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WO1990009798A1 - Cytokines immobilisees - Google Patents

Cytokines immobilisees Download PDF

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Publication number
WO1990009798A1
WO1990009798A1 PCT/US1990/001031 US9001031W WO9009798A1 WO 1990009798 A1 WO1990009798 A1 WO 1990009798A1 US 9001031 W US9001031 W US 9001031W WO 9009798 A1 WO9009798 A1 WO 9009798A1
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WIPO (PCT)
Prior art keywords
cytokine
immobilized
beads
cells
growth
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PCT/US1990/001031
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English (en)
Inventor
Gerald J. Vosika
Dennis A. Cornelius
Carl W. Gilbert
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Immunotherapeutics, Inc.
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Publication date
Application filed by Immunotherapeutics, Inc. filed Critical Immunotherapeutics, Inc.
Publication of WO1990009798A1 publication Critical patent/WO1990009798A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0018—Culture media for cell or tissue culture
    • C12N5/0025—Culture media for plant cell or plant tissue culture
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/16—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction
    • A61K9/167—Agglomerates; Granulates; Microbeadlets ; Microspheres; Pellets; Solid products obtained by spray drying, spray freeze drying, spray congealing,(multiple) emulsion solvent evaporation or extraction with an outer layer or coating comprising drug; with chemically bound drugs or non-active substances on their surface
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K17/00—Carrier-bound or immobilised peptides; Preparation thereof

Definitions

  • the present invention relates to cytokines that are immobilized on a solid support.
  • cytokines A number of biologically active mediators, generally termed cytokines, are produced by a variety of cells. Cytokines are produced on an obligatory basis for the maintenance of normal homeostasis, and also in response to pathological stimuli, such as immunological, infectious, and inflammatory processes. Those cytokines first described as products of lymphocytes are often referred to as “lymphokines,” and those cytokines originally described as products of monocytes have been termed “monokines.” Certain cytokines are also referred to as growth factors or colony stimulating factors, based on their effect on cell growth.
  • cytokines examples include: the lymphokines interleukin-1 (IL-1), interleukin-2 (IL-2), and
  • interleukin-3 IL-3
  • monokine gamma interferon the monokine gamma interferon
  • growth factors granulocyte-macrophage colony
  • GMCSF erythropoietin
  • EPO erythropoietin
  • cytokines serve as endogenous regulators (autocrines) and/or as intercellular
  • cytokines initially recognized by a single biological activity, have been shown to have multiple, overlapping biological activities, often acting synergistically to amplify the biological response.
  • the ultimate effect on the target cell includes regulation of growth, mobility,
  • Interleukin-1 also known as. lymphocyte activating factor, is produced by human monocytes, lymphocytes, endothelial cells, and fibroblasts. IL-1 promotes lymphocyte differentiation, as indicated by changes in phenotypic cell surface markers.
  • IL-1 stimulates T-lymphocyte functions and increases the production of lymphokines such as IL-2, colony-stimulating factors (CSF), B-cell growth factor (BCGF), gamma-interferon (y-IFN), and lymphocyte-derived chemotactic factors (LDCF), each with their own
  • IL-1 also augments the in vitro proliferation, differentiation, and antibody-producing functions of B-lymphocytes. These and other biological activities have made IL-1 a valuable lymphokine in a wide variety of in vivo and in vitro uses.
  • Interleukin-2 (IL-2) was first termed T-cell growth factor (TCGF) for its ability to induce TCGF
  • T-lymphocytes to proliferate and enable normal T-lymphocytes to be maintained continuously in culture.
  • IL-2 has been found useful in a wide variety of in vivo and in vitro applications. IL-2, when used as a vaccine adjuvant, overcomes genetic nonresponsiveness to malaria sporozoite peptides and enhances protection against Herpes simplex and rabies viruses. See M.
  • lymphokine a group of more selective T-cell populations known as lymphokine
  • In vitro lymphokine activated killer cells have been used in combination with the in vivo administration of interleukin-2 to achieve an improved antitumor effect.
  • the infusion of in vitro IL-2 activated killer cells and the concurrent administration of IL-2 has demonstrated antitumor activity in both animals and humans; such activity generally exceeding that observed with the use of IL-2 or lymphokine activated killer cells individually. See J. J. Mule et al., Science,
  • lymphocytes obtained from human malignancies have been induced by interleukin-2, in vitro, for periods of up to 60 days. These lymphocytes have demonstrated human antitumor activity in patients with lung cancer when administered without the concurrent intravenous administration of interleukin-2. See R. L. Kradin et al.,
  • Additional cytokines synthesized by T-cells include migration inhibition factor (inhibits the random migration of macrophages); leukocyte inhibition factor ( inhibits the random migration of neutrophils);
  • macrophage activation factor contributes the cytolytic activity of macrophages
  • fibroblast activation factor stimulates proliferation of fibroblasts
  • IL-3 interleukin-3 (IL-3) (activity similar to IL-3)
  • cytokine activity is believed to include the steps of: 1) binding of the cytokine to a specific cell surface receptor; 2) initiation of certain "cell surface activated” events; and 3) internalization of the
  • cytokine-receptor complex where internal interactions result in proliferation, growth, differentiation, and/or the expression of specialized cell function.
  • IL-2 the interaction of IL-2 with T-cells is believed to involve an initial interaction with a low affinity receptor, IL2Rb, resulting in induction of a second receptor molecule, IL2Ra, that forms a high affinity complex with IL-2.
  • IL2Rb a low affinity receptor
  • IL2Ra a second receptor molecule
  • association of IL-2 with the high affinity complex results in proliferation.
  • this process of activation and proliferation there is an internalization of the IL-2-receptor complex and a subsequent decrease in the number of surface IL-2 receptors. See K.
  • cytokines such as IL-2
  • IL-2 cytokine-like cytokine
  • modified cytokines that retain comparable, and in some cases, improved biological activity when compared with corresponding soluble or free cytokines, thus providing a biologically active cytokine that can be reused to stimulate biological activity and/or that can be used in significantly smaller quantities.
  • the present invention provides immobilized cytokines comprising cytokines firmly bound to a solid, preferably biologically compatible, insoluble
  • the bound cytokine retains substantially the activity of the free cytokine when bound to the support. Accordingly, the bound cytokine is able to be utilized repeatedly (reused) to stimulate biological activity, and/or used in significantly smaller total quantities than the corresponding soluble or free cytokine.
  • Cytokines useful in the present invention include, but are not limited to, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, tumor necrosis factor (TNF), gamma-interferon, alpha-interferon, beta-interferon, erythropoietin (EPO), granulocyte colony stimulating factor (GCSF), murine granulocyte colony stimulating factor (MuGCSF), granulocyte-macrophage colony
  • TNF tumor necrosis factor
  • EPO gamma-interferon
  • alpha-interferon alpha-interferon
  • beta-interferon beta-interferon
  • EPO erythropoietin
  • GCSF granulocyte colony stimulating factor
  • MuGCSF murine granulocyte colony stimulating factor
  • GMCSF murine granulocytemacrophage colony stimulating factor
  • ILGF-I insulinlike growth factor I
  • ILGF-II insulin-like growth factor II
  • TGF- ⁇ transformation growth factor beta
  • EGF epidermoid growth factor
  • PDGF platelet derived growth factor
  • FGFb fibroblast growth factor-basic
  • Preferred cytokines include those described in the Examples, and more preferrably IL-2, GMCSF, GCSF, EPO, TNF, FGFb, TGFb, EGF, and PDGF.
  • the cytokine is preferably bound to a biologically compatible, particulate support by means of covalent bonding, preferably by means of a linking arm.
  • the cytokine is preferably firmly bound to the support in a manner that allows the activity of the cytokine to be stabilized by the immobilization. The activity thus is durable and reusable.
  • substantially the activity of the free cytokine means that at least one of the one or more active sites of a cytokine remains active, and will produce significant biological activity as a bound cytokine.
  • a bound cytokine of the present invention may demonstrate one or more activities the same as, or similar to, that of the free cytokine.
  • one or more biological activities may be stabilized through immobilization.
  • at least one activity is preserved in the bound state, and in some cases may be enhanced by the binding of the cytokine to the support.
  • linking arm may be varied to optimize the biological activity of the bound cytokine.
  • Preferred linking arms comprise one or more linking groups selected from the group consisting of: (a) diamines, having the general formula NH 2 -R 1 -NH 2 , where R 1 is a C 2 -C 20 alkyl group; (b) amino acids, having the general formula NH 2 -R 2 -CO 2 H, where R 2 is a C 1 -C 20 alkyl group; and (c) dialdehydes, having the general formula OHC-R 3 -CHO, where R 3 is a C 1 -C 20 alkyl group.
  • Useful supports include, but are not limited to, fibers, microspheres, beads, particles,
  • cytokine refers to the natural or recombinant form of the cytokine, as well as to modified sequences, biologically active fragments or portions of cytokines, genetically or chemically modified forms of a cytokine, biologically equivalent synthetic ligands, or mixtures thereof, which exhibit a substantially equivalent profile of bioactivity, or a portion of the original profile of bioactivity.
  • the present invention also provides methods of using the immobilized cytokines, both in vitro and in vivo, for the proliferation, growth, differentiation and/or expression of specialized cell function,
  • cytokine-dependent cell line such as an IL-2 dependent cell line
  • Fig. 1 is a graphical depiction of the growth of CTLL-2 cells (DPM's ⁇ 10 -3 ) using IL-2 immobilized via a carboxyl group of the IL-2 as compared to IL-2
  • Fig. 2 is a graphical depiction of the concentration dependence of immobilized IL-2 ( ⁇ g IL-2 in initial coupling reaction) on the growth of CTLL-2 cells, a cytotoxic T-lymphocyte cell line, as determined by [ 3 H]-thymidine incorporation (DPM's ⁇ 10 -3 ).
  • Fig. 3 is a graphical depiction of the growth of CTLL-2 cells (DMP's x 10 -3 ) using immobilized IL-2 as a function of time (hours) relative to the growth of CTLL-2 cells using soluble IL-2.
  • Fig. 4 is a graphical depiction of the growth of human peripheral blood lymphocytes (PBL's in DPM's ⁇ 10 -3 ), using immobilized IL-2 as a function of time
  • Figs. 5A and 5B are graphical depictions of stimulation of granulopoiesis as measured by an increase in the white blood count of mice receiving soluble (5A) or immobilized (5B) MuGMCSF.
  • Fig. 6 is a graphical depiction of stimulation of granulopoiesis as measured by the increase in white blood count of cyclophosphamide-treated mice receiving soluble or immobilized recombinant murine GMCSF
  • Fig. 7 is a graphical depiction of the stability of covalent bound rMuGMCSF as measured by its retention in contrast to adsorbed rMuGMCSF following SDS washes.
  • Interleukin-2 (IL-2) is commercially available as T-cell growth factor (human interleukin-2
  • interleukin-3 (IL-3), natural sequence recombinant interleukin-4 (IL-4), and natural sequence recombinant interleukin-6 (IL-6) are commercially available from Amgen, Thousand Oaks, CA.
  • IL-3 natural sequence recombinant interleukin-4
  • IL-6 natural sequence recombinant interleukin-6
  • rHuGMCSF granulocyte-macrophage colony stimulating factor
  • rHuGCSF granulocyte colony stimulating factor
  • rHuEPO erythropoietin
  • rMuGMCSF murine granulocyte- macrophage colony stimulating factor
  • rHuIFN-gamma recombinant human epidermoid growth factor
  • rHuEGF recombinant human epidermoid growth factor
  • FGFb human platelet derived growth factor
  • rHuPDGF recombinant human insulin-like growth factor I
  • rHuILGF-II recombinant human insulin-like growth factor II
  • TGF-alpha transformation growth factor alpha
  • Transformation growth factor beta, porcine, (pTGF-beta) is commercially available from R & D Systems, Minneapolis, MN. Transformation growth factor beta is also commercially available from Collaborative Research, Bedford, MA. Recombinant interferon alpha is commercially available as RoferonTM from Roche Laboratories.
  • invention also includes binding the biologically active portions of cytokines to a suitable support.
  • IL-1 has multiple effects on lymphocyte populations, including its function as an autocrine growth factor for many T-cell clones.
  • IL-1 is also a potent stimulator of thymocyte proliferation, and of mitogen, nominal antigen plus la antigen, or alloantigen stimulated helper T-cells.
  • IL-1 increases interleukin-2 receptor expression and IL-2 secretion of human
  • peripheral T-cells in the presence of monoclonal
  • IL-1 acts as a cofactor for Con A activation of resting T-cells and is required for the proliferation of
  • lymphocytes which express a high affinity receptor for IL-1.
  • IL-1 is produced by human lung endothelium where it is believed to function in the development of
  • IL-1 is also a regulator of hematopoietic activity. IL-1 induces endothelial cells to release granulocyte-macrophage colony stimulating factor (GMCSF) and granulocyte colony stimulating factor (GCSF), thus providing the mechanism by which IL-1 modulates GMCSF and granulocyte colony stimulating factor (GCSF), thus providing the mechanism by which IL-1 modulates
  • IL-1 also releases GMCSF from monocytes and enhances growth factor dependent proliferation of human hematopoietic precursors.
  • IL-1 has demonstrated by its antitumor activity causing complete regression of relatively large
  • IL-1 has a direct cytotoxic effect in vitro on human A375 melanoma cells.
