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WO1997013859A1 - Cytokine hematopoietique et ses utilisations - Google Patents

Cytokine hematopoietique et ses utilisations Download PDF

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Publication number
WO1997013859A1
WO1997013859A1 PCT/US1996/016572 US9616572W WO9713859A1 WO 1997013859 A1 WO1997013859 A1 WO 1997013859A1 US 9616572 W US9616572 W US 9616572W WO 9713859 A1 WO9713859 A1 WO 9713859A1
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Prior art keywords
ebb
protein
ofthe
nucleic acid
cells
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PCT/US1996/016572
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WO1997013859A9 (fr
Inventor
Odile Devergne
Elliott D. Kieff
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Brigham And Women's Hospital, Inc.
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Priority claimed from US08/684,687 external-priority patent/US5830451A/en
Application filed by Brigham And Women's Hospital, Inc. filed Critical Brigham And Women's Hospital, Inc.
Publication of WO1997013859A1 publication Critical patent/WO1997013859A1/fr
Publication of WO1997013859A9 publication Critical patent/WO1997013859A9/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/54—Interleukins [IL]
    • C07K14/5434—IL-12
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • 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
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the fields of immunology and cytokines.
  • the present invention provides a novel purified complex, EBI3/p35, and further provides nucleic acids encoding this novel complex.
  • the EBI3/p35 complex, and nucleic acids operably encoding it may be formulated in pharmaceutical compositions and used in the treatment of various conditions.
  • antibodies to EBI3/p35 may be used in diagnostic tests.
  • Interleukin- 12 is a 70-kDa heterodimeric cytokine composed of two disulfide- linked glycosylated chains, p40 and p35.
  • the p35 subunit is structurally related to other alpha- helix rich haematopoietin cytokines, while the p40 subunit is a member ofthe haematopoietin receptor family.
  • IL12 was originally identified and purified from the culture cell supernatant of Epstein-Barr virus (EBV) transformed B lymphoblastoid cell lines, based on its ability to stimulate the maturation of cytotoxic lymphocytes (Cytotoxic Lymphocyte Maturation Factor) and the cytotoxicity of NK cells (Natural Killer cell Stimulatory Factor).
  • IL12 has pleiotropic effects on T and NK cells including: (i) induction of IFN- ⁇ production; (ii) proliferation; and (iii) enhancement of cytotoxic activity.
  • IL12 was shown to play a major role in the regulation of immune responses, by promoting the development of T helper type 1 (Thl ) responses, while inhibiting the development of Th2 cells (reviewed in Trichieri, (1993) Immunol. Today 14:335). Effects of IL12 on B lymphocyte differentiation (Jelinek et al., (1995) L Immunol. 154:1606) and on human haematopoiesis (Bellone et al., (1994) J. Immunol. 153:930) have also been reported.
  • IL12 is mainly produced by macrophages and other accessory cells (D'Andrea et al., (1992) J. Exp. Med. 176:1387). Human keratinocytes and keratinocyte cell lines have also been reported to express IL 12 (Aragane et al., ( 1994) J. Immunol. 153 :5366). All IL 12 expressing cell types analyzed so far produce the biologically active p70 heterodimer, together with a large excess ofthe free p40 subunit. No production of free p35 has been reported.
  • p35 appears to be ubiquitous and minimally regulated, whereas p40 expression is restricted to cell lines expressing the IL12 heterodimer and is inducible (D'Andrea et al., (1992) J. Exp. Med. 176:1387; D'Andrea et al., (1993) ____$______ 178:1041).
  • IL12 binding sites of three different affinities have been identified on activated T cells, suggesting that the IL12 receptor (IL12R) is composed of several subunits (Chua et al., (1994) Immunol. 153:128). Only one chain encoding a 100 kDa low affinity IL12R component has been cloned (Chua et al., (1994) J. Immunol. 153:128). This IL12 receptor subunit is a member ofthe haematopoietin receptor family, homologous to gpl 30.
  • homodimers ofthe p40 subunit can also bind the IL12R, and act in vitro as an antagonist for the IL12 heterodimer (Ling et al., (1995) J. Immunol. 154:1 16). However, there is no evidence that p40 homodimers exist in vivo.
  • the invention involves the discovery that EBI3, an Epstein Barr Virus induced protein, forms a heterodimer with the IL12 p35 subunit in vivo.
  • This complex has the ability to modulate the immune response and to influence the activity of cells, including hematopoietic, embryonal and neural cells. It therefore is believed useful in connection with treating a variety of conditions, including allergy, autoimmunity, host versus graft rejection, infectious disease, etc.
  • EBI3 is present in large quantities in placental syncytial trophoblasts, and thus it is also believed that the complex can be used to down regulate the maternal fetal immune response, decreasing the likelihood of spontaneous abortion. It further can be used to raise antibodies, which antibodies can be used inter alia, to isolate the complex, to identify tissues expressing the complex (including recombinant cells) and to diagnose pregnancy.
  • the EBI3/p35 protein complex comprises a heterodimer of a p35 subunit of IL12 and an EBB protein.
  • the EBI3 protein has the amino acid sequence disclosed as SEQ ID NO: 2.
  • the EBI3 protein is an allelomorph ofthe sequence disclosed as SEQ ID NO: 2.
  • the p35 subunit includes the amino acid sequence disclosed as SEQ ID NO: 4.
  • the p35 subunit may be an allelomorph ofthe sequence disclosed as SEQ ID NO: 4.
  • pharmaceutical preparations include substantially pure EBI3/p35 complex as described above, together with a pharmaceutically acceptable carrier. Such preparations preferably are sterile.
  • isolated nucleic acids comprising an expression vector including a first expression cassette and a second expression cassette in which the first cassette operably encodes a p35 subunit of IL12 and the second expression cassette operably encodes an EBI3 protein.
  • the second expression cassette includes (a) the complete coding region of SEQ ID NO: 1 ; (b) sequences which hybridize under stringent hybridization conditions to SEQ ID NO: 1 , or its complement, and which code for EBI3 protein; or (c) sequences which differ from (a) and (b) in codon sequence due to the degeneracy ofthe genetic code.
  • the first expression cassette likewise can include (a) the complete coding region of SEQ ID NO: 3; sequences which hybridize under stringent hybridization conditions to the coding region of SEQ ID NO: 3, or its complement, and which code for p35 subunit; or (c) sequences which differ from (a) and (b) in codon sequence due to the degeneracy ofthe genetic code.
  • the present invention also provides recombinant host cells including any ofthe above- described isolated nucleic acids which encode an EBI3/p35 cytokine. It is a further object to the invention to provide recombinant host cells which include two separate nucleic acids, one of which operably encodes an EBI3 protein and one of which operably encodes the IL12 p35 subunit. Expression of such nucleic acids in a recombinant host cell allows production ofthe EBI3/p35 heterodimer.
  • the invention also embraces not only substantially pure EBI3/p35, but also functional variants and fragments which are heterodimers as well as fusion proteins wherein the heterodimer or fragment thereof is manufactured as covalently attached EBI3-p35, or fragments thereof including at least a portion of EBI3 and a portion of p35 which form an active complex.
  • It is a particular object ofthe present invention to provide a method of diagnosing pregnancy and threatened spontaneous abortion comprising obtaining a fluid sample such as serum, urine or uterine secretion from a subject suspected of being pregnant; contacting the sample with an antibody to the EBI3/p35 heterodimer; and determining binding ofthe antibody and EBI3/p35 heterodimer within the sample as a measure of pregnancy
  • a method for modulating the immune system of a subject is provided.
  • An EBI3/p35 protein complex is administered to a subject in need of such immune modulation in an amount effective to modulate the immune response ofthe subject.
  • the EBI3/p35 protein complex may be substantially pure protein complex formulated as a pharmaceutical.
  • the EBI3/p35 protein complex also may be expressed in recombinant cells which are placed within the subject.
  • tissue transplants, including organ transplants may be genetically engineered to express EBI3/p35 so as to modulate the immune system ofthe host and reduce the chances of tissue or organ rejection.
  • the EBI3/p35 protein complex may be any ofthe embodiments that are described above.
  • the EBI3/p35 protein complex is administered to a pregnant woman or a woman with a history of infertility to reduce the chances of spontaneous abortion.
  • the EBI3/p35 protein complex is administered to a subject scheduled to have or who has had a tissue transplant, including but not limited to an organ transplant, in order to reduce the likelihood of tissue or organ transplant rejection.
  • the EBI3/p35 protein complex is administered to a subject that has an autoimmune condition to ameliorate the autoimmune condition.
  • One example is autoimmune disease due to NK or cytotoxic T-cell hyperactivity.
