KR20070018054A - Purified interleukin-15/Fc fusion protein and preparation thereof - Google Patents

Abstract

The present invention comprises the steps of: a) adding the composition to an affinity chromatography column and eluting the first IL-15 / Fc eluate from the column; and b) adding the eluate of step a) to an ion exchange chromatography column. Purifying the interleukin-15 / Fc fusion protein from the composition, comprising eluting a second IL-15 / Fc eluate from the composition; And purified interleukin-15 / Fc fusion proteins and compositions, particularly pharmaceutical compositions comprising such fusion proteins.

IL-15 / Fc fusion protein, purification, antagonist, antibody, affinity chromatography, ion exchange chromatography, recombinant plasmid

Description

Purified interleukin-15 / Fc fusion protein and preparation according to the present invention

The invention adds the composition to an affinity chromatography column and elutes the first IL-15 / Fc eluate from the column, and adds the eluate of step a) to the ion exchange chromatography column and removes the second from the column. Purifying the Interleukin-15 / Fc fusion protein from the composition comprising b) eluting the IL-15 / Fc eluate; And purified interleukin-15 / Fc fusion proteins and compositions, particularly pharmaceutical compositions comprising such fusion proteins.

Transplantation of organs or tissues is a standard method and in many cases has become a treatment for lifesaving from a number of fatal diseases. However, difficulties often arise with regard to rejection by the recipient organ caused by the immune response of the implant to foreign cell surface antigens. One method of inhibiting therapeutic rejection is to inhibit a humoral or cellular immune response using immunosuppressive agents, particularly antagonistic interleukin-15 (IL-15) or interleukin-2 (IL-2) antibodies or agonists. Various therapies using antibodies to IL-15 or IL-2 molecules have been previously described. Examples of effective IL-15 antagonists are fusion proteins consisting of N-terminal mutations or non-mutant IL-15 fragments and C-terminal Fc fragments (WO 97/41232; Kim et al. (1998) J. Immunol. 160: 5742-5748].

In order to successfully use these fusion proteins as medicaments, they need to be prepared in large quantities at very high purity and stored in a stable manner. Generally, proteins that are not naturally present are produced by genetic engineering methods. To this end, the protein is prepared in cell culture using a mammalian or bacterial cell line that has been genetically modified by introducing a recombinant plasmid containing the gene of the peptide of interest into the cell line to produce the protein. These cell lines are then cultured under suitable conditions in a complex culture medium containing, for example, sugars, amino acids, growth factors, salts, serums from different animals, and the like. It is then necessary to separate the protein of interest from the media components, metabolic products of the cells and other contaminants until the purity is reached to a degree sufficient for use as a therapeutic agent.

Methods of purifying proteins from cell cultures are well known to those skilled in the art. Some proteins are released directly into the surrounding medium by the cell line, while others remain in the cell. Proteins retained in cells need to first disrupt the cells, which is possible by a number of methods such as, for example, mechanical shearing, osmotic shock or enzyme treatment. In this case, the cell lysate contains the entire cell contents and an additional procedure is required to remove the subcellular fragments. Such additional procedures include, for example, differential centrifugation or filtration. If the protein is obtained directly from the supernatant, a step may also be required to remove some of the dead cells and the like. Thereafter, the protein is generally further purified by a combination of various chromatographic techniques. These techniques separate mixtures of proteins according to their size, charge, hydrophobicity, or affinity for a particular matrix. For each of these techniques, a number of column materials are commercially available, depending on the protein of interest. The target of any chromatography is a protein of interest that exhibits different migration properties from the contaminant on the column, eluting at a different time point than the contaminant.

It is an object of the present invention to provide as pure a form of IL-15 / Fc fusion protein as possible.

This object was achieved by a method of purifying IL-15 / Fc from a composition comprising the following steps:

Adding the composition to an affinity chromatography column and eluting a first IL-15 / Fc eluate from the column, and

Adding the eluate from step a) to an anion exchange chromatography column and eluting a second IL-15 / Fc eluate from the column. B).

IL-15 / Fc fusion protein (IL-15 / Fc) as used herein is a fusion protein comprising two fusion residues, namely the IL-15 component and the Fc component. Recombinant proteins comprising a fusion residue of an immunoglobulin in addition to the agonist protein are described, for example, in Capon et al. (US Pat. No. 5,428,130).

Preferred are fusion proteins consisting of an N-terminal mutation or a non-mutant IL-15 moiety and a C-terminal Fc moiety. Such proteins are described, for example, in WO 97/41232 and Kim et al. (1998, J. Immunol. 160: 5742-5748).

The IL-15 portion of the fusion protein mediates selective binding to, for example, an IL-15 receptor (IL-15R) expressed on activated T cells. Thus, the IL-15 moiety can be both naturally occurring IL-15 and mutants thereof.

In a more preferred embodiment, the IL-15 component is wild type IL-15. In this regard, IL-15 is any species, for example, mouse, rat, guinea pig, rabbit, cow, goat, sheep, horse, pig, dog, cat or monkey, preferably human May be IL-15. Also included are different splice variants and naturally occurring variants. Particular preference is given to mammalian, in particular human, nucleic acids or murine forms of such nucleic acids.

IL-15 mutants include IL-15 components having mutations such as, for example, one or more deletions, insertions or substitutions, or combinations thereof, as compared to naturally occurring IL-15. However, the IL-15 variant used must be able to bind the IL-15 / Fc fusion protein to IL-15R. This can be investigated, for example, by radiation ligand binding assays using membranes or cells with labeled IL-15 and IL-15 receptor (Carson WE et al., 1994, J. Exp Med. , 180 (4): 1395-1403].

In a preferred embodiment, the mutant may have a similar action to IL-15 (the IL-15 component with agonist action), the activity of which may be at the same level, reduced or even increased compared to IL-15. . A test system that can be used for an IL-15 / Fc fusion protein with an IL-15 component with agonist action is stimulation of murine CTLL-2 cell proliferation by the IL-15 component.

The IL-15 component has at least 10%, preferably at least 25%, more preferably at least 50%, even more preferably 100%, even more preferably 150% and most preferably at least 200% activity. , Agonist action according to the invention. The activity of an IL-15 component with agonist action refers to the percent stimulation of the response by the IL-15 component as compared to the stimulation with wild type IL-15 (wild type IL-15 is equivalent to 100% activity). . Only IL-15 components or fusion proteins can be used in this assay.

For IL-15 components with agonist action, conservative amino acid substitutions are preferred, in which one residue is replaced with another residue having similar properties. Typical substitutions are in aliphatic amino acid groups, in amino acid groups with aliphatic hydroxyl side chains, in amino acid groups with acidic radicals, in amino acid groups with amide derivatives, in amino acid groups with basic radicals or among amino acid groups with aromatic radicals. Is a substitution of. Typical conservative and semiconservative substitutions are as follows.

In another embodiment of the invention, IL-15 components having antagonism are used. This type of component inhibits the action of IL-15 or binding of IL-15 to IL-15R and allows complete or only partial inhibition. An assay system that can be used for IL-15 / Fc fusion proteins having an antagonistic IL-15 component is the assay system described in WO 97/41232 (BAF-B03 Cell Proliferation Assay). The IL-15 component inhibits at least 10%, preferably at least 25%, more preferably at least 50% and most preferably at least 95% of IL-15-mediated action or binding of IL-15 to IL-15R. In case, it has an antagonistic action according to the present invention. Only IL-15 components or fusion proteins can be used in this assay.

For IL-15 components with antagonistic action, non-conservative amino acid substitutions are preferred, in which one residue is replaced with another residue with different properties. It is further preferred to substitute within the region of the molecule responsible for interaction or signal transduction with IL-15-R.

In a preferred embodiment, the IL-15 component having an antagonistic action is characterized by a mutation in the IL-15 mutant or amino acid position 56 (aspartate; AAA21551) described in WO 97/41232. Having IL-15 component. Most preferred are point mutations introduced at amino acid positions 149 and / or 156 of interleukin-15, in particular mutants in which glutamine is substituted with aspartate (see WO 97/41232). .

In one aspect, it is also possible to combine the described mutations.

In one embodiment, the mutated IL-15 portion of the fusion protein is a wild type IL-15, preferably a human wild type IL-15 (e.g., database of the National Center for Biotechnology Information, Accession No. AAA21551), or any other natural variant (e.g., variants of Accession Nos. CAA63914 and CAA71044 in databases of the National Biotechnology Information Center) and at least 65%, preferably at least 70%, more preferably Preferably at least 85%, even more preferably at least 95%, and most preferably at least 99%.

The second unit of action of the IL-15 / Fc fusion protein is the Fc component. Fc portion refers to a constant (c = constant) fragment of an immunoglobulin, which can be made by papain cleavage and its amino acid sequence is highly conserved. Such Fc fragments are generally antibody fragments which do not bind any antigen. The Fc moiety according to the invention preferably also refers to an immunoglobulin fragment as defined above, and furthermore comprises also the hinge regions as well as the constant domains CH2 and CH3.

The Fc component is derived from the Fc portion of any antibody, for example IgA, IgD, IgG, IgE or IgM, preferably IgM or IgG, more preferably from the Fc portion of subclasses IgG1, IgG2, IgG3 and IgG4. will be.

In certain embodiments of the invention, the Fc portion of the fusion protein is an Fc fragment of immunoglobulin G (IgG) that lacks the light and heavy chains of the IgG-variable region.

Examples of IgG that can be used are IgG1, IgG2, IgG2a, IgG2b, IgG3 and IgG4. Human or murine IgG1 is preferred.

In the present invention, the entire Fc portion or only a portion thereof of the antibody can be used. However, some of the Fc moieties should preferably be designed in such a way that the IL-15 / Fc fusion protein has a longer circulating half-life than the IL-15 component without the immunoglobulin component in the blood. This can be examined by injecting the fusion protein and the IL-15 component into one or more experimental animals, for example by injecting it into the bloodstream and controlling the circulating half-life in the blood. Longer half-life is an increase in half-life, preferably at least 10%, more preferably at least 20%, even more preferably 50% and most preferably at least 100%.

The Fc portion may also be an Fc portion with one or more mutations. The mutated Fc can be mutated in the manner described above for the Il-15 moiety.

In one embodiment, the mutated Fc portion of the fusion protein is at least 65%, preferably at least 70%, more than the Fc portion of a rat or human wild-type immunoglobulin, preferably the Fc portion of human IgG1-Fc or other natural variant. Preferably at least 85%, even more preferably at least 95% and most preferably at least 99% are the same.

In a preferred embodiment of the invention, the Fc residue of the fusion protein is in its native form or has conservative amino acid substitutions and comprises an intact FcR- and / or complement-binding site. Fc residues of the fusion protein can mediate both activation of the complement system and binding to Fc receptor-expressing cells, thus depleting cells recognized by the IL-15 residues of the fusion protein. Introduction of mutations at amino acid positions that mediate complement activation and Fc-receptor binding, in particular non-conservative amino acid substitutions, can block these functions. Examples of these mutations are Fc receptor (FcR) or complement-binding sites (amino acid positions 214, 356, 358 and / or 435 in natural human IgG1, or Leu 235, Glu 318, Lys 320 and / or Lys 322 in native mouse IgG2A). Mutation of the binding site). Amino acid substitutions at these positions generally result in the dissolution of the Fc residue and the loss of the complement-activating action (WO 97/41232).

Further, for example, the position of the hinge region of human Fc residues, more preferably human IgG1 (position 167 of human IgG1), in order to prevent intermolecular linkage and thus aggregation of the expressed IL-15 / Fc fusion protein. It is preferable that the amino acid cysteine 4 is substituted with alanine.

In other preferred embodiments, the Fc moiety is the Fc moiety of human immunoglobulin IgG1 or murine immunoglobulin IgG2A and comprises the hinge region as well as the heavy chain regions CH2 and CH3.

In the IL-15 / Fc fusion protein, the IL-15 component is fused to the immunoglobulin component either directly or via a linker. The linker is preferably up to 25 amino acids, more preferably up to 15 amino acids, even more preferably up to 10 amino acids and most preferably 1, 2, 3, 4 or 5 It consists of amino acids.

In a preferred embodiment of the invention, the fusion protein is encoded by a nucleic acid at positions 905 through 2014 in SEQ ID NO: 1, or a nucleic acid in positions 1911 to 3020 in SEQ ID NO: 3, wherein the signal sequence (CD5) is in each case Is encoded by the first 74 nucleotides in. However, it may also be a protein having a sequence of SEQ ID NO: 4 or SEQ ID NO: 5. It also has an increased half-life in blood compared to the corresponding IL-15 / Fc fusion protein without the immunoglobulin component (for test systems, see above), and the corresponding IL-15 / Fc fusion protein is IL-15R. Proteins that bind to are at least about 60%, preferably about 75%, particularly preferably about 90% and especially about 95% identical to any sequence described above.

