FI117054B - Preparation for interleukin and DNA sequence encoding it - Google Patents

Description

1 11

The method of making interleukin-1S and its DNA sequence

FIELD OF THE INVENTION The invention relates generally to a so-called epithelial T cell factor ("ETF") polypeptide referred to herein as interl 15 ("IL-15"). Specifically, it relates to a cDNA sequence isolated from the IL-1 logical activity polypeptide ke 10, isolated IL-15 tide sequences and derivatives thereof, a method of making polypeptides using recombinant DNA, a method for inducing T cell proliferation and nucleation, and the like. IL-15 is included in compositions that confer anti-tumor immunity.

BACKGROUND OF THE INVENTION T cells, also referred to as charges, are a group of immune effector cells. T cells in peripheral tissues divide into two sizes on the basis that they express cell surface CD8 molecules, the expression of one of which excludes expression. Typical CD8 + T-sols; / t after activation to cytotoxic T cells j * * * [il 25 target antigen-containing target cells * · ·

• ·· Direct contact. Activated CD4H

·.: ** usually produce positive signals, es ·· ♦ The “helper” function for B cells allows the B cells to differentiate only into: 30 forming cells) and is therefore used ι «· - * **. mitvia help-T-snl nt-.

2 lymphocytes · IL-2 was originally detected in human! as a driver for long-term growth. In addition to T cell growth, there are killer (LA) 5 and cytotoxic T cell ("CTL") and ma] activation activated by natural tapj cells and lymphokines and stimulation of B cell growth.

IL-4 is a 15-20 kDa protein producing activated T cells, bone marrow stromal mast cells. The open reading frame of IL-4 encodes 140 µl of murine IL-4 and 153 amino acids of murine IL-4. IL-4 was originally defined as an activator of B cell and differentiation. Also included are activation and class of macrophages: induction of molecules, j of some T cell lines; 15 growth of human cell lines, the growth of human peripheral blood 1, and the formation of CTL cells and the growth factor of these hematopoietic progenitors from stem cells. IL-4 plays an important role in the regulation of NK cell activity by IL-2. Hui 4 has no activity against mouse cells. The IL-7 is a polypeptide of 20-25 kDa and 177 µi · ** 1 / length produced by bone marrow and ileum stromal cells. Although IL-7 was described as a * * * * ** a growth factor for progenitor B cells, it promotes it! ...

cell growth and pre-B cell growth. IL-7 induces m * · · V * proliferation of its peripheral blood T cells from cells, IL-2 receptor: 30, IL-2 formation, and upregulation of CD4 * and CD8 +. IL-7 also acts synergistically on IL-3 di. IL-9 was first identified as a helper growth factor. IL-9 stimulates erythroid cells from the thymus and enhances IL-3-induced proliferation of bone marrow mast cells. It also modulates IL-5 as a form of IgE and IgG produced by B cells. Murine IL-9 has an activity on human cells, whereas human IL-9 does not affect mouse cell. A 20 kDa oral 178 amino acid polypeptide that is macrophages and TH2-T helper cells but not TH1 and cells. Like IL-2, IL-4 and IL-7, IL-1 exhibits a variety of biological activities. IL-1 was determined by its ability to inhibit the production of cytokines by supported T cells. Both 15 and murine IL-10 are co-factors of tyrosocytes and T cell stimulating cofactors in association with IL-7 or IL-2 4. IL-10 stimulates mast cells and growth to IL-4 or IL-3 plus IL-4 in combination. IL-10 also induces IgG in B cells; 20 expression of MHC class II molecules and the viability of these cells in culture.

.V. IL-12 is constitutive or induces lymphoblastoid 1 * with lithium ester and calcium ionophore! lineages and is formed by st * * * 25 macrophages on LPS. IL-2 has a molecular weight of 70 kDa and • exceptionally heterodimeric residues from two of the two disulfide bonded • tt: proteins. The two glycoprotein subunits are 40 kDa and 328 amino acids in size: 30 peptides. The smaller glycoprotein subunit is. ***. of Hs is H nnhannn m H or non nnl vnonfl Λ 4 and acts synergistically with LAK cells. Unlike IL-2 and IL-7, but as well as very little or no sleep, 5 peripheral blood mononuclear cells increased

SUMMARY OF THE INVENTION The present invention provides an isolated and purified T cell growth factor, hereinafter referred to as "interleukin-15" ("IL-15"). This invention provided cDNA encoding a monkey IL-15 polypeptide containing a 483 bp 5'-coma region upstream of a 489 bp reading frame and a 303 bp 3'-toma region. . The human IL-15 polypeptide k15 in the cDNA sequence has a 316 bp 5T non-coding region upstream of a 489 bp reading frame and 397 bp 3'-half of the unidentified region. Nucleotide sequences and deduced acid sequences of monkey and human open frames are described in SEQUENCES ID NC Both monkey and human open reading frames eVe precursor polypeptide (SEQ ID NOs: 2 and 5).

I \ precursor polypeptide1 consists of 48 amino acids ml «i»

/ leader sequence and mature monkey or human IL

***** Of 25 coding sequences for peptides. Monkey and human IL-15 polypeptides are described in SEQ ID NOs: 3 and 6, respectively.

• J: The present invention further includes other 15 polypeptides encoded by the nucleotide sequence: which hybrid appears to be moderately restrictive.

. · * ·. for you in very strenuous circumstances SEKVG

5 manifestations. The present invention further includes nu sequences which, due to the degeneracy of the genetic code, encode the IL-15 polypeptides of the above described nucleotide and complementary sequences.

The present invention further provides compound molecules containing the above nucleotide sequences, for example misvectors or plasmids, transformed host cells suitable for the preparation of IL-15 polypeptides, and m recombinant IL-15 polypeptides using these cells.

Brief description of the drawings

Figure 1 shows the nucleotide sequence of the active monkey type o1515 and the deduced am sequence.

Figure 2 shows the active human nucleotide sequence and deduced amino acid of IL-15.

Figure 3 shows a purification scheme and a pr sequencing scheme suitable for the isolation of .I, .15 polypeptides.

Figure 4 shows the nucleotide sequence * * * encoding human type IL-15 * · * / natype IL-15

Vf 25 homology. Human sequence: 2. * Placed above the monkey sequence.

Figure 5 shows the amino acid sequences between the human type IL-15 V * type IL-15. The human sequence is shown in monkey sequence1; 30 dolls. In both types, the leader sequence (amine. ***. 48) is cleaved from the Drekursorinventide. of which 6 was determined under rising conditions using recombinant recombinant cytokine (expressed as y] ng / ml). Experimental results are reported as cpm values (x 10 -3) for thymidine collected in cells.

Figure 7 shows the lytic Akti of OTL cells; induction by rIL-15 and IL-2. Your antigen cytolytic T lymphocytes (CTL) were generated under conditions. Human peripheral blood mononuclear cell 10 from a single donor was stimulated with irradiated PBL from allogeneic donor and your cultures contained various concentrations of either IL-human rIL-15. Cultures were assayed for lytic activity against 51 Cr-labeled k 15 and blood from cells whose cells were initially used for stimulation. Mythical units were calculated using the fractional inverse of the response required to achieve 50% of the maximal specific 51 Cr : nv 20 from.

Figure 8 shows the lytic Akti .V. Of LAK cells. induction with rIL-15 and IL-2. Lymphokine * * *. · /. winning killer (LAK) cells were prepared in vi * · * ly ratios. Human PBLs were stimulated with irradiated autologous PBLs and cytolytic ak * * * 5 · * was assayed for lymphoblastoid Daudi and

Lytic units were calculated as the inverse of the proportion of reactive culture · * # * to 30% of maximal specific 51 Cr release. Figure 9 shows lytic activity of NK cells.

: ***: Induction using rIL-15 and IL-2. L

7 Monoclonal responses to CD16. Purified NK cells were grown for 3 days and lytic activity was determined against the K562 erythrole lineage.

5- Detailed Description of the Invention

Hinterleukin-15 "or" IL-15 "refers to polypeptides structurally similar to the polypeptides described in the invention and which are T lymphocytes for proliferation and differentiation. 10 distinguishable from IL-2, IL-4, IL-7, 1L-I0 and 1L-I2 are structurally based on the cells from which they are derived (Table 15 polypeptide is first formed in the epithelium of primates as a 162 amino acid precursor-IL15 peptide. This precursor contains 48 amino acids of its leader sequence which is deleted The 15 polypeptide is capable of acting as a signal for proliferation and localization of pre-cells or mature T cells.This protein may be caused by long-term promotion of T lymphocytes and T cell lines under in vitro conditions.