  • the EBI3/p35 protein complex is administered to a subject who has an infectious disease to ameliorate that condition.
  • One example involves infectious diseases which result in hyperactive cytotoxic T-cell responses that threaten organ destruction. It is even possible to supply a developing embryo or fetus, perhaps transiently, with recombinant nucleic acids encoding the EBI3/p35 protein complex to assist in preventing spontaneous abortion.
  • methods for modulating the activity of cells are provided.
  • the cells are contacted with an EBI3/p35 protein complex in an amount sufficient to modulate the growth, differentiation or development ofthe cells.
  • the cells for example, can be hematopoietic cells, embryonal cells or neural cells.
  • a protein complex can be supplied in substantially pure form or can be provided by recombinant expression.
  • the invention thus also embraces in vitro uses ofthe purified and isolated materials ofthe invention.
  • in vitro uses include, but are not limited to, making recombinant cells for transplantation into a host, making recombinant cells for expressing and purifying EBI3/p35 protein complex, and contacting cells such as a heterogeneous population of lymphocytes to selectively influence the growth, development, and/or differentiation of particular subsets of cells, such as by arresting cytotoxic T-cell or NK-cell development.
  • Fig. 1 contains computer digitized images of chemiluminescent immunoblots demonstrating that EBI3 coprecipitates with FLAG-tagged p35 from both the cell lysate (Fig. 1 A) or the cell culture supernatant (Fig. IB) of BJAB cells coexpressing both proteins.
  • Fig. 2 contains computer digitized images of chemiluminescent immunoblots showing that P 35 associates with EBI3FLAG (Fig. 2A) or EBI3 (Fig. 2B).
  • Fig. 3 contains computer digitized images of chemiluminescent immunoblots demonstrating that p35 and EBB can associate in solution as detected by EBB antiserum (Fig. 3 A) or IL 12 antiserum (Fig. 3B).
  • Fig. 4 contains computer digitized images of immunoprecipitates of radioactively labeled proteins showing the effect of EBB coexpression on p35 secretion in cell lysates (Fig. 4A) or cell culture supernatants (Fig. 4B) of BJAB cells.
  • Fig. 5 contains a computer digitized image of a chemiluminescent immunoblot showing the effect of p35 coexpression on EBB biosynthesis.
  • IL12 interleukin- 12
  • IL12 is a 70-75 kDa glycoprotein comprising two disulfide-linked subunits designated p35 and p40.
  • IL12 p35 subunit or simply “p35” means the approximately 35 kDa subunit of IL12 well known in the art and described in the references cited above.
  • SEQ ID NO: 4 An amino acid sequence of one human allele ofthe IL12 p35 subunit is reported in Wolf, et al. H991 . J. Immunol. 146:3074-3081 and is reproduced herein as SEQ ID NO: 4.
  • EBB Epstein Barr Virus induced protein number 3 described in the experimental section below.
  • the EBB protein has a predicted molecular mass of approximately 25.3 kDa and an apparent molecular mass of approximately 33 kDa and forms a heterodimer with the IL12 p35 subunit.
  • SEQ ID NO: 1 and SEQ ID NO: 2 The nucleotide sequence of one human EBB allele and the amino acid sequence ofthe corresponding EBB protein are reproduced herein as SEQ ID NO: 1 and SEQ ID NO: 2.
  • EBB/p35 means the heterodimer formed by the EBB protein and the IL12 p35 subunit.
  • substantially pure means that the polypeptides are essentially free of other substances with which they may be found in natural or in vivo systems to an extent practical and appropriate for their intended use.
  • the polypeptides are sufficiently pure and are sufficiently free from other biological constituents of their hosts cells so as to be useful in, for example, generating antibodies, sequencing, or producing pharmaceutical preparations.
  • substantially pure polypeptides may be produced in light ofthe nucleic acid and amino acid sequences disclosed herein. Because a substantially purified polypeptide of the invention may be admixed with a pharmaceutically acceptable carrier in a pharmaceutical preparation, the polypeptide may comprise only a small percentage by weight ofthe preparation. The polypeptide is nonetheless substantially pure in that it has been substantially separated from the substances with which it may be associated in living systems.
  • isolated means: (i) amplified in vitro by, for example, polymerase chain reaction (PCR); (ii) recombinantly produced by cloning; (iii) purified, as by cleavage and gel separation; or (iv) synthesized by, for example, chemical synthesis.
  • An isolated nucleic acid is one which is readily manipulable by recombinant DNA techniques well known in the art.
  • PCR polymerase chain reaction
  • An isolated nucleic acid may be substantially purified, but need not be.
  • a nucleic acid that is isolated within a cloning or expression vector is not pure in that it may comprise only a tiny percentage ofthe material in the cell in which it resides.
  • Such a nucleic acid is isolated, however, as the term is used herein because it is readily manipulable by standard techniques known to those of ordinary skill in the art.
  • a coding sequence and regulatory sequences are said to be "operably joined” when they are covalently linked in such a way as to place the expression or transcription ofthe coding sequence under the influence or control ofthe regulatory sequences. If it is desired that the coding sequences be translated into a functional protein, two DNA sequences are said to be operably joined if induction of a promoter in the 5' regulatory sequences results in the transcription ofthe coding sequence and if the nature ofthe linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability ofthe promoter region to direct the transcription ofthe coding sequences, or (3) interfere with the ability ofthe corresponding RNA transcript to be translated into a protein. Thus, a promoter region would be operably joined to a coding sequence if the promoter region were capable of effecting transcription of that DNA sequence such that the resulting transcript might be translated into the desired protein or polypeptide.
  • regulatory sequences needed for gene expression may vary between species or cell types, but shall in general include, as necessary, 5' non-transcribing and 5' non-translating sequences involved with initiation of transcription and translation respectively, such as a TATA box, capping sequence, CAAT sequence, and the like.
  • 5' non-transcribing regulatory sequences will include a promoter region which includes a promoter sequence for transcriptional control ofthe operably joined Gene. Promoters may be constitutive or inducible. Regulatory sequences may also include enhancer sequences or upstream activator sequences, as desired.
  • a "vector" may be any of a number of nucleic acids into which a desired sequence may be inserted by restriction and ligation for transport between different genetic environments or for expression in a host cell.
  • Vectors are typically composed of DNA although RNA vectors are also available.
  • Vectors include, but are not limited to, plasmids and phagemids.
  • a cloning vector is one which is able to replicate in a host cell, and which is further characterized by one or more endonuclease restriction sites at which the vector may be cut in a determinable fashion and into which a desired DNA sequence may be ligated such that the new recombinant vector retains its ability to replicate in the host cell.
  • replication ofthe desired sequence may occur many times as the plasmid increases in copy number within the host bacterium, or just a single time per host as the host reproduces by mitosis. In the case of phage, replication may occur actively during a lytic phase or passively during a lysogenic phase.
  • An expression vector is one into which a desired DNA sequence may be inserted by restriction and ligation such that it is operably joined to regulatory sequences and may be expressed as an RNA transcript. Vectors may further contain one or more marker sequences suitable for use in the identification of cells which have or have not been transformed or transfected with the vector.
  • Markers include, for example, genes encoding proteins which increase or decrease either resistance or sensitivity to antibiotics or other compounds, genes which encode enzymes whose activities are detectable by standard assays known in the art (e.g., ⁇ -galactosidase or alkaline phosphatase), and genes whicli visibly affect the phenotype of transformed or transfected cells, hosts, colonies or plaques.
  • Preferred vectors are those capable of autonomous replication and expression ofthe structural gene products present in the DNA segments to which they are operably joined.
  • stringent conditions refers to parameters known to those skilled in the art.
  • hybridization buffer 3.5 x SSC, 0.02% Ficoll, 0.02% polyvinyl pyrolidone, 0.02% bovine serum albumin (BSA), 25mM NaH 2 PO 4 (pH7), 0.5% SDS, 2mM EDTA).
  • SSC is 0.15M sodium chloride/0.15M sodium citrate, pH7; SDS is sodium dodecylsulphate; and EDTA is ethylene diamine tetra acetic acid.
  • a "therapeutically effective amount" ofthe cytokines or nucleic acids of the invention is a dosage large enough to produce the desired effect on the activity and/or proliferation of cells.
  • a therapeutically effective amount is not, however, a dosage so large as to cause adverse side effects, such as hyperviscosity syndromes, pulmonary edema, congestive heart failure, and the like.
  • a therapeutically effective amount may vary with the subject's age, condition, and sex, as well as the extent ofthe condition being treated, and can be determined by one of skill in the art. The dosage may be adjusted by the individual physician or veterinarian in the event of any complication.