The fusion protein can be prepared by introducing a nucleic acid encoding the fusion protein into a cell and then expressing the nucleic acid in the cell under suitable conditions. The fusion protein prepared in this way can then be recovered in the supernatant or the cell itself by the method according to the invention. This includes adding the composition containing the IL-15 / Fc fusion protein to an affinity chromatography column and eluting the first IL-15 / Fc eluate.

Affinity chromatography has a special form of adsorption chromatography that has a high affinity for the material to be removed and has a group that binds with high strength to the support so that the material can be preferentially adsorbed and separated from other materials. it means.

The support can be, for example, the solid phase, discontinuous phase or dispersed particles of the purification column. Possible examples of solid phases are porous glass columns or silica columns. In one aspect of the invention, the solid phase may be coated with a reagent (such as glycerol) to inhibit nonspecific attachment of contaminants to the solid phase. In a preferred embodiment, the support material used for chromatography is Sepharose, especially when Protein A and / or Protein G is used as ligand.

The binding partner to be separated comprises a) binding the ligand and the partner to be recovered under suitable conditions, b) washing the non-binding material, if necessary, and c) creating a condition that no longer enables binding of the two materials, eg For example, it can be recovered by separating the binding partner from the ligand by changing the pH or ionic strength of the buffer.

In a preferred embodiment, the affinity chromatography is Protein-A chromatography using Protein A, particularly preferably recombinant Protein A as ligand. Protein A is a bacterial cell wall protein of Staphylococcus aureus , which has a specific affinity for the Fc region of the G class of immunoglobulins (IgG). Protein A has a molecular weight of 42 kDa (recombinant protein A; approximately 32-45 kDa) and a high pH stability at pH 2-10. Protein A is immobilized on a solid phase (support material). Binding affinity to the Fc moiety is a function of pH, and after binding in the presence of neutral or slightly alkaline buffer, the immunoglobulin can be eluted with a falling pH gradient.

One example of a protein-A chromatography material is Prosep-A (BioProducing Ltd., UK) consisting of protein A bound to a glass of porous structure. Other suitable Protein-A formulations include Protein A Sepharose, such as Protein A Sepharose Fast Flow (Parmacia) and Toyopearl 650 Protein A (TosoHaas). to be.

Alternatively, affinity chromatography may also be protein-G chromatography, in which the ligand used is protein G. Protein G is a surface protein from Streptococci of the G group and has a different range of affinity than protein A. The affinity of an IgG antibody for Protein A is different from that for Protein G, so one skilled in the art can select a suitable affinity chromatography depending on the Fc moiety used.

In addition, antisera of heterologous species against the Fc portion or other IL-15 portion of the fusion protein can be used.

In order to carry out step a) of the process according to the invention, the preparative column can for example be equilibrated with a suitable buffer. The buffer is a buffer solution that stabilizes the pH with its acid-paired base. The buffer used to equilibrate the column is preferably an isotonic buffer whose pH is generally in the range of approximately 6-8. Possible examples of equilibration buffers are Tris 20 mmol / l, NaCl 25 mmol / l, EDTA 25 mmol / l, pH 7.5.

The composition containing the IL-15 / Fc fusion protein in a suitable buffer is then added to the column as loading buffer. In one embodiment, the compositions of equilibration buffer and loading buffer are the same. The column is then washed with a suitable wash buffer as needed, after which the IL-15 / Fc fusion protein is eluted with the selected elution buffer.

Any washing step that can be performed between loading of the column (adding the composition) and elution serves to remove contaminants that are non-specifically bound to the solid phase. The aim is to elute as few fusion proteins from the solid phase as possible. Generally, the washing step uses isotonic solutions such as, for example, Tris 20 mmol / l, NaCl 150 mmol / l, pH 7.4, or, alternatively, to low values, for example pH 5.5, to which the fusion protein is still bound This can be done by lowering the pH.

Elution buffer is used to elute the IL-15-Fc fusion protein from the column. The pH of the elution buffer is preferably low, thus disrupting the interaction between the ligand, in particular Protein A and the fusion protein. The pH of the elution buffer is preferably in the range of 2-5, more preferably 3-4. Examples of buffers controlling pH in this range are phosphate, acetate, citrate and ammonium buffers and mixtures thereof. Preferred buffers are citrate and acetate buffers, further preferably sodium citrate or sodium acetate buffer, most preferably 0.1 mol / l of citrate at pH 3.4. However, elution buffers or ligands, especially those with high pH (eg, above pH 9), may be used that interfere with the interaction between Protein A and the fusion protein.

Dilute the eluate of step a) as necessary and add it to an ion exchange chromatography column, a cation exchange chromatography column or preferably an anion exchange chromatography column in step b), and add the second IL-15 / Fc eluate to the column Elute from.

In a preferred embodiment of the invention, the pH of the eluate of step a) is increased by addition of base or buffer. This is preferably done immediately after elution to prevent possible inactivation of the fusion protein. This can be done, for example, by adding 1/10 of the volume of Tris 1 mol / l, pH 8.0.

In ion exchange chromatography, proteins are separated based on their charge. Proteins, in addition to radicals whose side chains are generally uncharged, can also carry acidic and basic radicals, and thus consist of various amino acids that contribute to the total charge of the protein. At low pH below the isoelectric point of the protein, the total charge is positive because of the protonation of the charged side chains, and at high pH the total charge is negative, due to deprotonation. Since proteins carry complex charge groups whose actual charge depends on the pH and also on the environment of the individual amino acids, charge separation is a powerful method of protein separation. In ion exchange chromatography, the pH is selected so that the selected protein can bind to the matrix. For anion exchangers, the pH is generally at least one unit of pH above the isoelectric point of the protein (the pH at which the protein has zero total charge), and for cation exchangers it is below the isoelectric point. Bonding to the matrix occurs through electrostatic interconnection. Proteins that do not bind to the matrix are washed with buffer. The bound protein is eluted with the addition of a salt, preferably using NaCl. For example, the presence of charged sodium and chlorine ions stops the electrostatic interaction of the protein with the matrix, thereby separating the protein from the matrix.

Anion exchangers or basic ion exchangers are ion exchangers in which the cationic groups are covalently bonded to the solid insoluble matrix, while the neutralizing anions are only ionically bound to substitute other anions. Chromatography with anion exchangers is generally used to purify negatively charged molecules. Examples of anion exchangers are aminoethyl, diethyl-aminoethyl, quaternary aminoethyl and quaternary ammonium groups coupled to polyacrylamide gel resins or hydrocarbon polymer resins (eg cellulose or dextrin).

Cation exchange chromatography also works according to similar principles. Here, the positively charged molecule to be purified is bound to a support matrix containing a negatively charged group (eg carboxy-methyl or sulfonic acid group). The counterions used are generally sodium or potassium ions, which are substituted with positively charged sample molecules. Examples of cation exchangers are carboxymethyl, sulfoethyl, sulfopropyl, phosphate or sulfonate groups coupled to polyacrylamide gel resins or hydrocarbon polymer resins (eg cellulose or dextrin).

The column form may be a conventional form of vertical flow, or other form with radial flow.

Prior to adding the eluate of step a), the resin is generally equilibrated with a buffer having a pH in the range of 5 to 10, preferably 6 to 9, more preferably 7 to 8. Complex buffers can maintain pH in this range. Although each of these buffers is suitable for use, Tris buffers are preferred at present, with buffers having Tris 20 mmol / l of pH 8.0 being more preferred.

After equilibration, the eluate of step a) is added to an ion exchange resin, preferably an anion exchange resin. The column is then washed, if necessary, followed by eluting the IL-15-Fc fusion protein by increasing ionic strength and / or changing pH. Any wash step generally uses the same buffer as the buffer in the equilibration step. However, wash buffers with different, for example, high molarity (eg, Tris 20 mmol / l at pH 8.0, 200 mmol / l NaCl) can also be used. The buffer concentration for chromatographic separation is generally at least 10 mmol / l, for example to ensure sufficient buffer capacity. The ionic strength of the buffer is generally kept low (<5 mS / cm) to avoid affecting the binding of the protein to the matrix. However, the ionic strength should not be so low as to denature or precipitate the protein. The buffer ions must have the same charge as the matrix, because the opposite charges affect the separation process and cause local pH disturbances. Elution is carried out using a linear gradient, stepwise increase in electrolyte concentration or isocratic. In general, the electrolyte used (eg, sodium chloride or any other electrolyte with the same action) has a final value of 0.5 mol / l or less, preferably 0.4 mol / l or less, more preferably 0.35 mol / l or less Obtained in molar concentrations.

The eluate of step a) having a preferred buffer concentration of 10 to 200 mmol / l is added to the equilibrated column. The pH of the eluate is titrated to 5-9, preferably 7.5-8.5. The pH can be adjusted using a suitable formulation, for example by adding NaOH or other suitable base. The column can then be washed with a buffer, preferably equilibration buffer or wash buffer. The buffer concentration is preferably 10 to 300 mmol / l. The pH is preferably in the range of 7.0 to 9.0, more preferably 7.75 to 8.25.

In a preferred embodiment of anion exchange chromatography, the column used is preferably a DEA-Sepharose column or a Q-Sepharose column, more preferably a Q-Sepharose column.

In a preferred embodiment, anion exchange chromatography is performed with FPLC (High Speed Protein Liquid Chromatography) equipment or an Akta purifier (eg Akta Pilot or BioPilot, Amersham, UK). For FPLC equipment, the ion exchange column is once equilibrated in starting buffer and then the protein mixture (in the sample loop) to be separated is added to the column. Proteins that do not bind to the column elute immediately. The buffer is mixed to slowly reduce the ionic strength of the buffer so that proteins are eluted one by one in the column and collected in the fraction collector.

In a preferred embodiment of the invention, the purification process according to the invention additionally comprises adding the eluate of step b) to a gel filtration column or a hydrophobic interaction chromatography column and removing the third IL-15 / Fc eluate from the column. Eluting step (c).

The eluate of step b) is purified here by gel filtration or hydrophobic interaction chromatography.

Gel filtration consists of gel beads in which the stationary phase is expanded and separated according to particle size (large particles move through the gel beads along the liquid phase, small particles remain in the pores and finally appear in the eluate). It is particularly suitable for separating proteins using. Elution is generally carried out isocratic, ie using only one buffer without gradients. The composition of the buffer generally does not affect the separation of the protein and depends on the requirements of the protein. Spherical polymers with porosity depending on their degree of crosslinking can be used as the gel matrix. Examples thereof are Sephadex, Sepharose, Biogel A, Sephacryl and Biogel P. Small proteins of suitable size can diffuse into the pores of these matrices. This slows their flow rate through the gel. If the proteins are too large, they flow out through the pores and pass through the gel at a higher flow rate. Thus high molecular weight proteins are eluted before low molecular weight proteins. The substance mixture is separated according to different Stokes' radii (typically proportional to the molecular weight of the molecule for spherical proteins). The application volume to achieve sufficient separation is preferably 5% or less, more preferably 1% or less of the column volume. The concentration may preferably be 50 mg / ml or less, more preferably 25 mg / ml and even more preferably 10 mg / ml.

The stationary phase of the gel filtration chromatography column preferably comprises column material fractionating in the range of 5000 to 600,000 kDa. Superdex 200, Superdex 75, Superose 6, Super Ross 12, Sephadex G 75, Sephacryl S-200 HR, Sephacryl S-300 HR or Sephacryl S-400 HR Is preferably used, more preferably Superdex 200 is used.

The column is generally equilibrated with an equilibration buffer (eg 50 mmol / l sodium phosphate, 150 mmol / l sodium chloride, pH 7.0) before applying the eluate of step b). The eluate of step b) is then added to the column. Very low salt concentrations can adversely affect the chromatographic separation properties of the column, so the ionic strength is> 20 mS / cm.

As an alternative to gel filtration, hydrophobic interaction chromatography (HIC) can also be performed. Hydrophobic interactions are very important biochemically. They are actually involved in stabilizing the three-dimensional tertiary structure of proteins. Hydrophobic interaction is the phenomenon in which two hydrophobic molecules spontaneously aggregate in a polar environment (eg water). Dissolution of salts and increasing the ionic strength of the medium also increase the hydrophobic interaction of the two nonpolar molecules. Proteins have a rather high proportion of hydrophobic surface structure. They can thus attach hydrophobic adsorbents at suitable high ionic strengths. The intensity of the interaction can be controlled by selecting the salt content and, in addition, the adsorbent. Examples of functional groups used are ethyl, butyl, propyl, octyl or else phenyl radicals. Adsorption is carried out in the presence of high salt concentrations; Correspondingly, elution is carried out using a falling salt gradient. The salt used is generally ammonium sulfate.

In certain embodiments of the invention, the composition of the eluate is filtered and / or concentrated before or after one or more of steps a) to c).