  • IL-1 has also shown synergistic effects with interleukin-2 (IL-2) in the production of lymphokine activated killer cells.
  • IL-2 interleukin-2
  • IL-1 as a regulator of lymphocyte function, hematopoiesis, and lymphocyte antitumor activity has made IL-1 a valuable cytokine in a wide variety of in vivo and in vitro
  • Interleukin-3 also known as multicolony stem cell activating factor, or as multicolony
  • IL-3 is a glycoprotein hematopoietic growth factor.
  • IL-3 has a broad range of activity, due to its ability to stimulate both early stem cells, common to many myeloid cell lineages, as well as
  • IL-3 binds to a 140 kilodalton cell surface phosphoprotein. In primates, continuous
  • IL-3 acts on early lineage cells that require a subsequent second factor to complete development. This hypothesis is consistent with tissue culture studies indicating that IL-3 is more effective in supporting colony formation by blast cells. In addition, IL-3 itself will not support in vitro colony formation, but requires a later acting factor, such as GMCSF. IL-3 acts synergistically with IL-6 to support early blast colony formation, with granulocyte colony stimulating factor (GCSF) to enhance neutrophil formation, and with GMCSF to enhance
  • IL-3 a valuable adjunct to hematopoietic cytokine therapy.
  • IL-3 Like other cytokines, IL-3 also has negative regulatory effects, as evidenced by its inhibition of lymphokine activated killer cells. To date, IL-3 has been the only cytokine implicated in the regulation of early B-cell development, as is evidenced by the IL-3 dependent clones from murine fetal liver or adult bone marrow which show characteristics of B-cell precursors. See, for example, R. E. Donahue et al., Science, 241, 1820 (1988); R. J. Isfort et al., Proc. Nat. Acad. Sci. USA, 85, 7982 (1988); D. Rennick et al., J. Immunol., 142, 161 (1989); and G. Gallagher et al.,
  • Interleukin-4 is also known as B-cell stimulatory factor-1 (BSF-1), B-cell differentiation factor (BCDF), and B-cell growth factor 1 (BCGF-1).
  • BSF-1 B-cell stimulatory factor-1
  • BCDF B-cell differentiation factor
  • BCGF-1 B-cell growth factor 1
  • IL-4 enhances immunoglobulin IgGl and IgE production in lipopolysaccharide activated cells, increases the expression of histocompatibility antigens on B-cells, and is required for the proliferation of anti-IgM activated B-cells.
  • IL-4 also augments the mitogen induced stimulation of human peripheral T-cells in the presence of dexamethasone, which inhibits IL-2 production. IL-4 also down modulates IL-2 induced human B-cell
  • IL-4 in conjunction with IL-2, however, augments the growth of tumor infiltrating lymphocytes to autologous human malignant melanoma. In addition to its effects on lymphoid cells, IL-4
  • Interleukin-6 (IL-6) is also known as B-cell stimulatory factor-2, interferon beta-2, and
  • IL-6 is a multifunctional cytokine, initially described as a T-cell lymphokine with antiviral activity. IL-6 has been demonstrated to be produced by a variety of cells including: monocytes, fibroblasts, hepatocytes, cardiac myxomas, brain glial cells, and vascular endothelxum. IL-6 activity is believed to include: regulation of fibroblast activity; acute phase protein production by hepatocytes; stimulation of human thymocytes and
  • T-lymphocytes in the presence of mitogen; proliferation and differentiation of murine T-lymphocytes into
  • cytotoxic cells maintenance of myeloma derived cell lines; autocrine signalling for human multiple myeloma, and inhibition of the growth of carcinoma and
  • leukemia/lymphoma cell lines See, for example,
  • Granulocyte-macrophage colony stimulating factor (GMCSF), granulocyte colony stimulating factor (GCSF), macrophage colony stimulating factor (MCSF) and multi-colony stimulating factor (IL-3) constitute a family of glycoproteins that have been recognized by their ability to stimulate and regulate the process of proliferation and differentiation of hematopoietic cells both in vivo and in vitro.
  • These individual cytokines are produced by one or more of the following cell sources: T-lymphocytes, monocytes, fibroblasts,
  • IL-1 also known as hemopoietin-1, participates in this regulatory network by enhancing the effects of IL-3, MCSF, GCSF, and GMCSF.
  • GMCSF results in a significant increase in neutrophils and neutrophils-eosinophils respectively, as well as an increase in bone marrow cellularity with immature cells, appearing in the blood.
  • Clinical side effects following the use of GMCSF in man have included fever, rash, myalgia, fatigue, gastrointestinal distress,
  • GCSF and GMCSF have included: restoration of hematopoiesis following myelo-suppressive cytotoxic chemotherapy; accelerated granulocyte recovery and decreased incidence of infection in patients with autologous bone marrow transplants; and improved circulating white cell, hemoglobin, and platelet count in patients with
  • GMCSF has been demonstrated in vivo to activate monocytes to a tumorcidal state suggesting another potential clinical application for this
  • cytokine cytokine.
  • GMCSF has also been demonstrated to stimulate the proliferation in vitro of osteogenic sarcoma cell lines, a breast cancer cell line, a simian virus SV-40 transformed bone marrow stromal cell line and normal bone marrow fibroblast precursors. See, for example: S. Vadhan-Raj et al., N. Engl. J. Med., 319, 1628
  • EPO Erythropoietin
  • Tumor Necrosis Factor also known as a multi-functional cytokine produced by TNF.
  • TNF-alpha a major factor in gram-negative endotoxin shock and induces a profound wasting (cachexia) syndrome in patients with cancer and chronic disease.
  • the range of activity includes stimulation of fibroblast growth, stimulation of osteoblast activity and bone reabsorption, promotion of angiogenesis, stimulation of collagenase and prostaglandin E 2 in synovial cells, and stimulation of procoagulant and platelet-activating factor in endothelial tissue.
  • TNF is an autocrine produced by macrophages. It functions as an immunomodulator, activating
  • TNF is chemotactic for macrophages, indicating its production at a site of inflammation both recruits and activates macrophages.
  • TNF participates in the cytokine network and induces the release of IL-1, GMCSF, platelet derived growth factor, and beta-2 interferon.
  • TNF is the mediator of endotoxin induced tumor regression.
  • TNF may be involved in the antitumor activity of IL-2, since IL-2 induces TNF in human peripheral blood monocytes. TNF, given systemically, induces regression of tumor in mice.
  • the direct anti-proliferative and tumor cytotoxic effects of TNF and IL-1 are believed synergistic.
  • Toxicities have included: fever, chills, fatigue, anorexia, hypotension, and tachycardia.
  • Several minor tumor responses have been noted to date. See, for example, B. Sherry and A. Cerami, J. Cell Biol., 107, 1269 (1988); J. J. Mule et al., Cancer
  • Interferon is a term originally assigned to a class of compounds discovered in the late 1950 's with anti-viral activity. Originally, three classes of interferon were designated as alpha, beta, and gamma; designating their original identification and isolation from leukocytes, fibroblasts, and lymphoid cells, respectively. As of 1988, at least 24 nonallelic genes, coding for structurally related forms of alphainterferon, had been described. These were divided into two subfamilies designated IFN-alpha I genes, which code for proteins of 165-166 amino acids, and IFN-alpha II genes, which code for protein of 172 amino acids. A single gene coding for what is commonly called
  • fibroblast interferon has been fully characterized in humans. However, fibroblast can produce more than one form of interferon and the more correct term for
  • fibroblast interferon is human interferon beta (HuIFN-beta).
  • HuIFN-beta has about 40% amino acid homology with HuIFN-alpha.
  • the human interferon gamma gene exists as a single copy with some individual to
  • Gamma-interferon has no homology to alpha- or beta-interferon.
  • INF-alpha and beta are also similar, but both differ from IFN-gamma. See E. DeMaeyer and J.
  • alpha and beta interferon The major biological activities of alpha and beta interferon are: antiviral effects; induction of monocytes to express major histocompatibility, complex class II antigens, and interleukin-1; antiproliferative effects; and regulation of human natural killer cell activity.
  • Interferon alpha and beta have an antitumor effect that involves a number of mechanisms including, among others, an antiproliferative effect, induction of differentiation, regulation of oncogene expression, and stimulation of the immune response.
  • human interferon alpha or beta may inhibit the maturation of monocytes to
  • a group of cytokines also termed growth factors have, among their biological activities, a positive or negative regulatory effect on wound healing and tissue repair including chemotactic activity, proliferation, growth and differentiation of epithelial cells and fibroblasts, stimulation of matrix formation and cartilage formation, and vascular formation
  • angiogenesis A large number of biologically active proteins have been described within this area and have been classified on taxonomical principles into families and species based on their biological effects and amino acid sequence homology (as shown below in Table 1).
  • cytokines Although this group of cytokines has been associated with tissue repair, they have other biological effects. In addition, other cytokines such as interleukin-1 and interleukin-3, which regulate immune responses, also have an effect on tissue repair.
  • Epidermal growth factor is a key representative member of a family of structurally related proteins including transformation growth factor (TGF) alpha, amphiregulin, and vaccinia growth factors.
  • TGF transformation growth factor
  • Human EGF was first isolated from urine and named urogastrone by its ability to inhibit gastric secretion (H. Gregory, Nature, 257, 324 (1975)).
  • Murine EGF isolated from the salivary gland is mitogenic for a large number of cell types including epithelial,
  • EGF is synthesized as a precursor protein which is processed into a 53-amino acid active protein.
  • Transformation growth factor alpha (TGF-alpha) binds to the same receptor as EGF and shares similar biological activity. See G. J. Todaro et
  • TGF-alpha like EGF is synthesized as a 160-amino acid precursor, which is proteolytically processed into a 50-amino acid biologically active residue. See R. Derynck et.al., Cell, 38, 287 (1984). TGF-alpha was originally recognized by its ability to synergize with TGF-beta to induce anchorage independent growth of normal rat kidney fibroblast. See M. A. Azano et al., Proc. Nat. Acad. Sci. USA, 80, 6264 (1983).
  • Platelet derived growth factor is purified from human blood platelets. See R. Ross and A. Vogel, Cell, 14, 203 (1978). It consists of two polypeptide chains: the A chain (124 amino acid
  • PDGF is a potent mitogen for cells of mesenchymal origin (e.g., smooth muscle and fibroblasts) but has no effect on epithelial or endothelial cells which lack PDGF receptors. See R. Ross, E. W. Raines, and
  • Platelet derived growth factor may also be obtained from porcine cells.
  • Transformation growth factor beta(s) were originally identified by their ability to act
  • TGF-betas are acid and heat-stable disulfide-linked homodimeric proteins of 112 amino acid residues which share 70% homology. See R. Derynck and J. A. Farrett et al.. Nature, 316, 701 (1985). Another member of the family, beta-3, has recently been
  • TGF-beta I has demonstrated major activity in wound healing.
  • Other biologically active proteins included in the TGF-beta family include: forms of gonadal proteins designated inhibin and activin that regulate pituitary secretion of follicle stimulating hormone; Mullerian inhibiting substances that cause regression of the female Mullerian ducts in the developing male embryo; and bone morphogenic proteins that are a group of polypeptides involved in the induction of cartilage and bone formation.
  • Fibroblast growth factors are single-chain proteins of 14-18 kilodaltons. Two well characterized forms are basic FGF, isolated form brain and pituitary, and acidic FGF, isolated from brain and retina. Basic FGF, in most systems, is more stable and has ten times the potency of acidic FGF. Both forms of FGF bind to the same receptor and are mitogenic for cells of mesodermal origin such as fibroblasts, vascular endothelial cells, vascular smooth muscle, myoblasts, chondrocytes and osteoblasts. See F. Esch and A. Baird et al., Proc. Natl. Acad. Sci. USA, 85, 6507 (1985). The products of the int-2 and hst proto-oncogenes are also included as members of the FBF family. (See
  • Insulin-like growth factor I also known as Somatomedia C
  • Insulin-like growth factor II also known as Somatomedia C
  • IGF-II represent a current nomenclature for a number of factors initially purified from serum and sharing the three biological activities of stimulating of sulfate incorporation into cartilage, insulin-like activity, and multiplication-stimulating activity.
  • the liver and fibroblasts are major sources of circulating
  • insulin-like growth factors but essentially all tissues have been shown to produce them.
  • Insulin-like growth factors among their biological activities, have also been shown to stimulate glucose metabolism, and stimulate DNA synthesis and cell proliferation of fibroblasts, sertoli cells, fetal brain cells, myoblasts, lens epithelium, pancreatic beta cells, lectin stimulating lymphocytes, and density arrested Balb/c 3T3 cells after being rendered
  • Cytokines react with cell surface receptors which themselves are complex and may consist of
  • cytokine may bind
  • the present invention also provides for immobilization of such cytokine fragments that may be directed to a particular subunit of the receptor. Immobilizing Supports
  • Support materials useful in the present invention are preferably biologically compatible, and may be nonbiodegradable or biodegradable as desired. It may be desirable that the support be biodegradable when the bound cytokine will be utilized in vivo, while insoluble support materials are useful in applications such as bioreactors.
  • Suitable supports include fibers, sheets, microspheres, particles, beads, membranes, and the like.
