NO318435B1 - Isolated DNA sequence from IL-15, isolated DNA sequence encoding a precursor polypeptide of the biologically active IL-15 polypeptide, recombinant expression vector, host cell, method of producing an IL-15 polypeptide, isolated biologically active IL-15 polypeptide preparation, pharmaceutical composition for stimulating T-lymphocyte proliferation, and using a polypeptide according to the invention for the preparation of a means for treating or preventing specific diseases. - Google Patents

Abstract

Described is a mammalian epithelial-derived T cell factor polypeptide ("ETT") referred to herein as interleukin-15 ("IL-15"), derivatives thereof, DNA sequences, recombinant DNA molecules, and transformed host cells that produce IL-15 polypeptides. More particularly, this invention provides isolated mammalian IL-15 polypeptides and derivatives thereof that regulate events of T lymphocyte proliferation.

Description

Field of the invention

Isolated DNA sequence from IL-15, isolated DNA sequence encoding a precursor polypeptide of the biologically active IL-15 polypeptide, recombinant expression vector, host cell, method for producing an IL-15 polypeptide, isolated,

biologically active IL-15 polypeptide preparation, pharmaceutical preparation for stimulating the proliferation of T-lymphocytes, as well as the use of a polypeptide according to the invention for the production of an agent for the treatment or prevention of specifically indicated diseases.

The background of the invention

T cells, also known as T lymphocytes, are a class of immune effector cells. In peripheral tissues, T cells can be divided into two general groups based on their mutually exclusive expression of CD4 and CD8 cell surface molecules. Typical CD8<+->T cells become cytotoxic T cells after activation and destruction

provides antigen-bearing target cells through direct cell contact. Activated CD4<*->T cells generally provide positive signals, e.g. "helper" function for B cells (it enables B-

cells to differentiate into antibody-producing cells), and cal-

therefore read helper T cells.

Six T-cell growth factors have previously been identified. The six are: interleukin (IL)-2, -4, -7, -9, -12 and cofactor IL-10. IL-2's open reading frame codes for a 15 kDa 153 amino acid polypeptide. IL-2 is produced by certain T cells and by large, granular lymphocytes. IL-2 was origi-

lig discovered as a factor that would support long-term growth of human T cells. In addition to T cell growth, its effects include activation of natural killer cells (NK

cells) and lymphocyte-activated killer cells (LAK cells), as well as cytotoxic T cells ("CTL"), macrophages and promoting B-cell growth.

IL-4 is a 15-20 kDa protein produced by activated T cells, bone marrow stromal cells and mast cells. The open IL-4 ise frame codes for 140 amino acid murine IL-4 and 153 amino acid human IL-4. Initially, IL-4 was defined as a factor that activated B-cell growth and differentiation. Its effects also include macrophage activation and induction of class II MHC molecules, growth of some T cell and mast cell lines, proliferation and CTL generation from human peripheral blood T cells, increase in immunoglobulin production by B cells and a cofactor in the growth of hematopoietic cells from stem cells. IL-4 plays an important role in the down-regulation of IL-2-induced NK cell and LAK cell activities. Human IL-4 is not active on murine cells.

IL-7 is a 20-25 kDa, 177 amino acid polypeptide produced by bone marrow and thymus stromal cells. Although originally described as a pre-B cell growth factor, IL-7 supports the growth of pro-B cells as well as pre-B cells. IL-7 also induces proliferation and CTL generation from human peripheral blood T cells, IL-2 receptor expression, IL-2 production and proliferation in CD4<*-> and CD8<+-> cells. IL-7 also synergizes with IL-2 and increases thymic T cell proliferation and induces proliferation of CD4" and CD8" thymocytes. IL-9 is a 30-40 kDa, 144 amino acid polypeptide produced by activated T-lymphocytes. IL-9 was first identified as a helper T cell growth factor. IL-9 stimulates erythroid development and increases IL-3-induced proliferation in bone marrow-derived mast cells. It also modulates IgE and igG production by B cells in the presence of IL-4. Murine IL-9 is active on human cells, while human IL-9 does not act on murine cells.

Human IL-10 is a 16-20 kDa, 178 amino acid polypeptide produced by macrophages and TH2, but not by TH1-T helper cells. In the same way as IL-2, IL-4 and IL-7, IL-10 has several different biological activities. IL-10 was discovered on the basis of its ability to inhibit cytokine production by activated T cells. Both human and murine IL-10 are growth stimulator cofactors for thymocytes and T cells in combination with IL-7 or IL-2 plus IL-4. IL-10 stimulates mast cell viability and growth in combination with IL-4 or IL-3 plus IL-4. IL-10 also induces the secretion of IgG and the expression of class II MHC molecules on B cells and increases their viability in culture. IL-12 is constitutive or induced by phorbol ester and calcium ionophore in lymphoblastoid cell lines and is produced by LPS-stimulated macrophages. IL-12 has a molecular weight of 70 kDa and an unusual heterodimer structure, in that it is formed from two disulfide-bonded glycoproteins. The larger of the two glycoprotein subunits is a 40 kDa 328 amino acid polypeptide. The smaller glycoprotein subunit is a 35 kDa 253 amino acid polypeptide. Both glycoprotein subunits are required for bioactivity. IL-12 induces the proliferation of activated T cells in both the CD4<+-> and CD8<+-> subsets, independently of IL-2. IL-12 also activates NK cell-mediated cytotoxicity and synergizes with IL-2, whereby LAK cells are produced. Unlike IL-2 and IL-7, but similarly to IL-4, IL-12 causes little or no proliferation of residual peripheral blood mononuclear cells.

Summary of the invention

The present invention comprises isolated DNA sequence, characterized in that the sequence comprises,

(a) DNA sequences encoding the polypeptides of the sequence wherein amino acid 52 is Leu or His, amino acid 57 is Ala or Thr, amino acid 58 is Ser or Asp, amino acid 73 is Ser or Ile and amino acid 80 is Val or Ile, ( b) variants of the sequences in (a) that encode a protein with corresponding biological activity, and (c) DNA sequences that hybridize detectably to the DNA sequences of (a) or (b), or their complementary strands, under very stringent conditions comprising hybridization and washing after hybridization at 55 °C or higher and at a salt concentration of 0.5 x SSC or lower, and which after expression encodes a polypeptide with similar biological activity to the sequences according to SEQ. ID NO. 3 or SEQ. ID NO. 6.

The invention also includes isolated DNA sequence which codes for a precursor polypeptide of the biologically active IL-15 polypeptide, characterized in that the DNA sequence is selected from the group consisting of: a DNA sequence which, after expression, codes for the polypeptide defined by SEQ ID NO. ID NO. 2, and

a DNA sequence which, after expression, codes for polypeptide defined by

SEQ. ID NO. 5.

A new T-cell growth factor, hereinafter referred to as "interleukin-15" ("IL-15"), has been isolated and purified. A cDNA sequence encoding a monkey IL-15 polypeptide was isolated and has a 483 bp 5' non-coding region preceding an open reading frame of 489 bp and a 303 bp 3' non-coding region area. A cDNA sequence encoding a human IL-15 polypeptide has a 316 bp 5' non-coding region preceding an open reading frame of 489 bp and a 397 bp 31 non-coding region. The nucleotide sequences and the deduced amino acid sequences for open reads from monkeys and humans are described in SEQ ID NO. 1 and 4. The open reading frames from both monkey and human code for a precursor polypeptide (SEQ ID NOS 2 and 5). The precursor polypeptides each comprise a 48 amino acid leader sequence and a sequence encoding mature monkey or human IL-15 polypeptides. The active monkey and human IL-15 polypeptides are described respectively in SEQ ID NO. 3 and 6.

The present invention further comprises other IL-15 polypeptides which are coded for by nucleotide sequences that hybridize under medium to very strong conditions to probes defined at nucleotides 145-489 in SEQ ID NO. 1 or 4, or to their complementary DNA or RNA strands, and which, after expression, encode polypeptides that stimulate T-lymphocytes to proliferate and differentiate. The invention further encompasses nucleotide sequences which, due to the degeneracy of the genetic code, encode IL-15 polypeptides encoded by the nucleotide sequences described above and sequences complementary thereto.