Various methods can be used to concentrate the protein in solution. This can be done, for example, by ultrafiltration. Here, the protein solution can pass through only small molecules (salts, solvents), and large molecules (eg, proteins) are compressed by pressing through the membranes where they remain. This reduces the volume of the solution, increasing the protein concentration. For filtration, for example, a tangentially-flow filtration system can be used. Suitable membranes for this are membranes containing proteins. Filtration can be carried out by the method of compression filtration in which the feed is pressed perpendicular to the membrane. As an alternative to this a cross-flow filtration method can be used in which the feed is directed tangentially across the membrane. The permeate passes through the membrane perpendicular to the flow direction. Continuous tangential skimming of the membrane achieves a purification effect that reduces the formation of the capping layer. For further improvement, the fabric layer between two parallel membranes can be aligned, which additionally causes turbulence.

In addition to concentration or alternatives thereto, the composition or eluate may be filtered. Filter materials that can be used can be cellulose nitrate, cellulose acetate, PVC, Teflon, or ceramic membranes (eg, composed of zirconium oxide). The filter may be a separate membrane or assembled into a membrane system (eg module). The module can be a tubular module, a spiral module or an annular module or a hollow fiber module.

In a preferred embodiment of the invention, the composition is clarified by filtration before step a) of the purification process. The filter used for the purification filtration has a pore size of about 5 μm or less, preferably 4 μm, more preferably 3 μm and most preferably 1 μm.

The pore size of the filter used for ultrafiltration is preferably 100,000 NMGT or less, more preferably 75,000 NMGT or less, even more preferably 50,000 NMGT or less and most preferably 30,000 NMGT or less.

The pore size of the filter used for sterile filtration is preferably 0.8 μm or less, more preferably 0.6 μm or less, even more preferably 0.4 μm or less and most preferably 0.22 μm or less.

In a preferred embodiment of the invention, the pH of the eluate of step a) is made acidic before step b). The acidic pH is at most 7.0, preferably at most 5.5, more preferably at most 4.0 and most preferably at most 3.5. PH values below 3.5 are suitable for virus inactivation. After virus inactivation, the pH is raised again by adding 1/10 of the volume of Tris 1 mol / l, for example pH 8.0. In addition to the pH reduction, the composition or any eluate may be filtered using a suitable filter to remove the virus. Suitable filters are known to those skilled in the art.

In a particularly preferred embodiment of the invention, the affinity chromatography column used in the purification method is a protein-A chromatography column and the anion exchange chromatography column used in the purification method is a Q-Sepharose column and is used in the purification method. The gel filtration column is a Superdex-200 column.

Further, if desired, using an IL-15 / Fc fusion protein having at least 90% purity, preferably at least 95% purity, most preferably at least 99% purity after purification, comprising the embodiments described above, Particular preference is given to a purification process using steps a) to c) above. Purity can be confirmed, for example, by HPLC-SEC, as described in the Examples.

The present invention further relates to a purified IL-15 / Fc fusion protein having a purity of at least 90%, preferably at least 95%, most preferably at least 99%, wherein the purity is as described in the Examples. As can be confirmed, for example, by HPLC-SEC.

In a further preferred embodiment, the average sialylation per N-glycan, ie the proportion of N-glycans occupied by sialic acid in the purified IL-15 / Fc fusion protein is at least 70%, more preferably 80% Or more, still more preferably 90% or more and most preferably 95% or more.

In another preferred embodiment, the average sialylation per antenna, ie, the proportion of antennas occupied by sialic acid in the purified IL-15 / Fc fusion protein is at least 50%, more preferably at least 70%, even more preferably At least 80% and most preferably at least 90%, the number of antennas corresponds to the number of branches of the N-glycans.

Average sialylation can also be measured by HPAEC-PAD (high performance anion exchange chromatography and pulsed amperometric detection) along with the number of antennas measured by HPLC. Sialic acid is the group name for N- and O-acylated neuramic acid derivatives. Because of their exposed position as the end group of the complex oligosaccharide chains of the glycoconjugates, sialic acid makes a significant contribution to the biological properties of the former. As high as possible sialylation is advantageous, as enzymatic cleavage of sialic acid in the glycoprotein causes a significant shortening of their plasma half-life. This allows the administration of IL-15 / Fc at low doses and / or at longer intervals. Purification methods according to the present invention provide suitable methods that can be used to prepare IL-15 / Fc fusion proteins having average sialylation of the above mentioned values.

N-acetylneuraminic acid is the most frequent sialic acid and an important component of glycoproteins. Like other sialic acids, it acts as a defense against inactivation, which is why high proportions of N-acetylneuraminic acid are advantageous.

In a further aspect of the invention, the combined N-glycolylneuraminic acid portion of N-acetylneuraminic acid, N-glycolylneuraminic acid and asialo-N-glycan in the purified IL-15 / Fc fusion protein is 20 % Or less, Preferably it is 15% or less, More preferably, it is 10% or less. N-glycolylneuraminic acid is a neuramic acid that is not generally present in humans, suggesting that it can act as an antigen. Therefore, their proportions should be as low as possible.

The amount of N-glycolylneuraminic acid as part of the sum of N-acetylneuraminic acid, N-glycolylneuraminic acid and asialo-N-glycans is described, for example, N-acetylneuraminic acid, N-glycolylneuramine. It can be measured by HPAEC-PAD (high performance anion exchange chromatography and pulsed amperometric detection) by measuring the area% of N-glycolylneuraminic acid in the total area of acid and asialo-N-glycan.

Basically, many different biological functions of protein glycosylation are described. Thus, it is possible for an oligosaccharide to exert a protective or masking function by preventing protein recognition by proteases, microorganisms and antibodies. In many cases, glycosylation plays a structural and stabilizing role by assisting in the correct folding of polypeptides and maintaining protein morphology in the rough endoplasmic reticulum. In addition, glycosylation of proteins can modulate interactions with ligands or receptors and plays a role in cell-cell and cell-matrix recognition.

It is therefore desirable to provide an IL-15 / Fc fusion protein with as much natural or cell-originated glycosylation as possible. The number of glycosylation sites, the number of sugar molecules and / or the number of antennas may serve as a measure for this.

The present invention further relates to compositions comprising purified IL-15 / Fc protein and also excipients and additives.

For example, suitable excipients and additives that provide for protein stabilization are well known to those skilled in the art (see, eg, Sucker H. et al., (1991) Pharmazeutische Technologie, 2nd Edition, Georg). Thieme Verlag, Stuttgart, Germany]). These include, for example, physiological saline, Ringer's dextrose, Ringer's lactate, demineralized water, stabilizers, antioxidants, complexing agents, antimicrobial compounds, protease inhibitors and / or inert gases. In a preferred embodiment, the excipients and additives are mannitol, sucrose and / or glycine.

In a preferred embodiment, the composition has a pH of 7.4 to 8.0, since liquid compositions containing IL-15 / Fc and having such pH have proven particularly stable.

In a further preferred embodiment, the composition according to the invention is a pharmaceutical composition. Pharmaceutical compositions are compositions intended to be used with and suitable for use as a medicament. Thus it includes pharmaceutically suitable excipients and additives. Examples thereof include aqua sterylissata (sterile water), substances which affect pH (e.g., organic and inorganic acids and bases and salts thereof), buffer substances for adjusting pH, isotonic agents (e.g. sodium chloride, Sodium bicarbonate, glucose and fructose), surfactants or surface-active substances and emulsifiers (e.g., partial fatty acid esters of polyoxyethylene sorbitan (Tween ^R ), or polyoxyethylene (Cremophor ^R ), for example). Fatty acid esters, fatty oils (e.g. peanut oil, soybean oil and castor oil), synthetic fatty acid esters (e.g. ethyl oleate, isopropyl myristate and neutral oil (Miglyol ^R ))}, and also polymerization excipients ( For example gelatin, dextran and polyvinylpyrrolidone), solubilization-increasing additives of organic solvents (e.g. propylene glycol, ethanol, N, N-dimethylacetamide and propylene glycol), or complexing agents (e.g. To Citrate and urea), preservatives (eg hydroxypropyl and methyl benzoate, benzyl alcohol), antioxidants (eg sodium sulfite) and stabilizers (eg EDTA, PVP), granules Cellulose esters (e.g. beeswax and / or Eudragit ^R , cellulose or Cremophor ^R based polymers), antioxidants, sweeteners (e.g. sucrose, xylitol or mannitol), as flavoring or sustained release preparations, flavoring agents , Fragrances, preservatives, colorants, buffers, direct tableting agents {e.g. microcrystalline cellulose, starch and starch hydrolysates (e.g. Celutab ^R ), lactose, polyethylene glycols, polyvinylpyrrolidone and dicalcium phosphate) }, Lubricants, fillers (e.g. lactose or starch), binders in the form of lactose, starch (e.g. wheat or corn or rice starch), cellulose derivatives (e.g. methylcellulose, hydroxypropyl cellulose A switch or diatomaceous earth), talkum, stearates (e.g., magnesium stearate, calcium stearate, talc, silicon digestion talc, stearic acid, cetyl alcohol or hydrogenated maintained).

The invention will be illustrated below by examples and figures which are not to be construed as limiting.

1 depicts a map of pcDNA3.1hCD5.6Ala7 expression constructs.

2-3 depict the sequence of the pcDNA3.1hCD5.6Ala7 expression construct (SEQ ID NO: 1).

4 depicts a map of the pMG10Ala7 expression construct.

5-6 depict the sequence of the pMG10Ala7 expression construct (SEQ ID NO: 2).

7A depicts the nucleic acid sequence (SEQ ID NO: 3) of human mutated IL-15 / Fc with a CD5 leader.

7B depicts the amino acid sequence (SEQ ID NO: 4) of human mutated IL-15 / Fc with a CD5 leader.

7C depicts the amino acid sequence (SEQ ID NO: 5) of murine mutated IL-15 / Fc with a CD5 leader.

FIG. 8 depicts IL-15 / Fc content in cell culture supernatants of CHO-K1 cells after transfection with the pcDNA3.1hCD5.6Ala7 plasmid comprising the leader sequence shown in each case.

9 depicts IL-15 / Fc content in cell culture supernatants of CHO-K1 cells after transfection with various expression constructs. Each bar represents the mean + SEM of two measurements in each case of two independent experiments. pcDNA3.1 is pcDNA3.1hCD5. Corresponds to the 6Ala7 vector. pVS8-Ala7 corresponds to the pSwitch plasmid (Valentis) with constructs for IL-15 / Fc constructs. pMG-Ala7 corresponds to the pMG plasmid (Invivogen) with constructs for IL-15 / Fc constructs. pCINeo-Ala7 corresponds to the pCI-Neo plasmid (Promega) with constructs for IL-15 / Fc constructs.

Example 1: Preparation of IL-15 / Fc in CHO-K1 Cells

To prepare a CHO-K1 producer cell line for IL-15 / Fc, an expression construct for IL-15 / Fc should be formed and optimized with respect to its secretory properties with the identity / integration of the fragment it contains and with a suitable resistance gene. .

a) starting material

Human IL-15 / Fc expression constructs (mutated IL-15 / human Fc) were provided by the Department of Immunology at the Institute of Beth Israel Deaconness Medical Center (Boston Harvard Medical School, USA).

Oligonucleotides were obtained from an organ [MWG-Biotech] (Ebersberg, Germany). Sequences of the relevant signal peptides were obtained from the gene library.

Restriction enzymes (BgIII, XBaI, BamHI, SmaI, BstXI, ApaI), Lipfectamine2000, other molecular biological reagents (T4-DNA ligase, T-polynucleotide kinase) and plasmid pSecTagA, pcDNA3.1 were manufactured by Invitrogen (Germany Karlsruhe)] or from Amersham-Pharmacia (NheI, Protein A Sepharose, Uppsala, Sweden).

Competent Lee. E. coli XL10-Gold cells were obtained from the manufacturer (Stratagene, Layola, USA). BCA kit (Pierce) was purchased from the manufacturer (KMF Laborchemie, St. Augustine, St.).

Plasmid-DNA purification kit (Endofree-Maxi kit, Endofree-Giga kit) was purchased from the manufacturer (Qiagen, Hilden, Germany).

Antibodies were produced by the manufacturer [BD-Pharmingen (mouse-anti-hIL-15; catalog no. 554712; Heidelberg, Germany) and the manufacturer [Dianova (goat-anti-mouse-POD; catalog no. 15-036-003); Goat-anti-human-POD; catalog number 109-036-008; Hamburg, Germany).

b) method / result

The starting plasmid contained in the pSecTagA vector backbone of the cDNA of the fusion protein containing the mutated human IL-15 was fused to the Fc portion (hinge region and CH2, CH3 region) of human IgG1. The structure of this plasmid is the same as that described by author Kim et al. (J. Immunol., 160: 5742-5748; 1998), except that the Fc portion cited in the following document is murine Igγ2a. Corresponds.