  • EBI3/p35 A Novel Cvtokine It is one object ofthe present invention to provide substantially pure preparations of novel complex comprising a heterodimer ofthe p35 subunit of interleukin- 12 and the EBB protein. This novel heterodimer is referred to as EBB/p35.
  • EBB a novel soluble cytokine receptor homologous to IL12 p40.
  • EBB was originally identified in and cloned from an EBV- infected Burkitt lymphoma cell cDNA library.
  • EBB was noted to have 27% identity to the IL12 p40 subunit and has conservative substitutions at many other residues.
  • EBB further resembles IL12 p40 in that both genes have a 3' untranslated Alu repeat sequence.
  • EBB lacks a membrane anchoring motif and is predicted to be secreted.
  • EBB is turned on in EBV-transformed B lymphocytes which must escape from NK and CD8 cytotoxic T cell destruction and is expressed at even higher levels in human placental syncytiotrophoblasts.
  • EBB EBB
  • IL12 p35 IL12 p35
  • EBB/p35 is naturally secreted from syncytial trophoblasts and some activated lymphocytes as a natural human cytokine.
  • EBB expression is turned on by EBV in latently infected cells which need to antagonize the effects of IL12 in increasing anti-EBV-infected cell cytotoxicity, and is naturally expressed at even higher levels in the placenta which needs to protect itself and the developing fetus from IL12 regulated NK and CD8 cytotoxic T cell activity, it appeared possible that the EBB/p35 heterodimer functions as a modulator of IL12 activities. This suggestion is also supported by the fact that, in the natural cytokine EBB/p35, more than 60% of the heteromeric protein is identical to IL12. In addition to having an effect on IL12 activity, EBB could modulate IL12 biosynthesis.
  • EBB could prevent p35 from associating with p40 in cells expressing both proteins.
  • EBB/p35 does not affect IL12 induced cell DNA synthesis or interferon release in human peripheral blood mononuclear cell assays and it does not bind to the previously identified IL12 receptors.
  • EBB is made in large quantities by syncytial trophoblasts, and these cells are the most proximal fetal cells to the maternal circulation, likely down regulating the maternal fetal immune response.
  • EBB/p35 complex thus, is still believed to act as a down regulator of cell-mediated NK and/or CD8 cytotoxicity and/or of Thl responses, therefore favoring fetal implantation or expansion of virus- infected cells.
  • the amino acid sequence of one human allelomorph ofthe EBB protein is reproduced herein as SEQ ID NO: 2.
  • functional allelomorphic variants of this EBB protein may exist in the human population and may be used in accordance with the present invention.
  • Such allelomorphic variants are obvious equivalents of the EBB protein described by SEQ ID NO: 2.
  • one of ordinary skill in the art may, without undue experimentation, produce variants ofthe EBB protein which are equivalent to the protein of SEQ ID NO: 2 by making, for example, conservative substitutions of one or more amino acids. Conservative substitutions include the replacement of an amino acid residue by another, chemically and biologically similar residue.
  • conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.
  • the first 20 amino acids ofthe EBB protein are highly hydrophobic and are predicted to be a cleavable signal peptide.
  • variants ofthe EBB protein in which all or some ofthe amino acids, including all or some ofthe first 20 amino acids, are omitted would be equivalents ofthe complete protein.
  • allelomorph ofthe IL12 p35 subunit protein is reproduced herein as SEQ ID NO: 4. This sequence is available in GenBank under accession number M65291 and was first reported in Wolf, et al. (1991) J. Immunol. 146:3074-3081. As will be obvious to one of ordinary skill in the art, functional allelomorphic variants ofthe p35 protein may exist in the human population and may be used in accordance with the present invention. Such allelomo ⁇ hic variants are obvious equivalents ofthe p35 protein described by SEQ ID NO: 4.
  • variants of the IL12 p35 subunit which are equivalent to the protein of SEQ ID NO: 4 by making, for example, conservative substitutions of one or more amino acids.
  • Conservative substitutions include the replacement of an amino acid residue by another, chemically and biologically similar residue.
  • conservative substitutions include the substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.
  • the nucleotide sequence ofthe IL12 gene has two potential start codons (ATG) which are in-frame with each other.
  • SEQ ID NO: 3 assumes that the first ATG sequence is the actual start codon.
  • the N-terminal residues ofthe IL12 p35 subunit appear to be a signal peptide and the mature peptide is believed to begin approximately at the residue numbered 57 in SEQ ID NO: 3 and SEQ ID NO: 4.
  • Variants ofthe p35 protein in which all or part ofthe signal peptide is omitted would be equivalents ofthe complete protein. Functional portions of any of the foregoing also are embraced by the invention.
  • EBB and p35 also can be produced as a fusion protein.
  • the two polypeptides can be fused by insertion of a short, flexible linker which permits sufficient rotational freedom ofthe polypeptides such that protein function is not hindered.
  • Substantially pure EBB/p35 can be obtained from host cells expressing the cytokine by standard methods of protein purification which are well known in the art. Such host cells may be produced using the isolated nucleic acids ofthe present invention described below. Nucleic Acids Encoding EBB/p35 and Expression Thereof It is another object ofthe present invention to provide isolated nucleic acids, and in particular, expression vectors, encoding the peptides of EBB/p35.
  • the nucleic acids encoding the EBB and IL12 p35 subunit may be separate molecules or vectors but, more preferably, both polypeptides are encoded by a single nucleic acid molecule or vector such that the single vector can transform or transfect a host cell and lead to expression of EBB/p35. That is, a DNA expression vector for expressing the heterodimeric EBB/p35 provides a system for independently cloning (inserting) the two translatable DNA sequences into two separate cassettes present in the vector, to form two separate cistrons for expressing the first and second polypeptides of the heterodimeric cytokine.
  • the DNA expression vector for expressing two cistrons is referred to as a dicistronic expression vector.
  • the dicistronic expression vector comprises a first cassette that includes upstream and downstream DNA regulatory sequences operably joined via a sequence of nucleotides adapted for directional ligation to an insert DNA.
  • the upstream translatable sequence preferably encodes the secretion signal as described above.
  • the cassette includes DNA regulatory sequences for expressing the first EBB/p35 polypeptide component that is produced when an insert translatable DNA sequence (insert DNA) is directionally inserted into the cassette via the sequence of nucleotides adapted for directional ligation.
  • the dicistronic expression vector also contains a second cassette for expressing the second EBI3/p35 polypeptide component.
  • the second cassette includes a second translatable DNA sequence that preferably encodes a secretion signal, as described above, operably joined at its 3' terminus via a sequence of nucleotides adapted for directional ligation to a downstream
  • the second translatable DNA sequence is operably joined at its 5' terminus to DNA regulatory sequences forming the 5' elements.
  • the second cassette is capable, upon insertion of a translatable DNA sequence (insert DNA), of expressing the second EBB/p35 polypeptide component comprising a secretion signal with a polypeptide coded by the insert DNA.
  • the EBB and p35 polypeptides are encoded by a single nucleic acid which encodes a fusion protein, as described above, operably joined to a single set of upstream and downstream regulatory sequences.
  • the coding regions for EBB and p35 are preferably joined by a short oligonucleotide sequence which encodes a short and flexible peptide linker between the EBB and p35 polypeptides.
  • linkeers of various lengths, flexibility, and conformation is well known to those of ordinary skill in the art.
  • the oligonucleotide encoding the peptide linker is selected to maintain the translation reading frame of the portion ofthe nucleic acid located downstream ofthe oligonucleotide.
  • the construction of a nucleic acid encoding a functional fusion protein including the EBB and p35 proteins will be known to one of ordinary skill in the art and may be accomplished without undue experimentation.
  • plasmid vectors that contain replication sites and control sequences derived from a species compatible with the host may be used.
  • suitable plasmid vectors include pBR322, pUC18, pUC19 and the like; suitable phage or bacteriophage vectors include ⁇ gtlO, ⁇ gtl 1 and the like; and suitable virus vectors include pMAM-neo, pKRC and the like.
  • the selected vector ofthe present invention has the capacity to autonomously replicate in the selected host cell.
  • Useful prokaryotic hosts include bacteria such as E. coli, Bacillus, Streptomyces, Pseudomonas, Salmonella, Serratia, and the like.
  • EBB/p35 in a prokaryotic cell, it is necessary to operably join the EBB sequence to a functional prokaryotic promoter.
  • promoters may be either constitutive or, more preferably, regulatable (i.e., inducible or derepressible).
  • constitutive promoters include the int promoter of bacteriophage ⁇ , the bla promoter ofthe ⁇ -lactamase gene sequence of pBR322, and the CAT promoter ofthe chloramphenicol acetyl transferase gene sequence of pPR325, and the like.
