CA2047730A1 - Immobilized cytokines - Google Patents

Abstract

The present invention provides immobilized cytokines comprising cytokines bound to a solid, preferably biologically inert, support. The bound cytokine, for example IL-2, retains comparable biological activity when bound to the support. Accordingly, the bound cytokine is able to be utilized repeatedly and/or in significantly smaller quantities, as compared to an individual soluble cytokine. Cytokines of the present invention include, but are not limited to, IL-1-alpha, IL-1 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.

Description

Wo9o~os7s8 PcT/US90/olo3l

2~47730 I

IMMOBILIZED CYTOXINES

Technical F~eld The present invention relates to cytokines that are immobilized on a solid support.

Backqround of the Inv~ntion 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 produc~s of lymphocytes are often referred to as "lymphokines,' and those cytokines originally described as products of monccytes have been termed "monokines." Certain cytokines are also re~erred to as growth factors or colony stimulating factors, based on their effect on cell growth.
Examples of cytokines include: the lymphokines interleuXin~ -1), interleukin-2 (I~-2), and interleukin-3 (IL-3) the monokine gamma interferon; and the growth factors granulocyte-macrophage colony stimulating factor (GMCSF) and erythropoietin (EPO).
Various cytokines serve as endogenous regulators ~autocrines) and/or as intercellular signals. ~any of these cytokines, initially reco~nized 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, differPntiation, and/or protein synthesis.
Interleukin-l (IL-1), also known as lymphocyte activating factor, is produced by human monocytes, lymphocytes, endothelial cells, and fibroblasts. IL-l promotes lymphocyte differentiation, as indicated ~y changes in phenotypic cell surface markers. In addition, IL-1 stimulates T-lymphocyte functions and '"" ' Wo90t09798 ~ 0 4 7 ~ O PCT/US9OJ01031 increases the production of lymphokines such as IL-2, colony-stimulating factors (CSF), B-cell growth factor (BCGF), qamma-interferon (~-IFN), and lymphocyte-derived chemotactic factors (LDCF), each with their own biological effects. IL-l also augments the in vitro proliferation, differentiation, and anti~ody-producing functions of B-lymphocytes. These and other biological activities have made IL-l 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 T-lymphocytes to proliferate and enable normal T-lympho-cytes to be maintained continuously in culture. ~ike IL-l, 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.
F. Good et al., J. Immunol., 141, 972 (l98a) and A. Weinberg et al., J. Immunol., 140, 294 (1988).
Among its biological acti~ities when used as a pharmacological agent, in vitro IL-2 results in the proliferation and differentiation of a group of more selective T-cell populations ~nown as lymphokine activated killer cells, tumor infiltrating lymphocytes, and cytotoxic T-cells. Such cells have been demon-strated in vitro to be cytotoxic to allogenic normal target cells and to both immunogenic and nonimmunogenic tumor cells. See S. A. Rosenberg, J. Nat. Can. Inst., ~0 75, 595 (198S); S. A. Rosenberg, J. Immunol., 121, 195 (1978); and S. A. Rosenberg et al., Science, 233, 1318 (1986).
_ vitro lymphokine activated killer cells have been used in combination with the in vivo administration of interleukin-2 to achieve an improved antitumor effect. ~he infusion of in vitro IL-2 activated killer cells and the concurrent administration of IL-2 has , ', , ~ . .
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WO~0/0979X PCT/US90/01031 47'730 :3 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, 225, 1437 (1984); R. ~afrenier, and S. A. Rosenberg, Cancer Res., 45, 37~5 (1985): S. A. Rosenberg et al., N. Enql. J. Med., 316, 889 (1987); J. J. Mule et al., J. Immunol., 136, 3899 (1986); H. W. West et al., N. En~l. J. Med., 316, 898 (1987); S. A. Rosenberg et al., N. Enql. J. Med., 313, 1485 (1985).
The growth of tumor infiltrating lymphocytes obtained from human malignancies has 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., Can. Immunol. Immunother., 24, 76 (1987).
Additional cytokines synthesized by T-cells include migration inhibition factor (inhibits the random migration of macrophages); leukocyte inhibition factor tinhibits the random migration of neutrophils);
macrophage activation factor (enhances the cytolytic activity of macrophages); fibro~last activation fator (stimulates prolifer~tion of fibroblasts); and interleukin-3 (IL-3) (activity similar to colony-stimulating factor).
Although the mechanistic details for cytokine activity are not known with certainty, the general mechanism for 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; a~d 3~ internalization of the cytokine-receptor complex where internal interactions result in proliferation, growth, differentiation, and/or the expression of specialized cell function.
Specifically, in the case of IL-2, the Wo ~09798 PCT/US90/01031 ~04~'73l) interaction of IL-2 with ~-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. Association of IL-2 with the high affinity complex results in proliferation. In 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.
A. Smith, Science, 240, 1169 (1988).
It has been suggested that cell surface associated events are sufficient for activity and that internalization of the receptor-ligand complex is not required, at least in cèrtain cases. Porcine insulin and murine alpha/beta interferon bound to Sepharose via cyanogen bromide activation have been reported to have biological activity, through cell surface associated events. See P. Cuatrecases, Proc. Nat. Acad. Sci. USA, 63, 450 (1969); H. Ankel et al., Proc. Nat. Acad. SC1.
USA, 70, 2360 (1973); and C. Chaney et al., Proc. Soc. Exp. Biol. Med., 147, 293 (1974).
The accuracy of these reports has been doubted by persons in the art, particularly because of the known instability of the particular covalent bond formed. See W. H. Scouten, Methods in Enzymoloqy, Klaus Mosbach, ed., Academic Press Pub., 135, 31 (1987). The question of the necessity of internalization has remained a debated issue. See E. DeMaeyer and J. DeMaeyer-Guignand, Interferon and Other RequlatorY
Cytokines, John Wiley and Sons Pub., 67-90 (1988).
The cost, availability, and toxicity of cytokines, such as IL-2, can be a limiting factor in the usefulness of the cytokine as a biologically active agent. Therefore, it would be desirable to be able to reuse and/or use less of a particular cytokine while retaining a substantial amount of their biological activity, with possibly decreas~d toxicity.

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wo ~/n9798 PCT/~S90/01031 Therefore, a continuing need exists for modified cytokines that retain comparable, and in some cases, improved biological activity when compared with corresponding soluble or free cytokines, thus providing a biological1y active cytokine ~hat can be reused to stimulate biological activity and/or that can be used in significantly smaller quantities.

Br~ef DescriPtion of the Invention The present invention pro~ides immobilized cytokines comprising cytokines firmly bound to a solid, preferably biologically compatible, insoluble immobilizing support. 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, I~-l, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, tumor necrosis factor (TNF), qamma-interferon, alPha-interferont beta-interferon, erythropoietin tEPO), granulocyte colony stimulating factor (GCSF), murine granulocyte colony stimulating factor (~uGCSF), granulocyte-macrophage colony stimulating factor ~GMCSF), murine granulocyte-macrophage colony stimulating factor (NuGMCSF), insulin-like growth factor I (ILGF-I), insulin-like growth factor Il (ILGF-II), transformation growth factor beta (TGF-~), epidermoid growth factor (EGF), platelet derived growth factor (PDGF), and fibroblast growth factor-basic (FGFb). 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 .- : . . .

Woso/()979R PCT/VS90/01031 20~773~ .

covalent ~onding, preferably by means of a linking arm.
The cytoki~e is preferably firmly bound to the support in a manner that allows the activity of the cytokine to be stabili~ed by the immobilization. The activity thus is durable and reusable.
As used herein, "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. In other words, because cytokines have multiple, often overlapping, biological or regulatory effects, a bound cytokine of the present invention may demonstrate one or more activities the same as, or similar to, that of the free cytokine. Thus, in demonstrating efficiency of an immobilized cyto~ine of the present invention, one or more biological activities may be stabilized through immo~ilization. Thus, Pt 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.
The structure or length of the linkin~ 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 - 25 consisting of: (a) diamines, having the general formula NH2-~1-NH2, where R1 is a C2-C20 alkyl group; (b) amino acids, having the general formula NH2-R2-CO2H, where R2 is a C1-C20 alkyl group; and (c) dialdehydes, having the general formula oHC-R3-CHo, where R3 iS a Cl-C20 alkyl group.
Useful supports include, but are not limited to, fibers, microspheres, beads, particles, membranes, sheets, and the like.
As used herein, "cytokine" refers to the natural or recombinant form of the cytokine, as well as to modified s~quences, biologically active fragments or portions of cytokines, genetically or chemically .. . . ~ . ..
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WOs~/09798 PCT/US9OtOtO31 2~ 31) modified forms of a cytokine, biologically equivalent synthetic ligands, or mixtures thereof, which exhibit a substantially equivalent profile of ~ioactivity, 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 prolifera~ion, growth, differentiati~n and/or expression of specialized cell function, including inducing growth of a cytokine-dependent cell line, such as an IL-2 dependent cell line, by contacting it with an effective amount of an immobilized cytokine of the invention.

SummarY of the Drawin~s Fig. l is a graphical depiction of the growth of CTLL-2 cells (DPM's x 10-3) using IL-2 immobilized via a carboxyl group of the IL-2 as c~mpared to IL-2 immobilized via an amino group of the IL-2.
Fig. 2 is a graphical depiction of the concentration dependence of immobilized IL-2 (yg IL-2 in initial coupling reaction) on the growth of CThL-2 cells, a cytotoxic T-lymphocyte cell line, as determined by ~3H]-thymidine incorporation (DP~'s x 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 x 10-3), using immobilized IL-2 as a function of time (hours) relative to the growth of PBL's using soluble ~L-2.
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 v~v~r~o ~ u~ ulu~l 2047~
~3 of granulopoiesis as measured by the increase in white blood count of cyclophosphamide-treated mice receiving soluble or imm~bilized recombinant murine GMCSF
(rMuGMCSF).
Fig. 7 is a graphical depiction of the sta~ility of cavalent bound rMuGMCSF as measured by its retention in contrast to adsorbed rMuGMCSF following SDS
washes.

Detailed Descri~tion of the Invention Cvtok~nes Interleukin-2 (IL-2) is commercially available as T-cell growth factor (human interleukin-2 recombinant; T3267) and as derived from cultured rat splenocytes (TD892) from Sigma Chemical Co., St. Louis, MO. Recombinant IL-2 (ala-125 analog and natural sequence) is also commercially available from Amgen, Thousand Oaks, CA. Natur;~l sequence recombinant interleukin-3 (IL-3), natural sequence recombinant 2C interleukin-4 (IL-4), and natural sequance recombinant interleu~in-6 (IL-6) are commercially a~aila~le from Amgen, Thousand Oaks, CA.
Recombinant human granulocyte-macrophage colony stimulating factor (rHuG~CSF), recombinant human granulocyte colony stimulating factor (rHu&CSF), recombinant human erythropoietin (rHuEPO), recombinant murine granulocyte- macrophage colony stimulating factor (rMuG~CSF), recombinant human gamma interferon (rHuIFN-gamma), and recombinant human epidermoid growth ~ -30 factor (rHuEGF) and fibroblast growth factor-basic . -(FGFb) all are available from Amgen, Thousand Oaks, CA.
Recombinant human platelet derived growth factor (rHuPDGF), recombinant human insulin-like growth ~actor : :
I (rHuILGF-I), recombinant human insulin-like growth factor II (rHuILGF-II), and transformation growth factor alpha (TGF-alpha) are commercially available from Bachem, Torrance, CA. Transformation growth factor .,, ... . " . . . . .
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, WO ~/0979X PCT/US90/01031 ';"73~3 beta, porcine, (pTGF-beta) is commercially available from R L D Systems, Minneapolis, MN. Transformation growth factor beta is also commercially available from Collaborative Research, Bedford, MA. Recombinant interferon alpha is commercially available as Roferon~
from Roche Laboratories.
The biologically active portions of certain cytokines have also been isolated. The present invention also includes binding the biologically active portions of cytokines to a suitable support.
IL-l 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 Ia 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 antibody to the antigen-receptor complex. In a~dition, IL-l 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-l is produced ~y human lung endothelium where it is believed to function in the development of inflammatory infiltrates.
IL-1 is also a regulator of hematopoietic activity. IL-l 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 granulocyte production and function during inflammation. IL-1 also releases GMCSF from monocytes and enhances growth factor dependent proliferation of human hematopoietic precursors.
IL-l has demonstrated by its antitumor activity causing complete regression of relatively large immunogenic murine sarcomas by augmenting an ongoing "
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WosO/n979X PCT/~S90/01031 204'7'73"

