoform in hematopoietic cells permits the generation of well-characterized anti-PP14 monoclonal antibodies with specificity for one or both isoforms.
  • Methods for producing both anti-PP14.1-specific and anti- PP14.2-specific antibodies are disclosed, and make use of the precise determination in the present invention of the amino acid sequence difference between the two isoforms. This knowledge permits the production and use as immuno- gens of peptides corresponding to junctional and internal amino acid sequences that distinguish between PP14.1 and PP14.2 polypeptides.
  • Such isoform-specific anti-PP14 antibodies are useful for detection of PP14 isoforms in the serum of patients, to determine which patients can benefit from PP14 blockade therapy, and to monitor therapeutic responses to such therapy.
  • - PP14 isoforms can serve as a marker for certain platelet disorders, such as disseminated intravascular coagulation, and PP14 isoform-specific or non-specific diagnostic assays are useful in this clinical setting.
  • PP14-isoform-specific anti- bodies allow for the discriminative analysis of hemato- poietic cell- and endometrial cell-derived PP14 poly- peptides.
  • This invention also allows production and use of a polypeptide derivative of a PP14 receptor (and/or anti- bodies with specificity for such a receptor) for blocking a functional interaction between a PP14 polypeptide and its receptor.
  • Methods for cloning receptors for known ligands such as ones based upon the use of readily puri ⁇ fied ligand:immunoglobulin Fc conjugates, are well-estab- lished and can be expeditiously carried out by those fam ⁇ iliar with the art.
  • this invention features any method by which the activity of either PP14 polypeptide isoform is reduced to thereby interfere with PP14-induced immunosuppression.
  • reduction of PP14 activity is useful for treatment of disease states (other than AIDS) characterized by elevated PP14 levels.
  • Those in the art will recognize that all of the various methods discussed above, as well as others (such as use of blocking PP14 peptides or blocking anti- idiotypic antibody mimics of PP14) , achieve the same end as one another, that is, the prevention of PP14-induced immunosuppression, by in some way affecting the end- function or production of PP14 in the body.
  • the invention also features therapeutic methods tar- geted at various immunological diseases. Unlike diseases that are the subject of PP14 blockade therapy, in which there is a need for reversal of immunosuppression, these other immunological diseases require exogenous immunosupp- ressive agents.
  • the PP14.2 polypeptide isoform provides a distinct immunosupp- ressive agent for pharmaceutical use which can be used independently of (or jointly with) PP14.1, or other immu- nosuppressive polypeptides.
  • PP14 therapeutic preparations having the hematopoietic PP14.2 polypeptide have advan ⁇ tages over PP14 therapeutic preparations consisting exclu ⁇ sively of the PP14.1 polypeptide, with respect to both stability (in blood and other tissue fluids) and thera ⁇ Commissionic efficacy for diseases described by Bolton, supra.
  • PP14 polypeptides can be used in known therapeutic methods which target cytokine circuits in the immune sys ⁇ tem, such as those predicated upon soluble interleukin-1 receptors for blocking interleukin-l:interleukin-l recep ⁇ tor interaction, for treating acute inflammatory condi ⁇ tions.
  • PP14.2 can also be used to suppress natural killer cell function.
  • the invention also features use of hematopoietic cells as a source of PP14 with immunosuppressive activity, providing the possibility of isolating both PP14.1 and PP14.2 polypeptides concurrently.
  • hematopoi ⁇ etic cells as well as other cells, can be used as cellu ⁇ lar transfection targets for the production of recombinant PP14.1 and PP14.2 polypeptides which will process and secrete the desired PP14 isoform in an appropriate fash ⁇ ion.
  • Both intact versions of these polypeptides, as well as polypeptide derivatives of these polypeptides having a desired biological activity can be readily generated.
  • a preferred composition includes a PP14 polypeptide sequence linked to a glycophosphatidylinositol (GPI) moiety.
  • GPI glycophosphatidylinositol
  • the invention also concerns production of PP14.1: PP14.2 heterodimers and PP14.2 homodimers. Dimers com ⁇ prising PP14.2 polypeptide are more stable than PP14.1: PP14.1 homodimers, and provide a more optimal therapeutic and diagnostic reagent. Standard cotransfection strate- gies can be effectively used to produce the heterodimers.
  • the invention features a method for treating a- patient suffering from a non-AIDS immunosuppression condition, by administering to the patient a reagent that specifically binds to at least one isoform of PP14, or to a receptor for a PP14 isoform, under physiological conditions, to thereby prevent or reduce binding of PP14 to its receptor.
  • non-AIDS immunosuppression is meant a condition which is caused by (or adversely affected by, or related to) an elevated level of a PP14 isoform. Provision of the reagent will allow the reagent to bind to the PP14 iso- form(s) , or its receptors, and thus neutralize the PP14 activity. Examples of such conditions include platelet disorders, such as disseminated intravascular coagulation, platelet-induced immunosuppression secondary to platelet transfusion, and thrombocytosis, and leukemia.
  • the reagent comprises, con ⁇ sists of, or consists essentially of, a compound selected from an antibody specific for PP14.1; an antibody (mono ⁇ clonal or polyclonal, which may be a humanized murine or non-primate antibody) specific for PP14.2, and antibody specific for PP14.1 and PP14.2, a receptor for a PP14 isoform, a portion of a receptor for a PP14 isoform, an antibody to a PP14 receptor, a polypeptide portion of a PP14 isoform, or any other reagent which competitively inhibits binding of PP14 to a PP14 receptor in vivo or in vitro, e.g..
  • a PP14 receptor antibody or anti-idiotypic antibody may be humanized, that is the framework may be derived from a human antibody and the complementarily determining regions from another organism, e.g.. a mouse or non-human primate.
  • the reagent prefer- ably competitively inhibits binding of PP14 and its receptor.
  • the polypeptide portion of PP14 includes that portion of the polypeptide defined by amino acids 33 through 54 of PP14.1 (i.e.. specific to PP14.1 and not PP14.2); the reagent may be (a) an anti-idiotypic antibody mimic of PP14 which competes with PP14 for bind ⁇ ing to (but does not- activate) a cellular receptor for PP14; (b) a soluble polypeptide derivative of a receptor for PP14 having the extracellular domain of a PP14 recep ⁇ tor free of the receptor's native transmembrane and cyto ⁇ plasmic domains; or (c) a soluble polypeptide derivative of a PP14 receptor:immunoglobulin Fc (or other useful tar ⁇ geting polypeptide) chimeric polypeptide.
  • the reagent may be (a) an anti-idiotypic antibody mimic of PP14 which competes with PP14 for bind ⁇ ing to (but does not- activate) a cellular
  • the invention features a method for cloning a PP14 receptor, by determining an amino acid sequence of the receptor and screening a library for a clone of the receptor using an oligonucleotide probe cor ⁇ responding to the amino acid sequence.
