Mitogens of leukocytes and inflamed tissues

Claim 2

Leukocyte derived mitogens characterized by the following properties: (a) biological activities in vivo and in vitro: selective induction of division and differentiation (mitosis) of bone marrow or tissue leukocytes; they are substantially free of other biological effects. (b) physico-chemical properties: electrophoretic migration in acrylamide matrices at a pH of 7.40 is anodic; soluble in aqueous media including in 10% ethanol at a pH value of 4.0 to 10; they adsorb reversibly in structure and biological activity on anion and cation exchangers, calcium phosphate gel and hydroxyapatite and can be subjected in native form to volume partition chromatography.

Claim 3

Mitogens according to claim 1 characterized in that they induce division and differentiation of bone marrow leukocytes.

Claim 4

Mitogen (monocyto-blastogen) according to claim 2 characterized in that it is obtainable from mononuclear leukocytes and possesses the following additional properties: (a) biological effects: specific stimulation of division and differentiation of bone marrow leukocytes; effective threshold dose in vitro <50 pmol/l (b) physico-chemical properties: molecular weight of the native protein (primary structure); approximately 25,000 dalton; insoluble in an ammonium sulfate solution at 90% saturation (3.6 mol/l); absorption spectrum (UV, visible and near IR-range) as given in FIG. 1; extinction coefficients according to the following Table I: TABLE I ______________________________________ wave length, nm E.sub.1 mg/ml, 1 cm (H.sbsb.2.sub.O, 20° C.) ± ______________________________________ 6% 248 (min) 0.23 260 0.36 276 (max) 0.56 280 0.55 290 0.38 400-1000 0 E.sub.280 /E.sub.260 1.53 ______________________________________

Claim 5

Mitogen (granulocyto-blastogen) according to claim 2, characterized in that it is obtainable from granulocytes and possesses the following additional properties: (a) biological activities in vivo: specific stimulation of division and differentiation of bone marrow leucocytes; effective threshold dose in vitro: <5 nmol/l (b) physico-chemical properties: molecular weight of the native protein (primary structure): approximately 85,000 dalton; insoluble in an ammonium sulfate solution at 90% saturation (3.6 mol/l); absorption spectrum (UV, visible and near IR-range) according to FIG. 2; extinction coefficient according to the following Table II TABLE II ______________________________________ wave length, nm E.sub.1 mg/ml, 1 cm (H.sbsb.2.sub.O, 20° C.) + ______________________________________ 6% 251 (min) 0.22 260 0.30 280 0.55 281 (max) 0.56 290 0.41 400-1000 0 E.sub.280 /E.sub.260 1.83 ______________________________________

Claim 6

Mitogens according to claims 1 or 2, characterized in that they induce the mitosis of the leukocytes of the tissue.

Claim 7

Mitogen (monocyto-histiomitogen) according to claim 5, characterized in that it is obtainable from mononuclear leukocytes and possesses the following additional properties: (a) biological activities: specific stimulation of mitosis of peritoneal macrophages; effective threshold dose in vitro <1 nmol/l (b) physico-chemical properties: molecular weight of the native protein (primary structure): approximately 13,000 dalton; insoluble in a 90% saturated ammonium sulfate solution (3.6 mol/l); absorption spectrum (UV, visible and near IR-range) as given in FIG. 3; extinction coefficient according to the following table III: TABLE III ______________________________________ wave length, nm E .sub.1 mg/ml, 1 cm (H.sub.2.sub.O, 20° C.) + ______________________________________ 6% 252 (min) 0.20 260 0.27 279 (max) 0.56 280 0.56 290 0.46 400-1000 0 E.sub.280 /E.sub.260 2.07 ______________________________________

Claim 8

Mitogen (lymphocyto-lymphomitogen) according to claim 5, characterized in that it is obtainable from lymphocytes and possesses the following additional properties: (a) biological activities: specific stimulation of mitosis of peripheral lymphocytes; effective threshold dose in vitro: <0,5 nmol/l (b) physico-chemical properties: molecular weight of the native protein (primary structure): approximately 17,000 dalton; insoluble in a 90% saturated ammonium sulfate solution (3.6 mol/l); absorption spectrum (UV, visible and near IR-range) as given in FIG. 4; extinction coefficient according to the following table IV TABLE IV ______________________________________ wave length, nm E.sub.1 mg/ml, 1 cm (H.sbsb.2.sub.O, 20° C.) + ______________________________________ 6% 252 (min) 0.32 260 0.41 280 0.78 282 (max) 0.79 290 0.61 400-1000 0 E.sub.280 /E.sub.260 1.90 ______________________________________

Claim 9

A pharmaceutical composition for specifically influencing the immune system, leucopoiesis and inflammatory processes of mammalians, comprising an effective amount of at least one leukocyte derived mitogen according to claim 1 with a suitable carrier therefor.