  • inducible prokaryotic promoters examples include the major right and left promoters of bacteriophage ⁇ (P L and P R ), the trp, ree A, lacZ, lad, and gal promoters of E. coli, the ⁇ -amylase (Ulmanen et al., J. Bacteriol. 162:176-182 (1985)) and the ⁇ - 28-specific promoters of B.
  • subtilis (Gilman et al., Gene sequence 32:11-20 (1984)), the promoters ofthe bacteriophages o ⁇ Bacillus (Gryczan, In: The Molecular Biology ofthe Bacilli, Academic Press, Inc., NY (1982)), and Streptomyces promoters (Ward et al., Mol. Gen. Genet. 203:468-478 (1986)).
  • Prokaryotic promoters are reviewed by Glick (J. Ind. Microbiol. 1 :277-282 (1987)); Cenatiempo (Biochimie 68:505-516 (1986)); and Gottesman (Ann. Rev. Genet. 18:415-442 (1984)).
  • Proper expression in a prokaryotic cell also requires the presence of a ribosome binding site upstream ofthe encoding sequence.
  • Such ribosome binding sites are disclosed, for example, by Gold et al. (Ann. Rev. Microbiol. 35:365-404 (1981)).
  • eukaryotic hosts include, for example, yeast, fungi, insect cells, and mammalian cells, either in vivo or in tissue culture.
  • Mammalian cells which may be useful as hosts include HeLa cells, cells of fibroblast origin such as VERO or CHO-K1, or cells of lymphoid origin, such as the hybridoma SP2/0-AG14 or the myeloma P3x63Sg8, and their derivatives.
  • Preferred mammalian host cells include SP2/0 and J558L, as well as neuroblastoma cell lines such as IMR 332 that may provide better capacities for correct post-translational processing.
  • Embryonic cells and mature cells of a transplantable organ also are useful according to some aspects ofthe invention.
  • plant cells are also available as hosts, and control sequences compatible with plant cells are available, such as the nopaline synthase promoter and polyadenylation signal sequences.
  • Another preferred host is an insect cell, for example in Drosophila larvae.
  • insect cells the Drosophila alcohol dehydrogenase promoter can be used (Rubin, Science 240:1453-1459 (1988)).
  • baculovirus vectors can be engineered to express large amounts of EBI3/p35 in insects cells (Jasny, Science 238:1653 (1987); Miller et al., In: Genetic Engineering (1986), Setiow, J.K., et al., eds., Plenum, Vol. 8, pp. 277-297).
  • yeast gene sequence expression systems may also be utilized which inco ⁇ orate promoter and termination elements from the genes coding for glycolytic enzymes which are produced in large quantities when the yeast are grown in media rich in glucose.
  • Known glycolytic gene sequences can also provide very efficient transcriptional control signals.
  • Yeast provide substantial advantages in that they can also carry out post-translational peptide modifications.
  • Yeast recognize leader sequences on cloned mammalian gene sequence products and secrete peptides bearing leader sequences (i.e., pre-peptides).
  • transcriptional and translational regulatory sequences may be employed, depending upon the nature of the host.
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, where the regulatory signals are associated with a particular gene sequence which has a high level of expression.
  • promoters from mammalian expression products such as actin, collagen, myosin, and the like, may be employed.
  • Transcriptional initiation regulatory signals may be selected which allow for repression or activation, so that expression ofthe gene sequences can be modulated.
  • regulatory signals which are temperature-sensitive so that by varying the temperature, expression can be repressed or initiated, or which are subject to chemical (such as metabolite) regulation.
  • EBB/p35 expression of EBB/p35 in eukaryotic hosts requires the use of eukaryotic regulatory regions. Such regions will, in general, include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Preferred eukaryotic promoters include, for example, the promoter ofthe mouse metallothionein I gene sequence (Hamer et al., J. Mol. Appl. Gen.
  • a vector is employed which is capable of integrating the desired gene sequences into the host cell chromosome.
  • Cells which have stably integrated the introduced DNA into their chromosomes can be selected by also introducing one or more markers which allow for selection of host cells which contain the expression vector.
  • the marker may, for example, provide for prototrophy to an auxotrophic host or may confer biocide resistance to, e.g., antibiotics, heavy metals, or the like.
  • the selectable marker gene sequence can either be directly linked to the DNA gene sequences to be expressed, or introduced into the same cell by co ⁇ transfection. Additional elements may also be needed for optimal synthesis of EBB or p35 mRNA. These elements may include splice signals, as well as transcription promoters, enhancers, and termination signals.
  • cDNA expression vectors inco ⁇ orating such elements include those described by Okayama, Molec. Cell. Biol 3:280 (1983).
  • the introduced sequence will be inco ⁇ orated into a plasmid or viral vector capable of autonomous replication in the recipient host.
  • a plasmid or viral vector capable of autonomous replication in the recipient host.
  • Any of a wide variety of vectors may be employed for this pu ⁇ ose. Factors of importance in selecting a particular plasmid or viral vector include: die ease with which recipient cells that contain the vector may be recognized and selected from those recipient cells which do not contain the vector; the number of copies ofthe vector which are desired in a particular host; and whether it is desirable to be able to "shuttle" the vector between host cells of different species.
  • Preferred prokaryotic vectors include plasmids such as those capable of replication in E.
  • coli such as, for example, pBR322, Col ⁇ l, pSClOl, pACYC 184, and ⁇ VX.
  • plasmids are, for example, disclosed by Sambrook, et al. (Molecular Cloning: A Laboratory Manual, second edition, edited by Sambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, 1989)).
  • Bacillus plasmids include pC194, pC221, pT127, and the like. Such plasmids are disclosed by Gryczan (In: The Molecular Biology ofthe Bacilli, Academic Press, NY ( 1982), pp. 307-329). Suitable
  • Streptomyces plasmids include pIJlOl (Kendall et al., J. Bacteriol. 169:4177-4183 (1987)), and streptomyces bacteriophages such as ⁇ C31 (Chater et al., In: Sixth International Symposium on Actinomycetales Biology, Akademiai Kaido, Budapest, Hungary (1986), pp. 45-54). Pseudomonas plasmids are reviewed by John et al. (Rev. Infect. Dis. 8:693-704 (1986)), and Izaki (Jpn. J. Bacteriol. 33:729-742 (1978)).
  • Preferred eukaryotic plasmids include, for example, BPV, ⁇ BV, SV40, 2-micron circle, and the like, or their derivatives.
  • Such plasmids are well known in the art (Botstein et al., Miami Wntr. Symp. 19:265-274 (1982); Broach, In: The Molecular Biology of the Yeast Saccharomyces: Life Cycle and Inheritance, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, p. 445- 470 (1981); Broach, Cell 28:203-204 (1982); Bollon et al., J. Clin. Hematol. Oncol. 10:39-48 (1980); Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol.
  • viral vectors are viral vectors.
  • the pox virus, he ⁇ es virus, adenovirus and various retroviruses may be employed.
  • the viral vectors may include either DNA or RNA viruses to cause expression ofthe insert DNA or insert RNA.
  • DNA or RNA encoding the EBB and/or p35 polypeptides may be directly injected into cells or may be impelled through cell membranes after being adhered to microparticles (see below).
  • nucleic acids encoding the EBB protein and the p35 subunit be included in a single dicistronic expression vector, this need not be the case. Rather, two separate expression vectors, one operably encoding the EBB protein and the other operably encoding the IL12 p35 subunit, can be constructed using regulatory sequences and vectors as described above. Appropriate host cells may then be co-transformed with these vectors such that both the EBB and p35 polypeptides are expressed in the same cell and are capable of associating to form the EBB/p35 heterodimer.
  • the recombinant cells ofthe present invention are either transformed with a single dicistronic vector operably encoding the EBB and p35 polypeptides or with two vectors, one operably encoding the EBB protein and the other operably encoding the p35 subunit.
  • the DNA construct(s) may be introduced into an appropriate host cell by any of a variety of suitable means, i.e., transformation, transfection, conjugation, protoplast fusion, eiectroporation, calcium phosphate-precipitation, direct microinjection, and the like.
  • recipient cells are grown in a selective medium, which selects for the growth of vector-containing cells.
  • Expression ofthe cloned gene sequence(s) results in the production of EBB/p35. This can take place in the transformed cells as such, or following the induction of these cells to differentiate (for example, by administration of bromodeoxyuracil to neuroblastoma cells or the like).
  • control sequences are dependent on the type of host cell used to express the gene.