  • the method is a method for cloning a complementary DNA corresponding to a PP14 recep ⁇ tor including providing a chimeric polypeptide having a PP14 polypeptide linked to the sequence of an Fc region of an immunoglobulin (or its equivalent) ; contacting the chimeric polypeptide with a cellular extract from a cell expressing a PP14 receptor under conditions suitable for forming a complex of the chimeric polypeptide bound to the PP14 receptor; precipitating complex by contacting protein A or protein G (or its equivalent) conjugated to an insol ⁇ uble matrix with the complex; recovering the PP14 receptor from the matrix; determining the amino acid sequence of a portion of the PP14 receptor; and screening a cDNA library for a PP14 receptor CDNA using an oligonucleotide probe corresponding to the amino acid sequence of the PP14 receptor.
  • the invention features a method for producing an antibody with specificity for a receptor for a PP14 polypeptide, by immunizing a host with a por ⁇ tion of a receptor for a PP14 polypeptide.
  • the invention features a method for blocking immunosuppression in a patient by administer ⁇ ing to the patient a reagent that blocks transcription of a PP14 gene and/or translation of a PP14 transcript.
  • an antisense oligonucleo ⁇ tide, ribozyme, or a triplex-forming nucleic acid is used to block transcription and/or translation of a PP14 poly ⁇ peptide.
  • the invention features a method for identifying a reagent that blocks transcription of PP14, by screening a chosen compound for its capacity to block a PP14 isoform promoter-driven transcription of a reporter gene.
  • the invention features a method for identifying a patient with a platelet disorder, by contacting a sample from the patient with an antibody or other reagent with specificity for PP14.1 and/or PP14.2, and determining the amount of reaction of the antibody with the sample, compared to the amount of reaction observed in a normal patient not having a disorder.
  • the invention features a method for preparing PP14.1-specific antibodies, by immunizing a host with a polypeptide having an antigenic portion of the polypeptide defined by amino acids 33 to 54 of PP14.1.
  • the invention features a method for preparing PP14.2-specific antibodies, by immunizing a host with a polypeptide having a sequence of amino acids overlapping the junctional site of amino acids 32-33 of PP14.2; a pharmaceutical or other composition including an antibody specific for PP14.1 or PP14.2 in a pharmaceutic- ally acceptable buffer; a pharmaceutical or other composi ⁇ tion, including a portion of a PP14 polypeptide which com ⁇ petitively inhibits the binding of a native PP14 polypep ⁇ tide to its receptor in a pharmaceutically acceptable buf ⁇ fer; a pharmaceutical or other composition, including an anti-idiotypic antibody mimic of PP14 which competes for binding to, but does not activate, a cellular receptor for PP14; a pharmaceutical or other composition including a soluble polypeptide derivative of a receptor for PP14 hav ⁇ ing the extracellular domain of a PP14 receptor free of the receptor's native transmembrane and cytoplasmic domains;
  • composition containing as its active reagent at least the noted compound in a buffer which is suitable for administration to a human or other animal.
  • the term is used in its art-recognized manner, and includes those standard reagents known in the art.
  • the invention features a method for treating a patient in need of immunosuppression by administering to the patient a PP14.2 polypeptide.
  • the patient suffers from an autoimmune disease, rheumatoid arthritis, an allergic dis ⁇ order, transplant rejection, or graft-versus-host disease.
  • the invention features a method for preparing a PP14 polypeptide by isolating PP14 from a hematopoietic cell, e.g.. a phorbol myristate acetate- induced cell, or a platelet; and the invention features the resulting purified PP14 polypeptide PP14.1, or PP14.2.
  • the invention features a method for preparing a PP14.2 polypeptide by introducing a trans- criptional cassette having a promoter transcriptionally linked to a portion of a coding sequence for PP14.2; a composition having a transcriptional cassette having a eukaryotic or prokaryotic promoter linked to a portion of a coding sequence for PP14.2; and a composition including a PP14 receptor-binding portion of a PP14 polypeptide linked to a glycosylphosphatidylinositol (GPI) moiety or its equivalent cell surface binding portion.
  • GPI glycosylphosphatidylinositol
  • the invention fea ⁇ tures a method for producing a PP14 polypeptide, by trans- fecting a cell with a transcriptional cassette including nucleic acid encoding a portion of a PP14 polypeptide linked to a cell targeting signal sequence (e.g.. glyco- sylphosphatidylinositol (GPI)), to express a PP14 poly ⁇ peptide linked to the signal sequence at the cell surface; and cleaving the signal sequence with a cleaving reagent, thereby releasing the PP14 polypeptide into the medium for subsequent recovery.
  • a cell targeting signal sequence e.g. glyco- sylphosphatidylinositol (GPI)
  • the invention also features a composition having a portion of a PP14 receptor-binding polypeptide linked to a portion of a member of a specific-binding pair (e.g. , streptavidin or avidin with a biotin-binding domain) ; and a purified or recombinant PP14 receptor, PP14 receptor antibody, anti-idiotypic antibody, and purified nucleic acids encoding therefore; and a cell having a transcrip- tional cassette including nucleic acid encoding a portion of a PP14 polypeptide linked to a cell surface resident specific signal sequence, such as a glycosylphosphatidyl- inositol (GPI) signal sequence, to express a PP14 polypep ⁇ tide linked to a GPI moiety at the cell surface.
  • a specific-binding pair e.g. , streptavidin or avidin with a biotin-binding domain
  • purified is meant that the composition is dif ⁇ ferent from that naturally occurring in nature in that the active ingredient is at higher purity relative to one or more other compounds naturally associated with it.
  • recombinant is meant produced by genetic engineering procedures, e.g. , it is expressed from a cloned gene or its equivalent.
  • the invention also features a composition including a heteromultimer of PP14.1 and PP14.2 polypeptide chains, or a homomultimer of PP14.2 polypeptide chains, and a method for producing such a heteromultimer by cotransfect- ing PP14.1 and PP14.2 transcriptional cassettes into a cell.
  • the invention features a method for suppressing natural killer cell activity by contacting such cells with a PP14.2 polypeptide; a PP14 polypeptide modified to target a cell surface (e.g.. an antigen- presenting cell) , e.g.. by covalent bonding of a polypep ⁇ tide moiety, such as GPI or its equivalent; and a method for coating an antigen-presenting cell by use of such a targeted PP14 polypeptide.
  • a PP14-GPI chim ⁇ eric polypeptide will target the cell surface of an anti ⁇ gen-presenting cell, or a PP14-streptavidin polypeptide will target a cell coated with biotin.
  • Such cells are useful for therapeutic or diagnostic procedures based upon detection of or binding to PP14, as discussed herein.
  • FIG. 1 is a RNA transfer blot demonstrating that PP14 mRNA expression is induction- and lineage-specific in leu- kemic cells differentiating along the megakaryocytic line ⁇ age.
  • Total RNA from uninduced and induced K562, HL-60, U937, and PLB-985 cell lines were probed with a purified PP14 probe.