T-cell response. IL-1 has a direct cytotoxic effect in vitro on human A375 melanoma cells. IL-l has also shown synergistic effects with interleukin-2 (IL-2) in the production of lymphokine activated Xiller cells This broad range of activity of IL-l as a regulator of lymphocyte function, hematopoiesis, and lymphocyte antitumor acti~ity has made ~L-1 a valua~le cytokine in a wide variety of in vivo and in vitro uses. See, for example, T. Hoang et al., J. Ex~. Ned., 168, 463 (1988); R. J. North et al., J. Ex~. Med., I68, 2031 (1988); B. Tartakovsky et al., J. Immunol., 141, 3863 (1988); A. H. Lichtman et al., Proc. Nat. Acad. Sci. USA, 85, 9699 (1988); B.
S. Bochner et al., J. Immunol., 139, 2297 (1987); and V.
C. Broudy et al., J.Immunol., 139, 464 (1987).
Interleukin-3 (IL-3), also known as multicolony stem cell activating factor, or as multicolony stimulating factor, 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 committed cells. IL-3 binds to a 140 kilodalton cell surface phosphoprotein. In primates, continuous infusion of ~L-3 results in a delayed, modest increase in the white cell count. However, IL-3 has a marked synergistic effect on the response to subsequent treatment with low doses of granulocyte-macrophage colony stimulating factor, suggesting that 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 (&CSF~ to enhance WO ~/0979X PCT/US90/01031 7'~30 neutrophil formation, and with GMCSF to enhance granulocyte and macrophage colony formation. This broad range of activity as a hematopoietic cytokine makes IL-3 a valuable adjunct to hematopoietic cytokine therapy.
S 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 tl988); R. J. Isfort et al., Proc. Nat. Acad. Sci.
USA, 85, 7982 (1988); D. Rennick et al., J. Immunol., I42, 161 (1989); and G. Gallagher et al., Clin. ExP. Immunol., 74, 166 (1988).
Interleukin-4 (IL-4) is also known as B-cell stimulatory factor-1 (~SF-1), B-cell differentiation factor (BCDF~, and B-cell growth factor 1 (BCGF-1). In the murine system, IL-4 enhances immunoglobulin IgGl and IgE production in lipopolysaccharide acti~ated cells, increases the expression of histocompatibility antigens on B-cells, and is required for the proliferation of anti-lgM activated B-cells.
In human studies a similar effect to that observed in the murine system on lymphocyte function has been described. High affinity receptors exist for IL-4 on both human hematopoietic and nonhematopoietic cells.
IL-4 can induce proliferation in unstimulated thymocytes, and the response is strongly augmented with mitogens. IL-4 also augments the mitogen induced `~ -stimulation of human peripheral T-cells in the presence of dexamethasone, which inhibits IL-2 production. I~-4 also down modulates IL-2 induced human B-cell proliferation, and inhibits I1-2 induced NR cell activation and proliferation. IL-4, in conjunction with IL-2, however, augments the growth of tumor infiltrating . . : .

W~ ~/09~98 20~7'~ PCT/US90/OtO31 lymphocytes to autologous human malignant melanoma. In addition to it~ effects on lymphoid cells, IL-4 interacts with GMCSF and EPO to enhance granulocyte-macrophage and erythroid cell forming units. See, f~r example, H. Spits et al., J. Immunol., 139, 1142 (1~87); Y. Kawakami et al., J. Exp. Med., 168, 2183 (1988); A. Nagler et al., J. Immunol., 141, 2349 (1988); A. Vazquez et al., J. Immunol., 142, 94 (1989);
T. DeFrance et al., J. ExP. Med., 16B, 1321 (1988);
S. Xarray et al., J. ExP. Med., 168, 85 (1988); and B. Brooks and R. C. Rees, Clin. Exp. Immunol., 74, 162 ~1988).
Interleukin-6 (IL-6) is also known as B-cell stimula~ory factor-2, interferon beta-2, and hybridoma-plasmacytoma growth factor. IL-6 is a multi-functional cytokine, initially described as a T-cell lymphokine with antiviral activity. IL-6 has been ~ --demonstrated to be produced by a ~ariety of cells including: monocytes, fibroblasts, hepatocytes, cardiac ~-myxomas, brain glial cells, and vascular endothelium.
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 mitog~n; 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, P. B. Sehgal et al., Science, 235, 731 (1987);
S. Shimizu et al., J. ExP. Med., 169, 339 (1989);
J. L. Ceuppens et al., J. Immunol., 141, 3868 (1988); G.
Tosato and S. E. Pike, J. Immunol., 141, 1556 (1988);
M. Lotz et al., J. ExP. Med., 167, 1253 (1988); and -L. Chen et al., Proc. Nat. Acad, Sci. VSA, 85, 8037 (1988).
Granulocyte-macrophage colony stimulating ,, .j . .

W090~0979~ ~r~ 3~ - PCTIUS90/01031 factor (GMCSF), yranulocyte 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, epithelial cells, or endothelial cells. Additionally, IL-1, also known as hemopoietin-l, participates in this regulatory network by enhancing the effects of IL-3, MCSF, GCSF, and GMCSF.
Studies in animals have shown that GMCSF, GCSF, and IL-3 increase the number of functional white blood cells, and that the effect is enhanced by IL-l.
Se~uential administration of IL-3 and GMCSF has resulted in an increased platelet count as well. Use of members of this group of cytokines in nonhuman primates has shown benefit in viral induced pancytopenia, chemotherapy, and irradiation therapy induced myelo-suppression, leukopenia following whole body irradiation or high dose cytotoxic chemotherapy, and autologous bone marrow transplant.
In man the administration of both GCSF and 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 G~CSF in man have included fever, rash, myalgia, fatigue, gastrointestinal dis~ress, thrombophlebitis, bone pain, pleuritis, pleural effusion, pericarditis, and pulmonary emboli. The only side effect noted with GCSF has been bone pain.
The demonstrated benefits of GCSF and GMCSF in man have included: restoration of hematopoiesis following myelo-suppressive cytotoxic chemotherapy;

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Woso/0979X 2~ 3~ PCT/US90/01031 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 myelodysplastic syndrome and aplastic anemia.
Administration of GNCSF to patients with AIDS associated leukopenia resulted in a significant increase in granulocytes and monocytes without increased viral production.
In addition to the hematopoietic effects descri~ed above, GMCSF has been demonstrated in vivo to activate monocytes to a tumorcidal state suggesting ~-another potential clinical application for this cytokine. GMCSF has also been demonstrated to stimulate lS 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. Enql. J. Med., 319, 1628 tl988); J. E. Groopman et al., N. Enal. J. Med., 317, 593 (1987); K. H. Grabstein et al., Science, 232, 506 (l986)`; S. Dedhar et al., Proc. Nat. Acad. Sci. USA, 85, 9253 (1988); and A. A. Jakubowski et al., N. Enql. J. Med., 320, 38 (1989~.
Erythropoietin (EPO) is the single cytokine required for the continued differentiation of the hematopoietic cells that produce mature red blood cells. In in vitro studies, the-combination of IL-3, G~CSF or GCSF with EP0 has been required for red cell production, suggesting that these cytokines are in~olved in the maintenance of the red cell precursor whereas EPO
is required for the terminal differentiation and maturation. See, for example, J. Suda et al., Blood, 67, 1002 (1986).
Tumor Necrosis Factor (TNF), also known as a multi-functional cytokine produced by monocytes-macrophages, is a particularly important WO90/0~798 PCT/US90/01031 ~7'~

mediator of inflammatory response. Two forms, ~NF-alpha tCachectin) and TNF-beta, exist. Among its effects TNF
is 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 osteo~last activity and bone reabsorption, promotion of angiogenesis, stimulation of collagenase and prostaglandin E2 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 macrophages and increasing their ability to specifically recognize and kill malignant cells. 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-l, GMCSF, platelet derived growth factor, and beta-2 interferon.
The major potential therapeutic effect of TNF
is its antitumor activity. TNF is the mediator of endotoxin induced tumor regre~sion. TNF may be involved in the antitumor ctivity 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-l are believed synergistic.
Initial clinical studies of TNF in man have included intravenous and intramuscular injections.
~oxicities 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 Immunol. Immunother., 26, 202 (1988~; Y. Ichir,ose et al., Cancer Immunol. Immunother., 27, 7 (1988);

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WO 90/09798 PCI/llS90/01031 2C~

P. B. Chapman et al., J. Clin. Onc., 5, 1942 (1987);
H. H Bartsch et al., Mol. Biother., 1, 21 (1988); and T. Steinmetz et al., J. ~iol. Resp. Mod., 7, 417 (1988).
Interferon (IFN~ 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 alpha-interferon, 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 nfibroblast 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). HuIF~-beta has about 40% amino acid homoloqy with HuIFN-alpha. The human interferon gamma gene exists as a single copy with some individual to individual allelism or difference in single amino acids. Gamma-interferon has no homoloqy to alPha- or beta-interferon.
Interferon, or virus-induced proteins with-anti-viral activity, have been identified from representatives of all vertebral classes except amphibia. ~he biological activity of various IFN-alpha su~types are relatively similar. The biological activity of INF-alpha and beta are also similar, but both differ from IPN-gamma. See E. DeNaeyer and J.
DeMaeyer-Guignard, Interferon and Other Requlatory CYtokines~ John ~iley and Sons, Pub., pp. 5-38 (1~88).
The major biological activities of alpha and beta interferon are: antiviral effects; induction of ~:~47~30 monocytes to express ma~or histocompatibility, complex class II antigens, and interleukin-l; antiproliferative effects; and regulation of human natural killer cell activity.
S Interferon alpha and beta have an antitumor effect that involves a number of mechanisms includin~, among others, an antiproliferative effect, induction of differentiation, regulation of oncogene expression, and stimulation of the immune response.
The exact biological effects may vary with the particular structural forms of alpha-interferon and with the sensiti~ity of the assay cell line. It is also possible to observe both positive and negative regulation, as for example, human interferon alpha or beta may inhibit the maturation of monocytes to macrophages. See E. DeMaeyer and J. DeMaeyer-Guidnand in Interferon and Other Re~ulatorY CYtokines, John Wiley and Sons, Pub., pp. 134-153 (1988).
A group of cytokines also termed growt~ 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 ~iological effects and amino acid sequence homology (as shown below in Table 1).
Although this group of cytokines has been associated with tissue repair, they have other biological effects.
In addition, other cytokines such as interleu~in-1 and interleukin-3, which regulate immune responses, also 3S have an effect on tissue repair.
Epidermal growth factor ( EGF ) is a key representative member of a family of structurally . .

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204>7'~30 .

related proteins including transformation growth factor (TGF) alpha, amphiregulin, and vaccinia growth factors.
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, fibroblasts, and endothelial cells (S. Nakagawa et al., Dif~erentiation, _ , 284 (198S)). It stimulates precocious eyelid opening and tooth eruption in newborn mice (S. Cohen, J. B. Chem., 237, 1555 (1962)), and is chemotactic for epithelial cells (J. Blay and K. D. Brown, J. Cell Physiol., 124, 107 (1985)). 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. ~odaro et al., Proc. Nat. Acad. Sci. USA, 77, 5258 (1980).
2~ 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 (PDGF) is purified from human blood platelets. See R. Ross and A. ~ogel, Cell, 14, 203 (1978). It consists of two polypeptide chains: the A chain ~124 amino acid residues); and the B chain (140 amino acid residues).
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 PD~F
receptors. See R. Ross, E. W. Raines, and D. F. Bowen-Pope, Cell, 45, 155 (1986). Platelet ~ ........... .

.
.