Even further, the invention provides for recombinant DNA molecules comprising the above nucleotide sequences, e.g. expression vectors or plasmids and transformed host cells that can be used in the production of IL-15 polypeptides, and methods for the production of recombinant IL-15 polypeptides using such molecules.

Brief description of the drawings

Figure 1 shows the nucleotide sequence and the deduced amino acid sequence of the active monkey species of IL-15. Figure 2 shows the nucleotide sequence and the deduced amino acid sequence of the active human species of IL-15. Figure 3 shows a purification and protein sequencing core that can be used when isolating IL-15 polypeptides. Figure 4 shows the homology between nucleotide sequences encoding human and monkey species of IL-15. The human sequence is shown above the monkey sequence. Figure 5 shows the homology between amino acid sequences of the human and monkey species of IL-15. The human sequence is shown above the monkey sequence. In both species, the leader sequence (amino acids 1-48) is cleaved from the precursor polypeptide, whereby the mature polypeptide is formed (amino acids 49-162). Figure 6 shows the biological activity of recombinant IL-15, IL-2 and IL-4 in vitro using CTLL-2 cells. The in vitro proliferative response of CTLL-2 cells was measured at increasing concentrations of recombinant cytokine (expressed as ng/ml). The data are expressed as cpm of <3>H-thymidine incorporated (X IO"<3>). Figure 7 shows the induction of lytic CTL activity by rIL-15 and IL-2. Antigen-specific cytolytic T lymphocytes (CTL ) were generated in vitro. Human peripheral blood mononuclear cells (PBL) from one donor were stimulated with irradiated PBL from an allogeneic donor in cultures containing different concentrations of either IL-2 or human rIL-15. Cultures were analyzed for on cytolytic activity against <51>Cr-labeled targets derived from the original stimulating donor Lytic units were calculated as the inverse of the fraction of the culture that responded, which is required to produce 50% of the maximal specific <51>Cr -release. Figure 8 shows the induction of LAK cell lytic activity by rIL-15 and IL-2. Lymphokine-activated killer (LAK) cells were generated in vitro. Human PBL were stimulated with irradiated autologous PBL,' and cytolytic activity was measured against Daudi lymphoblastoid cell line jen. Lytic units were calculated as the reciprocal of the fraction of the culture that responded, which is required to produce 30% of the maximum specific 51Cr release. Figure 9 shows the induction of NK cell lytic activity by rIL-15 and IL-2. Natural killer cells (NK cells) were isolated from whole human PBL isolated by antibody affinity to paramagnetic microspheres using monoclonal antibodies against CD16. The purified NK cells were cultured for 3 days, and cytolytic activity was measured against the K562 erythroleukemia cell line.

Detailed description of the invention

"Interleukin-15" or "IL-15" refers to mammalian polypeptides that are structurally similar to the polypeptides described herein and that stimulate T lymphocytes to proliferate and differentiate. IL-15 can be distinguished from IL-2, IL-4, IL-7, IL-9, IL-10 and IL-12 in structure and cellular origin (Table

1). In primates, IL-15 polypeptide is initially produced by epithelial cells as a 162 amino acid precursor IL-15 polypeptide. This precursor contains a 48 amino acid leader sequence that is removed from the precursor polypeptide so that the mature polypeptide is formed. The mature IL-15 polypeptide is capable of signaling proliferation and/or differentiation of progenitor or mature T cells. The protein can therefore be used to promote long-term cultivation in vitro of T-lymphocytes and T-cell lines.

"sIL-15" refers to a monkey species of IL-15. "hIL-15" refers to a human species of IL-15. "rIL-15" refers to recombinant IL-15. Both purified sIL-15 and rIL-15 will stimulate proliferation of CTLL-2 cells (Gillis and Smith, Nature 268:154 (1977); ATCC TIB 214). In the CTLL-2 proliferation assays, supernatants of cells transfected with recombinantly expressed precursor and fusions in the frame of mature forms of sIL-15 induced CTLL-2 cell proliferation. For other analyses, peripheral blood T cells ("PBT") and peripheral blood mononuclear leukocytes ("PBL") were isolated from human peripheral blood. We found that rIL-15 stimulated proliferation of PBT and PBL pre-cultured with phytohemagglutinin ("PHA"). rIL-15 also stimulated proliferation of PHA-activated CD4<+-> and CD8<*-> cells. rIL-15 stimulated proliferation of resting human T cells or resting murine T cell clones in the presence of anti-CD3 (T cell receptor) antibodies. Experiments with PHA-activated PBT show that rIL-15 exerts its growth-stimulating effects independently of IL-2 in that antibodies against IL-2 or against the IL-2 receptor do not inhibit IL-15.

The terms IL-15, sIL-15, and hIL-15 include analogs or subunits of naturally occurring mammalian polypeptides encoded by nucleic acids that bind to the nucleic acid sequences of Figures 1 and 2 (nucleotides 145 through 489 inclusive in SEQ ID NOS 1 and 4) under specified, stringent conditions and which induce proliferation and differentiation of T-lymphocytes, and stimulate proliferation of T-cell lines and isolated PBT.

"Recombinant DNA technology" or "recombinant" as used herein refers to techniques and methods for producing particular polypeptides from microbial (eg, bacterial, fungal or yeast) or mammalian cells or organisms ( eg transgenic organisms) that have been transformed or transfected with cloned or synthetic DNA sequences to enable the biosynthesis of heterologous peptides. Naturally occurring glycosylation patterns will only be achieved with mammalian cell expression systems. Yeast provides a distinct glycosylation pattern. Prokaryotic cell expression (e.g. E. coli) will generally yield polypeptides without glycosyl-

Eng.

"Biologically active" means that a particular IL-15 polypeptide is capable of stimulating T-lymphocyte proliferation and/or differentiation. In the case of sIL-15 and hIL-15, this biological activity also corresponds to stimulation of the proliferation of murine or primate, e.g. huraan T cell lines or PBT.

A "nucleotide sequence" refers to a polynucleotide in the form of a separate fragment or as a component of a larger DNA construct that has been derived from DNA isolated at least once in substantially pure form (ie, free of contaminating, endogenous materials) and in an amount or concentration that permits the identification, manipulation and recovery of its component nucleotide sequences by standard biochemical methods (such as those outlined in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY (1989)), such as a cloning vector. Such sequences are preferably provided in the form of an open reading frame that is uninterrupted by internal, untranslated sequences, or introns, which are usually present in eukaryotic genes. Sequences of untranslated DNA may be present 5' or 3' from an open reading frame where the same does not affect the manipulation or expression of the coding regions.

"Recombinant expression vector" refers to a plasmid comprising a transcription unit comprising a composition of (1) a genetic element or elements that have a regulatory role in gene expression, e.g. promoters or enhancers, (2) a structural or coding sequence that is transcribed into mRNA and translated into a polypeptide with biological IL-15 activity, and (3) appropriate transcription and transcription initiation and termination sequences. The different regulatory elements that can be used are discussed below (see techniques with recombinant DNA). Structural elements intended for use in yeast expression systems preferably comprise a leader sequence which enables extracellular secretion of translated polypeptide by means of a yeast host cell. Alternatively, the recombinant polypeptide in a bacterial expression system may comprise an N-terminal met-

ion residue. The N-terminal methionine residue can then be cleaved from the expressed, recombinant polypeptide, whereby a product is obtained which is suitable for further purification.

"Recombinant microbial expression system" refers to a substantially homogeneous monoculture of suitable host microorganisms, e.g. bacteria, such as E. coil, or yeast, such as S. cerevisiae, which have had a recombinant transcription unit stably integrated into chromosomal DNA, or which carry the recombinant transcription unit as a component of an intrinsic plasmid. In general, host cells constituting a recombinant microbial expression system are the origin of a single transformed stem cell. Recombinant, microbial expression systems will express heterologous polypeptides after induction of the regulatory elements bound to a structural nucleotide sequence to be expressed.

Transformed host cells are cells that have been transformed or transfected with a recombinant expression vector. Expressed mammalian IL-15 will be localized in the host cell and/or secreted into the culture supernatant, depending on the type of host cell and the gene construct inserted into the host cell.