  • K562 cells were induced along the megakaryo ⁇ cytic lineage for 0, 24, 48, and 72h with PMA (lOnM) , or along the erythroid lineage for 0, 24, 48, and 72h with hemin (50 ⁇ M) .
  • HL-60 cells were induced along the macro- phage lineage for 0 or 48h with PMA (lOnM) , or along the neutrophil lineage for 0 or 48h with DMSO (2%) .
  • U937 cells were induced along the macrophage lineage for 0 or 48h with PMA (lOnM)
  • PLB-985 were also induced along the macrophage lineage for 0 or 48h with PMA (lOnM) .
  • Peripheral blood mononuclear cells (PBL) stimulated with anti-CD3 antibody for 3 days serves as a negative control.
  • the approximately 800bp band corresponding to PP14 mRNA is indicated by an arrowhead.
  • Fig. 2 is a RNA transfer blot demonstrating the kine ⁇ tics of PP14 expression after PMA-induction and showing that PP14 mRNA is detectable after 6 hours of inducer treatment.
  • Fig. 3 is a copy of an autoradiogram showing PP14 immunoprecipitates from PMA-induced K562 cultured medium.
  • K562 cells were metabolically labeled by culturing in cys- teine and methionine free-medium with the addition of
  • Fig. 4 is a two-way mixed lymphocyte culture showing that a potent inhibitory effect of PMA-induced K562 condi ⁇ tioned medium upon alloantigen-stimulated proliferation can be neutralized with anti-PP14 antibodies.
  • K562 cells were induced with PMA (lOnM) for 24 or 48h to induce PP14 expression.
  • Conditioned medium was added to the mixed lymphocyte culture (MLC) at 50% v/v ratio with or without a rabbit polyclonal anti-PP14 antiserum (1:100 dilution). The MLC was harvested at days 5, 6, or 7, and incorporated 3 H-thymidine cpm were counted. Values represent the average of triplicate cultures, and the experiment was repeated two times with similar results.
  • MLC mixed lymphocyte culture
  • Figs. 5a-5c show a schematic representation of PP14.1 and PP14.2 mRNAs and highlight the nucleotide and amino acid sequence differences between the two.
  • Fig. 5a Exon-1 and exon-2, along with intron-1, of the PP14 gene are schematized, with differential shading of the two halves of exon-2; the upstream portion of exon-2 is elimi ⁇ nated from PP14.2 mRNA by alternative splicing. The dashed line indicates the stretch of sequence deleted by such alternative splicing.
  • Fig. 5b The nucleotide sequence of the portion of the PP14 gene involved in the alternative splicing event is shown.
  • the common splice donor (SD) site and the two alternative splice acceptor (SA) sites are highlighted by arrows, and the dinucleo- tides at the splice sites are boxed.
  • Fig. 5c The amino acid sequence of the portion of PP14.1 that is deleted in the PP14.2 polypeptide is boxed. Numbers indicate the boundaries of the deletion, based upon numbering of the PP14.1 amino acid sequence.
  • Fig. 6 is a reverse transcriptase-polymerase chain reaction (RT-PCR) analysis using PP14-specific primers able to distinguish between the PP14.1 and PP14.2 mRNA species in PMA-induced K562 cells of the megakaryocytic lineage and placenta cells.
  • PP14.2 mRNA is present in significant amounts only in K562 cells, not in placenta cells taken from 18, 28, 36 and 37 weeks old placenta.
  • Molecular weight numbers are included on the right-hand side.
  • Lane 5 spleen. Lane 6: small intestine. Lane7: liver. Lane 8: kidney. Lane 9: brain meninges. Lane 10: KM-102 bone marrow fibroblastoid stromal cells. Lane 11: placenta at 18 weeks. Lane 12: placenta at term. Lane 13: fibroblasts. Lane 14: primers alone. Lane M - molecular weight markers. Arrows indicate the relative portions of Ppl4.1 and PP14.2 as well as molecu ⁇ lar weight markers.
  • PP14 Platelet-derived PP14 Isoforms and Dimers Placental protein 14 (PP14) , originally named after the placental tissue it was thought to derive from, was later, shown to originate from associated endometrial tissue (Vaisse et al. , 9 DNA Cell. Biol. 401, 1990). Sub ⁇ sequent studies indicated that PP14 was present not only in the endometrial decidua and serum of pregnant women, but also in the seminal fluid of men. Applicant has dis ⁇ covered that PP14 is also produced in cells outside of the reproductive tract.
  • PP14 mRNA and protein has been local ⁇ ized not only to a human leukemic cell line induced to differentiate along the megakaryocytic lineage, but also to the end-cell of that lineage, namely, the platelet.
  • Cloning and hybridization analyses of PP14 mRNA, and immunoprecipitation analyses of PP14 protein have further established that whereas endometrial PP14 is composed of a single dominant species, hematopoietic PP14 (as detected in PMA-induced K562) is comprised of two codominant spe ⁇ cies.
  • One of the PP14 mRNA species comigrates with endo ⁇ metrial PP14 mRNA, whereas the second one is shorter.
  • the latter contains an internal deletion that is predicted to yield a 22 amino acid deletion in the encoded protein.
  • the undeleted and deleted variants have been here given the designations PP14.1 and PP14.2, respectively.
  • K562 (available from the American type culture col ⁇ lection) human myeloid leukemic cells, can be induced to differentiate along the megakaryocytic lineage by phorbol 12-myristate 13-acetate (PMA) , and provides a model cellu- lar system for addressing molecular issues in megakaryo- cytopoiesis.
  • Equivalent cell lines can be isolated by methods known in the art. Megakaryocytic markers known to be expressed by such cells include platelet glycoprotein Ilia (gpllla) , platelet-derived growth factor (PDGF) alpha and ⁇ chains, and transforming growth factor ⁇ (TGF8)
  • the human myelogenous leukemia cell line K562 (ATCC 243) was maintained in RPMI medium (Whittaker Bioproducts) supplemented with 10% heat-inactivated fetal calf serum
  • a phage cDNA library was constructed from a pool of three groups of K562 cells alternatively induced with PMA for 24, 48, and 72 hour. Such a pool was chosen to maxi ⁇ mize chances of finding different genes activated through ⁇ out the stochastic K562 differentiation program. Dupli ⁇ cate lifts of the "induced" K562 cDNA library were differ ⁇ entially screened with subtracted (induced minus uninduced K562) and nonsubtracted (uninduced K562) single-stranded probes.
  • RNA was isolated from cells using a standard guanidinium isothiocyanate/cesium chloride method. Poly (A)+ RNA was isolated using oligo (dT) cellulose. A cDNA library was constructed using 1.5 ⁇ g poly (A)+ RNA from K562 cells treated for 24, 48 and 72h (4.5 ⁇ g poly (A)+ RNA total) with PMA (lOOnM) . The library was constructed using the Uni-ZAP XR cloning kit according to manufac ⁇ turer's recommendations (Stratagene, La Jolla, CA) .
  • the DNA was UV cross- linked using the UV Stratalinker 1800 (Stratagene) and filters were allowed to dry.
  • Membranes for the primary screens were hybridized with non-subtracted single-stranded cDNA probe from untreated K562 cells (uninduced) and compared to duplicate lifts hybridized with subtracted single-stranded cDNA probe from PMA-treated K562 cells (induced) .
  • Secondary and tertiary screens were hybridized with single-stranded cDNA probe from untreated K562 cells and compared to duplicate lifts hybridized with non-subtracted single- stranded cDNA probe from PMA-treated K562 cells.
  • poly(A) + RNA from K562 cells treated with PMA for 24, 48, and 72 was pooled (l.O ⁇ g of poly (A) + RNA total) and reverse transcribed as described above, but with 250 ⁇ Ci ⁇ _- 32 P ⁇ dCTP.