.

wo90~n9798 ~IQ~ 7~ V PCT/US90/01031 derived growth factor may also be obtained from porcine cells.
Transformatio~ growth factor beta(s) were originally identified by their ability to act synergistically with EGF or TGF-alpha to induce anchorage independent growth of NRK cells. See M.
A. Anzano et al., Proc. Nat. Acad. Sci. USA, 80, 6264 (1983). They have subsequently been shown to have multiple biological effects including, chemotaxis, mitogenesis, growth inhi~ition and the induction or inhibition of differentiation depending upon other growth factors present. See M. B. Sporn and A.
B. Roberts et al., J. Cell Biol., 105, 1039 t1987). In their mature form, TGF-betas are acid and heat-sta~le disulfide-linked homodimeric proteins of 112 amino acid residues which share 70% homology. See R. Derynck and J. A. Farrett et al., Nature, 3l6, 701 (1985). Another member of the family, beta-3, has recently been described. See J. M. Wozney and V. Rosen et al;, Science, 242, 1582 (1988).
Although they share a variety of biological activities, different forms of TGF also possess unique biological activities for select target cells. See F. Rosa and A. B. Roberts et al., Science, 239, 783 (1988). 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 embrya; and bone morphogenic proteins that are a group of polypeptides involved in the induction of cartilage and bone formation. See ~.
M. Nozney and V. Rosen et al., Science, 242, 1528 (1988).
Fibroblast growth factors (FGF) are WO9Ot09798 204~ PCT/US90/0103t 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 C. Dickson and P. E. Gordon, Nature, 326, 833 (1987).
Insulin-like growth factor I ~ILG-I) also known as Somatomedia C, and Insulin-like growth factor II
(ILG-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 acti~ity, 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 ~lucose 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 3'r3 cells after being rendered ncompetent~ with Platelet ~erived Growth Factors. (See R. C. Baxter, Adv. Clin. Chem., 25, 50 ~1986)~.
Cytokines react with cell surface receptors which themselves are complex and may consist of subunits. Portions of the cytokine may bind preferentially to various subunits of the receptor resulting in different biological and/or regulatory W090/09798 PCT/US90tO1031 20 ~ 3~

effects. The present invention also provides for immobilization of such cytokine fragments that may be directed to a particular subunit of the receptor.

Immobilizin~ 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 ~ioreactors.
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 attachment of the cytokine. Accordingly, the support preferably includes a surface having appropriate functional groups which can covalently bind to a site on the cytoXine (e.g., an amino or carboxyl site), or to a suitable linking arm that can bind to a site on the cytokine. If the intended support does not have suitable functional groups for cytokine binding, such groups can be provided by appropriate chemical modification of the support surface. For example, a nonfunctionalized polystyrene support can be provided with a functionalized surface by suitable functionalization of the aromatic rings (e.g., via bromination).
Not all 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 a~ailability 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 ;~047730 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 sf 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 pro~ide a site for binding: (a3 directly to a site on the cytokine; or (b) to a suitable linking arm. Such functional groups include hydroxyl (-OH), amino (-NH2 or -NHR, wherein R is alkyl or aryl), carboxyl (-CO2H), 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, acrylonitrile, acrylic acid, methacrylic acid, esters of acrylic and methacrylic acid, vinyl acetate, fluoroalkene, acrylamide, and methacrylamide;
carbohydrate supports, such as, agarose, cross-linked agarose, dextran, cross-linked dextran, inulin, hyalur~nic acid, cellulose, cellulose derivatives such as carboxym~thyl cellulose (CMC), starch and starch wo ~/09798 PCT/US90/01031 2~ '73~

derivatives (e.g., starch microspheres); and insoluble protein materials, such as, gelatin, collagen, or albumin.
The surface of the immobilizing support of the present invention is preferably nonporousO The use of materials having a nonporous surface, such a~
substantially spherical polymeric beads or microspheres, allows for ~inding of the cytokine to the outer surface of the support, there~y 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 $o block or substantially hinder the migration of the cytokine into the interior of the spheres. For use as a sustained release, biodegradable formulation, 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 ym, 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 ym.

Cvtokine ~inkinq Grou~s The immobilized cyto~ines 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 activity of the bound cytokine. Suitable linking arms include one or more bifunctional linking groups such as: (l) diamines, having the formula NH2-Rl-NH2, where is a C2-C20 alkyl group; (2~ amino acids, having the general formula NH2-R2-C02H, where R2 is a Cl-C20 alXyl .. . ..

.~ .

., 204 ~

group; and (3) dialdehydes, having the formula oHC-R3-CHo, where R3 is a Cl-C20 alkyl group. Two or more linking groups may be coupled to provide additional length. Examples of suitable linking groups include 6-aminocaproic acid, l,6-diaminohexane, 1,12-diaminododecane, glutaraldehyde, and mixtures thereof.
In a preferred embodiment of the invention, 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 tha~ is covalently attached to the functional group. Following extensive washing of the lS support having a functionalized surface, the biologically active moiety (cytokine) is attached to an exposed functional group or to the linking arm.
Acceptable methods of attachment include; (l) 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.
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.

;' ' " -' - , . ' . ' W090~09798 ~ ~ 7~ PCT/US90/01031 2'i The stability of the following bonds linking a protein to an insoluble matrix are co~sidered relatively weak:
NH

1. Isourea (Polymer-0-C-NH-Protein) 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), l-cyano-4-N,N-dimethylamine pyridinium tetrafluoro-borate, and the like.

N-Protein Il 2. Imidocarbonate (Polymer-0-C-0-Polymer) This linkage is also formed as above for isourea, from the reaction of an amino group on the protein tchiefly the lysyl side chain) with polyhydroxylic matrices activated as above.

The stabilities of the following protein-insoluble matrix bonds are considered relati~ely strong:

l. Urethane (Polymer-0-l-NH-Protein) 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.
X
/ ~/ .
2. Triazine ether (Polymer-0-C -~ N
N
NH-protein I' " , '' . , . , : : .

Woso/09798 PCTtUS90/01031 ;~0~'73~

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.

The stability of the following pr~tein-insoluble matrix bonds are considered relatively very strong:

1. Amine (Polymer-NR-Protein) This linkage is formed in a variety of ways, including the reaction of a protein amino group with (l) polyhydroxylic matrices that have been activated with such reagents as tresyl chloride, sulfonyl chloride and the lS like, with oxiranes (epoxides) such as bisoxirane and epichlorohydrin and with t2) polyamino matrices that have been activated with such reagents as glutaraldehyde.

O O ' 2. Amide (Polymer-C-N~-Protein or Polymer-NX-C-Protein) 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 achie~ed 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 ylutamic acid side chain carboxyl groups.
It is preferred that 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 `
, - . , ': ' :- : ,::
: , .

Wo ~/09798 PCT/US90/01031 ~'~34'7'. 3~

of the linking arms, support, and cytokine to optimize biological activity.

Use of th~ sound Cvtokine S 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, macropha~es, 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 cyt~toxic 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 or secondary bone marrow failure due, for example, to a lack of erythropoietin in chronic renal failure and/or renal failure in patients on renal dialysis; (6) the treatment of hard and soft tissue wounds ~y æurface ap~lication of the ~ound cytokines or in-situ placement of the bound cytokines; and (7) treatment of osteoporosis by the in vivo intravenous administration of the bound cytokines. See S. Nakagawa and S. Yoshida et al., Differentiation, 29, 284 (1985), and J. Blay and K.D. Brown, J. Cell PhYsiol., 124, 107 (1985).
The present i~vention will be further described by reference to the following examples.

.' ' ' ' ` ' . ' . ' , WO 90~0g798 ~ ` PCI`IUS90/01031 ExamPle 1 Attachment of I~-2 to Rlue-Dved Polvstvrene Beads (~.64 Ym) Recombinant IL-2 (Amgen, ~housand Oaks, CA, ala-125 analog) was immobilized on 9.64 ~m blue-dyed polystyrene beads tPolysciences, Warrington, PA) using a bifunctional aldehyde in the following manner. A 0.25 ml-aliquot of a 2.5% aqueous suspension of 9.64 ym blue-dyed polystyrene beads was diluted with 1.0 ml of phosphate buffered saline tPBS, 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. 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 yg IL-2, activity 600,000 units). The reaction mixture was mixed overnight at room temperature, 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. This combined supernatant solution (ca. 1.0 ml) was preserved at 4C for the subsequent determination of residual I~-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 ,: :
- '' ~, .

.

WO9D/0979N ~3~ 3~ PCT/US90/OtO3l supernatant was discarded. 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 tO.88~), BSA ~1~), glycerol ~5%), and sodium azide ~O.l~) in 0.~2 M sodium phosphate (pH 7.40)). The beads were stored at 4C until used.
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.

Example 2 Attachmont of I~-2 to Blue-D~ed Poly~yrene Beads (0.93 ~mL
Recombinant IL-2 (Amgen, ala-125 analog, lOO yg 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 1 and kep~ at 4~C until used. An assay of the supernatant solution for I~-2 activity revealed an activity of 18,000 units (2.7~ of the activity of the criginal solution), indicating that 97.3~ of the IL-2 had been bound to the beads.

Example 3 Attachment of IL-2 to Blue-DYed Polvstyrene Particles (421 ~m~
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 . .
.. .. : ~ : , .

Wo~0/097~X PCT/US90/01031 Z04773~ :30 and coupled to recombinant IL-2 (O.2 ml of aqueous IL-2 solution, 200 ~g IL-2, activity l.5 x lO6 units) following the procedure described in Example l. Following coupling and processing as descri~ed in Example l, the ~eads were stored in l.0 ml of the storage buffer used in Example l at 4C. A determination of the IL-2 activity in the supernatant revealed an activity of 176,000 units (ll.7~
of the activity of the original solution), indicating that 88.3% of the IL-2 had been bound to the particles.
Example 4 Attachment of IL-2 to Blue-D~ed Polvst~rene Beads (9.64 ~m): Effect of ~L-2 Solution Concentration The effect of 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 l, except that the reactions were carried out at one-half the scale. The activated beads were then suspended in various amounts of PBS and 1~-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 l, suspended in 0.25 ml-portions of the storage buffer, and kept at 4C until used. The supernatant solutions obtained from the various coupling reactions were assayed for the residual IL-2 activity present. The results appear in ~able 2. The difference between the activity of each IL-2 solution used in the coupling reactions (before) and that recovered in the resultant supernatant (after) yielded the value of % IL-2 incorporated.

WO 90t097g8 PCI'/US90/01031 ^20~'730`

Example S
Attachment of IL-2 (Recombi nant Natural Sec~uence~
to Blue-Dyed PolvstYre-ne Beads (~ ~m~
Recombinant IL-2 (Amgen, natural sequence) was immobilized on 9.64 ~m blue-dyed polystyrene beads following a procedure similar to that described in Example 1. 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, ac~ivated with 0.5 ml of 8% glutaraldehyde/PBS, and suspended in a solution of recombinant IL-2 (O.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 ~entrifuged and the supernatant was carefully remo~ed 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 des~ribed in Example 1, suspended in 0.25 ml of the storage buffer, and kept at 4C 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 I~-2 had been bound to the beads.

EXamP19 6 Attachment of IL-2 to Polvbead~- Carboxvlate MicrosPheres (9.67 ym~
Recombinant I~-2 (Amgen, ala-125 analog~ was Lmmobilized on 9.67 ~m Polybead~ carboxylate microspheres (Polysciences, carboxylate modified polystyrene) using a water-soluble carbodiimide in the following manner. The pellèt obtained from a 0.25 ml-aliquot of a 2.5% aqueous suspension of Polybead~carboxylate microspheres was washed with three 1.0 ml-portions of P~S. The beads were .

,' '' . ' . ,." ,, " ~ , ' ~ ' .
, . . . . . . .
.
. .

wo9o/os79x PcTlus9o/olo3 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 (O.05 ml, 50 y~ IL-2, activity 375,000 units) was then added. After mixingovernight 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 0.2S ml of the storage buffer, and stored at 4OC 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.

Example 7 Attachment of IL-2 to PolYbea ~ Carboxylate Microspheres (9.67 ~m) Nith a 6-Aminocaproic Acid Linkinq Arm Recombinant IL-2 (Amgen, ala-125 analog) was immobilized on 9.67 ym Polybeads carboxylate microspheres with a 6-aminocaproic acid linking arm using a water-soluble carbodiimide in the following manner. The pellet obtained from a 0.25 ml-ali~uot 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-N~S, ~ierce 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-aminoc~proic acid in PBS. The resulting slurry was 3S mixed for 20 hours at room temperature and centrifuged.
The supernatant was discarded. The pellet was washed with three 0.5 ml-portivns of PBS, resuspended in 0.35 ml of OgO/09798 ~CTIUS90/01031 204 ~730 PBS, and treated with 0.05 ml of an aqueous solution of ~L-2 (so yg IL-2, activity 3?5,000 units) and 2.0 mg of EDCI. After vortexing to dissolve the reagents, the reaction mixture was ~ently mixed at room temperature overnight. ~he 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 4DC 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 15 beads.