Moderately stringent hybridization conditions refer, as defined here and as known to those skilled in the art, to conditions described e.g. in Sambrook et al., supra, vol. 2, pp. 8.46-8.49 and 9.47-9.55. Moderately strict conditions as defined by Sambrook et al., include e.g. overnight hybridization and post-hybridization washes at 55°C, 5 x SSC, 0.5% SDS. Very strict conditions include higher hybridization temperatures and post-hybridization washes, or lower salt concentrations.

IL-15 polypeptides

We have purified a monkey species of IL-15 (sIL-15) and sequenced the N-terminal peptide in mature sIL-15. Using the N-terminal amino acid sequence and PCR, we isolated a cDNA encoding sIL-15, and determined the nucleotide sequence and deduced amino acid sequence of mature sIL-15 (Figure 1), and the nucleotide sequence and deduced amino acid sequence of a precursor of sIL- 15 polypeptide (SEQ ID NO: 1 and SEQ ID NO: 2). The monkey precursor 1L-15 polypeptide sequence comprises a mature, active protein (SEQ ID NO: 3) following a 48 amino acid leader sequence. The leader sequence is amino acids 1-48 in SEQ ID NO. 2. Primate IL-15 stimulates proliferation of murine T-cell lines (eg, CTLL-2) and stimulates proliferation and differentiation of human PBT cells.

The present invention also encompasses other mammalian IL-15, including human IL-15, which has biological IL-15 activity and is encoded by nucleotide sequences that hybridize under medium to very stringent conditions to probes defined by SEQ ID NO. 5. A plasmid containing a recombinant clone of human IL-15 cDNA was deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD 20852, USA ("ATCC") in accordance with the Budapest Convention on the International Recognition of Deposits of microorganisms in connection with the processing of patent cases, 19 February 1993, under deposit no. ATCC 69245. The deposit was named "141-hETF" and comprised an E. coli strain containing plasmid hETF/pDC406 which contained a 316 bp 5<1->non-coding region in front of a 489 bp open reading frame and a 397 bp large 31 - non-coding region flanked by the Sal I adapters shown in SEQ ID NO. 7 and 8. All restrictions regarding the availability to the general public of the deposited material will be irrevocably removed after the grant of a patent.

The amino acid structure of IL-15 polypeptides described herein can be modified by forming covalent or aggregate conjugates with other chemical residues, such as glycosyl groups, lipids, phosphate, acetyl groups and the like, by . form amino acid sequence mutants. Covalent derivatives of mammalian IL-15 are prepared by attaching specific functional groups to mammalian IL-15 amino acid side chains or to the N-terminus or C-terminus of a mammalian IL-15 polypeptide. Other derivatives of mammalian IL-15 within the scope of this invention include covalent or aggregate conjugates of mammalian IL-15 or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. For example, the conjugated polypeptide may be a signal polypeptide sequence (or leader polypeptide sequence) in the N-terminal region of a mammalian IL-15 polypeptide for transport from its site of synthesis to a site within or outside the cell membrane or wall (e.g. the yeast-a-factor leader). By using common techniques, IL-15 polypeptides can further be expressed as polypeptide fusions comprising additional polypeptide sequences, such as Fc or other immunoglobulin sequences, linker sequences or other sequences that facilitate purification and identification of IL-15 polypeptides. Still further, IL-15 polypeptide fusions may include fusions with other cytokines to provide novel, poly-functional entities. Other cytokines include e.g. any of interleukins 1-13, tumor necrosis factor (TNF), granulocyte macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor (G-CSF), mast cell growth factor (MGF) and other cytokines that affect immune cell growth, differentiation or function.

The present invention further comprises IL-15 polypeptides with altered glycosylation. IL-15 polypeptides expressed in yeast or mammalian expression systems (eg, COS-7 cells (ATCC CRL 1651)) may be similar in molecular weight and glycosylation pattern to or significantly different from a naturally occurring IL-15 polypeptide. This depends on the choice of expression system. Expression of IL-15 polypeptides in bacterial expression systems, such as E. coli, yields non-glycosylated molecules.

Functional mutant analogs of human or other mammalian IL-15 can be synthesized, e.g. with inactivated N-glycosylation sites by means of oligonucleotide synthesis and ligation, or by site-specific mutagenesis techniques. IL-15 polypeptide derivatives can be expressed in homogeneous, reduced carbohydrate form using yeast expression systems. N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-*-£J where * is any amino acid except Pro, and £1 is Ser or Thr. An IL-15 mutant derivative referred to herein is a polypeptide that is substantially homologous to a sequence in a naturally occurring mammalian IL-15 but has an amino acid sequence that is different from a naturally occurring mammalian -IL-15 polypeptide due to a deletion, insertion or substitution.

Bioequivalent analogs of IL-15 polypeptides in IL-15 muteins can be constructed by making different substitutions of amino acid residues or sequences, or by dividing terminal or internal residues or sequences not needed for biological activity. For example, Cys residues can be split or replaced with other amino acids to prevent the formation of incorrect intramolecular disulfide bridges after renaturation. Other methods of mutagenesis include modification of dibasic amino acid residues to increase expression in yeast systems where KEX2 protease activity is present. In general, substitutions are made conservatively by substituting with an amino acid that has physiochemical characteristics similar to those of the naturally occurring residue.

Antisense or sense oligonucleotides comprise single-stranded nucleic acid sequences (either RNA or DNA) capable of binding to sense IL-15 mRNA or antisense IL-15 cDNA sequences. An antisense or sense oligonucleotide according to the present invention comprises a fragment of the nucleotide sequences in Figures 1 or 2, or a DNA or RNA complement to the nucleotide sequences in Figures 1 and 2. Such a fragment comprises at least approx. 14 nucleotides and is able to bind to IL-15 DNA. The ability to form an antisense or a sense oligonucleotide based on a cDNA sequence for IL-15 is described e.g. in Stein and Cohen, Cancer Res. 48:2659 (1988), and van der Krol et al., BioTechnigues 6:958

(1988).

Isolation and characterization of IL-15 from non-recombinant cell sources requires a mammalian cell line that produces IL-15 and a responder cell line that proliferates in response to IL-15 stimulation. A biological assay with regard to mammalian IL-15 can make use of a growth factor-dependent T cell line as a detector for factors that induce lymphoid cell proliferation. T cells isolated from blood samples taken from humans or from other mammals can also be used to analyze mammalian IL-15 polypeptides.

An IL-15-dependent cell line can be derived from murine

CTLL-2 cells. This cell line responds to purified, human, murine and recombinant IL-2 and murine IL-4, but not to IL-1, IL-3, human IL-4 or any of the other known growth factors.

One can utilize the monkey or human IL-15 cDNA sequences described herein to obtain cDNAs encoding other mammalian homologues of monkey or human IL-15 using cross-species hybridization techniques. Briefly, an oligonucleotide probe is formed from the nucleotide sequence in the protein-coding region of the sIL-15 cDNA as described in Figure 1, or SEQ. ID NO. 1, or hIL-15 cDNA as described in Figure 2 or SEQ ID NO. 4. This probe can be made using standard techniques, such as those described in Sambrook et al., supra. The monkey or human probe is used to screen a mammalian cDNA library or genomic DNA library under medium stringency conditions. Mammalian cDNA libraries can be made from mRNAs isolated from e.g. murine peripheral blood lymphocytes. Alternatively, other cDNA libraries or mRNAs isolated from different tissues or cell lines can be screened by Northern hybridization to determine a suitable source of mammalian IL-15 DNA or mRNA.

CV- 1/ EBNA IL- 15- cleaning hose

We have purified IL-15 to provide an isolated polypeptide preparation from a non-homogeneous protein solution, such as conditioned medium collected from cells expressing IL-15. IL-15 activity in crude samples of conditioned medium is not always detectable using currently available IL-15 bioassay techniques. At least one purification step is usually required before biological IL-15 activity is detectable using the biological assay techniques described herein.