An Igk leader already present in the pSecTagA vector was used for secretion of the fusion protein by intraskeletal cloning of the IL-15 / Fc moiety. To this end, the native signal sequence was removed from the native IL-15 sequence. However, due to cloning, 10 additional amino acids were introduced at the 3 'end of the Igk leader sequence and at the 5' end of the IL-15 cloning sequence, which remained in the secreted protein after processing of the protein. To remove these nonspecific amino acids and improve the secretory properties of the proteins, various leader sequences of other secreted or cell-surface proteins were tested: murine Igk (Coloma et al., J. Immun. Methods 152: 89 -104; 1992; Accession No. X91670), Human CD5 (Jones et al., Nature 323: 346-349; 1986); Accession No. X03491), CD4 (Hodge et. al. Hum. Immunol. 30: 99-104; 1991; Accession No. M35160), MCP-1 (see Yoshimura et al. Je. FEBS Lett. 244: 487-493; 1989); Accession No. M24545) and IL-2 (Taniguchi et al., Nature 302: 305-310; 1983; accession no. K02056) (accession number based on the National Biotechnology Information Center). After removal of the Igk leader and additional amino acids, the leader was cloned into the double stranded oligonucleotide and replaced with the signal peptide sequence. The entity is examined by sequencing. The resulting construct was then examined by transient transfection of HEK-293 cells using Lipfectamine2000. Protein-A-Sepharose Purification according to Moll and Vestweber's Method (Methods in Molecular Biology, 96: 77-84, 1999) It was then measured by BCA assay. The identity of the protein is confirmed by silver staining and Western blot methods of the SDS gel on the Fc or IL-15 moiety to confirm the presence of both components of the fusion protein. Optimal results were obtained with the described experiments when using a CD5 reader. The latter, where the leader itself is no longer present in the secreted fusion protein, was selected for further vector optimization.

It was further examined whether substituting the cDNA of the Fc portion with genomic DNA containing exon / intron structure contributes to improving protein expression. The presence of introns removed by the splice device of the nucleus can improve the transport of RNA from the nucleus and also RNA stability. Thus, the splice-donor and -recipient sites were inserted to bind the genomic Fc portion to the IL-15 cDNA sequence. The resulting plasmid was also modified using various leader sequences and examined as described above. However, protein analysis by Western blot proved to show various undesired splice variants, and decided to continue using the cDNA form of the Fc portion.

In conclusion, the plasmid obtained comprises a human CD5 leader and a cDNA Fc moiety.

Sequencing of the mutated IL-15 / Fc expression constructs indicated that the Fc portion contained three mutations that were already present in the original construct. Two of these mutations are related to amino acids in highly conserved positions. The third mutation was a Cys-Ala mutation at position 4 of the hinge region intentionally inserted to stop the formation of intramolecular and intermolecular cysteine bridges.

Fc cDNA was subcloned into RT-PCR to eliminate two unwanted mutations while maintaining Cys-Ala mutations. The RNA source used was a CHO-K1 cell line transfected with a construct encoding the VCAM-1-Fc fusion protein. The amplified Fc cDNA fragment was cloned into the CD5-mutIL-15 plasmid and the Fc portion was removed by BamHI / XbaI restriction cleavage.

The resulting plasmid was again analyzed based on unique restriction cleavage patterns and subsequent sequencing and named CD5-6Ala7. Since the use of Zeocin as a DNA-insertion agent can cause mutations, pcDNA3 removes the expression cassette for IL-15 / Fc from the original pSecTagA backbone and contains a neomycin resistance gene under the control of the SV40 promoter. Cloned to .1. The chains of both of the plasmids obtained were sequenced and showed perfect agreement with the IL-15 / Fc expression cassette.

The construct was retested for its protein expression by transient transfection of CHO-K1 cells and Western blot analysis of cell culture supernatants. As a positive control, transfection with the CD5-6Ala7 plasmid was performed in the same experiment.

To this end, the cells were seeded three times at a density of 5 × 10 ⁵ cells per well in a tissue culture plate with 6 wells the day before transfection. 2 µg of plasmid and 4 µl of lipofectamine2000 diluted in 250 µl of Optimem1 medium were used for transfection. Both solutions were mixed and incubated at room temperature for 30 minutes before the mixture was pipetted into the culture medium of the tissue culture plates.

After 2 days of transfection, the culture medium was removed and analyzed for its IL-15 / Fc content by Western blot for human IL-15 portions: 20 μl of cell culture supernatant and 5 μl of 5 × Laemmli buffer. Mix and incubate at 85 ° C. for 5 minutes. The sample was then developed to pass through a 12% polyacrylamide gel. The gel was then blotted using a semidry blotting chamber. The blot was treated overnight with blocking solution containing 5% dielectric fraction, 0.1% Tween20 in PBS. The blot was then incubated with monoclonal mouse-anti-human-IL-15 antibody diluted 1: 1000 in blocking solution for 4 hours. After three wash steps (PBS, 0.1% Tween20, 10 min), the blots were incubated with a second antibody goat-anti-mouse peroxidase (1: 5000 dilution) for an additional 2 hours at room temperature. After washing the blot three times again, Lumilight solution was dropped onto the blot surface and the X-ray film was exposed to the blot.

Specific Western blot signals within the signal range obtained after transfection with CD5-6Ala7 showed that the cell culture supernatants of all three equivalent transfections contained IL-15 / Fc as protein. It was therefore shown that the pcDNA3.1hCD5.6Ala7 plasmid (FIGS. 1-3) can be used for protein expression in CHO-K1 cells.

c) conclusion

An IL-15 / Fc plasmid, pcDNA3.1hCD5.6Ala7, containing an expression cassette comprising a CD5 leader was prepared with mutated human IL-15 fused to cDNA of human IgG1-Fc under the control of the CMV promoter. To screen stable eukaryotic cell clones, neomycin resistance genes were introduced. The plasmid was sequenced and showed only 100% consensus within the relevant coding region, while having only minor discrepancies (repeats of three base pairs) that had no correlation with the vector backbone. The function of this construct was confirmed by transient transfection of CHO-K1 cells.

Example 2

Transfection of eukaryotic cell lines (eg, CHO-K1 cells) using plasmids containing the DNA of the desired product is a standard method for preparing therapeutic proteins. Nevertheless, the low productivity level of stable cell clones produced by this method is a well known problem. Therefore, there are various strategies for increasing the productivity of existing cell lines. In addition to attempts to increase the number of plasmid copies in cells (eg, via the Methotrexat / DHFR system), the expression construct itself can be further modified. In addition to potent promoters (eg CMV promoters), the introduction of introns will probably result in better RNA stability and better transport of RNA from the nucleus, which is performed by the splice device of the cell. Nevertheless, testing should be performed to ensure that the combination of introns / transgenes is appropriate. To this end, various combinations of introns with human IL-15-Fc have been found to increase IL-15-Fc production by CHO-K1 cells.

a) substance

The plasmid used as starting material was the pcDNA3.1hCD5.6Ala7 plasmid. It is depicted schematically in FIG. 1. Its sequence is disclosed as SEQ ID NO: 1.

The test system used was DHO-K1 cells (manufacturer [DSM], Brunswick, Germany, Accession No. ACC110) or HEK-293 cells (manufacturer [Qbiogene], Grunberg, Germany, Accession No. AE80503, QBI-293A). this. E. coli cells (XL10-Gold, Strategene, Layola, USA) were also used. The cells were cultured under standard culture conditions (5% CO ₂ , 37 ° C., moisture). CHO-K1 cells were passed twice a week at a 1:20 ratio with HEK-293 cells passing at a 1: 6 ratio. The medium used was DMEM-F12 + 10% FKS + 1% PEN / Strep for CHO-K1 cells and DMEM + Glutamax + 10% FKS + 1% PEN / Strep for HEK-293 cells. Optimem medium was used for transfection. All media used the product of Invitrogen (Catalog No. 3313-028; No. 32430-027; No. 51598-018) from Karlsruhe, Germany. The plasmids used were pCl-Neo (Promega) comprising the CMV promoter and chimeric intron, the 5 'splice-donor position of the human beta-globin gene and the 3' splice-receptor position of the IgG heavy chain of the variable region. . pMG (Invivogen) is an extended CMV promoter comprising intron A from CMV. pSwitch (Valentis) is a synthetic intron, IVS8. In addition, the following enzymes and restriction enzymes were used: ApaI, EcoRV, XbaI, NruI, PacI, SmaI. XhoI, T4-DNA ligase, T4-DNA polymerase, alkaline phosphatase from calf intestine. These and other molecular-biological reagents (Lipofectamine 2000) were obtained from the manufacturer Invitrogen. NheI was obtained from the manufacturer Amersham-Pharmacia (Uppsala, Sweden) and the plasmid purification kit was obtained from the manufacturer (Qiagen, Hilden, Germany). "Expand High Fidelity PCR System" (Cat. No. 1 732 641) was obtained from the manufacturer (Roche, Mannheim, Germany).

b) method

i) The IL-15 / Fc insert of the pcDNA3.1hCD5.6Ala7 plasmid was isolated by NheI / ApaI cleavage. The plasmid was first linearized with ApaI restriction cleavage and the 5′-protruded ends were blunt terminated by treatment with T4-polymerase. The IL-15 / Fc insert was then separated by successive NheI cleavage. The fragment was linked with pcI Neo cleaved with NheI and SmaI.

ii) The CMV promoter of pcDNA3.1hCD5.6Ala7 was removed and replaced with an extended CMV promoter with intron A derived from pMG: pMG plasmids were cleaved with PacI and blunt ended by T4-polymerase treatment. . After the second XbaI treatment, the 1.7 kb fragment (including CMV promoter + Intron A) obtained by this method was purified by agarose gel electrophoresis. CMV promoter was removed from pcDNA3.1hCD5.6Ala7 with NheI and subsequent NruI restriction cleavage. The resulting fragment was linked to pMG-promoter overnight at 4 ° C.

iii) IVS8 introns were amplified by PCR and cloned between the 3 'end of the CMV promoter and the 5' end of the IL-15 insert in pcDNA3.1hCD5.6Ala7. The plasmid was linearized with NheI restriction cleavage and then treated with alkaline phosphatase from calf intestine. The intron was amplified by PCR using a primer containing the XbaI restriction cleavage site and using an extended high accuracy PCR system under the following conditions: The reaction mixture used was 2 μl of dNTPs (Qiagen, Taq core). Kit, 2 mmol / l}, 25 pmol primer, 5 μl 10 × buffer, 0.75 μl high accuracy Taq polymerase, 1 μl pSwitch-XhoI / EcoRV fragment, approximately 15 ng, and 50 μl volume of water lost. The PCR program (25 cycles) was as follows: 5 minutes at 95 ° C., 15 seconds at 94 ° C., 30 seconds at 55 ° C., 30 seconds at 72 ° C., 5 minutes at 72 ° C. PCR products were cleaved with XbaI, eluted from 0.8% agarose gel and linked with linearized plasmid.

The obtained plasmid was obtained from E. coli. E. coli XL10 Gold was transformed and the plasmid was analyzed by Miniprep. One clone of each of the plasmids exhibiting suitable restriction cleavage patterns was used for the production of continuous endotoxin free plasmids.

IL-15 / Fc expression was analyzed after transient transfection of HEK-293 or CHO-K1 cells. On the day before transfection, cells were seeded twice at a density of 5 × 10 ⁵ cells per well of cell culture plates with 6 wells. See Felgner et al. (Proc. Natl. Acad. Sci. USA, 84: 7413-7417; 1987) was performed by diluting 2 μg of plasmid and 4 μl of lipofectamine2000 in each case in 250 μl of Optime1 medium. It was. Both solutions were mixed and incubated at room temperature for 30 minutes before the mixture was pipetted into the cell culture medium of the cell culture plate. Two days after transfection, the culture medium was removed and the IL-15 / Fc content was measured using the ELISA test indicated in the Fc portion of IL-15 / Fc.

c) results

IL-15 / Fc secretion by HEK-293 cells transfected with various expression constructs was hardly affected by other vector components. In contrast, IL-15 / Fc expression by CHO-K1 cells was increased by a factor of 200-300 after inserting the intron into the IL-15 / Fc construct. The original construct, pcDNA3.1hCD5.6Ala7, with the intron inserted, resulted in almost undetectable (10 ng / ml or less) protein secretion levels and transfected the cells with pMG10AlaA7 (FIGS. 4-6; SEQ ID NO: 2). This resulted in IL-15 / Fc levels of approximately 300 ng / ml. ELISA data showing IL-15 / Fc-expressing levels in CHO-K1 cells are shown in FIG. 4. After transfection with the pMG construct, the pMG construct was selected to produce a stable CHO-K1-expressing cell line because the expression level was the highest.