  • “cell,” “cell line,” and “cell culture” may be used interchangeably and all such designations include progeny.
  • progeny include the primary subject cell and cultures derived therefrom, without regard to the number of transfers. It is also understood that all progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. However, as defined, mutant progeny have the same functionality as that ofthe originally transformed cell.
  • EBB or EBB/p35 in vivo in serum, urine or uterine secretion obtained from a pregnant or potentially pregnant woman provides an important new test to diagnose an early event in implantation ofthe developing placenta. Further, measurement ofthe levels of EBB and/or EBI3/p35 in serum or urine would be a useful indicator of threatened abortion, in which case the levels would be expected to fall. Given the presumed importance of foetal rejection in spontaneous abortion and the likely role of EBB/p35 as a natural defense against maternal rejection ofthe placenta and foetus, the measurement of EBB and/or EBB/p35 levels should be a physiopathologically significant parameter for spontaneous abortion.
  • the format ofthe diagnostic test ofthe present invention would use antibodies to measure EBB and/or EBB/p35 in serum, urine, or uterine secretions.
  • the antibodies may be generated to EBB itself or specifically to the EBB/p35 heterodimer. Because the p35 subunit is expressed in most tissues ofthe body, antibodies to p35 alone should not be used (except in a "sandwich” assay as described below).
  • Using the substantially pure protein ofthe present invention one of ordinary skill in the art may generate such antibodies without undue experimentation according to methods well known in the art. (See, e.g., Catty, D. (ed.) Antibodies. A Practical Approach. Vols. I and II, IRL Press, Washington, DC (1988); Kennett, R..
  • a sample of serum, urine, or uterine secretion may be obtained from a pregnant or potentially pregnant woman and may be contacted with the anti-EBB or anti- EBB/p35 antibodies ofthe invention. Binding ofthe antibodies to EBB or the EBB/p35 heterodimer may be detected by any ofthe standard means known in the art. Antibodies to a first EBB or EBB/p35 epitope may, for example, be immobilized on a substrate, contacted with the sample to allow binding ofthe antibodies to the epitope, and then washed to remove unbound materials. The presence of bound protein may then be assayed by standard means.
  • the assay may be ofthe "sandwich" type in which a first set of EBB or EBB/p35 antibodies is first contacted with the sample and then washed, and then labeled antibodies to a second EBB or EBB/p35 epitope are added. Binding ofthe second set of antibodies indicates the presence of EBB or EBB/p35 bound to the first set.
  • the labels used may be of any variety known in the art (e.g. enzymatic, radioactive, bioluminescent). In the sandwich-type assay, it is permissible that one set of antibodies be specific to the p35 subunit. Other antibody tests, including competitive binding tests, will be apparent to one of ordinary skill in the art.
  • antibody or “monoclonal antibody” (Mab) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab') 2 fragments) which are capable of binding an antigen.
  • Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding than an intact antibody.
  • EBI3/p35 may be administered to patients including, but not limited to, the following: (1) Pregnant or potentially pregnant women with a history of infertility, presumed or possibly due to failure of placental implantation; (2) Transplant recipients at risk for organ rejection or those experiencing organ rejection; (3) Patients with autoimmune diseases, for example, due to NK or cytotoxic T cell hyperactivity such as inflammatory bowel disease, lupus, rheumatoid arthritis, sarcoid, or multiple sclerosis; and (4) Patients with infectious disease for example, as results in hyperactive cytotoxic T cell responses that threaten organ destruction (e.g., tuberculous meningitis in which EBB/p35 would be useful parenterally, administered into the CSF).
  • cytotoxic T cell hyperactivity such as inflammatory bowel disease, lupus, rheumatoid arthritis, sarcoid, or multiple sclerosis.
  • the nucleic acids ofthe present invention may be administered to a patient such that the nucleic acids direct expression ofthe EBB/p35 cytokine by the patient's cells (or by transplanted cells placed within the patient).
  • nucleic acids of the invention may be used therapeutically in the following ways: (1) Expressed in uterine or placental tissue using expression vectors with constitutive or organ- specific promoters; (2) Expressed in an in vitro fertilized ovum using in vitro injected RNAs or appropriate DNA expression vectors; (3) Expressed in transplanted organs using in vitro (pre- transplant) or in vivo (post-transplant) RNA/DNA injection, infection, or transfection; and (4) Expressed in diseased or normal tissues in patients with autoimmune diseases or autoimmune manifestations of infectious diseases as described above.
  • DNA in expression cassettes could employ constitutive or tissue specific promoters. The latter would enable systemic administration and local organ specific expression.
  • EBB/p35 or nucleic acids encoding EBB/p35
  • Administration of EBB/p35, or nucleic acids encoding EBB/p35 may be via any suitable route including, oral, or parenteral.
  • the administration may be localized to the site or organ, for example, by injection to or perfusion ofthe site.
  • EBB/p35 or nucleic acids operably encoding EBB/p35 may be formulated with a pharmaceutically acceptable carrier as part of a pharmaceutical composition.
  • a pharmaceutical composition may include the cytokine or nucleic acids in combination with any standard physiologically and/or pharmaceutically acceptable carriers which are known in the art.
  • compositions should be sterile and contain a therapeutically effective amount of the cytokine or nucleic acids in a unit of weight or volume suitable for administration to a patient.
  • pharmaceutically acceptable means a non-toxic material that does not interfere with the effectiveness ofthe biological activity ofthe active ingredients.
  • physiologically acceptable refers to a non-toxic material that is compatible with a biological system such as a cell, cell culture, tissue, or organism. The characteristics ofthe carrier will depend on the route of administration. Physiologically and pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers, and other materials which are well known in the art.
  • the pharmaceutical composition ofthe invention may be in the form of a liposome in which the cytokine or nucleic acids are combined, in addition to other pharmaceutically acceptable carriers, with amphipathic agents such as lipids which exist in aggregated form as micelles, insoluble monolayers, liquid crystals, or lamellar layers which are in aqueous solution.
  • Suitable lipids for liposomal formulation include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like.
  • the composition When a therapeutically effective amount ofthe EBI3/p35 cytokine, or nucleic acids encoding the cytokine, is administered orally, the composition will be in the form of a tablet, capsule, powder, solution or elixir.
  • the pharmaceutical composition ofthe invention may additionally contain a solid carrier such as a gelatin or an adjuvant.
  • a liquid carrier such as water, petroleum, oils of animal or plant origin such as peanut oil, mineral oil, soybean oil, sesame oil, or synthetic oils may be added.
  • the liquid form ofthe pharmaceutical composition may further contain physiological saline solution, dextrose or other saccharide solution, or glycols such as ethylene glycol, propylene glycol or polyethylene glycol.
  • the oligonucleotides When a therapeutically effective amount ofthe EBB/p35 cytokine, or nucleic acids operably encoding the cytokine, is administered by intravenous, cutaneous or subcutaneous injection, the oligonucleotides will be in the form of a pyrogen-free, parenterally acceptable aqueous solution.
  • parenterally acceptable solutions having due regard to pH, isotonicity, stability, and the like, is within the skill in the art.
  • a preferred pharmaceutical composition for intravenous, cutaneous, or subcutaneous injection should contain, in addition to the active ingredient, an isotonic vehicle such as saline solution, Ringer's solution, dextrose solution, dextrose and sodium chloride solution, lactated Ringer's solution, or another vehicle as known in the art.
  • an isotonic vehicle such as saline solution, Ringer's solution, dextrose solution, dextrose and sodium chloride solution, lactated Ringer's solution, or another vehicle as known in the art.
  • the pharmaceutical composition ofthe present invention may also contain stabilizers, preservatives, buffers, antioxidants, or other additives known to those of skill in the art.
  • administering when the target cells are readily accessible, administration ofthe EBB/p35 cytokine or nucleic acids is localized to the region of the targeted cells in order to maximize the delivery ofthe active ingredient and to minimize the amount ofthe active ingredient needed per treatment.
  • administration is by direct injection at or perfusion ofthe site ofthe targeted cells, such as a placenta, uterus or transplant organ.
  • the nucleic acids ofthe invention may be adhered to small particles (e.g., microscopic gold beads) which are impelled through the membranes ofthe target cells (see, e.g., U.S. Pat. No. 5,149,655).
  • EBB/p35 protein complex ranging from 1 nanogram/kilogram to 100 milligrams/kilogram, depending upon the mode of administration and the particular condition being treated, will be effective.
  • the preferred range is believed to be between 500 nanograms and 500 micrograms/kilogram.
  • the absolute amount will depend on a variety of factors including the material selected for administration, whether the administration is in singular or multiple doses, individual patient parameters inlcuding age, physical condition, size and weight, and the condition being treated. These factors are well known to those of ordinary skill in the art and can be addressed with no more routine experimentation.