  • the single-stranded cDNA was annealed with a 30-fold excess of photobiotinylated poly (A) + RNA from untreated K562 cells and subtracted according to manufac ⁇ turer's suggestions (Invitrogen, San Diego, CA) .
  • Membranes were prehybridized at 42°C overnight in 50% deionized formamide, 1% sodium dodecyl sulfate (SDS) , 10% dextran sulfate (MW 300,000), 1M NaCl, and 150 ⁇ g/ml of denatured sheared salmon sperm DNA (Sigma) .
  • Single- stranded cDNA probe was added and incubated for 48h at 42°C in a rotating oven (Hybaid, Middlesex, UK) . Lifts were washed 2x in 2x SSC, 1% SDS at 22°C for 10 min. ; then 3x in O.lx SSC, 0.1% SDS at 65°C for 30 min.
  • Membranes were exposed to Kodak AR film as necessary using inten ⁇ sifying screens at -70°C. Approximately sixty-thousand cDNA clones were ana ⁇ lyzed, and 127 putative positives were selected after the first round of screening. Two of the 127 cDNA clones were identified as PP14, after analysis and sequencing of all the clones in a random fashion and subsequent sequence comparisons with the Genbank sequence database (Pearson et al., 85 Proc. Natl. Acad. Sci. USA 2444, 1988).
  • RNA (lO ⁇ g) was isolated as described above, heated to 65°C for 15 min. in 50% formamide, 6% formalde ⁇ hyde, lx EPPS (N- (2-hydroxyethyl) - piperazine-N'-3-pro- panesulfonic acid) buffer (lx EPPS buffer: 20mM EPPS (pH 8.2), lOmM Na-acetate (pH 5.2), 2 mM EDTA) and sepa ⁇ rated on 1.2% agarose gels containing lx EPPS buffer and 6% formaldehyde.
  • the RNA was passively transferred to Duralon UV membranes (Stratagene) and UV crosslinked. Membranes were prehybridized as described above.
  • Probe was generated by random priming 20ng of purified PP14 DNA. The DNA was denatured by heating to 100°C for 10 min., then incubated for 30 min. at 37°C in 50 ⁇ Ci ⁇ 32 P ⁇ dCTP (Amersham) , 0.2mM each dGTP, dTTP, dATP and 10U Klenow enzyme in a buffer containing random hexanucleotides (Boehringer Mannheim, Indianapolis, IN) . Probe was hybridized overnight at 42°C. Membranes were washed and exposed as described above.
  • PP14 mRNA was undetectable in uninduced K562 cells and became apparent within 6 hours of PMA treatment (Fig. 1) .
  • PP14 mRNA reached plateau levels at 24h and remained relatively constant out to at least 72h (Figs, l and 2) .
  • a single broad PP14 mRNA band corresponding to a length of approximately 0.8 kilobases was observed in all cases, coinciding with the size reported for endometrial PP14 mRNA.
  • Example 4 Cell Specificity of PP14
  • PMA-induction of K562 along the megakaryocytic lineage was associated with high levels of PP14 mRNA accumulation
  • hemin (50uM) -induction of K562 cells along the erythroid lineage demonstrated no similar PP14 activation.
  • HL-60 another bipotential leukemic line, was tested for PP14 inducibility.
  • PP14 does not seem to simply be a phorbol ester-responsive gene. More- over, these data suggest that PP14 is not promiscuously expressed in leukemic cells. Instead, these data are consistent with the notion that there is a specific asso ⁇ ciation between PP14 and the megakaryocytic lineage of PMA-induced K562 cells.
  • Example 5 PP14 Protein Expression
  • K562 cells were cultured at 5xl0 6 cells/ml and treated with PMA as described above. Cells were labeled with 0.5mCi 35 S-cysteine and 35 S-methionine (ICN Biomedicals, Inc., Irvine, CA) in cysteine- and methionine-free media supplemented with 10% FCS which had been extensively dia- lyzed in PBS. The next day, cells were collected by cen ⁇ trifugation and conditioned media were saved.
  • K562 cells were metabolically labeled with a combina ⁇ tion of 35 S-cysteine and 35 S-methionine.
  • Conditioned medium and cell lysis extracts were then immunoprecipitated using a mouse monoclonal anti-PP14 antibody, or a polyclonal antibody.
  • Significantly, two protein species were noted in the 28 kilodalton size range, with the upper one comigrating with purified endometrial PP14 (data not shown) .
  • the two PP14. isoforms were present in approxi- mately equivalent amounts.
  • PP14 protein parallels PP14 mRNA in its PMA-inducibility in K562 cells.
  • two-way mixed lymphocyte cultures were set up as follows.
  • the blood from two unrelated donors was col ⁇ lected in heparin (lOU/ml blood) .
  • Blood was diluted two times in lx PBS; 0.3 vol. ficoll-paque (Pharmacia, Upp ⁇ sala, Sweden) was under-layered and then centrifuged for 30 min. (1000 x g) .
  • the leukocyte layer was removed, washed three times with lx PBS, and lymphocytes counted in a hemocytometer.
  • Cells were cultured in 96 well plates at 2 xlO 5 cells/well in triplicate.
  • endometrial PP14 has been shown to be a potent suppressor of lymphocyte proliferation.
  • conditioned medium from PMA-induced K562 cells was added to two-way mixed lymphocyte cultures, and the effect on proliferation, as measured by 3 H-thymidine incorporation on days 5, 6, and 7, was monitored.
  • conditioned medium was added to the MLC at a 1:2 ratio.
  • positive control MLCs when lymphocytes from two unrelated individuals were cocultured, a high degree of proliferation, peaking on days 6 and 7, was detected (data not shown) .
  • TGF3 has been reported to be produced by K562 cells, and furthermore, since TGFS is known to inhibit lymphocyte proliferation, one can presume that the TGF / S that is present has not been cleaved into its active immunomodulatory form. Significantly, PP14 does not require proteolytic cleavage for functional activation.
  • Example 8 mRNA species of PP14 isoforms
  • RNA transfer blot analyses did not clearly resolve distinct PP14 mRNA species corresponding to the polypeptide iso- forms. Reexamination of weaker exposures of the RNA transfer blots, however, suggested the presence of a tight doublet hybridizing to the PP14 probe.
  • RT-PCR reverse transcriptase polymer- ase chain reaction
  • RNA was reverse transcribed as described above using a PP14 specific pri ⁇ mer (5' -GGATCCCATGCTCCAAGGGTTTATTAATAACCTCTGC-3' ; Seq. I.D. No. 1). Resulting product was PCR amplified with a 5' primer (5'-GGTACCGCTCCAGAGCTCAGAGCCACCCACAG-3' ; Seq. I.D. No. 2) and a 3' (5' -GTGCAGAACGATCTCCAGGTTG-3' ; Seq. I.D. No.
  • the initial two PP14 cDNA clones obtained through differential cDNA screening proved to be only partial clones encompassing the 3'-end of PP14 mRNA only.
  • PCR cloning of full-length PP14 cDNA was performed by annealing a PP143'-end specific primer (5'- CATGCTCCAAGGG TTTATTAATAACCTCTGC-3' ; Seq. I.D. No. 4) and reverse tran ⁇ scribing as described above.
  • the resulting product was used directly in a PCR (described above) using the 3'-end specific primer and a 5' -end specific primer (5'-AGCTCAGA GCCACCCACAGCCGCAG-3' ; Seq. I.D. No 5).
  • the PCR product was gel purified and cloned into a T-vector.
  • a full-length PP14 cDNA was RT-PCR cloned from PMA- induced K562 mRNA using primers based upon the 3'-end sequence of K562 PP14 mRNA (identical to the endometrial PP14 mRNA sequence) and the published 5' -end sequence of endometrial PP14 mRNA.
  • DNA sequencing was performed using the Tabor and Ri-chardson sequencing method, and the SEQUENASE ® sequencing kit as per the manufacture's recom ⁇ mendations (United States Biochemicals, Cleveland) .
  • PCR product using PP14 end primers was size- fractionated and visualized on agarose gels, two distinct PCR products were noted (Fig. 6) .