For this purpose, the plasmid was first subjected to single chain sequencing. Both chains of this construct were sequenced within the region comprising the IL-15 / Fc cassette, the newly inserted CMV promoter and the intron fragment. The plasmid contained an IL-15 / Fc cassette under the control of the CMV promoter. Intron A from CMV (MG plasmid) was placed between the promoter and the translation start position. This plasmid contained the BGHpolyA site downstream of the IL-15 / Fc fragment; The neomycin resistance gene was controlled by the SV40 promoter and also included the SV40polyA site. The plasmid is E. coli. Ampicillin-resistant genes for selection and amplification in E. coli.

d) discussion and conclusion

To increase protein yield of stable CHO-K1-IL-15 / Fc transfectants, expression plasmids were modified by introducing an intron between the promoter and the IL-15 / Fc cassette. The combination of intron-transgene-host cells strongly influences protein expression, so it is impossible to predict which intron is most effective at increasing IL-15 / Fc expression in the two cell types analyzed.

HEK-293 cells were little affected by the introduction of introns, while a large increase in IL-15 / Fc secretion was observed in CHO-K1 cells. The expression of IL-15 / Fc protein in CHO-K1 cells using plasmids containing intron A from the CMV promoter and pMG increased to a much greater extent compared to the original IL-15 / Fc expression vector. The plasmid can be used to prepare IL-15 / Fc producer cell lines that can be used to prepare IL-15 / Fc for preclinical and clinical research or for other industrial IL-15 / Fc production.

Example 3: Purification of IL-15 / Fc Fusion Proteins

c) purification and concentration

Approximately 3100 liters of supernatant from Example 2 (pMG10Ala7 plasmid) were clarified, concentrated and sterilized-filtered six times. This included clarifying the supernatant with a Profile Star filter (3 μm, 20 inches, Pall Corporation, East Hill, NY). The supernatant was then concentrated 10-15 times using a tangential-flow filtration system using 2.0 m ² total Biomax-30 membrane (Milipore, Billerica, Mass.). The inlet pressure was 2 to 2.5 bar and the outlet pressure was 1.5 bar. After concentration, the concentrates were prefiltered (Polysep II (0.2 μm, 10 inch, manufactured by Milipore, Billerica, Mass.)) And final filter {Durapore (0.22 μm, 10 inch, manufactured by Milipore, USA). Sterile-filtered through a filter system consisting of Billerica, Mass.). Concentration of six different runs took 4.5-8.5 hours.

b) r Protein-A chromatography

Approximately 240 liters of total collected output of the clarification and concentration steps were added to the rProtein-A column (2.6 liters). The flow rate during loading was 10 to 15.4 l / h (65-100 cm / h). The column was then washed with 10 column volumes of Tris 20 mmol / l, 150 mmol / l NaCl at pH 7.5, followed by 10 column volumes of 0.1 mol / l citrate at pH 5.0. IL-15 / Fc was eluted with 5 column volumes of citrate 0.1 mol / l at pH 3.4 and the column was stripped with 5 column volumes of 0.1 mol / l citrate at pH 3.0. IL-15 / Fc eluted with a single sharp peak. The eluate was yellow in color and contained particulate components. The eluate was immediately titrated to pH 3.5 using 1 mol / l citrate. Inactivation of the virus at low pH was carried out with constant stirring for 1 hour at room temperature. The treatment was terminated by addition of 1 mol / l of Tris and the pH adjusted to 8.0. Eluate (8.6 liters) was sterile-filtered using a Millipak-20 filter and stored in 10-liter Scott bottles at 2-8 ° C.

c) Q-Sepharose chromatography

The Q-Sepharose-FF column was packed to a height of 12.7 cm, corresponding to a column volume of 1.9 liters. After equilibration, the HETP (theoretical plate corresponding height) was 0.041 and the asymmetry was 1.1.

r The eluate of Protein-A chromatography was diluted 1: 5 using 50 mmol of Tris 20 mmol / l in a 50-liter Stedim bag. 42.2 kg of diluted r Protein-A eluate was added to a 1.9-liter Q-Sepharose column. The column was washed with 5 column volumes of Tris 20 mmol / l, NaCl 0.1 mmol / l, pH 8.0, followed by 5 column volumes of Tris 20 mmol / l, 0.2 mmol / l NaCl, pH 8.0. IL-15 / Fc was eluted with 5 column volumes of Tris 20 mmol / l, NaCl 0.35 mol / l, pH 8.0. The column was stripped into 5 column volumes of Tris 20 mmol / l, NaCl 1.0 mol / l, pH 8.0. Eluent (5.0 liters) of Q-Sepharose chromatography was sterile-filtered into a 10 liter Scott bottle using a Millipak-20 filter and stored at 2-8 ° C. The eluate was still slightly yellow, although much of the yellow contaminant had been removed.

Optical density measurements at 280 nm showed that IL-15 / Fc eluted at a single sharp peak at the end of the elution.

d) concentration of eluate from Q-Sepharose chromatography by ultrafiltration

The Q-Sepharose chromatographic eluate was concentrated to a final concentration of 8.85 g / l based on OD ₂₈₀ (optical density measured at ₂₈₀ nm). This involved the use of 0.1m ² Biomax 10-kD membranes. The inlet pressure was adjusted to 1.0 bar and the outlet pressure to 0.5 bar. The concentrate (1.9 liters) was sterile-filtered into 0.5 liter PETG bottles in 5 aliquots.

e) Superdex-200 chromatography

The column was charged to the column under a pressure of 3 bar. The column used had an HETP of 0.013 cm and an asymmetry of 3.0.

Five aliquots of the concentrate were purified by five successive runs through a 13.5 liter Superdex-200 column. The chromatogram shows small peaks at high molecular weights and major peaks at monomers IL-15 / Fc. Fractions were collected and the following fractions were obtained.

f) collection of superdex fractions

Fractions from Superdex-200 tablets were used in further preparation methods based on their purity as determined by HP-SEC analysis. Generally 5-11 fractions were collected for this purpose. The amount of IL-15 / Fc was calculated based on the peak area of the HP-SEC assay.

g) virus filtration

The collected Superdex fractions from all five runs were transferred to a Steady bag and collected and diluted to a final IL-15 / Fc concentration of 0.88 g / l using PBS pH 7.4. The diluted, the collection Superdex fraction through 1-m ² Plastic Nova (Planova) -35N and 1-m ² Plastic Nova -15N filter and filtered continuously. The working pressure was 0.5 bar for the Planova-35N filter. After filtration, the filter was washed with 3 liters of PBS (pH 7.4). The flow rate through the virus filter was 12 to 15 liters per hour during loading at 0.5 bar. During the wash, the flow rate was reduced to approximately 10 liters per hour because the wash was performed at 0.4 bar.

h) sterile filtration

16.4 kg of the final product was sterile filtered using a 0.22 μm Millipak filter and placed in 1 liter PETG bottle. The bottles were stored at 2-8 ° C. until further use.

Example 4: Characterization of IL-15 / Fc Fusion Proteins

a) SDS-PAGE / Western-Blot Analysis

Analysis on reduced SDS-PAGE using continuous silver staining showed that the IL-15 / Fc in the Q-eluate, the collected superdex fraction and the final product had similar migration properties as the IL-15 / Fc of the control group. It was. In a few gels, additional bands at low and high molecular weights were observed in the Q-eluate but were also present in the control. Two bands with high molecular weight were observed in several collected Superdex fractions. These bands were then detected in the control as well. Additional bands with high molecular weight are probably due to insufficient reduction of the sample prior to loading the gel, caused by long standing.

Subsequently, no additional bands were detected except for the main IL-15 / Fc band for the IL-15 / Fc final product on non-reduced SDS-PAGE using silver nitrate staining. Also non-reduced western blot using Fc follow-up detection only showed IL-15 / Fc band. This demonstrates that the quality of IL-15 / Fc corresponds at all process steps.

b) N-glycosylation of IL-15 / Fc

In the crude extract, the N-glycosylation properties of IL-15 / Fc were determined during the purification and purification steps of the purification intermediates and final product. The percent area of N-acetylneuraminic acid compared to the average sialylation and the sum of the regions of N-acetyl-neuraminic acid, N-glycolylneuraminic acid and asialo-N-glycans per natural N-glycans is HPAEC -PAD assay {HPLC: BioLC, detector PAD, column: CarboPacPA 100 Analytical (2 x 250 mm), buffer A for natural N-glycan measurement A: 0.1 mol / l NaOH; Buffer B: 0.1 mol / l NaOH, 0.6 mol / l sodium acetate, or buffer A for measuring asialo-N-glycans: 0.2 mol / l NaOH; Buffer B: measured with 0.2 mol / l NaOH, 0.6 mol / l} sodium acetate. The results are summarized in the table below.

When the sample was left for a long time, average sialylation and a decrease in the percent of N-acetylneuraminic acid were observed between clarification and concentration and rprotein-A chromatography. The saccharification of all other purification steps is corresponding.

c) host-cell proteins

Tests for host cell proteins were performed at NewLab AG on two different days using conventional ILA tests. Which respectively lead to the discovery of ³ and 2.1 x10 3.9 x10 ³ ng / ml host cell protein corresponding to 2.9 x10 ³ ppm and 5.3 x 10 ³ ppm. Further testing of host-cell proteins in the final product using the Cygnus test (Paper F015) yielded 240 ng / ml of foreign protein, corresponding to 3.3 × 10 ² ppm. Low values were also measured at short stand times.

d) N-terminal sequence

Terminal IL-15 / Fc sequences were determined in the final product. This test showed the N-terminal sequence of (Asn / Asp) ₁ -Trp ₂ -Val ₃ -Asn ₄ -Val ₅ -Ile ₆ -Ser ₇ -Asp ₈ -Leu ₉ -Lys ₁₀ . The amino acid sequence determined in this test is based on the expected N-terminal IL-15 / Fc sequence except that the N-terminal ASN is approximately 10% (sometimes even higher, but not more than 20%) deamidated. Correspondingly, consider 10% standard deamidation due to the Edman method. Long standing times result in approximately 45% deamination.

e) efficacy

The efficacy of IL-15 / Fc in the final product was determined by bioactivity test. This test detects the bioactivity of the IL-15 antagonist IL-15 / Fc by studying the proliferative-inhibitory action on IL-15-stimulated rat CTLL-2 cells as a dose function. ED ₅₀ was 1.65 ng / well.

f) protein-A residue

r Protein-A washed in the r Protein-A column was measured in the final product using r Protein-A-specific ELISA. This residue was found to be <0.001% here.

g) DNA

The residual DNA content in the final product was measured by Q-One. Less than 5 pg / 0.5 ml of residual DNA was found, corresponding to less than 14 pg of residual DNA per mg of IL-15 / Fc.

h) Geneticin

Geneticin was added during the preculture of the fermentation process. Therefore, it was investigated whether geneticin was sufficiently removed. Geneticin concentration was determined to be less than 30 ng / ml in the IL-15 / Fc final product.

i) characterization

The IL-15 / Fc product obtained by the above method had the following properties:

Product Concentration 0.70mg / ml

Product Purity 99%

pH 7.4

ED ₅₀ <0.1 μg / well

The identity of the product was confirmed by detecting both the IL-15 moiety and the Fc moiety. The bands in the reducing or non-reducing SDS-PAGE also corresponded to the control bands.

Example 5: Preparation of Formulations

a) effect of pH

IL-15 / Fc was diluted to 1 mg / ml with various buffer materials and additives as shown in the table. After their preparation, the buffer was filtered using a 0.2-μm filter (Acrodisc Syringe Filter, Pall). The finished formulations were stored in 10-ml glass containers (4 ml / vessel) at 40 ° C. and 75% relative humidity.

Purity of the IL-15 / Fc formulation was determined by HP-SEC. For this purpose a TSK-GEL G3000SWXL column (300 x 7.8 mm) and a TSK-GELSWXL column (40 x 6 mm) as guard columns were used. They were operated at 25 ° C. and a flow rate of 1 ml per minute. Injection volume was 50 μl. Detection was performed using a detector at 214 nm. At zero, the purity was only 85% because the IL-15 / Fc sample used contained approximately 15% aggregated product. Thus, 85% of the starting material is in active-dimeric form. At pH 4, the peak area of the IL-15 / Fc dimer decreased from approximately 85% to approximately 40% after 7 days. A decrease in purity was also observed at pH 5.5 and pH 6. Formulations at pH 8 and pH 7.4 proved to be stable at 40 ° C. over 7 days. This suggests a decrease in the stability of IL-15 / Fc with decreasing pH.