* «1 2 3 4 5 .1 / 1" sIL-15 "means monkey type IL-15

elm / 15 "refers to human-type IL-15." rIL

25 probe recombinant IL-15. Both refined sl 2 • · 1 3! 1 rIL-15 stimulate CTLL-2 cells [Gillis and Smit

: J; 268 (1977) 154; ATCC TIB 214]. S

4: Cell supernatants transfected with precursor form and ready-to-read mature forms expressed in compound 5 tplrni ilrnn ηνιιΊΊ a eaivaf ^ "raan__9 ·« 1 ie · 8 ("PBL"). RIL-15: In this invention, the proliferation of vi PBT and PBL cells was observed with st phytohemagglutinin ("PHA") in the present invention. rIL-15 stimulates the addition of PHA-activated CD4 + and CD8 * cells: 5 rIL-15 stimulates dormant human or dormant in the presence of antibodies directed against CD3 (T cell receptor) murine T cell clone. Experiments on PHA-activated bones show that rLL-10 growth is stimulated by its effects on IL-2 in a non-destructive manner that the antibodies directed against IL-2 or the receptor are not in 15.

Analogues or bonds of native mammalian polypeptides to the terms IL-15, sIL-15 and hIL-15 encoded by such nucleic acids,. relies on the nucleic acids of Figures 1 and 2 (ds 145-489 including SEQ ID NOs: 1 and 4, inclusive) under limiting conditions, and <lymphocyte proliferation and differentiation and vat T cell lines and isolated PBT cells * « where from.

• · «• M • • • • • ♦ • t · • · M ♦ •« «• • 0 0 • ♦ · il · ··· 000 4 I · • I 4 • ♦ • · · • · · 000 000 0 0 0 Λ 9

Table 1 Origin and structure of T cell growth factors

Amino-hap-Whole boy

Factor (kDa) Number * Material Source 5 _ _ _ _ IL-2 15 153 Activated Lymphocytes IL-4 15-20 153 Activated T-Lymphocytes, Bone marrow stromal cells, cells IL-7 25 177 Bone marrow and ear stroma IL-9 30 - 40,144 Activated T-lymphocytes 10 IL-10 16 - 20,178 Th2 lymphocytes · IL-12 ** 40 and 35,328 and 253 B-lymphoblastoids: lines • a VJ IL-15 15-17 162 Epithelial cells • I «» »• aa • aaa: .V * Number encoded by open reading frame hun aa a: *. * 15 in peptides.

** Two glycoproteins: T are required for activity.

a. "Recombinant DNA Technology" or "Compound] • aa. · * ·. 20 incorporates the techniques and, in order to enable the process, transformed or trans-cloned or synthesized DNA sequences into their native forms are obtained from time to time using mammalian cell expression systems. polypeptides without glycosylation are usually obtained in prokaryotic cells (e.g., coli).

"Biologically active" means that the specific IL-15 polypeptide is capable of stimulating proliferation and / or differentiation of lymphocytes and, in the case of hLL-15, also stimulating the proliferation of mice or primates, such as human lineages or PBTs, <15 "Nucleotide sequence "Means a leotide in the form of a single tin or a larger DNA construct in the form of a moth, such as a cloning vector, which is derived at least once from DNA isolated (i.e., from the endogenous material) and is present in an amount or The concentration of jc, V * is such that its components * · · nucleotide sequences can be identified, processed, and recovered by common biochemical methods [method • · *: · * in Sambrook, et al. ai., Molecular Clc • · * ··· * Laboratory Manual, 2nd ed «, Cold Sp ring Harbor Lal ···: Cold Spring Harbor, NY (1989) Outline Methods]. These sequences are obtained in the form of an e ·: 30 open reading frame, which is continuously interrupted in eukaryotic genes from a type 11 reading frame, where they do not interfere with processing or expression of the k regions.

"Recombinant expression vector" refers to a slide comprising 15 transcription units of an assembly comprising (1) a geneticist or elements that serve to regulate expression, such as promoter initiators, (2) structural or encoding mRNA and which undergoes 10 thio to a tide containing the biological activity of IL-15; and (3) the relevant transcription and initiation start and stop sequences. A number of different regulatory elements are discussed below (see section Recombinant DNA Mem 15 For use in yeast expression systems, the intentional elements preferably include a leader capable of secreting the yeast host cell into the polypeptide, which may alternatively be a bacterial polypeptide). il 20 system contains an N-terminal methionine The N-terminal methionine group can be further purified from the expressed recombinant polypeptide to provide a suitable product.

• «« *. * "System Microbial Expression System 25 provides a homogeneous culture of a suitable microorganism, e.g. bacterium + *: · 'E. coli, or yeast, such as S. cerevisiae, where only this V, and wherein the recombinant transcription unit is stably present in the chromosomal DNA or in each of the recombinant transcription units as a plasmid. Recombinant microbial expression systems are expressed after inducing structural nucleotide sequence regulation.

Transformed host cells are transformed or transf <5 expression cells. Expressed mammalian IL-15 is localized to the cell and / or secreted into the culture supernatant by the paternal type and the gene construct attached to the host cell. highly restrictive hybridization-10 means as defined herein, in the circumstances described in Sambrook, et al. above, Vol 2, 8.46 9.47 - 9.55. Moderately severe ratios as defined by Sambrook et al. Include, for example, overnight dressing and post-hybrid foundation washes at 55 ° C, 5 x SSC, and 0.5% SDS. . conditions that still use k20 hybridization temperatures and post-hybridisation temperatures or lower salt concentrations: \\ IL-15 polypeptides In this invention, purified monkey type ** ** mS15 (sIL-15) and sequenced mature N-term S: 25 peptide of sIL-15. The present invention has isolated sIL-15 * * * *; * cDNA by utilizing your N-terminal amino acid '' * and PCR and determined the mature sIL-15 nucleotide V and the deduced amino acid sequence (FIG.

sIL-15 polypeptide precursor nucleotide sequence: 30 deduced from this (SEQ ID NO: 2)

SEQ ID NO: 2). IL in monkey species

The proliferation of T cell lines (e.g., CTLL-2) stimulates the proliferation and proliferation of human PBT cells.

The present invention also encompasses other mammalian 515, including human IL-15, containing biological activity and encoded by nucleotides that hybridize to SEQ ID NO: 15. telematic probes under conditions that are extremely restrictive

10 limited circumstances. Human IL-15 cDNA

a plasmid containing the tele-clone was deposited as an American Type Culture Collection, 12301

Drive, Rockville, MD 20852 US ("ATCC *) Patent No. 69245 of February 19, 1993, under the terms of Agreement B 'of the International Agreement for the International Approval of Microorganisms for the Purpose of Purpose. The deposit was designated" 141-hE1 Containing E. coli containing plasmid pCD406 with a 316 bp non-coding 20 which had a 489 bp open reading frame; 397 bp 31-sided uncoded .V. above which areas were bordered by both • * ·. · /; All restrictions on the public availability of the deposited material as set forth in SEQ ID NOs: 7 and 8 are irrevocably removed after patenl * *: **.

*. .

* · «* The IL-15-poly: V: amino acid construct described in this invention can be modified either by valence or by aggregative conjugates to other IL-15 or lipid: ***, phage, acetyl and other corresponding 14 or mammalian IL- At the N-terminus of the 15 polypeptide: or at the C-terminus. Other hard aggregated conjugates of mammalian IL-15 or fragments thereof that are within the scope of this invention employing m racks or polypeptides, such as derivatives, are obtained by synthesizing recombinant DNA technology in an N-terminal or C-terminal na. For example, the conjugated polypeptide may be a signal (or leader) polypeptide sequence in the N-terminal region of 10 den of the IL-15 polypeptide for the purpose of transporting the polypeptide to its inner or outer cell membrane or cell wall (e.g., yeast? Factor 15). In addition, IL-15 polypeptides may be most commonly expressed as polypeptide fusions that further contain other polypeptide sequences, such as Fcs or other immunoglobulin sequences, cognates, or other sequences that promote purification and recognition of the peptides. Further polypeptide fusions may still include fusions with other polypeptides to provide new polyfunctional features. Other cytokines '/ mark any of the interleukins 1-13, t · i. · * ·' 25 erosive factor (TNF), granulocyte macrophage colony * · · • transfactor (GM-CSF), granulocyte colony stimulator (G-CSF), mast cell growth (M) ) and others • 9 * ·. ·: those that affect the growth, metabolism or function of immune cells.

The present invention further includes glycosyl. . . , -. ". . . . ....

15 similar or significantly different. This is a matter of what expression system to act on. When IL-15 polypeptides are expressed in expression systems such as E. coli, ni 5 yields non-glycosylated molecules.

Oligonucleotide synthesis and ligation by t mutagenesis methods can be used to synthesize inactivated N-glycosylation sites s 10 mutated analogs of human IL-15 or other mammalian IL-15. IL-15 polypeptides can be expressed in homogeneous form with lower carbohydrate levels than yeast expression systems. The N-glycosylation sites present in eukaryotic pDs have 15 specific amino acid triplets Asn-Φ-Ω, Φ is a Mike amino acid except pro and Ω is Ser or Thr. The enriched derivative of IL-1 in the present invention is pol which is a substantially homologous mammal. With respect to the native sequence of 15, but whose am 20 sequence differs from the native tide of mammalian IL-15, bioequivalent analogs of IL-15 polypeptides of the IL-15 muteins of deletion, insertion or substitution can be constructed.

ly, several deletions of different amino acid groups or sequences, or by deletion of terminal or internal groups or biological functions of biological activity.