  • the support preferably comprises a surface which is chemically compatible with the covalent
  • the support preferably includes a surface having appropriate
  • Suitable functional groups for cytokine binding can be provided by appropriate chemical
  • a nonfunctionalized polystyrene support can be provided with a functionalized surface by suitable
  • binding chemistries work equally well with each of the many various cytokines. Suitability of a particular binding chemistry used may, in part, depend upon the availability of reactive sites, and their proximity to the active site of the cytokine. Those skilled in the art can, however, reasonably predict a suitable approach from the amino acid sequence, the presence of reactive groups, and the active site. In applying the invention, those skilled in the art can also create genetically modified cytokines replacing amino acids with non-reactive amino acids, or vice versa to target linkage of the immobilization site. Those skilled in the art may also modify the codon of the cytokine to produce one with terminal reactive groups thereby providing a high probability of directing linkage of the immobilization site.
  • a functionalized surface includes reactive functional groups that provide a site for binding: (a) directly to a site on the cytokine; or (b) to a suitable linking arm.
  • Such functional groups include hydroxyl (-OH), amino (-NH 2 or -NHR, wherein R is alkyl or aryl), carboxyl (-CO 2 H), sulfhydryl (-SH), and halogens (-F, -Cl, -Br, -I).
  • a functionalized surface may be provided by a number of means in addition to chemical treatment of a surface. For example, blue-dyed polystyrene beads obtained from Polysciences, provide a functionalized surface despite polystyrene itself not having functional groups available for reaction. The blue dye is bound to, adsorbed on, or copolymerized with the polystyrene and provides free amino groups.
  • a wide variety of other methods for providing suitable functional groups are known.
  • Suitable particulate supports include inorganic supports, such as, glass, quartz, ceramics, zeolites, metals, and metal oxides; polymeric materials, including homopolymers, copolymers, and oligopolymers, derived from monomeric units comprising definite units such as styrene, divinylbenzene, ethylene, butadiene,
  • carbohydrate supports such as, agarose, cross-linked agarose, dextran, cross-linked dextran, inulin,
  • hyaluronic acid cellulose, cellulose derivatives such as carboxymethyl cellulose (CMC), starch and starch derivatives (e.g., starch microspheres); and insoluble protein materials, such as, gelatin, collagen, or
  • the surface of the immobilizing support of the present invention is preferably nonporous.
  • substantially spherical polymeric beads or microspheres allows for binding of the cytokine to the outer surface of the support, thereby providing the cytokine in a biologically available, unhindered position.
  • a surface is considered nonporous where the size of any pores in the material is sufficiently small so as to block or substantially hinder the migration of the cytokine into the interior of the spheres.
  • a porous surface may be preferred to permit high drug loading, with new active sites exposed as the support degrades.
  • the size and shapes of the support may be varied widely, depending on the particular cytokine and its intended use.
  • Polymeric spheres having a diameter of about 0.5-500 ⁇ m, and particularly about 1-75 ⁇ m, are preferable supports. Such supports are preferred, for example, for the in vitro growth of IL-2 dependent lymphocytes.
  • Other preferable supports include staple fibers having a diameter of about 5-200 ⁇ m.
  • the immobilized cytokines of the present invention preferably include a cytokine covalently bound, either directly or through a linking arm, to the support materials. It is believed that the length of the linking arm may be related to the biological
  • Suitable linking arms include one or more bifunctional linking groups such as: (1) diamines, having the formula NH 2 -R 1 -NH 2 , where R 1 is a C 2 -C 20 alkyl group; (2) amino acids, having the general formula NH 2 -R 2 -CO 2 H, where R 2 is a C 1 -C 20 alkyl group; and (3) dialdehydes, having the formula
  • linking groups may be coupled to provide additional length.
  • suitable linking groups include 6-aminocaproic acid, 1,6-diaminohexane,
  • 1,12-diaminododecane 1,12-diaminododecane, glutaraldehyde, and mixtures thereof.
  • the solid support includes a functionalized surface having a plurality of reactive, exposed functional groups.
  • the cytokine is thus directly covalently attached to a functional group on the surface, or to a linking arm of appropriate length that is covalently attached to the functional group.
  • cytokine biologically active moiety
  • Acceptable methods of attachment include: (1) the use of water-soluble carbodiimides in the reaction of a carboxyl group on the functionalized surface of the polymeric support and a free accessible amino group on the cytokine, believed to form a stable amide bond; (2) the use of bifunctional aldehydes (e.g., glutaraldehyde) as a linking arm, which can couple an amino group on the surface of the polymeric support and a free accessible amino group on the cytokine; and (3) the use of cyanogen bromide in the reaction of a hydroxyl group on the solid support with an amino group on a linking arm or on the cytokine.
  • bifunctional aldehydes e.g., glutaraldehyde
  • the stability of the immobilized cytokine will depend on the nature of the covalent bond(s) between the cytokine, either directly to the immobilizing surface, or through the linking arm (if present). Stable, firmly bound cytokines will demonstrate the desired biological activity through repeated uses. The stability of the following bonds linking a protein to an insoluble matrix are considered relatively weak:
  • This linkage is formed from the reaction of an amino group on the protein (chiefly the lysyl side chain amine) with polyhydroxylic matrices (e.g., agarose, cellulose, and dextran) that have been activated with such reagents as cyanogen bromide (CNBr),
  • polyhydroxylic matrices e.g., agarose, cellulose, and dextran
  • This linkage is also formed as above for isourea, from the reaction of an amino group on the protein (chiefly the lysyl side chain) with polyhydroxylic matrices activated as above.
  • This linkage is formed from the reaction of an amino group on the protein with polyhydroxylic matrices that have been activated with such reagents as 4-nitrophenyl chloroformate, N-hydroxysuccinimidyl chloroformate, carbonyl diimidazole, and the like.
  • This linkage is formed from the reaction of a protein amino group with a polyhydroxylic matrices that have been activated with such reagents as cyanuric chloride.
  • This linkage is formed in a variety of ways, including the reaction of a protein amino group with (1) polyhydroxylic matrices that have been activated with such reagents as tresyl chloride, sulfonyl chloride and the like, with oxiranes (epoxides) such as bisoxirane and epichlorohydrin and with (2) polyamino matrices that have been activated with such reagents as glutaraldehyde.
  • This linkage can be formed in a variety of ways, including the reaction of a protein amino group with an activated carboxyl group on an insoluble matrix. Activation of these carboxyl groups can be achieved via formation of "active" esters (e.g., N-hydroxysuccimimidyl, p-nitrophenol, or pentachlorophenol) or by reaction with carbodiimides. Conversely, an amide bond may also be formed by the reaction of an amino group on an insoluble matrix with a suitably activated (e.g., a water soluble carbodiimide) carboxyl group on the protein, especially the aspartic acid and glutamic acid side chain carboxyl groups.
  • a suitably activated e.g., a water soluble carbodiimide
  • the covalent attachment be directed to a single site on the cytokine, preferably a suitable distance from the biologically active site. This consideration may dictate the preferred choice of linking arms and the specific chemistry chosen in the attachment of the linking arms, support, and cytokine to optimize biological activity.
  • Bound cytokines of the present invention can be used to induce and regulate a variety of biological reactions, including for example: (1) in vitro growth and production of cellular blood components including stem cells, and cells in various stages of differentiation, including red cells, lymphocytes, macrophages, and/or neutrophils; (2) the in vitro growth and production of specialized effector cells, including lymphokine activated killer (LAK) cells, natural killer cells, subpopulations of lymphokine activated killer cells, tumor infiltrating lymphocytes, and/or cytotoxic T-cells; (3) the treatment of malignant disease by the in vivo intraperitoneal and/or intrapleural administration of the bound cytokines; (4) the treatment of malignant disease by the in vivo intravenous administration of the bound cytokines; (5) the treatment, preferably by intravenous administration or by in-situ placement of the bound cytokine, of refractory anemias, thrombocytopenias, and neutropenias associated with primary bone marrow failure
  • Recombinant IL-2 (Amgen, Thousand Oaks, CA, ala- 125 analog) was immobilized on 9.64 ⁇ m blue-dyed polystyrene beads (Polysciences, Warrington, PA) using a bifunctional aldehyde in the following manner.
  • a 0.25 mlaliquot of a 2.5% aqueous suspension of 9.64 ⁇ m blue-dyed polystyrene beads was diluted with 1.0 ml of phosphate buffered saline (PBS, pH 7.40) and centrifuged for 5 minutes in a microcentrifuge. The supernatant was carefully removed and discarded. The beads were washed twice by suspension in 1.0 ml-portions of PBS followed by centrifugation.
  • PBS phosphate buffered saline
  • the beads were then suspended in 0.75 ml of an 8% solution of glutaraldehyde in PBS. Activation was allowed to proceed for 5 hours at room temperature with gentle end-over-end mixing. The reaction mixture was centrifuged and the supernatant was discarded. The pellet, i.e., the agglomerated beads, was washed twice with 1.0 ml-portions of PBS. The pellet was then suspended in 0.4 ml of PBS and treated with 0.1 ml of an aqueous IL-2 solution (100 ⁇ g IL-2, activity 600,000 units). The reaction mixture was mixed overnight at room temperature, centrifuged, and the supernatant was carefully removed and saved.
  • an aqueous IL-2 solution 100 ⁇ g IL-2, activity 600,000 units
  • the pellet was resuspended in 0.5 ml of PBS, and the mixture was centrifuged. The supernatant was removed and added to the first supernatant. This combined supernatant solution (ca. 1.0 ml) was preserved at 4°C for the subsequent determination of residual IL-2 activity.
  • the beads were then processed in the following manner.
  • the beads were suspended in 0.5 ml of 0.5 M ethanolamine in PBS and mixed for 30 minutes at room temperature. The mixture was centrifuged, the supernatant was discarded, and the pellet was washed once with 0.5 ml of PBS.
  • the beads were suspended in 0.5 ml of 1% bovine serum albumin (BSA, Sigma, St. Louis, Mo) in PBS, mixed for 30 minutes at room temperature, and centrifuged. The supernatant was discarded.
  • BSA bovine serum albumin
  • the pellet was then washed twice with 0.5 ml-portions of the BSA/PBS solution and finally suspended in 0.5 ml of a storage buffer (sodium chloride (0.88%), BSA (1%), glycerol (5%), and sodium azide (0.1%) in 0.02 M sodium phosphate (pH 7.40)).
  • a storage buffer sodium chloride (0.88%), BSA (1%), glycerol (5%), and sodium azide (0.1%) in 0.02 M sodium phosphate (pH 7.40)
  • An assay of the supernatant solution for IL-2 activity revealed an activity of 50,400 units (8.4% of the activity of the original solution), indicating that 91.6% of the IL-2 had been bound to the beads.
  • Recombinant IL-2 (Amgen, ala-125 analog, 100 ⁇ g IL-2, activity 660,000 units) was immobilized on 0.93 ⁇ m blue-dyed polystyrene beads (Polysciences) using a bifunctional aldehyde following the procedure described in
  • Example 1 Because of the smaller bead size, however, longer centrifugation times (10 minutes) were required to effect the complete separation of the beads from the supernatant. Following the final washes, the beads were suspended in 0.5 ml of the storage buffer used in Example
  • Recombinant IL-2 (Amgen, ala-125 analog) was immobilized on blue-dyed polystyrene particles (Polysciences, 421 ⁇ m) using a bifunctional aldehyde in the following manner. Blue-dyed polystyrene particles (10 mg) were washed three times with 1.0 ml-portions of PBS (pH 7.40). They were then activated with glutaraldehyde and coupled to recombinant IL-2 (0.2 ml of aqueous IL-2 solution, 200 ⁇ g IL-2, activity 1.5 x 10 6 units) following the procedure described in Example 1.
  • Example 2 Following coupling and processing as described in Example 1, the beads were stored in 1.0 ml of the storage buffer used in Example 1 at 4°C. A determination of the IL-2 activity in the supernatant revealed an activity of 176,000 units (11.7% of the activity of the original solution), indicating that 88.3% of the IL-2 had been bound to the particles.
  • IL-2 (Amgen, ala-125 analog) concentration in the immobilization process was demonstrated in the following manner.
  • the pellets obtained from eight 0.125 ml-aliquots of a 2.5% aqueous suspension of blue-dyed polystyrene beads (9.64 ⁇ m) were washed with PBS and activated with glutaraldehyde as described in Example 1, except that the reactions were carried out at one-half the scale.
  • the activated beads were then suspended in various amounts of PBS and IL-2, as designated in Table 2, and allowed to react at room temperature overnight. Following this coupling reaction, the beads were processed according to the procedure described in Example 1, suspended in 0.25 ml-portions of the storage buffer, and kept at 4°C until used.
  • the pellet obtained from a 0.125 ml-aliquot of a 2.5% aqueous suspension of blue-dyed polystyrene beads was washed with three 0.5 ml-portions of PBS, activated with 0.5 ml of 8% glutaraldehyde/PBS, and suspended in a solution of recombinant IL-2 (0.032 ml of aqueous IL-2 solution, 32 ⁇ g IL-2, activity 60,000 units) in 0.4 ml of PBS. After allowing the reaction to proceed by mixing at room temperature overnight, the reaction mixture was centrifuged and the supernatant was carefully removed and preserved. The pellet was resuspended in 0.5 ml of PBS and the mixture was centrifuged.
  • the supernatant was removed and added to the first supernatant.