A non-homogeneous protein solution, e.g. conditioned medium, is prepared by growing the CV-1/EBNA line (C.J. McMahan et al., EMBO J-, 10(10):2821-2832 (1991); ATCC CRL 10478) of African green monkey kidney cells in a tissue culture ning medium. The medium is preferably Dulbecco's Modified High Glucose Essential Medium ("DMEM", Gibco). Preferably, a cultivation medium and a production medium are used. The culture medium used for the isolation of sIL-15 as described here was DMEM with a high glucose content (4,500 mg/l) supplemented with 7.5% fetal bovine serum, 50 u/ml penicillin, 50 pg/ml streptomycin, 3-4 .0 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids and 10 mM N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] ("HEPES") buffer. A serum-free production medium of DMEM without phenol red supplemented with 50 u/ml penicillin, 50 µg/ml streptomycin, 3-4.0 mM L-glutamine, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids and 10 mM HEPES buffer was developed for IL-15 production and purification. The CV-1/EBNA cells were adherent and could be grown in dishes, flasks, roller bottles or microcarriers.

Specifically, IL-15 was produced by growing CV-1/EBNA cells on microcarriers in a regulated bioreactor. Cell stocks were stored in roller bottle flasks. To initiate a production cycle, cells were trypsinized and inoculated into a spinner flask containing the culture medium described above and 5 g/l "Cytodex" 3 microcarriers (Pharmacia). Inoculation density at start ranged from 1.5 to 3.5 x 10<5> cells/ml. To ensure effective cell attachment to the microcarriers, the cells were kept in the spinner flask for 2-24 hours. Spinner flask cultures were incubated at 37°C and agitated at 25-40 RPM. After the fixation period, the culture was transferred to a regulated bioreactor. Bioreactor temperature, pH, oxygen and agitation set points were 37°C, 7.0, 20% saturation (relative to air) and 75-85 RPM, respectively. For routine observation of cell growth and condition, samples were observed using bright field microscopy Quantification of cell growth was achieved by counting released nuclei after treatment with a solution of 100 mM citric acid and 0.1% crystal violet.

The culture was supplemented with additional growth medium when ammonium levels reached 5.0 mM. This was repeated until the microcarriers were confluent. The culture medium was then replaced with the serum-free production medium described above. This procedure was carried out by allowing the microcarriers to settle to the bottom of the reactor, aerating the culture medium and replacing it with production medium. This was repeated until an approximately 3.125-fold dilution was achieved. 2 to 6 rounds of production can be expected. In each round, cells were allowed to produce for 4 to 7 days, after which 80% of the production medium was collected. This was repeated until the cells were completely detached from the microcarriers.

Approximately 64 liters of CV-1/EBNA conditioned medium was used to purify and provide protein for the N-amino acid sequence of sIL-15. As shown in Figure 3, the purification scheme included ultrafiltration, hydrophobic chromatography, anion exchange chromatography, reverse phase high performance liquid chromatography (RP-HPLC) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Protein was sequenced by blotting the SDS gel against a polyvinylidene fluoride ("PVDF" membrane) and determining an N-terminal amino acid sequence by Edman digestion directly from the PVDF membrane. N-terminal amino acid sequencing revealed the first 33 amino acids shown in SEQ ID NO. 3. Subsequent sequencing of a cDNA clone obtained from a CV-1/EBNA cDNA library yielded a DNA sequence encoding the polypeptide according to SEQ ID NO. 2. This clone comprises a relatively short 48 amino acid leader sequence from SEQ ID NO. 2 and a mature polypeptide represented by SEQ ID NO. 3.

Recombinant DNA techniques

Human, monkey, and other mammalian IL-15 polypeptides are preferably produced by recombinant DNA techniques. Such techniques include inserting a cDNA encoding a human or other mammalian IL-15 polypeptide, or a derivative thereof, into an expression vector.

Recombinant production of mammalian IL-15 polypeptides or derivatives thereof first requires isolation of a DNA clone (ie, cDNA) which upon expression encodes a mammalian IL-15 polypeptide or derivative thereof. cDNA clones are derived from primary cells or cell lines expressing mammalian IL-15 polypeptides. First, total cell mRNA is isolated, then a cDNA library is made from the mRNA using reverse transcription. A cDNA clone can be isolated and identified using the DNA sequence information provided herein to design a cross-species hybridization probe or PCR primer as described above.

The isolated cDNA is preferably in the form of an open reading frame uninterrupted by internal, untranslated sequences, or introns. Genomic DNA containing the relevant nucleotide sequences that code for expression of mammalian IL-15 polypeptides can also be used as a source of genetic information that can be used in the construction of coding sequences. The isolated cDNA can be mutated using techniques known in the art to yield IL-15 derivatives or analogs that exhibit biological IL-15 activity.

Recombinant expression vectors comprise synthetic or cDNA-derived DNA fragments encoding IL-15 or biologically active derivatives thereof. The DNA encoding an IL-15 or derivative thereof is operably linked to a suitable transcriptional or translational regulatory or structural nucleotide sequence, such as one derived from mammalian, microbial, viral or insect genes. Examples of regulator sequences include e.g. a genetic sequence with a regulatory role in gene expression (e.g. the transcripts promote or enhance), a possible operator sequence to regulate transcription, a sequence that codes for suitable ribosomal mRNA binding sites, and suitable sequences that regulate transcription and translation initiation and - termination. Nucleotide sequences are operatively linked when the regulatory sequence relates functionally to the structural gene. For example, a DNA sequence for a signal peptide (secretory leader) can be operably linked to a structural gene DNA sequence for a mammalian IL-15 or derivative thereof if the signal peptide is expressed as part of a precursor amino acid sequence and participates in the secretion of a mammalian IL-15 IL-15. Furthermore, a promoter nucleotide sequence is operatively linked to a coding sequence (e.g. structural gene DNA) if the promoter nucleotide sequence regulates the transcription of the structural gene nucleotide sequence. Even further, a ribosome binding site can be operably linked to a structural gene nucleotide coding sequence (eg, mammalian IL-15) if the ribosome binding site is located within the vector to aid translation.

Suitable host cells for expression of mammalian IL-15 or derivatives thereof include prokaryotes, yeast or higher eukaryotic cells under the control of appropriate promoters. Prokaryotes include gram-negative or gram-positive organisms, e.g. E. coli or bacilli. Suitable prokaryotic host cells for transformation include e.g. E. coli, Bacillus subtilis, Salmonella typhlmurium and various other species

within the genera Pseudomonas, Streptomyces and Staphylococcus. As discussed further below, examples of suitable host cells also include yeast, such as S. cerevisiae, a mammalian cell line, such as Chinese hamster ovary (CHO) cells, or insect cells. Cell-free translation systems can also be used to produce mammalian IL-15 or derivatives thereof using RNAs derived from the DNA constructs described herein. Suitable cloning and expression vectors for use with bacterial, fungal, yeast and mammalian cell hosts are described e.g. in Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985.

When" a mammalian IL-15 or derivative thereof is expressed in a yeast host cell, the nucleotide sequence (eg, the structural gene) which upon expression encodes a mammalian IL-15 or derivative thereof may comprise a leader sequence. The leader sequence may enable enhanced, extracellular secretion of translated polypeptide by a yeast host cell.

Mammalian IL-15 can be expressed in yeast host cells, preferably from the Saccharontyces genus (eg, S. cerevisiae). Other yeast genera, such as Pichla or Kluyveromyces, can also be used. Yeast vectors will often contain an origin of replication sequence from a 2 µ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation and sequences for transcription termination. Preferably, yeast vectors comprise an origin of replication sequence and selectable marker. Suitable promoter sequences for yeast vectors include promoters for metallothionein, 3-phospho-glycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7 :149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EP-A-73 657.

Yeast vectors can be put together, e.g. using DNA sequences from pBR322 for selection and replication in E. coli (Amp gene and origin of replication). Other yeast DNA sequences that can be included in a yeast expression construct include a glucose-repressible ADH2 promoter and an α-factor secretion leader. The ADH2 promoter has been described by Russell et al. (J. Biol. Chem. 258:2674, 1982) and Beier et al.

(Nature 300:724, 1982). The yeast α-factor leader sequence directs secretion of heterologous polypeptides. The α-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See e.g. Kurjan et al., Cell 30:933, 1982; and Bitter et al., Proe. Nati. Acad. Pollock. USA 81:5330, 1984. A leader sequence can be modified near its 3<*->end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene. Yeast transformation methods are known to those skilled in the art. One such method is described by Hinnen et al.. Proe. Nati. Acad. Seal. USA 75:1929, 1978. The method of Hinnen et al. selects with regard to Trp<+->transformants in a selective medium where the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 mg/ml adenine and 20 mg/ml uracil.