Example 8 Attachment of IL-2 to Poly~ ~ _CarboxYlate ~icrospheres (9.67 ~m~ With a 201,6-Diaminohexane/Glutaraldeh~de LinXinq Arm Recombinant IL-2 (Amgen, ala-125 analog) was immobilized on 9.67 ym 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 ~BS (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 .. . . .
:. .: j . . . - ~

. . .

.

Wo~o/n97Q8 PCT/US90/01031 ~ 7 ~ 0 34 suspended in 0.35 ml of PBS and treated with 0.05 ml of an aqueous solution of IL-2 (50 ~g IL-2, actiYity 375,000 units). The slurry was mixed overnight at room tempexature, centrifuged, and the supernatant was S 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 4OC 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.

Example 9 Attachment of IL-2 to Polybead~- CarboxYlate Microspheres (9.67 ym~ With a 1,12-Diaminododecane/Glutaraldeh~de Linkinq Arm Recombinant I~-2 (Amgen, ala-125 anaiog) was immobilized on ~.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 PRS (pH 7.40, 3 x 1.0 ml), suspended in 0.75 ml of 0.2 N 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 30 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 a~ueous IL-2 solution (100 yg I~-2, activity 750,000 units). The mixture was allowed to react overnight at room temperature. The slurry was centrifuged WO ~/09798 ;20~ PCT/US90/01031 and the supernatant was carefully removed and preserved.
The pellet was resuspended in 0.5 ml of PBS, and the mixture was centrifuged. ~he supernatant was removed and added to the first supernatant. The beads were then processed as described in Example 1, suspended in D.5 ml of the storage buffer, and stored at 4OC 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.

ExamPle 10 Attachment of IL-2 to Pol~ea ~ Carboxylate ~icrosPhere~ (65 ~ 25 ~m) With a 1,12-Diaminododecane/GlutaraldehYde Linkinq Arm Recombinant IL-2 (Amgen, ala-125 analog) was immobilized on 65 + 25 ym Polybead~ car~oxylate microspheres (Polysciences) with a 1,12-diaminododecane/glutaraldehyde linking arm using a water-soluble carbodiimide in the following manner. The pelletobtained from 0.50 ml of a 2.5% suspension of 65 ~ 25 ym car~oxylated poly~eads was washed with PBS (pH 7.40, 3 x l.0 ml), suspended in 1.0 ml of 0.2 ~ 1,12-diaminododecane in PBS (pH 6.00), and treated with lO mg of EDCI. After 2S mixing for 24 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 l.0 ml), and acti~ated with 1.0 ml of 8% glutaraldehyde in PBS as descri~ed in Example 8. After activation, the slurry was ~0 centrifuged, the supernatant ~as 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 I~-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 4C u~til - , .
, . ' ~

; .

WOgo/09798 20~ 7 7~ PCT/US90/01031 :3~
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.

Example 11 Attachment of IL-2 to Polybead~ Carboxylate ~icrospherès (9.67 ~m) With a 1,12-Diaminododecane Linking Arm via Free Carboxyl Gr~u~s on the Cytokine Recombinant I~-2 (Amgen, ala-125 analog) was immobilized on 9.67 ym Poly~ead~ carboxylate microspheres with a 1,12-diaminododecane linking arm via free carboxyl groups on IL-2 using a water-solu~le carbodiimide in the following manner. The pellet obtained from 0.25 ml of carboxylate microspheres (9.67 ym) was washed with PBS (pH
7.40, 3 x 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 P9S
(pH 7.40, 3 x 1.0 ml), resuspended in 0.4 ml of PBS, treated with 0.1 ml of an aqueous IL-2 s~lution (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 minutes at room tempera~ure. 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 4C until used. A determination of the IL-2 acti~ity present in the supernatant revealed 834 units (0.2~ of the original), indicating that 99.8~ of the IL-2 .

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WO ~J0979X PCTtUS90/01031 '73~
' . , had been bound to the beads.

Example 12 Attachment of Polyethylene Glycol-~odified IL~2 to Polybead~ CarboxYlate ~icrospheres (9.67 ~m) With a 1,12-Diaminododecane Linkinq Arm ~ ecombinant IL-2 ~Amgen, ala-125 analog) was reacted with a ten-fold molar excess of methoxypolyethylene glycolyl N-succinimidyl glutarate (~W
lC 4800) [Abuchowski et al., Cancer Biochem. Biophys., 1, 175 (1984)] following the procedure described by Katre and Knauf in International Patent Application Number PC~-/US86/01252 (International Publication Number W087/00056), incorporated herein by reference. The modified IL-2 was purified ~y size exclusion chromatography on a Bio-Gel P-10 column using PBS tpH
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. -~he 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 P3S, 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 care~ully 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 .- .

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WO90/~9~9~ PCT/US90/01031 ~134'~'730 ~

1% BSA/PB5 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 l.0 ml) and finally suspended in 0.5 ml of the storage buffer, and stored at 4C until used. A
determination of the IL-2 activity present in the supernatant revealed 509 units (0.2~ of the original), indicating that 9g.8~ of the IL-2 had been bound to the beads.
Example 13 Attachment of IL-2 to Polybead~ Amino Microspheres 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 l. After washing the beads with PBS (3 x 0.5 ml), they were suspended in 0.4 ml of PBS, and treated with O.l ml of an aqueous IL-2 solution (lO0 ~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 l, suspended in 0.5 ml of the storage buffer, and stored at 4C 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.l~ of the IL-2 had been bound to the beads.
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wo ~/09798 PCT/US90/01031 2~'7`~

Example 14 Attachment of IL-2 to Sephadex~ G-lO Particles (40-120 ym) With a 6-AminocaProic Acid SPacer Arm 5 Recom~inant IL-2 (Amgen, ala-125 analog) was immobilized on degradable Sephadex~ G-lO resin particles (Pharmacia, Piscataway, NJ, cross-linked dextran particles, 40-120 ~m) with a 6-aminocaproic acid linking arm in the following manner. A slurry of approximately 7.5 ml of wet, packed Sephadex~ G-lO resin in 7.5 ml of water was activated with l.5 g of cyanogen bromide (CNBr) according to the published procedure; see P. Cuatrecasas, J. Biol. Chem., 245, 3059 (1970), incorporated herein by reference. After activation, the resin was rapidly filtered, washed with lO0 ml of cold 0.2 M sodium borate buffer (pH 9.0), and added to 50 ml of l.0 M 6-aminocaproic acid in 0.2 ~ sodium borate (p~ 9.o). ~he mixture was mixed at room ~emperature for 20 hours. The resin was collected by filtration, washed with ca. 200 ml of ~2l and dried under high vacuum for 48 hours. A lO mg-portion of the dried resin was swollen for 24 hours in l.0 ml of PBS. The suspension was then centrifuged, the supernatant discarded, and the resin washed with PBS (3 x l.0 ml). The pellet was resuspended in 0.4 ml of PBS, treated with O.l ml of an aqueous solution of IL-2 (lO0 ~g IL-2, activity 750,000 units), followed by 3.0 mg of EDCI, and mixed overnight at room temperature. ~he resin was processed as described in Example l, suspended in 0.5 ml of the storage buffer, and stored at 4C until used.
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.

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~xamPle 15 Attachment of IL-2 to 5ephadex~ G-10 With a 1,6-Diaminohexane/Glutaraldehyde ~inkinq Arm Recombinant IL-2 tAmgen, ala-125 analog) was Lmmobilized on degradable Sephadex~ G-10 particles with a 1,6-hexanediamine/glutaraldehyde linking arm in the following manner. Wet, packed Sephadex~ G-lO resin (ca.
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.O M 1,6-hexanediamine in D.2 N
sodium borate (pH 9.0). The slurry was mixed at room temperature for 20 hours. The resin was collected by filtration, washed with 20~ ml of H~0, and dried under high vacuum for 48 hours. A 10 mg portion of thè dried resin was swollen and washed, as described in Example 12. The pallet was 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 activated resin was suspended in 0.4 ml of PBS, and treated with 0.1 ml of an aqueous solution of IL-2 ~100 yg 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 ~as 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. The resin was then processed as described in Example 1, suspended in 0.5 ml of the storage buffer, and stored at 4OC 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.

, W090t09798 PCT/US90/0103 ZQ~7'~ ~
~ .` ,: ., ExamPle 16 Attachment of IL-4 to Blue-DYed Polystyrene Beads (9.64 Recombinant IL-4 (Amgen, natural sequence) was immobilized on 9.64 ym blue-dyed polystyrene beads in the following manner. ~he pellet obtained from 0~25 ml of a 2.5~ suspension of blue-dyed beads was washed with PBS (pH
7.40, 3 x l.0 ml), and then activated with glutaraldehyde as described in Example 1. The beads were then suspended in 1.O ml of a commercial IL-4 formulation containing 10.0 ~g IL-4 (activity 2 x 105 units) and 0.025% human serum albumin (HSA) in PBS. The reaction mixture was mixed overnight 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 ~uffer and ~ept at 4OC 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 ~ quantifiable assay.
~xamPle 17 Attachment of IL-6 to Blue-DYed Polystyrene Beads (9.64 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 lO.0 yg IL-6 (activity 1-2 x ~05 units) and 0.025% HSA in PBS
The reaction mixture was mixed overnight at room temperature. Following the coupling reaction, the beads were processed as described in Example l, then suspended in 0.5`ml of storage buffer and kept at 4OC until used 3S An assay of the supernatant solution from the above coupling reaction for I~-6 activity could not be quantified due to the lack of a suitable indicator cell , .. .. . . . ..
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Example 19 Attachment of Murine Granulocyte-MacroPhaqe Colony 5 Stimulatinq Factor to Blue-Dyed Polystyrene Beads (0.93 ~ml 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 descri~ed in Example 2. The beads were then suspended in 0.5 ml of a commercial rMuGMCSF formulation containing 5.0 yg of the growth factor (activity 5 x 103 units) and 0.025% BSA in PBS. The reaction mixture was mixed overnight at room temperature. Following the final washes, the beads were suspended in 0.5 ml of the storage buffer and kept at 40C
until used. An assay of the supernatant solution for rMuGMCSF could not be quantified due to the unavailability of an indicator cell line.

Example l9 Attachment of ~uman Granulo~Yte-MacroPha~e ColonY
25 Stimulatinq Fac~or to Blue-Dyed PolYstyrene Beads ~0.93 ym) Recombinant human granulocyte-macrophage colony stimulating factor ~rHu&MCSF, 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 o~ernight at room temperature.
Following the final washes, the beads were suspended in ,:

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.. . ~ .

WO~o/0979X PCTIUS90/0103t ~0~'7'73 0.5 ml of storage buffer and kept at 4OC 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.
~xample 20 Attachment of I~-3 to Blue-Dyed PolYstYrene ~eads (9.64 ym~
Recombinant IL-3 (Am~en, natural sequence) was immobilized on 9.64 ym 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 I~-3 (activity 2 x 106 units) an~ 0.025~ ~SA in PBS. The reaction mixture was mixed overnight at room temperature.
Following processing, the beads were suspended in 0.5 ml of the storage buffer and kept at 4OC 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.

Example 21 Growth of C~LL-2 Cells ~sinq_~mmobilized IL-2 (Recombinant A~a-125 Analoq~
Samples of recombinant I~-2 (ala-125 analog) immobilized on 9.64 ym blue-dyed polystyrene beads; 0.93 ym blue-dyed polystyrene beads; 9.67 ym carboxylate polystyrene beads; 9.67 ym carboxylate polystyrene beads with 6-aminocaproic acid, 1,6-diaminohexane, and 1,12-diaminododecane linking arms; 65 ym carboxylate polystyrene beads with 1,12-diaminododecane linking arm;
5.29 ~m amino polystyrene beads; and Sephadex~ G-10 3S polydextran beads with 6-aminocaproic acid and 1,6-diaminohexane linking arms (see Examples 1, 2, 6, 7, 8, 9, 10, 13, 14, and 15) were examined to determine if Woso/0979R PCT/US90/01031 2C)477;~0 immobilized IL-2 would support in vitro growth of the IL-2 dependent cell line CTLL-2, a cytotoxic T-lymphocyte cell line.
The samples of beads comprising immobilized IL-2 were washed 3 times by su~pension and centrifugation in a Beckman Microfuge in RPMI-1640 tissue culture medium (Whittaker M. A. Bioproducts, Inc., Walkersville, ND) 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. Aliquots of the beads were added to individual wells in a 96-well flat-bottomed tissue culture plate ~Falcon #3075, Becton Dickinson & Co., Rutherford, NJ) followed by the addition of 1 x 104 CTLL-2 cells (an IL-2 growth dependent cell line (TIB-214) obtained from American ~ype CuI~ure Collection, Rockville, MD). 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 ~ortexed beads were used in the experiment. The IL-2 immobilized beads and the CTLL-2 cells were incubated for 48 hours in a 37C incubator with a 5% CO2 atmosphere. After 48 hours, 1 yCi of ~3H~-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 13H~-thymidine incorporation. The results are reported in ~a~le 3 and demonstrate that all the above-listed immobilized IL-2 combinations support CTLL-2 cell growth.