  • the two mRNA species were designated PP14.1 and PP14.2, with the latter corres ⁇ ponding to the smaller RNA species.
  • the size difference between these two PCR products was consistent with the 66 nucleotide deletion documented in the K562 PP14 mRNA that had been PCR-cloned.
  • PP14.1 mRNA var ⁇ iant could be readily detected in endometrial tissue.
  • Immunoprecipitation from platelets was performed from platelets isolated using a modified citrate buffer.
  • platelets were spun at 1,100 x g for 15 min. at 22°C. Reductive methylation of platelet extracts was performed by resus- pending the platelet pellet after the final spin in a modified lysis buffer containing 50mM sodium phosphate (pH 7.0), 1% NP-40,150mM NaCl, 2 ⁇ g/ml leupeptin, 2 ⁇ g/ml aprotinin, 20 ⁇ g/ml PMSF. NaCNBrH 3 (Sigma) was added to 50mM and [ 14 C] formaldehyde (Amersha ) was added to lOmM. The mixture was allowed to reductively methylate for lh at 37°C.
  • Example 10 Screening for PP14 by PCR The finding of PP14 mRNA and protein in hematopoietic cells of the megakaryocytic lineage prompted screening of additional tissue sites outside of the reproductive tract for the presence of PP14 mRNA. PCR analysis was used to optimize sensitivity. Total cellular RNA, isolated from autopsy tissues, was reverse-transcribed using PP14- specific primer (Seq. I.D. No. 1) and amplified using the same oligonucleotide as 3' primer, and a 5' primer (Seq. I.D. No. 2) designed to allow resolution of the two PP14 mRNA variants. As seen in Fig.
  • PP14.1 and PP14.2 were detected, and clearly resolved, in the PMA-induced K562 cells serving as controls (lanes 2 and 3) .
  • the placental tissue contaminated with PP14-containing con ⁇ taminant endometrial decidua
  • the panel of other tissues examined were all negative for PP14 transcript. These tissues included brain (lane 4) , spleen (lane 5) , small intestine (lane 6) , liver (lane 7) , kidney (lane 8) , and meninges (lane 9) .
  • KM-102 human bone marrow stromal cells; lane 10
  • dermal fibroblasts lane 11
  • PP14 mRNA expression seems to be restricted to the reproductive and hematopoi ⁇ etic systems.
  • PP14 is produced by cells of the platelet lineage leads to novel therapies for reversing platelet-driven immunosuppression.
  • PP14 was previously thought to be a beneficial molecule, in its suggested physiological role as a blocker of alloresponses in the female and male reproductive tracts.
  • the present inven- tion discloses that PP14 can also be a deleterious mole ⁇ cule, in its pathophysiological role as a potent general immunosuppressive agent released by platelets in certain clinical settings, such as coagulopathies.
  • This discovery provides the first motivation for developing methods for blocking PP14 and its immunosuppressive effects. Methods are disclosed for carrying out this PP14 blockade.
  • PP14 blockade therapies involve both methods for interfering with PP14 protein interaction with a PP14 receptor, and methods for preventing PP14 transcription and/or translation (i.e.. PP14 blockade therapies) .
  • the survey of human leukemic cell lines reported in this study indicates that PP14 is not promiscuously expressed in leukemic lines. In fact, even in K562, it is only expressed following chemical induction with one par ⁇ ticular chemical inducer. Nonetheless, it is possible that under in vivo conditions, myeloid leukemic cells that share K562's megakaryocytic differentiative potential, may be triggered into a more differentiated state wherein they can express PP14. This could occur spontaneously or in response to therapeutic agents.
  • PP14 by virtue of its potent immunosuppressive function, is expected to play a pathogenic role in blocking effec ⁇ tive anti-tumor immune responses.
  • the secreted PP14 is in its biologic ⁇ ally active form.
  • the present invention provides methods for detecting PP14.2 in serum of patients. Such methods can readily be applied to patients with leukemia or related diseases in order to determine which patients are candidates for PP14 blockade therapy.
  • PP14 Blockade Therapy Patients with elevated levels of one or both of the PP14 polypeptide isoforms in their serum are candidates for PP14 blockade therapy. Such elevated levels of PP14 may occur in a variety of disease conditions where plate ⁇ lets release excess PP14 into the bloodstream. For exam- pie, sepsis is often associated with disseminated intra- vascular coagulation,- a condition which leads to the release of platelet contents into blood as the platelets coagulate. PP14 that is released in this clinical setting contributes to generalized immunosuppression which, in turn, further aggravates the sepsis. Hence, PP14 blockade serves to interfere with this pathogenic cycle and aid recovery.
  • the first step is to identify a patient in need of PP14 blockade therapy. Preferred diagnostic methods for accomplishing this are described below. Generally, those individuals having higher than normal tissue or serum levels of PP14.1 or PP14.2 or both are treated by methods of this invention. Such levels can be determined by use of antibody-based assays.
  • PP14 blockade can have therapeutic benefit.
  • Methods for blocking PP14 action include standard approaches for blocking proteins to achieve therapeutic endpoints.
  • monoclonal and/or polyclonal antibodies can be used with specificity for the two isoforms of PP14 as PP14 blocking agents.
  • Antibodies with specificity for PP14 have previously been described, but, the only suggested therapeutic benefit was for treatment of an immune system disorder, the only given example of which was AIDS, and in the absence of knowledge about the two PP14 isoforms, the isoform-specificity of the antibodies remained unknown.
  • Knowledge of the PP14.2 isoform in the present invention permits the development of antibodies with PP14.1 or PP14.2-specificity and their therapeutic use.
  • this invention features methods to treat all other non-AIDS- diseases (or even other non- immune system disorders) characterized by excess amounts of PP14 in a patient or tissue. It also features use of PP14.2-specific antibodies for treatment of all diseases associated with elevated PP14 levels.
  • a preferred strategy for producing anti-PP14.1-speci- fie antibodies is to immunize with a peptide largely lim ⁇ ited to the amino acid sequence within the PP14.1-specific sequence that is absent from PP14.2.
  • pep- tides overlapping the junctions between this sequence and the rest of PP14.1 can be used, as well as other PP14.1 sequences.
  • antibodies with cross- reactivity to both PP14.1 and PP14.2 can be eliminated by conventional approaches.
  • a preferred strategy for producing anti-PP14.2-speci ⁇ fic antibodies is to immunize with a peptide spanning the junction corresponding to the site where the PP14.1 sequence insertion is present. Standard peptide immuniza- tion protocols can be employed.
  • PP14 peptides having only a portion of the native PP14 polypeptides can be used to competitively inhibit interaction of PP14 with a receptor for PP14 (a PP14 receptor) to thereby interfere with the nonspecific immu- nosuppression mediated by the native PP14 polypeptide.
  • Straightforward in vitro experiments based upon competi ⁇ tive peptide inhibition and/or site-specific mutagenesis can be used to localize the appropriate PP14 subsequences for effecting such blockade, and can be carried out based upon standard protocols by those familiar with the art.