Yeast host cells transformed using vectors containing the ADH2 promoter sequence can be cultured to induce expression in a "rich" medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone and 1% glucose supplemented with 80 mg/ml adenine and 80 mg/ml uracil. Derepression of the ADH2 promoter occurs when the medium is depleted of glucose.

Alternatively, in a prokaryotic host cell, such as E. coli, the mammalian IL-15 or derivative thereof may comprise an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in a prokaryotic host cell. The N-terminal Met can be cleaved from the expressed recombinant mammalian IL-15.

The recombinant expression vectors carrying the recombinant mammalian IL-15 structural gene nucleotide sequence or derivative thereof are transfected or transformed into a suitable host microorganism or mammalian cell line.

Expression vectors transfected into prokaryotic host cells generally comprise one or more phenotype selectable markers. A phenotype selectable marker is e.g. a gene encoding proteins conferring antibiotic resistance, or conferring an autotrophic requirement, and an origin of replication recognized by the host, to ensure amplification in the host. Other useful expression vectors for prokaryotic host cells include a selectable marker of bacterial origin, derived from commercially available plasmids. This selectable marker may comprise genetic elements from the cloning vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells. The pBR322 "backbone" segments are combined with an appropriate promoter and a mammalian IL-15 structural gene sequence. Other commercially available vectors include e.g. pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, WI, USA).

Promoter sequences are commonly used for recombinant prokaryotic host cell expression vectors. Common promoter sequences include B-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al.. Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al. , Nucl. Acids Res. 8:4057, 1980; and EPA 36, 776) and tac proteins (Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989)). A particularly useful prokaryotic host cell expression system makes use of a phage X PL promoter and a heat-labile cl857ts repressor sequence. Plasmid vectors available from the American Type Culture Collection that incorporate derivatives of the k PL promoter include plasmid pHUB2 (located in E. coli strain JMB9 (ATCC 37092)) and pPLc28 (located in E. coli RR1 (ATCC 53082)).

Mammalian or insect host cell culture systems will also be able to be used to express recombinant mammalian IL-15 polypeptide or derivatives thereof. Examples of suitable mammalian host cell lines include the C0S-7 line of monkey kidney cells

(Gluzman et al., Cell 23:175 (1981); ATCC CRL 1651), L cells, C127 cells, 3T3 cells (ATCC CCL 163), CHO cells, HeLa cells (ATCC CCL 2) and BHK - (ATCC CRL 10) cell lines. Suitable mammalian expression vectors include non-transcribed elements, such as an origin of replication, a promoter sequence, an enhancer linked to the structural gene, other 5'- or 3<1->flanking, non-transcribed sequences, such as ribosome-binding sites, a polyadenylation site, splice donors - and -acceptor sites, and transcription termination sequences.

Transcriptional and translational control sequences in mammalian host cell expression vectors can be provided by viral sources. For example, commonly used mammalian cell promoter and enhancer sequences are derived from polyora, adenovirus 2, simian virus 40 (SV4Q), and human cytomegalovirus. DNA sequences derived from the SV40 virus genome, e.g. SV40 origin, early and late promoter, enhancer, splicing and polyadenylation sites can be used to provide the other genetic elements required for expression of a structural gene sequence in a mammalian host cell. Viral early and late promoters are particularly useful as both are readily obtained from a viral genome as a fragment which may also contain a viral origin of replication (Fiers et al.. Nature 273:113, 1978). Smaller or larger SV40 fragments can also be used, provided that the approximately 250 sequence extending from the Hind 111 site toward the Bgl i site located in the SV40 viral origin of replication is included.

Exemplary mammalian expression vectors can be constructed as described by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983). Additional useful mammalian expression vectors are described in US Patent Application No. 07/480,694 filed February 14, 1990, and in US Patent Application No. 07/543,193 filed June 5, 1990.

Purification of recombinant pattedvr-IL-15

IL-15 polypeptides can be prepared by growing transformed host cells under culture conditions necessary to express mammalian IL-15 polypeptides or derivatives thereof. The resulting expressed polypeptides can then be purified from culture media or cell extracts. A mammalian IL-15 polypeptide or derivative thereof can be concentrated using a commercially available protein concentration filter, e.g. an "Amicon" or "Millipore Pellicon" ultrafiltration unit. With or without the concentration step, the culture medium can be applied to a purification matrix, such as a hydrophobic chromatography medium. Phenyl-Sepharose CL-4B (Pharmacia) is the preferred medium. Alternatively, an anion exchange resin can be used, e.g. a matrix or a substrate with protruding diethylaminoethyl groups (DEAE groups). The matrices can be acrylamide, agarose, dextran, cellulose or other types that are usually used in protein purification. Alternatively, gel filtration medium can be used. Recombinant IL-15 is stable in acidic, aqueous buffers, and a cation exchange resin can also be used.

Finally, one or more steps of reverse phase high performance liquid chromatography (RP-HPLC) can be employed using hydrophobic RP-HPLC media, e.g. silica gel with protruding methyl or other aliphatic groups, to further purify IL-15. Some or all of the previous purification steps can also be used in different combinations to provide an essentially homogeneous, recombinant protein. Alternatively, some or all of the steps used in the purification procedure described above for monkey IL-15 can also be used.

Recombinant protein produced in bacterial culture is usually isolated by initial disruption of the host cells, centrifugation, extraction from the cell pellet if it is an insoluble polypeptide, or from the supernatant if it is a soluble polypeptide, followed by one or more concentration, salting out, ion exchange or size exclusion chromatography step. Finally, RP-HPLC can be used for the final purification step. Microbial cells can be disrupted by any suitable method, including freeze-thaw cycles, sonication, mechanical disruption, or the use of cell lysing agents.

Transformed yeast host cells are preferably used to express IL-15 as a secreted polypeptide. This simplifies cleaning. Secreted recombinant polypeptide from a yeast-host cell mutant can be purified by methods analogous to those described by Urdal et al. (J. Chromatog. 296:171, 1984). Urdal et al. describes two sequence-

show reverse-phase HPLC steps for the purification of recombinant human IL-2 on a preparative HPLC column.

Administration of mammalian IL-15 polypeptide or derivative preparations

The present invention comprises the use of a polypeptide according to the invention v for the production of an agent for the treatment or prevention of anemia, carcinoma, melanoma, sarcoma, leukemia, lymphoma or for the treatment or prevention of infections caused by herpes viruses, including cytomegalovirus, polyoma virus, retrovirus, including HIV, influenza virus, hepatitis virus, including hepatitis A, hepatitis B, hepatitis C, hepatitis 5 or hepatitis D. For therapeutic use, purified IL-15 or a biologically active derivative thereof is administered to a patient, preferably a human, for treatment in a manner that suitable for indica-

the tion. Thus, e.g. IL-15 preparations administered to suppress a form of anemia are given by bolus injection

tion, continuous injection, prolonged release from implants or other suitable technique. Administration can be

by intravenous injection, subcutaneous injection or parenteral or intraperitoneal injection. Usually, a therapist will

ical IL-15 agent be administered in the form of a pharmaceutical preparation comprising purified polypeptide together with physiologically acceptable carriers, excipients or diluents. Such carriers will be non-toxic to patients at the dosages and concentrations used. Usually, the production of such preparations involves mixing a mammalian IL-15 polypeptide or derivative thereof with buffers, antioxidants, such

such as ascorbic acid, low molecular weight polypeptides (less than

about. 10 residues), proteins, amino acids, carbohydrates including glucose, sucrose or dextrans, such chelating agents

such as EDTA, glutathione and other stabilizers and excipients. Neutral buffered saline or saline solution mixed with conspecific serum albumin are exemplary suitable diluents.

The following examples are for illustrative purposes and

not as a limitation.

Example 1

Purification and sequencing for naturally occurring sIL-15

Ultrafiltration

Ultrafiltration was not absolutely necessary to purify IL-15. However, the process removes certain minor contaminating proteins and reduces the volume, thereby speeding up the purification process. The ultrafiltration step can be carried out by using either a YM10 or YM30 spiral cartridge, a hollow fiber cartridge or a disc membrane in different types of ultrafiltration apparatus. However, an "Amicon" ultrafiltration system with a YM30 spiral cartridge was preferred. No buffering was required before or after this step.

