Production of immune interferon and its mRNA

Claim 2

A process for the production of IFN-γ or its mRNA comprising incubating a viable cell suspension for a period of time at least sufficient for the production of IFN-δ, characterised in that said viable cell suspension includes (a) leukocytes, (b) an inducer for production of IFN-δ, and (c) a. modulator for said inducer selected from mezerein and 12,13-phorbol butyrate, said inducer and. said modulator being present in said suspension in amounts sufficient to stimulate production of IFN-δ

Claim 3

The process of Claim 1, wherein said inducer is a calcium ionophore selected from A-23187, 2,2-dimethyl-6,6,7,7,8,8,8-heptafluoro-3,5-octanedione (FOD), and ionomycin.

Claim 4

The process according to Claim 1 or 2, wherein said leukocytes comprise human peripheral blood leukocytes.

Claim 5

The process according to Claim 3, wherein, prior to said incubating step, said human leukocytes are suspended in a culture medium.

Claim 6

A process according to Claim 4, wherein said human leukocytes are suspended in a culture medium in a proportion of from 1.0 x 10 6 to 9.0 x 10 6 cells/ml. of cell suspension.

Claim 7

The process according to Claim 4, wherein said leukocytes are contained in buffy coats.

Claim 8

The process according to Claim 6, wherein, prior to said suspending step, erythrocytes are separated from said buffy coats.

Claim 9

A process according to Claim 4, wherein said modulator is mezerein, and is present in a minimum proportion of 0.7 ng./ml. of cell suspension.

Claim 10

A process according to Claim 8, wherein the mezerein is present in a proportion of 7-30 ng./ml. of cell suspension.

Claim 11

A process according to Claim 4, wherein said modulator is 12,.13-phorbol dibutyrate, and is present in a minimum proportion of 0.7 ng./ml. of cell suspension.

Claim 12

A process according to Claim 10, wherein the 12,13-phorbol dibutyrate is present in a proportion of 7-30 ng./ml. of cell suspension.

Claim 13

The process according to Claim 4, wherein said inducer is calcium ionophore A-23187, and is present in a proportion of 0.10-10.0 pg./ml. of cell suspension.

Claim 14

The process according to Claim 12, wherein the calcium ionophcre A-23!87 is present in a proportion of 0.10-2.0 µg./ml. of cell suspension.

Claim 15

The process according to Claim 4, wherein said inducer is calcium ionophere FOD and is present in a proportion of 0.10-300 µg.(ml. of cell suspension.

Claim 16

The process according to Claim 4, wherein said incubation period is at least 12 hours.

Claim 17

The process according to Claim 15, wherein said incubation period is 3-4 days.

Claim 18

The process according to Claim 4, wherein said cell suspension is incubated under relatively stationary conditions.

Claim 19

The process according to Claim 17, wherein said inducer is calcium ionophore A-23187 and is present in a proportion of 0.15-0.8 µg./ml. of cell suspension.

Claim 20

The process according to Claim 18, wherein the calcium ionophore A-23187 is present in a proportion of 0.25-0.6 µg./ml. of cell suspension.

Claim 21

The process according to Claim 4, wherein said cell suspension is agitated during incubation.

Claim 22

The process according to Claim 4, wherein subsequent to said incubation, the supernatant from said cell suspension, which contains IFN-δ, is separated from the cells.

Claim 23

The process according to Claim 21, including the step of purifying said IFN-δ

Claim 24

A process according to Claim 22, which comprises: (a) adsorbing said IFN-δ onto a column containing Controlled Pore Gas beads and eluting with ammonium sulfate; (b) adsorbing said IFN-δ containing eluate of step (a) onto a column containing Concanavalin A-Sepharose, lentil lectin-Sepharose or pea lectin-agarose and eluting with a buffer containing a. sugar; (c) adsorbing said IFN-δ containing eluate of step (b) onto a column containing Heparin-Sepharose or Procian Red-agarose and eluting with a buffer containing a high salt content; and (d) passing the IFN-δ containing eluate of step (c) through a gel-filtration column equilibrates in high salt and recovering purified IFN-δ.