In addition the formulations were subjected to non-reducing SDS-PAGE at 40 ° C. after 7 days of storage. The pH 4 formulation included two bands representing degradation products of approximately 70 and 50 kD. The pH 6 and pH 5.5 formulations also included two bands representing degradation products, but these bands had apparent molecular weights of approximately 60 and 80 kD. Differences in the apparent molecular weight of degradation products at pH 4, 5.5 and 6 indicate different degradation processes at different pH values.

Formulations at pH 7.4 and 8 showed no additional band below the main band. None of the formulations showed an increase in the band above the main band, indicating that aggregation did not increase in non-reducing SDS-PAGE during storage for 7 days at 40 ° C.

b) pH and additive effects

Various formulations were prepared and stored as described above under condition a). The following pH values and additives were used here:

For each formulation, osmoticity was measured at time t = 0 and pH at 40 ° C. was measured at time t = 4 weeks.

^* PH adjusted prior to the addition of arginine to

In all formulations, the pH of the formulation was as expected except for the formulation containing L-arginine. The L-arginine formulation had a much higher pH than expected pH due to the action of the amino acid L-arginine, which is strongly basic. When analyzing the stability results of the formulation, the extremely high pH of the L-arginine formulation should be taken into account.

All formulations except for those containing glycine at pH 7.4 were isotonic (osmotic from = 0.270 to 0.330 Osmol / kg). The glycine formulation at pH 7.4 was weak shelf life.

Various formulations were subjected to HP-SEC chromatography as described above. Some samples showed a change in peak shape (peak spreading or tailing). Although the shapes of the peaks were different, the total peak area was determined by the peak area of the dimer and used for purity measurement. Thus, the purity of these peaks is probably excessive. Relevant samples are marked with an asterisk in the table.

The results show that none of the formulations in citrate buffer at pH 6 are stable at 40 ° C. At least 20% loss of IL-15 / Fc dimer was observed. None of the additives had protective activity in formulations with citrate buffer at pH 6. Likewise, none of the formulations containing L-arginine (high pH) was stable at 40 ° C. The formulation at pH 7.4 containing sucrose and glycine was stable at 40 ° C. for 3 weeks. After 4 weeks the strong decrease in purity of the formulation with phosphate buffer and mannitol at pH 7.4 is due to microbial contamination. A slight but sustained decrease in purity (from 85-70%) was observed in the formulation with Tris buffer at pH 8. No differences between excipient sucrose, mannitol or glycine were observed in formulations with Tris buffer at pH 8. The same was observed in formulations with citrate buffer at pH 7.4.

^* = Shape of IL-15 / Fc main peak changed

nt = not checked

The purity of the individual formulations was also confirmed by RP-HPLC. For this purpose, a Vydac-214TP54 column (250 × 4.6 mm, 5 μm) was used, which was operated at 25 ° C. and a flow rate of 1 ml per minute. The eluent used was a linear concentration gradient of 100% water containing 0.1% TFA to 100% acetonitrile containing 0.1% TFA. Detection was performed at 214 nm. As in HP-SEC chromatography, the peak shape also changed. Affected samples are again marked with an asterisk ( ^* ) in the table. In general, RP-HPLC results indicate that formulations at pH 6 and formulations containing L-arginine are not stable at 40 ° C. Formulations at pH 7.4 and 8 containing sucrose and glycine and at pH 8 containing mannitol are stable for at least 3 weeks at 40 ° C. The strong decrease in purity of formulations containing mannitol at pH 7.4 may be due to microbial contamination.

^* = Shape of IL-15 / Fc main peak changed

nt = not checked

Samples were further studied by SDS-PAGE (reduced and non-reduced). For this purpose a gel containing 3-8% Tris acetate was used. In each case 0.5 μg of protein in 20 μl was applied to the gel, followed by electrophoresis for 80 minutes at 130 V, 110 mA, 23 W. The band was visualized by silver staining. Bands above the IL-15 / Fc main band showed aggregation and bands below the main band showed degradation. In samples stored at pH 6, degradation occurred after 1 week at 40 ° C., whereas after 2 weeks at pH 8, an increase in aggregation was observed. The two week formulation at pH 7.4 showed degradation, and after three weeks both degradation and aggregation appeared. IL-15 / Fc formulations at pH 7.4 or 8 containing sucrose or glycine, and IL-15 / Fc formulations at pH 8 containing mannitol were stable for 2 weeks at 40 ° C. Degradation in formulations containing mannitol at pH 7.4 was again due to microbial contamination.

Six formulations (corresponding to samples of pH 6, 7.4 and 8, containing sucrose and glycine in each case) were examined for their stability after 4 weeks and compared to T-free buffer without IL-15 / Fc. -Cell proliferation test. To this end, the biological activity of IL-15 / Fc was investigated by studying the proliferation-inhibitory action in IL-15-stimulated rat CTLL-2 cells as a dose function. To this end, CTLL-2 cells were stimulated at a constant concentration of IL-15 up to half and incubated with increasing IL-15 / Fc concentration. T-cell proliferation was detected by colorimetric assay (XTT test by Roche). Maximum half inhibition (ED ₅₀ ) was calculated across the active dose range using linear regression. Each sample was measured twice. The results were as follows.

These results indicate that IL-15 / Fc was biologically active in all formulations showing the highest activity in phosphate buffer at pH 7.4 containing sucrose or glycine. However, slightly lower activity, not significantly different, was observed at pH 8. In contrast, formulations stored at pH 6 resulted in significantly lower IL-15 / Fc bioactivity. The buffer formulation itself does not affect the bioassay.

Some formulations were also examined for their stability by HPAEC-PAD glycosylation assay. In this case, the ratio of the dialialo, trisialo and tetrasialo structures of the various formulations were studied after 4 weeks of storage at 40 ° C.

As the table above shows, all formulations tested corresponded to the relative proportions of di-, tri- and tetrasialo structures. Asialo and monosialo structures were different in various formulations. Formulations with sucrose include multiple peaks at these specific retention times compared to formulations without sucrose and the IL-15 / Fc control. Glycine formulations and IL-15 / Fc controls corresponded to the relative amounts of sialo and monosialo structures. The difference between the sucrose formulation and the glycine formulation is probably due to the presence of sucrose in the formulation, which means that the buffer of the control formulation containing sucrose (without IL-15 / Fc) also shows this extra peak at specific retention times. Because it included. It can be concluded that IL-15 / Fc formulations at pH 6, 7.4 or 8, in the presence of glycine or sucrose, generally do not affect the glycosylation properties of IL-15 / Fc. Even after 4 weeks of storage at 40 ° C., no change in glycosylation behavior was observed.

c) conclusion

Stability studies with IL-15 / Fc suggest that the stability of the formulations is highly pH-dependent in the range of pH 4-8. Lowering the pH within this range reduces stability. Formulations at pH near 10 are less stable than formulations at pH near 8. Best stability was observed at pH 7.4 and pH 8. No specific difference in stability was found in the IL-15 / Fc formulations containing sucrose, mannitol or glycine as an additive.

Example 6: Saccharification Aspects of Various Clones

In further experiments, glycosylation patterns of IL-15 / Fc fusion proteins with different expression levels were studied. Clone K 146 was made using pcDNA3.1 with the CMV promoter without introns, and all other clones were made with pMG10Ala7 with the CMV promoter and Intron A. The expression level in clone K 146 was approximately 3 to 5 pg / (cell * day) and the expression level in clones KN 10 and KN 13 was approximately 9 to 13 pg / (cell ^* day) and finally, clone KN. Expression levels at 110 and KN 120 were approximately 30-50 pg / (cell ^* day) of protein. Glycation data is summarized in the following table.