  • Preferred subsequences are generally those coinciding with hydrophilic amino acid sequence stretches.
  • anti-idiotypic antibodies that mimic PP14 in its capacity to bind to its receptor can be used for competitive blockade of a PP14 receptor. Methods for preparing such anti-idiotypic antibodies and their use are well-known to those familiar with the art, and are parti- cularly well-documented in the infectious disease literature.
  • the PP14 receptor instead of PP14 itself, can also be focussed upon for purposes of blocking the PP14:PP14 receptor interaction.
  • the purification and cloning of receptors in situations where the ligand is available in purified form, has become a straightforward exercise for those familiar with the art.
  • Prior to this invention the art provided no compelling therapeutic reason to purify and clone the PP14 receptor.
  • the issue of isoform-specificity for PP14 recep- tors could not be addressed.
  • PP14 is not simply a byproduct of pregnancy or restricted to a distal site of the male reproductive tract, but instead can be derived from platelets which contribute to pathophysiology, provides a compelling rea- son to purify and clone receptors for PP14. Structural characterization of the PP14 receptor allows, in turn, the generation of agents for blocking PP14:PP14 receptor interaction.
  • a preferred method for isolation and cloning the PP14 receptor involves generation and use of a PP14:immunoglob ⁇ ulin Fc chimeric polypeptide.
  • This chimeric polypeptide has the complete sequence of PP14, or a functional poly ⁇ peptide derivative thereof, linked to the Fc region of immunoglobulin Gl (IgGl) .
  • the latter serves as a useful tag for detecting and isolating the chimeric polypeptide, since the Fc region of immunoglobulin binds well to pro ⁇ tein A or protein G.
  • Analogous ligand:Fc chimeric poly ⁇ peptides have been used by a number of investigative groups to isolate specific receptors.
  • Hematopoietic cells such as monocytes and lymphocytes respond to PP14 and hence the mononuclear cell pool is a suitable cellular source from which to isolate PP14 receptors.
  • a detergent extract is prepared from said cells, and the PP14:immuno ⁇ globulin Fc chimera, produced by recombinant DNA methods in standard NOS cells or their equivalent, is added to the extract.
  • Protein A-sepharose chromatography is performed to isolate a complex in which the PP14 receptor complexed to the PP14:immunoglobulin Fc chimera. pH elution is used to recover the purified PP14 receptor.
  • Amino acid sequence for the amino-terminus, as well as for defined sub-peptides generated by peptidase cleavage, is deter ⁇ mined by conventional amino acid sequencing methods. Based upon this amino acid sequence, degenerate oligo- nucleotides encoding this amino acid sequence are syn ⁇ thesized using an oligonucleotide synthesizer. These degenerate oligonucleotides are then labeled and used as probes to screen a cDNA library from the same cells from which the receptor was originally purified. Hence, purif ⁇ ication and cloning of PP14 receptors can be carried out in a straightforward way using conventional methods.
  • PP14:Fc chimeras as a receptor trap
  • Alternative polypeptide tags can be appended to PP14 for detection and isolation purposes.
  • sequence of steps outlined here can be repeated for both PP14.1 and PP14.2, as well as heterodimeric PP14, poly ⁇ peptides in order to isolate and clone isoform-specific receptors.
  • native PP14 can be used in unmodi ⁇ fied form for binding to the receptor, and anti-PP14 antibodies can then be used to recover the PP14:PP14 receptor complex.
  • anti-PP14 receptor antibodies are infused into a patient in need of PP14- induced generalized or localized immunosuppression.
  • Anti- PP14 receptor antibodies can be generated using PP14 receptor, recombinant PP14 receptor or PP14 receptor pep- tides as immunogens. Methods for preparing monoclonal antibodies useful for human therapeutics are well- described in the scientific literature.
  • a soluble derivative of the PP14 receptor is infused into a patient in need of PP14-induced generalized immunosuppression.
  • This soluble derivative of the PP14 receptor can be readily generated through standard recombinant DNA methods. For example, a site-specific mutagenesis method can be employed to intro ⁇ quiz a stop codon at- the carboxy-terminus of the PP14 receptor extracellular domain.
  • This mutagenized coding sequence for a soluble PP14 receptor can be subcloned into any one of a number of available eukaryotic or prokaryotic expression vectors for quantitative production of this molecule.
  • An alternative therapeutic soluble derivative of PP14 receptor comprises the extracellular domain of the PP14 receptor linked to the Fc domain of immunoglobulin G. This latter molecule has the advantage of being a more stable molecule in vivo, as has been shown for other immunoglobulin Fc chimeric polypeptides, such as CD4: immunoglobulin Fc.
  • therapeutic methods can also be used which are based upon reducing levels of PP14 production. Such a reduction can be at transcriptional and post-transcriptional levels.
  • genetic therapeutic agents that interfere with PP14 expression by cells of the platelet lineage can be used.
  • a preferred agent for reducing PP14 production is an antisense PP14 oligonucleotide that has been covalently derivatized through known methods to enhance in vivo sta ⁇ bility and cell membrane penetration.
  • Isoform-specific antisense PP14 oligonucleotides can readily be designed based upon the PP14.1 and PP14.2 nucleotide sequence information disclosed herein, along with general guide ⁇ lines that are well-known to those familiar with the art for optimizing functional antisense oligonucleotides.
  • the antisense oligonucleotide can be directed against the 66 nucleotide stretch that is absent in PP14.2.
  • the antisense oligo ⁇ nucleotide can span the junctional site that overlaps the 66 nucleotide deletion of PP14.1 sequence such that the oligonucleotide binds only to this junction.
  • regu ⁇ latory polynucleotides can be readily designed, such as ones that incorporate ribozymes or that function as tri ⁇ plex-forming regulatory elements. There are well-estab ⁇ lished procedures for developing such agents and for screening their efficacies and toxicities. Other therapeutic methods can be developed that are based upon chemical agents that function as PP14 gene- specific transcriptional inhibitors. There are currently well-described methods for screening banks of chemicals for ones that will block transcription from a specific gene promoter.
  • One experimental approach involves gener ⁇ ating a reporter gene construct including the upstream sequence of the PP14 gene, with the PP14 promoter element, linked to a gene sequence encoding a readily detectable promoter, such as luciferase, beta-galactosidase, or chloramphenicol acetyltransferase.
  • This transcriptional cassette is stably transfected into a target cell that is capable of supporting active transcription from the PP14 promoter.
  • K562 cells are one example of a suitable cell line for this purpose.
  • Alternative cell lines with mega- karyocytic differentiation potential, as well as endo ⁇ metrial and testicular lines, can be readily identified.