Claim 25

A process according to Claim 22, which comprises: (a) (i) contacting said IFN-δ with Controll Pore Glass beads for a period of time sufficient to absorb said IFN-δ onto said beads in the presence of a neutral pH buffer; (ii) washing said beads with a chemically compatible buffer until the optical density of the eluant at 280 nm is O; (iii) washing said beads with a neutre. pH buffer solution until the optical density of e eluate at 280 nm is O; and (iv) eluting IFN-δ from said beads with an ammonium sulfate solution; (b) (i) contacting said eluate of step (a)(iv) with an adsorbent selected from the group consisting of Concanavalin A-Sepharose, lentil lectin-Sepharose and pea lectin-agarose for a period of time sufficient to adsorb said IFN-δ onto said adsorbent in the presence of a neutral pH buffer; (ii) washing said adsorbent with ammonium sulfate until the optical density of the eluant at 280 nm is 0; (iii) washing said adsorbent with a neutral pH buffer until the optical density of the eluate at 280 nm is 0; and (iv) eluting IFN-δ from said adsorbent with a buffer containing a sugar of the group consisting of alpha-methyl D-mannoside and 1-methyl D-glucoside; (c) (i) contacting said eluate of step (b)(iv) with an adsorbent selected from the group consisting of Heparin-Sepharose and Procian Red-agarose for a period of time sufficient to adsorb said IFN-δ onto said adsorbent in the presence of a neutral pH buffer; (ii) washing said adsorbent with a neutral pH buffer containing a sugar selected from the group consisting of alpha-methyl D-mannoside and I-methyl D-glucoside until the optical density of the eluate at 280 nm is O; (iii) washing said adsorbent with a neutral pH buffer until the optical density of the eluate is Q; and, (iv) eluting IFN-δ from said adsorbent with a neutral pH buffer containing a high concentra- tion of salt; and (d) (i) passing said eluate of step (c)(iv) through a gel-filtration column equilibrated in a neutral pH buffer containing a high concentration of salt; and, (ii) eluting IFN- δfrom said column with a neutral pH buffer.

Claim 26

The process of Claim 24, wherein homogeneous IFN-γ is recovered from the eluate of step (d)(ii).

Claim 27

The process according to Claim 24, wherein step (b) is carried out prior to step (a).

Claim 28

The process according to Claim 24, wherein the sequence of steps is from step (c) to step (b) to step (a) to step (d).

Claim 29

The process according to Claim 24, wherein. said IFN-γ in said supernatant is produced from ceil suspensions containing unlysed red blood cells, and, prior to beginning said purification steps, said supernatant has added thereto sufficient tris-(hydroxymethyl)-aminomethane to bring the concentration to SCO mM, and is buffered to pH 9.5 to 9.7.

Claim 30

The process according to Claim 24, wherein the final elution IFN-γ is further contacted with phenyl-Sepharose for a period of time sufficient to accomplish equilibrated adsorption of said IFN-γ to the phenyl-Sepharose, and the adsorbed IFN-γ is eluded with low salt buffers.

Claim 31

The process according to Claim 22, which comprises: (a) contacting said IFN-γ with a resin selected from Heparin-Sepharose and Procian-Red agarose for a peri.od of time sufficient to accomplish equilibrated adsorption of said IFN-γ on to the resin; (b) washing said resin with phosphate buffered saline solution containing a sugar selected from 0.1M to 2M alpha-methyl D-mannoside and 1-methyl D-glucoside until the optical density at 280 nm is 0; (c) washing with phosphate buffered saline solution until the optical density at 280 nm is 0; (d) eluting the adsorbed IFN-γ with phosphate buffered saline containing 2M sodium chloride; and (e) recovering substantially pure IFN-γ .

Claim 32

A process according to Claim 22, which comprises: (a) contacting said IFN-γ with Controlled Pore Glass beads for a period of time sufficient to adsorb said IFN-γ on to said beads in the presence of a neutral pH buffer; (b) washing said beads with a chemically compatible buffer until the optical density of the eluant at 280 nm is O; (c) washing said beads with a neutral pH buffered solution until the optical density of the eluant at 280 nm is O; (d) eluting IFN-γ from said beads with an ammonium sulfate solution; and (e) recovering substantially pure IFN-γ.