  • the beads were processed following the procedure described in Example 1, suspended in 0.25 ml of the storage buffer, and kept at 4°C until used.
  • a determination of the IL-2 activity in the supernatant revealed an activity of 5,700 units (9.5% of the activity of the original solution), indicating that 90.5% of the IL-2 had been bound to the beads.
  • Recombinant IL-2 (Amgen, ala-125 analog) was immobilized on 9.67 ⁇ m Polybead ® carboxylate microspheres (Polysciences, carboxylate modified polystyrene) using a water-soluble carbodiimide in the following manner.
  • the beads were suspended in 0.4 ml of PBS, and 3.0 mg of 1-ethyl-3-( 3-dimethylaminopropyl)carbodiimide-HCl (EDCI, Pierce Chemicals, Rockford, IL) was added and dissolved.
  • An aqueous solution of recombinant IL-2 (0.05 ml, 50 ⁇ g IL-2, activity 375,000 units) was then added. After mixing overnight at room temperature, the reaction mixture was centrifuged and the supernatant was carefully removed and preserved. The pellet was resuspended in 0.5 ml of PBS and the mixture was centrifuged. The supernatant was removed and added to the first supernatant.
  • the beads were then processed according to the method described in Example 1, suspended in 0.25 ml of the storage buffer, and stored at 4°C until used.
  • An assay for IL-2 activity in the supernatant revealed an activity of 570 units (0.2% of the activity of the original solution), indicating that 99.8% of the IL-2 had been bound to the beads.
  • Recombinant IL-2 (Amgen, ala-125 analog) was immobilized on 9.67 ⁇ m Polybead ® carboxylate microspheres with a 6-aminocaproic acid linking arm using a watersoluble carbodiimide in the following manner.
  • the pellet obtained from a 0.25 ml-aliquot of carboxylate microspheres was washed as described in Example 6, suspended in 0.5 ml of PBS, and treated with 3.0 mg of N-hydroxysulfosuccinimide (sulfo-NHS, Pierce Chemicals, Rockford, IL) and 3.0 mg of EDCI. After vortexing to dissolve the reagents, the reaction mixture was gently mixed for 30 minutes at room temperature.
  • the slurry was then centrifuged and the supernatant was discarded.
  • the pellet was suspended in 0.5 ml of a 0.5 M solution of 6-aminocaproic acid in PBS.
  • the resulting slurry was mixed for 20 hours at room temperature and centrifuged. The supernatant was discarded.
  • the pellet was washed with three 0.5 ml-portions of PBS, resuspended in 0.35 ml of PBS, and treated with 0.05 ml of an aqueous solution of IL-2 (50 ⁇ g IL-2, activity 375,000 units) and 2.0 mg of EDCI. After vortexing to dissolve the reagents, the reaction mixture was gently mixed at room temperature overnight.
  • the slurry was then centrifuged, and the supernatant was carefully removed and saved.
  • the pellet was resuspended in 0.6 ml of PBS, and the mixture was centrifuged.
  • the supernatant was removed and added to the first supernatant.
  • the beads were then processed as described in Example 1, suspended in 0.5 ml of the storage buffer, and stored at 4°C until used.
  • a determination of the IL-2 activity present in the supernatant revealed an activity of 460 units (0.1% of the original solution), indicating that 99.9% of the IL-2 had been bound to the beads.
  • Recombinant IL-2 (Amgen, ala-125 analog) was immobilized on 9.67 ⁇ m Polybead ® carboxylate microspheres with a 1,6-diaminohexane/glutaraldehyde linking arm using a water-soluble carbodiimide in the following manner.
  • the pellet obtained from a 0.25 ml-aliquot of carboxylate microspheres was washed with three 1.0 ml-portions of PBS (pH 7.40), suspended in 0.5 ml of a 0.5 M solution of 1,6-diaminohexane in PBS (pH 9.50) and treated with 3.0 mg of EDCI.
  • the slurry was vortexed to dissolve the reagents and mixed for 20 hours at room temperature. This reaction mixture was centrifuged, the supernatant was discarded, and the pellet was washed with three 0.5 ml-portions of PBS (pH 7.40). The pellet was then suspended in 0.5 ml of 8% glutaraldehyde in PBS and mixed for 4 hours at room temperature. The slurry was centrifuged, the supernatant was discarded, and the pellet was washed again with three 0.5 ml-portions of PBS .
  • the resulting pellet was then suspended in 0.35 ml of PBS and treated with 0.05 ml of an aqueous solution of IL-2 (50 ⁇ g IL-2, activity 375,000 units). The slurry was mixed overnight at room temperature, centrifuged, and the supernatant was carefully removed and preserved. The pellet was resuspended in 0.6 ml of PBS, and the mixture was centrifuged. The supernatant was removed and added to the first supernatant. The beads were then processed as described in Example 1, suspended in 0.5 ml of the storage buffer, and stored at 4°C until used. A determination of the IL-2 activity present in the supernatant revealed 50,000 units (13.3% of the original), indicating that 86.7% of the IL-2 had been bound to the beads.
  • Recombinant IL-2 (Amgen, ala-125 analog) was immobilized on 9.67 ⁇ m Polybead ® carboxylate microspheres with a 1,12-diaminododecane/glutaraldehyde linking arm using- a water-soluble carbodiimide in the following manner.
  • the pellet obtained from 0.25 ml of carboxylate microspheres was washed with PBS (pH 7.40, 3 x 1.0 ml), suspended in 0.75 ml of 0.2 M 1,12-diaminododecane in PBS (pH 7.0), and treated with 5.0 mg of EDCI. After mixing for 18 hours at room temperature, the reaction mixture was centrifuged and the supernatant was discarded.
  • the pellet was washed with PBS (pH 7.40, 3 x 1.0 ml), and activated with 1.0 ml of 8% glutaraldehyde in PBS as described in Example 8. After activation, the slurry was centrifuged, the supernatant was discarded, and the pellet was washed again with three 0.5 ml-portions of PBS. The resulting pellet was then suspended in 0.4 ml of PBS, treated with 0.1 ml of an aqueous IL-2 solution (100 ⁇ g IL-2, activity 750,000 units). The mixture was allowed to react overnight at room temperature. The slurry was centrifuged and the supernatant was carefully removed and preserved.
  • PBS pH 7.40, 3 x 1.0 ml
  • the pellet was resuspended in 0.5 ml of PBS, and the mixture was centrifuged. The supernatant was removed and added to the first supernatant.
  • the beads were then processed as described in Example 1, suspended in 0.5 ml of the storage buffer, and stored at 4°C until used. A determination of the IL-2 activity present in the supernatant showed an activity of 42,000 units (5.6% of the original), indicating that 94.4% of the IL-2 had been bound to the beads.
  • Recombinant IL-2 (Amgen, ala-125 analog) was immobilized on 65 ⁇ 25 ⁇ m Polybead ® carboxylate microspheres (Polysciences) with a 1,12-diaminododecane/glutaraldehyde linking arm using a watersoluble carbodiimide in the following manner.
  • the pellet obtained from 0.50 ml of a 2.5% suspension of 65 ⁇ 25 ⁇ m carboxylated polybeads was washed with PBS (pH 7.40, 3 x 1.0 ml), suspended in 1.0 ml of 0.2 M 1,12-diaminododecane in PBS (pH 6.00), and treated with 10 mg of EDCI.
  • the reaction mixture was centrifuged and the supernatant was discarded.
  • the pellet was washed with PBS (pH 7.40, 3 x 1.0 ml), and activated with 1.0 ml of 8% glutaraldehyde in PBS as described in Example 8. After activation, the slurry was centrifuged, the supernatant was discarded, and the pellet was washed again with three 0.5 ml-portions of PBS.
  • the resulting pellet was then suspended in 0.75 ml of PBS, and treated with 0.25 ml of an aqueous IL-2 solution (0.1025 mg IL-2, activity 900,000 units). The mixture was allowed to react by mixing overnight at room temperature.
  • the beads were processed as described in Example 1, suspended in 0.5 ml of the storage buffer, and stored at 4°C until used.
  • a determination of the IL-2 activity present in the supernatant revealed an activity of 144,450 units ( 16 . 0% of the original ) , indicating that 84% of the IL-2 had been bound to the beads.
  • Recombinant IL-2 (Amgen, ala-125 analog) was immobilized on 9.67 ⁇ m Polybead ® carboxylate microspheres with a 1,12-diaminododecane linking arm via free carboxyl groups on IL-2 using a water-soluble carbodiimide in the following manner.
  • the pellet obtained from 0.25 ml of carboxylate microspheres (9.67 ⁇ m) was washed with PBS (pH 7.40, 3 ⁇ 1.0 ml), and reacted with 1,12-diaminododecane/EDCI as described in Example 9. After mixing for 18 hours at room temperature, the reaction mixture was centrifuged and the supernatant was discarded.
  • the modified beads were then thoroughly washed with PBS (pH 7.40, 3 ⁇ 1.0 ml), resuspended in 0.4 ml of PBS, treated with 0.1 ml of an aqueous IL-2 solution (41 ⁇ g IL-2, activity 360,000 units) followed by 5.0 mg of EDCI, and mixed overnight at room temperature.
  • the reaction mixture was centrifuged and the supernatant was carefully removed and saved.
  • the pellet was resuspended in 0.5 ml of PBS and the mixture was centrifuged. The supernatant was removed and added to the first supernatant.
  • the beads were then suspended in 1.0 ml of 1% BSA/PBS and mixed for 30 minutes at room temperature.
  • the mixture was centrifuged and the supernatant was discarded.
  • the pellet was washed with the BSA/PBS solution (3 ⁇ 1.0 ml) and finally suspended in 0.5 ml of the storage buffer, and stored at 4°C until used.
  • a determination of the IL-2 activity present in the supernatant revealed 834 units (0.2% of the original), indicating that 99.8% of the IL-2 had been bound to the beads.
  • IL-2 Recombinant IL-2 (Amgen, ala-125 analog) was reacted with a ten-fold molar excess of methoxypolyethylene glycolyl N-succinxmidyl glutarate (MW 4800) [Abuchowski et al., Cancer Biochem. Biophys., 1, 175 (1984)] following the procedure described by Katre and Knauf in International Patent Application Number PCT/US86/01252 (International Publication Number WO87/00056), incorporated herein by reference.
  • the modified IL-2 was purified by size exclusion chromatography on a Bio-Gel P-10 column using PBS (pH 7.40) as the eluting solvent.
  • the purified column fraction used for this experiment contained 764,000 units of IL-2 activity per ml of buffer.
  • the modified IL-2 was immobilized on 9.67 ⁇ m Polybead ® carboxylate microspheres using a
  • 1,12-diaminododecane linking arm in the following manner.
  • the pellet obtained from 0.15 ml of carboxylate microspheres was reacted with 1,12-diaminododecane in the presence of EDCI following the procedure described in Example 9. After mixing for 18 hours at room temperature, the reaction mixture was centrifuged and the supernatant was discarded. The modified beads then were thoroughly washed with PBS (pH 7.40, 3 x 1.0 ml), resuspended in 0.3 ml of PBS, treated with 0.3 ml of the modified IL-2 solution (activity 229,000 units) followed by 5.0 mg of EDCI, and allowed to mix at room temperature overnight. The slurry was centrifuged and the supernatant was carefully removed and saved.
  • the pellet was resuspended in 0.5 ml of PBS and the mixture was centrifuged. The supernatant was removed and added to the first supernatant. The beads were then suspended in 1.0 ml of 1% BSA/PBS and mixed for 30 minutes at room temperature. The mixture was centrifuged and the supernatant was discarded. The pellet was washed with the BSA/PBS solution (3 x 1.0 ml) and finally suspended in 0.5 ml of the storage buffer, and stored at 4°C until used. A determination of the IL-2 activity present in the supernatant revealed 509 units (0.2% of the original), indicating that 99.8% of the IL-2 had been bound to the beads.
  • Recombinant IL-2 (Amgen, ala-125 analog) was immobilized on 5.29 ⁇ m Polybead ® amino microspheres (Polysciences, amino functionalized polystyrene) using a bifunctional aldehyde in the following manner.
  • the pellet obtained from a 0.25 ml-aliquot of Polybead amino microspheres was washed with PBS (3 x 0.5 ml), activated with 0.7 ml of 8% glutaraldehyde in PBS following the procedure described in Example 1.
  • Example 14 After washing the beads with PBS (3 x 0.5 ml), they were suspended in 0.4 ml of PBS, and treated with 0.1 ml of an aqueous IL-2 solution (100 ⁇ g IL-2, 750,000 units). The mixture was mixed overnight at room temperature. The reaction mixture was then centrifuged and the supernatant was carefully removed and preserved. The pellet was resuspended in 0.5 ml of PBS, and the mixture was centrifuged. The supernatant was removed and added to the first supernatant. The beads were processed as described in Example 1, suspended in 0.5 ml of the storage buffer, and stored at 4°C until used. A determination of the IL-2 activity present in the supernatant revealed 44,500 units (5.9% of the original solution), indicating that 94.1% of the IL-2 had been bound to the beads.
  • Example 14 A determination of the IL-2 activity present in the supernatant revealed 44,500 units (5.9% of the original solution), indicating that 94.1% of
  • Recombinant IL-2 (Amgen, ala-125 analog) was immobilized on degradable Sephadex ® G-10 resin particles
  • the pellet was resuspended in 0.4 ml of PBS, treated with 0.1 ml of an aqueous solution of IL-2 (100 ⁇ g IL-2, activity 750,000 units), followed by 3.0 mg of EDCI, and mixed overnight at room temperature.