Claim 10

A process for specifically influencing the immune system, the leucopoiesis and inflammatory processes of mammalians comprising administering an effective amount of at least one leukocyte derived division and differentiation inducing mitogen according to claim 1.

Claim 11

A process for producing leukocyte derived mitogens according to claim 1, comprising: (a) culturing a population of leukocytes in a suitable culture medium; (b) terminating the culturing; (c) separating said leukocytes from said medium to yield a culture solution; (d) adding a sufficient amount of a suitable salt to precipitate a first protein fraction from solution; (e) separating said first protein portion from solution; (f) adding a further amount of the salt to the solution to precipitate a second protein fraction therefrom; (g) separately purifying said first and second protein fractions by molecular sieve filtration, anion and cation exchange chromatography, chromatography on hydroxyapatite, zone precipitation chromatography, and recycling molecular sieve filtration to obtain said mitogens.

Claim 12

The process according to claim 10, wherein a mixed leukocyte population is cultured.

Claim 13

The process according to claim 10, wherein a specific leukocyte type is cultured.

Claim 14

The process according to claim 10, wherein the leukocytes are cultured in a fully synthetic cell culture medium containing serum albumin as the only protein.

Claim 15

The process according to claim 10, wherein leukocyte mitosis is induced during the culturing.

Claim 16

The process according to claim 14, wherein a polyvalent mitogen or endotoxin-mitogen is added or an immune reaction is promoted on the cell surface to induce leukocyte mitosis.

Claim 17

The process according to claim 14, wherein leukocyte mitosis is induced by the addition of a lectin.

Claim 18

The process according to claim 15, wherein a lectin from Canavalia ensiformis (Concanavalin A=CON) is used.

Claim 19

The process according to claim 13, wherein the cell culture medium has the composition given in Table V.

Claim 20

The process according to claim 18, wherein the leukocytes are cultured for approximately 40 hours at about 37° C. and a concentration of about 10 7 to 10 8 cells/ml of culture solution at a CO 2 -partial pressure of about 1%, while sufficient oxygen is supplied to the culture.

Claim 21

The process according to claim 19, wherein ammonium sulfate is used for precipitating the proteins.

Claim 22

The process according to claim 20, wherein the ammonium sulfate concentration of the culture solution is stepwise increased and the proteins precipitated after each ammonium sulfate addition are separated, thereby yielding several crude protein fractions having differing solubilities at different ammonium sulfate concentrations.

Claim 23

The process according to claim 21, wherein the ammonium sulfate concentration of the culture solution is adjusted stepwise to 35%, 45% and 90% saturation.

Claim 24

The process according to claim 19, wherein the supernatant liquid after separation of the protein precipitate is concentrated by ultrafiltration or dialysis.

Claim 25

The process according to claim 10, wherein at least two of said purification steps are performed in sequence.

Claim 26

The process according to claim 24, wherein at least three of said purification steps are performed in sequence.

Claim 27

A process for producing highly purified monocytoblastogen which comprises: (a) culturing a leukocyte population including mixed leukocytes or only monocytes in a suitable culture medium; (b) terminating culturing; (c) separating cells from the culture medium after termination of culturing to yield a culture solution; (d) adding ammonium sulfate to the culture solution to achieve up to a 90% saturation in order to precipitate proteins contained therein; (d) separating the precipitated proteins from the ammonium sulfate-containing supernatant; (f) redissolving said precipitated proteins; (g) purifying said proteins by anion exchange chromatography, preparative molecular seive filtration, cation exchange chromatography, chromatography on hydroxyapatite, zone precipitation chromatography and recycling molecular sieve filtration for removing the accompanying foreign proteins; and (h) isolating the substantially pure monocytoblastogen from the eluate of the recycling molecular sieve filtration by adding ammonium sulfate up to 80-90% saturation and separating the precipitate formed from the supernatant.