  • K562 cells transfected with the chimeric reporter the cells are distributed into multi-well tissue culture plates, and chemicals to be screened are added to individual wells, along with PMA to activate the PP14 pro ⁇ moter. Wells containing cells failing to express the reporter are identified. This represents a relatively rapid method for screening large numbers of chemicals for relevant drug candidates. Once a candidate is identified, procedures well-established in the field of pharmacology are used to study efficacy and toxicity and to optimize dosage regimens.
  • PP14 characterizes cells of the platelet lineage, and leukemic cells corresponding to this lineage also express
  • PP14 Leukemic cells producing this potent immunosuppres- sive molecule .in vivo may be protected from effective anti-leukemic T-cell responses. Moreover, PP14 derived from these cells will lead to generalized immunosuppres ⁇ sion in the patient, rendering the patient susceptible to other diseases, including infectious diseases. Hence, the discovery that certain leukemic cells produce PP14 pro ⁇ vides motivation for screening patients with leukemia for PP14 expression in blood and leukemic cells. Those patients who are PP14-positive, can be treated by PP14 blockade therapy, by one of the methods discussed above.
  • PP14.2 isoforms Patients with megakaryocytic leukemia or chronic myeloid leukemia cells with megakaryocytic differentiation poten ⁇ tial, are particularly suitable candidates for this mode of therapy.
  • the present invention discloses the existence of the PP14.2 isoform. This disclosure is significant in that it teaches those skilled in the art how to effectively block platelet-derived PP14, in that both isoforms must be tar ⁇ geted with antibody, PP14 peptide, or PP14 receptor deriv- atized therapeutic compositions. Cloning of the PP14.2 isoforms has provided critical sequence information which allows the generation of PP14 isoform-specific reagents. Such reagents can be used either in combination or inde ⁇ pendently.
  • therapeutic benefit may be obtained in a pregnant patient who is in need of ameliora ⁇ tion of platelet-induced generalized immunosuppression, as for example in the clinical context of a pregnancy-associ ⁇ ated coagulopathy. Since in such a patient, PP14.1 block ⁇ ade is undesirable due to potential adverse effects upon the developing fetus, selective PP14.2 blockade is prefer ⁇ able. A therapeutic benefit will be derived by decreasing the PP14 load in the patient, even if there is residual PP14.1 from endometrium and platelets in the patient.
  • PP14 Diagnostic Assays The disclosure of PP14 in cells of the platelet lin ⁇ eage has diagnostic implications. Monoclonal and/or poly- clonal antibodies, prepared by the methods described above, permit the simple development of ELISA assays for measuring PP14 expression in serum. PP14.1- and/or PP14.2-specific antibodies can be used for this purpose. A sensitive ELISA can be used for PP14 detection in serum samples. Two anti-PP14 antibodies with specificities for distinct, non-overlapping portions of PP14 protein, can be combined in a conventional double-antibody sandwich ELISA. The present invention teaches that two isoforms of PP14 are produced by platelets, and hence, informs those skilled in the art how to develop suitable diagnostic assays. The ability to distinguish between the PP14.1 and PP14.2 polypeptide isoforms is of particular utility when diagnosing platelet disorders in pregnant women, since the PP14 polypeptides deriving from platelet and endometrial cells can be resolved.
  • this assay can serve as a sensitive diagnostic tool for determining whether there is in fact a coagulopathy in a patient where the diagnosis is uncertain. According to the latter, PP14 detection can serve as a diagnostic test for clinical disorders such as disseminated intravascular coagulation which are often difficult to diagnose.
  • PP14.2 polypeptide is a useful diagnostic marker for platelets.
  • This invention provides a novel immunosuppressive agent that can be used for the treatment of patients with diseases where immunosuppression is the desired endpoint.
  • diseases include, but are not limited to, autoimmune diseases, rheumatoid arthritis, allergic disorders such as allergic dermatitis, transplant rejection, and graft- versus-host disease in the context of bone marrow trans- plantation.
  • the present invention provides PP14.2 as an alternative to PP14.1 for achieving immunosuppression in patients in need of this.
  • a patient is identified who is in need of immunosupp ⁇ ression.
  • a composition including a PP14 polypeptide, or a functional polypeptide derivative thereof, is adminis ⁇ tered to the patient.
  • the route of administration and the precise components of the pharmaceutical preparation are dictated by the particular disease entity being treated. For example, in the treatment of a systemic autoimmune disease, intravascular or intramuscular administration is preferable. In contrast, topical application is prefer ⁇ able for allergic dermatitis, whereas intra-articular injection may be necessary in the context of rheumatoid arthritis.
  • Methods for optimizing clinical protocols involving therapeutic polypeptides are well-established, and the methods to be used for PP14 polypeptides parallel these. For example, an amount of PP14 between 1 and 1000 ⁇ for kg animal per day is suitable.
  • PP14.1 and/or PP14.2 serum levels in treated patients can be monitored in order to provide one measure of therapeutic efficacy.
  • the present invention discloses a significant set of new potential sources for both small and large scale pro ⁇ duction of not only the novel PP14.2 isoform, but also the PP14.1 isoform.
  • the availability of first and second trimester endometrium from cases of abortion is limited by several factors.
  • platelets can be used as a source for native PP14.
  • the finding of PP14 polypeptide in PMA-induced K562 cells, which proliferate rapidly in cell culture provides another potential source for native (i.e. , non-recombi- nant) PP14.
  • both PP14 isoforms can be obtained from these sources, and these further serve as a source for functional dimeric molecules.
  • other cell lines corresponding to the megakaryocytic lineage are alternative sources for native PP14.
  • expression constructs for PP14.2 can be introduced into K562 or other megakaryocytic lines for purposes of PP14 overexpression.
  • Recombinant monomeric PP14.2, homodimeric PP14.2, and heterodimeric PP14.1: PP14.2 can all be produced in this way.
  • Such cells pro ⁇ vide optimal cellular backgrounds in which proper post- transcriptional processing of PP14 mRNA and protein can take place. Methods for gene transfer and the expression and purification of recombinant proteins are well- established in the art.
  • soluble PP14.1 and PP14.2 polypeptides can be devised for producing soluble PP14.1 and PP14.2 polypeptides.
  • One preferred method involves the expression of chimeric PP14.1:GPI and PP14.2:GPI polypeptides, including monomeric and hetero ⁇ dimeric forms of these polypeptides, on the surfaces of adherent cells and the recovery of soluble PP14.1 or PP14.2 polypeptides by cleavage of the GPI membrane anchor.
  • This type of method has been used successfully for the production of other soluble molecules and offers the particular advantage of being compatible with con ⁇ tinuous-flow cell culture systems.