  • the resin was processed as described in Example 1, suspended in 0.5 ml of the storage buffer, and stored at 4°C until used. A determination of the IL-2 activity present in the supernatant 852 units (0.1% of the original), indicating that 99.9% of the IL-2 had been bound to the resin.
  • Example 15 A determination of the IL-2 activity present in the supernatant 852 units (0.1% of the original), indicating that 99.9% of the IL-2 had been bound to the resin.
  • Recombinant IL-2 (Amgen, ala-125 analog) was immobilized on degradable Sephadex ® G-10 particles with a
  • Example 14 7.5 ml was activated with CNBr following the procedure described in Example 14. The washed activated resin was then added to 50 ml of 1.0 M 1,6-hexanediamine in 0.2 M sodium borate (pH 9.0). The slurry was mixed at room temperature for 20 hours. The resin was collected by filtration, washed with 200 ml of H 2 O, and dried under high vacuum for 48 hours. A 10 mg portion of the dried resin was swollen and washed, as described in Example 12. The pellet was activated with 1.0 ml of 8% glutaraldehyde in PBS as described in Example 8.
  • the slurry was centrifuged, the supernatant was discarded, and the pellet was washed again with three 0.5 ml-portions of PBS.
  • the activated resin was suspended in 0.4 ml of PBS, and treated with 0.1 ml of an aqueous solution of IL-2 (100 ⁇ g IL-2, activity 750,000 units). The mixture was allowed to react with mixing overnight at room temperature. The slurry was then centrifuged and the supernatant was carefully removed and preserved. The pellet was resuspended in 0.5 ml of PBS and the suspension was centrifuged. The supernatant was removed and added to the first supernatant.
  • Example 16 The resin was then processed as described in Example 1, suspended in 0.5 ml of the storage buffer, and stored at 4°C until used. A determination of the IL-2 activity remaining in the supernatant revealed an activity of 29,800 units (4.0% of the original), indicating that 96.0% of the IL-2 had been bound to the resin.
  • Example 16 A determination of the IL-2 activity remaining in the supernatant revealed an activity of 29,800 units (4.0% of the original), indicating that 96.0% of the IL-2 had been bound to the resin.
  • Recombinant IL-4 (Amgen, natural sequence) was immobilized on 9.64 ⁇ m blue-dyed polystyrene beads in the following manner.
  • the pellet obtained from 0.25 ml of a 2.5% suspension of blue-dyed beads was washed with PBS (pH 7.40, 3 x 1.0 ml), and then activated with glutaraldehyde as described in Example 1.
  • the beads were then suspended in 1.0 ml of a commercial IL-4 formulation containing 10.0 ⁇ g IL-4 (activity 2 x 10 5 units) and 0.025% human serum albumin (HSA) in PBS.
  • the reaction mixture was mixed overnight at room temperature.
  • the beads were processed as descrxbed in Example 1, then suspended in 0.5 ml of the storage buffer and kept at 4°C until used. A determination of the IL-4 activity present in the supernatant obtained from the above coupling reaction could not be measured due to the lack of a quantifiable assay.
  • Recombinant IL-6 (Amgen, natural sequence) was immobilized on 9.64 ⁇ m blue-dyed polystyrene beads in the following manner.
  • the pellet obtained from 0.25 ml of a 2.5% suspension of blue-dyed beads was washed with PBS (pH 7.40, 3 x 1.0 ml), and then activated with glutaraldehyde as described in Example 1.
  • the beads were then suspended in 1.0 ml of a commercial IL-6 formulation containing 10.0 ⁇ g IL-6 (activity 1-2 x 10 5 units) and 0.025% HSA in PBS. The reaction mixture was mixed overnight at room temperature.
  • the beads were processed as described in Example 1, then suspended in 0.5 ml of storage buffer and kept at 4°C until used.
  • An assay of the supernatant solution from the above coupling reaction for IL-6 activity could not be quantified due to the lack of a suitable indicator cell line .
  • Recombinant murine granulocyte-macrophage colony stimulating factor (rMuGMCSF, Amgen) was immobilized on 0.93 ⁇ m blue-dyed polystyrene beads in the following manner.
  • the pellet obtained from 0.25 ml of a 2.5% suspension of 0.93 ⁇ m blue-dyed beads was washed with PBS (pH 7.40, 3 x 1.0 ml), and then activated with glutaraldehyde following the procedure described in Example 2.
  • the beads were then suspended in 0.5 ml of a commercial rMuGMCSF formulation containing 5.0 ⁇ g of the growth factor (activity 5 x 10 3 units) and 0.025% BSA in PBS.
  • the reaction mixture was mixed overnight at room temperature.
  • the beads were suspended in 0.5 ml of the storage buffer and kept at 4°C until used.
  • An assay of the supernatant solution for rMuGMCSF could not be quantified due to the unavailability of an indicator cell line.
  • Recombinant human granulocyte-macrophage colony stimulating factor (rHuGMCSF, Amgen) was immobilized on 0.93 ⁇ m blue-dyed polystyrene beads in the following manner.
  • the pellet obtained from 0.125 ml of a 2.5% suspension of 0.93 ⁇ m blue-dyed beads was washed with PBS (pH 7.40, 3 x 1.0 ml), and then activated with glutaraldehyde as described in Example 2.
  • the beads were then suspended in 0.6 ml of a commercial rHuGMCSF formulation that contained 3.0 ⁇ g of the growth factor (activity 120,000 units) and 0.025% HSA in PBS.
  • the reaction mixture was mixed overnight at room temperature.
  • the beads were suspended in 0.5 ml of storage buffer and kept at 4°C until used.
  • An assay of the supernatant solution for GMCSF activity revealed 46 units (0.04% of the original), indicating that 99.96% of human GMCSF had been bound to the beads.
  • Recombinant IL-3 (Amgen, natural sequence) was immobilized on 9.64 ⁇ m blue-dyed polystyrene beads in the following manner.
  • the pellet obtained from 0.25 ml of a 2.5% suspension of blue-dyed beads was washed with PBS (pH 7.40, 3 x 1.0 ml), and activated with glutaraldehyde following the procedure described in Example 1.
  • the beads were then suspended on 0.4 ml of PBS, treated with 0.1 ml of a commercial IL-3 formulation containing 20 ⁇ g IL-3 (activity 2 x 10 6 units) and 0.025% HSA in PBS.
  • the reaction mixture was mixed overnight at room temperature.
  • the beads were suspended in 0.5 ml of the storage buffer and kept at 4°C until used.
  • An assay of the supernatant solution for IL-3 activity revealed 14,144 units (0.70% of the original), indicating that 99.3% of the IL-3 had been bound to the beads.
  • the samples of beads comprising immobilized IL-2 were washed 3 times by suspension and centrifugation in a Beckman Microfuge in RPMI-1640 tissue culture medium (Whittaker M. A. Bioproducts, Inc., Walkersville, MD) containing 4% antibiotics (Fungi-Bact Solution, Irvine Scientific, Santa Anna, CA).
  • the IL-2 immobilized beads were resuspended in RPMI-1640 medium and used for in vitro growth experiments.
  • the Sephadex ® G-10 beads with immobilized IL-2 were very irregularly shaped and settled so fast it was impossible to accurately determine a bead/cell number. Therefore, fixed volumes of freshly vortexed beads were used in the experiment.
  • the IL-2 immobilized beads and the CTLL-2 cells were incubated for 48 hours in a 37°C incubator with a 5% CO 2 atmosphere. After 48 hours, 1 ⁇ Ci of [ 3 H]-thymidine (ICN Biomedicals Inc., Irvine, CA) was added and the mixture was incubated for an additional 4 hours.
  • the cells were collected via a Skatron cell harvester and counted in a liquid scintillation counter to determine the amount of cell growth as determined by [ 3 H]-thymidine incorporation.
  • the results are reported in Table 3 and demonstrate that all the above-listed immobilized IL-2 combinations support CTLL-2 cell growth.
  • Recombinant natural sequence IL-2 immobilized on 9.64 ⁇ m blue-dyed polystyrene beads was examined to determine if it would support in vitro growth of the IL-2 dependent cell line CTLL-2.
  • Recombinant natural sequence IL-2 was immobilized on 9.64 ⁇ m beads as described in Example 5.
  • the IL-2 immobilized beads were washed and assayed as described in Example 21. The result is reported in Table 4 and demonstrates that immobilized recombinant natural sequence IL-2 supports CTLL-2 cell growth.
  • Recombinant IL-2 (ala-125 analog) immobilized on 9.67 ⁇ m carboxylate beads with a 1,12-diaminododecane spacer arm attached to the IL-2 via a carboxyl group was examined to determine if it supports in vitro growth of the IL-2 dependent cell line CTLL-2.
  • Recombinant IL-2 was immobilized on 9.67 ⁇ m carboxylate beads with a 1,12-diaminododecane spacer via carboxyl groups on the IL-2 molecule as described in Example 11. The immobilized IL-2 beads were washed and assayed as described in Example 21.
  • IL-2 Chemically modified (polyethylene glycol) recombinant IL-2 (ala-125 analog) immobilized on 9.67 ⁇ m carboxylate polystyrene beads with a 1,12-diaminododecane spacer group was examined to determine if it supports in vitro growth of the IL-2 dependent cell line CTLL-2.
  • IL-2 was chemically modified and immobilized according to the procedure outlined in Example 12. The immobilized chemically modified IL-2 beads were washed and assayed as described in Example 21. The results of the cell growth are shown in Table 6 and demonstrate that PEG-IL-2 beads support CTLL-2 growth.
  • CTLL-2 cells on immobilized recombinant IL-2 (ala-125 analog) was measured as a function of time and compared to the growth of CTLL-2 cells on soluble IL-2.
  • Recombinant IL-2 was immobilized on 9.64 ⁇ m blue-dyed polystyrene beads as described in Example 1. The beads were washed as described in Example 21. Aliquots of IL-2 immobilized beads (1, 5, and 10 beads/cell) were added to individual wells in a 96-well flat-bottomed tissue culture plate followed by the addition of 1 ⁇ 10 4 CTLL-2 cells (an IL-2 growth dependent cell line).
  • the beads containing immobilized IL-2 and the CTLL-2 cells were incubated for various times in a 37°C incubator with a 5% CO 2 atmosphere. At the end of each time period, 1 ⁇ Ci of [ 3 H]-thymidine was added and the mixture was incubated for an additional 4 hours. The cells were collected using a Skatron cell harvester and counted in a liquid scintillation counter to determine cell growth. The results are graphically presented in Figure 3 along with the results of an analysis using soluble IL-2 (100 units/ml and 1000 units/ml).
  • IL-2 immobilized on 9.64 ⁇ m blue-dyed polystyrene beads was prepared as described in Example 1, and washed as described in Example 21. These IL-2 immobilized beads were tested for their ability to be reused and to maintain long term cell cultures. Aliquots of IL-2 immobilized beads were added to sterile 1.5 ml screw cap microfuge tubes (Sarstedt Inc., Princeton, NJ), inoculated with 1 ⁇ 10 4 CTLL-2 cells, and incubated for 72 hours in a 37°C incubator with 5% CO 2 atmosphere. To several of the cultures, 1 ⁇ Ci of [ 3 H]-thymidine was added and the mixture was incubated for an additional 4 hours.
  • the cells were collected via Skatron cell harvester and counted in a liquid scintillation counter to determine cell growth. The remaining cultures were centrifuged for 5 minutes in a Beckman microfuge and the supernatant was removed and discarded. These cultures were then washed 5 times with 1 ml of RPMI-1640 tissue culture medium containing 4% antibiotics, stirred by vigorous vortexing, and centrifuged (this procedure eliminates over 90% of the cells). After the fifth washing, the IL-2 immobilized beads were resuspended in fresh medium, fresh CTLL-2 cells were added, and the 72 hour growth cycle was repeated. This procedure was repeated several times. The results are presented in Table 7, which demonstrates that IL-2 immobilized beads supported growth of CTLL-2 cells for four 72 hour growth cycles while soluble IL-2 could only support significant CTLL-2 growth for two cycles.
  • PBL's human peripheral blood lymphocytes
  • IL-2 immobilized recombinant IL-2
  • Recombinant IL-2 was immobilized on 9.64 ⁇ m blue-dyed polystyrene beads as described in Example 1.
  • the immobilized IL-2 beads were prepared as described in Example 21 and used in the following experiment.
  • PBL's were isolated from healthy donors by the following procedure.
  • Lymphocytes were isolated from heparinized blood after centrifugation over LeucoPREP (Becton Dickinson & Co.) cell separation medium.
  • the crude lymphocyte preparation was washed 3 times by centrifugation in RPMI-1640 tissue culture medium containing 4% antibiotics and 5% human AB serum (heat inactivated.
  • Human PBL's grown on immobilized recombinant IL- 2 were examined to determine if they exhibit lymphokine-activated killer (LAK) cell activity.
  • Human PBL's were isolated as described in Example 28, activated for 96 hours with IL-2 immobilized beads prepared as described in Example 1, and washed as described in Example 21.
  • the LAK cell killing activity was assayed using the cell targets K562, Raji, and Daudi.