Claim 28

A process for producing highly purified monocytoblastogen which comprises: (a) culturing a leukocyte population including mixed leukocytes or only monocytes in a suitable culture medium; (b) terminating culturing; (c) separating cells from the culture medium after termination of culturing to yield a culture solution; (d) adding ammonium sulfate to the culture solution to achieve up to a 90% saturation in order to precipitate proteins contained therein; (d) separating the precipitated proteins from the ammonium sulfate-containing supernatant; (f) redissolving said precipitated proteins; (g) purifying said proteins by anion exchange chromatography, preparative molecular seive filtration, cation exchange chromatography, chromatography on hydroxyapatite, zone precipitation chromatography and recycling molecular sieve filtration for removing the accompanying foreign proteins; and (h) isolating the substantially pure monocytoblastogen from the eluate of the recycling molecular sieve filtration by adding ammonium sulfate up to 80-90% saturation and separating the precipitate formed from the supernatant.

Claim 29

A process for producing highly purified granulocytoblastogen which comprises: (a) culturing a leukocyte population containing mixed leukocytes or only granulocytes in a suitable culture medium; (b) including the mitosis of the cells by CON during culturing; (c) terminating culturing; (d) separating cells from the culture medium to yield a culture solution; (e) adding sufficient ammonium sulfate to the culture solution to provide an ammonium sulfate concentration of up to a 90% saturation in order to precipitate proteins contained therein; (f) separating the precipitated proteins from the ammonium sulfate-containing supernatant; (g) redissolving said precipitated proteins; (h) purifying said proteins by anion exchange chromatography preparative molecular sieve filtration, cation exchange chromatography, chromatography on hydroxyapatite, zone precipitation chromatography, and recycling molecular sieve filtration; and (i) isolating the highly purified granulocytoblastogen from the eluate of the recycling molecular sieve filtration by adding ammonium sulfate up to 80-90% saturation and separating the precipitation formed from the supernatant.

Claim 30

A process for producing highly purified granulocytoblastogen which comprises: (a) culturing a leukocyte population containing mixed leukocytes or only granulocytes in a suitable culture medium; (b) including the mitosis of the cells by CON during culturing; (c) terminating culturing; (d) separating cells from the culture medium to yield a culture solution; (e) adding sufficient ammonium sulfate to the culture solution to provide an ammonium sulfate concentration of up to a 90% saturation in order to precipitate proteins contained therein; (f) separating the precipitated proteins from the ammonium sulfate-containing supernatant; (g) redissolving said precipitated proteins; (h) purifying said proteins by anion exchange chromatography preparative molecular sieve filtration, cation exchange chromatography, chromatography on hydroxyapatite, zone precipitation chromatography, and recycling molecular sieve filtration; and (i) isolating the highly purified granulocytoblastogen from the eluate of the recycling molecular sieve filtration by adding ammonium sulfate up to 80-90% saturation and separating the precipitation formed from the supernatant.

Claim 31

A process for producing highly purified monocytohistiomitogen, which comprises: (a) culturing a leukocyte population containing mixed leukocytes or only monocytes in a suitable culture medium; (b) including the mitosis of the cells by CON during culturing; (c) terminating culturing; (d) separating cells from the culture medium to yield a culture solution; (e) adding ammonium sulfate to the culture solution to achieve up to a 90% saturation in order to precipitate proteins contained therein; (f) separating the precipitated proteins from the ammonium sulfate containing supernatant; (g) redissolving said precipitated proteins; (h) purifying said proteins by anion exchange chromatography, preparative molecular sieve filtration, cation exchange chromatography, chromatography on hydroxyapatite, zone precipitation chromatography and recycling molecular sieve filtration for removing accompanying foreign proteins; and (i) isolating the highly purified monocytohistiomitogen from the eluate of the recycling molecular sieve filtration by adding up to 80-90% saturation and separating the precipitate formed from the supernatant.