Claim 33

The process according to Claim 21, wherein IFN-γ mRNA is extracted from said separated cells.

Claim 34

The process according to Claim 32, wherein said IFN-γ mRNA is extracted by the steps, comprising: (a) Lysing the cells and removing large contaminants; (b) precipitating remaining RNA in alcohol and then resuspending in a salt buffer; (c) passing the RNA suspension through a poly-(U) column; (d) eluting the mRNA from said column; and (e) size fractionating the eluted mRNA.

Claim 35

The process according to Claim 33, wherein said IFN-γ mRNA is extracted by the steps comprising: (a) lysing said cells in a solution comprising sodium acetate and SDS, or guanidine thiocyanate; (b) removing large strands of DNA and protein by phenol extraction or centrifuging with cesium chloride in a cellulose nitrate tube; (c) precipitating the remaining mRNA in ethanol and resuspending in a salt buffer; (d) passing the mRNA suspension through a poly-(U) Sepharose-4B column and washing with a buffer; (e) eluting the mRNA from said colum with fcrmamide; and (f) size fractionating the eluted mRNA in sucrose gradient.

Claim 36

The process according to Claim 34, wherein samples of the fractions of extracted IFN-γ mRNA are inserted into cells capable of expressing IFN-γ, said cells are allowed to express said IFN-γ , and quantitative expression of IFN-γ by said cells is measured.

Claim 37

A process for producing clones that express IFN-γ comprising: (a) adding reverse transcriptase to IFN- γmRNA from high IFN-γ activity fractions obtained in accordance with Claim 35, to transcribe the single-stranded complementary IFN-γ DNA; (b) adding DNA polymerase or reverse transcriptase to said single-strand DNA to produce double-stranded IFN-γ DNA; (c) making the IFN-f ds-DNA blunt-ended by incubation with nuclease; (d) selected a cloning vehicle carrying a phenotypical trait, said vehicle being compatible with a host cell; (e) cleaving said cloning veh'icle in the presence of restrictive endonuclease; (f) combining said cleaved vehicle with said IFN-γ double-stranded DNA in the presence of DNA ligase to form a recombinant vehicle; (g) adding complementary terminal codons to the blunt-ended IFN-γds-DNA and the cleaved cloning vehicle in the presence of terminal transferase; (h) transforming the host cell with the recombinant vehicle in the presence of calcium chloride; and (i) growing said host cells and then isolating the transformant containig the recombinant vehicles by means of the phenotypical trait.

Claim 38

The process for expressing IFN-γ comprising: (a) selecting the transformants of step (i) of claim 36 which demonstrate strong hybridization to mRNA from IFN-γ induced cells; and (b) removing IFN-γds-DNA from said transformants and inserting it in an optimal expression cloning vehicle.

Claim 39

Homogeneous IFN-γ.

Claim 40

Homogeneous IFN-γ.

Claim 41

Homogeneous human IFN-γ.

Claim 42

Homogeneous human IFN-γ.

Claim 43

The IFN-γ 1 species of said homogeneous human IFN-γof Claim 39 corresponding to an SDS-PAGL molecular weight band of 25,000 ± 3,000 daltons.

Claim 44

The dimer of the IFN-γ 1 species of said IFN- γof Claim 39 corresponding to an SDS-PAGE molecular weight band of 47,C00 ± 3,000 daltons.

Claim 45

The trimer of the IFN-γ 1 species of said IFN- γof Claim 39 corresponding to an SDS-PAGE molecular weight band of 63,000 ± 3,000 daltons.

Claim 46

The IFN-γ2 species of said homogeneous human IFN-γ of Claim 39 corresponding to an SDS-PACE molecular weight band of 20,000 3,000 daltons.

Claim 47

The recombinant cloning vehicle containing DNA segment coding for human IFN-γ.

Claim 48

The recombinant cloning vehicle containing DNA segment coding for human IFN-γ.

Claim 49

The host cell containing the recombinant cloning vehicle of Claim 44.