  • the assay for LAK cell killing used a 4 hour 51 Cr release assay that has been described in the literature. See T. L. Whiteside et al., Cancer Immunol. Immunother., 26, 1 (1988); H. F. Pross et al., J.
  • Recombinant IL-2 (ala-125 analog) immobilized on 9.64 ⁇ m blue-dyed polystyrene beads (Example 1) and 65 ⁇ m polystyrene beads (Example 10) were examined to determine if they stimulate murine lymphocytes in an ex vivo experiment to increase natural killing (NK) or lymphokineactivated killing (LAK) of a target cell line. That is, an ex vivo experiment was conducted to determine if the immobilized IL-2 beads could activate the host's immune system in the same manner that soluble IL-2 can activate LAK cell production in vivo. The experiment was performed as follows: Mature Balb/C male mice (groups of three, 17 weeks old) were injected i.p.
  • NK/LAK cell activity was assayed by a 4 hour 51 Cr release assay, also described in the above references.
  • the results of the ex vivo experiment are summarized in Table 9. This data indicates that soluble IL-2 activates murine splenocytes as expected, and immobilized IL-2 on 65 ⁇ m beads also activate LAK cells in the peritoneal cavity.
  • the LAK cell activity in the peritoneal cavity appears to be localized and may have a therapeutic value in the localized treatment of cancer.
  • Recombinant IL-4 was immobilized on 9.64 ⁇ m bluedyed polystyrene beads as described in Example 16.
  • the immobilized IL-4 beads were washed as described in Example 21, and used in a PHA (phytohaemagglutinin) costimulation experiment to induce T-cell proliferation.
  • Peripheral blood lymphocytes were obtained from healthy donors.
  • An enriched T-cell population was isolated from lymphocytes that were isolated from heparinized blood and separated over a Ficoll gradient (LSM, Lymphocyte Separation Medium, Organon Teknika Corp., Durham, NC).
  • Recombinant IL-6 was immobilized on 9.64 ⁇ m bluedyed polystyrene beads as described in Example 17.
  • the immobilized IL-6 beads were washed as described in Example 21, and used in a PHA (phytohaemagglutinin) costimulation experiment to induce T-cell proliferation.
  • Peripheral blood lymphocytes were obtained from healthy donors.
  • An enriched T-cell population was isolated from lymphocytes that were isolated from heparinized blood and separated over a Ficoll gradient (LSM, Lymphocyte Separation Medium).
  • the beads with immobilized rHuGMCSF were incubated with the AML-193 cells for 116 hours in a 37°C incubator with 5% CO 2 atmosphere. After 116 hours, 1 ⁇ Ci of [ 3 H]-thymidine was added and the mixture was incubated for an additional 4 hours. The cells were collected as described in Example 21. The results are reported in Table 12 and demonstrate that immobilized recombinant human GMCSF supports AML-193 cell growth.
  • Recombinant IL-3 immobilized on 9.64 ⁇ m blue-dyed polystyrene beads was examined to determine if it would support in vitro growth of an IL-3/GMCSF dependent cell line AML-193.
  • Recombinant IL-3 was immobilized on 9.64 ⁇ m blue-dyed beads as described in Example 20.
  • the immobilized IL-3 beads were washed as described in Example 21 and assayed as described in Example 33. The results are reported in Table 13 and demonstrate that immobilized recombinant IL-3 supports AML-193 cell growth.
  • Recombinant IL-1-beta (Amgen) was immobilized on 9.64 ⁇ m blue-dyed polystyrene beads in the following manner.
  • the pellet obtained from 0.15 ml of a 2.5% suspension of blue-dyed beads was washed with PBS (pH 7.40, 3 x 1.0 ml), then activated with glutaraldehyde as described in Example 1.
  • the beads were then suspended in 0.46 ml of PBS, treated with 0.04 ml of a commercial IL-1-beta formulation containing 8.0 ⁇ g IL-1-beta (activity 4 x 10 6 units) and 0.025% HSA in PBS.
  • the reaction mixture was mixed for 24 hours at room temperature. Following the coupling reaction, the beads were processed as described in Example 1, then suspended in 0.5 ml of the storage buffer and kept at 4°C until used.
  • Human sequence IL-1-alpha (R & D Systems) was immobilized on 9.64 ⁇ m blue-dyed polystyrene beads in the following manner.
  • the pellet obtained from 0.20 ml of a 2.5% suspension of blue-dyed beads was washed with phosphate buffered saline (PBS) (pH 7.40, 3 x 1.0 ml), and then activated with glutaraldehyde as described in Example 1.
  • PBS phosphate buffered saline
  • the activated beads were suspended in 0.42 ml of PBS, then treated with 0.08 ml of a formulation that contained 8.0 ⁇ g of the cytokine and 200 ⁇ g human serum albumin (HSA) in PBS.
  • the reaction mixture was mixed for 24 hours at room temperature.
  • Example 37 Following the coupling reaction; the beads were centrifuged, washed with PBS (0.5 ml), then treated with ethanolamine as described in Example 1. The beads were then washed (3 x 1.0 ml) with a solution containing 0.1% sodium dodecyl sulfate (SDS) in PBS in an effort to remove the last traces of any noncovalently bound cytokine. Following these washes, the beads were further processed as described in Example 1, then suspended in 0.5 ml of the storage buffer and kept at 4°C until used.
  • SDS sodium dodecyl sulfate
  • Recombinant human GCSF (rHuGCSF, Amgen) was immobilized on 9.64 ⁇ m blue-dyed polystyrene beads in the following manner.
  • the pellet obtained from 0.20 ml of a
  • rMuGMCSF Macrophage Colony Stimulating Factor
  • Recombinant murine GMCSF (Amgen) was immobilized on 0.93 ⁇ m blue-dyed polystyrene beads in the following manner. The pellet obtained from 0.25 ml of a 2.5% suspension of blue-dyed beads was washed with PBS (pH
  • Example 39 7.40, 3 x 1.0 ml), then activated with 8% glutaraldehyde as described in Examples 1 and 2.
  • the activated beads were then suspended in 0.50 ml of a commercial rMuGMCSF formulation containing 5.0 ⁇ g (activity 5 ⁇ 10 3 units.) of the growth factor and 0.025% BSA in PBS. The suspension was mixed for 24 hours at room temperature. Following the coupling reaction, the beads were processed as described in Examples 1 and 2, then suspended in 0.5 ml of the storage buffer and kept at 4°C until used.
  • a commercial rMuGMCSF formulation containing 5.0 ⁇ g (activity 5 ⁇ 10 3 units.) of the growth factor and 0.025% BSA in PBS.
  • the suspension was mixed for 24 hours at room temperature.
  • the beads were processed as described in Examples 1 and 2, then suspended in 0.5 ml of the storage buffer and kept at 4°C until used.
  • pellets obtained from two 0.2 ml-portions of a 2.5% suspension of 0.93 ⁇ m blue-dyed beads were washed with PBS (3 x 1.0 ml).
  • One pellet (labeled C) was then activated with 1.0 ml of 8.0% glutaraldehyde in PBS for 20 hours at room temperature as described in Examples 1 and 2.
  • the other pellet (labeled A) was suspended in 1.0 ml of PBS and also mixed for 20 hours. Both suspensions were centrifuged and the pellets washed with PBS (3 ⁇ 1.0 ml).
  • each pellet was then suspended in a 0.1 ml-portion of PBS and treated with 0.4 ml-portions (4.0 ⁇ g, activity 4000 units) of the commercial rMuGMCSF formulation used in Example 38.
  • the suspensions were then mixed overnight at room temperature, centrifuged, and the supernatants removed and saved.
  • the two pellets were again suspended in 0.5 ml portions of PBS, centrifuged, and the supernatants removed and added to the first supernatants (labeled A1 and C1, both ca. 1.0 ml).
  • the pellets were then treated with 1.0 ml portions of 0.5 M ethanolamine as described in Example 1.
  • the supernatants (labeled A2 and C2) were removed and saved.
  • the pellets were then suspended in 1.0 ml-portions of PBS, centrifuged, and the supernatants (labeled A3 and C3) were removed and saved.
  • the pellets were then suspended three times in 1.0 mlportions of 0.1% SDS/PBS, mixed for one hour, centrifuged, and the supernatants (labeled A4, A5, A6, C4, C5, and C6, respectively) were removed and saved.
  • the pellets were washed with 1.0 ml-portions of PBS, and the supernatants (labeled A7 and C7) were removed and saved.
  • the pellets were then treated with 1% BSA/PBS as described in Example 1, and the various supernatants (labeled A8, A9, A10, C8, C9, and C10, respectively) were removed and saved.
  • the beads were finally suspended in 0.5 ml of the storage buffer and, together with the supernatants, kept at 4°C until used.
  • Recombinant human TNF-alpha (Amgen) was immobilized on 9.64 ⁇ m blue-dyed beads in the following manner.
  • the pellet obtained from 0.2 ml of a 2.5% suspension of blue-dyed beads was washed with PBS (3 ⁇ 1.0 ml ) , then activated with glutaraldehyde as described in Example 1.
  • the washed beads were suspended in 0.46 ml PBS and treated with 0.04 ml of a commercial recombinant human TNF-alpha formulation containing 19.2 ⁇ g (activity 1.92 ⁇ 10 5 units) of the growth factor in a 0.04 M Tris/0.1 M NaCl buffer (pH 8.60).
  • the suspension was mixed for 24 hours at room temperature.
  • the beads were processed as described in Example 1, then suspended in 0.5 ml of the storage buffer and kept at 4°C until used.
  • Fibroblast Growth Factor Basic (Amgen) was immobilized on 2.85 ⁇ m blue-dyed beads in the following manner.
  • the pellet obtained from 0.2 ml of a 2.5% suspension of blue-dyed beads was washed with PBS (3 ⁇ 1.0 ml), then activated with glutaraldehyde as described in Example 1.
  • the washed beads were suspended in 0.44 ml PBS, then treated with 0.06 ml of a commercial FGFb formulation containing 30 ⁇ g of the growth factor in a 0.02 M sodium citrate/0.1 M sodium chloride buffer (pH 5.0).
  • the suspension was mixed for 24 hours at room temperature.
  • the beads were processed as described in Example 39, then suspended in 0.5 ml of the storage buffer, and together with the various supernatants, kept at 4°C until used.
  • TGF-beta-2 (R & D Systems) was immobilized on 2.85 ⁇ m blue-dyed beads in the following manner.
  • the pellet obtained from 0.2 ml of a 2.5% suspension of blue-dyed beads was washed with PBS (3 x 1.0 ml), then treated with glutaraldehyde as described in Example 1.
  • washed beads were suspended in 0.35 ml PBS, treated with 0.15 ml of a solution containing
  • the beads were processed as described in Example 39, then suspended in 0.5 ml of the storage buffer and, together with the various supernatants, kept at 4°C until used.
  • Recombinant human Interferon-alpha-2A (Roferon ® A, Roche Laboratories) was immobilized on 2.85 ⁇ m blue-dyed polystyrene beads in the following manner.
  • the pellet obtained from 0.2 ml of 2.5% suspension of blue-dyed beads was washed with PBS (3 ⁇ 1.0 ml), then treated with glutaraldehyde as described in Example 1.
  • the washed, activated beads were then suspended in 0.4 ml PBS and treated with 0.1 ml (activity 6 x 10 5 units) of a commercial recombinant human Interferon-alpha-2A aqueous formulation containing 0.9 mg sodium chloride, 0.5 mg HSA, and 0.3 mg phenol.
  • the suspension was mixed for 24 hours at room temperature. Following the coupling reaction, the beads were processed as described in Example 39, then suspended in 0.5 ml of the storage buffer and, together with the various supernatants, kept at 4°C until used.
  • Recombinant human EGF (rHuEGF, available from Amgen) was immobilized on 0.93 ⁇ m blue-dyed polystyrene beads in the following manner.
  • the pellet obtained from 0.2 ml of a 2.5% slurry of blue-dyed beads was washed with PBS (3 x 1.0 ml), then treated with glutaraldehyde as described in Examples 1 and 2.
  • the washed, activated beads were suspended in 0.35 ml PBS, then treated with 0.15 ml of a solution that contained 25.0 ⁇ g of the growth factor in PBS (pH 7.40).
  • the suspension was mixed for 18 hours at room temperature. Following the coupling reaction, the beads were processed as described in Examples 1 and 2, then suspended in 0.5 ml of the storage buffer and kept at 4°C until used.
  • rHuPDGF Red-Dyed Polystyrene Beads (2.85 ⁇ m)
  • rHuPDGF Recombinant human PDGF
  • Bachem was immobilized on 2.85 ⁇ m blue-dyed polystyrene beads in the following manner.
  • the pellet obtained from 0.2 ml of a 2.5% suspension of blue-dyed beads was washed with PBS (3 x 1.0 ml), then treated with glutaraldehyde as described in Example 1.
  • the washed beads were suspended in 0.35 ml PBS, and treated with 0.15 ml of a solution containing 15.0 ⁇ g of the growth factor in sterile water.
  • the suspension was mixed for 20 hours at room temperature.
  • the beads were processed as described in Example 1, then suspended in 0.5 ml of the storage buffer and kept at 4°C until used.