* ··· * ice. For example, Cys groups may be deleted; * · *: With other amino acids to prevent the formation of the wrong disulfide bridges of the molecule during m30 re-enrichment. Other mutagenesis approaches · *** · nvat afpn aml nnhamvtruhml are ιηηΗΊ filra 11 16 similar to those of the native group.

Antisense or sense oligonucleotides s: single-stranded nucleic acid sequences (either 5 DNA sequences) capable of binding to: an mRNA sequence or an antisense IL-15 cDNA sec1. mil sequence fragment or a fragment of RNA form complementary to the nucleotide sequences of Figures 1 or 2 or 10. Such a fragment contains at least about 14 nucleotides and is capable of binding to 15 DNA. The ability of the antisense or sense leotide to form the IL-15 cDNA sequence on the rope is described, for example, in Cohen, Cancer Res. 48: 2659 (1988) and van der Krol BioTechniques 6 (1988) 958.

Isolation and characterization of IL-15 non-; from a cellular material source, requires that a 20 mammalian cell line producing IL-15 be both responsive and that increases in response to IL-15. Biological levels of mammalian IL-15 * «* I '. can be lymphoid cell proliferation-inducing I * *; for growth detection, use a factor dependent on growth factor ** ** 9 25 cells. Amounts of mammalian IL-15 polypeptides:. * May also be used in T cells isolated from human or other mammalian blood samples.

* * * ·, ·! The IL-15 dependent cell line can be from murine CTLL2 cells. This cell line responds to pure human IL-2, mouse IL-2, and a combination of

• M

*** H-M Ύ * θΤ \ TT + mi iH-ha TT.-1 «to generate TT.-Q * non 17 nuclei by methods used in the inter-species hybrid. The embodiment kills features such that in Figure 1 or SEQUENCE! An oligonucleotide probe is prepared from the nu sequence of the cLNA of the sL-15 in the NO0: 1 form as described in SEQ ID NO: 5 or the SEQ ID NO: 4 cDNA. The monkey or human probe is used under restrictive conditions for screening for mammalian division or genomic DNA recorder Aston. The probe may be prepared by conventional methods as described in Sambrook, et al., supra. For example, mammalian cDNA can be prepared from mRNAs isolated from mouse peripheral lymphocytes. Alternatively, other cDNA libraries or mRNAs made from or derived from a variety of tissues may be screened to identify 1 source of Northern hybridizing mammalian IL-15 DNA or 1L-155 mRNA · 20 CV-1 / XL-15 derived from EBNA cells In the present invention, IL-15 is purified from a non-protein solution such as IL-15 lm; used to recover isolated polypeptide from a cell harvested spent culture. Before the biological activity of IL-15 is claimed using the biology methods described herein, typically one purification step is required.

: 30 Non-homogeneous protein solution, example · · · 18

Dulbecco's Modified Essential Medium ("DMEM") Most preferably, the growth medium and product are used. The medium used to isolate sIL-15 as described herein is high-glucose (4,500 mg / L) DMEM supplemented with 7, 5% fetal bovine serum, 50 nicillin, 50 pg / ml streptomycin, 3 to 4.0 n tamin, 1 mM sodium pyruvate, 0.1 mM non-essential amino acids and 10 mM N- [2-hydroxyethyl] pi] 10 For production and purification of ni-N '- [2-ethanesulfonic acid] ("HEPES") buffer, a serum tantomedium containing DMEM was omitted but supplemented with 50 µg / ml penicillin, 50 µg / ml streptomycin. 4.0 mM L-glutamine, 1 mM sodium pyruvate, 0 essential amino acids, and 10 mM HEPES buffer 1 / EBNA cells were adhesion dependent and could be grown in plates, flasks, roller bottles or microcarrier silicon. 15 hh produced by the CV-1 / EBNA cells controlled biorei .V. on the surface of microcarriers. The cell stock preparation * * was prepared in roller bottles. Uh! ; , * to start the cells were trypsinized and they; • * I; *; * 25 spinner bottles containing medium described and 5 g / L Cytodex · 3 ml] (Pharmacia). The cell density was from the beginning of growth: 1.5 to 3.5 x 105 cells / ml. To ensure e-adhesion to microcarriers, 30 spinner bottles were incubated for 2 to 24 hours with a * · *: * "· spinner bottle culture: 19 concentration and shaking settings were 37 at 20% saturation rate (air reports 75 to 85 rpm, stated wit. Samples for daily cell growth and fitness

Cell growth was quantified by counting the number of liberated after lysis with a solution containing 100 mM citric acid in crystal violet.

The culture was supplemented with a new growth medium when ammonia concentrations reached 5.0 mM. This was repeated until the cells growing on the surface of the microwells had grown to their ki. Subsequently, the culture medium was replaced by e 15 watt serum-free production medium. This amount was used by letting the microcarriers settle on the bottom, suctioning the culture medium and removing it on the production medium. This was repeated si | until 3,125-fold dilution was achieved: 20 detectable production cycles of 2 cycles, cells were allowed to produce 4-7 days, after which 80% of the production medium was assembled »I · • * · This was repeated until the cells were Olivi ♦ ·· I * completely from the surface of microcarriers.

About 64 liters of cultured medium for CV-1 / EBNA cells was purified to purify sIL-15 and to clarify the protein's N-amino acid sequence. The purification scheme included, as shown, ultrafiltration, hydrophobic chromium anion exchange chromatography, high performance ***** theophase liquid chromatography on iRP-HPLC ^ 1a sodium detergent from PVDF membrane. The first 33 amino acids shown in SEQ ID NO: 3 were resolved by N-terminal amino acidification. Later known cDNA clone from CV-1 / EBNA cDNA library

5 SEQUENCE IDENTIFICATION OF THE SEQ ID NO

a DNA sequence encoding a lane polypeptide. Täx contains a 48 amino acid leader sequence from SEQ ID NO: 2 and a mature polypeptide represented by SIN ID NO: 3.

10 Recombinant DNA Methods.

Human IL-15 polypeptides, IL of monkey

peptides and other mammalian IL-15 polypeptides are preferably recombinant. The invention encompasses that a cDNA encoding human IL-15 polypeptide 15 or another mammalian IL-15 polypeptide is inserted into an expression vector.

In the production of mammalian IL-15 polypeptides, or an example, by recombinant DNA techniques, the DNA clone

20 cDNA) which, when expressed, encodes mammalian IL

peptide or a derivative thereof. cDNA Clones ov, V. sin expressing mammalian IL-15 polypeptides * # ·. · /. cells or cell lines. First, total cellular mRNA is prepared and mRNA is prepared

; 25 cDNA library using reverse transcription. T

t «»: the DNA sequence information m m * * · ί · * presented in the seal can be isolated and identified by the cDNA clone used to design the thimerase or PCR primers used in the inter-species hybrid foundation as described above.

»« «

«1« —_ · _ *. . _ —Jm -i - | ·. , -1-1 J

21 uses genomic DNA which contains nucleotide sequences for the expression of mammalian IL-15 polypeptides. The isolated cDNA can be; methods known in the art for promoting biological activity of IL-15 included the preparation of 15 derivatives or analogs.

Recombinant expression vectors contain s or cDNA derived DNA fragments encoding IL-15 or biologically active derivatives thereof. The size of lL-I5 or a derivative thereof is operably linked to a sequence derived from a transcriptional regulatory or structural nucleotide sequence, such as a mammal, a microbe, a virus or an insect. Examples of the 15 sequences are, for example, the genetic sequence la has a regulatory function in gene expression (es transcription promoter or initiators), a ma operator sequence for transcription control, si encoding suitable ribosome binding in 20 mRNA, and suitable transcriptional and translational control and termination. Nucleotide .V. it is connected functionally when you »* *

! *. the gene is functionally linked to a structural gene. E

The I / S signal peptide (secretion leader) sequence may be operably linked to a mammalian * * * • · * 15 DNA sequence, or a derivative thereof, if the signal peptide is present; * · Φ: the amino acid sequence of the cursor and contributes to the secretion of ni IL-15. In addition, the sequence of the promoter nu: 30 is functionally encoded by it * · 9 ***** / AsfiRerlrilrs1! ralrennenoArrl n nrn 22 to the 15 coding sequence) if the si position of the ribosomes in the vector is located in the translational promoter.

Suitable host cells for expression of mammalian IL-15 derivatives, under the control of the appropriate prom, are prokaryotes, yeast, or higher oocytes. Prokaryotes include Gram-negative positive organisms such as E. Coli or set. Suitable prokaryotic host cells for transfo 10 are, for example, E. Coli. Bacillus subtilis, S typhimurium and several other species of the genus Pseudomonas, Streptoi Staphylococcus. Further, in more detail, yeast such as S. ce 15 mammalian cell line such as Chinese hamster Raunas (CHO cells) or insect cells are also suitable host cells. Cellular translation systems could also be used for the production of mammalian IL-2 derivatives using RNA mo 20 derived from the DNA constructs of the present invention. Relevant cloning and expression suitable for use in bacterial, s .V. in mammalian and cellular hosts, has been described as 4 hours * I ** in Pouwels, et al., Cloning Vectors, RL Manual, Elsevier, New York, 1985.

ft ft 4 25 When mammalian IL-15 or its leader. .