  • BL30, BL41, Louckes and BJAB are EBV(-) BL lines.
  • the BL41/B95-8 cell line was derived from BL41 by infection with the transforming B95-8 EBV (Calender, et al., (1990) Intl. J. Cancer 46:658-663.).
  • SLA, IB4 and W91 are LCLs generated by EBV transformation of primary human B lymphocytes.
  • P3HR1 is an EBV(+) BL cell line infected with the non-transforming P3HR1 EBV.
  • RHEK-1 is an adenovirus 12/SV40 hybrid virus transformed human keratinocyte line.
  • K562, U937 and HL60 are human myeloid leukemia cell lines with erythroid, monocytic and myeloblastic features respectively.
  • Jurkat and MOLT4 are human T cell leukemia lines.
  • TK143 is a human osteosarcoma line.
  • HeLa is a cervical carcinoma cell line.
  • COS7 is a SV40 transformed monkey kidney cell line.
  • Human peripheral blood mononuclear cells (PBMC) were isolated by ficoll separation of blood (Ficoll-Hypaque; Pharmacia, Vineland, NJ).
  • RNA preparation and analysis Cytoplasmic RNA was isolated from exponentially growing cells by acid phenol/guanidinium isothiocyanate extraction (Birkenbach, et al., (1993) J. Virol. 67:2209-2220). Total cellular RNA was purified from excised human spleen and tonsil, and from post-mortem bone marrow by acid phenol/guanidinium isothiocyanate extraction, followed by reprecipitation in 4 M LiCl. Polyadenylated IB4, BL41 and BL41/B95-8 RNA were purified by chromatography on oligodeoxythymidylate cellulose.
  • RNA samples (4-12 ⁇ g per lane) were denatured, size fractionated on 0.66 M formaldehyde, 1 % agarose gels and transferred to charged nylon membranes (GeneScreen Plus; New England Nuclear, Billerica, MA). Expression in other human tissues was analyzed using a commercially prepared blot (Multiple Tissue Northern; Clontech, Palo Alto, CA) containing 2 ⁇ g of polyadenylated heart, brain, placenta, lung, liver, kidney, skeletal muscle and pancreas RNA. The RNA blots were hybridized to 32 P-labeled cDNA probes as previously described (Birkenbach, et al., (1993) L Virol. 67:2209-2220).
  • the protein coding region ofthe EBB cDNA was cloned into the EcoRI site ofthe eukaryotic expression vector, pSG5 (Stratagene).
  • An ER/5-FLAG expression vector was constructed which included an in-frame fusion ofthe EBB open reading frame 5' with DNA encoding a kinase site (RRASVG) (S ⁇ Q ID NO: 5) and FLAG epitope (DYKDDDDK) (S ⁇ Q ID NO: 6), inserted into the ⁇ coRI site of pSG5.
  • RRASVG kinase site
  • DYKDDDDK FLAG epitope
  • Plasmid DNA was purified by isopycnic banding on CsCl gradients.
  • Approximately lxl 0 7 target cells were transfected with 40 to 50 ⁇ g purified plasmid DNA by eiectroporation at 200 V, 960 ⁇ F in 0.4 cm cuvettes (BioRad, Hercules, CA).
  • BJAB cells stably expressing ⁇ BB-FLAG protein were established by cotransfection ofthe pSG5 ⁇ BB-FLAG construct together with an expression vector carrying the hygromycin resistance gene. Transfectants were selected in 400 ⁇ g/ml hygromycin and cloned by limiting dilution.
  • Tissue samples of placenta or tonsil were frozen in dry ice/isopentane. Tissue sections or cell preparations were fixed 10 min in a 50:50 mixture of acetone:methanol at
  • Labeled cells were washed in ice-cold PBS and lysed in digitonin lysis buffer (1% digitonin, 10 mM triethanolamine pH 7.5, 150 mM NaCl) or NP40 lysis buffer (1% NP40, 20 mM Tris pH 7.4, 150 mM NaCl, 3% glycerol, 1.5 mM EDTA) containing 1 mg/ml BSA and protease inhibitors (1 mM PMSF, 1 ⁇ g/ml leupeptin, 1 ⁇ g/ml pepstatin).
  • Lysates were centrifuged for 30 min at 14,000xg and precleared with NRS bound to Protein A-Sepharose (Pharmacia) or normal mouse serum bound to protein G-Sepharose. Cleared extracts were incubated at 0°C for 1 h with primary antibodies. Immune complexes were bound to Sepharose beads and washed with lysis buffer.
  • 1.5xl0 7 EBB-Flag stably transfected BJAB cells were preincubated for 1.5h at 37 °C in 20 ml of Met/Cys free RPMI 1640 medium supplemented with 10% dialysed bovine serum (ICN), and pulsed for 10 min at 37°C in the same medium containing 2 mCi of 35 SMet Cys (ICN). Cells were then spun and diluted at a concentration of 10 6 per ml in regular RPMI 1640 medium supplemented with 10% bovine serum. At various times, aliquots of 3x10 7 cells were harvested, washed in cold PBS, and lysed in 1% NP40 lysis buffer.
  • ICN dialysed bovine serum
  • Cell lysates were immunoprecipitated with anti-Flag M2 antibody (International Biotechnologies, Inc., New Haven, CT) as described above.
  • immunoprecipitates were washed once in digitonin buffer containing 1 mg/ml BSA, four times in digitonin buffer, twice in 0.5 M LiCl, 0.1 M Tris pH 7.4, and once in 10 mM Tris, pH 7.4.
  • Immune complexes were eluted from protein A Sepharose by boiling for 5 minutes in 100 ⁇ L 50 mM Tris, pH 7.4, 0.5% SDS, 50 mM ⁇ -mercaptoethanol.
  • BJAB cells stably transfected with an EBB-Flag expression vector were washed in cold PBS, lysed by adding ice- cold 1% NP40 lysis buffer (1 ml/10 8 cells) containing 150 mM NaCl, 50 mM Tris pH 7.4, 1.5 mM EDTA, 3% glycerol, ImM PMSF, 1 ⁇ g/ml pepstatin, 1 ⁇ g/ml leupeptin, and rocked for 30 min at 4°C. After centrifugation at 14,000xg for 30 min the supernatant was rocked with anti- Flag M2 beads for 1.5 h at 4°C.
  • EBB EBV-induced cytokine receptor
  • the predicted protein shows structural features characteristic of members of the haematopoietin (cytokine) receptor family (Bazan (1990) Proc. Natl. Acad. Sci. OJSA1 87:6934; Cosman (1993) Cvtokine 5:95).
  • EBB is most closely homologous to the receptor for ciliary neurotrophic factor (CNTFR) (30% identity) and to the IL12 p40 subunit (27% identity), with conservative amino acid substitutions at many ofthe non-identical residues.
  • CNTFR ciliary neurotrophic factor
  • IL12 p40 subunit 27% identity
  • EBB further resembles IL12 p40 mRNA in having a 3 ' untranslated Alu repeat sequence (Gubler et al . ( 1991 ) Proc. Natl. Acad.
  • EBB EBB lacks obvious membrane anchoring sequences such as a hydrophobic or amphipathic segment or a GPI linkage consensus site. Sequencing of five additional clones isolated from the unamplified cDNA library failed to reveal alternative C- terminal domains which could convey membrane association.
  • the murine homologue of EBB has been isolated and similarly lacks a transmembrane domain. EBB is expressed in EBV-infected lymphocytes and placenta.
  • EBB RNA was undetectable in the EBV(-) control cell line BL41, while parallel blots hybridized with probes for glyceraldehyde phosphate dehydrogenase (GAPDH) and actin demonstrated that the BL41 lane contains as much or more RNA than the EBV-infected cell lanes.
  • GPDH glyceraldehyde phosphate dehydrogenase
  • EBB EBV-transformed LCLs
  • EBB RNA is detected at low levels in the non-transforming EBV mutant BL cell line, P3HR1, in the EBV(-) B cell line, Louckes, and several non-lymphoid cell lines, including K562, TK143, RHEK-1 and HeLa.
  • EBV-negative BJAB cell lines stably transfected with different EBV latent genes was analyzed.
  • Expression of EBNA-2 or EBNA-3C failed to induce higher EBB mRNA expression relative to parental BJAB, or vector-transfected BJAB/neo or BJAB/gpt control cell lines.
  • cells converted to expression of the EBV latent membrane protein 1 (LMP-1) had high levels of EBB mRNA, comparable to the level in EBV-transformed LCLs or EBV- infected BL41/B95-8 cells.