<110> F. Hoffmann-La Roche AG <120> Purified interleukin-15 / Fc fusion protein and preparation <130> C62388PC <150> EP 04008906.2 <151> 2004-04-14 <160> 5 <170> KopatentIn 1.71 <210> 1 <211> 6458 <212> DNA <213> Artificial sequence <220> <223> Plasmid pcDNA3.1hCD5.6Ala7 <400> 1 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgcg atgtacgggc cagatatacg cgttgacatt 240 gattattgac tagttattaa tagtaatcaa ttacggggtc attagttcat agcccatata 300 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 360 cccgcccatt gacgtcaata atgacgtatg ttcccatagt aacgccaata gggactttcc 420 attgacgtca atgggtggag tatttacggt aaactgccca cttggcagta catcaagtgt 480 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taaatggccc gcctggcatt 540 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 600 tcgctattac catggtgatg cggttttggc agtacatcaa tgggcgtgga tagcggtttg 660 actcacgggg atttccaagt ctccacccca ttgacgtcaa tgggagtttg ttttggcacc 720 aaaatcaacg ggactttcca aaatgtcgta acaactccgc cccattgacg caaatgggcg 780 gtaggcgtgt acggtgggag gtctatataa gcagagctct ctggctaact agagaaccca 840 ctgcttactg gcttatcgaa attaatacga ctcactatag ggagacccaa gctggctagc 900 caccatgccc atggggtctc tgcaaccgct ggccaccttg tacctgctgg ggatgctggt 960 cgcttcctgc ctcggaaact gggtgaatgt aataagtgat ttgaaaaaaa ttgaagatct 1020 tattcaatct atgcatattg atgctacttt atatacggaa agtgatgttc accccagttg 1080 caaagtaaca gcaatgaagt gctttctctt ggagttacaa gttatttcac ttgagtccgg 1140 agatgcaagt attcatgata cagtagaaaa tctgatcatc ctagcaaaca acagtttgtc 1200 ttctaatggg aatgtaacag aatctggatg caaagaatgt gaggaactgg aggaaaaaaa 1260 tattaaagaa tttttggaca gttttgtaca tattgtcgac atgttcatca acacttcgga 1320 tcccaaatct gctgacaaaa ctcacacatg cccaccgtgc ccagcacctg aactcctggg 1380 gggaccgtca gtcttcctct tccccccaaa acccaaggac accctcatga tctcccggac 1440 ccctgaggtc acgtgcgtgg tggtggacgt gagccacgaa gaccctgagg tcaagttcaa 1500 ctggtacgtg gacggcgtgg aggtgcataa tgccaagaca aagccgcggg aggagcagta 1560 caacagcacg taccgtgtgg tcagcgtcct caccgtcctg caccaggact ggctgaatgg 1620 caaggagtac aagtgcaagg tctccaacaa agccctccca gcccccatcg agaaaaccat 1680 ctccaaagcc aaagggcagc cccgagaacc acaggtgtac accctgcccc catcccggga 1740 tgagctgacc aagaaccagg tcagcctgac ctgcctggtc aaaggcttct atcccagcga 1800 catcgccgtg gagtgggaga gcaatgggca gccggagaac aactacaaga ccacgcctcc 1860 cgtgctggac tccgacggct ccttcttcct ctacagcaag ctcaccgtgg acaagagcag 1920 gtggcagcag gggaacgtct tctcatgctc cgtgatgcat gaggctctgc acaaccacta 1980 cacgcagaag agcctctccc tgtctccggg taaatgatct agagggcccg tttaaacccg 2040 ctgatcagcc tcgactgtgc cttctagttg ccagccatct gttgtttgcc cctcccccgt 2100 gccttccttg accctggaag gtgccactcc cactgtcctt tcctaataaa atgaggaaat 2160 tgcatcgcat tgtctgagta ggtgtcattc tattctgggg ggtggggtgg ggcaggacag 2220 caagggggag gattgggaag acaatagcag gcatgctggg gatgcggtgg gctctatggc 2280 ttctgaggcg gaaagaacca gctggggctc tagggggtat ccccacgcgc cctgtagcgg 2340 cgcattaagc gcggcgggtg tggtggttac gcgcagcgtg accgctacac ttgccagcgc 2400 cctagcgccc gctcctttcg ctttcttccc ttcctttctc gccacgttcg ccggctttcc 2460 ccgtcaagct ctaaatcggg ggctcccttt agggttccga tttagtgctt tacggcacct 2520 cgaccccaaa aaacttgatt agggtgatgg ttcacgtagt gggccatcgc cctgatagac 2580 ggtttttcgc cctttgacgt tggagtccac gttctttaat agtggactct tgttccaaac 2640 tggaacaaca ctcaacccta tctcggtcta ttcttttgat ttataaggga ttttgccgat 2700 ttcggcctat tggttaaaaa atgagctgat ttaacaaaaa tttaacgcga attaattctg 2760 tggaatgtgt gtcagttagg gtgtggaaag tccccaggct ccccagcagg cagaagtatg 2820 caaagcatgc atctcaatta gtcagcaacc aggtgtggaa agtccccagg ctccccagca 2880 ggcagaagta tgcaaagcat gcatctcaat tagtcagcaa ccatagtccc gcccctaact 2940 ccgcccatcc cgcccctaac tccgcccagt tccgcccatt ctccgcccca tggctgacta 3000 atttttttta tttatgcaga ggccgaggcc gcctctgcct ctgagctatt ccagaagtag 3060 tgaggaggct tttttggagg cctaggcttt tgcaaaaagc tcccgggagc ttgtatatcc 3120 attttcggat ctgatcaaga gacaggatga ggatcgtttc gcatgattga acaagatgga 3180 ttgcacgcag gttctccggc cgcttgggtg gagaggctat tcggctatga ctgggcacaa 3240 cagacaatcg gctgctctga tgccgccgtg ttccggctgt cagcgcaggg gcgcccggtt 3300 ctttttgtca agaccgacct gtccggtgcc ctgaatgaac tgcaggacga ggcagcgcgg 3360 ctatcgtggc tggccacgac gggcgttcct tgcgcagctg tgctcgacgt tgtcactgaa 3420 gcgggaaggg actggctgct attgggcgaa gtgccggggc aggatctcct gtcatctcac 3480 cttgctcctg ccgagaaagt atccatcatg gctgatgcaa tgcggcggct gcatacgctt 3540 gatccggcta cctgcccatt cgaccaccaa gcgaaacatc gcatcgagcg agcacgtact 3600 cggatggaag ccggtcttgt cgatcaggat gatctggacg aagagcatca ggggctcgcg 3660 ccagccgaac tgttcgccag gctcaaggcg cgcatgcccg acggcgagga tctcgtcgtg 3720 acccatggcg atgcctgctt gccgaatatc atggtggaaa atggccgctt ttctggattc 3780 atcgactgtg gccggctggg tgtggcggac cgctatcagg acatagcgtt ggctacccgt 3840 gatattgctg aagagcttgg cggcgaatgg gctgaccgct tcctcgtgct ttacggtatc 3900 gccgctcccg attcgcagcg catcgccttc tatcgccttc ttgacgagtt cttctgagcg 3960 ggactctggg gttcgaaatg accgaccaag cgacgcccaa cctgccatca cgagatttcg 4020 attccaccgc cgccttctat gaaaggttgg gcttcggaat cgttttccgg gacgccggct 4080 ggatgatcct ccagcgcggg gatctcatgc tggagttctt cgcccacccc aacttgttta 4140 ttgcagctta taatggttac aaataaagca atagcatcac aaatttcaca aataaagcat 4200 ttttttcact gcattctagt tgtggtttgt ccaaactcat caatgtatct tatcatgtct 4260 gtataccgtc gacctctagc tagagcttgg cgtaatcatg gtcatagctg tttcctgtgt 4320 gaaattgtta tccgctcaca attccacaca acatacgagc cggaagcata aagtgtaaag 4380 cctggggtgc ctaatgagtg agctaactca cattaattgc gttgcgctca ctgcccgctt 4440 tccagtcggg aaacctgtcg tgccagctgc attaatgaat cggccaacgc gcggggagag 4500 gcggtttgcg tattgggcgc tcttccgctt cctcgctcac tgactcgctg cgctcggtcg 4560 ttcggctgcg gcgagcggta tcagctcact caaaggcggt aatacggtta tccacagaat 4620 caggggataa cgcaggaaag aacatgtgag caaaaggcca gcaaaaggcc aggaaccgta 4680 aaaaggccgc gttgctggcg tttttccata ggctccgccc ccctgacgag catcacaaaa 4740 atcgacgctc aagtcagagg tggcgaaacc cgacaggact ataaagatac caggcgtttc 4800 cccctggaag ctccctcgtg cgctctcctg ttccgaccct gccgcttacc ggatacctgt 4860 ccgcctttct cccttcggga agcgtggcgc tttctcatag ctcacgctgt aggtatctca 4920 gttcggtgta ggtcgttcgc tccaagctgg gctgtgtgca cgaacccccc gttcagcccg 4980 accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggtaaga cacgacttat 5040 cgccactggc agcagccact ggtaacagga ttagcagagc gaggtatgta ggcggtgcta 5100 cagagttctt gaagtggtgg cctaactacg gctacactag aagaacagta tttggtatct 5160 gcgctctgct gaagccagtt accttcggaa aaagagttgg tagctcttga tccggcaaac 5220 aaaccaccgc tggtagcggt ggtttttttg tttgcaagca gcagattacg cgcagaaaaa 5280 aaggatctca agaagatcct ttgatctttt ctacggggtc tgacgctcag tggaacgaaa 5340 actcacgtta agggattttg gtcatgagat tatcaaaaag gatcttcacc tagatccttt 5400 taaattaaaa atgaagtttt aaatcaatct aaagtatata tgagtaaact tggtctgaca 5460 gttaccaatg cttaatcagt gaggcaccta tctcagcgat ctgtctattt cgttcatcca 5520 tagttgcctg actccccgtc gtgtagataa ctacgatacg ggagggctta ccatctggcc 5580 ccagtgctgc aatgataccg cgagacccac gctcaccggc tccagattta tcagcaataa 5640 accagccagc cggaagggcc gagcgcagaa gtggtcctgc aactttatcc gcctccatcc 5700 agtctattaa ttgttgccgg gaagctagag taagtagttc gccagttaat agtttgcgca 5760 acgttgttgc cattgctaca ggcatcgtgg tgtcacgctc gtcgtttggt atggcttcat 5820 tcagctccgg ttcccaacga tcaaggcgag ttacatgatc ccccatgttg tgcaaaaaag 5880 cggttagctc cttcggtcct ccgatcgttg tcagaagtaa gttggccgca gtgttatcac 5940 tcatggttat ggcagcactg cataattctc ttactgtcat gccatccgta agatgctttt 6000 ctgtgactgg tgagtactca accaagtcat tctgagaata gtgtatgcgg cgaccgagtt 6060 gctcttgccc ggcgtcaata cgggataata ccgcgccaca tagcagaact ttaaaagtgc 6120 tcatcattgg aaaacgttct tcggggcgaa aactctcaag gatcttaccg ctgttgagat 6180 ccagttcgat gtaacccact cgtgcaccca actgatcttc agcatctttt actttcacca 6240 gcgtttctgg gtgagcaaaa acaggaaggc aaaatgccgc aaaaaaggga ataagggcga 6300 cacggaaatg ttgaatactc atactcttcc tttttcaata ttattgaagc atttatcagg 6360 gttattgtct catgagcgga tacatatttg aatgtattta gaaaaataaa caaatagggg 6420 ttccgcgcac atttccccga aaagtgccac ctgacgtc 6458 <210> 2 <211> 7464 <212> DNA <213> Artificial sequence <220> <223> Plasmid pMG10 Ala7 <400> 2 gacggatcgg gagatctccc gatcccctat ggtgcactct cagtacaatc tgctctgatg 60 ccgcatagtt aagccagtat ctgctccctg cttgtgtgtt ggaggtcgct gagtagtgcg 120 cgagcaaaat ttaagctaca acaaggcaag gcttgaccga caattgcatg aagaatctgc 180 ttagggttag gcgttttgcg ctgcttcgta agctgcaata aacaatcatt attttcattg 240 gatctgtgtg ttggtttttt gtgtgggctt gggggagggg gaggccagaa tgactccaag 300 agctacagga aggcaggtca gagaccccac tggacaaaca gtggctggac tctgcaccat 360 aacacacaat caacagggga gtgagctgga tcgagctaga gtccgttaca taacttacgg 420 taaatggccc gcctggctga ccgcccaacg acccccgccc attgacgtca ataatgacgt 480 atgttcccat agtaacgcca atagggactt tccattgacg tcaatgggtg gagtatttac 540 ggtaaactgc ccacttggca gtacatcaag tgtatcatat gccaagtacg ccccctattg 600 acgtcaatga cggtaaatgg cccgcctggc attatgccca gtacatgacc ttatgggact 660 ttcctacttg gcagtacatc tacgtattag tcatcgctat taccatggtg atgcggtttt 720 ggcagtacat caatgggcgt ggatagcggt ttgactcacg gggatttcca agtctccacc 780 ccattgacgt caatgggagt ttgttttggc accaaaatca acgggacttt ccaaaatgtc 840 gtaacaactc cgccccattg acgcaaatgg gcggtaggcg tgtacggtgg gaggtctata 900 taagcagagc tcgtttagtg aaccgtcaga tcgcctggag acgccatcca cgctgttttg 960 acctccatag aagacaccgg gaccgatcca gcctccgcgg ccgggaacgg tgcattggaa 1020 cgcggattcc ccgtgccaag agtgacgtaa gtaccgccta tagagtctat aggcccaccc 1080 ccttggcttc ttatgcatgc tatactgttt ttggcttggg gtctatacac ccccgcttcc 1140 tcatgttata ggtgatggta tagcttagcc tataggtgtg ggttattgac cattattgac 1200 cactccccta ttggtgacga tactttccat tactaatcca taacatggct ctttgccaca 1260 actctcttta ttggctatat gccaatacac tgtccttcag agactgacac ggactctgta 1320 tttttacagg atggggtctc atttattatt tacaaattca catatacaac accaccgtcc 1380 ccagtgcccg cagtttttat taaacataac gtgggatctc cacgcgaatc tcgggtacgt 1440 gttccggaca tgggctcttc tccggtagcg gcggagcttc tacatccgag ccctgctccc 1500 atgcctccag cgactcatgg tcgctcggca gctccttgct cctaacagtg gaggccagac 1560 ttaggcacag cacgatgccc accaccacca gtgtgccgca caaggccgtg gcggtagggt 1620 atgtgtctga aaatgagctc ggggagcggg cttgcaccgc tgacgcattt ggaagactta 1680 aggcagcggc agaagaagat gcaggcagct gagttgttgt gttctgataa gagtcagagg 1740 taactcccgt tgcggtgctg ttaacggtgg agggcagtgt agtctgagca gtactcgttg 1800 ctgccgcgcg cgccaccaga cataatagct gacagactaa cagactgttc ctttccatgg 1860 gtcttttctg cagtcacccg ggggatcctt cgaacgtagc tctagccacc atgcccatgg 1920 ggtctctgca accgctggcc accttgtacc tgctggggat gctggtcgct tcctgcctcg 1980 gaaactgggt gaatgtaata agtgatttga aaaaaattga agatcttatt caatctatgc 2040 atattgatgc tactttatat acggaaagtg atgttcaccc cagttgcaaa gtaacagcaa 2100 tgaagtgctt tctcttggag ttacaagtta tttcacttga gtccggagat gcaagtattc 2160 atgatacagt agaaaatctg atcatcctag caaacaacag tttgtcttct aatgggaatg 2220 taacagaatc tggatgcaaa gaatgtgagg aactggagga aaaaaatatt aaagaatttt 2280 tggacagttt tgtacatatt gtcgacatgt tcatcaacac ttcggatccc aaatctgctg 2340 acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga ccgtcagtct 2400 tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct gaggtcacgt 2460 gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg tacgtggacg 2520 gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac agcacgtacc 2580 gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag gagtacaagt 2640 gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc aaagccaaag 2700 ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag ctgaccaaga 2760 accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc gccgtggagt 2820 gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg ctggactccg 2880 acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg cagcagggga 2940 acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg cagaagagcc 3000 tctccctgtc tccgggtaaa tgatctagag ggcccgttta aacccgctga tcagcctcga 3060 ctgtgccttc tagttgccag ccatctgttg tttgcccctc ccccgtgcct tccttgaccc 3120 tggaaggtgc cactcccact gtcctttcct aataaaatga ggaaattgca tcgcattgtc 3180 tgagtaggtg tcattctatt ctggggggtg gggtggggca ggacagcaag ggggaggatt 3240 gggaagacaa tagcaggcat gctggggatg cggtgggctc tatggcttct gaggcggaaa 3300 gaaccagctg gggctctagg gggtatcccc acgcgccctg tagcggcgca ttaagcgcgg 3360 cgggtgtggt ggttacgcgc agcgtgaccg ctacacttgc cagcgcccta gcgcccgctc 3420 ctttcgcttt cttcccttcc tttctcgcca cgttcgccgg ctttccccgt caagctctaa 3480 atcgggggct ccctttaggg ttccgattta gtgctttacg gcacctcgac cccaaaaaac 3540 ttgattaggg tgatggttca cgtagtgggc catcgccctg atagacggtt tttcgccctt 3600 tgacgttgga gtccacgttc tttaatagtg gactcttgtt ccaaactgga acaacactca 3660 accctatctc ggtctattct tttgatttat aagggatttt gccgatttcg gcctattggt 3720 taaaaaatga gctgatttaa caaaaattta acgcgaatta attctgtgga atgtgtgtca 3780 gttagggtgt ggaaagtccc caggctcccc agcaggcaga agtatgcaaa gcatgcatct 3840 caattagtca gcaaccaggt gtggaaagtc cccaggctcc ccagcaggca gaagtatgca 3900 aagcatgcat ctcaattagt cagcaaccat agtcccgccc ctaactccgc ccatcccgcc 3960 cctaactccg cccagttccg cccattctcc gccccatggc tgactaattt tttttattta 4020 tgcagaggcc gaggccgcct ctgcctctga gctattccag aagtagtgag gaggcttttt 4080 tggaggccta ggcttttgca aaaagctccc gggagcttgt atatccattt tcggatctga 4140 tcaagagaca ggatgaggat cgtttcgcat gattgaacaa gatggattgc acgcaggttc 4200 tccggccgct tgggtggaga ggctattcgg ctatgactgg gcacaacaga caatcggctg 4260 ctctgatgcc gccgtgttcc ggctgtcagc gcaggggcgc ccggttcttt ttgtcaagac 4320 cgacctgtcc ggtgccctga atgaactgca ggacgaggca gcgcggctat cgtggctggc 4380 cacgacgggc gttccttgcg cagctgtgct cgacgttgtc actgaagcgg gaagggactg 4440 gctgctattg ggcgaagtgc cggggcagga tctcctgtca tctcaccttg ctcctgccga 4500 gaaagtatcc atcatggctg atgcaatgcg gcggctgcat acgcttgatc cggctacctg 4560 cccattcgac caccaagcga aacatcgcat cgagcgagca cgtactcgga tggaagccgg 4620 tcttgtcgat caggatgatc tggacgaaga gcatcagggg ctcgcgccag ccgaactgtt 4680 cgccaggctc aaggcgcgca tgcccgacgg cgaggatctc gtcgtgaccc atggcgatgc 4740 ctgcttgccg aatatcatgg tggaaaatgg ccgcttttct ggattcatcg actgtggccg 4800 gctgggtgtg gcggaccgct atcaggacat agcgttggct acccgtgata ttgctgaaga 4860 gcttggcggc gaatgggctg accgcttcct cgtgctttac ggtatcgccg ctcccgattc 4920 gcagcgcatc gccttctatc gccttcttga cgagttcttc tgagcgggac tctggggttc 4980 gaaatgaccg accaagcgac gcccaacctg ccatcacgag atttcgattc caccgccgcc 5040 ttctatgaaa ggttgggctt cggaatcgtt ttccgggacg ccggctggat gatcctccag 5100 cgcggggatc tcatgctgga gttcttcgcc caccccaact tgtttattgc agcttataat 5160 ggttacaaat aaagcaatag catcacaaat ttcacaaata aagcattttt ttcactgcat 5220 tctagttgtg gtttgtccaa actcatcaat gtatcttatc atgtctgtat accgtcgacc 5280 tctagctaga gcttggcgta atcatggtca tagctgtttc ctgtgtgaaa ttgttatccg 5340 ctcacaattc cacacaacat acgagccgga agcataaagt gtaaagcctg gggtgcctaa 5400 tgagtgagct aactcacatt aattgcgttg cgctcactgc ccgctttcca gtcgggaaac 5460 ctgtcgtgcc agctgcatta atgaatcggc caacgcgcgg ggagaggcgg tttgcgtatt 5520 gggcgctctt ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga 5580 gcggtatcag ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca 5640 ggaaagaaca tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg 5700 ctggcgtttt tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt 5760 cagaggtggc gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc 5820 ctcgtgcgct ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct 5880 tcgggaagcg tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc 5940 gttcgctcca agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta 6000 tccggtaact atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca 6060 gccactggta acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag 6120 tggtggccta actacggcta cactagaaga acagtatttg gtatctgcgc tctgctgaag 6180 ccagttacct tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt 6240 agcggtggtt tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa 6300 gatcctttga tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg 6360 attttggtca tgagattatc aaaaaggatc ttcacctaga tccttttaaa ttaaaaatga 6420 agttttaaat caatctaaag tatatatgag taaacttggt ctgacagtta ccaatgctta 6480 atcagtgagg cacctatctc agcgatctgt ctatttcgtt catccatagt tgcctgactc 6540 cccgtcgtgt agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg 6600 ataccgcgag acccacgctc accggctcca gatttatcag caataaacca gccagccgga 6660 agggccgagc gcagaagtgg tcctgcaact ttatccgcct ccatccagtc tattaattgt 6720 tgccgggaag ctagagtaag tagttcgcca gttaatagtt tgcgcaacgt tgttgccatt 6780 gctacaggca tcgtggtgtc acgctcgtcg tttggtatgg cttcattcag ctccggttcc 6840 caacgatcaa ggcgagttac atgatccccc atgttgtgca aaaaagcggt tagctccttc 6900 ggtcctccga tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca 6960 gcactgcata attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag 7020 tactcaacca agtcattctg agaatagtgt atgcggcgac cgagttgctc ttgcccggcg 7080 tcaatacggg ataataccgc gccacatagc agaactttaa aagtgctcat cattggaaaa 7140 cgttcttcgg ggcgaaaact ctcaaggatc ttaccgctgt tgagatccag ttcgatgtaa 7200 cccactcgtg cacccaactg atcttcagca tcttttactt tcaccagcgt ttctgggtga 7260 gcaaaaacag gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga 7320 atactcatac tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg 7380 agcggataca tatttgaatg tatttagaaa aataaacaaa taggggttcc gcgcacattt 7440 ccccgaaaag tgccacctga cgtc 7464 <210> 3 <211> 1113 <212> DNA <213> Artificial sequence <220> <223> DNA for mutated IL-15 / Fc with CD5 leader <400> 3 atgcccatgg ggtctctgca accgctggcc accttgtacc tgctggggat gctggtcgct 60 tcctgcctcg gaaactgggt gaatgtaata agtgatttga aaaaaattga agatcttatt 120 caatctatgc atattgatgc tactttatat acggaaagtg atgttcaccc cagttgcaaa 180 gtaacagcaa tgaagtgctt tctcttggag ttacaagtta tttcacttga gtccggagat 240 gcaagtattc atgatacagt agaaaatctg atcatcctag caaacaacag tttgtcttct 300 aatgggaatg taacagaatc tggatgcaaa gaatgtgagg aactggagga aaaaaatatt 360 aaagaatttt tggacagttt tgtacatatt gtcgacatgt tcatcaacac ttcggatccc 420 aaatctgctg acaaaactca cacatgccca ccgtgcccag cacctgaact cctgggggga 480 ccgtcagtct tcctcttccc cccaaaaccc aaggacaccc tcatgatctc ccggacccct 540 gaggtcacgt gcgtggtggt ggacgtgagc cacgaagacc ctgaggtcaa gttcaactgg 600 tacgtggacg gcgtggaggt gcataatgcc aagacaaagc cgcgggagga gcagtacaac 660 agcacgtacc gtgtggtcag cgtcctcacc gtcctgcacc aggactggct gaatggcaag 720 gagtacaagt gcaaggtctc caacaaagcc ctcccagccc ccatcgagaa aaccatctcc 780 aaagccaaag ggcagccccg agaaccacag gtgtacaccc tgcccccatc ccgggatgag 840 ctgaccaaga accaggtcag cctgacctgc ctggtcaaag gcttctatcc cagcgacatc 900 gccgtggagt gggagagcaa tgggcagccg gagaacaact acaagaccac gcctcccgtg 960 ctggactccg acggctcctt cttcctctac agcaagctca ccgtggacaa gagcaggtgg 1020 cagcagggga acgtcttctc atgctccgtg atgcatgagg ctctgcacaa ccactacacg 1080 cagaagagcc tctccctgtc tccgggtaaa tga 1113 <210> 4 <211> 370 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of human CRB-15 with CD5 leader <400> 4 Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly 1 5 10 15 Met Leu Val Ala Ser Cys Leu Gly Asn Trp Val Asn Val Ile Ser Asp             20 25 30 Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr         35 40 45 Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met     50 55 60 Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp 65 70 75 80 Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn                 85 90 95 Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys             100 105 110 Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Asp Ser Phe Val         115 120 125 His Ile Val Asp Met Phe Ile Asn Thr Ser Asp Pro Lys Ser Ala Asp     130 135 140 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 145 150 155 160 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile                 165 170 175 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu             180 185 190 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His         195 200 205 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg     210 215 220 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 225 230 235 240 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu                 245 250 255 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr             260 265 270 Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu         275 280 285 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp     290 295 300 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 305 310 315 320 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp                 325 330 335 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His             340 345 350 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro         355 360 365 Gly lys     370 <210> 5 <211> 371 <212> PRT <213> Artificial sequence <220> <223> Amino acid sequence of murine IL-15 / Fc (human mutated IL-15,        murine IgG2A) with CD5 leader <400> 5 Met Pro Met Gly Ser Leu Gln Pro Leu Ala Thr Leu Tyr Leu Leu Gly 1 5 10 15 Met Leu Val Ala Ser Cys Leu Gly Asn Trp Val Asn Val Ile Ser Asp             20 25 30 Leu Lys Lys Ile Glu Asp Leu Ile Gln Ser Met His Ile Asp Ala Thr         35 40 45 Leu Tyr Thr Glu Ser Asp Val His Pro Ser Cys Lys Val Thr Ala Met     50 55 60 Lys Cys Phe Leu Leu Glu Leu Gln Val Ile Ser Leu Glu Ser Gly Asp 65 70 75 80 Ala Ser Ile His Asp Thr Val Glu Asn Leu Ile Ile Leu Ala Asn Asn                 85 90 95 Ser Leu Ser Ser Asn Gly Asn Val Thr Glu Ser Gly Cys Lys Glu Cys             100 105 110 Glu Glu Leu Glu Glu Lys Asn Ile Lys Glu Phe Leu Asp Ser Phe Val         115 120 125 His Ile Val Asp Met Phe Ile Asn Thr Ser Asp Pro Arg Gly Pro Thr     130 135 140 Ile Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly 145 150 155 160 Gly Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met                 165 170 175 Ile Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu             180 185 190 Asp Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val         195 200 205 His Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu     210 215 220 Arg Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly 225 230 235 240 Lys Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile                 245 250 255 Glu Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val             260 265 270 Tyr Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr         275 280 285 Leu Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu     290 295 300 Trp Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro 305 310 315 320 Val Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val                 325 330 335 Glu Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val             340 345 350 His Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr         355 360 365 Pro Gly Lys  