* · * Is expressed in a yeast host cell, the nucleotide (for example, a structural gene encoding mammalian 1L-15 or its leader) may contain a sequence. The yeast cell can, with the help of the leader sequence, secretion of the translation-derived polypeptide

Ml n _ - | Often containing the 2p yeast plasmid derived from the lysis initiation sequence, the autonomously ruptured sequence (ARS), the promoter region, the polyadenyl sequences, and the transcription termination sequences. Yeast 5 preferably contains an origin of replication selection marker. Suitable pro sequences for III vectors include the metallothionein promoter, 3-serate kinase [Hitzeman, et al., J. Biol. C (1980) 2073] promoter or the glycolite of others! 10 Hess, et al., J. Adv. Enzyme Reg. 7 (1968, Holland, et al., Biochem. 17, 4900 (1978) 4900), witches, glyceraldehyde-3-phosphate dehydrogenase, hesin, pyruvate decarboxylase, phospho-fructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate, pyruvate kinase, isomerase lucose isomerase and glucokinase, promoter suitable vectors and promoters for use in expression in yeast are more specifically described by Hitzeman, EP application 73 20 For example, DNA sequences derived from pBR322 and E. coli derived from pBR322 may be used * * *, · /: Gene and origin of replication). Other yeast * t # vectors that may be included in the yeast expression ...

, * * 25, are the glucose repressible ADH2-pr and the α-factor secretion leader. ADH2 pro «i · * ··· * is described by Russel, et al. [J. Biol. Chem. 2 V: 2674 and Beier, et al. [Nature 300: 724 (1982)]. The leader sequence of the factor directs the secretion of heterologous poly (ϊ): 30, the leader of the α-factor is inserted into the 4 **** strand of 4 ***** mnn'h'fwt. one or more restriction sites. Tämj fusion of the leader sequence to the structural gene.

Methods of yeast transformation are well known in the art. Natl. Acad. Sci. USA 75, 1929 (1978), describes a legal method. Hope, et al. ' In the method of Trp + transformants in selection medium, the composition of the selection medium is 0.67% yeast ty; nutrient, 0.5% casino acids, 2% glucose, 10 adenine and 20 mg / ml uracil.

Export transformed yeast host cells containing the ADH2 promoter sequence can be grown to induce growth in a "rich" medium, the rich medium being a medium containing 1 15 extracts, 2% peptone and 1% glucose and 80 mg / ml adenine and 80 mg / ml l . The ADH2 promoter is derepressed to the end of glucose from the medium.

In a prokaryotic host cell, such as E. coli, 20 mammalian IL-15 or a derivative thereof further contains an N-terminal methionine moiety having a. an attempt to promote recombinant polypeptide expression * * · in a karyotic host cell. N-terminal Met void / co-expressed mammalian recombinant IL-15 »ii ill 25 Mammalian recombinant IL-15 structural gene • · 1: * transduction vectors containing the tide sequence or a derivative thereof, or transfection from the host. . Other Applicable: Karyotic host cell expression vectors contain a bacterial selectable marker derived from globally available plasmids. This sele! The 5 keri may contain the cloning vector pBR322 (ATC genetic elements. PBR322 contains the ampicill tetracycline resistance genes and provides a simple means for the identification of the transformed gene. The "framework" portions of pBR322 are linked to the 10 promoter and the mammalian g. for example, pKK223-3 (Fharma <Chemicals, Uppsala, Sweden) and pGEM1 (Promegs Madison, WI, USA).

15 In the case of prokaryotic host cell combination markets, pro sequences are commonly used. Common promoter sequences o lactamase (penicillinase), lactose promoter [Chang, et al., Nature 275: 615 (1978); and Go 20 et al., Nature 281: 544 (1979)], tryptophan (trp) -p system [Goeddel, et al., Nucl. Acids Res. 4057; and EPA 36,776] and the tac promoter [Sambrook (Molecular Cloning: A Laboratory Manual, Cold Spri I. * Laboratory, 1989)]. In a particularly useful "*" 25 oocytic host cell expression system, the PL * promoter of the phage and the heat labile cl857ts-re sequence. Plasmid vectors available from the American Type Culture Collectii containing derivatives of the λ: η PL promoter are:

: Γ; PHUB2 [this is included in E. coli strain JMB9 (ATG

. · * ·. Ha r + -is eieälfvv P r> r »1 H RP1 · αοη f 1ΦΓΓ R

26 COS-7 cell lines [Gluzman, et al., Cell: 175; ATCC CRL 1651], L cells, C127 cells, 3T3-SO CCL 163), CHO cells, HeLa cells (ATCC CCL 2) and: CRL 10) cells. Suitable mammalian expressions include non-transcriptional genes such as origin of replication, promoter structural gene-associated initiator sequence, n-sequence surrounded by 5 'or 31-sided trans-participation sequences such as ribosome si 10 transcription sites, polyadenylation sites, N splicing do and translation control sequences obtained from viral starting materials. For example, the mammalian cell promoter and full sequence promoter sequences used in Route 15 are derived from polyoma, adenovir Simian Virus 40 (SV40) and DNA from the human cytomegalovirus SV40 virus genome, including SV40 origin, early and late motors, primers, splice sites, , may be used for such other g .V. to get the units you need i · «# · /. mammalian host for expression of the neg gene sequence Viral early and late promoters; It is useful because both of these are obtained from the * * *: · * viral genome as a fragment which may also contain the origin of * -i · * its replication (Fiers, et al., Ni: (1978) 113). Smaller fragments or SV40 fragments may also be used, provided that they contain a sequence of approximately 250 bp in length, extending over 1 "exemplified mammalian expression vectors. No. 07/480,694, dated February 14, 1990, and U.S. Patent Application No. 07 / 543,193 filed June 5, 1990.

Purification of recombinant mammalian IL-15 The IL-15 polypeptides can be prepared in cultured host cells in cultures necessary for expression of mammalian IL-15 tids or derivatives thereof. The expressed polypeptides obtained from T 10 may be: purified from culture medium or cell extracts, or the IL-15 polypeptide or derivative thereof may be centrifuged using a commercially available lineage concentration filter, for example an Am: 15 Millipore Pellieon ultrafiltration unit. The growth can be treated with either a concentration step without this step using a purification matrix such as high performance chromatography medium. Preferred medium is Sepharose * CL-4B (Pharmacia). Alternatively, 20 uses an anlon white resin, for example, a rix or substrate which has an outer surface covered with liaminoethyl (DEAE) groups. Matrix * * * be acrylamide, agarose, dextran, pulp! • ♦ · l. * Other matrix types commonly used in protein purification. Alternatively, you can use * * · * · 'filter media. Recombinant IL-15 is stable in b * ·! · * Aqueous buffers and may also use * · · resin.

Further purification of IL-15 can be used: 30 one or more high performance inverse ···. . _ «J. _Λ «vviT n \ j,, t. . . 28 also uses to obtain a remarkably homogeneous combination. Alternatively, some or all of the steps used to purify the monkey IL-15 described above may also be used.

The bacterial culture combination is usually isolated by first digestion of the father by centrifugation, extraction of the polypeptide from the knobs if insoluble, or supernatant if soluble followed by multiple concentration, desalting, ion exchange chromatography steps. Finally, RP-HPLC can be used for the final p steps. Your microbes are disrupted by any usable means, including sequential freeze-thaw ultrasonic treatment, mechanical disintegration, or the use of cellular agents.

Transformed yeast host cells used to express IL-15 secreted by polypeptide 20 simplify purification. Secreted combination derived from fermentation of yeast hosts. can be purified by methods such as those described by Urdal, €

Chromatog. 296: 171 (1984)]. Urdal, et al. illustrates .1 * 1 25 consecutive reverse phase HPLC steps for purification of human <RTIgt; IL-2 </RTI> in preparative HPLC.

: Administration of mammalian IL-15 polypeptide and its compositions. This invention provides a method of

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29 in a manner appropriate to the symptoms. Site inhibition of the anemia form may be accomplished by injection of the IL-15 composition as a bolus injection, infusion and continuous implantation using any other suitable method. An may be administered by intravenous injection, ih injection or parenteral or intraperitoneal infusion. Typically, the therapeutic agent is administered in a pharmaceutical composition containing the purified polypeptide in combination with physiologically acceptable carriers, excipients and additives. These carriers are present in the doses and concentrations used in the patients. Methods of making such compositions s 15 usually combining a mammalian IL-15 polypeptide derivative thereof with buffers, antibodies such as ascorbic acid, low molecular weight (more than about 10 groups) polypept proteins, amino acids, carbohydrates; 20 are glucose, sucrose, or dextrans, for substances such as EDTA, glutathione, and other, V, surfactants and additives. Examples of appropriate diluents are neutral buffered phys • ♦ *! / saline or physiological saline, which is y ***** to 25 serum albumin of the same type.