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Abstract

L'invention se rapporte à un procédé pour le traitement de patients souffrant d'une suppression immunitaire qui n'est pas le SIDA, ce procédé consistant à administrer un réactif qui se lie spécifiquement à une isoforme de PP14, ou un récepteur pour une isoforme de PP14 dans des conditions physiologiques. Un schéma de principe de la structure des gènes de PP14 et des informations de séquences partielles pour les gènes de PP14 et la protéine PP14.2 sont illustrés dans la figure.
PCT/US1993/009216 1992-09-30 1993-09-28 Therapie a base de pp14 Ceased WO1994007366A1 (fr)

Priority Applications (3)

Application Number Priority Date Filing Date Title
EP93922763A EP0662782A4 (fr) 1992-09-30 1993-09-28 Therapie a base de pp14.
JP6509255A JPH08502257A (ja) 1992-09-30 1993-09-28 Ｐｐ１４に基づく治療
AU51659/93A AU5165993A (en) 1992-09-30 1993-09-28 Pp14-based therapy

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US95480292A 1992-09-30 1992-09-30
US07/954,802 1992-09-30

Publications (1)

Publication Number Publication Date
WO1994007366A1 true WO1994007366A1 (fr) 1994-04-14

Family

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PCT/US1993/009216 Ceased WO1994007366A1 (fr) 1992-09-30 1993-09-28 Therapie a base de pp14

Country Status (5)

Country Link
EP (1) EP0662782A4 (fr)
JP (1) JPH08502257A (fr)
AU (1) AU5165993A (fr)
CA (1) CA2145762A1 (fr)
WO (1) WO1994007366A1 (fr)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0658624A3 (fr) * 1993-12-08 1996-08-21 Intermune Life Sciences Inc Gènes recombinants exprimant pp14.
EP1248552A4 (fr) * 2000-01-03 2004-11-17 Tr Associates L L C Proteines hybrides comprenant la pp14 et leurs procedes de fabrication et d'utilisation
WO2004093907A3 (fr) * 2003-04-23 2005-02-24 Tomoaki Hoshino Agent therapeutique destine a la dermatite et/ou l'alopecie et/ou les demangeaisons, methode d'evaluation de l'agent therapeutique destine a l'alopecie et/ou les demangeaisons, et nouvel animal transgenique

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5039521A (en) * 1989-01-11 1991-08-13 Hyal Pharmaceutical Corporation Immune cell proliferation inhibitors

Patent Citations (1)

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Publication number Priority date Publication date Assignee Title
US5039521A (en) * 1989-01-11 1991-08-13 Hyal Pharmaceutical Corporation Immune cell proliferation inhibitors

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
DNA and Cell Biology, Volume 9, No. 6, issued 1990, C. VAISSE et al., "Human Placental Protein 14 Gene: Sequence and Characterization of a Short Duplication", pages 401-413, see page 402, column 2, paragraphs 1-2, page 403, column 2, paragraphs 3-5, page 408, Figure 3. *
Journal of Immunological Methods, Volume 136, issued 1991, L. RIITTINEN et al., "Monoclonal Antibodies Against Endometrial Protein PP14 and Their Use for Purification and Radioimmunoassay of PP14", pages 85-90, see page 86, paragraph bridging columns 1-2. *
Proceedings of the National Academy of Sciences, Volume 85, issued December 1988, M. JULKUNEN et al., "Complete Amino Acid Sequence of Human Placental Protein 14: A Progesterone-Regulated Uterine Protein Homologous to Beta-Lactoglobulins", pages 8845-8849, see page 8845, Abstract. *
See also references of EP0662782A4 *
The Lancet, issued 14 March 1987, A.E. BOLTON et al., "Identification of Placental Protein 14 as an Immunosuppressive Factor in Human Reproduction", pages 593-595, see page 593, summary. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0658624A3 (fr) * 1993-12-08 1996-08-21 Intermune Life Sciences Inc Gènes recombinants exprimant pp14.
EP1248552A4 (fr) * 2000-01-03 2004-11-17 Tr Associates L L C Proteines hybrides comprenant la pp14 et leurs procedes de fabrication et d'utilisation
WO2004093907A3 (fr) * 2003-04-23 2005-02-24 Tomoaki Hoshino Agent therapeutique destine a la dermatite et/ou l'alopecie et/ou les demangeaisons, methode d'evaluation de l'agent therapeutique destine a l'alopecie et/ou les demangeaisons, et nouvel animal transgenique

Also Published As

Publication number Publication date
JPH08502257A (ja) 1996-03-12
EP0662782A1 (fr) 1995-07-19
EP0662782A4 (fr) 1995-10-25
AU5165993A (en) 1994-04-26
CA2145762A1 (fr) 1994-04-14

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