Example 22 Growth of CTLL-2 Cells ~sina Immobilized IL-2 (Recombinant Natural Sequence!
Recombinant natural sequence IL-2 immobilized on 9.64 ym blue-dyed polystyrene beads was examined to , ~
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, WO ~09~98 pcr/us9o/oto3l 2C~7'73'~

determine if it would support in vltro 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 i5 reported in Table 4 and demonstrates that immobilized recombinant natural sequence IL-2 supports CTLL-2 cell growth.

~xamPle 23 Growth of CTLL-2 Cells ~sinq Immobilized IL-2:
Carboxyl Group vs Amino GrouP Attachment 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. The growth of CTLL-2 cells using IL-2 immobilized via a carboxyl group on the IL-2 was compared to the growth of CTLL-2 cells using IL-2 immobilized via an amino group on the IL-2 (as described in Example 1). The results are reported in Table 5 and demonstrate that IL-2 attached via a carboxyl group to a bead supports CTLL-2 gr~wth and appears more active than IL-2 attached ~ia an amino group to a bead (see Figure 1).
~xamPle 24 Growth of CTLL-2 Cells ~inqLI~mobilized PolYeth~lene Glycul M~dified 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 WOsO/0979X PCT/US90/01031 . .
2047~3~ 46 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.

Exam10 Concentration Dependence of Immobilized Recombinant IL-2 on Growth of CTLL-2 Cells The effect of the concentration (units/ml or mg/ml) of recombinant IL-2 (ala-125 analog), immobilized on polystyrene beads, on the growth of CTLL-2 cells was examined. Recombinant IL-2 was immobilized on 9.64 ym blue-dyed polystyrene beads as described in Example 4.
These beads were washed and assayed as described in Example 21. Concentrations of 1 and 10 beads per cell were used. Under these conditions, growth of the CTLL-2 cells was determined to be concentration dependent (see Figure 2).

ExamPle 26 Gro~th of C~LL-2 Cells V6. Time sina Immobilized IL-2 The growth of CTLh-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 Ih-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 x 104 CTLL-2 cells (an I~-2 growth dependent cell line). The beads containing immobilized IL-2 and the CTLL-2 cells were incubated for various times in a 37C

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WO 90/0979~ PCr/US90/01031 ~347~3~

incubator with a 5% CO2 atmosphere. At the end of each time peri~d, 1 ~Ci of 13~ ] -thymidine was added and the mixture was incubated for an additional 4 hours. ~he 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). These results demonstrate that the growth of the CTLL-2 cells using immobilized IL-2 was comparable to or better than the growth of the C~LL-2 cells using the control, i.e., soluble IL-2. With one bead/cell, growth is not as dramatic in the 24 to 120 hour range as soluble IL-2, but growth remains steady up to 168 hours.
Esample 27 Growth of CTLL-2 Cells ~n Recycled IL-2 Immobilized Beads Recombinant IL-2 immobilized on 9.64 ym ~lue-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 x 104 CT~-2 cells, and incubated for 72 hours in a 37C incubator with 5% CO2 atmosphere. To several of the cultures, 1 ~Ci of C3H]-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 S
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 .. . .

., W~90/09798 PCT/US90/01031 2C~773{~

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.

E~ample 28 Growth of Human Peripheral Blood L~mphocytes on Immobilized Recombinant IL-2 The growth of human peripheral blood lymphocytes (PB~ s) on immobilized recombinant IL-2 (ala-125 analog) was examined. Recombinant IL-2 was immobilized on 9.64 ~m lS 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 ~0 blood after centrifugation over LeucoPREP (Becton Dickinson & Co.) cell separation medium. The crude lymphocyte preparation was washed 3 times ~y centrifugation in RPMI-1640 tissue culture medium containing 4% antibiotics and 5% human AB serum ~heat inactivated, North American Biologicals, Inc., Miami, FL).
2 x 105 PBL~s were added to various concentrations of IL-2 immobilized beads. The cells were incubated for various time periods in a 37C incubator with a 5~ CO2 atmosphere.
At the end of each time period, 1 ~Ci of t3H]-thymidine was added and the mixture wa~ incubated for an additional 4 hours. The cells were collected via Skatron cell har~ester and counted in a liquid scintillation counter to determine cell growth. The results are presented graphically in Figure 4. This example demonstrates that PBL's grow using immobilized I~-2, and that the growth of-the PBL's is equal to or better than control soluble IL-2, especially after 72 hours of culture.

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, W090/n9798 PCT/US90/01031 21~17'`~
qg Example 29 LAR Cell Activity Induced bY Soluble Recombinant IL-2 or Immobilized IL-2 Human PBL's grown on immo~ilized recombinant IL-2 (ala-125 analog) were examined to determine if they exhibit lymphokine-acti~ated ~iller (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 LAR cell killing activity was assayed using the cell targets K562, Raji, and Daudi.
The assay for LAK cell killing used a 4 hour 5~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. Clinical ImmunolooY, 1, 51 (1981). Normal NK (natural killer) cells isolated from fresh PBL~s killed K652 cells but did not kill Raji or Daudi cells when they were in an activated state. The results are reported in Table 8. IL-2 immobilized beads activated LAK cells killed X562, Raji, and Daudi cells.
Killing was equal to soluble IL-2 activated LAX cells.

ExamPle 30 NR/LAR Activity Induced by Immobilized IL-2 Recom~inant 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 (N~) or lymphokine-activated killing (hAX) of a target cell line. ~hat is, an ex vivo experiment was conducted to determine if the immobilized I~-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. with 200 ~1 of PBS, 50,000 , , .

W~Q0~0979X PCT/US90/01031 2~47'7~ 50 units recombinant soluble IL-2, 200,000 units IL-2 immobilized on 9.64 ym blue beads (Example l), or l00,000 units IL-2 immobilized on 65 ~m beads (Example l0). After 96 hours, cells from the peritoneal cavity and spleens were collected and assayed for NK/hAK cell activity.
Splenocytes were prepared from fresh spleens as descri~ed by M. EI. Zaroukian et al., Immunol. Invest., l5, 813 (1986) and C. W. Gilbert et al., J. Immunol., l40, 2B21 (1988). NR/LAX cell activity was assayed by a 4 hour 5LCr 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 6 ym beads also activate LAK cells in the peritoneal cavity.
The LAX cell activity in the peritoneal cavity appears to be localized and may have a therapeutic value in the localized treatment of cancer.

ExamPle 31 Growth of Human Peripheral Blood LYmphocYtes on Immo~ilized Recombinant IL-4 -Recombinant IL-4 was immobilized on 9.64 ym blue-dyed polystyrene beads as described in Example 16. The immobilized IL-4 beads were washed as described in Example 2l, and used in a PHA (phytohaemagglutinin) co-stimulation 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 ~e~nika Corp., Durham, NC).
Crude lymphocytes were incubated in plastic tissue culture flasks at 37C in RPMI-l640 medium containing 5~ heat inactivated human AB serum for 1 hour to remove monocytes and other adherent cells that interfere with the costimulation T-cell proliferation assay. This step was repeated twice. Nonadherent lymphocytes, enriched with T--- . . ~: ,,,, . "
: ' ' "'``'' - :. ~:
:

WO90/09798 2~ 3~ PCTtUS90/01031 'jl cellq were then used in a P~A co-stimulation proliferation assay. l x 105 cells were added to each well, in addition to soluble IL-4 (lO0 units/ml), PHA (0.05 ~g/ml), PHA plus soluble IL-4 (lO0 units/ml and l unit/ml), or PHA plus immobilized IL-4 on beads (0.5 and l bead/cell starting concentration), and incubated for 96 hours at 37OC. After 96 hours, the cultures were pulsed with ~3H]-thymidine for 4 hours to determine T-cell proliferation. The results are listed in Table lO and indicate that immobilized IL-4 beads stimulate T-cell proliferation over background suboptimal PHA levels.

Example 32 Growth of Human PeriPheral Blood Lymphocy~6 on Immobilized Recombinant IL-6 Recombinant IL-6 was immobilized on 9.64 ~m blue-dyed polystyxene beads as described in Example 17. The immobilized IL-6 beads were washed as described in Example 21, and used in a PHA (phytohaemagglutinin) co-stimulation 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 o~er a Ficoll gradient (LSM, Lymphocyte Separation Nedium). Crude lymphocytes were incubated in plastic tissue culture flasks at 37C in RPMI-1640 containing 5~ heat inacti~ated human AB serum for l hour to remove monocytes and other adherent cells that interfere with the costimulation T-cell proliferation assay. This step was repeated tw'ce. Nonadherent lymphocytes, enriched with T-cells were then used in a PHA
co-stimulation proliferation assay. l x 105 cells were added to each well, in addition to nothing, soluble I~-6 (100 units/ml), PHA (0.05 yg/ml), PHA plus soluble IL-6 (lO0 units/ml and l unit/ml), or PHA plus immobilized ~L-6 on beads (0.5 and l bead/cell starting concentration), and incubated for 96 hours at 37C. After 96 hours, the .~ . .

WO9o/0~798 PCT/US90/01031 cultures were pulsed with [3H]-thymidine for 4 hours to determine T-cell proliferation. The results are tabulated in Table 11 and indicate that immobilized IL-6 beads stimulate T-cell proliferation over background suboptimal PHA levels.

~xa~ple 33 Growth of AM~-193 Cells ~n Immobilized Recombinant ~uman GMCSF
Recombinant human GMCSF (rHuGMCSF) immobilized on 0.93 ym blue-dyed polystyrene beads was examined to determine if it would support in vitro growth of a GMCSF
dependent cell line AML-193. Recombinant human GMCSF was immobilized on 0.93 ym blue-dyed beads as described in Example 19. The immobilized recombinant human GMCSF beads were washed as described in Example 21. The growth assay for AML-193 cell line was as follows. Ali~uots 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 104 AML-193 cells (an IL-3/GMCSF dependent cell line obtained from American Type Culture Collection, Rockvi~lle, MD). The beads with immobilized rHuGMCSF were incubated with the ANL-193 cells for 116 hours in a 37C
incubator with 5~ C02 atmosphere. After 116 hours, 1 yCi of l3H~-thymidine was added and the mixture was incubated for an additional 4 hours. The cells were collected as described in ~xample 21. The results are reported in Table 12 and demonstrate that immobilized recombinant human GNCSF supports AML-193 cell growth.
E~ample 34 Growth of A~L-193 Cells on I~,obilized Re~ombinant IL-3 Reco~binant IL-3 immobilized on 9.64 ym blue-dyed polystyrene beads was examined to determine if it would 3S support in vitro growth of an IL-3/GMCSF dependent cell line AML-193. Recombinant IL-3 was Lmmobilized on 9.64 ym blue-dyed beads as described in Example 20. The WO ~/09798 PCT/US90/01031 Z0~73V

immobilized IL-3 beads wer~ washed as de~cribed in Example 21 and a~sayed as described in Example 33. The results are reported in ~able 13 and demonstrate that immobi~ized recombinant IL-3 supports AML-193 cell growth.
Example 35 Attachment of IL~ eta to Blue-DYed PolY~tYrene Beads l9.64 Ym) Recombinant IL-l-beta (Amgen) was immobilized on 9.64 ym 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 tpH
?.40, 3 x l.0 ml), then activated with glutaraldehyde as described in Example l. The beads were then suspended in 0.46 ml of PBS, treated with 0.04 ml of a commercial I~
beta formulation containing 8.0 ~g IL-l-beta (activity 4 x lO6 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 l, then suspended in 0.5 ml of the storage buffer and kept at 4C until used.