The following examples are given by way of illustration only and are not intended to limit the scope of the present invention: Example 1

Purification and sequencing of native sIL-15:: *: 30 Ultrafiltration • «« · «· _ _ | - _. * _. _. . _ 30 in filtration equipment using either YM10 or YM30 cartridge, hollow fiber cartridge or wafer

However, an Amicon ultrafiltration system using a YM30 coil cartridge was preferred. No buffer replacement was required before or after this

An inhomogeneous protein solution, unlike medium used for culture, was obtained by using CV-1 / EBNA cell cultures in serum-free: medium depleted of phenol red, in batches of 5 g / L Cytodex · 3

The material from eight to eight liters of bio (about 64 liters in total) was harvested to remove cells and microcarriers, filtered through a 0.22 micrometre cellulose acetate membrane slurry, and then concentrated to a volume of about 2 liters using a YM30 coil cartridge. YM30-conc

filtered before hydrophobic chromatography. T

minimizes contamination by removing bacterial particulate materials. Filters having a pore size of 0, micrometre and non-protein binding, preferably V, may be used. however, a 0.2 micron cellulose acetate * * · * \ filter.

• · »• ··

Hydrophobic Chromatography I From a hydrophobic chromatograph, I was able to transfer the protein to a small buffer which could be introduced into the anion: columns. In addition, it produced a 3- to 6-fold pu. Before or after this step, there was no fucking bumper replacement. Suitable for use 31

An amount of ammonium sulfate was added to the ultrafiltration concentrate to give a final concentration of 0.2 M. The ultrafiltration concentrate was concentrated in an amount of 1 M HEFES, h about 8.5, to a final concentration of about 20 mM. The concentrate was pumped onto a Phenyl Si-CL4B column and washed with a mixture of 0.2 M sulfate and 10 mM HEPES, pH about 10 fused proteins with 10 mM HEPES to remove unbound nickel. The eluted protein trap (this IL-15) was applied to an anion exchange column.

Anion exchange chromatography

Purification by Anion Exchange Chromatography $ 1 to $ 15 for additional purification without the need to change dial buffer. This lie contained the advantage that the combined Phenyl Sepharose * protein fraction was packed twice in anion exchange media. Binding proteins were eluted after 20 µl treatment with NaCl in HEPES for use in a variety of different anion I diodes and buffer systems with a pH of n <* * e · / · to about 9. then Mono-Q used after DEAE-Sephaoel * (Pharmacia) 1 * allowed the use of 25 anion exchange steps without dialysis of buffers. DEAE-Sephacel · was able to liquidize the p * «* '· * · * of some contaminating proteins by fast performance liquid chromatography (" FPLC ", Pharmacia). Using Mono-Q. Use * K K _.

32

NaCl was added from Phenyl Sepharose to obtain (For injected fractions, Laine had a final conductivity of about 1.2 millimeters per centimeter ("mS / cm") (less than about 0.1 M 5 this was pumped into a DEAE-Sephacel · column which < weighted with about 0.1 M NaCl 2 to about 10 mM HEPES and pH was about 8.5 * Bound proteins were eluted with an aromatic gradient at a concentration of 0.1 M to about 0.3 M NaCl 2 in 10 HEPES, pH about 8.5 The eluted α protein fractions (containing IL-15) were combined for treatment in a Mono-Q anion exchange column.

Combined active DEAE-Sephacel * fraction

was lost in such an amount of about 10 mM HEPES

The final conductivity of the 15 mixtures became less than 1.6 n (0.14 M NaCl). The diluted pooled fraction was then applied to a column of Mono-Q-FPLC for high-speed liquid chromatography equilibrated with about 0.14 M NaCl in n-HEPES pH 8.5. Bound p was eluted with a gradient of a NaCl concentration from 0.14 M to about 0.5 M in r.i /: HEPES pH about 8.5. Active ly (these contained IL-15) were combined to produce k i of l 25 by high performance reverse phase liquid (*) graph (RP-HPLC).

: ···: RP-HPLC

The native mammalian IL-15 polypeptide has a pH of about 7 to about 9 and about 30 to 2.5 acetonitrile ("AcN") of choice n 10. J__J__j r * · .____ j i_l__l ________ /. r-33 C4-RP-HPLC columns! 11a (Vydac1, 0.46 x 25 cm, 5 reversed phase columns (C8 or C18) did not work from the T1 alive C4 at high AcN concentration it was not recovered at all from C8 or C18 5 buffer systems which were attempted, ammonium acetate / methanol pH = pyridine acetate / propanol did not yield results.

RP-HPLC purification included preferential treatment of the combined active fr from Mono-Q in a Vydac ™ C4 matrix. In the first slide, the active mono-Q-combined compounds were pumped onto a C4-HPLC column at a flow rate of 1 mL / min and eluted with a gradient starting at

15 contained a mixture of 0.1% TF

in the final step, a mixture of o] TFA / 100% AcN and used to form g as follows: 0-45% AcN, 1% AcN per minute 20 45-60% AcN, 0.5% AcN per minute 60-100 % AcN, 2% AcN per minute Fractions of the active peak (these p. · /: IL-15) elute from about 48 to about 51% isells * «·

The active, determined by biological assay, were pooled, diluted with 0.1% TFA and H; :; * to lower the concentration of AcN and were * · ***** on the same C4 column.

* * · '· * * Active pooled fractions prepared from C4-TFA / AcN run were pumped back into C4-k5: «: 30 was washed with 0.1% TFA / H2O and» · » * «Πα1 1 aaI 1 e 14 ba! 1a 4 am ^ 4 11a 4aava a 1 34 Identification of IL-15 containing fractions Identified IL-15 containing fractions were pooled.

SDS-PAGE

Purified IL-15 can be visualized by 5 stained SDS-PAGE. Mammalian IL-15 protein bands isolated by SDS-PAGE are electroblotted and analyzed for their N-terminal amino acid sequences. 1L-15- can be identified by biological assay.

The reconstituted IL-15 prothelin fractions from the 10 C4-TFA / n-propanol HPLC run were dried by vacuum drying, suspended in SDS reconstitution buffer and run on SDS-pol amide gel. HPLC purified 1L-15 was subjected to SDS-PAGE run (Phastgel? 8-25%, Pharmacia lly in two adjacent channels.

Before attachment, 1 mm gel slices were cut from the Phastgel9 channel and directly subjected to biological assays. The remainder of the gel was applied and the silver-stained zones, i.e. the soul of the biological assays used. I do. IL-15 activity was equivalent to 15-17 kDa co-expressed IL-15 was resuspended in pel * 1 / SDS sample buffer at a specific activity of 25% run on a 14% SDS-polyacrylamide gel; * and stained with silver staining. The 15-17 kDa s # * * ** "'sIL-15 polypeptides had a purity of about 220 00

Median purity of sIL-15 polypeptide used for cultivation of CV1 / EBNA: purification chart: J 30 (Table 2). Table 2 shows purity and protein • * * 35 PVDF-bXotti 14% SDS polyacrylamide gel b for PVDF membrane (ProBlot ·, purchased from

Biosystems) using a constant current of about 60 V and 5 for about one hour. Protein bands stained by staining PVDF membrane with coma (0 solution in 10% acetic acid and 50% methane). The color can be highlighted in protein bands! sex removed from the membrane using the same solution 10 coma blue is omitted. The IL-15-Akti corresponding protein band was excised and the N-te protein sequence was directly determined from PVDF.

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• r- (::: no 2 · * · cfl IS 'O 2 ··· I fll ιΛ ^ ίΛ 37 1 Values in parentheses are estimates] from SDS-PAGE. Other protein values Quantity: Biorad Microprotein Assay (BSA- standard) amounts: 2 Activity was determined by biological CT! 5 assay 3 EM * unspecified sIL-15 polypeptide sequencing

From the RP-HPLC step above, the IL-15 preparation was analyzed by SDS-PAGE. KUJ

was stained with silver staining to visualize protein bands. Except for visible bands, assays performed on dense gel slices showed IL-15 activity as 15 for proteins with relative molecular weights <17 kDa. The N-terminal end of a 15-17 kDa polypeptide blotted on a PVDF membrane was sequenced by Editing in a Applied Biosystems protein isolator. The results revealed the identity of the first 33 amino acids shown in SEQ ID NO: 3 when the cDNA clone obtained from the monkey library subsequently .V. This resulted in a sequence encoding the polypeptide of <VENSION ID NO: 2. SEQUENCE: * * · β * β ·] polypeptide contains relatively short: * 4 4, 1! 25 nucleic acid leader sequence and mature poly:; * represented by SEQ ID NO: 3.