  • Recombinant human erythropoietin was obtained from Amgen as a liquid formulation containing 5000 units activity per ml of a solution comprised of 50% glycerol in 0.025 M HEPES buffer (pH 7.20).
  • the strip was covered and incubated at 35°C for 3 hours.
  • the supernatants A-D were then removed and saved for residual activity assays.
  • the wells were washed with buffer (2 ⁇ 0.1 ml), then treated with 0.2 ml portions of freshly prepared 1% BSA/PBS and again incubated at 35°C for one hour. These supernatants were discarded.
  • the wells were then thoroughly washed (3 x 0.2 ml) with RPMI-7640 tissue culture medium containing 1% Fungizone, then filled with the same media.
  • the strip was covered and kept at 4°C until used.
  • Recombinant IL-1-beta Polystyrene Beads Recombinant IL-1-beta immobilized on 9.63 ⁇ m blue-dyed polystyrene beads induces the murine lymphoma cell line LBRM.TG6 (American Type Culture Collection Co., Rockville, MD) to synthesize IL-2 which was then assayed in the IL-2 dependent CTLL-2 cell line.
  • the immobilized IL-1-beta beads were washed three times by suspension and centrifugation as described in Example 21.
  • IL-1-beta beads in conjunction with a suboptimal concentration of PHA [Phytoheamagglutinin P, Wellcome Foundation, Danford, England] (10 ⁇ g/ml) were added to 5 x 10 4 LBRM.TG6 cells [J. W. Larrick et al., J. Immunol. Methods, 79, 39 (1985] in 100 ⁇ l of Iscove's MEM, (Whittaker M.A. Bioproducts, Walkersville, MD) and incubated for 48 hours at 37°C in 5% CO 2 . The reaction was stopped by placing the LBRM.TG6 cells at 4°C for 24 hours.
  • PHA Physical Heamagglutinin P, Wellcome Foundation, Danford, England
  • CTLL-2 cell growth was dependent on IL-2 concentration and was measured by the up-take, and oxidation of the tetrazolium salt MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide) [T. Mosmann, J. Immunol Meth., 65, 55 (1983); and M.B. Hansen, S.E. Nielson and K. Berg, J.
  • MTT tetrazolium salt
  • IL-1-alpha beads in conjunction with a suboptimal concentration of PHA [Phytohemagglutinin P, Wellcome Foundation, Danford, England] (10 ⁇ g/ml) were added to 5 x 10 4 LBRM.TG6 cells in 100 ⁇ l of Iscove's MEM and incubated for 48 hours at 37°C in 5% CO 2 . The reaction was stopped by placing the LBRM.TG6 cells at 4°C for 24 hours. Next, 50 ⁇ l of the LBRM.TG6 cell supernatant was removed and added to 50 ⁇ l of fresh CTLL-2 cells. The released soluble IL-2 was assayed as described in Example 49. The results are reported in Table 15 and indicate that IL-1-alpha beads activate the release of soluble IL-2 from LBRM.TG6 cells, and that the IL-2 released by the LBRM.TG6 cells supports growth of IL-2 dependent CTLL-2 cells.
  • PHA Physical Hemagglutinin P, Wellcome Foundation, Danford, England
  • Recombinant human GCSF immobilized on 9.64 ⁇ m blue-dyed polystyrene beads was examined to determine if it would support in vitro growth of a growth factor (GCSF) dependent cell line AML-193, American Type Culture Collection.
  • GCSF growth factor
  • Recombinant human GCSF was immobilized on 9.64 ⁇ m blue-dyed polystyrene beads as described in Example 37.
  • the immobilized rHuGCSF beads were washed as described in Example 21.
  • the growth assay for AML-193 cell line was as follows. Aliquots of the washed beads were added to individual wells in a 96-well flat-bottomed tissue culture plate followed by the addition of 1 x 10 4 AML-193 cells as in Example 33.
  • rMuGMCSF Recombinant Murine GMCSF Immobilized on 0.93 ⁇ m Polystyrene Beads Stimulates Granulopoiesis in BDFl Mice
  • Recombinant murine GMCSF immobilized on 0.93 ⁇ m blue-dyed polystyrene beads stimulates granulopoiesis in BDFl mice.
  • Recombinant murine GMCSF was immobilized on 0.93 ⁇ m blue-dyed polystyrene beads as described in Example 38.
  • Immobilized rMuGMCSF beads like soluble rMuGMCSF, stimulates granulopoiesis in the peripheral blood of mice after an injection. Ishida et al., Acta. Haemat. , 8, 1 (1988) recently reported that GMCSF stimulates granulopoiesis in the peripheral blood of mice after a single injection of soluble GMCSF.
  • rMuGMCSF Recombinant Murine GMCSF Immobilized on 0.93 ⁇ m Polystyrene Beads Stimulates Granulopoiesis in
  • rMuGMCSF Recombinant murine GMCSF immobilized on 0.93 ⁇ m blue-dyed polystyrene beads stimulates granulopoiesis in cyclophosphamide-treated mice.
  • Recombinant murine GMCSF was immobilized on 0.93 ⁇ m bluedyed polystyrene beads as described in Example 38.
  • Immobilized rMuGMCSF beads like soluble rMuGMCSF stimulates granulopoiesis in the peripheral blood of mice after an injection. Ishida et al., Acta.
  • BDFl mice were injected with cyclophosphamide (250 mg/Kg weight) at zero time to deplete the neutrophil cell count. Twenty-four hours later, either soluble rMuGMCSF (2 units injected i.p. every 12 hours for 6 days; or 2 units injected i.v. on days 1, 3, and 5), or immobilized rMuGMCSF (2 units injected i.v. on days 1, 3, and 5) was administered.
  • Peripheral blood was drawn from the retro-orbital sinus of BDFl mice at day 0, 3, 5, 7, and 9 and the number of neutrophils (PMN)/ml was determined from a complete blood count. The results are shown in Figure 6 and indicate that immobilized rMuGMCSF is active in vivo. Furthermore, rMuGMCSF beads stimulate PMN production in numbers and rates comparable to soluble rMuGMCSF.
  • Recombinant murine GMCSF covalently attached to 0.93 ⁇ m blue-dyed polystyrene beads retain biological activity (i.e., promote the growth of DA1-E5 cells) while rMuGMCSF adsorbed to 0.93 ⁇ m blue-dyed polystyrene beads do not retain biological activity (i.e., DA1-E5 cells do not grow).
  • Covalent and adsorbed rMuGMCSF blue-dyed polystyrene beads were prepared and washed as described in Example 39. Beads were washed three times before the assays described in Example 21 were performed.
  • DA1-E5 cells an IL-3/GMCSF/EPO dependent cell line, obtained from Dr. Larry Gilbert, University of Alta, Edmonton, Alberta, Canada, were used to assay both soluble fractions of rMuGMCSF and immobilized rMuGMCSF bead fractions (covalently bound or adsorbed).
  • the rMuGMCSF assay is as follows. DA1-E5 cells (1x10 4 ) were incubated with either soluble rMuGMCSF or immobilized rMuGMCSF (covalent or adsorbed) for 48 hours as described in Example 21. Either MTT or 1 ⁇ Ci of [ 3 H]-thymidine was added. The mixture was incubated for an additional 4 hours.
  • Example 55 Cells were harvested as described in Example 21. When the polystyrene beads were washed with sodium dodecyl sulfate (SDS), the adsorbed rMuGMCSF was removed (Figure 7). These beads no longer retained any biological activity. Covalently linked rMuGMCSF, however, did not wash off with SDS. These beads retained biological activity. The results are listed in Table 17.
  • SDS sodium dodecyl sulfate
  • Table 18 indicate that immobilized rHuILGF-1 on polystyrene beads induce lymphocyte growth in a PHA co-stimulation assay in serum-free medium.
  • Recombinant human ILGF-II was immobilized on 9.63 ⁇ m blue-dyed polystyrene beads as described in Example 41.
  • the immobilized rHuILGF-II beads were washed as described in Example 21.
  • the assay performed was as described in Example 55. Results are summarized in Table 19 and show that immobilized rHuILGF-II beads induce lymphocyte growth in a PHA co-stimulation assay in serum-free medium.
  • TNF-alpha tumor necrosis factor alpha
  • Recombinant tumor necrosis factor alpha immobilized on 9.64 ⁇ m blue-dyed polystyrene beads kill murine LM cells in a 72 hour killing assay.
  • Recombinant TNF-alpha was immobilized on 9.64 ⁇ m blue-dyed polystyrene beads as described in Example 42.
  • the immobilized TNF-alpha beads were washed three times as described in Example 21. TNF-alpha killing was assayed using murine LM cells (American Type Culture Collection). The assay was as follows.
  • FGFb Fibroblast Growth Factor Basic
  • Immobilized FGFb stxmulates growth of Murine 3T3 cells in growth factor depleted medium.
  • Immobilized FGFb beads prepared according to Example 43 were washed three times by suspension and centrifugation as described in Example 21.
  • Murine 3T3 cells (American Type Culture Collection) were grown in 1,2-dimethoxyethane (DME) medium with antibiotics and 10% calf serum (CS) as described by Gospodarowcz, Nature, 249, 123 (1974), incorporated herein by reference.
  • 3T3 cells were isolated by trypsinization and plated at either 600 or 2000 cells/well (96-well plates) in DME medium plus 10% CS. 3T3 cells were incubated over night at 37°C.
  • TGF-beta-2 Transforming Growth Factor-beta-2 (TGF-beta-2) Immobilized on 2.85 ⁇ m Polystyrene Beads Induces Growth of NRK-49F Cells in Growth Factor Depleted Medium
  • Immobilized TGF-beta-2 stimulates the growth of NRK-49F cells in growth factor depleted medium.
  • Immobilized TGF-beta-2 beads prepared according to the method in Example 44 were washed three times by suspension and centrifugation as described in Example 21.
  • NRK-49F cells (American Type Culture Collection) were grown in DME medium with antibiotics and 10% calf serum (CS) as described by Assoin et al., J. Biol. Chem., 258, 7155 (1973), incorporated herein by reference.
  • NRK-49F cells were isolated by trypsinization and plated at a concentration of 5 ⁇ 10 3 cells/well (96-well plates) in DME plus 10% CS medium.
  • the cells were incubated over night at 37°C in 5% CO 2 , and then washed twice in DME medium containing 0.2% CS. The medium was replaced with 100 ⁇ l DME plus 0.2% CS and the cells were incubated as above for three to four days to deplete the medium of growth factors.
  • soluble TGF-beta-2a, immobilized TGF-beta-2a, or 10% CS was added to individual wells and the NRK-49F cells were incubated an additional 17 hours. Then 1 ⁇ Ci of [ 3 H]-thymidine was added to the wells and the cells were incubated for another 4 hours before harvesting as described in Example 21.
  • the results are listed in Table 22. They indicate that immobilized TGF-beta-2 beads stimulate NRK-49F cells to grow in growth factor depleted medium.
  • Immobilized recombinant Human Interferon-alpha-2A kills the interferon sensitive HeLa S3 cell line.
  • Recombinant human Interferon-alpha-2a (INF-alpha-2a) immobilized on 2.85 ⁇ m blue-dyed polystyrene beads inhibits [ 3 H]-thymidine uptake in a human epitheloid carcinoma cell line HeLa S3 (i.e., kills HeLa S3).
  • Recombinant INF-alpha-2a was immobilized on 2.85 ⁇ m bluedyed polystyrene beads as described in Example 45. The immobilized INF-alpha-2a beads were washed three times as described in Example 21.
  • INF-alpha-2a killing was assayed using a human epithelioid carcinoma cell line HeLa S3 (American Type Culture Collection). INF-alpha-2a blocks [ 3 H]-thymidine uptake which leads to cell death.
  • the assay was as follows. Aliquots of either soluble INF-alpha-2a or immobilized INF-alpha-2a were added to individual wells in a 96-well flat-bottomed tissue culture plate followed by the addition of 1 ⁇ 10 4 HeLa S3 cells.
  • the beads with the INF-alpha-2a or soluble INF-alpha-2a were incubated for either 48, or 72 hours, at which time 1 ⁇ Ci of [ 3 H]- thymidine was added to each well and the mixture incubated an additional 4 hours.
  • the cells were collected as described in Example 21. The results are reported in Table 23 and demonstrate that immobilized INF-alpha-2a inhibits thymidine uptake which leads to the death of the HeLa S3 tumor cells.
  • Recombinant human epidermal growth factor (rHuEGF) immobilized on 0.93 ⁇ m blue-dyed polystyrene beads induces NRK-49F cells to grow in the absence of serum.
  • Recombinant human EGF was immobilized on 0.93 ⁇ m blue-dyed polystyrene beads as described in Example 46.
  • the immobilized rHuEGF beads were washed three times as described in Experiment 21. Serum contains many growth factors that are required by cells to grow in vitro.
  • the assay procedure for the NRK-49F cells was as follows. NRK-49F cells were maintained in DMEM (Dulbecco's Modified Eagles Medium, Whittakar M.A.
  • NRK-49F cells are plated at 5 x 10 3 cells per well in 96-well flat-bottomed tissue culture plates and incubated for 24 hours in the 10% CS. The cells were then washed with serum-free medium and then replenished with serum-free DMEM. Aliquots of either soluble rHuEGF or immobilized rHuEGF were added to the individual wells. The beads with the rHuEGF or soluble rHuEGF were incubated for 24 or 48 hours. Growth was then measured by the addition of 1 ⁇ Ci of [ 3 H]-thymidine to each well and the mixture was incubated an additional 6 hours. The cells were collected as described in Example 21. The results are reported in Table 24 and demonstrate that immobilized rHuEGF will induce murine NRK-49F cells to grow.