Example 36 Attach~ent of IL-l-alpha to Blue-DYed Polystyrene Beads (9.64 ~m) Human sequence IL-l-alpha (R & D Systems) was immo~ilized 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 l.0 ml), and then activated with glutaraldehyde as described in Example l. The acti~ated beads were suspended in 0.42 ml of P8S, then treated with 0.08 ml of a formulation that contained 8.0 yg of the cytokine and 200 ~g human serum albumin (HSA) in PBS. The reaction mixture was mixed for 24 hours at room temperature. Following the coupling reaction, the beads were centrifuged, washed with P~S (0.5 ml), then , , . . ., - . . -- "~

.; . ., . . :
. .
' :

Wo90Jo~9~ PCTtUS90/~tO31 treated with ethanolamine as described in Example 1. The beads were then washed (3 x l.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 descxibed in Example 1, then sus-pended in 0.5 ml of the storage buffer and kept at 4C
until used.

lOE~amPle 37 Attachment of Recombinant Human GranulocYte Colony Stimulatinq Factor (r~uGCSF) to Blue-D7ed Polystvrene Beads (9.64 ~m) Recombinant human GCSF (rHuGCSF, Amgen) was immobilized on 9.64 ym 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 PBS (pH
7.40, 3 x 1.0 ml), then activated with glutaraldehyde as described in Example l. The activated beads were suspended in 0.3 ml PBS and treated with 0.2 ml of a commercial rHuGCSF formulation containing 0.5 yg (acti~ity 1 x 105 units) of the growth factor and 0.025~ HSA in 0.01 M sodium acetate (pH 5.4). The suspension was mixed overnight 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 4C until used.

Example 38 30Attachment of Recombinant ~urine GranulocYte Ma~rs:~phaqe ColonY Stimulatinq Factor t rMuG~CSF ) to ~lue-DYed PolYst~rene Bead~ ( O . 93 ~m) Recombinant murine GMCSF (Amgen) was immobilized on O.g3 ym blue-dyed polystyrene beads in the following manner. The pellet obtained from 0.25 ml of a 2.5 suspension of blue-dyed ~eads was washed with PBS (pH
7.40, 3 x 1.0 ml), then activated with 8% glutaraldehyde -: .
;, . ' .

W~O/n9?9~ PCT/US90/OtO3l

3~

as described in Examples 1 and 2. The activated beads were then suspended in O.50 ml of a commercial rMuG~CSF
formulation containing 5.0 yg (activity 5 x 103 units) of the growth factor and 0.025% BSA in PBS. The suspension was mixed for 24 hours at room tempera~ure. Following the coupling reaction, the beads were processed as described in Examples 1 and 2, then suspended in O.5 ml of the storage buffer and kept at 4C until used.

Example 39 C~valent Attachment/Adsorption of r~uGMCSF
to Blue-Dyed Polvstyrene Beads (O.93 ~m) The pellets obtained from two 0.2 ml-portions of a 2.5% suspensivn 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 -20 centrifuged and the pellets washed with PBS (3 x 1.0 mlJ.
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 rMuG~CSF formulation used in Example 38. ~he 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 tlabeled Al and C1, both ca. 1.0 ml). The pellets were then treated with 1.O ml- portions of O.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 ml-portions of 0.1~ SDS/PBS, mixed for one hour, centrifuged, and the supernatants (labeled A4, A5, A6, C4, C5, and C6, WO~o~os7s8 PCT/US90/01031 7'730 respectively) were removed and saved. The pellets were washed with l.0 ml-portions of PBS, and the supernatants (la~eled A7 and C7) were removed and saved. The pellets were then treated with 1% BSA/PBS as described in Example l, and the ~arious supernatants (labeled A8, A9, AlO, C8, C9, and ClO, 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 4C
until used.
Example 40 Attachment of Recombinant Human Insulin-like Growth Factor I (r~uILGF-I1 to Blue-DYed PolYstYrene Beads (9.64 ~m) Recombinant human insulin-like growth factor I
(rHuILGF-I, Somatomedin C, available from Bachem) was immobilized on 9.64 ym blue-dyed polystyrene beads in the following manner. The pellet ob~ained from 0.2 ml of a 2.5% suspension of blue-dyed beads was washed with PBS ~pH
7.40, 3 x l.0 ml) and then activated with l.0 ml of 8.0%
glutaraldehyde in PBS as described in Example l. The washed, activated beads were suspended in 0.42 ml PBS and treated with 0.08 ml of a solution that contained 20.0 ~g of the commercial growth factor in sterile water. ~he suspension was mixed for 20 hours at room temperature.
Following the coupling reaction, the beads were processed as described in Example l, then suspended in 0.5 ml of the storage buffer and kept at 4C until used.

~xample 41 Attachment of Recombinant ~uman Insulin-like Growth Fact~r ~I
(rHuILGF-II) to Blue-DYed Polyst~rene Beads (9.64 ~m) Recombinant human insulin-like growth factor II
(rHuILGF-II, a~ailable from Bachem) was immobilized on 9.64 ~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 l.0 -..

- ' '' .' - ~

Wo ~/09798 PCT/US90/01031 ZO L~ 7 ~3 ~

ml) and then activated with 1.0 ml of 8.0 glutaraldehyde/PBS as described in Example l. The washed, activated beads were then suspended in 0.45 ml PBS and treated with 0.05 ml of a solution that contained 12.5 ~g of the growth factor in sterile water. The suspension was mixed for 24 hours at room temperature. Following the coupling reaction, the beads were processed as descri~ed in Example 1, then suspended in 0.5 ml of the storage buffer and kept at 4C until used.
Example 42 Attachment of Recombinant Human Tumor Necrosi~ Factor (TNF-alpha/Cachectin) to Blue-~yed Polystyrene Beads (9.64 ~m) 15Recombinant 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 ~eads was washed with PBS (3 x 1.0 ml), then activated with glutaraldehyde as described in Example 1. Following ~he activation, 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 yg (acti~ity 1.92 x 105 units) of the growth factor in a 0.04 M Tris/0.1 N NaCl buffer (pH
8.60). The suspension 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 4C until used.

30Example 43 Attachment of Fibroblast Growth Factor Basic (FGFb) to Blue-Dyed Polys~yrene Beads (2.B5 ~m~
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 x 1.0 ml)~ then activated with glutaraldehyde as described in WO9(1/(19798 PCT/US90/01031 20~7730 -. ~` 58 Example l. Following activation, the washed beads were suspended in 0.44 ml P~S, then treated with 0.06 ml of a commercial FGFb formulation containing 30 yg of the growth factor in a 0.02 M sodium citrate/O.l M sodium chloride buffer (pH 5.0)~ 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 4C until used.
_xample 44 Attachment of Transformin~ Growth Factor-beta-2 (TGF-beta-2~ to Blue-Dyed Polystyrene Beads (2.85 ym) 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 2.54 suspension of blue-dyed beads was washed with PBS (3 x l.0 ml), then treated with glutaraldehyde as described in Example l.
Following activation, the washed beads were suspended in 0.35 ml PBS, treated with O.l5 ml of a solution containing 7.5 yg of the growth factor in O.Ol~ Triton X-lO0. The suspension was mixed for 18 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 4C until used.

~xamPle 45 Attachment of Recombinant ~uman Interferon-alpha-2A
(Rofero ~ A) to ~lue-Dved ~olYstyrene Beads (2.85 ym) Recombinant human Interferon-alpha-2A (Roferon~
A, Roche Laboratories) was immobilized on 2.85 ym 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 x l.0 ml), then treated with glutaraldehyde as described in Example l.
The washed, activated beads were then suspended in 0.4 ml .

.

~ '73 O
PBS and treated with O.l ml (activLty 6 x 105 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 ~uffer and, tcgether with the various supernatants, kept at 4C until used.

10~xample 46 Attachment of Recombinant Human Epidermal Growth ~actor (rHuEGF) to Blue-Dyed PolYstyrene Beads (O.g3 ym) Recombinant ~uman EGF (rHuEGF, available from Amgen) was immobilized on 0.93 ym 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 l.0 ml), then treated with glutaraldehyde as described in Examples l and 2. The washed, activated beads were suspended in 0.35 ml PBS, then treated with 0.l5 ml of a solution that contained 25.0 ~g of the growth factor in P~S ~p~ 7.40). The suspension was mixed for 18 hours at room temperature. Following the coupling reaction, the beads were processed as described in Examples l and 2, then suspended in 0.5 ml of the storage buffer and kept at 4C until used.

Example 47 Attachment of Recombinant ~uman Platelet Derived Growth Factor (rHuPDGF) to Blue-Dyed Polystyrene ~eads (2 85 ~m~
30Recombinant human PDGF (rHuPDGF, available from Bachem) was ir~mo~ilized on 2.85 ym blue-dy2d polystyrene ~eads 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 l.0 ml), then treated with glutaraldehyde as tescribed in Example l. Following activation, the washed beads were suspended in 0.35 ml PBS, and treated with 0.15 ml of a solution containing 15.0 yg of the growth factor WO ~/n9~98 PCT/US90/OtO31 in sterile water. The suspension was mixed for 20 hours at room temperature. Following the coupling xeaction, the beads were processed as described in Example l, then suspended in 0.5 ml of the storage buffer and kept at 4C
until used.

~xamPle 48 Attachment of Recombinant ~uman ~ythropoietin (rHuEPO) to Co-Bind~ Well striPs Recombinant human erythropoietin (rHuEPO) was obtained from Amgen as a li~uid formulation containing 5000 units activity per ml of a solution comprised of 50 glycerol in 0.025 M HEPES buffer ~pH 7.20). The Co-Bind~
well strips, strips whose surfaces have been chemically modified (i.e.t activated) to covalently bind proteins, were obtained from Micro Membranes, Inc., Newark, NJ.
Four wells of the 8-well strip were then filled as shown below:

rHuEPO Buffer, mls Well Units mls 50% qlYcerol in 0.025 N HEPES, pH ? . 20 A 200 0.04 0.16 B lOO 0.02 0.18 C 50 O.Ol O.l9 ~
25 D O 0.00 0.20 ~ -The strip was covered and incu~ated at 35C for 3 hours.
The supernatants A-D were then removed and saved for residual activity assays. The wells were washed with buffer (2 x O.l ml), then treated with 0.2 ml portions of freshly prepared 1% BSA/P~S and again incubated at 35C 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 l~ Fungizone, then filled with the same media. The strip was covered and kept at 4C
until used.

- . - . .
- - . . .

WO ~/09~98 20~ J1~7~1 PCT/~S90/OtO31 Example 49 Growth of C~LL-2 Cells Usin~ IL-2 Produced From L~RN.~G6 Cells StLmulated With Immobilized Recombinant IL-l-beta PolYstyrene Beads Recombinant IL-l-beta immobili~ed on 9.63 ym 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-l-beta beads were washed three times by suspension and centrifugation as described in Example 21. I~-l-beta beads in coniunction with a suboptimal concentration of PHA [Phytoheamagglutinin P, Wellcome Foundation, Danford, England] (lO ~g/ml) were added to 5 x 104 LBRM.TG6 cells [J. W. Larrick et al., J. Immunol. Methods, 79, 39 (1985~
in lOO yl of Iscove's MEM, (Whittaker M.A. Bioproducts, Walkersville, MD) and incubated for 48 hours at 37C in 5%
CO2. The reaction was stopped by placing the hBRM .TG6 cells at 4C for 24 hours. Next, a 50 yl-portion of the LBRM.TG6 cell supernatant was removed and added to 50 yl of fresh CT~L-2 cells. The released soluble IL-2 was assayed to determine if it would support CTLL-2 cell growth. 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-~iphenyl-tetrazolium bromide) ~T. Mosmann, J. Immunol Meth., 65, 55 tl~83); and ~.B.
Hansen, S.E. Nielson and X. Berg, J. Immunol Meth., ll9:
203-210t1989)~ The results are reported in Table 14 and indicate th~t IL-l-beta beads activate the release of soluble IL-2 from LBRM . TG6 cells, and that the I~-2 released by the LBRN. TG6 cells supports growth of IL-2 dependent CTLL-2 cells.

: .