  • Similar analyses of a series of transfectants in EBV-negative BL41 and Louckes cells also showed induction of EBI-3 gene expression by LMP-1.
  • EBB RNA In human lymphoid tissues, EBB RNA is present at low levels in normal unfractionated cells of tonsil and at significantly higher levels in spleen, but is undetectable in bone marrow and in resting peripheral blood mononuclear cells (PBMC). However, EBI-3 mRNA is induced in PBMC by stimulation with the B and T lymphocyte activating agent, pokeweed mitogen (PWM). In placenta EBB RNA levels were significantly higher than in lymphoid cells. EBB
  • EBB Tissue expression of EBB was further analysed by immunostaining with polyclonal antiserum. Staining of frozen tissue sections of human placenta demonstrated EBB in trophoblast cells lining placental villi with a diffuse cytoplasmic staining and perinuclear accentuation. Expression of EBB by trophoblasts has been confirmed by in situ hybridization with an EBB anti-sense RNA probe. In human tonsil, EBB was detected in scattered mononuclear cells of interfollicular zones. The EBB-positive cells showed a reticular cytoplasmic staining pattern and have abundant cytoplasm, and indented nuclei which were often larger than nuclei of neighboring lymphocytes.
  • EBB-positive cells fail to express the B cell marker, CD22.
  • the mo ⁇ hology and location of EBB -producing cells suggest these may be macrophages.
  • EBB positive cells were in perifollicular zones of periarteriolar sheaths and of lymphoid follicles, associated with sinusoids.
  • EBB-positive cells were significantly more abundant in spleen than in tonsil, consistent with the northern analysis.
  • EBB is present in the cytoplasm and on the plasma membranes of producing cells.
  • Live IB4 cells, or BJAB cells and COS7 cells transfected with a SV40 promoter EBB cDNA expression vector were stained in suspension using rabbit polyclonal EBB antiserum.
  • IB4 lymphocytes faint membrane fluorescence was observed. More intense plasma membrane staining was observed in both BJAB B lymphocytes and COS7 cells transfected with EBB cDNA.
  • An identical pattern was observed in ER/3-Flag transfected BJAB or COS7 cells stained with the anti-Flag M2 monoclonal antibody.
  • Fluorescence staining of live pSG5 vector transfected control cells was not detected with EBB antiserum or anti-Flag M2 antibody. Staining of IB4 or of ER/i-transfected BJAB or COS7 cells was not observed using NRS. Immunostaining of fixed IB4 cells or ER/3-transfected BJAB or COS7 cells revealed cytoplasmic fluorescence in a reticular pattern and weaker plasma membrane staining. No staining was observed in vector control transfected cells or with NRS. The more intense cytoplasmic staining indicates that most cell-associated EBB protein is retained in intracytoplasmic compartments. EBB is a secreted glycoprotein.
  • EBB antiserum identified a 33 kD protein in immunoblots of placenta and of EBV(+) BL41/B95-8 and IB4 cells, but not in EBV(-) BL41 cells.
  • a protein of identical size was detected in lysates of BJAB or COS7 cells transfected with EBB cDNA pSG5 expression vector, but was not present in control COS7 or BJAB cells transfected with the pSG5 expression vector alone. These results confirm that the EBB cDNA contains the complete reading frame.
  • 30 kD and 24-25 kD proteins were also detected in ER/J-transfected BJAB and COS7 cells. These are probably stable degradation products as they are not observed with short times of pulse labeling and immunoprecipitation.
  • EBB is 34 kD, slightly larger than cell-associated EBB.
  • no reactivity was detected in supernatants of pSG5 vector transfected control cells.
  • EBB associates with calnexin and a 60 kD protein. Proteins of 95 kD and 60 kD co- immunoprecipitated with EBB from digitonin or NP40 lysates of 35 S-methionine-labeled BJAB cells transfected with ER/5-Flag or EBB. Proteins of identical size were detected in anti-Flag
  • the 95 and 60 kD proteins were not precipitated with the M2 Mab or EBB Ab from vector transfected control BJAB or COS7 cells.
  • the 95 kD protein was also observed in immunoprecipitates from IB4 cells with EBB specific antiserum. The 95 kD protein was not substantially affected when immunoprecipitates were resolved in non-reducing conditions indicating that the 95 kD protein is not covalently associated with EBB by disulfide linkage.
  • p95 and p60 associated with EBB within 10 min.
  • a protein of 78 kD also associated with nascent EBB. However this association was transient.
  • both proteins were purified by immunoaffinity from BJAB cells stably transfected with ER/5:Flag, separated by SDS/PAG ⁇ , and protease digested.
  • the two peptide sequences were not identical to any sequences in the data bases.
  • a degenerate oligonucleotide probe complementary to the RNA sequence predicted to encode a portion ofthe first peptide was used to isolate p60 cDNA clones from the BL41/B95-8 cDNA library.
  • the predicted protein encoded by the p60 clones contains regions which match identically both peptide fragments, and has limited homology to the Drosophila Ref 2(P) protein (Dezelee et al. (1989) EMBO J. 8:3437-3446). However, no potential signal peptide for membrane translocation or transmembrane domain was identified.
  • p60 is a cytoplasmic protein which may associate with EBB via the cytosolic domain of calnexin or some other, as yet unidentified EBB-associated integral membrane protein.
  • the p35 subunit of IL12 associates in vivo with EBB to form a soluble non-covalent heterodimer.
  • coimmunoprecipitation experiments were performed in an EBV (-) B lymphoblast cell line (BJAB) transiently expressing EBB and a carboxyterminal FLAG-tagged p35, or an unrelated cytoplasmic FLAG-tagged control protein (FLAGLMP1CT).
  • BJAB cells were electroporated with pSG5 vector expressing the proteins indicated by a plus at the top of Fig. 1.
  • Digitonin lysates obtained 24 hours post-transfection or culture supernatants collected 48 hours post- transfection were subjected to immunoprecipitation with anti-FLAG M2 monoclonal antibody followed by protein G (Fig. 1 A, lanes 6-10) or anti-FLAG M2 affinity gel (Fig. IB, lanes 6-10).
  • Fig. 1 A lanes 6-10
  • Fig. IB anti-FLAG M2 affinity gel
  • COS cells were transiently transfected with a pSG5 p35 expression vector together with an EBBFLAG or EBB expression vector as indicated at the top of Figs. 2A and 2B, and lysed 48 hours post-transfection in 0.5% NP40 lysis buffer.
  • Cell lysates were submitted to immunoprecipitation with anti-FLAG M2 monoclonal antibody (Fig. 2A) or with affinity- purified EBB antiserum (Fig. 2B).
  • EBB specifically associates with p35 to form a novel soluble heterodimeric cytokine, EBB/p35.
  • EBB and p35 can associate in solution.
  • COS cells were electroporated with EBB and p35FLAG expression vectors as indicated at the top of Fig. 3.
  • COS cells were either cotransfected with EBB and p35FLAG expression vectors (lanes 3 and 8), or were independently transfected with EBB or p35FLAG expression vectors and then cocultivated (lanes 4-5, 9-10). After 48 hours (lanes 1, 2, 6, and 7) or 65 hours of culture (lanes 3-5, 8-10), cell culture supernatants were harvested and submitted to immunoprecipitation with anti-FLAG M2 affinity gel.
  • Immunoprecipitates were separated on an 11 % SDS-PAGE gel in reducing conditions, and subjected to western blot analysis using EBB antisera (Fig. 3 A) or IL12 antisera (Fig. 3B) followed by chemiluminescent detection.
  • EBB antisera Fig. 3 A
  • IL12 antisera Fig. 3B
  • the positions of molecular weight markers are indicated on the left of Figs. 3 A and B, and the positions of EBB and p35 are shown by arrows.
  • EBB coimmunoprecipitated along with p35FLAG In both cases, EBB coimmunoprecipitated along with p35FLAG. Thus, EBB/p35 association does not require intracellular coexpression of both proteins.
  • BJAB cells were electroporated with pSG5 vectors expressing the proteins indicated at the top of Fig. 4. Approximately 22 hours post-transfection, cells were labelled with 35 S-Met Cys for 18 hours. 0.5% NP40 solubilized cell extracts (Fig. 4A) or cell culture supernatants (Fig. 4B) were subjected to immunoprecipitation with anti-FLAG M2 antibody (Fig. 4A, lanes 1, 3-6), anti- FLAG M2 affinity gel (Fig.
  • FIG. 4B lanes 1, 3-6) or with affinity-purified rabbit EBB antiserum (Figs. 4A and B, lane 2) and the immunoprecipitates separated on a 10% SDS-PAGE gel in reducing conditions.