A non-homogeneous protein solution, i.e. conditioned medium, was obtained by growing CV-1/EBNA cell cultures in serum-free, phenol red-free DMEM in bioreactors with 5 g/l "Cytodex" 3 microcarriers. 8x8 liter bioreactors (total approx. 64 liters) were harvested, centrifuged to remove cells and microcarriers, filtered through a 0.22 µm cellulose acetate membrane filter and then concentrated to a final volume of approx. 2 liters using a YM30 spiral cartridge. Preferably, the YM30 concentrate is filtered before undergoing hydrophobic chromatography. This step will minimize contamination by removing bacteria and other particles. Any filter with a pore size from 0.1 to 0.45 µm that does not bind protein can be used; however, a 0.22 µm cellulose acetate membrane filter was preferred.

Hydrophobic chromatography

Hydrophobic chromatography provided a rapid way of transferring the protein into a low salt buffer that could be applied to anion exchange columns. In addition, it provided three to six times of purification. Even further, no buffering was required before or after this step. Various hydrophobic columns are suitable. A phenyl "Sepharose" CL-4B column (Pharmacia) was preferred. As an alternative to the hydrophobic chromatography step described here, a diafiltration or dialysis step can be used.

Preferably, ammonium sulfate was added to the ultrafiltration concentrate until a final concentration of approx.

0.2 M. The ultrafiltration concentrate was buffered with 1 M HEPES at approx. pH 8.5 until a final concentration of approx. 20 mM. The concentrate was then pumped onto a phenyl-Sepharose CL-4B column and washed with 0.2 M ammonium sulfate, 10 mM HEPES, at approx. pH 8.5 to remove unbound protein. Bound proteins were eluted with 10 mM HEPES at approx. pH 8.5. The eluted protein peak (including IL-15) was passed to an anion exchange column.

Anion exchange chromatography

Anion exchange chromatography purification enabled further purification without requiring dialysis or buffer exchange. This step preferably involved two passes of the phenyl-Sepharose protein mixture over columns containing an anion exchange medium. After each passage, the bound proteins were eluted with NaCl in HEPES. Although various anion exchange media and buffers exist with a pH of approx. 8 to approx. 9 was suitable, DEAE-"Sephacel" (Pharmacia) followed by Mono Q, enabled sequential anion exchange steps without buffer exchanges or dialysis. DEAE-"Sephacel" provided removal of some contaminating proteins prior to application to a fast-acting Mono Q higher resolution chromatography ("FPLC", Pharmacia). The NaCl concentration used was dependent on the selected anion exchanger and the pH of the selected buffer. Other salts could be replaced with NaCl to elute the protein from the anion exchange gel.

Most preferably, NaCl was added to the phenyl "Sepharose" mixture until a final conductivity of approx. 1.2 mil-liSiemens/centimeter ("mS/cm") (less than about 0.1 M NaCl) and pumped onto a DEAE "Sephacel" column equilibrated with about 0.1 M NaCl for approx. 10 mM HEPES at approx. pH 8.5. Bound proteins were eluted with a linear gradient from approx. 0.1 to approx. 0.3 M NaCl for approx. 10 mM HEPES at approx. pH 8.5. The active protein fractions that eluted (including IL-15) were pooled for input to eh Mono Q anion exchange column.

The active DEAE-"Sephacel" mixture was diluted with approx. 10 mM HEPES until a final conductivity less than 1.6 mS/cm (less than 0.14 M NaCl). The diluted mixture was then pumped onto a Mono Q FPLC fast liquid chromatography column equilibrated with approx. 0.14 M NaCl for approx. 10 mM HEPES at approx. pH 8.5. Bound proteins were eluted with a gradient from approx. 0.14 M to approx. 0.5 M NaCl for approx. 10 mM HEPES at approx. pH 8.5. The active fractions (including IL-15) were pooled for input to reverse phase high performance liquid chromatography (RP-HPLC).

RP-HPLC

The naturally occurring mammalian IL-15 polypeptide is stable from approx. pH 7 to approx. 9 and at approx. pH 2.5 in 0.1%

Trifluoroacetic acid ("TFA") in acetonitrile ("AcN"). IL-15 activity was not recovered when acidic aqueous buffers were used; many HPLC buffer systems were thus eliminated, and cation-exchange chromatography could not be included in the purification scheme. C4-RP-HPLC columns ("Vydac" 0.46 x 25 cm, 5 pm) gave the greatest purification. Other reverse phase columns (C8 or C18) did not work; the protein eluted from C4 at a high AcN concentration and was not recovered from C8 or C18 at all. Other buffer systems that were tried (ie, ammonium acetate/methanol, pH 7, and pyridine acetate/propanol) were not successful.

The RP-HPLC purification preferably involved two passes of the active Mono Q mixture through the "Vydac" C4 matrix. In the first pass, the active Mono Q mixture was pumped onto a C4-HPLC column at approx. 1 ml/min. and eluted with a gradient of 0.1% TFA/H 2 O to 0.1% TFA/100% AcN at the following gradient:

0 to 45% AcN, 1% AcN/minute

45 to 60% AcN, 0.5% AcN/minute

60 to 100% AcN, 2% AcN/minute.

Active top fractions (including IL-15) elute between approx. 48 and approx. 51% AcN. Active fractions determined by biological analysis were pooled, diluted with 0.1% tfa/h 2 O to reduce AcN concentration, and fed to the same C4 column.

The active, diluted mixture from the C4-TFA/AcN pre-screen was pumped back to the C4 column, washed with 0.1% TFA/H2O and eluted with a linear gradient of 0.1% TFA/H2O (buffer A) to 0.1% TFA/60% n-propanol (buffer B) at approx.

0.5 ml/min. The gradient was run at approx. 0.5% buffer B/min. Fractions were biologically analyzed to identify the IL-

15-containing fractions. IL-15-containing fractions were pooled.

SDS-PAGE

- Purified IL-15 can be visualized using silver-stained SDS-PAGE. Bands of purified mammalian IL-15 protein isolated by SDS-PAGE can be electroblotted and analyzed to determine their N-terminal amino acid sequences. The IL-15 protein band can be identified by biological analysis.

Fractions with purified IL-15 protein from the C4-TFA/n-propanol HPLC experiment were quickly placed under vacuum until dryness, resuspended in reducing SDS sample buffer and run on a polyacrylamide SDS gel. Preferably, HPLC-purified IL-15 was run on SDS-PAGE ("Phastgel" 8-25%, Pharmacia) in two adjacent fields. Before fixation and staining, approximately 1 mm pieces of gel were cut from one field in "Phastgel" and put directly into biological assays. The remaining gel was developed and the silver colored bands compared with pieces taken for biological analyses. The IL-15 activity corresponded to 15-17 kDa. For specific activity determination, purified IL-15 was resuspended in reducing SDS sample buffer, run on a 14% polyacrylamide-SDS gel (Novex) and silver stained. The purity of the sIL-15 polypeptides corresponding to 15-17 kDa was approximately 222,000-fold greater than the sIL-15 polypeptide purity in the CV-1/EBNA-conditioned medium at the beginning of the purification schedule (Table 2). In addition to purity and protein data, Table 2 shows the activity of the naturally occurring sIL-15 polypeptide in a CTLL-2 biological assay (described below in Example 2) performed after each step of the purification process.