. . ` - ~ . ~.

wo vo~ns7sx PCI/US90/01031 047~73V
Example so Recombinant IL-l-alpha Immobilized on Pol~styrene ~eads Induces LBRM.TG6 Cell~ to Produce IL-2 Recombinant IL-l-alpha immobilized on 9.64 ym blue-dyed polystyrene beads induces the murine lymphoma cell line L~RN.TG6, American Type Culture Collection, to synthesize IL-2 which was then assayed in the IL-2 dependent CTLL-2 cell line. ~uman sequence IL-1-alpha was immobilized on 9.64 ym blue-dyed polystyrene beads as described in Example 36. The immobilized IL-1-alpha beads were washed three times as described in Example 21. IL-~-alpha beads in conjunction with a suboptimal concentration of PHA [Phytohemagglutinin P, Nellcome Foundation, Danford, England] (lO ~g/ml) were added to 5 x 104 LBRM.TG6 cells in 100 ~l of Isco~e~s ~EM and incubated for 48 hours at 37~C in 5% CO2. The reaction was stopped by placing the LBRM.TG6 cells at 4C for 24 hours. Next, 50 yl of the ~BRM.TG6 cell supernatant was removed and added to 50 yl of fresh CTLL-2 cells. T~e released soluble IL-2 was assayed as described in Example 49. The results are reported in Table 15 and indicate that IL-l-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.

~ample 51 Growth of AML-193 Cell~ on Immobilized Recombinant ~uman GCSF
Recombinant human GCSF (rHuGCSF) 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 ~ype Culture Collection. Recombinant human GCSF was immobilized on 9.64 ym blue-dyed polystyrene beads as described in Example 37. The immobilized rHuGCSF beads WO ~/n9?9N 21~4 t! 7~0 PCT/US90/01031 were washed as described ln Example 21. The growth assay for AML-193 cell line was as follows. Ali~uots 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 104 AML-193 cells as in Example 33. The beads with the immobilized rHuGCSF were incubated with ~he AML-198 cells for 116 hours in a 37OC incubator with a 5%
CO2 atmosphere. ~3H]-thymidine (1 yCi) was then added to each well 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 16 and demonstrate that immobilized recombinant human GCSF supports AML-193 cell growth.

ExamPle 52 Recombinant Murine GMCSF (rMuGMCSFl I~ obilized on 0.93 ~m Polvstyrene Beads Stimulates Granulopoiesis in BDFl Mice Recombinant murine GMCSF (rMuGMCSF) immobilized on 0.93 ~m blue-dyed polystyrene beads stimulates granulopoiesis in BDFl mice. Recombinant murine GNCSF was immobilized on 0.93 ym blue-dyed polystyrene beads as described in Example 38. Immobilized rMuGMCSF beads, like soluble rMuGNCSF, 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. These experiments were repeated using immobilized rNuGMCSF to determine if immobilized rMuGMCSF is active in vi~o. BDF1 mice were injected with either soluble r~uGMCSF (20 units i.p.) or immobilized rMuGMCSF (50 units i.v.). Peripheral blood was drawn from the retro-orbital sinus of BDF1 mice at 0, 6, 12, 24, 48, 72, and 96 hours and the number of neutrophils (PM~)/ml was determined from a complete blood count. The results are shown in Figure 5 and indicate that immobilized rMuG~CSF is active in vivo. Furthermore, the results indicate that the beads stimulate PNN

WO 90~0g798 PCI`/US90/01031 production in num~ers (about a 2-fold increase) and rates (maximum i~ 12 hours with decline to initial levels within 24 to 48 hours) compara~le to soluble rMuGMCSF.

5~xample 53 Recombinant ~urine G~CSF irMuG~CSF~ Immobilized on O.93 ~m PolYstyrene Beads Stimulate~ GranuloPoie~is in Cvclopho~phamide-Treated BDFl Mice Recombinant murine GMCSF ~rMuG~CSF) immobilized on 0.93 ~m blue-dyed polystyrene beads stimulates granulopoiesis in cyclophosphamide-treated mice.
Recombinant murine G~CSF was lmmobilized on 0.93 ~m blue-dyed polystyrene beads as described in Example 3~.
Immobilized rMuGMCSF beads like soluble rMuGMCSF
1~ 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 their lymphocyte population was depleted by a single injection of cyclophosphamide. Repeated doses of GMCSF help these mice to recover lymphocyte numbers 2 to 3 days faster than untreated mice. Since immobilized rMuGNCSF shows in vivo activity (Example 52), cyclophosphamide-treated mice were given either soluble rMuGMCSF or immobilized rMuGMCSF to determine the effects rMuGMCSF on neutrophil counts. The experimental protocol was as follows. BDF1 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 in~ected i.p. e~ery 12 hours for 6 days; or 2 units injected i.v.
on days l, 3, and 5), or Lmmobilized rMuGMCSF ~2 units injecte~ 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 (PNN)/ml was determined from a complete blood csunt. The results are hown in ~igure 6 and indicate that immobilized rNuGNCSF is active in vivo. Furthermore, ,,: - , -: ~' :: :
:: :' ,, WO ~/0979R ;?0 ~7 ~3 0 PCT/US90/01031 rMuGMCSF beads stimulate PMN production in numbers and rates comparable to solu~le rMuGMCSF.

~xample 545 Covalently Linked rMuGMCSF Polystvrene Beads Retain Cytokine ActivitY While Adsorbed rMuGMCSF
Polystyrene Beads Do Not Retain Cytokine Activity Recombinant murine GMCSF (rNuGMCSF) covalently attached to 0.93 ym blue-dyed polystyrene beads retain biological activity (i.e., promote the growth of DAl-E5 cells) while rNuGMCSF adsorbed to 0.93 ym blue-dyed polystyrene beads do not retain biological activity (i.e., DAl-E5 cells do not grow). Covalent and adsor~ed 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.
~Al-E5 cells an I~-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 rNuG~CSF and immobilized r~uGMCSF bead fractions (covalently bound or adsorbed). The rNuGMCSF
assay~is as follows. DAl-E5 cells (lx104) were incubated with either soluble rMuG~CSF or immobilized rMuGMCSF
(covalent or adsorbed) f or 48 hours as descri~ed in Example 21. Either MT~r or 1 ~Ci of [3H]-thymidine was added. The mixture was incubated for an additional 4 hours. Cells were harvested as described in Ex,~mple 2l.
When the polystyrene beads were washed with sodium dodecyl sulfate (SDS), the adsor~ed r~uGMCSF was remo~ed (Figure 7). These beads no longer retained any bi~logical activity. Covalently linked rMuGMCSF, however, did not wash off with SDS. These beads retained biological activity. The results are listed in Table 17.

,. . ' ::

' ~ ' WO9OJ~979~ pcrtus9otolo31 ~xample 55 Recombinant Human Insulin-Like Growth Factor-I
~rHulLÆF-I~ Immobilized ~n 9.63 ym Blue-dYed Polystyrene Beads Stimulate~ a Crude Lymphocytic Preparation to Proliferate in Serum-Free Medium Recombinant human ILGF-I was immobilized on 9.63 ~m blue-dyed polystyrene beads as described in Example 40. Immobilized rHuILGF-I beads were washed as described in Example 21. Schimpff et al., Acta Endocrinolo~ica, 102, 21 (1983) disclose that ILGF-I in conjunction with lectin co-stimulation can induce lymphocytes to grow in serum-free medium. Recombinant human ILGF-I activity was assayed as follows: 1 x 105 lymphocytes were added to individual wells of 96-well flat-bottomed tissue culture plates containing 100 ~1 of RPMI-1640 tissue culture medium, 5 ~g/ml PHA, O.25% low endotoxin BSA and either sol~ble rHuILGF-I or immo~ilized rHulLGF-I beads. The mixture was incubated for 48 hours at 37C. Next ~ Ci/well of [3H]-thymidine was added and the mixture was incubated for another 18 hours. The cells were harvested as described in Example 21. The results are summarized in Table 18. They indicate that immobilized rHuILGF-1 on polystyrene beads induce lymphocyte growth in a PHA
co-stimulation assay in serum-free medium.
ExamPle_56 Re~ombinant Human Insulin-Like Growth Factor II
(rHuILGF-II) I ,~ ,obilized on 9.63 ~m Blue-Dyed PolYstyrene Beads Stimulates a Crude L~mPho~Le Pre~aration to Proliferate in Serum-Free ~dium Recombinant human ILGF-II was immobilized on 9.63 ~m blue-dyed polystyrene beads as described in E~ample 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.

WO90~0979~ PCT/US90/01031 70~'7'~J3~
~7 ~xample 57 Immobilized Recombinant Human Tumor Necrosis Factor (TNF-alpha) Rills Murine LN Cells Recombinant tumor necrosis factor alpha (TNF-alpha) immobilized on 9.64 ~m blue-dyed polystyrene beads kill murine LM cells in a 72 hour killing assay.
Recom~inant TN~-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 asæayed using murine LM cells (American Type Culture Collection).
The assay was as follows. Aliquots of either soluble TNF-alpha or immobilized TNF-alpha were added to indi~idual wells in a 96-well flat-bottomed tissue culture plate followed by the addi~ion of 1 x 104 ~M cells. ~he mixtures were incubated for 72 hours. Killing was then assayed ~y either the addition of 1 ~Ci of [3H]-thymidine to each well or MTT and the mixture incubated an additional 4 hours.
The thymidine-labeled cells were collected as descri~ed in Example 21. The MTT-labeled cells were fixed in isopropyl alcohol and the amount of MTT uptake measured by reading the a~sorbance at 590 nm. The results are reported in Table 20 and demonstrate that immobilized TNF-alpha inhibits both thymidine uptake, MTT uptake, and oxidation, which indicates cell death.

Example 58 Fibroblast Growth Factor Basic (FGFb~ Immobilized on 2.85 ~m PolvstYrene Beads Induces Gr~wth of rine 3T3 Cell~ in Growth Factor Depleted Medium Immobilized FGFb stimulates 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 (DNE) medium WO ~/09798 PCT/US90/01031 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 S plates) in DME medium plus 10% CS. 3~3 cells were incubated over night at 37C. The next morning, the wells were washed three times, resuspended in DME medium containing 0.4% CS, and incu~ated an additional 24 hours to deplete the cells and medium of growth factors. After 24 hours in DNE medium containing 0.4~ CS, either soluble FGFb, immobilized FGFb, or 10% CS was added to individual wells and the 3T3 cells incubated an additional 24 to 48 hours. The cells were then labeled with l ~Ci/well of [3H]-thymidine and incubated for an additional 16 hours.
lS ~he results are displayed in Table 21. They indicate that immobilized FGFb beads stimulated 3~3 cell growth to levels comparable to soluble FGFb.

~xample 59 20Transforminq Growth Factor-beta-2 (TGF-beta-2) Immobilized on 2.85 ~m Pol~stYrene Beads Induces Growth of NRK-4gF Cells in Growth Factor De~leted Medium Immobilized TGF-beta-2 stimulates the growth of NRK-49F cells in growth factor depleted medium.
Immobilized ~GF-beta-2 beads prepared according to the method in Example 44 were washed three times by suspension and centrifugation as described in Example 21. NRX-49F
cells (American Type Culture Collection~ were grown in DME
medium with antibiotics and 10% calf serum (CS) as described by As~oin et al., J. ~iol. Chem., 258, 7155 (1973), incorporated herein by reference. NRK-49F cells were isolated by trypsinization and plated at a co~centration of 5 x 103 cells/well (96-well plates) in DNE
plus 10% CS medium. ~he cells were incubated over night at 37C in 5% CO2, and then washed twice in DME medium containing 0.2% CS. The medium was replaced with lO0 ~l DME plus 0.2% CS and the cells were incubated as above for ,:

WO'~/0979R PCT~US90/01031 20~7730 three to four days to deplete the medium of growth factors. Whe~ the NRK-49F cells had reached about 75%
confluency, soluble TGF-beta-2a, immobilized TGF-beta-2a, or lO~ CS was added to individual wells and the NR~-49F
cells were incubated an additional 17 hours. Then l ~Ci of [3H]-thymidine was added to the wells and the cells were incubated for another 4 hours before harvesting as described in Example 2l. The results are listed in Table 22. 'rhey indicate that immobilized ~GF-beta-2 beads stimulate NRK-49F cells to grow in growth factor depleted medium.