Claim 32

A process for producing highly purified monocytohistiomitogen, which comprises: (a) culturing a leukocyte population containing mixed leukocytes or only monocytes in a suitable culture medium; (b) including the mitosis of the cells by CON during culturing; (c) terminating culturing; (d) separating cells from the culture medium to yield a culture solution; (e) adding ammonium sulfate to the culture solution to achieve up to a 90% saturation in order to precipitate proteins contained therein; (f) separating the precipitated proteins from the ammonium sulfate containing supernatant; (g) redissolving said precipitated proteins; (h) purifying said proteins by anion exchange chromatography, preparative molecular sieve filtration, cation exchange chromatography, chromatography on hydroxyapatite, zone precipitation chromatography and recycling molecular sieve filtration for removing accompanying foreign proteins; and (i) isolating the highly purified monocytohistiomitogen from the eluate of the recycling molecular sieve filtration by adding up to 80-90% saturation and separating the precipitate formed from the supernatant.

Claim 33

A process for producing highly purified lymphocyto lumphomitogen, which comprises: (a) culturing a leukocyte population containing mixed leukocytes or only lymphotyces in a suitable culture medium; (b) inducing the mitosis of the cells by CON during culturing; (c) terminating culturing; (d) separating cells from the culture medium to yield a culture solution; (e) adding ammonium sulfate to the culture solution up to a 90% saturation in order to precipitate proteins concentrated therein; (f) separating the precipitated proteins from the ammonium sulfate-containing supernatant; (g) redissolving said proteins; (h) purifying said proteins by anion exchange chromatography, preparative molecular sieve filtration, cation exchange chromatography, chromatography on hydroxyapatite, zone precipitation chromatography and recycling molecular sieve filtration for removing accompanying foreign proteins; and (i) isolating highly purified lymphocytolymphomitogen from the eluate of the recycling molecular sieve filtration by adding ammonium sulfate up to 80-90% saturation and separating the precipitate formed from the supernatant.

Claim 34

A process for producing highly purified lymphocyto lumphomitogen, which comprises: (a) culturing a leukocyte population containing mixed leukocytes or only lymphotyces in a suitable culture medium; (b) inducing the mitosis of the cells by CON during culturing; (c) terminating culturing; (d) separating cells from the culture medium to yield a culture solution; (e) adding ammonium sulfate to the culture solution up to a 90% saturation in order to precipitate proteins concentrated therein; (f) separating the precipitated proteins from the ammonium sulfate-containing supernatant; (g) redissolving said proteins; (h) purifying said proteins by anion exchange chromatography, preparative molecular sieve filtration, cation exchange chromatography, chromatography on hydroxyapatite, zone precipitation chromatography and recycling molecular sieve filtration for removing accompanying foreign proteins; and (i) isolating highly purified lymphocytolymphomitogen from the eluate of the recycling molecular sieve filtration by adding ammonium sulfate up to 80-90% saturation and separating the precipitate formed from the supernatant.

Claim 35

Highly purified lymphocytolymphomitogen.

Claim 36

Highly purified lymphocytolymphomitogen.

Claim 37

Highly purified monocytohistiomitogen.

Claim 38

Highly purified monocytohistiomitogen.

Claim 39

Highly purified granulocytoblastogen.

Claim 40

Highly purified granulocytoblastogen.

Claim 41

Highly purified monocytoblastogen.

Claim 42

Highly purified monocytoblastogen.

Claim 43

Highly purified lymphocytolymphomitogen derived from leukocyte culture, the soluble portion of leukocytes or inflamed tissue homogenate.

Claim 44

Highly purified lymphocytolymphomitogen derived from leukocyte culture, the soluble portion of leukocytes or inflamed tissue homogenate.

Claim 45

Highly purified monocytohistiomitogen derived from leukocyte culture, the soluble portion of leukocytes or inflamed tissue homogenate.

Claim 46

Highly purified monocytohistiomitogen derived from leukocyte culture, the soluble portion of leukocytes or inflamed tissue homogenate.

Claim 47

Highly purified granulocytoblastogen derived from leukocyte culture, the soluble portion of leukocytes or inflamed tissue homogenate.

Claim 48

Highly purified granulocytoblastogen derived from leukocyte culture, the soluble portion of leukocytes or inflamed tissue homogenate.

Claim 49

Highly purified monocytoblastogen derived from leukocyte culture, the soluble portion of leukocytes or inflamed tissue homogenate.

Claim 50

Highly purified monocytoblastogen derived from leukocyte culture, the soluble portion of leukocytes or inflamed tissue homogenate.