EXAMPLE 2 '* 1 Biological Assay CTLL2 Cells Provide Rapid Biological Detection of IL-15 Polypeptides Detected by 38 (Avanzi et al., Br. J. Haematol. 69: 355 (1988) (Kitamura, et al., J. Cell Physiol 140 (1989) CTLL-2 cells were preferably cultured in DMEM containing glucose containing about 30 ng / ml IL-2, 5% f bovine serum (FBS) 5 x 10-5 M 2-mercaptoethanol penicillin, 50 µg / ml streptomycin and 3-4, tamin at 37 ° C, 10% CO 2, and 97% teas CTLL-2 is a factor dependent cell! 10 needs to grow IL-2 Therefore, CT was washed for assay to remove IL-2 with high glucose DMEM: 1 supplemented with 5% FBS, 5x10 5 captoethanol, 50 µg / ml penicillin, 50 µg / ml 15 mycine and 3 to 4.0 mM glutamine Samples to be assayed were titrated in DMEM containing 5 '96 well flat bottom microtiter washings. CTLL-2 cells were added to wells (one assay volume of 100 μΐ), 2000 cells / well and incubated January 20 for about 24 hours at 37 ° C for 10 -% isessi plates were treated with 3 H-thymidine (25 μ .V. at a dose of 0.5 pCi per well after about 5 hours ai * 4 J / · they were harvested (96-well Inotec * »· 1 / bone harvester) and cpm values determined. * I 25 Matrix 96 gas-filled proportional calculation *« * • tele). Units were calculated on the basis of cpm "··· * in these one unit corresponds to the microliter *. * * Is obtained at 50% of maximum stimulation.

Example 3

Preparation of j / 30 sIL-15 cDNA Clone. Λ 39 amino acids (Asn-Trp-VaX-Asn-Val-Ile) were used to design the primer, which was a Degener mixture that encoded all possible uses of the codons of the six non-amino acid residues and which was shown in SEQ ID NO: 3r. where Y is T or C? B is A, T or C; and N is A, 10 T. The N-terminal end acid sequences of the mature monkey mature protein 26-31 (for the design of a second primer of Tyr-Thr-Glu-Ser-Asp-Valti, a degenerative mixture that encoded position 3 s for Val except for the complementary form of all 15 amino acids of the amino acids 26 and 5? -ACRTCNGAYTCNGTRTA-3f and 5'-ACRTCRCTYTCNGTRTA-3 '20 as set forth in SEQ ID NO: 11, respectively, where R is 1 * Polyadenylated RNA molecules originating from 24 hours, 37 hours and 72 hours of phorbol * · · ·: 25 static 13-acetate (PMA) stimulated CV-1 • · ·: · cells were used. As separate templates, I do not prepare cDNA for synthesis. A portion of the first strand * * · thiode mixture was added to commercially available reaction mixtures containing 30 oligonucleotides.

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Ma «t **** i i. - 40 at 55 ° C, 53 ° C and 51 ° C, 2 were used where the coupling temperature was 49 ° C.

After amplification, the samples were purified by agarose gel electrophoresis. Herein, a 5 92 bp DNA fragment from a gel channel derived from two separate sections attached to CV-1 / EBNA cells. The 92 bp DNA fragment was purified using Eluon (Schleicher & Schuell, Keene, NH), 10h pBluescript SK '(Stratagene, La Jolla), and k DNA sequencing by the dideoxy method.

The hybridization probe was prepared with a var subcloned 92 base pair DNA fragment using random primers. Hybridization was used to screen a portion of the plasmid library for insertion sequences prepared from polyadenylated CV-1 / EBNA RNA. As a result, clone C85.SXL-15 was obtained which contains an open I20 containing the nucleotide sequence set forth in SEQ ID NO.

The nu sequence of the SLL-15 polypeptide coding region is described in SEQ ID NO: 1. This α 4 was derived from that C85.sIL-15 insertion sequence 1 / was linked by an Sal I linker expression vector; V 25 [McMahan, CJ, et al., EMBO J. 10 (10) (199: • 2832). Sal I. A CV-1 / EBNA cell line was prepared with polyadenylated mRNA and cDNA molecules using standard ** procedures. The CV-1 / EBNA cell line is a production line. Haymerle, et al., Nucleic Acid Res. 1-ti a ^ d ^ m 41 (SEQ ID NOs: 7 and 8, respectively, sequenced) and these were cloned into the pDC406 vector containing isolates containing about 500 Plasmid single isolates. , plated and labeled using hybridization DNA probe fragment A DNA probe fragment was prepared using amplification of sIL-15 sequences of C cell line cDNA.

Example 4 10 Cloning of human IL-15 The sIL-15 probe was prepared by providing s la random primers using the sIL-15 cDNA from the isolated, purified and radioactive bat Sal I fragment (about 15). x 10 6 cpm was hybridized in Northern blots from a variety of sources including human mRNAs on IMTLH cell lines IMTLH was derived from 20 transformations of human bone marrow stromal cell culture with pSVNeo. in about 40 formamide for about 18 hours. Hybridization) »» * e · / · the material was washed in 6 x SSC at about 10 * »1 * 22 eC, then washed in 2 x SSC for about 30 minutes I drive. Autoradiography revealed p • ft 4 * · * its signal on the IMTLH channel.

This example dealt with IMTLH cDNA · · ·

Southern blots with probes to identify a human IL-15 cDNA:? IMTLH Letter A A A ·. . _ & A A ^ .A. .. 4. . . . Then, approximately 4,000 "141" was washed with probes using conventional colonization techniques using human IL-15 cDNA to identify the clone. Only one

5 I41.hETF, was shown to encode human I

The degree of identity between the human and monkey IL-15 open frame nucleotide sequences is about 96% between amino acid sequences and 5).

EXAMPLE 5 Stimulation of CTLL-2 Reproduction cDNA mo (nucleotides 145-489 encoding the mature forms of rIL-15 IL15, including the boundaries in VENTS ID N0: 1 and 4) was inserted downstream of the 15 heterologous secretion signal sIL-hIL. The secretion signal is to a large extent a single hydrophobic amino acid region (this is located at the N-terminal end of the polypeptide and? 20 of the polypeptide between the N-terminal end of the parental peptide and the N-terminal end of the mature e protein). cleavage [von Heijne, Eur. J.

116, 419 (1981)]. Secretion signal sequence used 1 / ren II-7 signal sequence. Plasmid * 25 prepared were fused to C0S-7 cells. Supernatants from transfected cells stimulate additional CTLL-2 * * #

In addition, the precursor form coding region of hIL-15 (* * * V * ID NO: 3) was ligated to pDC406 and transfected with EBNA cells. pDC406: hIL-15 (e.g., super «1« -. u, * j U 1 ^ li 1,. ^.,..., _. derived from cells transfected with hET: 30).

43

Example 6 Induction of CTLL-2 proliferation by rIL-15:?

Monkey recombinant IL-15 (monkey rIL-15) was retained by the yeast super ™ 5 expressing the sIL-15 cDNA purification method by ultrafiltration and ion exchange step. The bioavailability of purified monkey rIL-15 was compared to that of purified recombinant II IL-4. The purity of all column 10 strains was confirmed by amino acid analysis as the biological active ingredient of the three purified proteins; were run in vitro using CTLL-: CTLL-2 cultures prepared with 100 µl of harvested tissue culture medium containing said 15 µl and containing 2000 cells per culture. CT beads were transiently treated with 0.5 pCi [din per culture for the last 4 hours, cells were harvested on a glass fiber filter]. Bioactivity was determined by gas jet ionization. The in vitro proliferation response of CTLL-2 cells was determined using rising concentrations; m-cytokine (expressed in ng / ml). Ko: Figure 6 shows the accumulated 3H-t * »i / cpm values in cells (x 10 * 3).

!; *: * Example 7 i * * * f Induction of CTL, LAK, and NK Cell Activity by rIL-15 '· * * Antigen-specific cytolytic T-lymph <(CTL) was prepared in vitro. Peripheral vein from one * 30 donors * ··. ____,, y ___ * ... - .........

44 cytokines. Cultures were cultured in Widmer, M.B., 1 J. Exp. Med. 166, 1447 (1987), were harvested after 7 days and moistened with cytolytic activity against 51Cr-equipped target cells from the alpha donor used. Cell lysis: Multiple amounts of reactive PBL were cultured in 1000-labeled target cell in 200 µl of medium in v-wells and pooled for 4 hours of incubation. The myths were counted as the inverse of the responding culture required to obtain 50 wells. specific release of 51 Cr. Experimental results for CTL treated cells are summarized in Figure 7.

Lymphokine-activated killer cells (LAK were assayed for CTL cells as described above under culture conditions except that irradiated autologous PBL-l, v. Cytol. · 1 · · · against the Daudi cell line Lytic units 1 • »« J.1 In the case of the LAK assay, you are the inverse of the 25 reactants required to produce • · 1 *; 1 of the specific specific release of s1Cr.

the test results for the cells treated with them are summarized in Table 8.

Natural killer cells were isolated from the tornβίβ2 tower from human PBL cells isolated from Λ Λ.