Claims (17)

a) adding the liquid composition to an affinity chromatography column, eluting the first IL-15 / Fc eluate from the column, and b) adding the eluate of step a) to an ion exchange chromatography column and eluting a second IL-15 / Fc eluate from the column A method of purifying the IL-15 / Fc fusion protein from a liquid composition comprising a. The method of claim 1 wherein the ion exchange chromatography column is an anion exchange chromatography column or a cation exchange chromatography column. The process of claim 1 or 2, further comprising adding the eluate of step b) to a gel filtration column or a hydrophobic interaction chromatography column and eluting a third IL-15 / Fc eluate from the column. How to include. The method of claim 1 or 2, wherein the composition is filtered and / or concentrated before or after any one or more of steps a) and b). The method of claim 3, wherein the composition is filtered and / or concentrated before or after any one or more of steps a), b) and c). The process according to any one of claims 1 to 5, wherein the pH of the eluate of step a) is acidified before step b). The method according to claim 1, wherein the affinity chromatography column is a protein-A chromatography column or a protein-G chromatography column, preferably a protein-A chromatography column. The process according to claim 2, wherein the anion exchange chromatography column is a DEA-Sepharose column or a Q-Sepharose column, preferably a Q-Sepharose column. 9. The gel filtration column according to claim 3, wherein the gel filtration column is a Sephadex, Sepharose, Biogel-A, Sephacryl or Biogel-P column, preferably Method that is a Superdex-200 column. The method according to claim 1, wherein after purification, the IL-15 / Fc fusion protein has at least 90% purity, preferably at least 95% purity, most preferably at least 99% purity. Purified IL-15 / Fc fusion protein having at least 90% purity, preferably at least 95% purity, most preferably at least 99% purity. 12. The purified IL-15 / Fc of claim 11, wherein the average sialylation per N-glycan is at least 70%, more preferably at least 80%, even more preferably at least 90% and most preferably at least 95%. Fusion protein. The purified IL-15 / according to claim 11 or 12, wherein the average sialylation per antenna is at least 50%, more preferably at least 70%, even more preferably at least 80% and most preferably at least 90%. Fc fusion protein. The N-glycolylneuraminic acid portion according to any one of claims 11 to 13, wherein the N-glycolylneuraminic acid portion of the sum of N-acetylneuraminic acid, N-glycolylneuraminic acid and asialo-N-glycan is 20% or less Purified IL-15 / Fc fusion protein, preferably at most 15%, more preferably at most 10%. A composition comprising the purified IL-15 / Fc fusion protein according to any one of claims 11 to 14 and also excipients and additives. The composition of claim 15 having a pH of 7.4 to 8.0. The composition of claim 15 or 16 which is a pharmaceutical composition.

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