  • Numbers at the left of Figs. 4A and B indicate the position of protein molecular weight standards (in kilodaltons). The positions of EBB and p35 are indicated on the right of Figs. 4A and B.
  • CORRESPONDENCE ADDRESS (A) ADDRESSEE: WOLF, GREENFIELD & SACKS, P.C.
  • GAATTCCGCA GCC ATG ACC CCG CAG CTT CTC CTG GCC CTT GTC CTC TGG 49
  • ATC CTC AGG GCT GTG CGG CCC CGA GCC AGG TAC TAC GTC CAA GTG GCG 625 He Leu Arg Ala Val Arg Pro Arg Ala Arg Tyr Tyr Val Gin Val Ala 190 195 200
  • CTCCAGACAG CACCTGGGTC
  • CTCGCCACCC TAAGCCCCGG GACACCTGTT GGAGGGCGGA 780
  • CTACTCTCTC CTTTACCTT ACCTTTACCA CAGTGCAGGG CTGACTGAAC TGTCACTGTG 960
  • AAACCAGTCC GGGCCACACA GCAAGACCCC ATCTCTAAAA AATTAATATA AATATAAAAT 1140
  • AAGCTATTTC TCTACCAAAG TGTTTGTGGA AACAAACATG TAAGCATAAC TTATTTTAAA 1180

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Abstract

L'invention porte sur une nouvelle cytokine hématopoïétique hétérodimérique constituée à partir de protéine 3 induite par le virus Epstein Barr (EBI3) et de la sous-unité p35 de l'interleukine-12 (IL12). L'invention concerne également: des préparations sensiblement pures de cette cytokine EBI3/p35, et des anticorps dirigés contre celle-ci; des acides nucléiques isolés codant la cytokine EBI3/p35, ainsi que des cellules hôtes recombinées transformées par ces acides nucléiques. L'invention porte en outre sur des procédés utilisés pour traiter des patients au moyen de la cytokine EBI3/p35 ou d'acides nucléiques codant cette cytokine. L'invention concerne enfin des analyses diagnostiques servant à déceler une grossesse ou un avortement spontané potentiel au moyen d'anticorps dirigés contre ladite cytokine.
PCT/US1996/016572 1995-10-11 1996-10-11 Cytokine hematopoietique et ses utilisations WO1997013859A1 (fr)

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US509295P 1995-10-11 1995-10-11
US60/005,092 1995-10-11
US08/684,687 US5830451A (en) 1996-07-19 1996-07-19 Haematopoietic cytokine Epstein Barr virus-induced protein
US08/684,687 1996-07-19

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0894854A3 (fr) * 1997-07-29 2000-01-26 Smithkline Beecham Corporation Nouveau variant d'épissage du récepteur couplé à la protéine G induit par le virus d'Epstein-Barr
WO2002012282A3 (fr) * 2000-08-03 2003-01-09 Inst Genetics Llc Proteine ebi-3-alt et molecules d'acides nucleiques, nouvelles, et utilisations associees
WO2004003017A1 (fr) * 2002-06-26 2004-01-08 Akzo Nobel N.V. Cytokines aviaires, du type il-12, comportant une ou plusieurs sous-unites p40 et/ou p35
WO2005090400A1 (fr) * 2004-03-18 2005-09-29 The University Court Of The University Of Glasgow Cytokine immunosuppressive
WO2005079848A3 (fr) * 2004-02-17 2005-12-15 Schering Corp Procedes de modulation de l'activite de la cytokine, et reactifs connexes
WO2008036973A3 (fr) * 2006-09-22 2008-06-19 St Jude Childrens Res Hospital Modulation de l'activité régulatrice des lymphocytes t via l'interleukine 35
WO2010101870A1 (fr) 2009-03-03 2010-09-10 St. Jude Children's Research Hospital Compositions et méthodes de génération de lymphocytes t régulateurs induits par l'interleukine-35
WO2011028390A1 (fr) 2009-08-24 2011-03-10 St. Jude Children's Research Hospital Compositions et procédés de potentialisation d'interleukine-35
WO2011063198A2 (fr) 2009-11-20 2011-05-26 St. Jude Children's Research Hospital Procédés et compositions de modulation de l'activité du complexe du récepteur de l'interleukine 35
CN105688190A (zh) * 2016-01-27 2016-06-22 武汉大学 白细胞介素35在制备抗病毒药物的用途

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Title
BIOCHIMICA ET BIOPHYSICA ACTA, 1995, Vol. 1270, ZARLENGA et al., "Enzymatic Amplification and Molecular Cloning of cDNA Encoding the Small and Large Subunits of Bovine Interleukin 12", pages 215-217. *
PROC. NATL. ACAD. SCI. U.S.A., May 1991, Vol. 88, GUBLER et al., "Coexpression of two Distinct Genes is Required to Generate Secreted Bioactive Cytotoxic Lymphocyte Maturation Factor", pages 4143-4147. *
THE JOURNAL OF IMMUNOLOGY, 01 June 1992, Vol. 148, No. 11, SCHOENHAUT et al., "Cloning and Expression of Murine IL-12", pages 3433-3440. *
THE JOURNAL OF IMMUNOLOGY, 01 May 1991, Vol. 146, No. 9, WOLF et al., "Cloning of cDNA for Natural Killer Cell Stimulatory Factor, a Heterodimeric Cytokine with Multiple Biologic Effects on T and Natural Killer Cells", pages 3074-3081. *
THE JOURNAL OF IMMUNOLOGY, 1995, Vol. 155, VILLINGER et al., "Comparative Sequence Analysis of Cytokine Genes From Human and Nonhuman Primates", pages 3946-3954. *

Cited By (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0894854A3 (fr) * 1997-07-29 2000-01-26 Smithkline Beecham Corporation Nouveau variant d'épissage du récepteur couplé à la protéine G induit par le virus d'Epstein-Barr
WO2002012282A3 (fr) * 2000-08-03 2003-01-09 Inst Genetics Llc Proteine ebi-3-alt et molecules d'acides nucleiques, nouvelles, et utilisations associees
WO2004003017A1 (fr) * 2002-06-26 2004-01-08 Akzo Nobel N.V. Cytokines aviaires, du type il-12, comportant une ou plusieurs sous-unites p40 et/ou p35
US7347996B1 (en) 2002-06-26 2008-03-25 Intevert International B.V. Avian cytokines, such as IL-12, comprising a p40 and/or p35 subunit and vaccines
WO2005079848A3 (fr) * 2004-02-17 2005-12-15 Schering Corp Procedes de modulation de l'activite de la cytokine, et reactifs connexes
WO2005090400A1 (fr) * 2004-03-18 2005-09-29 The University Court Of The University Of Glasgow Cytokine immunosuppressive
US9217135B2 (en) 2006-09-22 2015-12-22 St. Jude Children's Research Hospital T effector cells modulated via interleukin 35 and methods of culturing same
AU2007298571B2 (en) * 2006-09-22 2012-06-07 St. Jude Children's Research Hospital Modulating regulatory T cell activity via Interleukin 35
US8784807B2 (en) 2006-09-22 2014-07-22 St. Jude Children's Research Hospital Method of inhibiting regulatory T-cell activity by administering an antibody that inhibits interleukin 35
WO2008036973A3 (fr) * 2006-09-22 2008-06-19 St Jude Childrens Res Hospital Modulation de l'activité régulatrice des lymphocytes t via l'interleukine 35
US9518113B2 (en) 2006-09-22 2016-12-13 St. Jude Children's Research Hospital Monoclonal antibodies to interleukin 35 and methods of use thereof to inhibit regulatory T cell function
WO2010101870A1 (fr) 2009-03-03 2010-09-10 St. Jude Children's Research Hospital Compositions et méthodes de génération de lymphocytes t régulateurs induits par l'interleukine-35
WO2011028390A1 (fr) 2009-08-24 2011-03-10 St. Jude Children's Research Hospital Compositions et procédés de potentialisation d'interleukine-35
WO2011063198A2 (fr) 2009-11-20 2011-05-26 St. Jude Children's Research Hospital Procédés et compositions de modulation de l'activité du complexe du récepteur de l'interleukine 35
US10392431B2 (en) 2009-11-20 2019-08-27 St. Jude Children's Research Hospital Polynucleotide encoding IL-35 receptor
CN105688190A (zh) * 2016-01-27 2016-06-22 武汉大学 白细胞介素35在制备抗病毒药物的用途
CN105688190B (zh) * 2016-01-27 2020-03-10 武汉大学 白细胞介素35在制备抗病毒药物的用途

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