PVDF blot incr

The 14% polyacrylamide-SDS gel was blotted onto a PVDF membrane ("ProBlot" from Applied Biosystems) using constant amperage, at approx. 60 V setting for approx. 1 hour. Protein bands were visualized by staining the PVDF membrane with Coomassie blue (0.1% in 10% acetic acid, 50% methanol). The membrane can be destained using the same solution without the Coomassie blue dye to highlight the protein bands. The protein band corresponding to IL-15 activity was excised, and N-terminal protein sequencing was performed directly from the PVDF membrane.

sIL-15 polypeptide sequence

The IL-15 preparation resulting from the preceding RP-HPLC step was analyzed by SDS-PAGE. Each gel was silver stained to indicate the presence of protein bands. Biological analysis of unstained gel pieces corresponding to visible bands indicated that IL-15 activity was associated with proteins with molecular weights in the range of 15-17 kDa. The N-terminus of the 15-17 kDa polypeptide, blotted onto a PVDF membrane, was sequenced by Edman digestion in an Applied Biosystems protein sequencer. The results indicated the identity of the first 33 amino acids shown in SEQ ID NO. 3. Subsequent sequencing of a cDNA clone obtained from a monkey library gave a sequence which codes for the polypeptide according to SEQ ID NO. 2. The polypeptide according to SEQ ID NO. 2 comprises a relatively short 48 amino acid leader sequence and a mature polypeptide represented by SEQ ID NO. 3. 1

Example 2

Biological analysis

CTLL-2 cells provided a rapid and sensitive bioassay for the detection of IL-15 polypeptides. Other cell lines that also proliferate in response to IL-15 with varying sensitivity are CTLL-2.4 (Valentine et al., Eur. J. Immunol, 21(4):913 (1991)), 32D (Greenberger, Federation Proceedings 42:2762 (1983)), BAF-B03 (Hatakeyama et al., Cell, 59:837

(1989)), M07e (Avanzi et al., Br. J. Haematol. 69:359 (1988)) and TF1 (Kitamura et al., J. Cell. Physiol., 140:323 (1989)). Preferably, CTLL-2 cells were cultured in high glucose DMEM supplemented with ca. 30 ng/ml IL-2, 5% fetal bovine serum {FBS), 5 x 10-<5> M 2-mercaptoethanol, 50 u/ml penicillin, 50 ug/ml streptomycin and 3-4.0 mM L-glutamine at 37 "C, 10% CO2, and 97% humidity. CTLL-2 is a factor-dependent cell line that requires IL-2 for growth. Accordingly, for analysis, CTLL-2 cells were washed twice with high-glucose DMEM supplemented with 5% FBS, 5 x 10"s M 2 -mercaptoethanol, 50 u/ml penicillin, 50 ug/ml streptomycin, 3-4.0 mM L-glutamine to remove IL-2. IL-15 samples to be analyzed were titrated in DMEM 5% FBS in 96-well flat-bottomed microtiter plates. Washed CTLL-2 cells were added (final assay volume 100 µl, 2000 cells/well), and the plates were incubated for approx. 24 hours at 37 °C and 10% C02. The plates were pulsed with <3>H-thymidine (25 Ci/mMol) at 0.5 uCi/well for approx. 5 h, then harvested ("Inotech" 96-well cell harvester) and CPM counted ("Packard Matrix" 96 "gas proportional counting system). Units were calculated from CPM where 1 unit is equal to the number of microliters that gives 50% maximal stimulation.

Example 3

Preparation of sIL-15 cDNA clone

The sequence of the N-terminal 31 amino acids of purified IL-15 polypeptide (amino acids 1-31 of SEQ ID NO: 3) was used to design synthetic oligonucleotide primers for PCR amplification of IL-15-specific DNA sequences. The first six amino acids at the N-terminus (Asn-Trp-Val-Asn-Val-Ile) were used to design one primer, a degenerate mixture encoding all possible codon usages in the first six amino acid residues:

as shown in SEQ ID NO. 9 where Y is T or C; H is A, T or C; and N is A, C, G or T. The amino acid sequences of the mature monkey N terminus 26-31 (Tyr-Thr-Glu-Ser-Asp-Val) were used to design a second primer, a degenerate mixture encoding for a complement of all possible codon uses of amino acids 26-31 where the 3-position for Val is avoided: as shown respectively in SEQ ID NO. 10 and 11, where Y and N are as defined above, and R is A or G. Polyadenylated RNAs from CV-1/EBNA cells stimulated for 24 hours, 37 hours, and 72 hours with 10 ng/ml phorbol 12- myri-stat 13-acetate ("PMA"), were used as separate templates for first-strand cDNA synthesis. An aliquot of first strand reaction mixtures was added to commercially available PCR reaction mixtures containing the oligonucleotide primers. This mixture was subjected to 31 cycles of PCR amplification in 100 µl reaction mixtures in standard buffer with primers at 1 µM concentration. The cycles were programmed as follows: denaturation at 94 °C for 0.5 min, annealing step for 0.5 min and extension step at 72 °C for 0.75 min. Two cycles of annealing at each of 55, 53 and 51 °C were followed by 25 cycles at an annealing temperature of 49 °C.

After amplification, samples were purified and subjected to agarose gel electrophoresis. This yielded a 92 base pair DNA fragment that was excised from gel fields from two separate reaction mixtures involving CV-1/EBNA cells. The 92-bp DNA fragment was purified using an "Elutip-D" column (Schleicher & Schuell, Keene, NH), cloned into "pBluescript SK""

(Stratagene, La Jolla, CA) and used for dideoxy DNA sequencing.

A hybridization probe was prepared by random priming of the subcloned 92 base pair DNA fragment. The hybridization probe was used to screen a portion of a plasmid library containing cDNA inserts prepared from CV-1/EBNA polyadenylated RNA. This resulted in the isolation of clone C85.SIL-15 which has an open reading frame comprising the nucleotide sequence shown in SEQ ID NO. 1.

The nucleotide sequence of the polypeptide coding region in sIL-15 is illustrated in SEQ ID NO. 1. This sequence was derived from inserted C85.SIL-15 which was Sal I ligated into the Sal I site in expression vector pDC406 (C.J. McMahan et al., EMBO J., 10(10):2821-2832 (1991)) . Polyadenylated mRNA was prepared from a CV-1/EBNA cell line, and cDNAs were prepared using standard techniques. The CV-1/EBNA line is a producer of sIL-15. cDNA ends were ligated with Sal I adapters (Haymerle et al., Nucleic Acid Res., 14:8615-24 (1986)):

(SEQ ID NO: 7 and 8, respectively) and cloned into vector pDC406. A mixture consisting of approximately 500 individual plasmid-containing isolates was plated and screened by hybridization against a DNA probe fragment. The DNA probe fragment was prepared using PCR amplification of slL-15 sequences from CV-1/EBNA cell line cDNA.

Example 4

Cloning of human IL-15

A sIL-15 probe was prepared from an isolated, purified and radiolabeled Sal I fragment (about 1.37 kb) containing sIL-15 cDNA by random priming. The specific activity of the probe was approximately 1 x 10<6> cpm/ng. After Northern blots, the probe was hybridized against human RNAs from various sources, including an IMTLH cell line. The IMTLH cell line was derived from a stable transformation of a human bone marrow stromal cell culture with pSV3Neo. The probe was hybridized to human RNAs at approx. 42 °C for approx. 40% formamide for approx. 18 hours. Hybridization was followed by washing in 6 x SSC for approx. 10 min at 22'C, followed by washing in 2 x SSC at 42'C for approximately 30 min. Autoradiography revealed a positive signal in the IMTLH field.

We probed Southern blots of Sal i-digested library mixtures of the iMTLH cDNA library to identify a mixture containing a human IL-15 cDNA. Using the method of Haymerle et al., Nucleic Acid Res., 14:8615-24 (1986) above used for the CV-1/EBNA library, the IMTLH library was constructed in expression vector pDC406. Mixture "141", a mixture of approximately 1,000 different cDNA clones, was identified as positive. Approximately 4,000 colonies of "141" were then plated and probed by standard colony hybridization methods to identify a clone containing the human IL-15 cDNA. Only a single clone, l41.hETF, was found to encode human IL-15. There is approximately 96% nucleotide sequence identity and approximately 96% amino acid sequence identity between human and monkey IL-15 open reading frame sequences (Figures 4 and 5).

Example 5

rIL-15 stimulates CTLL-2 proliferation

cDNAs encoding mature forms of IL-15 (nucleotides 145 through 489 of SEQ ID NOS 1 and 4) were inserted downstream of a heterologous patted<y>rsecretion signal to create rIL-15- expression plasmid for sIL-15 and hIL-15. The secretion signal is a largely hydrophobic stretch of amino acids (usually at the N-terminus of a polypeptide) that directs secretion and cleavage of the polypeptide between the C-terminus of the signal peptide and the N-terminus of the mature, secreted protein (von Heijne, Eur. J. Biochem., 116:419 (1981)). The secretion signal sequence used was a murine IL-7 signal sequence. The resulting plasmids were transfected into COS-7 cells. Supernatants of the transfected cell cultures stimulated CTLL-2 proliferation. In addition, the coding region for the precursor form of hIL-15 (SEQ ID NO: 3) was inserted into pDC406 and transfected into CV-1/EBNA cells. Supernatants from cells transfected with pDC406:hlL-15 (ie, hETF/pDC406), but not those transfected with empty vector, stimulated CTLL-2 proliferation.