Example 60 Tmmobilized Recombinant Human Interferon-alpha-2a ills the Interferon Sensitive HeLa S3 Cell Line Immobilized recom~inant H~man Interferon-alpha-2A kills the interferon sensitive HeLa S3 cell line.
Recombinant human Interferon-alpha-2a (INF-alpha-2a) immobilized on 2.85 ym blue-dyed polystyrene beads inhibits [3H]-thymidine uptake in a human epitheloid carcinoma cell line HeLa S3 (i.e.r kills HeLa S3).
Recombinant INF-alpha-2a was immobilized on 2.85 ~m blue-dyed 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 [3H]-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 indi~idual wells in a 96-well flat-bottomed tissue culture plate followed by the addition of l x 104 HeLa S3 cells. The beads with the IN~-alpha-2a or soluble INF-alpha-2a ~ere incubated for either 48, or 72 hours, at which time l yCi of [3H]--thymidine was added to each well and the mixture incl~batedan additional 4 hours. The cells were collected as described in Example 2l. The results are reported in , ", ' ' ', ' ,... ...
, 20~ 3~ 70 Table 23 and demonstrate that immobilized INF-alpha-2a inhibits thymidine uptake which leads to the death of the HeLa S3 tumor cells, S~xample 6l ' Recombinant Human ~pidermal Growth Factor Immobilized on 0.93 ~m Polvstvrene Beads Induces NRR-49F Cells to Grow in the Absence of Serum Recombinant human epidermal growth factor (rHuEGF) immobilized on 0.93 ym blue-dyed polystyrene ~eads induces NRK-49F cells to grow in the absence of serum. Recombinant human EGF was immobilized on 0.93 ym 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 ~itro. The assay procedure for the NRK-49F cells was as follows.
NRK~49F cells were maintained in DMEM (Dulbecco's Modified Eagles ~edium, Whittakar M.A. Bioproducts) medium plus 10%
calf serum (CS). NRX-49F cells are plated at 5 x 1~3 cells per well in 96-well flat-bottomed tissue culture plates and incubated for 24 hours in the 10% CS. ~he 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 ~eads with the rHuEGF or soluble rHuEGF were incubated for 24 or 48 hours. Growth was then measured by the `
addition of l ~Ci of ~3H~-thymidine to each well and the mixture was incubated an additional 6 hours. ~he cells were collected as described in Example 21. The results are reported in Table 24 and demonstrate that immobilized rHuEGF will induce murine NRX-49F cells to grow.

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Wo90/0979X PCT/US90/01031 73~

Example 62 ~ecombinant Platelet-Derived Growth Factor Immobilized on 2.85 ym Pol~stYIene Beads Induces ~urine 3T3 Cell~
to Grow in the Absence of Serum 5Recombinant platelet-derived growth factor (rHuPDGF) immobilized on 2.85 ~m blue-dyed polystyrene beads induce murine 3T3 cells to grow in the absence of serum. Recom~inant human PDGF was immohilized on 2.87 ym blue-dyed polystyrene beads as described in Example 47.
The immobilized rHuPDGF beads were washed three times as described in Experiment 2l. Serum contains many growth factors that are required by cells to grow in vitro. Most cells will not grow if they are ~epleted of these growth factors. Murine Swiss 3T3 is such a cell line, which is lS 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 l x 104 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 serum-free DMEN. 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 yCi of [3H]-thymidine to each well and the mixture was incubated an additional 6 hours.
The cells were collected as described in Example 2l. The results are reported in Table 25 and demonstrate that immobilized rHuPDGF will induce murine 3T3 cells to grow.

~xample 63 Growth of DAl-~5 Cells on Recombinant Human ErYthropoietin I~mobilized on Co-Bind~ Polystyrene Plates Recombinant human erythropoietin (rHuEPO) immobilized on Co-Bind~ polystyrene plates induces growth :

.

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2 ~ 47 ~ 3 0 72 of EPO/IL-3 dependent DAl-E5 cells (see Example 54).
Recombinant human E~O was immobilized on C~-Bind~
polystyrene plates as described in Example 48. Wells containing Immobilized rHuEPO were washed five times with lX PBS, followed by washing five times with Iscove's ME~
containing lO~ heat-inactivated serum, then filled with 0.050 ml of IMDM with 10% serum. DAl-E5 cell growth was assayed as follows. l x 104 cells were added to wells containing immobilized rHuEPO or soluble rHuEPO. ~he cells were incubated for 48 hours before either NTT or l ~Ci [3H3-thymidine was added to each well and the mixture was then incubated an additional 4 hours. The results are reported in Table 26 and demonstrate that immobilized rHuEPO will induce growth in EPO/IL-3 dependent DAl-E5 cells.

Example 64 Attachment of Recombinant Human Gamma-Interferon to Co-~ind~ Well StriPs Recombinant human qamma-interferon (rHuIFN-gamma) was obtained from Genzyme, Boston, MA, as a liquid formulation that contained 1 x 106 U/ml (2.5 x 107 U/mg).
An aliquot (O.02 mls, 2 x 104 U) of this solution was diluted to 2.0 ml with PBS to give a stoc~ solution that was l x 104 U/ml. Four wells of the 8-well strip were then filled as shown below.
rHuIFN-~amma PBS, mls Well mlsunits -O.1 1000 0.1 B 0.05 500 0.15 C O . 01100 0 . 19 .
D 0.00~ 50 0.195 After filling the wells, the strips were covered and incubated at 37C for 3 hours, then processed exactly as described in Example 48. After washing thoroughly with PBS, the wells were filled with PBS, the strips were WO ~/09798 PCT/US90/0~031 204~3~

covered and kept at 4C until used.

Example 65 Bioloqical Activity of Recombinant Human Gamma-Interferon Human peripheral blood monocytes were isolated from blood drawn into a heparini7ed syringe and isolated by gradient centrifugation on 46~ Percoll (Pharmacia, NewarX, NJ). The monocytes were isolated from the interface, washed three times in phosphate buffered saline and resuspended in ~PMI-1640 media containing 5~ human AB
sera to a concentration of 1 x 106 cells per ml. Co-bind~
strips containing 4 wells qamma-interferon immobilized as in Example 67 were washed three times with phosphate buffered saline, washed three times with RP~ 1640 media containing 2% Fungi-Bact, and wiped with a sterile gauze.
To each well was added 1 x 105 mon~cytes in a volume of 0.1 ml. Soluble qamma-interferon was added to wells which did not contain the immobilized ~amma-interferon. The cultures were incubated for 24 hours after which 0.1 ml of -20 the media was removed and assayed for tumor necrosis factor production using commercially available Elisa kits. ~he results shown in Table 27 demonstrate that immobilized qamma-interferon is biologically active.
The invention has been described with reference to various specific and preferred embodiments and techniques. However, it should be understood that many variations and modifications may be made while remaining within the spirit and scope of the invention.

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Claims (30)

WHAT IS CLAIMED IS:

1. An immobilized cytokine comprising a cytokine bound to a solid support, said immobilized cytokine having substantially the biological activity demonstrated by the free cytokine, and wherein said immobilized cytokine is reusable.

2. The immobilized cytokine of claim 1 wherein said cytokine is covalently bound to said solid support.

3. The immobilized cytokine of claim 2 wherein said cytokine is covalently bound to said solid support using a urethane, triazine ether, amine, or amide linkage.

4. The immobilized cytokine of claim 3 wherein said cytokine is covalently bound to said solid support using an amine or amide linkage.

5. The immobilized cytokine of claim 4 further including a linking arm wherein said cytokine is bound to said solid support by said linking arm and said linking arm comprises one or more linking groups selected from the group consisting of:
(a) diamines, having the general formula NH2-R1-NH2, where R1 is a C2-C20 alkyl group;
(b) amino acids, having the general formula NH2-R2-CO2H, where R2 is a C1-C20 alkyl group;
and (c) dialdehydes, having the general formula OHC-R3-CHO, where R3 is a C1-C20 alkyl group.

6. The immobilized cytokine of claim 5 wherein said linking arm comprises one or more linking groups selectedfrom the group consisting of 6-aminocaproic acid, 1,6-diaminohexane, 1,12-diaminododecane, glutaraldehyde, and mixtures thereof.

7. The immobilized cytokine of claim 1 wherein said cytokine is selected from the group consisting of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, tumor necrosis factor, gamma-interferon, alpha-interferon, beta-interferon, erythropoietin, granulocyte colony stimulating factor, murine granulocyte colony stimulating factor, granulocyte-macrophage colony stimulating factor, murine granulocyte-macrophage colony stimulating factor, insulin--like growth factor I, insulin-like growth factor II, transformation growth factor beta, epidermoid growth factor, platelet derived growth factor, and fibroblast growth factor basic.

8. The immobilized cytokine of claim 7 wherein said cytokine is selected from the group consisting of IL-2, GMCSF, GCSF, EPO, TNF, FGFb, TGFb, PDGF.

9. The immobilized cytokine of claim 8 wherein said cytokine is a polyethylene glycol-modified IL-2 or an ala-125 IL-2 analogue.

10. The immobilized cytokine of claim 1 wherein said solid support is nonporous.

11. The immobilized cytokine of claim 10 wherein said solid support is a substantially spherical bead having a diameter of about 0.5-500 µm.

12. The immobilized cytokine of claim 11 wherein said spherical bead has a diameter of about 1-75 µm.

13. The immobilized cytokine of claim 12 wherein said solid support is a staple fiber having a diameter of about 5-200 µm.

14. The immobilized cytokine of claim 10 wherein said support is selected from the group consisting of inorganic supports, including glass, quartz, ceramics, zeolites, metals, and metal oxides; polymeric materials including homopolymers, copolymers, or oligopolymers derived from monomeric units selected from the group consisting of styrene, divinylbenzene, ethylene, butadiene, acrylonitrile, acrylic acid, methacrylic acid, esters of acrylic and methacrylic acid, vinyl acetate, fluoro-alkenes, acrylamide and methacrylamide; carbohydrate supports, including agarose, cross-linked agarose, dextran, inulin, hyaluronic acid, cellulose, cellulose derivatives, starch and starch derivatives; and insoluble protein materials, including gelatin, collagen and albumin.

15. The immobilized cytokine of claim 14 wherein said support comprises a homopolymer, copolymer, or oligopolymer derived from monomeric units selected from the group consisting of styrene, divinylbenzene, ethylene, butadiene, acrylonitrile, acrylic acid, methacrylic acid, esters of acrylic and methacrylic acid, vinyl acetate, fluoroalkenes, acrylamide and methacrylamide.

16. The immobilized cytokine of claim 10 wherein said support includes a functionalized surface having a plurality of functional groups selected from the group consisting of hydroxyl, amino, carboxyl, sulfhydryl, and halogen.

17. A method for the in vitro growth of a cytokine dependent cell line comprising inducing growth of said cell line by contacting it with an effective amount of a cytokine bound to a solid support.

18. The method of claim 17 wherein said dependent cell line is CTTL-2 and said cytokine is IL-2.

19. The method of claim 17 wherein said dependent cell line is AML-193 and said cytokine is selected from the group consisting of HuGMCSF, HuGCSF, and IL-3.

20. The method of claim 17 wherein said dependent cell line is Balb/c 3T3 and said cytokine is PDGF or FGF-beta.

21. The method of claim 17 wherein said dependent cell line is NRK-49F and said cytokine is TFG-beta or EGF.

22. The method of claim 17 wherein dependent cell line is DA1-E5 and said cytokine is erythropoietin.

23. A method for the in vitro growth of cellular blood components comprising inducing growth of said components by contacting said components with an effective amount of a cytokine bound to a solid support.

24. The method of claim 23 wherein said cellular blood components are human peripheral blood lymphocytes.

25. A method for the in vitro growth of effector cells selected from the group consisting of lymphokine activated killer cells, natural killer cells, tumor infiltrating lymphocytes, and cytotoxic T-cells comprising inducing growth of said cells by contacting said cells with an effective amount of a cytokine bound to a solid support.

26. A method for the in vivo stimulation of the natural killer or lymphokine activated killer cells in the immune system of a host comprising injecting an effective amount of a cytokine bound to a solid support.

27. A method for the in vivo stimulation of hematopoietic cell growth of a host comprising injecting an effective amount of a cytokine bound to a solid support.

28. A method of claim 27 wherein said hematopoietic cells are granulocyte macrophages and said cytokine is GMCSF.

29. A method for the stabilization of a cytokine and substantially reducing proteolytic degregation in vivo, comprising attaching said cytokine to a solid support prior to introduction into the host.

30. A method for preventing the systemic absorption of cytokines, and the toxicity caused by the absorption of cytokines, comprising attaching said cytokine to a solid support, prior to introduction into the host.