  • Example 62 The results are reported in Table 24 and demonstrate that immobilized rHuEGF will induce murine NRK-49F cells to grow.
  • rHuPDGF rHuPDGF immobilized on 2.85 ⁇ m blue-dyed polystyrene beads induce murine 3T3 cells to grow in the absence of serum.
  • Recombinant human PDGF was immobilized on 2.87 ⁇ m blue-dyed polystyrene beads as described in Example 47.
  • the immobilized rHuPDGF beads were washed three times as described in Experiment 21.
  • Serum contains many growth factors that are required by cells to grow in vitro. Most cells will not grow if they are depleted of these growth factors.
  • Murine Swiss 3T3 is such a cell line, which is available from American Type Culture Collection. The assay procedure was as follows. Swiss 3T3 cells were maintained in DMEM medium plus 10% calf serum (CS).
  • the 3T3 cells are plated at 1 x 10 4 cells per well in 96-well flat-bottomed tissue culture plates and grown to confluency. The medium was then changed to 2% CS, and the 3T3 cells remained viable but did not grow. Before growth factors were added, the cells were washed free of the 2% CS with serum-free DMEM, and then replenished with serumfree DMEM. Aliquots of either soluble rHuPDGF or immobilized rHuPDGF were added to the individual wells. The cells were incubated for 16 hours. Growth was measured by the addition of 1 ⁇ Ci of [ 3 H]-thymidine to each well and the mixture was incubated an additional 6 hours. The cells were collected as described in Example 21. The results are reported in Table 25 and demonstrate that immobilized rHuPDGF will induce murine 3T3 cells to grow.
  • Recombinant human erythropoietin (rHuEPO) immobilized on Co-BindTM polystyrene plates induces growth of EPO/lL-3 dependent DA1-E5 cells (see Example 54).
  • Recombinant human EPO was immobilized on Co-BindTM polystyrene plates as described in Example 48.
  • Wells containing Immobilized rHuEPO were washed five times with 1X PBS, followed by washing five times with Iscove's MEM containing 10% heat-inactivated serum, then filled with 0.050 ml of IMDM with 10% serum.
  • DA1-E5 cell growth was assayed as follows.
  • Recombinant human gamma-interferon (rHuIFN-gamma) was obtained from Genzyme, Boston, MA, as a liquid formulation that contained 1 ⁇ 10 6 U/ml (2.5 ⁇ 10 7 U/mg). An aliquot (0.02 mis, 2 ⁇ 10 4 U) of this solution was diluted to 2.0 ml with PBS to give a stock solution that was 1 ⁇ 10 4 U/ml. Four wells of the 8-well strip were then filled as shown below.
  • Human peripheral blood monocytes were isolated from blood drawn into a heparinized syringe and isolated by gradient centrifugation on 46% Percoll (Pharmacia, Newark, NJ). The monocytes were isolated from the interface, washed three times in phosphate buffered saline and resuspended in RPMI-1640 media containing 5% human AB sera to a concentration of 1 ⁇ 10 6 cells per ml.
  • Co-bindTM strips containing 4 wells gamma-interferon immobilized as in Example 67 were washed three times with phosphate buffered saline, washed three times with RPMI-1640 media containing 2% Fungi-Bact, and wiped with a sterile gauze.
  • EGF Epidermoid Growth Factor
  • Transformation Growth Factor Alpha TGF-alpha
  • VEF Vaccinia Growth Factor
  • SFGF Shape Fibroma Growth Factor
  • MEF Myxoma Growth Factor
  • BMP's Bone Morphogenic Proteins
  • FGF Fibroblast Growth Factor
  • IGF-1 Insulin-like Growth Factor 1
  • Soluble IL-2 1 (Control) 72,042 ⁇ 9574 --- 100 units 100%
  • Soluble IL-2 1 (Control) 109,900 ⁇ 10,550 --- 100 units 100%
  • Soluble IL-2 1 (Control) 134,190 ⁇ 15,700 --- 100 units 100%
  • IL-2 concentration was 1000 units/ml
  • Soluble IL-2 concentration was 1000 units/ml
  • Soluble IL-2 concentration was 1000 units/ml
  • Soluble IL-2 concentration was 1000 units/ml
  • Control is fresh IL-2 immobilized beads or soluble IL-2 set up at
  • Soluble IL-2 90 22 62 (100 units/ml)
  • Soluble IL-6 (100 units/ml) 175 ⁇ 263 ----
  • Soluble IL-3 concentration v/as 1000 units/ml.
  • Soluble IL-2 (10 units/ml) 0.122 ⁇ 0.003 100%
  • DPM's Incorporation Control Incorporation Control (DPM's) Soluble rHuILGF-I
  • DPM's Incorporation Control Incorporation Control
  • Assay is a 72-hour killing assay that does not use Actinomycin D that inhibits DNA synthesis. MTT measures oxidation via the mitochondria to give insoluble blue crystals.
  • DPM's Incorporation Control Incorporation Control (DPM's) Soluble rHuEGF

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Abstract

L'invention concerne des cytokines immobilisées comprenant des cytokines liées à un solide, de préférence un support biologiquement inerte. La cytokine liée, par exemple IL-2, conserve une activité biologique comparable lorsqu'elle est liée au support. Par conséquent on peut utiliser la cytokine liée de manière répétée et/ou en des quantités significativement inférieures, par rapport à une cytokine individuelle soluble. Les cytokines de l'invention comprennent, sans s'y limiter, IL-1-alpha, IL1-beta, rIL-2, IL-2, IL-3, IL-4, IL-6, MuGMCSF, HuGMCSF, HuGCSF, HuEPO, alpha-interferon, gamma-interferon, TNF-alpha, HuILGF-I, HuILGF-II, FGFB, TGF-beta-II, HuEGF, HuPDGF.
PCT/US1990/001031 1989-02-24 1990-02-23 Cytokines immobilisees WO1990009798A1 (fr)

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US48225990A 1990-02-16 1990-02-16
US482,259 1990-02-16

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Cited By (15)

* Cited by examiner, † Cited by third party
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EP0503583A1 (fr) * 1991-03-12 1992-09-16 Takeda Chemical Industries, Ltd. Composition à libération prolongée à base d'erythropoiètine
WO1994023701A1 (fr) * 1993-04-21 1994-10-27 Institut Pasteur Vecteur particulaire et composition pharmaceutique le contenant
US5422340A (en) * 1989-09-01 1995-06-06 Ammann; Arthur J. TGF-βformulation for inducing bone growth
US5591713A (en) * 1991-03-12 1997-01-07 Takeda Chemical Industries, Ltd. Water-soluble composition for sustained-release
US5824778A (en) * 1988-12-22 1998-10-20 Kirin-Amgen, Inc. Chemically-modified G-CSF
WO1999026674A3 (fr) * 1997-11-24 1999-09-16 Herbert P Jennissen Procede pour immobiliser des molecules mediatrices sur des materiaux d'implants inorganiques et metalliques
US6083521A (en) * 1993-08-27 2000-07-04 Novartis Ag Polymeric matrices and their uses in pharmaceutical compositions
EP1307216A1 (fr) * 2000-07-12 2003-05-07 Gryphon Therapeutics, Inc. Chemokines synthetiques bioactives a polymeres modifies et procedes de fabrication et d'utilisation
WO2003020320A3 (fr) * 2001-08-31 2003-10-30 Fraunhofer Ges Forschung Nanoparticules comportant le facteur de necrose des tumeurs tnf a activite biologique, immobilise dessus
EP1092979A3 (fr) * 1999-10-13 2004-01-21 A-Fem Medical Corporation Dispositifs fabriqués par liaison covalente des molécules sur une phase solide activée
US6759060B2 (en) 1993-03-02 2004-07-06 Biovector Therapeutics, S.A. Synthetic particulate vectors and preparation process
US6956027B2 (en) 1994-10-12 2005-10-18 Amgen Inc. N-terminally chemically modified protein compositions and methods
US7090835B2 (en) 1994-10-12 2006-08-15 Amgen, Inc. N-terminally chemically modified protein compositions and methods
US9095640B2 (en) 2000-08-01 2015-08-04 Morphoplant Gmbh Bioactive implant and method of use
US11066644B2 (en) 2018-02-01 2021-07-20 Nkmax Co., Ltd. Method of producing natural killer cells and composition for treating cancer

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JPH0363299A (ja) * 1989-08-01 1991-03-19 Chugai Pharmaceut Co Ltd 修飾エリスロポエチン
JP2006016323A (ja) * 2004-06-30 2006-01-19 Hiroshima Industrial Promotion Organization 生理活性バイオマテリアル

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US4240662A (en) * 1979-08-10 1980-12-23 Anderson Bernice J Folding shampoo chair
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US3639213A (en) * 1969-12-02 1972-02-01 Baxter Laboratories Inc Streptokinase chemically bonded to a carbohydrate matrix
US4240662A (en) * 1979-08-10 1980-12-23 Anderson Bernice J Folding shampoo chair
US4609546A (en) * 1982-06-24 1986-09-02 Japan Chemical Research Co., Ltd. Long-acting composition
JPS6153300A (ja) * 1984-08-23 1986-03-17 Takeda Chem Ind Ltd 固定化インタ−ロイキン−２
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Cited By (24)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5824778A (en) * 1988-12-22 1998-10-20 Kirin-Amgen, Inc. Chemically-modified G-CSF
US5422340A (en) * 1989-09-01 1995-06-06 Ammann; Arthur J. TGF-βformulation for inducing bone growth
EP0503583A1 (fr) * 1991-03-12 1992-09-16 Takeda Chemical Industries, Ltd. Composition à libération prolongée à base d'erythropoiètine
US5591713A (en) * 1991-03-12 1997-01-07 Takeda Chemical Industries, Ltd. Water-soluble composition for sustained-release
US6759060B2 (en) 1993-03-02 2004-07-06 Biovector Therapeutics, S.A. Synthetic particulate vectors and preparation process
US5891475A (en) * 1993-04-21 1999-04-06 Institut Pasteur Particulate vector and pharmaceutical composition containing it
FR2704145A1 (fr) * 1993-04-21 1994-10-28 Pasteur Institut Vecteur particulaire et composition pharmaceutique le contenant.
WO1994023701A1 (fr) * 1993-04-21 1994-10-27 Institut Pasteur Vecteur particulaire et composition pharmaceutique le contenant
US6083521A (en) * 1993-08-27 2000-07-04 Novartis Ag Polymeric matrices and their uses in pharmaceutical compositions
US6262127B1 (en) 1993-08-27 2001-07-17 Novartis Ag Polymeric matrices and their uses in pharmaceutical compositions
US7662933B2 (en) 1994-10-12 2010-02-16 Amgen Inc. N-terminally chemically modified protein compositions and methods
US7090835B2 (en) 1994-10-12 2006-08-15 Amgen, Inc. N-terminally chemically modified protein compositions and methods
US6956027B2 (en) 1994-10-12 2005-10-18 Amgen Inc. N-terminally chemically modified protein compositions and methods
US7255872B2 (en) 1997-11-24 2007-08-14 Morphoplant Gmbh Method for immobilization of mediator molecules on metallic and ceramic implant materials
US6635269B1 (en) * 1997-11-24 2003-10-21 Morphoplant Gmbh Immobilizing mediator molecules via anchor molecules on metallic implant materials containing oxide layer
WO1999026674A3 (fr) * 1997-11-24 1999-09-16 Herbert P Jennissen Procede pour immobiliser des molecules mediatrices sur des materiaux d'implants inorganiques et metalliques
EP1092979A3 (fr) * 1999-10-13 2004-01-21 A-Fem Medical Corporation Dispositifs fabriqués par liaison covalente des molécules sur une phase solide activée
EP1307216A4 (fr) * 2000-07-12 2005-01-12 Gryphon Therapeutics Inc Chemokines synthetiques bioactives a polymeres modifies et procedes de fabrication et d'utilisation
EP1307216A1 (fr) * 2000-07-12 2003-05-07 Gryphon Therapeutics, Inc. Chemokines synthetiques bioactives a polymeres modifies et procedes de fabrication et d'utilisation
US9095640B2 (en) 2000-08-01 2015-08-04 Morphoplant Gmbh Bioactive implant and method of use
WO2003020320A3 (fr) * 2001-08-31 2003-10-30 Fraunhofer Ges Forschung Nanoparticules comportant le facteur de necrose des tumeurs tnf a activite biologique, immobilise dessus
US7368295B2 (en) 2001-08-31 2008-05-06 Fraunhofer-Gesellschaft Zur Foderung Der Angewandten Forschung E.V. Nanoparticles comprising biologically active TNF which is immobilized on the same
US11066644B2 (en) 2018-02-01 2021-07-20 Nkmax Co., Ltd. Method of producing natural killer cells and composition for treating cancer
US12098388B2 (en) 2018-02-01 2024-09-24 Nkmax Co., Ltd. Method of producing natural killer cells and composition for treating cancer

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CA2047730A1 (fr) 1990-08-25
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EP0460101A1 (fr) 1991-12-11

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