45 against the K562 erythroleukemia cell line. In the case of NK, the lytic units were calculated as the inverse of the part of the culture needed to produce 30% of the maximal specific 51Cr v 5. Losses on cytokine treated NK cells are summarized in Figure 9.

Because IL-2 and IL-15 were at activity marks 6 and 7, one of ordinary skill in the art would expect that IL-15 would stimulate the activity of CTL 10 cells and NK cells and increase its population capable of killing cells and virus-infected cells. As described above, administration of an IL-15 polypeptide, an effective amount of IL-15 prepared in a suitable diluent or carrier 15 is administered to patients with noma, melanoma, sarcoma, leukemia or 1, or to patients with Herpesviridae virus including cytomegalovirus, Polyoraaviridae virus Influenza viruses including HIV, Hepadnaviridae, hepatitis A, hepatitis C, hepatitis delta or hepatitis D virus. infect.

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Sequencing (1) GENERAL INFORMATION: (i) SEARCH: Grabstein, Kenneth Anderson, Dirk Eisenman, June Fung, Victor Rauch, Charles (ii) NAME OF THE INVENTION: Interleukin-15 (iii) NUMBER OF SEQUENCES: 12 (iv) EXCHANGE OF LETTERS (A) TO: Immunex Corporatio (B) STREET: 51 University Street (C) CITY: Seattle (D) STATE OR COUNTY: Washington

(E) COUNTRY: USA

(F) POST NUMBER OR CODE: 98101 (v) MACHINE CODE FORMAT: (A) TOOL TYPE: Floppy (B) COMPUTER: IBM PC compatible

(C) OPERATING SYSTEM: PC-DOS / MS-DOS

(D) SOFTWARE: Patent Release # 1.0, # 1.25 (vi) CURRENT APPLICATION DETAILS: (A) APPLICATION NUMBER: (B) DATE OF APPLICATION:; V; (C) CLASSIFICATION: «· l / A (viii) AGENT INFORMATION:, v [(A) NAME: Launer, Charlene * .V (B) REGISTRATION NUMBER: 33 035 (C) REFERENCE / LIST NUMBER: 2811 *«:., 0 (ix) TELECOMMUNICATIONS: (A) PHONE: 206-587-0430 • ti 9 (2) SEQ ID NO: 1 DETAILS: \ j .: (i) SEQUENCE FEATURES: ··· / & ^ DTi * rnrc «aqq a 47 1 (ix) CHARACTERISTICS;

(A) NAME / EXPLANATION: CDS

(B) POSITION: 1..489 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1:

ATG AGA ATT TCG AAA CCA CAT TTG AGA ATT TCC ATC CAG TGC TAC

Met Arg Ile Sei Lys Pro His Leu Arg Sei Ile See Ile Gin Cys Tyr 15 10 15

CTG TGT TTA CTT CTA AAG AGT CAT TTT CTA ACT GAA GCT GGC ATT CAT

Leu Cys Leu Leu Leu Lys Ser His Phe Leu Thr Glu Lower Gly Ile His 20 25 30

GTC TTC ATT TTG GGC TGT TTC AGT GCA GGG CTC CCT AAA ACA GAA GCC

Vai Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala 35 40 45

AAC TGG GTG AAT GTA ATA AGT GAT TTG AAA AAA ATT GAA GAT CTT ATT

Asn Trp Or Asn Vai Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile 50 55 60

CAA TCT ATG CAT ATT GAT GCT ACT TTA TAT ACA GAA AGT GAT GTT CAC

Gin Ser Het His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Vai His 65 70 75 80

CCC AGT TGC AAG GTA ACA GCA ATG AAG TGC TTT CTC TTG GAG TTG CAA

Pro Ser Cys Lys Or Thr Lower Met Lys Cys Phe Leii Leu Glu Leu Gin 85 90 95

GTT ATT TCA CAT GAG TCC GGA GAT ACA GAT ATT CAT GAT ACA GTA GAA

Vai Ile Ser His Glu Ser Gly Asp Thr Asp Ile His Asp Thr Vai Glu 100 105 110

AAT CTT ATC ATC CTA GCA AAC AAC ATC TTG TCT TCT AAT GGG AAT ATA

Asn Leu Ile Ile Leu Area Asn Asn Ile Leu Ser Ser Asn Gly Asn Ile 115 120 125 • · e »*

: \ j ACA GAA TCT GGA TGC AAA GAA TGT GAG GAA CTA GAG GAA AAA AAT ATT

* * · Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile JY: 130 135 140 * ·

: * · *: AAA GAA TTT TTG CAG AGT TTT GTA CAT ATT GTC CAA ATG TTC ATC AAC

* Lys Glu Phe Leu Gin Ser Phe Or His Ile Or Gin Met Phe Ile Asn 145 150 155 160 «« ·

Y i ACT TCT TGA

Thr Ser. (2) SEQ ID NO: 2 INFORMATION:::: «« «

mi f 4 \ CI2IA7DMCC TfcJ ΑΜΤΜλ TCDT TDOIDPOI

48

Het Arg Ile Sec Lye Pro His Leu Arg Ser Ile Ser He Gin Cys Ty r 1 5 10 15

Leu Cys Leu Leu Leu Lys Ser His Phe Leu Thr Glu Lower Gly He His 20 2S 30

Or Phe He Leu Gly Cys Phe Ser Lower Glv Leu Pro Lvs Thr Glu Lower 35 40 45

Asn Trp Or Asn Val He Ser Asp Leu Lys Lys He Glu Asp Leu He 50 55 60

Gin Ser Met His He Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His 65 70 75 80

Pro Ser Cys Lys Val Thr Lower Met Lys Cys Phe Leu Leu Glu Leu Gin 85 90 95

Val He Ser His Glu Ser Gly Asp Thr Asp He His Asp Thr Val Glu 100 105 110

Asn Leu Ile He Leu Area Asn Asn He Leu $ er Ser Asn Gly Asn He 115 120 125

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn lie 130 13S 140

Lys Glu Phe Leu Gin Ser Phe Val His lie Val Gin Met Phe lie Asn 145 150 155 160

Thr Ser ¢ 2) SEQ ID NO: 3 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 114 amino acids 1 (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECYL TYPE: protein

: V: (iii) HYPOTHETICAL (yes / no): NO

«« I · ·

• »I

jV. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: * · * 8 · • «* * · \ Asn Trp Vai Asn Vai Ile Ser Asp Leu Lys Lys Ile Glu Asp Le 1 -5 10 15 * 88 8 8» V · Gin Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Va 20 25 30: Ser Cys Lys Vai Thr Ala Met Lys Cys Phe Leu Leu Glu Le · *: 35 40 45. (2) SEQ ID NO: 4 DETAILS: 49 (i) SEQUENCE FEATURES; (A) LENGTH: 489 bp (B) TYPE: Nucleic Acid (C) YARN: Single-stranded (D) TOPOLOGY: Linear

(ii) MOLECYLYL TYPE: cDNA

(ix) FEATURES:

(A) NAME / EXPLANATION; CDS

(B) POSITION: 1 * * 489 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4:

ATG AGA ATT TCG AAA CCA CAT TTG AGA ATT TCC ATC CAG TGC T

Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gin Cys T

15 10 15

TTG TGT TTA CTT CTA AAC AGT CAT TTT CTA ACT GAA GCT GGC ATT C

Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile H

20 25 30

GTC TTC ATT TTG GGC TGT TTC AGT GCA GGG CTT CCT AAA ACA GAA G

Vai Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu A

35 40 45

AAC TGG GTG AAT GTA ATA AGT GAT TTG AAA AAA ATT GAA GAT CTT A

Asn Trp Or Asn Vai Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu I

50 55 60

β · β · β CAA TCT ATG CAT ATT GAT GCT ACT TTA TAT ACG GAA AGT GAT GTT C

·, · ** Gin Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Vai H

: 65 70 75 -

• M

• ♦

• V. CCC AGT TGC AAA GTA ACA GCA ATG AAG TGC TTT CTC TTG GAG TTA C

* · '· * Pro Ser Cys Lys Vai Thr Lower Met Lys Cys Phe Leu Leu Glu Leu G

85 90 95 • e

I; GTT ATT TCA CTT GAG TCC GGA GAT GCA AGT ATT CAT GAT ACA GTA G

*** Vai Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Vai G

::: 100 105 no

AAT CTG ATC ATC CTA GCA AAC AAC AGT TTG TCT TCT AAT GGG AAT G

Asn Leu Ile Ile Leu Area Asn Asn Ser Leu Ser Ser Asn Gly Asn V

: .i .: 115 120 125 (2) SEQ ID NO: 5 DETAILS: 50 (i) SEQUENCE FEATURES: (A) LENGTH: 162 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECYL TYPE: protein ) SEQUENCE DESCRIPTION: SEQ ID NO: 5:

Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gin Cy 15 10 1

Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly II 20 25 30

Vai Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr G1 '35 40 45

Asn Trp Or Asn Vai Ile Ser Asp Leu Lys Lys Ile Glu Asp Le 50 55 60

Gin Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Va 65 70 75

Pro Ser Cys Lys Vai Thr Ala Met Lys Cys Phe Leu Leu Glu Le 85 90 9

Vai Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Va 100 105 110

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly As 115 120 125 • V; Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys As; \ 130 135 140 • · · •

l. * Lys Glu Phe Leu Gin Ser Phe Vil His Ile Vai Gin Met Phe II

V .: 145 150 155 f V Thr Ser • · * • «« ·· »··· 5.J: (2) SEQ ID NO: 6 DETAILS :. (i) SEQUENCE FEATURES::. · .ί (A) LENGTH: 114 amino acids! ··· ίΒΊ TYPE: aminohaDDo 51

Asn Trp Or Asn Vai Ile Ser Asp Leu Lys Lys Ile Glu Asp Le 1 5 10 1 £

Gin Ser Met His lie Asp Ala Thr Leu Tvr Thr Glu Ser Asp Vc 20 25 30

Pro Ser Cys Lys Val Thr Lower Met Lys Cys Phe Leu Leu Glu Le 35 40 “45

Vai He Ser Leu Glu Ser Gly Asp Ala Ser He His Asp Thr V <50 55 60

Asn Leu Ile He Leu Area Asn Asn Ser Leu Ser Ser Asn Gly A * 65 70 75

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Αί Θ5 90 9i

Lys Glu Phe Leu Gin Ser Phe Val His He Val Gin Met Phe I] 100 105 110

Thr Ser (2) SEQ ID NO: 7 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 24 base pairs (B) TYPE: Nucleic acid (C) SPECTACULARITY; single-stranded (D) TOPOLOGY: linear

(ii) MOLECYL TYPE: cDNA: V: (iii) HYPOTHETICAL (yes / no); NO

• e

: Λ: (iv) ANTI-SENSE: NO

• (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: • · ψ • · e

TCGACTGGAA CGAGACGACC TGCT

• e ♦ • * »♦ (2) SEQ ID NO: 8 DETAILS:: · <: ·! (i) SEQUENCE FEATURES: / »\ DTfPTirrC · ΟΠ ^ 52 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: agcaggtcgt ctcgttccag (2) SEQUENCE NO: 9 DETAILS: (1) SEQ ID NO: 18 TYPE: Nucleic Acid (C) YARNITY: Single-stranded (D) TOPOLOGY: Linear

(11) MOLECYL TYPE: cDNA (lii) HYPOTHETICAL (Yes / No): NO (en) ANTI-SENSE: NO

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: AAYTGGGTNA AYGTNATH

(2) SEQ ID NO: 10 DETAILS: (1) SEQUENCE FEATURES: (A) LENGTH: 17 base pairs (B) TYPE: Nucleic Acid (C) DURABILITY: Single-stranded (D) TOPOLOGY: Linear

(ii) MOLECYLYL TYPE: cDNA: V; (lii) HYPOTHETICAL (yes / no): NO

9 9

V ·! (iv) ANTI-SENSE: KYLLX

• ψ 9 9 · • · 9 9 9: v: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: • * • · · • · ·

!!! ACRTCNGAYT CNGTRTA

(2) DETAILS OF SEQ ID NO: 11: (i) SEQUENCE FEATURES; (A) LENGTH: 17 base pairs (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 53

ACRTCRCTYT CNGTRTA

(2) SEQ ID NO: 12 DETAILS: (i) SEQUENCE FEATURES: (A) LENGTH: 114 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECYL TYPE: protein

(iii) HYPOTHETICAL (yes / no): NO

(xl) SEQUENCE DESCRIPTION: SEQ ID NO: 12: Α5Π Trp Vai Asn Vai Ile Ser Asp Leu Lys Ile Glu Asp L <15 10 1;

Gin Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Vi 20 25 30

Pro Ser Cys Lys Vai Thr Lower Met Lys Cys Phe Leu Leu Glu L · 35 40 45

Vai Ile Ser Xaa Glu Ser Gly Asp Xaa Xaa Ile His Asp Thr V 50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Xaa Leu Ser Ser Asn Gly A; % 65 70 75 * t 4 • # · I, 1 2 3 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys A, 85 90 9 • e »

: Lys Glu Phe Leu Gin Ser Phe Vai His Ile Vai Gin Met Phe I

100 105 110 • »• i 1

Thr Ser • 4 f · »2 • · # 3 444 • e

Claims (15)

An isolated DNA sequence encoding a peptide exhibiting the biological activity of deuterleukin-15 (IL-15) characterized in that the venom is: (a) DNA sequence encoding a mammalian polypeptide which includes the sequence; Asn Trp Vai Asn Val lie Ser Asp Leu Lys Lys lie Glu Asp L «15 1Ö 15 Gin Ser With His lie Asp Ala Thr Leu Tyr Thr Glu Ser Asp Va 20 25 30 Pro Ser Cys Lys Val Thr Ala With Lys Cys Phe Leu Leu Glu L € 35 40 45 Val Tie Ser Xaa Glu Ser Gly Asp Xaa Xaa lie His Asp Thr Va 50 55 60 Asn Leu lie lie Leu Ala Asn Asn Xaa Leu Ser Ser Asn Gly As 65 70 75 Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys As 85 "90 95 Lys Glu Phe Leu Gin Ser Phe Val His He Val Gin With Phe II ea 100 105 110" "" I *, Thr Ser "" 4 \ *: ϊ where amino acid 52 is Leu or His, amino acid 5 'Γ · or Thr, amino acid 58 is Ser or Asp, amino acid 4; Ser or Ile and amino acid 80 is Val or Ile; e * <4 (b) any DNA sequence which on a detected "v" hybridizes to DNA sequences according to (a) or tJ complementary strands under highly stringent B1 SEQUENCE ID NO: 1; (b) any of the DNA sequences which hybridize in a manner to DNA sequences of (. 5 to their complementary strands under high stride conditions and which, upon expression, encode a po with mammalian IL-15 biological activity; or (c) any of the DNA sequences, sort of degenerate genetic code, encode a polyp; 10 is encoded by any of the preceding DNA sequences. An isolated DNA sequence according to claim 1, characterized in that said DNA sequence, which encodes polypeptide, which exhibits mammalian IL-15 activity, comprises a DNA sequence which is: (a) nucleotides 145-489 of DNA sequence SEQ ID NO: 4; (b) any of the DNA sequences which in a hybrid manner hybridize to DNA sequences of (, to their complementary strands under high stride conditions) and which, upon expression, encode a po with mammalian IL-15 biological activity or (c) any of the DNA sequences, which, on the basis of the degeneracy of the genetic code, encode a polyp * * 1 · encoded by any of the preceding DNA sequences. A recombinant expression vector, characterized in that it comprises a DNA sequence 2 * · for a mammalian IL-15 biological act polypeptide • A recombinant expression vector as characterized in that said DNA sequence, so for a polypeptide exhibiting mammalian IL-15 aces: (c) any of the DNA sequences which hybridize in a manner to DNA sequences of {(b) or their complementary strings are unc string conditions and, as in expression, in a polypeptide with mammalian IL-15 biological activates (d) any of the DNA sequences, which, on the basis of the degenerative genetic code, encode a polyp encoded by someone of predecessor DNA sequences. A host cell transformed or trans with an expression vector according to claim 4. 7. A host cell according to claim 6, wherein the host cell is E. coli, Pseudomonas, lice, Streptomyces, yeast, no fungus, no insect or any mammalian cell. A host cell according to claim 7, wherein the host cell is E. coli. 9. A host cell according to claim 7, wherein the host cell is Saccharomyces cerevisiae 10. A host cell according to claim 7, characterized in that the host cell is a mammalian cell. 11. A host cell according to claim 10, characterized in that the host cell is a Chinese hamster tree. A host cell transformed or transduced with an expression vector according to claim 5. 17. A host cell according to claim 16, characterized in that the host cell is a Chinese hamster tree 18. A method for producing an polypeptide, characterized in that the method 5 takes up the culture of a host cell according to claim 6, which promotes expression, and regenerates the culture of a polypeptide that exhibits IL-biolivity. A method for producing a polypeptide, characterized in that the method takes the culture of a host cell according to claim 7, which promotes expression and recovery: the cultivation of a polypeptide exhibiting IL-15 bioactivity. A method according to claim 18 e characterized in that the IL-15 polypeptide comprises polypeptide encoded by an isolated DNA sequence of claim 1, a polypeptide defined by SEQ ID NO: 1 polypeptide defined by SEQ ID NO: 2, a PC 20 defined by SEQ ID NO: 6 or a polypeptide row of SEQ ID NO: 5. 21. An isolated DNA sequence encoding for the kurs «cursor polypeptide of the biologically active IL-15 time, characterized in that said DNA sequence A DNA sequence which, upon expression, encodes the peptide defined by SEQ ID NO: 2; or a DNA sequence which, upon expression, encodes the f: peptide defined by SEQ ID NO: 5. 22. An isolated DNA sequence according to claim 30 characterized in that said DNA sequence, st. * ··. ___1_________________ J ...