Example 6

Induction of CTLL-2 proliferation by means of rIL-15

Recombinant monkey IL-15 (monkey rIL-15) was purified from the supernatant of yeast expressing the sIL-15 cDNA by a modification of the purification method described above in Example 1, where the ultrafiltration and ion exchange steps were omitted. The purified monkey riL-15 was compared with purified, recombinant IL-2 and IL-4 with regard to biological activity. The purity of each of the three proteins was confirmed by amino acid analysis. Each of the three purified recombinant proteins was assayed for biological activity in vitro using CTLL-2 cells. CTLL-2 cultures contained the indicated cytokine at 2,000 cells/culture in 100 µl of supplemented culture medium. The CTLL-2 cultures were pulsed with 0.5 uCi [3 H]thymidine/culture for at least 4 hours of a 24-hour culture period, harvested on glass fiber filters, and radioactivity was measured by avalanche gasification. The proliferative response in vitro of CTLL-2 cells was measured at increasing concentrations of recombinant cytokine (expressed as ng/ml). The data are expressed as cpm of <3>H-thymidine incorporated (X IO"<3>) in Figure 6.

Example 7

Induction of CTL-. LAK- and NK-lvetic activity of rIL-15

Antigen-specific cytolytic T lymphocytes (CTL) were generated in vitro. Human peripheral blood mononuclear cells (PBL) from one donor (5 x 10<5>/culture) were stimulated with irradiated PBL (5 x 10<5>/culture) from an allogeneic donor in cultures containing different concentrations of either IL-2 or human rIL-15 or no cytokine. Cultivations were performed as described by M.B. Widmer et al. in J. Exp. Med., 166:1447 (1987), harvested after 7 days and assayed for cytolytic activity against <51>Cr-labeled targets derived from the original stimulating donor. The lysis assay contained various numbers of responding PBLs cultured with 1,000 labeled targets in 200 µl medium in V-bottomed wells, and the supernatants were collected after 4 hours of incubation. Lytic units were calculated as the inverse of the fraction of the responding culture required to produce 50% of the maximal specific <51>Cr release. The data for the cytokine-treated CTL are summarized in Figure 7.

Lymphokine-activated killer cells (LAK cells) were generated under identical culture conditions as the CTLs described above, except that the human PBLs were not stimulated with irradiated PBLs from an allogeneic donor. Instead, it was replaced with irradiated, autologous PBL, and cytolytic activity was measured against the Daudi lymphoblastoid cell line. For the LAK assay, lytic units were calculated as the inverse of the fraction of the culture that responded, which was required to produce 30% of the maximal specific <51>Cr release. The data for the cytokine-treated LAK cells are summarized in Figure 8.

Natural killer cells (NK cells) were isolated from whole human PBL isolated by antibody affinity to paramagnetic microbeads with MACS (Miltenyi Biotec, Sunnyvale, CA) using monoclonal antibodies against CD16. The purified NK cells were cultured for 3 days, and cytolytic activity was measured against the K562 erythroleukemia cell line. For the NK assay, lytic units were calculated as the inverse of the fraction of the culture that responded and was required to produce 30% of the maximal specific <51>Cr release. The data for the cytokine-treated NK cells are summarized in Figure 9.

Because of the similarity in activity between IL-2 and IL-15 in Examples 6 and 7, those skilled in the art would expect that IL-15 stimulates the activity of CTL, LAK and NK cells and expands the population of T cells that can destroy tumor cells and virus-infected cells. As described above in the section on administration of the IL-15 polypeptide, an effective amount of IL-15 in a suitable diluent or carrier can be administered to patients with carcinomas, melanomas, leukemic sarcomas or lymphomas, or patients infected with herpes virus, including cytomegalovirus, polyomavirus, retrovirus. Including HIV, influenza virus, hepadnavirus, hepatitis A, hepatitis B, hepatitis C, hepatitis delta or hepatitis D.

Claims (15)

1. Isolated DNA sequence, characterized in that the sequence comprises, (a) DNA sequences that code for the polypeptides according to the sequence: wherein amino acid 52 is Leu or His, amino acid 57 is Ala or Thr, amino acid 58 is Ser or Asp, amino acid 73 is Ser or Ile and amino acid 80 is Val or Ile, (b) variants of the sequences in (a) which encode proteins with corresponding biological activity, and (c) its DNA sequences demonstrably hybridize to the DNA sequences according to (a) or (b), or their complementary strands, under very stringent conditions including hybridization and washing after hybridization at 55 °C or higher and at a salt concentration of 0.5 x SSC or lower, and which, after expression, codes for a polypeptide with similar biological activity to the sequences according to SEQ ID NO. ID NO. 3 or SEQ. ID NO. 6. 2. Isolated DNA sequence according to claim 1, characterized in that the sequence comprises, (a) nucleotides 145 - 489 in the DNA sequence according to SEQ. ID NO. 1 or SEQ. ID NO. 4, (b) DNA sequences that detectably hybridize to the DNA sequences of (a) or their complementary strands under very stringent conditions, including hybridization and washing after hybridization at 55 °C or higher, and at a salt concentration of 0.5 x SSC or lower, and which, after expression, codes for a polypeptide with similar biological activity to the sequences according to SEQV. ID NO. 3 or SEQ. ID NO. 6, and (c) DNA sequences which, due to the degeneracy of the genetic code, encode a polypeptide encoded by any of the DNA sequences under (a) and (b). 3. Isolated DNA sequence encoding a precursor polypeptide of the biologically active IL-15 polypeptide, characterized in that the DNA sequence is selected from the group consisting of: a DNA sequence which, after expression, codes for the polypeptide defined by SEQ. ID NO. 2, and a DNA sequence which, after expression, codes for polypeptide defined by SEQ. ID NO. 5. 4. Isolated DNA sequence according to claim 3, characterized in that the DNA sequence which codes for a precursor polypeptide of the biologically active IL-15 polypeptide is selected from the group consisting of: the DNA sequence according to SEQ. ID NO. 1, and the DNA sequence according to SEQ. ID NO. 4. 5. Recombinant expression vector, characterized in that it comprises the DNA sequences according to any of claims 1-2. 6. Host cell, characterized in that it is transformed or transfected with an expression vector according to claim 5. 7. Host cell according to claim 6, characterized in that the host cell is selected from the group consisting of E. coli, Pseudomonas, Bacillus, Streptromyces, yeast, fungi, insect cells and mammalian cells. 8. Host cell according to claim 7, characterized in that the host cell is Saccharomyces cerevisiae. 9. Host cell according to claim 7, characterized in that the host cell is an ovary cell from a Chinese hamster. 10. Method for producing an IL-15 polypeptide, characterized in that it comprises culturing a host cell according to claim 6 under conditions that promote expression, and recovering a polypeptide exhibiting biological IL-15 activity from the culture. 11. Isolated biologically active IL-15 polypeptide preparation, characterized in that it comprises an amino acid sequence which is coded for by an isolated DNA sequence according to claim 1. 12. Isolated polypeptide of the biologically active IL-15 polypeptide according to claim 11, characterized in that it comprises the polypeptide defined by SEQ. ID NO. 2. 13. Isolated polypeptide of the biologically active IL-15 polypeptide according to claim 11, characterized in that it comprises the polypeptide defined by SEQ. ID NO. 5. 14. Pharmaceutical preparation for stimulating the proliferation of T-lymphocytes, characterized in that it comprises the isolated IL-15 polypeptide according to claim 11, and a physiologically acceptable carrier, excipient or diluent. 15. Use of a polypeptide according to claims 11-13 for the preparation of an agent for the treatment or prevention of anemia, carcinoma, melanoma, sarcoma, leukemia, lymphoma or pretreatment or prevention of infections caused by herpes viruses, including cytomegalovirus, polyomavirus, retrovirus, including HIV, influenza virus, hepatitis virus, including hepatitis A, hepatitis B, hepatitis C, hepatitis 5 or hepatitis D.